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Sample records for human immortalized keratinocytes

  1. Development of a full-thickness human gingiva equivalent constructed from immortalized keratinocytes and fibroblasts

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    J.K. Buskermolen; C.M.A. Reijnders; S.W. Spiekstra; T. Steinberg; C.J. Kleverlaan; A.J. Feilzer; A.D. Bakker; S. Gibbs

    2016-01-01

    Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines

  2. Immortalization of human foreskin keratinocytes by various human papillomavirus DNAs corresponds to their association with cervical carcinoma

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    Woodworth, C.D.; Doniger, J.; DiPaolo, J.A.

    1989-01-01

    Normal human foreskin keratinocytes cotransfected with the neomycin resistance gene and recombinant human papillomavirus (HPV) DNAs (types 16, 18, 31, and 33) that have a high or moderate association with cervical malignancy acquired immortality and contained integrated and transcriptionally active viral genomes. Only transcripts from the intact E6 and E7 genes were detected in at least one cell line, suggesting that one or both of these genes are responsible for immortalization. Recombinant HPV DNAs with low or no oncogenic potential for cervical cancer (HPV1a, -5, -6b, and -11) induced small G418-resistant colonies that senesced as did the nontransfected cells. These colonies contained only episomal virus DNA; therefore, integration of HPV sequences is important for immortalization of keratinocytes. This study suggests that the virus-encoded immortalization function contributes to the pathogenesis of cervical carcinoma.

  3. Development of a Full-Thickness Human Gingiva Equivalent Constructed from Immortalized Keratinocytes and Fibroblasts.

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    Buskermolen, Jeroen K; Reijnders, Christianne M A; Spiekstra, Sander W; Steinberg, Thorsten; Kleverlaan, Cornelis J; Feilzer, Albert J; Bakker, Astrid D; Gibbs, Susan

    2016-08-01

    Organotypic models make it possible to investigate the unique properties of oral mucosa in vitro. For gingiva, the use of human primary keratinocytes (KC) and fibroblasts (Fib) is limited due to the availability and size of donor biopsies. The use of physiologically relevant immortalized cell lines would solve these problems. The aim of this study was to develop fully differentiated human gingiva equivalents (GE) constructed entirely from cell lines, to compare them with the primary cell counterpart (Prim), and to test relevance in an in vitro wound healing assay. Reconstructed gingiva epithelium on a gingiva fibroblast-populated collagen hydrogel was constructed from cell lines (keratinocytes: TERT or HPV immortalized; fibroblasts: TERT immortalized) and compared to GE-Prim and native gingiva. GE were characterized by immunohistochemical staining for proliferation (Ki67), epithelial differentiation (K10, K13), and basement membrane (collagen type IV and laminin 5). To test functionality of GE-TERT, full-thickness wounds were introduced. Reepithelialization, fibroblast repopulation of hydrogel, metabolic activity (MTT assay), and (pro-)inflammatory cytokine release (enzyme-linked immunosorbent assay) were assessed during wound closure over 7 days. Significant differences in basal KC cytokine secretion (IL-1α, IL-18, and CXCL8) were only observed between KC-Prim and KC-HPV. When Fib-Prim and Fib-TERT were stimulated with TNF-α, no differences were observed regarding cytokine secretion (IL-6, CXCL8, and CCL2). GE-TERT histology, keratin, and basement membrane protein expression very closely represented native gingiva and GE-Prim. In contrast, the epithelium of GE made with HPV-immortalized KC was disorganized, showing suprabasal proliferating cells, limited keratinocyte differentiation, and the absence of basement membrane proteins. When a wound was introduced into the more physiologically relevant GE-TERT model, an immediate inflammatory response (IL-6, CCL2, and

  4. Human papillomavirus type 59 immortalized keratinocytes express late viral proteins and infectious virus after calcium stimulation.

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    Lehr, Elizabeth E; Qadadri, Brahim; Brown, Calla R; Brown, Darron R

    2003-09-30

    Human papillomavirus type 59 (HPV 59) is an oncogenic type related to HPV 18. HPV 59 was recently propagated in the athymic mouse xenograft system. A continuous keratinocyte cell line infected with HPV 59 was created from a foreskin xenograft grown in an athymic mouse. Cells were cultured beyond passage 50. The cells were highly pleomorphic, containing numerous abnormally shaped nuclei and mitotic figures. HPV 59 sequences were detected in the cells by DNA in situ hybridization in a diffuse nuclear distribution. Southern blots were consistent with an episomal state of HPV 59 DNA at approximately 50 copies per cell. Analysis of the cells using a PCR/reverse blot strip assay, which amplifies a portion of the L1 open reading frame, was strongly positive. Differentiation of cells in monolayers was induced by growth in F medium containing 2 mM calcium chloride for 10 days. Cells were harvested as a single tissue-like sheet, and histologic analysis revealed a four-to-six cell-thick layer. Transcripts encoding involucrin, a cornified envelope protein, and the E1/E4 and E1/E4/L1 viral transcripts were detected after several days of growth in F medium containing 2 mM calcium chloride. The E1/E4 and L1 proteins were detected by immunohistochemical analysis, and virus particles were seen in electron micrographs in a subset of differentiated cells. An extract of differentiated cells was prepared by vigorous sonication and was used to infect foreskin fragments. These fragments were implanted into athymic mice. HPV 59 was detected in the foreskin xenografts removed 4 months later by DNA in situ hybridization and PCR/reverse blot assay. Thus, the complete viral growth cycle, including production on infectious virus, was demonstrated in the HPV 59 immortalized cells grown in a simple culture system.

  5. PCB153 reduces telomerase activity and telomere length in immortalized human skin keratinocytes (HaCaT) but not in human foreskin keratinocytes (NFK)

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    Senthilkumar, P.K. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Robertson, L.W. [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA (United States); Ludewig, G., E-mail: Gabriele-ludewig@uiowa.edu [Interdisciplinary Graduate Program in Human Toxicology, The University of Iowa, Iowa City, IA (United States); Department of Occupational and Environmental Health, The University of Iowa, Iowa City, IA (United States)

    2012-02-15

    Polychlorinated biphenyls (PCBs), ubiquitous environmental pollutants, are characterized by long term-persistence in the environment, bioaccumulation, and biomagnification in the food chain. Exposure to PCBs may cause various diseases, affecting many cellular processes. Deregulation of the telomerase and the telomere complex leads to several biological disorders. We investigated the hypothesis that PCB153 modulates telomerase activity, telomeres and reactive oxygen species resulting in the deregulation of cell growth. Exponentially growing immortal human skin keratinocytes (HaCaT) and normal human foreskin keratinocytes (NFK) were incubated with PCB153 for 48 and 24 days, respectively, and telomerase activity, telomere length, superoxide level, cell growth, and cell cycle distribution were determined. In HaCaT cells exposure to PCB153 significantly reduced telomerase activity, telomere length, cell growth and increased intracellular superoxide levels from day 6 to day 48, suggesting that superoxide may be one of the factors regulating telomerase activity, telomere length and cell growth compared to untreated control cells. Results with NFK cells showed no shortening of telomere length but reduced cell growth and increased superoxide levels in PCB153-treated cells compared to untreated controls. As expected, basal levels of telomerase activity were almost undetectable, which made a quantitative comparison of treated and control groups impossible. The significant down regulation of telomerase activity and reduction of telomere length by PCB153 in HaCaT cells suggest that any cell type with significant telomerase activity, like stem cells, may be at risk of premature telomere shortening with potential adverse health effects for the affected organism. -- Highlights: ► Human immortal (HaCaT) and primary (NFK) keratinocytes were exposed to PCB153. ► PCB153 significantly reduced telomerase activity and telomere length in HaCaT. ► No effect on telomere length and

  6. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer

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    Chen, Yi; Pirisi, Lucia [Department of Pathology, Microbiology and Immunology, School of Medicine, University of South Carolina, Columbia, SC (United States); Creek, Kim E., E-mail: creekk@sccp.sc.edu [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC (United States)

    2013-09-15

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer. - Highlights: • Ski oncoprotein levels increase during progression of HPV16-transformed cells. • Ski and phospho-Ski protein levels are cell cycle dependent. • Ski knock-down in HPV16-transformed keratinocytes inhibited cell proliferation. • Cervical cancer samples overexpress Ski.

  7. HPV16 E7 protein and hTERT proteins defective for telomere maintenance cooperate to immortalize human keratinocytes.

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    Jonathan Miller

    Full Text Available Previous studies have shown that wild-type human telomerase reverse transcriptase (hTERT protein can functionally replace the human papillomavirus type 16 (HPV-16 E6 protein, which cooperates with the viral E7 protein in the immortalization of primary keratinocytes. In the current study, we made the surprising finding that catalytically inactive hTERT (hTERT-D868A, elongation-defective hTERT (hTERT-HA, and telomere recruitment-defective hTERT (hTERT N+T also cooperate with E7 in mediating bypass of the senescence blockade and effecting cell immortalization. This suggests that hTERT has activities independent of its telomere maintenance functions that mediate transit across this restriction point. Since hTERT has been shown to have a role in gene activation, we performed microarray studies and discovered that E6, hTERT and mutant hTERT proteins altered the expression of highly overlapping sets of cellular genes. Most important, the E6 and hTERT proteins induced mRNA and protein levels of Bmi1, the core subunit of the Polycomb Group (PcG complex 1. We show further that Bmi1 substitutes for E6 or hTERT in cell immortalization. Finally, tissue array studies demonstrated that expression of Bmi1 increased with the severity of cervical dysplasia, suggesting a potential role in the progression of cervical cancer. Together, these data demonstrate that hTERT has extra-telomeric activities that facilitate cell immortalization and that its induction of Bmi1 is one potential mechanism for mediating this activity.

  8. Ski protein levels increase during in vitro progression of HPV16-immortalized human keratinocytes and in cervical cancer.

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    Chen, Yi; Pirisi, Lucia; Creek, Kim E

    2013-09-01

    We compared the levels of the Ski oncoprotein, an inhibitor of transforming growth factor-beta (TGF-β) signaling, in normal human keratinocytes (HKc), HPV16 immortalized HKc (HKc/HPV16), and differentiation resistant HKc/HPV16 (HKc/DR) in the absence and presence of TGF-β. Steady-state Ski protein levels increased in HKc/HPV16 and even further in HKc/DR, compared to HKc. TGF-β treatment of HKc, HKc/HPV16, and HKc/DR dramatically decreased Ski. TGF-β-induced Ski degradation was delayed in HKc/DR. Ski and phospho-Ski protein levels are cell cycle dependent with maximal Ski expression and localization to centrosomes and mitotic spindles during G2/M. ShRNA knock down of Ski in HKc/DR inhibited cell proliferation. More intense nuclear and cytoplasmic Ski staining and altered Ski localization were found in cervical cancer samples compared to adjacent normal tissue in a cervical cancer tissue array. Overall, these studies demonstrate altered Ski protein levels, degradation and localization in HPV16-transformed human keratinocytes and in cervical cancer.

  9. Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes.

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    Merkley, Mark A; Hildebrandt, Ellen; Podolsky, Robert H; Arnouk, Hilal; Ferris, Daron G; Dynan, William S; Stöppler, Hubert

    2009-08-23

    Infection with high-risk type human papilloma viruses (HPVs) is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE). The median within-group coefficient of variation was 19-21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23%) of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2%) were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1); and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27). This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

  10. Large-scale analysis of protein expression changes in human keratinocytes immortalized by human papilloma virus type 16 E6 and E7 oncogenes

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    Arnouk Hilal

    2009-08-01

    Full Text Available Abstract Background Infection with high-risk type human papilloma viruses (HPVs is associated with cervical carcinomas and with a subset of head and neck squamous cell carcinomas. Viral E6 and E7 oncogenes cooperate to achieve cell immortalization by a mechanism that is not yet fully understood. Here, human keratinocytes were immortalized by long-term expression of HPV type 16 E6 or E7 oncoproteins, or both. Proteomic profiling was used to compare expression levels for 741 discrete protein features. Results Six replicate measurements were performed for each group using two-dimensional difference gel electrophoresis (2D-DIGE. The median within-group coefficient of variation was 19–21%. Significance of between-group differences was tested based on Significance Analysis of Microarray and fold change. Expression of 170 (23% of the protein features changed significantly in immortalized cells compared to primary keratinocytes. Most of these changes were qualitatively similar in cells immortalized by E6, E7, or E6/7 expression, indicating convergence on a common phenotype, but fifteen proteins (~2% were outliers in this regulatory pattern. Ten demonstrated opposite regulation in E6- and E7-expressing cells, including the cell cycle regulator p16INK4a; the carbohydrate binding protein Galectin-7; two differentially migrating forms of the intermediate filament protein Cytokeratin-7; HSPA1A (Hsp70-1; and five unidentified proteins. Five others had a pattern of expression that suggested cooperativity between the co-expressed oncoproteins. Two of these were identified as forms of the small heat shock protein HSPB1 (Hsp27. Conclusion This large-scale analysis provides a framework for understanding the cooperation between E6 and E7 oncoproteins in HPV-driven carcinogenesis.

  11. A method for the immortalization of newborn mouse skin keratinocytes

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    Brianna O Hammiller

    2015-07-01

    Full Text Available Isolation and culture of mouse primary epidermal keratinocytes is a common technique that allows for easy genetic and environmental manipulation. However, due to their limited lifespan in culture, experiments utilizing primary keratinocytes require large numbers of animals, and are time consuming and expensive. To avoid these issues, we developed a method for the immortalization of primary mouse epidermal keratinocytes. Upon isolation of newborn epidermal keratinocytes according to established methods, the cells were cultured long-term in keratinocyte growth factor-containing medium. The cells senesced within a few weeks and eventually, small, slowly growing colonies emerged. After they regained confluency, the cells were passaged and slowly refilled the dish. With several rounds of subculture, the cells adapted to culture conditions, were easily subcultured, maintained normal morphology, and were apparently immortal. The immortalized cells retained the ability to differentiate with increased calcium concentrations, and were maintained to high passage numbers, while maintaining a relatively stable karyotype. Analysis of multiple immortalized cell lines as well as primary keratinocyte cultures, revealed increased numbers of chromosomes, especially in the primary keratinocytes, and chromosomal aberrations in most of the immortalized cultures and in the primary keratinocytes. Orthotopic grafting of immortalized keratinocytes together with fibroblasts onto nude mouse hosts produced skin while v-rasHa infection of the immortalized keratinocytes prior to grafting produced squamous cell carcinoma. In summary, this method of cell line generation allows for decreased use of animals, reduces the expense and time involved in research, and provides a useful model for cutaneous keratinocyte experimentation.

  12. Development of a full-thickness human skin equivalent in vitro model derived from TERT-immortalized keratinocytes and fibroblasts

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    C.M.A. Reijnders; A. van Lier; S. Roffel; D. Kramer; R.J. Scheper; S. Gibbs

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve th

  13. Development of a Full-Thickness Human Skin Equivalent In Vitro Model Derived from TERT-Immortalized Keratinocytes and Fibroblasts

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    Reijnders, Christianne M. A.; van Lier, Amanda; Roffel, Sanne; Kramer, Duco; Scheper, Rik J.; Gibbs, Susan

    2015-01-01

    Currently, human skin equivalents (HSEs) used for in vitro assays (e.g., for wound healing) make use of primary human skin cells. Limitations of primary keratinocytes and fibroblasts include availability of donor skin and donor variation. The use of physiologically relevant cell lines could solve th

  14. Role of gp91phox Homolog Nox1 in Induction of Premalignant Spindle Phenotypes of HPV 16 E6/E7—Immortalized Human Keratinocytes

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    Walee Chamulitrat

    2010-01-01

    Full Text Available The NADPH oxidase (Nox family of superoxide- and hydrogen peroxide—producing proteins has been recognized as important for signal transduction that regulates receptor-mediated functions, including cytoskeleton remodeling, cell proliferation, migration, differentiation, and cell death. Nox1 was the first of the Nox catalytic subunits to be cloned and shown to induce tumorigenic conversion of mouse fibroblasts. While Nox1 has been shown to be expressed in human colon and prostate cancers, however, limited studies have demonstrated the involvement of Nox1 in an early step of cell transformation. The aim of this review is to provide an overview on the role of Nox1 in cancer, as well as the contribution of our studies to demonstrate the involvement of Nox1 on neoplastic progression of human keratinocytes beyond the immortalization step. The generation of spindle phenotypes concomitant with anchorage-independent growth and invasiveness will be highlighted and discussed in relation to the possible role of Nox1 in epithelial-mesenchymal transition. Understanding these mechanisms may provide insight into Nox1 and redox signaling components as potential therapeutic targets to inhibit tumor progression.

  15. A Polyphenol-Enriched Fraction of Rose Oil Distillation Wastewater Inhibits Cell Proliferation, Migration and TNF-α-Induced VEGF Secretion in Human Immortalized Keratinocytes.

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    Wedler, Jonas; Rusanov, Krasimir; Atanassov, Ivan; Butterweck, Veronika

    2016-07-01

    Water steam distillation of rose flowers separates the essential oil from the polyphenol-containing rose oil distillation wastewater. Recently, a strategy was developed to separate rose oil distillation wastewater into a polyphenol depleted water fraction and a polyphenol-enriched fraction [RF20-(SP-207)]. The objective of the present study was to investigate RF20-(SP-207) and fraction F(IV), augmented in quercetin and ellagic acid, for possible antiproliferative effects in immortalized human keratinocytes (HaCaT) since rose petals are known to contain compounds with potential antiproliferative activity.RF20-(SP-207) revealed dose-dependent antiproliferative activity (IC50 of 9.78 µg/mL). In a nontoxic concentration of 10 µg/mL, this effect was stronger than that of the two positive controls LY294002 (10 µM, PI3 K-inhibitor, 30 % inhibition) and NVP-BEZ235 (100 nM, dual PI3 K/mTOR inhibitor, 30 % inhibition) and clearly exceeded the antiproliferative action of quercetin (50 µM, 25 % inhibition) and ellagic acid (1 µM, 15 % inhibition). Time-lapse microscopy detected a significant impairment of cell migration of RF20-(SP-207) and F(IV). At concentrations of 10 µg/mL of both, extract and fraction, cell migration was strongly suppressed (51 % and 28 % gap closure, respectively, compared to 95 % gap closure 24 hours after control treatment). The suppression of cell migration was comparable to the positive controls LY294002, NVP-BEZ235, and quercetin. Furthermore, basal and TNF-α-stimulated VEGF-secretion was significantly reduced by RF20-(SP-207) and F(IV) at 10 µg/mL (44 % vs. untreated control).In conclusion, RF20-(SP-207) showed promising antiproliferative and antimigratory effects and could be developed as a supportive, therapy against hyperproliferation-involved skin diseases.

  16. H-ras expression in immortalized keratinocytes produces an invasive epithelium in cultured skin equivalents.

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    Melville B Vaughan

    Full Text Available BACKGROUND: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in vitro skin equivalent. METHODOLOGY/PRINCIPAL FINDINGS: Previously described cdk4 and hTERT immortalized foreskin keratinocytes were engineered to express ectopically introduced H-ras. Skin equivalents, composed of normal fibroblast-contracted collagen gels overlaid with keratinocytes (immortal or immortal expressing H-ras, were prepared and incubated for 3 weeks. Harvested tissues were processed and sectioned for histology and antibody staining. Antigens specific to differentiation (involucrin, keratin-14, p63, basement-membrane formation (collagen IV, laminin-5, and epithelial to mesenchymal transition (EMT; e-cadherin, vimentin were studied. Results showed that H-ras keratinocytes produced an invasive, disorganized epithelium most apparent in the lower strata while immortalized keratinocytes fully stratified without invasive properties. The superficial strata retained morphologically normal characteristics. Vimentin and p63 co-localization increased with H-ras overexpression, similar to basal wound-healing keratinocytes. In contrast, the cdk4 and hTERT immortalized keratinocytes differentiated similarly to normal unimmortalized keratinocytes. CONCLUSIONS/SIGNIFICANCE: The use of isogenic derivatives of stable immortalized keratinocytes with specified genetic alterations may be helpful in developing more robust in vitro models of cancer progression.

  17. Citric acid induces cell-cycle arrest and apoptosis of human immortalized keratinocyte cell line (HaCaT) via caspase- and mitochondrial-dependent signaling pathways.

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    Ying, Tsung-Ho; Chen, Chia-Wei; Hsiao, Yu-Ping; Hung, Sung-Jen; Chung, Jing-Gung; Yang, Jen-Hung

    2013-10-01

    Citric acid is an alpha-hydroxyacid (AHA) widely used in cosmetic dermatology and skincare products. However, there is concern regarding its safety for the skin. In this study, we investigated the cytotoxic effects of citric acid on the human keratinocyte cell line HaCaT. HaCaT cells were treated with citric acid at 2.5-12.5 mM for different time periods. Cell-cycle arrest and apoptosis were investigated by 4,6-diamidino-2-phenylindole dihydrochloride (DAPI) staining, flow cytometry, western blot and confocal microscopy. Citric acid not only inhibited proliferation of HaCaT cells in a dose-dependent manner, but also induced apoptosis and cell cycle-arrest at the G2/M phase (before 24 h) and S phase (after 24 h). Citric acid increased the level of Bcl-2-associated X protein (BAX) and reduced the levels of B-cell lymphoma-2 (BCL-2), B-cell lymphoma-extra large (BCL-XL) and activated caspase-9 and caspase-3, which subsequently induced apoptosis via caspase-dependent and caspase-independent pathways. Citric acid also activated death receptors and increased the levels of caspase-8, activated BH3 interacting-domain death agonist (BID) protein, Apoptosis-inducing factor (AIF), and Endonuclease G (EndoG). Therefore, citric acid induces apoptosis through the mitochondrial pathway in the human keratinocyte cell line HaCaT. The study results suggest that citric acid is cytotoxic to HaCaT cells via induction of apoptosis and cell-cycle arrest in vitro.

  18. Antioxidant and Anti-Inflammatory Effects of Selected Natural Compounds Contained in a Dietary Supplement on Two Human Immortalized Keratinocyte Lines

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    Elena Fasano

    2014-01-01

    Full Text Available Several advantages may derive from the use of dietary supplements containing multiple natural antioxidants and/or anti-inflammatory agents. At present, however, there is scarce information on the properties and potential of combined supplements. To fill the gap, the antioxidant and anti-inflammatory activities exerted by a combination of seven natural components (coenzyme Q10, krill oil, lipoic acid, resveratrol, grape seed oil, α-tocopherol, and selenium contained in a dietary supplement used for the prevention of skin disorders were investigated in vitro. Each component was administered, alone or in combination, to human keratinocytes, and the inhibition of Reactive Oxygen Species production and lipid peroxidation as well as the ability to reduce inflammatory cytokine secretion and to modulate Nuclear Factor-κB pathway was evaluated. The combination exhibited high antioxidant activity and in specific conditions the combination’s efficiency was higher than that of the most powerful components administered individually. Moreover, the combination showed remarkable anti-inflammatory properties. It reduced more efficiently than each component the secretion of Monocyte Chemoattractant Protein-1, a crucial cytokine for the development of chronic inflammation in skin, and inhibited Nuclear Factor-κB molecular pathway. Overall, our findings suggest that the combined formulation may have the potential to powerfully inhibit oxidative stress and inflammation at skin level.

  19. Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes.

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    Härle-Bachor, C; Boukamp, P

    1996-06-25

    Cellular senescence is defined by the limited proliferative capacity of normal cultured cells. Immortal cells overcome this regulation and proliferate indefinitively. One step in the immortalization process may be reactivation of telomerase activity, a ribonucleoprotein complex, which, by de novo synthesized telomeric TTAGGG repeats, can prevent shortening of the telomeres. Here we show that immortal human skin keratinocytes, irrespective of whether they were immortalized by simian virus 40, human papillomavirus 16, or spontaneously, as well as cell lines established from human skin squamous cell carcinomas exhibit telomerase activity. Unexpectedly, four of nine samples of intact human skin also were telomerase positive. By dissecting the skin we could show that the dermis and cultured dermal fibroblasts were telomerase negative. The epidermis and cultured skin keratinocytes, however, reproducibly exhibited enzyme activity. By separating different cell layers of the epidermis this telomerase activity could be assigned to the proliferative basal cells. Thus, in addition to hematopoietic cells, the epidermis, another example of a permanently regenerating human tissue, provides a further exception of the hypothesis that all normal human somatic tissues are telomerase deficient. Instead, these data suggest that in addition to contributing to the permanent proliferation capacity of immortal and tumor-derived keratinocytes, telomerase activity may also play a similar role in the lifetime regenerative capacity of normal epidermis in vivo.

  20. Comparative Cytotoxicity of Glycyrrhiza glabra Roots from Different Geographical Origins Against Immortal Human Keratinocyte (HaCaT), Lung Adenocarcinoma (A549) and Liver Carcinoma (HepG2) Cells.

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    Basar, Norazah; Oridupa, Olayinka Ayotunde; Ritchie, Kenneth J; Nahar, Lutfun; Osman, Nashwa Mostafa M; Stafford, Angela; Kushiev, Habibjon; Kan, Asuman; Sarker, Satyajit D

    2015-06-01

    Glycyrrhiza glabra L. (Fabaceae), commonly known as 'liquorice', is a well-known medicinal plant. Roots of this plant have long been used as a sweetening and flavouring agent in food and pharmaceutical products, and also as a traditional remedy for cough, upper and lower respiratory ailments, kidney stones, hepatitis C, skin disorder, cardiovascular diseases, diabetes, gastrointestinal ulcers and stomach ache. Previous pharmacological and clinical studies have revealed its antitussive, antiinflammatory, antiviral, antimicrobial, antioxidant, immunomodulatory, hepatoprotective and cardioprotective properties. While glycyrrhizin, a sweet-tasting triterpene saponin, is the principal bioactive compound, several bioactive flavonoids and isoflavonoids are also present in the roots of this plant. In the present study, the cytotoxicity of the methanol extracts of nine samples of the roots of G. glabra, collected from various geographical origins, was assessed against immortal human keratinocyte (HaCaT), lung adenocarcinoma (A549) and liver carcinoma (HepG2) cell lines using the in vitro 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazoliumbromide cell toxicity/viability assay. Considerable variations in levels of cytotoxicity were observed among various samples of G. glabra.

  1. Micronucleus formation in human keratinocytes is dependent on radiation quality and tissue architecture.

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    Snijders, Antoine M; Mannion, Brandon J; Leung, Stanley G; Moon, Sol C; Kronenberg, Amy; Wiese, Claudia

    2015-01-01

    The cytokinesis-block micronucleus (MN) assay was used to assess the genotoxicity of low doses of different types of space radiation. Normal human primary keratinocytes and immortalized keratinocytes grown in 2D monolayers each were exposed to graded doses of 0.3 or 1.0 GeV/n silicon ions or similar energies of iron ions. The frequencies of induced MN were determined and compared to γ-ray data. RBE(max) values ranged from 1.6 to 3.9 for primary keratinocytes and from 2.4 to 6.3 for immortalized keratinocytes. At low radiation doses ≤ 0.4 Gy, 0.3 GeV/n iron ions were the most effective at inducing MN in normal keratinocytes. An "over-kill effect" was observed for 0.3 GeV/n iron ions at higher doses, wherein 1.0 GeV/n iron ions were most efficient in inducing MN. In immortalized keratinocytes, 0.3 GeV/n iron ions produced MN with greater frequency than 1.0 GeV/n iron ions, except at the highest dose tested. MN formation was higher in immortalized keratinocytes than in normal keratinocytes for all doses and radiation qualities investigated. MN induction was also assessed in human keratinocytes cultured in 3D to simulate the complex architecture of human skin. RBE values for MN formation in 3D were reduced for normal keratinocytes exposed to iron ions, but were elevated for immortalized keratinocytes. Overall, MN induction was significantly lower in keratinocytes cultured in 3D than in 2D. Together, the results suggest that tissue architecture and immortalization status modulate the genotoxic response to space radiation, perhaps via alterations in DNA repair fidelity. © 2014 Wiley Periodicals, Inc.

  2. Characterization of global transcription profile of normal and HPV-immortalized keratinocytes and their response to TNF treatment

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    Colo Anna

    2008-06-01

    Full Text Available Abstract Background Persistent infection by high risk HPV types (e.g. HPV-16, -18, -31, and -45 is the main risk factor for development of cervical intraepithelial neoplasia and cervical cancer. Tumor necrosis factor (TNF is a key mediator of epithelial cell inflammatory response and exerts a potent cytostatic effect on normal or HPV16, but not on HPV18 immortalized keratinocytes. Moreover, several cervical carcinoma-derived cell lines are resistant to TNF anti-proliferative effect suggesting that the acquisition of TNF-resistance may constitute an important step in HPV-mediated carcinogenesis. In the present study, we compared the gene expression profiles of normal and HPV16 or 18 immortalized human keratinocytes before and after treatment with TNF for 3 or 60 hours. Methods In this study, we determined the transcriptional changes 3 and 60 hours after TNF treatment of normal, HPV16 and HPV18 immortalized keratinocytes by microarray analysis. The expression pattern of two genes observed by microarray was confirmed by Northern Blot. NF-κB activation was also determined by electrophoretic mobility shift assay (EMSA using specific oligonucleotides and nuclear protein extracts. Results We observed the differential expression of a common set of genes in two TNF-sensitive cell lines that differs from those modulated in TNF-resistant ones. This information was used to define genes whose differential expression could be associated with the differential response to TNF, such as: KLK7 (kallikrein 7, SOD2 (superoxide dismutase 2, 100P (S100 calcium binding protein P, PI3 (protease inhibitor 3, skin-derived, CSTA (cystatin A, RARRES1 (retinoic acid receptor responder 1, and LXN (latexin. The differential expression of the KLK7 and SOD2 transcripts was confirmed by Northern blot. Moreover, we observed that SOD2 expression correlates with the differential NF-κB activation exhibited by TNF-sensitive and TNF-resistant cells. Conclusion This is the first in

  3. Altered chromosome 6 in immortal human fibroblasts.

    Science.gov (United States)

    Hubbard-Smith, K; Patsalis, P; Pardinas, J R; Jha, K K; Henderson, A S; Ozer, H L

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene.

  4. Altered chromosome 6 in immortal human fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Hubbard-Smith, K.; Pardinas, J.R.; Jha, K.K.; Ozer, H.L. (New Jersey Medical School, Newark, NJ (United States)); Patsalis, P.; Henderson, A.S. (City Univ. of New York, NY (United States))

    1992-05-01

    Human diploid fibroblasts have a limited life span in vitro, and spontaneous immortalization is an extremely rare event. We have used transformation of human diploid fibroblasts by an origin-defective simian virus 40 genome to develop series of genetically matched immortal cell lines to analyze immortalization. Comparison of a preimmortal transformant (SVtsA/HF-A) with its uncloned and cloned immortalized derivatives (AR5 and HAL) has failed to reveal any major alteration involving the simian virus 40 genome. Karyotypic analysis, however, demonstrated that all of the immortal cell lines in this series have alterations of chromosome 6 involving loss of the portion distal to 6q21. The karyotypic analysis was corroborated by DNA analyses. Southern analysis demonstrated that only one copy of three proto-oncogene loci (ros1, c-myb, and mas1) on 6q was retained in immortal cells. Polymerase chain reaction analysis of the microsatellite polymorphism at 6q22 (D6S87) showed loss of heterozygosity. In addition, elevated expression of c-myb (6q22-23) was observed. We hypothesize that the region at and/or distal to 6q21 plays a role in immortalization, consistent with the presence of a growth suppressor gene. 66 refs., 6 figs., 2 tabs.

  5. Maintenance of telomeres in SV40-transformed pre-immortal and immortal human fibroblasts.

    Science.gov (United States)

    Small, M B; Hubbard, K; Pardinas, J R; Marcus, A M; Dhanaraj, S N; Sethi, K A

    1996-09-01

    Shortening of telomeres has been hypothesized to contribute to cellular senescence and may play a role in carcinogenesis of human cells. Furthermore, activation of telomerase has frequently been demonstrated in tumor-derived and in vitro immortalized cells. In this study, we have assessed these phenomena during the life span of simian virus 40 (SV40)-transformed preimmortal and immortal human fibroblasts. We observed progressive reduction in telomere length in preimmortal transformed cells with extended proliferative capacity, with the most dramatic shortening at late passage. Telomere lengths became stabilized (or increased) in immortal fibroblasts accompanied, in one case, by the activation of telomerase. However, an independent immortal cell line that displayed stable telomeres did not have detectable telomerase activity. Furthermore, we found significant telomerase activity in two preimmortal derivatives. Our results provide further evidence for maintenance of telomeres in immortalized human fibroblasts, but they suggest a lack of causal relationship between telomerase activation and immortalization.

  6. Human keratinocytes are vanilloid resistant.

    Directory of Open Access Journals (Sweden)

    László Pecze

    Full Text Available BACKGROUND: Use of capsaicin or resiniferatoxin (RTX as analgesics is an attractive therapeutic option. RTX opens the cation channel inflammatory pain/vanilloid receptor type 1 (TRPV1 permanently and selectively removes nociceptive neurons by Ca(2+-cytotoxicity. Paradoxically, not only nociceptors, but non-neuronal cells, including keratinocytes express full length TRPV1 mRNA, while patient dogs and experimental animals that underwent topical treatment or anatomically targeted molecular surgery have shown neither obvious behavioral, nor pathological side effects. METHODS: To address this paradox, we assessed the vanilloid sensitivity of the HaCaT human keratinocyte cell line and primary keratinocytes from skin biopsies. RESULTS: Although both cell types express TRPV1 mRNA, neither responded to vanilloids with Ca(2+-cytotoxicity. Only ectopic overproduction of TRPV1 rendered HaCaT cells sensitive to low doses (1-50 nM of vanilloids. The TRPV1-mediated and non-receptor specific Ca(2+-cytotoxicity ([RTX]>15 microM could clearly be distinguished, thus keratinocytes were indeed resistant to vanilloid-induced, TRPV1-mediated Ca(2+-entry. Having a wider therapeutic window than capsaicin, RTX was effective in subnanomolar range, but even micromolar concentrations could not kill human keratinocytes. Keratinocytes showed orders of magnitudes lower TRPV1 mRNA level than sensory ganglions, the bona fide therapeutic targets in human pain management. In addition to TRPV1, TRPV1b, a dominant negative splice variant was also noted in keratinocytes. CONCLUSION: TRPV1B expression, together with low TRPV1 expression, may explain the vanilloid paradox: even genuinely TRPV1 mRNA positive cells can be spared with therapeutic (up to micromolar doses of RTX. This additional safety information might be useful for planning future human clinical trials.

  7. Specific chromosomal imbalances in human papillomavirus-transfected cells during progression toward immortality

    Science.gov (United States)

    Solinas-Toldo, Sabina; Dürst, Matthias; Lichter, Peter

    1997-01-01

    High risk human papillomaviruses (HPVs) known to be closely associated with cervical cancer, such as HPV16 and HPV18, have the potential to immortalize human epithelial cells in culture. Four lines of HPV-transfected keratinocytes were analyzed by comparative genomic hybridization at different time points after transfection. A number of chromosomal imbalances was found to be highly characteristic for the cultures progressing toward immortality. Whereas several of these were new and previously not found as recurrent aberrations in cervical tumors, some were identical to chromosomal changes observed during cervical carcinogenesis. The data put new emphasis on the studied cell system as a relevant model for HPV-induced pathogenesis. PMID:9108068

  8. Traceless Targeting and Isolation of Gene-Edited Immortalized Keratinocytes from Epidermolysis Bullosa Simplex Patients

    Directory of Open Access Journals (Sweden)

    Magomet Aushev

    2017-09-01

    Full Text Available Epidermolysis bullosa simplex (EBS is a blistering skin disease caused by dominant-negative mutations in either KRT5 or KRT14, resulting in impairment of keratin filament structure and epidermal fragility. Currently, nearly 200 mutations distributed across the entire length of these genes are known to cause EBS. Genome editing using programmable nucleases enables the development of ex vivo gene therapies for dominant-negative genetic diseases. A clinically feasible strategy involves the disruption of the mutant allele while leaving the wild-type allele unaffected. Our aim was to develop a traceless approach to efficiently disrupt KRT5 alleles using TALENs displaying unbiased monoallelic disruption events and devise a strategy that allows for subsequent screening and isolation of correctly modified keratinocyte clones without the need for selection markers. Here we report on TALENs that efficiently disrupt the KRT5 locus in immortalized patient-derived EBS keratinocytes. Inactivation of the mutant allele using a TALEN working at sub-optimal levels resulted in restoration of intermediate filament architecture. This approach can be used for the functional inactivation of any mutant keratin allele regardless of the position of the mutation within the gene and is furthermore applicable to the treatment of other inherited skin disorders.

  9. Isolation, cultivation and transfection of human keratinocytes.

    Science.gov (United States)

    Zare, Sona; Zarei, Mohammad Ali; Ghadimi, Tayyeb; Fathi, Fardin; Jalili, Ali; Hakhamaneshi, Mohammad Saeed

    2014-04-01

    Human keratinocytes could be used in the repair of damaged skin, in tissue engineering applications, gene therapy and recently, the generation of iPS cells. We isolated human keratinocytes from foreskin and subsequently cultured them on fibronectin, collagen type I, gelatin and laminin-coated dishes that contained three different types of serum-free medium (epilife, KSM or CnT). We developed improved conditions for efficient transfection of these human keratinocytes by testing three common transfection methods and a GFP plasmid vector. The isolated cells showed typical keratinocyte morphology and expressed the epithelial cell specific antigen, cytokeratin 14. Collagen type 1, epilife medium and lipofectamin 2000 gave the best results for isolation and transfection of human keratinocytes. Our protocol can be used as a reproducible, simple and efficient method for isolation, cultivation and genetic manipulation of human keratinocytes, which may be useful in cell and gene therapy applications.

  10. Radiosensitivity of cultured human and mouse keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Parkinson, E.K.; Hume, W.J.; Potten, C.S.

    1986-10-01

    Clonogenic survival assays after ..gamma..-radiation in vitro were performed on freshly isolated and subcultured keratinocytes from mouse skin, mouse tongue and human skin. Survival curves were constructed by fitting the data to a multi-target model of cell survival. When subcultured, keratinocytes from all sites produced survival curves which showed a reduced shoulder region and an increased D/sub 0/ when compared with their freshly isolated counterparts. Freshly isolated human skin keratinocytes were more radiosensitive than mouse keratinocytes from either skin or tongue.

  11. Differential regulation of iron chelator-induced IL-8 synthesis via MAP kinase and NF-κB in immortalized and malignant oral keratinocytes

    Directory of Open Access Journals (Sweden)

    Lee Suk-Keun

    2007-09-01

    Full Text Available Abstract Background Interleukin-8 (IL-8 is a cytokine that plays an important role in tumor progression in a variety of cancer types; however, its regulation is not well understood in oral cancer cells. In the present study, we examined the expression and mechanism of IL-8 in which it is involved by treating immortalized (IHOK and malignant human oral keratinocytes (HN12 cells with deferoxamine (DFO. Methods IL-8 production was measured by an enzyme-linked immunoabsorbent assay and reverse transcriptase-polymerase chain reaction (RT-PCR analysis. Electrophoretic mobility shift assays was used to determine NF-κB binding activity. Phosphorylation and degradation of the I-κB were analyized by Western blot. Results IHOK cells incubated with DFO showed increased expression of IL-8 mRNA, as well as higher release of the IL-8 protein. The up-regulation of DFO-induced IL-8 expression was higher in IHOK cells than in HN12 cells and was concentration-dependent. DFO acted additively with IL-1β to strongly up-regulate IL-8 in IHOK cells but not in HN12 cells. Accordingly, selective p38 and ERK1/2 inhibitors for both kinases abolished DFO-induced IL-8 expression in both IHOK and HN12 cells. Furthermore, DFO induced the degradation and phosphorylation of IκB, and activation of NF-κB. The IL-8 inducing effects of DFO were mediated by a nitric oxide donor (S-nitrosoglutathione, and by pyrrolidine dithiocarbamate, an inhibitor of NF-κB, as well as by wortmannin, which inhibits the phosphatidylinositol 3-kinase-dependent activation of NAD(PH oxidase. Conclusion This results demonstrate that DFO-induced IL-8 acts via multiple signaling pathways in immortalized and malignant oral keratinocytes, and that the control of IL-8 may be an important target for immunotheraphy against human oral premalignant lesions.

  12. SV40-mediated immortalization of human fibroblasts.

    Science.gov (United States)

    Ozer, H L; Banga, S S; Dasgupta, T; Houghton, J; Hubbard, K; Jha, K K; Kim, S H; Lenahan, M; Pang, Z; Pardinas, J R; Patsalis, P C

    1996-01-01

    We have identified a multistep mechanism by which the DNA virus SV40 overcomes cellular senescence. Expression of SV40 T antigen is required for both transient extension of life span and unlimited life span or immortalization. These effects are mediated through inactivation of function of growth suppressors pRB and p53 via complex formation with T antigen. However, immortalization additionally requires inactivation of a novel growth suppressor gene, which has recently been identified to be on the distal portion of the long arm of chromosome 6, designated SEN6. We propose that SEN6 is responsible for cellular senescence in fibroblasts and other cells.

  13. The Effect of Lipoaspirates on Human Keratinocytes.

    Science.gov (United States)

    Kim, Bong-Sung; Gaul, Charel; Paul, Nora E; Dewor, Manfred; Stromps, Jan-Philipp; Hwang, Soo Seok; Nourbakhsh, Mahtab; Bernhagen, Jürgen; Rennekampff, Hans-Oliver; Pallua, Norbert

    2016-09-01

    One increasingly important trend in plastic, reconstructive, and aesthetic surgery is the use of fat grafts to improve cutaneous wound healing. In clinical practice, lipoaspirates (adipose tissue harvested by liposuction) are re-injected in a procedure called lipofilling. Previous studies, however, mainly evaluated the regenerative effect of isolated adipocytes, adipose-derived stem cells, and excised en bloc adipose tissue on keratinocytes, whereas no study to date has examined the effect of lipoaspirates. The authors aimed to investigate differences in the regenerative property of en bloc adipose tissue and lipoaspirates on keratinocytes. Human keratinocytes, lipoaspirates, and en bloc adipose tissue from 36 healthy donors were isolated. In vitro proliferation, differentiation, migration, stratification, and wound healing of keratinocyte monolayers were measured. Furthermore, secreted levels of VEGF, bFGF, IGF-1, MMP-9, and MIF were detected by ELISA. Migration, proliferation, and wound healing of keratinocytes were increased by lipoaspirates. Interestingly, the effect of lipoaspirates on keratinocyte proliferation was significantly higher than by en bloc adipose tissue after 5 days. The differentiation of keratinocytes was equally attenuated by lipoaspirates and en bloc adipose tissue. Stratification of keratinocyte layers was enhanced by lipoaspirates and en bloc fat when compared to controls. Lipoaspirates secrete higher levels of bFGF, whereas higher levels of VEGF and IGF-1 are released by en bloc adipose tissue. We show that lipoaspirates and en bloc adipose tissue have a regenerative effect on keratinocytes. One reason for the higher effect of lipoaspirates on keratinocyte proliferation may be the secretion of different cytokines. © 2016 The American Society for Aesthetic Plastic Surgery, Inc. Reprints and permission: journals.permissions@oup.com.

  14. H-Ras Expression in Immortalized Keratinocytes Produces an Invasive Epithelium in Cultured Skin Equivalents

    OpenAIRE

    Vaughan, Melville B.; Ruben D Ramirez; Andrews, Capri M.; Woodring E Wright; Jerry W Shay

    2009-01-01

    BACKGROUND: Ras proteins affect both proliferation and expression of collagen-degrading enzymes, two important processes in cancer progression. Normal skin architecture is dependent both on the coordinated proliferation and stratification of keratinocytes, as well as the maintenance of a collagen-rich basement membrane. In the present studies we sought to determine whether expression of H-ras in skin keratinocytes would affect these parameters during the establishment and maintenance of an in...

  15. KGF-transfected cells can stimulate growth and proliferation of human cultured keratinocytes in vitro

    Institute of Scientific and Technical Information of China (English)

    1998-01-01

    Objective: To establish two stably KGF-transfected, immortalized cell lines. Methods: HaCaT-keratinocytes and KMST-6-fibroblasts were transfected by liposome mediated gene transfer. Transfection effectivity, gene integration and configuration of the transgenic protein were investigated by ELISA, DANN-PCR and β-Gal-staining. Results: Most effective GF producing clones were tested by a colorimetric XTT-test. Conclusion: This is a significant acceleration of cell proliferation and mitosis of human keratinocytes in an Air Liquid Interface (ALI) test system.

  16. H(+)/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport.

    Science.gov (United States)

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko; Akagawa, Mitsugu; Tsuji-Naito, Kentaro

    2016-07-08

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H(+) gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. Copyright © 2016 DHC corporation. Published by Elsevier Inc. All rights reserved.

  17. Karyotypic analysis of gene transformed human keratinocyte line

    Institute of Scientific and Technical Information of China (English)

    1999-01-01

    @@ INTRODUCTION In order to solve the difficult problem of long term in vitro culture of human keratinocytes, the technique of gene transfer was utilized to transform human keratinocytes with simian virus 40 (SV40).

  18. Genes involved in immortalization of human mammary cells

    Energy Technology Data Exchange (ETDEWEB)

    Stampfer, Martha R.; Yaswen, Paul

    2001-09-27

    Breast cancer progression is characterized by inappropriate cell growth. Normal cells cease growth after a limited number of cell divisions--a process called cellular senescence-while tumor cells may acquire the ability to proliferate indefinitely (immortality). Inappropriate expression of specific oncogenes in a key cellular signaling pathway (Ras, Raf) can promote tumorigenicity in immortal cells, while causing finite lifespan cells to undergo a rapid senescence-like arrest. We have studied when in the course of transformation of cultured human mammary epithelial cells (HMEC), the response to overexpressed oncogenic Raf changes from being tumor-suppressive to tumor enhancing, and what are the molecular underpinnings of this response. Our data indicate: (1) HMEC acquire the ability to maintain growth in the presence of oncogenic Raf not simply as a consequence of overcoming senescence, but as a result of a newly discovered step in the process of immortal transformation uncovered by our lab, termed conversion. Immortal cells that have not undergone conversion (e.g., cells immortalized by exogenous introduction of the immortalizing enzyme, telomerase) remain growth inhibited. (2) Finite lifespan HMEC growth arrest in response to oncogenic Raf using mediators of growth inhibition that are very different from those used in response to oncogenic Raf by rodent cells and certain other human cell types, including the connective tissue cells from the same breast tissue. While many diverse cell types appear to have in common a tumor-suppressive response to this oncogenic signal, they also have developed multiple mechanisms to elicit this response. Understanding how cancer cells acquire the crucial capacity to be immortal and to abrogate normal tumor-suppressive mechanisms may serve both to increase our understanding of breast cancer progression, and to provide new targets for therapeutic intervention. Our results indicate that normal HMEC have novel means of enforcing a Raf

  19. Staphylococcal superantigens stimulate immortalized human adipocytes to produce chemokines.

    Directory of Open Access Journals (Sweden)

    Bao G Vu

    Full Text Available BACKGROUND: Human adipocytes may have significant functions in wound healing and the development of diabetes through production of pro-inflammatory cytokines after stimulation by gram-negative bacterial endotoxin. Diabetic foot ulcers are most often associated with staphylococcal infections. Adipocyte responses in the area of the wound may play a role in persistence and pathology. We studied the effect of staphylococcal superantigens (SAgs on immortalized human adipocytes, alone and in the presence of bacterial endotoxin or staphylococcal α-toxin. METHODOLOGY/PRINCIPAL FINDINGS: Primary non-diabetic and diabetic human preadipocytes were immortalized by the reverse transcriptase component of telomerase (TERT and the E6/E7 genes of human papillomavirus. The immortal cells were demonstrated to have properties of non-immortalized pre-adipocytes and could be differentiated into mature and functional adipocytes. Differentiated adipocytes exposed to staphylococcal SAgs produced robust levels of cytokines IL-6 and IL-8, but there were no significant differences in levels between the non-diabetic and diabetic cells. Cytokine production was increased by co-incubation of adipocytes with SAgs and endotoxin together. In contrast, α-toxin alone was cytotoxic at high concentrations, but, at sub-cytotoxic doses, did not stimulate production of IL-6 and IL-8. CONCLUSIONS/SIGNIFICANCE: Endotoxin has been proposed to contribute to diabetes through enhanced insulin resistance after chronic exposure and stimulation of adipocytes to produce cytokines. Our data indicate staphylococcal SAgs TSST-1 and SEB alone and in combination with bacterial endotoxin also stimulate adipocytes to produce cytokines and thus may contribute to the inflammatory response found in chronic diabetic ulcers and in the systemic inflammation that is associated with the development and persistence of diabetes. The immortal human pre-adipocytes reported here will be useful for studies to

  20. Streptococci-human papilloma virus interaction with ethanol exposure leads to keratinocyte damage.

    Science.gov (United States)

    Schwartz, Joel; Pavlova, Sylvia; Kolokythas, Antonia; Lugakingira, Mulokozi; Tao, Lin; Miloro, Michael

    2012-08-01

    Ethanol, human papilloma virus (HPV), and poor oral hygiene are risk factors that have been attributed to oral carcinogenesis. Streptococci sp and HPV infections are common in the head and neck, often associated with sexual activity. Although HPV is linked to head and neck squamous cell carcinoma, it is unclear whether there is a similar role for Streptococci sp. This cell study examines whether Streptococci sp and HPV-16 with exposure to ethyl alcohol (ETOH) can act as cofactors in the malignant transformation of oral keratinocytes. ETOH (0.1%-20% vol/vol) was used to investigate Streptococci sp attachment with immortalized E6-expressing HPV/HOK-16B cells, human oral buccal keratinocytes, and foreskin keratinocytes. Streptococci sp (Streptococci mutans [LT11]) and various strains of acetaldehyde (AA) producer and nonproducer Streptococcus salivarius (110-1, 109-2, 101-7, and 107-1) and a lactic acid producer bacterium, Lactobacillus rhamnosus (24-1 and 25-2), were examined for interactions with keratinocytes by use of a green dye (percent of cells with colonies after 24 hours). Carcinogens, AA, malondialdehyde, DNA damage, and proliferation (5'-bromo-2-deoxyuridine) among keratinocytes were also quantified. AA and malondialdehyde production from permissible Streptococci sp significantly increased with attachment to keratinocytes, whereas L rhamnosus did not significantly attach to keratinocytes. This attachment was associated with enhanced levels of AA adduct formation, proliferation (5'-bromo-2-deoxyuridine incorporation), and enhanced migration through integrin-coated basement membrane by HPV oral keratinocytes, which are characteristics of a malignant phenotype. These cell studies suggest that oral Streptococci sp and HPV (HPV-16) cooperate to transform oral keratinocytes after low-level ETOH (1%) exposure. These results appear to suggest a significant clinical interaction, but further validation is warranted. Copyright © 2012 American Association of Oral and

  1. H{sup +}/peptide transporter (PEPT2) is expressed in human epidermal keratinocytes and is involved in skin oligopeptide transport

    Energy Technology Data Exchange (ETDEWEB)

    Kudo, Michiko; Katayoshi, Takeshi; Kobayashi-Nakamura, Kumiko [DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025 (Japan); Akagawa, Mitsugu [Department of Biological Chemistry, Division of Applied Life Science, Graduate School of Life and Environmental Sciences, Osaka Prefecture University, 1-1 Gakuen-cho, Naka-ku, Sakai 599-8531 (Japan); Tsuji-Naito, Kentaro, E-mail: knaito@dhc.co.jp [DHC Corporation Laboratories, Division 2, 2-42 Hamada, Mihama-ku, Chiba 261-0025 (Japan)

    2016-07-08

    Peptide transporter 2 (PEPT2) is a member of the proton-coupled oligopeptide transporter family, which mediates the cellular uptake of oligopeptides and peptide-like drugs. Although PEPT2 is expressed in many tissues, its expression in epidermal keratinocytes remains unclear. We investigated PEPT2 expression profile and functional activity in keratinocytes. We confirmed PEPT2 mRNA expression in three keratinocyte lines (normal human epidermal keratinocytes (NHEKs), immortalized keratinocytes, and malignant keratinocytes) by reverse transcription-polymerase chain reaction (RT-PCR) and quantitative real-time RT-PCR. In contrast to PEPT1, PEPT2 expression in the three keratinocytes was similar or higher than that in HepG2 cells, used as PEPT2-positive cells. Immunolocalization analysis using human skin showed epidermal PEPT2 localization. We studied keratinocyte transport function by measuring the oligopeptide content using liquid chromatography/tandem mass spectrometry. Glycylsarcosine uptake in NHEKs was pH-dependent, suggesting that keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. We also performed a skin-permeability test of several oligopeptides using skin substitute, suggesting that di- and tripeptides pass actively through the epidermis. In conclusion, PEPT2 is expressed in keratinocytes and involved in skin oligopeptide uptake. -- Highlights: •PEPT2 is expressed in keratinocytes, which are more common than other skin cells. •Immunolocalization analysis using human skin revealed epidermal PEPT2 localization. •Keratinocytes could absorb small peptides in the presence of an inward H{sup +} gradient. •Di- and tripeptide pass actively through the epidermis.

  2. Immortalization of human alveolar epithelial cells to investigate nanoparticle uptake.

    Science.gov (United States)

    Kemp, Sarah J; Thorley, Andrew J; Gorelik, Julia; Seckl, Michael J; O'Hare, Michael J; Arcaro, Alexandre; Korchev, Yuri; Goldstraw, Peter; Tetley, Teresa D

    2008-11-01

    Primary human alveolar type 2 (AT2) cells were immortalized by transduction with the catalytic subunit of telomerase and simian virus 40 large-tumor antigen. Characterization by immunochemical and morphologic methods demonstrated an AT1-like cell phenotype. Unlike primary AT2 cells, immortalized cells no longer expressed alkaline phosphatase, pro-surfactant protein C, and thyroid transcription factor-1, but expressed increased caveolin-1 and receptor for advanced glycation end products (RAGE). Live cell imaging using scanning ion conductance microscopy showed that the cuboidal primary AT2 cells were approximately 15 microm and enriched with surface microvilli, while the immortal AT1 cells were attenuated more than 40 microm, resembling these cells in situ. Transmission electron microscopy highlighted the attenuated morphology and showed endosomal vesicles in some immortal AT1 cells (but not primary AT2 cells) as found in situ. Particulate air pollution exacerbates cardiopulmonary disease. Interaction of ultrafine, nano-sized particles with the alveolar epithelium and/or translocation into the cardiovasculature may be a contributory factor. We hypothesized differential uptake of nanoparticles by AT1 and AT2 cells, depending on particle size and surface charge. Uptake of 50-nm and 1-microm fluorescent latex particles was investigated using confocal microscopy and scanning surface confocal microscopy of live cells. Fewer than 10% of primary AT2 cells internalized particles. In contrast, 75% immortal AT1 cells internalized negatively charged particles, while less than 55% of these cells internalized positively charged particles; charge, rather than size, mattered. The process was rapid: one-third of the total cell-associated negatively charged 50-nm particle fluorescence measured at 24 hours was internalized during the first hour. AT1 cells could be important in translocation of particles from the lung into the circulation.

  3. Immortalization of human articular chondrocytes and induction of their phenotype

    Institute of Scientific and Technical Information of China (English)

    何清义; 李起鸿; 杨柳; 许建中

    2003-01-01

    Objective To immortalize human articular chondrocytes (HACs) using gene transfection and to maintain stable phenotype of transformed HACs after induction.Methods HACs were transfected with the retroviral vector pLXSN encoding human papillomavirus 16E7 (HPV16E7), and the transformed clones were sorted and proliferated. Karyotype analysis, clone forming tests and nude mice tumor forming tests were applied to check the characteristics of the transformation. Type Ⅱ collagen of transformed chondrocytes was inducted with free serum medium (FSM) supplemented with nutridoma-sp and ascorbate. Results Immortalized HACs were isolated with fifty passages achieved. The HPV16E7 transformed cells were confirmed to be benign. Induction of FSM with nutridoma-sp and ascorbate promoted type Ⅱ collagen of transformed chondrocytes to the high levels of normal chondrocytes. Conclusion HACs transformed with HPV16E7 survive for long periods in vitro, and type Ⅱ collagen can maintain stability after induction.

  4. Immortalization of human normal and NF1 neurofibroma Schwann cells.

    Science.gov (United States)

    Li, Hua; Chang, Lung-Ji; Neubauer, Debbie R; Muir, David F; Wallace, Margaret R

    2016-10-01

    Neurofibromas, which are benign Schwann cell tumors, are the hallmark feature in the autosomal dominant condition neurofibromatosis 1 (NF1) and are associated with biallelic loss of NF1 gene function. There is a need for effective therapies for neurofibromas, particularly the larger, plexiform neurofibromas. Tissue culture is an important tool for research. However, it is difficult to derive enriched human Schwann cell cultures, and most enter replicative senescence after 6-10 passages, impeding cell-based research in NF1. Through exogenous expression of human telomerase reverse transcriptase and murine cyclin-dependent kinase (mCdk4), normal (NF1 wild-type), neurofibroma-derived Schwann cells heterozygous for NF1 mutation, and neurofibroma-derived Schwann cells homozygous for NF1 mutation were immortalized, including some matched samples from the same NF1 patient. Initial experiments employed retroviral vectors, while subsequent work utilized lentiviral vectors carrying these genes because of improved efficiency. Expression of both transgenes was required for immortalization. Molecular and immunohistochemical analysis indicated that these cell lines are of Schwann cell lineage and have a range of phenotypes, many of which are consistent with their primary cultures. This is the first report of immortalization and detailed characterization of multiple human NF1 normal nerve and neurofibroma-derived Schwann cell lines, which will be highly useful research tools to study NF1 and other Schwann tumor biology and conditions.

  5. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts.

    Science.gov (United States)

    Ramos-Jerz, Maria Del R; Villanueva, Socorro; Jerz, Gerold; Winterhalter, Peter; Deters, Alexandra M

    2013-01-01

    Methanolic avocado (Persea americana Mill., Lauraceae) seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK) and normal human dermal fibroblasts (NHDF). The methanol-water partition (M) from avocado seeds and HSCCC fraction 3 (M.3) were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

  6. Persea americana Mill. Seed: Fractionation, Characterization, and Effects on Human Keratinocytes and Fibroblasts

    Directory of Open Access Journals (Sweden)

    Maria del R. Ramos-Jerz

    2013-01-01

    Full Text Available Methanolic avocado (Persea americana Mill., Lauraceae seed extracts were separated by preparative HSCCC. Partition and HSCCC fractions were principally characterized by LC-ESI-MS/MS analysis. Their in vitro influence was investigated on proliferation, differentiation, cell viability, and gene expression on HaCaT and normal human epidermal keratinocytes (NHEK and normal human dermal fibroblasts (NHDF. The methanol-water partition (M from avocado seeds and HSCCC fraction 3 (M.3 were mostly composed of chlorogenic acid and its isomers. Both reduced NHDF but enhanced HaCaT keratinocytes proliferation. HSCCC fraction M.2 composed of quinic acid among chlorogenic acid and its isomers inhibited proliferation and directly induced differentiation of keratinocytes as observed on gene and protein level. Furthermore, M.2 increased NHDF proliferation via upregulation of growth factor receptors. Salidrosides and ABA derivatives present in HSCCC fraction M.6 increased NHDF and keratinocyte proliferation that resulted in differentiation. The residual solvent fraction M.7 contained among low concentrations of ABA derivatives high amounts of proanthocyanidins B1 and B2 as well as an A-type trimer and stimulated proliferation of normal cells and inhibited the proliferation of immortalized HaCaT keratinocytes.

  7. Integrin α3β1 signaling through MEK/ERK determines alternative polyadenylation of the MMP-9 mRNA transcript in immortalized mouse keratinocytes.

    Science.gov (United States)

    Missan, Dara S; Mitchell, Kara; Subbaram, Sita; DiPersio, C Michael

    2015-01-01

    Integrin α3β1 is highly expressed in both normal and tumorigenic epidermal keratinocytes where it regulates genes that control cellular function and extracellular matrix remodeling during normal and pathological tissue remodeling processes, including wound healing and development of squamous cell carcinoma (SCC). Previous studies identified a role for α3β1 in immortalized and transformed keratinocytes in the regulation of genes that promote tumorigenesis, invasion, and pro-angiogenic crosstalk to endothelial cells. One such gene, matrix metalloproteinase-9 (MMP-9), is induced by α3β1 through a post-transcriptional mechanism of enhanced mRNA stability. In the current study, we sought to investigate the mechanism through which α3β1 controls MMP-9 mRNA stability. First, we utilized a luciferase reporter assay to show that AU-rich elements (AREs) residing within the 3'-untranslated region (3'-UTR) of the MMP-9 mRNA renders the transcript unstable in a manner that is independent of α3β1. Next, we cloned a truncated variant of the MMP-9 mRNA which is generated through usage of an alternative, upstream polyadenylation signal and lacks the 3'-UTR region containing the destabilizing AREs. Using an RNase protection assay to distinguish "long" (full-length 3'-UTR) and "short" (truncated 3'-UTR) MMP-9 mRNA variants, we demonstrated that the shorter, more stable mRNA that lacks 3'-UTR AREs was preferentially generated in α3β1-expressing keratinocytes compared with α3β1-deficient (i.e., α3-null) keratinocytes. Moreover, we determined that α3β1-dependent alternative polyadenylation was acquired by immortalized keratinocytes, as primary neonatal keratinocytes did not display α3β1-dependent differences in the long and short transcripts. Finally, pharmacological inhibition of the extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) pathway in α3β1-expressing keratinocytes caused a shift towards long variant expression, while Raf-1

  8. Protective effect of N-acetyl cysteine against chemical hypoxia-induced injury to an immortal human skin keratinocyte line HaCaT%N-乙酰半胱氨酸对化学性缺氧引起HaCaT细胞损伤的保护作用

    Institute of Scientific and Technical Information of China (English)

    张美芬; 杨春涛; 杨战利; 孟金兰; 曾凡钦; 韩艳芳; 陈培熹; 冯鉴强

    2010-01-01

    Objective To estimate the influences of N-acetyl cysteine (NAC) on a chemical hypoxiamimetic agent CoCl2 induced-injury to, and expressions of inflammatory factors by, an immortal human skin keratinocyte line HaCaT. Methods HaCaT cells were treated with CoCl2 of 2000 μmol/L for 4 hours to set up a chemical hypoxia-induced cell model of skin injury. NAC of various concentrations ( 1000, 2000, 3000 μmol/L)was used to pretreat HaCaT cells for 2 hours prior to the establishment of cell model. After these treatments,cell viability was detected by cell counting kit 8 (CCK-8), the levels of interleukin 6 and 8 (IL-6 and -8) and tumor necrosis factor α (TNF-α) in culture supernatant by ELISA kits, mitochondrial membrane potential (MMP) by rhodamine 123 (Rh123) staining and photofluorography, intracellular reduced glutathione (GSH)content by glutathione detection kit. Results An obvious decline was observed in HaCaT cell viability after pretreatment with various concentrations of NAC for 2 hours. The treatment with CoCl2 of 2000 μmol/L for 4 hours induced an elevation in the supernatant levels of IL-6, IL-8 and TNF-α and a decrease in GSH content and MMP, while the pretreatment with NAC for 2 hours retarded the CoCl2-induced increase in IL-6 and IL-8 levels as well as decrease in GSH content and MMP. Conclusion The reactive oxygen species (ROS) scavenger NAC can protect against CoCl2-induced injury to and inflammatory reaction in HaCaT cells, which may be associated with a decrement in oxidative stress.%目的 探讨N-乙酰半胱氨酸(NAC)能否保护人皮肤永生化角质形成细胞(HaCaT)对抗化学性低氧模拟剂氯化钴(CoCl2)诱导的损伤及对炎症因子的影响.方法 用2000 μmol/L CoCl2处理HaCaT细胞,建立化学性低氧诱导皮肤细胞损伤的实验模型.应用CCK-8比色法检测细胞存活率;ELISA试剂盒检测细胞培养基中IL-6、IL-8和TNF-α的水平;罗丹明123(Rh123)染色/荣光显微镜照相术检测线粒体膜电

  9. Methicillin-resistant Staphylococcus aureus adaptation to human keratinocytes.

    Science.gov (United States)

    Soong, Grace; Paulino, Franklin; Wachtel, Sarah; Parker, Dane; Wickersham, Matthew; Zhang, Dongni; Brown, Armand; Lauren, Christine; Dowd, Margaret; West, Emily; Horst, Basil; Planet, Paul; Prince, Alice

    2015-04-21

    Skin is the most common site of Staphylococcus aureus infection. While most of these infections are self-limited, recurrent infections are common. Keratinocytes and recruited immune cells participate in skin defense against infection. We postulated that S. aureus is able to adapt to the milieu within human keratinocytes to avoid keratinocyte-mediated clearance. From a collection of S. aureus isolated from chronically infected patients with atopic dermatitis, we noted 22% had an agr mutant-like phenotype. Using several models of human skin infection, we demonstrate that toxin-deficient, agr mutants of methicillin-resistant S. aureus (MRSA) USA300 are able to persist within keratinocytes by stimulating autophagy and evading caspase-1 and inflammasome activation. MRSA infection induced keratinocyte autophagy, as evidenced by galectin-8 and LC3 accumulation. Autophagy promoted the degradation of inflammasome components and facilitated staphylococcal survival. The recovery of more than 58% agr or RNAIII mutants (P keratinocytes compared to control keratinocytes reflected the survival advantage for mutants no longer expressing agr-dependent toxins. Our results illustrate the dynamic interplay between S. aureus and keratinocytes that can result in the selection of mutants that have adapted specifically to evade keratinocyte-mediated clearance mechanisms. Human skin is a major site of staphylococcal infection, and keratinocytes actively participate in eradication of these pathogens. We demonstrate that methicillin-resistant Staphylococcus aureus (MRSA) is ingested by keratinocytes and activates caspase-1-mediated clearance through pyroptosis. Toxin-deficient MRSA mutants are selected within keratinocytes that fail to induce caspase-1 activity and keratinocyte-mediated clearance. These intracellular staphylococci induce autophagy that enhances their intracellular survival by diminishing inflammasome components. These findings suggest that S. aureus mutants, by exploiting

  10. Differentiation and apoptosis in human immortalized sebocytes.

    Science.gov (United States)

    Wróbel, Anna; Seltmann, Holger; Fimmel, Sabine; Müller-Decker, Karin; Tsukada, Miki; Bogdanoff, Birgit; Mandt, Nathalie; Blume-Peytavi, Ulrike; Orfanos, Constantin E; Zouboulis, Christos C

    2003-02-01

    Increased cell volume, accumulation of lipid droplets in the cytoplasm, and nuclear degeneration are phenomena indicating terminal differentiation of human sebocytes followed by holocrine secretion and cell death. The molecular pathways of natural and induced sebocyte elimination are still unknown, however. In this study, SZ95 sebocytes were found to exhibit DNA fragmentation after a 6 h culture followed by increased lactate dehydrogenase release after 24 h, indicating cell damage. With the help of morphologic studies and using Oil Red detection of cellular lipids, cell enlargement, accumulation of lipid droplets in the cytoplasm, and nuclear fragmentation could be observed under treatment with arachidonic acid. Staurosporine, a potent inhibitor of phospholipid Ca2+-dependent protein kinase, increased externalized phosphatidylserine levels on SZ95 sebocytes, detected by annexin V/propidium iodide flow cytometry, as early as after 1 h, whereas dose-dependent reduction of bcl-2 mRNA and protein expression, enhanced DNA fragmentation, and increased caspase 3 levels, detected by caspase 3 inhibitor/propidium iodide flow cytometry, were found after 6 h of treatment. SZ95 sebocyte death was detected as early as after 6 h of SZ95 sebocyte treatment with high staurosporine concentrations (10(-6)-10(-5) M). 5Alpha-dihydrotestosterone (10(-8)-10(-5) M) did not affect externalized phosphatidylserine levels and DNA fragmentation in SZ95 sebocytes but slightly decreased lactate dehydrogenase cell release. Neither acitretin nor 13-cis retinoic acid (10(-8)-10(-5) M) affected externalized phosphatidylserine levels, DNA fragmentation, and lactate dehydrogenase cell release, despite the increased caspase 3 levels under treatment with 13-cis retinoic acid. The combined staurosporine and 13-cis retinoic acid treatment enhanced DNA fragmentation in SZ95 sebocytes to the same magnitude as in cells only treated with staurosporine. In conclusion, SZ95 sebocytes in vitro undergo apoptosis

  11. Network signatures of cellular immortalization in human lymphoblastoid cell lines

    Energy Technology Data Exchange (ETDEWEB)

    Shim, Sung-Mi; Jung, So-Young; Nam, Hye-Young; Kim, Hye-Ryun; Lee, Mee-Hee; Kim, Jun-Woo; Han, Bok-Ghee [National Biobank of Korea, Center for Genome Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of); Jeon, Jae-Pil, E-mail: jaepiljeon@hanmail.net [Division of Brain Diseases, Center for Biomedical Science, Korea National Institute of Health, Osong 363-951 (Korea, Republic of)

    2013-11-15

    Highlights: •We identified network signatures of LCL immortalization from transcriptomic profiles. •More than 41% of DEGs are possibly regulated by miRNAs in LCLs. •MicroRNA target genes in LCLs are involved in apoptosis and immune-related functions. •This approach is useful to find functional miRNA targets in specific cell conditions. -- Abstract: Human lymphoblastoid cell line (LCL) has been used as an in vitro cell model in genetic and pharmacogenomic studies, as well as a good model for studying gene expression regulatory machinery using integrated genomic analyses. In this study, we aimed to identify biological networks of LCL immortalization from transcriptomic profiles of microRNAs and their target genes in LCLs. We first selected differentially expressed genes (DEGs) and microRNAs (DEmiRs) between early passage LCLs (eLCLs) and terminally differentiated late passage LCLs (tLCLs). The in silico and correlation analysis of these DEGs and DEmiRs revealed that 1098 DEG–DEmiR pairs were found to be positively (n = 591 pairs) or negatively (n = 507 pairs) correlated with each other. More than 41% of DEGs are possibly regulated by miRNAs in LCL immortalizations. The target DEGs of DEmiRs were enriched for cellular functions associated with apoptosis, immune response, cell death, JAK–STAT cascade and lymphocyte activation while non-miRNA target DEGs were over-represented for basic cell metabolisms. The target DEGs correlated negatively with miR-548a-3p and miR-219-5p were significantly associated with protein kinase cascade, and the lymphocyte proliferation and apoptosis, respectively. In addition, the miR-106a and miR-424 clusters located in the X chromosome were enriched in DEmiR–mRNA pairs for LCL immortalization. In this study, the integrated transcriptomic analysis of LCLs could identify functional networks of biologically active microRNAs and their target genes involved in LCL immortalization.

  12. Six1 overexpression at early stages of HPV16-mediated transformation of human keratinocytes promotes differentiation resistance and EMT

    Energy Technology Data Exchange (ETDEWEB)

    Xu, Hanwen [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 (United States); Pirisi, Lucia [Department of Pathology, Microbiology and Immunology, University of South Carolina School of Medicine, University of South Carolina, Columbia, SC 29208 (United States); Creek, Kim E., E-mail: creekk@sccp.sc.edu [Department of Drug Discovery and Biomedical Sciences, South Carolina College of Pharmacy, University of South Carolina, Columbia, SC 29208 (United States)

    2015-01-01

    Previous studies in our laboratory discovered that SIX1 mRNA expression increased during in vitro progression of HPV16-immortalized human keratinocytes (HKc/HPV16) toward a differentiation-resistant (HKc/DR) phenotype. In this study, we explored the role of Six1 at early stages of HPV16-mediated transformation by overexpressing Six1 in HKc/HPV16. We found that Six1 overexpression in HKc/HPV16 increased cell proliferation and promoted cell migration and invasion by inducing epithelial–mesenchymal transition (EMT). Moreover, the overexpression of Six1 in HKc/HPV16 resulted in resistance to serum and calcium-induced differentiation, which is the hallmark of the HKc/DR phenotype. Activation of MAPK in HKc/HPV16 overexpressing Six1 is linked to resistance to calcium-induced differentiation. In conclusion, this study determined that Six1 overexpression resulted in differentiation resistance and promoted EMT at early stages of HPV16-mediated transformation of human keratinocytes. - Highlights: • Six1 expression increases during HPV16-mediated transformation. • Six1 overexpression causes differentiation resistance in HPV16-immortalized cells. • Six1 overexpression in HPV16-immortalized keratinocytes activates MAPK. • Activation of MAPK promotes EMT and differentiation resistance. • Six1 overexpression reduces Smad-dependent TGF-β signaling.

  13. Differential gene expression in SV40-mediated immortalization of human fibroblasts.

    Science.gov (United States)

    Pardinas, J; Pang, Z; Houghton, J; Palejwala, V; Donnelly, R J; Hubbard, K; Small, M B; Ozer, H L

    1997-06-01

    Normal human diploid fibroblasts (HF) have a limited life span, undergo senescence, and rarely, if ever, spontaneously immortalize in culture. Introduction of the gene for T antigen encoded by the DNA virus SV40 extends the life span of HF and increases the frequency of immortalization; however, immortalization requires both T-dependent and T-independent functions. We previously generated independent SV40-transformed non-immortal (pre-immortal) HF cell lines from which we then obtained immortal sublines as part of a multifaceted approach to identify functions responsible for immortalization. In this study we undertook a search for cellular mRNAs which are differentially expressed upon immortalization. A lambda cDNA library was prepared from a pre-immortal SV40-transformed HF (HF-C). We screened the library with a subtracted probe enriched for sequences present in HF-C and reduced in immortal AR5 cells. A more limited screen was also employed for sequences overexpressed in AR5 using a different strategy. Alterations in the level of mRNAs in AR5 encoding functions relevant to signal transduction pathways were identified; however, most cDNAs encoded novel sequences. In an effort to clarify which of the altered mRNAs are most relevant to immortalization, we performed Northern analysis with RNA prepared from three paired sets of independent pre-immortal and immortal (4 cell lines) SV40-transformants using eight cloned cDNAs which show reduced expression in AR5. Three of these were reduced in additional immortal cell lines as well; one, J4-4 (unknown function) is reduced in all the immortal cell lines tested; a second, J4-3 (possible PP2C type phosphatase) is reduced in 2 of the 3 matched sets; and a third, J2-2 (unknown function) is reduced in 2 unrelated immortal cell lines. Although the roles of these genes are as yet unclear, their further analysis should extend our understanding of the molecular bases for immortalization. In particular, the patterns of expression of

  14. Motoneuron differentiation of immortalized human spinal cord cell lines.

    Science.gov (United States)

    Li, R; Thode, S; Zhou, J; Richard, N; Pardinas, J; Rao, M S; Sah, D W

    2000-02-01

    Human motoneuron cell lines will be valuable tools for spinal cord research and drug discovery. To create such cell lines, we immortalized NCAM(+)/neurofilament(+) precursors from human embryonic spinal cord with a tetracycline repressible v-myc oncogene. Clonal NCAM(+)/neurofilament(+) cell lines differentiated exclusively into neurons within 1 week. These neurons displayed extensive processes, exhibited immunoreactivity for mature neuron-specific markers such as tau and synaptophysin, and fired action potentials upon current injection. Moreover, a clonal precursor cell line gave rise to multiple types of spinal cord neurons, including ChAT(+)/Lhx3(+)/Lhx4(+) motoneurons and GABA(+) interneurons. These neuronal restricted precursor cell lines will expedite the elucidation of molecular mechanisms that regulate the differentiation, maturation and survival of specific subsets of spinal cord neurons, and the identification and validation of novel drug targets for motoneuron diseases and spinal cord injury.

  15. Vitamin D derivatives enhance cytotoxic effects of H2O2 or cisplatin on human keratinocytes.

    Science.gov (United States)

    Piotrowska, Anna; Wierzbicka, Justyna; Ślebioda, Tomasz; Woźniak, Michał; Tuckey, Robert C; Slominski, Andrzej T; Żmijewski, Michał A

    2016-06-01

    Although the skin production of vitamin D is initiated by ultraviolet radiation type B (UVB), the role vitamin D plays in antioxidative or pro-oxidative responses remains to be elucidated. We have used immortalized human HaCaT keratinocytes as a model of proliferating epidermal cells to test the influence of vitamin D on cellular response to H2O2 or the anti-cancer drug, cisplatin. Incubation of keratinocytes with 1,25(OH)2D3 or its low calcemic analogues, 20(OH)D3, 21(OH)pD or calcipotriol, sensitized cells to ROS resulting in more potent inhibition of keratinocyte proliferation by H2O2 in the presence of vitamin D compounds. These results were supported by cell cycle and apoptosis analyses, and measurement of the mitochondrial transmembrane potentials (MMP), however some unique properties of individual secosteroids were observed. Furthermore, in HaCaT keratinocytes treated with H2O2, 1,25(OH)2D3, 21(OH)pD and calcipotriol stimulated the expression of SOD1 and CAT genes, but not SOD2, indicating a possible role of mitochondria in ROS-modulated cell death. 1,25(OH)2D3 also showed a short-term, protective effect on HaCaT keratinocytes, as exemplified by the inhibition of apoptosis and the maintenance of MMP. However, with prolonged incubation with H2O2 or cisplatin, 1,25(OH)2D3 caused an acceleration in the death of the keratinocytes. Therefore, we propose that lead vitamin D derivatives can protect the epidermis against neoplastic transformation secondary to oxidative or UV-induced stress through activation of vitamin D-signaling. Furthermore, our data suggest that treatment with low calcemic vitamin D analogues or the maintenance of optimal level of vitamin D by proper supplementation, can enhance the anticancer efficacy of cisplatin. Copyright © 2016 Elsevier Inc. All rights reserved.

  16. Rho Kinase Inhibitor Y-27632 Prolongs the Life Span of Adult Human Keratinocytes, Enhances Skin Equivalent Development, and Facilitates Lentiviral Transduction

    Science.gov (United States)

    Rodijk-Olthuis, Diana; Jansen, Patrick A.M.; van Vlijmen-Willems, Ivonne M.J.J.; van Erp, Piet E.; Joosten, Irma; Zeeuwen, Patrick L.J.M.

    2012-01-01

    The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases, Y-27632, was recently reported to immortalize neonatal human foreskin keratinocytes. Here, we investigated the potential use of Y-27632 to expand human adult keratinocytes and evaluated its effects on HSE development and in vitro gene delivery assays. Y-27632 was found to significantly increase the life span of human adult keratinocytes (up to five to eight passages). The epidermal morphology of HSEs generated from high-passage, Y-27632-treated keratinocytes resembled the native epidermis and was improved by supplementing Y-27632 during the submerged phase of HSE development. In addition, Y-27632-treated keratinocytes responded normally to inflammatory stimuli, and could be used to generate HSEs with a psoriatic phenotype, upon stimulation with relevant cytokines. Furthermore, Y-27632 significantly enhanced both lentiviral transduction efficiency of primary adult keratinocytes and epidermal morphology of HSEs generated thereof. Our study indicates that Y-27632 is a potentially powerful tool that is used for a variety of applications of adult human keratinocytes. PMID:22519508

  17. Decorin gene expression and its regulation in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Velez-DelValle, Cristina; Marsch-Moreno, Meytha; Castro-Munozledo, Federico [Department of Cell Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Apdo. Postal 14-740, Mexico D.F. 07000 (Mexico); Kuri-Harcuch, Walid, E-mail: walidkuri@gmail.com [Department of Cell Biology, Centro de Investigacion y de Estudios Avanzados del IPN, Apdo. Postal 14-740, Mexico D.F. 07000 (Mexico)

    2011-07-22

    Highlights: {yields} We showed that cultured human diploid epidermal keratinocytes express and synthesize decorin. {yields} Decorin is found intracytoplasmic in suprabasal cells of cultures and in human epidermis. {yields} Decorin mRNA expression in cHEK is regulated by pro-inflammatory and proliferative cytokines. {yields} Decorin immunostaining of psoriatic lesions showed a lower intensity and altered intracytoplasmic arrangements. -- Abstract: In various cell types, including cancer cells, decorin is involved in regulation of cell attachment, migration and proliferation. In skin, decorin is seen in dermis, but not in keratinocytes. We show that decorin gene (DCN) is expressed in the cultured keratinocytes, and the protein is found in the cytoplasm of differentiating keratinocytes and in suprabasal layers of human epidermis. RT-PCR experiments showed that DCN expression is regulated by pro-inflammatory and proliferative cytokines. Our data suggest that decorin should play a significant role in keratinocyte terminal differentiation, cutaneous homeostasis and dermatological diseases.

  18. Immortalized human hepatocytes as a tool for the study of hepatocytic (de-)differentiation

    NARCIS (Netherlands)

    Schippers, IJ; Moshage, H; Roelofsen, H; Muller, M; Heymans, HSA; Ruiters, M; Kuipers, F

    Primary human hepatocytes were immortalized by stable transfection with a recombinant plasmid containing the early region of simian virus (SV) 40. The cells were cultured in serum-free, hormonally defined medium during the immortalization procedure. Foci of dividing cells were seen after 3 months.

  19. Alcohol metabolism by oral streptococci and interaction with human papillomavirus leads to malignant transformation of oral keratinocytes.

    Science.gov (United States)

    Tao, Lin; Pavlova, Sylvia I; Gasparovich, Stephen R; Jin, Ling; Schwartz, Joel

    2015-01-01

    Poor oral hygiene, ethanol consumption, and human papillomavirus (HPV) are associated with oral and esophageal cancers. However, the mechanism is not fully known. This study examines alcohol metabolism in Streptococcus and its interaction with HPV-16 in the malignant transformation of oral keratinocytes. The acetaldehyde-producing strain Streptococcus gordonii V2016 was analyzed for adh genes and activities of alcohol and aldehyde dehydrogenases. Streptococcus attachment to immortalized HPV-16 infected human oral keratinocytes, HOK (HPV/HOK-16B), human oral buccal keratinocytes, and foreskin keratinocytes was studied. Acetaldehyde, malondialdehyde, DNA damage, and abnormal proliferation among keratinocytes were also quantified. We found that S. gordonii V2016 expressed three primary alcohol dehydrogenases, AdhA, AdhB, and AdhE, which all oxidize ethanol to acetaldehyde, but their preferred substrates were 1-propanol, 1-butanol, and ethanol, respectively. S. gordonii V2016 did not show a detectable aldehyde dehydrogenase. AdhE is the major alcohol dehydrogenase in S. gordonii. Acetaldehyde and malondialdehyde production from permissible Streptococcus species significantly increased the bacterial attachment to keratinocytes, which was associated with an enhanced expression of furin to facilitate HPV infection and several malignant phenotypes including acetaldehyde adduct formation, abnormal proliferation, and enhanced migration through integrin-coated basement membrane by HPV-infected oral keratinocytes. Therefore, expression of multiple alcohol dehydrogenases with no functional aldehyde dehydrogenase contributes to excessive production of acetaldehyde from ethanol by oral streptococci. Oral Streptococcus species and HPV may cooperate to transform oral keratinocytes after ethanol exposure. These results suggest a significant clinical interaction, but further validation is warranted.

  20. Paracrine Activity from Adipose-Derived Stem Cells on In Vitro Wound Healing in Human Tympanic Membrane Keratinocytes.

    Science.gov (United States)

    Ong, Huan Ting; Redmond, Sharon L; Marano, Robert J; Atlas, Marcus D; von Unge, Magnus; Aabel, Peder; Dilley, Rodney J

    2017-03-15

    Stem cell therapies for tympanic membrane repair have shown initial experimental success using mesenchymal stem cells in rat models to promote healing; however, the mechanisms providing this benefit are not known. We investigated in vitro the paracrine effects of human adipose-derived stem cells (ADSCs) on wound healing mechanisms for human tympanic membrane-derived keratinocytes (hTM) and immortalized human keratinocytes (HaCaT). ADSC conditioned media (CMADSC) were assessed for paracrine activity on keratinocyte proliferation and migration, with hypoxic conditions for ADSC culture used to generate contrasting effects on cytokine gene expression. Keratinocytes cultured in CMADSC showed a significant increase in cell number compared to serum-free cultures and further significant increases in hypoxic CMADSC. Assessment of ADSC gene expression on a cytokine array showed a range of wound healing cytokines expressed and under stringent hypoxic and serum-free conditions was upregulated (VEGF A, MMP9, Tissue Factor, PAI-1) or downregulated (CXCL5, CCL7, TNF-α). Several of these may contribute to the activity of conditioned media on the keratinocytes with potential applications in TM perforation repair. VEGFA protein was confirmed by immunoassay to be increased in conditioned media. Together with gene regulation associated with hypoxia in ADSCs, this study has provided several strong leads for a stem cell-derived approach to TM wound healing.

  1. Cyclooxygenases in human and mouse skin and cultured human keratinocytes: association of COX-2 expression with human keratinocyte differentiation

    Science.gov (United States)

    Leong, J.; Hughes-Fulford, M.; Rakhlin, N.; Habib, A.; Maclouf, J.; Goldyne, M. E.

    1996-01-01

    Epidermal expression of the two isoforms of the prostaglandin H-generating cyclooxygenase (COX-1 and COX-2) was evaluated both by immunohistochemistry performed on human and mouse skin biopsy sections and by Western blotting of protein extracts from cultured human neonatal foreskin keratinocytes. In normal human skin, COX-1 immunostaining is observed throughout the epidermis whereas COX-2 immunostaining increases in the more differentiated, suprabasilar keratinocytes. Basal cell carcinomas express little if any COX-1 or COX-2 immunostaining whereas both isozymes are strongly expressed in squamous cell carcinomas deriving from a more differentiated layer of the epidermis. In human keratinocyte cultures, raising the extracellular calcium concentration, a recognized stimulus for keratinocyte differentiation, leads to an increased expression of both COX-2 protein and mRNA; expression of COX-1 protein, however, shows no significant alteration in response to calcium. Because of a recent report that failed to show COX-2 in normal mouse epidermis, we also looked for COX-1 and COX-2 immunostaining in sections of normal and acetone-treated mouse skin. In agreement with a previous report, some COX-1, but no COX-2, immunostaining is seen in normal murine epidermis. However, following acetone treatment, there is a marked increase in COX-1 expression as well as the appearance of significant COX-2 immunostaining in the basal layer. These data suggest that in human epidermis as well as in human keratinocyte cultures, the expression of COX-2 occurs as a part of normal keratinocyte differentiation whereas in murine epidermis, its constitutive expression is absent, but inducible as previously published.

  2. Human Keratinocytes Radioprotection with Mentha Longifolia

    Science.gov (United States)

    Rizzo, Angela Maria; Berselli, P.; Zava, S.; Negroni, M.; Corsetto, P.; Montorfano, G.; Bertolotti, A.; Ranza, E.; Ottolenghi, A.; Berra, B.

    Antioxidants are suggested to act as radioprotectors, and dietary supplements based on antiox-idants have been proposed for astronauts involved in long-term space missions. Plant extracts with antioxidant properties may be used in dietetic supplements for astronauts; in fact recent nutritional guidelines suggest that "fruits and vegetables may become as important on space-going vessels as limes were on the sea-going vessels of old". Mint presents a large variety of biological properties, such as antiallergenic, antibacterial, anti-inflammatory, antitumor, an-tiviral, gastrointestinal protective, hepatoprotective, chemopreventive activities, most of which are attributable to its antioxidant activity. The aim of the present study is to evaluate the antioxidant properties and protective bio-efficacy of a phenol enriched Mentha longifolia ex-tract on gamma rays stressed human keratinocytes (NCTC2544). We assessed first the in vitro antioxidant activity (ABTS and DPPH), and then evaluated different stress markers in order to investigate various oxidative stress targets: cell viability (MTT); retained proliferating ca-pability (CA); DNA damage (histone H2AX) and protein damage (HSP70 induction). Results indicate that this Mint extract has a higher antioxidant activity respect to fresh extracts, that could be responsible of its really interesting radio-protective effects.

  3. Inducible immortality in hTERT-human mesenchymal stem cells.

    Science.gov (United States)

    Piper, Samantha L; Wang, Miqi; Yamamoto, Akira; Malek, Farbod; Luu, Andrew; Kuo, Alfred C; Kim, Hubert T

    2012-12-01

    Human mesenchymal stem cells (hMSCs) are attractive candidates for tissue engineering and cell-based therapy because of their multipotentiality and availability in adult donors. However, in vitro expansion and differentiation of these cells is limited by replicative senescence. The proliferative capacity of hMSCs can be enhanced by ectopic expression of telomerase, allowing for long-term culture. However, hMSCs with constitutive telomerase expression demonstrate unregulated growth and even tumor formation. To address this problem, we used an inducible Tet-On gene expression system to create hMSCs in which ectopic telomerase expression can be induced selectively by the addition of doxycycline (i-hTERT hMSCs). i-hTERT hMSCs have inducible hTERT expression and telomerase activity, and are able to proliferate significantly longer than wild type hMSCs when hTERT expression is induced. They stop proliferating when hTERT expression is turned off and can be rescued when expression is re-induced. They retain multipotentiality in vitro even at an advanced age. We also used a selective inhibitor of telomere elongation to show that the mechanism driving immortalization of hMSCs by hTERT is dependent upon maintenance of telomere length. Thanks to their extended lifespan, preserved multipotentiality and controlled growth, i-hTERT hMSCs may prove to be a useful tool for the development and testing of novel stem cell therapies. Copyright © 2012 Orthopaedic Research Society.

  4. The catalytic and the RNA subunits of human telomerase are required to immortalize equid primary fibroblasts.

    Science.gov (United States)

    Vidale, Pamela; Magnani, Elisa; Nergadze, Solomon G; Santagostino, Marco; Cristofari, Gael; Smirnova, Alexandra; Mondello, Chiara; Giulotto, Elena

    2012-10-01

    Many human primary somatic cells can be immortalized by inducing telomerase activity through the exogenous expression of the human telomerase catalytic subunit (hTERT). This approach has been extended to the immortalization of cell lines from several mammals. Here, we show that hTERT expression is not sufficient to immortalize primary fibroblasts from three equid species, namely donkey, Burchelli's zebra and Grevy's zebra. In vitro analysis of a reconstituted telomerase composed by hTERT and an equid RNA component of telomerase (TERC) revealed a low activity of this enzyme compared to human telomerase, suggesting a low compatibility of equid and human telomerase subunits. This conclusion was also strengthened by comparison of human and equid TERC sequences, which revealed nucleotide differences in key regions for TERC and TERT interaction. We then succeeded in immortalizing equid fibroblasts by expressing hTERT and hTERC concomitantly. Expression of both human telomerase subunits led to telomerase activity and telomere elongation, indicating that human telomerase is compatible with the other equid telomerase subunits and proteins involved in telomere metabolism. The immortalization procedure described herein could be extended to primary cells from other mammals. The availability of immortal cells from endangered species could be particularly useful for obtaining new information on the organization and function of their genomes, which is relevant for their preservation.

  5. Thermolysin in human cultured keratinocyte isolation

    Directory of Open Access Journals (Sweden)

    A. Gragnani

    Full Text Available BACKGROUND: When treating extensively burned patients using cultured epidermal sheets, the main problem is the time required for its production. Conventional keratinocyte isolation is usually done using Trypsin. We used a modification of the conventional isolation method in order to improve this process and increase the number of colonies from the isolated epidermal cell population. PURPOSE: To compare the action of trypsin and thermolysin in the keratinocyte isolation using newborn foreskin. METHODS: This method used thermolysin as it selectively digests the dermo-epidermal junction. After dermis separation, the epidermis was digested by trypsin in order to obtain a cell suspension. RESULTS: Compared to the conventional procedure, these experiments demonstrated that in the thermolysin group, the epidermis was easily detached from the dermis, there was no fibroblast contamination and there were a larger number of keratinocyte colonies which had a significant statistical difference. CONCLUSION: The number of colonies in the thermolysin group was significantly greater than in the trypsin group.

  6. TELOMERE AND TELOMERASE MODULATION BY BERGAMOT POLYPHENOLIC FRACTION IN EXPERIMENTAL PHOTOAGEING IN HUMAN KERATINOCYTES.

    Science.gov (United States)

    Nisticò, S; Ehrlich, J; Gliozzi, M; Maiuolo, J; Del Duca, E; Muscoli, C; Mollace, V

    2015-01-01

    Photoageing represents the addition of extrinsic chronic ultraviolet radiation-induced damage on intrinsic ageing and accounts for most age-associated changes in skin appearance. In this study, we evaluated the effect of 38% BPF, a highly concentrated extract of the bergamot fruit (Citrus bergamia) on UVB-induced photoageing by examining inflammatory cytokine expression, telomere length/telomerase alterations and cellular viability in human immortalized HaCaT keratinocytes. Our results suggest that 38% BPF protects HaCaT cells against UVB-induced oxidative stress and markers of photoageing in a dose-dependent manner and could be a useful supplement in skin care products. Together with antioxidant properties, BPF, a highly concentrated extract of the bergamot fruit, appears to modulate basic cellular signal transduction pathways leading to anti-proliferative, anti-aging and immune modulating responses.

  7. Exposure to Carbon Nanotube Material: Assessment of Nanotube Cytotoxicity Using Human Keratinocyte Cells

    Science.gov (United States)

    Shvedova, Anna A.; Castranova, Vincent; Kisin, Elena R.; Schwegler-Berry, Diane; Murray, Ashley R.; Gandelsman, Vadim Z.; Maynard, Andrew; Baron, Paul

    2003-01-01

    Carbon nanotubes are new members of carbon allotropes similar to fullerenes and graphite. Because of their unique electrical, mechanical, and thermal properties, carbon nanotubes are important for novel applications in the electronics, aerospace, and computer industries. Exposure to graphite and carbon materials has been associated with increased incidence of skin diseases, such as carbon fiber dermatitis, hyperkeratosis, and naevi. We investigated adverse effects of single-wall carbon nanotubes (SWCNT) using a cell culture of immortalized human epidermal keratinocytes (HaCaT). After 18 h of exposure of HaCaT to SWCNT, oxidative stress and cellular toxicity were indicated by formation of free radicals, accumulation of peroxidative products, antioxidant depletion, and loss of cell viability. Exposure to SWCNT also resulted in ultrastructural and morphological changes in cultured skin cells. These data indicate that dermal exposure to unrefined SWCNT may lead to dermal toxicity due to accelerated oxidative stress in the skin of exposed workers.

  8. Transcriptional Dynamics of Immortalized Human Mesenchymal Stem Cells during Transformation.

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    Masao Takeuchi

    Full Text Available Comprehensive analysis of alterations in gene expression along with neoplastic transformation in human cells provides valuable information about the molecular mechanisms underlying transformation. To further address these questions, we performed whole transcriptome analysis to the human mesenchymal stem cell line, UE6E7T-3, which was immortalized with hTERT and human papillomavirus type 16 E6/E7 genes, in association with progress of transformation in these cells. At early stages of culture, UE6E7T-3 cells preferentially lost one copy of chromosome 13, as previously described; in addition, tumor suppressor genes, DNA repair genes, and apoptosis-activating genes were overexpressed. After the loss of chromosome 13, additional aneuploidy and genetic alterations that drove progressive transformation, were observed. At this stage, the cell line expressed oncogenes as well as genes related to anti-apoptotic functions, cell-cycle progression, and chromosome instability (CIN; these pro-tumorigenic changes were concomitant with a decrease in tumor suppressor gene expression. At later stages after prolong culture, the cells exhibited chromosome translocations, acquired anchorage-independent growth and tumorigenicity in nude mice, (sarcoma and exhibited increased expression of genes encoding growth factor and DNA repair genes, and decreased expression of adhesion genes. In particular, glypican-5 (GPC5, which encodes a cell-surface proteoglycan that might be a biomarker for sarcoma, was expressed at high levels in association with transformation. Patched (Ptc1, the cell surface receptor for hedgehog (Hh signaling, was also significantly overexpressed and co-localized with GPC5. Knockdown of GPC5 expression decreased cell proliferation, suggesting that it plays a key role in growth in U3-DT cells (transformants derived from UE6E7T-3 cells through the Hh signaling pathway. Thus, the UE6E7T-3 cell culture model is a useful tool for assessing the functional

  9. The effect of the plasma needle on the human keratinocytes related to the wound healing process

    Science.gov (United States)

    Korolov, Ihor; Fazekas, Barbara; Széll, Márta; Kemény, Lajos; Kutasi, Kinga

    2016-01-01

    In the present study we aim to verify the influence of a non-thermal atmospheric pressure plasma on the wound healing process. In this process the major contributors are the keratinocytes, which migrate to fill in the gap created by the wound. Therefore, we performed the direct treatment of HPV-immortalized human keratinocytes, protected by a layer of phosphate buffered saline (PBS) solution, with the glow discharge generated in flowing helium by a plasma needle. To mimick a wound, a 4 mm scratch was performed on the cell culture (scratch assay). We conducted two types of experiments: (i) cell proliferation and (ii) wound-healing model experiments. The plasma needle configuration, the plasma treatment conditions and the thickness of the protecting PBS layer were set based on viability experiments. The proliferation studies showed that short, 5-10 s, and low power treatments, such as 18 W and 20 W input power, could positively influence the cell proliferation when keratinocytes were protected by PBS. On the other hand, the plasma treatment of cell medium covered keratinocytes resulted in the decrease of proliferation. The wound-healing model (scratch assay) studies showed, that there was a maximum in the wound reduction as a function of the input power and treatment time, namely, at 18 W and 5 s. Furthermore, the wound reduction strongly depended on the treated cell—PBS interaction time. To mimic an infected wound, the scratch assay was covered with a 1× {{10}9} cfu ml-1 Propionibacterium acnes suspension. The plasma treatment of this infected assay resulted in closing of the scratch, while in the non-treated assay the wound did not close at all.

  10. Biocompatibility Evaluation of Dental Luting Cements Using Cytokine Released from Human Oral Fibroblasts and Keratinocytes

    Directory of Open Access Journals (Sweden)

    Jae-Sung Kwon

    2015-10-01

    Full Text Available Dental luting cements are commonly used in dentistry for cementation of prosthetic restoration. Many previous studies focused on the measurement of the cell viability as the method of cytotoxicity evaluation during biocompatibility study for the material. In this study, the biocompatibility of various dental luting cements were evaluated using the new method of cytokine release measurement in order to better simulate inflammatory reactions in animal or clinical model using two different oral cells; immortalized human gingival fibroblast and immortalized human oral keratinocytes. Cells were exposed to extractions of various commercially available dental luting cements for different durations. Cytokines of IL-1α and IL-8 were measured from the supernatants of the cells and the results were then compared to the conventional MTT viability test. The result from the conventional cell viability study showed a relatively simple and straight forward indication that only one of the dental luting cements tested in this study was cytotoxic with increasing duration of exposure for both cells. Meanwhile, the result from the cytokine measurement study was much more complex at the time point they were measured, type of cells used for the study and the type of cytokines measured, all of which influenced the interpretation of the results. Hence, the better understanding of the cytokine release would be required for the application in biocompatibility evaluation.

  11. Transcriptional repression in normal human keratinocytes by wild-type and mutant p53.

    Science.gov (United States)

    Alvarez-Salas, L M; Velazquez, A; Lopez-Bayghen, E; Woodworth, C D; Garrido, E; Gariglio, P; DiPaolo, J A

    1995-05-01

    Wild-type p53 is a nuclear phosphoprotein that inhibits cell proliferation and represses transcriptionally most TATA box-containing promoters in transformed or tumor-derived cell lines. This study demonstrates that p53 alters transcription of the long control region (LCR) of human papillomavirus type 18 (HPV-18). Wild-type and mutant p53 143Val to Ala repressed the HPV-18 LCR promoter in normal human keratinocytes, the natural host cell for HPV infections. Repression by wild-type p53 was also observed in C-33A cells and in an HPV-16-immortalized cell line with an inducible wild-type p53. However, when C-33A cells were cotransfected with the HPV-18 LCR and mutant 143Val to Ala, repression did not occur. Mutant p53 135Cys to Ser did not induce repression in either normal human keratinocytes or in the C-33A line; although like 143Val to Ala, it is thought to affect the DNA binding activity of the wild-type protein. The ability of mutant p53 143Val to Ala to inactivate the HPV early promoter in normal cells (by approximately 60% reduction) suggests that this mutant may be able to associate with wild-type p53 and interact with TATA box-binding proteins. Therefore, these results demonstrate that the transcriptional activities of p53 mutants may be dependent upon the cell type assayed and the form of its endogenous p53. Furthermore, normal human keratinocytes represent an alternative model for determining the activities of p53 mutants.

  12. Ultraviolet B light-induced apoptosis in human keratinocytes enriched with epidermal stem cells and normal keratinocytes

    Institute of Scientific and Technical Information of China (English)

    MEI Xue-ling; LIAN Shi

    2011-01-01

    Background The stem-cell compartment is the primary target for the accumulation of oncogenic mutations.Overexposure to solar ultraviolet radiation is responsible for the development and progression of >90% of skin cancers.Ultraviolet B (UVB) light-induced keratinocyte apoptosis is a strong preventive mechanism against carcinogenesis. The aim of this study was to isolate keratinocytes enriched with putative human epidermal stem cells and to investigate their apoptotic induction by UVB.Methods Keratinocytes enriched with putative human epidermal stem cells were isolated by adherence to collagen Ⅳ and the expressions of β1-integrin and p63 were investigated. Keratinocytes enriched with putative human epidermal stem cells and normal keratinocytes were irradiated with UVB at 0-80 mJ/cm2. The apoptotic response was investigated with phase-contrast microscopy, Hoechst 33342 staining, flow cytometry of annexin V/PI, and procaspase-3 Western blotting.Results Keratinocyte enriched with stem cells expressed high levels of p63 protein and β1-integrin and low level of pan-keratin (C11). In comparison to non-irradiated cells, significant apoptosis of keratinocyte enriched with stem cells was found with 40 and 80 mJ/cm2 UVB. However, significant apoptosis of normal keratinocytes was only found for 80 mJ/cm2 UVB.Conclusions Human epidermal stem cells can undergo apoptosis in response to UVB radiation and are more susceptible than other keratinocytes. The method could be used in vitro studies of human epidermal stem cells.

  13. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha

    2008-01-01

    There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged...

  14. Human keratinocytes synthesize and secrete the extracellular matrix protein, thrombospondin.

    Science.gov (United States)

    Wikner, N E; Dixit, V M; Frazier, W A; Clark, R A

    1987-02-01

    Thrombospondin (TSP) a glycoprotein originally identified as the endogenous lectin of platelets, is also synthesized by fibroblasts, endothelial cells, pneumocytes, smooth muscle cells, and macrophages. Thrombospondin is subdivided into functional domains which bind specifically to heparin, fibronectin, collagen, and to specific cellular receptors. It is found within the basement membranes of kidney, lung, smooth muscle, and skin. Thus TSP may serve as an important link between cells and matrices. Thrombospondin also has been reported at the epidermal-dermal junction. We wished to determine whether human keratinocytes synthesize and secrete TSP. Pure human keratinocytes were grown in defined medium without fibroblast feeder layers. Immunofluorescent staining with either rabbit polyclonal or mouse monoclonal antibodies to human platelet TSP yielded specific granular staining within the cytoplasm of keratinocytes. Culture media and cellular lysates were harvested from cultures metabolically labeled with [35S]methionine. Trichloroacetic acid precipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and autoradiography revealed a major labeled band comigrating with purified platelet TSP in both the media and the cellular lysates. Immunoprecipitation with either the polyclonal or the monoclonal anti-TSP antibodies followed by SDS-PAGE and autoradiography identified this band as TSP. Thus keratinocytes in culture synthesize and secrete TSP. Thrombospondin may play an important role in epidermal interactions with extracellular matrix.

  15. Effect of silver nanoparticles on human primary keratinocytes.

    Science.gov (United States)

    Szmyd, Radoslaw; Goralczyk, Anna Grazyna; Skalniak, Lukasz; Cierniak, Agnieszka; Lipert, Barbara; Filon, Francesca Larese; Crosera, Matteo; Borowczyk, Julia; Laczna, Eliza; Drukala, Justyna; Klein, Andrzej; Jura, Jolanta

    2013-01-01

    Silver nanoparticles (AgNPs) have many biological applications in biomedicine, biotechnology and other life sciences. Depending on the size, shape and the type of carrier, AgNPs demonstrate different physical and chemical properties. AgNPs have strong antimicrobial, antiviral and antifungal activity, thus they are used extensively in a range of medical settings, particularly in wound dressings but also in cosmetics. This study was undertaken to examine the potential toxic effects of 15 nm polyvinylpyrrolidone-coated AgNPs on primary normal human epidermal keratinocytes (NHEK). Cells were treated with different concentrations of AgNPs and then cell viability, metabolic activity and other biological and biochemical aspects of keratinocytes functioning were studied. We observed that AgNPs decrease keratinocyte viability, metabolism and also proliferatory and migratory potential of these cells. Moreover, longer exposure resulted in activation of caspase 3/7 and DNA damage. Our studies show for the first time, that AgNPs may present possible danger for primary keratinocytes, concerning activation of genotoxic and cytotoxic processes depending on the concentration.

  16. Cytotoxicity of HBD3 for dendritic cells, normal human epidermal keratinocytes, hTERT keratinocytes, and primary oral gingival epithelial keratinocytes in cell culture conditions.

    Science.gov (United States)

    Leelakanok, Nattawut; Fischer, Carol L; Bates, Amber M; Guthmiller, Janet M; Johnson, Georgia K; Salem, Aliasger K; Brogden, Kim A; Brogden, Nicole K

    2015-12-03

    Human β-defensin 3 (HBD3) is a prominent host defense peptide. In our recent work, we observed that HBD3 modulates pro-inflammatory agonist-induced chemokine and cytokine responses in human myeloid dendritic cells (DCs), often at 20.0 μM concentrations. Since HBD3 can be cytotoxic in some circumstances, it is necessary to assess its cytotoxicity for DCs, normal human epidermal keratinocytes (NHEKs), human telomerase reverse transcriptase (hTERT) keratinocytes, and primary oral gingival epithelial (GE) keratinocytes in different cell culture conditions. Cells, in serum free media with resazurin and in complete media with 10% fetal bovine serum and resazurin, were incubated with 5, 10, 20, and 40 μM HBD3. Cytotoxicity was determined by measuring metabolic conversion of resazurin to resorufin. The lethal dose 50 (LD50, mean μM±Std Err) values were determined from the median fluorescent intensities of test concentrations compared to live and killed cell controls. The LD50 value range of HBD3 was 18.2-35.9 μM in serum-free media for DCs, NHEKs, hTERT keratinocytes, and GE keratinocytes, and >40.0 μM in complete media. Thus, HBD3 was cytotoxic at higher concentrations, which must be considered in future studies of HBD3-modulated chemokine and cytokine responses in vitro. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  17. Tissue distribution and engraftment of human mesenchymal stem cells immortalized by human telomerase reverse transcriptase gene

    DEFF Research Database (Denmark)

    Bentzon, J F; Stenderup, K; Hansen, F D

    2005-01-01

    Engraftment of mesenchymal stem cells (MSC) in peripheral tissues for replenishing of local stem cell function has been proposed as a therapeutic approach to degenerative diseases. We have previously reported the development of an immortalized human telomerase reverse transcriptase transduced MSC...... that infused cells were efficiently arrested in microvasculature during first-pass, but only for a fraction of the infused cells was arrest followed by vascular emigration and tissue engraftment. Few engrafted cells in lungs, heart, and kidney glomeruli remained after 4 weeks. These observations are consistent...

  18. Immortalization capacity of HPV types is inversely related to chromosomal instability

    DEFF Research Database (Denmark)

    Schütze, Denise M; Krijgsman, Oscar; Snijders, Peter J F

    2016-01-01

    High-risk human papillomavirus (hrHPV) types induce immortalization of primary human epithelial cells. Previously we demonstrated that immortalization of human foreskin keratinocytes (HFKs) is HPV type dependent, as reflected by the presence or absence of a crisis period before reaching immortality....... This study determined how the immortalization capacity of ten hrHPV types relates to DNA damage induction and overall genomic instability in HFKs.Twenty five cell cultures obtained by transduction of ten hrHPV types (i.e. HPV16/18/31/33/35/45/51/59/66/70 E6E7) in two or three HFK donors each were studied.......All hrHPV-transduced HFKs showed an increased number of double strand DNA breaks compared to controls, without exhibiting significant differences between types. However, immortal descendants of HPV-transduced HFKs that underwent a prior crisis period (HPV45/51/59/66/70-transduced HFKs) showed...

  19. In Vitro Toxicity of Aluminum Nanoparticles in Human Keratinocytes

    Science.gov (United States)

    2008-03-01

    absorption of aluminum has been reported after application of underarm deodorant (Becaria, 2002:311). Two adult volunteers were dermally exposed to one...NPs can be classified as a sensitizer agent. Since sensitization has been identified in persons exposed to aluminum in deodorant , vaccines, and...IN VITRO TOXICITY OF ALUMINUM NANOPARTICLES IN HUMAN KERATINOCYTES THESIS Stephanie McCormack-Brown, Major, USAF, BSC AFIT/GIH/ENV

  20. Generation and evaluation of a human corneal model cell system for ophthalmologic issues using the HPV16 E6/E7 oncogenes as uniform immortalization platform.

    Science.gov (United States)

    Schulz, Simon; Steinberg, Thorsten; Beck, David; Tomakidi, Pascal; Accardi, Rosita; Tommasino, Massimo; Reinhard, Thomas; Eberwein, Philipp

    2013-01-01

    The present study aimed at employing the human papillomavirus type 16 (HPV16) E6/E7 gene platform, to create a uniform authentic in vitro model cell system of the human cornea for ophthalmologic issues and here especially for prospective biomaterial evaluations for therapeutic regenerative approaches. Therefore, HPV16 E6/E7 genes were employed as uniform platform to immortalize primary human corneal keratinocytes (IHCK), fibroblasts (IHCF), and endothelial (IHCE) cells. qPCR revealed that E6/E7 mRNA transcription persisted at rising passages and FISH detection of the chromosome portfolio 1, 8, 10 and 18 showed fairly the disomic cytogenetic status. Hot spot passages proved oscillation of aneuploidies in the entire passage spectrum under study, while hot spot aneuploidies annotated prevalence for distinct chromosomes. Though IIF revealed general endurance, tissue-innate corneal biomarkers were modulated, i.e. expressed in a temporal-confluence, temporal-spatial or passage-dependent manner. In summary, by the fairly normal chromosomal status, and expression of tissue-innate biomarkers, we created for the first time a uniform authentic in vitro model cell system of the human cornea, by application of the HPV16 E6/E7 immortalization platform only. This system renders a precious tool for prospective iterative in vitro studies on issues such as corneal tissue homeostasis, pharmaceutical generics, and/or evaluation of new biomaterials for clinical corneal applications. Copyright © 2013 International Society of Differentiation. Published by Elsevier B.V. All rights reserved.

  1. Efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with lentiviral vector

    Science.gov (United States)

    2013-01-01

    Introduction The development of an appropriate procedure for lentiviral gene transduction into keratinocyte stem cells is crucial for stem cell biology and regenerative medicine for genetic disorders of the skin. However, there is little information available on the efficiency of lentiviral transduction into human keratinocyte stem/progenitor cells and the effects of gene transduction procedures on growth potential of the stem cells by systematic assessment. Methods In this study, we explored the conditions for efficient expansion of human keratinocyte stem/progenitor cells carrying a transgene with a lentiviral vector, by using the culture of keratinocytes on a feeder layer of 3 T3 mouse fibroblasts. The gene transduction and expansion of keratinocytes carrying a transgene were analyzed by Western blotting, quantitative PCR, and flow cytometry. Results Polybrene (hexadiamine bromide) markedly enhanced the efficiency of lentiviral gene transduction, but negatively affected the maintenance of the keratinocyte stem/progenitor cells at a concentration higher than 5 μg/ml. Rho-assiciated kinase (ROCK) inhibitor Y-27632, a small molecule which enhanced keratinocyte proliferation, significantly interfered with the lentiviral transduction into cultured human keratinocytes. However, a suitable combination of polybrene and Y-27632 effectively expanded keratinocytes carrying a transgene. Conclusions This study provides information for effective expansion of cultured human keratinocyte stem/progenitor cells carrying a transgene. This point is particularly significant for the application of genetically modified keratinocyte stem/progenitor stem cells in regenerative medicine. PMID:24406242

  2. Transcriptional responses of human epidermal keratinocytes to Oncostatin-M.

    Science.gov (United States)

    Finelt, Nika; Gazel, Alix; Gorelick, Steven; Blumenberg, Miroslav

    2005-08-21

    Oncostatin-M (OsM) plays an important role in inflammatory and oncogenic processes in skin, including psoriasis and Kaposi sarcoma. However, the molecular responses to OsM in keratinocytes have not been explored in depth. Here we show the results of transcriptional profiling in OsM-treated primary human epidermal keratinocytes, using high-density DNA microarrays. We find that OsM strongly and specifically affects the expression of many genes, in particular those involved with innate immunity, angiogenesis, adhesion, motility, tissue remodeling, cell cycle and transcription. The timing of the responses to OsM comprises two waves, early at 1h, and late at 48 h, with much fewer genes regulated in the intervening time points. Secreted cytokines and growth factors and their receptors, as well as nuclear transcription factors, are primary targets of OsM regulation, and these, in turn, effect the secondary changes.

  3. Transcriptional network of p63 in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Silvia Pozzi

    Full Text Available p63 is a transcription factor required for the development and maintenance of ectodermal tissues in general, and skin keratinocytes in particular. The identification of its target genes is fundamental for understanding the complex network of gene regulation governing the development of epithelia. We report a list of almost 1000 targets derived from ChIP on chip analysis on two platforms; all genes analyzed changed in expression during differentiation of human keratinocytes. Functional annotation highlighted unexpected GO terms enrichments and confirmed that genes involved in transcriptional regulation are the most significant. A detailed analysis of these transcriptional regulators in condition of perturbed p63 levels confirmed the role of p63 in the regulatory network. Rather than a rigid master-slave hierarchical model, our data indicate that p63 connects different hubs involved in the multiple specific functions of the skin.

  4. The People Paradox: Self-Esteem Striving, Immortality Ideologies, and Human Response to Climate Change

    Directory of Open Access Journals (Sweden)

    Janis L. Dickinson

    2009-06-01

    Full Text Available In 1973, Ernest Becker, a cultural anthropologist cross-trained in philosophy, sociology, and psychiatry, invoked consciousness of self and the inevitability of death as the primary sources of human anxiety and repression. He proposed that the psychological basis of cooperation, competition, and emotional and mental health is a tendency to hold tightly to anxiety-buffering cultural world views or "immortality projects" that serve as the basis for self-esteem and meaning. Although he focused mainly on social and political outcomes like war, torture, and genocide, he was increasingly aware that materialism, denial of nature, and immortality-striving efforts to control, rather than sanctify, the natural world were problems whose severity was increasing. In this paper I review Becker's ideas and suggest ways in which they illuminate human response to global climate change. Because immortality projects range from belief in technology and materialism to reverence for nature or belief in a celestial god, they act both as barriers to and facilitators of sustainable practices. I propose that Becker's cross-disciplinary "science of man," and the predictions it generates for proximate-level determinants of social behavior, add significantly to our understanding of and potential for managing the people paradox, i.e., that the very things that bring us symbolic immortality often conflict with our prospects for survival. Analysis of immortality projects as one of the proximate barriers to addressing climate change is both cautionary and hopeful, providing insights that should be included in the cross-disciplinary quest to uncover new pathways toward rational, social change.

  5. Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties.

    Science.gov (United States)

    Raymon, H K; Thode, S; Zhou, J; Friedman, G C; Pardinas, J R; Barrere, C; Johnson, R M; Sah, D W

    1999-07-01

    A renewable source of human sensory neurons would greatly facilitate basic research and drug development. We had established previously conditionally immortalized human CNS cell lines that can differentiate into functional neurons (). We report here the development of an immortalized human dorsal root ganglion (DRG) clonal cell line, HD10.6, with a tetracycline-regulatable v-myc oncogene. In the proliferative condition, HD10.6 cells have a doubling time of 1.2 d and exhibit a neuronal precursor morphology. After differentiation of clone HD10.6 for 7 d in the presence of tetracycline, v-myc expression was suppressed, and >50% of the cells exhibited typical neuronal morphology, stained positively for neuronal cytoskeletal markers, and fired action potentials in response to current injection. Furthermore, this cell line was fate-restricted to a neuronal phenotype; even in culture conditions that promote Schwann cell or smooth muscle differentiation of neural crest stem cells, HD10.6 differentiated exclusively into neurons. Moreover, differentiated HD10.6 cells expressed sensory neuron-associated transcription factors and exhibited capsaicin sensitivity. Taken together, these data indicate that we have established an immortalized human DRG cell line that can differentiate into sensory neurons with nociceptive properties. The cell line HD10.6 represents the first example of a human sensory neuronal line and will be valuable for basic research, as well as for the discovery of novel drug targets and clinical candidates.

  6. C/EBPδ gene targets in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Serena Borrelli

    Full Text Available C/EBPs are a family of B-Zip transcription factors--TFs--involved in the regulation of differentiation in several tissues. The two most studied members--C/EBPα and C/EBPβ--play important roles in skin homeostasis and their ablation reveals cells with stem cells signatures. Much less is known about C/EBPδ which is highly expressed in the granular layer of interfollicular epidermis and is a direct target of p63, the master regular of multilayered epithelia. We identified C/EBPδ target genes in human primary keratinocytes by ChIP on chip and profiling of cells functionally inactivated with siRNA. Categorization suggests a role in differentiation and control of cell-cycle, particularly of G2/M genes. Among positively controlled targets are numerous genes involved in barrier function. Functional inactivation of C/EBPδ as well as overexpressions of two TF targets--MafB and SOX2--affect expression of markers of keratinocyte differentiation. We performed IHC on skin tumor tissue arrays: expression of C/EBPδ is lost in Basal Cell Carcinomas, but a majority of Squamous Cell Carcinomas showed elevated levels of the protein. Our data indicate that C/EBPδ plays a role in late stages of keratinocyte differentiation.

  7. Protein composition of catalytically active human telomerase from immortal cells

    DEFF Research Database (Denmark)

    Cohen, Scott B; Graham, Mark E; Lovrecz, George O

    2007-01-01

    Telomerase is a ribonucleoprotein enzyme complex that adds 5'-TTAGGG-3' repeats onto the ends of human chromosomes, providing a telomere maintenance mechanism for approximately 90% of human cancers. We have purified human telomerase approximately 10(8)-fold, with the final elution dependent on th...

  8. Protective effect of trehalose-loaded liposomes against UVB-induced photodamage in human keratinocytes

    Science.gov (United States)

    EMANUELE, ENZO; BERTONA, MARCO; SANCHIS-GOMAR, FABIAN; PAREJA-GALEANO, HELIOS; LUCIA, ALEJANDRO

    2014-01-01

    Trehalose, a naturally occurring non-reducing disaccharide, is known to act as a major protein stabilizer that can reduce ultraviolet B (UVB)-induced corneal damage when topically applied to the eye. However, due to the low skin permeability of trehalose, which makes the development of topical formulations difficult, its use as a skin photoprotective agent has been limited. Previous findings demonstrated that liposomes may significantly improve the intracellular delivery of trehalose. Therefore, the present study aimed to assess the protective effects of trehalose-loaded liposomes against UVB-induced photodamage using the immortalized human keratinocyte cell line, HaCaT. The effects were also compared to those of the common skin photoprotective compounds, including L-carnosine, L-(+)-ergothioneine, L-ascorbic acid and DL-α-tocopherol. The levels of cyclobutane pyrimidine dimers, 8-hydroxy-2′-deoxyguanosine and protein carbonylation in HaCaT cells were used as biological markers of UVB-induced damage. Compared to other compounds, trehalose-loaded liposomes showed the highest efficacy in reducing the levels of the three markers following UVB irradiation of HaCaT cells (all P<0.001 when compared to each of the four other photoprotective compounds). Therefore, these findings indicate that there may be a clinical application for trehalose-loaded liposomes, and further studies should be performed to assess the potential usefulness in skin photoprotection and the prevention of non-melanoma skin cancer. PMID:25054023

  9. A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes

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    Xiangshan Zhao

    2011-01-01

    Full Text Available Introduction: Emerging evidence suggests a direct role of cancer stem cells (CSCs in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs immortalized with human telomerase reverse transcriptase (hTERT possess properties of mammary stem / progenitor cells. Materials and Methods: In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with g-radiation, share with hTERT, the ability to maintain mammary stem / progenitor cells. Results: The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC. Conclusions: Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers.

  10. Steroid synthesis by primary human keratinocytes; implications for skin disease

    Energy Technology Data Exchange (ETDEWEB)

    Hannen, Rosalind F., E-mail: r.f.hannen@qmul.ac.uk [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom); Michael, Anthony E. [Centre for Developmental and Endocrine Signalling, Academic Section of Obstetrics and Gynaecology, Division of Clinical Developmental Sciences, 3rd Floor, Lanesborough Wing, St. George' s, University of London, Cranmer Terrace, Tooting, London SW17 0RE (United Kingdom); Jaulim, Adil [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom); Bhogal, Ranjit [Life Science, Unilever R and D Colworth House, Sharnbrook, Bedfordshire MK44 1LQ (United Kingdom); Burrin, Jacky M. [Centre for Endocrinology, William Harvey Research Institute, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London EC1M 6BQ (United Kingdom); Philpott, Michael P. [Centre for Cutaneous Research, Institute of Cell and Molecular Science, Barts and The London School of Medicine and Dentistry, Queen Mary University of London, London E1 2AT (United Kingdom)

    2011-01-07

    Research highlights: {yields} Primary keratinocytes express the steroid enzymes required for cortisol synthesis. {yields} Normal primary human keratinocytes can synthesise cortisol. {yields} Steroidogenic regulators, StAR and MLN64, are expressed in normal epidermis. {yields} StAR expression is down regulated in eczema and psoriatic epidermis. -- Abstract: Cortisol-based therapy is one of the most potent anti-inflammatory treatments available for skin conditions including psoriasis and atopic dermatitis. Previous studies have investigated the steroidogenic capabilities of keratinocytes, though none have demonstrated that these skin cells, which form up to 90% of the epidermis are able to synthesise cortisol. Here we demonstrate that primary human keratinocytes (PHK) express all the elements required for cortisol steroidogenesis and metabolise pregnenolone through each intermediate steroid to cortisol. We show that normal epidermis and cultured PHK express each of the enzymes (CYP11A1, CYP17A1, 3{beta}HSD1, CYP21 and CYP11B1) that are required for cortisol synthesis. These enzymes were shown to be metabolically active for cortisol synthesis since radiometric conversion assays traced the metabolism of [7-{sup 3}H]-pregnenolone through each steroid intermediate to [7-{sup 3}H]-cortisol in cultured PHK. Trilostane (a 3{beta}HSD1 inhibitor) and ketoconazole (a CYP17A1 inhibitor) blocked the metabolism of both pregnenolone and progesterone. Finally, we show that normal skin expresses two cholesterol transporters, steroidogenic acute regulatory protein (StAR), regarded as the rate-determining protein for steroid synthesis, and metastatic lymph node 64 (MLN64) whose function has been linked to cholesterol transport in steroidogenesis. The expression of StAR and MLN64 was aberrant in two skin disorders, psoriasis and atopic dermatitis, that are commonly treated with cortisol, suggesting dysregulation of epidermal steroid synthesis in these patients. Collectively these data

  11. Establishment and transformation of telomerase-immortalized human small airway epithelial cells by heavy ions

    Science.gov (United States)

    Zhao, Y. L.; Piao, C. Q.; Hei, T. K.

    Previous studies from this laboratory have identified a number of causally linked genes including the novel tumor suppressor Betaig-h3 that were differentially expressed in radiation induced tumorigenic BEP2D cells. To extend these studies using a genomically more stable bronchial cell line, we show here that ectopic expression of the catalytic subunit of telomerase (hTERT) in primary human small airway epithelial (SAE) cells resulted in the generation of several clonal cell lines that have been continuously in culture for more than 250 population doublings and are considered immortal. Comparably-treated control SAE cells infected with only the viral vector senesced after less than 10 population doublings. The immortalized clones demonstrated anchorage dependent growth and are non-tumorigenic in nude mice. These cells show no alteration in the p53 gene but a decrease in p16 expression. Exponentially growing SAEh cells were exposed to graded doses of 1 GeV/nucleon of 56Fe ions accelerated at the Brookhaven National Laboratory. Irradiated cells underwent gradual phenotypic alterations after extensive in vitro cultivation. Transformed cells developed through a series of successive steps before becoming anchorage independent in semisolid medium. These findings indicate that hTERT-immortalized cells, being diploid and chromosomal stable, should be a useful model in assessing mechanism of radiation carcinogenesis.

  12. Arsenite suppression of BMP signaling in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Phillips, Marjorie A.; Qin, Qin [Department of Environmental Toxicology, University of California, Davis, CA 95616-8588 (United States); Hu, Qin; Zhao, Bin [State Key Laboratory of Environmental Chemistry and Ecotoxicology, Research Center for Eco-Environmental Sciences, Chinese Academy of Sciences, Beijing 100085 (China); Rice, Robert H., E-mail: rhrice@ucdavis.edu [Department of Environmental Toxicology, University of California, Davis, CA 95616-8588 (United States)

    2013-06-15

    Arsenic, a human skin carcinogen, suppresses differentiation of cultured keratinocytes. Exploring the mechanism of this suppression revealed that BMP-6 greatly increased levels of mRNA for keratins 1 and 10, two of the earliest differentiation markers expressed, a process prevented by co-treatment with arsenite. BMP also stimulated, and arsenite suppressed, mRNA for FOXN1, an important transcription factor driving early keratinocyte differentiation. Keratin mRNAs increased slowly after BMP-6 addition, suggesting they are indirect transcriptional targets. Inhibition of Notch1 activation blocked BMP induction of keratins 1 and 10, while FOXN1 induction was largely unaffected. Supporting a requirement for Notch1 signaling in keratin induction, BMP increased levels of activated Notch1, which was blocked by arsenite. BMP also greatly decreased active ERK, while co-treatment with arsenite maintained active ERK. Inhibition of ERK signaling mimicked BMP by inducing keratin and FOXN1 mRNAs and by increasing active Notch1, effects blocked by arsenite. Of 6 dual-specificity phosphatases (DUSPs) targeting ERK, two were induced by BMP unless prevented by simultaneous exposure to arsenite and EGF. Knockdown of DUSP2 or DUSP14 using shRNAs greatly reduced FOXN1 and keratins 1 and 10 mRNA levels and their induction by BMP. Knockdown also decreased activated Notch1, keratin 1 and keratin 10 protein levels, both in the presence and absence of BMP. Thus, one of the earliest effects of BMP is induction of DUSPs, which increases FOXN1 transcription factor and activates Notch1, both required for keratin gene expression. Arsenite prevents this cascade by maintaining ERK signaling, at least in part by suppressing DUSP expression. - Highlights: • BMP induces FOXN1 transcription. • BMP induces DUSP2 and DUSP14, suppressing ERK activation. • Arsenite suppresses levels of phosphorylated Smad1/5 and FOXN1 and DUSP mRNA. • These actions rationalize arsenite suppression of keratinocyte

  13. Stanniocalcin-1 regulates re-epithelialization in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Bonnie H Y Yeung

    Full Text Available Stanniocalcin-1 (STC1, a glycoprotein hormone, is believed to be involved in various biological processes such as inflammation, oxidative responses and cell migration. Riding on these emerging evidences, we hypothesized that STC1 may participate in the re-epithelialization during wound healing. Re-epithelialization is a critical step that involves keratinocyte lamellipodia (e-lam formation, followed by cell migration. In this study, staurosporine (STS treatment induced human keratinocyte (HaCaT e-lam formation on fibronectin matrix and migration via the activation of focal adhesion kinase (FAK, the surge of intracellular calcium level [Ca²⁺]i and the inactivation of Akt. In accompanied with these migratory features, a time- and dose-dependent increase in STC1 expression was detected. STC1 gene expression was found not the downstream target of FAK-signaling as illustrated by FAK inhibition using PF573228. The reduction of [Ca²⁺]i by BAPTA/AM blocked the STS-mediated keratinocyte migration and STC1 gene expression. Alternatively the increase of [Ca²⁺]i by ionomycin exerted promotional effect on STS-induced STC1 gene expression. The inhibition of Akt by SH6 and GSK3β by lithium chloride (LiCl could respectively induce and inhibit the STS-mediated e-lam formation, cell migration and STC1 gene expression. The STS-mediated e-lam formation and cell migration were notably hindered or induced respectively by STC1 knockdown or overexpression. This notion was further supported by the scratched wound assay. Collectively the findings provide the first evidence that STC1 promotes re-epithelialization in wound healing.

  14. In Search of Rationality in Human Longevity and Immortality.

    Science.gov (United States)

    Bhar, Gopal C

    2016-01-01

    The human body is machine-like, but self-moving, self-regulating, and self-adjusting, governed by willpower and intelligence. Aging of the body is basically a maintenance problem and so it could perhaps be postponed by thorough and frequent maintenance. Aging brings on a cascade of ills and health problems leading to deterioration of physical, mental, emotional, and social dimensions of life. This paper deals with solution of the problem philosophically in the light of Indian scriptures without entering into traditional bioethical issues. With a meaningful reason for existence, life can be extended. Examining the scientific perspectives on aging, some common manipulations for its extension are discussed. These are calorie restriction, vitamin and antioxidant treatment, exercise and hormonal interventions, etc. Finally, the question of longevity is explored through pursuance of eternal value-based activity and spirituality in the tradition of Indian heritage.

  15. Bradykinin promotes migration and invasion of human immortalized trophoblasts

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    Lisboa Francisco

    2011-07-01

    Full Text Available Abstract Having demonstrated that the bradykinin B2 receptor (B2R is expressed in cells that participate in trophoblast invasion in humans and guinea-pigs, we investigated the role of bradykinin (BK on cell migration and invasion in the HTR-8/SVneo trophoblast cell line using wound healing and invasion assays. First, we documented that HTR-8/SVneo cells expressed kallikrein, B2R, B1R, MMP-2 and MMP-9 using immunocytochemistry. Incubation with BK (10.0 microMol/L for 18 hours increased the migration index 3-fold in comparison to controls or to cells preincubated with the B2R antagonist HOE-140. BK (10.0 microMol/L incubation yielded a similar number of proliferating and viable cells as controls, therefore the enhanced closure of the wound cannot be attributed to proliferating cells. Incubation with BK (10.0 microMol/L for 18 hours increased the invasion index 2-fold in comparison to controls or to cells preincubated with the antagonist of the B2R. Neither the B1R ligand Lys-des-Arg9 BK, nor its antagonist Lys-(des-Arg9-Leu8, modified migration and invasion. Further support for the stimulatory effect of B2R activation on migration and invasion is provided by the 3-fold increase in the number of filopodia per cell versus controls or cells preincubated with the B2R antagonist. Bradykinin had no effect on the cellular protein content of the B2R, nor the MMP-9 and MMP-2 gelatinase activity in the culture media varied after incubation with BK. This study adds bradykinin-acting on the B2R-to the stimuli of trophoblast migration and invasion, an effect that should be integrated to other modifications of the kallikrein-kinin system in normal and pathological pregnancies.

  16. Immortalized pathological human myoblasts: towards a universal tool for the study of neuromuscular disorders

    Directory of Open Access Journals (Sweden)

    Mamchaoui Kamel

    2011-11-01

    Full Text Available Abstract Background Investigations into both the pathophysiology and therapeutic targets in muscle dystrophies have been hampered by the limited proliferative capacity of human myoblasts. Isolation of reliable and stable immortalized cell lines from patient biopsies is a powerful tool for investigating pathological mechanisms, including those associated with muscle aging, and for developing innovative gene-based, cell-based or pharmacological biotherapies. Methods Using transduction with both telomerase-expressing and cyclin-dependent kinase 4-expressing vectors, we were able to generate a battery of immortalized human muscle stem-cell lines from patients with various neuromuscular disorders. Results The immortalized human cell lines from patients with Duchenne muscular dystrophy, facioscapulohumeral muscular dystrophy, oculopharyngeal muscular dystrophy, congenital muscular dystrophy, and limb-girdle muscular dystrophy type 2B had greatly increased proliferative capacity, and maintained their potential to differentiate both in vitro and in vivo after transplantation into regenerating muscle of immunodeficient mice. Conclusions Dystrophic cellular models are required as a supplement to animal models to assess cellular mechanisms, such as signaling defects, or to perform high-throughput screening for therapeutic molecules. These investigations have been conducted for many years on cells derived from animals, and would greatly benefit from having human cell models with prolonged proliferative capacity. Furthermore, the possibility to assess in vivo the regenerative capacity of these cells extends their potential use. The innovative cellular tools derived from several different neuromuscular diseases as described in this report will allow investigation of the pathophysiology of these disorders and assessment of new therapeutic strategies.

  17. Rotation is the primary motion of paired human epidermal keratinocytes.

    Science.gov (United States)

    Tate, Sota; Imai, Matome; Matsushita, Natsuki; Nishimura, Emi K; Higashiyama, Shigeki; Nanba, Daisuke

    2015-09-01

    Collective motion of keratinocytes is involved in morphogenesis, homeostasis, and wound healing of the epidermis. Yet how the collective motion of keratinocytes emerges from the behavior of individual cells is still largely unknown. The aim of this study was to find the cellular behavior that links single and collective motion of keratinocytes. We investigated the behavior of two-cell colonies of HaCaT keratinocytes by a combination of time-lapse imaging and image processing. The two-cell colonies of HaCaT cells were formed as a contacted pair of keratinocyte clones. Image analysis and cell culture experiments revealed that the rotational speed of two-cell colonies was positively associated with their proliferative capacity. α6 integrin was required for the rotational motion of two-cell keratinocyte colonies. We also confirmed that two-cell colonies of keratinocytes predominantly exhibited the rotational, but not translational, motion, two modes of motion in a contact pair of rotating objects. The rotational motion is the primary motion of two-cell keratinocyte colonies and its speed is positively associated with their proliferative capacity. This study suggests that the assembly of rotating keratinocytes generates the collective motion of proliferative keratinocytes during morphogenesis and wound healing of the epidermis. Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.

  18. Benzo (a) pyrene induced tumorigenesity of human immortalized oral epithelial cells: transcription profiling

    Institute of Scientific and Technical Information of China (English)

    LI Jin-zhong; PAN Hong-ya; ZHENG Jia-wei; ZHOU Xiao-jian; ZHANG Ping; CHEN Wan-tao; ZHANG Zhi-yuan

    2008-01-01

    Background The present study was designed to examine and analyze the global gene expression changes during the tumorigenesis of a human immortalized oral epithelial cell line, and search for the possible genes that may play a role in the carcinogenesis of oral cancer associated with benzo (a) pyrene.Methods The human immortalized oral epithelial cells, which have been established through transfection of E6/E7 genasof human papillomavirus type 16 and proved to be non-tumorigenic in nude mice, were treated with benzo (a) pyrene.Tumorigenesity of the treated cells were examined through nude mice subcutaneous injection. The global gene expression profiles of immortalized cells and the tumorigenic cells were acquired through hybridization of a microarray of Affymetrix U133 plus 2.0. The data were analyzed using Spring 7.0 software and treated statistically using one-way analysis of variance (ANOVA). The differentially expressed genes were classified using a Venn diagram and annotated with gene ontology. Several highlighted genes were validated in cells using a real-time polymerase chain reaction.Results There were 883 differentially expressed genes during the tumorigenesis and most of them changed expression in the early stage of tumorigenesis. These genes mainly involved in macromolecule metabolism and signal transduction,possessed the molecular function of transition metal ion binding, nucleotide binding and kinase activity; their protein products were mainly integral to membranes or localized in the nucleus and cytoskeleton. The expression patterns of IGFBP3, S100A8, MAP2K, KRT6B, GDF15, MET were validated in cells using a real-time polymerase chain reaction; the expression of IGFBP3 was further validated in clinical oral cancer specimens.Concluslona This study provides the global transcription profiling associated with the tumorigenesis of oral epithelial cells exposed to benzo (a) pyrene; IGFBP3 may play a potential role in the initiation of oral cancer related to

  19. Human papillomavirus (HPV) upregulates the cellular deubiquitinase UCHL1 to suppress the keratinocyte's innate immune response

    NARCIS (Netherlands)

    Karim, R.; Tummers, B.; Meyers, C.; Biryukov, J.L.; Alam, S.; Backendorf, C.; Jha, V.; Offringa, R.; van Ommen, G.J.; Melief, C.J.; Guardavaccaro, D.; Boer, J.M.; van der Burg, S.H.

    2013-01-01

    Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity

  20. Human papillomavirus (HPV) upregulates the cellular deubiquitinase UCHL1 to suppress the keratinocyte's innate immune response

    NARCIS (Netherlands)

    Karim, R.; Tummers, B.; Meyers, C.; Biryukov, J.L.; Alam, S.; Backendorf, C.; Jha, V.; Offringa, R.; van Ommen, G.J.; Melief, C.J.; Guardavaccaro, D.; Boer, J.M.; van der Burg, S.H.

    2013-01-01

    Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV) may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs) but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity

  1. Human keratinocytes produce the complement inhibitor factor H: synthesis is regulated by interferon-gamma.

    NARCIS (Netherlands)

    Timar, K.K.; Pasch, M.C.; Bosch, N.H. van den; Jarva, H.; Junnikkala, S.; Meri, S.; Bos, J.D.; Asghar, S.S.

    2006-01-01

    Locally synthesized complement is believed to play an important role in host defense and inflammation at organ level. In the epidermis, keratinocytes have so far been shown to synthesize two complement components, C3 and factor B. Here, we studied the synthesis of factor H by human keratinocytes. We

  2. Herpes Simplex Virus 1 Enters Human Keratinocytes by a Nectin-1-Dependent, Rapid Plasma Membrane Fusion Pathway That Functions at Low Temperature.

    Science.gov (United States)

    Sayers, Charlotte L; Elliott, Gillian

    2016-11-15

    Herpes simplex virus 1 (HSV-1) infects humans through stratified epithelia that are composed primarily of keratinocytes. The route of HSV-1 entry into keratinocytes has been the subject of limited investigation, but it is proposed to involve pH-dependent endocytosis, requiring the gD-binding receptor nectin-1. Here, we have utilized the nTERT human keratinocyte cell line as a new model for dissecting the mechanism of HSV-1 entry into the host. Although immortalized, these cells nonetheless retain normal growth and differentiation properties of primary cells. Using short interfering RNA (siRNA) depletion studies, we confirm that, despite nTERT cells expressing high levels of the alternative gD receptor HVEM, HSV-1 requires nectin-1, not HVEM, to enter these cells. Strikingly, virus entry into nTERT cells occurred with unusual rapidity, such that maximum penetration was achieved within 5 min. Moreover, HSV-1 was able to enter keratinocytes but not other cell types at temperatures as low as 7°C, conditions where endocytosis was shown to be completely inhibited. Transmission electron microscopy of early entry events at both 37°C and 7°C identified numerous examples of naked virus capsids located immediately beneath the plasma membrane, with no evidence of virions in cytoplasmic vesicles. Taken together, these results imply that HSV-1 uses the nectin-1 receptor to enter human keratinocyte cells via a previously uncharacterized rapid plasma membrane fusion pathway that functions at low temperature. These studies have important implications for current understanding of the relationship between HSV-1 and its relevant in vivo target cell. The gold standard of antiviral treatment for any human virus infection is the prevention of virus entry into the host cell. In the case of HSV-1, primary infection in the human begins in the epidermis of the skin or the oral mucosa, where the virus infects keratinocytes, and it is therefore important to understand the molecular events

  3. Immortalization of Human Precartilaginous Stem Cells by Transfecting SV40Tag

    Institute of Scientific and Technical Information of China (English)

    Junfang WANG; Huang FANG; Renyun XIA; Anming CHEN; Hao CHENG

    2009-01-01

    Immortalized human precartilaginous stem cells (IPSCs) were established to provide stable cell resource for the study of the molecular mechanism of gene targeting on the differentiation of PSCs.Plasmid pCMVSV40T/PUR containing simian virus 40 large T antigen gene (SV40Tag) was transfected into human PSCs by using lipofectin transfection.Colonies were isolated by puromycin selection and expanded by multiple passages,lmmunohistochemistry,RT-PCR and Southern blotting were used to identify the transfected cells and to detect the expression and integration of SV40Tag in expanded cell lines.The positive colonies were isolated and subcultured,designated immortalized precartilaginous stem cells (IPSCs),which were confirmed as fibroblast growth factor receptor-3 (FGFR-3) positive cells by immunohistochemistry and RT-PCR.SV40Tag cDNA was found in cultured IPSCs of passage 8 by Southern blotting,and the expressions of SV40Tag mRNA and protein were confirmed by RT-PCR.These findings suggested that IPSCs strain with SV40Tag was constructed successfully.

  4. Immortality of cancers

    Science.gov (United States)

    Duesberg, Peter; McCormack, Amanda

    2013-01-01

    Immortality is a common characteristic of cancers, but its origin and purpose are still unclear. Here we advance a karyotypic theory of immortality based on the theory that carcinogenesis is a form of speciation. Accordingly, cancers are generated from normal cells by random karyotypic rearrangements and selection for cancer-specific reproductive autonomy. Since such rearrangements unbalance long-established mitosis genes, cancer karyotypes vary spontaneously but are stabilized perpetually by clonal selections for autonomy. To test this theory we have analyzed neoplastic clones, presumably immortalized by transfection with overexpressed telomerase or with SV40 tumor virus, for the predicted clonal yet flexible karyotypes. The following results were obtained: (1) All immortal tumorigenic lines from cells transfected with overexpressed telomerase had clonal and flexible karyotypes; (2) Searching for the origin of such karyotypes, we found spontaneously increasing, random aneuploidy in human fibroblasts early after transfection with overexpressed telomerase; (3) Late after transfection, new immortal tumorigenic clones with new clonal and flexible karyotypes were found; (4) Testing immortality of one clone during 848 unselected generations showed the chromosome number was stable, but the copy numbers of 36% of chromosomes drifted ± 1; (5) Independent immortal tumorigenic clones with individual, flexible karyotypes arose after individual latencies; (6) Immortal tumorigenic clones with new flexible karyotypes also arose late from cells of a telomerase-deficient mouse rendered aneuploid by SV40 virus. Because immortality and tumorigenicity: (1) correlated exactly with individual clonal but flexible karyotypes; (2) originated simultaneously with such karyotypes; and (3) arose in the absence of telomerase, we conclude that clonal and flexible karyotypes generate the immortality of cancers. PMID:23388461

  5. Expression of cellular genes in HPV16-immortalized and cigarette smoke condensate-transformed human endocervical cells.

    Science.gov (United States)

    Yang, X; Nakao, Y; Pater, M M; Tang, S C; Pater, A

    1997-09-01

    We studied the molecular mechanism of successive multistep cervical carcinogenic progression with our previously established in vitro model system. This system was composed of primary human endocervical cells (HEN), two lines of HEN immortalized by HPV16 and their counterparts subsequently malignantly transformed by cigarette smoke condensate (CSC). The expression was examined of diverse cellular genes associated with oncogenesis and senescence, especially for cervical cancer. Consistent results were seen for the pairs of immortalized and malignantly transformed lines. Immortalization of HEN by HPV16 resulted in enhanced expression of H-ras, c-myc, B-myb, p53, p16INK4 and PCNA mRNA; enhanced expression of p16 and PCNA proteins; decreased expression of WAF1/p21/Cip1/Sid1 and fibronectin mRNA; and decreased p53 protein. On the other hand, the CSC-transformed counterparts of HPV16-immortalized cells had up-regulated levels of B-myb, p53 and WAF1 mRNA and p53 protein. Our results indicate that the differential activation or inactivation of multiple cellular genes is important for the immortalization, as well as the transformation, of human cervical cells. Further, we suggest that our in vitro model system is useful for investigating the molecular mechanism of multistep cervical carcinogenesis.

  6. Asymmetric migration of human keratinocytes under mechanical stretch and cocultured fibroblasts in a wound repair model.

    Directory of Open Access Journals (Sweden)

    Dongyuan Lü

    Full Text Available Keratinocyte migration during re-epithelization is crucial in wound healing under biochemical and biomechanical microenvironment. However, little is known about the underlying mechanisms whereby mechanical tension and cocultured fibroblasts or keratinocytes modulate the migration of keratinocytes or fibroblasts. Here we applied a tensile device together with a modified transwell assay to determine the lateral and transmembrane migration dynamics of human HaCaT keratinocytes or HF fibroblasts. A novel pattern of asymmetric migration was observed for keratinocytes when they were cocultured with non-contact fibroblasts, i.e., the accumulative distance of HaCaT cells was significantly higher when moving away from HF cells or migrating from down to up cross the membrane than that when moving close to HF cells or when migrating from up to down, whereas HF migration was symmetric. This asymmetric migration was mainly regulated by EGF derived from fibroblasts, but not transforming growth factor α or β1 production. Mechanical stretch subjected to fibroblasts fostered keratinocyte asymmetric migration by increasing EGF secretion, while no role of mechanical stretch was found for EGF secretion by keratinocytes. These results provided a new insight into understanding the regulating mechanisms of two- or three-dimensional migration of keratinocytes or fibroblasts along or across dermis and epidermis under biomechanical microenvironment.

  7. Quantitative assessment of the relative antineoplastic potential of the n-butanolic leaf extract of Annona muricata Linn. in normal and immortalized human cell lines.

    Science.gov (United States)

    George, V Cijo; Kumar, D R Naveen; Rajkumar, V; Suresh, P K; Kumar, R Ashok

    2012-01-01

    Natural products have been the target for cancer therapy for several years but there is still a dearth of information on potent compounds that may protect normal cells and selectively destroy cancerous cells. The present study was aimed to evaluate the cytotoxic potential of n-butanolic leaf extract of Annona muricata L. on WRL-68 (normal human hepatic cells), MDA-MB-435S (human breast carcinoma cells) and HaCaT (human immortalized keratinocyte cells) lines by XTT assay. Prior to cytotoxicity testing, the extract was subjected to phytochemical screening for detecting the presence of compounds with therapeutic potential. Their relative antioxidant properties were evaluated using the reducing power and DPPH* radical scavenging assay. Since most of the observed chemo-preventive potential invariably correlated with the amount of total phenolics present in the extract, their levels were quantified and identified by HPLC analysis. Correlation studies indicated a strong and significant (PAnnona muricata. Isolation of the active metabolites from the extract is in prospect.

  8. Functional genomic and radiosensitivity of human keratinocytes: from differentiated to stem cells

    Energy Technology Data Exchange (ETDEWEB)

    Lamartine, J.; Rachidi, W.; Franco, N.; Lemaitre, G.; Vaigot, P.; Le Minter, P.; Waksman, G.; Martin, M.T. [Evry Univ. Lab. of Genomic and Radiobiology of Keratinocytes, Service de Genomique Fonctionnelle, CEA, 91 (France)

    2006-07-01

    Despite improvements in radiation techniques, patients can still experience radiation toxicity on the skin. Keratinocytes from the basal layer of the epidermis have been long proposed by ICRP as the main target of ionizing radiation in human skin, both for early and late effects of radiation. But the exact roles of these cells in radiation skin damage are still largely unknown., This is why a new program was started to define the radiosensitivity of human keratinocytes according to their differentiation. In a first study was characterized the response of differentiated keratinocytes to low and high doses of gamma radiation (1). To examine whether the response to low doses was different from that induced by high doses, cultured primary keratinocytes isolated from adult normal skin were irradiated with single doses of 1 c Gray or 2 c Gy. A major finding of this study was the identification of an important number of low dose specific genes (140), most of which were modulated at 48 h. Clustering analysis also revealed low dose specific profiles. These results show for the first time that low dose ionizing irradiation is able to induce specific transcriptional responses in human keratinocytes. Then came the part to characterize the radiosensitivity of human basal keratinocytes. The results show for the first time that keratinocytes stem cells from human epidermis are more resistant than proliferative basal keratinocytes. In summary, using cellular biology and functional genomics, we are improving the knowledge on the effects of ionizing radiation on human epidermis, one of the main target tissue of radiation in the human body. (N.C.)

  9. Human epidermal keratinocyte cell response on integrin-specific artificial extracellular matrix proteins.

    Science.gov (United States)

    Tjin, Monica Suryana; Chua, Alvin Wen Choong; Ma, Dong Rui; Lee, Seng Teik; Fong, Eileen

    2014-08-01

    Cell-matrix interactions play critical roles in regulating cellular behavior in wound repair and regeneration of the human skin. In particular, human skin keratinocytes express several key integrins such as alpha5beta1, alpha3beta1, and alpha2beta1 for binding to the extracellular matrix (ECM) present in the basement membrane in uninjured skin. To mimic these key integrin-ECM interactions, artificial ECM (aECM) proteins containing functional domains derived from laminin 5, type IV collagen, fibronectin, and elastin are prepared. Human skin keratinocyte cell responses on the aECM proteins are specific to the cell-binding domain present in each construct. Keratinocyte attachment to the aECM protein substrates is also mediated by specific integrin-material interactions. In addition, the aECM proteins are able to support the proliferation of keratinocyte stem cells, demonstrating their promise for use in skin tissue engineering.

  10. A long-term hepatitis B viremia model generated by transplanting nontumorigenic immortalized human hepatocytes in Rag-2-deficient mice

    NARCIS (Netherlands)

    Parashar, B; Moshage, H; Tanaka, KE; Engelhardt, D; Rabbani, E; Roy-Chowdhury, N; Roy-Chowdhury, J

    2000-01-01

    Development of new therapies for human hepatitis B virus infection (HBV) would be greatly facilitated by the availability of a suitable small-animal model for HBV virus production in vivo. To develop a murine model for HBV production, we established an immortalized, cloned liver cell line by transfe

  11. Promoting effect of lactoferrin on barrier function and epithelial differentiation of human keratinocytes.

    Science.gov (United States)

    Uchida, Ryo; Aoki, Reiji; Aoki-Yoshida, Ayako; Tajima, Atsushi; Takayama, Yoshiharu

    2017-02-01

    The purpose of this study was to elucidate the effects of bovine lactoferrin on keratinocyte differentiation and barrier function. Addition of bovine lactoferrin to differentiating HaCaT human keratinocytes led to increased transepithelial electrical resistance (TER), a marker of epithelial barrier function. This elevation was followed by upregulation of two differentiation markers, involucrin and filaggrin. The expression level of sterol regulatory element-binding protein-1 was also enhanced by bovine lactoferrin. The lactoferrin-induced upregulation of involucrin and filaggrin expression were confirmed in normal human epidermal keratinocytes (NHEK). Treatment with SB203580, a p38 mitogen-activated protein kinase (MAPK) α inhibitor, impaired the upregulation of involucrin and filaggrin expression in response to lactoferrin. The elevation of p38 MAPK phosphorylation was further enhanced by lactoferrin in the initial stage of differentiation of HaCaT keratinocytes. The findings suggest that bovine lactoferrin promotes epithelial differentiation by a p38-MAPK-dependent mechanism.

  12. Reliable and versatile immortal muscle cell models from healthy and myotonic dystrophy type 1 primary human myoblasts.

    Science.gov (United States)

    Pantic, Boris; Borgia, Doriana; Giunco, Silvia; Malena, Adriana; Kiyono, Tohru; Salvatori, Sergio; De Rossi, Anita; Giardina, Emiliano; Sangiuolo, Federica; Pegoraro, Elena; Vergani, Lodovica; Botta, Annalisa

    2016-03-01

    Primary human skeletal muscle cells (hSkMCs) are invaluable tools for deciphering the basic molecular mechanisms of muscle-related biological processes and pathological alterations. Nevertheless, their use is quite restricted due to poor availability, short life span and variable purity of the cells during in vitro culture. Here, we evaluate a recently published method of hSkMCs immortalization, relying on ectopic expression of cyclin D1 (CCND1), cyclin-dependent kinase 4 (CDK4) and telomerase (TERT) in myoblasts from healthy donors (n=3) and myotonic dystrophy type 1 (DM1) patients (n=2). The efficacy to maintain the myogenic and non-transformed phenotype, as well as the main pathogenetic hallmarks of DM1, has been assessed. Combined expression of the three genes i) maintained the CD56(NCAM)-positive myoblast population and differentiation potential; ii) preserved the non-transformed phenotype and iii) maintained the CTG repeat length, amount of nuclear foci and aberrant alternative splicing in immortal muscle cells. Moreover, immortal hSkMCs displayed attractive additional features such as structural maturation of sarcomeres, persistence of Pax7-positive cells during differentiation and complete disappearance of nuclear foci following (CAG)7 antisense oligonucleotide (ASO) treatment. Overall, the CCND1, CDK4 and TERT immortalization yields versatile, reliable and extremely useful human muscle cell models to investigate the basic molecular features of human muscle cell biology, to elucidate the molecular pathogenetic mechanisms and to test new therapeutic approaches for DM1 in vitro.

  13. Immortalization protocols used in cell culture models of human breast morphogenesis

    DEFF Research Database (Denmark)

    Gudjonsson, T; Villadsen, R; Rønnov-Jessen, L

    2004-01-01

    of the tissue of origin. In recent years, we have sought to establish immortalized primary breast cells, which retain crucial characteristics of their original in situ tissue pattern. This review discusses various approaches to immortalization of breast-derived epithelial and stromal cells and the application...

  14. Effects of titanium dioxide nanoparticles on human keratinocytes.

    Science.gov (United States)

    Wright, Clayton; Iyer, Anand Krishnan V; Wang, Liying; Wu, Nianqiang; Yakisich, Juan S; Rojanasakul, Yon; Azad, Neelam

    2017-01-01

    Titanium dioxide (TiO2) is a ubiquitous whitening compound widely used in topical products such as sunscreens, lotions and facial creams. The damaging health effects of TiO2 inhalation has been widely studied in rats, mice and humans showing oxidative stress increase, DNA damage, cell death and inflammatory gene upregulation in lung and throat cells; however, the effects on skin cells from long-term topical use of various products remain largely unknown. In this study, we assessed the effect of specific TiO2 nanoparticles (H2TiO7) on a human keratinocyte cell line (HaCaT). We performed a comparative analysis using three TiO2 particles varying in size (Fine, Ultrafine and H2TiO7) and analyzed their effects on HaCaTs. There is a clear dose-dependent increase in superoxide production, caspase 8 and 9 activity, and apoptosis in HaCaTs after treatment with all three forms of TiO2; however, there is no consistent effect on cell viability and proliferation with either of these TiO2 particles. While there is data suggesting UV exposure can enhance the carcinogenic effects of TiO2, we did not observe any significant effect of UV-C exposure combined with TiO2 treatment on HaCaTs. Furthermore, TiO2-treated cells showed minimal effects on VEGF upregulation and Wnt signaling pathway thereby showing no potential effect on angiogenesis and malignant transformation. Overall, we report here an increase in apoptosis, which may be caspase 8/Fas-dependent, and that the H2TiO7 nanoparticles, despite their smaller particle size, had no significant enhanced effect on HaCaT cells as compared to Fine and Ultrafine forms of TiO2.

  15. Study of HLA-DR synthesis in cultured human keratinocytes.

    Science.gov (United States)

    Wikner, N E; Huff, J C; Norris, D A; Boyce, S T; Cary, M; Kissinger, M; Weston, W L

    1986-11-01

    Within the normal human epidermis only Langerhans and indeterminate cells express HLA-DR. Human keratinocytes (HK), however, may also express HLA-DR in certain disease states characterized by mononuclear cell infiltrates. Previous studies have shown that HK synthesize HLA-DR in response to stimulation by interferon gamma (INF-gamma). The purposes of this study were to define conditions under which cultured HK might express HLA-DR and to compare the HLA-DR synthesis of HK with that of monocytes. HLA-DR expression by HK as determined by indirect immunofluorescence of HK cultures was absent under standard low calcium conditions and remained absent with the addition of calcium, serum, mitogens, and supernatants from Pam-212 cells containing epidermal thymocyte-activating factor. HLA-DR expression in HK was induced by cocultivation with concanavalin A-stimulated peripheral blood mononuclear cells (PBMC), but not unstimulated PBMC. This effect was time-dependent and directly related to the number of PBMC. HLA-DR expression was also induced in a time- and dose-dependent manner by addition of supernatant from stimulated PBMC (SS) or by addition of recombinant INF-gamma but not by addition of interleukin (IL)-1 or IL-2. Induction by either SS or INF-gamma was blocked by an antiserum to INF-gamma. As determined by a semiquantitative immunoprecipitation technique, HLA-DR synthesis by HK was directly related to INF-gamma concentration. The pattern of HLA-DR peptides produced by HK was similar to that of monocytes, but the relative quantity synthesized was far less than that of monocytes.

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

    Science.gov (United States)

    Khanna, Sugandha; Darbre, Philippa D

    2013-05-01

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

  17. Remifentanil protects human keratinocytes against hypoxia-reoxygenation injury through activation of autophagy.

    Science.gov (United States)

    Kwon, Jae-Young; Park, Bong-Soo; Kim, Yong-Ho; Kim, Yong-Deok; Kim, Cheul-Hong; Yoon, Ji-Young; Yoon, Ji-Uk

    2015-01-01

    The proliferation, differentiation, and migration of keratinocytes are essential in the early stages of wound healing. Hypoxia-Reoxygenation (H/R) injury to keratinocytes can occur in various stressful environments such as surgery, trauma, and various forms of ulcers. The effects of remifentanil on human keratinocytes under hypoxia-reoxygenation have not been fully studied. Therefore, we investigated the effects of remifentanil on the proliferation, apoptosis, and autophagic activation of human keratinocytes during hypoxic-reoxygenation. Human keratinocytes were cultured under 1% oxygen tension for 24 h. The cells were then treated with various concentrations of remifentanil (0.01, 0.1, 0.5, and 1 ng/mL) for 2 h. Thereafter, the cells were reoxygenated for 12 h at 37°C. We measured cell viability via MTT assay. Using quantitative real-time PCR and western blot analysis, we measured the expression levels of proteins associated with apoptosis and autophagy. Quantification of apoptotic cells was performed using flow cytometer analysis and autophagic vacuoles were observed under a fluorescence microscope. Remifentanil treatment brought about an increase in the proliferation of human keratinocytes damaged by hypoxia-reoxygenation and decreased the apoptotic cell death, enhancing autophagic activity. However, the autophagy pathway inhibitor 3-MA inhibited the protective effect of remifentanil in hypoxia-reoxygenation injury. In conclusion, the current study demonstrated that remifentanil treatment stimulated autophagy and reduced apoptotic cell death in a hypoxia-reoxygenation model of human keratinocytes. Our results provide additional insights into the relationship between apoptosis and autophagy.

  18. Remifentanil protects human keratinocytes against hypoxia-reoxygenation injury through activation of autophagy.

    Directory of Open Access Journals (Sweden)

    Jae-Young Kwon

    Full Text Available The proliferation, differentiation, and migration of keratinocytes are essential in the early stages of wound healing. Hypoxia-Reoxygenation (H/R injury to keratinocytes can occur in various stressful environments such as surgery, trauma, and various forms of ulcers. The effects of remifentanil on human keratinocytes under hypoxia-reoxygenation have not been fully studied. Therefore, we investigated the effects of remifentanil on the proliferation, apoptosis, and autophagic activation of human keratinocytes during hypoxic-reoxygenation. Human keratinocytes were cultured under 1% oxygen tension for 24 h. The cells were then treated with various concentrations of remifentanil (0.01, 0.1, 0.5, and 1 ng/mL for 2 h. Thereafter, the cells were reoxygenated for 12 h at 37°C. We measured cell viability via MTT assay. Using quantitative real-time PCR and western blot analysis, we measured the expression levels of proteins associated with apoptosis and autophagy. Quantification of apoptotic cells was performed using flow cytometer analysis and autophagic vacuoles were observed under a fluorescence microscope. Remifentanil treatment brought about an increase in the proliferation of human keratinocytes damaged by hypoxia-reoxygenation and decreased the apoptotic cell death, enhancing autophagic activity. However, the autophagy pathway inhibitor 3-MA inhibited the protective effect of remifentanil in hypoxia-reoxygenation injury. In conclusion, the current study demonstrated that remifentanil treatment stimulated autophagy and reduced apoptotic cell death in a hypoxia-reoxygenation model of human keratinocytes. Our results provide additional insights into the relationship between apoptosis and autophagy.

  19. Cytokine secretion profiles of human keratinocytes during Trichophyton tonsurans and Arthroderma benhamiae infections.

    Science.gov (United States)

    Shiraki, Yumi; Ishibashi, Yoshio; Hiruma, Masataro; Nishikawa, Akemi; Ikeda, Shigaku

    2006-09-01

    Dermatophytes cause intractable superficial infections in humans. Arthroderma benhamiae, a zoophilic dermatophyte, triggers severe inflammatory responses in humans, while Trichophyton tonsurans, an anthropophilic dermatophyte, triggers minimal ones. Cytokines and other factors derived from keratinocytes play important roles in inflammatory and immune responses in the skin. The authors performed an in vitro investigation to determine the human keratinocyte cytokine profiles during dermatophyte infection. The human keratinocyte cell line PHK16-0b was infected with A. benhamiae or T. tonsurans for 24 h, and the cytokines secreted were analysed using a human cytokine antibody array. Marked differences were observed in the cytokine profiles of the cells infected with the two dermatophytes. A. benhamiae infection resulted in the secretion of a broad spectrum of cytokines, including proinflammatory cytokines, chemokines, and immunomodulatory cytokines. In contrast, T. tonsurans-infected keratinocytes secreted only limited cytokines, including eotaxin-2, interleukin (IL)-8 and IL-16. cDNA microarray analysis confirmed that A. benhamiae infection upregulated genes encoding IL-1beta, IL-2, IL-4, IL-6, IL-10, IL-13, IL-15, IL-16, IL-17 and interferon (IFN)-gamma, while T. tonsurans infection upregulated only a few genes, such as those encoding IL-1beta and IL-16. RT-PCR demonstrated that infection by both dermatophytes enhanced IL-8 mRNA expression in keratinocytes. These results suggest that A. benhamiae-induced secretion of several cytokines from keratinocytes may be involved in a severe inflammatory response, and that the limited cytokine secretion from keratinocytes in response to T. tonsurans infection may result in a minimal inflammatory response in the skin. These cytokine profiles may aid in proving the clinical features of dermatophytosis.

  20. Exome-wide Mutation Profile in Benzo[a]pyrene-derived Post-stasis and Immortal Human Mammary Epithelial Cells

    Science.gov (United States)

    Severson, Paul L.; Vrba, Lukas; Stampfer, Martha R.; Futscher, Bernard W.

    2014-01-01

    Genetic mutations are known to drive cancer progression and certain tumors have mutation signatures that reflect exposures to environmental carcinogens. Benzo[a]pyrene (BaP) has a known mutation signature and has proven capable of inducing changes to DNA sequence that drives normal pre-stasis human mammary epithelial cells (HMEC) past a first tumor suppressor barrier (stasis) and towards immortality. We analyzed normal, pre-stasis HMEC, three independent BaP-derived post-stasis HMEC strains (184Aa, 184Be, 184Ce) and two of their immortal derivatives(184A1 and 184BE1) by whole exome sequencing. The independent post-stasis strains exhibited between 93 and 233 BaP-induced mutations in exons. Seventy percent of the mutations were C:G>A:T transversions, consistent with the known mutation spectrum of BaP. Mutations predicted to impact protein function occurred in several known and putative cancer drivers including p16, PLCG1, MED12, TAF1 in 184Aa; PIK3CG, HSP90AB1, WHSC1L1, LCP1 in 184Be and FANCA, LPP in 184Ce. Biological processes that typically harbor cancer driver mutations such as cell cycle, regulation of cell death and proliferation, RNA processing, chromatin modification and DNA repair were found to have mutations predicted to impact function in each of the post-stasis strains. Spontaneously immortalized HMEC lines derived from two of the BaP-derived post-stasis strains shared greater than 95% of their BaP-induced mutations with their precursor cells. These immortal HMEC had 10 or fewer additional point mutations relative to their post-stasis precursors, but acquired chromosomal anomalies during immortalization that arose independent of BaP. The results of this study indicate that acute exposures of HMEC to high dose BaP recapitulate mutation patterns of human tumors and can induce mutations in a number of cancer driver genes. PMID:25435355

  1. Effects of human papillomavirus (HPV type 16 oncoproteins on the expression of involucrin in human keratinocytes

    Directory of Open Access Journals (Sweden)

    Gyöngyösi Eszter

    2012-02-01

    Full Text Available Abstract Background The human papillomavirus (HPV life cycle is closely linked to keratinocyte differentiation. Oncogenic HPV infection has been shown to hamper the normal differentiation of keratinocytes; however, the underlying mechanisms responsible for this phenomenon are yet to be clarified. Here, we aimed to study the effects of HPV16 E6 and E7 oncogenes on the expression of involucrin (IVL, an established marker of keratinocyte differentiation, in human foreskin keratinocyte (HFK cells. Results The differentiation of HFK cells by serum and high calcium significantly increased both the mRNA and the protein levels of IVL. The E6 and E7 oncoproteins of HPV16 together caused strong down-regulation of IVL mRNA and protein both in proliferating and in differentiating HFK cells. To study the effects of HPV oncogenes on the IVL promoter, we made transient transfection assays and luciferase tests and found that HPV 16 E6 but not E7 repressed IVL promoter activity in proliferating HFK cells. The inhibitory effect of HPV 16 E6 on the human IVL promoter could be localised to the proximal regulatory region (PRR of the gene. Conclusions These results suggest that the down-regulation of IVL promoter activity by HPV 16 E6 significantly contribute to the inhibition of endogenous IVL expression by the HPV 16 oncoproteins. In contrast, the down-regulation of endogenous IVL expression by HPV16 E7 is probably not caused by a direct and specific effect of E7 on the IVL promoter.

  2. Human Papilloma Viral DNA Replicates as a Stable Episome in Cultured Epidermal Keratinocytes

    Science.gov (United States)

    Laporta, Robert F.; Taichman, Lorne B.

    1982-06-01

    Human papilloma virus (HPV) is poorly understood because systems for its growth in tissue culture have not been developed. We report here that cultured human epidermal keratinocytes could be infected with HPV from plantar warts and that the viral DNA persisted and replicated as a stable episome. There were 50-200 copies of viral DNA per cell and there was no evidence to indicate integration of viral DNA into the cellular genome. There was also no evidence to suggest that viral DNA underwent productive replication. We conclude that cultured human epidermal keratinocytes may be a model for the study of certain aspects of HPV biology.

  3. Human keratinocytes restrict chikungunya virus replication at a post-fusion step

    Energy Technology Data Exchange (ETDEWEB)

    Bernard, Eric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Hamel, Rodolphe [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Neyret, Aymeric [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Ekchariyawat, Peeraya [Laboratoire Maladies Infectieuses et Vecteurs: Ecologie, Génétique, Evolution, Contrôle, UMR 5290 CNRS/IRD/UM1, Montpellier (France); Molès, Jean-Pierre [INSERM U1058, UM1, CHU Montpellier (France); Simmons, Graham [Blood Systems Research Institute, San Francisco, CA 94118 (United States); Chazal, Nathalie [Centre d' étude d’agents Pathogènes et Biotechnologies pour la Santé, CPBS CNRS- UMR5236/UM1/UM2, Montpellier (France); Desprès, Philippe [Unité Interactions Moléculaires Flavivirus-Hôtes, Institut Pasteur, Paris (France); and others

    2015-02-15

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enhanced transcription of type I/II and type III interferon genes. Concomitantly, we show that despite viral particles internalization into Rab5-positive endosomes and efficient fusion of virus and cell membranes, keratinocytes poorly replicate CHIKV as attested by absence of nonstructural proteins and genomic RNA synthesis. Accordingly, human keratinocytes behave as an antiviral defense against CHIKV infection rather than as a primary targets for initial replication. This picture significantly differs from that reported for Dengue and West Nile mosquito-borne viruses. - Highlights: • Human keratinocytes support endocytosis of CHIKV and fusion of viral membranes. • CHIKV replication is blocked at a post entry step in these cells. • Infection upregulates type-I, –II and –III IFN genes expression. • Keratinocytes behave as immune sentinels against CHIKV.

  4. A transcriptional roadmap to the senescence and differentiation of human oral keratinocytes.

    Science.gov (United States)

    Jang, Da Hyun; Bhawal, Ujjal K; Min, Hong-Ki; Kang, Hyun Ki; Abiko, Yoshimitsu; Min, Byung-Moo

    2015-01-01

    Human epithelial cells undergo morphological and molecular changes leading to terminal differentiation and replicative senescence after a finite number of cell divisions during serial subculture. However, the target genes and their functional significance in the senescence and differentiation in normal human oral keratinocytes have been poorly defined. Here, we demonstrated normal human oral keratinocytes transcriptional signature profiling to senescence and differentiation. Using microarray analysis, our findings indicated that the gene expression profiles induced by serial subculture are distinct classes of gene. The greatest number of these altered genes was identified as being related to biological pathways of transport, cell proliferation, cell cycle, defense and immune response, cell death, transcription, apoptosis, and inflammatory response, suggesting that the serial subculture is able to induce a multitude of specific gene expression changes during senescence and differentiation. Several highly upregulated genes (IL-1β, S100A8, S100A9, MMP1, MMP9, IL-8, BHLHB2, HES1, and TWIST1) in response to the serial subculture in normal human oral keratinocytes were observed. In vitro and in vivo studies also exhibited a close relationship between senescence and differentiation of primary oral keratinocytes and expression of inflammatory molecules. These results suggest a new approach to determine the biological events underlying the pathogenesis of oral keratinocyte aging. © The Author 2014. Published by Oxford University Press on behalf of The Gerontological Society of America. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  5. Genomic instability and tumorigenic induction in immortalized human bronchial epithelial cells by heavy ions

    Science.gov (United States)

    Hei, T. K.; Piao, C. Q.; Wu, L. J.; Willey, J. C.; Hall, E. J.

    1998-11-01

    Carcinogenesis is postulated to be a progressive multistage process characterized by an increase in genomic instability and clonal selection with each mutational event endowing a selective growth advantage. Genomic instability as manifested by the amplification of specific gene fragments is common among tumor and transformed cells. In the present study, immortalized human bronchial (BEP2D) cells were irradiated with graded doses of either 1GeV/nucleon 56Fe ions or 150 keV/μm alpha particles. Transformed cells developed through a series of successive steps before becoming tumorigenic in nude mice. Tumorigenic cells showed neither ras mutations nor deletion in the p16 tumor suppressor gene. In contrast, they harbored mutations in the p53 gene and over-expressed cyclin D1. Genomic instability among transformed cells at various stage of the carcinogenic process was examined based on frequencies of PALA resistance. Incidence of genomic instability was highest among established tumor cell lines relative to transformed, non-tumorigenic and control cell lines. Treatment of BEP2D cells with a 4 mM dose of the aminothiol WR-1065 significantly reduced their neoplastic transforming response to 56Fe particles. This model provides an opportunity to study the cellular and molecular mechanisms involved in malignant transformation of human epithelial cells by heavy ions.

  6. Characterization of lipid metabolism in insulin-sensitive adipocytes differentiated from immortalized human mesenchymal stem cells.

    Science.gov (United States)

    Prawitt, Janne; Niemeier, Andreas; Kassem, Moustapha; Beisiegel, Ulrike; Heeren, Joerg

    2008-02-15

    There is a great demand for cell models to study human adipocyte function. Here we describe the adipogenic differentiation of a telomerase-immortalized human mesenchymal stem cell line (hMSC-Tert) that maintains numerous features of terminally differentiated adipocytes even after prolonged withdrawal of the peroxisome proliferator activated receptor gamma (PPARgamma) agonist rosiglitazone. Differentiated hMSC-Tert developed the characteristic monolocular phenotype of mature adipocytes. The expression of adipocyte specific markers was highly increased during differentiation. Most importantly, the presence of the PPARgamma agonist rosiglitazone was not required for the stable expression of lipoprotein lipase, adipocyte fatty acid binding protein and perilipin on mRNA and protein levels. Adiponectin expression was post-transcriptionally down-regulated in the absence of rosiglitazone. Insulin sensitivity as measured by insulin-induced phosphorylation of Akt and S6 ribosomal protein was also independent of rosiglitazone. In addition to commonly used adipogenic markers, we investigated further PPARgamma-stimulated proteins with a role in lipid metabolism. We observed an increase of lipoprotein receptor (VLDLR, LRP1) and apolipoprotein E expression during differentiation. Despite this increased expression, the receptor-mediated endocytosis of lipoproteins was decreased in differentiated adipocytes, suggesting that these proteins may have an additional function in adipose tissue beyond lipoprotein uptake.

  7. Genome-wide transcriptional reorganization associated with senescence-to-immortality switch during human hepatocellular carcinogenesis.

    Science.gov (United States)

    Yildiz, Gokhan; Arslan-Ergul, Ayca; Bagislar, Sevgi; Konu, Ozlen; Yuzugullu, Haluk; Gursoy-Yuzugullu, Ozge; Ozturk, Nuri; Ozen, Cigdem; Ozdag, Hilal; Erdal, Esra; Karademir, Sedat; Sagol, Ozgul; Mizrak, Dilsa; Bozkaya, Hakan; Ilk, Hakki Gokhan; Ilk, Ozlem; Bilen, Biter; Cetin-Atalay, Rengul; Akar, Nejat; Ozturk, Mehmet

    2013-01-01

    Senescence is a permanent proliferation arrest in response to cell stress such as DNA damage. It contributes strongly to tissue aging and serves as a major barrier against tumor development. Most tumor cells are believed to bypass the senescence barrier (become "immortal") by inactivating growth control genes such as TP53 and CDKN2A. They also reactivate telomerase reverse transcriptase. Senescence-to-immortality transition is accompanied by major phenotypic and biochemical changes mediated by genome-wide transcriptional modifications. This appears to happen during hepatocellular carcinoma (HCC) development in patients with liver cirrhosis, however, the accompanying transcriptional changes are virtually unknown. We investigated genome-wide transcriptional changes related to the senescence-to-immortality switch during hepatocellular carcinogenesis. Initially, we performed transcriptome analysis of senescent and immortal clones of Huh7 HCC cell line, and identified genes with significant differential expression to establish a senescence-related gene list. Through the analysis of senescence-related gene expression in different liver tissues we showed that cirrhosis and HCC display expression patterns compatible with senescent and immortal phenotypes, respectively; dysplasia being a transitional state. Gene set enrichment analysis revealed that cirrhosis/senescence-associated genes were preferentially expressed in non-tumor tissues, less malignant tumors, and differentiated or senescent cells. In contrast, HCC/immortality genes were up-regulated in tumor tissues, or more malignant tumors and progenitor cells. In HCC tumors and immortal cells genes involved in DNA repair, cell cycle, telomere extension and branched chain amino acid metabolism were up-regulated, whereas genes involved in cell signaling, as well as in drug, lipid, retinoid and glycolytic metabolism were down-regulated. Based on these distinctive gene expression features we developed a 15-gene

  8. Keratinocyte growth factor and the expression of wound-healing-related genes in primary human keratinocytes from burn patients.

    Science.gov (United States)

    Chomiski, Verônica; Gragnani, Alfredo; Bonucci, Jéssica; Correa, Silvana Aparecida Alves; Noronha, Samuel Marcos Ribeiro de; Ferreira, Lydia Masako

    2016-08-01

    To evaluate the effect of keratinocyte growth factor (KGF) treatment on the expression of wound-healing-related genes in cultured keratinocytes from burn patients. Keratinocytes were cultured and divided into 4 groups (n=4 in each group): TKB (KGF-treated keratinocytes from burn patients), UKB (untreated keratinocytes from burn patients), TKC (KGF-treated keratinocytes from controls), and UKC (untreated keratinocytes from controls). Gene expression analysis using quantitative polymerase chain reaction (qPCR) array was performed to compare (1) TKC versus UKC, (2) UKB versus UKC, (3) TKB versus UKC, (4) TKB versus UKB, (5) TKB versus TKC, and (6) UKB versus TKC. Comparison 1 showed one down-regulated and one up-regulated gene; comparisons 2 and 3 resulted in the same five down-regulated genes; comparison 4 had no significant difference in relative gene expression; comparison 5 showed 26 down-regulated and 7 up-regulated genes; and comparison 6 showed 25 down-regulated and 11 up-regulated genes. There was no differential expression of wound-healing-related genes in cultured primary keratinocytes from burn patients treated with keratinocyte growth factor.

  9. Chitin modulates innate immune responses of keratinocytes.

    Directory of Open Access Journals (Sweden)

    Barbara Koller

    Full Text Available BACKGROUND: Chitin, after cellulose the second most abundant polysaccharide in nature, is an essential component of exoskeletons of crabs, shrimps and insects and protects these organisms from harsh conditions in their environment. Unexpectedly, chitin has been found to activate innate immune cells and to elicit murine airway inflammation. The skin represents the outer barrier of the human host defense and is in frequent contact with chitin-bearing organisms, such as house-dust mites or flies. The effects of chitin on keratinocytes, however, are poorly understood. METHODOLOGY/PRINCIPAL FINDINGS: We hypothesized that chitin stimulates keratinocytes and thereby modulates the innate immune response of the skin. Here we show that chitin is bioactive on primary and immortalized keratinocytes by triggering production of pro-inflammatory cytokines and chemokines. Chitin stimulation further induced the expression of the Toll-like receptor (TLR TLR4 on keratinocytes at mRNA and protein level. Chitin-induced effects were mainly abrogated when TLR2 was blocked, suggesting that TLR2 senses chitin on keratinocytes. CONCLUSIONS/SIGNIFICANCE: We speculate that chitin-bearing organisms modulate the innate immune response towards pathogens by upregulating secretion of cytokines and chemokines and expression of MyD88-associated TLRs, two major components of innate immunity. The clinical relevance of this mechanism remains to be defined.

  10. Geraniin supplementation increases human keratinocyte proliferation in serum-free culture

    Directory of Open Access Journals (Sweden)

    Indra Kusuma

    2013-04-01

    Full Text Available Background Various products used in cellular therapy utilize tissue culture techniques requiring keratinocyte culture. An efficient and clinically acceptable keratinocyte culture system requires supplements with mitogenic activity. Geraniin is a phytochemical with the potential as a supplement for expansion culture of keratinocytes. The objective of the present study was to verify the mitogenic activity of geraniin on human keratinocytes. Methods This was an experimental study using two samples of human foreskin obtained by circumcision of a male child. Epidermal keratinocytes were isolated from the foreskin samples and were divided into paired groups, comprising intervention and control groups. The intervention groups were cultured with geraniin supplementation, whereas the control groups with standard supplements, without the addition of geraniin. Mitochondrial activity of the cells was evaluated by means of the 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium-bromide (MTT proliferation assay. Absorbance values in each of the groups was measured at 450 nm. Data analysis was performed with the paired t-test. Results Geraniin supplementation significantly increased the keratinocyte proliferation rates at dosages of 0.8 to 3.1 µM. An increase of 57% in the proliferation rate was obtained at a dosage of 1.6 µM, while at a dosage of 12.5 µM toxic effects were starting to appear. Geraniin presumably causes increased cellular energy status, resulting in increased proliferation rates. Conclusion The findings in this study provide evidence in support of the utilization of geraniin as a supplement for expansion culture of keratinocytes. Further studies may presumably identify the molecules acting as geraniin receptors and the intracellular mechanisms underlying the increase in proliferation rates.

  11. Immortality, but not oncogenic transformation, of primary human cells leads to epigenetic reprogramming of DNA methylation and gene expression.

    Science.gov (United States)

    Gordon, Katrina; Clouaire, Thomas; Bao, Xun X; Kemp, Sadie E; Xenophontos, Maria; de Las Heras, Jose Ignacio; Stancheva, Irina

    2014-04-01

    Tumourigenic transformation of normal cells into cancer typically involves several steps resulting in acquisition of unlimited growth potential, evasion of apoptosis and non-responsiveness to growth inhibitory signals. Both genetic and epigenetic changes can contribute to cancer development and progression. Given the vast genetic heterogeneity of human cancers and difficulty to monitor cancer-initiating events in vivo, the precise relationship between acquisition of genetic mutations and the temporal progression of epigenetic alterations in transformed cells is largely unclear. Here, we use an in vitro model system to investigate the contribution of cellular immortality and oncogenic transformation of primary human cells to epigenetic reprogramming of DNA methylation and gene expression. Our data demonstrate that extension of replicative life span of the cells is sufficient to induce accumulation of DNA methylation at gene promoters and large-scale changes in gene expression in a time-dependent manner. In contrast, continuous expression of cooperating oncogenes in immortalized cells, although essential for anchorage-independent growth and evasion of apoptosis, does not affect de novo DNA methylation at promoters and induces subtle expression changes. Taken together, these observations imply that cellular immortality promotes epigenetic adaptation to highly proliferative state, whereas transforming oncogenes confer additional properties to transformed human cells.

  12. Human Keratinocyte Growth and Differentiation on Acellular Porcine Dermal Matrix in relation to Wound Healing Potential

    Directory of Open Access Journals (Sweden)

    Robert Zajicek

    2012-01-01

    Full Text Available A number of implantable biomaterials derived from animal tissues are now used in modern surgery. Xe-Derma is a dry, sterile, acellular porcine dermis. It has a remarkable healing effect on burns and other wounds. Our hypothesis was that the natural biological structure of Xe-Derma plays an important role in keratinocyte proliferation and formation of epidermal architecture in vitro as well as in vivo. The bioactivity of Xe-Derma was studied by a cell culture assay. We analyzed growth and differentiation of human keratinocytes cultured in vitro on Xe-Derma, and we compared the results with formation of neoepidermis in the deep dermal wounds treated with Xe-Derma. Keratinocytes cultured on Xe-Derma submerged in the culture medium achieved confluence in 7–10 days. After lifting the cultures to the air-liquid interface, the keratinocytes were stratified and differentiated within one week, forming an epidermis with basal, spinous, granular, and stratum corneum layers. Immunohistochemical detection of high-molecular weight cytokeratins (HMW CKs, CD29, p63, and involucrin confirmed the similarity of organization and differentiation of the cultured epidermal cells to the normal epidermis. The results suggest that the firm natural structure of Xe-Derma stimulates proliferation and differentiation of human primary keratinocytes and by this way improves wound healing.

  13. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage.

    Science.gov (United States)

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-07-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death.

  14. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage

    Science.gov (United States)

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Shin, Jennifer H; Park, Yeunsoo; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-01-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repressed UVB-facilitated programmed cell death in the keratinocytes, as evidenced by a reduction in apoptotic body formation, and nuclear fragmentation. Additionally, isorhamnetin suppressed the ability of UVB light to trigger mitochondrial dysfunction. Taken together, these results indicate that isorhamnetin has the potential to protect human keratinocytes against UVB-induced cell damage and death. PMID:26157553

  15. Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Xie, Xin [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China); Dai, Hui [Department of Cardiology, The First Affiliated Hospital of Harbin Medical University, Harbin, 150001, Heilongjiang Province (China); Zhuang, Binyu [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China); Chai, Li; Xie, Yanguang [Institute of Dermatology of Heilongjiang Province, Harbin, 150001, Heilongjiang Province (China); Li, Yuzhen, E-mail: liyuzhen@medmail.com.cn [Department of Dermatology, The Second Affiliated Hospital of Harbin Medical University, Harbin, 150086, Heilongjiang Province (China)

    2016-04-08

    The effects and the underlying mechanisms of hydrogen sulfide (H{sub 2}S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H{sub 2}S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H{sub 2}S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagic vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H{sub 2}S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation. - Highlights: • Exogenous H{sub 2}S promotes keratinocyte proliferation and differentiation. • The effects of H{sub 2}S on proliferation and differentiation is modulated by autophagy. • Exogenous H{sub 2}S has no effect on keratinocyte apoptosis.

  16. Immortalization of human umbilical vein endothelial cells with telomerase reverse transcriptase and simian virus 40 large T antigen

    Institute of Scientific and Technical Information of China (English)

    BIAN Chang; ZHAO Kui; TONG Guo-xin; ZHU Yong-liang; CHEN Peng

    2005-01-01

    Objective: To establish normally conditionally-immortalized human umbilical vein endothelial cells (HUVECs) by ectopic expression of the human telomerase catalytic enzyme (hTERT) and simian virus 40 large T (SV40 LT) antigen. Methods:Primary HUVECs were transfected with recombinant retrovirus containing hTERT or SV40 LT respectively. Subsequently drug resistant cell clones were screened and expanded for further studies. Endothelial cell biomarkers were confirmed by examination.Results: The morphological phenotype of the transfected cells was similar to the non-transfected cells. Von Willebrand factor,hTERT and SV40 LT could be detected in transfected HUVECs. Moreover, higher telomerase activity in transfected cells was maintained for over 50 population doublings compared with only low level of endogenous telomerase transiently at early population doublings in primary HUVECs. When exposed to TNF-α (tumor necrosis factor-α), the expression of E-selectin in transfected cells was significantly up-regulated, but no alteration of endothelial lipase was found. Conclusion: Ectopic coexpression of hTERT and SV40 LT can effectively immortalize HUVECs without tumorigenicity in vitro. Immortalized HUVECs may be an ideal target of further molecular function studies.

  17. Zeno's Paradox of Immortality.

    Science.gov (United States)

    Olshansky, S Jay; Carnes, Bruce A

    2013-01-01

    Scientists who speculate on the future of human longevity have a broad range of views ranging from the promise of immortality, to radical life extension, to declines in life expectancy. Among those who contend that radical life extension is already here, or on the horizon, or immortality is forthcoming, elements of their reasoning appear surprisingly close, if not identical, to a famous mathematical paradox posed by the ancient Greek mathematician known as Zeno. Here we examine the underlying assumptions behind the views that much longer life expectancies are forthcoming or have already arrived, and place their line of reasoning within the context of a new Zeno paradox described here as The Paradox of Immortality. Copyright © 2012 S. Karger AG, Basel.

  18. Establishment of a novel method for primary culture of normal human cervical keratinocytes

    Institute of Scientific and Technical Information of China (English)

    LIU Yu-zhen; L(U) Xiu-ping; PAN Zi-xuan; ZHANG Wei; CHEN Zhao-ri; WANG Hui; LIU Hua

    2013-01-01

    Background Cervical keratinocytes are recovered at a low numbers and frequently associated with contaminating human fibroblasts which rapidly overgrow the epithelial cells in culture with medium supplemented with 10% fetal bovine serum (FBS).However,it is difficult to initiate keratinocyte cultures with serum-free keratinocyte growth medium alone because cell attachment can be poor.Therefore,the culture of these cells is extremely difficult.In this study,we described a modified culture medium and coated culture plastics for growing normal human cervical epithelial cells in vitro.Methods Normal cervical epithelial tissue pieces were obtained and digested with type Ⅰ collagenase to dissociate the cells and a single cell suspension produced.The cells were cultured on plastic tissue culture substrate alone or substrate coated with collagen type Ⅰ from rat tail,with modified keratinocyte serum-free medium (K-SFM) supplemented with 5% FBS.After attachment,the medium were replaced with K-SFM without FBS.The expression of basal keratins of the ectocervical epithelium,K5,K14 and K19 were assayed by immunofiuorescence with monoclonal antibodies to identify the cell purity.Results Our results indicate that cells attached to the culture plastic more quickly in K-SFM supplemented with 5%FBS than in K-SFM alone,as well as to tissue culture plastic coated with collagen type Ⅰ than plastic alone.The modified medium composed of K-SFM and 5% FBS combined with a specific tissue culture plastic coated with collagen type Ⅰ from rat tail was the best method for culture of normal cervical epithelial cells.K5,K14 and K19 were assayed and keratinocyte purity was nearly 100%.Conclusion A novel,simple and effective method can be used to rapidly obtain highly purified keratinocytes from normal human cervical epithelium.

  19. Exogenous hydrogen sulfide promotes cell proliferation and differentiation by modulating autophagy in human keratinocytes.

    Science.gov (United States)

    Xie, Xin; Dai, Hui; Zhuang, Binyu; Chai, Li; Xie, Yanguang; Li, Yuzhen

    2016-04-01

    The effects and the underlying mechanisms of hydrogen sulfide (H2S) on keratinocyte proliferation and differentiation are still less known. In the current study, we investigated the effects and the underlying mechanisms of exogenous H2S on keratinocyte proliferation and differentiation. Human keratinocytes (HaCaT cells) were treated with various concentrations (0.05, 0.25, 0.5 and 1 mM) of sodium hydrosulfide (NaHS, a donor of H2S) for 24 h. A CCK-8 assay was used to assess cell viability. Western blot analysis was performed to determine the expression levels of proteins associated with differentiation and autophagy. Transmission electron microscopy was performed to observe autophagic vacuoles, and flow cytometry was applied to evaluate apoptosis. NaHS promoted the viability, induced the differentiation, and enhanced autophagic activity in a dose-dependent manner in HaCaT cells but had no effect on cell apoptosis. Blockage of autophagy by ATG5 siRNA inhibited NaHS-induced cell proliferation and differentiation. The current study demonstrated that autophagy in response to exogenous H2S treatment promoted keratinocyte proliferation and differentiation. Our results provide additional insights into the potential role of autophagy in keratinocyte proliferation and differentiation.

  20. Generation of Integration-free Human Induced Pluripotent Stem Cells Using Hair-derived Keratinocytes.

    Science.gov (United States)

    Hung, Sandy S C; Pébay, Alice; Wong, Raymond C B

    2015-08-20

    Recent advances in reprogramming allow us to turn somatic cells into human induced pluripotent stem cells (hiPSCs). Disease modeling using patient-specific hiPSCs allows the study of the underlying mechanism for pathogenesis, also providing a platform for the development of in vitro drug screening and gene therapy to improve treatment options. The promising potential of hiPSCs for regenerative medicine is also evident from the increasing number of publications (>7000) on iPSCs in recent years. Various cell types from distinct lineages have been successfully used for hiPSC generation, including skin fibroblasts, hematopoietic cells and epidermal keratinocytes. While skin biopsies and blood collection are routinely performed in many labs as a source of somatic cells for the generation of hiPSCs, the collection and subsequent derivation of hair keratinocytes are less commonly used. Hair-derived keratinocytes represent a non-invasive approach to obtain cell samples from patients. Here we outline a simple non-invasive method for the derivation of keratinocytes from plucked hair. We also provide instructions for maintenance of keratinocytes and subsequent reprogramming to generate integration-free hiPSC using episomal vectors.

  1. Improvement of human keratinocyte migration by a redox active bioelectric dressing.

    Science.gov (United States)

    Banerjee, Jaideep; Das Ghatak, Piya; Roy, Sashwati; Khanna, Savita; Sequin, Emily K; Bellman, Karen; Dickinson, Bryan C; Suri, Prerna; Subramaniam, Vish V; Chang, Christopher J; Sen, Chandan K

    2014-01-01

    Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED) which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i) generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii) phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii) reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization.

  2. Improvement of human keratinocyte migration by a redox active bioelectric dressing.

    Directory of Open Access Journals (Sweden)

    Jaideep Banerjee

    Full Text Available Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization.

  3. Improvement of Human Keratinocyte Migration by a Redox Active Bioelectric Dressing

    Science.gov (United States)

    Banerjee, Jaideep; Das Ghatak, Piya; Roy, Sashwati; Khanna, Savita; Sequin, Emily K.; Bellman, Karen; Dickinson, Bryan C.; Suri, Prerna; Subramaniam, Vish V.; Chang, Christopher J.; Sen, Chandan K.

    2014-01-01

    Exogenous application of an electric field can direct cell migration and improve wound healing; however clinical application of the therapy remains elusive due to lack of a suitable device and hence, limitations in understanding the molecular mechanisms. Here we report on a novel FDA approved redox-active Ag/Zn bioelectric dressing (BED) which generates electric fields. To develop a mechanistic understanding of how the BED may potentially influence wound re-epithelialization, we direct emphasis on understanding the influence of BED on human keratinocyte cell migration. Mapping of the electrical field generated by BED led to the observation that BED increases keratinocyte migration by three mechanisms: (i) generating hydrogen peroxide, known to be a potent driver of redox signaling, (ii) phosphorylation of redox-sensitive IGF1R directly implicated in cell migration, and (iii) reduction of protein thiols and increase in integrinαv expression, both of which are known to be drivers of cell migration. BED also increased keratinocyte mitochondrial membrane potential consistent with its ability to fuel an energy demanding migration process. Electric fields generated by a Ag/Zn BED can cross-talk with keratinocytes via redox-dependent processes improving keratinocyte migration, a critical event in wound re-epithelialization. PMID:24595050

  4. Low electromagnetic field (50 Hz) induces differentiation on primary human oral keratinocytes (HOK).

    Science.gov (United States)

    Manni, Vanessa; Lisi, Antonella; Rieti, Sabrina; Serafino, Annalucia; Ledda, Mario; Giuliani, Livio; Sacco, Donatella; D'Emilia, Enrico; Grimaldi, Settimio

    2004-02-01

    This work concerns the effect of low frequency electromagnetic fields (ELF) on biochemical properties of human oral keratinocytes (HOK). Cells exposed to a 2 mT, 50 Hz, magnetic field, showed by scanning electron microscopy (SEM) modification in shape and morphology; these modifications were also associated with different actin distribution, revealed by phalloidin fluorescence analysis. Moreover, exposed cells had a smaller clonogenic capacity, and decreased cellular growth. Indirect immunofluorescence with fluorescent antibodies against involucrin and beta-catenin, both differentiation and adhesion markers, revealed an increase in involucrin and beta-catenin expression. The advance in differentiation was confirmed by a decrease of expression of epidermal growth factor (EGF) receptor in exposed cells, supporting the idea that exposure to electromagnetic field carries keratinocytes to higher differentiation level. These observations support the hypothesis that 50 Hz electromagnetic fields may modify cell morphology and interfere in differentiation and cellular adhesion of normal keratinocytes.

  5. Toxicity Assessment of Six Titanium Dioxide Nanoparticles in Human Epidermal Keratinocytes

    Science.gov (United States)

    Toxicity Assessment of Six Titanium Dioxide Nanoparticles in Human Epidermal Keratinocytes Nanoparticle uptake in cells may be an important determinant of their potential cytotoxic and inflammatory effects. Six commercial TiO2 NP (A=Alfa Aesar,10nm, A*=Alfa Aesar 32nm, B=P25 27...

  6. Toxicity Assessment of Six Titanium Dioxide Nanoparticles in Human Epidermal Keratinocytes

    Science.gov (United States)

    Toxicity Assessment of Six Titanium Dioxide Nanoparticles in Human Epidermal Keratinocytes Nanoparticle uptake in cells may be an important determinant of their potential cytotoxic and inflammatory effects. Six commercial TiO2 NP (A=Alfa Aesar,10nm, A*=Alfa Aesar 32nm, B=P25 27...

  7. Comparison of rat epidermal keratinocyte organotypic culture (ROC) with intact human skin

    DEFF Research Database (Denmark)

    Pappinen, Sari; Hermansson, Martin; Kuntsche, Judith

    2008-01-01

    The present report is a part of our continuing efforts to explore the utility of the rat epidermal keratinocyte organotypic culture (ROC) as an alternative model to human skin in transdermal drug delivery and skin irritation studies of new chemical entities and formulations. The aim of the presen...

  8. Melatonin and its metabolites ameliorate ultraviolet B-induced damage in human epidermal keratinocytes.

    Science.gov (United States)

    Janjetovic, Zorica; Nahmias, Zachary P; Hanna, Sherie; Jarrett, Stuart G; Kim, Tae-Kang; Reiter, Russel J; Slominski, Adrzej T

    2014-08-01

    We investigated the protective effects of melatonin and its metabolites: 6-hydroxymelatonin (6-OHM), N1-acetyl-N2-formyl-5-methoxykynuramine (AFMK), N-acetylserotonin (NAS), and 5-methoxytryptamine (5-MT) in human keratinocytes against a range of doses (25, 50, and 75 mJ/cm2) of ultraviolet B (UVB) radiation. There was significant reduction in the generation of reactive oxygen species (50-60%) when UVB-exposed keratinocytes were treated with melatonin or its derivatives. Similarly, melatonin and its metabolites reduced the nitrite and hydrogen peroxide levels that were induced by UVB as early as 30 min after the exposure. Moreover, melatonin and its metabolites enhanced levels of reduced glutathione in keratinocytes within 1 hr after UVB exposure in comparison with control cells. Using proliferation assay, we observed a dose-dependent increase in viability of UVB-irradiated keratinocytes that were treated with melatonin or its derivatives after 48 hr. Using the dot-blot technique and immunofluorescent staining we also observed that melatonin and its metabolites enhanced the DNA repair capacity of UVB-induced pyrimidine photoproducts (6-4)or cyclobutane pyrimidine dimers generation in human keratinocytes. Additional evidence for induction of DNA repair in cells exposed to UVB and treated with the indole compounds was shown using the Comet assay. Finally, melatonin and its metabolites further enhanced expression of p53 phosphorylated at Ser-15 but not at Ser-46 or its nonphosphorylated form. In conclusion, melatonin, its precursor NAS, and its metabolites 6-OHM, AFMK, 5-MT, which are endogenously produced in keratinocytes, protect these cells against UVB-induced oxidative stress and DNA damage. © 2014 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  9. Arsenic compromises conducting airway epithelial barrier properties in primary mouse and immortalized human cell cultures.

    Directory of Open Access Journals (Sweden)

    Cara L Sherwood

    Full Text Available 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.

  10. Two type XVII collagen (BP180) mRNA transcripts in human keratinocytes : a long and a short form

    NARCIS (Netherlands)

    Molnar, K; van der Steege, G; Jonkman, MF; Nijenhuis, Albertine; Husz, S; van der Meer, JB; Pas, HH

    We have analysed BP180 mRNA expression in normal human keratinocytes. Here we report the presence in normal keratinocytes of two COL17A1 transcripts which differ by 0.6 kb in length. Both mRNAs hybridized on Northern blot with probes directed to sequences encoding intracellular and extracellular

  11. Investigation on etretin effects on expression of Fas/FasL ligand and apoptosis in cultured human keratinocytes

    Institute of Scientific and Technical Information of China (English)

    Ping Liu; Shunsheng Tan; Yanping Xi; Zhenping Cao

    2005-01-01

    Objective: To further illuminate a possibme mechanism of Fas/FasL in the treatment of psoriasis, the expression of it and apoptosis of KC were investigated. Methods: With cell culture,immunocytochemistry, the expression of Fas/FasL protein after the treatment with etretin was observed in cultured human normal keratinocytes. Then, the state of apoptosis in cultured keratinocyte after stimuwasn't involved in apoptosis in cultured normol human keratinocytes. But during limited period, the apoptosis of KC could be induced by etretin, thus it can antagonize benign proliferate of keratinocytes. Our data showed up-regulation of the expression of Fas/FasL and apoptosis in cultured human keratinocytes stimulated by etretin, and its function may be involved in the therapeutic machanism of psoriasis.

  12. Transcriptional profiling of ectoderm specification to keratinocyte fate in human embryonic stem cells.

    Science.gov (United States)

    Tadeu, Ana Mafalda Baptista; Lin, Samantha; Hou, Lin; Chung, Lisa; Zhong, Mei; Zhao, Hongyu; Horsley, Valerie

    2015-01-01

    In recent years, several studies have shed light into the processes that regulate epidermal specification and homeostasis. We previously showed that a broad-spectrum γ-secretase inhibitor DAPT promoted early keratinocyte specification in human embryonic stem cells triggered to undergo ectoderm specification. Here, we show that DAPT accelerates human embryonic stem cell differentiation and induces expression of the ectoderm protein AP2. Furthermore, we utilize RNA sequencing to identify several candidate regulators of ectoderm specification including those involved in epithelial and epidermal development in human embryonic stem cells. Genes associated with transcriptional regulation and growth factor activity are significantly enriched upon DAPT treatment during specification of human embryonic stem cells to the ectoderm lineage. The human ectoderm cell signature identified in this study contains several genes expressed in ectodermal and epithelial tissues. Importantly, these genes are also associated with skin disorders and ectodermal defects, providing a platform for understanding the biology of human epidermal keratinocyte development under diseased and homeostatic conditions.

  13. Determination of Drug Toxicity Using 3D Spheroids Constructed From an Immortal Human Hepatocyte Cell Line

    DEFF Research Database (Denmark)

    Fey, S. J.; Wrzesinski, Krzysztof

    2012-01-01

    Numerous publications have documented that the immortal cells grown in three-dimensional (3D) cultures possess physiological behavior, which is more reminiscent of their parental organ than when the same cells are cultivated using classical two-dimensional (2D) culture techniques. The goal...

  14. Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells

    DEFF Research Database (Denmark)

    Mortensen, O V; Thomassen, M; Larsen, M B

    1999-01-01

    were found to possess the additional 379 bp fragment. The integrity of the promoter was furthermore confirmed by genomic Southern blotting. The promoter activity was analyzed by reporter gene assays in neuronal and non-neuronal serotonergic cell lines. In immortalized serotonergic raphe neurons, RN46A...

  15. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor.

    Science.gov (United States)

    Bae, Ok-Nam; Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung; Kim, Eun-Sun; Jeong, Tae Cheon; Chun, Young-Jin; Lee, Ai-Young; Noh, Minsoo

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD.

  16. Comparison of cytotoxicity, genotoxicity and immunological inflammatory biomarker activity of several endodontic sealers against immortalized human pulp cells.

    Science.gov (United States)

    Martinho, F C; Camargo, S E A; Fernandes, A M M; Campos, M S; Prado, R F; Camargo, C H R; Valera, M C

    2017-04-25

    To establish an SV40 T-Ag-transfected cell line of human pulp-derived cells in order to compare the cytotoxicity, genotoxicity and to investigate the activities of immunological biomarkers of several endodontic sealers. Primary human pulp cells and transfected cells were cultured. Cell morphology and proliferation were analysed, and the expression of cell-specific gene transcripts and proteins was detected by RT-PCR and immunohistochemistry. Transfection of human pulp-derived cells resulted in an immortalized cell line retaining phenotypic characteristics from the primarily cells tested. The SV40 T-Ag-transfected cells were cultured and stimulated by sealers (Apexit Plus, Real Seal, AH Plus, and EndoREZ) to evaluate the cytotoxicity and genotoxicity by MTT and MTN assays, respectively. Immunological inflammatory biomarkers (IL6, IL8 and TNF-α) were determined by ELISA assay. The differences between median values were statistically analysed using Kruskal-Wallis and Dunn's tests at 5% significance level. The cytotoxicity assay revealed that multimethacrylate (Real Seal) was the most cytotoxic sealer (P root canal sealers tested were able to stimulate the immortalized pulp cells to produce IL-6, IL-8 and TNF-α, with differences in relation to the control group (P sealers tested (P epoxy resin-based sealer (AHPlus), single-methacrylate sealer (EndoREZ) and calcium hydroxide-based sealer (Apexit Plus), regardless of the cytokine investigated (all P > 0.05). A SV40 T-Ag-transfected cell line of human pulp-derived cells was established. The methacrylate resin-based sealer (Real Seal) exhibited the greatest cytoxicity and inflammatory potential against immortalized pulp cells compared to an epoxy resin-based sealer (AH Plus), a methacrylate-based sealer (EndoRez) and a calcium hydroxide-based sealer (Apexit). © 2017 International Endodontic Journal. Published by John Wiley & Sons Ltd.

  17. Candida albicans phospholipomannan triggers inflammatory responses of human keratinocytes through Toll-like receptor 2.

    Science.gov (United States)

    Li, Min; Chen, Qing; Shen, Yongnian; Liu, Weida

    2009-07-01

    The Toll-like receptors (TLRs) play an important role in the recognition of Candida albicans components and activation of innate immunity. Phospholipomannan (PLM), a glycolipid, is expressed at the surface of C. albicans cell wall, which acts as a member of the pathogen-associated molecular patterns family. In this study, we sought to clarify whether C. albicans-native PLM could induce an inflammation response in human keratinocytes and to determine the underlying mechanisms. Exposure of cultured human primary keratinocytes to PLM led to the increased gene expression and secretion of proinflammatory cytokines (IL-6) and chemokines (IL-8). PLM hydrolysed with beta-d-mannoside mannohydrolase failed to induce gene expression and secretion of IL-6 and IL-8. PLM up-regulated the mRNA and protein levels of TLR2, whereas the mRNA level of TLR4 was not altered. Keratinocytes challenged with PLM resulted in the activation of NF-kappaB and mitogen-activated protein kinase (MAPKs) including p38. Anti-TLR2 neutralizing antibody, NFkappaB and p38MAPK inhibitors blocked the PLM-induced secretion of IL-6, IL-8 in keratinocytes, but no such effect was observed in pretreatment with anti-TLR4-neutralizing antibody and lipopolysaccharide inhibitor (polymyxin B). These data suggest C. albicans-native PLM may contribute to the inflammatory responses of cutaneous candidiasis in the TLR2-NF-kappaB and p38MAPK signalling pathway dependent manner.

  18. Enhancing effect of tazarotene on the HLA-DR expression of cultured human keratinocytes induced by interferon-gamma

    Institute of Scientific and Technical Information of China (English)

    ZHANG Jian-gan; TAN Sheng-shun

    2005-01-01

    Objective: To investigate the effect of tazarotene on the expression of HLA-DR induced by IFN-γ. Methods: (1) Keratinocytes from normal human skin were cultured in vitro;(2) Tazarotene, IFN-γ and the combination of the two compounds were incubated with the keratinocytes in medium, respectively. The expression of HLA-DR in keratinocytes was determined using immunocytochemistry techniques at 24h after incubation. Results: (1) There was rare expression of HLA-DR in normal human keratinocytes; (2) 10-6mol/L tazarotene failed to induce the expression of HLA-DR in keratinocytes at 24h after incubation; (3) 500 U/ml IFN-γ obviously induced the HLA-DR expression in keratinocytes at 24h after treatment; (4) After 24h, 10-7-10-5 mol/L tazarotene had a significantly enhancing effect on the expression of HLA-DR induced by IFN-γ (P<0.005). Conclusion: Tazarotene up-regulates the expression of HLA-DR in keratinocytes cultured in vitro when combined with IFN-γ . Therefore, the reduction of HLA-DR positive keratinocytes in psoriatic lesions may be attributed to not direct interaction of tazarotene in combination with IFN-γ but other pathways.

  19. Human keratinocytes restrict chikungunya virus replication at a post-fusion step

    OpenAIRE

    Bernard, E.; Hamel, Rodolphe; Neyret, A.; Ekchariyawat, P.; Molès, J.P.; Simmons, G; Chazal, N; Desprès, P; Missé, Dorothée; Briant, L

    2015-01-01

    Transmission of chikungunya virus (CHIKV) to humans is initiated by puncture of the skin by a blood-feeding Aedes mosquito. Despite the growing knowledge accumulated on CHIKV, the interplay between skin cells and CHIKV following inoculation still remains unclear. In this study we questioned the behavior of human keratinocytes, the predominant cell population in the skin, following viral challenge. We report that CHIKV rapidly elicits an innate immune response in these cells leading to the enh...

  20. Artichoke compound cynarin differentially affects the survival, growth and stress response of normal, immortalized and cancerous human cells

    DEFF Research Database (Denmark)

    Gezer, Ceren; Yücecan, Sevinç; Rattan, Suresh Inder Singh

    2015-01-01

    Cynarin (CYN) is the main derivative of caffeoylquinic acid, found in leaves and heads of artichoke. Potential health-beneficial effects of CYN include as being choloretic-cholesterol lowering, hepatoprotective, anti-atherosclerotic, and antioxidative. We have tested the effects of various doses...... of CYN on the proliferative potential, survival, morphology, and stress response (SR) markers haemoxygenase-1 (HO-1) and heat shock protein-70 (HSP70) in normal human skin fibroblasts (FSF-1), telomerase-immortalized mesenchymal stem cells (hTERT-MSC) and cervical cancer cells, HeLa. Effects of CYN...

  1. Artichoke compound cynarin differentially affects the survival, growth and stress response of normal, immortalized and cancerous human cells

    DEFF Research Database (Denmark)

    Gezer, Ceren; Yücecan, Sevinç; Rattan, Suresh Inder Singh

    2015-01-01

    Cynarin (CYN) is the main derivative of caffeoylquinic acid, found in leaves and heads of artichoke. Potential health-beneficial effects of CYN include as being choloretic-cholesterol lowering, hepatoprotective, anti-atherosclerotic, and antioxidative. We have tested the effects of various doses...... of CYN on the proliferative potential, survival, morphology, and stress response (SR) markers haemoxygenase-1 (HO-1) and heat shock protein-70 (HSP70) in normal human skin fibroblasts (FSF-1), telomerase-immortalized mesenchymal stem cells (hTERT-MSC) and cervical cancer cells, HeLa. Effects of CYN...

  2. Paracoccidioides brasiliensis interacts with dermal dendritic cells and keratinocytes in human skin and oral mucosa lesions.

    Science.gov (United States)

    Silva, Wellington Luiz Ferreira da; Pagliari, Carla; Duarte, Maria Irma Seixas; Sotto, Mirian N

    2016-05-01

    Paracoccidioidomycosis (PCM) is a systemic disease caused by the fungus Paracoccidioides brasiliensis and Paracoccidioides lutzii. In PCM the skin and oral mucosa are often affected. Dendritic cells and keratinocytes of the integument play a role in innate and adaptive immune response against pathogens, due to their function as antigen presenting cells. Aiming to verify the interaction of P. brasiliensis with these cell populations, we studied 52 skin and 47 oral mucosa samples taken from patients with proven diagnosis of PCM. The biopsies were subjected to immunohistochemical and/or immunofluorescence staining with anti-factor XIIIa (marker of dermal dendrocytes), anti-CD207 (marker of mature Langerhans cells), anti-pan cytokeratins (AE1-AE3) and anti-P. brasiliensis antibodies. Analyses with confocal laser microscopy were also performed for better visualization of the interaction between keratinocytes and the fungi. In sum, 42% of oral mucosa samples displayed yeast forms in Factor XIIIa dermal dendrocytes cytoplasm. Langerhans cells in skin and oral mucosa samples did not show yeast cells in their cytoplasm. In sum, 54% of skin and 60% of mucosal samples displayed yeast cells in the cytoplasm of keratinocytes. The parasitism of keratinocytes may represent a possible mechanism of evasion of the fungus to local immune mechanisms. Factor XIIIa dendrocytes and keratinocytes may be acting as antigen-presenting cells to fulfill the probably impaired function of Langerhans cells in skin and oral mucosa of human PCM. © The Author 2015. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  3. Vanillin protects human keratinocyte stem cells against ultraviolet B irradiation.

    Science.gov (United States)

    Lee, Jienny; Cho, Jae Youl; Lee, Sang Yeol; Lee, Kyung-Woo; Lee, Jongsung; Song, Jae-Young

    2014-01-01

    Ultraviolet-B (UVB) irradiation is one of major factors which induce cellular damages in the epidermis. We investigated protective effects and mechanisms of vanillin, a main constituent of vanilla beans, against UVB-induced cellular damages in keratinocyte stem cells (KSC). Here, vanillin significantly attenuated UVB irradiation-induced cytotoxicity. The vanillin effects were also demonstrated by the results of the senescence-associated β-galactosidase and alkaline comet assays. In addition, vanillin induced production of pro-inflammatory cytokines. Attempts to elucidate a possible mechanism underlying the vanillin-mediated effects revealed that vanillin significantly reduced UVB-induced phosphorylation of ataxia telangiectasia mutated (ATM), serine threonine kinase checkpoint kinase 2 (Chk2), tumor suppressor protein 53 (p53), p38/mitogen-activated protein kinase (p38), c-Jun N-terminal kinase/stress-activated protein kinase (JNK), S6 ribosomal protein (S6RP), and histone 2A family member X (H2A.X). UVB-induced activation of p53 luciferase reporter was also significantly inhibited by vanillin. In addition, while ATM inhibitor had no effect on the vanillin effects, mouse double minute 2 homolog (MDM2) inhibitor significantly attenuated suppressive effects of vanillin on UVB-induced activation of p53 reporter in KSC. Taken together, these findings suggest that vanillin protects KSC from UVB irradiation and its effects may occur through the suppression of downstream step of MDM2 in UVB irradiation-induced p53 activation.

  4. Model of human epidermis reconstructed in vitro with keratinocytes and melanocytes on dead de-epidermized human dermis

    Directory of Open Access Journals (Sweden)

    Jussara Rehder

    Full Text Available CONTEXT: Recent progress in the field of epithelial culture techniques has allowed the development of culture systems in which the reconstructed epidermis presents characteristics of morphological differentiation similar to those seen in vivo. Human epidermis reconstructed in vitro may be used as the best alternative for the in vitro testing of the toxicology and efficiency of products for topical use, as well as in the treatment of skin burns and chronic skin ulcers. OBJECTIVE: To demonstrate a method for obtaining human epidermis reconstructed in vitro, using keratinocytes and melanocytes cultivated on dead de-epidermized human dermis. TYPE OF STUDY: Experimental/laboratory. SETTING: Skin Cell Culture Laboratory of the Faculdade de Ciências Médicas, Universidade Estadual de Campinas, Campinas, São Paulo, Brazil. PROCEDURE: Human keratinocytes and melanocytes cultured in vitro were grown on a biological matrix (dead de-epidermized human dermis and the system was kept at an air-liquid interface, in a suitable culturing medium, until a stratified human epidermis was formed, maintaining the histological characteristics of the epidermis in vivo. RESULTS: It was histologically demonstrated that it is possible to reproduce a differentiated epidermis through keratinocytes and melanocytes cultured on dead de-epidermized human dermis, thus obtaining a correctly positioned human epidermis reconstructed in vitro with functional keratinocytes and melanocytes that is similar to in vivo epidermis. CONCLUSIONS: It is possible to obtain a completely differentiated human epidermis reconstructed in vitro from keratinocyte and melanocyte cultures on a dead de-epidermized human dermis.

  5. Fluorescent immortalized human adipose derived stromal cells (hASCs-TS/GFP+) for studying cell drug delivery mediated by microvesicles.

    Science.gov (United States)

    Coccè, Valentina; Balducci, Luigi; Falchetti, Maria Laura; Pascucci, Luisa; Ciusani, Emilio; Brini, Anna Teresa; Sisto, Francesca; Piovani, Giovanna; Alessandri, Giulio; Parati, Eugenio; Cabeza, Laura; Pessina, Augusto

    2017-03-27

    A new tool for the drug delivery is based on the use of Mesenchymal Stromal Cells (MSCs) loaded in vitro with anti-cancer drugs. Unfortunately, the restricted lifespan of MSCs represents a significant limitation to produce them in high amounts and for long time studies. Immortalized MSCs from adipose tissue (hASC) have been generated as good source of cells with stable features. These cells could improve the development of standardized procedures for both in vitro and preclinical studies. Furthermore they facilitate procedures for preparing large amounts of secretome containing microvesicles (MVs). We used human adipose tissue derived MSCs immortalized with hTERT+SV40 (TS) genes and transfected with GFP (hASCs-TS/GFP+). This line was investigate for its ability to uptake and release anticancer drugs. Microvesicles associated to paclitaxel (MVs/PTX) were isolated, quantified, and tested on pancreatic cancer cells. The line hASCs-TS/GFP+ maintained the main mesenchymal characters and was able to uptake and release, in active form, both paclitaxel and gemcitabine. From paclitaxel loaded hASCs-TS/GFP+ cells were isolated microvesicles in sufficient amount to inhibit "in vitro" the proliferation of pancreatic tumor cells. Our study suggests that human immortalized MSCs could be used for a large scale production of cells for mediated drug delivery. Moreover, the secretion of drug-associated MVs could represent a new way for producing new drug formulation by "biogenesis". In the context of the "advanced cell therapy procedure", the MVs/PTX production would be less resource and time consuming and it could possibly contribute to simplification of GMP procedures. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  6. Immortality in Empedocles

    OpenAIRE

    Long, Alex

    2017-01-01

    Most of the work for the paper was undertaken in Toronto during a Leverhulme Research Fellowship. The paper examines Empedocles’ attributions of immortality. I argue that Empedocles does not withhold immortality from the gods but rather has an unorthodox conception of what immortality is. Immortality does not mean, or imply, endless duration. A god’s immortality is its continuity, as one and the same organism, over a long but finite period. This conception of divine immortality then influe...

  7. Replacement of murine fibroblasts by human fibroblasts irradiated in obtaining feeder layer for the culture of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Yoshito, Daniele; Sufi, Bianca S.; Santin, Stefany P.; Mathor, Monica B. [Instituto de Pesquisas Energeticas e Nucleares (IPEN/CNEN-SP), Sao Paulo, SP (Brazil); Altran, Silvana C.; Isaac, Cesar [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Medicina. Lab. de Microcirurgia Plastica; Esteves-Pedro, Natalia M. [Universidade Sao Paulo (USP), Sao Paulo, SP (Brazil). Fac. de Ciencias Farmaceuticas. Lab. de Controle Biologico; Herson, Marisa R. [DonorTissue Bank of Victoria (Australia)

    2011-07-01

    Human autologous epithelia cultivated in vitro, have been used successfully in treating damage to skin integrity. The methodology allowed the cultivation of these epithelia was described by Rheinwald and Green in 1975, this methodology consisted in seeding keratinocytes onto a feeder layer composed of lineage 3T3 murine fibroblasts, the proliferation rate is controlled through the action of ionizing radiation. However, currently there is a growing concern about the possibility of transmitting prions and murine viruses to transplanted patients. Taking into account this concern, in this present work, we replaced the feeder layer originally composed of murine fibroblasts by human fibroblasts. To obtain this new feeder layer was necessary to standardize the enough irradiation dose to inhibit the replication of human fibroblasts and the verification of effectiveness of the development of keratinocytes culture on a feeder layer thus obtained. According to the obtained results we can verify that the human fibroblasts irradiated at various tested doses (60, 70, 100, 200, 250 and 300 Gy) had their mitotic activity inactivated by irradiation, allowing the use of any of these doses to confection of the feeder layer, since these fibroblasts irradiated still showed viable until fourteen days of cultivation. In the test of colony formation efficiency was observed that keratinocytes seeded on irradiated human fibroblasts were able to develop satisfactorily, preserving their clonogenic potential. Therefore it was possible the replacement of murine fibroblasts by human fibroblasts in confection of the feeder layer, in order to eliminate this xenobiotic component of the keratinocytes culture. (author)

  8. Chemical allergens stimulate human epidermal keratinocytes to produce lymphangiogenic vascular endothelial growth factor

    Energy Technology Data Exchange (ETDEWEB)

    Bae, Ok-Nam [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Ahn, Seyeon; Jin, Sun Hee; Hong, Soo Hyun; Lee, Jinyoung [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of); Kim, Eun-Sun [College of Pharmacy, Institute of Pharmaceutical Science and Technology, Hanyang University, Ansan 426-791 (Korea, Republic of); Jeong, Tae Cheon [College of Pharmacy, Yeungnam University, Gyeongsan 712-749 (Korea, Republic of); Chun, Young-Jin [College of Pharmacy, Chung-Ang University, Seoul 156-756 (Korea, Republic of); Lee, Ai-Young, E-mail: leeay@duih.org [Department of Dermatology, Dongguk University Ilsan Hospital, Goyang 410-773 (Korea, Republic of); Noh, Minsoo, E-mail: minsoo@alum.mit.edu [College of Pharmacy, Natural Products Research Institute, Seoul National University, Seoul 151-742 (Korea, Republic of)

    2015-03-01

    Allergic contact dermatitis (ACD) is a cell-mediated immune response that involves skin sensitization in response to contact with various allergens. Angiogenesis and lymphangiogenesis both play roles in the allergic sensitization process. Epidermal keratinocytes can produce vascular endothelial growth factor (VEGF) in response to UV irradiation and during wound healing. However, the effect of haptenic chemical allergens on the VEGF production of human keratinocytes, which is the primary contact site of toxic allergens, has not been thoroughly researched. We systematically investigated whether immune-regulatory cytokines and chemical allergens would lead to the production of VEGF in normal human keratinocytes (NHKs) in culture. VEGF production significantly increased when NHKs were treated with IFNγ, IL-1α, IL-4, IL-6, IL-17A, IL-22 or TNFα. Among the human sensitizers listed in the OECD Test Guideline (TG) 429, we found that CMI/MI, DNCB, 4-phenylenediamine, cobalt chloride, 2-mercaptobenzothiazole, citral, HCA, cinnamic alcohol, imidazolidinyl urea and nickel chloride all significantly upregulated VEGF production in NHKs. In addition, common human haptenic allergens such as avobenzone, formaldehyde and urushiol, also induced the keratinocyte-derived VEGF production. VEGF upregulation by pro-inflammatory stimuli, IFNγ, DNCB or formaldehyde is preceded by the production of IL-8, an acute inflammatory phase cytokine. Lymphangiogenic VEGF-C gene transcription was significantly increased when NHKs were treated with formaldehyde, DNCB or urushiol, while transcription of VEGF-A and VEGF-B did not change. Therefore, the chemical allergen-induced VEGF upregulation is mainly due to the increase in lymphangiogenic VEGF-C transcription in NHKs. These results suggest that keratinocyte-derived VEGF may regulate the lymphangiogenic process during the skin sensitization process of ACD. - Highlights: • Pro-inflammatory cytokines induced VEGF production in normal human

  9. TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin

    Energy Technology Data Exchange (ETDEWEB)

    De Abrew, K. Nadira [Molecular and Environmental Toxicology Center, University of Wisconsin—Madison, Madison, WI 53706 (United States); Thomas-Virnig, Christina L.; Rasmussen, Cathy A. [Department of Pathology, University of Wisconsin—Madison, Madison, WI 53706 (United States); Bolterstein, Elyse A. [Molecular and Environmental Toxicology Center, University of Wisconsin—Madison, Madison, WI 53706 (United States); Schlosser, Sandy J. [Department of Pathology, University of Wisconsin—Madison, Madison, WI 53706 (United States); Allen-Hoffmann, B. Lynn, E-mail: blallenh@wisc.edu [Molecular and Environmental Toxicology Center, University of Wisconsin—Madison, Madison, WI 53706 (United States); Department of Pathology, University of Wisconsin—Madison, Madison, WI 53706 (United States)

    2014-05-01

    The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial–stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin. - Highlights: • TCDD causes hyperkeratosis and basement membrane changes in a model of human skin. • TCDD induces MMP-10 expression in organotypic cultures

  10. Enhanced constitutive invasion activity in human nontumorigenic keratinocytes exposed to a low level of barium for a long time.

    Science.gov (United States)

    Thang, Nguyen D; Yajima, Ichiro; Ohnuma, Shoko; Ohgami, Nobutaka; Kumasaka, Mayuko Y; Ichihara, Gaku; Kato, Masashi

    2015-02-01

    We have recently demonstrated that exposure to barium for a short time (≤4 days) and at a low level (5 µM = 687 µg/L) promotes invasion of human nontumorigenic HaCaT cells, which have characteristics similar to those of normal keratinocytes, suggesting that exposure to barium for a short time enhances malignant characteristics. Here we examined the effect of exposure to low level of barium for a long time, a condition mimicking the exposure to barium through well water, on malignant characteristics of HaCaT keratinocytes. Constitutive invasion activity, focal adhesion kinase (FAK) protein expression and activity, and matrix metalloproteinase 14 (MMP14) protein expression in primary cultured normal human epidermal keratinocytes, HaCaT keratinocytes, and HSC5 and A431 human squamous cell carcinoma cells were augmented following an increase in malignancy grade of the cells. Constitutive invasion activity, FAK phosphorylation, and MMP14 expression levels of HaCaT keratinocytes after treatment with 5 µM barium for 4 months were significantly higher than those of control untreated HaCaT keratinocytes. Taken together, our results suggest that exposure to a low level of barium for a long time enhances constitutive malignant characteristics of HaCaT keratinocytes via regulatory molecules (FAK and MMP14) for invasion. © 2013 Wiley Periodicals, Inc.

  11. Human papillomavirus (HPV upregulates the cellular deubiquitinase UCHL1 to suppress the keratinocyte's innate immune response.

    Directory of Open Access Journals (Sweden)

    Rezaul Karim

    Full Text Available Persistent infection of basal keratinocytes with high-risk human papillomavirus (hrHPV may cause cancer. Keratinocytes are equipped with different pattern recognition receptors (PRRs but hrHPV has developed ways to dampen their signals resulting in minimal inflammation and evasion of host immunity for sustained periods of time. To understand the mechanisms underlying hrHPV's capacity to evade immunity, we studied PRR signaling in non, newly, and persistently hrHPV-infected keratinocytes. We found that active infection with hrHPV hampered the relay of signals downstream of the PRRs to the nucleus, thereby affecting the production of type-I interferon and pro-inflammatory cytokines and chemokines. This suppression was shown to depend on hrHPV-induced expression of the cellular protein ubiquitin carboxyl-terminal hydrolase L1 (UCHL1 in keratinocytes. UCHL1 accomplished this by inhibiting tumor necrosis factor receptor-associated factor 3 (TRAF3 K63 poly-ubiquitination which lead to lower levels of TRAF3 bound to TANK-binding kinase 1 and a reduced phosphorylation of interferon regulatory factor 3. Furthermore, UCHL1 mediated the degradation of the NF-kappa-B essential modulator with as result the suppression of p65 phosphorylation and canonical NF-κB signaling. We conclude that hrHPV exploits the cellular protein UCHL1 to evade host innate immunity by suppressing PRR-induced keratinocyte-mediated production of interferons, cytokines and chemokines, which normally results in the attraction and activation of an adaptive immune response. This identifies UCHL1 as a negative regulator of PRR-induced immune responses and consequently its virus-increased expression as a strategy for hrHPV to persist.

  12. NOVEL NON-CALCEMIC SECOSTEROIDS THAT ARE PRODUCED BY HUMAN EPIDERMAL KERATINOCYTES PROTECT AGAINST SOLAR RADIATION

    Science.gov (United States)

    Slominski, Andrzej T.; Janjetovic, Zorica; Kim, Tae-Kang; Wasilewski, Piotr; Rosas, Sofia; Hanna, Sherie; Sayre, Robert M.; Dowdy, John C.; Li, Wei; Tuckey, Robert C.

    2015-01-01

    CYP11A1 hydroxylates the side chain of vitamin D3 (D3) in a sequential fashion [D3→20S(OH)D3→20,23(OH)2D3→ 17,20,23(OH)3D3], in an alternative to the classical pathway of activation [D3→25(OH)D3→1,25(OH)2D3]. The products/intermediates of the pathway can be further modified by the action of CYP27B1. The CYP11A1-derived products are biologically active with functions determined by the lineage of the target cells. This pathway can operate in epidermal keratinocytes. To further define the role of these novel secosteroids we tested them for protective effects against UVB-induced damage in human epidermal keratinocytes, melanocytes and HaCaT keratinocytes, cultured in vitro. The secosteroids attenuated ROS, H2O2 and NO production by UVB-irradiated keratinocytes and melanocytes, with an efficacy similar to 1,25(OH)2D3, while 25(OH)D3 had lower efficacy. These attenuations were also seen to some extent for the 20(OH)D3 precursor, 20S-hydroxy-7-dehydrocholesterol. These effects were accompanied by upregulation of genes encoding enzymes responsible for defence against oxidative stress. Using immunofluorescent staining we observed that the secosteroids reduced the generation cyclobutane pyrimidine dimers in response to UVB and enhanced expression of p53 phosphorylated at Ser-15, but not at Ser-46. Additional evidence for protection against DNA damage in cells exposed to UVB and treated with secosteroids was provided by the Comet assay where DNA fragmentation was markedly reduced by 20(OH)D3 and 20,23(OH)2D3. In conclusion, novel secosteroids that can be produced by the action of CYP11A1 in epidermal keratinocytes have protective effects against UVB radiation. PMID:25617667

  13. Effect of 1,24R-dihydroxyvitamin D3 on the growth of human keratinocytes.

    LENUS (Irish Health Repository)

    Matsumoto, K

    1990-02-01

    The effect of 1,24R-dihydroxyvitamin D3 (1,24R(OH)2D3), a synthetic analogue of a biologically active form of vitamin D3 (1,25-dihydroxyvitamin D3, 1,25(OH)2D3), on the growth of human keratinocytes cultured in serum-free medium was investigated. The growth of cultured normal human keratinocytes was inhibited by 65% by 10(-8)M 1,24R(OH)2D3 and by 90% by 10(-7)M 1,24(OH)2D3. It inhibited cell growth almost completely at 10(-6)M. The DNA synthesis of keratinocytes was also inhibited with 1,24R(OH)2D3 by 27% at 10(-8)M, 59% at 10(-7)M, and 92% at 10(-6)M. The inhibition of cell growth and DNA synthesis were more remarkable by 1,24R(OH)2D3 than by 1,25(OH)2D3. 1,24R(OH)2D3 also inhibited the growth of keratinocytes derived from patients with psoriasis vulgaris; the growth inhibitory effect was again more remarkable with 1,24R(OH)2D3 than with 1,25(OH)2D3. The viability and protein synthesis of keratinocytes were not affected by 1,24R(OH)2D3, suggesting that the growth inhibitory effect is due to its biological activity, not to cytotoxicity. The binding of [3H]-labeled 1,25(OH)2D3 to its receptor in the cytosolic fraction of cultured keratinocytes was competitively substituted by unlabeled 1,24R(OH)2D3 as well as 1,25(OH)2D3, suggesting that 1,24R(OH)2D3 binds to the 1,25(OH)2D3 receptor. It was found that the affinity of 1,24R(OH)2D3 for the receptor was slightly higher than that of 1,25(OH)2D3. These results demonstrate that 1,24R(OH)2D3 functions as a potent growth inhibitor in vitro in human keratinocytes from both normal and psoriatic epidermis, and it possesses a higher affinity for the 1,25(OH)2D3 receptor in cultured human keratinocytes. The difference in affinity of 1,24R(OH)2D3 for the 1,25(OH)2D3 receptor correlates with its greater inhibition of keratinocyte growth than 1,25(OH)2D3. 1,24R(OH)2D3 may be useful in the treatment of psoriasis.

  14. COP1 contributes to UVB-induced signaling in human keratinocytes.

    Science.gov (United States)

    Kinyó, Agnes; Kiss-László, Zsuzsanna; Hambalkó, Szabolcs; Bebes, Attila; Kiss, Mária; Széll, Márta; Bata-Csörgo, Zsuzsanna; Nagy, Ferenc; Kemény, Lajos

    2010-02-01

    UVB irradiation has been shown to trigger a broad range of changes in gene expression in human skin; however, factors governing these events are still not well understood. In this study, we show that human constitutive photomorphogenic protein-1 (huCOP1), an E3 ligase, contributes to the orchestration of UVB response of keratinocytes. Accordingly, our data show that (i) huCOP1 protein is expressed both in the nucleus and in the cytoplasm of cultured keratinocytes, (ii) UVB reduces the levels of the huCOP1 mRNA and protein, and (iii) induces changes in the subcellular localization of huCOP1. Finally, we show that gene-specific silencing of huCOP1 induces the accumulation of the tumor suppressor p53 protein, which is further increased after UVB irradiation.

  15. Tumourigenesis driven by the human papillomavirus type 16 Asian-American e6 variant in a three-dimensional keratinocyte model.

    Directory of Open Access Journals (Sweden)

    Robert Jackson

    Full Text Available Infection with a transforming human papillomavirus (HPV such as type 16 (of species Alphapapillomavirus 9 causes ano-genital and oral tumours via viral persistence in human squamous cell epithelia. Epidemiological studies showed that the naturally occurring HPV16 Asian-American (AA variant (sublineage D2/D3 is found more often than the European Prototype (EP (sublineage A1 in high-grade cervical neoplasia and tumours compared to non-cancer controls. Just three amino acid changes within the early gene, E6, of HPV16 AA have been linked to this augmented tumourigenicity. The AAE6 variant's greater immortalizing and transforming potential over EPE6 has recently been confirmed in retrovirally-transduced keratinocytes expressing the E6 gene only. However, the tumourigenic role of the full-length viral genome of HPV16 has not yet been addressed with regard to these E6 variants. To investigate this process in the context of these two HPV16 E6 genotypes, an organotypic tissue culture model was used to simulate the HPV infectious life cycle. The AAE6 variant demonstrated an enhanced ability over EPE6 to drive the viral life cycle toward tumourigenesis, as evidenced phenotypically-by a more severe grade of epithelial dysplasia with higher proliferation and deregulated differentiation, and molecularly-by high viral oncogene E6 and E7 expression, but lack of productive viral life cycle markers. In contrast, EPE6 had low E6 and E7 but high E1∧E4 expression, indicative of a productive life cycle. We suggest increased viral integration into the host genome for AAE6 as one possible mechanism for these observed differences from EPE6. Additionally, we found downstream effects on immortalization and host innate immune evasion. This study highlights how minor genomic variations in transforming viruses can have a significant affect on their tumourigenic ability.

  16. Transforming growth factor-beta stimulates the expression of fibronectin by human keratinocytes.

    Science.gov (United States)

    Wikner, N E; Persichitte, K A; Baskin, J B; Nielsen, L D; Clark, R A

    1988-09-01

    Transforming growth factor beta (TGF-beta) is a 25-kD protein which has regulatory activity over a variety of cell types. It is distinct from epidermal growth factor (EGF) and EGF analogs, and exerts its action via a distinct receptor. Its effect on proliferation or differentiation can be positive or negative depending on the cell type and the presence of other growth factors. It also modulates the expression of cellular products. TGF-beta causes fibroblasts to increase their production of the extracellular matrix components, fibronectin and collagen. Human keratinocytes (HK) are known to have TGF-beta receptors. We wished to study the effect of TGF-beta on the production of extracellular matrix proteins by human keratinocytes in culture. Human keratinocytes were grown in serum-free defined medium (MCDB-153) to about 70% confluence. Following a 16-h incubation in medium lacking EGF and TGF-beta, cells were incubated for 12 h in medium containing varying concentrations of EGF and TGF-beta. Cells were then labeled with 35S-methionine for 10 h in the same conditions. Labeled proteins from the medium were analyzed by SDS-PAGE and autoradiography. TGF-beta at 10 ng/ml induced a sixfold increase in the secretion of fibronectin, as well as an unidentified 50-kD protein. Thrombospondin production was also increased, but not over a generalized twofold increase in the production of all other proteins. EGF, at 10 ng/ml, caused a smaller additive effect. TGF-beta may be an important stimulator of extracellular matrix production by human keratinocytes.

  17. Ectopic PDX-1 Expression Directly Reprograms Human Keratinocytes along Pancreatic Insulin-Producing Cells Fate

    Science.gov (United States)

    Chernichovski, Ellad; Nakar, Odelia; Winkler, Eyal; Mazkereth, Ram; Orenstein, Arie; Bar-Meir, Eran; Ravassard, Philippe; Meivar-Levy, Irit; Ferber, Sarah

    2011-01-01

    Background Cellular differentiation and lineage commitment have previously been considered irreversible processes. However, recent studies have indicated that differentiated adult cells can be reprogrammed to pluripotency and, in some cases, directly into alternate committed lineages. However, although pluripotent cells can be induced in numerous somatic cell sources, it was thought that inducing alternate committed lineages is primarily only possible in cells of developmentally related tissues. Here, we challenge this view and analyze whether direct adult cell reprogramming to alternate committed lineages can cross the boundaries of distinct developmental germ layers. Methodology/Principal Findings We ectopically expressed non-integrating pancreatic differentiation factors in ectoderm-derived human keratinocytes to determine whether these factors could directly induce endoderm-derived pancreatic lineage and β-cell-like function. We found that PDX-1 and to a lesser extent other pancreatic transcription factors, could rapidly and specifically activate pancreatic lineage and β-cell-like functional characteristics in ectoderm-derived human keratinocytes. Human keratinocytes transdifferentiated along the β cell lineage produced processed and secreted insulin in response to elevated glucose concentrations. Using irreversible lineage tracing for KRT-5 promoter activity, we present supporting evidence that insulin-positive cells induced by ectopic PDX-1 expression are generated in ectoderm derived keratinocytes. Conclusions/Significance These findings constitute the first demonstration of human ectoderm cells to endoderm derived pancreatic cells transdifferentiation. The study represents a proof of concept which suggests that transcription factors induced reprogramming is wider and more general developmental process than initially considered. These results expanded the arsenal of adult cells that can be used as a cell source for generating functional endocrine

  18. In Vitro Cytotoxicity and Phototoxicity Assessment of Acylglutamate Surfactants Using a Human Keratinocyte Cell Line

    OpenAIRE

    Abhay Kyadarkunte; Milind Patole; Varsha Pokharkar

    2014-01-01

    In the current study, human keratinocyte cell line was used as in vitro cell culture model to elucidate the effects of the fatty acid chain length of acylglutamate (amino acid-based surfactant) namely, sodium cocoyl glutamate, sodium lauroyl glutamate, and sodium myristoyl glutamate on their cytotoxicity and the ultraviolet B induced phototoxicity. The endpoint used to assess toxicity was a tetrazolium-based assay whereas, the phototoxic potential of acylglutamate surfactants was predicted u...

  19. Isorhamnetin Protects Human Keratinocytes against Ultraviolet B-Induced Cell Damage

    OpenAIRE

    Han, Xia; Piao, Mei Jing; Kim, Ki Cheon; Madduma Hewage, Susara Ruwan Kumara; Yoo, Eun Sook; Koh, Young Sang; Kang, Hee Kyoung; Jennifer H. Shin; PARK, YEUNSOO; Yoo, Suk Jae; Chae, Sungwook; Hyun, Jin Won

    2015-01-01

    Isorhamnetin (3-methylquercetin) is a flavonoid derived from the fruits of certain medicinal plants. This study investigated the photoprotective properties of isorhamnetin against cell damage and apoptosis resulting from excessive ultraviolet (UV) B exposure in human HaCaT keratinocytes. Isorhamnetin eliminated UVB-induced intracellular reactive oxygen species (ROS) and attenuated the oxidative modification of DNA, lipids, and proteins in response to UVB radiation. Moreover, isorhamnetin repr...

  20. Xenobiotic metabolism capacities of human skin in comparison with a 3D-epidermis model and keratinocyte-based cell culture as in vitro alternatives for chemical testing: phase II enzymes.

    Science.gov (United States)

    Götz, Christine; Pfeiffer, Roland; Tigges, Julia; Ruwiedel, Karsten; Hübenthal, Ulrike; Merk, Hans F; Krutmann, Jean; Edwards, Robert J; Abel, Josef; Pease, Camilla; Goebel, Carsten; Hewitt, Nicola; Fritsche, Ellen

    2012-05-01

    The 7th Amendment to the EU Cosmetics Directive prohibits the use of animals in cosmetic testing for certain endpoints, such as genotoxicity. Therefore, skin in vitro models have to replace chemical testing in vivo. However, the metabolic competence neither of human skin nor of alternative in vitro models has so far been fully characterized, although skin is the first-pass organ for accidentally or purposely (cosmetics and pharmaceuticals) applied chemicals. Thus, there is an urgent need to understand the xenobiotic-metabolizing capacities of human skin and to compare these activities to models developed to replace animal testing. We have measured the activity of the phase II enzymes glutathione S-transferase, UDP-glucuronosyltransferase and N-acetyltransferase in ex vivo human skin, the 3D epidermal model EpiDerm 200 (EPI-200), immortalized keratinocyte-based cell lines (HaCaT and NCTC 2544) and primary normal human epidermal keratinocytes. We show that all three phase II enzymes are present and highly active in skin as compared to phase I. Human skin, therefore, represents a more detoxifying than activating organ. This work systematically compares the activities of three important phase II enzymes in four different in vitro models directly to human skin. We conclude from our studies that 3D epidermal models, like the EPI-200 employed here, are superior over monolayer cultures in mimicking human skin xenobiotic metabolism and thus better suited for dermatotoxicity testing. © 2012 John Wiley & Sons A/S.

  1. In Vitro Cytotoxicity and Phototoxicity Assessment of Acylglutamate Surfactants Using a Human Keratinocyte Cell Line

    Directory of Open Access Journals (Sweden)

    Abhay Kyadarkunte

    2014-07-01

    Full Text Available In the current study, human keratinocyte cell line was used as in vitro cell culture model to elucidate the effects of the fatty acid chain length of acylglutamate (amino acid-based surfactant namely, sodium cocoyl glutamate, sodium lauroyl glutamate, and sodium myristoyl glutamate on their cytotoxicity and the ultraviolet B induced phototoxicity. The endpoint used to assess toxicity was a tetrazolium-based assay whereas, the phototoxic potential of acylglutamate surfactants was predicted using two models namely, the Photo-Irritation Factor and Mean Photo Effect. The results of this study showed that the fatty acid chain length of acylglutamate greatly influences toxic effects on human keratinocyte cells. In addition, all the acylglutamate surfactants tested on human keratinocyte cells demonstrated significantly less cytotoxicity (when irradiated and non-irradiated with ultraviolet B light; p < 0.05 and no phototoxic potential was observed in any of the acylglutamate surfactants, when compared with the positive control chlorpromazine. In conclusion, the in vitro studies confirm the suitability of sodium lauroyl glutamate destined for the synthesis and stabilization of lipid nanoparticles.

  2. Biological properties of differently-aged human keratinocytes:population doubling time growth curve and cell cycle analysis

    Institute of Scientific and Technical Information of China (English)

    2009-01-01

    Objective To explore the biological properties of keratinocytes from differently-aged healthy human beings. Methods Keratinocytes from fetus,teenager and middle-aged groups were separated and cultured. The population doubling time (PDT) and cell growth curve in different cells were compared,and the cell cycles were analyzed by flow cytometry. Results ① In primary culture of keratinocytes,the adherence time in middle-aged group was longer than that in fetus and teenager groups. However,all cell morphology sh...

  3. A Sensitive Sensor Cell Line for the Detection of Oxidative Stress Responses in Cultured Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Ute Hofmann

    2014-06-01

    Full Text Available In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells expressing green fluorescent protein (GFP under the control of the stress-inducible HSP70B’ promoter were constructed. Exposure of HaCaT sensor cells to 25 µM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ~300-fold induction of transcript level of the gene coding for heat shock protein HSP70B’. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B’ gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B’ promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 µM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (µTAS that may be utilized in dermatology, toxicology, pharmacology and drug screenings.

  4. A sensitive sensor cell line for the detection of oxidative stress responses in cultured human keratinocytes.

    Science.gov (United States)

    Hofmann, Ute; Priem, Melanie; Bartzsch, Christine; Winckler, Thomas; Feller, Karl-Heinz

    2014-06-25

    In the progress of allergic and irritant contact dermatitis, chemicals that cause the generation of reactive oxygen species trigger a heat shock response in keratinocytes. In this study, an optical sensor cell line based on cultured human keratinocytes (HaCaT cells) expressing green fluorescent protein (GFP) under the control of the stress-inducible HSP70B' promoter were constructed. Exposure of HaCaT sensor cells to 25 µM cadmium, a model substance for oxidative stress induction, provoked a 1.7-fold increase in total glutathione and a ~300-fold induction of transcript level of the gene coding for heat shock protein HSP70B'. An extract of Arnica montana flowers resulted in a strong induction of the HSP70B' gene and a pronounced decrease of total glutathione in keratinocytes. The HSP70B' promoter-based sensor cells conveniently detected cadmium-induced stress using GFP fluorescence as read-out with a limit of detection of 6 µM cadmium. In addition the sensor cells responded to exposure of cells to A. montana extract with induction of GFP fluorescence. Thus, the HaCaT sensor cells provide a means for the automated detection of the compromised redox status of keratinocytes as an early indicator of the development of human skin disorders and could be applied for the prediction of skin irritation in more complex in vitro 3D human skin models and in the development of micro-total analysis systems (µTAS) that may be utilized in dermatology, toxicology, pharmacology and drug screenings.

  5. Impact of AQP3 inducer treatment on cultured human keratinocytes, ex vivo human skin and volunteers.

    Science.gov (United States)

    Garcia, N; Gondran, C; Menon, G; Mur, L; Oberto, G; Guerif, Y; Dal Farra, C; Domloge, N

    2011-10-01

    One of the main functions of the skin is to protect the organism against environmental threats, such as thermal stress. Aquaporin-3 (AQP3) facilitates water and glycerol transport across cell membranes and therefore regulates osmotic balance in different situations of stress. This mechanism seems to be particularly important for the resistance of different organisms to cold stress. Consequently, we were interested in investigating the effect of cold and osmotic stress on AQP3 expression in normal human keratinocytes. We developed a new active ingredient to stimulate aquaporins in skin and demonstrated the partial restoration of AQP3 expression in keratinocytes transfected with AQP3 siRNA. Moreover, we examined the effect of cold stress on cell morphology and the impact of a pre-treatment with the active ingredient. Our results indicated that induction of AQP3 helped maintain a correct organization of the actin cytoskeleton, preserving cell morphology and preventing cells from rounding. Immunofluorescent staining revealed cytoplasmic localization of AQP3 and its translocation to the cell membrane following osmotic stress. Histological ex vivo studies of skin under different conditions, such as cold environment and tape-stripping, indicated that increase in AQP3 expression appears to be involved in skin protection and showed that the pattern of AQP3 expression was more enhanced in the active ingredient-treated samples. In vivo confocal microscopy by Vivascope showed a generally healthier appearance of the skin in the treated areas. These results attest to the potential value of the active ingredient in optimizing environmental stress resistance and protecting the skin from stratum corneum damage.

  6. Dual oxidase 2 is essential for house dust mite-induced pro-inflammatory cytokine production in human keratinocytes.

    Science.gov (United States)

    Ko, Eunbi; Choi, Hyun; Park, Kkot-Nara; Park, Ju-Yearl; Lee, Tae Ryong; Shin, Dong Wook; Bae, Yun Soo

    2015-12-01

    House dust mites (HDMs) are known to trigger chronic inflammation through Toll-like receptors (TLRs) and their signalling cascades. In this study, we found that TLR2 ligation by HDMs induced the activation of dual oxidase 2 (Duox2) and nuclear factor-κB (NF-κB), leading to the production of pro-inflammatory cytokines in human keratinocytes. Stimulation of human keratinocytes with HDMs resulted in increases in interleukin-8 (IL-8) and chemokine (C-C motif) ligand 20 (CCL20) levels. However, pro-inflammatory cytokine production was abolished in keratinocytes transfected with TLR2 siRNA, indicating that HDM-induced cytokine production was mediated via TLR2 signalling. We also examined the function of Duox1/2 isozymes, which are primarily expressed in keratinocytes, in HDM-mediated pro-inflammatory cytokine production. Human keratinocytes transfected with control siRNA or Duox1 siRNA showed no inhibition of IL-8 or CCL20 production in response to HDMs, whereas the silencing of Duox2 expression resulted in a failure to induce cytokine production. Moreover, the phosphorylation and nuclear localization of RelA/p65, a component of NF-κB, were induced by HDMs in human keratinocytes. Transfection of human keratinocytes with TLR2 siRNA or Duox2 siRNA resulted in the complete abolishment of RelA/p65 nuclear localization in response to HDMs. Taken together, these results indicate that the HDM-dependent TLR2-Duox2 signalling axis indeed promotes NF-κB activation, which induces IL-8 and CCL20 production and mediates epidermal keratinocyte inflammation. © 2015 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  7. Long-term subculture of human keratinocytes under an anoxic condition.

    Science.gov (United States)

    Kino-oka, Masahiro; Agatahama, Yuka; Haga, Yuki; Inoie, Masukazu; Taya, Masahito

    2005-07-01

    The serial subculturing of human keratinocyte cells under the anoxic and normoxic conditions was examined. The cumulative number of population doublings in the subcultures under the former condition increased 2.1-fold while maintaining an appreciable growth rate of cells, as compared with that under the latter condition. Moreover, the migration ability, which was estimated by the rotation rate of paired cells, was maintained accompanied by fully developed filopodia of F-actin filaments under the anoxic condition, despite of the poor development of stress fibers at the center of the cellular body. The cells passaged under the anoxic condition possessed the sufficient clonogenic potential to form epithelial sheets, supporting the view that the long-term subculture of keratinocytes under the anoxic condition can be applied for cell expansion in the practical production of epithelial sheets.

  8. Conditionally immortalized human pancreatic stellate cell lines demonstrate enhanced proliferation and migration in response to IGF-I

    Energy Technology Data Exchange (ETDEWEB)

    Rosendahl, Ann H., E-mail: ann.rosendahl@med.lu.se [Lund University, Department of Clinical Sciences Lund, Division of Surgery, Lund (Sweden); Lund University and Skåne University Hospital, Department of Clinical Sciences Lund, Division of Oncology and Pathology, Lund (Sweden); Gundewar, Chinmay; Said Hilmersson, Katarzyna [Lund University, Department of Clinical Sciences Lund, Division of Surgery, Lund (Sweden); Ni, Lan; Saleem, Moin A. [University of Bristol, School of Clinical Sciences, Children' s Renal Unit and Academic Renal Unit, Bristol (United Kingdom); Andersson, Roland [Lund University, Department of Clinical Sciences Lund, Division of Surgery, Lund (Sweden)

    2015-01-15

    Pancreatic stellate cells (PSCs) play a key role in the dense desmoplastic stroma associated with pancreatic ductal adenocarcinoma. Studies on human PSCs have been minimal due to difficulty in maintaining primary PSC in culture. We have generated the first conditionally immortalized human non-tumor (NPSC) and tumor-derived (TPSC) pancreatic stellate cells via transformation with the temperature-sensitive SV40 large T antigen and human telomerase (hTERT). These cells proliferate at 33°C. After transfer to 37°C, the SV40LT is switched off and the cells regain their primary PSC phenotype and growth characteristics. NPSC contained cytoplasmic vitamin A-storing lipid droplets, while both NPSC and TPSC expressed the characteristic markers αSMA, vimentin, desmin and GFAP. Proteome array analysis revealed that of the 55 evaluated proteins, 27 (49%) were upregulated ≥3-fold in TPSC compared to NPSC, including uPA, pentraxin-3, endoglin and endothelin-1. Two insulin-like growth factor binding proteins (IGFBPs) were inversely expressed. Although discordant IGFBP-2 and IGFBP-3 levels, IGF-I was found to stimulate proliferation of both NPSC and TPSC. Both basal and IGF-I stimulated motility was significantly enhanced in TPSC compared to NPSC. In conclusion, these cells provide a unique resource that will facilitate further study of the active stroma compartment associated with pancreatic cancer. - Highlights: • Generation of human conditionally immortalized human pancreatic stellate cell lines. • Temperature-sensitive SV40LT allows switch to primary PSC phenotype characteristics. • Proteome profiling revealed distinct expression patterns between TPSC and NPSC. • Enhanced IGF-I-stimulated proliferation and motility by TPSC compared to NPSC.

  9. Entry Pathways of Herpes Simplex Virus Type 1 into Human Keratinocytes Are Dynamin- and Cholesterol-Dependent

    Science.gov (United States)

    Hsu, Mei-Ju; Rixon, Frazer J.; Knebel-Mörsdorf, Dagmar

    2011-01-01

    Herpes simplex virus type 1 (HSV-1) can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s). Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol. PMID:22022400

  10. Entry pathways of herpes simplex virus type 1 into human keratinocytes are dynamin- and cholesterol-dependent.

    Directory of Open Access Journals (Sweden)

    Elena Rahn

    Full Text Available Herpes simplex virus type 1 (HSV-1 can enter cells via endocytic pathways or direct fusion at the plasma membrane depending on the cell line and receptor(s. Most studies into virus entry have used cultured fibroblasts but since keratinocytes represent the primary entry site for HSV-1 infection in its human host, we initiated studies to characterize the entry pathway of HSV-1 into human keratinocytes. Electron microscopy studies visualized free capsids in the cytoplasm and enveloped virus particles in vesicles suggesting viral uptake both by direct fusion at the plasma membrane and by endocytic vesicles. The ratio of the two entry modes differed in primary human keratinocytes and in the keratinocyte cell line HaCaT. Inhibitor studies further support a role for endocytosis during HSV-1 entry. Infection was inhibited by the cholesterol-sequestering drug methyl-β-cyclodextrin, which demonstrates the requirement for host cholesterol during virus entry. Since the dynamin-specific inhibitor dynasore and overexpression of a dominant-negative dynamin mutant blocked infection, we conclude that the entry pathways into keratinocytes are dynamin-mediated. Electron microscopy studies confirmed that virus uptake is completely blocked when the GTPase activity of dynamin is inhibited. Ex vivo infection of murine epidermis that was treated with dynasore further supports the essential role of dynamin during entry into the epithelium. Thus, we conclude that HSV-1 can enter human keratinocytes by alternative entry pathways that require dynamin and host cholesterol.

  11. In vitro human keratinocyte migration rates are associated with SNPs in the KRT1 interval.

    Directory of Open Access Journals (Sweden)

    Heng Tao

    Full Text Available Efforts to develop effective therapeutic treatments for promoting fast wound healing after injury to the epidermis are hindered by a lack of understanding of the factors involved. Re-epithelialization is an essential step of wound healing involving the migration of epidermal keratinocytes over the wound site. Here, we examine genetic variants in the keratin-1 (KRT1 locus for association with migration rates of human epidermal keratinocytes (HEK isolated from different individuals. Although the role of intermediate filament genes, including KRT1, in wound activated keratinocytes is well established, this is the first study to examine if genetic variants in humans contribute to differences in the migration rates of these cells. Using an in vitro scratch wound assay we observe quantifiable variation in HEK migration rates in two independent sets of samples; 24 samples in the first set and 17 samples in the second set. We analyze genetic variants in the KRT1 interval and identify SNPs significantly associated with HEK migration rates in both samples sets. Additionally, we show in the first set of samples that the average migration rate of HEK cells homozygous for one common haplotype pattern in the KRT1 interval is significantly faster than that of HEK cells homozygous for a second common haplotype pattern. Our study demonstrates that genetic variants in the KRT1 interval contribute to quantifiable differences in the migration rates of keratinocytes isolated from different individuals. Furthermore we show that in vitro cell assays can successfully be used to deconstruct complex traits into simple biological model systems for genetic association studies.

  12. Low dose chronic treatment of human keratinocytes with inorganic arsenic causes hyperproliferation and altered protein phosphorylation

    Energy Technology Data Exchange (ETDEWEB)

    Steinberg, M.L. [City College of New York, NY (United States); Su, L.; Snow, E.T. [New York Univ. Medical Center, Tuxedo, NY (United States)]|[City College of New York, NY (United States)

    1997-10-01

    Chronic exposure to arsenate [As(V)] or arsenite [As(III)] causes hyperproliferation of normal and SV40-transformed human epidermal keratinocytes. Line 327 SV40-infected human keratinocytes were grown in the presence of either As(III) or As(V) (0.01 to 10 {mu}M) in complete medium for seven days prior to harvesting and counting. Both As(III) and As(V) were cytotoxic at micromolar concentrations, however submicromolar arsenic caused a significant increase in cell growth. Cell numbers in cultures exposed to As(V) were increased more than 186% relative to controls, and an even larger stimulation in cell growth was observed after treatment with 50 nM As(III). Normal non-SV40 T-antigen. Preliminary cell cycle analysis using unselected, log-phase cultures of arsenic-treated keratinocytes shows an increased proportion of cells in S- and G2/M-phase. Isoelectric focusing of phosphotyrosine-containing proteins from cells labeled with {sup 32}P-inorganic phosphate showed that the hyperproliferation of keratinocytes grown in low concentrations of arsenic is accompanied by altered tyrosine-specific protein phosphorylation. A number of phosphorylated proteins were observed in As-treated cells that were not observed in the controls; and minor bands at IEPs of 3.0, 4.2, 7.2, 7.5 and 8.2. These results, together with the lack of direct enzyme inhibition by arsenic shown by Su et al., this volume, suggest that arsenic-induced skin lesions and carcinogenesis may be the result of altered cell cycle control rather than DNA damage or reduced DNA repair.

  13. Accumulation and altered localization of telomere-associated protein TRF2 in immortally transformed and tumor-derived human breast cells

    Energy Technology Data Exchange (ETDEWEB)

    Nijjar, Tarlochan; Bassett, Ekaterina; Garbe, James; Takenaka, Yasuhiro; Stampfer, Martha R.; Gilley, David; Yaswen, Paul

    2004-12-23

    We have used cultured human mammary epithelial cells (HMEC) and breast tumor-derived lines to gain information on defects that occur during breast cancer progression. HMEC immortalized by a variety of agents (the chemical carcinogen benzo(a)pyrene, oncogenes c-myc and ZNF217, and/or dominant negative p53 genetic suppressor element GSE22) displayed marked up regulation (10-15 fold) of the telomere binding protein, TRF2. Up-regulation of TRF2 protein was apparently due to differences in post-transcriptional regulation, as mRNA levels remained comparable in finite life span and immortal HMEC. TRF2 protein was not up-regulated by the oncogenic agents alone in the absence of immortalization, nor by expression of exogenously introduced hTERT genes. We found TRF2 levels to be at least 2-fold higher than in control cells in 11/15 breast tumor cell lines, suggesting that elevated TRF2 levels are a frequent occurrence during the transformation of breast tumor cells in vivo. The dispersed distribution of TRF2 throughout the nuclei in some immortalized and tumor-derived cells indicated that not all the TRF2 was associated with telomeres in these cells. The process responsible for accumulation of TRF2 in immortalized HMEC and breast tumor-derived cell lines may promote tumorigenesis by contributing to the cells ability to maintain an indefinite life span.

  14. Caspase-1 activity is required for UVB-induced apoptosis of human keratinocytes.

    Science.gov (United States)

    Sollberger, Gabriel; Strittmatter, Gerhard E; Grossi, Serena; Garstkiewicz, Martha; Auf dem Keller, Ulrich; French, Lars E; Beer, Hans-Dietmar

    2015-05-01

    Caspase-1 has a crucial role in innate immunity as the protease activates the proinflammatory cytokine prointerleukin(IL)-1β. Furthermore, caspase-1 induces pyroptosis, a lytic form of cell death that supports inflammation. Activation of caspase-1 occurs in multi-protein complexes termed inflammasomes, which assemble upon sensing of stress signals. In the skin and in skin-derived keratinocytes, UVB irradiation induces inflammasome-dependent IL-1 secretion and sunburn. Here we present evidence that caspase-1 and caspase-4 are required for UVB-induced apoptosis. In UVB-irradiated human primary keratinocytes, apoptosis occurs significantly later than inflammasome activation but depends on caspase-1 activity. However, it proceeds independently of inflammasome activation. By a proteomics approach, we identified the antiapoptotic Bap31 as a putative caspase-1 substrate. Caspase-1-dependent apoptosis is possibly a recent process in evolution as it was not detected in mice. These results suggest a protective role of caspase-1 in keratinocytes during UVB-induced skin cancer development through the induction of apoptosis.

  15. Effects of Arsenic on Cell Proliferation and Its Related Gene Expression in Human Epidermal Keratinocyte

    Institute of Scientific and Technical Information of China (English)

    顾军; 毕新岭; 米庆胜; 文军慧

    2002-01-01

    Objective:To study the effects of low concentration of arsenic (As2O3) on DNA synthesisand related transcription factor gene E2F1 expression in keratinocyte. Methods: Human epidermal kerati-nocyte (cell line HaCaT) cultured in vitro was used. After treatment with various concentrations of arse-nic, DNA synthesis and E2F1 expression in HaCaT cells were detected by using 3 H-TdR method and RT-PCR. Results: Arsenic caused a modest increase of keratinocyte DNA synthesis when the concentrationreached the range within 0.5-16 nmol/L, but the amount of incorporated 3 H-TdR decreased and returnedto baseline level when the concentration of arsenic increased to over 16 nmol/L. RT-PCR analysis showedthe level of E2F1 mRNA was elevated in HaCaT cells with the increase of DNA synthesis. Conclusion:Ar-senic of a certain concentration could increase DNA synthesis and enhance E2F1 expression in HaCaT cellline, which might be one of the pathological mechanisms of skin disease related to arsenic.

  16. Sensitive Tumorigenic Potential Evaluation of Adult Human Multipotent Neural Cells Immortalized by hTERT Gene Transduction.

    Directory of Open Access Journals (Sweden)

    Kee Hang Lee

    Full Text Available Stem cells and therapeutic genes are emerging as a new therapeutic approach to treat various neurodegenerative diseases with few effective treatment options. However, potential formation of tumors by stem cells has hampered their clinical application. Moreover, adequate preclinical platforms to precisely test tumorigenic potential of stem cells are controversial. In this study, we compared the sensitivity of various animal models for in vivo stem cell tumorigenicity testing to identify the most sensitive platform. Then, tumorigenic potential of adult human multipotent neural cells (ahMNCs immortalized by the human telomerase reverse transcriptase (hTERT gene was examined as a stem cell model with therapeutic genes. When human glioblastoma (GBM cells were injected into adult (4-6-week-old Balb/c-nu, adult NOD/SCID, adult NOG, or neonate (1-2-week-old NOG mice, the neonate NOG mice showed significantly faster tumorigenesis than that of the other groups regardless of intracranial or subcutaneous injection route. Two kinds of ahMNCs (682TL and 779TL were primary cultured from surgical samples of patients with temporal lobe epilepsy. Although the ahMNCs were immortalized by lentiviral hTERT gene delivery (hTERT-682TL and hTERT-779TL, they did not form any detectable masses, even in the most sensitive neonate NOG mouse platform. Moreover, the hTERT-ahMNCs had no gross chromosomal abnormalities on a karyotype analysis. Taken together, our data suggest that neonate NOG mice could be a sensitive animal platform to test tumorigenic potential of stem cell therapeutics and that ahMNCs could be a genetically stable stem cell source with little tumorigenic activity to develop regenerative treatments for neurodegenerative diseases.

  17. Telomerase-immortalized human mammary stem/progenitor cells with ability to self-renew and differentiate.

    Science.gov (United States)

    Zhao, Xiangshan; Malhotra, Gautam K; Lele, Subodh M; Lele, Manjiri S; West, William W; Eudy, James D; Band, Hamid; Band, Vimla

    2010-08-10

    There is increasing evidence that breast and other cancers originate from and are maintained by a small fraction of stem/progenitor cells with self-renewal properties. Whether such cancer stem/progenitor cells originate from normal stem cells based on initiation of a de novo stem cell program, by reprogramming of a more differentiated cell type by oncogenic insults, or both remains unresolved. A major hurdle in addressing these issues is lack of immortal human stem/progenitor cells that can be deliberately manipulated in vitro. We present evidence that normal and human telomerase reverse transcriptase (hTERT)-immortalized human mammary epithelial cells (hMECs) isolated and maintained in Dana-Farber Cancer Institute 1 (DFCI-1) medium retain a fraction with progenitor cell properties. These cells coexpress basal (K5, K14, and vimentin), luminal (E-cadherin, K8, K18, or K19), and stem/progenitor (CD49f, CD29, CD44, and p63) cell markers. Clonal derivatives of progenitors coexpressing these markers fall into two distinct types--a K5(+)/K19(-) type and a K5(+)/K19(+) type. We show that both types of progenitor cells have self-renewal and differentiation ability. Microarray analyses confirmed the differential expression of components of stem/progenitor-associated pathways, such as Notch, Wnt, Hedgehog, and LIF, in progenitor cells compared with differentiated cells. Given the emerging evidence that stem/progenitor cells serve as precursors for cancers, these cellular reagents represent a timely and invaluable resource to explore unresolved questions related to stem/progenitor origin of breast cancer.

  18. The expressions of ABCC4 and ABCG2 xenobiotic transporters in human keratinocytes are proliferation-related.

    Science.gov (United States)

    Bebes, Attila; Kis, Kornélia; Nagy, Tünde; Kurunczi, Anita; Polyánka, Hilda; Bata-Csörgo, Zsuzsanna; Kemény, Lajos; Dobozy, Attila; Széll, Márta

    2012-01-01

    Xenobiotic transporters of the ATP-binding cassette (ABC) protein superfamily play important roles in maintaining the biochemical barrier of various tissues, but their precise functions in the skin are not yet known. Screening of the expressions of the known xenobiotic transporter genes in two in vitro keratinocyte differentiation models revealed that the ABCC4 and ABCG2 transporters are highly expressed in proliferating keratinocytes, their expressions decreasing along with differentiation. Abrogation of the ABCC4 and ABCG2 protein functions by siRNA-mediated silencing and chemical inhibition did not affect the proliferation of HaCaT cells. In contrast, disruption of the ABCG2 function had no effect on normal human epidermal keratinocyte proliferation, while the inhibition of ABCC-type transporters by probenecid resulted in a striking decrease in the proliferation of the cells. These results indicate that, besides their possible therapy-modulating effects, xenobiotic transporters may contribute significantly to other keratinocyte functions, such as cell proliferation.

  19. Mu-opiate receptor and Beta-endorphin expression in nerve endings and keratinocytes in human skin.

    Science.gov (United States)

    Bigliardi-Qi, M; Sumanovski, L T; Büchner, S; Rufli, T; Bigliardi, P L

    2004-01-01

    We have previously shown that human epidermal keratinocytes express a functionally active micro-opiate receptor, which adds a new dimension to the recently developed research in neuroimmunodermatology and neurogenic inflammation in skin diseases. Human keratinocytes specifically bind and also produce beta-endorphin, the endogenous micro-opiate receptor ligand. Using confocal imaging microscopy, we could now demonstrate that micro-opiate receptors are not only expressed in keratinocytes, but also on unmyelinated peripheral nerve fibers in the dermis and epidermis. Some of the peripheral nerve fibers also express the ligand beta-endorphin. The keratinocytes positive for beta-endorphin staining are clustered around the terminal ends of the unmyelinated nerve fibers. Therefore the opiate receptor system seems to be crucial in the direct communication between nerves and skin. The keratinocytes can influence the unmyelinated nerve fibers in the epidermis directly via secreting beta-endorphin. On the other hand, nerve fibers can also secrete beta-endorphin and influence the migration, differentiation and probably also the cytokine production pattern of keratinocytes.

  20. The activation of cultured keratinocytes by cholesterol depletion during reconstruction of a human epidermis is reminiscent of monolayer cultures.

    Science.gov (United States)

    De Vuyst, Évelyne; Giltaire, Séverine; Lambert de Rouvroit, Catherine; Chrétien, Aline; Salmon, Michel; Poumay, Yves

    2015-05-01

    Transient cholesterol depletion from plasma membranes of human keratinocytes has been shown to reversibly activate signalling pathways in monolayer cultures. Consecutive changes in gene expression have been characterized in such conditions and were interestingly found to be similar to transcriptional changes observed in keratinocytes of atopic dermatitis (AD) patients. As an inflammatory skin disease, AD notably results in altered histology of the epidermis associated with a defective epidermal barrier. To further investigate whether the activation of keratinocytes obtained by cholesterol depletion could be responsible for some epidermal alterations reported in AD, this study was undertaken to analyse cholesterol depletion in stratified cultures of keratinocytes, i.e. a reconstructed human epidermis (RHE). RHE contains heterogeneous populations of keratinocytes, either proliferating or progressively differentiating and stratifying towards the creation of a cornified barrier. Cholesterol depletion induced in this model was found reversible and resulted in activation of signalling pathways similar to those previously identified in monolayers. In addition, selected changes in the expression of several genes suggested that keratinocytes in RHE respond to cholesterol depletion as monolayers. However, preserved histology and barrier function indicate that some additional activation, likely from the immune system, is required to obtain epidermal alterations such as the ones found in AD.

  1. Ecto-nucleoside triphosphate diphosphohydrolase 2 modulates local ATP-induced calcium signaling in human HaCaT keratinocytes.

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    Chia-Lin Ho

    Full Text Available Keratinocytes are the major building blocks of the human epidermis. In many physiological and pathophysiological conditions, keratinocytes release adenosine triphosphate (ATP as an autocrine/paracrine mediator that regulates cell proliferation, differentiation, and migration. ATP receptors have been identified in various epidermal cell types; therefore, extracellular ATP homeostasis likely determines its long-term, trophic effects on skin health. We investigated the possibility that human keratinocytes express surface-located enzymes that modulate ATP concentration, as well as the corresponding receptor activation, in the pericellular microenvironment. We observed that the human keratinocyte cell line HaCaT released ATP and hydrolyzed extracellular ATP. Interestingly, ATP hydrolysis resulted in adenosine diphosphate (ADP accumulation in the extracellular space. Pharmacological inhibition by ARL 67156 or gene silencing of the endogenous ecto-nucleoside triphosphate diphosphohydrolase (NTPDase isoform 2 resulted in a 25% reduction in both ATP hydrolysis and ADP formation. Using intracellular calcium as a reporter, we found that although NTPDase2 hydrolyzed ATP and generated sustainable ADP levels, only ATP contributed to increased intracellular calcium via P2Y2 receptor activation. Furthermore, knocking down NTPDase2 potentiated the nanomolar ATP-induced intracellular calcium increase, suggesting that NTPDase2 globally attenuates nucleotide concentration in the pericellular microenvironment as well as locally shields receptors in the vicinity from being activated by extracellular ATP. Our findings reveal an important role of human keratinocyte NTPDase2 in modulating nucleotide signaling in the extracellular milieu of human epidermis.

  2. Pseudomonas-derived ceramidase induces production of inflammatory mediators from human keratinocytes via sphingosine-1-phosphate.

    Directory of Open Access Journals (Sweden)

    Ami Oizumi

    Full Text Available Ceramide is important for water retention and permeability barrier functions in the stratum corneum, and plays a key role in the pathogenesis of atopic dermatitis (AD. A Pseudomonas aeruginosa-derived neutral ceramidase (PaCDase isolated from a patient with AD was shown to effectively degrade ceramide in the presence of Staphylococcus aureus-derived lipids or neutral detergents. However, the effect of ceramide metabolites on the functions of differentiating keratinocytes is poorly understood. We found that the ceramide metabolite sphingosine-1-phosphate (S1P stimulated the production of inflammatory mediators such as TNF-α and IL-8 from three-dimensionally cultured human primary keratinocytes (termed "3D keratinocytes", which form a stratum corneum. PaCDase alone did not affect TNF-α gene expression in 3D keratinocytes. In the presence of the detergent Triton X-100, which damages stratum corneum structure, PaCDase, but not heat-inactivated PaCDase or PaCDase-inactive mutant, induced the production of TNF-α, endothelin-1, and IL-8, indicating that this production was dependent on ceramidase activity. Among various ceramide metabolites, sphingosine and S1P enhanced the gene expression of TNF-α, endothelin-1, and IL-8. The PaCDase-enhanced expression of these genes was inhibited by a sphingosine kinase inhibitor and by an S1P receptor antagonist VPC 23019. The TNF-α-binding antibody infliximab suppressed the PaCDase-induced upregulation of IL-8, but not TNF-α, mRNA. PaCDase induced NF-κB p65 phosphorylation. The NF-κB inhibitor curcumin significantly inhibited PaCDase-induced expression of IL-8 and endothelin-1. VPC 23019 and infliximab inhibited PaCDase-induced NF-κB p65 phosphorylation and reduction in the protein level of the NF-κB inhibitor IκBα. Collectively, these findings suggest that (i 3D keratinocytes produce S1P from sphingosine, which is produced through the hydrolysis of ceramide by PaCDase, (ii S1P induces the production

  3. Novel non-calcemic secosteroids that are produced by human epidermal keratinocytes protect against solar radiation.

    Science.gov (United States)

    Slominski, Andrzej T; Janjetovic, Zorica; Kim, Tae-Kang; Wasilewski, Piotr; Rosas, Sofia; Hanna, Sherie; Sayre, Robert M; Dowdy, John C; Li, Wei; Tuckey, Robert C

    2015-04-01

    CYP11A1 hydroxylates the side chain of vitamin D3 (D3) in a sequential fashion [D3→20S(OH)D3→20,23(OH)2D3→17,20,23(OH)3D3], in an alternative to the classical pathway of activation [D3→25(OH)D3→1,25(OH)2D3]. The products/intermediates of the pathway can be further modified by the action of CYP27B1. The CYP11A1-derived products are biologically active with functions determined by the lineage of the target cells. This pathway can operate in epidermal keratinocytes. To further define the role of these novel secosteroids we tested them for protective effects against UVB-induced damage in human epidermal keratinocytes, melanocytes and HaCaT keratinocytes, cultured in vitro. The secosteroids attenuated ROS, H2O2 and NO production by UVB-irradiated keratinocytes and melanocytes, with an efficacy similar to 1,25(OH)2D3, while 25(OH)D3 had lower efficacy. These attenuations were also seen to some extent for the 20(OH)D3 precursor, 20S-hydroxy-7-dehydrocholesterol. These effects were accompanied by upregulation of genes encoding enzymes responsible for defense against oxidative stress. Using immunofluorescent staining we observed that the secosteroids reduced the generation cyclobutane pyrimidine dimers in response to UVB and enhanced expression of p53 phosphorylated at Ser-15, but not at Ser-46. Additional evidence for protection against DNA damage in cells exposed to UVB and treated with secosteroids was provided by the Comet assay where DNA fragmentation was markedly reduced by 20(OH)D3 and 20,23(OH)2D3. In conclusion, novel secosteroids that can be produced by the action of CYP11A1 in epidermal keratinocytes have protective effects against UVB radiation. This article is part of a special issue entitled '17th Vitamin D Workshop'. Copyright © 2015 Elsevier Ltd. All rights reserved.

  4. Regulation of oxidative-stress responsive genes by arecoline in human keratinocytes.

    Science.gov (United States)

    Thangjam, G S; Kondaiah, P

    2009-10-01

    Arecoline, an arecanut alkaloid present in the saliva of betel quid chewers, has been implicated in the pathogenesis of a variety of inflammatory oral diseases, including oral submucous fibrosis and periodontitis. To understand the molecular basis of arecoline action in epithelial changes associated with these diseases, we investigated the effects of arecoline on human keratinocytes with respect to cell growth regulation and the expression of stress-responsive genes. Human keratinocyte cells (of the HaCaT cell line) were treated with arecoline, following which cell viability was assessed using the Trypan Blue dye-exclusion assay, cell growth and proliferation were analyzed using the MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) and 5-bromo-2-deoxyuridine incorporation assays, cell cycle arrest and generation of reactive oxygen species were examined using flow cytometry, and gene expression changes were investigated using the reverse transcription-polymerase chain reaction technique. The role of oxidative stress, muscarinic acetylcholine receptor and mitogen-activated protein kinase (MAPK) pathways were studied using specific inhibitors. Western blot analysis was performed to study p38 MAPK activation. Arecoline induced the generation of reactive oxygen species and cell cycle arrest at the G1/G0 phase in HaCaT cells without affecting the expression of p21/Cip1. Arecoline-induced epithelial cell death at higher concentrations was caused by oxidative trauma without eliciting apoptosis. Sublethal concentrations of arecoline upregulated the expression of the following stress-responsive genes: heme oxygenase-1; ferritin light chain; glucose-6-phosphate dehydrogenase; glutamate-cysteine ligase catalytic subunit; and glutathione reductase. Additionally, there was a dose-dependent induction of interleukin-1alfa mRNA by arecoline via oxidative stress and p38 MAPK activation. Our data highlight the role of oxidative stress in arecoline-mediated cell death

  5. Transcriptome analysis of airborne PM2.5-induced detrimental effects on human keratinocytes.

    Science.gov (United States)

    Kim, Hyoung-June; Bae, Il-Hong; Son, Eui Dong; Park, Juyearl; Cha, Nari; Na, Hye-Won; Jung, Changjo; Go, You-Seak; Kim, Dae-Yong; Lee, Tae Ryong; Shin, Dong Wook

    2017-05-05

    Ambient air pollution is becoming more severe worldwide, posing a serious threat to human health. Fine airborne particles of particulate matter (PM2.5) show higher cytotoxicity than other coarse fractions. Indeed, PM2.5 induces cardiovascular or respiratory damage; however, few studies have evaluated the detrimental effect of PM2.5 to normal human skin. We used a next-generation sequencing-based (RNA-Seq) method with transcriptome and Gene Ontology (GO) enrichment analysis to determine the harmful influences of PM2.5 on human normal epidermal keratinocytes. DAVID analysis showed that the most significantly enriched GO terms were associated with epidermis-related biological processes such as "epidermis development (GO: 0008544)" and "keratinocyte differentiation (GO: 0030216)", suggesting that PM2.5 has some deleterious effects to the human epidermis. In addition, Ingenuity Pathway Analysis predicted inflammation-related signaling as one of the major PM2.5-induced signaling pathways, and pro-inflammatory cytokines as upstream regulators with symptoms similar to psoriasis as downstream effects. PM2.5 caused considerable changes in the expression of pro-inflammatory cytokines and psoriatic skin disease-related genes, might lead to epidermal dysfunctions. Our results might help to understand the mechanism of air pollution-induced skin barrier perturbation and contribute to the development of a new strategy for the prevention or recovery of the consequent damage. Copyright © 2017 Elsevier B.V. All rights reserved.

  6. UVB exposure of a humanized skin model reveals unexpected dynamic of keratinocyte proliferation and Wnt inhibitor balancing.

    Science.gov (United States)

    Michalczyk, Teresa; Biedermann, Thomas; Böttcher-Haberzeth, Sophie; Klar, Agnes S; Meuli, Martin; Reichmann, Ernst

    2017-07-17

    We developed human dermo-epidermal skin substitutes which are presently applied in phase I and II clinical trials. Here we used these very same skin equivalents containing melanocytes, named MelSkin, as an experimental skin model. We investigated the effects of UVB irradiation on the skin grafts transplanted on immune-compromised rats. The irradiation induces a strong wound healing response going along with massive proliferation of basal keratinocytes, basically quiescent under non-irradiated, homeostatic conditions. As a consequence of UVB irradiation, the initially clearly defined basal keratinocyte (mono)layer expands into about three layers of keratinocytes, all of which still express the basal keratinocyte marker Keratin15. In contrast, epidermal melanocytes remain quiescent under these circumstances. Moreover, the Wnt inhibitors Dickkopf 3 and Wif1 are downregulated upon UVB irradiation in basal keratinocytes, whereas melanocytes continue to express Wnt inhibitors. These findings suggest that there is 1) a suprabasal population, proliferating in the homeostatic state, hence maintaining the integrity of the epidermis, and 2) a basal, usually quiescent keratinocyte population that is induced to massively proliferate upon irradiation. Importantly, the finding that MelSkin responds in a physiological fashion to UVB is of paramount importance in light of the planned clinical application. This article is protected by copyright. All rights reserved.

  7. Biological properties of differently-aged human keratinocytes:population doubling time growth curve and cell cycle analysis

    Institute of Scientific and Technical Information of China (English)

    Hui-qun Ma; Jie Feng; Lech Chyczewski; Jacek Niklinski

    2009-01-01

    Objective To explore the biological properties of keratinocytes from differently-aged healthy human beings. Methods Keratinocytes from fetus, teenager and middle-aged groups were separated and cultured. The population doubling time (PDT) and cell growth curve in different cells were compared, and the cell cycles were analyzed by flow cytometry. Results ① In primary culture of keratinocytes, the adherence time in middle-aged group was longer than that in fetus and teenager groups. However, all cell morphology showed no obvioas differences. In subculture of kecatinocytes, with donator's age increasing, time of cell adherence prolonged, passage number decreused and differences in cell morphology were obrioas. ② The average PDT of keratinocytes was shorter in fetus group than in teenager and middle-aged groups. Bat difference in cell growth curve between different passages was not observed. ③ Keratinocytes showed G2/M period in fetus group but G0/G1 period in teenager and middle-aged groups mainly. Conclusion As age increases, the biological properties of keratinocytes change obviously.

  8. Human epidermal keratinocytes death and expression of protein markers of apoptosis after ionizing radiation exposure

    Directory of Open Access Journals (Sweden)

    Sharon Wong

    2013-12-01

    Full Text Available Purpose: Knowledge of the pathophysiology of the irradiated skin is important to understand the tolerance and cosmetic response of the human skin to radiation. There are limited studies on the effect of radiotherapy dosage and fraction size in inducing apoptotic cell death in human skin. The expression of apoptotic biomarkers within a controlled population in different fractionation schemes has also never been studied. This study aims to investigate radiation induced apoptotic cell death in human skin cells after fractionated radiation exposure and the expression of unique biomarkers that reflect cell death or biology using multiplexed immunoassays.Methods: Breast skin biopsies were obtained from a single individual and divided into small pieces. Each piece was irradiated under different radiotherapy treatment fractionation schedules to a total dose of 50Gy. The irradiated skin tissues were analysed using Tunnel, immunohistochemistry and Western blot assays for expression of apoptotic keratinocytes and biomarkers (p53, p21, and PCNA. Haematoxylin and eosin (H&E immunostaining was performed to study the morphological changes in the skin cells. Results: Radiation is mostly absorbed by the epidermal layers and observed to damage the epidermal keratinocytes leading to the activation of apoptotic proteins. Apoptotic proteins (p53, p21 and PCNA were confirmed to be up-regulated in radiation exposed skin cells as compared to normal skin cells with no radiation. There is strong correlation of apoptotic protein expressions with increased radiation dosage and dose fractionation. Statistical analysis with ANOVA revealed a significant increase of PCNA and p21 expression with increased radiation dosage and dose fractionation (p < 0.05. Immunohistochemically, 14 % (range 10.71% to 17.29% of the keratinocytes were positive for PCNA and 22.5% (range 18.28% to 27.2% for p21 after 2Gy of irradiation.  The most widespread, intense and uniform staining for PCNA

  9. 1,25-Dihydroxyvitamin D3 production by human keratinocytes. Kinetics and regulation.

    OpenAIRE

    Bikle, D D; Nemanic, M K; Gee, E; Elias, P

    1986-01-01

    Human foreskin keratinocytes in vitro metabolize 25-hydroxyvitamin D3 to a number of metabolites, including 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). This metabolite remains mostly within the cell and does not accumulate in the medium under the conditions of these experiments. With time, 1,25(OH)2D3 is catabolized, and more polar metabolites appear in both the cells and the medium. The production of 1,25(OH)2D3 has an apparent Michaelis constant (Km) for 25-hydroxyvitamin D3 of 5.4 X 10(-8) M. ...

  10. Shuttling of the autoantigen La between nucleus and cell surface after uv irradiation of human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bachmann, M.; Chang, S.; Slor, H.; Kukulies, J.; Mueller, W.E. (Universitaet, Mainz (Germany, F.R.))

    1990-12-01

    During the past years we have established that the nuclear autoantigen La shuttles between the nucleus and the cytoplasm in tumor cells after inhibition of transcription or virus infection. We reinvestigated this shuttling using primary human keratinocytes from both healthy donors and patients with xeroderma pigmentosum. Ultraviolet irradiation resulted in both an inhibition of transcription and a translocation of La protein from the nucleus to the cytoplasm. After a prolonged inhibition of transcription La protein relocated into the nucleus and assembled with nuclear storage regions. The uv-induced shuttling included a translocation to the cell surface, where La protein colocalized with epidermal growth factor receptors.

  11. Epithelial cells derived from human embryonic stem cells display p16INK4A senescence, hypermotility, and differentiation properties shared by many P63+ somatic cell types

    DEFF Research Database (Denmark)

    Dabelsteen, Sally; Hercule, Paula; Barron, Patricia

    2009-01-01

    Human embryonic stem (hES) cells can generate cells expressing p63, K14, and involucrin, which have been proposed to be keratinocytes. Although these hES-derived, keratinocyte-like (hESderK) cells form epithelioid colonies when cultured in a fibroblast feeder system optimal for normal tissue......(+)/K14(+) urothelial and tracheobronchial epithelial cells. Primary and immortalized lines of these cell types had growth requirements and hypermotility responses similar to keratinocytes and bmi1 expression facilitated their immortalization by engineering to express the catalytic subunit of telomerase...

  12. Hyperthermia-induced micronucleus formation in a human keratinocyte cell line

    Energy Technology Data Exchange (ETDEWEB)

    Hintzsche, Henning; Riese, Thorsten [Universitaet Wuerzburg, Institut fuer Pharmakologie und Toxikologie, Versbacher Str. 9, 97078 Wuerzburg (Germany); Stopper, Helga, E-mail: Stopper@toxi.uni-wuerzburg.de [Universitaet Wuerzburg, Institut fuer Pharmakologie und Toxikologie, Versbacher Str. 9, 97078 Wuerzburg (Germany)

    2012-10-15

    Elevated temperature can cause biological effects in vitro and in vivo. Many studies on effects of hypo- and hyperthermia have been conducted, but only few studies systematically investigated the formation of genomic damage in the micronucleus test in human cells in vitro as a consequence of different temperatures. In the present study, HaCaT human keratinocytes were exposed to different temperatures from 37 Degree-Sign C to 42 Degree-Sign C for 24 h in a regular cell culture incubator. Micronucleus frequency as a marker of genomic damage was elevated in a temperature-dependent and statistically significant manner. Apoptosis occurred at temperatures of 39 Degree-Sign C or higher. Cell proliferation was unaffected up to 40 Degree-Sign C and decreased at 41 Degree-Sign C and 42 Degree-Sign C. Expression of the heat shock protein Hsp70 was elevated, particularly at temperatures of 40 Degree-Sign C and higher. These findings are in agreement with several in vivo studies and some in vitro studies looking at single, specific temperatures, but a systematically investigated temperature-dependent increase of genomic damage in human keratinocytes in vitro is demonstrated for the first time here.

  13. Asian dust storm particles induce a broad toxicological transcriptional program in human epidermal keratinocytes.

    Science.gov (United States)

    Choi, Hyun; Shin, Dong Wook; Kim, Wonnyon; Doh, Seong-Jae; Lee, Soo Hwan; Noh, Minsoo

    2011-01-15

    Exposure to airborne dust particles originated from seasonal Asian dust storms in Chinese and Mongolian deserts results in increased incidence of a range of diseases including asthma, contact dermatitis and conjunctivitis. The areas affected by Asian dust particles extend from East China to the west coast of North America. In order to study toxicological mechanisms in human skin, we evaluated the effects of dust particles collected during Asian dust storms (Asian dust particles) on gene expression in human epidermal keratinocytes (HEK). In HEK, exposure to Asian dust particles significantly increased gene expressions of cytochrome P450 1A1 (CYP1A1), CYP1A2, and CYP1B1, which is an indication of aryl hydrocarbon receptor (AHR) activation. In addition, Asian dust particles increased gene transcription of the cytokines IL-6, IL-8, and GM-CSF, which have broad pro-inflammatory and immunomodulatory properties. Asian dust particles significantly up-regulated expression of caspase 14 in HEK, suggesting that Asian dust particles directly affect keratinocyte differentiation. We also demonstrated that protein extract of pollen, a material frequently adsorbed onto Asian dust particles, potentially contributes to the increased transcription of IL-6, CYP1A1, CYP1A2, and CYP1B1. Taken together, these studies suggest that Asian dust particles can exert toxicological effects on human skin through the activation of the cellular detoxification system, the production of pro-inflammatory and immunomodulatory cytokines, and changes in the expression of proteins essential in normal epidermal differentiation.

  14. A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use.

    Science.gov (United States)

    Jubin, K; Martin, Y; Lawrence-Watt, D J; Sharpe, J R

    2011-12-01

    Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

  15. Staphylococcus aureus Biofilm and Planktonic cultures differentially impact gene expression, mapk phosphorylation, and cytokine production in human keratinocytes

    Directory of Open Access Journals (Sweden)

    Olerud John E

    2011-06-01

    Full Text Available Abstract Background Many chronic diseases, such as non-healing wounds are characterized by prolonged inflammation and respond poorly to conventional treatment. Bacterial biofilms are a major impediment to wound healing. Persistent infection of the skin allows the formation of complex bacterial communities termed biofilm. Bacteria living in biofilms are phenotypically distinct from their planktonic counterparts and are orders of magnitude more resistant to antibiotics, host immune response, and environmental stress. Staphylococcus aureus is prevalent in cutaneous infections such as chronic wounds and is an important human pathogen. Results The impact of S. aureus soluble products in biofilm-conditioned medium (BCM or in planktonic-conditioned medium (PCM on human keratinocytes was investigated. Proteomic analysis of BCM and PCM revealed differential protein compositions with PCM containing several enzymes involved in glycolysis. Global gene expression of keratinocytes exposed to biofilm and planktonic S. aureus was analyzed after four hours of exposure. Gene ontology terms associated with responses to bacteria, inflammation, apoptosis, chemotaxis, and signal transduction were enriched in BCM treated keratinocytes. Several transcripts encoding cytokines were also upregulated by BCM after four hours. ELISA analysis of cytokines confirmed microarray results at four hours and revealed that after 24 hours of exposure, S. aureus biofilm induced sustained low level cytokine production compared to near exponential increases of cytokines in planktonic treated keratinocytes. The reduction in cytokines produced by keratinocytes exposed to biofilm was accompanied by suppressed phosphorylation of MAPKs. Chemical inhibition of MAPKs did not drastically reduce cytokine production in BCM-treated keratinocytes suggesting that the majority of cytokine production is mediated through MAPK-independent mechanisms. Conclusions Collectively the results indicate that S

  16. Triterpenoid α-amyrin stimulates proliferation of human keratinocytes but does not protect them against UVB damage

    DEFF Research Database (Denmark)

    Biskup, Edyta; Gołębiowski, Marek; Gniadecki, Robert;

    2012-01-01

    Rhaponticum carthamoides plants ("maral root") are widely used in Siberian folk medicine. The present study reports for the first time the presence of pentacyclic terpenoid, α-amyrin, in methanol extract from leaves of this plant. α-Amyrin induced proliferation of human keratinocytes (HaCaT) by a......Rhaponticum carthamoides plants ("maral root") are widely used in Siberian folk medicine. The present study reports for the first time the presence of pentacyclic terpenoid, α-amyrin, in methanol extract from leaves of this plant. α-Amyrin induced proliferation of human keratinocytes (Ha...

  17. Growth regulation of primary human keratinocytes by prostaglandin E receptor EP2 and EP3 subtypes.

    Science.gov (United States)

    Konger, R L; Malaviya, R; Pentland, A P

    1998-02-04

    We examined the contribution of specific EP receptors in regulating cell growth. By RT-PCR and northern hybridization, adult human keratinocytes express mRNA for three PGE2 receptor subtypes associated with cAMP signaling (EP2, EP3, and small amounts of EP4). In actively growing, non-confluent primary keratinocyte cultures, the EP2 and EP4 selective agonists, 11-deoxy PGE1 and 1-OH PGE1, caused complete reversal of indomethacin-induced growth inhibition. The EP3/EP2 agonist (misoprostol), and the EP1/EP2 agonist (17-phenyl trinor PGE2), showed less activity. Similar results were obtained with agonist-induced cAMP formation. The ability of exogenous dibutyryl cAMP to completely reverse indomethacin-induced growth inhibition support the conclusion that growth stimulation occurs via an EP2 and/or EP4 receptor-adenylyl cyclase coupled response. In contrast, activation of EP3 receptors by sulprostone, which is virtually devoid of agonist activity at EP2 or EP4 receptors, inhibited bromodeoxyuridine uptake in indomethacin-treated cells up to 30%. Although human EP3 receptor variants have been shown in other cell types to markedly inhibit cAMP formation via a pertussis toxin sensitive mechanisms, EP3 receptor activation and presumably growth inhibition was independent of adenylyl cyclase, suggesting activation of other signaling pathways.

  18. Effect of JP-8 jet fuel exposure on protein expression in human keratinocyte cells in culture.

    Science.gov (United States)

    Witzmann, F A; Monteiro-Riviere, N A; Inman, A O; Kimpel, M A; Pedrick, N M; Ringham, H N; Riviere, J E

    2005-12-30

    Dermal exposure to jet fuel is a significant occupational hazard. Previous studies have investigated its absorption and disposition in skin, and the systemic biochemical and immunotoxicological sequelae to exposure. Despite studies of JP-8 jet fuel components in murine, porcine or human keratinocyte cell cultures, proteomic analysis of JP-8 exposure has not been investigated. This study was conducted to examine the effect of JP-8 administration on the human epidermal keratinocyte (HEK) proteome. Using a two-dimensional electrophoretic approach combined with mass spectrometric-based protein identification, we analyzed protein expression in HEK exposed to 0.1% JP-8 in culture medium for 24 h. JP-8 exposure resulted in significant expression differences (p<0.02) in 35 of the 929 proteins matched and analyzed. Approximately, a third of these alterations were increased in protein expression, two-thirds declined with JP-8 exposure. Peptide mass fingerprint identification of effected proteins revealed a variety of functional implications. In general, altered proteins involved endocytotic/exocytotic mechanisms and their cytoskeletal components, cell stress, and those involved in vesicular function.

  19. Additive Effects of Millimeter Waves and 2-Deoxyglucose Co-Exposure on the Human Keratinocyte Transcriptome

    Science.gov (United States)

    Soubere Mahamoud, Yonis; Aite, Meziane; Martin, Catherine; Zhadobov, Maxim; Sauleau, Ronan; Le Dréan, Yves

    2016-01-01

    Millimeter Waves (MMW) will be used in the next-generation of high-speed wireless technologies, especially in future Ultra-Broadband small cells in 5G cellular networks. Therefore, their biocompatibilities must be evaluated prior to their massive deployment. Using a microarray-based approach, we analyzed modifications to the whole genome of a human keratinocyte model that was exposed at 60.4 GHz-MMW at an incident power density (IPD) of 20 mW/cm2 for 3 hours in athermic conditions. No keratinocyte transcriptome modifications were observed. We tested the effects of MMWs on cell metabolism by co-treating MMW-exposed cells with a glycolysis inhibitor, 2-deoxyglucose (2dG, 20 mM for 3 hours), and whole genome expression was evaluated along with the ATP content. We found that the 2dG treatment decreased the cellular ATP content and induced a high modification in the transcriptome (632 coding genes). The affected genes were associated with transcriptional repression, cellular communication and endoplasmic reticulum homeostasis. The MMW/2dG co-treatment did not alter the keratinocyte ATP content, but it did slightly alter the transcriptome, which reflected the capacity of MMW to interfere with the bioenergetic stress response. The RT-PCR-based validation confirmed 6 MMW-sensitive genes (SOCS3, SPRY2, TRIB1, FAM46A, CSRNP1 and PPP1R15A) during the 2dG treatment. These 6 genes encoded transcription factors or inhibitors of cytokine pathways, which raised questions regarding the potential impact of long-term or chronic MMW exposure on metabolically stressed cells. PMID:27529420

  20. The Effect of Secretory Factors of Adipose-Derived Stem Cells on Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Soo-Wan Nam

    2012-01-01

    Full Text Available The beneficial effects of adipose-derived stem cell conditioned medium (ADSC-CM on skin regeneration have been reported. Although the mechanism of how ADSC-CM promotes skin regeneration is unclear, ADSC-CM contained various growth factors and it is an excellent raw material for skin treatment. ADSC-CM produced in a hypoxia condition of ADSC—in other words, Advanced Adipose-Derived Stem cell Protein Extract (AAPE—has great merits for skin regeneration. In this study, human primary keratinocytes (HKs, which play fundamental roles in skin tissue, was used to examine how AAPE affects HK. HK proliferation was significantly higher in the experimental group (1.22 μg/mL than in the control group. DNA gene chip demonstrated that AAPE in keratinocytes (p < 0.05 notably affected expression of 290 identified transcripts, which were associated with cell proliferation, cycle and migration. More keratinocyte wound healing and migration was shown in the experimental group (1.22 μg/mL. AAPE treatment significantly stimulated stress fiber formation, which was linked to the RhoA-ROCK pathway. We identified 48 protein spots in 2-D gel analysis and selected proteins were divided into 64% collagen components and 30% non-collagen components as shown by the MALDI-TOF analysis. Antibody array results contained growth factor/cytokine such as HGF, FGF-1, G-CSF, GM-CSF, IL-6, VEGF, and TGF-β3 differing from that shown by 2-D analysis. Conclusion: AAPE activates HK proliferation and migration. These results highlight the potential of the topical application of AAPE in the treatment of skin regeneration.

  1. Additive Effects of Millimeter Waves and 2-Deoxyglucose Co-Exposure on the Human Keratinocyte Transcriptome.

    Science.gov (United States)

    Soubere Mahamoud, Yonis; Aite, Meziane; Martin, Catherine; Zhadobov, Maxim; Sauleau, Ronan; Le Dréan, Yves; Habauzit, Denis

    2016-01-01

    Millimeter Waves (MMW) will be used in the next-generation of high-speed wireless technologies, especially in future Ultra-Broadband small cells in 5G cellular networks. Therefore, their biocompatibilities must be evaluated prior to their massive deployment. Using a microarray-based approach, we analyzed modifications to the whole genome of a human keratinocyte model that was exposed at 60.4 GHz-MMW at an incident power density (IPD) of 20 mW/cm2 for 3 hours in athermic conditions. No keratinocyte transcriptome modifications were observed. We tested the effects of MMWs on cell metabolism by co-treating MMW-exposed cells with a glycolysis inhibitor, 2-deoxyglucose (2dG, 20 mM for 3 hours), and whole genome expression was evaluated along with the ATP content. We found that the 2dG treatment decreased the cellular ATP content and induced a high modification in the transcriptome (632 coding genes). The affected genes were associated with transcriptional repression, cellular communication and endoplasmic reticulum homeostasis. The MMW/2dG co-treatment did not alter the keratinocyte ATP content, but it did slightly alter the transcriptome, which reflected the capacity of MMW to interfere with the bioenergetic stress response. The RT-PCR-based validation confirmed 6 MMW-sensitive genes (SOCS3, SPRY2, TRIB1, FAM46A, CSRNP1 and PPP1R15A) during the 2dG treatment. These 6 genes encoded transcription factors or inhibitors of cytokine pathways, which raised questions regarding the potential impact of long-term or chronic MMW exposure on metabolically stressed cells.

  2. Upregulation of FOXM1 induces genomic instability in human epidermal keratinocytes

    Directory of Open Access Journals (Sweden)

    Philpott Michael P

    2010-02-01

    Full Text Available Abstract Background The human cell cycle transcription factor FOXM1 is known to play a key role in regulating timely mitotic progression and accurate chromosomal segregation during cell division. Deregulation of FOXM1 has been linked to a majority of human cancers. We previously showed that FOXM1 was upregulated in basal cell carcinoma and recently reported that upregulation of FOXM1 precedes malignancy in a number of solid human cancer types including oral, oesophagus, lung, breast, kidney, bladder and uterus. This indicates that upregulation of FOXM1 may be an early molecular signal required for aberrant cell cycle and cancer initiation. Results The present study investigated the putative early mechanism of UVB and FOXM1 in skin cancer initiation. We have demonstrated that UVB dose-dependently increased FOXM1 protein levels through protein stabilisation and accumulation rather than de novo mRNA expression in human epidermal keratinocytes. FOXM1 upregulation in primary human keratinocytes triggered pro-apoptotic/DNA-damage checkpoint response genes such as p21, p38 MAPK, p53 and PARP, however, without causing significant cell cycle arrest or cell death. Using a high-resolution Affymetrix genome-wide single nucleotide polymorphism (SNP mapping technique, we provided the evidence that FOXM1 upregulation in epidermal keratinocytes is sufficient to induce genomic instability, in the form of loss of heterozygosity (LOH and copy number variations (CNV. FOXM1-induced genomic instability was significantly enhanced and accumulated with increasing cell passage and this instability was increased even further upon exposure to UVB resulting in whole chromosomal gain (7p21.3-7q36.3 and segmental LOH (6q25.1-6q25.3. Conclusion We hypothesise that prolonged and repeated UVB exposure selects for skin cells bearing stable FOXM1 protein causes aberrant cell cycle checkpoint thereby allowing ectopic cell cycle entry and subsequent genomic instability. The aberrant

  3. Normal human epidermal keratinocytes treated with 7-dehydrocholesterol express increased levels of heat shock protein.

    Science.gov (United States)

    Mammone, Thomas; Muizzuddin, Neelam; Goyarts, Earl; Gan, David; Giacomoni, Paolo; Marenus, Ken; Maes, Daniel

    2004-01-01

    Human skin, and its isolated cells, respond to insults with a variety of repair and protective mechanisms. One such mechanism is the production of heat shock proteins (HSPs). Heat shock proteins help the other cellular proteins fold correctly into their active three-dimensional structures. Therefore, they can enhance the survival of cells under harsh, denaturing conditions. In order to develop a means of promoting the heat shock response to prepare the skin to withstand insult, we are investigating materials that appear to protect the skin biologically. One such material is vitamin D3 and its precursors. We have observed that keratinocytes treated with 7-dehydrocholesterol (7-DHC), a precursor of vitamin D3, have increased levels of protein and mRNA for heat shock proteins. In addition, we observed that topically applied 7-DHC increases the minimal dose of UVB required to induce erythema. These data suggest that 7-DHC can induce heat shock proteins in skin keratinocytes and that they will be more resistant to UVB insult.

  4. Saponins from Tribulus terrestris L. protect human keratinocytes from UVB-induced damage.

    Science.gov (United States)

    Sisto, Margherita; Lisi, Sabrina; D'Amore, Massimo; De Lucro, Raffaella; Carati, Davide; Castellana, Donatello; La Pesa, Velia; Zuccarello, Vincenzo; Lofrumento, Dario D

    2012-12-05

    Chronic exposure to solar UVB radiation damages skin, increasing the risk to develop cancer. Hence the identification of compounds with a photoprotective efficacy is essential. This study examined the role of saponins derived from Tribulus terrestris L. (TT) on the modulation of apoptosis in normal human keratinocytes (NHEK) exposed to physiological doses of UVB and to evaluate their antitumoral properties. In NHEK, TT saponins attenuate UVB-induced programmed cell death through inhibition of intrinsic apoptotic pathway. In squamous cell carcinomas (SCC) TT saponins do not make the malignant keratinocytes more resistant to UVB and determine an enhanced apoptotic response. The photoprotective effect of TT saponins is tightly correlated to the enhancement of NER genes expression and the block of UVB-mediated NF-κB activation. Collectively, our study shows experimental evidence that TT has a preventive efficacy against UVB-induced carcinogenesis and the molecular knowledge on the mechanisms through which TT saponins regulate cell death suggests great potential for TT to be developed into a new medicine for cancer patients.

  5. Phevalin (aureusimine B) production by Staphylococcus aureus biofilm and impacts on human keratinocyte gene expression.

    Science.gov (United States)

    Secor, Patrick R; Jennings, Laura K; James, Garth A; Kirker, Kelly R; Pulcini, Elinor Delancey; McInnerney, Kate; Gerlach, Robin; Livinghouse, Tom; Hilmer, Jonathan K; Bothner, Brian; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2012-01-01

    Staphylococcus aureus biofilms are associated with chronic skin infections and are orders of magnitude more resistant to antimicrobials and host responses. S. aureus contains conserved nonribosomal peptide synthetases that produce the cyclic dipeptides tyrvalin and phevalin (aureusimine A and B, respectively). The biological function of these compounds has been speculated to be involved in virulence factor gene expression in S. aureus, protease inhibition in eukaryotic cells, and interspecies bacterial communication. However, the exact biological role of these compounds is unknown. Here, we report that S. aureus biofilms produce greater amounts of phevalin than their planktonic counterparts. Phevalin had no obvious impact on the extracellular metabolome of S. aureus as measured by high-performance liquid chromatography-mass spectrometry and nuclear magnetic resonance. When administered to human keratinocytes, phevalin had a modest effect on gene expression. However, conditioned medium from S. aureus spiked with phevalin amplified differences in keratinocyte gene expression compared to conditioned medium alone. Phevalin may be exploited as potential biomarker and/or therapeutic target for chronic, S. aureus biofilm-based infections.

  6. Analyses of the secondary particle radiation and the DNA damage it causes to human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lebel E.; Rusek A.; Sivertz, M.; Yip, K.; Thompson, K.; Tafrov, S.

    2011-11-22

    High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.

  7. Analyses of the Secondary Particle Radiation and the DNA Damage it Causes to Human Keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Lebel E. A.; Tafrov S.; Rusek, A.; Sivertz, M. B.; Yip, K.; Thompson, K. H.

    2011-11-01

    High-energy protons, and high mass and energy ions, along with the secondary particles they produce, are the main contributors to the radiation hazard during space explorations. Skin, particularly the epidermis, consisting mainly of keratinocytes with potential for proliferation and malignant transformation, absorbs the majority of the radiation dose. Therefore, we used normal human keratinocytes to investigate and quantify the DNA damage caused by secondary radiation. Its manifestation depends on the presence of retinol in the serum-free media, and is regulated by phosphatidylinositol 3-kinases. We simulated the generation of secondary radiation after the impact of protons and iron ions on an aluminum shield. We also measured the intensity and the type of the resulting secondary particles at two sample locations; our findings agreed well with our predictions. We showed that secondary particles inflict DNA damage to different extents, depending on the type of primary radiation. Low-energy protons produce fewer secondary particles and cause less DNA damage than do high-energy protons. However, both generate fewer secondary particles and inflict less DNA damage than do high mass and energy ions. The majority of cells repaired the initial damage, as denoted by the presence of 53BPI foci, within the first 24 hours after exposure, but some cells maintained the 53BP1 foci longer.

  8. Cysteine Prevents the Reduction in Keratin Synthesis Induced by Iron Deficiency in Human Keratinocytes.

    Science.gov (United States)

    Miniaci, Maria Concetta; Irace, Carlo; Capuozzo, Antonella; Piccolo, Marialuisa; Di Pascale, Antonio; Russo, Annapina; Lippiello, Pellegrino; Lepre, Fabio; Russo, Giulia; Santamaria, Rita

    2016-02-01

    L-cysteine is currently recognized as a conditionally essential sulphur amino acid. Besides contributing to many biological pathways, cysteine is a key component of the keratin protein by its ability to form disulfide bridges that confer strength and rigidity to the protein. In addition to cysteine, iron represents another critical factor in regulating keratins expression in epidermal tissues, as well as in hair follicle growth and maturation. By focusing on human keratinocytes, the aim of this study was to evaluate the effect of cysteine supplementation as nutraceutical on keratin biosynthesis, as well as to get an insight on the interplay of cysteine availability and cellular iron status in regulating keratins expression in vitro. Herein we demonstrate that cysteine promotes a significant up-regulation of keratins expression as a result of de novo protein synthesis, while the lack of iron impairs keratin expression. Interestingly, cysteine supplementation counteracts the adverse effect of iron deficiency on cellular keratin expression. This effect was likely mediated by the up-regulation of transferrin receptor and ferritin, the main cellular proteins involved in iron homeostasis, at last affecting the labile iron pool. In this manner, cysteine may also enhance the metabolic iron availability for DNA synthesis without creating a detrimental condition of iron overload. To the best of our knowledge, this is one of the first study in an in vitro keratinocyte model providing evidence that cysteine and iron cooperate for keratins expression, indicative of their central role in maintaining healthy epithelia.

  9. Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes.

    Directory of Open Access Journals (Sweden)

    Xiaoru Zhang

    Full Text Available Ultraviolet radiation (UVR from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR. A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.

  10. Solar Simulated Ultraviolet Radiation Induces Global Histone Hypoacetylation in Human Keratinocytes.

    Science.gov (United States)

    Zhang, Xiaoru; Kluz, Thomas; Gesumaria, Lisa; Matsui, Mary S; Costa, Max; Sun, Hong

    2016-01-01

    Ultraviolet radiation (UVR) from sunlight is the primary effector of skin DNA damage. Chromatin remodeling and histone post-translational modification (PTM) are critical factors in repairing DNA damage and maintaining genomic integrity, however, the dynamic changes of histone marks in response to solar UVR are not well characterized. Here we report global changes in histone PTMs induced by solar simulated UVR (ssUVR). A decrease in lysine acetylation of histones H3 and H4, particularly at positions of H3 lysine 9, lysine 56, H4 lysine 5, and lysine 16, was found in human keratinocytes exposed to ssUVR. These acetylation changes were highly associated with ssUVR in a dose-dependent and time-specific manner. Interestingly, H4K16ac, a mark that is crucial for higher order chromatin structure, exhibited a persistent reduction by ssUVR that was transmitted through multiple cell divisions. In addition, the enzymatic activities of histone acetyltransferases were significantly reduced in irradiated cells, which may account for decreased global acetylation. Moreover, depletion of histone deacetylase SIRT1 in keratinocytes rescued ssUVR-induced H4K16 hypoacetylation. These results indicate that ssUVR affects both HDAC and HAT activities, leading to reduced histone acetylation.

  11. TCDD induces dermal accumulation of keratinocyte-derived matrix metalloproteinase-10 in an organotypic model of human skin.

    Science.gov (United States)

    De Abrew, K Nadira; Thomas-Virnig, Christina L; Rasmussen, Cathy A; Bolterstein, Elyse A; Schlosser, Sandy J; Allen-Hoffmann, B Lynn

    2014-05-01

    The epidermis of skin is the first line of defense against the environment. A three dimensional model of human skin was used to investigate tissue-specific phenotypes induced by the environmental contaminant, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). Continuous treatment of organotypic cultures of human keratinocytes with TCDD resulted in intracellular spaces between keratinocytes of the basal and immediately suprabasal layers as well as thinning of the basement membrane, in addition to the previously reported hyperkeratinization. These tissue remodeling events were preceded temporally by changes in expression of the extracellular matrix degrading enzyme, matrix metalloproteinase-10 (MMP-10). In organotypic cultures MMP-10 mRNA and protein were highly induced following TCDD treatment. Q-PCR and immunoblot results from TCDD-treated monolayer cultures, as well as indirect immunofluorescence and immunoblot analysis of TCDD-treated organotypic cultures, showed that MMP-10 was specifically contributed by the epidermal keratinocytes but not the dermal fibroblasts. Keratinocyte-derived MMP-10 protein accumulated over time in the dermal compartment of organotypic cultures. TCDD-induced epidermal phenotypes in organotypic cultures were attenuated by the keratinocyte-specific expression of tissue inhibitor of metalloproteinase-1, a known inhibitor of MMP-10. These studies suggest that MMP-10 and possibly other MMP-10-activated MMPs are responsible for the phenotypes exhibited in the basement membrane, the basal keratinocyte layer, and the cornified layer of TCDD-treated organotypic cultures. Our studies reveal a novel mechanism by which the epithelial-stromal microenvironment is altered in a tissue-specific manner thereby inducing structural and functional pathology in the interfollicular epidermis of human skin.

  12. Nuclear DNA damage-triggered NLRP3 inflammasome activation promotes UVB-induced inflammatory responses in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Hasegawa, Tatsuya, E-mail: tatsuya.hasegawa@to.shiseido.co.jp; Nakashima, Masaya; Suzuki, Yoshiharu

    2016-08-26

    Ultraviolet (UV) radiation in sunlight can result in DNA damage and an inflammatory reaction of the skin commonly known as sunburn, which in turn can lead to cutaneous tissue disorders. However, little has been known about how UV-induced DNA damage mediates the release of inflammatory mediators from keratinocytes. Here, we show that UVB radiation intensity-dependently increases NLRP3 gene expression and IL-1β production in human keratinocytes. Knockdown of NLRP3 with siRNA suppresses UVB-induced production of not only IL-1β, but also other inflammatory mediators, including IL-1α, IL-6, TNF-α, and PGE{sub 2}. In addition, inhibition of DNA damage repair by knockdown of XPA, which is a major component of the nucleotide excision repair system, causes accumulation of cyclobutane pyrimidine dimer (CPD) and activation of NLRP3 inflammasome. In vivo immunofluorescence analysis confirmed that NLRP3 expression is also elevated in UV-irradiated human epidermis. Overall, our findings indicate that UVB-induced DNA damage initiates NLRP3 inflammasome activation, leading to release of various inflammatory mediators from human keratinocytes. - Highlights: • UVB radiation induces NLRP3 inflammasome activation in human keratinocytes. • NLRP3 knockdown suppresses production of UVB-induced inflammatory mediators. • UVB-induced DNA damage triggers NLRP3 inflammasome activation. • NLRP3 expression in human epidermis is elevated in response to UV radiation.

  13. Expression of human cell cycle regulators in the primary cell line of the African savannah elephant (loxodonta africana) increases proliferation until senescence, but does not induce immortalization.

    Science.gov (United States)

    Fukuda, Tomokazu; Iino, Yuuka; Onuma, Manabu; Gen, Bando; Inoue-Murayama, Miho; Kiyono, Tohru

    2016-01-01

    The African savannah elephant (Loxodonta africana) is one of the critically endangered animals. Conservation of genetic and cellular resources is important for the promotion of wild life-related research. Although primary cultured cells are a useful model for the physiology and genomics of the wild-type animals, their distribution is restricted due to the limited number of cell divisions allowed in them. Here, we tried to immortalize a primary cell line of L. africana with by overexpressing human mutant form of cyclin-dependent kinase 4 (CDK4R24C), cyclin D, and telomerase (TERT). It has been shown before that the combination of human CDK4R24C, cyclin D, and TERT induces the efficient cellular immortalization of cells derived from humans, bovine, swine, and monkeys. Interestingly, although the combination of these three genes extended the cellular proliferation of the L. africana-derived cells, they did not induce cellular immortalization. This study suggest that control of cellular senescence in L. africana-derived cells would be different molecular mechanisms compared to those governing human, bovine, swine, and monkey cells.

  14. Characterization of a novel telomerase-immortalized human endometrial stromal cell line, St-T1b

    Directory of Open Access Journals (Sweden)

    Brosens Jan J

    2009-07-01

    Full Text Available Abstract Background Coordinated differentiation of the endometrial compartments in the second half of the menstrual cycle is a prerequisite for the establishment of pregnancy. Endometrial stromal cells (ESC decidualize under the influence of ovarian progesterone to accommodate implantation of the blastocyst and support establishment of the placenta. Studies into the mechanisms of decidualization are often hampered by the lack of primary ESC. Here we describe a novel immortalized human ESC line. Methods Primary ESC were immortalized by the transduction of telomerase. The resultant cell line, termed St-T1b, was characterized for its morphological and biochemical properties by immunocytochemistry, RT-PCR and immunoblotting. Its progestational response was tested using progesterone and medroxyprogesterone acetate with and without 8-Br-cAMP, an established inducer of decidualization in vitro. Results St-T1b were positive for the fibroblast markers vimentin and CD90 and negative for the epithelial marker cytokeratin-7. They acquired a decidual phenotype indistinguishable from primary ESC in response to cAMP stimulation. The decidual response was characterized by transcriptional activation of marker genes, such as PRL, IGFBP1, and FOXO1, and enhanced protein levels of the tumor suppressor p53 and the metastasis suppressor KAI1 (CD82. Progestins alone had no effect on St-T1b cells, but medroxyprogesterone acetate greatly enhanced the cAMP-stimulated expression of IGFBP-1 after 3 and 7 days. Progesterone, albeit more weakly, also augmented the cAMP-induced IGFBP-1 production but only after 7 days of treatment. The cell line remained stable in continuous culture for more than 150 passages. Conclusion St-T1b express the appropriate phenotypic ESC markers and their decidual response closely mimics that of primary cultures. Decidualization is efficiently induced by cAMP analog and enhanced by medroxyprogesterone acetate, and, to a lesser extent, by natural

  15. Human epidermal keratinocytes death and expression of protein markers of apoptosis after ionizing radiation exposure

    Directory of Open Access Journals (Sweden)

    Sharon Wong

    2013-12-01

    Full Text Available Purpose: Knowledge of the pathophysiology of the irradiated skin is important to understand the tolerance and cosmetic response of the human skin to radiation. There are limited studies on the effect of radiotherapy dosage and fraction size in inducing apoptotic cell death in human skin. The expression of apoptotic biomarkers within a controlled population in different fractionation schemes has also never been studied. This study aims to investigate radiation induced apoptotic cell death in human skin cells after fractionated radiation exposure and the expression of unique biomarkers that reflect cell death or biology using multiplexed immunoassays. Methods: Breast skin biopsies were obtained from a single individual and divided into small pieces. Each piece was irradiated under different radiotherapy treatment fractionation schedules to a total dose of 50Gy. The irradiated skin tissues were analysed using Tunnel, immunohistochemistry and Western blot assays for expression of apoptotic keratinocytes and biomarkers (p53, p21, and PCNA. Haematoxylin and eosin (H&E immunostaining was performed to study the morphological changes in the skin cells. Results: Radiation is mostly absorbed by the epidermal layers and observed to damage the epidermal keratinocytes leading to the activation of apoptotic proteins. Apoptotic proteins (p53, p21 and PCNA were confirmed to be up-regulated in radiation exposed skin cells as compared to normal skin cells with no radiation. There is strong correlation of apoptotic protein expressions with increased radiation dosage and dose fractionation. Statistical analysis with ANOVA revealed a significant increase of PCNA and p21 expression with increased radiation dosage and dose fractionation (p < 0.05. Immunohistochemically, 14 % (range 10.71% to 17.29% of the keratinocytes were positive for PCNA and 22.5% (range 18.28% to 27.2% for p21 after 2Gy of irradiation. The most widespread, intense and uniform staining for PCNA and

  16. Chromium III histidinate exposure modulates antioxidant gene expression in HaCaT human keratinocytes exposed to oxidative stress

    Science.gov (United States)

    While the toxicity of hexavalent chromium is well established, trivalent Cr (Cr(III)) is an essential nutrient involved in insulin and glucose homeostasis. Recently, antioxidant effects of chromium (III) histidinate (Cr(III)His) were reported in HaCaT human keratinocytes exposed to oxidative stress...

  17. EphA2 proteomics in human keratinocytes reveals a novel association with afadin and epidermal tight junctions.

    Science.gov (United States)

    Perez White, Bethany E; Ventrella, Rosa; Kaplan, Nihal; Cable, Calvin J; Thomas, Paul M; Getsios, Spiro

    2017-01-01

    EphA2 is a receptor tyrosine kinase that helps to maintain epidermal tissue homeostasis. A proximity-dependent biotin identification (BioID) approach was used to identify proteins in close proximity to EphA2 within primary human keratinocytes and three-dimensional (3D) reconstituted human epidermis (RHE) cultures to map a putative protein interaction network for this membrane receptor that exhibits a polarized distribution in stratified epithelia. Although a subset of known EphA2 interactors were identified in the BioID screen, >97% were uniquely detected in keratinocytes with over 50% of these vicinal proteins only present in 3D human epidermal culture. Afadin (AFDN), a cytoskeletal and junction-associated protein, was present in 2D and 3D keratinocyte cultures, and validated as a so-far-unknown EphA2-interacting protein. Loss of EphA2 protein disrupted the subcellular distribution of afadin and occludin in differentiated keratinocytes, leading to impairment of tight junctions. Collectively, these studies illustrate the use of the BioID approach in order to map receptor interaction networks in 3D human epithelial cultures, and reveal a positive regulatory role for EphA2 in the organization of afadin and epidermal tight junctions. © 2017. Published by The Company of Biologists Ltd.

  18. EFFECT OF ARSENICALS ON THE EXPRESSION OF CELL CYCLE PROTEINS AND EARLY SIGNALING EVENTS IN PRIMARY HUMAN KERATINOCYTES.

    Science.gov (United States)

    Effect of Arsenicals on the Expression of Cell Cycle Proteins and Early Signaling Events in Primary Human Keratinocytes.Mudipalli, A, Owen R. D. and R. J. Preston, Environmental Carcinogenesis Division, USEPA, RTP, NC 27711.Environmental exposure to arsenic is a m...

  19. Basement membrane reconstruction in human skin equivalents is regulated by fibroblasts and/or exogenously activated keratinocytes

    NARCIS (Netherlands)

    El Ghalbzouri, A; Jonkman, MF; Dijkman, R; Ponec, M

    2005-01-01

    This study was undertaken to examine the role fibroblasts play in the formation of the basement membrane (BM) in human skin equivalents. For this purpose, keratinocytes were seeded on top of fibroblast-free or fibroblast-populated collagen matrix or de-epidermized dermis and cultured in the absence

  20. The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways

    DEFF Research Database (Denmark)

    Celis, J E; Rasmussen, H H; Gromov, P

    1995-01-01

    The master two-dimensional (2-D) gel database of human keratinocytes currently lists 3154 cellular proteins (2224 isoelectric focusing, IEF; and 930 nonequilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to post-translational modifications. 1082 polypeptides have been ident...

  1. Transcriptional profiling defines the effects of nickel in human epidermal keratinocytes.

    Science.gov (United States)

    Gazel, Alix; Rosdy, Martin; Tornier, Carine; De Fraissinette, Anne De Brugerolle; Blumenberg, Miroslav

    2008-12-01

    Nickel is a ubiquitous and virtually unavoidable environmental pollutant and occupational hazard, but its molecular and cellular effects are not well understood. Human epidermal keratinocytes are the sentinel and the primary target for nickel. We treated with nickel salts skin equivalents containing differentiating epidermal keratinocytes grown on air-liquid interface in standard cell culture conditions. We identified the transcriptional profiles affected by nickel in reconstructed human epidermis (RHE) using DNA microarrays. The Ni-regulated genes were determined at two time points, immediate-early, 30 min after treatment, and late, at 6 h. Using in silico data analysis, we determined that 134 genes are regulated by nickel; of these, 97 are induced and 37 suppressed. Functional categories of regulated genes suggest that Ni inhibits apoptosis, promotes cell cycle and induces synthesis of extracellular matrix proteins and extracellular proteases. Importantly, Ni also regulates a set of secreted signaling proteins, inducing VEGF, amphiregulin, PGF, GDF15, and BST2, while suppressing IL-18, galectin-3, and LITAF. These secreted proteins may be important in Ni-caused allergic reactions. Ni induced inhibitors of the NFkappaB signaling pathway, and suppressed its activators. Correspondingly, NFkappaB binding sites were found to be overrepresented in the Ni-suppressed genes, whereas cFOS/AP1 binding sites were common in the Ni-induced genes. Significant parallels were found between the Ni-regulated genes and the genes regulated by TGFbeta, EGF, glucocorticoids, or Oncostatin-M. The comprehensive identification of Ni-regulated genes in human epidermal equivalents significantly advances our understanding of the molecular effects of nickel in skin.

  2. Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity.

    Science.gov (United States)

    Mauro, Marcella; Crosera, Matteo; Pelin, Marco; Florio, Chiara; Bellomo, Francesca; Adami, Gianpiero; Apostoli, Piero; De Palma, Giuseppe; Bovenzi, Massimo; Campanini, Marco; Filon, Francesca Larese

    2015-07-17

    Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs) have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1) and needle-abraded human skin (experiment 2). Co3O4NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled-Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm⁻²), while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm⁻²) and those with intact skin (1.08 ± 0.20 ng·cm⁻²). To further investigate Co3O4NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co3O4NPs and cytotoxicity evaluated by MTT, Alamarblue and propidium iodide (PI) uptake assays. The results indicate that a long exposure time (i.e., seven days) was necessary to induce a concentration-dependent cell viability reduction (EC50 values: 1.3 × 10-4 M, 95% CL = 0.8-1.9 × 10⁻⁴ M, MTT essay; 3.7 × 10⁻⁵ M, 95% CI = 2.2-6.1 × 10⁻⁵ M, AlamarBlue assay) that seems to be associated to necrotic events (EC50 value: 1.3 × 10⁻⁴ M, 95% CL = 0.9-1.9 × 10⁻⁴ M, PI assay). This study demonstrated that Co3O4NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure.

  3. Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity

    Directory of Open Access Journals (Sweden)

    Marcella Mauro

    2015-07-01

    Full Text Available Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1 and needle-abraded human skin (experiment 2. Co3O4NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled–Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm−2, while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm−2 and those with intact skin (1.08 ± 0.20 ng·cm−2. To further investigate Co3O4NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co3O4NPs and cytotoxicity evaluated by MTT, Alamarblue® and propidium iodide (PI uptake assays. The results indicate that a long exposure time (i.e., seven days was necessary to induce a concentration-dependent cell viability reduction (EC50 values: 1.3 × 10−4 M, 95% CL = 0.8–1.9 × 10−4 M, MTT essay; 3.7 × 10−5 M, 95% CI = 2.2–6.1 × 10−5 M, AlamarBlue® assay that seems to be associated to necrotic events (EC50 value: 1.3 × 10−4 M, 95% CL = 0.9–1.9 × 10−4 M, PI assay. This study demonstrated that Co3O4NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure.

  4. Arsenic exposure disrupts epigenetic regulation of SIRT1 in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Herbert, Katharine J. [School of Health Sciences, University of Tasmania, Launceston, TAS 7250 (Australia); Holloway, Adele [Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS 7000 (Australia); Cook, Anthony L. [School of Health Sciences, University of Tasmania, Launceston, TAS 7250 (Australia); Chin, Suyin P. [Menzies Research Institute Tasmania, University of Tasmania, Hobart, TAS 7000 (Australia); Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au [School of Health Sciences, University of Tasmania, Launceston, TAS 7250 (Australia)

    2014-11-15

    Arsenic is an environmental toxin which increases skin cancer risk for exposed populations worldwide; however the underlying biomolecular mechanism for arsenic-induced carcinogenesis is complex and poorly defined. Recent investigations show that histone deacetylase and DNA methyltransferase activity is impaired, and epigenetic patterns of gene regulation are consistently altered in cancers associated with arsenic exposure. Expression of the histone deacetylase SIRT1 is altered in solid tumours and haematological malignancies; however its role in arsenic-induced pathology is unknown. In this study we investigated the effect of arsenic on epigenetic regulation of SIRT1 and its targeting microRNA, miR-34a in primary human keratinocytes. Acetylation of histone H4 at lysine 16 (H4K16) increased in keratinocytes exposed to 0.5 μM arsenite [As(III)]; and this was associated with chromatin remodelling at the miR-34a promoter. Moreover, although SIRT1 protein initially increased in these As(III)-exposed cells, after 24 days expression was not significantly different from untreated controls. Extended exposure to low-dose As(III) (0.5 μM; > 5 weeks) compromised the pattern of CpG methylation at SIRT1 and miR-34a gene promoters, and this was associated with altered expression for both genes. We have found that arsenic alters epigenetic regulation of SIRT1 expression via structural reorganisation of chromatin at the miR-34a gene promoter in the initial 24 h of exposure; and over time, through shifts in miR-34a and SIRT1 gene methylation. Taken together, this investigation demonstrates that arsenic produces cumulative disruptions to epigenetic regulation of miR-34a expression, and this is associated with impaired coordination of SIRT1 functional activity. - Highlights: • Submicromolar arsenic concentrations disrupt SIRT1 activity and expression in human keratinocytes. • Arsenic-induced chromatin remodelling at the miR-34a gene promoter is associated with hyperacetylation

  5. Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity

    Science.gov (United States)

    Mauro, Marcella; Crosera, Matteo; Pelin, Marco; Florio, Chiara; Bellomo, Francesca; Adami, Gianpiero; Apostoli, Piero; De Palma, Giuseppe; Bovenzi, Massimo; Campanini, Marco; Larese Filon, Francesca

    2015-01-01

    Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs) have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1) and needle-abraded human skin (experiment 2). Co3O4NPs at a concentration of 1000 mg/L in physiological solution were used as donor phase. Cobalt content was evaluated by Inductively Coupled–Mass Spectroscopy. Co permeation through the skin was demonstrated after 24 h only when damaged skin protocol was used (57 ± 38 ng·cm−2), while no significant differences were shown between blank cells (0.92 ± 0.03 ng cm−2) and those with intact skin (1.08 ± 0.20 ng·cm−2). To further investigate Co3O4NPs toxicity, human-derived HaCaT keratinocytes were exposed to Co3O4NPs and cytotoxicity evaluated by MTT, Alamarblue® and propidium iodide (PI) uptake assays. The results indicate that a long exposure time (i.e., seven days) was necessary to induce a concentration-dependent cell viability reduction (EC50 values: 1.3 × 10−4 M, 95% CL = 0.8–1.9 × 10−4 M, MTT essay; 3.7 × 10−5 M, 95% CI = 2.2–6.1 × 10−5 M, AlamarBlue® assay) that seems to be associated to necrotic events (EC50 value: 1.3 × 10−4 M, 95% CL = 0.9–1.9 × 10−4 M, PI assay). This study demonstrated that Co3O4NPs can penetrate only damaged skin and is cytotoxic for HaCat cells after long term exposure. PMID:26193294

  6. Hydrogen sulfide inhibits IL-8 expression in human keratinocytes via MAP kinase signaling

    National Research Council Canada - National Science Library

    Mirandola, Prisco; Gobbi, Giuliana; Micheloni, Cristina; Vaccarezza, Mauro; Di Marcantonio, Daniela; Ruscitti, Francesca; de Panfilis, Giuseppe; Vitale, Marco

    2011-01-01

    .... Psoriasis is now considered a genetically programmed, immune-mediated, inflammatory disease, in which intralesional T lymphocytes trigger keratinocytes to proliferate and perpetuate the disease process. Interleukin (IL...

  7. Human eccrine sweat gland cells turn into melanin-uptaking keratinocytes in dermo-epidermal skin substitutes.

    Science.gov (United States)

    Böttcher-Haberzeth, Sophie; Biedermann, Thomas; Pontiggia, Luca; Braziulis, Erik; Schiestl, Clemens; Hendriks, Bart; Eichhoff, Ossia M; Widmer, Daniel S; Meuli-Simmen, Claudia; Meuli, Martin; Reichmann, Ernst

    2013-02-01

    Recently, Biedermann et al. (2010) have demonstrated that human eccrine sweat gland cells can develop a multilayered epidermis. The question still remains whether these cells can fulfill exclusive and very specific functional properties of epidermal keratinocytes, such as the incorporation of melanin, a feature absent in sweat gland cells. We added human melanocytes to eccrine sweat gland cells to let them develop into an epidermal analog in vivo. The interaction between melanocytes and sweat gland-derived keratinocytes was investigated. The following results were gained: (1) macroscopically, a pigmentation of the substitutes was seen 2-3 weeks after transplantation; (2) we confirmed the development of a multilayered, stratified epidermis with melanocytes distributed evenly throughout the basal layer; (3) melanocytic dendrites projected to suprabasal layers; and (4) melanin was observed to be integrated into former eccrine sweat gland cells. These skin substitutes were similar or equal to skin substitutes cultured from human epidermal keratinocytes. The only differences observed were a delay in pigmentation and less melanin uptake. These data suggest that eccrine sweat gland cells can form a functional epidermal melanin unit, thereby providing striking evidence that they can assume one of the most characteristic keratinocyte properties.

  8. Cloning and Expression of Human Keratinocyte Growth Factor in Escherichia coli for Recombinant Drug Production

    Directory of Open Access Journals (Sweden)

    Ebrahimzadeh

    2014-09-01

    Full Text Available Background Keratinocyte growth factor (KGF is a member of fibroblast growth factor (FGF family which induces proliferation and differentiation in a wide variety of epithelial tissues. KGF plays an important role in protection, repair of various types of epithelial cells, and re-epithelialization of wounds. Therefore, in patients with hematologic malignancies receiving high doses of chemotherapy and radiotherapy, treatment with KGF decreases the incidence and duration of severe oral mucositis. Objectives The aim of this study was to express the recombinant form of human keratinocyte growth factor in Escherichia coli. Materials and Methods KGF gene was amplified by PCR and cloned into the expression vector pET28a(+. The recombinant vectors were transformed into E. coli BL21(DE3 as expression host and expression of the desired protein was induced by IPTG. The expression was evaluated at RNA and protein levels by reverse transcriptase PCR (RT-PCR and SDS-PAGE analyses, respectively and the expressed protein was confirmed through western blotting. Results Cloning was confirmed by PCR and restriction digestion. RT-PCR and SDS-PAGE represented expression of KGF in E. coli. The optimized expression was achieved 16 hours after induction with 0.3 mM IPTG at 37°C in luria broth (LB containing kanamycin. The 18 kDa protein was confirmed by western blotting, using anti-His antibodies. Conclusions The result of the present study indicated that E. coli expression system was suitable for overexpression of recombinant human KGF and the expressed protein can be considered as a homemade product.

  9. Mitochondrial and glycolytic activity of UV-irradiated human keratinocytes and its stimulation by a Saccharomyces cerevisiae autolysate.

    Science.gov (United States)

    Schütz, Rolf; Kuratli, Karin; Richard, Nathalie; Stoll, Clarissa; Schwager, Joseph

    2016-06-01

    Cutaneous aging is correlated with mitochondrial dysfunction and a concomitant decline in energy metabolism that can be accelerated by extrinsic factors such as UV radiation (UVR). In this study we compared cellular bioenergetics of normal and UV-irradiated primary human epidermal keratinocytes. Moreover, we investigated the influence of a Saccharomyces cerevisiae autolysate (SCA) on stressed keratinocytes to regain cellular homeostasis. Cellular metabolism was assessed by extracellular flux analysis which measures oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) as well as by ATP quantification. The expression level of ten mitochondria related genes in normal and UVR-stimulated (60mJ/cm(2) UVB) keratinocytes was quantified by real-time PCR and the impact of SCA addition was determined. Sublethal UV stress increased mitochondrial dysfunction in keratinocytes which resulted in reduced viability, uncoupled oxidative phosphorylation, and down-regulated mitochondrial gene expression. Particularly, gene expression of SHDA, UPC2, BID, and ATP5A1 was reduced about twofold within 4h. Treatment of keratinocytes with SCA shifted cellular metabolism towards a more energetic status by increasing the respiratory rate and glycolysis. SCA also stimulated cellular ATP production after short (4h) and prolonged (22h) incubations and induced the expression of genes related to mitochondrial function towards normal expression levels upon UV irradiation. The decreased respiratory capacity of UV-irradiated keratinocytes was partially compensated by the addition of SCA which enhanced glycolytic activity and thereby increased cellular resistance to environmental stress. Copyright © 2016 Elsevier B.V. All rights reserved.

  10. Human melanocytes mitigate keratinocyte-dependent contraction in an in vitro collagen contraction assay.

    Science.gov (United States)

    Rakar, Jonathan; Krammer, Markus P; Kratz, Gunnar

    2015-08-01

    Scarring is an extensive problem in burn care, and treatment can be especially complicated in cases of hypertrophic scarring. Contraction is an important factor in scarring but the contribution of different cell types remains unclear. We have investigated the contractile behavior of keratinocytes, melanocytes and fibroblasts by using an in vitro collagen gel assay aimed at identifying a modulating role of melanocytes in keratinocyte-mediated contraction. Cells were seeded on a collagen type I gel substrate and the change in gel dimensions were measured over time. Hematoxylin & Eosin-staining and immunohistochemistry against pan-cytokeratin and microphthalmia-associated transcription factor showed that melanocytes integrated between keratinocytes and remained there throughout the experiments. Keratinocyte- and fibroblast-seeded gels contracted significantly over time, whereas melanocyte-seeded gels did not. Co-culture assays showed that melanocytes mitigate the keratinocyte-dependent contraction (significantly slower and 18-32% less). Fibroblasts augmented the contraction in most assays (approximately 6% more). Non-contact co-cultures showed some influence on the keratinocyte-dependent contraction. Results show that mechanisms attributable to melanocytes, but not fibroblasts, can mitigate keratinocyte contractile behavior. Contact-dependent mechanisms are stronger modulators than non-contact dependent mechanisms, but both modes carry significance to the contraction modulation of keratinocytes. Further investigations are required to determine the mechanisms involved and to determine the utility of melanocytes beyond hypopigmentation in improved clinical regimes of burn wounds and wound healing.

  11. Inhibition of inflammatory and proliferative responses of human keratinocytes exposed to the sesquiterpene lactones dehydrocostuslactone and costunolide.

    Directory of Open Access Journals (Sweden)

    Claudia Scarponi

    Full Text Available The imbalance of the intracellular redox state and, in particular, of the glutathione (GSH/GSH disulfide couple homeostasis, is involved in the pathogenesis of a number of diseases. In many skin diseases, including psoriasis, oxidative stress plays an important role, as demonstrated by the observation that treatments leading to increase of the local levels of oxidant species ameliorate the disease. Recently, dehydrocostuslactone (DCE and costunolide (CS, two terpenes naturally occurring in many plants, have been found to exert various anti-inflammatory and pro-apoptotic effects on different human cell types. These compounds decrease the level of the intracellular GSH by direct interaction with it, and, therefore, can alter cellular redox state. DCE and CS can trigger S-glutathionylation of various substrates, including the transcription factor STAT3 and JAK1/2 proteins. In the present study, we investigated on the potential role of DCE and CS in regulating inflammatory and proliferative responses of human keratinocytes to cytokines. We demonstrated that DCE and CS decreased intracellular GSH levels in human keratinocytes, as well as inhibited STAT3 and STAT1 phosphorylation and activation triggered by IL-22 or IFN-γ, respectively. Consequently, DCE and CS decreased the IL-22- and IFN-γ-induced expression of inflammatory and regulatory genes in keratinocytes, including CCL2, CXCL10, ICAM-1 and SOCS3. DCE and CS also inhibited proliferation and cell-cycle progression-related gene expression, as well as they promoted cell cycle arrest and apoptosis. In parallel, DCE and CS activated the anti-inflammatory EGFR and ERK1/2 molecules in keratinocytes, and, thus, wound healing in an in vitro injury model. In light of our findings, we can hypothesize that the employment of DCE and CS in psoriasis could efficiently counteract the pro-inflammatory effects of IFN-γ and IL-22 on keratinocytes, revert the apoptosis-resistant phenotype, as well as inhibit

  12. Immunomodulatory effects of a set of amygdalin analogues on human keratinocyte cells.

    Science.gov (United States)

    Baroni, A; Paoletti, I; Greco, R; Satriano, R A; Ruocco, E; Tufano, M A; Perez, J J

    2005-11-01

    Peptide T (PT) is an octapeptide shown to resolve psoriatic lesions. Our previous investigations suggest that keratinocytes play an important role in conditioning the therapeutic effects of the PT in psoriasis. However, peptides are not good therapeutic agents, because they exhibit poor absorption, are easily metabolized and are immunogenic. Using computational methods, the natural product amygdalin was identified as peptidomimetic of PT. However, amygdalin exhibits a toxic profile due to its cyanide group. To overcome this deleterious effect, we synthesized analogues lacking the cyanide group. Human keratinocytes were treated with PT or with three different peptidomimetics of PT. To study its effects on the expression of HSP-70, TGF-beta, alpha-v integrin, ICAM-1 and cytokines, we analysed the protein levels by Western blot and ELISA. Our results show that the different peptidomimetics of PT tested exhibit a similar biological behaviour in regard to the overexpression of HSP-70, TGF-beta and alpha-v integrin than the native peptide. TNF-alpha is overexpressed by PT and SVT-03018; between the other two analogs, SVT-03016 do not produce any significant change in regard to the control, while SVT-03017 shows only a moderate increase in regard to control. SVT-03018 provokes a remarkable upregulation of IL-10, stronger than SVT-03016, SVT-03017 and PT. All the other three analogues reduce comparably to the PT, the expression of ICAM-1 and do not increase the release of proinflammatory cytokines. The results highlighted that the three analogues of amygdalin with the cyanide group removed exhibit the same biological effects of PT. Therefore, they can be considered peptidomimetics, suggesting their possible use in the treatment of psoriasis.

  13. Impairment of human keratinocyte mobility and proliferation by advanced glycation end products-modified BSA.

    Science.gov (United States)

    Zhu, Ping; Yang, Chuan; Chen, Li-Hong; Ren, Meng; Lao, Guo-Juan; Yan, Li

    2011-07-01

    The migration and proliferation of keratinocytes is critical to wound re-epithelialization and defects in this function are associated with the clinical phenomenon of chronic non-healing wounds. Advanced glycation end products (AGEs) occur through non-enzymatic glycation of long-lived proteins in diabetes and play important roles in diabetic complications. However, specific roles for AGEs in keratinocyte migration and proliferation, and the underlying molecular mechanisms, have not been fully established. The aim of the current study was to elucidate the interaction between AGE-modified bovine serum albumin (AGE-BSA) and keratinocytes. As a result, we found that AGE-BSA had no effect on the viability of keratinocytes for up to 48 h of incubation with 50 μg/ml of AGE-BSA. AGE-BSA (but not non-glycated BSA) exerted a concentration-dependent suppression of keratinocyte migration at a range of concentrations. The expression of matrix metalloproteinase-9 (MMP-9) was significantly up-regulated in keratinocytes incubated with increasing AGE-BSA, but tissue inhibitor of metalloproteinases-1 (TIMP-1) expression was down-regulated. AGE-BSA also profoundly depressed phospho-focal adhesion kinase-Tyr397 (p-FAK) and α2β1 integrin expression, while total-FAK expression levels remained constant, in keratinocytes. The proliferative capacity of keratinocytes was diminished after 72 h AGE-BSA incubation. Taken together, these findings suggested that in the presence of AGE-BSA, keratinocytes lose their migratory and proliferation abilities. These data also indicated that, in the context of the chronic hyperglycemia in diabetes, the effects of AGE-BSA on keratinocyte migration might be mediated through MMP-9/TIMP-1, p-FAK and α2β1 integrin.

  14. Cadmium regulation of apoptotic and stress response genes in tumoral and immortalized epithelial cells of the human breast.

    Science.gov (United States)

    Sirchia, Rosalia; Longo, Alessandra; Luparello, Claudio

    2008-10-01

    Cadmium (Cd) is a widely-disseminated metal which can be imported and accumulated in living cells thereby drastically interfering with their biological mechanisms. Increasing interest has been recently focused on the elucidation of the cellular and molecular aspects of Cd-dependent regulation of gene expression and signal transduction pathways in different model system. Concerning breast cancer, very limited studies have been produced so far on the role played by Cd on estrogen receptor-negative human breast cancer cells, that are expected to be insensitive to the already-proven metallo-estrogenic effect exerted by Cd on the estrogen receptor-positive cell counterparts. Here, we have examined the effects of long-term (96 h) exposure of estrogen receptor-negative MDA-MB231 malignant adenocarcinoma cells to CdCl(2) at 5 microM concentration, corresponding to the IC(50) for this time of incubation, by evaluating the expression levels of genes coding for stress response factors (e.g. heat shock proteins and metallothioneins), and for apoptosis-related factors and enzymes. In parallel, we tested the gene expression pattern of immortalized HB2 breast epithelial cells, taken as non-tumoral counterpart, after the same exposure to the metal which instead did not exert any change in their cell number with respect to controls. Our cumulative results indicate that, whilst HB2 cells appear to activate defense mechanisms against metal stress principally via metallothionein massive up-regulation and appearance of the spliced form of XBP-1 message, MDA-MB231 cells seem to couple the onset of a protective reaction (e.g. up-regulation of hsp27 and metallothioneins) to the switching-on of new intracellular pathways directing cells to a kind of death which shares several aspects with the apoptotic program, such as down-regulation of Bcl-2 and over-expression of Dap kinase and several caspases.

  15. Separation and identification of differentially expressed nuclear matrix proteins between human esophageal immortalized and carcinomatous cell lines

    Institute of Scientific and Technical Information of China (English)

    Xing-Dong Xiong; En-Min Li; Li-Yan Xu; Hai-Bin Chen; Ling Chen; Wei-Jia Cai; Ya-Li Han; Zhong-Ying Shen; Yi Zeng

    2003-01-01

    AIM: To separate and identify differentially expressed nuclear matrix proteins (NMPs) between the immortalized human esophageal epithelial cell line (SHEE) and the malignantly transformed esophageal carcinoma cell line (SHEEC), and to provide new ways for finding specific markers and the pathogenesis of esophageal carcinoma.METHODS: SHEE and SHEEC cell lines were used to extract NMPs. The quality of NMPs was monitored by Western blot analysis including DNA topoisomerase Ⅱα, proliferation cell nuclear antigen (PCNA) and histone. NMPs of SHEE and SHEEC were analyzed by two-dimensional electrophoresis (2-DE), silver staining and PDQuest6.2 image analysis software. Three spots in which the differentially expressed NMlPs were more obvious, were selected and analyzed with matrix-assisted laser desorption/ionization time of flying mass spectrometry (MALDI- TOF-MS) and database search.RESULTS: Western blot analysis revealed that DNA topoisomerase Ⅱα and PCNA were detected, and the majority of histones were deleted in NMPs of SHEE and SHEEC. After 2-DE image analysis by PDQuest6.2 software, the 2-DE maps were detected with an average of 106±7.1 spots in SHEE and 132±5.0 spots in SHEEC. Most of them were matched one another (r=0.72), only 16 protein spots were found differing in intensity. Three NMPs including cytoskeletal tropomyosin,FK506bindingprotein6,similartoretinoblastoma binding protein 8 were preliminarily identified by MALDI- TOF-MS.CONCLUSION: These differentially expressed NMPs may play an important role during malignant transformation from SHEE to SHEEC. Their separation and identification will contribute to searching for specific markers and probing into the pathogenesis of esophageal carcinoma.

  16. Persistent Exposure to Porphyromonas gingivalis Promotes Proliferative and Invasion Capabilities, and Tumorigenic Properties of Human Immortalized Oral Epithelial Cells

    Science.gov (United States)

    Geng, Fengxue; Liu, Junchao; Guo, Yan; Li, Chen; Wang, Hongyang; Wang, Hongyan; Zhao, Haijiao; Pan, Yaping

    2017-01-01

    Recent epidemiological studies revealed a significant association between oral squamous cell carcinoma (OSCC) and Porphyromonas gingivalis, a major pathogen of periodontal disease. As a keystone pathogen of periodontitis, P. gingivalis is known not only to damage local periodontal tissues, but also to evade the host immune system and eventually affect systemic health. However, its role in OSCC has yet to be defined. To explore the underlying effect of chronic P. gingivalis infection on OSCC and to identify relevant biomarkers as promising targets for therapy and prevention, we established a novel model by exposing human immortalized oral epithelial cells (HIOECs) to P. gingivalis at a low multiplicity of infection (MOI) for 5–23 weeks. The P. gingivalis infected HIOECs were monitored for tumor biological alteration by proliferation, wound healing, transwell invasion, and gelatin zymography assays. Microarray and proteomic analyses were performed on HIOECs infected with P. gingivalis for 15 weeks, and some selected data were validated by quantitative real-time PCR and (or) western blot on cells infected for 15 and 23 weeks. Persistent exposure to P. gingivalis caused cell morphological changes, increased proliferation ability with higher S phase fraction in the cell cycle, and promoted cell migratory and invasive properties. In combining results of bioinformatics analyses and validation assays, tumor-related genes such as NNMT, FLI1, GAS6, lncRNA CCAT1, PDCD1LG2, and CD274 may be considered as the key regulators in tumor-like transformation in response to long-time exposure of P. gingivalis. In addition, some useful clinical biomarkers and novel proteins were also presented. In conclusion, P. gingivalis could promote tumorigenic properties of HIOECs, indicating that chronic P. gingivalis infection may be considered as a potential risk factor for oral cancer. The key regulators detected from the present model might be used in monitoring the development of OSCC with

  17. The Effect of Calcipotriol on the Expression of Human β Defensin-2 and LL-37 in Cultured Human Keratinocytes

    Directory of Open Access Journals (Sweden)

    Beom Joon Kim

    2009-01-01

    Full Text Available Background. Vitamin D has been reported to regulate innate immunity by controlling the expression of antimicrobial peptides (AMPs. Objective. We investigated the effect of calcipotriol on the expression of AMPs in human cultured keratinocytes. Methods. Keratinocytes were treated with lipopolysaccharide (LPS, TNF-α, Calcipotriol and irradiated with UVB, cultured, and harvested. To assess the expression of human beta defensin-2 and LL-37 in the control group, not exposed to any stimulants, the experimental group was treated with LPS, TNF-α, or UVB, and another group was treated again with calcipotriol; reverse transcriptase-polymerase chain reaction, Western blotting, and immunohistochemical staining were performed. Results. In the experimental group treated with LPS, UVB irradiation, and TNF-α, the expression of β-defensin and LL-37 was increased more than in the control group and then decreased in the experimental group treated with calcipotriol. Conclusions. Calcipotriol suppressed HBD-2 and LL-37, which were stimulated by UVB, LPS, and TNF-α.

  18. Immortalization of Werner syndrome and progeria fibroblasts

    Energy Technology Data Exchange (ETDEWEB)

    Saito, H.; Moses, R.E. (Baylor College of Medicine, Houston, TX (USA))

    1991-02-01

    Human fibroblast cells from two different progeroid syndromes, Werner syndrome (WS) and progeria, were established as immortalized cell lines by transfection with plasmid DNA containing the SV40 early region. The lineage of each immortalized cell line was confirmed by VNTR analysis. Each of the immortalized cell lines maintained its original phenotype of slow growth. DNA repair ability of these cells was also studied by measuring sensitivity to killing by uv or the DNA-damaging drugs methyl methansulfonate, bleomycin, and cis-dichlorodiamine platinum. The results showed that both WS and progeria cells have normal sensitivity to these agents.

  19. The Asian-American variant of human papillomavirus type 16 exhibits higher activation of MAPK and PI3K/AKT signaling pathways, transformation, migration and invasion of primary human keratinocytes.

    Science.gov (United States)

    Hochmann, Jimena; Sobrinho, João S; Villa, Luisa L; Sichero, Laura

    2016-05-01

    Asian-American (AA) HPV-16 variants are associated with higher risk of cancer. Abnormal activation of intracellular signaling play a critical role in cancer development and progression. Our aim was to elucidate mechanisms underlying the higher oncogenic potential attributed to AA variant. We evaluated activation of MAPK and PI3K/AKT pathways in primary human keratinocytes (PHKs) transduced with E6/E7 of three HPV-16 variants: E-P, AA, E-350G. Phenotypes examined included migration, anchorage independent growth and invasion. AA PHKs presented the highest levels of active proteins involved in all cascades analyzed: MAPK-ERK, MAPK-p38 and PI3K-AKT. AA PHKs were more efficient in promoting anchorage independent growth, and in stimulating cell migration and invasion. MEK1 inhibition decreased migration. The mesenchymal phenotype marker vimentin was increased in AA PHKs. Our results suggest that MEK1, ERK2, AKT2 hyperactivation influence cellular behavior by means of GSK-3b inactivation and EMT induction prompting AA immortalized PHKs to more efficiently surpass carcinogenesis steps.

  20. Inhibition of aryl hydrocarbon receptor signaling and induction of NRF2-mediated antioxidant activity by cinnamaldehyde in human keratinocytes.

    Science.gov (United States)

    Uchi, Hiroshi; Yasumatsu, Mao; Morino-Koga, Saori; Mitoma, Chikage; Furue, Masutaka

    2017-01-01

    Dioxins and other environmental pollutants are toxic and remain in biological tissues for a long time leading to various levels of oxidative stress. Although the toxicity of these agents has been linked to activation of the aryl hydrocarbon receptor (AHR), no effective treatment has been developed. To explore novel phytochemicals that inhibit AHR activation in keratinocytes. Keratinocytes were used in this study because the skin is one of the organs most affected by dioxin and other environmental pollutants. HaCaT cells, which are a human keratinocyte cell line, and normal human epidermal keratinocytes were stimulated with benzo[a]pyrene to induce AHR activation, and the effects of traditional Japanese Kampo herbal formulae were analyzed. Quantification of mRNA, western blotting, immunofluorescence localization of molecules, siRNA silencing, and visualization of oxidative stress were performed. Cinnamomum cassia extract and its major constituent cinnamaldehyde significantly inhibited the activation of AHR. Cinnamaldehyde also activated the NRF2/HO1 pathway and significantly alleviated the production of reactive oxygen species in keratinocytes. The inhibition of AHR signaling and the activation of antioxidant activity by cinnamaldehyde operated in a mutually independent manner as assessed by siRNA methods In addition, AHR signaling was effectively inhibited by traditional Kampo formulae containing C. cassia. Cinnamaldehyde has two independent biological activities; namely, an inhibitory action on AHR activation and an antioxidant effect mediated by NRF2/HO1 signaling. Through these dual functions, cinnamaldehyde may be beneficial for the treatment of disorders related to oxidative stress such as dioxin intoxication, acne, and vitiligo. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  1. Modulation of the pro-inflammatory cytokines and matrix metalloproteinases production in co-cultivated human keratinocytes and melanocytes.

    Science.gov (United States)

    Decean, H; Perde-Schrepler, M; Tatomir, C; Fischer-Fodor, E; Brie, I; Virag, P

    2013-10-01

    The human epidermis exerts immunoregulatory functions through the variety of cytokines and other molecules elaborated by keratinocytes and melanocytes. Their constitutive production is very low; however, considerably increased upon stimulation. In vivo, keratinocytes and melanocytes have a typical exposure in the skin, referred as melanocyte epidermal unit. In the present study we co-cultivated these cells in vitro proposing to elucidate some communication links in close cell-to-cell association. We assessed the amounts of IL-6, IL-8, and matrix metalloproteinases (MMP-2 and MMP-9) in individually and co-cultured cells, exposed or not to UVB radiation. Normal human epidermal keratinocytes and melanocytes were grown in specific media and supplements. Cells were exposed to UVB radiation (100 mJ/cm(2)) to create comparable stress to the environmental one. Cytokines were determined with ELISA and confirmed with Western blot and metalloproteinases with gel zimography. Pure cultures of keratinocytes and melanocytes released low amounts of cytokines and metalloproteinases, these secretions being enhanced by UVB irradiation. In co-cultures, the cell-to-cell proximity triggered signals which markedly augmented the cytokines' secretions, whereas metalloproteinases were down-regulated. UVB irradiation did not influence either of these secretions in co-cultures. Concurrently with the highest levels of the pro-inflammatory cytokines, MMP-9 was up-regulated creating pro-inflammatory conditions and premises for changes in cellular survival, differentiation and phenotype. A complex network of interactions occurred between keratinocytes and melanocytes in co-cultures, resulting in modulated pro-inflammatory cytokines and metalloproteinases productions. Therefore, any disturbances in the microenvironmental signaling system and its molecular constituents may result in inflammation or even tumorigenesis in the epidermis.

  2. Functional analysis of a novel human serotonin transporter gene promoter in immortalized raphe cells

    DEFF Research Database (Denmark)

    Mortensen, O V; Thomassen, M; Larsen, M B

    1999-01-01

    To investigate the structural basis for genetic regulation of the human serotonin transporter gene, a 1.8 kb fragment upstream to the cap site was cloned and sequenced. The promoter possesses a polymorphic repeat region with 16 and 14 repeats, respectively. Both were cloned and characterized....... The promoter sequence revealed an internal 379 bp fragment not reported in previous publications. This novel fragment contains consensus sequences for several transcription factors including SpI and GATA. DNA from 48 unrelated individuals was PCR amplified, in this region, to test for allelic variations. All...

  3. MicroRNA-4739 regulates osteogenic and adipocytic differentiation of immortalized human bone marrow stromal cells via targeting LRP3

    DEFF Research Database (Denmark)

    Elsafadi, Mona; Manikandan, Muthurangan; Alajez, Nehad M

    2017-01-01

    3 (LRP3) in regulating the osteogenic and adipogenic differentiation of immortalized hBMSCs. Gene expression profiling revealed significantly higher LRP3 levels in the highly osteogenic hBMSC clone imCL1 than in the less osteogenic clone imCL2, as well as a significant upregulation of LRP3 during...

  4. cDNA microarray analysis of human keratinocytes cells of patients submitted to chemoradiotherapy and oral photobiomodulation therapy: pilot study.

    Science.gov (United States)

    Antunes, Heliton S; Wajnberg, Gabriel; Pinho, Marcos B; Jorge, Natasha Andressa Nogueira; de Moraes, Joyce Luana Melo; Stefanoff, Claudio Gustavo; Herchenhorn, Daniel; Araújo, Carlos M M; Viégas, Celia Maria Pais; Rampini, Mariana P; Dias, Fernando L; de Araujo-Souza, Patricia Savio; Passetti, Fabio; Ferreira, Carlos G

    2017-08-24

    Oral mucositis is an acute toxicity that occurs in patients submitted to chemoradiotherapy to treat head and neck squamous cell carcinoma. In this study, we evaluated differences in gene expression in the keratinocytes of the oral mucosa of patients treated with photobiomodulation therapy and tried to associate the molecular mechanisms with clinical findings. From June 2009 to December 2010, 27 patients were included in a randomized double-blind pilot study. Buccal smears from 13 patients were obtained at days 1 and 10 of chemoradiotherapy, and overall gene expression of samples from both dates were analyzed by complementary DNA (cDNA) microarray. In addition, samples from other 14 patients were also collected at D1 and D10 of chemoradiotherapy for subsequent validation of cDNA microarray findings by qPCR. The expression array analysis identified 105 upregulated and 60 downregulated genes in our post-treatment samples when compared with controls. Among the upregulated genes with the highest fold change, it was interesting to observe the presence of genes related to keratinocyte differentiation. Among downregulated genes were observed genes related to cytotoxicity and immune response. The results indicate that genes known to be induced during differentiation of human epidermal keratinocytes were upregulated while genes associated with cytotoxicity and immune response were downregulated in the laser group. These results support previous clinical findings indicating that the lower incidence of oral mucositis associated with photobiomodulation therapy might be correlated to the activation of genes involved in keratinocyte differentiation.

  5. Pulmonary malformation in transgenic mice expressing human keratinocyte growth factor in the lung.

    Science.gov (United States)

    Simonet, W S; DeRose, M L; Bucay, N; Nguyen, H Q; Wert, S E; Zhou, L; Ulich, T R; Thomason, A; Danilenko, D M; Whitsett, J A

    1995-01-01

    Expression of human keratinocyte growth factor (KGF/FGF-7) was directed to epithelial cells of the developing embryonic lung of transgenic mice disrupting normal pulmonary morphogenesis during the pseudoglandular stage of development. By embryonic day 15.5(E15.5), lungs of transgenic surfactant protein C (SP-C)-KGF mice resembled those of humans with pulmonary cystadenoma. Lungs were cystic, filling the thoracic cavity, and were composed of numerous dilated saccules lined with glycogen-containing columnar epithelial cells. The normal distribution of SP-C proprotein in the distal regions of respiratory tubules was disrupted. Columnar epithelial cells lining the papillary structures stained variably and weakly for this distal respiratory cell marker. Mesenchymal components were preserved in the transgenic mouse lungs, yet the architectural relationship of the epithelium to the mesenchyme was altered. SP-C-KGF transgenic mice failed to survive gestation to term, dying before E17.5. Culturing mouse fetal lung explants in the presence of recombinant human KGF also disrupted branching morphogenesis and resulted in similar cystic malformation of the lung. Thus, it appears that precise temporal and spatial expression of KGF is likely to play a crucial role in the control of branching morphogenesis during fetal lung development. Images Fig. 1 Fig. 2 Fig. 3 PMID:8618921

  6. The Antimicrobial Peptide Human Beta-Defensin-3 Is Induced by Platelet-Released Growth Factors in Primary Keratinocytes

    Directory of Open Access Journals (Sweden)

    Andreas Bayer

    2017-01-01

    Full Text Available Platelet-released growth factors (PRGF and its related clinically used formulations (e.g., Vivostat Platelet-Rich Fibrin (PRF® contain a variety of chemokines, cytokines, and growth factors and are therefore used to support healing of chronic, hard-to-heal, or infected wounds. Human beta-defensin-3 (hBD-3 is an antimicrobial peptide inducibly expressed in human keratinocytes especially upon wounding. The potent antimicrobial activity of hBD-3 together with its wound closure-promoting activities suggests that hBD-3 may play a crucial role in wound healing. Therefore, we analyzed the influence of PRGF on hBD-3 expression in human primary keratinocytes in vitro. In addition, we investigated the influence of Vivostat PRF on hBD-3 expression in artificially generated human skin wounds in vivo. PRGF treatment of primary keratinocytes induced a significant, concentration- and time-dependent increase in hBD-3 gene expression which was partially mediated by the epidermal growth factor receptor (EGFR. In line with these cell culture data, in vivo experiments revealed an enhanced hBD-3 expression in experimentally produced human wounds after the treatment with Vivostat PRF. Thus, the induction of hBD-3 may contribute to the beneficial effects of thrombocyte concentrate lysates in the treatment of chronic or infected wounds.

  7. Calcium and Vitamin D increase mRNA levels for the growth control hIK1 channel in human epidermal keratinocytes but functional channels are not observed

    Directory of Open Access Journals (Sweden)

    Rossie Sandra

    2004-06-01

    Full Text Available Abstract Background Intermediate-conductance, calcium-activated potassium channels (IKs modulate proliferation and differentiation in mesodermal cells by enhancing calcium influx, and they contribute to the physiology of fluid movement in certain epithelia. Previous reports suggest that IK channels stimulate proliferative growth in a keratinocyte cell line; however, because these channels indirectly promote calcium influx, a critically unique component of the keratinocyte differentiation program, an alternative hypothesis is that they would be anti-proliferative and pro-differentiating. This study addresses these hypotheses. Methods Real-time PCR, patch clamp electrophysiology, and proliferation assays were used to determine if human IK1 (hIK1 expression and function are correlated with either proliferation or differentiation in cultured human skin epidermal keratinocytes, and skin biopsies grown in explant culture. Results hIK1 mRNA expression in human keratinocytes and skin was increased in response to anti-proliferative/pro-differentiating stimuli (elevated calcium and Vitamin D. Correspondingly, the hIK1 agonist 1-EBIO inhibited keratinocyte proliferation suggesting that the channel could be anti-proliferative and pro-differentiating. However, this proliferative inhibition by 1-EBIO was not reversed by a panel of hIK1 blockers, calling into question the mechanism of 1-EBIO action. Subsequent patch clamp electrophysiological analysis failed to detect hIK1 channel currents in keratinocytes, even those expressing substantial hIK1 mRNA in response to calcium and Vitamin D induced differentiation. Identical electrophysiological recording conditions were then used to observe robust IK1 currents in fibroblasts which express IK1 mRNA levels comparable to those of keratinocytes. Thus, the absence of observable hIK1 currents in keratinocytes was not a function of the electrophysiological techniques. Conclusion Human keratinocyte differentiation is

  8. Interaction of Mycobacterium leprae with the HaCaT human keratinocyte cell line: new frontiers in the cellular immunology of leprosy.

    Science.gov (United States)

    Lyrio, Eloah C D; Campos-Souza, Ivy C; Corrêa, Luiz C D; Lechuga, Guilherme C; Verícimo, Maurício; Castro, Helena C; Bourguignon, Saulo C; Côrte-Real, Suzana; Ratcliffe, Norman; Declercq, Wim; Santos, Dilvani O

    2015-07-01

    Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae affecting the skin and peripheral nerves. Despite M. leprae invasion of the skin and keratinocytes importance in innate immunity, the interaction of these cells in vitro during M. leprae infection is poorly understood. Conventional and fluorescence optical microscopy, transmission electronic microscopy, flow cytometry and ELISA were used to study the in vitro interaction of M. leprae with the HaCaT human keratinocyte cell line. Keratinocytes uptake of M. leprae is described, and modulation of the surface expression of CD80 and CD209, cathelicidin expression and TNF-α and IL-1β production of human keratinocytes are compared with dendritic cells and macrophages during M. leprae interaction. This study demonstrated that M. leprae interaction with human keratinocytes enhanced expression of cathelicidin and greatly increased TNF-α production. The highest spontaneous expression of cathelicidin was by dendritic cells which are less susceptible to M. leprae infection. In contrast, keratinocytes displayed low spontaneous cathelicidin expression and were more susceptible to M. leprae infection than dendritic cells. The results show, for the first time, an active role for keratinocytes during infection by irradiated whole cells of M. leprae and the effect of vitamin D on this process. They also suggest that therapies which target cathelicidin modulation may provide novel approaches for treatment of leprosy.

  9. Effect of lead on IL-8 production and cell proliferation in human oral keratinocytes

    Institute of Scientific and Technical Information of China (English)

    Thaweboon Srosiri; Poomsawat Sopee; Thaweboon Boonyanit

    2010-01-01

    Objective:To investigate the effect of lead on the production of IL-8 and cell proliferation in normal human oral keratinocytes (NHKs). Methods: NHKs were prepared as outgrowths from normal human buccal mucosa. The cells were treated with three concentrations of lead glutamate (4.5í10-5M, 4.5í10-6M and 4.5í10-7M). NHKs grown in glutamic acid were used as control. The amounts of IL-8 secreted in the culture supernatants were evaluated at 12 and 24 h using enzyme-linked immunospecific assay (ELISA). Cell proliferation was determined by the MTT colorimetric assay. Three cultures were used for each experiment, and three independent experiments were performed. Analysis of variance and Duncan’s multiple range tests were used for statistical analysis. Results:An elevation of IL-8 in culture supernatants of NHKs treated with lead at all concentrations at 12 and 24 h after exposure in a dose-dependent manner was revealed. A significant increase in cell numbers was observed only at 24 h exposed to 4.5í10-5M lead glutamate. Conclusions: The capacity of NHKs, to secrete IL-8, enhanced by lead glutamate, is demonstrated here. Induction of cell proliferation is revealed only after exposure to high lead concentration. The elevation of secreted IL-8 is a probable initial sign for the acute inflammatory response and may be involved in the pathogenesis of lead stomatitis.

  10. Triterpenoid α-amyrin stimulates proliferation of human keratinocytes but does not protect them against UVB damage

    DEFF Research Database (Denmark)

    Biskup, Edyta; Gołębiowski, Marek; Gniadecki, Robert

    2012-01-01

    Rhaponticum carthamoides plants ("maral root") are widely used in Siberian folk medicine. The present study reports for the first time the presence of pentacyclic terpenoid, α-amyrin, in methanol extract from leaves of this plant. α-Amyrin induced proliferation of human keratinocytes (HaCaT) by a......Rhaponticum carthamoides plants ("maral root") are widely used in Siberian folk medicine. The present study reports for the first time the presence of pentacyclic terpenoid, α-amyrin, in methanol extract from leaves of this plant. α-Amyrin induced proliferation of human keratinocytes (Ha......CaT) by about 18% while other extract components were ineffective. A panel of biochemical and cell-based assays testing the antioxidative and cytoprotective activites of α-amyrin indicated no antioxidative activity of this compound. α-Amyrin did not protect HaCaT cells against the damage caused by UVB radiation....

  11. PAI-1 transcriptional regulation during the G0 --> G1 transition in human epidermal keratinocytes.

    Science.gov (United States)

    Qi, Li; Allen, Rosalie R; Lu, Qi; Higgins, Craig E; Garone, Rosemarie; Staiano-Coico, Lisa; Higgins, Paul J

    2006-10-01

    Plasminogen activator inhibitor type-1 (PAI-1) is the major negative regulator of the plasmin-dependent pericellular proteolytic cascade. PAI-1 gene expression is normally growth state regulated but frequently elevated in chronic fibroproliferative and neoplastic diseases affecting both stromal restructuring and cellular migratory activities. Kinetic modeling of cell cycle transit in synchronized human keratinocytes (HaCaT cells) indicated that PAI-1 transcription occurred early after serum stimulation of quiescent (G0) cells and prior to entry into a cycling G1 condition. PAI-1 repression (in G0) was associated with upstream stimulatory factor-1 (USF-1) occupancy of two consensus E box motifs (5'-CACGTG-3') at the PE1 and PE2 domains in the PF1 region (nucleotides -794 to -532) of the PAI-1 promoter. Chromatin immunoprecipitation (ChIP) analysis established that the PE1 and PE2 site E boxes were occupied by USF-1 in quiescent cells and by USF-2 in serum-activated, PAI-1-expressing keratinocytes. This reciprocal and growth state-dependent residence of USF family members (USF-1 vs. USF-2) at PE1/PE2 region chromatin characterized the G0 --> G1 transition period and the transcriptional status of the PAI-1 gene. A consensus E box motif was required for USF/E box interactions, as a CG --> AT substitution at the two central nucleotides inhibited formation of USF/probe complexes. The 5' flanking sites (AAT or AGAC) in the PE2 segment were not necessary for USF binding. USF recognition of the PE1/PE2 region E box sites required phosphorylation with several potential involved residues, including T153, maping to the USF-specific region (USR). A T153A substitution in USF-1 did not repress serum-induced PAI-1 expression whereas the T153D mutant was an effective suppressor. As anticipated from the ChIP results, transfection of wild-type USF-2 failed to inhibit PAI-1 induction. Collectively, these data suggest that USF family members are important regulators of PAI-1 gene

  12. 20-Hydroxycholecalciferol, product of vitamin D3 hydroxylation by P450scc, decreases NF-kappaB activity by increasing IkappaB alpha levels in human keratinocytes.

    Directory of Open Access Journals (Sweden)

    Zorica Janjetovic

    Full Text Available The side chain of vitamin D3 is hydroxylated in a sequential manner by cytochrome P450scc (CYP11A1 to form 20-hydroxycholecalciferol, which can induce growth arrest and differentiation of both primary and immortalized epidermal keratinocytes. Since nuclear factor-kappaB (NF-kappaB plays a pivotal role in the regulation of cell proliferation, differentiation and apoptosis, we examined the capability of 20-hydroxycholecalciferol to modulate the activity of NF-kappaB, using 1,25-dihydroxycholecalciferol (calcitriol as a positive control. 20-hydroxycholecalciferol inhibits the activation of NFkappaB DNA binding activity as well as NF-kappaB-driven reporter gene activity in keratinocytes. Also, 20-hydroxycholecalciferol induced significant increases in the mRNA and protein levels of the NF-kappaB inhibitor protein, IkappaB alpha, in a time dependent manner, while no changes in total NF-kappaB-p65 mRNA or protein levels were observed. Another measure of NF-kappaB activity, p65 translocation from the cytoplasm into the nucleus was also inhibited in extracts of 20-hydroxycholecalciferol treated keratinocytes. Increased IkappaB alpha was concomitantly observed in cytosolic extracts of 20-hydroxycholecalciferol treated keratinocytes, as determined by immunoblotting and immunofluorescent staining. In keratinocytes lacking vitamin D receptor (VDR, 20-hydroxycholecalciferol did not affect IkappaB alpha mRNA levels, indicating that it requires VDR for its action on NF-kappaB activity. Comparison of the effects of calcitrol, hormonally active form of vitamin D3, with 20-hydrocholecalciferol show that both agents have a similar potency in inhibiting NF-kappaB. Since NF-kappaB is a major transcription factor for the induction of inflammatory mediators, our findings indicate that 20-hydroxycholecalciferol may be an effective therapeutic agent for inflammatory and hyperproliferative skin diseases.

  13. EGF receptor signaling blocks aryl hydrocarbon receptor-mediated transcription and cell differentiation in human epidermal keratinocytes

    OpenAIRE

    Sutter, Carrie Hayes; Yin, Hong; Li, Yunbo; Mammen, Jennifer S.; Bodreddigari, Sridevi; Stevens, Gaylene; Cole, Judith A; Sutter, Thomas R.

    2009-01-01

    Dioxin is an extremely potent carcinogen. In highly exposed people, the most commonly observed toxicity is chloracne, a pathological response of the skin. Most of the effects of dioxin are attributed to its activation of the aryl hydrocarbon receptor (AHR), a transcription factor that binds to the Ah receptor nuclear translocator (ARNT) to regulate the transcription of numerous genes, including CYP1A1 and CYP1B1. In cultures of normal human epidermal keratinocytes dioxin accelerates cell diff...

  14. Cobalt Oxide Nanoparticles: Behavior towards Intact and Impaired Human Skin and Keratinocytes Toxicity

    OpenAIRE

    Marcella Mauro; Matteo Crosera; Marco Pelin; Chiara Florio; Francesca Bellomo; Gianpiero Adami; Piero Apostoli; Giuseppe Palma; Massimo Bovenzi; Marco Campanini; Francesca Larese Filon

    2015-01-01

    Skin absorption and toxicity on keratinocytes of cobalt oxide nanoparticles (Co3O4NPs) have been investigated. Co3O4NPs are commonly used in industrial products and biomedicine. There is evidence that these nanoparticles can cause membrane damage and genotoxicity in vitro, but no data are available on their skin absorption and cytotoxicity on keratinocytes. Two independent 24 h in vitro experiments were performed using Franz diffusion cells, using intact (experiment 1) and needle-abraded huma...

  15. Impact on Autophagy and Ultraviolet B Induced Responses of Treatment with the MTOR Inhibitors Rapamycin, Everolimus, Torin 1, and pp242 in Human Keratinocytes.

    Science.gov (United States)

    Xu, Song; Li, Li; Li, Min; Zhang, Mengli; Ju, Mei; Chen, Xu; Gu, Heng

    2017-01-01

    The mechanistic target of Rapamycin (MTOR) protein is a crucial signaling regulator in mammalian cells that is extensively involved in cellular biology. The function of MTOR signaling in keratinocytes remains unclear. In this study, we detected the MTOR signaling and autophagy response in the human keratinocyte cell line HaCaT and human epidermal keratinocytes treated with MTOR inhibitors. Moreover, we detected the impact of MTOR inhibitors on keratinocytes exposed to the common carcinogenic stressors ultraviolet B (UVB) and UVA radiation. As a result, keratinocytes were sensitive to the MTOR inhibitors Rapamycin, everolimus, Torin 1, and pp242, but the regulation of MTOR downstream signaling was distinct. Next, autophagy induction only was observed in HaCaT cells treated with Rapamycin. Furthermore, we found that MTOR signaling was insensitive to UVB but sensitive to UVA radiation. UVB treatment also had no impact on the inhibition of MTOR signaling by MTOR inhibitors. Finally, MTOR inhibition by Rapamycin, everolimus, or pp242 did not affect the series of biological events in keratinocytes exposed to UVB, including the downregulation of BiP and PERK, activation of Histone H2A and JNK, and cleavage of caspase-3 and PARP. Our study demonstrated that MTOR inhibition in keratinocytes cannot always induce autophagy, and the MTOR pathway does not play a central role in the UVB triggered cellular response.

  16. Impact on Autophagy and Ultraviolet B Induced Responses of Treatment with the MTOR Inhibitors Rapamycin, Everolimus, Torin 1, and pp242 in Human Keratinocytes

    Science.gov (United States)

    Xu, Song; Li, Li; Li, Min; Zhang, Mengli

    2017-01-01

    The mechanistic target of Rapamycin (MTOR) protein is a crucial signaling regulator in mammalian cells that is extensively involved in cellular biology. The function of MTOR signaling in keratinocytes remains unclear. In this study, we detected the MTOR signaling and autophagy response in the human keratinocyte cell line HaCaT and human epidermal keratinocytes treated with MTOR inhibitors. Moreover, we detected the impact of MTOR inhibitors on keratinocytes exposed to the common carcinogenic stressors ultraviolet B (UVB) and UVA radiation. As a result, keratinocytes were sensitive to the MTOR inhibitors Rapamycin, everolimus, Torin 1, and pp242, but the regulation of MTOR downstream signaling was distinct. Next, autophagy induction only was observed in HaCaT cells treated with Rapamycin. Furthermore, we found that MTOR signaling was insensitive to UVB but sensitive to UVA radiation. UVB treatment also had no impact on the inhibition of MTOR signaling by MTOR inhibitors. Finally, MTOR inhibition by Rapamycin, everolimus, or pp242 did not affect the series of biological events in keratinocytes exposed to UVB, including the downregulation of BiP and PERK, activation of Histone H2A and JNK, and cleavage of caspase-3 and PARP. Our study demonstrated that MTOR inhibition in keratinocytes cannot always induce autophagy, and the MTOR pathway does not play a central role in the UVB triggered cellular response.

  17. An ascorbic acid-enriched tomato genotype to fight UVA-induced oxidative stress in normal human keratinocytes.

    Science.gov (United States)

    Petruk, Ganna; Raiola, Assunta; Del Giudice, Rita; Barone, Amalia; Frusciante, Luigi; Rigano, Maria Manuela; Monti, Daria Maria

    2016-10-01

    UVA radiations contribute up to 95% of the total UV exposure and are known to induce cell damage, leading to apoptosis. Since the benefic effects of ascorbic acid on human health are well known, a new tomato genotype (named DHO4), highly rich in ascorbic acid, has been recently obtained. Here, we compared the effects of ascorbic acid and hydrophilic DHO4 extracts in protecting human keratinocytes exposed to UVA stress. Keratinocytes were pre-incubated with ascorbic acid or with extracts from the ascorbic acid enriched tomato genotype and irradiated with UVA light. Then, ROS production, intracellular GSH and lipid peroxidation levels were quantified. Western blots were carried out to evaluate mitogen-activated protein kinases cascade, activation of caspase-3 and inflammation levels. We demonstrated that ROS, GSH and lipid peroxidation levels were not altered in cell exposed to UVA stress when cells were pre-treated with ascorbic acid or with tomato extracts. In addition, no evidence of apoptosis and inflammation were observed in irradiated pre-treated cells. Altogether, we demonstrated the ability of an ascorbic acid enriched tomato genotype to counteract UVA-oxidative stress on human keratinocytes. This protective effect is due to the high concentration of vitamin C that acts as free radical scavenger. This novel tomato genotype may be used as genetic material in breeding schemes to produce improved varieties with higher antioxidant levels.

  18. Tumor necrosis factor beta and ultraviolet radiation are potent regulators of human keratinocyte ICAM-1 expression

    Energy Technology Data Exchange (ETDEWEB)

    Krutmann, J.; Koeck, A.S.; Schauer, E.; Parlow, F.; Moeller, A.K.; Kapp, A.; Foerster, E.S.; Schoepf, E.L.; Luger, T.A. (Univ. of Freiburg (Germany, F.R.))

    1990-08-01

    Intercellular adhesion molecule-1 (ICAM-1) functions as a ligand of leukocyte function-associated antigen-1 (LFA-1), as well as a receptor for human picorna virus, and its regulation thus affects various immunologic and inflammatory reactions. The weak, constitutive ICAM-1 expression on human keratinocytes (KC) can be up-regulated by cytokines such as interferon-gamma (IFN gamma) and tumor necrosis factor alpha (TNF alpha). In order to further examine the regulation of KC ICAM-1 expression, normal human KC or epidermoid carcinoma cells (KB) were incubated with different cytokines and/or exposed to ultraviolet (UV) radiation. Subsequently, ICAM-1 expression was monitored cytofluorometrically using a monoclonal anti-ICAM-1 antibody. Stimulation of cells with recombinant human (rh) interleukin (IL) 1 alpha, rhIL-4, rhIL-5, rhIL-6, rh granulocyte/macrophage colony-stimulating factor (GM-CSF), rh interferon alpha (rhIFN alpha), and rh transforming growth factor beta (TGF beta) did not increase ICAM-1 surface expression. In contrast, rhTNF beta significantly up-regulated ICAM-1 expression in a time- and dose-dependent manner. Moreover, the combination of rhTNF beta with rhIFN gamma increased the percentage of ICAM-1-positive KC synergistically. This stimulatory effect of rhTNF beta was further confirmed by the demonstration that rhTNF beta was capable of markedly enhancing ICAM-1 mRNA expression in KC. Finally, exposure of KC in vitro to sublethal doses of UV radiation (0-100 J/m2) prior to cytokine (rhIFN tau, rhTNF alpha, rhTNF beta) stimulation inhibited ICAM-1 up-regulation in a dose-dependent fashion. These studies identify TNF beta and UV light as potent regulators of KC ICAM-1 expression, which may influence both attachment and detachment of leukocytes and possibly viruses to KC.

  19. Selective peroxidation and externalization of phosphatidylserine in normal human epidermal keratinocytes during oxidative stress induced by cumene hydroperoxide.

    Science.gov (United States)

    Shvedova, Anna A; Tyurina, Julia Y; Kawai, Kazuaki; Tyurin, Vladimir A; Kommineni, Choudari; Castranova, Vincent; Fabisiak, James P; Kagan, Valerian E

    2002-06-01

    Reactive oxygen species not only modulate important signal transduction pathways, but also induce DNA damage and cytotoxicity in keratinocytes. Hydrogen peroxide and organic peroxides are particularly important as these chemicals are widely used in dermally applied cosmetics and pharmaceuticals, and also represent endogenous metabolic intermediates. Lipid peroxidation is of fundamental interest in the cellular response to peroxides, as lipids are extremely sensitive to oxidation and lipid-based signaling systems have been implicated in a number of cellular processes, including apoptosis. Oxidation of specific phospholipid classes was measured in normal human epidermal keratinocytes exposed to cumene hydroperoxide after metabolic incorporation of the fluorescent oxidation-sensitive fatty acid, cis-parinaric acid, using a fluorescence high-performance liquid chromatography assay. In addition, lipid oxidation was correlated with changes in membrane phospholipid asymmetry and other markers of apoptosis. Although cumene hydroperoxide produced significant oxidation of cis-parinaric acid in all phospholipid classes, one phospholipid, phosphatidylserine, appeared to be preferentially oxidized above all other species. Using fluorescamine derivatization and annexin V binding it was observed that specific oxidation of phosphatidylserine was accompanied by phosphatidylserine translocation from the inner to the outer plasma membrane surface where it may serve as a recognition signal for interaction with phagocytic macrophages. These effects occurred much earlier than any detectable changes in other apoptotic markers such as caspase-3 activation, DNA fragmentation, or changes in nuclear morphology. Thus, normal human epidermal keratinocytes undergo profound lipid oxidation with preference for phosphatidylserine followed by phosphatidylserine externalization upon exposure to cumene hydroperoxide. It is therefore likely that normal human epidermal keratinocytes exposed to similar

  20. Vitamin C Compound Mixtures Prevent Ozone-Induced Oxidative Damage in Human Keratinocytes as Initial Assessment of Pollution Protection.

    Directory of Open Access Journals (Sweden)

    Giuseppe Valacchi

    Full Text Available One of the main functions of cutaneous tissues is to protect our body from the outdoor insults. Ozone (O3 is among the most toxic stressors to which we are continuously exposed and because of its critical location, the skin is one of the most susceptible tissues to the oxidative damaging effect of O3. O3 is not able to penetrate the skin, and although it is not a radical per se, the damage is mainly a consequence of its ability to induce oxidative stress via the formation of lipid peroxidation products.In this study we investigated the protective effect of defined "antioxidant" mixtures against O3 induced oxidative stress damage in human keratinocytes and understand their underlying mechanism of action.Results showed that the mixtures tested were able to protect human keratinocytes from O3-induced cytotoxicity, inhibition of cellular proliferation, decrease the formation of HNE protein adducts, ROS, and carbonyls levels. Furthermore, we have observed the decreased activation of the redox sensitive transcription factor NF-kB, which is involved in transcribing pro-inflammatory cytokines and therefore constitutes one of the main players associated with O3 induced skin inflammation. Cells exposed to O3 demonstrated a dose dependent increase in p65 subunit nuclear expression as a marker of NF-kB activation, while pre-treatment with the mixtures abolished NF-kB nuclear translocation. In addition, a significant activation of Nrf2 in keratinocytes treated with the mixtures was also observed.Overall this study was able to demonstrate a protective effect of the tested compounds versus O3-induced cell damage in human keratinocytes. Pre-treatment with the tested compounds significantly reduced the oxidative damage induced by O3 exposure and this protective effect was correlated to the abolishment of NF-kB nuclear translocation, as well as activation of Nrf2 nuclear translocation activating the downstream defence enzymes involved in cellular detoxification

  1. In vitro evaluation of the effects of yttria-alumina-silica microspheres on human keratinocyte cells.

    Science.gov (United States)

    Radu, T; Chiriac, M T; Popescu, O; Simon, V; Simon, S

    2013-02-01

    The behavior of yttria-alumina-silica spray-dried microspheres was investigated in vitro on a human keratinocyte cell line, first to exclude their cytotoxicity. The HaCaT cells were chosen due to their well-characterized phenotype and their phagocytic ability. Microscopic analysis and cell viability tests showed no negative effect of the microspheres on cells morphology and behavior. Scanning electron microscopy and transmission electron microscopy results evidenced the cellular internalization of the microspheres at 48 h after their incubation with cultured cells. The shape, size distribution, structure, composition, and chemical states of the elements on samples surface were analyzed by SEM, transmission electron microscopy, x-ray diffraction, and x-ray photoelectron spectroscopy, because these properties could influence their internalization by cells. The yttrium distribution on the microspheres surface was indicated by fluorescence microscopy imaging. The microspheres dimension and shape inside the cells was in accordance with their dimension and shape before incubation. The microspheres seemed captured and engulfed by the cells in native form and appeared resistant to degradation over the first 48 h. Most of the analyzed cells took up more microspheres, suggesting that the microspheres were actively phagocytosed by the cells and accumulated within the cytoplasm. X-ray photoelectron spectroscopy results on Al and Si atomic environments denoted Al-O-Si crosslinks, which improve the surface protection to corrosion. Copyright © 2012 Wiley Periodicals, Inc.

  2. Gene Expression Profiling of Human Epidermal Keratinocytes in Simulated Microgravity and Recovery Cultures

    Institute of Scientific and Technical Information of China (English)

    Jade Q. Clement; Shareen M. Lacy; Bobby L. Wilson

    2008-01-01

    Simulated microgravity (SMG) bioreactors and DNA microarray technology are powerful tools to identify "space genes" that play key roles in cellular response to microgravity. We applied these biotechnology tools to investigate SMG and post-SMG recovery effects on human epidermal keratinocytes by exposing cells to SMG for 3,4,9, and 10d using the high aspect ratio vessel bioreactor followed by recovery culturing for 15,50, and 60d in normal gravity. As a result, we identified 162 differentially expressed genes, 32 of which were "center genes" that were most consistently affected in the time course experiments. Eleven of the center genes were from the integrated stress response pathways and were coordinately down regulated. Another seven of the center genes, which are all metallothionein MT-Ⅰ and MT-Ⅱ isoforms, were coordinately up-regulated. In addition, HLA-G, a key gene in cellular immune response suppression, was found to be significantly upregulated during the recovery phase. Overall, more than 80% of the differentially expressed genes from the shorter exposures (≤4d) recovered in 15d; for longer (≥9d) exposures, more than 50d were needed to recover to the impact level of shorter exposures. The data indicated that shorter SMG exposure duration would lead to quicker and more complete recovery from the microgravity effect.

  3. Non-thermal dielectric-barrier discharge plasma damages human keratinocytes by inducing oxidative stress

    Science.gov (United States)

    KIM, KI CHEON; PIAO, MEI JING; HEWAGE, SUSARA RUWAN KUMARA MADDUMA; HAN, XIA; KANG, KYOUNG AH; JO, JIN OH; MOK, YOUNG SUN; SHIN, JENNIFER H.; PARK, YEUNSOO; YOO, SUK JAE; HYUN, JIN WON

    2016-01-01

    The aim of this study was to identify the mechanisms through which dielectric-barrier discharge plasma damages human keratinocytes (HaCaT cells) through the induction of oxidative stress. For this purpose, the cells were exposed to surface dielectric-barrier discharge plasma in 70% oxygen and 30% argon. We noted that cell viability was decreased following exposure of the cells to plasma in a time-dependent manner, as shown by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. The levels of intracellular reactive oxygen species (ROS) were determined using 2′,7′-dichlorodihydro-fluorescein diacetate and dihydroethidium was used to monitor superoxide anion production. Plasma induced the generation of ROS, including superoxide anions, hydrogen peroxide and hydroxyl radicals. N-acetyl cysteine, which is an antioxidant, prevented the decrease in cell viability caused by exposure to plasma. ROS generated by exposure to plasma resulted in damage to various cellular components, including lipid membrane peroxidation, DNA breaks and protein carbonylation, which was detected by measuring the levels of 8-isoprostane and diphenyl-1-pyrenylphosphine assay, comet assay and protein carbonyl formation. These results suggest that plasma exerts cytotoxic effects by causing oxidative stress-induced damage to cellular components. PMID:26573561

  4. Immunochemistry of a keratinocyte-fibroblast co-culture model for reconstruction of human skin.

    Science.gov (United States)

    Fleischmajer, R; MacDonald, E D; Contard, P; Perlish, J S

    1993-09-01

    Our purpose was to determine differentiation markers of an in vitro co-culture model in which fibroblasts grown in a three-dimensional nylon mesh were recombined with human keratinocytes. The cultures were kept for 5 weeks and then processed for electron microscopy and immunochemistry. The specimens revealed an epidermis, a basal lamina, an anchoring zone, and a dermis. Epidermal differentiation was confirmed by the presence of K10-keratin, trichohyalin, and filaggrin. The basal lamina contained Type IV collagen, laminin, nidogen, and heparan sulfate. Type IV collagen, laminin, and nidogen were also noted in the extracellular matrix. Type VI collagen was present in the anchoring zone and also gave a reticulated pattern in the rest of the dermis. There was a heavy signal for tenascin and fibronectin throughout the dermis. Osteonectin was restricted to the epidermis and dermal fibroblasts. Fibrillin stained at the anchoring zone and dermis but elastin and vitronectin were negative, suggesting early formation of elastic fibrils. Collagen fibrils stained for Types I, III, and V, as well as the amino propeptide of Types I and III procollagen, suggesting newly synthesized collagen. Decorin was present throughout the dermis. The model described appears suitable for in vitro reconstruction of the skin and may be useful to study the development of various supramolecular skin structures.

  5. Stages of Cell Cannibalism--Entosis--in Normal Human Keratinocyte Culture.

    Science.gov (United States)

    Garanina, A S; Khashba, L A; Onishchenko, G E

    2015-11-01

    Entosis is a type of cell cannibalism during which one cell penetrates into another cell and usually dies inside it. Researchers mainly pay attention to initial and final stages of entosis. Besides, tumor cells in suspension are the primary object of studies. In the present study, we investigated morphological changes of both cells-participants of entosis during this process. The substrate-dependent culture of human normal keratinocytes HaCaT was chosen for the work. A combination of light microscopy and scanning electron microscopy was used to prove that one cell was completely surrounded by the plasma membrane of another cell. We investigated such "cell-in-cell" structures and described the structural and functional changes of both cells during entosis. The outer cell nucleus localization and shape were changed. Gradual degradation of the inner cell nucleus and of the junctions between the inner and the outer cells was revealed. Moreover, repeated redistribution of the outer cell membrane organelles (Golgi apparatus, lysosomes, mitochondria, and autophagosomes), rearrangement of its cytoskeleton, and change in the lysosomal, autophagosomal, and mitochondrial state in both entotic cells were observed during entosis. On the basis of these data, we divided entosis into five stages that make it possible to systematize description of this type of cell death.

  6. Increase of integrin α6+p63+ cells after ultraviolet B irradiation in normal human keratinocytes

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    Gyeong-hun Park

    2009-12-01

    Full Text Available Epidermal stem cells (SC are believed to be resistant to environmental damage for the purpose of self renewal. Most promising SC markers include integrin a6 and p63. The aim of our study was to determine whether the integrin a6+p63+ cell fraction representative of the epidermal progenitor or SC is increased after ultraviolet B (UVB irradiation and to clarify the hypothesis that epidermal SC are resistant to high-dose UVB damage. We irradiated early passage normal human keratinocytes (NHK with 0, 25, 50, and 100 mJ/cm2 UVB. The percentage of cell death was calculated. Real-time reverse transcriptase-polymerase chain reaction (RT-PCR and western blotting analyses were performed to identify integrin a6 and p63, and flow cytometry analysis with integrin a6 and p63 antibodies was done. After 50 and 100 mJ/cm2 UVB, integrin a6+p63+cells were found to be much increased by fluorescence-activated cell sorting. Expression of integrin a6 and p63 was increased in NHK after UVB irradiation, which was shown with real-time RT-PCR and western blotting analyses. We concluded that an increase of integrin a6+p63+ cells after high-dose UVB may suggest that the putative progenitor or SC are resistant to UVB irradiation.

  7. A mint purified extract protects human keratinocytes from short-term, chemically induced oxidative stress.

    Science.gov (United States)

    Berselli, Patrizia Valeria Rita; Zava, Stefania; Montorfano, Gigliola; Corsetto, Paola Antonia; Krzyzanowska, Justyna; Oleszek, Wieslaw; Berra, Bruno; Rizzo, Angela Maria

    2010-11-10

    Oxidative stress is strictly correlated to the pathogenesis of many diseases, and a diet rich in fruits and vegetables, or adequately integrated, is currently considered to be a protective and preventive factor. This study aimed to analyze the efficacy of a 1 h preincubation with the highest nontoxic dose of a characterized Mentha longifolia extract (80 μg/mL) in protecting human keratinocytes (NCTC2544) from chemically induced oxidative stress (500 μM H2O2 for 2, 16, and 24 h). As reference synthetic pure compounds rosmarinic acid (360.31 μg/mL), a major mint phenolic constituent, and resveratrol (31.95 mg/mL), a well-known antioxidant, were used. Cellular viability was significantly protected by mint, which limited protein and DNA damage, decreased lipid peroxidation, and preserved glutathione and superoxide dismutase activity in the shorter phases of oxidative stress induction, in extents comparable to or better than those of pure compounds. These data suggest that mint use as only a flavoring has to be revised, taking into consideration its enrichment in foodstuff and cosmetics.

  8. Basic Red 51, a permitted semi-permanent hair dye, is cytotoxic to human skin cells: Studies in monolayer and 3D skin model using human keratinocytes (HaCaT).

    Science.gov (United States)

    Zanoni, Thalita B; Tiago, Manoela; Faião-Flores, Fernanda; de Moraes Barros, Silvia B; Bast, Aalt; Hageman, Geja; de Oliveira, Danielle Palma; Maria-Engler, Silvya S

    2014-06-01

    The use of hair dyes is closely associated with the increase of cancer, inflammation and other skin disorders. The recognition that human skin is not an impermeable barrier indicates that there is the possibility of human systemic exposure. The carcinogenic potential of hair dye ingredients has attracted the attention of toxicologists for many decades, mainly due to the fact that some ingredients belong to the large chemical family of aromatic amines. Herein, we investigated the cytotoxicity of Basic Red 51 (BR51) in immortalized human keratinocytes (HaCaT). BR51 is a temporary hair dye that belongs to the azo group (NN); the cleavage of this bond may result in the release of toxic aromatic amines. The half maximal effective concentration (EC50) in HaCaT cells is 13μg/mL. BR51 induced a significant decrease on expression of p21 in a dose dependent manner. p53 was not affected, whereas BR51 decreased procaspase 8 and cleaved procaspase 9. These results proved that caspase 3 is fully involved in BR51-induced apoptosis. The dye was also able to stop this cell cycle on G2 in sub-toxic doses. Moreover, we reconstructed a 3D artificial epidermis using HaCaT cells; using this model, we observed that BR51 induced cell injury and cells were undergoing apoptosis, considering the fragmented nuclei. Subsequently, BR51 induced reactive oxygen species (ROS) leading to an increase on the levels of 8-oxo-dG. In conclusion, we provide strong evidence that consumer and/or professional exposure to BR51 poses risk to human health.

  9. Gracilaria bursa-pastoris (Gmelin) Silva extract attenuates ultraviolet B radiation-induced oxidative stress in human keratinocytes.

    Science.gov (United States)

    Piao, M J; Kim, K C; Zheng, J; Yao, C W; Cha, J W; Kang, H K; Yoo, E S; Koh, Y S; Ko, M H; Lee, N H; Hyun, Jin Won

    2014-01-01

    The purpose of this study was to assess the protective effects of an ethanol extract derived from the red alga Gracilaria bursa-pastoris (Gmelin) Silva (GBE) on ultraviolet B (UVB)-irradiated human HaCaT keratinocytes. GBE exhibited scavenging activity against intracellular reactive oxygen species that were induced by either hydrogen peroxide or UVB radiation. In addition, both the superoxide anion and the hydroxyl radical were scavenged by GBE in cell-free systems. GBE absorbed light in the UVB range (280-320 nm) of the electromagnetic spectrum and lessened the extent of UVB-induced oxidative damage to cellular lipids, proteins, and DNA. Finally, GBE-treated keratinocytes showed a reduction in UVB-induced apoptosis, as exemplified by fewer apoptotic bodies. These results suggest that GBE exerts cytoprotective actions against UVB-stimulated oxidative stress by scavenging ROS and absorbing UVB rays, thereby attenuating injury to cellular constituents and preventing cell death.

  10. Incorporation and distribution of dihomo-gamma-linolenic acid, arachidonic acid, and eicosapentaenoic acid in cultured human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Punnonen, K.; Puustinen, T.; Jansen, C.T.

    1986-02-01

    Human keratinocytes in culture were labelled with /sup 14/C-dihomo-gamma-linolenic acid, /sup 14/C-arachidonic acid or /sup 14/C-eicosapentaenoic acid. All three eicosanoid precursor fatty acids were effectively incorporated into the cells. In phospholipids most of the radioactivity was recovered, in neutral lipids a substantial amount, and as free unesterified fatty acids only a minor amount. Most of the radioactivity was found in phosphatidylethanolamine which was also the major phospholipid as measured by phosphorous assay. The incorporation of dihomo-gamma-linolenic acid and arachidonic acid into lipid subfractions was essentially similar. Eicosapentaenoic acid was, however, much less effectively incorporated into phosphatidylinositol + phosphatidylserine and, correspondingly, more effectively into triacylglycerols as compared to the two other precursor fatty acids. Once incorporated, the distribution of all three precursor fatty acids was relatively stable, and only minor amounts of fatty acids were released into the culture medium during short term culture (two days). Our study demonstrates that eicosanoid precursor fatty acids are avidly taken up by human keratinocytes and esterified into membrane lipids. The clinical implication of this finding is that dietary manipulations might be employed to cause changes in the fatty acid composition of keratinocytes.

  11. Role of VEGF receptors in normal and psoriatic human keratinocytes: evidence from irradiation with different UV sources.

    Directory of Open Access Journals (Sweden)

    Jian-Wei Zhu

    Full Text Available Vascular endothelial growth factor (VEGF promotes angiogenesis and plays important roles both in physiological and pathological conditions. VEGF receptors (VEGFRs are high-affinity receptors for VEGF and are originally considered specific to endothelial cells. We previously reported that VEGFRs were also constitutively expressed in normal human keratinocytes and overexpressed in psoriatic epidermis. In addition, UVB can activate VEGFRs in normal keratinocytes, and the activated VEGFR-2 signaling is involved in the pro-survival mechanism. Here, we show that VEGFRs were also upregulated and activated by UVA in normal human keratinocytes via PKC, and interestingly, both the activated VEGFR-1 and VEGFR-2 protected against UVA-induced cell death. As VEGFRs were over-expressed in psoriatic epidermis, we further investigated whether narrowband UVB (NB-UVB phototherapy or topical halomethasone monohydrate 0.05% cream could affect their expression. Surprisingly, the over-expressed VEGFRs in psoriatic epidermis were significantly attenuated by both treatments. During NB-UVB therapy, VEGFRs declined first in the basal, and then gradually in the upper psoriatic epidermis. VEGFRs were activated in psoriatic epidermis, their activation was enhanced by NB-UVB, but turned undetectable after whole therapy. This process was quite different from that by halomethasone, in which VEGFRs and phospho-VEGFRs decreased in a gradual, homogeneous manner. Our findings further suggest that UV-induced activation of VEGFRs serves as a pro-survival signal for keratinocytes. In addition, VEGFRs may be involved in the pathological process of psoriasis, and UV phototherapy is effective for psoriasis by directly modulating the expression of VEGFRs.

  12. Ubiquitin/proteasome pathway regulates levels of retinoic acid receptor gamma and retinoid X receptor alpha in human keratinocytes.

    Science.gov (United States)

    Boudjelal, M; Wang, Z; Voorhees, J J; Fisher, G J

    2000-04-15

    Repeated exposure of human skin to solar UV radiation leads to premature aging (photoaging) and skin cancer. UV-induced skin damage can be ameliorated by all-trans retinoic acid treatment. The actions of retinoic acid in skin keratinocytes are mediated primarily by nuclear retinoic acid receptor gamma (RARgamma) and retinoid X receptor alpha (RXRalpha). We found that exposure of cultured primary human keratinocytes to UV irradiation (30 mJ/cm2) substantially reduced (50-90%) RARgamma and RXRalpha mRNA and protein within 8 h. The rates of disappearance of RARgamma and RXRalpha proteins after UV exposure or treatment with the protein synthesis inhibitor cycloheximide were similar. UV irradiation did not increase the rate of breakdown of RARgamma or RXRalpha but rather reduced their rate of synthesis. The addition of proteasome inhibitors MG132 and LLvL, but not the lysosomal inhibitor E64, prevented loss of RARgamma and RXRalpha proteins after exposure of keratinocytes to either UV radiation or cycloheximide. Soluble extracts from nonirradiated or UV-irradiated keratinocytes possessed similar levels of proteasome activity that degraded RARgamma and RXRalpha proteins in vitro. Furthermore, RARgamma and RXRalpha were polyubiquitinated in intact cells. RXRalpha was found to contain two proline, glutamate/aspartate, serine, and threonine (PEST) motifs, which confer rapid turnover of many short-lived regulatory proteins that are degraded by the ubiquitin/proteasome pathway. However, the PEST motifs in RXRalpha did not function to regulate its stability, because deletion of the PEST motifs individually or together did not alter ubiquitination or proteasome-mediated degradation of RXRalpha. These results demonstrate that loss of RARgamma and RXRalpha proteins after UV irradiation results from degradation via the ubiquitin/proteasome pathway. Taken together, the data here indicate that ubiquitin/proteasome-mediated breakdown is an important mechanism regulating the levels of

  13. Regulation of migratory activity of human keratinocytes by topography of multiscale collagen-containing nanofibrous matrices.

    Science.gov (United States)

    Fu, Xiaoling; Xu, Meng; Liu, Jie; Qi, Yanmei; Li, Shaohua; Wang, Hongjun

    2014-02-01

    Nanofibrous matrices hold great promise in skin wound repair partially due to their capability of recapturing the essential attributes of native extracellular matrix (ECM). With regard to limited studies on the effect of nanofibrous matrices on keratinocytes, the present study was aimed to understand how the topographical feature of nanofibrous matrices regulates keratinocyte motility by culturing keratinocytes on polycaprolactone (PCL)/collagen nanofibrous matrices (rough surface with fiber diameters of 331 ± 112 nm) or the matrices coated with a thin layer of collagen gel to form a secondary ultrafine fibrous network (smooth surface with ultrafine fiber diameters of 55 ± 26 nm). It was found that the PCL/collagen nanofibrous matrices alone did not stimulate cell migration, while collagen gel coating could significantly increase cell motility. Further studies demonstrated that the ultrafine fibrous network of collagen gel coating significantly activated integrin β1, Rac1 and Cdc42, facilitated the deposition of laminin-332 (formerly called laminin-5), and promoted the expression of active matrix metalloproteinases (MMPs) (i.e., MMP-2 and 9). Neutralization of integrin β1 activity abrogated the gel coating-induced keratinocyte migration. These findings provide important evidence on the role of topographical features of nanofibrous matrices in regulating the phenotypic alteration of keratinocytes and suggest the possible utility of collagen-containing nanofibrous matrices for skin regeneration especially in re-epithelialization.

  14. Characterization of vitamin C-induced cell sheets formed from primary and immortalized human corneal stromal cells for tissue engineering applications.

    Science.gov (United States)

    Grobe, Gesa Maria; Reichl, Stephan

    2013-01-01

    The purpose of this study was to compare the ability of primary human corneal stromal cells (HuFib cells) and SV40-immortalized human corneal keratocytes (HCK cells) to synthesize their own extracellular matrix induced by vitamin C supplementation. Therefore, the amount of collagen secreted and resulting biomechanical properties based on the culture duration were assessed. Cells were cultivated for several weeks with or without vitamin C. The amount of collagen secreted by the cells was quantified based on the culture duration. Cell viability was simultaneously determined via the MTT assay. Collagen secretion was increased as a result of vitamin C supplementation. The effect was stronger in primary cells. In addition, vitamin C supplementation had a positive effect on HuFib cell viability. Vitamin C supplementation induced the formation of detachable cell sheets in both primary and immortalized cells. The biomechanical properties of the sheets were evaluated using a static material testing machine, and the ultrastructure of the cell sheets was examined using scanning electron microscopy. The cell sheets formed from HuFib cells had a higher percentage of light transmission between 400 and 800 nm and were superior in terms of E-modulus and ultimate strength testing. Indirect immunofluorescence and Western blot confirmed the presence of collagen type I in the HuFib and HCK cell cultures. Stimulating secretion of the extracellular matrix in corneal stromal cells is a promising approach for corneal stroma reconstruction for tissue engineering applications. Copyright © 2013 S. Karger AG, Basel.

  15. A Novel Controllable Hydrogen Sulfide-Releasing Molecule Protects Human Skin Keratinocytes Against Methylglyoxal-Induced Injury and Dysfunction

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    Chun-tao Yang

    2014-09-01

    Full Text Available Background/Aim: Delayed wound healing is a common skin complication of diabetes, which is associated with keratinocyte injury and dysfunction. Levels of methylglyoxal (MGO, an α-dicarbonyl compound, are elevated in diabetic skin tissue and plasma, while levels of hydrogen sulfide (H2S, a critical gaseous signaling molecule, are reduced. Interestingly, the gas has shown dermal protection in our previous study. To date, there is no evidence demonstrating whether MGO affects keratinocyte viability and function or H2S donation abolishes these effects and improves MGO-related impairment of wound healing. The current study was conducted to examine the effects of MGO on the injury and function in human skin keratinocytes and then to evaluate the protective action of a novel H2S-releasing molecule. Methods: An N-mercapto-based H2S donor (NSHD-1 was synthesized and its ability to release H2S was observed in cell medium and cells, respectively. HaCaT cells, a cell line of human skin keratinocyte, were exposed to MGO to establish an in vitro diabetic wound healing model. NSHD-1 was added to the cells before MGO exposure and the improvement of cell function was observed in respect of cellular viability, apoptosis, oxidative stress, mitochondrial membrane potential (MMP and behavioral function. Results: Treatment with MGO decreased cell viability, induced cellular apoptosis, increased intracellular reactive oxygen species (ROS content and depressed MMP in HaCaT cells. The treatment also damaged cell behavioral function, characterized by decreased cellular adhesion and migration. The synthesized H2S-releasing molecule, NSHD-1, was able to increase H2S levels in both cell medium and cells. Importantly, pretreatment with NSHD-1 inhibited MGO-induced decreases in cell viability and MMP, increases in apoptosis and ROS accumulation in HaCaT cells. The pretreatment was also able to improve adhesion and migration function. Conclusion: These results demonstrate that

  16. Differential cytotoxicity of long-chain bases for human oral gingival epithelial keratinocytes, oral fibroblasts, and dendritic cells

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    Leslie A. Mehalick

    2015-12-01

    Full Text Available Long-chain bases, found in the oral cavity, have potent antimicrobial activity against oral pathogens. In an article associated with this dataset, Poulson and colleagues determined the cytotoxicities of long-chain bases (sphingosine, dihydrosphingosine, and phytosphingosine for human oral gingival epithelial (GE keratinocytes, oral gingival fibroblasts (GF, dendritic cells (DC, and squamous cell carcinoma (SCC cell lines [1]. Poulson and colleagues found that GE keratinocytes were more resistant to long-chain bases as compared to GF, DC, and SCC cell lines [1]. In this study, we assess the susceptibility of DC to lower concentrations of long chain bases. 0.2–10.0 µM long-chain bases and GML were not cytotoxic to DC; 40.0–80.0 µM long-chain bases, but not GML, were cytotoxic for DC; and 80.0 µM long-chain bases were cytotoxic to DC and induced cellular damage and death in less than 20 mins. Overall, the LD50 of long-chain bases for GE keratinocytes, GF, and DC were considerably higher than their minimal inhibitory concentrations for oral pathogens, a finding important to pursuing their future potential in treating periodontal and oral infections.

  17. Typha latifolia L. fruit polysaccharides induce the differentiation and stimulate the proliferation of human keratinocytes in vitro.

    Science.gov (United States)

    Gescher, Kirsten; Deters, Alexandra M

    2011-09-01

    In Northern America Typha latifolia L. (Typhaceae) fruits are used for more than 4000 years for treatment of skin disorders, burns and as wound dressing to absorb the ichors. The following studies attempted to characterize water-soluble polysaccharides from aqueous Typha latifolia extracts and to investigate the influence of the polymers on cell physiology of human dermal fibroblasts (NHDF) and epidermal keratinocytes (NHEK). Water-soluble raw polysaccharides (RPS) were isolated from Typha latifolia fruits and fractionated by anion exchange chromatography (AEC) and size exclusion chromatography (GPC). Fractions obtained were characterized concerning monosaccharide composition by HPAEC-PAD. The bioactivity of the polysaccharides was investigated on cell viability, proliferation, differentiation and gene expression NHDF of NHEK. RPS was fractionated into 5 heterodisperse fractions (TL1-TL5). The polysaccharides were composed mainly of glucose (more than 50% in RPS and TL4), galactose, xylose, mannose, glucuronic acid, galacturonic acid, arabinose, ribose, fucose, rhamnose, and fructose with differing amounts concerning to RPS and AEC-fractions. Proteins were detected in the RPS (10%) and to a less extend in TL1-TL3 (1-3%). TL1-TL3 significantly increased the proliferation of keratinocytes, whereas TL4 was shown to be a potent inductor of the early differentiation process of keratinocytes. Gene expression analysis supported these results since Smad3 and PKC-α, known to be part of signal pathways leading to cell differentiation, were significantly up regulated. Effects on fibroblasts were not observed, indicating cell specific activity of the polysaccharides. The results clearly indicate a rationale for the traditional use of Typha latifolia fruits extracts for wound healing to the strong stimulatory activity of the polysaccharides on keratinocytes proliferation and early differentiation, major activities necessary for potent wound-healing agents. Copyright © 2011

  18. Cytotoxicity and chromosome aberrations in normal human oral keratinocytes induced by chemical carcinogens: Comparison of inter-individual variations.

    Science.gov (United States)

    Tsutsui, T; Kawamoto, Y; Suzuki, N; Gladen, B C; Barrett, J C

    1991-01-01

    Normal human keratinocytes from the oral cavity were cultured in vitro in serum-free medium. Cultures from different individuals were established, and the responses of the cells to different chemicals were compared. The cells, grown at clonal densities, were treated separately with an alkylating agent (N-methyl-N'-nitro-N-nitrosoguanidine; MNNG), two arsenical salts (sodium arsenate or sodium arsenite), sodium fluoride or two polyaromatic hydrocarbons (benzo[a]pyrene or 7,12-dimethylbenz[a]-anthracene). There were no significant differences in the colony-forming efficiencies (22.8 +/- 4.2%) of control (untreated) cells from five different individuals. At selected doses, each of the chemicals reduced the colony-forming efficiencies of the treated cells. The cytotoxicity of most of the chemicals did not differ significantly among cells derived from different individuals, with the exception of sodium arsenate at two doses and sodium fluoride at the highest dose tested. Induction of chromosome aberrations by MNNG, sodium arsenite, sodium arsenate and sodium flouride was analysed with cells derived from up to nine individuals. There was little difference in the inducibilities of chromosome aberrations among cultured keratinocytes from different donors. Treatment of cells from nine donors with one dose of sodium fluoride revealed a statistically significant inter-individual variation. These findings provide a model system to study the effects of carcinogens on the target cells for oral cancers. The results can be compared with findings for cells from other epithelial tissues, since the culture conditions support the growth of keratinocytes regardless of origin. Little inter-individual variation was observed in the response of oral keratinocytes to the chemicals examined.

  19. Expression of parathyroid hormone-related protein during immortalization of human peripheral blood mononuclear cells by HTLV-1: Implications for transformation

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    Nadella Kiran S

    2008-06-01

    Full Text Available Abstract Background Adult T-cell leukemia/lymphoma (ATLL is initiated by infection with human T-lymphotropic virus type-1 (HTLV-1; however, additional host factors are also required for T-cell transformation and development of ATLL. The HTLV-1 Tax protein plays an important role in the transformation of T-cells although the exact mechanisms remain unclear. Parathyroid hormone-related protein (PTHrP plays an important role in the pathogenesis of humoral hypercalcemia of malignancy (HHM that occurs in the majority of ATLL patients. However, PTHrP is also up-regulated in HTLV-1-carriers and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP patients without hypercalcemia, indicating that PTHrP is expressed before transformation of T-cells. The expression of PTHrP and the PTH/PTHrP receptor during immortalization or transformation of lymphocytes by HTLV-1 has not been investigated. Results We report that PTHrP was up-regulated during immortalization of lymphocytes from peripheral blood mononuclear cells by HTLV-1 infection in long-term co-culture assays. There was preferential utilization of the PTHrP-P2 promoter in the immortalized cells compared to the HTLV-1-transformed MT-2 cells. PTHrP expression did not correlate temporally with expression of HTLV-1 tax. HTLV-1 infection up-regulated the PTHrP receptor (PTH1R in lymphocytes indicating a potential autocrine role for PTHrP. Furthermore, co-transfection of HTLV-1 expression plasmids and PTHrP P2/P3-promoter luciferase reporter plasmids demonstrated that HTLV-1 up-regulated PTHrP expression only mildly, indicating that other cellular factors and/or events are required for the very high PTHrP expression observed in ATLL cells. We also report that macrophage inflammatory protein-1α (MIP-1α, a cellular gene known to play an important role in the pathogenesis of HHM in ATLL patients, was highly expressed during early HTLV-1 infection indicating that, unlike PTHrP, its expression was

  20. The Effects of Antifungal Azoles on Inflammatory Cytokine Production in Human Keratinocytes

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    K Zomorodian

    2008-04-01

    Full Text Available ABSTRACT: Introduction & Objective: Azoles drugs are being used successfully in treatment of fungal infections. Recently, immunosuppressive effects of some of these agents have been reported. Keratinocytes, as the major cells of the skin, have an important role in innate immunity against pathogenic agents. Considering the scanty of information about the effects of azoles on immune responces, this study was conducted to assess the expression and secretion of inflammatory cytokines in keratinocytes following treatment with azole drugs. Materials & Methods: This is an exprimental study conducted in in molecular biology division in Tehran University of Medical Sciences and Immunodermatology Department in Vienna Medical University. Primery keratinocytes were cultured and treated with different concentrations of fluconazole, itraconazole, ketoconazole and griseofulvin. Secreted IL1, IL6 and TNF-α by keratinocytes in culture supernatant were measured by quantitative enzyme immunoassay technique. Moreover, expression of the genes encoding IL1 and IL8 was evaluated by Real Time-PCR. Results: Treatment of keratinocytes with different concentrations of fluconazole and low concentration of ketoconazole resulted in decrease in IL1 secretion, but Itraconazole and griseofulvin did not show such an effect at the same concentrations. In addition, none of the examined drugs had an effect on secretion level of IL6 and TNF-α. Quantitative analysis of IL1 and IL8 encoding genes revealed that transcription on these genes might be suppressed following treatment with fluconazole or ketoconazole. Conclusion: Fluconazole and ketoconazole might modulate the expression and secretion of IL1 and IL8 and affect the direction of immune responses induced by keratinocytes

  1. La aportación de Étienne Gilson al problema de la inmortalidad del alma humana en Cayetano / The Contribution of Étienne Gilson to the Problem of the Immortality of the Human Soul in Cajetan

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    Ceferino P. D. Muñoz

    2013-12-01

    Full Text Available In his article the author reviews Cajetan’s different positions on the prob-lem of the immortality of the human soul, and investigates possible reasons which led the Cardinal to dissent with the position of Thomas Aquinas. For this purpose, he invokes selected interpretations of distinguished scholars, with special reference to the approach of Étienne Gilson. Against the back-ground of his analyses the authors attempts to give a synthesis which, as he hopes, can become a modest contribution to contemporary comments on the thought of this great French medievalist about the problem of the immortality of the human soul in Cajetan.

  2. Potential Wound Healing Activities of Galla Rhois in Human Fibroblasts and Keratinocytes.

    Science.gov (United States)

    Park, Hyo-Hyun; Park, Na-Young; Kim, Sun-Gun; Jeong, Kyu-Tae; Lee, Eu-Jin; Lee, Eunkyung

    2015-01-01

    Wound healing is a complex process orchestrated by the regeneration of the epithelium and the remodeling of the extracellular matrix through processes like collagen deposition. Galla Rhois has been widely used in traditional Korean medicine for its various pharmacological effects, including an anticoccidial effect, however, little is known about its healing activity. The purpose of this study was to determine the effects of Galla Rhois ethanol extract (GRE) on wound healing activities, including H2O2-induced oxidative stress, cell migration, and lactate dehydrogenase (LDH) release assays using human keratinocyte (HaCaT) and dermal fibroblasts (CCD-986SK). In addition, total soluble collagen deposition and collagen gene expression for Type I and III collagen were evaluated in CCD-986SK. Total tannin and flavonoid contents for GRE were measured. GRE induced a significant increase in the number and migration of cells, along with a decrease in cell death and LDH release. In addition, it also induced the over-expression of collagen Type I and III mRNA and caused increased synthesis of total soluble collagen. The contents of total tannin and flavonoid for GRE were 55.7% ([Formula: see text][Formula: see text]mg/g) and 62.9% ([Formula: see text][Formula: see text]mg/g), respectively. The results suggest that GRE can cause accelerated wound healing by increasing cell survival, proliferation, migration, and collagen synthesis along with a potential anti-oxidant property. This evidence provides novel insight into natural therapy for tissue injury.

  3. The expression of O(6) -methylguanine-DNA methyltransferase in human oral keratinocytes stimulated with arecoline.

    Science.gov (United States)

    Lee, Shiuan-Shinn; Tsai, Chung-Hung; Yu, Cheng-Chia; Ho, Yung-Chuan; Hsu, Hsin-I; Chang, Yu-Chao

    2013-09-01

    O(6) -methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that can protect cells from carcinogenic effects of alkylating agents by removing adducts from the O(6) position of guanine. Evidences indicated that areca quid chewing may increase the risk of oral squamous cell carcinoma (OSCC). This study was to investigate the role of MGMT expression in OSCCs and the normal oral tissues. Thirty-two OSCCs from areca quid chewers and ten normal oral tissue biopsy samples without areca quid chewing were analyzed by the immunohistochemistry for MGMT. Primary human oral keratinocytes (HOKs) were challenged with arecoline, the major alkaloid of areca nut, by Western blot. Nicotine, an important component of cigarette smoke, was added to find the possible regulatory mechanisms. Significant association was observed between low MGMT expression and advanced clinical stage of OSCCs and lymph node metastasis (P = 0.03). MGMT expression was significantly higher in patients only chewing areca quid than patients both chewing areca quid and smoking (P = 0.028). Arecoline was found to elevate MGMT expression in a dose- and time-dependent manner. The addition of nicotine was found to enhance arecoline-induced MGMT expression. Our results indicate that MGMT could be used clinically as a predictive marker for tumor processing, the potential for lymph node metastasis as well as advanced clinical stage. MGMT expression was significantly upregulated by arecoline in HOKs. Nicotine has a synergistic effect of arecoline-induced MGMT expression. The cigarette smoking may act synergistically in the pathogenesis of OSCC in areca quid chewers via the upregulation of MGMT. © 2012 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Nicotinamide attenuates aquaporin 3 overexpression induced by retinoic acid through inhibition of EGFR/ERK in cultured human skin keratinocytes.

    Science.gov (United States)

    Song, Xiuzu; Xu, Aie; Pan, Wei; Wallin, Brittany; Kivlin, Rebecca; Lu, Shan; Cao, Cong; Bi, Zhigang; Wan, Yinsheng

    2008-08-01

    The most common adverse effects that are related to all-trans retinoic acid (atRA) treatment are irritation and dryness of the skin. atRA therapy is reported to impair barrier function as achieved by trans-epidermal water loss (TEWL). Treatment with nicotinamide prior to initiation of atRA therapy provides additional barrier protection and thus reduces susceptibility of retinoic acid. Our previous studies showed that atRA upregulates aquaporin 3 (AQP3) in cultured human skin keratinocytes and fibroblasts. Others have demonstrated that in atopic dermatitis, overexpression of AQP3 is linked to elevated TEWL and that nicotinamide treatment reduces skin TEWL. In this study, we observed that while atRA upregulates AQP3 expression in cultured human skin keratinocytes (HaCaT cells), nicotinamide attenuates the effect of atRA in a concentration-dependent manner. atRA treatment induces EGFR and ERK activation. PD153035, an EGFR inhibitor, and U0126, an ERK inhibitor, inhibit atRA-induced upregulation of AQP3. Nicotinamide also inhibits atRA-induced activation of EGFR/ERK signal transduction and decreases water permeability by downregulating AQP3 expression. Collectively, our results indicate that the effect of atRA on AQP3 expression is at least partly mediated by EGFR/ERK signaling in cultured human skin keratinocytes. Nicotinamide attenuates atRA-induced AQP3 expression through inhibition of EGFR/ERK signal transduction and eventually decreases water permeability and water loss. Our study provides insights into the molecular mechanism through which nicotinamide reverses the side effects of dryness in human skin after treatment with atRA.

  5. The Parkinsonism-associated protein DJ-1/Park7 prevents glycation damage in human keratinocyte.

    Science.gov (United States)

    Advedissian, Tamara; Deshayes, Frédérique; Poirier, Françoise; Viguier, Mireille; Richarme, Gilbert

    2016-04-22

    Reducing sugars and dicarbonyls form covalent adducts with proteins through a nonenzymatic process known as glycation, which inactivates proteins, is increased in diabetic patients and is associated with diabetic complications, including retinopathy, cataracts, nephropathy, neuropathy, cardiomyopathy and skin defects. We recently characterized DJ-1/Park7 as a protein deglycase that repairs proteins from glycation by glyoxal and methylglyoxal, two major glycating agents which are responsible for up to 65% of glycation events. In this study, we investigated the ability of DJ-1 to prevent protein glycation in keratinocytes. Glycation of collagen and keratinocyte proteins was tested by measuring ultraviolet absorption and fluorescence emission. Protein glycation in HaCaT keratinocytes was investigated by immunodetection with anti-advanced glycation endproduct antibodies, after DJ-1 depletion or overexpression. In vitro, DJ-1 prevented glycation of collagen and keratinocyte protein extracts. In cell culture, DJ-1 depletion by small interfering RNAs resulted in a 3-fold increase in protein glycation levels. Moreover, protein glycation levels were decreased several-fold in cells overexpressing DJ-1 after addition of the Nrf2 inducer sulforaphane or after transfection with a DJ-1 plasmid. Thus, the DJ-1 deglycase plays a major role in preventing protein glycation in eukaryotic cells and might be important for preventing skin glycation.

  6. Activated protein C: A regulator of human skin epidermal keratinocyte function.

    Science.gov (United States)

    McKelvey, Kelly; Jackson, Christopher John; Xue, Meilang

    2014-05-26

    Activated protein C (APC) is a physiological anticoagulant, derived from its precursor protein C (PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor (EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal keratinocytes. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-2 activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of keratinocyte monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal growth factor receptor or Tie2. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APC's function in skin epidermis/keratinocytes and its therapeutical potential in skin inflammatory conditions.

  7. Activated protein C: A regulator of human skin epidermal keratinocyte function

    Institute of Scientific and Technical Information of China (English)

    Kelly; McKelvey; Christopher; John; Jackson; Meilang; Xue

    2014-01-01

    Activated protein C(APC) is a physiological anticoagulant, derived from its precursor protein C(PC). Independent of its anticoagulation, APC possesses strong anti-inflammatory, anti-apoptotic and barrier protective properties which appear to be protective in a number of disorders including chronic wound healing. The epidermis is the outermost skin layer and provides the first line of defence against the external environment. Keratinocytes are the most predominant cells in the epidermis and play a critical role in maintaining epidermal barrier function. PC/APC and its receptor, endothelial protein C receptor(EPCR), once thought to be restricted to the endothelium, are abundantly expressed by skin epidermal keratinocytes. These cells respond to APC by upregulating proliferation, migration and matrix metalloproteinase-2 activity and inhibiting apoptosis/inflammation leading to a wound healing phenotype. APC also increases barrier function of keratinocyte monolayers by promoting the expression of tight junction proteins and re-distributing them to cell-cell contacts. These cytoprotective properties of APC are mediated through EPCR, protease-activated receptors, epidermal growth factor receptor or Tie2. Future preventive and therapeutic uses of APC in skin disorders associated with disruption of barrier function and inflammation look promising. This review will focus on APC’s function in skin epidermis/keratinocytes and its therapeutical potential in skin inflammatory conditions.

  8. Combined introduction of Bmi-1 and hTERT immortalizes human adipose tissue-derived stromal cells with low risk of transformation

    Energy Technology Data Exchange (ETDEWEB)

    Tatrai, Peter, E-mail: peter.tatrai@biomembrane.hu [Institute of Enzymology, Research Center for Natural Sciences, Hungarian Academy of Sciences, Karolina ut 29, H-1113 Budapest (Hungary); Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Szepesi, Aron, E-mail: aron.szepesi@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Matula, Zsolt, E-mail: matula.zsolt@gmail.com [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Szigeti, Anna, E-mail: anna.szigeti@biomembrane.hu [Creative Cell Ltd., Puskas Tivadar utca 13, H-1119 Budapest (Hungary); Buchan, Gyoengyi, E-mail: buchan@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Madi, Andras, E-mail: madi@med.unideb.hu [Department of Biochemistry and Molecular Biology, Medical and Health Science Center, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Stem Cell, Apoptosis and Genomics Research Group of the Hungarian Academy of Sciences, University of Debrecen, Egyetem ter 1, H-4032 Debrecen (Hungary); Uher, Ferenc, E-mail: uher@biomembrane.hu [Stem Cell Laboratory, Hungarian National Blood Transfusion Service, Dioszegi ut 64, H-1113 Budapest (Hungary); and others

    2012-05-25

    Highlights: Black-Right-Pointing-Pointer We immortalized human adipose stromal cells (ASCs) with hTERT, Bmi-1, and SV40T. Black-Right-Pointing-Pointer hTERT-only ASCs are prone to transformation, while Bmi-only ASCs become senescent. Black-Right-Pointing-Pointer SV40T introduced along with hTERT abrogates proliferation control and multipotency. Black-Right-Pointing-Pointer hTERT combined with Bmi-1 yields stable phenotype up to 140 population doublings. -- Abstract: Adipose tissue-derived stromal cells (ASCs) are increasingly being studied for their usefulness in regenerative medicine. However, limited life span and donor-dependent variation of primary cells such as ASCs present major hurdles to controlled and reproducible experiments. We therefore aimed to establish immortalized ASC cell lines that provide steady supply of homogeneous cells for in vitro work while retain essential features of primary cells. To this end, combinations of human telomerase reverse transcriptase (hTERT), murine Bmi-1, and SV40 large T antigen (SV40T) were introduced by lentiviral transduction into ASCs. The resulting cell lines ASC{sup hTERT}, ASC{sup Bmi-1}, ASC{sup Bmi-1+hTERT} and ASC{sup SV40T+hTERT} were tested for transgene expression, telomerase activity, surface immunomarkers, proliferation, osteogenic and adipogenic differentiation, karyotype, tumorigenicity, and cellular senescence. All cell lines have maintained expression of characteristic surface immunomarkers, and none was tumorigenic. However, ASC{sup Bmi-1} had limited replicative potential, while the rapidly proliferating ASC{sup SV40T+hTERT} acquired chromosomal aberrations, departed from MSC phenotype, and lost differentiation capacity. ASC{sup hTERT} and ASC{sup hTERT+Bmi-1}, on the other hand, preserved all essential MSC features and did not senesce after 100 population doublings. Notably, a subpopulation of ASC{sup hTERT} also acquired aberrant karyotype and showed signs of transformation after long-term culture

  9. SIRT1 inhibition restores apoptotic sensitivity in p53-mutated human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Herbert, Katharine J.; Cook, Anthony L., E-mail: Anthony.Cook@utas.edu.au; Snow, Elizabeth T., E-mail: elizabeth.snow@utas.edu.au

    2014-06-15

    Mutations to the p53 gene are common in UV-exposed keratinocytes and contribute to apoptotic resistance in skin cancer. P53-dependent activity is modulated, in part, by a complex, self-limiting feedback loop imposed by miR-34a-mediated regulation of the lysine deacetylase, SIRT1. Expression of numerous microRNAs is dysregulated in squamous and basal cell carcinomas; however the contribution of specific microRNAs to the pathogenesis of skin cancer remains untested. Through use of RNAi, miRNA target site blocking oligonucleotides and small molecule inhibitors, this study explored the influence of p53 mutational status, SIRT1 activity and miR-34a levels on apoptotic sensitivity in primary (NHEK) and p53-mutated (HaCaT) keratinocyte cell lines. SIRT1 and p53 are overexpressed in p53-mutated keratinocytes, whilst miR-34a levels are 90% less in HaCaT cells. HaCaTs have impaired responses to p53/SIRT1/miR-34a axis manipulation which enhanced survival during exposure to the chemotherapeutic agent, camptothecin. Inhibition of SIRT1 activity in this cell line increased p53 acetylation and doubled camptothecin-induced cell death. Our results demonstrate that p53 mutations increase apoptotic resistance in keratinocytes by interfering with miR-34a-mediated regulation of SIRT1 expression. Thus, SIRT1 inhibitors may have a therapeutic potential for overcoming apoptotic resistance during skin cancer treatment. - Highlights: • Impaired microRNA biogenesis promotes apoptotic resistance in HaCaT keratinocytes. • TP53 mutations suppress miR-34a-mediated regulation of SIRT1 expression. • SIRT1 inhibition increases p53 acetylation in HaCaTs, restoring apoptosis.

  10. Human keratinocyte caspase-14 expression is altered in human epidermal 3D models by dexamethasone and by natural products used in cosmetics.

    Science.gov (United States)

    Kataoka, Saori; Hattori, Kenji; Date, Akira; Tamura, Hiroomi

    2013-10-01

    Caspase-14 is a cysteinyl-aspartate-specific proteinase that is specifically expressed in epidermal keratinocytes. Dysregulation of caspase-14 expression is implicated in impaired skin barrier formation. To elucidate the regulation of caspase-14 in differentiated keratinocytes, we characterized the expression of caspase-14 in normal human epidermal keratinocytes (NHEKs) and two types of three-dimensional (3D) human epidermis culture models, EPI-200 and EPI-201, via RT-PCR and immunoblot analyses. Caspase-14 expression was absent in subconfluent NHEKs, but was present in confluent NHEKs as well as those induced to differentiate by calcium. Caspase-14 expression levels in the 3D epidermis models were almost equal to that in the Ca(2+)-treated differentiated NHEKs. Despite the presence of caspase-14 expression in these models, caspase-14 activity was found only in the mature 3D skin model, EPI-200. This was confirmed by detection of a 17 kDa cleaved fragment of caspase-14 present only in the EPI-200 model. Since glucocorticoid (GC) receptor is required for skin barrier competence, we investigated whether the GC dexamethasone (Dex) and various natural components of common skin moisturizers affect caspase-14 expression in keratinocytes. Dex decreased caspase-14 expression in undifferentiated, but not differentiated, NHEKs. Conversely, Dex increased caspase-14 expression in both 3D skin models, although it did not alter caspase protease activity. Similar to treatment with Dex, treatment of the premature 3D skin mode, EPI-201 with a Galactomyces ferment filtrate markedly increased expression of caspase-14. Further, these results suggest that the effect of Dex, or lack thereof, on caspase-14 expression is dependent on the stage of keratinocyte differentiation.

  11. Flavonoid myricetin inhibits TNF-α-stimulated production of inflammatory mediators by suppressing the Akt, mTOR and NF-κB pathways in human keratinocytes.

    Science.gov (United States)

    Lee, Da Hee; Lee, Chung Soo

    2016-08-05

    Flavonoid myricetin has been shown to exhibit anti-inflammatory and anti-oxidant effects. Nevertheless, the effect of myricetin on the TNF-α-stimulated production of inflammatory mediators in keratinocytes has not been studied. Using human keratinocytes, we examined the effect of myricetin on the TNF-α-stimulated production of inflammatory mediators in relation to the Akt, mTOR and NF-κB pathways, which regulate the transcription genes involved in immune and inflammatory responses. TNF-α stimulated production of the inflammatory mediators and reactive oxygen species in keratinocytes, and activation of the Akt, mTOR and NF-κB pathways in HaCaT cells and primary keratinocytes. Myricetin, Akt inhibitor, Bay 11-7085 (an inhibitor of NF-κB activation), rapamycin (mTOR inhibitor) and N-acetylcysteine attenuated TNF-α-induced activation of Akt, mTOR and NF-κB. Myricetin and N-acetylcysteine attenuated the TNF-α-stimulated production of cytokines and chemokines, and production of reactive oxygen species in keratinocytes. The results show that myricetin may reduce TNF-α-stimulated inflammatory mediator production in keratinocytes by suppressing the activation of the Akt, mTOR and NF-κB pathways. The effect of myricetin appears to be associated with inhibition of the production of reactive oxygen species. Further, myricetin appears to attenuate the proinflammatory mediator-induced inflammatory skin diseases.

  12. Propolis Inhibits UVA-Induced Apoptosis of Human Keratinocyte HaCaT Cells by Scavenging ROS

    OpenAIRE

    2016-01-01

    Propolis is a resinous material collected by honeybees from several plant sources. This research aimed at showing its protective effect against UVA-induced apoptosis of human keratinocyte HaCaT cells. Using Hoechst staining, it was demonstrated that propolis (5 and 10 μg/mL) significantly inhibited the apoptosis of HaCaT cells induced by UVA-irradiation. Propolis also showed the protective effect against loss of mitochondrial membrane potential induced by UVA-irradiaiton in HaCaT cells. Propo...

  13. Peroxisome Proliferator-Activated Receptor-alpha Is a Functional Target of p63 in Adult Human Keratinocytes

    DEFF Research Database (Denmark)

    Pozzi, Silvia; Boergesen, Michael; Sinha, Satrajit;

    2009-01-01

    healing process, is a target of p63 in human keratinocytes. Silencing of p63 by RNA interference and transient transfections showed that p63 represses PPARalpha through a functional region of promoter B. Chromatin immunoprecipitation analyses indicate that p63 is bound to this region, in the absence......p63 is a master switch in the complex network of signaling pathways controlling the establishment and maintenance of stratified epithelia. We provide evidence that peroxisome proliferator-activated receptor-alpha (PPARalpha), a ligand-activated nuclear receptor that participates in the skin wound...

  14. A telomerase immortalized human proximal tubule cell line with a truncation mutation (Q4004X in polycystin-1.

    Directory of Open Access Journals (Sweden)

    Brittney-Shea Herbert

    Full Text Available 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. 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.

  15. A p38(MAPK)/HIF-1 pathway initiated by UVB irradiation is required to induce Noxa and apoptosis of human keratinocytes.

    Science.gov (United States)

    Nys, Kris; Van Laethem, An; Michiels, Carine; Rubio, Noemi; Piette, Jacques G; Garmyn, Maria; Agostinis, Patrizia

    2010-09-01

    The signal transduction pathways leading to apoptosis of human keratinocytes responding to UVB irradiation are complex and not completely understood. Previously, we reported that in UVB-irradiated keratinocytes, p38(MAPK) instigates Bcl-2-associated X protein (Bax) activation and mitochondrial apoptosis. However, the molecular mechanism underlying the pro-apoptotic function of p38(MAPK) remained unclear. Here, we show that in UVB-treated human primary keratinocytes the activation of p38(MAPK) is necessary to upregulate Noxa, a BH3-only pro-apoptotic dominantly induced by UVB and required for apoptosis. Whereas p53-silencing was marginally cytoprotective and poorly affected Noxa expression, p38(MAPK) inhibition in p53-silenced keratinocytes or in p53(-/-) cells could still efficiently prevent Noxa induction and intrinsic apoptosis after UVB, indicating that p38(MAPK) signals mainly through p53-independent mechanisms. Furthermore, p38(MAPK) was required for the induction and activation of hypoxia-inducible factor 1 (HIF-1) in response to UVB, and HIF-1 knockdown reduced Noxa expression and apoptosis. In UVB-irradiated keratinocytes, Noxa targeted the anti-apoptotic myeloid cell leukemia sequence 1 (Mcl-1) for degradation, and small-interfering RNA (siRNA)-mediated knockdown of Noxa or p38(MAPK) inhibition restored levels of Mcl-1 and abolished apoptosis. Thus, the pro-apoptotic mechanisms orchestrated by p38(MAPK) in human keratinocytes in response to UVB involve an HIF-1/Noxa axis, which prompts the downregulation of anti-apoptotic Mcl-1, thereby favoring Bax-mediated mitochondrial apoptosis of UVB-damaged keratinocytes.

  16. Resveratrol prevents oxidative stress-induced senescence and proliferative dysfunction by activating the AMPK-FOXO3 cascade in cultured primary human keratinocytes.

    Science.gov (United States)

    Ido, Yasuo; Duranton, Albert; Lan, Fan; Weikel, Karen A; Breton, Lionel; Ruderman, Neil B

    2015-01-01

    The aging process is perceived as resulting from a combination of intrinsic factors such as changes in intracellular signaling and extrinsic factors, most notably environmental stressors. In skin, the relationship between intrinsic changes and keratinocyte function is not clearly understood. Previously, we found that increasing the activity of AMP-activated protein kinase (AMPK) suppressed senescence in hydrogen peroxide (H2O2)-treated human primary keratinocytes, a model of oxidative stress-induced cellular aging. Using this model in the present study, we observed that resveratrol, an agent that increases the activities of both AMPK and sirtuins, ameliorated two age-associated phenotypes: cellular senescence and proliferative dysfunction. In addition, we found that treatment of keratinocytes with Ex527, a specific inhibitor of sirtuin 1 (SIRT1), attenuated the ability of resveratrol to suppress senescence. In keeping with the latter observation, we noted that compared to non-senescent keratinocytes, senescent cells lacked SIRT1. In addition to these effects on H2O2-induced senescence, resveratrol also prevented the H2O2-induced decrease in proliferation (as indicated by 3H-thymidine incorporation) in the presence of insulin. This effect was abrogated by inhibition of AMPK but not SIRT1. Compared to endothelium, we found that human keratinocytes expressed relatively high levels of Forkhead box O3 (FOXO3), a downstream target of both AMPK and SIRT1. Treatment of keratinocytes with resveratrol transactivated FOXO3 and increased the expression of its target genes including catalase. Resveratrol's effects on both senescence and proliferation disappeared when FOXO3 was knocked down. Finally, we performed an exploratory study which showed that skin from humans over 50 years old had lower AMPK activity than skin from individuals under age 20. Collectively, these findings suggest that the effects of resveratrol on keratinocyte senescence and proliferation are regulated by

  17. Nicotinamide enhances repair of ultraviolet radiation-induced DNA damage in human keratinocytes and ex vivo skin.

    Science.gov (United States)

    Surjana, Devita; Halliday, Gary M; Damian, Diona L

    2013-05-01

    Nicotinamide (vitamin B3) protects from ultraviolet (UV) radiation-induced carcinogenesis in mice and from UV-induced immunosuppression in mice and humans. Recent double-blinded randomized controlled Phase 2 studies in heavily sun-damaged individuals have shown that oral nicotinamide significantly reduces premalignant actinic keratoses, and may reduce new non-melanoma skin cancers. Nicotinamide is a precursor of nicotinamide adenine dinucleotide (NAD(+)), an essential coenzyme in adenosine triphosphate (ATP) production. Previously, we showed that nicotinamide prevents UV-induced ATP decline in HaCaT keratinocytes. Energy-dependent DNA repair is a key determinant of cellular survival after exposure to DNA-damaging agents such as UV radiation. Hence, in this study we investigated whether nicotinamide protection from cellular energy loss influences DNA repair. We treated HaCaT keratinocytes with nicotinamide and exposed them to low-dose solar-simulated UV (ssUV). Excision repair was quantified using an assay of unscheduled DNA synthesis. Nicotinamide increased both the proportion of cells undergoing excision repair and the repair rate in each cell. We then investigated ssUV-induced cyclobutane pyrimidine dimers (CPDs) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8oxoG) formation and repair by comet assay in keratinocytes and with immunohistochemistry in human skin. Nicotinamide reduced CPDs and 8oxoG in both models and the reduction appeared to be due to enhancement of DNA repair. These results show that nicotinamide enhances two different pathways for repair of UV-induced photolesions, supporting nicotinamide's potential as an inexpensive, convenient and non-toxic agent for skin cancer chemoprevention.

  18. Treatment of Hypertrophic Scar in Human with Autologous Transplantation of Cultured Keratinocytes and Fibroblasts along with Fibrin Glue

    Directory of Open Access Journals (Sweden)

    Ehsan Taghiabadi

    2015-04-01

    Full Text Available Objective: Hypertrophic scar involves excessive amounts of collagen in dermal layer and may be painful. Nowadays, we can’t be sure about effectiveness of procedure for hypertrophic scar management. The application of stem cells with natural scaffold has been the best option for treatment of burn wounds and skin defect, in recent decades. Fibrin glue (FG was among the first of the natural biomaterials applied to enhance skin deformity in burn patients. This study aimed to identify an efficient, minimally invasive and economical transplantation procedure using novel FG from human cord blood for treatment of hypertrophic scar and regulation collagen synthesis. Materials and Methods: In this case series study, eight patients were selected with hypertrophic scar due to full-thickness burns. Human keratinocytes and fibroblasts derived from adult skin donors were isolated and cultured. They were tested for the expression of cytokeratin 14 and vimentin using immunocytochemistry. FG was prepared from pooled cord blood. Hypertrophic scars were extensively excised then grafted by simply placing the sheet of FG containing autologous fibroblast and keratinocytes. Histological analyses were performed using Hematoxylin and eosin (H&E and Masson’s Trichrome (MT staining of the biopsies after 8 weeks. Results: Cultured keratinocytes showed a high level of cytokeratin 14 expression and also fibroblasts showed a high level of vimentin. Histological analyses of skin biopsies after 8 weeks of transplantation revealed re-epithelialization with reduction of hypertrophic scars in 2 patients. Conclusion: These results suggest may be the use of FG from cord blood, which is not more efficient than previous biological transporters and increasing hypertrophic scar relapse, but could lead to decrease pain rate.

  19. Subcellular Raman Microspectroscopy Imaging of Nucleic Acids and Tryptophan for Distinction of Normal Human Skin Cells and Tumorigenic Keratinocytes.

    Science.gov (United States)

    Piredda, Paola; Berning, Manuel; Boukamp, Petra; Volkmer, Andreas

    2015-07-07

    At present, tumor diagnostic imaging is commonly based on hematoxylin and eosin or immunohistochemical staining of biopsies, which requires tissue excision, fixation, and staining with exogenous marker molecules. Here, we report on label-free tumor imaging using confocal spontaneous Raman scattering microspectroscopy, which exploits the intrinsic vibrational contrast of endogenous biomolecular species. We present a chemically specific and quantitative approach to monitoring normal human skin cells (keratinocytes and fibroblasts) as well as the human HaCaT in vitro skin carcinogenesis model and the tumor-derived MET in vivo skin cancer progression model. Mapping the amplitudes of two spectrally well isolated Raman bands at 752 and 785 cm(-1) allowed for direct visualization of the distributions representative of tryptophan-rich proteins and nucleic acids, respectively, with subcellular spatial resolution. Using these Raman markers, it was feasible to discriminate between normal human epidermal keratinocytes (NHEK) and dermal fibroblasts (NHDF) and to confine all tumorigenic cells from both the NHEK and NHDF. First evidence for the successful application of the proposed intracellular nucleic acid and tryptophan Raman signatures for skin cancer diagnosis was further demonstrated in an organotypic cutaneous squamous cell carcinomas model, allowing for the identification of tumor cells and their surrounding stroma in the tissue context.

  20. The effect of 648 nm diode laser irradiation on second messengers in senescent human keratinocytes

    Science.gov (United States)

    Hawkins Evans, D.; Abrahamse, H.

    2009-02-01

    Background/purpose: Stress induced premature senescence (SIPS) is defined as the long-term effect of subcytotoxic stress on proliferative cell types. Cells in SIPS display differences at the level of protein expression which affect energy metabolism, defense systems, redox potential, cell morphology and transduction pathways. This study aimed to determine the effect of laser irradiation on second messengers in senescent cells and to establish if that effect can be directly linked to changes in cellular function such as cell viability or proliferation. Materials and Methods: Human keratinocyte cell cultures were modified to induce premature senescence using repeated sub-lethal stresses of 200 uM H2O2 or 5% OH every day for four days with two days recovery. SIPS was confirmed by senescence-associated β-galactosidase staining. Control conditions included normal, repeated stress of 500 uM H2O2 to induce apoptosis and 200 uM PBN as an anti-oxidant or free radical scavenger. Cells were irradiated with 1.5 J/cm2 on day 1 and 4 using a 648 nm diode laser (3.3 mW/cm2) and cellular responses were measured 1 h post irradiation. The affect on second messengers was assessed by measuring cAMP, cGMP, nitric oxide and intracellular calcium (Ca2+) while functional changes were assessed using cell morphology, ATP cell viability, LDH membrane integrity and WST-1 cell proliferation. Results: Results indicate an increase in NO and a decrease in cGMP and Ca2+ in 200 uM H2O2 irradiated cells while PBN irradiated cells showed a decrease in cAMP and an increase in ATP viability and cell proliferation. Conclusion: Laser irradiation influences cell signaling which ultimately changes the biological function of senescent cells. If laser therapy can stimulate the biological function of senescent cells it may be beneficial to conditions such as immune senescence, skin ageing, muscle atrophy, premature ageing of arteries in patients with advanced heart disease, neurodegenerative disorders and

  1. Roughness threshold for cell attachment and proliferation on plasma micro-nanotextured polymeric surfaces: the case of primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts

    Science.gov (United States)

    Bourkoula, A.; Constantoudis, V.; Kontziampasis, D.; Petrou, P. S.; Kakabakos, S. E.; Tserepi, A.; Gogolides, E.

    2016-08-01

    Poly(methyl methacrylate) surfaces have been micro-nanotextured in oxygen plasmas with increasing ion energy, leading to micro-nanotopography characterized by increased root mean square roughness, correlation length and fractal dimension. Primary human skin fibroblasts and mouse immortalized 3T3 fibroblasts were cultured on these surfaces and the number of adhering cells, their proliferation rate and morphology (cytoplasm and nucleus area) were evaluated as a function of roughness height, correlation length, and fractal dimension. A roughness threshold behavior was observed for both types of cells leading to dramatic cell number decrease above this threshold, which is almost similar for the two types of cells, despite their differences in size and stiffness. The results are discussed based on two theoretical models, which are reconciled and unified when the elastic moduli and the size of the cells are taken into account.

  2. Amygdalin analogues inhibit IFN-γ signalling and reduce the inflammatory response in human epidermal keratinocytes.

    Science.gov (United States)

    Paoletti, Iole; De Gregorio, Vincenza; Baroni, Adone; Tufano, Maria Antonietta; Donnarumma, Giovanna; Perez, Juan Jesus

    2013-12-01

    Peptide T (PT), an octapeptide fragment located in the V2 region of the HIV-1 gp120-coating protein, appears to be beneficial in the treatment of psoriasis. Our previous investigations suggest that keratinocytes play a key role in conditioning the therapeutic effects of PT in psoriasis. The aim of this study was to explore the effects of PT and the peptidomimetic natural products, Dhurrin and Prunasin, on the expression of the IL-6, IL-8, IL-23, HSP70 and ICAM-1 on IFN-γ and TNF-α-NHEK activated cells. Moreover, we analysed the interference of PT and its analogues through STAT-3 activation. Our results show that the analogues tested exhibit the beneficial biological effects of PT, suggesting the primary role of keratinocytes upon which PT and the peptidomimetics act directly, by reducing proinflammatory responses. Its reduction appears to be important for therapeutic approach in psoriasis pathogenesis.

  3. Science, self, and immortality.

    Science.gov (United States)

    Haught, John F

    2011-10-01

    The following considers the concept of scientific naturalism in relation to life after death and contrasts three alternative perspectives on immortality of the soul, including naturalistic fatalism, otherworldly optimism, and long-suffering hope. © 2011 New York Academy of Sciences.

  4. Functional pharmacological evidence for EP2 and EP4 prostanoid receptors in immortalized human trabecular meshwork and non-pigmented ciliary epithelial cells.

    Science.gov (United States)

    Crider, J Y; Sharif, N A

    2001-02-01

    The aim of these studies was to characterize the molecular pharmacology of the prostanoid receptors positively coupled to stimulation of adenylyl cyclase activity in immortalized human trabecular meshwork (TM-3) cells and to compare these results with that of the receptors in immortalized human nonpigmented epithelial (NPE) cells. In general, the TM-3 and NPE cells showed a similar profile with respect to their responses to various prostaglandin (PG) receptor agonists. The rank order of potency (EC50; means +/- SEM) for these compounds in the TM-3 cells was: PGE2 (124 +/- 21 nM) > 13,14-dihydro-PGE1 (430 +/- 110 nM) = PGE1 (522 +/- 345 nM) > 11-deoxy-PGE1 (1063 +/- 118 nM) = 16,16-dimethyl-PGE2 (1776 +/- 460 nM) = butaprost (1920 +/- 527 nM) > PGD2 = PGI2 = PGF2alpha (n = 3 - 12). While the agonist profile indicated the presence of EP2 receptors, the effects of the EP4 receptor antagonists suggested the additional expression of EP4 receptors in both of these cells. Thus, the EP4 receptor antagonist, AH23848B, at a concentration of 30 microM, caused a dextral shift in the PGE2 concentration-response curves in both TM-3 and NPE cells coupled with a 20-28% decrease in the maximal response of PGE2, indicating apparent noncompetitive antagonism profiles. The antagonist potency of AH23848B in these cells was: Kb = 38.4 +/- 14.8 microM and 23.5 +/- 4.5 microM; -log Kb = 4.7. The other EP4 receptor antagonist, AH22921 (-log Kb = 4.1 - 4.7), was weaker than AH23848B. Taken together, these pharmacological studies have shown than TM-3 and NPE cells apparently contain functional EP2 and EP4 prostanoid receptors positively coupled to adenylyl cyclase.

  5. Adiponectin Suppresses UVB-Induced Premature Senescence and hBD2 Overexpression in Human Keratinocytes.

    Science.gov (United States)

    Kim, MinJeong; Park, Kui Young; Lee, Mi-Kyung; Jin, Taewon; Seo, Seong Jun

    2016-01-01

    Recent studies have revealed that adiponectin can suppress cellular inflammatory signaling pathways. This study aimed to elucidate the effect of adiponectin on the unregulated production of hBD2 in UVB-induced premature senescent keratinocytes. We constructed an in vitro model of premature senescent keratinocytes through repeated exposure to low energy UVB. After repeated low energy UVB exposure, there was significant generation of reactive oxygen species (ROS) and induction of senescence-associated markers, including senescence associated beta-galactosidase activity and expression of p16INK4a and histone H2AX. In addition, the present clinical study showed higher expression of hBD2 in sun-exposed skin of elderly group, and the overexpression of hBD2 was observed by c-Fos activation in vitro. Adiponectin has the ability to scavenge ROS and consequently inhibit MAPKs and SA-markers in UVB-exposed keratinocytes. An inhibitor study demonstrated that adiponectin downregulated hBD2 mRNA expression through suppression of the AP-1 transcription factor components c-Fos via inactivation of p38 MAPK. Collectively, the dysregulated production of hBD2 by the induction of oxidative stress was attenuated by adiponectin through the suppression of p38 and JNK/SAPK MAPK signaling in UVB-mediated premature senescent inducible conditions. These results suggest the feasibility of adiponectin as an anti-photoaging and anti-inflammatory agent in the skin.

  6. Response of Primary Human Bone Marrow Mesenchymal Stromal Cells and Dermal Keratinocytes to Thermal Printer Materials In Vitro.

    Science.gov (United States)

    Schmelzer, Eva; Over, Patrick; Gridelli, Bruno; Gerlach, Jörg C

    Advancement in thermal three-dimensional printing techniques has greatly increased the possible applications of various materials in medical applications and tissue engineering. Yet, potential toxic effects on primary human cells have been rarely investigated. Therefore, we compared four materials commonly used in thermal printing for bioengineering, namely thermally printed acrylonitrile butadiene styrene, MED610, polycarbonate, and polylactic acid, and investigated their effects on primary human adult skin epidermal keratinocytes and bone marrow mesenchymal stromal cells (BM-MSCs) in vitro. We investigated indirect effects on both cell types caused by potential liberation of soluble substances from the materials, and also analyzed BM-MSCs in direct contact with the materials. We found that even in culture without direct contact with the materials, the culture with MED610 (and to a lesser extent acrylonitrile butadiene styrene) significantly affected keratinocytes, reducing cell numbers and proliferation marker Ki67 expression, and increasing glucose consumption, lactate secretion, and expression of differentiation-associated genes. BM-MSCs had decreased metabolic activity, and exhibited increased cell death in direct culture on the materials. MED610 and acrylonitrile butadiene styrene induced the strongest expression of genes associated to differentiation and estrogen receptor activation. In conclusion, we found strong cell-type-specific effects of the materials, suggesting that materials for applications in regenerative medicine should be carefully selected not only based on their mechanical properties but also based on their cell-type-specific biological effects.

  7. Metabolic activation of pyrrolizidine alkaloids leading to phototoxicity and photogenotoxicity in human HaCaT keratinocytes.

    Science.gov (United States)

    Wang, Chia-Chi; Xia, Qingsu; Li, Meng; Wang, Shuguang; Zhao, Yuewei; Tolleson, William H; Yin, Jun-Jie; Fu, Peter P

    2014-01-01

    Pyrrolizidine alkaloids, produced by a large number of poisonous plants with wide global distribution, are associated with genotoxicity, tumorigenicity, and hepatotoxicity in animals and humans. Mammalian metabolism converts pyrrolizidine alkaloids to reactive pyrrolic metabolites (dehydropyrrolizidine alkaloids) that form covalent protein and DNA adducts. Although a mechanistic understanding is currently unclear, pyrrolizidine alkaloids can cause secondary (hepatogenous) photosensitization and induce skin cancer. In this study, the phototoxicity of monocrotaline, riddelliine, dehydromonocrotaline, dehydroriddelliine, and dehydroretronecine (DHR) in human HaCaT keratinocytes under ultraviolet A (UVA) irradiation was determined. UVA irradiation of HaCaT cells treated with dehydromonocrotaline, dehydroriddelline, and DHR resulted in increased release of lactate dehydrogenase and enhanced photocytotoxicity proportional to the UVA doses. UVA-induced photochemical DNA damage also increased proportionally with dehydromonocrotaline and dehydroriddelline. UVA treatment potentiated the formation of 8-hydroxy-2'-deoxyguanosine DNA adducts induced by dehydromonocrotaline in HaCaT skin keratinocytes. Using electron spin resistance trapping, we found that UVA irradiation of dehydromonocrotaline and dehydroriddelliine generates reactive oxygen species (ROS), including hydroxyl radical, singlet oxygen, and superoxide, and electron transfer reactions, indicating that cytotoxicity and genotoxicity of these compounds could be mediated by ROS. Our results suggest that dehydropyrrolizidine alkaloids formed or delivered to the skin cause pyrrolizidine alkaloid-induced secondary photosensitization and possible skin cancer.

  8. Human papillomavirus types detected in skin warts and cancer differ in their transforming properties but commonly counteract UVB induced protective responses in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Shterzer, Naama; Heyman, Dariya; Shapiro, Beny; Yaniv, Abraham; Jackman, Anna [Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Serour, Francis [Department of Pediatric Surgery, The E. Wolfson Medical Center, Holon (Israel); Chaouat, Malka [Laboratory of Experimental Surgery, Hadassah University Hospital, Ein Karem, Jerusalem (Israel); Gonen, Pinhas [Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv (Israel); Tommasino, Massimo [International Agency for Research on Cancer, World Health Organization, Lyon (France); Sherman, Levana [Department of Clinical Microbiology and Immunology, Sackler School of Medicine, Tel-Aviv University, Tel-Aviv (Israel)

    2014-11-15

    In the present study, E6E7 and E6 proteins of human papillomaviruses (HPVs) associated with skin warts and cancer were compared for their transforming and carcinogenic abilities in primary human keratinocytes (PHKs). We show that E6E7 of cancer associated beta HPV types, notably 49 and 24, were able to extend the life span and enhance the clonogenic efficiency of PHKs when maintained in serum free/low calcium medium. Activities of the beta HPV E6E7 were lower than those of HPV16 E6E7. In contrast, E6 proteins from HPV types detected in skin warts or cancer, notably 10, 49 and 38, attenuated UVB induced protective responses in PHKs including cell death, proliferation arrest and accumulation of the proapoptotic proteins, p53, bax or bak. Together, this investigation revealed functional differences and commonalities between HPVs associated with skin warts and cancer, and allowed the identification of specific properties of beta HPVs supporting their involvement in skin carcinogenesis. - Highlights: • Primary keratinocytes were used to evaluate transforming and carcinogenic abilities of cutaneous HPVs. • E6E7 of cancer associated β HPV types transform primary human keratinocytes. • E6 proteins of cancer and wart associated HPVs inhibit UVB induced cell death. • E6s of cancer and wart associated HPVs attenuate UVB induced proliferation arrest. • E6s of cancer and wart associated HPVs attenuate UVB induced apoptosis signaling.

  9. Conditionally immortalized human proximal tubular epithelial cells isolated from the urine of a healthy subject express functional calcium-sensing receptor.

    Science.gov (United States)

    Di Mise, Annarita; Tamma, Grazia; Ranieri, Marianna; Svelto, Maria; Heuvel, Bert van den; Levtchenko, Elena N; Valenti, Giovanna

    2015-06-01

    The calcium-sensing receptor (CaSR) is a G protein-coupled receptor, which plays an essential role in regulating Ca(2+) homeostasis. Here we show that conditionally immortalized proximal tubular epithelial cell line (ciPTEC) obtained by immortalizing and subcloning cells exfoliated in the urine of a healthy subject expresses functional endogenous CaSR. Immunolocalization studies of polarized ciPTEC revealed the apical localization of the receptor. By Western blotting of ciPTEC lysates, both monomeric and dimeric forms of CaSR at 130 and ∼250 kDa, respectively, were detected. Functional studies indicated that both external calcium and the positive CaSR allosteric modulator, NPS-R568, induced a significant increase in cytosolic calcium, proving a high sensitivity of the endogenous receptor to its agonists. Calcium depletion from the endoplasmic reticulum using cyclopiazonic acid abolished the increase in cytosolic calcium elicited by NPS-R568, confirming calcium exit from intracellular stores. Activation of CaSR by NPS-R significantly reduced the increase in cAMP elicited by forskolin (FK), a direct activator of adenylate cyclase, further confirming the functional expression of the receptor in this cell line. CaSR expressed in ciPTEC was found to interact with Gq as a downstream effector, which in turn can cause release of calcium from intracellular stores via phospholipase C activation. We conclude that human proximal tubular ciPTEC express functional CaSR and respond to its activation with a release of calcium from intracellular stores. These cell lines represent a valuable tool for research into the disorder associated with gain or loss of function of the CaSR by producing cell lines from patients. Copyright © 2015 the American Physiological Society.

  10. Anandamide Suppresses Proinflammatory T Cell Responses In Vitro through Type-1 Cannabinoid Receptor-Mediated mTOR Inhibition in Human Keratinocytes.

    Science.gov (United States)

    Chiurchiù, Valerio; Rapino, Cinzia; Talamonti, Emanuela; Leuti, Alessandro; Lanuti, Mirko; Gueniche, Audrey; Jourdain, Roland; Breton, Lionel; Maccarrone, Mauro

    2016-11-01

    The endocannabinoid system comprises cannabinoid receptors 1 and 2 (CB1 and CB2), their endogenous ligands, anandamide (AEA) and 2-arachidonoylglycerol, and metabolic enzymes of these ligands. The endocannabinoid system has recently been implicated in the regulation of various pathophysiological processes of the skin that include immune competence and/or tolerance of keratinocytes, the disruption of which might promote the development of skin diseases. Recent evidence showed that CB1 in keratinocytes limits the secretion of proinflammatory chemokines, suggesting that this receptor might also regulate T cell dependent inflammatory diseases of the skin. In this article, we sought to investigate the cytokine profile of IFN-γ-activated keratinocytes, and found that CB1 activation by AEA suppressed production and release of signature TH1- and TH17-polarizing cytokines, IL-12 and IL-23, respectively. We also set up cocultures between a conditioned medium of treated keratinocytes and naive T cells to disclose the molecular details that regulate the activation of highly proinflammatory TH1 and TH17 cells. AEA-treated keratinocytes showed reduced an induction of IFN-γ-producing TH1 and IL-17-producing TH17 cells, and these effects were reverted by pharmacological inhibition of CB1 Further analyses identified mammalian target of rapamycin as a proinflammatory signaling pathway regulated by CB1, able to promote either IL-12 and IL-23 release from keratinocytes or TH1 and TH17 polarization. Taken together, these findings demonstrate that AEA suppresses highly pathogenic T cell subsets through CB1-mediated mammalian target of rapamycin inhibition in human keratinocytes. Thus, it can be speculated that the latter pathway might be beneficial to the physiological function of the skin, and can be targeted toward inflammation-related skin diseases. Copyright © 2016 by The American Association of Immunologists, Inc.

  11. Human papillomavirus type 16 E6 and E 7 proteins alter NF-kB in cultured cervical epithelial cells and inhibition of NF-kB promotes cell growth and immortalization

    Energy Technology Data Exchange (ETDEWEB)

    Vandermark, Erik R.; Deluca, Krysta A.; Gardner, Courtney R.; Marker, Daniel F.; Schreiner, Cynthia N.; Strickland, David A.; Wilton, Katelynn M. [Department of Biology, Clarkson University, Potsdam, NY 13699-5805 (United States); Mondal, Sumona [Department of Mathematics, Clarkson University, Potsdam, NY 13699-5805 (United States); Woodworth, Craig D., E-mail: woodworth@clarkson.edu [Department of Biology, Clarkson University, Potsdam, NY 13699-5805 (United States)

    2012-03-30

    The NF-kB family of transcription factors regulates important biological functions including cell growth, survival and the immune response. We found that Human Papillomavirus type 16 (HPV-16) E7 and E6/E7 proteins inhibited basal and TNF-alpha-inducible NF-kB activity in human epithelial cells cultured from the cervical transformation zone, the anatomic region where most cervical cancers develop. In contrast, HPV-16 E6 regulated NF-kB in a cell type- and cell growth-dependent manner. NF-kB influenced immortalization of cervical cells by HPV16. Inhibition of NF-kB by an IkB alpha repressor mutant increased colony formation and immortalization by HPV-16. In contrast, activation of NF-kB by constitutive expression of p65 inhibited proliferation and immortalization. Our results suggest that inhibition of NF-kB by HPV-16 E6/E7 contributes to immortalization of cells from the cervical transformation zone.

  12. Universal mortality law and immortality

    Science.gov (United States)

    Azbel', Mark Ya.

    2004-10-01

    Well-protected human and laboratory animal populations with abundant resources are evolutionarily unprecedented. Physical approach, which takes advantage of their extensively quantified mortality, establishes that its dominant fraction yields the exact law, which is universal for all animals from yeast to humans. Singularities of the law demonstrate new kinds of stepwise adaptation. The law proves that universal mortality is an evolutionary by-product, which at any given age is reversible, independent of previous life history, and disposable. Life expectancy may be extended, arguably to immortality, by minor biological amendments in the animals. Indeed, in nematodes with a small number of perturbed genes and tissues it increased 6-fold (to 430 years in human terms), with no apparent loss in health and vitality. The law relates universal mortality to specific processes in cells and their genetic regulation.

  13. Angiogenic peptide (AG)-30/5C activates human keratinocytes to produce cytokines/chemokines and to migrate and proliferate via MrgX receptors.

    Science.gov (United States)

    Kiatsurayanon, Chanisa; Niyonsaba, François; Chieosilapatham, Panjit; Okumura, Ko; Ikeda, Shigaku; Ogawa, Hideoki

    2016-09-01

    In addition to their antimicrobial activities, antimicrobial peptides, also known as host defense peptides (HDPs) activate keratinocytes; promote wound healing; and improve the skin barrier. AG-30/5C is a novel angiogenic HDP that activates various functions of fibroblasts and endothelial cells, including cytokine/chemokine production and wound healing. To investigate whether AG-30/5C activates human keratinocytes and to examine the underlying mechanisms. Production of cytokines/chemokines was assessed by ELISA. Expression of Mas-related G-protein coupled receptors X (MrgXs) in keratinocytes was determined by real-time PCR and Western blot. MAPK and NF-κB activation was analysed by Western blot. Cell migration was assessed by chemotaxis microchamber and in vitro wound closure assay, whereas cell proliferation was analysed using an XTT assay. We found that AG-30/5C was more efficient than its parent peptide AG-30 in increasing the production of various cytokines/chemokines and promoting keratinocyte migration and proliferation. Furthermore, MrgX3 and MrgX4 receptors were constitutively expressed in keratinocytes at higher levels than MrgX1 and MrgX2, and were up-regulated upon stimulation with TLR ligands. Because MrgX3 and MrgX4 siRNAs suppressed AG-30/5C-mediated cytokine/chemokine production, keratinocyte migration and proliferation, we propose that AG-30/5C utilizes these MrgXs to stimulate keratinocytes. In addition, AG-30/5C-induced activation of keratinocytes was controlled by MAPK and NF-κB pathways, as evidenced by the inhibitory effects of ERK-, JNK-, p38- and NF-κB-specific inhibitors. Indeed, we confirmed that AG-30/5C enhanced phosphorylation of MAPKs and IκB. Our findings provide novel evidence that AG-30/5C may be a useful therapeutic agent for wound healing by activating human keratinocytes. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

  14. Suppression of Ultraviolet B Exposure-Mediated Activation of NF-κB in Normal Human Keratinocytes by Resveratrol

    Directory of Open Access Journals (Sweden)

    Vaqar Mustafa Adhami

    2003-01-01

    Full Text Available Chemoprevention by naturally occurring agents is a newer dimension in the management of neoplasia, including skin cancer. Solar ultraviolet (UV radiation is the major cause of skin cancer. We recently demonstrated that resveratrol (3,5,4'-trihydroxystilbene, a polyphenolic antioxidant found in grapes and red wine, imparts protection from UVB-mediated cutaneous damages in SKH-1 hairless mice. The mechanism of action of resveratrol is not clearly understood. Here, we investigated the involvement of nuclear factor kappa B (NF-κB, which is known to play a critical role in skin biology and the development of skin cancer, as the mechanism of chemoprevention of UV damage by resveratrol. In the normal human epidermal keratinocytes, resveratrol blocked UVB-mediated (40 mJ/cm2 activation of NF-κB in a dose-dependent (5, 10, and 25μM resveratrol for 24 hours as well as time-dependent (5μ/M resveratrol for 12, 24, and 48 hours fashion. Resveratrol treatment of keratinocytes also inhibited UVB-mediated 1 phosphorylation and degradation of IκBα, and 2 activation of IKKα. We suggest that NF-κB pathway plays a critical role in the chemopreventive effects of resveratrol against the adverse effects of UV radiation including photocarcinogenesis.

  15. Silver nanoparticles exert a long-lasting antiproliferative effect on human keratinocyte HaCaT cell line.

    Science.gov (United States)

    Zanette, Caterina; Pelin, Marco; Crosera, Matteo; Adami, Gianpiero; Bovenzi, Massimo; Larese, Francesca Filon; Florio, Chiara

    2011-08-01

    For their antibacterial activity, silver nanoparticles (Ag NPs) are largely used in various commercially available products designed to come in direct contact with the skin. In this study we investigated the effects of Ag NPs on skin using the human-derived keratinocyte HaCaT cell line model. Ag NPs caused a concentration- and time-dependent decrease of cell viability, with IC(50) values of 6.8 ± 1.3 μM (MTT assay) and 12 ± 1.2 μM (SRB assay) after 7 days of contact. A 24h treatment, followed by a 6 day recovery period in Ag NPs-free medium, reduced cell viability with almost the same potency (IC(50)s of 15.3 ± 4.6 and 35 ± 20 μM, MTT and SRB assays, respectively). Under these conditions, no evidence of induction of necrotic events (propidium iodide assay) was found. Apocynin, NADPH-oxidase inhibitor, or N(G)-monomethyl-L-argynine, nitric oxide synthase inhibitor, did not prevent NPs-induced reduction of cell viability. TEM analysis of cells exposed to NPs for 24h revealed alteration of nuclear morphology but only a marginal presence of individual NPs inside the cells. These results demonstrate that on HaCaT keratinocytes a relatively short time of contact with Ag NPs causes a long-lasting inhibition of cell growth, not associated with consistent Ag NPs internalization.

  16. Regulation of haptoglobin expression in a human keratinocyte cell line HaCaT by inflammatory cytokines and dexamethasone

    Institute of Scientific and Technical Information of China (English)

    XIA Li-xin; XIAO Ting; CHEN Hong-duo; LI Ping; WANG Ya-kun; WANG He

    2008-01-01

    Background Haptoglobin(Hp)is one of the acute-phase proteins. Recent studies have demonstrated that Hp exerts immunoregulatory and anti-inflammatory actions and may be one of the inhibitory factors of immune reactions in the skin. In this study we investigated the regulation of Hp expression in a human keratinocyte cell line HaCaT by various cytokines and glucocorticod. Methods HaCaT cells were cultured with IL-6(50 ng/ml), TNF-α(20 ng/ml), IFN-Y(20 ng/ml)or IL-4(20 ng/ml)with or without 1 μmol/L dexamethasone in 6-well plates for 12, 24 and 48 hours. Both the cells and the supernatants were collected to detect the changes of Hp expression by reverse-transcription PCR, ELISA and immunohistochemistry. Results The results showed that Hp expression were elevated at both the mRNA and protein level by the combination of IL-6, TNF-α or IL-4 with dexamethasone, whereas the three cytokines alone did not upregulate the Hp expression. IFN-Y showed no effect on the Hp expression in HaCaT cells. Conclusions These findings suggest that different inflammatory cytokines as well as glucocorticoid may be involved in the regulation of Hp expression in keratinocytes, and this may be one of the negative feedback mechanisms in inflammatory skin diseases.

  17. Phospholipase C-gamma1 is required for subculture-induced terminal differentiation of normal human oral keratinocytes.

    Science.gov (United States)

    Oh, Ju-Eun; Kook, Joong-Ki; Park, Kyung-Hee; Lee, Gene; Seo, Byoung-Moo; Min, Byung-Moo

    2003-04-01

    Serial subculture of primary normal human oral keratinocytes (NHOKs) to the post-mitotic stage induces terminal differentiation, which is in part linked to elevated levels of phospholipase C (PLC)-gamma1. Therefore, PLC-gamma1 may be involved in the signal transduction system that leads to the calcium regulation of subculture-induced keratinocyte differentiation. To test this hypothesis, the expression of PLC-gamma1 in primary NHOKs was blocked by transfecting cells with the antisense PLC-gamma1 cDNA construct. These cells demonstrated dramatic reductions in PLC-gamma1 protein and in the differentiation markers involucrin and transglutaminase following calcium exposure and an increase (15-20%) in in vitro life span versus empty vector-transfected cells. In addition, we established the ability of antisense PLC-gamma1 to block the serial subculture-induced rise in intracellular calcium. Similar observations were made following treatment with the specific PLC inhibitor U73122. These results indicate that the terminal differentiation of NHOKs by serial subculture is associated with PLC-gamma1, which mediates calcium regulation by mobilizing intracellular calcium.

  18. IFI16 and cGAS cooperate in the activation of STING during DNA sensing in human keratinocytes.

    Science.gov (United States)

    Almine, Jessica F; O'Hare, Craig A J; Dunphy, Gillian; Haga, Ismar R; Naik, Rangeetha J; Atrih, Abdelmadjid; Connolly, Dympna J; Taylor, Jordan; Kelsall, Ian R; Bowie, Andrew G; Beard, Philippa M; Unterholzner, Leonie

    2017-02-13

    Many human cells can sense the presence of exogenous DNA during infection though the cytosolic DNA receptor cyclic GMP-AMP synthase (cGAS), which produces the second messenger cyclic GMP-AMP (cGAMP). Other putative DNA receptors have been described, but whether their functions are redundant, tissue-specific or integrated in the cGAS-cGAMP pathway is unclear. Here we show that interferon-γ inducible protein 16 (IFI16) cooperates with cGAS during DNA sensing in human keratinocytes, as both cGAS and IFI16 are required for the full activation of an innate immune response to exogenous DNA and DNA viruses. IFI16 is also required for the cGAMP-induced activation of STING, and interacts with STING to promote STING phosphorylation and translocation. We propose that the two DNA sensors IFI16 and cGAS cooperate to prevent the spurious activation of the type I interferon response.

  19. Zinc oxide nanoparticles-induced epigenetic change and G2/M arrest are associated with apoptosis in human epidermal keratinocytes

    Directory of Open Access Journals (Sweden)

    Gao F

    2016-08-01

    Full Text Available Fei Gao, Ningjie Ma, Hong Zhou, Qing Wang, Hao Zhang, Pu Wang, Haoli Hou, Huan Wen, Lijia Li State Key Laboratory of Hybrid Rice, College of Life Sciences, Wuhan University, Wuhan, People’s Republic of China Abstract: As an engineered nanomaterial, zinc oxide nanoparticles (ZnO NPs are used frequently in biological applications and can make contact with human skin. Here, we systematically investigated the effects of ZnO NPs on non-tumorigenic human epidermal keratinocytes, which were used as a test model for this in vitro study, at the epigenetic and molecular levels. Our results showed that ZnO NPs induced cell cycle arrest at the G2/M checkpoint before the viability of human epidermal keratinocytes was reduced, which was associated with the chromatin changes at the epigenetic level, including increased methylation of histone H3K9 and decreased acetylation of histone H4K5 accompanied by chromatin condensation at 24 hours. The mRNA expression of the methyltransferase genes G9a and GLP was also increased upon treatment with ZnO NPs, and the acetyltransferase genes GCN5, P300, and CBP were downregulated. Reactive oxygen species were found to be more abundant after treatment with ZnO NPs for 6 hours, and DNA damage was observed at 24 hours. Transmission electron microscopy and flow cytometry confirmed that ZnO NPs were absorbed into the cell when they were added to the medium. Apoptotic human epidermal keratinocytes were detected, and the expression of the proapoptotic genes Bax, Noxa, and Puma increased significantly, while the expression of the antiapoptotic gene Bcl-xl decreased 24 hours after exposure to ZnO NPs. These findings suggest that the ZnO NPs induced cell cycle arrest at G2/M, which was associated with epigenetic changes and accompanied by p53-Bax mitochondrial pathway-mediated apoptosis. Keywords: ZnO nanoparticle, cell cycle G2/M arrest, histone modification, p53-Bax mitochondrial apoptosis pathway, reactive oxygen species

  20. Rho kinase inhibitor y-27632 prolongs the life span of adult human keratinocytes, enhances skin equivalent development, and facilitates lentiviral transduction.

    NARCIS (Netherlands)

    Bogaard, E.H.J. van den; Rodijk-Olthuis, D.; Jansen, P.A.M.; Vlijmen-Willems, I.M.J.J. van; Erp, P.E.J. van; Joosten, I.; Zeeuwen, P.L.J.M.; Schalkwijk, J.

    2012-01-01

    The use of tissue-engineered human skin equivalents (HSE) for fundamental research and industrial application requires the expansion of keratinocytes from a limited number of skin biopsies donated by adult healthy volunteers or patients. A pharmacological inhibitor of Rho-associated protein kinases,

  1. Symbiotic Plant Peptides Eliminate Candida albicans Both In Vitro and in an Epithelial Infection Model and Inhibit the Proliferation of Immortalized Human Cells

    Directory of Open Access Journals (Sweden)

    Lilla Ördögh

    2014-01-01

    Full Text Available The increasing number of multidrug-resistant microbes now emerging necessitates the identification of novel antimicrobial agents. Plants produce a great variety of antimicrobial peptides including hundreds of small, nodule-specific cysteine-rich NCR peptides that, in the legume Medicago truncatula, govern the differentiation of endosymbiotic nitrogen fixing bacteria and, in vitro, can display potent antibacterial activities. In this study, the potential candidacidal activity of 19 NCR peptides was investigated. Cationic NCR peptides having an isoelectric point above 9 were efficient in killing Candida albicans, one of the most common fungal pathogens of humans. None of the tested NCR peptides were toxic for immortalized human epithelial cells at concentrations that effectively killed the fungus; however, at higher concentrations, some of them inhibited the division of the cells. Furthermore, the cationic peptides successfully inhibited C. albicans induced human epithelial cell death in an in vitro coculture model. These results highlight the therapeutic potential of cationic NCR peptides in the treatment of candidiasis.

  2. The Alteration of the Epidermal Basement Membrane Complex of Human Nevus Tissue and Keratinocyte Attachment after High Hydrostatic Pressurization

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    Naoki Morimoto

    2016-01-01

    Full Text Available We previously reported that human nevus tissue was inactivated after high hydrostatic pressure (HHP higher than 200 MPa and that human cultured epidermis (hCE engrafted on the pressurized nevus at 200 MPa but not at 1000 MPa. In this study, we explore the changes to the epidermal basement membrane in detail and elucidate the cause of the difference in hCE engraftment. Nevus specimens of 8 mm in diameter were divided into five groups (control and 100, 200, 500, and 1000 MPa. Immediately after HHP, immunohistochemical staining was performed to detect the presence of laminin-332 and type VII collagen, and the specimens were observed by transmission electron microscopy (TEM. hCE was placed on the pressurized nevus specimens in the 200, 500, and 1000 MPa groups and implanted into the subcutis of nude mice; the specimens were harvested at 14 days after implantation. Then, human keratinocytes were seeded on the pressurized nevus and the attachment was evaluated. The immunohistochemical staining results revealed that the control and 100 MPa, 200 MPa, and 500 MPa groups were positive for type VII collagen and laminin-332 immediately after HHP. TEM showed that, in all of the groups, the lamina densa existed; however, anchoring fibrils were not clearly observed in the 500 or 1000 MPa groups. Although the hCE took in the 200 and 500 MPa groups, keratinocyte attachment was only confirmed in the 200 MPa group. This result indicates that HHP at 200 MPa is preferable for inactivating nevus tissue to allow its reuse for skin reconstruction in the clinical setting.

  3. Induction of interleukin-6 production by ultraviolet radiation in normal human epidermal keratinocytes and in a human keratinocyte cell line is mediated by DNA damage

    NARCIS (Netherlands)

    Petit-Frère, C.; Clingen, P.H.; Grewe, M.; Krutmann, J.; Roza, L.; Arlett, C.F.; Green, M.H.L.

    1998-01-01

    The sunburn reaction is the most common consequence of human exposure to ultraviolet radiation (UVR), and is mediated at least in part by interleukin- 6 (IL-6). The aim of this study was to determine if DNA is a major chromophore involved in the induction of IL-6 following UV irradiation of a human

  4. Cdc42 is crucial for the maturation of primordial cell junctions in keratinocytes independent of Rac1

    DEFF Research Database (Denmark)

    Du, Dan; Pedersen, Esben; Wang, Zhipeng

    2008-01-01

    immortalized and primary keratinocytes form only punctate primordial cell contacts in vitro, which cannot mature into belt-like junctions. This defect was independent of enhanced degradation of beta-catenin, but correlated to an impaired activation and localization of aPKCzeta in the Cdc42-null keratinocytes...

  5. Triggering apoptotic death of human epidermal keratinocytes by malic Acid: involvement of endoplasmic reticulum stress- and mitochondria-dependent signaling pathways.

    Science.gov (United States)

    Hsiao, Yu-Ping; Lai, Wan-Wen; Wu, Shi-Bei; Tsai, Chung-Hung; Tang, Sheau-Chung; Chung, Jing-Gung; Yang, Jen-Hung

    2015-01-09

    Malic acid (MA) has been commonly used in cosmetic products, but the safety reports in skin are sparse. To investigate the biological effects of MA in human skin keratinocytes, we investigated the potential cytotoxicity and apoptotic effects of MA in human keratinocyte cell lines (HaCaT). The data showed that MA induced apoptosis based on the observations of DAPI staining, DNA fragmentation, and sub-G1 phase in HaCaT cells and normal human epidermal keratinocytes (NHEKs). Flow cytometric assays also showed that MA increased the production of mitochondrial superoxide (mito-SOX) but decreased the mitochondrial membrane potential. Analysis of bioenergetics function with the XF 24 analyzer Seahorse extracellular flux analyzer demonstrated that oxygen consumption rate (OCR) was significantly decreased whereas extracellular acidification rate (ECAR) was increased in MA-treated keratinocytes. The occurrence of apoptosis was proved by the increased expressions of FasL, Fas, Bax, Bid, caspases-3, -8, -9, cytochrome c, and the declined expressions of Bcl-2, PARP. MA also induced endoplasmic reticulum stress associated protein expression such as GRP78, GADD153, and ATF6α. We demonstrated that MA had anti-proliferative effect in HaCaT cell through the inhibition of cell cycle progression at G0/G1, and the induction of programmed cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways.

  6. Triggering Apoptotic Death of Human Epidermal Keratinocytes by Malic Acid: Involvement of Endoplasmic Reticulum Stress- and Mitochondria-Dependent Signaling Pathways

    Directory of Open Access Journals (Sweden)

    Yu-Ping Hsiao

    2015-01-01

    Full Text Available Malic acid (MA has been commonly used in cosmetic products, but the safety reports in skin are sparse. To investigate the biological effects of MA in human skin keratinocytes, we investigated the potential cytotoxicity and apoptotic effects of MA in human keratinocyte cell lines (HaCaT. The data showed that MA induced apoptosis based on the observations of DAPI staining, DNA fragmentation, and sub-G1 phase in HaCaT cells and normal human epidermal keratinocytes (NHEKs. Flow cytometric assays also showed that MA increased the production of mitochondrial superoxide (mito-SOX but decreased the mitochondrial membrane potential. Analysis of bioenergetics function with the XF 24 analyzer Seahorse extracellular flux analyzer demonstrated that oxygen consumption rate (OCR was significantly decreased whereas extracellular acidification rate (ECAR was increased in MA-treated keratinocytes. The occurrence of apoptosis was proved by the increased expressions of FasL, Fas, Bax, Bid, caspases-3, -8, -9, cytochrome c, and the declined expressions of Bcl-2, PARP. MA also induced endoplasmic reticulum stress associated protein expression such as GRP78, GADD153, and ATF6α. We demonstrated that MA had anti-proliferative effect in HaCaT cell through the inhibition of cell cycle progression at G0/G1, and the induction of programmed cell death through endoplasmic reticulum stress- and mitochondria-dependent pathways.

  7. Protective Effect of Garlic on Cellular Senescence in UVB-Exposed HaCaT Human Keratinocytes.

    Science.gov (United States)

    Kim, Hye Kyung

    2016-07-29

    Ultraviolet (UV) irradiation generates reactive oxygen species (ROS) in the cells, which induces the cellular senescence and photoaging. The present study investigated the protective effects of garlic on photo-damage and cellular senescence in UVB-exposed human keratinocytes, HaCaT cells. An in vitro cell free system was used to examine the scavenging activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) free radicals and nitric oxide (NO). The effect of garlic extract on ROS formation, MMP-1 protein and mRNA expressions, cytokines such as interleukin (IL)-1β and IL-6, senescence associated-β-galactosidase (SA-β-gal) activity, and silent information regulator T1 (SIRT1) activity were determined in UVB-irradiated HaCaT cells. Garlic exhibited strong DPPH radical and NO scavenging activity in cell free system exhibiting IC50 values of 2.50 mg/mL and 4.38 mg/mL, respectively. Garlic pretreatment attenuated the production of UVB-induced intracellular ROS. MMP-1 level, which has been known to be induced by ROS, was dramatically elevated by UVB irradiation, and UVB-induced MMP-1 mRNA and protein expressions were significantly reduced by garlic treatment (50 µg/mL) comparable to those of UV-unexposed control cells. UV-induced pro-inflammatory cytokine productions (IL-6 and IL-1β) were significantly inhibited by pretreatment with garlic in a dose-dependent manner. SA-β-gal activity, a classical biomarker of cellular senescence, and SIRT1 activity, which has attracted attention as an anti-aging factor in recent years, were ameliorated by garlic treatment in UV-irradiated HaCaT cells. The present study provides the first evidence of garlic inhibiting UVB-induced photoaging as a result of augmentation of cellular senescence in HaCaT human keratinocytes.

  8. Tungsten carbide cobalt nanoparticles exert hypoxia-like effects on the gene expression level in human keratinocytes

    Science.gov (United States)

    2010-01-01

    Background Tungsten carbide (WC) and tungsten carbide cobalt (WC-Co) nanoparticles are of occupational health relevance because of the increasing usage in hard metal industries. Earlier studies showed an enhanced toxic potential for WC-Co compared to WC or cobalt ions alone. Therefore, we investigated the impact of these particles, compared to cobalt ions applied as CoCl2, on the global gene expression level in human keratinocytes (HaCaT) in vitro. Results WC nanoparticles exerted very little effects on the transcriptomic level after 3 hours and 3 days of exposure. In contrast, WC-Co nanoparticles caused significant transcriptional changes that were similar to those provoked by CoCl2. However, CoCl2 exerted even more pronounced changes in the transcription patterns. Gene set enrichment analyses revealed that the differentially expressed genes were related to hypoxia response, carbohydrate metabolism, endocrine pathways, and targets of several transcription factors. The role of the transcription factor HIF1 (hypoxia inducible factor 1) is particularly highlighted and aspects of downstream events as well as the role of other transcription factors related to cobalt toxicity are considered. Conclusions This study provides extensive data useful for the understanding of nanoparticle and cobalt toxicity. It shows that WC nanoparticles caused low transcriptional responses while WC-Co nanoparticles are able to exert responses similar to that of free cobalt ions, particularly the induction of hypoxia-like effects via interactions with HIF1α in human keratinocytes. However, the enhanced toxicity of WC-Co particles compared to CoCl2 could not be explained by differences in gene transcription. PMID:20105288

  9. Arabinogalactan Proteins from Baobab and Acacia Seeds Influence Innate Immunity of Human Keratinocytes in vitro.

    Science.gov (United States)

    Zahid, Abderrakib; Despres, Julie; Benard, Magalie; Nguema-Ona, Eric; Leprince, Jerome; Vaudry, David; Rihouey, Christophe; Vicré-Gibouin, Maité; Driouich, Azeddine; Follet-Gueye, Marie-Laure

    2016-10-13

    Plant derived arabinogalactan proteins (AGP) were repeatedly confirmed as immunologically as well as dermatologically active compounds. However little is currently known regarding their potential activity towards skin innate immunity. Here, we extracted and purified AGP from acacia (Acacia senegal) and baobab (Adansonia digitata) seeds to investigate their biological effects on the HaCaT keratinocyte cell line in an in vitro system. While AGP from both sources did not exhibit any cytotoxic effect, AGP from acacia seeds enhanced cell viability Moreover, real-time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that AGP extracted from both species induced a substantial overexpression of hBD-2, TLR-5, and IL1-α genes. These data suggest that plant AGP, already known to control plant defensive processes, could also modulate skin innate immune responses. This article is protected by copyright. All rights reserved.

  10. Human T-Lymphotropic virus (HTLV type I in vivo integration in oral keratinocytes

    Directory of Open Access Journals (Sweden)

    Martha C Domínguez

    2011-03-01

    Full Text Available Although the infection of HTLV-1 to cell components of the mouth have been previously reported, there was not until this report, a detailed study to show the characteristics of such infection. From 14 Tropical Spastic Paraparesis/ HTLV-1-Associated Myelopathy (HAM/TSP patients and 11 asymptomatic carrier individuals (AC coming from HTLV-1 endemic areas of southwest Pacific of Colombia, infected oral mucosa cells were primary cultured during five days. These cell cultures were immunophenotyped by dual color fluorescence cell assortment using different lymphocyte CD markers and also were immunohistochemically processed using a polyclonal anti-keratin antibody. Five days old primary cultures were characterized as oral keratinocytes, whose phenotype was CD3- /CD4-/CD8-/CD19-/CD14-/CD45-/A575-keratin+. From DNA extracted of primary cultures LTR, pol, env and tax HTLV-1 proviral DNA regions were differentially amplified by PCR showing proviral integration. Using poly A+ RNA obtained of these primary cultures, we amplify by RT-PCR cDNA of tax and pol in 57.14% (8/14 HAM/TSP patients and 27.28% (3/11 AC. Tax and pol poly A+ RNA were expressed only in those sIgA positive subjects. Our results showed that proviral integration and viral gene expression in oral keratinocytes are associated with a HTLV-1 specific local mucosal immune response only in those HTLV-1 infected individuals with detectable levels of sIgA in their oral fluids. Altogether the results gave strong evidence that oral mucosa infection would be parte of the systemic spreading of HTLV-1 infection.

  11. Effects of Prisma® Skin dermal regeneration device containing glycosaminoglycans on human keratinocytes and fibroblasts.

    Science.gov (United States)

    Belvedere, Raffaella; Bizzarro, Valentina; Parente, Luca; Petrella, Francesco; Petrella, Antonello

    2017-08-10

    Prisma® Skin is a new pharmaceutical device developed by Mediolanum Farmaceutici S.p.a. It includes alginates, hyaluronic acid and mainly mesoglycan. The latter is a natural glycosaminoglycan preparation containing chondroitin sulfate, dermatan sulfate, heparan sulfate and heparin and it is used in the treatment of vascular disease. Glycosaminoglycans may contribute to the re-epithelialization in the skin wound healing, as components of the extracellular matrix. Here we describe, for the first time, the effects of Prisma® Skin in in vitro cultures of adult epidermal keratinocytes and dermal fibroblasts. Once confirmed the lack of cytotoxicity by mesoglycan and Prisma® Skin, we have shown the increase of S and G2 phases of fibroblasts cell cycle distribution. We further report the strong induction of cell migration rate and invasion capability on both cell lines, two key processes of wound repair. In support of these results, we found significant cytoskeletal reorganization, following the treatments with mesoglycan and Prisma® Skin, as confirmed by the formation of F-actin stress fibers. Additionally, together with a significant reduction of E-cadherin, keratinocytes showed an increase of CD44 expression and the translocation of ezrin to the plasma membrane, suggesting the involvement of CD44/ERM (ezrin-radixin-moesin) pathway in the induction of the analyzed processes. Furthermore, as showed by immunofluorescence assay, fibroblasts treated with mesoglycan and Prisma® Skin exhibited the increase of Fibroblast Activated Protein α and a remarkable change in shape and orientation, two common features of reactive stromal fibroblasts. In all experiments Prisma® Skin was slightly more potent than mesoglycan. In conclusion, based on these findings we suggest that Prisma® Skin may be able to accelerate the healing process in venous skin ulcers, principally enhancing re-epithelialization and granulation processes.

  12. Incoming human papillomavirus 16 genome is lost in PML protein-deficient HaCaT keratinocytes.

    Science.gov (United States)

    Bienkowska-Haba, Malgorzata; Luszczek, Wioleta; Keiffer, Timothy R; Guion, Lucile G M; DiGiuseppe, Stephen; Scott, Rona S; Sapp, Martin

    2017-05-01

    Human papillomaviruses (HPVs) target promyelocytic leukemia (PML) nuclear bodies (NBs) during infectious entry and PML protein is important for efficient transcription of incoming viral genome. However, the transcriptional down regulation was shown to be promoter-independent in that heterologous promoters delivered by papillomavirus particles were also affected. To further investigate the role of PML protein in HPV entry, we used small hairpin RNA to knockdown PML protein in HaCaT keratinocytes. Confirming previous findings, PML knockdown in HaCaT cells reduced HPV16 transcript levels significantly following infectious entry without impairing binding and trafficking. However, when we quantified steady-state levels of pseudogenomes in interphase cells, we found strongly reduced genome levels compared with parental HaCaT cells. Because nuclear delivery was comparable in both cell lines, we conclude that viral pseudogenome must be removed after successful nuclear delivery. Transcriptome analysis by gene array revealed that PML knockdown in clonal HaCaT cells was associated with a constitutive interferon response. Abrogation of JAK1/2 signaling prevented genome loss, however, did not restore viral transcription. In contrast, knockdown of PML protein in HeLa cells did not affect HPV genome delivery and transcription. HeLa cells are transformed by HPV18 oncogenes E6 and E7, which have been shown to interfere with the JAK/Stat signaling pathway. Our data imply that PML NBs protect incoming HPV genomes. Furthermore, they provide evidence that PML NBs are key regulators of the innate immune response in keratinocytes. Promyelocytic leukemia nuclear bodies (PML NBs) are important for antiviral defense. Many DNA viruses target these subnuclear structures and reorganize them. Reorganization of PML NBs by viral proteins is important for establishment of infection. In contrast, HPVs require the presence of PML protein for efficient transcription of incoming viral genome. Our

  13. Borges, immortality and the circular ruins.

    Science.gov (United States)

    Bronstein, Catalina

    2002-06-01

    The author explores ideas surrounding immortality and death focusing on the interplay between their development in two stories by Borges ('The circular ruins' and 'The immortal') and their manifestation in a patient. With the help of Borges's stories, the author addresses the desperate necessity experienced by some individuals to search for immortality. This is not just an expression of the universal wish to live forever but, at a deeper level, arises from the impossibility of bearing the mental pain of experiencing ordinary human vulnerability and loss - death being the ultimate expression of such vulnerability. It is suggested that the relentless pursuit of immortality in such individuals expresses an omnipotent phantasy of ridding the self of the emotional pain and fear that arises through being alive. It leads to a denial of the emotional significance of passage of time, of separation and sexual differences. In actuality, the individual's state of not feeling approximates to a complete loss of human identity and emotional death, with no place for any meaningful others. The individual him/herself becomes a 'mere image', living in a delusional world peopled by him/herself and his/ her projections, and ending up trapped inside the circular ruins he/she has generated. The horror experienced at the stark awareness of the individual's emotional death and the wish to re-establish contact with the good internal objects that have been attacked sets in motion the long process of searching for the recovery of a sense of temporality (that would still include the wish for immortality) and, with it, a sense of identity.

  14. GATA3 is a master regulator of the transcriptional response to low-dose ionizing radiation in human keratinocytes

    Energy Technology Data Exchange (ETDEWEB)

    Bonin, F.; Molina, M.; Berthier-Vergnes, O.; Lamartine, J. [Universite de Lyon, Lyon, F-69003 (France); Universite Lyon 1, Lyon, F-69003 (France); CNRS, UMR5534, Centre de Genetique Moleculaire et Cellulaire, Villeurbanne, F-69622 (France); Malet, C.; Ginestet, C. [Centre Leon Berard, Service de Radiotherapie, Lyon F-69008 (France); Martin, M.T. [Laboratoire de Genomique et Radiobiologie de la Keratinopoiese, CEA, IRCM, Evry F-91000 (France)

    2009-07-01

    Background: The general population is constantly exposed to low levels of radiation through natural, occupational or medical irradiation. Even if the biological effects of low-level radiation have been intensely debated and investigated, the molecular mechanisms underlying the cellular response to low doses remain largely unknown. Results: The present study investigated the role of GATA3 protein in the control of the cellular and molecular response of human keratinocytes exposed to a 1 cGy dose of X-rays. Chromatin immunoprecipitation showed GATA3 to be able to bind the promoter of 4 genes responding to a 1 cGy exposure. To go further into the role of GATA3 after ionizing radiation exposure, we studied the cellular and molecular consequences of radiation in GATA3 knock-down cells. Knockdown was obtained by lentiviral-mediated expression of an shRNA targeting the GATA3 transcript in differentiated keratinocytes. First, radiosensitivity was assessed: the toxicity, in terms of immediate survival (with XTT test), associated with 1 cGy radiation was found to be increased in GATA3 knock-down cells. The impact of GATA3 knock-down on the transcriptome of X-ray irradiated cells was also investigated, using oligonucleotide micro-arrays to assess changes between 3 h and 72 h post-irradiation in normal vs GATA3 knock-down backgrounds; transcriptome response was found to be completely altered in GATA3 knock-down cells, with a strong induction/repression peak 48 h after irradiation. Functional annotation revealed enrichment in genes known to be involved in chaperone activity, TGF{beta} signalling and stress response. Conclusion: Taken together, these data indicate that GATA3 is an important regulator of the cellular and molecular response of epidermal cells to very low doses of radiation. (authors)

  15. Broad-spectrum sunscreens prevent the secretion of proinflammatory cytokines in human keratinocytes exposed to ultraviolet A and phototoxic lomefloxacin

    Energy Technology Data Exchange (ETDEWEB)

    Reinhardt, P.; Cybulski, M. [Lasers and Electro-Optics Div., Consumer and Clinical Radiation Protection Bureau, Product Safety Program, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario (Canada)], E-mail: pascale_reinhardt@hc-sc.gc.ca; Miller, S.M.; Ferrarotto, C.; Wilkins, R. [Radiobiology Div., Consumer and Clinical Radiation Protection Bureau, Product Safety Program, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario (Canada); Deslauriers, Y. [Lasers and Electro-Optics Div., Consumer and Clinical Radiation Protection Bureau, Product Safety Program, Healthy Environments and Consumer Safety Branch, Health Canada, Ottawa, Ontario (Canada)

    2006-02-15

    The combination of phototoxic drugs and ultraviolet (UV) radiation can trigger the release of proinflammatory cytokines. The present study measured the ability of sunscreens to prevent cytokine secretion in human keratinocytes following cotreatment of these cells with a known photoreactive drug and UVA. Keratinocytes were treated for 1 h with increasing concentrations of lomefloxacin (LOM) or norfloxacin (NOR), exposed to 15 J/cm{sup 2} UVA, and incubated for 24 h. NOR, owing to the absence of a fluorine atom in position 8, was non-phototoxic and used as a negative control. Cell viability and the release of 3 cytokines were assessed, namely interleukin-1{alpha} (IL-1{alpha}), interleukin-6 (IL-6), and tumour necrosis factor-{alpha} (TNF-{alpha}). The measurement of these cytokines may be a useful tool for detecting photoreactive compounds. To measure their ability to prevent cytokine secretion, various sunscreens were inserted between the UVA source and the cells. Treatment with NOR, NOR plus UVA, or LOM had no effect on the cells. LOM plus UVA, however, had an effect on cell viability and on cytokine secretion. IL-1{alpha} levels increased with LOM concentration. The release of TNF-{alpha} and IL-6 followed the same pattern at lower concentrations of LOM but peaked at 15 {mu}mol/L and decreased at higher concentrations. Sunscreens protected the cells from the effects of LOM plus UVA, as cell viability and levels of cytokines remained the same as in the control cells. In conclusion, the application of broad-spectrum sunscreen by individuals exposed to UVA radiation may prevent phototoxic reactions initiated by drugs such as LOM. (author)

  16. T Helper 1 and T Helper 2 Cytokines Differentially Modulate Expression of Filaggrin and its Processing Proteases in Human Keratinocytes

    Institute of Scientific and Technical Information of China (English)

    Zheng-Hong Di; Lei Ma; Rui-Qun Qi; Xiao-Dong Sun; Wei Huo; Li Zhang; Ya-Ni Lyu

    2016-01-01

    Background: Atopic dermatitis (AD) is characterized by defective skin barrier and imbalance in T helper 1/T helper 2 (Th 1/Th2) cytokine expression.Filaggrin (FLG) is the key protein to maintaining skin barrier function.Recent studies indicated that Th1/Th2 cytokines influence FLG expression in keratinocytes.However, the role ofThl/Th2 cytokines on FLG processing is not substantially documented.Our aim was to investigate the impact ofThl/Th2 cytokines on FLG processing.Methods: HaCaT cells and normal human keratinocytes were cultured in low and high calcium media and stimulated by either interleukin (IL)-4, 13 or interferon-γ(IFN-γ).FLG, its major processing proteases and key protease inhibitor lymphoepithelial Kazal-type-related inhibitor (LEKTI) were measured by both real-time quantitative polymerase chain reaction and Western blotting.Their expression was also evaluated in acute and chronic AD lesions by immunohistochemistry.Results: IL-4/13 significantly reduced, while IFN-γsignificantly up-regulated FLG expression.IL-4/13 significantly increased, whereas IFN-γsignificantly decreased the expression ofkallikreins 5 and 7, matriptase and channel-activating serine protease 1.On the contrary, IL-4/13 significantly decreased, while IFN-γincreased the expression of LEKTI and caspase-14.Similar trends were observed in AD lesions.Conclusions: Our results suggested that Th1/Th2 cytokines differentially regulated the expression of major FLG processing enzymes.The imbalance between Th1 and Th2 polarized immune response seems to extend to FLG homeostasis, through the network of FLG processing enzymes.

  17. Integrin-blocking antibodies delay keratinocyte re-epithelialization in a human three-dimensional wound healing model.

    Directory of Open Access Journals (Sweden)

    Christophe Egles

    Full Text Available The alpha6beta4 integrin plays a significant role in tumor growth, angiogenesis and metastasis through modulation of growth factor signaling, and is a potentially important therapeutic target. However, alpha6beta4-mediated cell-matrix adhesion is critical in normal keratinocyte attachment, signaling and anchorage to the basement membrane through its interaction with laminin-5, raising potential risks for targeted therapy. Bioengineered Human Skin Equivalent (HSE, which have been shown to mimic their normal and wounded counterparts, have been used here to investigate the consequences of targeting beta4 to establish toxic effects on normal tissue homeostasis and epithelial wound repair. We tested two antibodies directed to different beta4 epitopes, one adhesion-blocking (ASC-8 and one non-adhesion blocking (ASC-3, and determined that these antibodies were appropriately localized to the basal surface of keratinocytes at the basement membrane interface where beta4 is expressed. While normal tissue architecture was not altered, ASC-8 induced a sub-basal split at the basement membrane in non-wounded tissue. In addition, wound closure was significantly inhibited by ASC-8, but not by ASC-3, as the epithelial tongue only covered 40 percent of the wound area at 120 hours post-wounding. These results demonstrate beta4 adhesion-blocking antibodies may have adverse effects on normal tissue, whereas antibodies directed to other epitopes may provide safer alternatives for therapy. Taken together, we conclude that these three-dimensional tissue models provide a biologically relevant platform to identify toxic effects induced by candidate therapeutics, which will allow generation of findings that are more predictive of in vivo responses early in the drug development process.

  18. Cox2 and β-Catenin/T-cell Factor Signaling Intestinalize Human Esophageal Keratinocytes When Cultured under Organotypic Conditions

    Directory of Open Access Journals (Sweden)

    Jianping Kong

    2011-09-01

    Full Text Available The incidence of esophageal adenocarcinoma (EAC is rising in the United States. An important risk factor for EAC is the presence of Barrett esophagus (BE. BE is the replacement of normal squamous esophageal epithelium with a specialized columnar epithelium in response to chronic acid and bile reflux. However, the emergence of BE from squamous keratinocytes has not yet been demonstrated. Our research has focused on this. Wnt and cyclooxygenase 2 (Cox2 are two pathways whose activation has been associated with BE and progression to EAC, but their role has not been tested experimentally. To explore their contribution, we engineered a human esophageal keratinocyte cell line to express either a dominant-active Wnt effector CatCLef or a Cox2 complementary DNA. In a two-dimensional culture environment, Cox2 expression increases cell proliferation and migration, but neither transgene induces known BE markers. In contrast, when these cells were placed into three-dimensional organotypic culture conditions, we observed more profound effects. CatCLef-expressing cells were more proliferative, developed a thicker epithelium, and upregulated Notch signaling and several BE markers including NHE2. Cox2 expression also increased cell proliferation and induced a thicker epithelium. More importantly, we observed cysts form within the epithelium, filled with intestinal mucins including Muc5B and Muc17. This suggests that Cox2 expression in a three-dimensional culture environment induces a lineage of mucin-secreting cells and supports an important causal role for Cox2 in BE pathogenesis. We conclude that in vitro modeling of BE pathogenesis can be improved by enhancing Wnt signaling and Cox2 activity and using three-dimensional organotypic culture conditions.

  19. Ageing and immortality.

    Science.gov (United States)

    Rose, M R; Mueller, L D

    2000-01-01

    The concept of the force of natural selection was developed to explain the evolution of ageing. After ageing, however, comes a period in which mortality rates plateau and some individual organisms could, in theory, live forever. This late-life immortality has no presently agreed upon explanation. Two main theories have been offered. The first is heterogeneity within ageing cohorts, such that only extremely robust individuals survive ageing. This theory can be tested by comparisons of more and less robust cohorts. It can also be tested by fitting survival data to its models. The second theory is that late-life plateaus in mortality reflect the inevitable late-life plateau in the force of natural selection. This theory can be tested by changing the force of natural selection in evolving laboratory populations, particularly the age at which the force plateaus. This area of research has great potential for elucidating the overall structure of life-history evolution, particularly the interrelationship between the three life-history phases of development, ageing and immortality. PMID:11127912

  20. HMGB1/RAGE axis promotes autophagy and protects keratinocytes from ultraviolet radiation-induced cell death.

    Science.gov (United States)

    Mou, Kuanhou; Liu, Wei; Han, Dan; Li, Pan

    2017-03-01

    The primary cause of skin cancer is ultraviolet (UV) light from the sun. Keratinocytes are the predominant cell type in the epidermis and form a barrier against environmental damage, especially from UV light irradiation. Autophagy is a self-digestion mechanism for energy homeostasis at critical times during development and as a response to stress. High-mobility group protein 1 (HMGB1) is a highly conserved nuclear protein that is associated with cell autophagy. We investigated the role of HMGB1 in keratinocytes exposed to UV irradiation and its regulation of keratinocyte autophagy. Specimens of UV-exposed human skin were assayed immunohistochemically for HMGB1. HaCaT immortalized human keratinocytes were used to investigate the mechanism of HMGB1 translocation induced by UV irradiation. Levels of cytosolic reactive oxygen species (ROS) were determined by H2DCF assay, apoptosis was assayed by flow cytometry and western-blot after lentivirus-mediated shRNA targeting of HMGB1 in keratinocytes by UV irradiation. Phosphorylated-Erk1/2 expression was assayed by western blotting. HMGB1 and its receptor (receptor for advanced glycation end products, RAGE) were both expressed by HaCaT cells, and HMGB1 was transferred from the nucleus to the cytoplasm after UV irradiation in both HaCaT and human skin keratinocytes. Knockdown of HMGB1 expression by lentivirus-mediated shRNA limited UV-induced autophagy and led to increased apoptosis of HaCaT cells. Pharmacological inhibition of HMGB1 cytoplasmic translocation by agents such as ethyl pyruvate limits starvation-induced autophagy. UV irradiation led to phosphorylation of Erk1/2 in HaCaT cells. Inhibition of RAGE and Erk1/2 limited HaCaT cell autophagy. Autocrine HMGB1 modulated HaCaT autophagy via a RAGE/HMGB1/extracellular signal-regulated Erk1/2-dependent pathway to protect keratinocytes from apoptosis during UV irradiation. Copyright © 2016 Japanese Society for Investigative Dermatology. Published by Elsevier B.V. All

  1. Human papillomavirus E6E7-mediated adenovirus cell killing: selectivity of mutant adenovirus replication in organotypic cultures of human keratinocytes.

    Science.gov (United States)

    Balagué, C; Noya, F; Alemany, R; Chow, L T; Curiel, D T

    2001-08-01

    Replication-competent adenoviruses are being investigated as potential anticancer agents. Exclusive virus replication in cancer cells has been proposed as a safety trait to be considered in the design of oncolytic adenoviruses. From this perspective, we have investigated several adenovirus mutants for their potential to conditionally replicate and promote the killing of cells expressing human papillomavirus (HPV) E6 and E7 oncoproteins, which are present in a high percentage of anogenital cancers. For this purpose, we have employed an organotypic model of human stratified squamous epithelium derived from primary keratinocytes that have been engineered to express HPV-18 oncoproteins stably. We show that, whereas wild-type adenovirus promotes a widespread cytopathic effect in all infected cells, E1A- and E1A/E1B-deleted adenoviruses cause no deleterious effect regardless of the coexpression of HPV18 E6E7. An adenovirus deleted in the CR2 domain of E1A, necessary for binding to the pRB family of pocket proteins, shows no selectivity of replication as it efficiently kills all normal and E6E7-expressing keratinocytes. Finally, an adenovirus mutant deleted in the CR1 and CR2 domains of E1A exhibits preferential replication and cell killing in HPV E6E7-expressing cultures. We conclude that the organotypic keratinocyte culture represents a distinct model to evaluate adenovirus selectivity and that, based on this model, further modifications of the adenovirus genome are required to restrict adenovirus replication to tumor cells.

  2. β1 integrin signaling in asymmetric migration of keratinocytes under mechanical stretch in a co-cultured wound repair model.

    Science.gov (United States)

    Lü, Dongyuan; Li, Zhan; Gao, Yuxin; Luo, Chunhua; Zhang, Fan; Zheng, Lu; Wang, Jiawen; Sun, Shujin; Long, Mian

    2016-12-28

    Keratinocyte (KC) migration in re-epithelization is crucial in repairing injured skin. But the mechanisms of how mechanical stimuli regulate the migration of keratinocytes have been poorly understood. Human immortalized keratinocyte HaCaT cells were co-cultured with skin fibroblasts on PDMS membranes and transferred to the static stretch device developed in-house for additional 6 day culture under mechanical stretch to mimic surface tension in skin. To detect the expression of proteins on different position at different time points and the effect of β1 integrin mechanotransduction on HaCaT migration, Immunofluorescence, Reverse transcription-polymerase chain reaction, Flow cytometry, Western blotting assays were applied. Mechanical receptor of β1 integrin that recognizes its ligand of collagen I was found to be strongly associated with migration of HaCaT cells since the knockdown of β1 integrin via RNA silence eliminated the key protein expression dynamically. Here the expression of vinculin was lower but that of Cdc42 was higher for the cells at outward edge than those at inward edge, respectively, supporting that the migration capability of keratinocytes is inversely correlated with the formation of focal adhesion complexes but positively related to the lamellipodia formation. This asymmetric expression feature was further confirmed by high or low expression of PI3K for outward- or inward-migrating cells. And ERK1/2 phosphorylation was up-regulated by mechanical stretch. We reported here, a novel mechanotransduction signaling pathways were β1 integrin-dependent pattern of keratinocytes migration under static stretch in an in vitro co-culture model. These results provided an insight into underlying molecular mechanisms of keratinocyte migration under mechanical stimuli.

  3. Human amniotic epithelial stem cells promote wound healing by facilitating migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways.

    Science.gov (United States)

    Zhao, Bin; Liu, Jia-Qi; Zheng, Zhao; Zhang, Jun; Wang, Shu-Yue; Han, Shi-Chao; Zhou, Qin; Guan, Hao; Li, Chao; Su, Lin-Lin; Hu, Da-Hai

    2016-07-01

    Wound healing is a highly orchestrated physiological process consisting in a complex interaction of cellular and biochemical events. Human amniotic epithelial stem cells (HAESCs) have been shown to be an attractive resource for wound healing because they are primitive stem cells. However, the exact effects of amnion-derived stem cells on the migration or proliferation of keratinocytes and their potential mechanism are not fully understood. We have found that HAESCs accelerate the migration of keratinocytes and induce a remarkable increase in the activity of phospho-ERK, phospho-JNK, and phospho-AKT, the blockade of which by their specific inhibitors significantly inhibits migration induced by HAESC-conditioned medium (CM). Furthermore, the co-culture of keratinocytes with HAESCs up-regulates the expression levels of cell proliferation proteins Cyclin D1, Cyclin D3 and Mdm2. In vivo animal experiments have shown that HAESC-CM improves wound healing, whereas blockade with ERK, JNK and AKT inhibitors significantly impairs wound healing. Taken together, these results reveal, for the first time, that HAESCs promote wound healing by facilitating the migration and proliferation of keratinocytes via ERK, JNK and AKT signaling pathways and might be a potential therapy in skin wound healing.

  4. FOXM1 allows human keratinocytes to bypass the oncogene-induced differentiation checkpoint in response to gain of MYC or loss of p53

    Science.gov (United States)

    Molinuevo, R; Freije, A; de Pedro, I; Stoll, S W; Elder, J T; Gandarillas, A

    2017-01-01

    Tumour suppressor p53 or proto-oncogene MYC is frequently altered in squamous carcinomas, but this is insufficient to drive carcinogenesis. We have shown that overactivation of MYC or loss of p53 via DNA damage triggers an anti-oncogenic differentiation-mitosis checkpoint in human epidermal keratinocytes, resulting in impaired cell division and squamous differentiation. Forkhead box M1 (FOXM1) is a transcription factor recently proposed to govern the expression of a set of mitotic genes. Deregulation of FOXM1 occurs in a wide variety of epithelial malignancies. We have ectopically expressed FOXM1 in keratinocytes of the skin after overexpression of MYC or inactivation of endogenous p53. Ectopic FOXM1 rescues the proliferative capacity of MYC- or p53-mutant cells in spite of higher genetic damage and a larger cell size typical of differentiation. As a consequence, differentiation induced by loss of p53 or MYC is converted into increased proliferation and keratinocytes displaying genomic instability are maintained within the proliferative compartment. The results demonstrate that keratinocyte oncogene-induced differentiation is caused by mitosis control and provide new insight into the mechanisms driving malignant progression in squamous cancer. PMID:27452522

  5. Long-term exposure to cigarette smoke extract induces hypomethylation at the RUNX3 and IGF2-H19 loci in immortalized human urothelial cells.

    Directory of Open Access Journals (Sweden)

    Li-Mei Chen

    Full Text Available Cigarette smoking is the single most important epidemiological risk factor for bladder cancer but it is not known whether exposure of urothelial cells to the systemic soluble contents of cigarette smoke is directly causative to bladder cancer and the associated epigenetic changes such as tumor suppressor gene hypermethylation. We undertook this study to investigate if long-term treatment of human urothelial cells with cigarette smoke extract (CSE results in tumor suppressor gene hypermethylation, a phenotype that was previously associated with long-term constant CSE treatment of airway epithelial cells. We chronically treated an immortalized human urothelial cell line UROtsa with CSE using a cyclic daily regimen but the cells were cultured in CSE-free medium between daily treatments. Bisulfite sequencing and real-time PCR array-based methylation profiling were employed to evaluate methylation changes at tumor suppressor gene loci in the chronically CSE-treated cells versus the passage-matched untreated control cells. The RUNX3 tumor suppressor gene promoter was hypomethylated with a significant increase in proportion of the completely unmethylated haplotype after the long-term CSE treatment; whereas RUNX3 promoter hypermethylation was previously reported for bladder cancers of smokers. Hypomethylation induced by the long-term CSE treatment was also observed for the IGF2-H19 locus. The methylation status at the PRSS8/prostasin and 16 additional loci however, was unaffected by the chronic CSE treatment. Transient CSE treatment over 1 daily regimen resulted in transcriptional down-regulation of RUNX3 and H19, but only the H19 transcription was down-regulated in the chronically CSE-treated urothelial cells. Transcription of a key enzyme in one-carbon metabolism, dihydrofolate reductase (DHFR was greatly reduced by the long-term CSE treatment, potentially serving as a mechanism for the hypomethylation phenotype via a reduced supply of methyl donor

  6. The Immortal Fire Within

    Science.gov (United States)

    Sheehan, William

    2007-12-01

    Preface; Key to abbreviations in notes; 1. Through rugged ways; 2. Ardent and faithful work with a telescope; 3. Mars; his moons and his heavens; 4. A seeker of comets; 5. Vanderbilt astronomer; 6. In the realm of the nebulae; 7. Go west, young man!; 8. Hanging fire; 9. On Mt. Hamilton; 10. A year of wonders; 11. The young rebel; 12. 'I am tired here'; 13. Immortality; 14. Travels and travails; 15. Barnard and Mars; 16. Nature's true artisan; 17. A tide in his affairs; 18. Yerkes observatory; 19. Disappointments and triumphs; 20. The comet and Milky Way photographs; 21. Comet tales; 22. Observer of all that shines - or obscures; 23. Eclipse and decline; 24. Ad astra; Index.

  7. Suppressive effects of antimycotics on tumor necrosis factor-alpha-induced CCL27, CCL2, and CCL5 production in human keratinocytes.

    Science.gov (United States)

    Kanda, Naoko; Watanabe, Shinichi

    2006-08-14

    Antimycotic agents are reported to improve cutaneous symptoms of atopic dermatitis or psoriasis vulgaris. Keratinocytes in these lesions excessively produce chemokines, CCL27, CCL2, or CCL5 which trigger inflammatory infiltrates. Tumor necrosis factor-alpha (TNF-alpha) induces production of these chemokines via activating nuclear factor-kappaB (NF-kappaB). We examined in vitro effects of antimycotics on TNF-alpha-induced CCL27, CCL2, and CCL5 production in human keratinocytes. Antimycotics ketoconazole and terbinafine hydrochloride suppressed TNF-alpha-induced CCL27, CCL2, and CCL5 secretion and mRNA expression in keratinocytes in parallel to the inhibition of NF-kappaB activity while fluconazole was ineffective. Anti-prostaglandin E2 (PGE2) antiserum or antisense oligonucleotides against PGE2 receptor EP2 or EP3 abrogated inhibitory effects of ketoconazole and terbinafine hydrochloride on TNF-alpha-induced NF-kappaB activity and CCL27, CCL2, and CCL5 production, indicating the involvement of endogenous PGE2 in the inhibitory effects. Prostaglandin H2, a precursor of PGE2 can be converted to thromboxane A2. Ketoconazole, terbinafine hydrochloride and thromboxane A2 synthase (EC 5.3.99.5) inhibitor, carboxyheptyl imidazole increased PGE2 release from keratinocytes and reduced that of thromboxane B2, a stable metabolite of thromboxane A2. Carboxyheptyl imidazole also suppressed TNF-alpha-induced NF-kappaB activity and CCL27, CCL2, and CCL5 production. These results suggest that ketoconazole and terbinafine hydrochloride may suppress TNF-alpha-induced NF-kappaB activity and CCL27, CCL2, and CCL5 production by increasing PGE2 release from keratinocytes. These antimycotics may suppress thromboxane A2 synthesis and redirect the conversion of PGH2 toward PGE2. These antimycotics may alleviate inflammatory infiltration in atopic dermatitis or psoriasis vulgaris by suppressing chemokine production.

  8. Discrimination of skin sensitizers from non-sensitizers by interleukin-1α and interleukin-6 production on cultured human keratinocytes.

    Science.gov (United States)

    Jung, Daun; Che, Jeong-Hwan; Lim, Kyung-Min; Chun, Young-Jin; Heo, Yong; Seok, Seung Hyeok

    2016-09-01

    In vitro testing methods for classifying sensitizers could be valuable alternatives to in vivo sensitization testing using animal models, such as the murine local lymph node assay (LLNA) and the guinea pig maximization test (GMT), but there remains a need for in vitro methods that are more accurate and simpler to distinguish skin sensitizers from non-sensitizers. Thus, the aim of our study was to establish an in vitro assay as a screening tool for detecting skin sensitizers using the human keratinocyte cell line, HaCaT. HaCaT cells were exposed to 16 relevant skin sensitizers and 6 skin non-sensitizers. The highest dose used was the dose causing 75% cell viability (CV75) that we determined by an MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The levels of extracellular production of interleukin-1α (IL-1α) and IL-6 were measured. The sensitivity of IL-1α was 63%, specificity was 83% and accuracy was 68%. In the case of IL-6, sensitivity: 69%, specificity: 83% and accuracy: 73%. Thus, this study suggests that measuring extracellular production of pro-inflammatory cytokines IL-1α and IL-6 by human HaCaT cells may potentially classify skin sensitizers from non-sensitizers. Copyright © 2015 John Wiley & Sons, Ltd.

  9. Trichloroethylene-mediated cytotoxicity in human epidermal keratinocytes is mediated by the rapid accumulation of intracellular calcium: Interception by naringenin.

    Science.gov (United States)

    Ali, F; Khan, A Q; Khan, R; Sultana, S

    2016-02-01

    Industrial solvents pose a significant threat to the humankind. The mechanisms of their toxicity still remain in debate. Trichloroethylene (TCE) is a widespread industrial solvent responsible for severe liver dysfunction, cutaneous toxicity in occupationally exposed humans. We utilized an in vitro system of human epidermal keratinocyte (HaCaT) cells in this study to avoid complex cell and extracellular interactions. We report the cytotoxicity of organic solvent TCE in HaCaT and its reversal by a natural flavanone, naringenin (Nar). The cytotoxicity was attributed to the rapid intracellular free calcium (Ca(2+)) release, which might lead to the elevation of protein kinase C along with robust free radical generation, instability due to energy depletion, and sensitization of intracellular stress signal transducer nuclear factor κB. These effects were actually seen to induce significant amount of genomic DNA fragmentation. Furthermore, all these effects of TCE were effectively reversed by the treatment of Nar, a natural flavanone. Our studies identify intracellular Ca as a unique target used by organic solvents in the cytotoxicity and highlight the Ca(2+) ion stabilizer properties of Nar.

  10. The human keratinocyte two-dimensional protein database (update 1994): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases

    DEFF Research Database (Denmark)

    Celis, J E; Rasmussen, H H; Olsen, E

    1994-01-01

    The master two-dimensional (2-D) gel database of human keratinocytes currently lists 3087 cellular proteins (2168 isoelectric focusing, IEF; and 919 none-quilibrium pH gradient electrophoresis, NEPHGE), many of which correspond to posttranslational modifications, 890 polypeptides have been...... identified (protein name, organelle components, etc.) using one or a combination of procedures that include (i) comigration with known human proteins, (ii) 2-D gel immunoblotting using specific antibodies (iii) microsequencing of Coomassie Brilliant Blue stained proteins, (iv) mass spectrometry and (v...... in the database. We also report a database of proteins recovered from the medium of noncultured, unfractionated keratinocytes. This database lists 398 polypeptides (309 IEF; 89 NEPHGE) of which 76 have been identified. The aim of the comprehensive databases is to gather, through a systematic study...

  11. Gene expression studies on human keratinocytes transduced with human growth hormone gene for a possible utilization in gene therapy; Estudos da expressao genica mediante utilizacao de queratinocitos humanos normais transduzidos com o gene do hormonio de crscimento humano. Possivel utilizacao em terapia genica

    Energy Technology Data Exchange (ETDEWEB)

    Mathor, Monica Beatriz

    1994-12-31

    Taking advantage of the recent progress in the DNA-recombinant techniques and of the potentiality of normal human keratinocytes primary culture to reconstitute the epidermis, it was decided to genetically transform these keratinocytes to produce human growth hormone under controllable conditions that would be used in gene therapy at this hormone deficient patients. The first step to achieve this goal was to standardize infection of keratinocytes with retrovirus producer cells containing a construct which included the gene of bacterial b-galactosidase. The best result was obtained cultivating the keratinocytes for 3 days in a 2:1 mixture of retrovirus producer cells and 3T3-J2 fibroblasts irradiated with 60 Gy, and splitting these infected keratinocytes on 3T3-J2 fibroblasts feeder layer. Another preliminary experiment was to infect normal human keratinocytes with interleukin-6 gene (hIL-6) that, in pathologic conditions, could be reproduced by keratinocytes and secreted to the blood stream. Thus, we verify that infected keratinocytes secrete an average amount of 500 ng/10{sup 6} cell/day of cytokin during the in vitro life time, that certify the stable character of the injection. These keratinocytes, when grafted in mice, secrete hIL-6 to the blood stream reaching levels of 40 pg/ml of serum. After these preliminary experiments, we construct a retroviral vector with the human growth hormone gene (h GH) driven by human metallothionein promoter (h PMT), designated DChPMTGH. Normal human keratinocytes were infected with DChPMTGH producer cells, following previously standardized protocol, obtaining infected keratinocytes secreting to the culture media 340 ng h GH/10{sup 6} cell/day without promoter activation. This is the highest level of h GH secreted in human keratinocytes primary culture described in literature. The h GH value increases approximately 10 times after activation with 100 {mu}M Zn{sup +2} for 8-12 hours. (author). 158 refs., 42 figs., 6 tabs.

  12. Integrative Responses to IL-17 and TNF-α in Human Keratinocytes Account for Key Inflammatory Pathogenic Circuits in Psoriasis

    National Research Council Canada - National Science Library

    Chiricozzi, Andrea; Guttman-Yassky, Emma; Suárez-Fariñas, Mayte; Nograles, Kristine E; Tian, Suyan; Cardinale, Irma; Chimenti, Sergio; Krueger, James G

    2011-01-01

    .... We sought to investigate the effects of TNF-α and IL-17 on keratinocyte (KC) gene profile, to identify genes that might be coregulated by these cytokines and determine how synergistically activated genes relate to the psoriasis transcriptome...

  13. Sonoporation delivery of monoclonal antibodies against human papillomavirus 16 E6 restores p53 expression in transformed cervical keratinocytes

    National Research Council Canada - National Science Library

    Togtema, Melissa; Pichardo, Samuel; Jackson, Robert; Lambert, Paul F; Curiel, Laura; Zehbe, Ingeborg

    2012-01-01

    .... Therapies targeted at blocking the HPV16 E6 protein and its deleterious effects on the tumour suppressor pathways of the cell can reverse the malignant phenotype of affected keratinocytes while sparing uninfected cells...

  14. Low-Dose Methylmercury-Induced Genes Regulate Mitochondrial Biogenesis via miR-25 in Immortalized Human Embryonic Neural Progenitor Cells

    Directory of Open Access Journals (Sweden)

    Xinjin Wang

    2016-12-01

    Full Text Available Mitochondria are essential organelles and important targets for environmental pollutants. The detection of mitochondrial biogenesis and generation of reactive oxygen species (ROS and p53 levels following low-dose methylmercury (MeHg exposure could expand our understanding of underlying mechanisms. Here, the sensitivity of immortalized human neural progenitor cells (ihNPCs upon exposure to MeHg was investigated. We found that MeHg altered cell viability and the number of 5-ethynyl-2′-deoxyuridine (EdU-positive cells. We also observed that low-dose MeHg exposure increased the mRNA expression of cell cycle regulators. We observed that MeHg induced ROS production in a dose-dependent manner. In addition, mRNA levels of peroxisome-proliferator-activated receptor gammacoactivator-1α (PGC-1α, mitochondrial transcription factor A (TFAM and p53-controlled ribonucleotide reductase (p53R2 were significantly elevated, which were correlated with the increase of mitochondrial DNA (mtDNA copy number at a concentration as low as 10 nM. Moreover, we examined the expression of microRNAs (miRNAs known as regulatory miRNAs of p53 (i.e., miR-30d, miR-1285, miR-25. We found that the expression of these miRNAs was significantly downregulated upon MeHg treatment. Furthermore, the overexpression of miR-25 resulted in significantly reducted p53 protein levels and decreased mRNA expression of genes involved in mitochondrial biogenesis regulation. Taken together, these results demonstrated that MeHg could induce developmental neurotoxicity in ihNPCs through altering mitochondrial functions and the expression of miRNA.

  15. Protective Effect of Cyanidin-3-O-Glucoside against Ultraviolet B Radiation-Induced Cell Damage in Human HaCaT Keratinocytes.

    Science.gov (United States)

    Hu, Yunfeng; Ma, Yuetang; Wu, Shi; Chen, Tianfeng; He, Yong; Sun, Jianxia; Jiao, Rui; Jiang, Xinwei; Huang, Yadong; Deng, Liehua; Bai, Weibin

    2016-01-01

    Ultraviolet radiation is the major environmental harmful factor that has emotional impact on human skin. The aim of the present study was to determine the mechanism of protection of cyanidin-3-O-glucoside against ultraviolet B (UVB)-induced damage to human HaCaT keratinocytes. Our results show that cyanidin-3-O-glucoside decreased the levels of intracellular reactive oxygen species generated by UVB treatment. Cyanidin-3-O-glucoside also decreased the UVB-augmented levels of the DNA damage indicators phospho-p53 and phospho-ATM/ATR. In addition, cyanidin-3-O-glucoside protected keratinocytes from UVB-induced injury by overturning the disruption of mitochondrial membrane potential and reversing apoptosis. The expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2) was attenuated in UVB-exposed cells but restored in UVB/cyanidin-3-O-glucoside-treated cells. Furthermore, expression of the proapoptotic proteins Bcl-2-associated X (Bax) and the key apoptosis executer cleaved caspase-3 were increased in UVB-irradiated cells and decreased in UVB/cyanidin-3-O-glucoside-treated cells. For these reasons, the results demonstrate that cyanidin-3-O-glucoside protects human keratinocytes against UVB-induced oxidative stress and apoptosis. Our study provides a theoretical basis for the use of cyanidin-3-O-glucoside in the fight against light damage.

  16. Protective effect of cyanidin-3-O-glucoside against ultraviolet B radiation-induced cell damage in human HaCaT keratinocytes

    Directory of Open Access Journals (Sweden)

    Yunfeng Hu

    2016-09-01

    Full Text Available Ultraviolet radiation is the major environmental harmful factor that has emotional impact on human skin. The aim of the present study was to determine the mechanism of protection of cyanidin-3-O-glucoside against ultraviolet B (UVB -induced damage to human HaCaT keratinocytes. Our results show that cyanidin-3-O-glucoside decreased the levels of intracellular reactive oxygen species generated by UVB treatment. cyanidin-3-O-glucoside also decreased the UVB-augmented levels of the DNA damage indicators phospho-p53 and phospho-ATM/ATR. In addition, cyanidin-3-O-glucoside protected keratinocytes from UVB-induced injury by overturning the disruption of mitochondrial membrane potential and reversing apoptosis. The expression of anti-apoptotic protein B-cell lymphoma 2 (Bcl-2 was attenuated in UVB-exposed cells but restored in UVB/cyanidin-3-O-glucoside-treated cells. Furthermore, expression of the proapoptotic proteins Bcl-2-associated X (Bax and the key apoptosis executer cleaved caspase-3 were increased in UVB-irradiated cells and decreased in UVB/cyanidin-3-O-glucoside-treated cells. For these reasons, the results demonstrate that cyanidin-3-O-glucoside protects human keratinocytes against UVB-induced oxidative stress and apoptosis. Our study provides a theoretical basis for the use of cyanidin-3-O-glucoside in the fight against light damage .

  17. The comparison of two methods to obtain human oral keratinocytes in primary culture; Comparacao de dois metodos de obtencao celular para cultura primaria de queratinocitos bucais humanos

    Energy Technology Data Exchange (ETDEWEB)

    Klingbeil, Maria Fatima Guarizo

    2006-07-01

    The therapeutic procedures frequently used in oral treatments for the pathological diseases are surgical, resulting in failures of the mucosal continuity.The possibility to obtain transplantable oral epithelia from an in vitro cell culture opens new utilization perspectives not only to where it comes from, but also as a reconstructive material for other parts of the human body, such as: urethra, epithelia corneo-limbal, cornea, ocular surface. Many researchers still use controversial methods for obtaining cells. It was therefore evaluated and compared the efficiency in both methods: enzymatic and direct explant to obtain oral keratinocytes from human oral mucosa. Fragments of intra oral epithelial tissues from healthy human subjects, undergoing dental surgeries, were donated to the research project. The keratinocytes were cultivated over a feeder-layer from a previously irradiated 3T3 Swiss albino fibroblasts. In this study it was compared the time needed in the cell obtention, the best cell amount between both methods, the life-span, the cell capacity to form an in vitro epithelia and its morphologic structure. The results in the assessment of both methods have shown the possibility to obtain keratinocytes from a small oral fragment, but at the same time we may verify the advantages and peculiar restrictions for each one of both analyzed methods. (author)

  18. A novel method for banking stem cells from human exfoliated deciduous teeth: lentiviral TERT immortalization and phenotypical analysis

    OpenAIRE

    Yin, Zhanhai; Wang, Qi; Li, Ye; Wei, Hong; Shi, Jianfeng; Li, Ang

    2016-01-01

    Background Stem cells from human exfoliated deciduous teeth (SHED) have recently attracted attention as novel multipotential stem cell sources. However, their application is limited due to in vitro replicative senescence. Ectopic expression of telomerase reverse transcriptase (TERT) is a promising strategy for overcoming this replicative senescence. Nevertheless, its potential application and the phenotype as well as tumorigenicity have never been assessed in SHED. Methods TERT expression was...

  19. Characterization of immortalized human umbilical and iliac vein endothelial cell lines after transfection with SV40 large T-antigen.

    Science.gov (United States)

    van Leeuwen, E B; Veenstra, R; van Wijk, R; Molema, G; Hoekstra, A; Ruiters, M H; van der Meer, J

    2000-01-01

    Most in vitro studies of human endothelial cells have relied on cells derived from human umbilical veins (HUVEC); however, heterogeneity of primary cultured endothelial cells can make critical interpretation of results difficult. Several endothelial cell lines have been produced to serve as a more constant source of endothelial cells. In this study, we characterized the endothelial cell lines EVLB3 and EVLC2 derived from HUVEC, and EVLK1 and EVLK2 derived from human iliac vein endothelial cells (HIVEC). These cell lines maintained the typical endothelial cell cobblestone morphology and appeared to be growth factor independent. They lost PECAM-1 and von Willebrand factor, GP96 was reduced to the level of vascular smooth muscle cells (SMC), but aSMC-actin was far less than in vascular SMC. Antigen levels of tissue-type plasminogen activator (tPA) and plasminogen activator inhibitor (PAI-1) were comparable with young endothelial cells, and mRNA was present for tPA, PAI-1, tissue factor (TF), tissue factor pathway inhibitor and thrombomodulin. This study revealed that mRNA and protein expression of coagulation and fibrinolytic factors was influenced by the stage of cell confluence. No differences could be detected between the endothelial cell lines derived from HUVEC and HIVEC. These cell lines may be a useful tool for studies on cellular interactions of fibrinolytic components or exploring the regulation of TF expression.

  20. Calcium spirulan derived from Spirulina platensis inhibits herpes simplex virus 1 attachment to human keratinocytes and protects against herpes labialis.

    Science.gov (United States)

    Mader, Julia; Gallo, Antonio; Schommartz, Tim; Handke, Wiebke; Nagel, Claus-Henning; Günther, Patrick; Brune, Wolfram; Reich, Kristian

    2016-01-01

    Chronic infections with herpes simplex virus (HSV) type 1 are highly prevalent in populations worldwide and cause recurrent oral lesions in up to 40% of infected subjects. We investigated the antiviral activity of a defined Spirulina platensis microalga extract and of purified calcium spirulan (Ca-SP), a sulfated polysaccharide contained therein. The inhibitory effects of HSV-1 were assessed by using a plaque reduction assay and quantitative PCR in a susceptible mammalian epithelial cell line and confirmed in human keratinocytes. Time-of-addition and attachment experiments and fluorescence detection of the HSV-1 tegument protein VP16 were used to analyze the mechanism of HSV-1 inhibition. Effects of Ca-SP on Kaposi sarcoma-associated herpesvirus/human herpes virus 8 replication and uptake of the ORF45 tegument protein were tested in human retinal pigment epithelial cells. In an observational trial the prophylactic effects of topically applied Ca-SP were compared with those of systemic and topical nucleoside analogues in 198 volunteers with recurrent herpes labialis receiving permanent lip makeup. Ca-SP inhibited HSV-1 infection in vitro with a potency at least comparable to that of acyclovir by blocking viral attachment and penetration into host cells. Ca-SP also inhibited entry of Kaposi sarcoma-associated herpesvirus/human herpes virus 8. In the clinical model of herpes exacerbation, the prophylactic effect of a Ca-SP and microalgae extract containing cream was superior to that of acyclovir cream. These data indicate a potential clinical use of Ca-SP containing Spirulina species extract for the prophylactic treatment of herpes labialis and suggest possible activity of Ca-SP against infections caused by other herpesviruses. Copyright © 2015 American Academy of Allergy, Asthma & Immunology. Published by Elsevier Inc. All rights reserved.

  1. Immortality versus resurrection in the Christian tradition.

    Science.gov (United States)

    Murphy, Nancey

    2011-10-01

    For those in contemporary society who believe in an afterlife, there are a number of views available. The most common may be based on belief in an immortal soul. However, the early Christian account was, instead, bodily resurrection. As Christianity moved throughout the Mediterranean world, apologists and theologians adapted their teaching on human nature and the afterlife to Greek and Roman philosophies. By the time of Augustine (d. 430), the doctrines of body-soul dualism and immortality of the soul were firmly entrenched in Christian teaching. The incorporation of the concept of an immortal soul into Christian accounts of life after death produced a hybrid account. The body dies, the soul (at least of those who were to be saved) travels to heaven. At the end of history, there would be a general resurrection, and the souls would be reunited with their bodies, although the bodies would be in a transformed, indestructible state. This hybrid account of life after death went largely uncontested until the twentieth century. In this essay, I describe this history and argue for a return to the early Christian view of humans as a unity, not a duality, and for belief in resurrection of the body as the appropriate expectation for eternal life. This would not only be truer to Christian sources, but, valuable, I believe, in focusing Christian attention on the need to care for the environment. © 2011 New York Academy of Sciences.

  2. EGFR signaling downstream of EGF regulates migration, invasion, and MMP secretion of immortalized cells derived from human ameloblastoma.

    Science.gov (United States)

    da Rosa, Marina Rolo Pinheiro; Falcão, Aline Semblano Carreira; Fuzii, Hellen Thais; da Silva Kataoka, Maria Sueli; Ribeiro, André L R; Boccardo, Enrique; de Siqueira, Adriane Sousa; Jaeger, Ruy G; de Jesus Viana Pinheiro, João; de Melo Alves Júnior, Sérgio

    2014-11-01

    Ameloblastoma is an odontogenic tumor characterized by local invasiveness and frequent recurrence. The surrounding stroma, composed of different cell types and extracellular matrix (ECM), may influence ameloblastoma invasive behavior. Furthermore, tumor and stromal cells secrete matrix metalloproteases (MMPs), which, in turn, can modulate the matrix and promote the release of ECM-bound growth factors. Among these growth factors, epidermal growth factor (EGF) and its receptor, EGFR, have already been shown to stimulate MMP synthesis, suggesting that an interdependent mechanism, involving MMP activity and growth factors release, may contribute to tumor invasiveness. The aim of this study was to evaluate the effects of the EGF/EGFR signaling pathway on migration, invasion, and MMP activity, in a primary cell line derived from human ameloblastoma. We established and characterized a primary cell line (AME-1) from a human ameloblastoma sample. This cell line was transduced with human papillomavirus type 16 (HPV16) E6/E7 oncogenes, generating the AME-HPV continuous cell line. EGF, MMP2, and MMP9 expression in ameloblastoma biopsies and in the AME-HPV cell line was analyzed by immunohistochemistry and immunofluorescence, respectively. Migratory activity of EGF-treated AME-HPV cells was investigated using monolayer wound assays and Transwell chambers. EGF-induced invasion was assessed in Boyden chambers coated with Matrigel. Conditioned medium from EGF-treated cells was subjected to zymography. EGFR expression in AME-HPV cells was silenced by small interfering RNA (siRNA), to verify the relationship between this receptor and MMP secretion. Ameloblastoma samples and AME-HPV cells expressed EGF, EGFR, MMP2, and MMP9. AME-HPV cells treated with EGF showed increased rates of migration and invasion, as well as enhanced MMP2 and MMP9 activity. EGFR knockdown decreased MMP2 and MMP9 levels in AME-HPV cells. EGFR signaling downstream of EGF probably regulates migration, invasion

  3. Chitosan-shelled oxygen-loaded nanodroplets abrogate hypoxia dysregulation of human keratinocyte gelatinases and inhibitors: New insights for chronic wound healing

    Energy Technology Data Exchange (ETDEWEB)

    Khadjavi, Amina [Dipartimento di Neuroscienze, Università di Torino, Torino (Italy); Magnetto, Chiara [Istituto Nazionale di Ricerca Metrologica (INRIM), Torino (Italy); Panariti, Alice [Dipartimento di Scienze della Salute, Università di Milano Bicocca, Monza (Italy); Argenziano, Monica [Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, Torino (Italy); Gulino, Giulia Rossana [Dipartimento di Oncologia, Università di Torino, Torino (Italy); Rivolta, Ilaria [Dipartimento di Scienze della Salute, Università di Milano Bicocca, Monza (Italy); Cavalli, Roberta [Dipartimento di Scienza e Tecnologia del Farmaco, Università di Torino, Torino (Italy); Giribaldi, Giuliana [Dipartimento di Oncologia, Università di Torino, Torino (Italy); Guiot, Caterina [Dipartimento di Neuroscienze, Università di Torino, Torino (Italy); Prato, Mauro, E-mail: mauro.prato@unito.it [Dipartimento di Neuroscienze, Università di Torino, Torino (Italy); Dipartimento di Scienze della Sanità Pubblica e Pediatriche, Università di Torino, Torino (Italy)

    2015-08-01

    Background: : In chronic wounds, efficient epithelial tissue repair is hampered by hypoxia, and balances between the molecules involved in matrix turn-over such as matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) are seriously impaired. Intriguingly, new oxygenating nanocarriers such as 2H,3H-decafluoropentane-based oxygen-loaded nanodroplets (OLNs) might effectively target chronic wounds. Objective: : To investigate hypoxia and chitosan-shelled OLN effects on MMP/TIMP production by human keratinocytes. Methods: : HaCaT cells were treated for 24 h with 10% v/v OLNs both in normoxia or hypoxia. Cytotoxicity and cell viability were measured through biochemical assays; cellular uptake by confocal microscopy; and MMP and TIMP production by enzyme-linked immunosorbent assay or gelatin zymography. Results: : Normoxic HaCaT cells constitutively released MMP-2, MMP-9, TIMP-1 and TIMP-2. Hypoxia strongly impaired MMP/TIMP balances by reducing MMP-2, MMP-9, and TIMP-2, without affecting TIMP-1 release. After cellular uptake by keratinocytes, nontoxic OLNs abrogated all hypoxia effects on MMP/TIMP secretion, restoring physiological balances. OLN abilities were specifically dependent on time-sustained oxygen diffusion from OLN core. Conclusion: : Chitosan-shelled OLNs effectively counteract hypoxia-dependent dysregulation of MMP/TIMP balances in human keratinocytes. Therefore, topical administration of exogenous oxygen, properly encapsulated in nanodroplet formulations, might be a promising adjuvant approach to promote healing processes in hypoxic wounds. - Highlights: • Hypoxia impairs MMP9/TIMP1 and MMP2/TIMP2 balances in HaCaT human keratinocytes. • Chitosan-shelled oxygen-loaded nanodroplets (OLNs) are internalised by HaCaT cells. • OLNs are not toxic to HaCaT cells. • OLNs effectively counteract hypoxia effects on MMP/TIMP balances in HaCaT cells. • OLNs appear as promising and cost-effective therapeutic tools for hypoxic

  4. Loss of viability and induction of apoptosis in human keratinocytes exposed to Staphylococcus aureus biofilms in vitro.

    Science.gov (United States)

    Kirker, Kelly R; Secor, Patrick R; James, Garth A; Fleckman, Philip; Olerud, John E; Stewart, Philip S

    2009-01-01

    Bacteria colonizing chronic wounds are believed to exist as polymicrobial, biofilm communities; however, there are few studies demonstrating the role of biofilms in chronic wound pathogenesis. This study establishes a novel method for studying the effect of biofilms on the cell types involved in wound healing. Cocultures of Staphylococcus aureus biofilms and human keratinocytes (HK) were created by initially growing S. aureus biofilms on tissue culture inserts then transferring the inserts to existing HK cultures. Biofilm-conditioned medium (BCM) was prepared by culturing the insert-supported biofilm in cell culture medium. As a control planktonic-conditioned medium (PCM) was also prepared. Biofilm, BCM, and PCM were used in migration, cell viability, and apoptosis assays. Changes in HK morphology were followed by brightfield and confocal microscopy. After only 3 hours exposure to BCM, but not PCM, HK formed dendrite-like extensions and displayed reduced viability. After 9 hours, there was an increase in apoptosis (p< or =0.0004). At 24 hours, biofilm-, BCM-, and PCM-exposed HK all exhibited reduced scratch closure (p< or =0.0001). The results demonstrated that soluble products of both S. aureus planktonic cells and biofilms inhibit scratch closure. Furthermore, S. aureus biofilms significantly reduced HK viability and significantly increased HK apoptosis compared with planktonic S. aureus.

  5. Metabolic effects of TiO2 nanoparticles, a common component of sunscreens and cosmetics, on human keratinocytes.

    Science.gov (United States)

    Tucci, P; Porta, G; Agostini, M; Dinsdale, D; Iavicoli, I; Cain, K; Finazzi-Agró, A; Melino, G; Willis, A

    2013-03-21

    The long-term health risks of nanoparticles remain poorly understood, which is a serious concern given their prevalence in the environment from increased industrial and domestic use. The extent to which such compounds contribute to cellular toxicity is unclear, and although it is known that induction of oxidative stress pathways is associated with this process, the proteins and the metabolic pathways involved with nanoparticle-mediated oxidative stress and toxicity are largely unknown. To investigate this problem further, the effect of TiO2 on the HaCaT human keratinocyte cell line was examined. The data show that although TiO2 does not affect cell cycle phase distribution, nor cell death, these nanoparticles have a considerable and rapid effect on mitochondrial function. Metabolic analysis was performed to identify 268 metabolites of the specific pathways involved and 85 biochemical metabolites were found to be significantly altered, many of which are known to be associated with the cellular stress response. Importantly, the uptake of nanoparticles into the cultured cells was restricted to phagosomes, TiO2 nanoparticles did not enter into the nucleus or any other cytoplasmic organelle. No other morphological changes were detected after 24-h exposure consistent with a specific role of mitochondria in this response.

  6. Effect of extremely low-frequency electromagnetic fields on antioxidant activity in the human keratinocyte cell line NCTC 2544.

    Science.gov (United States)

    Calcabrini, Cinzia; Mancini, Umberto; De Bellis, Roberta; Diaz, Anna Rita; Martinelli, Maddalena; Cucchiarini, Luigi; Sestili, Piero; Stocchi, Vilberto; Potenza, Lucia

    2016-03-22

    Some epidemiological studies have suggested possible associations between exposure to extremely low-frequency electromagnetic fields (ELF-EMFs) and various diseases. Recently, ELF-EMF has been considered as a therapeutic agent. To support ELF-EMF use in regenerative medicine, in particular in the treatment of skin injuries, we investigated whether significant cell damage occurs after ELF-EMF exposure. Reactive oxygen species (ROS) production was evaluated in the human keratinocyte exposed for 1 H to 50 Hz ELF-EMF in a range of field strengths from 0.25 to 2 G. Significant ROS increases resulted at 0.5 and 1 G and under these flux densities ROS production, glutathione content, antioxidant defense activity, and lipid peroxidation markers were assessed for different lengths of time. Analyzed parameters of antioxidant defense and membrane integrity showed a different trend at two selected magnetic fluxes, with a greater sensitivity of the cells exposed to 0.5 G, especially after 1 H. All significant alterations observed in the first 4 H of exposure reverted to controls 24 H after suggesting that under these conditions, ELF-EMF induces a slight oxidative stress that does not overwhelm the metabolic capacity of the cells or have a cytotoxic effect.

  7. Sonoporation delivery of monoclonal antibodies against human papillomavirus 16 E6 restores p53 expression in transformed cervical keratinocytes.

    Directory of Open Access Journals (Sweden)

    Melissa Togtema

    Full Text Available High-risk types of human papillomavirus (HPV, such as HPV16, have been found in nearly all cases of cervical cancer. Therapies targeted at blocking the HPV16 E6 protein and its deleterious effects on the tumour suppressor pathways of the cell can reverse the malignant phenotype of affected keratinocytes while sparing uninfected cells. Through a strong interdisciplinary collaboration between engineering and biology, a novel, non-invasive intracellular delivery method for the HPV16 E6 antibody, F127-6G6, was developed. The method employs high intensity focused ultrasound (HIFU in combination with microbubbles, in a process known as sonoporation. In this proof of principle study, it was first demonstrated that sonoporation antibody delivery into the HPV16 positive cervical carcinoma derived cell lines CaSki and SiHa was possible, using chemical transfection as a baseline for comparison. Delivery of the E6 antibody using sonoporation significantly restored p53 expression in these cells, indicating the antibody is able to enter the cells and remains active. This delivery method is targeted, non-cytotoxic, and non-invasive, making it more easily translatable for in vivo experiments than other transfection methods.

  8. Release of inflammatory mediators (PGE2, IL-6) by fenofibric acid-photosensitized human keratinocytes and fibroblasts.

    Science.gov (United States)

    Terencio, M C; Guillén, I; Gómez-Lechón, M J; Miranda, M A; Castell, J V

    1998-09-01

    Ultraviolet-A radiation has weak effects on the release of inflammatory mediators by skin cells due to the poor overlap between UVA wavelengths and the absorption spectra of the relevant chromophores of key biomolecules. However, this situation could be very different in the presence of a photosensitizing drug. To investigate this issue, we have irradiated human skin cells (keratinocytes and fibroblasts) in the presence of fenofibric acid (the active phototoxic metabolite of fenofibrate). The results of this research show a dual effect on the production/release of inflammatory mediators: the synthesis of the proinflammatory cytokine interleukin-6 becomes strongly inhibited at photosensitizer concentrations that clearly stimulate the production of prostaglandins (PGE2) by skin cells. We have found evidences showing that the de novo synthesis of cytokines is inhibited in photosensitized cells due to the fact that cellular mRNA is degraded. Interestingly, when the medium taken from irradiated cultures is added to nonexposed cells, a significant stimulation of cytokine synthesis is observed that can be inhibited by anti-PGE2 antibodies. These observations may be relevant in vivo, where prostaglandins released by photosensitized skin cells could stimulate cytokine synthesis by underlying, nonirradiated cells.

  9. Mechanical response of a living human epidermal keratinocyte sheet as measured in a composite diaphragm inflation experiment.

    Science.gov (United States)

    Selby, John C; Shannon, Mark A

    2007-01-01

    Sheets of normal human epidermal keratinocytes (NHEKs) were reconstituted in vitro on tensed but highly compliant, freestanding polydimethylsiloxane (PDMS) membranes, 5.0 mm in diameter and 10 mum thick. NHEK-PDMS composite diaphragm (CD) specimens were then subjected to cyclical axisymmetric inflation tests to investigate epithelial sheet rheology under conditions of physiologically severe deformations (~50% nominal polar biaxial strains). Because the compliance of the specially formulated PDMS membrane was greater than that of the attached cell layer, the finite load-deformation behavior (mechanical response) of the living NHEK sheet was inferred from differences between the mechanical behavior of the CD specimen and the response of the underlying PDMS membrane measured prior to cell culture. In these composite diaphragm inflation (CDI) experiments, interconnected NHEKs exhibited rheological behaviors that were suggestive of a viscoelastic-plastic stress response. Remarkably, specimens returned to quiescent culture following a sequence of inflation tests recovered at least 80% of their original ability to store elastic strain energy, evidence of biological adaptation and recovery or restitutio ad integrum. Unlike methodologies that assay the morphological or biochemical response of cultured cells to an applied mechanostimulus, CDI experiments can be used to probe the load-bearing functions of desmosomes and adherens junctions within a living epithelial sheet, as well as to assess the rheological behaviors of the intermediate filament and microfilament networks that these cell-cell junctions serve to interconnect.

  10. Cytogenotoxicity of selected organophosphate insecticides on HaCaT keratinocytes and NL-20 human bronchial cells.

    Science.gov (United States)

    Arteaga-Gómez, Eduardo; Rodríguez-Levis, Alejandra; Cortés-Eslava, Josefina; Arenas-Huertero, Francisco; Valencia-Quintana, Rafael; Gómez-Arroyo, Sandra

    2016-02-01

    Organophosphate insecticides (OI) are widely used. To humans the main routes of exposure are skin and inhalation. For this, keratinocytes (HaCaT) and bronchial cells (NL-20) were used as cell culture models to evaluate the effects of OI. The aim of this study was to evaluate the effect of four OI on HaCaT and NL-20 cells: azinphos-methyl, (AM); parathion-methyl (PM); omethoate (OM); and methamidophos (MET). Cells were exposed to 0.1, 1 and 10 μg/μL of each. Results showed a decrease in cell viability in both cell lines. Viability of the NL-20 cell line decreased with the three concentrations of OM. All differences were significant (p insecticides except MET, induced cell death. MET caused DNA damage in HaCaT cells at all concentrations. Differences were significant (p insecticide. Quantitative real time-polymerase chain reaction (RT-qPCR) showed an increase of BN1 gene in HaCaT by effect of AM and MET at 1 μg/μL. In conclusion, all the insecticides induced different levels of cyto and genotoxic effects in both cell lines.

  11. Bmi-1 extends the life span of normal human oral keratinocytes by inhibiting the TGF-{beta} signaling

    Energy Technology Data Exchange (ETDEWEB)

    Kim, Reuben H., E-mail: rkim@dentistry.ucla.edu [UCLA School of Dentistry, Los Angeles, CA 90095 (United States); UCLA Dental Research Institute, Los Angeles, CA 90095 (United States); UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA 90095 (United States); Lieberman, Mark B.; Lee, Rachel [UCLA School of Dentistry, Los Angeles, CA 90095 (United States); Shin, Ki-Hyuk [UCLA School of Dentistry, Los Angeles, CA 90095 (United States); UCLA Dental Research Institute, Los Angeles, CA 90095 (United States); UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA 90095 (United States); Mehrazarin, Shebli; Oh, Ju-Eun [UCLA School of Dentistry, Los Angeles, CA 90095 (United States); Park, No-Hee [UCLA School of Dentistry, Los Angeles, CA 90095 (United States); UCLA Dental Research Institute, Los Angeles, CA 90095 (United States); UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA 90095 (United States); David Geffen School of Medicine at UCLA, Los Angeles, CA 90095 (United States); Kang, Mo K., E-mail: mkang@dentistry.ucla.edu [UCLA School of Dentistry, Los Angeles, CA 90095 (United States); UCLA Dental Research Institute, Los Angeles, CA 90095 (United States); UCLA Jonsson Comprehensive Cancer Center, Los Angeles, CA 90095 (United States)

    2010-10-01

    We previously demonstrated that Bmi-1 extended the in vitro life span of normal human oral keratinocytes (NHOK). We now report that the prolonged life span of NHOK by Bmi-1 is, in part, due to inhibition of the TGF-{beta} signaling pathway. Serial subculture of NHOK resulted in replicative senescence and terminal differentiation and activation of TGF-{beta} signaling pathway. This was accompanied with enhanced intracellular and secreted TGF-{beta}1 levels, phosphorylation of Smad2/3, and increased expression of p15{sup INK4B} and p57{sup KIP2}. An ectopic expression of Bmi-1 in NHOK (HOK/Bmi-1) decreased the level of intracellular and secreted TGF-{beta}1 induced dephosphorylation of Smad2/3, and diminished the level of p15{sup INK4B} and p57{sup KIP2}. Moreover, Bmi-1 expression led to the inhibition of TGF-{beta}-responsive promoter activity in a dose-specific manner. Knockdown of Bmi-1 in rapidly proliferating HOK/Bmi-1 and cancer cells increased the level of phosphorylated Smad2/3, p15{sup INK4B}, and p57{sup KIP2}. In addition, an exposure of senescent NHOK to TGF-{beta} receptor I kinase inhibitor or anti-TGF-{beta} antibody resulted in enhanced replicative potential of cells. Taken together, these data suggest that Bmi-1 suppresses senescence of cells by inhibiting the TGF-{beta} signaling pathway in NHOK.

  12. In Vitro Growth of Human Keratinocytes and Oral Cancer Cells into Microtissues: An Aerosol-Based Microencapsulation Technique

    Directory of Open Access Journals (Sweden)

    Wai Yean Leong

    2017-05-01

    Full Text Available Cells encapsulation is a micro-technology widely applied in cell and tissue research, tissue transplantation, and regenerative medicine. In this paper, we proposed a growth of microtissue model for the human keratinocytes (HaCaT cell line and an oral squamous cell carcinoma (OSCC cell line (ORL-48 based on a simple aerosol microencapsulation technique. At an extrusion rate of 20 μL/min and air flow rate of 0.3 L/min programmed in the aerosol system, HaCaT and ORL-48 cells in alginate microcapsules were encapsulated in microcapsules with a diameter ranging from 200 to 300 μm. Both cell lines were successfully grown into microtissues in the microcapsules of alginate within 16 days of culture. The microtissues were characterized by using a live/dead cell viability assay, field emission-scanning electron microscopy (FE-SEM, fluorescence staining, and cell re-plating experiments. The microtissues of both cell types were viable after being extracted from the alginate membrane using alginate lyase. However, the microtissues of HaCaT and ORL-48 demonstrated differences in both nucleus size and morphology. The microtissues with re-associated cells in spheroids are potentially useful as a cell model for pharmacological studies.

  13. On the immortality of television sets: "function" in the human genome according to the evolution-free gospel of ENCODE.

    Science.gov (United States)

    Graur, Dan; Zheng, Yichen; Price, Nicholas; Azevedo, Ricardo B R; Zufall, Rebecca A; Elhaik, Eran

    2013-01-01

    A recent slew of ENCyclopedia Of DNA Elements (ENCODE) Consortium publications, specifically the article signed by all Consortium members, put forward the idea that more than 80% of the human genome is functional. This claim flies in the face of current estimates according to which the fraction of the genome that is evolutionarily conserved through purifying selection is less than 10%. Thus, according to the ENCODE Consortium, a biological function can be maintained indefinitely without selection, which implies that at least 80 - 10 = 70% of the genome is perfectly invulnerable to deleterious mutations, either because no mutation can ever occur in these "functional" regions or because no mutation in these regions can ever be deleterious. This absurd conclusion was reached through various means, chiefly by employing the seldom used "causal role" definition of biological function and then applying it inconsistently to different biochemical properties, by committing a logical fallacy known as "affirming the consequent," by failing to appreciate the crucial difference between "junk DNA" and "garbage DNA," by using analytical methods that yield biased errors and inflate estimates of functionality, by favoring statistical sensitivity over specificity, and by emphasizing statistical significance rather than the magnitude of the effect. Here, we detail the many logical and methodological transgressions involved in assigning functionality to almost every nucleotide in the human genome. The ENCODE results were predicted by one of its authors to necessitate the rewriting of textbooks. We agree, many textbooks dealing with marketing, mass-media hype, and public relations may well have to be rewritten.

  14. Tumorigenic heterogeneity in cancer stem cells evolved from long-term cultures of telomerase-immortalized human mesenchymal stem cells

    DEFF Research Database (Denmark)

    Burns, Jorge S; Abdallah, Basem M; Guldberg, Per

    2005-01-01

    Long-term cultures of telomerase-transduced adult human mesenchymal stem cells (hMSC) may evolve spontaneous genetic changes leading to tumorigenicity in immunodeficient mice (e.g., hMSC-TERT20). We wished to clarify whether this unusual phenotype reflected a rare but dominant subpopulation...... or if the stem cell origin allowed most cells to behave as cancer stem cells. Cultures of the hMSC-TERT20 strain at population doubling 440 were highly clonogenic (94%). From 110 single-cell clones expanded by 20 population doublings, 6 underwent detailed comparison. Like the parental population, each clone had...... tumorigenicity correlated with good viability plus capillary morphogenesis on serum starvation and high cyclin D1 expression. Thus, hMSC-TERT20 clones represent cancer stem cells with hierarchical tumorigenicity, providing new models to explore the stem cell hypothesis for cancer....

  15. Characterization of serum antibodies from women immunized with Gardasil: A study of HPV-18 infection of primary human keratinocytes.

    Science.gov (United States)

    Wang, Hsu-Kun; Wei, Qing; Moldoveanu, Zina; Huh, Warner K; Vu, Huong Lan; Broker, Thomas R; Mestecky, Jiri; Chow, Louise T

    2016-06-01

    The prevalent human papillomaviruses (HPVs) infect human epithelial tissues. Infections by the mucosotropic HPV genotypes cause hyperproliferative ano-genital lesions. Persistent infections by high-risk (HR) HPVs such as HPV-16, HPV-18 and related types can progress to high grade intraepithelial neoplasias and cancers. Prophylactic HPV vaccines are based on DNA-free virus-like particles (VLPs) composed of the major capsid protein L1 of HPV-16, -18, -6 and -11 (Gardasil) or HPV-16 and -18 (Cervarix). Sera from vaccinated animals effectively prevent HPV pseudovirions to infect cell lines and mouse cervical epithelia. Both vaccines have proven to be highly protective in people. HPV pseudovirions are assembled in HEK293TT cells from matched L1 and L2 capsid proteins to encapsidate a reporter gene. Pseudovirions and genuine virions have structural differences and they infect cell lines or primary human keratinocytes (PHKs) with different efficiencies. In this study, we show that sera and isolated IgG from women immunized with Gardasil prevent authentic HPV-18 virions from infecting PHKs, whereas non-immune sera and purified IgG thereof are uniformly ineffective. Using early passage PHKs, neutralization is achieved only if immune sera are added within 2-4h of infection. We attribute the timing effect to a conformational change in HPV virions, thought to occur upon initial binding to heparan sulfate proteoglycans (HSPG) on the cell surface. This interpretation is consistent with the inability of immune IgG bound to or taken up by PHKs to neutralize the virus. Interestingly, the window of neutralization increases to 12-16h in slow growing, late passage PHKs, suggestive of altered cell surface molecules. In vivo, this window might be further lengthened by the time required to activate the normally quiescent basal cells to become susceptible to infection. Our observations help explain the high efficacy of HPV vaccines.

  16. Inhibitors of cysteine cathepsin and calpain do not prevent ultraviolet-B-induced apoptosis in human keratinocytes and HeLa cells

    DEFF Research Database (Denmark)

    Bang, Bo; Baadsgaard, Ole; Skov, Lone

    2004-01-01

    Caspases, members of the cysteine protease family, execute UVB-induced apoptosis in several cell lines and keratinocytes. Several researchers investigating UVB-induced apoptosis have demonstrated a dose-dependent protective effect of the synthetic peptide caspase inhibitor zVAD-fmk. However, z......VAD-fmk displays a dose-dependent protective effect against UVB-induced apoptosis, even at doses higher than those required to block all known proapoptotic caspases. In addition, it is known that zVAD-fmk also inhibits other cysteine proteases including cathepsins and calpains, and these proteases have recently....... This was done by investigating the effect of the irreversible cysteine protease inhibitor zFA-fmk, the cathepsin B inhibitor CA-074-Me and the calpain inhibitor ALLN on the viability of UVB-irradiated human keratinocytes and HeLa cells. At concentrations of 10 microM and above zVAD-fmk conferred partial dose...

  17. Transforming growth factor-beta modulates plasminogen activator activity and plasminogen activator inhibitor type-1 expression in human keratinocytes in vitro.

    Science.gov (United States)

    Wikner, N E; Elder, J T; Persichitte, K A; Mink, P; Clark, R A

    1990-11-01

    Transforming growth factor beta (TGF-beta) is a multifunctional mediator with effects on cellular growth, differentiation, and extracellular matrix (ECM) metabolism. Because TGF-beta stimulates fibronectin expression in cultured human keratinocytes, we wished to determine whether it might also affect ECM degradation through the plasminogen activator (PA)-plasminogen activator inhibitor (PAI) system. Immunofluorescence of human keratinocytes using a monospecific antiserum to type 1 PAI (PAI-1) showed enhanced cellular and ECM staining when they were cultured in the presence of TGF-beta. The antiserum also identified an Mr 50,000 protein in conditioned media that was markedly enhanced by TGF-beta. A corresponding stimulation of PAI-1 mRNA was demonstrated by quantitative RNA blot analysis. Total plasminogen activating activity of conditioned medium was markedly decreased by TGF-beta. Zymography showed this to be at least partially due to decreased secreted urokinase activity. TGF-beta may play an important role in stabilizing the provisional matrix synthesized by keratinocytes in healing wounds.

  18. Microarray-Based Comparisons of Ion Channel Expression Patterns: Human Keratinocytes to Reprogrammed hiPSCs to Differentiated Neuronal and Cardiac Progeny

    Directory of Open Access Journals (Sweden)

    Leonhard Linta

    2013-01-01

    Full Text Available Ion channels are involved in a large variety of cellular processes including stem cell differentiation. Numerous families of ion channels are present in the organism which can be distinguished by means of, for example, ion selectivity, gating mechanism, composition, or cell biological function. To characterize the distinct expression of this group of ion channels we have compared the mRNA expression levels of ion channel genes between human keratinocyte-derived induced pluripotent stem cells (hiPSCs and their somatic cell source, keratinocytes from plucked human hair. This comparison revealed that 26% of the analyzed probes showed an upregulation of ion channels in hiPSCs while just 6% were downregulated. Additionally, iPSCs express a much higher number of ion channels compared to keratinocytes. Further, to narrow down specificity of ion channel expression in iPS cells we compared their expression patterns with differentiated progeny, namely, neurons and cardiomyocytes derived from iPS cells. To conclude, hiPSCs exhibit a very considerable and diverse ion channel expression pattern. Their detailed analysis could give an insight into their contribution to many cellular processes and even disease mechanisms.

  19. 3,4,5-Tricaffeoylquinic Acid Attenuates TRAIL-induced Apoptosis in Human Keratinocytes by Suppressing Apoptosis-related Protein Activation.

    Science.gov (United States)

    Lee, Da Hee; Nam, Yoon Jeong; Lee, Min Sung; Sohn, Dong Suep; Shin, Yong Kyoo; Lee, Chung Soo

    2015-10-01

    Caffeoyl derivatives exhibit antiinflammatory and antioxidant effects. However, the effect of 3,4,5-tricaffeoylquinic acid on the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in keratinocytes that may be involved in skin diseases has not been studied. In this respect, we investigated the effect of 3,4,5-tricaffeoylquinic acid on TRAIL-induced apoptosis in human keratinocytes. 3,4,5-Tricaffeoylquinic acid and oxidant scavengers attenuated the decrease in the cytosolic levels of Bid, Bcl-2, and survivin proteins; the increase in the levels of cytosolic Bax, p53, and phosphorylated p53; the increase in the levels of phosphorylated p38; the increase in the mitochondrial levels of the voltage-dependent anion channel; loss of the mitochondrial transmembrane potential; the release of cytochrome c; activation of caspases (8, 9, and 3); cleavage of poly [ADP-ribose] polymerase-1; production of reactive oxygen species; the depletion of glutathione (GSH); nuclear damage; and cell death in keratinocytes treated with TRAIL. These results suggest that 3,4,5-tricaffeoylquinic acid may reduce TRAIL-induced apoptosis in human keratinocytes by suppressing the activation of the caspase-8 and Bid pathways and the mitochondria-mediated cell death pathway. The effect appears to be associated with the inhibitory effect on the production of reactive oxygen species and depletion of GSH. 3,4,5-Tricaffeoylquinic acid appears to be effective in the prevention of TRAIL-induced apoptosis-mediated skin diseases. Copyright © 2015 John Wiley & Sons, Ltd.

  20. Eupafolin ameliorates COX-2 expression and PGE2 production in particulate pollutants-exposed human keratinocytes through ROS/MAPKs pathways.

    Science.gov (United States)

    Lee, Chiang-Wen; Lin, Zih-Chan; Hsu, Lee-Fen; Fang, Jia-You; Chiang, Yao-Chang; Tsai, Ming-Horng; Lee, Ming-Hsueh; Li, Shu-Yu; Hu, Stephen Chu-Sung; Lee, I-Ta; Yen, Feng-Lin

    2016-08-02

    Eupafolin is a major bioactive compound derived from the methanolic extract of the medicinal herb Phyla nodiflora, which has been used in traditional Chinese medicine to treat various inflammatory diseases. Recently, particulate air pollutants have been shown to induce inflammation of the skin. In this study, we seek to determine whether eupafolin can inhibit the production of inflammatory mediators in a human skin keratinocyte cell line exposed to particulate air pollutants (particulate matter, PM), and determine the molecular mechanisms involved. Human keratinocyte HaCaT cells were treated with PM in the presence or absence of eupafolin. Cyclooxygenase-2 (COX-2) protein and gene expression levels were determined by Western blotting, RT-PCR and luciferase activity assay. Prostaglandin E2 (PGE2) production was evaluated by the enzyme immunoassay method. Generation of intracellular reactive oxygen species (ROS) was measured by the dichlorofluorescin (DCFH) oxidation assay, and nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was determined by a chemiluminescence assay. For in vivo studies, COX-2 expression in the skin of BALB/c nude mice was analyzed by immunohistochemistry. Eupafolin inhibited PM-induced COX-2 protein and gene expression and PGE2 production in HaCaT cells. In addition, eupafolin suppressed PM-induced intracellular ROS generation, NADPH oxidase activity, MAPK (ERK, JNK and p38) activation and NK-κB activation. In vivo studies showed that topical treatment with eupafolin inhibited COX-2 expression in the epidermal keratinocytes of PM-treated mice. Eupafolin exerts anti-inflammatory and antioxidant effects on skin keratinocytes exposed to particulate air pollutants, and may have potential use in the treatment or prevention of air pollutant-induced inflammatory skin diseases in the future. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  1. UVB-irradiated human keratinocytes and interleukin-1αindirectly increase MAP kinase/AP-1 activation and MMP-1 production in UVA-irradiated dermal fibroblasts

    Institute of Scientific and Technical Information of China (English)

    WANG Xiao-yong; BI Zhi-gang

    2006-01-01

    Background Solar ultraviolet (UV) irradiation induces the production of matrix metalloproteinases (MMPs) by activating cellular signalling transduction pathways. MMPs are responsible for the degradation and/or inhibition of synthesis of collagenous extracellular matrix in connective tissues. We mimicked the action of environmental ultraviolet on skin and investigated the effects of UVB-irradiated human keratinocytes HaCaT and IL-1α on mitogen activated protein (MAP) kinase activation, c-Jun and c-Fos (AP-1 is composed of Jun and Fos proteins)mRNA expression and MMP-1 production in UVA-irradiated dermal fibroblasts.Methods Following UVA irradiation, the culture medium of fibroblasts was replaced by culture medium from UVB-irradiated HaCaT, or replaced by the complete culture medium with interleukin (IL)-1α. MAP kinase activity expression in fibroblasts was detected by Western blot. c-Jun and c-Fos mRNA expressions were determined by reverse transcriptional polymerase chain reaction (RT-PCR); MMP-1 production in culture medium was detected by enzyme-linked immunosorbent assay (ELISA).Results Culture medium from UVB-irradiated keratinocytes increased MAP kinase activity and c-Jun mRNA expression in UVA-irradiated fibroblasts. IL-1α increased MAP kinase activity and c-Jun mRNA expression,IL-1 α also increased c-Fos mRNA expression. Both culture media from UVB-irradiated human keratinocytes and externally applied IL-1 α increased MMP-1 production in UVA-irradiated fibroblasts.Conclusions UVB-irradiated keratinocytes and IL-1α indirectly promote MMP-1 production in UVA-irradiated fibroblasts by increasing MAP kinase/AP-1 activity. IL-1 may play an important role in the paracrine activation and dermal collagen excessive degradation leading to skin photoaging.

  2. Investigation of the in vitro therapeutic efficacy of nilotinib in immortalized human NF2-null vestibular schwannoma cells.

    Directory of Open Access Journals (Sweden)

    Nesrin Sabha

    Full Text Available Vestibular schwannomas (VS are a common posterior fossa brain tumor, and though benign can cause significant morbidity, particularly loss of hearing, tinnitus, vertigo and facial paralysis. The current treatment options for VS include microsurgical resection, stereotactic radiosurgery or close surveillance monitoring, with each treatment option carrying associated complications and morbidities. Most importantly, none of these options can definitively reverse hearing loss or tinnitus. Identification of a novel medical therapy, through the use of targeted molecular inhibition, is therefore a highly desirable treatment strategy that may minimize complications arising from both tumor and treatment and more importantly be suitable for patients whose options are limited with respect to surgical or radiosurgical interventions. In this study we chose to examine the effect of Nilotinib on VS. Nilotinib (Tasigna® is a second-generation receptor tyrosine kinase (RTK inhibitor with a target profile similar to that of imatinib (Gleevec®, but increased potency, decreased toxicity and greater cellular and tissue penetration. Nilotinib targets not only the BCR-ABL oncoprotein, but also platelet-derived growth factor (PDGF receptor signalling. In this preclinical study, the human NF2-null schwannoma cell line HEI-193 subjected to nilotinib inhibition demonstrated decreased viability, proliferation and anchorage-independent growth, and increased apoptosis. A daily dose of nilotinib for 5 days inhibited HEI-I93 proliferation at a clinically-relevant concentration in a dose-dependent manner (IC(50 3-5 µmol/L in PDGF-stimulated cells. These anti-tumorigenic effects of nilotinib were correlated to inhibited activation of PDGFR-α and PDGFR-β and major downstream signalling pathways. These experiments support a therapeutic potential for Nilotinib in VS.

  3. MEETING AT CAMBRIDGE, MA: GENE EXPRESSION IN NORMAL HUMAN KERATINOCYTES MODULATED BY TRIVALENT ARSENICALS

    Science.gov (United States)

    Arsenic exposure has been correlated with the development of several human cancers including those found in the skin, lung, liver, kidney and urinary bladder. Humans are generally exposed to inorganic forms of arsenic, which may be inhaled or ingested. Arsenic forms mono- and d...

  4. MEETING AT SAN DIEGO, CA: GENE EXPRESSION IN NORMAL HUMAN KERATINOCYTES MODULATED BY TRIVALENT ARSENICALS

    Science.gov (United States)

    Arsenic exposure has been correlated with the development of several human cancers including those found in the skin, lung, liver, kidney and urinary bladder. Humans are generally exposed to inorganic forms of arsenic, which may be inhaled or ingested. Arsenic forms mono- and di-...

  5. Astaxanthin and withaferin A block paracrine cytokine interactions between UVB-exposed human keratinocytes and human melanocytes via the attenuation of endothelin-1 secretion and its downstream intracellular signaling.

    Science.gov (United States)

    Niwano, Takao; Terazawa, Shuko; Nakajima, Hiroaki; Wakabayashi, Yuki; Imokawa, Genji

    2015-06-01

    Paracrine interactions between keratinocytes and melanocytes via cytokines play an essential role in regulating pigmentation in epidermal hyperpigmentary disorders. There is an urgent need for a human epidermal model in which melanogenic paracrine interactions between UVB-exposed keratinocytes and melanocytes can be precisely evaluated because human epidermal equivalents consisting of multilayered keratinocytes and melanocytes have significant limitations in this respect. To resolve this challenge, we established a co-culture system with cell inserts using human keratinocytes and human melanocytes that serves as an appropriate new model for UVB-induced hyperpigmentation. Using that new model, we examined the blocking effects of two natural chemicals, astaxanthin and withaferin A, on paracrine cytokine interactions between UVB-exposed keratinocytes and melanocytes and characterized their mechanisms of action. RT-PCR analysis showed that co-culture of human keratinocytes that had been exposed to UVB significantly stimulated human melanocytes to increase their expression of genes encoding microphthalmia-associated transcription factor, tyrosinase and tyrosinase-related protein 1. The catalytic activity of tyrosinase was also increased. ELISA assays revealed that UVB significantly increased the secretion of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1 but not α-melanocyte stimulating hormone. The addition of an endothelin-1 neutralizing antibody significantly abrogated the increase of tyrosinase activity. Post-irradiation treatment with astaxanthin or withaferin A significantly abolished the up-regulation of tyrosinase activity induced by UVB. Treatment with astaxanthin or withaferin A significantly reduced the increased levels of interleukin-1α, interleukin-6/8, granulocyte macrophage stimulatory factor and endothelin-1. Withaferin A but not astaxanthin also significantly abrogated the endothelin-1-stimulated activity

  6. Inflammatory responses of a human keratinocyte cell line to 10 nm citrate- and PEG-coated silver nanoparticles

    Science.gov (United States)

    Bastos, V.; Brown, D.; Johnston, H.; Daniel-da-Silva, A. L.; Duarte, I. F.; Santos, C.; Oliveira, H.

    2016-07-01

    Silver nanoparticles (AgNPs) are among the most commonly used engineered NPs and various commercially available products are designed to come in direct contact with the skin (wound dressings, textiles, creams, among others). Currently, there is limited understanding of the influence of coatings on the toxicity of AgNPs and in particular their ability to impact on AgNP's mediated inflammatory responses. As AgNPs are often stabilized by different coatings, including citrate and polyethyleneglycol (PEG), in this study we investigate the influence of citrate (Cit10) or PEG (PEG10) coatings to 10 nm AgNP on skin, using human HaCaT keratinocytes. AgNPs cytotoxicity and inflammatory response (nuclear factor (NF)-κB induction and cytokine production) of HaCaT were assessed after in vitro exposure to 10 and 40 µg/mL after 4, 24, and 48 h. Results showed that although both types of coated AgNPs decreased cell proliferation and viability, Cit10 AgNPs were more toxic. NF-κB inhibition was observed for the highest concentration (40 µg/mL) of PEG10 AgNPs, and the putative link to early apoptotic pathways observed in these cells is discussed. No production of IL-1β, IL-6, IL-10, and TNFα was stimulated by AgNPs. Furthermore, Cit10 and PEG10 AgNPs decreased the release of MCP-1 by HaCaT cells after 48 h of exposure. As cytokines are vital for the immunologic regulation in the human body, and it is demonstrated that they may interfere with NPs, more research is needed to understand how different AgNPs affect the immune system.

  7. Keratinocyte-specific stat3 heterozygosity impairs development of skin tumors in human papillomavirus 8 transgenic mice.

    Science.gov (United States)

    De Andrea, Marco; Rittà, Massimo; Landini, Manuela M; Borgogna, Cinzia; Mondini, Michele; Kern, Florian; Ehrenreiter, Karin; Baccarini, Manuela; Marcuzzi, Gian Paolo; Smola, Sigrun; Pfister, Herbert; Landolfo, Santo; Gariglio, Marisa

    2010-10-15

    Human papillomaviruses (HPV) of the genus β are thought to play a role in human skin cancers, but this has been difficult to establish using epidemiologic approaches. To gain insight into the transforming activities of β-HPV, transgenic mouse models have been generated that develop skin tumors. Recent evidence suggests a central role of signal transducer and activator of transcription 3 (Stat3) as a transcriptional node for cancer cell-autonomous initiation of a tumor-promoting gene signature associated with cell proliferation, cell survival, and angiogenesis. Moreover, high levels of phospho-Stat3 have been detected in tumors arising in HPV8-CER transgenic mice. In this study, we investigate the in vivo role of Stat3 in HPV8-induced skin carcinogenesis by combining our established experimental model of HPV8-induced skin cancer with epidermis-restricted Stat3 ablation. Stat3 heterozygous epidermis was less prone to tumorigenesis than wild-type epidermis. Three of the 23 (13%) Stat3(+/-):HPV8 animals developed tumors within 12 weeks of life, whereas 54.3% of Stat3(+/+):HPV8 mice already exhibited tumors in the same observation period (median age for tumor appearance, 10 weeks). The few tumors that arose in the Stat3(+/-):HPV8 mice were benign and never progressed to a more malignant phenotype. Collectively, these results offer direct evidence of a critical role for Stat3 in HPV8-driven epithelial carcinogenesis. Our findings imply that targeting Stat3 activity in keratinocytes may be a viable strategy to prevent and treat HPV-induced skin cancer.

  8. The IL-8 release from cultured human keratinocytes, mediated by antibodies to bullous pemphigoid autoantigen 180, is inhibited by dapsone

    Science.gov (United States)

    Schmidt, E; Reimer, S; Kruse, N; Bröcker, E-B; Zillikens, D

    2001-01-01

    Bullous pemphigoid (BP) is a subepidermal blistering disease associated with autoantibodies to the hemidesmosomal 180 kD BP autoantigen (BP180). However, the binding of autoantibodies to BP180 alone is not sufficient for blister formation in this disease and the infiltration of neutrophils into the skin is required. Dapsone and nicotinamide inhibit neutrophil chemotaxis and are used effectively in treating BP. IL-8 is a known chemoattractant for neutrophils and has been implicated in the inflammatory process of both human and experimental murine BP. We have recently shown that antibodies to BP180 mediate a dose and time-dependent release of IL-6 and IL-8 from cultured normal human epidermal keratinocytes (NHEK). In the present study, we addressed the question whether dapsone or nicotinamide influence this cytokine release. We demonstrate that dapsone, but not nicotinamide, in its pharmacological range, inhibits the IL-8, but not the IL-6 release from NHEK, induced by anti-BP180 IgG, in a dose-dependent fashion as detected by ELISA. IL-8 mRNA levels, as determined by RT-PCR, were the same in cells treated with BP IgG alone compared to cells treated with BP IgG plus dapsone. This observation suggests that dapsone inhibits the BP IgG-induced IL-8 release from cultured NHEK by mechanisms at the post-transcriptional level. Our findings contribute to the understanding how dapsone leads to a reduced influx of neutrophils into BP lesions and, finally, to the cessation of blister formation in this disease. PMID:11359455

  9. Acquisition of high-level chromosomal instability is associated with integration of human papillomavirus type 16 in cervical keratinocytes.

    Science.gov (United States)

    Pett, Mark R; Alazawi, William O F; Roberts, Ian; Dowen, Sally; Smith, David I; Stanley, Margaret A; Coleman, Nicholas

    2004-02-15

    Whereas two key steps in cervical carcinogenesis are integration of high-risk human papillomavirus (HR-HPV) and acquisition of an unstable host genome, the temporal association between these events is poorly understood. Chromosomal instability is induced when HR-HPV E7 oncoprotein is overexpressed from heterologous promoters in vitro. However, it is not known whether such events occur at the "physiologically" elevated levels of E7 produced by deregulation of the homologous HR-HPV promoter after integration. Indeed, an alternative possibility is that integration in vivo is favored in an already unstable host genome. We have addressed these issues using the unique human papillomavirus (HPV) 16-containing cervical keratinocyte cell line W12, which was derived from a low-grade squamous intraepithelial lesion and thus acquired HPV16 by "natural" infection. Whereas W12 at low passage contains HPV16 episomes only, long-term culture results in the emergence of cells containing integrated HPV16 only. We show that integration of HPV16 in W12 is associated with 3' deletion of the E2 transcriptional repressor, resulting in deregulation of the homologous promoter of the integrant and an increase in E7 protein levels. We further demonstrate that high-level chromosomal instability develops in W12 only after integration and that the forms of instability observed correlate with the physical state of HPV16 DNA and the level of E7 protein. Whereas intermediate E7 levels are associated with numerical chromosomal abnormalities, maximal levels are associated with both numerical and structural aberrations. HR-HPV integration is likely to be a critical event in cervical carcinogenesis, preceding the development of chromosomal abnormalities that drive malignant progression.

  10. Effects of the novel compound DK223 ([1E,2E-1,2-Bis(6-methoxy-2H-chromen-3-yl)methylene]hydrazine) on migration and proliferation of human keratinocytes and primary dermal fibroblasts.

    Science.gov (United States)

    Ho, Manh Tin; Kang, Hyun Sik; Huh, Jung Sik; Kim, Young Mee; Lim, Yoongho; Cho, Moonjae

    2014-07-23

    Wound healing plays an important role in protecting the human body from external infection. Cell migration and proliferation of keratinocytes and dermal fibroblasts are essential for proper wound healing. Recently, several studies have demonstrated that secondary compounds produced in plants could affect skin cells migration and proliferation. In this study, we identified a novel compound DK223 ([1E,2E-1,2-bis(6-methoxy-2H-chromen-3-yl)methylene]hydrazine) that concomitantly induced human keratinocyte migration and dermal fibroblast proliferation. We evaluated the regulation of epithelial and mesenchymal protein markers, such as E-cadherin and Vimentin, in human keratinocytes, as well as extracellular matrix (ECM) secretion and metalloproteinase families in dermal fibroblasts. DK223 upregulated keratinocyte migration and significantly increased the epithelial marker E-cadherin in a time-dependent manner. We also found that reactive oxygen species (ROS) increased significantly in keratinocytes after 2 h of DK223 exposure, returning to normal levels after 24 h, which indicated that DK223 had an early shock effect on ROS production. DK223 also stimulated fibroblast proliferation, and induced significant secretion of ECM proteins, such as collagen I, III, and fibronectin. In dermal fibroblasts, DK223 treatment induced TGF-β1, which is involved in a signaling pathway that mediates proliferation. In conclusion, DK223 simultaneously induced both keratinocyte migration via ROS production and fibroblast proliferation via TGF-β1 induction.

  11. Effects of the Novel Compound DK223 ([1E,2E-1,2-Bis(6-methoxy-2H-chromen-3-ylmethylene]hydrazine on Migration and Proliferation of Human Keratinocytes and Primary Dermal Fibroblasts

    Directory of Open Access Journals (Sweden)

    Manh Tin Ho

    2014-07-01

    Full Text Available Wound healing plays an important role in protecting the human body from external infection. Cell migration and proliferation of keratinocytes and dermal fibroblasts are essential for proper wound healing. Recently, several studies have demonstrated that secondary compounds produced in plants could affect skin cells migration and proliferation. In this study, we identified a novel compound DK223 ([1E,2E-1,2-bis(6-methoxy-2H-chromen-3-ylmethylene]hydrazine that concomitantly induced human keratinocyte migration and dermal fibroblast proliferation. We evaluated the regulation of epithelial and mesenchymal protein markers, such as E-cadherin and Vimentin, in human keratinocytes, as well as extracellular matrix (ECM secretion and metalloproteinase families in dermal fibroblasts. DK223 upregulated keratinocyte migration and significantly increased the epithelial marker E-cadherin in a time-dependent manner. We also found that reactive oxygen species (ROS increased significantly in keratinocytes after 2 h of DK223 exposure, returning to normal levels after 24 h, which indicated that DK223 had an early shock effect on ROS production. DK223 also stimulated fibroblast proliferation, and induced significant secretion of ECM proteins, such as collagen I, III, and fibronectin. In dermal fibroblasts, DK223 treatment induced TGF-β1, which is involved in a signaling pathway that mediates proliferation. In conclusion, DK223 simultaneously induced both keratinocyte migration via ROS production and fibroblast proliferation via TGF-β1 induction.

  12. Immortality in the Christian Physicalistic Theology: A Critical Survey

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    Hasan Ahmadizade

    2017-07-01

    Full Text Available Physicalistic Theology is a term that has no exact definition in theologian views. In the 20th century some of Christian thinkers on theology, like Nancy Murphy and Peter van Inwagen, by accepting a Physicalistic approach on human being, tried to analyze the Christian beliefs about human identity and his immortality. This approach today is called Physicalistic Theology. According to this approach, human is not but this physical body itself and so we can simply analyze the immortality problem. In this article we try to by an analytic and descriptive method, analyze the immortality of human according to the view of Physicalistic Theology. We will analyze the most important reasoning of Physicalistic Theology that is: no-interaction between the material and the immaterial, interaction between the person and the body, and the physicalism in Christian beliefs. One of the conclusions of this article is that according to Physicalistic view, the person that at some time has not been in the world, must exists any time to destroyed forever because the Christians believe to things that cannot justify rationally. The problem of immortality is one of these matters. Physicalistic Theology try to prove the immortality based on the miracles and the absolute power of God.

  13. APOPTOSIS GENE EXPRESSION IN HUMAN EPDERMAL KERATINOCYTES TREATED WITH SODIUM ARSENITE USING REAL TIME PCR ARRAY

    Science.gov (United States)

    Arsenic exposure via contaminated drinking water is a great public health concern worldwide. Chronic arsenic exposure has been associated with human skin, lung and bladder cancer and other chronic effects. We have previous reported that sodium arsenite stimulated cell proliferati...

  14. Triggering of toll-like receptors 2 and 4 by Aspergillus fumigatus conidia in immortalized human corneal epithelial cells to induce inflammatory cytokines

    Institute of Scientific and Technical Information of China (English)

    ZHAO Jie; WU Xin-yi

    2008-01-01

    Background Cornea epithelial cells play early and crucial roles in the initiation of ocular surface responses to pathogens.Participation of toll-like receptor(TLR)2 and TLR4,which are major forms of fungi receptors,may be involved in Aspergillus fumigatus induced immune responses.The obiective of the present study was to examine whether inactive Aspergillus fumigatus conidia induce NF-κB activation and production of proinflammaory cytokines,and whether the expression of TLR2 and TLR4 were amplified by conidia in cultured immortalized human corneal epithelial cells (THCEs).This may contribute to our knowledge of the mechanism by which the host cornea can successfully defend against invasive fungi.Methods Aspergillus fumigatus conidia were used to challenge THCE cells.THCE cells were harvested after 0.5,1,2or 4 hours incubation.Real-time quantitative PCR was performed to determine the expression of TLR2,TLR4,TNF-α and IL-8.Western blotting was performed to determine the expression of NF-κB.Enzyme-linked immunosorbent assay (EUSA)was performed to determine the expression of TNF-α and IL-8.And the release of TNF-α and IL-8 in the cell supematant were also assessed by ELISA with or without pretreatment with TLR2 and TLR4 neutralizing antibodies.Results Aspergillus fumigatus conidia elicited the expression of TLR2,TLR4,TNF-α and IL-8 mRNA in THCEs.Exposure of THCE cells to Aspergillus fumigatus conidia resulted in NF-κB activation,which increased at 30 minutes (increased from 11.35±2.74 in the controls to 19.12±3.48,P<0.05)and thereafter increased steadily up to 4 hours after challenge(P<0.01).Concomitant with NF-κB acfivation,secretion of TNF-α and IL-8 in conidia-challenged cells was increased in a time-dependent manner.Incubation of THCE cells with TLR2 antibody or TLR4 antibody before conidia challenge resulted in jnhibifion of conidia-induced TNF-α and IL-8 secretion(P<0.05),TLR2 antibody and TLR4 antibody together significantly increased

  15. Establishment of Immortalized Human Lymphocytes Cell Lines with Multiple SNP Genotyping in Chinese Han Population%中国汉族人群代谢酶基因分型永生化淋巴细胞库的建立

    Institute of Scientific and Technical Information of China (English)

    张娟; 熊梦祯; 尹立红; 浦跃朴

    2011-01-01

    目的 建立汉族人群代谢酶基因分型的永生化淋巴细胞库,为单核苷酸多态(SNP)结构对毒物诱导的基因功能表达的影响建立研究平台.方法 用EB病毒(Epstein-Barr Virus,EBV)转化中国汉族健康成人外周血B淋巴细胞,通过对中国汉族人群CYP2E1和NQ01的单核苷酸多态(SNP)位点信息分析,选择中国人群特异性CYP2EI、NQ01基因SNP位点CYP2E1rs2070673,CYP2E1rs2031921,CYP2E13813866,NQO1rs 1800566,NQO1rs10517,应用适配器连接介导的等位基因特异性扩增法(ALM-SAS)对上述位点进行基因分型.将供体信息和细胞株基因分型的信息录入,构建中国汉族基因分型的永生化淋巴细胞库.结果 通过对细胞株进行上述5个SNP位点的基因分型,获得每株细胞的基因分型信息,获得CYP2E1rs2070673,CYP2E1rs2031921,CYP2E13813866,NQO1rs 1800566,NQO1rs10517不同基因分型的永生化淋巴细胞株.结论 通过永生化细胞库的建立、基因分型,初步建立中国的汉族人群代谢酶基因分型永生化淋巴细胞库,该细胞库的进一步建设,可以为研究中国人群环境毒物和药物代谢酶基因结构和功能的关系提供技术平台.%Objective To establish immortalized human lymphocytes cell lines with multiple SNP genotyping of Chinese Han population which could afford a platform to study gene function expression influenced by SNPs structure induced by chemical Methods EBV (Epstein-Bart Virus) were used to immortalize peripheral B lymphocytes derived from Chinese Hah population. After analyzing CYP2EI、NQOI SNP database of Chinese population, the five Chinese specific SNP sites: CYP2EIrs2070673,CYP2Elrs2031921, CYP2E13813866,NQOlrs 1800566, NQOl rs10517 were genotyped by an adapter-ligation mediated allele specific amplification (ALM-ASA) method. The information of providers and genotypes of cell lines were input to establish the database of immortalized human lymphocytes cell lines with multiple SNP genotyping

  16. Protective effect of C. sativa leaf extract against UV mediated-DNA damage in a human keratinocyte cell line.

    Science.gov (United States)

    Almeida, I F; Pinto, A S; Monteiro, C; Monteiro, H; Belo, L; Fernandes, J; Bento, A R; Duarte, T L; Garrido, J; Bahia, M F; Sousa Lobo, J M; Costa, P C

    2015-03-01

    Toxic effects of ultraviolet (UV) radiation on skin include protein and lipid oxidation, and DNA damage. The latter is known to play a major role in photocarcinogenesis and photoaging. Many plant extracts and natural compounds are emerging as photoprotective agents. Castanea sativa leaf extract is able to scavenge several reactive species that have been associated to UV-induced oxidative stress. The aim of this work was to analyze the protective effect of C. sativa extract (ECS) at different concentrations (0.001, 0.01, 0.05 and 0.1 μg/mL) against the UV mediated-DNA damage in a human keratinocyte cell line (HaCaT). For this purpose, the cytokinesis-block micronucleus assay was used. Elucidation of the protective mechanism was undertaken regarding UV absorption, influence on (1)O₂ mediated effects or NRF2 activation. ECS presented a concentration-dependent protective effect against UV-mediated DNA damage in HaCaT cells. The maximum protection afforded (66.4%) was achieved with the concentration of 0.1 μg/mL. This effect was found to be related to a direct antioxidant effect (involving (1)O₂) rather than activation of the endogenous antioxidant response coordinated by NRF2. Electrochemical studies showed that the good antioxidant capacity of the ECS can be ascribed to the presence of a pool of different phenolic antioxidants. No genotoxic or phototoxic effects were observed after incubation of HaCaT cells with ECS (up to 0.1 μg/mL). Taken together these results reinforce the putative application of this plant extract in the prevention/minimization of UV deleterious effects on skin.

  17. MicroRNA-181b negatively regulates the proliferation of human epidermal keratinocytes in psoriasis through targeting TLR4.

    Science.gov (United States)

    Feng, Cheng; Bai, Ming; Yu, Nan-Ze; Wang, Xiao-Jun; Liu, Zeng

    2017-02-01

    Our study aims to explore the role of microRNA-181b (miR-181b) and TLR in the regulation of cell proliferation of human epidermal keratinocytes (HEKs) in psoriasis. Twenty-eight patients diagnosed with psoriasis vulgaris were selected as a case group with their lesional and non-lesional skin tissues collected. A control group consisted of 20 patients who underwent plastic surgery with their healthy skin tissues collected. Real-time quantitative fluorescence polymerase chain reaction (RT-qPCR), in situ hybridization and immunohistochemistry were used to detect the expressions of miR-181b and TLR4 in HEKs of healthy skin, psoriatic lesional skin and non-lesional skin respectively. The 3' untranslated region (3'UTR) of TLR4 combined with miR-181b was verified by a dual-luciferase reporter assay. Western blotting and bromodeoxyuridine were applied for corresponding detection of TLR4 expression and cell mitosis. The expression of miR-181b in HEKs of psoriatic lesional skin was less than healthy skin and psoriatic non-lesional skin. In psoriatic lesional and non-lesional skin, TLR4-positive cell rates and the number of positive cells per square millimetre were higher than healthy skin. The dual-luciferase reporter assay verified that miR-181b targets TLR4. HEKs transfected with miR-181b mimics had decreased expression of TLR4, along with the decrease of mitotic indexes and Brdu labelling indexes. However, HEKs transfected with miR-181b inhibitors showed increased TLR4 expression, mitotic indexes and Brdu labelling indexes. HEKs transfected with both miR-181b inhibitors and siTLR4 had decreased mitotic indexes and Brdu labelling indexes. These results indicate that miR-181b can negatively regulate the proliferation of HEKs in psoriasis by targeting TLR4.

  18. Deoxynivalenol induced mouse skin tumor initiation: Elucidation of molecular mechanisms in human HaCaT keratinocytes.

    Science.gov (United States)

    Mishra, Sakshi; Tewari, Prachi; Chaudhari, Bhushan P; Dwivedi, Premendra D; Pandey, Haushila P; Das, Mukul

    2016-11-01

    Among food contaminants, mycotoxins are toxic to both human and animal health. Our prior studies suggest that Deoxynivalenol (DON), a mycotoxin, behaves as a tumor promoter by inducing edema, hyperplasia, ODC activity and activation of MAPK's in mouse skin. In this study, topical application of DON, 336 and 672 nmol significantly enhanced ROS levels, DNA damage and apoptosis with concomitant downregulation of Ki-67, cyclin D, cyclin E, cyclin A and cyclin-dependent kinases (CDK4 and CDK2) thereby resulting in tumor initiation in mouse skin. Further, the elucidation of molecular mechanisms of tumor initiation by DON (0.42-3.37 nmol/ml) in HaCaT keratinocytes, revealed (i) enhanced ROS generation with cell cycle phase arrest in G0/G1 phase, (ii) increase in levels of 8-OxoG (6-24 hr) and γH2AX protein, (iii) significant enhancement in oxidative stress marker enzymes LPO, GSH, GR with concomitant decrease in antioxidant enzymes catalase, GPx, GST, SOD and mitochondrial membrane potential after DON (1.68 nmol) treatment, (iv) suppression of Nrf2 translocation to nucleus, enhanced phosphorylation with subsequent activation ERK1/2, p38 and JNK MAPK's following DON (1.68 nmol) treatment, (v) overexpression of c-jun, c-fos proteins, upregulation of Bax along with downregulation of Bcl-2 proteins, (vi) increase in cytochrome-c, caspase-9, caspase-3 and poly ADP ribose polymerase levels leads to apoptosis. Pretreatment of superoxide dismutase, mannitol and ethanol to HaCaT cells resulted in significant reduction in ROS levels and apoptosis indicating the role of superoxide and hydroxyl radicals in DON induced apoptosis as an early event and skin tumor initiation as a late event.

  19. Proteomic Analysis of Arsenic-Induced Oxidative Stress in Human Epidermal Keratinocytes

    Science.gov (United States)

    Chronic exposure to inorganic arsenic (IAs) has been associated with the development of several human cancers, including those found in the skin, lung, urinary bladder, liver, prostate and kidney. The precise mechanisms by which arsenic causes cancer are unknown. Defining the mod...

  20. Gene Expression of Normal Human Epidermal Keratinocytes Modulated by Trivalent Arsenicals

    Science.gov (United States)

    Chronic exposure to inorganic arsenic (iAs) is associated with the development of benign and malignant human skin lesions including nonmelanoma skin cancers. The precise arsenical form(s) responsible for this carcinogenic effect are unknown, although trivalent inorganic arsenic (...

  1. Modulation of TLR2 protein expression by a miR-105 in human oral keratinocytes

    Science.gov (United States)

    Mammalian biological processes such as inflammation, involve regulation of hundreds of genes controlling onset and termination. MicroRNAs (miRNAs) can translationally repress target mRNAs and can regulate innate immune responses. Our model system comprised primary human keratinoc...

  2. Association of a genetic polymorphism (-44 C/G SNP in the human DEFB1 gene with expression and inducibility of multiple β-defensins in gingival keratinocytes

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    Dommisch Henrik

    2009-08-01

    Full Text Available Abstract Background Human β-defensins (hBDs are antimicrobial peptides with a role in innate immune defense. Our laboratory previously showed that a single nucleotide polymorphism (SNP in the 5' untranslated region of the hBD1 gene (DEFB1, denoted -44 (rs1800972, is correlated with protection from oral Candida. Because this SNP alters the putative mRNA structure, we hypothesized that it alters hBD1 expression. Methods Transfection of reporter constructs and evaluation of antimicrobial activity and mRNA expression levels in keratinocytes from multiple donors were used to evaluate the effect of this SNP on constitutive and induced levels of expression. Results Transfection of CAT reporter constructs containing the 5' untranslated region showed that the -44 G allele yielded a 2-fold increase in CAT protein compared to other common haplotypes suggesting a cis effect on transcription or translation. The constitutive hBD1 mRNA level in human oral keratinocytes was significantly greater in cells from donors with the -44 GG genotype compared to those with the common CC genotype. Surprisingly, the hBD3 mRNA level as well as antimicrobial activity of keratinocyte extracts also correlated with the -44 G allele. Induced levels of hBD1, hBD2, and hBD3 mRNA were evaluated in keratinocytes challenged with Toll-like receptor 2 and 4 ligands, interleukin-1β, TNFα, and interferon-γ (IFNγ. In contrast to constitutive expression levels, IFNγ-induced keratinocyte hBD1 and hBD3 mRNA expression was significantly greater in cells with the common CC genotype, but there was no clear correlation of genotype with hBD2 expression. Conclusion The DEFB1 -44 G allele is associated with an increase in overall constitutive antimicrobial activity and expression of hBD1 and hBD3 in a manner that is consistent with protection from candidiasis, while the more common C allele is associated with IFNγ inducibility of these β-defensins and is likely to be more protective in

  3. In vivo production of novel vitamin D2 hydroxy-derivatives by human placentas, epidermal keratinocytes, Caco-2 colon cells and the adrenal gland

    Science.gov (United States)

    Slominski, Andrzej T.; Kim, Tae-Kang; Shehabi, Haleem Z.; Tang, Edith; Benson, Heather A. E.; Semak, Igor; Lin, Zongtao; Yates, Charles R.; Wang, Jin; Li, Wei; Tuckey, Robert C.

    2014-01-01

    We investigated the metabolism of vitamin D2 to hydroxyvitamin D2 metabolites ((OH)D2) by human placentas ex-utero, adrenal glands ex-vivo and cultured human epidermal keratinocytes and colonic Caco-2 cells, and identified 20(OH)D2, 17,20(OH)2D2, 1,20(OH)2D2, 25(OH)D2 and 1,25(OH)2D2 as products. Inhibition of product formation by 22R-hydroxycholesterol indicated involvement of CYP11A1 in 20- and 17-hydroxylation of vitamin D2, while use of ketoconazole indicated involvement of CYP27B1 in 1α-hydroxylation of products. Studies with purified human CYP11A1 confirmed the ability of this enzyme to convert vitamin D2 to 20(OH)D2 and 17,20(OH)2D2. In placentas and Caco-2 cells, production of 20(OH)D2 was higher than 25(OH)D2 while in human keratinocytes the production of 20(OH)D2 and 25(OH)D2 were comparable. HaCaT keratinocytes showed high accumulation of 1,20(OH)2D2 relative to 20(OH)D2 indicating substantial CYP27B1 activity. This is the first in vivo evidence for a novel pathway of vitamin D2 metabolism initiated by CYP11A1 and modified by CYP27B1, with the product profile showing tissue- and cell-type specificity. PMID:24382416

  4. DeltaNp63alpha repression of the Notch1 gene supports the proliferative capacity of normal human keratinocytes and cervical cancer cells.

    Science.gov (United States)

    Yugawa, Takashi; Narisawa-Saito, Mako; Yoshimatsu, Yuki; Haga, Kei; Ohno, Shin-ichi; Egawa, Nagayasu; Fujita, Masatoshi; Kiyono, Tohru

    2010-05-15

    The p53 family member p63 is a master regulator of epithelial development. One of its isoforms, DeltaNp63alpha, is predominantly expressed in the basal cells of stratified epithelia and plays a fundamental role in control of regenerative potential and epithelial integrity. In contrast to p53, p63 is rarely mutated in human cancers, but it is frequently overexpressed in squamous cell carcinomas (SCC). However, its functional relevance to tumorigenesis remains largely unclear. We previously identified the Notch1 gene as a novel transcriptional target of p53. Here, we show that DeltaNp63alpha functions as a transcriptional repressor of the Notch1 gene through the p53-responsive element. Knockdown of p63 caused upregulation of Notch1 expression and marked reduction in proliferation and clonogenicity of both normal human keratinocytes and cervical cancer cell lines overexpressing DeltaNp63alpha. Concomitant silencing of Notch1 significantly rescued this phenotype, indicating the growth defect induced by p63 deficiency to be, at least in part, attributable to Notch1 function. Conversely, overexpression of DeltaNp63alpha decreased basal levels of Notch1, increased proliferative potential of normal human keratinocytes, and inhibited both p53-dependent and p53-independent induction of Notch1 and differentiation markers upon genotoxic stress and serum exposure, respectively. These results suggest that DeltaNp63alpha maintains the self-renewing capacity of normal human keratinocytes and cervical cancer cells partly through transcriptional repression of the Notch1 gene and imply a novel pathogenetical significance of frequently observed overexpression of DeltaNp63alpha together with p53 inactivation in SCCs.

  5. Keratinocyte-targeted expression of human laminin γ2 rescues skin blistering and early lethality of laminin γ2 deficient mice.

    Directory of Open Access Journals (Sweden)

    Tracy L Adair-Kirk

    Full Text Available Laminin-332 is a heterotrimeric basement membrane component comprised of the α3, ß3, and γ2 laminin chains. Laminin-332 modulates epithelial cell processes, such as adhesion, migration, and differentiation and is prominent in many embryonic and adult tissues. In skin, laminin-332 is secreted by keratinocytes and is a key component of hemidesmosomes connecting the keratinocytes to the underlying dermis. In mice, lack of expression of any of the three Laminin-332 chains result in impaired anchorage and detachment of the epidermis, similar to that seen in human junctional epidermolysis bullosa, and death occurs within a few days after birth. To bypass the early lethality of laminin-332 deficiency caused by the knockout of the mouse laminin γ2 chain, we expressed a dox-controllable human laminin γ2 transgene under a keratinocyte-specific promoter on the laminin γ2 (Lamc2 knockout background. These mice appear similar to their wild-type littermates, do not develop skin blisters, are fertile, and survive >1.5 years. Immunofluorescence analyses of the skin showed that human laminin γ2 colocalized with mouse laminin α3 and ß3 in the basement membrane zone underlying the epidermis. Furthermore, the presence of "humanized" laminin-332 in the epidermal basement membrane zone rescued the alterations in the deposition of hemidesmosomal components, such as plectin, collagen type XVII/BP180, and integrin α6 and ß4 chains, seen in conventional Lamc2 knockout mice, leading to restored formation of hemidesmosomes. These mice will be a valuable tool for studies of organs deficient in laminin-332 and the role of laminin-332 in skin, including wound healing.

  6. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation.

    Science.gov (United States)

    Bennett, Darin C; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K K; McElwee, Kevin J; Cheng, Kimberly M

    2015-09-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51×faster), ostrich oil (1.46×faster), and rhea oil (1.64×faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35×slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions.

  7. Ratite oils promote keratinocyte cell growth and inhibit leukocyte activation

    Science.gov (United States)

    Bennett, Darin C.; Leung, Gigi; Wang, Eddy; Ma, Sam; Lo, Blanche K. K.; McElwee, Kevin J.; Cheng, Kimberly M.

    2015-01-01

    Traditionally, native Australian aborigines have used emu oil for the treatment of inflammation and to accelerate wound healing. Studies on mice suggest that topically applied emu oil may have anti-inflammatory properties and may promote wound healing. We investigated the effects of ratite oils (6 emu, 3 ostrich, 1 rhea) on immortalized human keratinocytes (HaCaT cells) in vitro by culturing the cells in media with oil concentrations of 0%, 0.5%, and 1.0%. Peking duck, tea tree, and olive oils were used as comparative controls. The same oils at 0.5% concentration were evaluated for their influence on peripheral blood mononuclear cell (PBMC) survival over 48 hr and their ability to inhibit IFNγ production in PBMCs activated by phytohemagglutinin (PHA) in ELISpot assays. Compared to no oil control, significantly shorter population doubling time durations were observed for HaCaT cells cultured in emu oil (1.51 × faster), ostrich oil (1.46 × faster), and rhea oil (1.64 × faster). Tea tree oil demonstrated significant antiproliferative activity and olive oil significantly prolonged (1.35 × slower) cell population doubling time. In contrast, almost all oils, particularly tea tree oil, significantly reduced PBMC viability. Different oils had different levels of inhibitory effect on IFNγ production with individual emu, ostrich, rhea, and duck oil samples conferring full inhibition. This preliminary investigation suggests that emu oil might promote wound healing by accelerating the growth rate of keratinocytes. Combined with anti-inflammatory properties, ratite oil may serve as a useful component in bandages and ointments for the treatment of wounds and inflammatory skin conditions. PMID:26217022

  8. The illusion of cell immortality

    Science.gov (United States)

    Hayflick, L

    2000-01-01

    Normal cultured cell populations are mortal but cells that are immortal are abnormal and most have properties of cancer cells. Nevertheless, this distinction becomes blurred because the terms ‘mortality’ and ‘immortality’ are subject to enormous variations in understanding. Forty years ago we showed that cell mortality and immortality are inextricably linked to longevity determination, ageing and cancer. We suggested that a counting mechanism existed in normal cells and that has now been identified as telomere attrition. This replicometer, in combination with the discovery of the enzyme telomerase, has gone very far in explaining why most normal somatic cells have a finite capacity to replicate both in vivo and in vitro and how immortal cancer cells circumvent this inevitability. It is suggested that telomere attrition may be better understood as a direct measure of longevity determination and to only have an indirect association with age changes. © 2000 Cancer Research Campaign PMID:10970682

  9. Human atopic dermatitis skin-derived T cells can induce a reaction in mouse keratinocytes in vivo

    DEFF Research Database (Denmark)

    Martel, Britta C; Blom, Lars; Dyring-Andersen, Beatrice

    2015-01-01

    In atopic dermatitis (AD), the inflammatory response between skin infiltrating T cells and keratinocytes is fundamental to the development of chronic lesional eczema. The aim of this study was to investigate whether skin-derived T cells from AD patients could induce an inflammatory response in mice...

  10. A Molecular Approach to Promote Wound Healing in the Sulfur Mustard-Exposed Human Keratinocyte Model

    Science.gov (United States)

    2006-11-01

    three NOS Isoforms It has been reported that all major NOS isoforms, that is, NOS1, NOS2 (iNOS), and NOS3 , are present in the human skin, but...The level of NO produced by NOS1 or NOS3 is very low and considered to only have a role in the maintenance of normal skin function (in barrier...All three major NOSs, nNOS (NOS1), iNOS (NOS2), and eNOS ( NOS3 ) are found in the skin. The amount of NO produced by the constitutive nNOS and eNOS

  11. Differential response of human adipose tissue-derived mesenchymal stem cells, dermal fibroblasts, and keratinocytes to burn wound exudates: potential role of skin-specific chemokine CCL27.

    Science.gov (United States)

    van den Broek, Lenie J; Kroeze, Kim L; Waaijman, Taco; Breetveld, Melanie; Sampat-Sardjoepersad, Shakun C; Niessen, Frank B; Middelkoop, Esther; Scheper, Rik J; Gibbs, Susan

    2014-01-01

    Many cell-based regenerative medicine strategies toward tissue-engineered constructs are currently being explored. Cell-cell interactions and interactions with different biomaterials are extensively investigated, whereas very few studies address how cultured cells will interact with soluble wound-healing mediators that are present within the wound bed after transplantation. The aim of this study was to determine how adipose tissue-derived mesenchymal stem cells (ASC), dermal fibroblasts, and keratinocytes will react when they come in contact with the deep cutaneous burn wound bed. Burn wound exudates isolated from deep burn wounds were found to contain many cytokines, including chemokines and growth factors related to inflammation and wound healing. Seventeen mediators were identified by ELISA (concentration range 0.0006-9 ng/mg total protein), including the skin-specific chemokine CCL27. Burn wound exudates activated both ASC and dermal fibroblasts, but not keratinocytes, to increase secretion of CXCL1, CXCL8, CCL2, and CCL20. Notably, ASC but not fibroblasts or keratinocytes showed significant increased secretion of vascular endothelial growth factor (5-fold) and interleukin-6 (253-fold), although when the cells were incorporated in bi-layered skin substitute (SS) these differences were less pronounced. A similar discrepancy between ASC and dermal fibroblast mono-cultures was observed when recombinant human-CCL27 was used instead of burn wound exudates. Although CCL27 did not stimulate the secretion of any of the wound-healing mediators by keratinocytes, these cells, in contrast to ASC or dermal fibroblasts, showed increased proliferation and migration. Taken together, these results indicate that on transplantation, keratinocytes are primarily activated to promote wound closure. In contrast, dermal fibroblasts and, in particular, ASC respond vigorously to factors present in the wound bed, leading to increased secretion of angiogenesis/granulation tissue formation

  12. In Vitro Endothelialization Test of Biomaterials Using Immortalized Endothelial Cells.

    Directory of Open Access Journals (Sweden