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Sample records for epidermal cell area

  1. 25 YEARS OF EPIDERMAL STEM CELLS

    PubMed Central

    Ghadially, Ruby

    2012-01-01

    This is a chronicle of concepts in the field of epidermal stem cell biology and a historic look at their development over time. The last 25 years have seen the evolution of epidermal stem cell science, from first fundamental studies to a sophisticated science. The study of epithelial stem cell biology was aided by the ability to visualize the distribution of stem cells and their progeny through lineage analysis studies. The excellent progress we have made in understanding epidermal stem cell biology is discussed in this article. The challenges we still face in understanding epidermal stem cell include defining molecular markers for stem and progenitor subpopulations, determining the locations and contributions of the different stem cell niches, and mapping regulatory pathways of epidermal stem cell proliferation and differentiation. However, our rapidly evolving understanding of epidermal stem cells has many potential uses that promise to translate into improved patient therapy. PMID:22205306

  2. Epidermal Stem Cells in Orthopaedic Regenerative Medicine

    PubMed Central

    Li, Jin; Zhen, Gehua; Tsai, Shin-Yi; Jia, Xiaofeng

    2013-01-01

    In the last decade, great advances have been made in epidermal stem cell studies at the cellular and molecular level. These studies reported various subpopulations and differentiations existing in the epidermal stem cell. Although controversies and unknown issues remain, epidermal stem cells possess an immune-privileged property in transplantation together with easy accessibility, which is favorable for future clinical application. In this review, we will summarize the biological characteristics of epidermal stem cells, and their potential in orthopedic regenerative medicine. Epidermal stem cells play a critical role via cell replacement, and demonstrate significant translational potential in the treatment of orthopedic injuries and diseases, including treatment for wound healing, peripheral nerve and spinal cord injury, and even muscle and bone remodeling. PMID:23727934

  3. Mechanotransduction in epidermal Merkel cells

    PubMed Central

    Nakatani, Masashi; Maksimovic, Srdjan; Baba, Yoshichika; Lumpkin, Ellen A.

    2014-01-01

    The cellular and molecular basis of vertebrate touch reception remains least understood among the traditional five senses. Somatosensory afferents that innervate the skin encode distinct tactile qualities, such as flutter, slip and pressure. Gentle touch is thought to be transduced by somatosensory afferents whose tactile end organs selectively filter mechanical stimuli. These tactile end organs comprise afferent terminals in association with non-neuronal cell types such as Merkel cells, keratinocytes and Schwann cells. An open question is whether these non-neuronal cells serve primarily as passive mechanical filters or whether they actively participate in mechanosensory transduction. This question has been most extensively studied in Merkel cells, which are epidermal cells that complex with sensory afferents in regions of high tactile acuity such as fingertips, whisker follicles, and touch domes. Merkel cell-neurite complexes mediate slowly adapting type I (SAI) responses, which encode sustained pressure and represent object features with high fidelity. How Merkel cells contribute to unique SAI firing patterns has been debated for decades; however, three recent studies in rodent models provide some direct answers. First, whole-cell recordings demonstrate that Merkel cells are touch-sensitive cells with fast, mechanically activated currents that require Piezo2. Second, optogenetics and intact recordings show that Merkel cells mediate sustained SAI firing. Finally, loss-of-function studies in transgenic mouse models reveal that SAI afferents are also touch sensitive. Together, these studies identify molecular mechanisms of mechanotransduction in Merkel cells, reveal unexpected functions for these cells in touch and support a revised, two-receptor site model of mechanosensory transduction. PMID:25053537

  4. Cell motion predicts human epidermal stemness

    PubMed Central

    Toki, Fujio; Tate, Sota; Imai, Matome; Matsushita, Natsuki; Shiraishi, Ken; Sayama, Koji; Toki, Hiroshi; Higashiyama, Shigeki

    2015-01-01

    Image-based identification of cultured stem cells and noninvasive evaluation of their proliferative capacity advance cell therapy and stem cell research. Here we demonstrate that human keratinocyte stem cells can be identified in situ by analyzing cell motion during their cultivation. Modeling experiments suggested that the clonal type of cultured human clonogenic keratinocytes can be efficiently determined by analysis of early cell movement. Image analysis experiments demonstrated that keratinocyte stem cells indeed display a unique rotational movement that can be identified as early as the two-cell stage colony. We also demonstrate that α6 integrin is required for both rotational and collective cell motion. Our experiments provide, for the first time, strong evidence that cell motion and epidermal stemness are linked. We conclude that early identification of human keratinocyte stem cells by image analysis of cell movement is a valid parameter for quality control of cultured keratinocytes for transplantation. PMID:25897083

  5. In vitro transformation of Syrian hamster epidermal cells by N-methyl-N'-nitro-N-nitrosoguanidine

    SciTech Connect

    Sun, N.C.; Sun, C.R.Y.; Chao, L.; Fung, W.P.; Tennant, R.W.; Hsie, A.W.

    1981-05-01

    The selection of Syrian hamster epidermal cells which do not terminally differentiate has provided a quantitative focus assay for in vitro chemical transformation. One-day-old Syrian hamster epidermal cells plated at 5 x 10/sup 6//100-mm dish were treated for 5 hr with various concentrations of N-methyl-N-nitro-N'-nitrosoguanidine. After 4 weeks, the normal epidermal cells began to terminally differentiate to keratinized squamous cells and died, but transformed epidermal colonies grew to higher cell densities and appeared as darker areas against a lightly stained normal cell background. Transformed epidermal foci were isolated and subcultured for at least 15 passages, whereas normal epidermal cells could not be subcultured under the same conditions. The transformed cells assumed the typical cobblestone-like morphology of epithelial cells, retained desmosomes and tonofilaments, and were able to use citrulline in place of arginine. Argininosuccinate synthetase (EC 6.3.4.5) activity was significantly higher in the epidermal cells than in fibroblasts. The injection of 5 x 10/sup 6/ cells of two transformed epidermal cell lines into athymic nude mice resulted in the formation of tumors which were identified as keratinizing squamous carcinomas.

  6. [Biology of epidermal stem cells: impact on medicine].

    PubMed

    Pikuła, Michał; Trzonkowski, Piotr

    2009-01-01

    The epidermis is a self-renewing tissue which regenerates constantly. It consists mainly of keratinocytes of various degree of differentiation, from the proliferative basal layer to the terminally differentiated horny layer. Keratinocytes are specialized cells responsible for cohesion, barrier functions, and immunological reactions. The maintenance of homeostasis in the epidermis is possible via the self-renewing ability of the epidermal stem-cell population, which gives rise to differentiated keratinocytes. It is believed that epidermal stem cells play an important role in cellular regeneration, wound healing, and the pathogenesis of skin cancers. Epidermal stem cells reside in the basal layer of the epidermis, the bulge region of the hair follicle, and the germinal hair follicle matrix. Epidermal stem cells are relatively quiescent, slow-cycling cells defined by their great proliferative potential and unlimited capacity for self-renewal. Adult human epidermal stem cells can be activated and expanded in vitro under appropriate conditions. Cultured human keratinocytes and epidermal stem cells may be then transplanted as a biological dressing in burn injuries, chronic wounds, and various skin diseases. Additionally, epidermal stem cells have become a target for gene therapy and drug testing. In this review the fundamental characteristics of epidermal stem cells and the signaling pathways involved in the regulation of their proliferation and differentiation are discussed. The possibilities of using epidermal stem cells in medicine are also presented. PMID:19837987

  7. Metabolic profiling of Arabidopsis thaliana epidermal cells

    PubMed Central

    Ebert, Berit; Zöller, Daniela; Erban, Alexander; Fehrle, Ines; Hartmann, Jürgen; Niehl, Annette; Kopka, Joachim; Fisahn, Joachim

    2010-01-01

    Metabolic phenotyping at cellular resolution may be considered one of the challenges in current plant physiology. A method is described which enables the cell type-specific metabolic analysis of epidermal cell types in Arabidopsis thaliana pavement, basal, and trichome cells. To achieve the required high spatial resolution, single cell sampling using microcapillaries was combined with routine gas chromatography-time of flight-mass spectrometry (GC-TOF-MS) based metabolite profiling. The identification and relative quantification of 117 mostly primary metabolites has been demonstrated. The majority, namely 90 compounds, were accessible without analytical background correction. Analyses were performed using cell type-specific pools of 200 microsampled individual cells. Moreover, among these identified metabolites, 38 exhibited differential pool sizes in trichomes, basal or pavement cells. The application of an independent component analysis confirmed the cell type-specific metabolic phenotypes. Significant pool size changes between individual cells were detectable within several classes of metabolites, namely amino acids, fatty acids and alcohols, alkanes, lipids, N-compounds, organic acids and polyhydroxy acids, polyols, sugars, sugar conjugates and phenylpropanoids. It is demonstrated here that the combination of microsampling and GC-MS based metabolite profiling provides a method to investigate the cellular metabolism of fully differentiated plant cell types in vivo. PMID:20150518

  8. Optimal allocation of leaf epidermal area for gas exchange.

    PubMed

    de Boer, Hugo J; Price, Charles A; Wagner-Cremer, Friederike; Dekker, Stefan C; Franks, Peter J; Veneklaas, Erik J

    2016-06-01

    A long-standing research focus in phytology has been to understand how plants allocate leaf epidermal space to stomata in order to achieve an economic balance between the plant's carbon needs and water use. Here, we present a quantitative theoretical framework to predict allometric relationships between morphological stomatal traits in relation to leaf gas exchange and the required allocation of epidermal area to stomata. Our theoretical framework was derived from first principles of diffusion and geometry based on the hypothesis that selection for higher anatomical maximum stomatal conductance (gsmax ) involves a trade-off to minimize the fraction of the epidermis that is allocated to stomata. Predicted allometric relationships between stomatal traits were tested with a comprehensive compilation of published and unpublished data on 1057 species from all major clades. In support of our theoretical framework, stomatal traits of this phylogenetically diverse sample reflect spatially optimal allometry that minimizes investment in the allocation of epidermal area when plants evolve towards higher gsmax . Our results specifically highlight that the stomatal morphology of angiosperms evolved along spatially optimal allometric relationships. We propose that the resulting wide range of viable stomatal trait combinations equips angiosperms with developmental and evolutionary flexibility in leaf gas exchange unrivalled by gymnosperms and pteridophytes. PMID:26991124

  9. Epidermal growth factor signaling in transformed cells

    PubMed Central

    Lindsey, Stephan; Langhans, Sigrid A.

    2016-01-01

    Members of the epidermal growth factor receptor (EGFR/ErbB) family play a critical role in normal cell growth and development. However, many ErbB family members, especially EGFR, are aberrantly expressed or deregulated in tumors and are thought to play crucial roles in cancer development and metastatic progression. In this chapter, we provide an overview of key mechanisms contributing to aberrant EGFR/ErbB signaling in transformed cells which results in many phenotypic changes associated with the earliest stages of tumor formation, including several hallmarks of epithelial-to-mesenchymal transition (EMT). These changes often occur through interaction with other major signaling pathways important to tumor progression resulting in a multitude of transcriptional changes that ultimately impact cell morphology, proliferation and adhesion, all of which are crucial for tumor progression. The resulting mesh of signaling networks will need to be taken into account as new regimens are designed for targeting EGFR for therapeutic intervention. As new insights into the molecular mechanisms of the cross-talk of EGFR signaling with other signaling pathways and their role in therapeutic resistance to anti-EGFR therapies are gained a continual reassessment of clinical therapeutic regimes and strategies will be required. Understanding the consequences and complexity of EGF signaling and how it relates to tumor progression is critical for the development of clinical compounds and establishing clinical protocols for the treatment of cancer. PMID:25619714

  10. Specification of epidermal cell fate in plant shoots.

    PubMed

    Takada, Shinobu; Iida, Hiroyuki

    2014-01-01

    Land plants have evolved a single layer of epidermal cells, which are characterized by mostly anticlinal cell division patterns, formation of a waterproof coat called cuticle, and unique cell types such as stomatal guard cells and trichomes. The shoot epidermis plays important roles not only to protect plants from dehydration and pathogens but also to ensure their proper organogenesis and growth control. Extensive molecular genetic studies in Arabidopsis and maize have identified a number of genes that are required for epidermal cell differentiation. However, the mechanism that specifies shoot epidermal cell fate during plant organogenesis remains largely unknown. Particularly, little is known regarding positional information that should restrict epidermal cell fate to the outermost cell layer of the developing organs. Recent studies suggested that certain members of the HD-ZIP class IV homeobox genes are possible master regulators of shoot epidermal cell fate. Here, we summarize the roles of the regulatory genes that are involved in epidermal cell fate specification and discuss the possible mechanisms that limit the expression and/or activity of the master transcriptional regulators to the outermost cell layer in plant shoots. PMID:24616724

  11. Anti-epidermal-cell-surface pemphigus antibody detaches viable epidermal cells from culture plates by activation of proteinase.

    PubMed Central

    Farb, R M; Dykes, R; Lazarus, G S

    1978-01-01

    Immunoglobulin from pemphigus patients binds to the surface of mouse epidermal cells in culture. Cells incubated with the pemphigus antibody are easily detached from culture plates whereas cells incubated with serum from normal patients remain on the plate. Pemphigus antibody-mediated cell detachment is blocked by the addition of the proteinase inhibitors soybean trypsin inhibitor and alpha2-macroglobulin to the culture media. Detachable cells are viable, and activation of the complement cascade is not necessary for cell detachment. The anti-cell-surface antibody of pemphigus appears to disrupt adhesion between viable epidermal cells by activation of proteinase. Images PMID:272663

  12. Do epidermal lens cells facilitate the absorptance of diffuse light?

    PubMed

    Brodersen, Craig R; Vogelmann, Thomas C

    2007-07-01

    Many understory plants rely on diffuse light for photosynthesis because direct light is usually scattered by upper canopy layers before it strikes the forest floor. There is a considerable gap in the literature concerning the interaction of direct and diffuse light with leaves. Some understory plants have well-developed lens-shaped epidermal cells, which have long been thought to increase the absorption of diffuse light. To assess the role of epidermal cell shape in capturing direct vs. diffuse light, we measured leaf reflectance and transmittance with an integrating sphere system using leaves with flat (Begonia erythrophylla, Citrus reticulata, and Ficus benjamina) and lens-shaped epidermal cells (B. bowerae, Colocasia esculenta, and Impatiens velvetea). In all species examined, more light was absorbed when leaves were irradiated with direct as opposed to diffuse light. When leaves were irradiated with diffuse light, more light was transmitted and more was reflected in both leaf types, resulting in absorptance values 2-3% lower than in leaves irradiated with direct light. These data suggest that lens-shaped epidermal cells do not aid the capture of diffuse light. Palisade and mesophyll cell anatomy and leaf thickness appear to have more influence in the capture and absorption of light than does epidermal cell shape. PMID:21636475

  13. [Wound treatment with autogenous epidermal cell expansion culture].

    PubMed

    Bonnekoh, B; Müller, R P; Mahrle, G; Steigleder, G K

    1988-11-11

    Sheets of autologous epidermal cells grown by expansion culture were used to cover small skin defects in seven patients with postoperative necroses, necroses due to temporal arteritis, varicose ulcers or after tangential excision of tattoos. Several transplantation techniques were used: backing of the cultured epithelia with vaseline gauze, Surfasoft, Adaptic, Silastic foil, culturing directly from Petriperm-foil. Meshed Silastic-foil proved to give the best support. Optimal take of the in-vitro epithelia (more than 80% of their surface area) was achieved only for fresh dermal wound-beds. The take was only moderate on chronic granulation tissue, but the transplants reduced the formation of fibrinous-necrotic material and favoured the formation of fresh granulation tissue. PMID:3181024

  14. Water Relations of Leaf Epidermal Cells of Tradescantia virginiana12

    PubMed Central

    Tomos, Alun Deri; Steudle, Ernst; Zimmermann, Ulrich; Schulze, Ernst-Detlev

    1981-01-01

    Water-relation parameters (cell turgor pressure [P], volumetric elastic modulus [ε] and hydraulic conductivity [Lp]) of individual leaf epidermal cells of Tradescantia virginiana have been determined with the pressure-probe technique. Turgor was 4.5 ± 2.1 [41] bar (mean ± sd; in brackets the number of cells) and ranged from 0.9 to 9.6 bar. By vacuum infiltration with nutrient solution, it was raised to 7.5 ± 1.5 [5] bar (range: 5.3-8.8 bar). There was a large variability in the absolute value of ε of individual cells. ε ranged from 40 to 360 bar; mean ± sd: 135 ± 83 bar; n = 50 cells. ε values of individual cells seemed to be rather independent of changes in cell turgor. A critical assessment of the errors incurred in determining ε by the technique is included. The half-times of water exchange of individual cells ranged from 1 to 35 seconds, which gave values of 0.2 to 11 × 10−6 centimeters per second per bar for Lp (mean ± sd: 3.1 ± 2.3 × 10−6 centimeters per second per bar; n = 39 cells). The large range in Lp and ε is believed to be due to the difficulties in determining the effective surface area of water exchange of the cells. Lp is not influenced by active salt pumping driven by respiration energy inasmuch as it was not altered by 0.1 millimolar KCN. The temperature dependence of Lp (T½) was measured for the first time in individual higher-plant cells. Lp increased by a factor of 2 to 4, when the temperature was increased by 10 C. The activation energy of water exchange was found to be between 50 and 186 kilojoules per mole. Within the large range of variation it was found that T½, Lp, and ε did not change under various experimental conditions (intact and excised tissue, water content and turgidity, age, etc.). Similar results were obtained for the epidermal cells of Tradescantia andersoniana. The measurements suggest that the entire epidermis would respond very rapidly (i.e. with a half-time of 1 to 30 s) to a demand for water from the

  15. Epidermal Th22 and Tc17 cells form a localized disease memory in clinically healed psoriasis.

    PubMed

    Cheuk, Stanley; Wikén, Maria; Blomqvist, Lennart; Nylén, Susanne; Talme, Toomas; Ståhle, Mona; Eidsmo, Liv

    2014-04-01

    Psoriasis is a common and chronic inflammatory skin disease in which T cells play a key role. Effective treatment heals the skin without scarring, but typically psoriasis recurs in previously affected areas. A pathogenic memory within the skin has been proposed, but the nature of such site-specific disease memory is unknown. Tissue-resident memory T (TRM) cells have been ascribed a role in immunity after resolved viral skin infections. Because of their localization in the epidermal compartment of the skin, TRM may contribute to tissue pathology during psoriasis. In this study, we investigated whether resolved psoriasis lesions contain TRM cells with the ability to maintain and potentially drive recurrent disease. Three common and effective therapies, narrowband-UVB treatment and long-term biologic treatment systemically inhibiting TNF-α or IL-12/23 signaling were studied. Epidermal T cells were highly activated in psoriasis and a high proportion of CD8 T cells expressed TRM markers. In resolved psoriasis, a population of cutaneous lymphocyte-associated Ag, CCR6, CD103, and IL-23R expressing epidermal CD8 T cells was highly enriched. Epidermal CD8 T cells expressing the TRM marker CD103 responded to ex vivo stimulation with IL-17A production and epidermal CD4 T cells responded with IL-22 production after as long as 6 y of TNF-α inhibition. Our data suggest that epidermal TRM cells are retained in resolved psoriasis and that these cells are capable of producing cytokines with a critical role in psoriasis pathogenesis. We provide a potential mechanism for a site-specific T cell-driven disease memory in psoriasis. PMID:24610014

  16. Cell Fate Determination and the Switch from Diffuse Growth to Planar Polarity in Arabidopsis Root Epidermal Cells

    PubMed Central

    Balcerowicz, Daria; Schoenaers, Sébastjen; Vissenberg, Kris

    2015-01-01

    Plant roots fulfill important functions as they serve in water and nutrient uptake, provide anchorage of the plant body in the soil and in some species form the site of symbiotic interactions with soil-living biota. Root hairs, tubular-shaped outgrowths of specific epidermal cells, significantly increase the root’s surface area and aid in these processes. In this review we focus on the molecular mechanisms that determine the hair and non-hair cell fate of epidermal cells and that define the site on the epidermal cell where the root hair will be initiated (=planar polarity determination). In the model plant Arabidopsis, trichoblast and atrichoblast cell fate results from intra- and intercellular position-dependent signaling and from complex feedback loops that ultimately regulate GL2 expressing and non-expressing cells. When epidermal cells reach the end of the root expansion zone, root hair promoting transcription factors dictate the establishment of polarity within epidermal cells followed by the selection of the root hair initiation site at the more basal part of the trichoblast. Molecular players in the abovementioned processes as well as the role of phytohormones are discussed, and open areas for future experiments are identified. PMID:26779192

  17. Identification of Candidate Transcriptional Regulators of Epidermal Transfer Cell Development in Vicia faba Cotyledons.

    PubMed

    Arun-Chinnappa, Kiruba S; McCurdy, David W

    2016-01-01

    Transfer cells (TCs) are anatomically-specialized cells formed at apoplasmic-symplasmic bottlenecks in nutrient transport pathways in plants. TCs form invaginated wall ingrowths which provide a scaffold to amplify plasma membrane surface area and thus increase the density of nutrient transporters required to achieve enhanced nutrient flow across these bottlenecks. Despite their importance to nutrient transport in plants, little is known of the transcriptional regulation of wall ingrowth formation. Here, we used RNA-Seq to identify transcription factors putatively involved in regulating epidermal TC development in cotyledons of Vicia faba. Comparing cotyledons cultured for 0, 3, 9, and 24 h to induce trans-differentiation of epidermal TCs identified 43 transcription factors that showed either epidermal-specific or epidermal-enhanced expression, and 10 that showed epidermal-specific down regulation. Members of the WRKY and ethylene-responsive families were prominent in the cohort of transcription factors showing epidermal-specific or epidermal-enhanced expression, consistent with the initiation of TC development often representing a response to stress. Members of the MYB family were also prominent in these categories, including orthologs of MYB genes involved in localized secondary wall deposition in Arabidopsis thaliana. Among the group of transcription factors showing down regulation were various homeobox genes and members of the MADs-box and zinc-finger families of poorly defined functions. Collectively, this study identified several transcription factors showing expression characteristics and orthologous functions that indicate likely participation in transcriptional regulation of epidermal TC development in V. faba cotyledons. PMID:27252730

  18. Epidermal Merkel cells in psoriatic lesions: immunohistochemical investigations on neuroendocrine antigen expression.

    PubMed

    Wollina, U; Mahrle, G

    1992-05-01

    Biopsy specimens from lesional psoriatic skin and from normal controls were investigated by immunohistochemistry for the presence of epidermal Merkel cells (MC). MC were defined as epidermal cells expressing simple-type keratins, i.e. nos. 8, 18, and 19. A significant number of MC could be found at the bottom of the rete ridges of psoriatic lesions (about 19.6 MC per square mm skin surface area) and of normal skin (about 14.0 MC per square mm surface area). In contrast to normal skin, MC of psoriatic lesions were positive for synaptophysin (21.7% of simple-type keratin positive epidermal cells, i.e. MC), pancreatic polypeptide (14.8%), somatostatin (7.0%), and chromogranin A (less than 3%). The immunostaining was rather faint though significantly different from normal skin. The findings suggest that in psoriasis, epidermal MC show variations of the expression of neuropeptides compared to normal skin. Since some of the neuropeptides are thought to be involved in hyperproliferation and/or skin immunology, our findings might suggest a functional activity of epidermal MC in psoriatic lesions different from normal controls. PMID:1498093

  19. Fate by Chance, not by Choice: Epidermal Stem Cells Go Live.

    PubMed

    Gonzalez-Celeiro, Meryem; Zhang, Bing; Hsu, Ya-Chieh

    2016-07-01

    The skin epidermis is constantly renewed by epidermal stem cells. In a recent Science paper, Rompolas et al. utilize live imaging to track epidermal stem cells over their lifetimes. Their findings provide new insights into epidermal stem cell behaviors and unravel how newly generated cells are integrated into pre-existing tissues. PMID:27392221

  20. Why do so many petals have conical epidermal cells?

    PubMed Central

    Whitney, Heather M.; Bennett, K. M. Veronica; Dorling, Matthew; Sandbach, Lucy; Prince, David; Chittka, Lars; Glover, Beverley J.

    2011-01-01

    Background The conical epidermal cells found on the petals of most Angiosperm species are so widespread that they have been used as markers of petal identity, but their function has only been analysed in recent years. This review brings together diverse data on the role of these cells in pollination biology. Scope The published effects of conical cells on petal colour, petal reflexing, scent production, petal wettability and pollinator grip on the flower surface are considered. Of these factors, pollinator grip has been shown to be of most significance in the well-studied Antirrhinum majus/bumble-bee system. Published data on the relationship between epidermal cell morphology and floral temperature were limited, so an analysis of the effects of cell shape on floral temperature in Antirrhinum is presented here. Statistically significant warming by conical cells was not detected, although insignificant trends towards faster warming at dawn were found, and it was also found that flat-celled flowers could be warmer on warm days. The warming observed is less significant than that achieved by varying pigment content. However, the possibility that the effect of conical cells on temperature might be biologically significant in certain specific instances such as marginal habitats or weather conditions cannot be ruled out. Conclusions Conical epidermal cells can influence a diverse set of petal properties. The fitness benefits they provide to plants are likely to vary with pollinator and habitat, and models are now required to understand how these different factors interact. PMID:21470973

  1. Estimating the Size of Onion Epidermal Cells from Diffraction Patterns

    NASA Astrophysics Data System (ADS)

    Groff, Jeffrey R.

    2012-10-01

    Bioscience and premedical profession students are a major demographic served by introductory physics courses at many colleges and universities. Exposing these students to biological applications of physical principles will help them to appreciate physics as a useful tool for their future professions. Here I describe an experiment suitable for introductory physics where principles of wave optics are applied to probe the size of onion epidermal cells. The epidermis tissue is composed of cells of relatively uniform size and shape (Fig. 1) so the tissue acts like a one-dimensional transmission diffraction grating. The diffraction patterns generated when a laser beam passes through the tissue (Fig. 2) are analyzed and an estimate of the average width of individual onion epidermal cells is calculated. The results are compared to direct measurements taken using a light microscope. The use of microscopes and plant-cell tissue slides creates opportunities for cross-discipline collaboration between physics and biology instructors.

  2. Spatiotemporal coordination of stem cell commitment during epidermal homeostasis.

    PubMed

    Rompolas, Panteleimon; Mesa, Kailin R; Kawaguchi, Kyogo; Park, Sangbum; Gonzalez, David; Brown, Samara; Boucher, Jonathan; Klein, Allon M; Greco, Valentina

    2016-06-17

    Adult tissues replace lost cells via pools of stem cells. However, the mechanisms of cell self-renewal, commitment, and functional integration into the tissue remain unsolved. Using imaging techniques in live mice, we captured the lifetime of individual cells in the ear and paw epidermis. Our data suggest that epidermal stem cells have equal potential to either divide or directly differentiate. Tracking stem cells over multiple generations reveals that cell behavior is not coordinated between generations. However, sibling cell fate and lifetimes are coupled. We did not observe regulated asymmetric cell divisions. Lastly, we demonstrated that differentiating stem cells integrate into preexisting ordered spatial units of the epidermis. This study elucidates how a tissue is maintained by both temporal and spatial coordination of stem cell behaviors. PMID:27229141

  3. Spatiotemporal coordination of stem cell commitment during epidermal homeostasis

    PubMed Central

    Rompolas, Panteleimon; Mesa, Kailin R.; Kawaguchi, Kyogo; Park, Sangbum; Gonzalez, David; Brown, Samara; Boucher, Jonathan; Klein, Allon M.; Greco, Valentina

    2016-01-01

    Adult tissues replace lost cells via pools of stem cells. However, the mechanisms of cell self-renewal, commitment, and functional integration into the tissue remain unsolved. Using imaging techniques in live mice, we captured the lifetime of individual cells in the ear and paw epidermis. Our data suggest that epidermal stem cells have equal potential to either divide or directly differentiate. Tracking stem cells over multiple generations reveals that cell behavior is not coordinated between generations. However, sibling cell fate and lifetimes are coupled. We did not observe regulated asymmetric cell divisions. Lastly, we demonstrated that differentiating stem cells integrate into preexisting ordered spatial units of the epidermis. This study elucidates how a tissue is maintained by both temporal and spatial coordination of stem cell behaviors. PMID:27229141

  4. Identification of Candidate Transcriptional Regulators of Epidermal Transfer Cell Development in Vicia faba Cotyledons

    PubMed Central

    Arun-Chinnappa, Kiruba S.; McCurdy, David W.

    2016-01-01

    Transfer cells (TCs) are anatomically-specialized cells formed at apoplasmic-symplasmic bottlenecks in nutrient transport pathways in plants. TCs form invaginated wall ingrowths which provide a scaffold to amplify plasma membrane surface area and thus increase the density of nutrient transporters required to achieve enhanced nutrient flow across these bottlenecks. Despite their importance to nutrient transport in plants, little is known of the transcriptional regulation of wall ingrowth formation. Here, we used RNA-Seq to identify transcription factors putatively involved in regulating epidermal TC development in cotyledons of Vicia faba. Comparing cotyledons cultured for 0, 3, 9, and 24 h to induce trans-differentiation of epidermal TCs identified 43 transcription factors that showed either epidermal-specific or epidermal–enhanced expression, and 10 that showed epidermal-specific down regulation. Members of the WRKY and ethylene-responsive families were prominent in the cohort of transcription factors showing epidermal-specific or epidermal–enhanced expression, consistent with the initiation of TC development often representing a response to stress. Members of the MYB family were also prominent in these categories, including orthologs of MYB genes involved in localized secondary wall deposition in Arabidopsis thaliana. Among the group of transcription factors showing down regulation were various homeobox genes and members of the MADs-box and zinc-finger families of poorly defined functions. Collectively, this study identified several transcription factors showing expression characteristics and orthologous functions that indicate likely participation in transcriptional regulation of epidermal TC development in V. faba cotyledons. PMID:27252730

  5. Heterogeneity and plasticity of epidermal stem cells

    PubMed Central

    Schepeler, Troels; Page, Mahalia E.; Jensen, Kim B.

    2014-01-01

    The epidermis is an integral part of our largest organ, the skin, and protects us against the hostile environment. It is a highly dynamic tissue that, during normal steady-state conditions, undergoes constant turnover. Multiple stem cell populations residing in autonomously maintained compartments facilitate this task. In this Review, we discuss stem cell behaviour during normal tissue homeostasis, regeneration and disease within the pilosebaceous unit, an integral structure of the epidermis that is responsible for hair growth and lubrication of the epithelium. We provide an up-to-date view of the pilosebaceous unit, encompassing the heterogeneity and plasticity of multiple discrete stem cell populations that are strongly influenced by external cues to maintain their identity and function. PMID:24961797

  6. Short communication: Initial evidence supporting existence of potential rumen epidermal stem and progenitor cells.

    PubMed

    Yohe, T T; Tucker, H L M; Parsons, C L M; Geiger, A J; Akers, R M; Daniels, K M

    2016-09-01

    The bovine rumen epidermis is a keratinized multilayered tissue that experiences persistent cell turnover. Because of this constant cell turnover, epidermal stem cells and their slightly more differentiated daughter cells, epidermal progenitor cells, must exist in the stratum basale of rumen epidermis. To date, these 2 epidermal cell populations and any unique cellular markers they may possess remain completely uncharacterized in the bovine rumen. An important first step in this new research area is the demonstration of the relative abundance and existence of markers for these cells in rumen tissue. A related second step is to document rumen epidermal proliferative responses to an extrinsic signal such as nutrient concentration within the rumen. The objectives of this experiment were to evaluate the extrinsic effect of diet on (1) gene expression of 6 potential rumen epidermal stem or progenitor cell markers and (2) rumen epidermal cell proliferation within the stratum basale. Twelve preweaned Holstein heifers were fed either a restricted diet (R) or an enhanced diet (EH). Animals on R received a milk replacer (MR) diet fed at 0.44kg of powder dry matter (DM)/d (20.9% crude protein, 29.8% fat, DM basis) and EH received MR at 1.08kg of powder dry matter/d (28.9% crude protein, 26.2% fat, DM basis). All calves had access to a 20% crude protein starter and were weaned during wk 7 of the experiment. Lifetime DM intake was 0.73kg of DM/calf per day for R (5.88 Mcal of net energy/calf per day) and 1.26kg of DM/calf per day for EH (10.68 Mcal of net energy/calf per day). Twenty-four hours before slaughter heifers received an intravenous dose of 5-bromo-2'-deoxyuridine to label proliferating cells. Heifers were slaughtered at 8 wk of age, and rumen samples from the ventral sac region were obtained and stored in RNA preservative and processed for routine histology. Quantitative real-time reverse transcriptase PCR was used to analyze relative abundance of genes. Candidate

  7. Effects of Nitrogen on Mesophyll Cell Division and Epidermal Cell Elongation in Tall Fescue Leaf Blades 1

    PubMed Central

    MacAdam, Jennifer W.; Volenec, Jeffrey J.; Nelson, Curtis J.

    1989-01-01

    Leaf elongation rate (LER) in grasses is dependent on epidermal cell supply (number) and on rate and duration of epidermal cell elongation. Nitrogen (N) fertilization increases LER. Longitudinal sections from two genotypes of tall fescue (Festuca arundinacea Schreb.), which differ by 50% in LER, were used to quantify the effects of N on the components of epidermal cell elongation and on mesophyll cell division. Rate and duration of epidermal cell elongation were determined by using a relationship between cell length and displacement velocity derived from the continuity equation. Rate of epidermal cell elongation was exponential. Relative rates of epidermal cell elongation increased by 9% with high N, even though high N increased LER by 89%. Duration of cell elongation was approximately 20 h longer in the high- than in the low-LER genotype regardless of N treatment. The percentage of mesophyll cells in division was greater in the high- than in the low-LER genotype. This increased with high N in both genotypes, indicating that LER increased with cell supply. Division of mesophyll cells adjacent to abaxial epidermal cells continued after epidermal cell division stopped, until epidermal cells had elongated to a mean length of 40 micrometers in the high-LER and a mean length of 50 micrometers in the low-LER genotype. The cell cycle length for mesophyll cells was calculated to be 12 to 13 hours. Nitrogen increased mesophyll cell number more than epidermal cell number: in both genotypes, the final number of mesophyll cells adjacent to each abaxial epidermal cell was 10 with low N and 14 with high N. A spatial model is used to describe three cell development processes relevant to leaf growth. It illustrates the overlap of mesophyll cell division and epidermal cell elongation, and the transition from epidermal cell elongation to secondary cell wall deposition. PMID:16666581

  8. Growth of melanocytes in human epidermal cell cultures

    SciTech Connect

    Staiano-Coico, L.; Hefton, J.M.; Amadeo, C.; Pagan-Charry, I.; Madden, M.R.; Cardon-Cardo, C. )

    1990-08-01

    Epidermal cell cultures were grown in keratinocyte-conditioned medium for use as burn wound grafts; the melanocyte composition of the grafts was studied under a variety of conditions. Melanocytes were identified by immunohistochemistry based on a monoclonal antibody (MEL-5) that has previously been shown to react specifically with melanocytes. During the first 7 days of growth in primary culture, the total number of melanocytes in the epidermal cultures decreased to 10% of the number present in normal skin. Beginning on day 2 of culture, bipolar melanocytes were present at a mean cell density of 116 +/- 2/mm2; the keratinocyte to melanocyte ratio was preserved during further primary culture and through three subpassages. Moreover, exposure of cultures to mild UVB irradiation stimulated the melanocytes to proliferate, suggesting that the melanocytes growing in culture maintained their responsiveness to external stimuli. When the sheets of cultured cells were enzymatically detached from the plastic culture flasks before grafting, melanocytes remained in the basal layer of cells as part of the graft applied to the patient.

  9. Microtubules CLASP to Adherens Junctions in epidermal progenitor cells.

    PubMed

    Shahbazi, Marta N; Perez-Moreno, Mirna

    2014-01-01

    Cadherin-mediated cell adhesion at Adherens Junctions (AJs) and its dynamic connections with the microtubule (MT) cytoskeleton are important regulators of cellular architecture. However, the functional relevance of these interactions and the molecular players involved in different cellular contexts and cellular compartments are still not completely understood. Here, we comment on our recent findings showing that the MT plus-end binding protein CLASP2 interacts with the AJ component p120-catenin (p120) specifically in progenitor epidermal cells. Absence of either protein leads to alterations in MT dynamics and AJ functionality. These findings represent a novel mechanism of MT targeting to AJs that may be relevant for the maintenance of proper epidermal progenitor cell homeostasis. We also discuss the potential implication of other MT binding proteins previously associated to AJs in the wider context of epithelial tissues. We hypothesize the existence of adaptation mechanisms that regulate the formation and stability of AJs in different cellular contexts to allow the dynamic behavior of these complexes during tissue homeostasis and remodeling. PMID:24522006

  10. Microtubules CLASP to Adherens Junctions in epidermal progenitor cells

    PubMed Central

    Shahbazi, Marta N; Perez-Moreno, Mirna

    2014-01-01

    Cadherin-mediated cell adhesion at Adherens Junctions (AJs) and its dynamic connections with the microtubule (MT) cytoskeleton are important regulators of cellular architecture. However, the functional relevance of these interactions and the molecular players involved in different cellular contexts and cellular compartments are still not completely understood. Here, we comment on our recent findings showing that the MT plus-end binding protein CLASP2 interacts with the AJ component p120-catenin (p120) specifically in progenitor epidermal cells. Absence of either protein leads to alterations in MT dynamics and AJ functionality. These findings represent a novel mechanism of MT targeting to AJs that may be relevant for the maintenance of proper epidermal progenitor cell homeostasis. We also discuss the potential implication of other MT binding proteins previously associated to AJs in the wider context of epithelial tissues. We hypothesize the existence of adaptation mechanisms that regulate the formation and stability of AJs in different cellular contexts to allow the dynamic behavior of these complexes during tissue homeostasis and remodeling. PMID:24522006

  11. Epidermal cell growth-dependent arylhydrocarbon-hydroxylase (AHH) activity in vitro.

    PubMed

    Thiele, B; Merk, H F; Bonnekoh, B; Mahrle, G; Steigleder, G K

    1987-01-01

    Cytochrome P-450-dependent arylhydrocarbon-hydroxylase (AHH) activity and inducibility by benzanthracene (BA) was measured in cultured guinea pig and human epidermal cells. Basal AHH-activity (AHHb) in guinea pig epidermal cells was much higher than in human epidermal cells. AHHb in guinea pig epidermal cells was directly related to the labeling index and decreased to the original level between the 5th and 7th day of cell culturing. On the other hand, the induction-ratio of AHH reached its maximum level when the number of cells began to rise (proliferation phase) and remained high at day 7 of the cell culture. These results suggest a cell growth dependent activity and inducibility of carcinogen-metabolizing enzymes, such as AHH, in isolated epidermal cells. PMID:3435181

  12. Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals.

    PubMed

    Mochizuki-Kawai, Hiroko; Niki, Tomoko; Shibuya, Kenichi; Ichimura, Kazuo

    2015-01-01

    In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals. PMID:26605547

  13. Programmed Cell Death Progresses Differentially in Epidermal and Mesophyll Cells of Lily Petals

    PubMed Central

    Mochizuki-Kawai, Hiroko; Niki, Tomoko; Shibuya, Kenichi; Ichimura, Kazuo

    2015-01-01

    In the petals of some species of flowers, programmed cell death (PCD) begins earlier in mesophyll cells than in epidermal cells. However, PCD progression in each cell type has not been characterized in detail. We separately constructed a time course of biochemical signs and expression patterns of PCD-associated genes in epidermal and mesophyll cells in Lilium cv. Yelloween petals. Before visible signs of senescence could be observed, we found signs of PCD, including DNA degradation and decreased protein content in mesophyll cells only. In these cells, the total proteinase activity increased on the day after anthesis. Within 3 days after anthesis, the protein content decreased by 61.8%, and 22.8% of mesophyll cells was lost. A second peak of proteinase activity was observed on day 6, and the number of mesophyll cells decreased again from days 4 to 7. These biochemical and morphological results suggest that PCD progressed in steps during flower life in the mesophyll cells. PCD began in epidermal cells on day 5, in temporal synchrony with the time course of visible senescence. In the mesophyll cells, the KDEL-tailed cysteine proteinase (LoCYP) and S1/P1 nuclease (LoNUC) genes were upregulated before petal wilting, earlier than in epidermal cells. In contrast, relative to that in the mesophyll cells, the expression of the SAG12 cysteine proteinase homolog (LoSAG12) drastically increased in epidermal cells in the final stage of senescence. These results suggest that multiple PCD-associated genes differentially contribute to the time lag of PCD progression between epidermal and mesophyll cells of lily petals. PMID:26605547

  14. Host cell reactivation studies with epidermal cells of mice sensitive and resistant to carcinogenesis

    SciTech Connect

    Strickland, J.E.; Strickland, A.G.

    1984-03-01

    Primary epidermal cells from AKR, BALB/c, CD-1, and SENCAR mice, listed in order of least to most sensitive to epidermal carcinogenesis by initiation and promotion protocols, were found to be equally competent to ''reactivate'' herpes simplex virus type 1 irradiated by germicidal ultraviolet radiation. Nontumorigenic BALB/c epidermal cell lines selected in vitro for resistance to terminal differentiation after in vivo or in vitro treatment with initiating doses of carcinogens showed virus survival curves similar to those of primary cells. Similarly, primary cultures which were allowed to grow to confluency following a single treatment with the tumor promoter 12-O-tetradecanoylphorbol-13-acetate (100 ng/ml) retained normal host cell reactivation. Host cell reactivation studies with mouse dermal fibroblasts could not be done because of the failure of the herpes simplex virus to infect these cells and produce plaques. These results demonstrate that survival of ultraviolet light-damaged virus in primary epidermal cells in culture is unrelated to whether the cells are derived from mice sensitive or resistant to epidermal carcinogenesis. Furthermore, virus survival is not changed by tumor promoter treatment or by treatment with initiating doses of carcinogens which results in differentiation-resistant cells.

  15. Vertebrate epidermal cells are broad-specificity phagocytes that clear sensory axon debris.

    PubMed

    Rasmussen, Jeffrey P; Sack, Georgeann S; Martin, Seanna M; Sagasti, Alvaro

    2015-01-14

    Cellular debris created by developmental processes or injury must be cleared by phagocytic cells to maintain and repair tissues. Cutaneous injuries damage not only epidermal cells but also the axonal endings of somatosensory (touch-sensing) neurons, which must be repaired to restore the sensory function of the skin. Phagocytosis of neuronal debris is usually performed by macrophages or other blood-derived professional phagocytes, but we have found that epidermal cells phagocytose somatosensory axon debris in zebrafish. Live imaging revealed that epidermal cells rapidly internalize debris into dynamic phosphatidylinositol 3-monophosphate-positive phagosomes that mature into phagolysosomes using a pathway similar to that of professional phagocytes. Epidermal cells phagocytosed not only somatosensory axon debris but also debris created by injury to other peripheral axons that were mislocalized to the skin, neighboring skin cells, and macrophages. Together, these results identify vertebrate epidermal cells as broad-specificity phagocytes that likely contribute to neural repair and wound healing. PMID:25589751

  16. Effects of Wnt3a on proliferation and differentiation of human epidermal stem cells

    SciTech Connect

    Jia Liwei; Zhou Jiaxi; Peng Sha; Li Juxue; Cao Yujing; Duan Enkui

    2008-04-11

    Epidermal stem cells maintain development and homeostasis of mammalian epidermis throughout life. However, the molecular mechanisms involved in the proliferation and differentiation of epidermal stem cells are far from clear. In this study, we investigated the effects of Wnt3a and Wnt/{beta}-catenin signaling on proliferation and differentiation of human fetal epidermal stem cells. We found both Wnt3a and active {beta}-catenin, two key members of the Wnt/{beta}-catenin signaling, were expressed in human fetal epidermis and epidermal stem cells. In addition, Wnt3a protein can promote proliferation and inhibit differentiation of epidermal stem cells in vitro culture. Our results suggest that Wnt/{beta}-catenin signaling plays important roles in human fetal skin development and homeostasis, which also provide new insights on the molecular mechanisms of oncogenesis in human epidermis.

  17. Skin mucus of Cyprinus carpio inhibits cyprinid herpesvirus 3 binding to epidermal cells

    PubMed Central

    2011-01-01

    Cyprinid herpesvirus 3 (CyHV-3) is the aetiological agent of a mortal and highly contagious disease in common and koi carp. The skin is the major portal of entry of CyHV-3 in carp after immersion in water containing the virus. In the present study, we used in vivo bioluminescence imaging to investigate the effect of skin mucus removal and skin epidermis lesion on CyHV-3 entry. Physical treatments inducing removal of the mucus up to complete erosion of the epidermis were applied on a defined area of carp skin just before inoculation by immersion in infectious water. CyHV-3 entry in carp was drastically enhanced on the area of the skin where the mucus was removed with or without associated epidermal lesion. To investigate whether skin mucus inhibits CyHV-3 binding to epidermal cells, tail fins with an intact mucus layer or without mucus were inoculated ex vivo. While electron microscopy examination revealed numerous viral particles bound on the fins inoculated after mucus removal, no particle could be detected after infection of mucus-covered fins. Finally, anti-CyHV-3 neutralising activity of mucus extract was tested in vitro. Incubation of CyHV-3 with mucus extract reduced its infectivity in a dose dependent manner. The present study demonstrates that skin mucus removal and epidermal lesions enhance CyHV-3 entry in carp. It highlights the role of fish skin mucus as an innate immune protection against viral epidermal entry. PMID:21816061

  18. Langerhans Cells Facilitate UVB-induced Epidermal Carcinogenesis

    PubMed Central

    Lewis, Julia M.; Bürgler, Christina D.; Freudzon, Marianna; Golubets, Kseniya; Gibson, Juliet F.; Filler, Renata B.; Girardi, Michael

    2015-01-01

    Ultraviolet B (UVB) light is considered the major environmental inducer of human keratinocyte DNA mutations, including within the tumor-suppressor gene p53, and chronic exposure is associated with cutaneous squamous cell carcinoma (SCC) formation. Langerhans cells (LC) comprise a dendritic network within the suprabasilar epidermis, yet the role of LC in UVB-induced carcinogenesis is largely unknown. Herein, we show that LC-intact epidermis develops UVB-induced tumors more readily than LC-deficient epidermis. While levels of epidermal cyclopyrimidine dimers (CPD) following acute UVB exposure are equivalent in the presence or absence of LC, chronic UVB-induced p53 mutant clonal islands expand more readily in association with LC which remain largely intact and are preferentially found in proximity to the expanding mutant keratinocyte populations. The observed LC facilitation of mutant p53 clonal expansion is completely αβ and γδ T-cell independent, and is associated with increased intraepidermal expression of interleukin (IL)-22 and the presence of group 3 innate lymphoid cells (ILC3). These data demonstrate that LC play a key role in UVB-induced cutaneous carcinogenesis, and suggest that LC locally stimulate keratinocyte proliferation and innate immune cells that provoke tumor outgrowth. PMID:26053049

  19. Targeting epidermal growth factor receptor for head and neck squamous cell carcinoma: still lost in translation?

    PubMed Central

    Chapman, Christopher H.; Saba, Nabil F.

    2016-01-01

    The epidermal growth factor receptor (EGFR) is preferentially expressed in head and neck squamous cell carcinoma (HNSCC), and is a promising therapeutic target. Yet other than cetuximab, no agent targeting EGFR has been approved for this disease, and none has shown benefit over the standard of care. Several randomized trials of antibody and small molecule agents have found no new indication for these agents, despite their initial promise. In this review, we examine the major clinical evidence and discuss potential future developments of translational science in this area, including use of these agents in risk-stratified subgroups, inhibition of downstream/parallel targets, and combination with immunotherapy. PMID:27004227

  20. Epigenetic Regulation of Epidermal Stem Cell Biomarkers and Their Role in Wound Healing

    PubMed Central

    Saldanha, Sabita N.; Royston, Kendra J.; Udayakumar, Neha; Tollefsbol, Trygve O.

    2015-01-01

    As an actively renewable tissue, changes in skin architecture are subjected to the regulation of stem cells that maintain the population of cells responsible for the formation of epidermal layers. Stems cells retain their self-renewal property and express biomarkers that are unique to this population. However, differential regulation of the biomarkers can initiate the pathway of terminal cell differentiation. Although, pockets of non-clarity in stem cell maintenance and differentiation in skin still exist, the influence of epigenetics in epidermal stem cell functions and differentiation in skin homeostasis and wound healing is clearly evident. The focus of this review is to discuss the epigenetic regulation of confirmed and probable epidermal stem cell biomarkers in epidermal stratification of normal skin and in diseased states. The role of epigenetics in wound healing, especially in diseased states of diabetes and cancer, will also be conveyed. PMID:26712738

  1. CD133 Is a Marker For Long-Term Repopulating Murine Epidermal Stem Cells

    PubMed Central

    Charruyer, A; Strachan, LR; Yue, L; Toth, AS; Mancianti, ML; Ghadially, R

    2012-01-01

    Maintenance, repair and renewal of the epidermis are thought to depend on a pool of dedicated epidermal stem cells. Like for many somatic tissues, isolation of a nearly pure population of stem cells is a primary goal in cutaneous biology. We used a quantitative transplantation assay, using injection of keratinocytes into subcutis combined with limiting dilution analysis, to assess the long-term repopulating ability of putative murine epidermal stem populations. Putative epidermal stem cell populations were isolated by FACS sorting. The CD133+ population and the subpopulation of CD133+ cells that exhibits high mitochondrial membrane potential (DΨmhi), were enriched for long-term repopulating epidermal stem cells vs. unfractionated cells (3.9 and 5.2-fold, respectively). Evidence for self-renewal capacity was obtained by serial transplantation of long-term epidermal repopulating units derived from CD133+ and CD133+ΔΨmhi keratinocytes. CD133+ keratinocytes were multipotent and produced significantly more hair follicles than CD133− cells. CD133+ cells were a subset of the previously described integrin α6+CD34+ bulge cell population and 28.9±8.6% were label retaining cells. Thus, murine keratinocytes within the CD133+ and CD133+ΔΨmhi populations contain epidermal stem cells that regenerate epidermis for the long-term, are self-renewing, multipotent, and label-retaining cells. PMID:22763787

  2. Gloss, colour and grip: multifunctional epidermal cell shapes in bee- and bird-pollinated flowers.

    PubMed

    Papiorek, Sarah; Junker, Robert R; Lunau, Klaus

    2014-01-01

    Flowers bear the function of filters supporting the attraction of pollinators as well as the deterrence of floral antagonists. The effect of epidermal cell shape on the visual display and tactile properties of flowers has been evaluated only recently. In this study we quantitatively measured epidermal cell shape, gloss and spectral reflectance of flowers pollinated by either bees or birds testing three hypotheses: The first two hypotheses imply that bee-pollinated flowers might benefit from rough surfaces on visually-active parts produced by conical epidermal cells, as they may enhance the colour signal of flowers as well as the grip on flowers for bees. In contrast, bird-pollinated flowers might benefit from flat surfaces produced by flat epidermal cells, by avoiding frequent visitation from non-pollinating bees due to a reduced colour signal, as birds do not rely on specific colour parameters while foraging. Moreover, flat petal surfaces in bird-pollinated flowers may hamper grip for bees that do not touch anthers and stigmas while consuming nectar and thus, are considered as nectar thieves. Beside this, the third hypothesis implies that those flower parts which are vulnerable to nectar robbing of bee- as well as bird-pollinated flowers benefit from flat epidermal cells, hampering grip for nectar robbing bees. Our comparative data show in fact that conical epidermal cells are restricted to visually-active parts of bee-pollinated flowers, whereas robbing-sensitive parts of bee-pollinated as well as the entire floral surface of bird-pollinated flowers possess on average flat epidermal cells. However, direct correlations between epidermal cell shape and colour parameters have not been found. Our results together with published experimental studies show that epidermal cell shape as a largely neglected flower trait might act as an important feature in pollinator attraction and avoidance of antagonists, and thus may contribute to the partitioning of flower

  3. Gloss, Colour and Grip: Multifunctional Epidermal Cell Shapes in Bee- and Bird-Pollinated Flowers

    PubMed Central

    Papiorek, Sarah; Junker, Robert R.; Lunau, Klaus

    2014-01-01

    Flowers bear the function of filters supporting the attraction of pollinators as well as the deterrence of floral antagonists. The effect of epidermal cell shape on the visual display and tactile properties of flowers has been evaluated only recently. In this study we quantitatively measured epidermal cell shape, gloss and spectral reflectance of flowers pollinated by either bees or birds testing three hypotheses: The first two hypotheses imply that bee-pollinated flowers might benefit from rough surfaces on visually-active parts produced by conical epidermal cells, as they may enhance the colour signal of flowers as well as the grip on flowers for bees. In contrast, bird-pollinated flowers might benefit from flat surfaces produced by flat epidermal cells, by avoiding frequent visitation from non-pollinating bees due to a reduced colour signal, as birds do not rely on specific colour parameters while foraging. Moreover, flat petal surfaces in bird-pollinated flowers may hamper grip for bees that do not touch anthers and stigmas while consuming nectar and thus, are considered as nectar thieves. Beside this, the third hypothesis implies that those flower parts which are vulnerable to nectar robbing of bee- as well as bird-pollinated flowers benefit from flat epidermal cells, hampering grip for nectar robbing bees. Our comparative data show in fact that conical epidermal cells are restricted to visually-active parts of bee-pollinated flowers, whereas robbing-sensitive parts of bee-pollinated as well as the entire floral surface of bird-pollinated flowers possess on average flat epidermal cells. However, direct correlations between epidermal cell shape and colour parameters have not been found. Our results together with published experimental studies show that epidermal cell shape as a largely neglected flower trait might act as an important feature in pollinator attraction and avoidance of antagonists, and thus may contribute to the partitioning of flower

  4. Epidermal Merkel cells are mechanosensory cells that tune mammalian touch receptors.

    PubMed

    Maksimovic, Srdjan; Nakatani, Masashi; Baba, Yoshichika; Nelson, Aislyn M; Marshall, Kara L; Wellnitz, Scott A; Firozi, Pervez; Woo, Seung-Hyun; Ranade, Sanjeev; Patapoutian, Ardem; Lumpkin, Ellen A

    2014-05-29

    Touch submodalities, such as flutter and pressure, are mediated by somatosensory afferents whose terminal specializations extract tactile features and encode them as action potential trains with unique activity patterns. Whether non-neuronal cells tune touch receptors through active or passive mechanisms is debated. Terminal specializations are thought to function as passive mechanical filters analogous to the cochlea's basilar membrane, which deconstructs complex sounds into tones that are transduced by mechanosensory hair cells. The model that cutaneous specializations are merely passive has been recently challenged because epidermal cells express sensory ion channels and neurotransmitters; however, direct evidence that epidermal cells excite tactile afferents is lacking. Epidermal Merkel cells display features of sensory receptor cells and make 'synapse-like' contacts with slowly adapting type I (SAI) afferents. These complexes, which encode spatial features such as edges and texture, localize to skin regions with high tactile acuity, including whisker follicles, fingertips and touch domes. Here we show that Merkel cells actively participate in touch reception in mice. Merkel cells display fast, touch-evoked mechanotransduction currents. Optogenetic approaches in intact skin show that Merkel cells are both necessary and sufficient for sustained action-potential firing in tactile afferents. Recordings from touch-dome afferents lacking Merkel cells demonstrate that Merkel cells confer high-frequency responses to dynamic stimuli and enable sustained firing. These data are the first, to our knowledge, to directly demonstrate a functional, excitatory connection between epidermal cells and sensory neurons. Together, these findings indicate that Merkel cells actively tune mechanosensory responses to facilitate high spatio-temporal acuity. Moreover, our results indicate a division of labour in the Merkel cell-neurite complex: Merkel cells signal static stimuli, such as

  5. Epidermal Viral Immunity Induced by CD8α+ Dendritic Cells But Not by Langerhans Cells

    NASA Astrophysics Data System (ADS)

    Allan, Rhys S.; Smith, Chris M.; Belz, Gabrielle T.; van Lint, Allison L.; Wakim, Linda M.; Heath, William R.; Carbone, Francis R.

    2003-09-01

    The classical paradigm for dendritic cell function derives from the study of Langerhans cells, which predominate within skin epidermis. After an encounter with foreign agents, Langerhans cells are thought to migrate to draining lymph nodes, where they initiate T cell priming. Contrary to this, we show here that infection of murine epidermis by herpes simplex virus did not result in the priming of virus-specific cytotoxic T lymphocytes by Langerhans cells. Rather, the priming response required a distinct CD8α+ dendritic cell subset. Thus, the traditional view of Langerhans cells in epidermal immunity needs to be revisited to accommodate a requirement for other dendritic cells in this response.

  6. Epidermal Micrografts Produced via an Automated and Minimally Invasive Tool Form at the Dermal/Epidermal Junction and Contain Proliferative Cells That Secrete Wound Healing Growth Factors

    PubMed Central

    Osborne, Sandra N.; Schmidt, Marisa A.; Derrick, Kathleen; Harper, John R.

    2015-01-01

    ABSTRACT OBJECTIVE: The aim of this scientific study was to assess epidermal micrografts for formation at the dermal-epidermal (DE) junction, cellular outgrowth, and growth factor secretion. Epidermal harvesting is an autologous option that removes only the superficial epidermal layer of the skin, considerably limiting donor site damage and scarring. Use of epidermal grafting in wound healing has been limited because of tedious, time-consuming, and inconsistent methodologies. Recently, a simplified, automated epidermal harvesting tool (CelluTome Epidermal Harvesting System; Kinetic Concepts Inc, San Antonio, Texas) that applies heat and suction concurrently to produce epidermal micrografts has become commercially available. The new technique of epidermal harvesting was shown to create viable micrografts with minimal patient discomfort and no donor-site scarring. DESIGN: This study was a prospective institutional review board–approved healthy human study. SETTING: This study was conducted at the multispecialty research facility, Clinical Trials of Texas, Inc, in San Antonio, Texas. PATIENTS: The participants were 15 healthy human volunteers. RESULTS: Epidermal micrografts formed at the DE junction, and migratory basal layer keratinocytes and melanocytes were proliferative in culture. Basement membrane–specific collagen type IV was also found to be present in the grafts, suggesting that the combination of heat and vacuum might cause partial delamination of the basement membrane. Viable basal cells actively secreted key growth factors important for modulating wound healing responses, including vascular endothelial growth factor, hepatocyte growth factor, granulocyte colony-stimulating factor, platelet-derived growth factor, and transforming growth factor α. CONCLUSIONS: Harvested epidermal micrografts retained their original keratinocyte structure, which is critical for potential re-epithelialization and repigmentation of a wound environment. PMID:26258460

  7. Role of Pin1 in UVA-induced cell proliferation and malignant transformation in epidermal cells

    SciTech Connect

    Han, Chang Yeob; Hien, Tran Thi; Lim, Sung Chul; Kang, Keon Wook

    2011-06-24

    Highlights: {yields} Pin1 expression is enhanced by low energy UVA irradiation in both skin tissues of hairless mice and JB6 C141 epidermal cells. {yields} UVA irradiation increases activator protein-1 activity and cyclin D1 in a Pin1-dependent manner. {yields} UVA potentiates EGF-inducible, anchorage-independent growth of epidermal cells, and this is suppressed by Pin1 inhibition or by anti-oxidant. -- Abstract: Ultraviolet A (UVA) radiation ({lambda} = 320-400 nm) is considered a major cause of human skin cancer. Pin1, a peptidyl prolyl isomerase, is overexpressed in most types of cancer tissues and plays an important role in cell proliferation and transformation. Here, we demonstrated that Pin1 expression was enhanced by low energy UVA (300-900 mJ/cm{sup 2}) irradiation in both skin tissues of hairless mice and JB6 C141 epidermal cells. Exposure of epidermal cells to UVA radiation increased cell proliferation and cyclin D1 expression, and these changes were blocked by Pin1 inhibition. UVA irradiation also increased activator protein-1 (AP-1) minimal reporter activity and nuclear levels of c-Jun, but not c-Fos, in a Pin1-dependent manner. The increases in Pin1 expression and in AP-1 reporter activity in response to UVA were abolished by N-acetylcysteine (NAC) treatment. Finally, we found that pre-exposure of JB6 C141 cells to UVA potentiated EGF-inducible, anchorage-independent growth, and this effect was significantly suppressed by Pin1inhibition or by NAC.

  8. Mechanosensory calcium-selective cation channels in epidermal cells

    NASA Technical Reports Server (NTRS)

    Ding, J. P.; Pickard, B. G.

    1993-01-01

    This paper explores the properties and likely functions of an epidermal Ca(2+)-selective cation channel complex activated by tension. As many as eight or nine linked or linkable equivalent conductance units or co-channels can open together. Open time for co-channel quadruplets and quintuplets tends to be relatively long with millimolar Mg2+ (but not millimolar Ca2+) at the cytosolic face of excised plasma membrane. Sensitivity to tension is regulated by transmembrane voltage and temperature. Under some circumstances channel activity is sychronized in rhythmic pulses. Certain lanthanides and a cytoskeleton-disturbing herbicide that inhibit gravitropic reception act on the channel system at low concentrations. Specifically, ethyl-N-phenylcarbamate promotes tension-dependent activity at micromolar levels. With moderate suction, Gd3+ provided at about 0.5 micromole at the extracellular face of the membrane promotes for several seconds but may then become inhibitory. Provision at 1-2 micromoles promotes and subsequently inhibits more vigorously (often abruptly and totally), and at high levels inhibits immediately. La3+, a poor gravitropic inhibitor, acts similarly but much more gradually and only at much higher concentrations. These properties, particularly these susceptibilities to modulation, indicate that in vivo the mechanosensitive channel must be mechanosensory and mechanoregulatory. It could serve to transduce the shear forces generated in the integrated wall-membrane-cytoskeleton system during turgor changes and cell expansion as well as transducing the stresses induced by gravity, touch and flexure. In so far as such transduction is modulated by voltage and temperature, the channels would also be sensors for these modalities as long as the wall-membrane-cytoskeleton system experiences mechanical stress.

  9. c-Rel in Epidermal Homeostasis: A Spotlight on c-Rel in Cell Cycle Regulation.

    PubMed

    Lorenz, Verena N; Schön, Michael P; Seitz, Cornelia S

    2016-06-01

    To maintain proper skin barrier function, epidermal homeostasis requires a subtly governed balance of proliferating and differentiating keratinocytes. While differentiation takes place in the suprabasal layers, proliferation, including mitosis, is usually restricted to the basal layer. Only recently identified as an important regulator of epidermal homeostasis, c-Rel, an NF-κB transcription factor subunit, affects the viability and proliferation of epidermal keratinocytes. In human keratinocytes, decreased expression of c-Rel causes a plethora of dysregulated cellular functions including impaired cell viability, increased apoptosis, and abnormalities during mitosis and cell cycle regulation. On the other hand, c-Rel shows aberrant expression in many epidermal tumors. Here, in the context of its role in different cell types and compared with other NF-κB subunits, we discuss the putative function of c-Rel as a regulator of epidermal homeostasis and mitotic progression. In addition, implications for disease pathophysiology with perturbed c-Rel function and abnormal homeostasis, such as epidermal carcinogenesis, will be discussed. PMID:27032306

  10. Hair matrix germinative epidermal cells confer follicle-inducing capabilities on dermal sheath and high passage papilla cells.

    PubMed

    Reynolds, A J; Jahoda, C A

    1996-10-01

    Low passage cultured dermal papilla cells from adult rats stimulate complete hair follicle neogenesis when re-implanted into heterotypic skin. In contrast, cultured sheath cells are non-inductive despite sharing other behavioural characteristics (a common lineage and in situ proximity) with papilla cells. However, since sheath cells can behave inductively in amputated follicles after regenerating the papilla, this poses the question of what influences the sheath to papilla cell transition? During reciprocal tissue interactions specific epidermal cues are crucial to skin appendage development, and while in vivo assays to date have focussed on dermal interactive influence, our aim was to investigate epidermal potential. We have previously observed that hair follicle epidermal cells display exceptional interactive behaviour when combined with follicle dermal cells in vitro. Thus in the present study, hair follicle germinative, outer root sheath or skin basal epidermal cells were separately combined with each of three non-inductive dermal cell types (high passage papilla, low passage sheath or fibroblast) and then implanted into small ear skin wounds. The sheath/germinative and papilla/germinative cell implants repeatedly induced giant vibrissa-type follicles and fibres. In complete contrast, any single cell type and all other forms of recombination were consistently non-inductive. Hence, the adult germinative epidermal cells enable non-inductive adult dermal cells to stimulate hair follicle neogenesis, effectively, by altering their 'status', causing the sheath cells to 'specialise' and the 'aged' papilla cells to 'rejuvenate'. PMID:8898222

  11. Regenerative and reparative effects of human chorion-derived stem cell conditioned medium on photo-aged epidermal cells.

    PubMed

    Li, Qiankun; Chen, Yan; Ma, Kui; Zhao, Along; Zhang, Cuiping; Fu, Xiaobing

    2016-04-17

    Epidermal cells are an important regenerative source for skin wound healing. Aged epidermal cells have a low ability to renew themselves and repair skin injury. Ultraviolet (UV) radiation, particularly UVB, can cause photo-aging of the skin by suppressing the viability of human epidermal cells. A chorion-derived stem cell conditioned medium (CDSC-CNM) is thought to have regenerative properties. This study aimed to determine the regenerative effects of CDSC-CNM on UVB-induced photo-aged epidermal cells. Epidermal cells were passaged four times and irradiated with quantitative UVB, and non-irradiated cells served as a control group. Cells were then treated with different concentrations of CDSC-CNM. Compared to the non-irradiated group, the proliferation rates and migration rates of UVB-induced photo-aged epidermal cells significantly decreased (p < 0.05) with increasing intracellular radical oxygen species (ROS) generation and DNA damage. After treatment with CDSC-CNM, photo-aged epidermal cells significantly improved their viability, and their ROS generation and DNA damage decreased. The secretory factors in CDSC-CNM, including epidermal growth factor (EGF), transforming growth factor-β (TGF-β), interleukin (IL)-6, and IL-8 and the related signaling pathway protein levels, increased compared to the control medium (CM). The potential regenerative and reparative effects of CDSC-CNM indicate that it may be a candidate material for the treatment of prematurely aged skin. The functions of the secretory factors and the mechanisms of CDSC-CNM therapy deserve further attention. PMID:27097375

  12. Single-cell gene expression profiling reveals functional heterogeneity of undifferentiated human epidermal cells

    PubMed Central

    Tan, David W. M.; Jensen, Kim B.; Trotter, Matthew W. B.; Connelly, John T.; Broad, Simon; Watt, Fiona M.

    2013-01-01

    Human epidermal stem cells express high levels of β1 integrins, delta-like 1 (DLL1) and the EGFR antagonist LRIG1. However, there is cell-to-cell variation in the relative abundance of DLL1 and LRIG1 mRNA transcripts. Single-cell global gene expression profiling showed that undifferentiated cells fell into two clusters delineated by expression of DLL1 and its binding partner syntenin. The DLL1+ cluster had elevated expression of genes associated with endocytosis, integrin-mediated adhesion and receptor tyrosine kinase signalling. Differentially expressed genes were not independently regulated, as overexpression of DLL1 alone or together with LRIG1 led to the upregulation of other genes in the DLL1+ cluster. Overexpression of DLL1 and LRIG1 resulted in enhanced extracellular matrix adhesion and increased caveolin-dependent EGFR endocytosis. Further characterisation of CD46, one of the genes upregulated in the DLL1+ cluster, revealed it to be a novel cell surface marker of human epidermal stem cells. Cells with high endogenous levels of CD46 expressed high levels of β1 integrin and DLL1 and were highly adhesive and clonogenic. Knockdown of CD46 decreased proliferative potential and β1 integrin-mediated adhesion. Thus, the previously unknown heterogeneity revealed by our studies results in differences in the interaction of undifferentiated basal keratinocytes with their environment. PMID:23482486

  13. Determining the Contribution of Epidermal Cell Shape to Petal Wettability Using Isogenic Antirrhinum Lines

    PubMed Central

    Whitney, Heather M.; Poetes, Rosa; Steiner, Ullrich; Chittka, Lars; Glover, Beverley J.

    2011-01-01

    The petal epidermis acts not only as a barrier to the outside world but also as a point of interaction between the flower and potential pollinators. The presence of conical petal epidermal cells has previously been shown to influence the attractiveness of the flower to pollinating insects. Using Antirrhinum isogenic lines differing only in the presence of a single epidermal structure, conical cells, we were able to investigate how the structure of the epidermis influences petal wettability by measuring the surface contact angle of water drops. Conical cells have a significant impact on how water is retained on the flower surface, which may have indirect consequences for pollinator behaviour. We discuss how the petal epidermis is a highly multifunctional one and how a battery of methods, including the use of isogenic lines, is required to untangle the impacts of specific epidermal properties in an ecological context. PMID:21423738

  14. Persistence of skin-resident memory T cells within an epidermal niche.

    PubMed

    Zaid, Ali; Mackay, Laura K; Rahimpour, Azad; Braun, Asolina; Veldhoen, Marc; Carbone, Francis R; Manton, Jonathan H; Heath, William R; Mueller, Scott N

    2014-04-01

    Barrier tissues such as the skin contain various populations of immune cells that contribute to protection from infections. These include recently identified tissue-resident memory T cells (TRM). In the skin, these memory CD8(+) T cells reside in the epidermis after being recruited to this site by infection or inflammation. In this study, we demonstrate prolonged persistence of epidermal TRM preferentially at the site of prior infection despite sustained migration. Computational simulation of TRM migration within the skin over long periods revealed that the slow rate of random migration effectively constrains these memory cells within the region of skin in which they form. Notably, formation of TRM involved a concomitant local reduction in dendritic epidermal γδ T-cell numbers in the epidermis, indicating that these populations persist in mutual exclusion and may compete for local survival signals. Accordingly, we show that expression of the aryl hydrocarbon receptor, a transcription factor important for dendritic epidermal γδ T-cell maintenance in skin, also contributes to the persistence of skin TRM. Together, these data suggest that skin tissue-resident memory T cells persist within a tightly regulated epidermal T-cell niche. PMID:24706879

  15. Persistence of skin-resident memory T cells within an epidermal niche

    PubMed Central

    Zaid, Ali; Mackay, Laura K.; Rahimpour, Azad; Braun, Asolina; Veldhoen, Marc; Carbone, Francis R.; Manton, Jonathan H.; Heath, William R.; Mueller, Scott N.

    2014-01-01

    Barrier tissues such as the skin contain various populations of immune cells that contribute to protection from infections. These include recently identified tissue-resident memory T cells (TRM). In the skin, these memory CD8+ T cells reside in the epidermis after being recruited to this site by infection or inflammation. In this study, we demonstrate prolonged persistence of epidermal TRM preferentially at the site of prior infection despite sustained migration. Computational simulation of TRM migration within the skin over long periods revealed that the slow rate of random migration effectively constrains these memory cells within the region of skin in which they form. Notably, formation of TRM involved a concomitant local reduction in dendritic epidermal γδ T-cell numbers in the epidermis, indicating that these populations persist in mutual exclusion and may compete for local survival signals. Accordingly, we show that expression of the aryl hydrocarbon receptor, a transcription factor important for dendritic epidermal γδ T-cell maintenance in skin, also contributes to the persistence of skin TRM. Together, these data suggest that skin tissue-resident memory T cells persist within a tightly regulated epidermal T-cell niche. PMID:24706879

  16. [Epidermal cell cultures--significance for wound coverage in the human].

    PubMed

    Bonnekoh, B; Thiele, B; Mahrle, G; Steigleder, G K

    1986-10-15

    Epithelial sheets can be cultivated from isolated epidermal cells; in this way, it is possible to increase the cell number considerably. H. Green and co-workers were the first to make use of such epithelia for the autologous covering of burn wounds. We modified this method and report on our experiences with this technique in a patient with small skin defects. PMID:2432733

  17. Examination of endothelial cell-induced epidermal regeneration in a mice-based chimney wound model.

    PubMed

    Seo, Joseph; Park, Soon-Jung; Choi, Jong-Jin; Kang, Sun-Woong; Lim, Joa-Jin; Lee, Hye-Jin; Kim, Jong-Soo; Yang, Heung-Mo; Kim, Sung-Joo; Kim, Eun-Young; Park, Se-Pil; Moon, Sung-Hwan; Chung, Hyung-Min

    2016-07-01

    As wound contraction in the cutaneous layer occurs rapidly in mice, mechanical means are typically used to deliberately expose the wound to properly investigate healing by secondary intention. Previously, silicon rings and splinting models were attempted to analyze histological recovery but prevention of surrounding epidermal cell migration and subsequent closure was minimal. Here, we developed an ideal chimney wound model to evaluate epidermal regeneration in murine under hESC-EC transplantation through histological analysis encompassing the three phases of regeneration: migration, proliferation, and remodeling. Human embryonic stem cell derived endothelial cells (hESC-EC) were transplanted due to possessing a well-known therapeutic effect in angiogenesis which also enhances epidermal repair to depict the process of regeneration. Following a standard 1 mm biopsy punch, a chimney manufactured by modifying a 1.7 mL microtube was simply inserted into the excisional wound to complete the modeling process. Under this model, the excisional wound remained fully exposed for 14 days and even after 4 weeks, only a thin transparent layer of epidermal tissue covered the wound site. This approach is able to more accurately depict epidermal repair in relation to histology while also being a user-friendly and cost-effective way to mimic human recovery in rodents and evaluate epithelial repair induced by a form of therapy. PMID:27237949

  18. Genetics Home Reference: epidermal nevus

    MedlinePlus

    ... primarily of a specific cell type called a keratinocyte. One group of epidermal nevi, called keratinocytic or nonorganoid epidermal nevi, includes nevi that involve only keratinocytes. Keratinocytic epidermal nevi are typically found on the ...

  19. SNAI2 controls the undifferentiated state of human epidermal progenitor cells.

    PubMed

    Mistry, Devendra S; Chen, Yifang; Wang, Ying; Zhang, Kang; Sen, George L

    2014-12-01

    The transcription factor, SNAI2, is an inducer of the epithelial to mesenchymal transition (EMT) which mediates cell migration during development and tumor invasion. SNAI2 can also promote the generation of mammary epithelial stem cells from differentiated luminal cells when overexpressed. How SNAI2 regulates these critical and diverse functions is unclear. Here, we show that the levels of SNAI2 expression are important for epidermal cell fate decisions. The expression of SNAI2 was found to be enriched in the basal layer of the interfollicular epidermis where progenitor cells reside and extinguished upon differentiation. Loss of SNAI2 resulted in premature differentiation whereas gain of SNAI2 expression inhibited differentiation. SNAI2 controls the differentiation status of epidermal progenitor cells by binding to and repressing the expression of differentiation genes with increased binding leading to further transcriptional silencing. Thus, the levels of SNAI2 binding to genomic targets determine the differentiation status of epithelial cells with increased levels triggering EMT and dedifferentiation, moderate (physiological) levels promoting epidermal progenitor function, and low levels leading to epidermal differentiation. PMID:25100569

  20. A rapid procedure for flow cytometric DNA analysis in cultures of normal and transformed epidermal cells.

    PubMed

    Tennenbaum, T; Giloh, H; Fusenig, N E; Kapitulnik, J

    1988-06-01

    A simple, rapid, and highly reproducible procedure for flow cytometric DNA analysis has been adapted for studying cell cycle kinetics in epidermal cell cultures. The preparation of cell nuclei and their staining with the fluorescent dye propidium iodide were performed directly on the culture dish, without prior suspension and fixation of the cells. Singly dispersed nuclei were produced by mild trypsinization of cells in the presence of the nonionic detergent Nonidet P-40 and spermine. The culture dishes could be kept frozen for prolonged periods of time before trypsinization and staining, without affecting either the recovery of nuclei or the cell cycle distribution profiles. This remarkable stability of cell nuclei greatly simplified the analysis of multiple samples in cell cycle kinetic studies. This method was used to analyze the cell cycle distribution in cultures of normal and transformed mouse epidermal cells, human colon carcinoma cells, primary bovine aortic endothelial cells, and fibroblastic and myogenic cell lines. This procedure should be very useful in studying growth kinetics, differentiation, and transformation of epidermal as well as other adherent cell types. PMID:2453587

  1. Increased symplasmic permeability in barley root epidermal cells correlates with defects in root hair development.

    PubMed

    Marzec, M; Muszynska, A; Melzer, M; Sas-Nowosielska, H; Kurczynska, E U

    2014-03-01

    It is well known that the process of plant cell differentiation depends on the symplasmic isolation of cells. Before starting the differentiation programme, the individual cell or group of cells should restrict symplasmic communication with neighbouring cells. We tested the symplasmic communication between epidermal cells in the different root zones of parental barley plants Hordeum vulgare L., cv. 'Karat' with normal root hair development, and two root hairless mutants (rhl1.a and rhl1.b). The results clearly show that symplasmic communication was limited during root hair differentiation in the parental variety, whereas in both root hairless mutants epidermal cells were still symplasmically connected in the corresponding root zone. This paper is the first report on the role of symplasmic isolation in barley root cell differentiation, and additionally shows that a disturbance in the restriction of symplasmic communication is present in root hairless mutants. PMID:23927737

  2. Increased symplasmic permeability in barley root epidermal cells correlates with defects in root hair development

    PubMed Central

    Marzec, M; Muszynska, A; Melzer, M; Sas-Nowosielska, H; Kurczynska, E U; Wick, S

    2014-01-01

    It is well known that the process of plant cell differentiation depends on the symplasmic isolation of cells. Before starting the differentiation programme, the individual cell or group of cells should restrict symplasmic communication with neighbouring cells. We tested the symplasmic communication between epidermal cells in the different root zones of parental barley plants Hordeum vulgare L., cv. ‘Karat’ with normal root hair development, and two root hairless mutants (rhl1.a and rhl1.b). The results clearly show that symplasmic communication was limited during root hair differentiation in the parental variety, whereas in both root hairless mutants epidermal cells were still symplasmically connected in the corresponding root zone. This paper is the first report on the role of symplasmic isolation in barley root cell differentiation, and additionally shows that a disturbance in the restriction of symplasmic communication is present in root hairless mutants. PMID:23927737

  3. Epidermal cell proliferation in guinea pigs with experimental dermatophytosis

    SciTech Connect

    Tagami, H.

    1985-08-01

    To elucidate the mechanisms underlying the self-healing process of experimental dermatophytosis produced in guinea pigs by an occlusive method with Trichophyton mentagrophytes, epidermal proliferative activity was evaluated by the in vivo tritiated thymidine-labeling technique performed at various intervals after the first and second infections. Determination of labeling indices disclosed that an increased epidermal proliferation correlated well with the severity of inflammatory changes, i.e., a peak activity was noted after 10 days in primary infection and at 2 days in reinfection, respectively, and was followed by subsequent spontaneous lesion clearance after 10 days. Application of a heat-killed spore suspension produced inflammatory changes with enhanced epidermopoiesis, similar to those induced by reinoculation of living spores, only in immune animals. The present results indicate that the dermatitic changes occurring in experimental dermatophytosis increase epidermopoiesis which facilitates elimination of the fungus from the stratum corneum and that host immune activity, particularly contact sensitivity to fungal antigen, exerts a crucial role to induce these changes.

  4. Wdr1-mediated cell shape dynamics and cortical tension are essential for epidermal planar cell polarity

    PubMed Central

    Pasolli, H. Amalia; Chai, Sophia; Nikolova, Maria; Stokes, Nicole; Fuchs, Elaine

    2015-01-01

    During mouse development, core planar cell polarity (PCP) proteins become polarized in the epidermal plane to guide angling/morphogenesis of hair follicles. How PCP is established is poorly understood. Here, we identify a key role for Wdr1 (also known as Aip1), an F-actin-binding protein that enhances cofilin/destrin-mediated F-actin disassembly. We show that cofilin and destrin function redundantly in developing epidermis, but their combined depletion perturbs cell adhesion, cytokinesis, apicobasal polarity and PCP. Although Wdr1 depletion accentuates single-loss-of-cofilin/destrin phenotypes, alone it resembles core PCP mutations. Seeking a mechanism, we find that Wdr1 and cofilin/destrin-mediated actomyosin remodelling are essential for generating or maintaining cortical tension within the developing epidermal sheet and driving the cell shape and planar orientation changes that accompany establishment of PCP in mammalian epidermis. Our findings suggest intriguing evolutionary parallels but mechanistic modifications to the distal wing hinge-mediated mechanical forces that drive cell shape change and orient PCP in the Drosophila wing disc. PMID:25915128

  5. Association between vascular-poor area of primary tumors and epidermal growth factor receptor gene status in advanced lung adenocarcinoma.

    PubMed

    Togashi, Yosuke; Masago, Katsuhiro; Kubo, Takeshi; Fujimoto, Daichi; Sakamori, Yuichi; Nagai, Hiroki; Kim, Young Hak; Togashi, Kaori; Mishima, Michiaki

    2012-12-01

    Mutation of the epidermal growth factor receptor gene (EGFR mutation) is a very important marker in the treatment for non-small cell lung cancer. Since signaling from this receptor induces tumor-associated angiogenesis, we hypothesized that lung cancers with EGFR mutations tend to develop locally with increased angiogenesis. Thus, the association between vascular-poor area of primary tumors and EGFR status was retrospectively investigated in advanced lung adenocarcinomas. To assess vascular-poor area, contrast-enhanced computed tomography scans taken before initial treatment for lung cancer were analyzed, together with primary tumor location (peripheral or central) and size. We analyzed 178 patients with advanced lung adenocarcinoma. EGFR mutations were detected in 95 of the 178 patients (53.4 %). EGFR mutation was found to be significantly related to women (P = 0.0070), never-smokers (P < 0.0001), and tumors without vascular-poor area (P < 0.0001). Based on a multivariate analysis, presence of EGFR mutations was independently associated with never-smokers (P = 0.0046), lack of vascular-poor area (P = 0.0001), and tumor size >30 mm (P = 0.0080). EGFR mutations were found in 41 of 51 never-smokers without vascular-poor area (80.4 %), 19 of 36 never-smokers with vascular-poor area (52.8 %), 19 of 37 current or former-smokers without vascular-poor area (51.4 %), and 16 of 54 current or former-smokers with vascular-poor area (29.6 %). This study showed an association between vascular-poor area of primary tumors and EGFR status. As a consequence, evaluation using a combination of smoking status and vascular-poor area allows us to predict presence of EGFR mutations at a high frequency. PMID:22492281

  6. Establishment of epidermal cell lines derived from the skin of the Atlantic bottlenose dolphin (Tursiops truncatus).

    PubMed

    Yu, Jin; Kindy, Mark S; Ellis, Blake C; Baatz, John E; Peden-Adams, Margie; Ellingham, Tara J; Wolff, Daynna J; Fair, Patricia A; Gattoni-Celli, Sebastiano

    2005-12-01

    The Atlantic bottlenose dolphin (Tursiops truncatus), a marine mammal found off the Atlantic coast, has become the focus of considerable attention because of an increasing number of mortality events witnessed in this species over the last several years along the southeastern United States. Assessment of the impact of environmental stressors on bottlenose dolphins (BND) has been difficult because of the protected status of these marine mammals. The studies presented herein focused on establishing epidermal cell cultures and cell lines as tools for the in vitro evaluation of environmental stressors on BND skin. Epidermal cell cultures were established from skin samples obtained from Atlantic BND and subjected to karyotype analysis. These cultures were further characterized using immunohistochemical methods demonstrating expression of cytokeratins. By two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), we observed that the proteomic profile of BND skin tissue samples shared distinct similarities with that of skin-derived cultures. Epidermal cell cultures were transfected with a plasmid encoding the SV40 small t- and large T-antigens, as well as the neomycin-resistance gene. Five neomycin-resistant clones were isolated and expanded, and all of them proliferated at a faster rate than nontransfected BND epidermal cultures, which exhibited signs of senescence. Cell lysates prepared from two transfected clones were shown to express, by Western blot analysis, both SV40 tumor antigens. These experimental results are consistent with the concept that transfected clones expressing SV40 tumor antigens represent immortalized BND cell lines. Epidermal cell lines derived from Tursiops truncatus will provide a unique tool for studying key features of the interaction occurring between dolphins and the environment in which they live at their most crucial interface: the skin. PMID:16281302

  7. Mast Cells Regulate Epidermal Barrier Function and the Development of Allergic Skin Inflammation.

    PubMed

    Sehra, Sarita; Serezani, Ana P M; Ocaña, Jesus A; Travers, Jeffrey B; Kaplan, Mark H

    2016-07-01

    Atopic dermatitis is a chronic inflammatory skin disease characterized by infiltration of eosinophils, T helper cells, and mast cells. The role of mast cells in atopic dermatitis is not completely understood. To define the effects of mast cells on skin biology, we observed that mast cells regulate the homeostatic expression of epidermal differentiation complex and other skin genes. Decreased epidermal differentiation complex gene expression in mice that genetically lack mast cells (Kit(W-sh/W-sh) mice) is associated with increased uptake of protein antigens painted on the skin by dendritic cells (DCs) compared with similarly treated wild-type mice, suggesting a protective role for mast cells in exposure to nominal environmental allergens. To test this further, we crossed Kit(W-sh/W-sh) mice with signal transducer and activator of transcription 6 (i.e., Stat6) VT transgenic mice that develop spontaneous atopic dermatitis-like disease that is dependent on T helper cell 2 cytokines and is associated with high serum concentrations of IgE. We observed that Stat6VT × Kit(W-sh/W-sh) mice developed more frequent and more severe allergic skin inflammation than Stat6VT transgenic mice that had mast cells. Together, these studies suggest that mast cells regulate epidermal barrier function and have a potential protective role in the development of atopic dermatitis-like disease. PMID:27021404

  8. A Theoretical Model of Jigsaw-Puzzle Pattern Formation by Plant Leaf Epidermal Cells

    PubMed Central

    Higaki, Takumi; Kutsuna, Natsumaro; Akita, Kae; Takigawa-Imamura, Hisako; Yoshimura, Kenji; Miura, Takashi

    2016-01-01

    Plant leaf epidermal cells exhibit a jigsaw puzzle–like pattern that is generated by interdigitation of the cell wall during leaf development. The contribution of two ROP GTPases, ROP2 and ROP6, to the cytoskeletal dynamics that regulate epidermal cell wall interdigitation has already been examined; however, how interactions between these molecules result in pattern formation remains to be elucidated. Here, we propose a simple interface equation model that incorporates both the cell wall remodeling activity of ROP GTPases and the diffusible signaling molecules by which they are regulated. This model successfully reproduces pattern formation observed in vivo, and explains the counterintuitive experimental results of decreased cellulose production and increased thickness. Our model also reproduces the dynamics of three-way cell wall junctions. Therefore, this model provides a possible mechanism for cell wall interdigitation formation in vivo. PMID:27054467

  9. A Theoretical Model of Jigsaw-Puzzle Pattern Formation by Plant Leaf Epidermal Cells.

    PubMed

    Higaki, Takumi; Kutsuna, Natsumaro; Akita, Kae; Takigawa-Imamura, Hisako; Yoshimura, Kenji; Miura, Takashi

    2016-04-01

    Plant leaf epidermal cells exhibit a jigsaw puzzle-like pattern that is generated by interdigitation of the cell wall during leaf development. The contribution of two ROP GTPases, ROP2 and ROP6, to the cytoskeletal dynamics that regulate epidermal cell wall interdigitation has already been examined; however, how interactions between these molecules result in pattern formation remains to be elucidated. Here, we propose a simple interface equation model that incorporates both the cell wall remodeling activity of ROP GTPases and the diffusible signaling molecules by which they are regulated. This model successfully reproduces pattern formation observed in vivo, and explains the counterintuitive experimental results of decreased cellulose production and increased thickness. Our model also reproduces the dynamics of three-way cell wall junctions. Therefore, this model provides a possible mechanism for cell wall interdigitation formation in vivo. PMID:27054467

  10. In vivo ultraviolet-exposed human epidermal cells activate T suppressor cell pathways that involve CD4+CD45RA+ suppressor-inducer T cells

    SciTech Connect

    Baadsgaard, O.; Salvo, B.; Mannie, A.; Dass, B.; Fox, D.A.; Cooper, K.D. )

    1990-11-01

    In vivo UV exposure of human epidermis abrogates the function of CD1+DR+ Langerhans cells and induces the appearance of CD1-DR+ Ag-presenting macrophages. Epidermal cells from UV-exposed skin, in contrast to epidermal cells from normal skin, potently activate autologous CD4+ T cells, and, in particular, the CD45RA+ (2H4+) (suppressor-inducer) subset. We therefore determined whether UV-exposure in humans leads to a T cell response in which suppression dominates. Autologous blood T cells were incubated with epidermal cell suspensions from in vivo UV-irradiated skin. After activation, repurified T cells were transferred in graded numbers to autologous mononuclear cells (MNC) stimulated with PWM and the resultant IgG production analyzed by ELISA. Relative to T cells activated by unirradiated control epidermal cells, T cells activated by UV-exposed epidermal cells demonstrated enhanced capacity to suppress IgG production (n = 6; p less than or equal to 0.03). Within the T cell population, CD8+ cells stimulated by UV-exposed epidermal cells could be directly activated to suppress PWM-stimulated MNC Ig production if IL-2 was provided in the reaction mixture. The suppressive activity was also transferable with purified CD4+ T cells stimulated by UV-exposed epidermal cells (n = 10; p less than or equal to 0.01), and was radiosensitive. Suppression was decreased when PWM-stimulated MNC were depleted of CD8+ T cells before mixing with CD4+ T cells activated by UV-exposed epidermal cells, suggesting indirect induction of CD8+ Ts cells contained within the responding MNC populations. Indeed, physical depletion of CD45RA+ cells resulted in total abrogation of the suppressor function contained in the CD4+ T cells. Activation of suppressor function was critically dependent on DR+ APC contained in UV-exposed epidermis.

  11. Proteins deposited in the dermis are rapidly captured and presented by epidermal Langerhans cells

    PubMed Central

    Flacher, Vincent; Tripp, Christoph H.; Stoitzner, Patrizia; Haid, Bernhard; Ebner, Susanne; Koch, Franz; Park, Chae Gyu; Steinman, Ralph M.; Idoyaga, Juliana; Romani, Nikolaus

    2010-01-01

    Antigen-presenting cells can capture antigens that are deposited in the skin, including vaccines given subcutaneously. These include different dendritic cells (DC) such as epidermal Langerhans cells (LC), dermal DC and dermal langerin+ DC. To evaluate access of dermal antigens to skin DC, we used mAb to two C-type lectin endocytic receptors, DEC-205/CD205 and langerin/CD207. When applied to murine and human skin explant cultures, these mAb were efficiently taken up by epidermal LC. Additionally, anti-DEC-205 targeted langerin+ CD103+ and langerin− CD103− mouse dermal DC. Unexpectedly, intradermal injection of either mAb, but not isotype control, resulted in strong and rapid labelling of LC in situ, implying that large molecules can diffuse through the basement membrane into the epidermis. Epidermal LC targeted in vivo by ovalbumin-coupled anti-DEC-205 potently presented antigen to CD4+ and CD8+ T cells. Thus, epidermal LC play a major role in uptake of lectin-binding ligands under standard vaccination conditions. PMID:19890348

  12. What is the influence of ordinary epidermal cells and stomata on the leaf plasticity of coffee plants grown under full-sun and shady conditions?

    PubMed

    Pompelli, M F; Martins, S C V; Celin, E F; Ventrella, M C; Damatta, F M

    2010-11-01

    Stomata are crucial in land plant productivity and survival. In general, with lower irradiance, stomatal and epidermal cell frequency per unit leaf area decreases, whereas guard-cell length or width increases. Nevertheless, the stomatal index is accepted as remaining constant. The aim of this paper to study the influence of ordinary epidermal cells and stomata on leaf plasticity and the influence of these characteristics on stomata density, index, and sizes, in the total number of stomata, as well as the detailed distribution of stomata on a leaf blade. As a result, a highly significant positive correlation (R²(a) = 0.767 p ≤ 0.001) between stomatal index and stomatal density, and with ordinary epidermal cell density (R²(a) = 0.500 p ≤ 0.05), and a highly negative correlation between stomatal index and ordinary epidermal cell area (R²(a) = -0.571 p ≤ 0.001), were obtained. However in no instance was the correlation between stomatal index or stomatal density and stomatal dimensions taken into consideration. The study also indicated that in coffee, the stomatal index was 19.09% in shaded leaves and 20.08% in full-sun leaves. In this sense, variations in the stomatal index by irradiance, its causes and the consequences on plant physiology were discussed. PMID:21180918

  13. CD166-mediated epidermal growth factor receptor phosphorylation promotes the growth of oral squamous cell carcinoma.

    PubMed

    Jia, Guodong; Wang, Xu; Yan, Ming; Chen, Wantao; Zhang, Ping

    2016-08-01

    CD166 has been considered a relatively specific marker of stem cells and cancer stem cells, and the altered expression of CD166 has also been reported as a prognostic marker of several other types of cancer. However, the molecular functions of CD166 in these cancer cells are largely unknown. In this study, we found that CD166 significantly enhanced epidermal growth factor receptor (EGFR) phosphorylation and prolonged epidermal growth factor (EGF)/EGFR signalling activation. In addition, EGF stimulation in CD166-overexpressing oral squamous carcinoma cells led to enhanced colony formation, invasion capacity and cytoskeletal re-organization in vitro and elevated tumourigenesis in vivo. Taken together, the results of our study identify CD166 as an intriguing therapeutic target for patients suffering from oral squamous cell carcinoma (OSCC). PMID:27424177

  14. Epidermal Cells Expressing Putative Cell Markers in Nonglabrous Skin Existing in Direct Proximity with the Distal End of the Arrector Pili Muscle

    PubMed Central

    Rufaut, N. W.; Jones, L.; Sinclair, R.

    2016-01-01

    Inconsistent with the view that epidermal stem cells reside randomly spread along the basal layer of the epidermal rete ridges, we found that epidermal cells expressing stem cell markers in nonglabrous skin exist in direct connection with the distal end of the arrector pili muscle. The epidermal cells that express stem cell markers consist of a subpopulation of basal keratinocytes located in a niche at the lowermost portion of the rete ridges at the distal arrector pili muscle attachment site. Keratinocytes in the epidermal stem cell niche express K15, MCSP, and α6 integrin. α5 integrin marks the distal end of the APM colocalized with basal keratinocytes expressing stem cell markers located in a well-protected and nourished environment at the lowermost point of the epidermis; these cells are hypothesized to participate directly in epidermal renewal and homeostasis and also indirectly in wound healing through communication with the hair follicle bulge epithelial stem cell population through the APM. Our findings, plus a reevaluation of the literature, support the hierarchical model of interfollicular epidermal stem cell units of Fitzpatrick. This new view provides insights into epidermal control and the possible involvement of epidermal stem cells in nonmelanoma skin carcinogenesis. PMID:27375744

  15. Epidermal stem cells: markers, patterning and the control of stem cell fate.

    PubMed Central

    Watt, F M

    1998-01-01

    Within the epidermis, proliferation takes place in the basal layer of keratinocytes that are attached to an underlying basement membrane. Cells that leave the basal layer undergo terminal differentiation as they move towards the tissue surface. The basal layer contains two types of proliferative keratinocyte: stem cells, which have unlimited self-renewal capacity, and transit amplifying cells, those daughters of stem cells that are destined to withdraw from the cell cycle and terminally differentiate after a few rounds of division. Stem cells express higher levels of the beta 1-integrin family of extracellular matrix receptors than transit amplifying cells and this can be used to isolate each subpopulation of keratinocyte and to determine its location within the epidermis. Variation in the levels of E-cadherin, beta-catenin and plakoglobin within the basal layer suggests that stem cells may also differ from transit amplifying cells in intercellular adhesiveness. Stem cells have a patterned distribution within the epidermal basal layer and patterning is subject to autoregulation. Constitutive expression of the transcription factor c-Myc promotes terminal differentiation by driving keratinocytes from the stem cell compartment into the transit amplifying compartment. PMID:9684280

  16. Effects of Telomerase and Telomere Length on Epidermal Stem Cell Behavior

    NASA Astrophysics Data System (ADS)

    Flores, Ignacio; Cayuela, María L.; Blasco, María A.

    2005-08-01

    A key process in organ homeostasis is the mobilization of stem cells out of their niches. We show through analysis of mouse models that telomere length, as well as the catalytic component of telomerase, Tert, are critical determinants in the mobilization of epidermal stem cells. Telomere shortening inhibited mobilization of stem cells out of their niche, impaired hair growth, and resulted in suppression of stem cell proliferative capacity in vitro. In contrast, Tert overexpression in the absence of changes in telomere length promoted stem cell mobilization, hair growth, and stem cell proliferation in vitro. The effects of telomeres and telomerase on stem cell biology anticipate their role in cancer and aging.

  17. Isolation of genes predominantly expressed in guard cells and epidermal cells of Nicotiana glauca.

    PubMed

    Smart, L B; Cameron, K D; Bennett, A B

    2000-04-01

    Guard cells are specialized and metabolically active cells which arise during the differentiation of the epidermis. Using Nicotiana glauca epidermal peels as a source of purified guard cells, we have constructed a cDNA library from guard cell RNA. In order to isolate genes that are predominantly expressed in guard cells, we performed a differential screen of this library, comparing the hybridization of a radiolabeled cDNA probe synthesized from guard cell RNA to that from a mesophyll cell cDNA probe. Sixteen clones were isolated based on their greater level of hybridization with the guard cell probe. Of these, eight had high homology to lipid transfer protein (LTP), two were similar to glycine-rich protein (GRP), and one displayed high homology to proline-rich proteins from Arabidopsis thaliana (AtPRP2, AtPRP4) and from potato guard cells (GPP). Northern analysis confirmed that one or more NgLTP genes, NgGRP1, and NgGPP1 are all differentially expressed, with highest levels in guard cells, and low or undetectable levels in mesophyll cells and in roots. In addition, all are induced to some degree in drought-stressed guard cells. NgLTP and NgGRP1 expression was localized by in situ hybridization to the guard cells and pavement cells in the epidermis. NgGRP1 expression was also detected in cells of the vasculature. Genomic Southern analysis indicated that LTP is encoded by a family of highly similar genes in N. glauca. This work has identified members of a subset of epidermis- and guard cell-predominant genes, whose protein products are likely to contribute to the unique properties acquired by guard cells and pavement cells during differentiation. PMID:10890533

  18. Transient Expression of P-type ATPases in Tobacco Epidermal Cells.

    PubMed

    Poulsen, Lisbeth R; Palmgren, Michael G; López-Marqués, Rosa L

    2016-01-01

    Transient expression in tobacco cells is a convenient method for several purposes such as analysis of protein-protein interactions and the subcellular localization of plant proteins. A suspension of Agrobacterium tumefaciens cells carrying the plasmid of interest is injected into the intracellular space between leaf epidermal cells, which results in DNA transfer from the bacteria to the plant and expression of the corresponding proteins. By injecting mixes of Agrobacterium strains, this system offers the possibility to co-express a number of target proteins simultaneously, thus allowing for example protein-protein interaction studies. In this chapter, we describe the procedure to transiently express P-type ATPases in tobacco epidermal cells, with focus on subcellular localization of the protein complexes formed by P4-ATPases and their β-subunits. PMID:26695049

  19. Three-dimensional culture of epidermal cells on ordered cellulose scaffolds.

    PubMed

    Seyama, Tomoko; Suh, Eun Young; Kondo, Tetsuo

    2013-06-01

    An ordered cellulose film scaffold, termed a nematic ordered cellulose (NOC) template, had unique surface properties and successfully induced the establishment of a three-dimensional (3D), hierarchical structure of epidermal cells by cell attachment and subsequent culture. Initially, the scaffold surface properties were characterized through contact angle measurements and atomic force microscopy to evaluate appropriate hydrophobicity and orientation of molecular chains for 3D culture. The template surfaces exhibited higher hydrophobicity, in the range of 70-75°, than usual cellulose films and appeared suitable for surface cell adhesion. In fact, epidermal cells successfully attached and proliferated favorably on the NOC templates, similar to development in normal culture flasks. Furthermore, the NOC film, as a semipermeable template, was also employed to allow 3D proliferation of epidermal cell layers in the perpendicular direction. The template proved to be suitable as a 3D cell culture device, resulting in the proposal that the construction processes of these 3D cell layers followed the basic concept of skin formation. PMID:23624420

  20. Colorimetric growth assay for epidermal cell cultures by their crystal violet binding capacity.

    PubMed

    Bonnekoh, B; Wevers, A; Jugert, F; Merk, H; Mahrle, G

    1989-01-01

    The application of a simple, rapid, and inexpensive colorimetric growth assay was tested for human epidermal cells subcultured in uncoated plastic dishes. Cell layers were incubated with a crystal violet (CV) solution (0.2% with ethanol 2% in 0.5 M Tris-Cl buffer, pH 7.8) for 10 min at room temperature. After rinsing with 0.5 M Tris-Cl (pH 7.8) the cell layer was dried and decolorized with a sodium-dodecylsulfate solution (0.5% with ethanol 50% in 0.5 M Tris-Cl, pH 7.8) for 60 min at 37 degrees C. The extinction of the supernatant was read at the absorption maximum of 586 nm. The protein content of attached cells as classical parameter for quantifying cell growth was strongly related to CV extinction with a correlation coefficient of r = 0.98. Furthermore, the subcellular protein binding qualities of CV were analyzed. The water-soluble protein fraction of cultured epidermal cells was separated by sodium-dodecylsulfate polyacrylamide gel electrophoresis and stained with CV. We found a staining pattern which was qualitatively very similar to that of Coomassie blue, however less intense. Keratin electrophoresis revealed an affinity of CV to the 48, 50, and 56 kD cytokeratins. In conclusion, this CV assay is a reliable and simple method for the monitoring of epidermal cell growth in cultures. PMID:2482013

  1. Single cell-type comparative metabolomics of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum

    PubMed Central

    Barkla, Bronwyn J.; Vera-Estrella, Rosario

    2015-01-01

    One of the remarkable adaptive features of the halophyte Mesembryanthemum crystallinum are the specialized modified trichomes called epidermal bladder cells (EBC) which cover the leaves, stems, and peduncle of the plant. They are present from an early developmental stage but upon salt stress rapidly expand due to the accumulation of water and sodium. This particular plant feature makes it an attractive system for single cell type studies, with recent proteomics and transcriptomics studies of the EBC establishing that these cells are metabolically active and have roles other than sodium sequestration. To continue our investigation into the function of these unusual cells we carried out a comprehensive global analysis of the metabolites present in the EBC extract by gas chromatography Time-of-Flight mass spectrometry (GC-TOF) and identified 194 known and 722 total molecular features. Statistical analysis of the metabolic changes between control and salt-treated samples identified 352 significantly differing metabolites (268 after correction for FDR). Principal components analysis provided an unbiased evaluation of the data variance structure. Biochemical pathway enrichment analysis suggested significant perturbations in 13 biochemical pathways as defined in KEGG. More than 50% of the metabolites that show significant changes in the EBC, can be classified as compatible solutes and include sugars, sugar alcohols, protein and non-protein amino acids, and organic acids, highlighting the need to maintain osmotic homeostasis to balance the accumulation of Na+ and Cl− ions. Overall, the comparison of metabolic changes in salt treated relative to control samples suggests large alterations in M. crystallinum epidermal bladder cells. PMID:26113856

  2. Differential Requirements of TCR Signaling in Homeostatic Maintenance and Function of Dendritic Epidermal T Cells.

    PubMed

    Zhang, Baojun; Wu, Jianxuan; Jiao, Yiqun; Bock, Cheryl; Dai, Meifang; Chen, Benny; Chao, Nelson; Zhang, Weiguo; Zhuang, Yuan

    2015-11-01

    Dendritic epidermal T cells (DETCs) are generated exclusively in the fetal thymus and maintained in the skin epithelium throughout postnatal life of the mouse. DETCs have restricted antigenic specificity as a result of their exclusive usage of a canonical TCR. Although the importance of the TCR in DETC development has been well established, the exact role of TCR signaling in DETC homeostasis and function remains incompletely defined. In this study, we investigated TCR signaling in fully matured DETCs by lineage-restricted deletion of the Lat gene, an essential signaling molecule downstream of the TCR. We found that Lat deletion impaired TCR-dependent cytokine gene activation and the ability of DETCs to undergo proliferative expansion. However, linker for activation of T cells-deficient DETCs were able to maintain long-term population homeostasis, although with a reduced proliferation rate. Mice with Lat deletion in DETCs exhibited delayed wound healing accompanied by impaired clonal expansion within the wound area. Our study revealed differential requirements for TCR signaling in homeostatic maintenance of DETCs and in their effector function during wound healing. PMID:26408667

  3. Genetically Induced Cell Death in Bulge Stem Cells Reveals Their Redundancy for Hair and Epidermal Regeneration

    PubMed Central

    Driskell, Iwona; Oeztuerk-Winder, Feride; Humphreys, Peter; Frye, Michaela

    2015-01-01

    Adult mammalian epidermis contains multiple stem cell populations in which quiescent and more proliferative stem and progenitor populations coexist. However, the precise interrelation of these populations in homeostasis remains unclear. Here, we blocked the contribution of quiescent keratin 19 (K19)-expressing bulge stem cells to hair follicle formation through genetic ablation of the essential histone methyltransferase Setd8 that is required for the maintenance of adult skin. Deletion of Setd8 eliminated the contribution of bulge cells to hair follicle regeneration through inhibition of cell division and induction of cell death, but the growth and morphology of hair follicles were unaffected. Furthermore, ablation of Setd8 in the hair follicle bulge blocked the contribution of K19-postive stem cells to wounded epidermis, but the wound healing process was unaltered. Our data indicate that quiescent bulge stem cells are dispensable for hair follicle regeneration and epidermal injury in the short term and support the hypothesis that quiescent and cycling stem cell populations are equipotent. Stem Cells 2015;33:988–998 PMID:25447755

  4. Proliferative and toxic effects of ultraviolet light and inflammation on epidermal pigment cells

    SciTech Connect

    Nordlund, J.J.; Ackles, A.E.; Traynor, F.F.

    1981-10-01

    The ear of the mouse is useful for studying the effects of ultraviolet light on epidermal pigment cells. The quantity of light penetrating into the skin causing an inflammatory response can be assessed easily by measuring with an engineering calipers the swelling of the ear. The inflammatory response of the ear exhibits a linear relationship to the dose of light delivered. We observed that doses of shortwave ultraviolet light which are noninflammatory when repeated at daily intervals induce moderate to severe inflammation. Small doses of psoralen and prolonged exposure to UVA (PUVA) were more inflammatory than larger amounts of psoralen and short exposure to light. Doses of shortwave ultraviolet light and PUVA which produce only a minimal inflammation of the skin stimulate the proliferation of epidermal melanocytes. In contrast, PUVA in doses sufficiently large to cause a marked inflammatory reaction in the skin seems injurious to pigment cells and kills them or causes only a minimal proliferative response. The inflammatory reaction itself does not seem to stimulate or inhibit the proliferation of melanocytes. Prostaglandins A, E, and F2 alpha have no effect on the proliferation of epidermal pigment cells. In contrast, dimethyl sulfoxide (DMSO) and allergic contact dermatitis increase the numerical density of pigment cells. Steroids may block the function of the enzyme tyrosinase. Our experiments indicate that pigment cells, like many other varieties of cells, are susceptible to injury and can be killed at least by large doses of PUVA.

  5. The Androgen Receptor Antagonizes Wnt/β-Catenin Signaling in Epidermal Stem Cells

    PubMed Central

    Kretzschmar, Kai; Cottle, Denny L; Schweiger, Pawel J; Watt, Fiona M

    2015-01-01

    Activation of Wnt/β-catenin signaling in adult mouse epidermis leads to expansion of the stem cell compartment and redirects keratinocytes in the interfollicular epidermis and sebaceous glands (SGs) to differentiate along the hair follicle (HF) lineages. Here we demonstrate that during epidermal development and homeostasis there is reciprocal activation of the androgen receptor (AR) and β-catenin in cells of the HF bulb. AR activation reduced β-catenin-dependent transcription, blocked β-catenin-induced induction of HF growth, and prevented β-catenin-mediated conversion of SGs into HFs. Conversely, AR inhibition enhanced the effects of β-catenin activation, promoting HF proliferation and differentiation, culminating in the formation of benign HF tumors and a complete loss of SG identity. We conclude that AR signaling has a key role in epidermal stem cell fate selection by modulating responses to β-catenin in adult mouse skin. PMID:26121213

  6. Human epidermal neural crest stem cells as a source of Schwann cells

    PubMed Central

    Sakaue, Motoharu; Sieber-Blum, Maya

    2015-01-01

    We show that highly pure populations of human Schwann cells can be derived rapidly and in a straightforward way, without the need for genetic manipulation, from human epidermal neural crest stem cells [hEPI-NCSC(s)] present in the bulge of hair follicles. These human Schwann cells promise to be a useful tool for cell-based therapies, disease modelling and drug discovery. Schwann cells are glia that support axons of peripheral nerves and are direct descendants of the embryonic neural crest. Peripheral nerves are damaged in various conditions, including through trauma or tumour-related surgery, and Schwann cells are required for their repair and regeneration. Schwann cells also promise to be useful for treating spinal cord injuries. Ex vivo expansion of hEPI-NCSC isolated from hair bulge explants, manipulating the WNT, sonic hedgehog and TGFβ signalling pathways, and exposure of the cells to pertinent growth factors led to the expression of the Schwann cell markers SOX10, KROX20 (EGR2), p75NTR (NGFR), MBP and S100B by day 4 in virtually all cells, and maturation was completed by 2 weeks of differentiation. Gene expression profiling demonstrated expression of transcripts for neurotrophic and angiogenic factors, as well as JUN, all of which are essential for nerve regeneration. Co-culture of hEPI-NCSC-derived human Schwann cells with rodent dorsal root ganglia showed interaction of the Schwann cells with axons, providing evidence of Schwann cell functionality. We conclude that hEPI-NCSCs are a biologically relevant source for generating large and highly pure populations of human Schwann cells. PMID:26251357

  7. Neuropilin 1 expression correlates with differentiation status of epidermal cells and cutaneous squamous cell carcinomas.

    PubMed

    Shahrabi-Farahani, Shokoufeh; Wang, Lili; Zwaans, Bernadette M M; Santana, Jeans M; Shimizu, Akio; Takashima, Seiji; Kreuter, Michael; Coultas, Leigh; D'Amore, Patricia A; Arbeit, Jeffrey M; Akslen, Lars A; Bielenberg, Diane R

    2014-07-01

    Neuropilins (NRPs) are cell surface receptors for vascular endothelial growth factor (VEGF) and SEMA3 (class 3 semaphorin) family members. The role of NRPs in neurons and endothelial cells has been investigated, but the expression and role of NRPs in epithelial cells is much less clear. Herein, the expression and localization of NRP1 was investigated in human and mouse skin and squamous cell carcinomas (SCCs). Results indicated that NRP1 mRNA and protein was expressed in the suprabasal epithelial layers of the skin sections. NRP1 staining did not overlap with that of keratin 14 (K14) or proliferating cell nuclear antigen, but did co-localize with staining for keratin 1, indicating that differentiated keratinocytes express NRP1. Similar to the expression of NRP1, VEGF-A was expressed in suprabasal epithelial cells, whereas Nrp2 and VEGFR2 were not detectable in the epidermis. The expression of NRP1 correlated with a high degree of differentiation in human SCC specimens, human SCC xenografts, and mouse K14-HPV16 transgenic SCC. UVB irradiation of mouse skin induced Nrp1 upregulation. In vitro, Nrp1 was upregulated in primary keratinocytes in response to differentiating media or epidermal growth factor-family growth factors. In conclusion, the expression of NRP1 is regulated in the skin and is selectively produced in differentiated epithelial cells. NRP1 may function as a reservoir to sequester VEGF ligand within the epithelial compartment, thereby modulating its bioactivity. PMID:24791743

  8. Epidermal Notch1 recruits RORγ+ group 3 innate lymphoid cells to orchestrate normal skin repair

    PubMed Central

    Li, Zhi; Hodgkinson, Tom; Gothard, Elizabeth J.; Boroumand, Soulmaz; Lamb, Rebecca; Cummins, Ian; Narang, Priyanka; Sawtell, Amy; Coles, Jenny; Leonov, German; Reboldi, Andrea; Buckley, Christopher D.; Cupedo, Tom; Siebel, Christian; Bayat, Ardeshir; Coles, Mark C.; Ambler, Carrie A.

    2016-01-01

    Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ+ ILC3s into wounded dermis; RORγ+ ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ+ ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair. PMID:27099134

  9. Epidermal Notch1 recruits RORγ(+) group 3 innate lymphoid cells to orchestrate normal skin repair.

    PubMed

    Li, Zhi; Hodgkinson, Tom; Gothard, Elizabeth J; Boroumand, Soulmaz; Lamb, Rebecca; Cummins, Ian; Narang, Priyanka; Sawtell, Amy; Coles, Jenny; Leonov, German; Reboldi, Andrea; Buckley, Christopher D; Cupedo, Tom; Siebel, Christian; Bayat, Ardeshir; Coles, Mark C; Ambler, Carrie A

    2016-01-01

    Notch has a well-defined role in controlling cell fate decisions in the embryo and the adult epidermis and immune systems, yet emerging evidence suggests Notch also directs non-cell-autonomous signalling in adult tissues. Here, we show that Notch1 works as a damage response signal. Epidermal Notch induces recruitment of immune cell subsets including RORγ(+) ILC3s into wounded dermis; RORγ(+) ILC3s are potent sources of IL17F in wounds and control immunological and epidermal cell responses. Mice deficient for RORγ(+) ILC3s heal wounds poorly resulting from delayed epidermal proliferation and macrophage recruitment in a CCL3-dependent process. Notch1 upregulates TNFα and the ILC3 recruitment chemokines CCL20 and CXCL13. TNFα, as a Notch1 effector, directs ILC3 localization and rates of wound healing. Altogether these findings suggest that Notch is a key stress/injury signal in skin epithelium driving innate immune cell recruitment and normal skin tissue repair. PMID:27099134

  10. Prevention of skin tumorigenesis and impairment of epidermal cell proliferation by targeted aquaporin-3 gene disruption.

    PubMed

    Hara-Chikuma, Mariko; Verkman, A S

    2008-01-01

    Aquaporin-3 (AQP3) is a water/glycerol-transporting protein expressed strongly at the plasma membranes of basal epidermal cells in skin. We found that human skin squamous cell carcinoma strongly overexpresses AQP3. A novel role for AQP3 in skin tumorigenesis was discovered using mice with targeted AQP3 gene disruption. We found that AQP3-null mice were remarkably resistant to the development of skin tumors following exposure to a tumor initiator and phorbol ester promoter. Though tumor initiator challenge produced comparable apoptotic responses in wild-type and AQP3-null mice, promoter-induced cell proliferation was greatly impaired in the AQP3-null epidermis. Reductions of epidermal cell glycerol, its metabolite glycerol-3-phosphate, and ATP were found in AQP3 deficiency without impairment of mitochondrial function. Glycerol supplementation corrected the reduced proliferation and ATP content in AQP3 deficiency, with cellular glycerol, ATP, and proliferative ability being closely correlated. Our data suggest involvement of AQP3-facilitated glycerol transport in epidermal cell proliferation and tumorigenesis by a novel mechanism implicating cellular glycerol as a key determinant of cellular ATP energy. AQP3 may thus be an important determinant in skin tumorigenesis and hence a novel target for tumor prevention and therapy. PMID:17967887

  11. Sensitivity of human granulosa cell tumor cells to epidermal growth factor receptor inhibition.

    PubMed

    Andersson, Noora; Anttonen, Mikko; Färkkilä, Anniina; Pihlajoki, Marjut; Bützow, Ralf; Unkila-Kallio, Leila; Heikinheimo, Markku

    2014-04-01

    Epidermal growth factor receptor (EGFR) is implicated in the progression of many human cancers, but its significance in ovarian granulosa cell tumor (GCT) pathobiology remains poorly understood. We assessed the EGFR gene copy number, surveyed the mRNA and protein expression patterns of EGFR in 90 adult GCTs, and assessed the in vitro sensitivity of GCT cells to EGFR inhibition. Low-level amplification of EGFR gene was observed in five GCTs and high-level amplification in one sample. EGFR mRNA was robustly expressed in GCTs. Most tumors expressed both unphosphorylated and phosphorylated EGFR protein, but the protein expression did not correlate with clinical parameters, including the risk of recurrence. Small-molecule EGFR inhibitors reduced the EGF-induced activation of EGFR and its downstream signaling molecules at nanomolar doses, but cell viability was reduced, and caspase-3/7 was activated in GCT cells only at micromolar doses. Based on the present results, EGFR is active and abundantly expressed in the majority of GCTs, but probably has only minor contribution to GCT cell growth. Given the high doses of EGFR inhibitors required to reduce GCT cell viability in vitro, they are not likely to be effective for GCT treatment as single agents; they should rather be tested as part of combination therapies for these malignancies. PMID:24463098

  12. Correction of junctional epidermolysis bullosa by transplantation of genetically modified epidermal stem cells.

    PubMed

    Mavilio, Fulvio; Pellegrini, Graziella; Ferrari, Stefano; Di Nunzio, Francesca; Di Iorio, Enzo; Recchia, Alessandra; Maruggi, Giulietta; Ferrari, Giuliana; Provasi, Elena; Bonini, Chiara; Capurro, Sergio; Conti, Andrea; Magnoni, Cristina; Giannetti, Alberto; De Luca, Michele

    2006-12-01

    The continuous renewal of human epidermis is sustained by stem cells contained in the epidermal basal layer and in hair follicles. Cultured keratinocyte stem cells, known as holoclones, generate sheets of epithelium used to restore severe skin, mucosal and corneal defects. Mutations in genes encoding the basement membrane component laminin 5 (LAM5) cause junctional epidermolysis bullosa (JEB), a devastating and often fatal skin adhesion disorder. Epidermal stem cells from an adult patient affected by LAM5-beta3-deficient JEB were transduced with a retroviral vector expressing LAMB3 cDNA (encoding LAM5-beta3), and used to prepare genetically corrected cultured epidermal grafts. Nine grafts were transplanted onto surgically prepared regions of the patient's legs. Engraftment was complete after 8 d. Synthesis and proper assembly of normal levels of functional LAM5 were observed, together with the development of a firmly adherent epidermis that remained stable for the duration of the follow-up (1 year) in the absence of blisters, infections, inflammation or immune response. Retroviral integration site analysis indicated that the regenerated epidermis is maintained by a defined repertoire of transduced stem cells. These data show that ex vivo gene therapy of JEB is feasible and leads to full functional correction of the disease. PMID:17115047

  13. Meis1 Regulates Epidermal Stem Cells and Is Required for Skin Tumorigenesis

    PubMed Central

    Okumura, Kazuhiro; Saito, Megumi; Isogai, Eriko; Aoto, Yoshimasa; Hachiya, Tsuyoshi; Sakakibara, Yasubumi; Katsuragi, Yoshinori; Hirose, Satoshi; Kominami, Ryo; Goitsuka, Ryo; Nakamura, Takuro; Wakabayashi, Yuichi

    2014-01-01

    Previous studies have shown that Meis1 plays an important role in blood development and vascular homeostasis, and can induce blood cancers, such as leukemia. However, its role in epithelia remains largely unknown. Here, we uncover two roles for Meis1 in the epidermis: as a critical regulator of epidermal homeostasis in normal tissues and as a proto-oncogenic factor in neoplastic tissues. In normal epidermis, we show that Meis1 is predominantly expressed in the bulge region of the hair follicles where multipotent adult stem cells reside, and that the number of these stem cells is reduced when Meis1 is deleted in the epidermal tissue of mice. Mice with epidermal deletion of Meis1 developed significantly fewer DMBA/TPA-induced benign and malignant tumors compared with wild-type mice, suggesting that Meis1 plays a role in both tumor development and malignant progression. This is consistent with the observation that Meis1 expression increases as tumors progress from benign papillomas to malignant carcinomas. Interestingly, we found that Meis1 localization was altered to neoplasia development. Instead of being localized to the stem cell region, Meis1 is localized to more differentiated cells in tumor tissues. These findings suggest that, during the transformation from normal to neoplastic tissues, a functional switch occurs in Meis1. PMID:25013928

  14. Epidermal cells are the primary phagocytes in the fragmentation and clearance of degenerating dendrites in Drosophila

    PubMed Central

    Xiao, Hui; Wang, Denan; Franc, Nathalie C.; Jan, Lily Yeh; Jan, Yuh-Nung

    2014-01-01

    SUMMARY During developmental remodeling, neurites destined for pruning often degenerate on-site. Physical injury also induces degeneration of neurites distal to the injury site. Prompt clearance of degenerating neurites is important for maintaining tissue homeostasis and preventing inflammatory responses. Here we show that in both dendrite pruning and dendrite injury of Drosophila sensory neurons, epidermal cells rather than hemocytes are the primary phagocytes in clearing degenerating dendrites. Epidermal cells act via Draper-mediated recognition to facilitate dendrite degeneration and to engulf and degrade degenerating dendrites. Using multiple dendritic membrane markers to trace phagocytosis, we show that two members of the CD36 family, croquemort (crq) and debris buster (dsb), act at distinct stages of phagosome maturation for dendrite clearance. Our finding reveals the physiological importance of coordination between neurons and their surrounding epidermis, for both dendrite fragmentation and clearance. PMID:24412417

  15. Effects of topical pimecrolimus 1% on high-dose ultraviolet B-irradiated epidermal Langerhans cells.

    PubMed

    Yin, ZhiQiang; Xu, JiaLi; Zhang, ZhiHong; Luo, Dan

    2012-12-01

    Some studies reported no changes in the number of epidermal Langerhans cells (LC) that were observed in mice treated with pimecrolimus, and low-dose stimulated solar radiation (once)-induced changers in LC are minimally affected by pimecrolimus. This study is to investigate the effects of topical pimecrolimus 1% on high-dose ultraviolet B (UVB)-irradiated epidermal LC. Forty human foreskin tissues were randomly divided into 4 groups of 10 tissues each: Group A, control; Group B, pimecrolimus 1% (once)-only; Group C, 180 mJ/cm(2) UVB (once)-only; Group D, UVB+pimecrolimus. Each tissue was cut into 4 pieces corresponding to 4 time points. All the tissues were cultured at 37 °C. After being treated, the tissues were collected respectively and processed for immunohistochemical staining and immunofluorescence staining. For UVB-only group, epidermal CD1a(+) LC number at 18h decreased from 39.6 ± 8.30 to 22.3 ± 2.26/5 high magnification, compared to CD1a(+) LC number at 0 h (P<0.01). The CD1a(+) LC number of UVB-only group was significantly less than other groups at 18 h, 24h and 48 h (P<0.05, respectively). Similar results were obtained with immunofluorescence staining for CD 1a and immunohistochemical staining for Langerin. The numbers of epidermal HLA-DR(+) LC had no significant differences among all groups at different time points. Our study found a single 180 mJ/cm(2) UVB irradiation significantly reduced epidermal LC numbers at 18 h, 24h and 48 h, however, topical pimecrolimus could reverse these changes. UVB plus pimecrolimus treatment did not affect human LC maturation. PMID:23079131

  16. Skin Stem Cells: At the Frontier Between the Laboratory and Clinical Practice. Part 1: Epidermal Stem Cells.

    PubMed

    Pastushenko, I; Prieto-Torres, L; Gilaberte, Y; Blanpain, C

    2015-11-01

    Stem cells are characterized by their ability to self-renew and differentiate into the different cell lineages of their tissue of origin. The discovery of stem cells in adult tissues, together with the description of specific markers for their isolation, has opened up new lines of investigation, expanding the horizons of biomedical research and raising new hope in the treatment of many diseases. In this article, we review in detail the main characteristics of the stem cells that produce the specialized cells of the skin (epidermal, mesenchymal, and melanocyte stem cells) and their potential implications and applications in diseases affecting the skin. Part I deals with the principal characteristics and potential applications of epidermal stem cells in dermatology. PMID:26189363

  17. Epidermal growth factor receptor in non-small cell lung cancer

    PubMed Central

    2015-01-01

    Following the identification of a group of patients in the initial tyrosine kinase inhibitor (TKI) trials for lung cancer, there has been detailed focus on which patients may benefit from inhibitor therapy. This article reviews the background, genetics and prevalence of epidermal growth factor mutations in non-small cell lung cancer (NSCLC). Additionally, the prevalence in unselected patients is compared against various other reviews. PMID:25870793

  18. Substance P combined with epidermal stem cells promotes wound healing and nerve regeneration in diabetes mellitus.

    PubMed

    Zhu, Fei-Bin; Fang, Xiang-Jing; Liu, De-Wu; Shao, Ying; Zhang, Hong-Yan; Peng, Yan; Zhong, Qing-Ling; Li, Yong-Tie; Liu, De-Ming

    2016-03-01

    Exogenous substance P accelerates wound healing in diabetes, but the mechanism remains poorly understood. Here, we established a rat model by intraperitoneally injecting streptozotocin. Four wounds (1.8 cm diameter) were drilled using a self-made punch onto the back, bilateral to the vertebral column, and then treated using amniotic membrane with epidermal stem cells and/or substance P around and in the middle of the wounds. With the combined treatment the wound-healing rate was 100% at 14 days. With prolonged time, type I collagen content gradually increased, yet type III collagen content gradually diminished. Abundant protein gene product 9.5- and substance P-immunoreactive nerve fibers regenerated. Partial nerve fiber endings extended to the epidermis. The therapeutic effects of combined substance P and epidermal stem cells were better than with amniotic membrane and either factor alone. Our results suggest that the combination of substance P and epidermal stem cells effectively contributes to nerve regeneration and wound healing in diabetic rats. PMID:27127492

  19. Substance P combined with epidermal stem cells promotes wound healing and nerve regeneration in diabetes mellitus

    PubMed Central

    Zhu, Fei-bin; Fang, Xiang-jing; Liu, De-wu; Shao, Ying; Zhang, Hong-yan; Peng, Yan; Zhong, Qing-ling; Li, Yong-tie; Liu, De-ming

    2016-01-01

    Exogenous substance P accelerates wound healing in diabetes, but the mechanism remains poorly understood. Here, we established a rat model by intraperitoneally injecting streptozotocin. Four wounds (1.8 cm diameter) were drilled using a self-made punch onto the back, bilateral to the vertebral column, and then treated using amniotic membrane with epidermal stem cells and/or substance P around and in the middle of the wounds. With the combined treatment the wound-healing rate was 100% at 14 days. With prolonged time, type I collagen content gradually increased, yet type III collagen content gradually diminished. Abundant protein gene product 9.5- and substance P-immunoreactive nerve fibers regenerated. Partial nerve fiber endings extended to the epidermis. The therapeutic effects of combined substance P and epidermal stem cells were better than with amniotic membrane and either factor alone. Our results suggest that the combination of substance P and epidermal stem cells effectively contributes to nerve regeneration and wound healing in diabetic rats. PMID:27127492

  20. A sensitive electrochemiluminescence cytosensor for quantitative evaluation of epidermal growth factor receptor expressed on cell surfaces.

    PubMed

    Tang, Yanjuan; Zhang, Shaolian; Wen, Qingqing; Huang, Hongxing; Yang, Peihui

    2015-06-30

    A sensitive electrochemiluminescence (ECL) strategy for evaluating the epidermal growth factor receptor (EGFR) expression level on cell surfaces was designed by integrating the specific recognition of EGFR expressed on MCF-7 cell surfaces with an epidermal growth factor (EGF)-funtionalized CdS quantum dots (CdSQDs)-capped magnetic bead (MB) probe. The high sensitivity of ECL probe of EGF-funtionalized CdSQD-capped-MB was used for competitive recognition with EGFR expressed on cell surfaces with recombinant EGFR protein. The changes of ECL intensity depended on both the cell number and the expression level of EGFR receptor on cell surfaces. A wide linear response to cells ranging from 80 to 4×10(6)cellsmL(-1) with a detection limit of 40cellsmL(-1) was obtained. The EGF-cytosensor was used to evaluate EGFR expression levels on MCF-7 cells, and the average number of EGFR receptor on single MCF-7 cells was 1.35×10(5) with the relative standard deviation of 4.3%. This strategy was further used for in-situ and real-time evaluating EGFR receptor expressed on cell surfaces in response to drugs stimulation at different concentration and incubation time. The proposed method provided potential applications in the detection of receptors on cancer cells and anticancer drugs screening. PMID:26041531

  1. Asymmetric growth of root epidermal cells is related to the differentiation of root hair cells in Hordeum vulgare (L.)

    PubMed Central

    Marzec, Marek

    2013-01-01

    The root epidermis of most vascular plants harbours two cell types, namely trichoblasts (capable of producing a root hair) and atrichoblasts. Here, in vivo analysis, confocal laser-scanning microscopy, transmission electron microscopy, histological analysis, and three-dimensional reconstruction were used to characterize the cell types present in the barley root epidermis and their distribution in the tissue. Both trichoblasts and atrichoblasts were present in the wild-type cultivars and could be distinguished from one another at an early stage. Trichoblast/atrichoblast differentiation depended on asymmetric cell expansion after a period of symmetrical cell division. After asymmetric growth, only the shorter epidermal cells could produce root hairs, whereas the longer cells became atrichoblasts. Moreover, the root epidermis did not develop root hairs at all if the epidermal cells did not differentiate into two asymmetric cell types. The root hairless phenotype of bald root barley (brb) and root hairless 1.b (rhl1.b) mutants was caused by a mutation in a gene related to the asymmetric expansion of the root epidermal cells. Additionally, the results showed that the mechanism of trichoblast/atrichoblast differentiation is not evolutionally conserved across the subfamilies of the Poaceae; in the Pooideae subfamily, both asymmetric division and asymmetric cell expansion have been observed. PMID:24043851

  2. Effects of epidermal growth factor on neural crest cells in tissue culture

    SciTech Connect

    Erickson, C.A.; Turley, E.A.

    1987-04-01

    Epidermal growth factor (EGF) stimulates the release of hyaluronic acid (HA) and chondroitin sulfate proteoglycan (CSPG) from quail trunk neural crest cultures in a dose-dependent fashion. It also promotes the expression of cell-associated heparan sulfate proteoglycan (HSPG) as detected by immunofluorescence and immunoprecipitation of the /sup 3/H-labeled proteoglycan. Furthermore, EGF stimulates (/sup 3/H)thymidine incorporation into total cell DNA. These results raise the possibility that EGF or an analogous growth factor is involved in regulation of neural crest cell morphogenesis.

  3. Mechanisms of Chemical Cooperative Carcinogenesis by Epidermal Langerhans Cells

    PubMed Central

    Lewis, Julia M.; Bürgler, Christina D.; Fraser, Juliet A.; Liao, Haihui; Golubets, Kseniya; Kucher, Cynthia L.; Zhao, Peter Y.; Filler, Renata B.; Tigelaar, Robert E.; Girardi, Michael

    2014-01-01

    Cutaneous squamous cell carcinoma (SCC) is the most prevalent invasive malignancy with metastatic potential. The epidermis is exposed to a variety of environmental DNA-damaging chemicals, principal among which are polyaromatic hydrocarbons (PAH) ubiquitous in the environment, tobacco smoke, and broiled meats. Langerhans cells (LC) comprise a network of dendritic cells situated adjacent to basal, suprabasal, and follicular infundibular keratinocytes that when mutated can give rise to SCC, and LC-intact mice are markedly more susceptible than LC-deficient mice to chemical carcinogenesis provoked by initiation with the model PAH, 7,12-dimethylbenz[a]anthracene (DMBA). LC rapidly internalize and depot DMBA as numerous membrane-independent cytoplasmic foci. Repopulation of LC-deficient mice using fetal liver LC-precursors restores DMBA-induced tumor susceptibility. LC expression of p450 enzyme CYP1B1 is required for maximal rapid induction of DNA-damage within adjacent keratinocytes and their efficient neoplastic transformation; however, effects of tumor progression also attributable to the presence of LC were revealed as CYP1B1-independent. Thus, LC make multifaceted contributions to cutaneous carcinogenesis, including via the handling and metabolism of chemical mutagens. Such findings suggest a cooperative carcinogenesis role for myeloid-derived cells resident within cancer susceptible epithelial tissues principally by influencing early events in malignant transformation. PMID:25233073

  4. Corrective transduction of human epidermal stem cells in laminin-5-dependent junctional epidermolysis bullosa.

    PubMed

    Dellambra, E; Vailly, J; Pellegrini, G; Bondanza, S; Golisano, O; Macchia, C; Zambruno, G; Meneguzzi, G; De Luca, M

    1998-06-10

    Laminin-5 is composed of three distinct polypeptides, alpha3, beta3, and gamma2, which are encoded by three different genes, LAMA3, LAMB3, and LAMC2, respectively. We have isolated epidermal keratinocytes from a patient presenting with a lethal form of junctional epidermolysis bullosa characterized by a homozygous mutation of the LAMB3 gene, which led to complete absence of the beta3 polypeptide. In vitro, beta3-null keratinocytes were unable to synthesize laminin-5 and to assemble hemidesmosomes, maintained the impairment of their adhesive properties, and displayed a decrease of their colony-forming ability. A retroviral construct expressing a human beta3 cDNA was used to transduce primary beta3-null keratinocytes. Clonogenic beta3-null keratinocytes were transduced with an efficiency of 100%. Beta3-transduced keratinocytes were able to synthesize and secrete mature heterotrimeric laminin-5. Gene correction fully restored the keratinocyte adhesion machinery, including the capacity of proper hemidesmosomal assembly, and prevented the loss of the colony-forming ability, suggesting a direct link between adhesion to laminin-5 and keratinocyte proliferative capacity. Clonal analysis demonstrated that holoclones expressed the transgene permanently, suggesting stable correction of epidermal stem cells. Because cultured keratinocytes are used routinely to make autologous grafts for patients suffering from large skin or mucosal defects, the full phenotypic reversion of primary human epidermal stem cells defective for a structural protein opens new perspectives in the long-term treatment of genodermatoses. PMID:9650620

  5. Low calcium culture condition induces mesenchymal cell-like phenotype in normal human epidermal keratinocytes

    SciTech Connect

    Takagi, Ryo; Yamato, Masayuki; Murakami, Daisuke; Sugiyama, Hiroaki; Okano, Teruo

    2011-08-26

    Highlights: {yields} Normal human epidermal keratinocytes serially cultured under low calcium concentration were cytokeratin and vimentin double positive cells. {yields} The human keratinocytes expressed some epithelial stem/progenitor cell makers, mesenchymal cell markers, and markers of epithelial-mesenchymal transition. {yields} Mesenchymal cell-like phenotype in the keratinocytes was suppressed under high-calcium condition. -- Abstract: Epithelial-mesenchymal transition (EMT) is an important cellular phenomenon in organ developments, cancer invasions, and wound healing, and many types of transformed cell lines are used for investigating for molecular mechanisms of EMT. However, there are few reports for EMT in normal human epithelial cells, which are non-transformed or non-immortalized cells, in vitro. Therefore, normal human epidermal keratinocytes (NHEK) serially cultured in low-calcium concentration medium (LCM) were used for investigating relations between differentiation and proliferation and mesenchymal-like phenotype in the present study, since long-term cultivation of NHEK is achieved in LCM. Interestingly, NHEK serially cultured in LCM consisted essentially of cytokeratin-vimentin double positive cells (98%), although the NHEK exhibited differentiation under high-calcium culture condition with 3T3 feeder layer. The vimentin expression was suppressed under high-calcium condition. These results may indicate the importance of mesenchymal-like phenotype for serially cultivation of NHEK in vitro.

  6. Effect of microtubule-associated protein-4 on epidermal cell migration under different oxygen concentrations.

    PubMed

    Chen, Xin; Zhou, Xin; Mao, Tong-Chun; Shi, Xiao-Hua; Fan, Dong-Li; Zhang, Yi-Ming

    2016-06-01

    After skin trauma, regional epidermal cell migration mediates the re-epithelialization of the wound surface, which is an important step for wound healing, yet the underlying molecular regulatory mechanism is unclear. In the current study, HaCaT cells were maintained under different oxygen concentrations (1%, 21%, 40% and 65%). Technologies including immunofluorescence staining, wound scratch, transwell invasion, western blot and low-expression lentiviral vector were utilized to observe the changes in microtubule dynamics and the microtubule-associated protein (MAP)4 expression. MAP4's effect on cell migration under different oxygen concentrations was also studied. The results showed that under hyperoxic (40% and 65%) and hypoxic (1%) conditions, HaCaT cells were able to regulate cell microtubule dynamics by MAP4, thus promoting cell migration. On the other hand, MAP4 silencing through targeted shRNA attenuated HaCaT cell migration under the above oxygen concentrations. These results imply that MAP4 plays an important role in epidermal cell migration under different oxygen concentrations. PMID:26602869

  7. Cell type-specific responses to salinity - the epidermal bladder cell transcriptome of Mesembryanthemum crystallinum.

    PubMed

    Oh, Dong-Ha; Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar; Lee, Sang-Yeol; Bohnert, Hans J; Dassanayake, Maheshi

    2015-08-01

    Mesembryanthemum crystallinum (ice plant) exhibits extreme tolerance to salt. Epidermal bladder cells (EBCs), developing on the surface of aerial tissues and specialized in sodium sequestration and other protective functions, are critical for the plant's stress adaptation. We present the first transcriptome analysis of EBCs isolated from intact plants, to investigate cell type-specific responses during plant salt adaptation. We developed a de novo assembled, nonredundant EBC reference transcriptome. Using RNAseq, we compared the expression patterns of the EBC-specific transcriptome between control and salt-treated plants. The EBC reference transcriptome consists of 37 341 transcript-contigs, of which 7% showed significantly different expression between salt-treated and control samples. We identified significant changes in ion transport, metabolism related to energy generation and osmolyte accumulation, stress signalling, and organelle functions, as well as a number of lineage-specific genes of unknown function, in response to salt treatment. The salinity-induced EBC transcriptome includes active transcript clusters, refuting the view of EBCs as passive storage compartments in the whole-plant stress response. EBC transcriptomes, differing from those of whole plants or leaf tissue, exemplify the importance of cell type-specific resolution in understanding stress adaptive mechanisms. PMID:25944243

  8. Model system for plant cell biology: GFP imaging in living onion epidermal cells

    NASA Technical Reports Server (NTRS)

    Scott, A.; Wyatt, S.; Tsou, P. L.; Robertson, D.; Allen, N. S.

    1999-01-01

    The ability to visualize organelle localization and dynamics is very useful in studying cellular physiological events. Until recently, this has been accomplished using a variety of staining methods. However, staining can give inaccurate information due to nonspecific staining, diffusion of the stain or through toxic effects. The ability to target green fluorescent protein (GFP) to various organelles allows for specific labeling of organelles in vivo. The disadvantages of GFP thus far have been the time and money involved in developing stable transformants or maintaining cell cultures for transient expression. In this paper, we present a rapid transient expression system using onion epidermal peels. We have localized GFP to various cellular compartments (including the cell wall) to illustrate the utility of this method and to visualize dynamics of these compartments. The onion epidermis has large, living, transparent cells in a monolayer, making them ideal for visualizing GFP. This method is easy and inexpensive, and it allows for testing of new GFP fusion proteins in a living tissue to determine deleterious effects and the ability to express before stable transformants are attempted.

  9. Communication is key: Reducing DEK1 activity reveals a link between cell-cell contacts and epidermal cell differentiation status.

    PubMed

    Galletti, Roberta; Ingram, Gwyneth C

    2015-01-01

    Plant epidermis development requires not only the initial acquisition of tissue identity, but also the ability to differentiate specific cell types over time and to maintain these differentiated states throughout the plant life. To set-up and maintain differentiation, plants activate specific transcriptional programs. Interfering with these programs can prevent differentiation and/or force differentiated cells to lose their identity and re-enter a proliferative state. We have recently shown that the Arabidopsis Defective Kernel 1 (DEK1) protein is required both for the differentiation of epidermal cells and for the maintenance of their fully differentiated state. Defects in DEK1 activity lead to a deregulation of the expression of epidermis-specific differentiation-promoting HD-ZIP IV transcription factors. Here we propose a working model in which DEK1, by maintaining cell-cell contacts, and thus communication between neighboring cells, influences HD-ZIP IV gene expression and epidermis differentiation. PMID:27064205

  10. Lewisy Promotes Migration of Oral Cancer Cells by Glycosylation of Epidermal Growth Factor Receptor

    PubMed Central

    Lin, Wei-Ling; Lin, Yi-Shiuan; Shi, Guey-Yueh; Chang, Chuan-Fa; Wu, Hua-Lin

    2015-01-01

    Aberrant glycosylation changes normal cellular functions and represents a specific hallmark of cancer. Lewisy (Ley) carbohydrate upregulation has been reported in a variety of cancers, including oral squamous cell carcinoma (OSCC). A high level of Ley expression is related to poor prognosis of patients with oral cancer. However, it is unclear how Ley mediates oral cancer progression. In this study, the role of Ley in OSCC was explored. Our data showed that Ley was upregulated in HSC-3 and OC-2 OSCC cell lines. Particularly, glycosylation of epidermal growth factor receptor (EGFR) with Ley was found in OC-2 cells, and this modification was absent upon inhibition of Ley synthesis. The absence of Ley glycosylation of EGFR weakened phosphorylation of AKT and ERK in response to epidermal growth factor (EGF). Additionally, EGF-triggered cell migration was reduced, but cell proliferation was not affected. Ley modification stabilized EGFR upon ligand activation. Conversely, absence of Ley glycosylation accelerated EGFR degradation. In summary, these results indicate that increased expression of Ley in OSCC cells is able to promote cell migration by modifying EGFR which in turn stabilizes EGFR expression and downstream signaling. Targeting Ley on EGFR could have a potential therapeutic effect on oral cancer. PMID:25799278

  11. Cell population kinetic parameters for acute epidermal reactions in man

    SciTech Connect

    Cohen, L.

    1986-11-01

    Cell population kinetic parameters for acute reactions in squamous epithelium were estimated using available data on skin tolerance doses. Roughly equivalent doses for kilovoltage radiation delivered in equal daily fractions, as reported by F. Ellis (Br. J. Radiol. 15, 348-350 (1942)) and by R. Paterson (The Treatment of Malignant Disease by Radium and X-Rays. Edward Arnold, London, 1948), were combined with data for nonstandard fractionation at longer intervals of 1 or 2 weeks. By analyzing the combined data set, well-determined parameters could be derived. The data show that repopulation, with a potential cell doubling time of about 7 days, must occur in irradiated human skin, though this may possibly be limited to no more than seven doublings. The parameters derived are distinctly different from those associated with late-reacting dose-limiting tissues. The main difference is the steeper initial slope of the computed survival curve, that is a larger J parameter (multitarget model) or a larger alpha component (linear-quadratic model).

  12. Amphiregulin enhances regulatory T cell-suppressive function via the epidermal growth factor receptor.

    PubMed

    Zaiss, Dietmar M W; van Loosdregt, Jorg; Gorlani, Andrea; Bekker, Cornelis P J; Gröne, Andrea; Sibilia, Maria; van Bergen en Henegouwen, Paul M P; Roovers, Rob C; Coffer, Paul J; Sijts, Alice J A M

    2013-02-21

    Epidermal growth factor receptor (EGFR) is known to be critically involved in tissue development and homeostasis as well as in the pathogenesis of cancer. Here we showed that Foxp3(+) regulatory T (Treg) cells express EGFR under inflammatory conditions. Stimulation with the EGF-like growth factor Amphiregulin (AREG) markedly enhanced Treg cell function in vitro, and in a colitis and tumor vaccination model we showed that AREG was critical for efficient Treg cell function in vivo. In addition, mast cell-derived AREG fully restored optimal Treg cell function. These findings reveal EGFR as a component in the regulation of local immune responses and establish a link between mast cells and Treg cells. Targeting of this immune regulatory mechanism may contribute to the therapeutic successes of EGFR-targeting treatments in cancer patients. PMID:23333074

  13. Expression of an Exogenous Growth Hormone Gene by Transplantable Human Epidermal Cells

    NASA Astrophysics Data System (ADS)

    Morgan, Jeffrey R.; Barrandon, Yann; Green, Howard; Mulligan, Richard C.

    1987-09-01

    Retrovirus-mediated gene transfer was used to introduce a recombinant human growth hormone gene into cultured human keratinocytes. The transduced keratinocytes secreted biologically active growth hormone into the culture medium. When grafted as an epithelial sheet onto athymic mice, these cultured keratinocytes reconstituted an epidermis that was similar in appearance to that resulting from normal cells, but from which human growth hormone could be extracted. Transduced epidermal cells may prove to be a general vehicle for the delivery of gene products by means of grafting.

  14. Protein profiling of epidermal bladder cells from the halophyte Mesembryanthemum crystallinum.

    PubMed

    Barkla, Bronwyn J; Vera-Estrella, Rosario; Pantoja, Omar

    2012-09-01

    Plant epidermal trichomes are as varied in morphology as they are in function. In the halophyte Mesembryanthemum crystallinum, specialized trichomes called epidermal bladder cells (EBC) line the surface of leaves and stems, and increase dramatically in size and volume upon plant salt-treatment. These cells have been proposed to have roles in plant defense and UV protection, but primarily in sodium sequestration and as water reservoirs. To gain further understanding into the roles of EBC, a cell-type-specific proteomics approach was taken in which precision single-cell sampling of cell sap from individual EBC was combined with shotgun peptide sequencing (LC-MS/MS). Identified proteins showed diverse biological functions and cellular locations, with a high representation of proteins involved in H(+)-transport, carbohydrate metabolism, and photosynthesis. The proteome of EBC provides insight into the roles of these cells in ion and water homeostasis and raises the possibility that they are photosynthetically active and functioning in Crassulacean acid metabolism. PMID:22848050

  15. Vanadate induces apoptosis in epidermal JB6 P+ cells via hydrogen peroxide-mediated reactions.

    PubMed

    Ye, J; Ding, M; Leonard, S S; Robinson, V A; Millecchia, L; Zhang, X; Castranova, V; Vallyathan, V; Shi, X

    1999-12-01

    Apoptosis is a physiological mechanism for the control of DNA integrity in mammalian cells. Vanadium induces both DNA damage and apoptosis. It is suggested that vanadium-induced apoptosis serves to eliminate DNA-damaged cells. This study is designed to clarify a role of reactive oxygen species in the mechanism of apoptosis induced by vanadium. We established apoptosis model with murine epidermal JB6 P+ cells in the response to vanadium stimulation. Apoptosis was detected by a cell death ELISA assay and morphological analysis. The result shows that apoptosis induced by vanadate is dose-dependent, reaching its saturation level at a concentration of 100 microM vanadate. Vanadyl (IV) can also induce apoptosis albeit with lesser potency. A role of reactive oxygen species was analyzed by multiple reagents including specific scavengers of different reactive oxygen species. The result shows that vanadate-induced apoptosis is enhanced by NADPH, superoxide dismutase and sodium formate, but was inhibited by catalase and deferoxamine. Cells exposed to vanadium consume more molecular oxygen and at the same time, produce more H2O2 as measured by the change in fluorescence of scopoletin in the presence of horseradish peroxidase. This change in oxygen consumption and H2O2 production is enhanced by NADPH. Taken together, these results show that vanadate induces apoptosis in epidermal cells and H2O2 induced by vanadate plays a major role in this process. PMID:10705990

  16. Ultrastructure of the Epidermal Cell Wall and Cuticle of Tomato Fruit (Solanum lycopersicum L.) during Development1[OPEN

    PubMed Central

    Segado, Patricia; Domínguez, Eva

    2016-01-01

    The epidermis plays a pivotal role in plant development and interaction with the environment. However, it is still poorly understood, especially its outer epidermal wall: a singular wall covered by a cuticle. Changes in the cuticle and cell wall structures are important to fully understand their functions. In this work, an ultrastructure and immunocytochemical approach was taken to identify changes in the cuticle and the main components of the epidermal cell wall during tomato fruit development. A thin and uniform procuticle was already present before fruit set. During cell division, the inner side of the procuticle showed a globular structure with vesicle-like particles in the cell wall close to the cuticle. Transition between cell division and elongation was accompanied by a dramatic increase in cuticle thickness, which represented more than half of the outer epidermal wall, and the lamellate arrangement of the non-cutinized cell wall. Changes in this non-cutinized outer wall during development showed specific features not shared with other cell walls. The coordinated nature of the changes observed in the cuticle and the epidermal cell wall indicate a deep interaction between these two supramolecular structures. Hence, the cuticle should be interpreted within the context of the outer epidermal wall. PMID:26668335

  17. Epidermal growth factor increases LRF/Pokemon expression in human prostate cancer cells.

    PubMed

    Aggarwal, Himanshu; Aggarwal, Anshu; Agrawal, Devendra K

    2011-10-01

    Leukemia/lymphoma related factor/POK erythroid myeloid ontogenic factor (LRF/Pokemon) is a member of the POK family of proteins that promotes oncogenesis in several forms of cancer. Recently, we found higher LRF expression in human breast and prostate carcinomas compared to the corresponding normal tissues. The aim of this study was to examine the regulation of LRF expression in human prostate cells. Epidermal growth factor (EGF) and its receptors mediate several tumorigenic cascades that regulate cell differentiation, proliferation, migration and survival of prostate cancer cells. There was significantly higher level of LRF expression in the nucleus of LNCaP and PC-3 cells than RWPE-1 cells. A significant increase in LRF expression was observed with increasing doses of EGF in more aggressive and androgen-sensitive prostate cancer cells suggesting that EGF signaling pathway is critical in upregulating the expression of LRF/Pokemon to promote oncogenesis. PMID:21640721

  18. Basic helix-loop-helix transcription factors and epidermal cell fate determination in Arabidopsis

    PubMed Central

    Zhao, Hongtao; Li, Xia; Ma, Ligeng

    2012-01-01

    Cell fate determination is an important process in multicellular organisms. Plant epidermis is a readily-accessible, well-used model for the study of cell fate determination. Our knowledge of cell fate determination is growing steadily due to genetic and molecular analyses of root hairs, trichomes, and stomata, which are derived from the epidermal cells of roots and aerial tissues. Studies have shown that a large number of factors are involved in the establishment of these cell types, especially members of the basic helix-loop-helix (bHLH) superfamily, which is an important family of transcription factors. In this mini-review, we focus on the role of bHLH transcription factors in cell fate determination in Arabidopsis. PMID:23073001

  19. A GFP-MAP4 reporter gene for visualizing cortical microtubule rearrangements in living epidermal cells

    PubMed

    Marc; Granger; Brincat; Fisher; Kao; McCubbin; Cyr

    1998-11-01

    Microtubules influence morphogenesis by forming distinct geometrical arrays in the cell cortex, which in turn affect the deposition of cellulose microfibrils. Although many chemical and physical factors affect microtubule orientation, it is unclear how cortical microtubules in elongating cells maintain their ordered transverse arrays and how they reorganize into new geometries. To visualize these reorientations in living cells, we constructed a microtubule reporter gene by fusing the microtubule binding domain of the mammalian microtubule-associated protein 4 (MAP4) gene with the green fluorescent protein (GFP) gene, and transient expression of the recombinant protein in epidermal cells of fava bean was induced. The reporter protein decorates microtubules in vivo and binds to microtubules in vitro. Confocal microscopy and time-course analysis of labeled cortical arrays along the outer epidermal wall revealed the lengthening, shortening, and movement of microtubules; localized microtubule reorientations; and global microtubule reorganizations. The global microtubule orientation in some cells fluctuates about the transverse axis and may be a result of a cyclic self-correcting mechanism to maintain a net transverse orientation during cellular elongation. PMID:9811799

  20. Response of mouse epidermal cells to single doses of heavy-particles

    NASA Technical Reports Server (NTRS)

    Leith, J. T.; Schilling, W. A.; Welch, G. P.

    1972-01-01

    The survival of mouse epidermal cells to heavy-particles has been studied In Vivo by the Withers clone technique. Experiments with accelerated helium, lithium and carbon ions were performed. The survival curve for the helium ion irradiations used a modified Bragg curve method with a maximum tissue penetration of 465 microns, and indicated that the dose needed to reduce the original cell number to 1 surviving cell/square centimeters was 1525 rads with a D sub o of 95 rads. The LET at the basal cell layer was 28.6 keV per micron. Preliminary experiments with lithium and carbon used treatment doses of 1250 rads with LET's at the surface of the skin of 56 and 193 keV per micron respectively. Penetration depths in skin were 350 and 530 microns for the carbon and lithium ions whose Bragg curves were unmodified. Results indicate a maximum RBE for skin of about 2 using the skin cloning technique. An attempt has been made to relate the epidermal cell survival curve to mortality of the whole animal for helium ions.

  1. Transdifferentiation of Umbilical Cord-Derived Mesenchymal Stem Cells Into Epidermal-Like Cells by the Mimicking Skin Microenvironment.

    PubMed

    Chen, Deyun; Hao, Haojie; Tong, Chuan; Liu, Jiejie; Dong, Liang; Ti, Dongdong; Hou, Qian; Liu, Huiling; Han, Weidong; Fu, Xiaobing

    2015-06-01

    Human umbilical cord-derived mesenchymal stem cells (UC-MSCs) are multipotent, primitive, and have been widely used for skin tissue engineering. Their transdifferentiation is determined by the local microenvironment. In this study, we investigated the potential epidermal differentiation of UC-MSCs and the formation of epidermis substitutes in a 3-dimensional (3D) microenvironment, which was fabricated by UC-MSCs embedded into collagen-chitosan scaffolds (CCSs) combined with an air-liquid interface (ALI) culture system. Using fluorescence microscope, we observed that UC-MSCs were spindle-shaped and evenly distributed in the scaffold. Methyl thiazolyl blue tetrazolium bromide assay and Live/Dead assay indicated that the CCSs have good biocompatibility with UC-MSCs. Immunohistochemistry and western blotting assay showed that UC-MSCs on the surface of the CCSs were positive for the epidermal markers cytokeratin 19 and involucrin at 14 days. In addition, hematoxylin-eosin staining indicated that multilayered epidermis substitutes were established. The constructed epidermis substitutes were applied to treat full-thickness wounds in rats and proved to promote wound healing. In conclusion, manipulating the 3D microenvironment is a novel method for inducing the epidermal differentiation of MSCs to engineer epidermal substitutes, which provides an alternative strategy for skin tissue engineering. PMID:25700709

  2. IL-31-Driven Skin Remodeling Involves Epidermal Cell Proliferation and Thickening That Lead to Impaired Skin-Barrier Function

    PubMed Central

    Singh, Brijendra; Jegga, Anil G.; Shanmukhappa, Kumar S.; Edukulla, Ramakrishna; Khurana, Gurjit H.; Medvedovic, Mario; Dillon, Stacey R.; Madala, Satish K.

    2016-01-01

    Interleukin-31 (IL-31) is a type 2 helper T-cell-derived cytokine that has recently been shown to cause severe inflammation and tissue remodeling in multiple chronic diseases of the skin and lungs. IL-31 is upregulated in allergic and inflammatory diseases, including atopic dermatitis, asthma, cutaneous T-cell lymphomas, and allergic rhinitis, as well as autoimmune diseases such as systemic erythematosus. Overexpression of IL-31 in T cells causes severe inflammation, with histological features similar to skin lesions of patients with atopic dermatitis. However, the molecular mechanisms involved in IL31-driven pathological remodeling in skin diseases remain largely unknown. Here, we studied the role of IL-31 in skin damage as a result of intradermal administration of recombinant IL-31 into mice. Notably, IL-31 was sufficient to increase epidermal basal-cell proliferation and thickening of the epidermal skin layer. Our findings demonstrate a progressive increase in transepidermal water loss with chronic administration of IL-31 into the skin. Further, analysis of the skin transcriptome indicates a significant increase in the transcripts involved in epidermal-cell proliferation, epidermal thickening, and mechanical integrity. In summary, our findings demonstrate an important role for IL-31 signaling in epidermal cell proliferation and thickening that together may lead to impaired skin-barrier function in pathological remodeling of the skin. PMID:27556734

  3. The expression of peripheral benzodiazepine receptors in human skin: the relationship with epidermal cell differentiation.

    PubMed

    Stoebner, P E; Carayon, P; Penarier, G; Fréchin, N; Barnéon, G; Casellas, P; Cano, J P; Meynadier, J; Meunier, L

    1999-06-01

    The peripheral benzodiazepine receptor (PBR) is a protein of mitochondrial outer membranes utilizing porphyrins as endogenous ligands. PBR is part of a heteromeric receptor complex involved in the formation of mitochondrial permeability transition pores and in the early events of apoptosis. PBR may function as an oxygen-dependent signal generator; recent data indicate that these receptors may preserve the mitochondria of haematopoietic cell lines from damage caused by oxygen radicals. To identify PBRs in human skin, we used a specific monoclonal antibody directed against the C-terminus fragment of the human receptor. PBR immunoreactivity was found in keratinocytes, Langerhans cells, hair follicles and dermal vascular endothelial cells. Interestingly, confocal microscopic examination of skin sections revealed that PBR expression was strongly upregulated in the superficial differentiated layers of the epidermis. Ultrastructurally, PBRs were distributed throughout the cytoplasm but were selectively expressed on the mitochondrial membranes of epidermal cells. The elevated level of PBRs in the spinous layer was not associated with an increased number of mitochondria nor with an increased amount of mRNA as assessed by in situ hybridization on microautoradiographed skin sections. The present work provides, for the first time, evidence of PBR immunoreactivity in human skin. This mitochondrial receptor may modulate apoptosis in the epidermis; its increased expression in differentiated epidermal layers may represent a novel mechanism of natural skin protection against free radical damage generated by ultraviolet exposure. PMID:10354064

  4. Transcriptional mechanisms link epithelial plasticity to adhesion and differentiation of epidermal progenitor cells

    PubMed Central

    Lee, Briana; Villarreal-Ponce, Alvaro; Fallahi, Magid; Ovadia, Jeremy; Sun, Peng; Yu, Qian-Chun; Ito, Seiji; Sinha, Satrajit; Nie, Qing; Dai, Xing

    2014-01-01

    During epithelial tissue morphogenesis, developmental progenitor cells undergo dynamic adhesive and cytoskeletal remodeling to trigger proliferation and migration. Transcriptional mechanisms that restrict such mild form of epithelial plasticity to maintain lineage-restricted differentiation in committed epithelial tissues are poorly understood. Here we report that simultaneous ablation of transcriptional repressor-encoding Ovol1 and Ovol2 results in expansion and blocked terminal differentiation of embryonic epidermal progenitor cells. Conversely, mice overexpressing Ovol2 in their skin epithelia exhibit precocious differentiation accompanied by smaller progenitor cell compartments. We show that Ovol1/2-deficient epidermal cells fail to undertake α-catenin–driven actin cytoskeletal reorganization and adhesive maturation, and exhibit changes that resemble epithelial-to-mesenchymal transition (EMT). Remarkably, these alterations as well as defective terminal differentiation are reversed upon depletion of EMT-promoting transcriptional factor Zeb1. Collectively, our findings reveal Ovol-Zeb1-α-catenin sequential repression and highlight functions of Ovol as gatekeepers of epithelial adhesion and differentiation by inhibiting progenitor-like traits and epithelial plasticity. PMID:24735878

  5. In Situ Measurement of Epidermal Cell Turgor, Leaf Water Potential, and Gas Exchange in Tradescantia virginiana L. 1

    PubMed Central

    Shackel, Kenneth A.; Brinckmann, Enno

    1985-01-01

    A combined system has been developed in which epidermal cell turgor, leaf water potential, and gas exchange were determined for transpiring leaves of Tradescantia virginiana L. Uniform and stable values of turgor were observed in epidermal cells (stomatal complex cells were not studied) under stable environmental conditions for both upper and lower epidermises. The changes in epidermal cell turgor that were associated with changes in leaf transpiration were larger than the changes in leaf water potential, indicating the presence of transpirationally induced within-leaf water potential gradients. Estimates of 3 to 5 millimoles per square meter per second per megapascal were obtained for the value of within-leaf hydraulic conductivity. Step changes in atmospheric humidity caused rapid changes in epidermal cell turgor with little or no initial change in stomatal conductance, indicating little direct relation between stomatal humidity response and epidermal water status. The significance of within-leaf water potential gradients to measurements of plant water potential and to current hypotheses regarding stomatal response to humidity is discussed. PMID:16664210

  6. Effect of Storage Temperature on Cultured Epidermal Cell Sheets Stored in Xenobiotic-Free Medium

    PubMed Central

    Jackson, Catherine; Aabel, Peder; Eidet, Jon R.; Messelt, Edward B.; Lyberg, Torstein; von Unge, Magnus; Utheim, Tor P.

    2014-01-01

    Cultured epidermal cell sheets (CECS) are used in regenerative medicine in patients with burns, and have potential to treat limbal stem cell deficiency (LSCD), as demonstrated in animal models. Despite widespread use, short-term storage options for CECS are limited. Advantages of storage include: flexibility in scheduling surgery, reserve sheets for repeat operations, more opportunity for quality control, and improved transportation to allow wider distribution. Studies on storage of CECS have thus far focused on cryopreservation, whereas refrigeration is a convenient method commonly used for whole skin graft storage in burns clinics. It has been shown that preservation of viable cells using these methods is variable. This study evaluated the effect of different temperatures spanning 4°C to 37°C, on the cell viability, morphology, proliferation and metabolic status of CECS stored over a two week period in a xenobiotic–free system. Compared to non-stored control, best cell viability was obtained at 24°C (95.2±9.9%); reduced cell viability, at approximately 60%, was demonstrated at several of the temperatures (12°C, 28°C, 32°C and 37°C). Metabolic activity was significantly higher between 24°C and 37°C, where glucose, lactate, lactate/glucose ratios, and oxygen tension indicated increased activation of the glycolytic pathway under aerobic conditions. Preservation of morphology as shown by phase contrast and scanning electron micrographs was best at 12°C and 16°C. PCNA immunocytochemistry indicated that only 12°C and 20°C allowed maintenance of proliferative function at a similar level to non-stored control. In conclusion, results indicate that 12°C and 24°C merit further investigation as the prospective optimum temperature for short-term storage of cultured epidermal cell sheets. PMID:25170754

  7. Analysis of the Actin–Myosin II System in Fish Epidermal Keratocytes: Mechanism of Cell Body Translocation

    PubMed Central

    Svitkina, Tatyana M.; Verkhovsky, Alexander B.; McQuade, Kyle M.; Borisy, Gary G.

    1997-01-01

    While the protrusive event of cell locomotion is thought to be driven by actin polymerization, the mechanism of forward translocation of the cell body is unclear. To elucidate the mechanism of cell body translocation, we analyzed the supramolecular organization of the actin–myosin II system and the dynamics of myosin II in fish epidermal keratocytes. In lamellipodia, long actin filaments formed dense networks with numerous free ends in a brushlike manner near the leading edge. Shorter actin filaments often formed T junctions with longer filaments in the brushlike area, suggesting that new filaments could be nucleated at sides of preexisting filaments or linked to them immediately after nucleation. The polarity of actin filaments was almost uniform, with barbed ends forward throughout most of the lamellipodia but mixed in arc-shaped filament bundles at the lamellipodial/cell body boundary. Myosin II formed discrete clusters of bipolar minifilaments in lamellipodia that increased in size and density towards the cell body boundary and colocalized with actin in boundary bundles. Time-lapse observation demonstrated that myosin clusters appeared in the lamellipodia and remained stationary with respect to the substratum in locomoting cells, but they exhibited retrograde flow in cells tethered in epithelioid colonies. Consequently, both in locomoting and stationary cells, myosin clusters approached the cell body boundary, where they became compressed and aligned, resulting in the formation of boundary bundles. In locomoting cells, the compression was associated with forward displacement of myosin features. These data are not consistent with either sarcomeric or polarized transport mechanisms of cell body translocation. We propose that the forward translocation of the cell body and retrograde flow in the lamellipodia are both driven by contraction of an actin–myosin network in the lamellipodial/cell body transition zone. PMID:9334344

  8. A theoretical approach to the relationship between wettability and surface microstructures of epidermal cells and structured cuticles of flower petals

    PubMed Central

    Taneda, Haruhiko; Watanabe-Taneda, Ayako; Chhetry, Rita; Ikeda, Hiroshi

    2015-01-01

    Background and Aims The epidermal surface of a flower petal is composed of convex cells covered with a structured cuticle, and the roughness of the surface is related to the wettability of the petal. If the surface remains wet for an excessive amount of time the attractiveness of the petal to floral visitors may be impaired, and adhesion of pathogens may be promoted. However, it remains unclear how the epidermal cells and structured cuticle contribute to surface wettability of a petal. Methods By considering the additive effects of the epidermal cells and structured cuticle on petal wettability, a thermodynamic model was developed to predict the wetting mode and contact angle of a water droplet at a minimum free energy. Quantitative relationships between petal wettability and the geometries of the epidermal cells and the structured cuticle were then estimated. Measurements of contact angles and anatomical traits of petals were made on seven herbaceous species commonly found in alpine habitats in eastern Nepal, and the measured wettability values were compared with those predicted by the model using the measured geometries of the epidermal cells and structured cuticles. Key Results The model indicated that surface wettability depends on the height and interval between cuticular steps, and on a height-to-width ratio for epidermal cells if a thick hydrophobic cuticle layer covers the surface. For a petal epidermis consisting of lenticular cells, a repellent surface results when the cuticular step height is greater than 0·85 µm and the height-to-width ratio of the epidermal cells is greater than 0·3. For an epidermis consisting of papillate cells, a height-to-width ratio of greater than 1·1 produces a repellent surface. In contrast, if the surface is covered with a thin cuticle layer, the petal is highly wettable (hydrophilic) irrespective of the roughness of the surface. These predictions were supported by the measurements of petal wettability made on flowers of

  9. Epidermal growth factor receptors in non-small cell lung cancer.

    PubMed Central

    Veale, D.; Ashcroft, T.; Marsh, C.; Gibson, G. J.; Harris, A. L.

    1987-01-01

    The epidermal growth factor receptor is homologous to the oncogene erb-beta and is the receptor for a class of tumour growth factors (TGF-alpha). The clinical correlations with its expression were studied in 77 non-small cell lung cancers (NSCLC). They were stained for epidermal growth factor receptor (EGFr) by means of an indirect immunoperoxidase technique using a monoclonal antibody against the receptor. Normal lung tissue and normal bronchus were stained for comparison. Cancer tissue showed significantly increased staining compared to normal lung (P less than 0.05). Staining for EGFr in 40 squamous carcinomas was significantly stronger than in 37 specimens of other types of NSCLC (P less than 0.05), and staining in stage three NSCLC was stronger than in stage 1 and 2 (P less than 0.05). These results suggest that the presence of a high intensity of staining for EGF receptor is associated with spread of human non-small cell lung cancer and this receptor may be a suitable target for therapy. Images Figure 1 Figure 2 PMID:3038157

  10. Regulation of human epidermal stem cell proliferation and senescence requires polycomb- dependent and -independent functions of Cbx4.

    PubMed

    Luis, Nuno Miguel; Morey, Lluis; Mejetta, Stefania; Pascual, Gloria; Janich, Peggy; Kuebler, Bernd; Cozutto, Luca; Roma, Guglielmo; Nascimento, Elisabete; Frye, Michaela; Di Croce, Luciano; Benitah, Salvador Aznar

    2011-09-01

    Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex 1 (PRC1)-associated protein, maintains human epidermal stem cells as slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its antisenescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors such as Ezh2, Dnmt1, and Bmi1 to prevent the active stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation. PMID:21885019

  11. Recurrent exposure to nicotine differentiates human bronchial epithelial cells via epidermal growth factor receptor activation

    SciTech Connect

    Martinez-Garcia, Eva; Irigoyen, Marta; Anso, Elena; Martinez-Irujo, Juan Jose; Rouzaut, Ana

    2008-05-01

    Cigarette smoking is the major preventable cause of lung cancer in developed countries. Nicotine (3-(1-methyl-2-pyrrolidinyl)-pyridine) is one of the major alkaloids present in tobacco. Besides its addictive properties, its effects have been described in panoply of cell types. In fact, recent studies have shown that nicotine behaves as a tumor promoter in transformed epithelial cells. This research focuses on the effects of acute repetitive nicotine exposure on normal human bronchial epithelial cells (NHBE cells). Here we show that treatment of NHBE cells with recurrent doses of nicotine up to 500 {mu}M triggered cell differentiation towards a neuronal-like phenotype: cells emitted filopodia and expressed neuronal markers such as neuronal cell adhesion molecule, neurofilament-M and the transcription factors neuronal N and Pax-3. We also demonstrate that nicotine treatment induced NF-kB translocation to the nucleus, phosphorylation of the epidermal growth factor receptor (EGFR), and accumulation of heparin binding-EGF in the extracellular medium. Moreover, addition of AG1478, an inhibitor of EGFR tyrosine phosphorylation, or cetuximab, a monoclonal antibody that precludes ligand binding to the same receptor, prevented cell differentiation by nicotine. Lastly, we show that differentiated cells increased their adhesion to the extracellular matrix and their protease activity. Given that several lung pathologies are strongly related to tobacco consumption, these results may help to better understand the damaging consequences of nicotine exposure.

  12. The effect of the state of differentiation on labeling of epidermal cell surface glycoproteins

    SciTech Connect

    Brysk, M.M.; Snider, J.M.

    1982-05-01

    Epidermal cells were grown in a medium in which the Ca++ concentration controlled the stage of differentiation. Cell surface molecules of differentiated and undifferentiated cells were compared by lactoperoxidase-catalyzed iodination, by the interaction with /sup 125/I-lectins, and by the metabolic incorporation of L-(/sup 3/H)-fucose. Molecular weights of the labeled components were determined by SDS-PAGE and autoradiography. After lactoperoxidase iodination, most of the radioactivity was found in polypeptide bands of 79,000, 65,000 and 56,000 daltons. The 79,000 band is the most intense for undifferentiated cells but disappears as differentiation proceeds. The 56,000 band is present in normal cells at all stages of differentiation but is absent from neoplastic cells. Glycoproteins reacted with /sup 125/I-lectins were found at 180,000, 130,000 and 85,000 daltons. The 130,000 band was the most prominent for differentiated cells labeled with wheat germ agglutinin but was essentially absent from the undifferentiated cells. With Ricinus communis agglutinin, this band was weaker for undifferentiated than for differentiated cells but was the most intense for both. After metabolic incorporation of tritiated fucose, radioactive glycoproteins were found at 130,000 and 85,000 daltons, with comparable intensities for differentiated and undifferentiated cells.

  13. [Nitric oxide and electrogenic metals (Ca, Na, K) in epidermal cells].

    PubMed

    Petukhov, V I; Baumane, L K; Dmitriev, E V; Vanin, A F

    2015-01-01

    Using atomic emission spectrometry and EPR analysis metal-ligand homeostasis (MLH) has been studied in epidermal cells of 954 liquidators of the Chernobyl accident and 947 healthy individuals. A possible association of the redox status with the quantitative changes in the MLH, which could be used as discriminators of oxidative/nitrosative stress, attracts special interest. Characteristic features of oxidative stress mainly related to electrogenic metals (Ca, K, Na), were found not only among the liquidators examined, but also in some healthy individuals (18.1%); this suggests the presence of oxidative/nitrosative stress of non-radiation origin. Correlation between intracellular production of nitric oxide (NO) with quantitative changes in the electrogenic metals may indicate the possible involvement of NO in the generation of an electric potential of the cell. PMID:26350742

  14. Epidermal wound repair is regulated by the planar cell polarity signaling pathway.

    PubMed

    Caddy, Jacinta; Wilanowski, Tomasz; Darido, Charbel; Dworkin, Sebastian; Ting, Stephen B; Zhao, Quan; Rank, Gerhard; Auden, Alana; Srivastava, Seema; Papenfuss, Tony A; Murdoch, Jennifer N; Humbert, Patrick O; Parekh, Vishwas; Boulos, Nidal; Weber, Thomas; Zuo, Jian; Cunningham, John M; Jane, Stephen M

    2010-07-20

    The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects, and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3(-)(/-) mice, we identified RhoGEF19, a homolog of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerization, cellular polarity, and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling and broadly implicate this pathway in epidermal repair. PMID:20643356

  15. Epidermal wound repair is regulated by the planar cell polarity signaling pathway

    PubMed Central

    Caddy, Jacinta; Wilanowski, Tomasz; Darido, Charbel; Dworkin, Sebastian; Ting, Stephen B.; Zhao, Quan; Rank, Gerhard; Auden, Alana; Srivastava, Seema; Papenfuss, Tony A.; Murdoch, Jennifer N.; Humbert, Patrick O.; Boulos, Nidal; Weber, Thomas; Zuo, Jian; Cunningham, John M.; Jane, Stephen M.

    2010-01-01

    SUMMARY The mammalian PCP pathway regulates diverse developmental processes requiring coordinated cellular movement, including neural tube closure and cochlear stereociliary orientation. Here, we show that epidermal wound repair is regulated by PCP signaling. Mice carrying mutant alleles of PCP genes Vangl2, Celsr1, PTK7, and Scrb1, and the transcription factor Grhl3, interact genetically, exhibiting failed wound healing, neural tube defects and disordered cochlear polarity. Using phylogenetic analysis, ChIP, and gene expression in Grhl3−/− mice, we identified RhoGEF19, a homologue of a RhoA activator involved in PCP signaling in Xenopus, as a direct target of GRHL3. Knockdown of Grhl3 or RhoGEF19 in keratinocytes induced defects in actin polymerisation, cellular polarity and wound healing, and re-expression of RhoGEF19 rescued these defects in Grhl3-kd cells. These results define a role for Grhl3 in PCP signaling, and broadly implicate this pathway in epidermal repair. PMID:20643356

  16. aPKCλ controls epidermal homeostasis and stem cell fate through regulation of division orientation

    PubMed Central

    Niessen, Michaela T.; Scott, Jeanie; Zielinski, Julia G.; Vorhagen, Susanne; Sotiropoulou, Panagiota A.; Blanpain, Cédric

    2013-01-01

    The atypical protein kinase C (aPKC) is a key regulator of polarity and cell fate in lower organisms. However, whether mammalian aPKCs control stem cells and fate in vivo is not known. Here we show that loss of aPKCλ in a self-renewing epithelium, the epidermis, disturbed tissue homeostasis, differentiation, and stem cell dynamics, causing progressive changes in this tissue. This was accompanied by a gradual loss of quiescent hair follicle bulge stem cells and a temporary increase in proliferating progenitors. Lineage tracing analysis showed that loss of aPKCλ altered the fate of lower bulge/hair germ stem cells. This ultimately led to loss of proliferative potential, stem cell exhaustion, alopecia, and premature aging. Inactivation of aPKCλ produced more asymmetric divisions in different compartments, including the bulge. Thus, aPKCλ is crucial for homeostasis of self-renewing stratifying epithelia, and for the regulation of cell fate, differentiation, and maintenance of epidermal bulge stem cells likely through its role in balancing symmetric and asymmetric division. PMID:24019538

  17. aPKCλ controls epidermal homeostasis and stem cell fate through regulation of division orientation.

    PubMed

    Niessen, Michaela T; Scott, Jeanie; Zielinski, Julia G; Vorhagen, Susanne; Sotiropoulou, Panagiota A; Blanpain, Cédric; Leitges, Michael; Niessen, Carien M

    2013-09-16

    The atypical protein kinase C (aPKC) is a key regulator of polarity and cell fate in lower organisms. However, whether mammalian aPKCs control stem cells and fate in vivo is not known. Here we show that loss of aPKCλ in a self-renewing epithelium, the epidermis, disturbed tissue homeostasis, differentiation, and stem cell dynamics, causing progressive changes in this tissue. This was accompanied by a gradual loss of quiescent hair follicle bulge stem cells and a temporary increase in proliferating progenitors. Lineage tracing analysis showed that loss of aPKCλ altered the fate of lower bulge/hair germ stem cells. This ultimately led to loss of proliferative potential, stem cell exhaustion, alopecia, and premature aging. Inactivation of aPKCλ produced more asymmetric divisions in different compartments, including the bulge. Thus, aPKCλ is crucial for homeostasis of self-renewing stratifying epithelia, and for the regulation of cell fate, differentiation, and maintenance of epidermal bulge stem cells likely through its role in balancing symmetric and asymmetric division. PMID:24019538

  18. Application of Microneedles to Skin Induces Activation of Epidermal Langerhans Cells and Dermal Dendritic Cells in Mice.

    PubMed

    Takeuchi, Asuka; Nomoto, Yusuke; Watanabe, Mai; Kimura, Soichiro; Morimoto, Yasunori; Ueda, Hideo

    2016-08-01

    An adequate immune response to percutaneous vaccine application is generated by delivery of sufficient amounts of antigen to skin and by administration of toxin adjuvants or invasive skin abrasion that leads to an adjuvant effect. Microneedles penetrate the stratum corneum, the outermost layer of the skin, and enable direct delivery of vaccines from the surface into the skin, where immunocompetent dendritic cells are densely distributed. However, whether the application of microneedles to the skin activates antigen-presenting cells (APCs) has not been demonstrated. Here we aimed to demonstrate that microneedles may act as a potent physical adjuvant for successful transcutaneous immunization (TCI). We prepared samples of isolated epidermal and dermal cells and analyzed the expression of major histocompatibility complex (MHC) class II and costimulatory molecules on Langerhans or dermal dendritic cells in the prepared samples using flow cytometry. The expression of MHC class II and costimulatory molecules demonstrated an upward trend in APCs in the skin after the application of 500- and 300-µm microneedles. In addition, in the epidermal cells, application of microneedles induced more effective activation of Langerhans cells than did an invasive tape-stripping (positive control). In conclusion, the use of microneedles is likely to have a positive effect not only as an antigen delivery system but also as a physical technique inducing an adjuvant-like effect for TCI. PMID:27251665

  19. Does salinity reduce growth in maize root epidermal cells by inhibiting their capacity for cell wall acidification?

    PubMed

    Zidan, I; Azaizeh, H; Neumann, P M

    1990-05-01

    The reduction in growth of maize (Zea mays L.) seedling primary roots induced by salinization of the nutrient medium with 100 millimolar NaCl was accompanied by reductions in the length of the root tip elongation zone, the length of fully elongated epidermal cells, and the apparent rate of cell production: Each was partially restored when calcium levels in the salinized growth medium were increased from 0.5 to 10.0 millimolar. We investigated the possibility that the inhibition of elongation growth by salinity might be associated with an inhibition of cell wall acidification, such as that which occurs when root growth is inhibited by IAA. A qualitative assay of root surface acidification, using bromocresol purple pH indicator in agar, showed that salinized roots, with and without extra calcium, produced a zone of surface acidification which was similar to that produced by control roots. The zone of acidification began 1 to 2 millimeters behind the tip and coincided with the zone of cell elongation. The remainder of the root alkalinized its surface. Kinetics of surface acidification were assayed quantitatively by placing a flat tipped pH electrode in contact with the elongation zone. The pH at the epidermal surfaces of roots grown either with 100 millimolar NaCl (growth inhibitory), or with 10 millimolar calcium +/- NaCl (little growth inhibition), declined from 6.0 to 5.1 over 30 minutes. We conclude that NaCl did not inhibit growth by reducing the capacity of epidermal cells to acidify their walls. PMID:16667468

  20. Salinity Stiffens the Epidermal Cell Walls of Salt-Stressed Maize Leaves: Is the Epidermis Growth-Restricting?

    PubMed Central

    Zörb, Christian; Mühling, Karl H.; Kutschera, Ulrich; Geilfus, Christoph-Martin

    2015-01-01

    As a result of salt (NaCl)-stress, sensitive varieties of maize (Zea mays L.) respond with a strong inhibition of organ growth. The reduction of leaf elongation investigated here has several causes, including a modification of the mechanical properties of the cell wall. Among the various tissues that form the leaf, the epidermis plays a special role in controlling organ growth, because it is thought to form a rigid outer leaf coat that can restrict elongation by interacting with the inner cell layers. This study was designed to determine whether growth-related changes in the leaf epidermis and its cell wall correspond to the overall reduction in cell expansion of maize leaves during an osmotic stress-phase induced by salt treatment. Two different maize varieties contrasting in their degree of salt resistance (i.e., the hybrids Lector vs. SR03) were compared in order to identify physiological features contributing to resistance towards salinity. Wall loosening-related parameters, such as the capacity of the epidermal cell wall to expand, β-expansin abundance and apoplastic pH values, were analysed. Our data demonstrate that, in the salt-tolerant maize hybrid which maintained leaf growth under salinity, the epidermal cell wall was more extensible under salt stress. This was associated with a shift of the epidermal apoplastic pH into a range more favourable for acid growth. The more sensitive hybrid that displayed a pronounced leaf growth-reduction was shown to have stiffer epidermal cell walls under stress. This may be attributable to the reduced abundance of cell wall-loosening β-expansin proteins following a high salinity-treatment in the nutrient solution (100 mM NaCl, 8 days). This study clearly documents that salt stress impairs epidermal wall-loosening in growth-reduced maize leaves. PMID:25760715

  1. Growth and differentiation in cultured human thyroid cells: effects of epidermal growth factor and thyrotropin.

    PubMed

    Errick, J E; Ing, K W; Eggo, M C; Burrow, G N

    1986-01-01

    Human thyroid cells were grown and subcultured in vitro to examine their responses to known hormones and growth factors, and to serum. The cells were obtained from surgical specimens and were either neoplastic or nonneoplastic. The effects of culture conditions on cell growth were measured by changes in cell numbers and by stimulation of [3H]thymidine incorporation. The results showed that serum (0.5%) was essential for cell proliferation, and that a mixture of insulin (10 micrograms/ml), transferrin (5 micrograms/ml), hydrocortisone (10 micrograms/ml), somatostatin (10 ng/ml), and glycyl-histidyl-lysine (10 ng/ml) enhanced the effect of serum. Maximum growth of the cells was obtained when epidermal growth factor was present at 10(-9) M. Differentiation was measured by production of thyroglobulin, which was found to be stimulated by thyrotropin. This system provides a means to study the hormonal control of growth and differentiation in human thyroid cells. PMID:3511027

  2. Epidermal growth factor receptor expression in primary cultured human colorectal carcinoma cells.

    PubMed Central

    Tong, W. M.; Ellinger, A.; Sheinin, Y.; Cross, H. S.

    1998-01-01

    In situ hybridization on human colon tissue demonstrates that epidermal growth factor receptor (EGFR) mRNA expression is strongly increased during tumour progression. To obtain test systems to evaluate the relevance of growth factor action during carcinogenesis, primary cultures from human colorectal carcinomas were established. EGFR distribution was determined in 2 of the 27 primary cultures and was compared with that in well-defined subclones derived from the Caco-2 cell line, which has the unique property to differentiate spontaneously in vitro in a manner similar to normal enterocytes. The primary carcinoma-derived cells had up to three-fold higher total EGFR levels than the Caco-2 subclones and a basal mitotic rate at least fourfold higher. The EGFR affinity constant is 0.26 nmol l(-1), which is similar to that reported in Caco-2 cells. The proliferation rate of Caco-2 cells is mainly induced by EGF from the basolateral cell surface where the majority of receptors are located, whereas primary cultures are strongly stimulated from the apical side also. This corresponds to a three- to fivefold higher level of EGFR at the apical cell surface. This redistribution of EGFR to apical plasma membranes in advanced colon carcinoma cells suggests that autocrine growth factors in the colon lumen may play a significant role during tumour progression. Images Figure 1 Figure 2 PMID:9667648

  3. Epidermal Growth Factor Receptor Mutation Enhances Expression of Cadherin-5 in Lung Cancer Cells

    PubMed Central

    Hung, Ming-Szu; Chen, I-Chuan; Lung, Jr-Hau; Lin, Paul-Yann; Li, Ya-Chin; Tsai, Ying-Huang

    2016-01-01

    Epidermal growth factor receptor (EGFR) activation has been shown to play a critical role in tumor angiogenesis. In this study, we investigate the correlation between EGFR mutations and cadherin-5 (CDH5), which is an angiogenic factor, in lung cancer cells. Increased expression CDH5 is observed in lung cancer cells with EGFR mutations. Stable lung cancer cell lines expressing mutant (exon 19 deletion E746-A750, and exon 21 missense mutation L858R) and wild type EGFR genes are established. A significantly higher expression of CDH5 is observed in exon 19 deletion stable lung cancer cells and mouse xenografts. Further studies show that expression of CDH5 is decreased after the inhibition of EGFR and downstream Akt pathways in lung cancer cells with EGFR mutation. In addition, mutant EGFR genes potentiates angiogenesis in lung cancer cells, which is inhibited by CDH5 siRNA, and potentiates migration and invasion in lung cancer cells. Our study shows that mutant EGFR genes are associated with overexpression of CDH5 through increased phosphorylation of EGFR and downstream Akt pathways. Our result may provide an insight into the association of mutant EGFR and CDH5 expression in lung cancer and aid further development of target therapy for NSCLC in the future. PMID:27362942

  4. Skin dose rate conversion factors after contamination with radiopharmaceuticals: influence of contamination area, epidermal thickness and percutaneous absorption.

    PubMed

    Covens, P; Berus, D; Caveliers, V; Struelens, L; Vanhavere, F; Verellen, D

    2013-06-01

    Skin contamination with radiopharmaceuticals can occur during biomedical research and daily nuclear medicine practice as a result of accidental spills, after contact with bodily fluids of patients or by inattentively touching contaminated materials. Skin dose assessment should be carried out by repeated quantification to map the course of the contamination together with the use of appropriate skin dose rate conversion factors. Contamination is generally characterised by local spots on the palmar surface of the hand and complete decontamination is difficult as a result of percutaneous absorption. This specific issue requires special consideration as to the skin dose rate conversion factors as a measure for the absorbed dose rate to the basal layer of the epidermis. In this work we used Monte Carlo simulations to study the influence of the contamination area, the epidermal thickness and the percutaneous absorption on the absorbed skin dose rate conversion factors for a set of 39 medical radionuclides. The results show that the absorbed dose to the basal layer of the epidermis can differ by up to two orders of magnitude from the operational quantity Hp(0.07) when using an appropriate epidermal thickness in combination with the effect of percutaneous absorption. PMID:23519114

  5. Nitric oxide stimulates the proliferation of neural stem cells bypassing the epidermal growth factor receptor.

    PubMed

    Carreira, Bruno Pereira; Morte, Maria Inês; Inácio, Angela; Costa, Gabriel; Rosmaninho-Salgado, Joana; Agasse, Fabienne; Carmo, Anália; Couceiro, Patrícia; Brundin, Patrik; Ambrósio, António Francisco; Carvalho, Caetana Monteiro; Araújo, Inês Maria

    2010-07-01

    Nitric oxide (NO) was described to inhibit the proliferation of neural stem cells. Some evidence suggests that NO, under certain conditions, can also promote cell proliferation, although the mechanisms responsible for a potential proliferative effect of NO in neural stem cells have remained unaddressed. In this work, we investigated and characterized the proliferative effect of NO in cell cultures obtained from the mouse subventricular zone. We found that the NO donor NOC-18 (10 microM) increased cell proliferation, whereas higher concentrations (100 microM) inhibited cell proliferation. Increased cell proliferation was detected rapidly following exposure to NO and was prevented by blocking the mitogen-activated kinase (MAPK) pathway, independently of the epidermal growth factor (EGF) receptor. Downstream of the EGF receptor, NO activated p21Ras and the MAPK pathway, resulting in a decrease in the nuclear presence of the cyclin-dependent kinase inhibitor 1, p27(KIP1), allowing for cell cycle progression. Furthermore, in a mouse model that shows increased proliferation of neural stem cells in the hippocampus following seizure injury, we observed that the absence of inducible nitric oxide synthase (iNOS(-/-) mice) prevented the increase in cell proliferation observed following seizures in wild-type mice, showing that NO from iNOS origin is important for increased cell proliferation following a brain insult. Overall, we show that NO is able to stimulate the proliferation of neural stem cells bypassing the EGF receptor and promoting cell division. Moreover, under pathophysiological conditions in vivo, NO from iNOS origin also promotes proliferation in the hippocampus. PMID:20506358

  6. Parabens and Human Epidermal Growth Factor Receptor Ligand Cross-Talk in Breast Cancer Cells

    PubMed Central

    Pan, Shawn; Yuan, Chaoshen; Tagmount, Abderrahmane; Rudel, Ruthann A.; Ackerman, Janet M.; Yaswen, Paul; Vulpe, Chris D.; Leitman, Dale C.

    2015-01-01

    Background: Xenoestrogens are synthetic compounds that mimic endogenous estrogens by binding to and activating estrogen receptors. Exposure to estrogens and to some xenoestrogens has been associated with cell proliferation and an increased risk of breast cancer. Despite evidence of estrogenicity, parabens are among the most widely used xenoestrogens in cosmetics and personal-care products and are generally considered safe. However, previous cell-based studies with parabens do not take into account the signaling cross-talk between estrogen receptor α (ERα) and the human epidermal growth factor receptor (HER) family. Objectives: We investigated the hypothesis that the potency of parabens can be increased with HER ligands, such as heregulin (HRG). Methods: The effects of HER ligands on paraben activation of c-Myc expression and cell proliferation were determined by real-time polymerase chain reaction, Western blots, flow cytometry, and chromatin immunoprecipitation assays in ERα- and HER2-positive human BT-474 breast cancer cells. Results: Butylparaben (BP) and HRG produced a synergistic increase in c-Myc mRNA and protein levels in BT-474 cells. Estrogen receptor antagonists blocked the synergistic increase in c-Myc protein levels. The combination of BP and HRG also stimulated proliferation of BT-474 cells compared with the effects of BP alone. HRG decreased the dose required for BP-mediated stimulation of c-Myc mRNA expression and cell proliferation. HRG caused the phosphorylation of serine 167 in ERα. BP and HRG produced a synergistic increase in ERα recruitment to the c-Myc gene. Conclusion: Our results show that HER ligands enhanced the potency of BP to stimulate oncogene expression and breast cancer cell proliferation in vitro via ERα, suggesting that parabens might be active at exposure levels not previously considered toxicologically relevant from studies testing their effects in isolation. Citation: Pan S, Yuan C, Tagmount A, Rudel RA, Ackerman JM

  7. Outcome of Burns Treated With Autologous Cultured Proliferating Epidermal Cells: A Prospective Randomized Multicenter Intrapatient Comparative Trial.

    PubMed

    Gardien, Kim L M; Marck, Roos E; Bloemen, Monica C T; Waaijman, Taco; Gibbs, Sue; Ulrich, Magda M W; Middelkoop, Esther

    2016-01-01

    Standard treatment for large burns is transplantation with meshed split skin autografts (SSGs). A disadvantage of this treatment is that healing is accompanied by scar formation. Application of autologous epidermal cells (keratinocytes and melanocytes) may be a suitable therapeutic alternative, since this may enhance wound closure and improve scar quality. A prospective, multicenter randomized clinical trial was performed in 40 adult patients with acute full thickness burns. On two comparable wound areas, conventional treatment with SSGs was compared to an experimental treatment consisting of SSGs in combination with cultured autologous epidermal cells (ECs) seeded in a collagen carrier. The primary outcome measure was wound closure after 5-7 days. Secondary outcomes were safety aspects and scar quality measured by graft take, scar score (POSAS), skin colorimeter (DermaSpectrometer) and elasticity (Cutometer). Wound epithelialization after 5-7 days was significantly better for the experimental treatment (71%) compared to the standard treatment (67%) (p = 0.034, Wilcoxon), whereas the take rates of the grafts were similar. No related adverse events were recorded. Scar quality was evaluated at 3 (n = 33) and 12 (n = 28) months. The POSAS of the observer after 3 and 12 months and of the patient after 12 months were significantly better for the experimental area. Improvements between 12% and 23% (p ≤ 0.010, Wilcoxon) were detected for redness, pigmentation, thickness, relief, and pliability. Melanin index at 3 and 12 months and erythema index at 12 months were closer to normal skin for the experimental treatment than for conventional treatment (p ≤ 0.025 paired samples t-test). Skin elasticity showed significantly higher elasticity (p = 0.030) in the experimental area at 3 months follow-up. We showed a safe application and significant improvements of wound healing and scar quality in burn patients after treatment with ECs versus SSGs only

  8. Contact-stimulated proliferation of cultured mouse epidermal cells by 3T3 feeder layers: inhibition of proliferation by 12-O-tetradecanoylphorbol-13-acetate (TPA)

    SciTech Connect

    Miller, D.R.; Hamby, K.M.; Slaga, T.J.

    1982-07-01

    Mouse epidermal cells can be subcultured at 31/sup 0/C onto an irradiated BALB/c 3T3 clone A31 feeder layer. A31 cells (supposedly derived from embryonic fibroblasts) were found to be specifically required for the optimal production of keratinizing epidermal colonies in secondary culture. This effect was not transmitted through the medium nor by the culture surface, since A31 cells plated on one end of a flask did not stimulate epidermal cell proliferation at the other end, even if the other end had previously held A31 cells. Epidermal cell contact with metabolizing A31 cells was probably necessary for the effect; fixed or freeze-thawed A31 cells were ineffective. The tumor promoter 12-O-tetradecanoylphorbol-13-acetate, recently shown to interfere with contact-mediated transfer of label (metabolic cooperation) between Swiss 3T3 cells and cells of an established epidermal line in vitro, also blocked epidermal colony formation. The A31-epidermal cell interaction is apparently not a typical mesenchymal-epithelial interaction, since the basement membrane would prevent this contact in intact skin.

  9. The inflammatory response after an epidermal burn depends on the activities of mouse mast cell proteases 4 and 5.

    PubMed

    Younan, George; Suber, Freeman; Xing, Wei; Shi, Tong; Kunori, Yuichi; Abrink, Magnus; Pejler, Gunnar; Schlenner, Susan M; Rodewald, Hans-Reimer; Moore, Francis D; Stevens, Richard L; Adachi, Roberto; Austen, K Frank; Gurish, Michael F

    2010-12-15

    A second-degree epidermal scald burn in mice elicits an inflammatory response mediated by natural IgM directed to nonmuscle myosin with complement activation that results in ulceration and scarring. We find that such burn injury is associated with early mast cell (MC) degranulation and is absent in WBB6F1-Kit(W)/Kit(Wv) mice, which lack MCs in a context of other defects due to a mutation of the Kit receptor. To address further an MC role, we used transgenic strains with normal lineage development and a deficiency in a specific secretory granule component. Mouse strains lacking the MC-restricted chymase, mouse MC protease (mMCP)-4, or elastase, mMCP-5, show decreased injury after a second-degree scald burn, whereas mice lacking the MC-restricted tryptases, mMCP-6 and mMCP-7, or MC-specific carboxypeptidase A3 activity are not protected. Histologic sections showed some disruption of the epidermis at the scald site in the protected strains suggesting the possibility of topical reconstitution of full injury. Topical application of recombinant mMCP-5 or human neutrophil elastase to the scalded area increases epidermal injury with subsequent ulceration and scarring, both clinically and morphologically, in mMCP-5-deficient mice. Restoration of injury requires that topical administration of recombinant mMCP-5 occurs within the first hour postburn. Importantly, topical application of human MC chymase restores burn injury to scalded mMCP-4-deficient mice but not to mMCP-5-deficient mice revealing nonredundant actions for these two MC proteases in a model of innate inflammatory injury with remodeling. PMID:21076070

  10. Epidermal growth factor receptor tyrosine kinase inhibitors for non-small cell lung cancer

    PubMed Central

    Asami, Kazuhiro; Atagi, Shinji

    2014-01-01

    First-generation epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), including gefitinib and erlotinib, have proven to be highly effective agents for advanced non-small cell lung cancer (NSCLC) in patients harboring an activating EGFR mutation such as the exon 19 deletion mutation and L858R. Although those reversible small molecular targeted agents provide a significant response and survival benefit, all responders eventually acquire resistance. Second-generation EGFR-targeting agents, such as irreversible EGFR/HER2 tyrosine kinase inhibitors and pan-HER TKIs, may improve survival further and be useful for patients who acquired resistance to first-generation EGFR-TKIs. This review discusses novel therapeutic strategies for EGFR-mutated advanced NSCLC using first- and second-generation EGFR-TKIs. PMID:25302168

  11. Gefitinib in the treatment of nonsmall cell lung cancer with activating epidermal growth factor receptor mutation

    PubMed Central

    Nurwidya, Fariz; Takahashi, Fumiyuki; Takahashi, Kazuhisa

    2016-01-01

    Lung cancer is still the main cause of cancer-related deaths worldwide, with most patients present with advanced disease and poor long-term prognosis. The aim of lung cancer treatment is to slow down the progression of the disease, to relieve the patients from the lung cancer symptoms and whenever possible, to increase the overall survival. The discovery of small molecule targeting tyrosine kinase of epidermal growth factor receptor opens a new way in the management of advanced nonsmall cell lung cancer (NSCLC). This review will discuss several Phase II and III trials evaluated the clinical efficacy of gefitinib as monotherapy in pretreated patients with advanced NSCLC, as well as both monotherapy and combined with chemotherapy in chemotherapy-naive patients. PMID:27433059

  12. Axonal transport of herpes simplex virions to epidermal cells: evidence for a specialized mode of virus transport and assembly.

    PubMed Central

    Penfold, M E; Armati, P; Cunningham, A L

    1994-01-01

    To examine the transmission of herpes simplex virus (HSV) from axon to epidermal cell, an in vitro model was constructed consisting of human fetal dorsal root ganglia cultured in the central chamber of a dual-chamber tissue culture system separated from autologous skin explants in an exterior chamber by concentric steel cylinders adhering to the substratum through silicon grease and agarose. Axons grew through the agarose viral diffusion barrier and terminated on epidermal cells in the exterior chamber. After inoculation of HSV onto dorsal root ganglia, anterograde axonal transport of glycoprotein and nucleocapsid antigen was observed by confocal microscopy to appear in exterior chamber axons within 12 h and in epidermal cells within 16 h, moving at 2-3 mm/h. Although both enveloped and unenveloped nucleocapsids were observed in the neuronal soma by transmission electron microscopy, only nucleocapsids were observed in the axons, closely associated with microtubules. Nodule formation at the surface of HSV-infected axons, becoming more dense at the axon terminus on epidermal cells, and patches of axolemmal HSV glycoprotein D expression were observed by scanning (immuno)electron microscopy, probably representing virus emerging from the axolemma. These findings strongly suggest a specialized mode of viral transport, assembly, and egress in sensory neurons: microtubule-associated intermediate-fast anterograde axonal transport of unenveloped nucleocapsids with separate transport of glycoproteins to the distal regions of the axon and assembly prior to virus emergence at the axon terminus. Images PMID:7517552

  13. A novel component of epidermal cell-matrix and cell-cell contacts: transmembrane protein type XIII collagen.

    PubMed

    Peltonen, S; Hentula, M; Hägg, P; Ylä-Outinen, H; Tuukkanen, J; Lakkakorpi, J; Rehn, M; Pihlajaniemi, T; Peltonen, J

    1999-10-01

    Type XIII collagen is a short chain collagen which has recently been shown to be a transmembrane protein. The purpose of this study was to elucidate the presence and localization of type XIII collagen in normal human skin and cultured keratinocytes. Expression of type XIII collagen was demonstrated in normal human skin and epidermis at the RNA level using reverse transcription followed by polymerase chain reaction and at the protein level using western blotting and indirect immunofluorescence labeling. Immunolabeling of epidermis revealed type XIII collagen both in the cell-cell contact sites and in the dermal-epidermal junction. In cultured keratinocytes type XIII collagen epitopes were detected in focal contacts and in intercellular contacts. The results of this study show very little colocalization of type XIII collagen and desmosomal components at the light microscopic level. Thus, these results suggest that type XIII collagen is unlikely to be a component of desmosomes. Instead, the punctate labeling pattern of type XIII collagen at the cell-cell contact sites and high degree of colocalization with E-cadherin suggests that type XIII collagen is very likely to be closely associated with adherens type junctions, and may, in fact, be a component of these junctions. PMID:10504453

  14. Effects of Epidermal Cell Shape and Pigmentation on Optical Properties of Antirrhinum Petals at Visible and Ultraviolet Wavelengths.

    PubMed Central

    Gorton, H. L.; Vogelmann, T. C.

    1996-01-01

    We used the Mixta+ and mixta- lines of Antirrhinum majus as a model system to investigate the effects of epidermal cell shape and pigmentation on tissue optical properties in the visible and ultraviolet (UV) spectral regions. Adaxial epidermal cells of Mixta+ flowers have a conical-papillate shape; in the mixta- line the cells are slightly domed. Mixta+ cells contained significantly more anthocyanin and other flavonoids than mixta- cells when plants were grown under either high- or low-UV conditions. Mixta+ cells focused light (3.5-4.7 times incident) within their pigmented interiors, whereas mixta- cells focused light (2.1-2.7 times incident) in the unpigmented mesophyll. UV light penetrated the epidermis (commonly 20-50% transmittance at 312 nm) mainly through the unpigmented peripheral regions of the cells that were similar for the two lines, so that overall penetration through Mixta+ and mixta- epidermises was equal. However, maximum UV absorption in the central region of epidermal cells was slightly greater in Mixta+ than mixta-, and intact Mixta+ flowers reflected less light in the spectral regions with intermediate flavonoid absorbance. In both cases, about 50 to 75% of the difference could be attributed to cell shape and resulting changes in the optical pathlength or focusing. PMID:12226425

  15. Ambivalent Effects of Atorvastatin on Angiogenesis, Epidermal Cell Proliferation and Tumorgenesis in Animal Models

    PubMed Central

    Garjani, Alireza; Rezazadeh, Hassan; Andalib, Sina; Ziaee, Mojtaba; Doustar, Yousef; Soraya, Hamid; Garjani, Mehraveh; Khorrami, Arash; Asadpoor, Mostafa; Maleki-Dizaji, Nasrin

    2012-01-01

    Background: A growing body of preclinical data indicates that statins may possess antineoplastic properties; however, some studies have raised the possibility that statins may also have carcinogenic potential. Methods: An air pouch model was used for angiogenesis. Single or multiple applications of croton oil on the back of Swiss albino mice with or without initiation by dimethylbenz(a)antheracene (DMBA) were used to evaluate the skin tumorgenesis, ultrastructural and histological alterations. Results: Atorvastatin (orally, 10 mg/kg/day) produced a significant (P<0.05) reduction in angiogenesis. Concurrent administration of mevalonate reversed the anti-angiogenic effect of atorvastatin. However, local injection of atorvastatin (200 µg) into the pouches induced a significant (P<0.5) increase in angiogenesis that was not reversed by co-administration of mevalonate. The disturbance of cell polarity, inflammatory response, thickness of epidermal layer, and mitotic index induced by croton oil were inhibited markedly and dose-dependently (P<0.001) by pre-treatment with atorvastatin. In spite of the strong anti-inflammatory and anti-proliferative effects of atorvastatin on epidermal cell proliferation, it was identified that the same doses of atorvastatin in DMBA-initiated and croton oil-promoted skin tumorgenesis in mice increased the incidence of tumors and their conversion into malignant carcinoma. Conclusion: The reasons for these discrepancies remain unclear, and could be related to ambivalent effects of atorvastatin on angiogenesis or to specific differences in the experimental conditions. It is suggested that the pro-angiogenic effect of the drug, which could be responsible for promotion of skin tumors, is independent of the 3-hydroxy-3-methyl-glutaryl-coenzyme A reductase inhibition that can be mediated directly by atorvastatin. PMID:22801278

  16. INSULIN INDUCED EPIDERMAL GROWTH FACTOR ACTIVATION IN VASCULAR SMOOTH MUSCLE CELLS IS ADAM-DEPENDENT

    PubMed Central

    Roztocil, Elisa; Nicholl, Suzanne M.; Davies, Mark G.

    2008-01-01

    Background With the rise in metabolic syndrome, understanding the role of insulin signaling within the cells of vasculature has become more important but yet remains poorly defined. The study examines the role of insulin actions on a pivotal cross-talk receptor, Epidermal Growth Factor Receptor (EGFR). EGFR is transactivated by both G-protein-coupled receptors and receptor linked tyrosine kinases and is key to many of their responses. Objective To determine the pathway of EGFR transactivation by insulin in human coronary smooth muscle cells (VSMC) Methods VSMC were cultured in vitro. Assays of EGFR phosphorylation were examined in response to insulin in the presence and absence of the plasmin inhibitors (e-aminocaproic acid and aprotinin) matrix metalloprotease (MMP) inhibitor GM6001, the ADAM (A Disintegrin And Metalloproteinase Domain) inhibitors TAPI-0 and TAPI-1, Heparin binding epidermal growth factor (HB-EGF) inhibitor, CRM197, HB-EGF inhibitory antibodies, EGF inhibitory antibodies and the EGFR inhibitor AG1478. Results Insulin induced time-dependent EGFR phosphorylation, which was inhibited by AG1478 in a concentration dependent manner. Application of the plasmin inhibitors did not block the response. EGFR phosphorylation by insulin was blocked by inhibition of MMP activity and the ligand HB-EGF. The presence of the ADAM inhibitors, TAPI-0 and TAPI-1 significantly decreased EGFR activation. EGFR phosphorylation by EGF was not interrupted by inhibition of plasmin, MMPs TAPIs, or HB-EGF. Direct blockade of the EGFR prevented activation by both insulin and EGF. Conclusion Insulin can induce transactivation of EGFR by an ADAM-mediated, HB-EGF dependent process. This is the first description of crosstalk via ADAM between insulin and EGFR in vascular SMC. Targeting a pivotal cross-talk receptor such as EGFR, which can be transactivated by both G-protein-coupled receptors and receptor tyrosine kinases is an attractive molecular target. PMID:18656632

  17. Emodin Suppresses Maintenance of Stemness by Augmenting Proteosomal Degradation of Epidermal Growth Factor Receptor/Epidermal Growth Factor Receptor Variant III in Glioma Stem Cells

    PubMed Central

    Kim, Jeongyub; Lee, Jong-Seon; Jung, Jieun; Lim, Inhye; Lee, Ji-Yun

    2015-01-01

    There is a growing body of evidence that small subpopulations of cells with stem cell-like characteristics within most solid tumors are responsible for the malignancy of aggressive cancer cells and that targeting these cells might be a good therapeutic strategy to reduce the risk of tumor relapse after therapy. Here, we examined the effects of emodin (1,3,8-trihydroxy-6-methylanthraquinone), an active component of the root and rhizome of Rheum palmatum that has several biological activities, including antitumor effects, on primary cultured glioma stem cells (GSCs). Emodin inhibited the self-renewal activity of GSCs in vitro as evidenced by neurosphere formation, limiting dilution, and soft agar clonogenic assays. Emodin inhibited the maintenance of stemness by suppressing the expression of Notch intracellular domain, nonphosphorylated β-catenin, and phosphorylated STAT3 proteins. In addition, treatment with emodin partially induced apoptosis, reduced cell invasiveness, and sensitized GSCs to ionizing radiation. Intriguingly, emodin induced proteosomal degradation of epidermal growth factor receptor (EGFR)/EGFR variant III (EGFRvIII) by interfering with the association of EGFR/EGFRvIII with heat shock protein 90, resulting in the suppression of stemness pathways. Based on these data, we propose that emodin could be considered as a potent therapeutic adjuvant that targets GSCs. PMID:25229646

  18. Epoc-1: a POU-domain gene expressed in murine epidermal basal cells and thymic stromal cells.

    PubMed

    Yukawa, K; Yasui, T; Yamamoto, A; Shiku, H; Kishimoto, T; Kikutani, H

    1993-11-15

    POU-domain transcription factors are known as developmental regulators which control organ development and cell phenotypes. In order to clarify the roles of POU-domain transcription factors in cell differentiation, we cloned a novel POU family gene, Epoc-1, from a murine thymus cDNA library. The amino acid (aa) sequence of the POU-specific domain of Epoc-1 is almost identical to those of Oct-1 and Oct-2. However, within the POU-homeodomain, 13 out of 60 aa differ between Epoc-1 and Oct-2. Recombinant Epoc-1 products were found to bind specifically to the octamer sequence. Epoc-1 was found to be expressed in skin, thymus, stomach and testis. In situ hybridization experiments and RNase protection assays indicated that Epoc-1 is expressed in the epidermal basal cells of the skin, which contain stem cells unipotent for keratinocyte differentiation and in thymic stromal elements. These results suggest that Epoc-1 might be one of the developmental regulators which controls epidermal development and thymic organogenesis. PMID:8224904

  19. Intracellular processing of epidermal growth factor by early wound healing cells

    SciTech Connect

    Seyfer, A.E.; Nassaux, P.; Emory, R.; Wray, H.L.; Schaudies, R.P. )

    1990-01-01

    Epidermal growth factor (EGF) is a potent 53-amino-acid residue polypeptide that has been implicated in normal wound healing. Although past studies have shown that locally applied EGF accelerates wound healing, these studies have not examined intracellular events related to the processing of the growth factor. The objective of this study was to characterize both initial and later postbinding intracellular processing of EGF by a responsive cell line (osteoblasts) that is important in the healing of wounds. Cloned mouse calvarial osteoblasts (MC-3TC-E1) were incubated with radiolabeled EGF, with and without preincubation with nonlabeled EGF, for specific time intervals. Cell-associated radioactivity was characterized by nondenaturing polyacrylamide gel electrophoresis. Results showed that EGF is processed as three distinct species and that the relative proportions of these species are altered at later time periods when compared with initial processing. The patterns, similar to those reported for human fibroblasts, indicate a possible common pathway for the mitogenic signal in cells associated with the early events of wound healing. In addition, these data represent the first direct evidence that preexposure of cells to nonlabeled EGF alters the processing of radiolabeled EGF. This is significant, because cells must be exposed to EGF for 5 to 8 hours to elicit a growth response. Such data may help to explain the lag phase of wound healing.

  20. Osmotic induction of fluid-phase endocytosis in onion epidermal cells.

    PubMed

    Oparka, K J; Prior, D A; Harris, N

    1990-03-01

    A transient plasmolysis/deplasmolysis (plasmolytic cycle) of onion epidermal cells has been shown to induce the formation of fluid-phase endocytic vesicles. Plasmolysis in the presence of the membrane-impermeant fluorescent probes Lucifer Yellow CH (LYCH) and Cascade Blue hydrazide resulted in the uptake of these probes by fluid-phase endocytosis. Following deplasmolysis, many of the dye-containing vesicles left their parietal positions within the cell and underwent vigorous streaming in the cytoplasm. Vesicles were observed to move within transvacuolar strands and their movements were recorded over several hours by video-microscopy. Within 2 h of deplasmolysis several of the larger endocytic vesicles had clustered around the nuclear membrane, apparently lodged in the narrow zone of cytoplams surrounding the nucleus. In further experiments LYCH was endocytically loaded into the cells during the first plasmolytic cycle and Cascade Blue subsequently loaded during a second plasmolytic cycle. This resulted in the introduction of two populations of endocytic vesicles into the cells, each containing a different probe. Both sets of vesicles underwent cytoplasmic streaming. The data are discussed in the light of previous observations of fluid-phase endocytosis in plant cells. PMID:24202101

  1. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition.

    PubMed

    Hata, Aaron N; Niederst, Matthew J; Archibald, Hannah L; Gomez-Caraballo, Maria; Siddiqui, Faria M; Mulvey, Hillary E; Maruvka, Yosef E; Ji, Fei; Bhang, Hyo-eun C; Krishnamurthy Radhakrishna, Viveksagar; Siravegna, Giulia; Hu, Haichuan; Raoof, Sana; Lockerman, Elizabeth; Kalsy, Anuj; Lee, Dana; Keating, Celina L; Ruddy, David A; Damon, Leah J; Crystal, Adam S; Costa, Carlotta; Piotrowska, Zofia; Bardelli, Alberto; Iafrate, Anthony J; Sadreyev, Ruslan I; Stegmeier, Frank; Getz, Gad; Sequist, Lecia V; Faber, Anthony C; Engelman, Jeffrey A

    2016-03-01

    Although mechanisms of acquired resistance of epidermal growth factor receptor (EGFR)-mutant non-small-cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here we observe that acquired resistance caused by the EGFR(T790M) gatekeeper mutation can occur either by selection of pre-existing EGFR(T790M)-positive clones or via genetic evolution of initially EGFR(T790M)-negative drug-tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug-tolerant cells had a diminished apoptotic response to third-generation EGFR inhibitors that target EGFR(T790M); treatment with navitoclax, an inhibitor of the anti-apoptotic factors BCL-xL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor-resistant patient tumors. These findings provide evidence that clinically relevant drug-resistant cancer cells can both pre-exist and evolve from drug-tolerant cells, and they point to therapeutic opportunities to prevent or overcome resistance in the clinic. PMID:26828195

  2. Epidermal growth factor promotes proliferation of dermal papilla cells via Notch signaling pathway.

    PubMed

    Zhang, Haihua; Nan, Weixiao; Wang, Shiyong; Zhang, Tietao; Si, Huazhe; Wang, Datao; Yang, Fuhe; Li, Guangyu

    2016-08-01

    The effect of epidermal growth factor (EGF) on the development and growth of hair follicle is controversial. In the present study, 2-20 ng/ml EGF promoted the growth of mink hair follicles in vitro, whereas 200 ng/ml EGF inhibited follicle growth. Further, dermal papilla (DP) cells, a group of mesenchymal cells that govern hair follicle development and growth, were isolated and cultured in vitro. Treatment with or forced expression of EGF accelerated proliferation and induced G1/S transition in DP cells. Moreover, EGF upregulated the expression of DP mesenchymal genes, such as alkaline phosphatase (ALP) and insulin-like growth factor (IGF-1), as well as the Notch pathway molecules including Notch1, Jagged1, Hes1 and Hes5. In addition, inhibition of Notch signaling pathway by DAPT significantly reduced the basal and EGF-enhanced proliferation rate, and also suppressed cell cycle progression. We also show that the expression of several follicle-regulatory genes, such as Survivin and Msx2, were upregulated by EGF, and was inhibited by DAPT. In summary, our study demonstrates that the concentration of EGF is critical for the switch between hair follicle growth and inhibition, and EGF promotes DP cell proliferation via Notch signaling pathway. PMID:27109378

  3. Phosphatidylinositol kinase is activated in membranes derived from cells treated with epidermal growth factor.

    PubMed Central

    Walker, D H; Pike, L J

    1987-01-01

    The ability of epidermal growth factor (EGF) to stimulate phosphatidylinositol (PtdIns) kinase activity in A431 cells was examined. The incorporation of 32P from [gamma-32P]ATP into PtdIns by A431 membranes was increased in membranes prepared from cells that had been pretreated with EGF. Demonstration of a stimulation of the PtdIns kinase activity by EGF required the use of subconfluent cultures and was dependent on the inclusion of protease inhibitors in the buffers used to prepare the membranes. Stimulation of the PtdIns kinase activity was rapid. The activation peaked 2 min after the addition of EGF and declined slowly thereafter. Half-maximal stimulation of the PtdIns kinase occurred at 7 nM EGF. Kinetic analyses of the reaction indicated that treatment of the cells with EGF resulted in a decrease in the Km for PtdIns with no change in the Vmax. The kinetic parameters for the utilization of ATP were unchanged in the EGF-treated membranes compared to the control membranes. Pretreatment of the cells with the phorbol ester phorbol 12-myristate 13-acetate blocked the ability of EGF to stimulate PtdIns kinase activity. These findings demonstrate that a PtdIns kinase activity in A431 cells is regulated by EGF and provide a good system for examining the mechanism by which EGF stimulates the activity of this intracellular enzyme. PMID:2823265

  4. Oak ellagitannins suppress the phosphorylation of the epidermal growth factor receptor in human colon carcinoma cells.

    PubMed

    Fridrich, Diana; Glabasnia, Arne; Fritz, Jessica; Esselen, Melanie; Pahlke, Gudrun; Hofmann, Thomas; Marko, Doris

    2008-05-14

    The ellagitannins castalagin and vescalagin, and the C-glycosides grandinin and roburin E as well as ellagic acid were found to potently inhibit the growth of human colon carcinoma cells (HT29) in vitro. In a cell-free system these compounds were identified as potent inhibitors of the protein tyrosine kinase activity of the epidermal growth factor receptor (EGFR) with IC 50 values in the low nanomolar range. To address the question of whether the interference with the activity of the isolated EGFR also plays a role within intact cells, effects on the phosphorylation status of the EGFR, as a measure for its activity, were determined in HT29 cells. As exemplified for castalagin and grandinin, both the nonglycosylated and the glycosylated ellagitannins effectively suppressed EGFR phosphorylation, but only at concentrations > or =10 microM, thus, in a concentration range where growth inhibition was observed. These results indicate that the suppression of EGFR-mediated signaling might contribute to the growth inhibitory effects of these compounds present in oak-matured wines and spirits such as whiskey. In contrast, despite substantial growth inhibitory properties, ellagic acid did not significantly affect EGFR phosphorylation in HT29 cells up to 100 microM. PMID:18419129

  5. Sequential cultivation of human epidermal keratinocytes and dermal mesenchymal like stromal cells in vitro.

    PubMed

    Mahabal, Shyam; Konala, Vijay Bhaskar Reddy; Mamidi, Murali Krishna; Kanafi, Mohammad Mahboob; Mishra, Suniti; Shankar, Krupa; Pal, Rajarshi; Bhonde, Ramesh

    2016-08-01

    Human skin has continuous self-renewal potential throughout adult life and serves as first line of defence. Its cellular components such as human epidermal keratinocytes (HEKs) and dermal mesenchymal stromal cells (DMSCs) are valuable resources for wound healing applications and cell based therapies. Here we show a simple, scalable and cost-effective method for sequential isolation and propagation of HEKs and DMSCs under defined culture conditions. Human skin biopsy samples obtained surgically were cut into fine pieces and cultured employing explant technique. Plated skin samples attached and showed outgrowth of HEKs. Gross microscopic examination displayed polygonal cells with a granular cytoplasm and H&E staining revealed archetypal HEK morphology. RT-PCR and immunocytochemistry authenticated the presence of key HEK markers including trans-membrane protein epithelial cadherin (E-cadherin), keratins and cytokeratin. After collection of HEKs by trypsin-EDTA treatment, mother explants were left intact and cultured further. Interestingly, we observed the appearance of another cell type with fibroblastic or stromal morphology which were able to grow up to 15 passages in vitro. Growth pattern, expression of cytoskeletal protein vimentin, surface proteins such as CD44, CD73, CD90, CD166 and mesodermal differentiation potential into osteocytes, adipocytes and chondrocytes confirmed their bonafide mesenchymal stem cell like status. These findings albeit preliminary may open up significant opportunities for novel applications in wound healing. PMID:25698160

  6. Is there a role for epidermal growth factor receptor tyrosine kinase inhibitors in epidermal growth factor receptor wild-type non-small cell lung cancer?

    PubMed Central

    Arriola, Edurne; Taus, Álvaro; Casadevall, David

    2015-01-01

    Non-small cell lung cancer (NSCLC) is the most common type of lung cancer with a world-wide annual incidence of around 1.3 million. The majority of patients are diagnosed with advanced disease and survival remains poor. However, relevant advances have occurred in recent years through the identification of biomarkers that predict for benefit of therapeutic agents. This is exemplified by the efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors for the treatment of EGFR mutant patients. These drugs have also shown efficacy in unselected populations but this point remains controversial. Here we have reviewed the clinical data that demonstrate a small but consistent subgroup of EGFR wild-type patients with NSCLC that obtain a clinical benefit from these drugs. Moreover, we review the biological rationale that may explain this benefit observed in the clinical setting. PMID:26266101

  7. Selective early innervation of a subset of epidermal cells in Xenopus may be mediated by chondroitin sulfate proteoglycans.

    PubMed

    Somasekhar, T; Nordlander, R H

    1997-04-18

    The epidermis of early Xenopus embryos is innervated by the Rohon-Beard (RB) neurons lying within the spinal cord and by extramedullary (EM) neurons lying outside of the cord. We have examined the innervation patterns of the three epidermal cell types using wholemount preparations of skin double-labelled with the HNK-1 antibody as a marker for neurons and with antibodies to chondroitin sulfate proteoglycan (CSPG). Cells of one of the three epidermal cell types, here termed conical cells, are innervated well before the other two. In wholemounts of embryonic skin incubated with antibodies to chondroitin-6-sulfate (C6S), all epidermal cells except conical cells show CSPG immunoreactivity in their basal lamina. Double-labelling of skin preparations with HNK-1 and anti-C6S confirmed that these conical cells which lack C6S immunoreactivity are the first to be innervated by RB axons. It is proposed that C6S-bearing proteoglycan initially inhibits innervation of cells whose basal lamina contain the proteoglycan, thus favoring innervation of the conical cells which lack it. PMID:9125474

  8. The Antiaging Properties of Andrographis paniculata by Activation Epidermal Cell Stemness.

    PubMed

    You, Jiyoung; Roh, Kyung-Baeg; Li, Zidan; Liu, Guangrong; Tang, Jian; Shin, Seoungwoo; Park, Deokhoon; Jung, Eunsun

    2015-01-01

    Andrographis paniculata (A. paniculata, Chuanxinlian), a medicinal herb with an extremely bitter taste that is native to China and other parts of Southeast Asia, possesses immense therapeutic value; however, its therapeutic properties have rarely been applied in the field of skin care. In this study, we investigated the effect of an A. paniculata extract (APE) on human epidermal stem cells (EpSCs), and confirmed its anti-aging effect through in vitro, ex vivo, and in vivo study. An MTT assay was used to determine cell proliferation. A flow cytometric analysis, with propidium iodide, was used to evaluate the cell cycle. The expression of integrin β1 (CD29), the stem cell marker, was detected with antibodies, using flow cytometry in vitro, and immunohistochemical assays in ex vivo. Type 1 collagen and VEGF (vascular endothelial growth factor) were measured using an enzyme-linked immunosorbent assay (ELISA). During the clinical study, skin hydration, elasticity, wrinkling, sagging, and dermal density were evaluated before treatment and at four and eight weeks after the treatment with the test product (containing the APE) on the face. The proliferation of the EpSCs, treated with the APE, increased significantly. In the cell cycle analysis, the APE increased the G2/M and S stages in a dose-dependent manner. The expression of integrin β1, which is related to epidermal progenitor cell expansion, was up-regulated in the APE-treated EpSCs and skin explants. In addition, the production of VEGF in the EpSCs increased significantly in response to the APE treatment. Consistent with these results, the VEGF and APE-treated EpSCs conditioned medium enhanced the Type 1 collagen production in normal human fibroblasts (NHFs). In the clinical study, the APE improved skin hydration, dermal density, wrinkling, and sagging significantly. Our findings revealed that the APE promotes a proliferation of EpSCs, through the up-regulation of the integrin β1 and VEGF expression. The VEGF

  9. Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

    SciTech Connect

    Nagata, Yosuke Ohashi, Kazuya; Wada, Eiji; Yuasa, Yuki; Shiozuka, Masataka; Nonomura, Yoshiaki; Matsuda, Ryoichi

    2014-08-01

    Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermal growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and its

  10. Formation of embryogenic cell clumps from carrot epidermal cells is suppressed by 5-azacytidine, a DNA methylation inhibitor.

    PubMed

    Yamamoto, Nozomi; Kobayashi, Hatsumi; Togashi, Takashi; Mori, Yukiko; Kikuchi, Koji; Kuriyama, Kyoko; Tokuji, Yoshihiko

    2005-01-01

    Using a direct somatic embryogenesis system in carrot, we examined the role of DNA methylation in the change of cellular differentiation state, from somatic to embryogenic. 5-Azacytidine (aza-C), an inhibitor of DNA methylation suppressed the formation of embryogenic cell clumps from epidermal carrot cells. Aza-C also downregulated the expression of DcLEC1c, a LEC1-like embryonic gene in carrot, during morphogenesis of embryos. A carrot DNA methyltransferase gene, Met1-5 was expressed transiently after the induction of somatic embryogenesis by 2,4-dichlorophenoxyacetic acid (2,4-D), before the formation of embryogenic cell clumps. These findings suggested the significance of DNA methylation in acquiring the embryogenic competence in somatic cells in carrot. PMID:15700420

  11. UV Radiation Induces the Epidermal Recruitment of Dendritic Cells that Compensate for the Depletion of Langerhans Cells in Human Skin.

    PubMed

    Achachi, Amine; Vocanson, Marc; Bastien, Philippe; Péguet-Navarro, Josette; Grande, Sophie; Goujon, Catherine; Breton, Lionel; Castiel-Higounenc, Isabelle; Nicolas, Jean-François; Gueniche, Audrey

    2015-08-01

    UVR causes skin injury and inflammation, resulting in impaired immune function and increased skin cancer risk. Langerhans cells (LCs), the immune sentinels of the epidermis, are depleted for several days following a single UVR exposure and can be reconstituted from circulating monocytes. However, the differentiation pathways leading to the recovery of a normal pool of LCs is still unclear. To study the dynamic changes in human skin with UV injury, we exposed a cohort of 29 healthy human volunteers to a clinically relevant dose of UVR and analyzed sequential epidermal biopsies for changes in leukocyte and dendritic cell (DC) subsets. UV-induced depletion of CD1a(high) LC was compensated by sequential appearance of various epidermal leukocytes. CD14(+) monocytes were recruited as early as D1 post exposure, followed by recruitment of two inflammatory DC subsets that may represent precursors of LCs. These CD1a(low) CD207(-) and the heretofore unknown CD1a(low) CD207(+) DCs appeared at day 1 and day 4 post UVR, respectively, and were endowed with T-cell-activating properties similar to those of LCs. We conclude that recruitment of monocytes and inflammatory DCs appear as a physiological response of the epidermis in order to repair UVR-induced LC depletion associated with immune suppression. PMID:25806853

  12. Trafficking of epidermal growth factor receptor ligands in polarized epithelial cells.

    PubMed

    Singh, Bhuminder; Coffey, Robert J

    2014-01-01

    A largely unilamellar epithelial layer lines body cavities and organ ducts such as the digestive tract and kidney tubules. This polarized epithelium is composed of biochemically and functionally separate apical and basolateral surfaces. The epidermal growth factor receptor (EGFR) signaling pathway is a critical regulator of epithelial homeostasis and is perturbed in a number of epithelial disorders. It is underappreciated that in vivo EGFR signaling is most often initiated by cell-surface delivery and processing of one of seven transmembrane ligands, resulting in release of the soluble form that binds EGFR. In polarized epithelial cells, EGFR is restricted largely to the basolateral surface, and apical or basolateral ligand delivery therefore has important biological consequences. In vitro approaches have been used to study the biosynthesis, cell-surface delivery, proteolytic processing, and release of soluble EGFR ligands in polarized epithelial cells. We review these results, discuss their relevance to normal physiology, and demonstrate the pathophysiological consequences of aberrant trafficking. These studies have uncovered a rich diversity of apico-basolateral trafficking mechanisms among the EGFR ligands, provided insights into the pathogenesis of an inherited magnesium-wasting disorder of the kidney (isolated renal hypomagnesemia), and identified a new mode of EGFR ligand signaling via exosomes. PMID:24215440

  13. Trafficking of Epidermal Growth Factor Receptor Ligands in Polarized Epithelial Cells

    PubMed Central

    Singh, Bhuminder; Coffey, Robert J.

    2014-01-01

    A largely unilamellar epithelial layer lines body cavities and organ ducts such as the digestive tract and kidney tubules. This polarized epithelium is composed of biochemically and functionally separate apical and basolateral surfaces. The epidermal growth factor receptor (EGFR) signaling pathway is a critical regulator of epithelial homeostasis and is perturbed in a number of epithelial disorders. It is underappreciated that in vivo EGFR signaling is most often initiated by cell-surface delivery and processing of one of seven transmembrane ligands, resulting in release of the soluble form that binds EGFR. In polarized epithelial cells, EGFR is restricted largely to the basolateral surface, and apical or basolateral ligand delivery therefore has important biological consequences. In vitro approaches have been used to study the biosynthesis, cell-surface delivery, proteolytic processing, and release of soluble EGFR ligands in polarized epithelial cells. We review these results, discuss their relevance to normal physiology, and demonstrate the pathophysiological consequences of aberrant trafficking. These studies have uncovered a rich diversity of apico-basolateral trafficking mechanisms among the EGFR ligands, provided insights into the pathogenesis of an inherited magnesium-wasting disorder of the kidney (isolated renal hypomagnesemia), and identified a new mode of EGFR ligand signaling via exosomes. PMID:24215440

  14. Synergistic action of auxin and ethylene on root elongation inhibition is caused by a reduction of epidermal cell length.

    PubMed

    Alarcón, M Victoria; Lloret, Pedro G; Salguero, Julio

    2014-01-01

    Auxin and ethylene have been largely reported to reduce root elongation in maize primary root. However the effects of auxin are greater than those caused by ethylene. Although auxin stimulates ethylene biosynthesis through the specific increase of ACC synthase, the auxin inhibitory effect on root elongation is not mediated by the auxin-induced increase of ethylene production. Recently it has been demonstrated that root inhibition by the application of the synthetic auxin NAA (1-naphtalenacetic acid) is increased if combined with the ethylene precursor ACC (1-aminocyclopropane-1-carboxilic acid) when both compounds are applied at very low concentrations.   Root elongation is basically the result of two processes: a) cell divisions in the meristem where meristematic cells continuously generate new cells and b) subsequently polarized growth by elongation along the root axis as cells leave the meristem and enter the root elongation zone. Our results indicate that exogenous auxin reduced both root elongation and epidermal cell length. In a different way, ethylene at very low concentrations only inhibited root elongation without affecting significantly epidermal cell length. However, these concentrations of ethylene increased the inhibitory effect of auxin on root elongation and cell length. Consequently the results support the hypothesis that ethylene acts synergistically with auxin in the regulation of root elongation and that inhibition by both hormones is due, at least partially, to the reduction of cell length in the epidermal layer. PMID:24598313

  15. IL-1β-dependent activation of dendritic epidermal T cells in contact hypersensitivity1

    PubMed Central

    Nielsen, Morten M.; Lovato, Paola; MacLeod, Amanda S.; Witherden, Deborah A.; Skov, Lone; Dyring-Andersen, Beatrice; Dabelsteen, Sally; Woetmann, Anders; Ødum, Niels; Havran, Wendy L.; Geisler, Carsten; Bonefeld, Charlotte M.

    2014-01-01

    Substances that penetrate the skin surface can act as allergens and induce a T cell-mediated inflammatory skin disease called contact hypersensitivity (CHS). IL-17 is a key cytokine in CHS and was originally thought to be produced solely by CD4+ T cells. However, it is now known that several cell types including γδ T cells can produce IL-17. Here, we determine the role of γδ T cells, especially the dendritic epidermal T cells (DETC), in CHS. By use of a well-established model for CHS where dinitroflourobenzen (DNFB) is used as allergen, we found that γδ T cells are important players in CHS. Thus, an increased number of IL-17 producing DETC appear in the skin following exposure to DNFB in WT mice, and DNFB-induced ear-swelling is reduced by approximately 50% in TCRδ−/− mice compared to WT mice. In accordance, DNFB-induced ear-swelling was reduced by approximately 50% in IL-17−/− mice. We show that DNFB triggers DETC activation and IL-1β production in the skin, and that keratinocytes produce IL-1β when stimulated with DNFB. We find that DETC activated in vitro by incubation with anti-CD3 and IL-1β produce IL-17. Importantly, we demonstrate that the IL-1 receptor antagonist anakinra significantly reduces CHS responses as measured by decreased ear-swelling, inhibition of local DETC activation and a reduction in the number of IL-17+ γδ T cells and DETC in the draining lymph nodes. Taken together, we show that DETC become activated and produce IL-17 in an IL-1β-dependent manner during CHS suggesting a key role for DETC in CHS. PMID:24600030

  16. Modulation of cultured porcine granulosa cell responsiveness to follicle stimulating hormone and epidermal growth factor

    SciTech Connect

    Buck, P.A.

    1986-01-01

    Ovarian follicular development is dependent upon the coordinated growth and differentiation of the granulosa cells which line the follicle. Follicle stimulating hormone (FSH) induces granulosa cell differentiation both in vivo and in vitro. Epidermal growth factor (EGF) stimulates granulosa cell proliferation in vitro. The interaction of these two effectors upon selected parameters of growth and differentiation was examined in monolayer cultures of porcine granulose cells. Analysis of the EGF receptor by /sup 125/I-EGF binding revealed that the receptor was of high affinity with an apparent dissociation constant of 4-6 x 10/sup -10/ M. The average number of receptors per cell varied with the state of differentiation both in vivo and in vitro; highly differentiated cells bound two-fold less /sup 125/I-EGF and this effect was at least partially induced by FSH in vitro. EGF receptor function was examined by assessing EGF effects on cell number and /sup 3/H-thymidine incorporation. EGF stimulated thymidine incorporation in both serum-free and serum-supplemented culture, but only in serum-supplemented conditions was cell number increased. EGF receptor function was inversely related to the state of differentiation and was attenuated by FSH. The FSH receptor was examined by /sup 125/I-FSH binding. EGF increased FSH receptor number, and lowered the affinity of the receptor. The function of these receptors was assessed by /sup 125/I-hCG binding and progesterone radioimmunoassay. If EGF was present continuously in the cultures. FSH receptor function was attenuated regardless of FSH receptor number. A preliminary effort to examine the mechanism of this interaction was performed by analyzing hormonally controlled protein synthesis with /sup 35/S-methionine labeling, SDS polyacrylamide gel electrophoresis and fluorography. FSH promoted the expression of a 27,000 dalton protein. This effect was attenuated by EGF.

  17. Stem cell factor rescues dark epidermal pigmentation in discreet anatomic locations in albino and fair-skinned mice

    PubMed Central

    Vanover, Jillian C.; Spry, Malinda L.; Hamilton, Laura; Wakamatsu, Kazumasa; Ito, Shosuke; D’Orazio, John A.

    2016-01-01

    We previously reported a transgenic animal model of variant pigmentation based on epidermal expression of stem cell factor (SCF) and well-characterized coat color genes bred into the C57Bl/6 background. In this system, constitutive expression of SCF by epidermal keratinocytes results in the maintenance of epidermal melanocytes in the interfollicular basal epidermal layer and subsequent pigmentation of the epidermis itself. In this report, we describe extending this animal model by developing a compound mutant transgenic amelanotic animal defective at both the melanocortin 1 receptor (Mc1r) and tyrosinase (Tyr) loci. We have observed SCF-dependent pigment deposition in specific anatomic regions regardless of tyrosinase (Tyr) or Mc1r genetic status. Thus, in the presence of K14-Scf, tyrosinase-null animals (previously thought incapable of synthesizing melanin) exhibited progressive robust epidermal pigmentation with age in the ears and tails. Furthermore, in the presence of the K14-Scf transgene, Tyr-defective animals demonstrated tyrosinase activity, suggesting that the c2j Tyr promoter defect is leaky and that Tyr expression can be rescued in part by SCF in the ears and tail. Lastly, we found that UV sensitivity of K14-Scf congenic animals differing only at the Mc1r or Tyr loci depended mainly on the amount of eumelanin present in the skin. These findings suggest that c-kit signaling can overcome the c2j Tyr promoter mutation in the ears and tails of aging animals but that UV resistance depends on accumulation of epidermal eumelanin. PMID:19682281

  18. Rapid and dynamic subcellular reorganization following mechanical stimulation of Arabidopsis epidermal cells mimics responses to fungal and oomycete attack

    PubMed Central

    Hardham, Adrienne R; Takemoto, Daigo; White, Rosemary G

    2008-01-01

    Background Plant cells respond to the presence of potential fungal or oomycete pathogens by mounting a basal defence response that involves aggregation of cytoplasm, reorganization of cytoskeletal, endomembrane and other cell components and development of cell wall appositions beneath the infection site. This response is induced by non-adapted, avirulent and virulent pathogens alike, and in the majority of cases achieves penetration resistance against the microorganism on the plant surface. To explore the nature of signals that trigger this subcellular response and to determine the timing of its induction, we have monitored the reorganization of GFP-tagged actin, microtubules, endoplasmic reticulum (ER) and peroxisomes in Arabidopsis plants – after touching the epidermal surface with a microneedle. Results Within 3 to 5 minutes of touching the surface of Arabidopsis cotyledon epidermal cells with fine glass or tungsten needles, actin microfilaments, ER and peroxisomes began to accumulate beneath the point of contact with the needle. Formation of a dense patch of actin was followed by focusing of actin cables on the site of contact. Touching the cell surface induced localized depolymerization of microtubules to form a microtubule-depleted zone surrounding a dense patch of GFP-tubulin beneath the needle tip. The concentration of actin, GFP-tubulin, ER and peroxisomes remained focused on the contact site as the needle moved across the cell surface and quickly dispersed when the needle was removed. Conclusion Our results show that plant cells can detect the gentle pressure of a microneedle on the epidermal cell surface and respond by reorganizing subcellular components in a manner similar to that induced during attack by potential fungal or oomycete pathogens. The results of our study indicate that during plant-pathogen interactions, the basal defence response may be induced by the plant's perception of the physical force exerted by the pathogen as it attempts to

  19. Role of epidermal Langerhans' cells in the induction of protective immunity to Schistosoma mansoni in guinea-pigs.

    PubMed Central

    Sato, H; Kamiya, H

    1995-01-01

    Percutaneous exposure of guinea-pigs to attenuated or normal larvae of Schistosoma mansoni induced proliferative T-cell responses in the skin-draining lymph nodes (SLN). The responses elicited by attenuated larvae were stronger and more prolonged [2-12 days post-infection (p.i.)] than those by normal larvae (3-8 days p.i.). The former were coincident with greater and more sustained increases in numbers of SLN dendritic cells. During this event, epidermal Langerhans' cells (LC) showed marked changes in their distribution and morphology. Resident LC were similarly exhausted by either attenuated or normal larvae between 12 hr and 1 day p.i., but thereafter more blood-borne LC were recruited around the former, since reaggregation of LC around these persisted larvae was more frequent and intensive, and enhanced replenishment of epidermal LC was achieved by 8 days p.i. When the skin depleted of epidermal LC by short-wavelength ultraviolet (UVC) irradiation was exposed to attenuated larvae, consequent T-cell responses were delayed. Excision of the whole exposed skin on day 4 p.i. also reduced T-cell responses to marginal levels. These results indicate that during the afferent phase of immunity to S. mansoni, efficient T-cell responses in the SLN need an active involvement of not only resident LC but also blood-borne LC as immunostimulatory cells. Images Figure 3 Figure 5 PMID:7750999

  20. An epidermal stem cells niche microenvironment created by engineered human amniotic membrane.

    PubMed

    Ji, Shi-zhao; Xiao, Shi-chu; Luo, Peng-fei; Huang, Guo-feng; Wang, Guang-yi; Zhu, Shi-hui; Wu, Min-juan; Xia, Zhao-fan

    2011-11-01

    How to amplify epidermal stem cells (ESCs) rapidly is a challenging crux in skin tissue engineering research. The present study describes the preparation of 3D micronized (300-600 μm) amniotic membrane (mAM) by means of repeated freeze-thawing cycles to deplete cell components and homogenized with a macrohomogenizer in liquid nitrogen. This newly prepared mAM not only possessed the characteristics of a microcarrier but completely retained the basement membrane structure and abundant active substances such as NGF, HGF, KGF, bFGF, TGF-β1 and EGF in the AM matrix. The result showed that mAM combined with rotary cell culture system (RCCS) was able to amplify ESCs quickly. The relative cell viability at day 7 and 14 was significantly higher than that of the conventional 2D plate culture (326 ± 28% and 535 ± 47% versus 232 ± 21% and 307 ± 32%, P < 0.05). In addition, the new method was able to prevent cell differentiation effectively and retain the characteristics of stem cells. When mAM loaded with ESCs (ESC-mAM) was further transplanted to full-thickness skin defects in nude mice, ESCs survived well and formed a new epidermis. Four weeks after transplantation, papilla-like structures were observed, and collagen fibers were well and regularly arranged in the newly formed dermal layer. In conclusion, the mAM as a novel natural microcarrier possesses an intact basement membrane structure and bioactivities. It not only provides the microenvironment similar to the stem cell niche within the human body favorable for ex vivo culture and amplification of ESCs but can be used as the dermal scaffold in constructing a skin substitute containing ESCs for the repair of full-thickness skin defects. PMID:21803416

  1. Epidermal growth factor precursor in mouse lactating mammary gland alveolar cells

    SciTech Connect

    Brown, C.F.; Teng, C.T.; Pentecost, B.T.; DiAugustine, R.P. )

    1989-07-01

    Previous studies have demonstrated that high levels of epidermal growth factor (EGF) occur in human and rodent milk and that oral administration of this polypeptide stimulates rodent gastrointestinal development. It is not known whether EGF in milk originates from cells of the lactating mammary gland or is sequestered from an extramammary source. In the present study, prepro-EGF mRNA (approximately 4.7 kilobases) was detected in the CD-1 mouse mammary gland throughout the period of lactation; by comparison, negligible levels of this EGF transcript were found in the gland during pregnancy. Low levels of EGF immunoreactivity (4-5 ng/g wet wt tissue) were extracted from lactating (day 18) mammary glands with dilute acetic acid. Immunolocalization was evident with antisera to either EGF or two other regions of the EGF precursor in essentially all alveolar cells of the lactating gland. The most prominent staining with antiserum to EGF was observed along the luminal borders of cells; this pattern of cellular staining required proteolytic pretreatment of tissue sections. Western blot analyses of cell membranes isolated from the day 16 lactating mammary gland revealed an EGF-immunoreactive band at about 145K, which was equivalent in size to the EGF precursor found in mouse kidney cell membranes. Despite these findings, labeling of lactating mammary gland mince with L-(35S)methionine and cysteine for up to 4 h did not reveal any specific bands in immunoprecipitates. These cumulative findings suggest that the precursor form of EGF occurs in alveolar cells of lactating mammary gland and that this protein is translocated to the cell membrane.

  2. Amplification of Coronary Arteriogenic Capacity of Multipotent Stromal Cells by Epidermal Growth factor

    PubMed Central

    Belmadani, Souad; Matrougui, Khalid; Kolz, Chris; Pung, Yuh Fen; Palen, Desiree; Prockop, Darwin J; Chilian, William M

    2009-01-01

    Objective We determined if increasing adherence of multipotent stromal cells (MSCs) would amplify their effects on coronary collateral growth (CCG). Methods and Results Adhesion was established in cultured coronary endothelials cells (CECS) or MSCs treated with epidermal growth factor (EGF). EGF increased MSCs adhesion to CECs, and increased intercellular adhesion molecule (ICAM-1) or vascular cell adhesion molecule (VCAM-1) expression. Increased adherence was blocked by EGF receptor antagonism or antibodies to the adhesion molecules. To determine if adherent MSCs, treated with EGF, would augment CCG, repetitive episodes of myocardial ischemia (RI) were introduced and CCG was measured from the ratio of collateral-dependent (CZ) and normal zone (NZ) flows. CZ/NZ was increased by MSCs without treatment vs RI-control and was further increased by EGF-treated MSCs. EGF-treated MSCs significantly improved myocardial function vs RI or RI+ MSCs demonstrating that the increase in collateral flow was functionally significant. Engraftment of MSCs into myocardium was also increased by EGF treatment. Conclusions These results reveal the importance of EGF in MSCs adhesion to endothelium and suggest that MSCs may be effective therapies for the stimulation of coronary collateral growth when interventions are employed to increase their adhesion and homing (in vitro EGF treatment) to the jeopardized myocardium. PMID:19342596

  3. Autoradiographic localization of epidermal growth factor receptors to all major uterine cell types

    SciTech Connect

    Lin, T.H.; Mukku, V.R.; Verner, G.; Kirkland, J.L.; Stancel, G.M.

    1988-03-01

    We have recently studied the structure and function of the uterine epidermal growth factor (EGF) receptor, its hormonal regulation, and its possible role in estrogen-induced uterine DNA synthesis. Since the uterus is composed of multiple cell types, we sought, in the work reported here, to localize EGF binding in this organ by autoradiography. Prior to the actual autoradiography, we performed a companion series of experiments to insure that EGF binding to uterine tissue in situ represented a true receptor interaction. Uteri from immature female rats were incubated in vitro with 125I-EGF at 25 degrees C. Tissue binding was maximal within 120 min and remained constant for at least an additional 120 min. This binding of labeled EGF was largely abolished by excess unlabeled EGF but not by other growth factors, indicating that binding was to specific receptors. The binding of 125I-EGF was saturable and reached a plateau at 4-8 nM; specific binding was half-maximal at 1-2 nM EGF. In situ cross-linking studies revealed that 125I-EGF was bound predominantly to a 170,000 MW EGF receptor similar to that seen in isolated uterine membranes. Incubation of uteri with 125I-EGF followed by autoradiography revealed binding to epithelial cells, stroma, and myometrium. These results provide evidence for the presence of specific EGF receptors in all major uterine cell types of the immature rat.

  4. The Potential of Menstrual Blood-Derived Stem Cells in Differentiation to Epidermal Lineage: A Preliminary Report

    PubMed Central

    Faramarzi, Hossein; Mehrabani, Davood; Fard, Maryam; Akhavan, Maryam; Zare, Sona; Bakhshalizadeh, Shabnam; Manafi, Amir; Kazemnejad, Somaieh; Shirazi, Reza

    2016-01-01

    BACKGROUND Menstrual blood-derived stem cells (MenSCs) are a novel source of stem cells that can be easily isolated non-invasively from female volunteered donor without ethical consideration. These mesenchymal-like stem cells have high rate of proliferation and possess multi lineage differentiation potency. This study was undertaken to isolate the MenSCs and assess their potential in differentiation into epidermal lineage. METHODS About 5-10 ml of menstrual blood (MB) was collected using sterile Diva cups inserted into vagina during menstruation from volunteered healthy fertile women aged between 22-30 years. MB was transferred into Falcon tubes containing phosphate buffered saline (PBS) without Ca2+ or Mg2+ supplemented with 2.5 µg/ml fungizone, 100 µg/mL streptomycin, 100 U/mL penicillin and 0.5 mM EDTA. Mononuclear cells were separated using Ficoll-Hypaque density gradient centrifugation and washed out in PBS. The cell pellet was suspended in DMEM-F12 medium supplemented with 10% FBS and cultured in tissue culture plates. The isolated cells were co-cultured with keratinocytes derived from the foreskin of healthy newborn male aged 2-10 months who was a candidate for circumcision for differentiation into epidermal lineage. RESULTS The isolated MenSCs were adhered to the plate and exhibited spindle-shaped morphology. Flow cytometric analysis revealed the expression of mesenchymal markers of CD10, CD29, CD73, and CD105 and lack of hematopoietic stem cells markers. An early success in derivation of epidermal lineage from MenSCs was visible. CONCLUSION The MenSCs are a real source to design differentiation to epidermal cells that can be used non-invasively in various dermatological lesions and diseases. PMID:27308237

  5. Differentiation of ionic currents in CNS progenitor cells: dependence upon substrate attachment and epidermal growth factor.

    PubMed

    Feldman, D H; Thinschmidt, J S; Peel, A L; Papke, R L; Reier, P J

    1996-08-01

    Multipotential progenitor cells grown from central nervous system (CNS) tissues in defined media supplemented with epidermal growth factor (EGF), when attached to a suitable substratum, differentiate to express neural and glial histochemical markers and morphologies. To assess the functional characteristics of such cells, expression of voltage-gated Na+ and K+ currents (INa, IK) was studied by whole-cell patch clamp methods in progenitors raised from postnatal rat forebrain. Undifferentiated cells were acutely dissociated from proliferative "spheres," and differentiated cells were studied 1-25 days after plating spheres onto polylysine/laminin-treated coverslips. INa and IK were detected together in 58%, INa alone in 11%, and IK alone in 19% of differentiated cells recorded with K(+)-containing pipettes. With internal Cs+ (to isolate INa), INa up to 45 pA/pF was observed in some cells within 1 day after plating. I Na ranged up to 150 pA/pF subsequently. Overall, 84% of cells expressed I Na, with an average of 38 pA/pF. INa had fast kinetics, as in neurons, but steadystate inactivation curves were strongly negative, resembling those of glial INa. Inward tail currents sensitive to [K+]out were observed upon repolarization after the 10-ms test pulse with internal Cs+, indicating the expression of K+ channels in 82% of cells. In contrast to the substantial currents observed in differentiating cells, little or no INa or Ik-tail currents were detected in recordings from cells acutely dissociated from spheres. Thus, in the presence of EGF, ionic currents develop early during differentiation induced by attachment to an appropriate substratum. Cells switched from EGF to basic fibroblast growth factor (bFGF) when plated onto coverslips showed greatly reduced proliferation and developed less neuron-like morphologies than cells plated in the presence of EGF. INa was observed in only 53% of bFGF-treated cells, with an average of 9 pA/pF. Thus, in contrast to reports that b

  6. The carboxy-terminus of p63 links cell cycle control and the proliferative potential of epidermal progenitor cells

    PubMed Central

    Suzuki, Daisuke; Sahu, Raju; Leu, N. Adrian; Senoo, Makoto

    2015-01-01

    The transcription factor p63 (Trp63) plays a key role in homeostasis and regeneration of the skin. The p63 gene is transcribed from dual promoters, generating TAp63 isoforms with growth suppressive functions and dominant-negative ΔNp63 isoforms with opposing properties. p63 also encodes multiple carboxy (C)-terminal variants. Although mutations of C-terminal variants have been linked to the pathogenesis of p63-associated ectodermal disorders, the physiological role of the p63 C-terminus is poorly understood. We report here that deletion of the p63 C-terminus in mice leads to ectodermal malformation and hypoplasia, accompanied by a reduced proliferative capacity of epidermal progenitor cells. Notably, unlike the p63-null condition, we find that p63 C-terminus deficiency promotes expression of the cyclin-dependent kinase inhibitor p21Waf1/Cip1 (Cdkn1a), a factor associated with reduced proliferative capacity of both hematopoietic and neuronal stem cells. These data suggest that the p63 C-terminus plays a key role in the cell cycle progression required to maintain the proliferative potential of stem cells of many different lineages. Mechanistically, we show that loss of Cα, the predominant C-terminal p63 variant in epithelia, promotes the transcriptional activity of TAp63 and also impairs the dominant-negative activity of ΔNp63, thereby controlling p21Waf1/Cip1 expression. We propose that the p63 C-terminus links cell cycle control and the proliferative potential of epidermal progenitor cells via mechanisms that equilibrate TAp63 and ΔNp63 isoform function. PMID:25503409

  7. Quercetin 3-O-glucoside suppresses epidermal growth factor-induced migration by inhibiting EGFR signaling in pancreatic cancer cells.

    PubMed

    Lee, Jungwhoi; Han, Song-I; Yun, Jeong-Hun; Kim, Jae Hoon

    2015-12-01

    Pancreatic cancer is one of the most dangerous cancers and is associated with a grave prognosis. Despite increased knowledge of the complex signaling networks responsible for progression of pancreatic cancer, many challenging therapies have fallen short of expectations. In this study, we examined the anti-migratory effect of quercetin 3-O-glucoside in epidermal growth factor-induced cell migration by inhibiting EGF receptor (EGFR) signaling in several human pancreatic cancer cell lines. Treatment with quercetin, quercetin 3-O-glucoside, and quercetin 7-O-glucoside differentially suppressed epidermal growth factor-induced migration activity of human pancreatic cancer cells. In particular, quercetin 3-O-glucoside strongly inhibited the infiltration activity of pancreatic cancer cells in a dose-dependent manner. Furthermore, quercetin 3-O-glucoside exerted the anti-migratory effect even at a relatively low dose compared with other forms of quercetin. The anti-tumor effects of quercetin 3-O-glucoside were mediated by selectively inhibiting the EGFR-mediated FAK, AKT, MEK1/2, and ERK1/2 signaling pathway. Combinatorial treatment with quercetin 3-O-glucoside plus gemcitabine showed the synergistic anti-migratory effect on epidermal growth factor-induced cell migration in human pancreatic cancer cell lines. These results suggest that quercetin 3-O-glucoside has potential for anti-metastatic therapy in human pancreatic cancer. PMID:26109002

  8. Polarity of Water Transport across Epidermal Cell Membranes in Tradescantia virginiana1[W][OPEN

    PubMed Central

    Wada, Hiroshi; Fei, Jiong; Knipfer, Thorsten; Matthews, Mark A.; Gambetta, Greg; Shackel, Kenneth

    2014-01-01

    Using the automated cell pressure probe, small and highly reproducible hydrostatic pressure clamp (PC) and pressure relaxation (PR) tests (typically, applied step change in pressure = 0.02 MPa and overall change in volume = 30 pL, respectively) were applied to individual Tradescantia virginiana epidermal cells to determine both exosmotic and endosmotic hydraulic conductivity (LpOUT and LpIN, respectively). Within-cell reproducibility of measured hydraulic parameters depended on the method used, with the PR method giving a lower average coefficient of variation (15.2%, 5.8%, and 19.0% for half-time, cell volume [Vo], and hydraulic conductivity [Lp], respectively) than the PC method (25.4%, 22.0%, and 24.2%, respectively). Vo as determined from PC and PR tests was 1.1 to 2.7 nL and in the range of optically estimated Vo values of 1.5 to 4.9 nL. For the same cell, Vo and Lp estimates were significantly lower (about 15% and 30%, respectively) when determined by PC compared with PR. Both methods, however, showed significantly higher LpOUT than LpIN (LpOUT/LpIN ≅ 1.20). Because these results were obtained using small and reversible hydrostatically driven flows in the same cell, the 20% outward biased polarity of water transport is most likely not due to artifacts associated with unstirred layers or to direct effects of externally applied osmotica on the membrane, as has been suggested in previous studies. The rapid reversibility of applied flow direction, particularly for the PR method, and the lack of a clear increase in LpOUT/LpIN over a wide range of Lp values suggest that the observed polarity is an intrinsic biophysical property of the intact membrane/protein complex. PMID:24495955

  9. Epidermal growth factor receptor is overexpressed in neuroblastoma tissues and cells.

    PubMed

    Zheng, Chao; Shen, Ruling; Li, Kai; Zheng, Na; Zong, Yuqing; Ye, Danrong; Wang, Qingcheng; Wang, Zuopeng; Chen, Lian; Ma, Yangyang

    2016-08-01

    Neuroblastoma is the most common abdominal malignant tumor in childhood. Immunotoxin (IT) that targets the tumor cell surface receptor is a new supplementary therapeutic treatment approach. The purpose of this study is to detect the expression of epidermal growth factor receptor (EGFR) in neuroblastoma cell lines and tissues, and to explore if IT therapy can be used to treat refractory neuroblastoma. The EGFR expression in human neuroblastoma tissue samples was detected by immunohistochemistry staining. The positive rate of EGFR expression was 81.0% in neuroblastoma tissue and 50.0% in gangliocytoma, respectively, but without statistical significance between them (P > 0.05). The positive rate of EGFR expression in favorable type and unfavorable type was 62.5% and 92.3%, respectively, but they were not statistically different (P > 0.05). Results from pre-chemotherapy and post-chemotherapy samples showed that there was no significant statistical difference (P > 0.05) between them in the EGFR expression. Furthermore, the EGFR expression levels in five neuroblastoma cell lines were measured using cell-based ELISA assay and western blot analysis. The results showed that the expression of EGFR was higher in KP-N-NS and BE(2)-C than those in other cell lines. Our results revealed that there are consistent and widespread expressions of EGFR in neuroblastoma tissues as well as in neuroblastoma cell lines, suggesting that it is possible to develop future treatment strategies of neuroblastoma by targeting at the EGFR. PMID:27353319

  10. IgG and IgA with potential microbial-binding activity are expressed by normal human skin epidermal cells.

    PubMed

    Jiang, Dongyang; Ge, Jing; Liao, Qinyuan; Ma, Junfan; Liu, Yang; Huang, Jing; Wang, Chong; Xu, Weiyan; Zheng, Jie; Shao, Wenwei; Lee, Gregory; Qiu, Xiaoyan

    2015-01-01

    The innate immune system of the skin is thought to depend largely on a multi-layered mechanical barrier supplemented by epidermis-derived antimicrobial peptides. To date, there are no reports of antimicrobial antibody secretion by the epidermis. In this study, we report the expression of functional immunoglobulin G (IgG) and immunoglobulin A (IgA), previously thought to be only produced by B cells, in normal human epidermal cells and the human keratinocyte line HaCaT. While B cells express a fully diverse Ig, epidermal cell-expressed IgG or IgA showed one or two conservative VHDJH rearrangements in each individual. These unique VDJ rearrangements in epidermal cells were found neither in the B cell-derived Ig VDJ databases published by others nor in our positive controls. IgG and IgA from epidermal cells of the same individual had different VDJ rearrangement patterns. IgG was found primarily in prickle cells, and IgA was mainly detected in basal cells. Both epidermal cell-derived IgG and IgA showed potential antibody activity by binding pathogens like Staphylococcus aureus, the most common pathogenic skin bacteria, but the microbial-binding profile was different. Our data indicates that normal human epidermal cells spontaneously express IgG and IgA, and we speculate that these Igs participate in skin innate immunity. PMID:25625513

  11. Preparation of Epidermal Peels and Guard Cell Protoplasts for Cellular, Electrophysiological, and -Omics Assays of Guard Cell Function.

    PubMed

    Zhu, Mengmeng; Jeon, Byeong Wook; Geng, Sisi; Yu, Yunqing; Balmant, Kelly; Chen, Sixue; Assmann, Sarah M

    2016-01-01

    Bioassays are commonly used to study stomatal phenotypes. There are multiple options in the choice of plant materials and species used for observation of stomatal and guard cell responses in vivo. Here, detailed procedures for bioassays of stomatal responses to abscisic acid (ABA) in Arabidopsis thaliana are described, including ABA promotion of stomatal closure, ABA inhibition of stomatal opening, and ABA promotion of reaction oxygen species (ROS) production in guard cells. We also include an example of a stomatal bioassay for the guard cell CO2 response using guard cell-enriched epidermal peels from Brassica napus. Highly pure preparations of guard cell protoplasts can be produced, which are also suitable for studies on guard cell signaling, as well as for studies on guard cell ion transport. Small-scale and large-scale guard cell protoplast preparations are commonly used for electrophysiological and -omics studies, respectively. We provide a procedure for small-scale guard cell protoplasting from A. thaliana. Additionally, a general protocol for large-scale preparation of guard cell protoplasts, with specifications for three different species, A. thaliana, B. napus, and Vicia faba is also provided. PMID:26577784

  12. p53 Acts as a Co-Repressor to Regulate Keratin 14 Expression during Epidermal Cell Differentiation

    PubMed Central

    Chao, Chung-Faye; Lu, Mei-Hua; Lin, Hwang-Chi; Chiou, Shih-Hwa; Tao, Pao-Luh; Chen, Jang-Yi

    2012-01-01

    During epidermal cell differentiation, keratin 14 (K14) expression is down-regulated, p53 expression varies, and the expression of the p53 target genes, p21 and 14-3-3σ, increases. These trends suggest that the relative transcriptional activity of p53 is increased during epidermal cell differentiation. To determine the relationship between K14 and p53, we constructed K14 promoters of various sizes and found that wild-type p53 could repress the promoter activity of all of the K14 promoter constructs in H1299 cells. K14-p160 contains an SP1 binding site mutation that prevents p53 from repressing K14 expression. Using a DNA affinity precipitation assay, we confirmed that p53 forms a complex with SP1 at the SP1 binding site between nucleotides -48 and -43 on the K14 promoter. Thus, our data indicate that p53 acts as a co-repressor to down-regulate K14 expression by binding to SP1. Next, we used a 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced epidermal cell differentiation model to examine the inhibition of K14 expression caused by increased p53 activity. Human ovarian teratocarcinoma C9 cells were treated with TPA to induce differentiation. Over-expression of the dominant negative p53 mutant ΔTAp53, which inhibits p53 activity, prevented the TPA-induced K14 down-regulation in C9 cells. Furthermore, treatment of normal primary human foreskin keratinocytes (PHFK) with the p53 inhibitor pifithrin-α (PFT-α) showed that the inhibition of p53 activity relieves K14 repression during epidermal cell differentiation. Finally, we found that TPA induces the phosphorylation of p53 at residue 378, which enhances the affinity of p53 to bind to Sp1 and repress K14 expression. PMID:22911849

  13. Transient expression of minimum linear gene cassettes in onion epidermal cells via direct transformation.

    PubMed

    Cheng, Yun-Qing; Yang, Jun; Xu, Feng-Ping; An, Li-Jia; Liu, Jian-Feng; Chen, Zhi-Wen

    2009-12-01

    A new method without any special devices for direct transformation of linear gene cassettes was developed. Its feasibility was verified through 5'-fluorescent dye (fluorescein isothiocyanate, FITC)-labeled fluorescent tracing and transient expression of a gus reporter gene. Minimal linear gene cassettes, containing necessary regulation elements and a gus reporter gene, was prepared by polymerase chain reaction and dissolved in transformation buffer solution to 100 ng/mL. The basic transformation solution used was Murashige and Skoog basal salt mixture (MS) liquid medium. Hypertonic pretreatment of explants and transformation cofactors, including Ca(2+), surfactant assistants, Agrobacterium LBA4404 cell culture on transformation efficiency were evaluated. Prior to the incubation of the explants and target linear cassette in each designed transformation solution for 3 h, the onion low epidermal explants were pre-cultured in darkness at 27 degrees C for 48 h and then transferred to MS solid media for 72 h. FITC-labeled linear DNA was used to trace the delivery of DNA entry into the cell and the nuclei. By GUS staining and flow-cytometry-mediated fluorescent detection, a significant increase of the ratios of fluorescent nuclei as well as expression of the gus reporter gene was observed by each designed transformation solution. This potent and feasible method showed prospective applications in plant transgenic research. PMID:19255730

  14. Nitric oxide promotes epidermal stem cell migration via cGMP-Rho GTPase signalling

    PubMed Central

    Zhan, Rixing; He, Weifeng; Wang, Fan; Yao, Zhihui; Tan, Jianglin; Xu, Rui; Zhou, Junyi; Wang, Yuzhen; Li, Haisheng; Wu, Jun; LUO, Gaoxing

    2016-01-01

    The migration and reepithelization of epidermal stem cells (ESCs) are the most critical processes in wound healing. The gaseous messenger nitric oxide (NO) has multiple biological effects, but its actions on ESCs are poorly understood. In this study, an NO donor, S-nitroso-N-acetylpenicillamine (SNAP), was found to facilitate the in vitro migration of human ESCs (huESCs) in both live-imaging and scratch models. In addition, pull-down assays demonstrated that SNAP could activate the small GTPases RhoA and Rac1 of the Rho family, but not Cdc42. Moreover, the effects of SNAP on the migration and F-actin polymerization of ESCs could be blocked by inhibitors of cGMP, PKG, RhoA or Rac1, and by a specific siRNA of RhoA or Rac1, but not by a Cdc42 inhibitor or siRNA. Furthermore, the roles of NO in ESC migration via cGMP-Rho GTPase signalling in vivo were confirmed by tracing 5-bromo-2-deoxyuridine (BrdU)-labelled cells in a superficial, partial-thickness scald mouse model. Thus, the present study demonstrated that the NO donor SNAP could promote huESC migration in vitro. Furthermore, NO was found to induce ESC migration via cGMP-Rho GTPase RhoA and Rac1 signalling, but not Cdc42 signalling, both in vivo and in vitro. PMID:27469024

  15. Epidermal stem cells (ESCs) accelerate diabetic wound healing via the Notch signalling pathway

    PubMed Central

    Yang, Rong-Hua; Qi, Shao-Hai; Shu, Bin; Ruan, Shu-Bin; Lin, Ze-Peng; Lin, Yan; Shen, Rui; Zhang, Feng-Gang; Chen, Xiao-Dong; Xie, Ju-Lin

    2016-01-01

    Chronic, non-healing wounds are a major complication of diabetes. Recently, various cell therapies have been reported for promotion of diabetic wound healing. Epidermal stem cells (ESCs) are considered a powerful tool for tissue therapy. However, the effect and the mechanism of the therapeutic properties of ESCs in the diabetic wound healing are unclear. Herein, to determine the ability of ESCs to diabetic wound healing, a dorsal skin defect in a streptozotocin (STZ)-induced diabetes mellitus (DM) mouse model was used. ESCs were isolated from mouse skin. We found that both the mRNA and protein levels of a Notch ligand Jagged1 (Jag1), Notch1 and Notch target gene Hairy Enhancer of Split-1 (Hes1) were significantly increased at the wound margins. In addition, we observed that Jag1 was high expressed in ESCs. Overexpression of Jag1 promotes ESCs migration, whereas knockdown Jag1 resulted in a significant reduction in ESCs migration in vitro. Importantly, Jag1 overexpression improves diabetic wound healing in vivo. These results provide evidence that ESCs accelerate diabetic wound healing via the Notch signalling pathway, and provide a promising potential for activation of the Notch pathway for the treatment of diabetic wound. PMID:27129289

  16. Epidermal stem cells (ESCs) accelerate diabetic wound healing via the Notch signalling pathway.

    PubMed

    Yang, Rong-Hua; Qi, Shao-Hai; Shu, Bin; Ruan, Shu-Bin; Lin, Ze-Peng; Lin, Yan; Shen, Rui; Zhang, Feng-Gang; Chen, Xiao-Dong; Xie, Ju-Lin

    2016-08-01

    Chronic, non-healing wounds are a major complication of diabetes. Recently, various cell therapies have been reported for promotion of diabetic wound healing. Epidermal stem cells (ESCs) are considered a powerful tool for tissue therapy. However, the effect and the mechanism of the therapeutic properties of ESCs in the diabetic wound healing are unclear. Herein, to determine the ability of ESCs to diabetic wound healing, a dorsal skin defect in a streptozotocin (STZ)-induced diabetes mellitus (DM) mouse model was used. ESCs were isolated from mouse skin. We found that both the mRNA and protein levels of a Notch ligand Jagged1 (Jag1), Notch1 and Notch target gene Hairy Enhancer of Split-1 (Hes1) were significantly increased at the wound margins. In addition, we observed that Jag1 was high expressed in ESCs. Overexpression of Jag1 promotes ESCs migration, whereas knockdown Jag1 resulted in a significant reduction in ESCs migration in vitro Importantly, Jag1 overexpression improves diabetic wound healing in vivo These results provide evidence that ESCs accelerate diabetic wound healing via the Notch signalling pathway, and provide a promising potential for activation of the Notch pathway for the treatment of diabetic wound. PMID:27129289

  17. Optical characterization of epidermal cells and their relationship to DNA recovery from touch samples

    PubMed Central

    Stanciu, Cristina E.; Philpott, M. Katherine; Kwon, Ye Jin; Bustamante, Eduardo E.; Ehrhardt, Christopher J.

    2015-01-01

    The goal of this study was to investigate the relative contributions of different cellular and genetic components to biological samples created by touch or contact with a surface – one of the most challenging forms of forensic evidence. Touch samples were generated by having individuals hold an object for five minutes and analyzed for quantity of intact epidermal cells, extracellular DNA, and DNA from pelleted cell material after elution from the collection swab. Comparisons were made between samples where individuals had washed their hands immediately prior to handling and those where hand washing was not controlled. The vast majority (84-100%) of DNA detected in these touch samples was extracellular and was uncorrelated to the number of epidermal cells detected. Although little to no extracellular or cell pellet-associated DNA was detected when individuals washed their hands prior to substrate handling, we found that a significant number of epidermal cells (between ~5x10 3 and ~1x10 5) could still be recovered from these samples, suggesting that other types of biological information may be present even when no amplifiable nuclear DNA is present. These results help to elucidate the biological context for touch samples and characterize factors that may contribute to patterns of transfer and persistence of genetic material in forensic evidence. PMID:26870321

  18. Microenvironmental stiffness enhances glioma cell proliferation by stimulating epidermal growth factor receptor signaling.

    PubMed

    Umesh, Vaibhavi; Rape, Andrew D; Ulrich, Theresa A; Kumar, Sanjay

    2014-01-01

    The aggressive and rapidly lethal brain tumor glioblastoma (GBM) is associated with profound tissue stiffening and genomic lesions in key members of the epidermal growth factor receptor (EGFR) pathway. Previous studies from our laboratory have shown that increasing microenvironmental stiffness in culture can strongly enhance glioma cell behaviors relevant to tumor progression, including proliferation, yet it has remained unclear whether stiffness and EGFR regulate proliferation through common or independent signaling mechanisms. Here we test the hypothesis that microenvironmental stiffness regulates cell cycle progression and proliferation in GBM tumor cells by altering EGFR-dependent signaling. We began by performing an unbiased reverse phase protein array screen, which revealed that stiffness modulates expression and phosphorylation of a broad range of signals relevant to proliferation, including members of the EGFR pathway. We subsequently found that culturing human GBM tumor cells on progressively stiffer culture substrates both dramatically increases proliferation and facilitates passage through the G1/S checkpoint of the cell cycle, consistent with an EGFR-dependent process. Western Blots showed that increasing microenvironmental stiffness enhances the expression and phosphorylation of EGFR and its downstream effector Akt. Pharmacological loss-of-function studies revealed that the stiffness-sensitivity of proliferation is strongly blunted by inhibition of EGFR, Akt, or PI3 kinase. Finally, we observed that stiffness strongly regulates EGFR clustering, with phosphorylated EGFR condensing into vinculin-positive focal adhesions on stiff substrates and dispersing as microenvironmental stiffness falls to physiological levels. Our findings collectively support a model in which tissue stiffening promotes GBM proliferation by spatially and biochemically amplifying EGFR signaling. PMID:25000176

  19. [Transplanted epidermal neural crest stem cell in a peripheral nerve gap].

    PubMed

    Zhang, Lu; Zhang, Jieyuan; Li, Bingcang; Liu, Zheng; Liu, Bin

    2014-04-01

    Neural crest stem cells originated from hair follicle (epidermal neural crest stem cell, EPI-NCSC) are easy to obtain and have potentials to differentiate into various tissues, which make them eminent seed cells for tissue engineering. EPI-NCSC is now used to repair nerve injury, especially, the spinal cord injury. To investigate their effects on repairing peripheral nerve injury, EPI-NCSC from a GFP-SD rat were primarily cultured on coated dishes and on a poly lactic acid coglycolic acid copolymer (PLGA) membrane. Methyl thiazolyl tetrazolium (MTT) assay showed that the initial adhesion rate of EPI-NCSC was 89.7% on PLGA membrane, and the relative growth rates were 89.3%, 87.6%, 85.6%, and 96.6% on the 1st, 3rd, 5th, 7th day respectively. Cell cycles and DNA ploidy analysis demonstrated that cell cycles and proliferation indexes of cultured EPI-NCSC had the same variation pattern on coated dishes and PLGA membrane. Then cultured EPI-NCSC were mixed with equal amount of extracellular matrix and injected into a PLGA conduit to connect a 10 mm surgery excision gap of rat sciatic nerve, Dulbecco's Modified Eagle's medium (DMEM) was used to substitute EPI-NCSC in the control group. After four weeks of transplantation, the defected sciatic nerve achieved a histological restoration, the sensory function of rat hind limb was partly recovered and the sciatic nerve index was also improved. The above results showed that a PLGA conduit filled with EPI-NCSC has a good repair effect on the peripheral nerve injury. PMID:25195250

  20. The influence of stromal cells on the pigmentation of tissue-engineered dermo-epidermal skin grafts.

    PubMed

    Biedermann, Thomas; Böttcher-Haberzeth, Sophie; Klar, Agnieszka S; Widmer, Daniel S; Pontiggia, Luca; Weber, Andreas D; Weber, Daniel M; Schiestl, Clemens; Meuli, Martin; Reichmann, Ernst

    2015-03-01

    It has been shown in vitro that melanocyte proliferation and function in palmoplantar skin is regulated by mesenchymal factors derived from fibroblasts. In this study, we investigated in vivo the influence of mesenchymal-epithelial interactions in human tissue-engineered skin substitutes reconstructed from palmar- and nonpalmoplantar-derived fibroblasts. Tissue-engineered dermo-epidermal analogs based on collagen type I hydrogels were populated with either human palmar or nonpalmoplantar fibroblasts and seeded with human nonpalmoplantar-derived melanocytes and keratinocytes. These skin substitutes were transplanted onto full-thickness skin wounds of immunoincompetent rats. Four weeks after transplantation the development of skin color was measured and grafts were excised and analyzed with regard to epidermal characteristics, in particular melanocyte number and function. Skin substitutes containing palmar-derived fibroblasts in comparison to nonpalmoplantar-derived fibroblasts showed (a) a significantly lighter pigmentation; (b) a reduced amount of epidermal melanin granules; and (c) a distinct melanosome expression. However, the number of melanocytes in the basal layer remained similar in both transplantation groups. These findings demonstrate that human palmar fibroblasts regulate the function of melanocytes in human pigmented dermo-epidermal skin substitutes after transplantation, whereas the number of melanocytes remains constant. This underscores the influence of site-specific stromal cells and their importance when constructing skin substitutes for clinical application. PMID:25300246

  1. The Influence of Stromal Cells on the Pigmentation of Tissue-Engineered Dermo-Epidermal Skin Grafts

    PubMed Central

    Biedermann, Thomas; Böttcher-Haberzeth, Sophie; Klar, Agnieszka S.; Widmer, Daniel S.; Pontiggia, Luca; Weber, Andreas D.; Weber, Daniel M.; Schiestl, Clemens; Meuli, Martin

    2015-01-01

    It has been shown in vitro that melanocyte proliferation and function in palmoplantar skin is regulated by mesenchymal factors derived from fibroblasts. In this study, we investigated in vivo the influence of mesenchymal–epithelial interactions in human tissue-engineered skin substitutes reconstructed from palmar- and nonpalmoplantar-derived fibroblasts. Tissue-engineered dermo-epidermal analogs based on collagen type I hydrogels were populated with either human palmar or nonpalmoplantar fibroblasts and seeded with human nonpalmoplantar-derived melanocytes and keratinocytes. These skin substitutes were transplanted onto full-thickness skin wounds of immunoincompetent rats. Four weeks after transplantation the development of skin color was measured and grafts were excised and analyzed with regard to epidermal characteristics, in particular melanocyte number and function. Skin substitutes containing palmar-derived fibroblasts in comparison to nonpalmoplantar-derived fibroblasts showed (a) a significantly lighter pigmentation; (b) a reduced amount of epidermal melanin granules; and (c) a distinct melanosome expression. However, the number of melanocytes in the basal layer remained similar in both transplantation groups. These findings demonstrate that human palmar fibroblasts regulate the function of melanocytes in human pigmented dermo-epidermal skin substitutes after transplantation, whereas the number of melanocytes remains constant. This underscores the influence of site-specific stromal cells and their importance when constructing skin substitutes for clinical application. PMID:25300246

  2. Attenuation fluctuations and local dermal reflectivity are indicators of immune cell infiltrate and epidermal hyperplasia in skin inflammation

    NASA Astrophysics Data System (ADS)

    Phillips, Kevin G.; Wang, Yun; Choudhury, Niloy; Levitz, David; Swanzey, Emily; Lagowski, James; Kulesz-Martin, Molly; Jacques, Steven

    2012-02-01

    Psoriasis is a common inflammatory skin disease resulting from genetic and environmental alterations of cutaneous immune responses responsible for skin homeostasis. While numerous therapeutic targets involved in the immunopathogenesis of psoriasis have been identified, the in vivo dynamics of psoriasis remains under investigated. To elucidate the spatial-temporal morphological evolution of psoriasis we undertook in vivo time course focus-tracked optical coherence tomography (OCT) imaging to non-invasively document dermal alterations due to immune cell infiltration and epidermal hyperplasia in an Imiquimod (IMQ) induced model of psoriasis-like inflammation in DBA2/C57Bl6 hybrid mice. Quantitative appraisal of dermal architectural changes was achieved through a three parameter fit of OCT axial scans in the dermis of the form A(z) = ρ exp(-mu;z +ɛ(z)). Ensemble averaging of the fit parameters over 2000 axial scans per mouse in each treatment arm revealed that the local dermal reflectivity ρ, decreased significantly in response to 6 day IMQ treatment (p = 0.0001), as did the standard deviation of the attenuation fluctuation std(ɛ(z)), (p = 0.04), in comparison to cream controls and day 1 treatments. No significant changes were observed in the average dermal attenuation rate, μ. Our results suggest these label-free OCT-based metrics can be deployed to investigate new therapeutic targets in animal models as well as aid in clinical staging of psoriasis in conjunction with the psoriasis area and severity index.

  3. Cellular processes involved in human epidermal cells exposed to extremely low frequency electric fields.

    PubMed

    Collard, J-F; Hinsenkamp, M

    2015-05-01

    We observed on different tissues and organisms a biological response after exposure to pulsed low frequency and low amplitude electric or electromagnetic fields but the precise mechanism of cell response remains unknown. The aim of this publication is to understand, using bioinformatics, the biological relevance of processes involved in the modification of gene expression. The list of genes analyzed was obtained after microarray protocol realized on cultures of human epidermal explants growing on deepidermized human skin exposed to a pulsed low frequency electric field. The directed acyclic graph on a WebGestalt Gene Ontology module shows six categories under the biological process root: "biological regulation", "cellular process", "cell proliferation", "death", "metabolic process" and "response to stimulus". Enriched derived categories are coherent with the type of in vitro culture, the stimulation protocol or with the previous results showing a decrease of cell proliferation and an increase of differentiation. The Kegg module on WebGestalt has highlighted "cell cycle" and "p53 signaling pathway" as significantly involved. The Kegg website brings out interactions between FoxO, MAPK, JNK, p53, p38, PI3K/Akt, Wnt, mTor or NF-KappaB. Some genes expressed by the stimulation are known to have an exclusive function on these pathways. Analyses performed with Pathway Studio linked cell proliferation, cell differentiation, apoptosis, cell cycle, mitosis, cell death etc. with our microarrays results. Medline citation generated by the software and the fold change variation confirms a diminution of the proliferation, activation of the differentiation and a less well-defined role of apoptosis or wound healing. Wnt and DKK functional classes, DKK1, MACF1, ATF3, MME, TXNRD1, and BMP-2 genes proposed in previous publications after a manual analysis are also highlighted with other genes after Pathway Studio automatic procedure. Finally, an analysis conducted on a list of genes

  4. Regulation of epidermal cell interleukin-6 production by UV light and corticosteroids

    SciTech Connect

    Kirnbauer, R.; Koeck, A.N.; Neuner, P.; Foerster, E.K.; Krutmann, J.; Urbanski, A.; Schauer, E.; Ansel, J.C.; Schwarz, T.; Luger, T.A. )

    1991-04-01

    Epidermal cells (EC) are well known as a source of cytokines, including interleukin (IL)-6. In the present study, we investigated whether ultraviolet (UV) light and corticosteroids (CS) affect IL-6 production by normal (HNK) or malignant (KB) human keratinocytes. Supernatants derived from UVB (100 J/m2)- but not from UVA (100-1500 kJ/m2)-exposed EC (HNK and KB) contained significantly increased levels of IL-6 activity. This was also confirmed by Western blot analysis, resulting in specific bands at 23 kD and 27 kD. Northern blot analysis revealed an enhanced IL-6 mRNA expression after UVB exposure. Addition of hydrocortisone, prednisolone, or dexamethasone immediately after UVB irradiation significantly blocked UVB or IL-1-induced IL-6 mRNA expression and production by EC. The suppressive effect was observed at doses in the physiologic (10(-7)-10(-9) M) as well as pharmacologic (10(-5)-10(-7) M) range. In contrast, the nonactive steroid prednisone did not affect EC IL-6 mRNA expression. These findings indicate that increased IL-6 production by EC after UVB irradiation may mediate local and systemic inflammatory reactions following extensive sun exposure. Thus, the therapeutic effect of corticosteroids observed in various inflammatory diseases may be partly due to their downregulating capacity of IL-6 production.

  5. Crosstalk between Src and major vault protein in epidermal growth factor-dependent cell signalling.

    PubMed

    Kim, Euikyung; Lee, Seunghwan; Mian, Md Firoz; Yun, Sang Uk; Song, Minseok; Yi, Kye-Sook; Ryu, Sung Ho; Suh, Pann-Ghill

    2006-02-01

    Vaults are highly conserved, ubiquitous ribonucleoprotein (RNP) particles with an unidentified function. For the three protein species (TEP1, VPARP, and MVP) and a small RNA that comprises vault, expression of the unique 100-kDa major vault protein (MVP) is sufficient to form the basic vault structure. To identify and characterize proteins that interact with the Src homology 2 (SH2) domain of Src and potentially regulate Src activity, we used a pull-down assay using GST-Src-SH2 fusion proteins. We found MVP as a Src-SH2 binding protein in human stomach tissue. Interaction of Src and MVP was also observed in 253J stomach cancer cells. A subcellular localization study using immunofluorescence microscopy shows that epidermal growth factor (EGF) stimulation triggers MVP translocation from the nucleus to the cytosol and perinuclear region where it colocalizes with Src. We found that the interaction between Src and MVP is critically dependent on Src activity and protein (MVP) tyrosyl phosphorylation, which are induced by EGF stimulation. Our results also indicate MVP to be a novel substrate of Src and phosphorylated in an EGF-dependent manner. Interestingly, purified MVP inhibited the in vitro tyrosine kinase activity of Src in a concentration-dependent manner. MVP overexpression downregulates EGF-dependent ERK activation in Src overexpressing cells. To our knowledge, this is the first report of MVP interacting with a protein tyrosine kinase involved in a distinct cell signalling pathway. It appears that MVP is a novel regulator of Src-mediated signalling cascades. PMID:16441665

  6. Multiple requirements for SHPTP2 in epidermal growth factor-mediated cell cycle progression.

    PubMed Central

    Bennett, A M; Hausdorff, S F; O'Reilly, A M; Freeman, R M; Neel, B G

    1996-01-01

    Using transient overexpression and microinjection approaches, we examined SHPTP2's function in growth factor signaling. Overexpression of catalytically inactive SHPTP2 (PTP2CS) but not catalytically inactive SHPTP1, inhibited mitogen-activated protein (MAP) kinase activation and Elk-1 transactivation following epidermal growth factor (EGF) stimulation of 293 cells. An SHPTP2 mutant with both C-terminal tyrosyl phosphorylation sites converted to phenylalanine (PTP2YF) was also without effect; moreover, PTP2YF rescued PTP2CS-induced inhibition of EGF-induced Elk-1 transactivation. PTP2CS did not inhibit transactivation by activated Ras, suggesting that SHPTP2 acts upstream of or parallel to Ras. Neither PTP2CS nor PTP2YF inhibited platelet-derived growth factor (PDGF)-induced Elk-1 transactivation. Thus, protein-tyrosine phosphatase activity, but not tyrosyl phosphorylation of SHPTP2, is required for the immediate-early responses to EGF but not to PDGF. To determine whether SHPTP2 is required later in the cell cycle, we assessed S-phase entry in NIH 3T3 cells microinjected with anti-SHPTP2 antibodies or with a glutathione S-transferase (GST) fusion protein encoding both SH2 domains (GST-SH2). Microinjection of anti-SHPTP2 antibodies prior to stimulation inhibited EGF- but no PDGF- or serum-induced S-phase entry. Anti-SHPTP2 antibodies or GST-SH2 fusion protein could inhibit EGF-induced S-phase entry for up to 8 h after EGF addition. Although MAP kinase activation was detected shortly after EGF stimulation, no MAP kinase activation was detected around the restriction point. Therefore, SHPTP2 is absolutely required for immediate-early and late events induced by some, but not all, growth factors, and the immediate-early and late signal transduction pathways regulated by SHPTP2 are distinguishable. PMID:8622663

  7. Lysine-specific demethylase 1 mediates epidermal growth factor signaling to promote cell migration in ovarian cancer cells.

    PubMed

    Shao, Genbao; Wang, Jie; Li, Yuanxia; Liu, Xiuwen; Xie, Xiaodong; Wan, Xiaolei; Yan, Meina; Jin, Jie; Lin, Qiong; Zhu, Haitao; Zhang, Liuping; Gong, Aihua; Shao, Qixiang; Wu, Chaoyang

    2015-01-01

    Epigenetic abnormalities play a vital role in the progression of ovarian cancer. Lysine-specific demethylase 1 (LSD1/KDM1A) acts as an epigenetic regulator and is overexpressed in ovarian tumors. However, the upstream regulator of LSD1 expression in this cancer remains elusive. Here, we show that epidermal growth factor (EGF) signaling upregulates LSD1 protein levels in SKOV3 and HO8910 ovarian cancer cells overexpressing both LSD1 and the EGF receptor. This effect is correlated with a decrease in the dimethylation of H3K4, a major substrate of LSD1, in an LSD1-dependent manner. We also show that inhibition of PI3K/AKT, but not MEK, abolishes the EGF-induced upregulation of LSD1 and cell migration, indicating that the PI3K/PDK1/AKT pathway mediates the EGF-induced expression of LSD1 and cell migration. Significantly, LSD1 knockdown or inhibition of LSD1 activity impairs both intrinsic and EGF-induced cell migration in SKOV3 and HO8910 cells. These results highlight a novel mechanism regulating LSD1 expression and identify LSD1 as a promising therapeutic target for treating metastatic ovarian cancer driven by EGF signaling. PMID:26489763

  8. Hdac1 and Hdac2 act redundantly to control p63 and p53 functions in epidermal progenitor cells

    PubMed Central

    LeBoeuf, Matthew; Terrell, Anne; Trivedi, Sohum; Sinha, Satrajit; Epstein, Jonathan A.; Olson, Eric N.; Morrisey, Edward E.; Millar, Sarah E.

    2010-01-01

    Summary Epidermal and hair follicle development from surface ectodermal progenitor cells require coordinated changes in gene expression. Histone deacetylases alter gene expression programs through modification of chromatin and transcription factors. We find that deletion of ectodermal Hdac1 and Hdac2 results in dramatic failure of hair follicle specification and epidermal proliferation and stratification, phenocopying loss of the key ectodermal transcription factor p63. While expression of p63 and its positively regulated basal cell targets is maintained in Hdac1/2 deficient ectoderm, targets of p63-mediated repression, including p21, 14-3-3σ and p16/INK4a, are ectopically expressed, and HDACs bind and are active at their promoter regions in normal undifferentiated keratinocytes. Mutant embryos display increased levels of acetylated p53, which opposes p63 functions, and p53 is required for HDAC inhibitor-mediated p21 expression in keratinocytes. Our data identify critical requirements for HDAC1/2 in epidermal development, and indicate that HDAC1/2 directly mediate repressive functions of p63, and suppress p53 activity. PMID:21093383

  9. Althaea rosea Cavanil and Plantago major L. suppress neoplastic cell transformation through the inhibition of epidermal growth factor receptor kinase.

    PubMed

    Choi, Eun-Sun; Cho, Sung-Dae; Shin, Ji-Ae; Kwon, Ki Han; Cho, Nam-Pyo; Shim, Jung-Hyun

    2012-10-01

    For thousands of years in Asia, Althaea rosea Cavanil (ARC) and Plantago major L. (PML) have been used as powerful non-toxic therapeutic agents that inhibit inflammation. However, the anticancer mechanisms and molecular targets of ARC and PML are poorly understood, particularly in epidermal growth factor (EGF)-induced neoplastic cell transformation. The aim of this study was to evaluate the chemopreventive effects and mechanisms of the methanol extracts from ARC (MARC) and PML (MPML) in EGF-induced neoplastic cell transformation of JB6 P+ mouse epidermal cells using an MTS assay, anchorage-independent cell transformation assay and western blotting. Our results showed that MARC and MPML significantly suppressed neoplastic cell transformation by inhibiting the kinase activity of the EGF receptor (EGFR). The activation of EGFR by EGF was suppressed by MARC and MPML treatment in EGFR(+/+) cells, but not in EGFR(-/-) cells. In addition, MARC and MPML inhibited EGF-induced cell proliferation in EGFR-expressing murine embryonic fibroblasts (EGFR(+/+)). These results strongly indicate that EGFR targeting by MARC and MPML may be a good strategy for chemopreventive or chemotherapeutic applications. PMID:22767187

  10. Ultraviolet Radiation-Induced Skin Aging: The Role of DNA Damage and Oxidative Stress in Epidermal Stem Cell Damage Mediated Skin Aging

    PubMed Central

    Panich, Uraiwan; Sittithumcharee, Gunya; Rathviboon, Natwarath

    2016-01-01

    Skin is the largest human organ. Skin continually reconstructs itself to ensure its viability, integrity, and ability to provide protection for the body. Some areas of skin are continuously exposed to a variety of environmental stressors that can inflict direct and indirect damage to skin cell DNA. Skin homeostasis is maintained by mesenchymal stem cells in inner layer dermis and epidermal stem cells (ESCs) in the outer layer epidermis. Reduction of skin stem cell number and function has been linked to impaired skin homeostasis (e.g., skin premature aging and skin cancers). Skin stem cells, with self-renewal capability and multipotency, are frequently affected by environment. Ultraviolet radiation (UVR), a major cause of stem cell DNA damage, can contribute to depletion of stem cells (ESCs and mesenchymal stem cells) and damage of stem cell niche, eventually leading to photoinduced skin aging. In this review, we discuss the role of UV-induced DNA damage and oxidative stress in the skin stem cell aging in order to gain insights into the pathogenesis and develop a way to reduce photoaging of skin cells. PMID:27148370

  11. Stomatal Spacing Safeguards Stomatal Dynamics by Facilitating Guard Cell Ion Transport Independent of the Epidermal Solute Reservoir.

    PubMed

    Papanatsiou, Maria; Amtmann, Anna; Blatt, Michael R

    2016-09-01

    Stomata enable gaseous exchange between the interior of the leaf and the atmosphere through the stomatal pore. Control of the pore aperture depends on osmotic solute accumulation by, and its loss from the guard cells surrounding the pore. Stomata in most plants are separated by at least one epidermal cell, and this spacing is thought to enhance stomatal function, although there are several genera that exhibit stomata in clusters. We made use of Arabidopsis (Arabidopsis thaliana) stomatal patterning mutants to explore the impact of clustering on guard cell dynamics, gas exchange, and ion transport of guard cells. These studies showed that stomatal clustering in the Arabidopsis too many mouths (tmm1) mutant suppressed stomatal movements and affected CO2 assimilation and transpiration differentially between dark and light conditions and were associated with alterations in K(+) channel gating. These changes were consistent with the impaired dynamics of tmm1 stomata and were accompanied by a reduced accumulation of K(+) ions in the guard cells. Our findings underline the significance of spacing for stomatal dynamics. While stomatal spacing may be important as a reservoir for K(+) and other ions to facilitate stomatal movements, the effects on channel gating, and by inference on K(+) accumulation, cannot be explained on the basis of a reduced number of epidermal cells facilitating ion supply to the guard cells. PMID:27406168

  12. Cell Density-Dependent Upregulation of PDCD4 in Keratinocytes and Its Implications for Epidermal Homeostasis and Repair

    PubMed Central

    Wang, Tao; Long, Shuang; Zhao, Na; Wang, Yu; Sun, Huiqin; Zou, Zhongmin; Wang, Junping; Ran, Xinze; Su, Yongping

    2015-01-01

    Programmed cell death 4 (PDCD4) is one multi-functional tumor suppressor inhibiting neoplastic transformation and tumor invasion. The role of PDCD4 in tumorigenesis has attracted more attention and has been systematically elucidated in cutaneous tumors. However, the normal biological function of PDCD4 in skin is still unclear. In this study, for the first time, we find that tumor suppressor PDCD4 is uniquely induced in a cell density-dependent manner in keratinocytes. To determine the potential role of PDCD4 in keratinocyte cell biology, we show that knockdown of PDCD4 by siRNAs can promote cell proliferation in lower cell density and partially impair contact inhibition in confluent HaCaT cells, indicating that PDCD4 serves as an important regulator of keratinocytes proliferation and contact inhibition in vitro. Further, knockdown of PDCD4 can induce upregulation of cyclin D1, one key regulator of the cell cycle. Furthermore, the expression patterns of PDCD4 in normal skin, different hair cycles and the process of wound healing are described in detail in vivo, which suggest a steady-state regulatory role of PDCD4 in epidermal homeostasis and wound healing. These findings provide a novel molecular mechanism for keratinocytes’ biology and indicate that PDCD4 plays a role in epidermal homeostasis. PMID:26703592

  13. Epidermal growth factor-like domain 7 promotes cell invasion and angiogenesis in pancreatic carcinoma.

    PubMed

    Shen, Xiaochun; Han, Ye; Xue, Xiaofeng; Li, Wei; Guo, Xiaobo; Li, Pu; Wang, Yunliang; Li, Dechun; Zhou, Jin; Zhi, Qiaoming

    2016-02-01

    Epidermal growth factor-like domain 7 (EGFL7), also known as vascular endothelial stain, was firstly identified as a modulator of smooth muscle cell migration. Though the expression of EGFL7 was reported to be up-regulated during tumorigenesis, the clinical and biological functions of EGFL7 in pancreatic carcinoma (PC) were still not fully elucidated. In this study, we found that the serum EGFL7 level in PC tissues was statistically higher than that in normal subjects (p<0.001), and its level in non-resectable patients was also higher than that in resectable ones (p=0.013). Among these resectable PC patients, the postoperative EGFL7 expression was significantly down-regulated when tumors were resected (p=0.018). Using the immunohistochemistry method, our results demonstrated that the positive expression of EGFL7 was significantly associated with the TNM stage (p=0.024), lymph node metastasis (p=0.003) and local invasion (p=0.022), and the EGFL7 expression closely correlated to the micro-vessel density (MVD) in PC tissues by Spearman analysis (r=0.941, p=0.000). In vitro, EGFL7 was silenced by the small interference RNA in PC cells, and our data indicated that down-regulation of EGFL7 did not influence the cycle progression, proliferation, colony formation and apoptosis of PC cells (p>0.05), whereas inhibition of EGFL7 expression could decrease PaCa-2 cell invasion (p<0.05). More interestingly, by tubular formation, Chick embryo chorioallantoic membrane (CAM) and ELISA assays, our results revealed that silencing EGFL7 expression represented a strong inhibiting effect on tubular formation of micro-vessels through down-regulating the protein levels of VEGF and Ang-2 (p<0.05). Our results raised the possibility of using EGFL7as a potential prognostic biomarker and therapy target of PC, and down-regulation of EGFL7 might be considered to be a potentially important molecular treatment strategy for patients with PC. PMID:26796281

  14. Reconstruction of damaged cornea by autologous transplantation of epidermal adult stem cells

    PubMed Central

    Yang, Xueyi; Moldovan, Nicanor I.; Zhao, Qingmei; Mi, Shengli; Zhou, Zhenhui; Chen, Dan; Gao, Zhimin; Tong, Dewen

    2008-01-01

    Purpose It is crucial for the treatment of severe ocular surface diseases such as Stevens-Johnson syndrome (SJS) and ocular cicatricial pemphigoid (OCP) to find strategies that avoid the risks of allograft rejection and immunosuppression. Here, we report a new strategy for reconstructing the damaged corneal surface in a goat model of total limbal stem cell deficiency (LSCD) by autologous transplantation of epidermal adult stem cells (EpiASC). Methods EpiASC derived from adult goat ear skin by explant culture were purified by selecting single cell-derived clones. These EpiASC were cultivated on denuded human amniotic membrane (HAM) and transplanted onto goat eyes with total LSCD. The characteristics of both EpiASC and reconstructed corneal epithelium were identified by histology and immunohistochemistry. The clinical characteristic of reconstructed corneal surface was observed by digital camera. Results Ten LSCD goats (10 eyes) were treated with EpiASC transplantation, leading to the restoration of corneal transparency and improvement of postoperative visual acuity to varying degrees in 80.00% (8/10) of the experimental eyes. The corneal epithelium of control groups either with HAM transplantation only or without any transplantation showed irregular surfaces, diffuse vascularization, and pannus on the entire cornea. The reconstructed corneal epithelium (RCE) expressed CK3, CK12, and PAX-6 and had the function of secreting glycocalyx-like material (AB-PAS positive). During the follow-up period, all corneal surfaces remained transparent and there were no serious complications. We also observed that the REC expressed CK1/10 weakly at six months after operation but not at 12 months after operation, suggesting that the REC was derived from grafted EpiASC. Conclusions Our results showed that EpiASC repaired the damaged cornea of goats with total LSCD and demonstrated that EpiASC can be induced to differentiate into corneal epithelial cell types in vivo, which at least in

  15. Epidermal growth factor receptor is required for estradiol-stimulated bovine satellite cell proliferation.

    PubMed

    Reiter, B C; Kamanga-Sollo, E; Pampusch, M S; White, M E; Dayton, W R

    2014-07-01

    The objective of this study was to assess the role of the epidermal growth factor receptor (EGFR) in estradiol-17β (E2)-stimulated proliferation of cultured bovine satellite cells (BSCs). Treatment of BSC cultures with AG1478 (a specific inhibitor of EGFR tyrosine kinase activity) suppresses E2-stimulated BSC proliferation (P < 0.05). In addition, E2-stimulated proliferation is completely suppressed (P < 0.05) in BSCs in which EGFR expression is silenced by treatment with EGFR small interfering RNA (siRNA). These results indicate that EGFR is required for E2 to stimulate proliferation in BSC cultures. Both AG1478 treatment and EGFR silencing also suppress proliferation stimulated by LR3-IGF-1 (an IGF1 analogue that binds normally to the insulin-like growth factor receptor (IGFR)-1 but has little or no affinity for IGF binding proteins) in cultured BSCs (P < 0.05). Even though EGFR siRNA treatment has no effect on IGFR-1β mRNA expression in cultured BSCs, IGFR-1β protein level is substantially reduced in BSCs treated with EGFR siRNA. These data suggest that EGFR silencing results in post-transcriptional modifications that result in decreased IGFR-1β protein levels. Although it is clear that functional EGFR is necessary for E2-stimulated proliferation of BSCs, the role of EGFR is not clear. Transactivation of EGFR may directly stimulate proliferation, or EGFR may function to maintain the level of IGFR-1β which is necessary for E2-stimulated proliferation. It also is possible that the role of EGFR in E2-stimulated BSC proliferation may involve both of these mechanisms. PMID:24906928

  16. AZD9291 in epidermal growth factor receptor inhibitor—resistant non-small-cell lung cancer

    PubMed Central

    2016-01-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in advanced EGFR mutant non-small cell lung cancer have an objective response rate (ORR) of approximately 60–70% and a median progression free-survival (PFS) of approximately 10-13 months. Studies of tumor biopsies performed after progression on EGFR TKI revealed that 50-60% of EGFR mutant NSCLC developed an EGFR exon 20 T790M mutation as a mechanism of acquired resistance. AZD9291 is a third generation irreversible EGFR TKI with activity against the activating EGFR mutation, the T790M acquired resistance mutation, and relative sparing of the wild-type EGFR. AZD9291 was investigated in a phase I trial with expansion cohorts in patients with disease progression after EGFR TKI. Patients with and without detectable T790M mutations were enrolled in the trial. The ORR in patients with centrally confirmed and without detectable T790M mutations was 61% (95% CI, 52–70%) and 21% (95% CI, 12–34%), respectively. The PFS observed in patients with centrally confirmed and without detectable T790M mutations was 9.6 months (95% CI, 8.3 to not reached) and 2.8 months (95% CI, 2.1–4.3 months), respectively. At the dose for further investigation, 80 mg daily, the rate of all grade 3-5 drug related adverse events was 11%, and the rates of grade 3 diarrhea and rash were 1% and 0%, respectively. The identification of the T790M resistance mutation and the subsequent development of an agent against the mechanism of resistance provide a template for future drug development for acquired resistance to targeted therapy. PMID:26958499

  17. AZD9291 in epidermal growth factor receptor inhibitor-resistant non-small-cell lung cancer.

    PubMed

    Stinchcombe, Thomas E

    2016-02-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in advanced EGFR mutant non-small cell lung cancer have an objective response rate (ORR) of approximately 60-70% and a median progression free-survival (PFS) of approximately 10-13 months. Studies of tumor biopsies performed after progression on EGFR TKI revealed that 50-60% of EGFR mutant NSCLC developed an EGFR exon 20 T790M mutation as a mechanism of acquired resistance. AZD9291 is a third generation irreversible EGFR TKI with activity against the activating EGFR mutation, the T790M acquired resistance mutation, and relative sparing of the wild-type EGFR. AZD9291 was investigated in a phase I trial with expansion cohorts in patients with disease progression after EGFR TKI. Patients with and without detectable T790M mutations were enrolled in the trial. The ORR in patients with centrally confirmed and without detectable T790M mutations was 61% (95% CI, 52-70%) and 21% (95% CI, 12-34%), respectively. The PFS observed in patients with centrally confirmed and without detectable T790M mutations was 9.6 months (95% CI, 8.3 to not reached) and 2.8 months (95% CI, 2.1-4.3 months), respectively. At the dose for further investigation, 80 mg daily, the rate of all grade 3-5 drug related adverse events was 11%, and the rates of grade 3 diarrhea and rash were 1% and 0%, respectively. The identification of the T790M resistance mutation and the subsequent development of an agent against the mechanism of resistance provide a template for future drug development for acquired resistance to targeted therapy. PMID:26958499

  18. The regeneration of epidermal cells of Saintpaulia leaves as a new plant-tissue system for cellular radiation biology.

    PubMed

    Engels, F M; van der Laan, F M; Leenhouts, H P; Chadwick, K H

    1980-09-01

    Investigation of the nucleus of epidermal cells of the petioles of Saintpaulia leaves by cytofluorimetry revealed that all cells are in a non-cycling pre DNA synthesis phase. Cultivation of dissected leaves results in a synchronous regeneration process of a defined number of cells. Five days after onset of cultivation the cells reach the first mitosis. The nuclear development during the regeneration process is described. Irradiation of the leaves results in a directly visible inhibition of this regenerating capability which is used to quantify cell survival in a tissue. The data show that the radiation response has a similar shape to that of the survival of single cells in culture. This response can be observed before the first mitosis of the cells and its application as a new plant tissue system for cellular radiation research is discussed. PMID:7012060

  19. [The effect of salicylic acid on epidermal cell proliferation kinetics in psoriasis. Autoradiographic in vitro-investigations(author's transl)].

    PubMed

    Pullmann, H; Lennartz, K J; Steigleder, G K

    1975-01-01

    Salicylic acid in the therapeutic concentrations from 0.5 to 10% does not affect the rate of proliferation of psoriatic epidermal cells. In 18 patients suffering from psoriasis the H3-I (H3-thymidine labelling index) was determined using autoradiographic in vitro labelling techniques. In 12 of these patients double-labelling with C14-and H3-thymidine was used to determine the H3-I, the DNA-synthesis time (ts) and the duration of the cell-cycle (tc). No significant changes were observed following external application of salicyclic acid in white Vaseline in concentrations of 0.5, 2 and 10% for one week. PMID:1119844

  20. Effects of immunomodulatory drugs on TNF-α and IL-12 production by purified epidermal langerhans cells and peritoneal macrophages

    PubMed Central

    2011-01-01

    Background Langerhans cells constitute a special subset of immature dendritic cells localized in the epidermis that play a key role in the skin's immune response. The production of cytokines is a key event in both the initiation and the regulation of immune responses, and different drugs can be used to remove or modify their production by DC and, therefore, alter immune responses in a broad spectrum of diseases, mainly in human inflammatory and autoimmune diseases. In the present study, we examined the effects of prednisone, thalidomide, cyclosporine A, and amitriptyline, drugs used in a variety of clinical conditions, on the production of TNF-α, IL-10, and IL-12 by purified epidermal Langerhans cells and peritoneal macrophages in BALB/c mice. Findings All drugs inhibited TNF-α production by Langerhans cells after 36 hours of treatment at two different concentrations, while prednisone and thalidomide decreased IL-12 secretion significantly, amitriptyline caused a less pronounced reduction and cyclosporine A had no effect. Additionally, TNF-α and IL-12 production by macrophages decreased, but IL-10 levels were unchanged after all treatments. Conclusions Our results demonstrate that these drugs modulate the immune response by regulating pro-inflammatory cytokine production by purified epidermal Langerhans cells and peritoneal macrophages, indicating that these cells are important targets for immunosuppression in various clinical settings. PMID:21276247

  1. Label-free and dynamic evaluation of cell-surface epidermal growth factor receptor expression via an electrochemiluminescence cytosensor.

    PubMed

    Qiu, Youyi; Wen, Qingqing; Zhang, Lin; Yang, Peihui

    2016-04-01

    A label-free electrochemiluminescence (ECL) cytosensor was developed for dynamically evaluating of epidermal growth factor receptor (EGFR) expression on MCF-7 cancer cells based on the specific recognition of epidermal growth factor (EGF) with its receptor (EGFR). EGF-cytosensor was fabricated by in-situ electro-polymerization of polyaniline as substrate, using CdS quantum dots (CdS QDs) as ECL probe and gold nanoparticles (AuNPs) as a carrier for loading of EGF. AuNPs and CdS QDs were jointly attached on polyaniline surface to provide a sensitive and stable sensing interface, as well as a simple and label-free mode for ECL assay. Electron microscopy, atomic force microscopy (AFM) and electrochemical methods were employed to characterize the multilayer construction process of the sensing interface. The proposed EGF-cytosensor exhibited excellent analytical performance for MCF-7 cancer cells, ranging from 12 to 1.2 × 10(6) cells mL(-1), with a low detection limit of 12 cells mL(-1). Also, it was successfully applied in evaluating EGFR expression of cells surface, which was stimulated by some inhibitors or activator, and the results were confirmed by using flow cytometry and laser scanning confocal microscopy analysis. The proposed ECL cytosensor has potential applications in monitoring the dynamic variation of receptor molecules expression on cell surfaces in response to external stimulation by drugs and screening anti-cancer therapeutic agents. PMID:26838410

  2. BAG-1 enhances cell-cell adhesion, reduces proliferation and induces chaperone-independent suppression of hepatocyte growth factor-induced epidermal keratinocyte migration

    SciTech Connect

    Hinitt, C.A.M.; Wood, J.; Lee, S.S.; Williams, A.C.; Howarth, J.L.; Glover, C.P.; Uney, J.B.; Hague, A.

    2010-08-01

    Cell motility is important in maintaining tissue homeostasis, facilitating epithelial wound repair and in tumour formation and progression. The aim of this study was to determine whether BAG-1 isoforms regulate epidermal cell migration in in vitro models of wound healing. In the human epidermal cell line HaCaT, endogenous BAG-1 is primarily nuclear and increases with confluence. Both transient and stable p36-Bag-1 overexpression resulted in increased cellular cohesion. Stable transfection of either of the three human BAG-1 isoforms p36-Bag-1 (BAG-1S), p46-Bag-1 (BAG-1M) and p50-Bag-1 (BAG-1L) inhibited growth and wound closure in serum-containing medium. However, in response to hepatocyte growth factor (HGF) in serum-free medium, BAG-1S/M reduced communal motility and colony scattering, but BAG-1L did not. In the presence of HGF, p36-Bag-1 transfectants retained proliferative response to HGF with no change in ERK1/2 activation. However, the cells retained E-cadherin localisation at cell-cell junctions and exhibited pronounced cortical actin. Point mutations in the BAG domain showed that BAG-1 inhibition of motility is independent of its function as a chaperone regulator. These findings are the first to suggest that BAG-1 plays a role in regulating cell-cell adhesion and suggest an important function in epidermal cohesion.

  3. RNA interference for epidermal growth factor receptor enhances the radiosensitivity of esophageal squamous cell carcinoma cell line Eca109

    PubMed Central

    ZHANG, HEPING; LI, JIANCHENG; CHENG, WENFANG; LIU, DI; CHEN, CHENG; WANG, XIAOYING; LU, XUJING; ZHOU, XIFA

    2015-01-01

    The present study investigated the effects of small interfering RNAs (siRNAs) specific to the epidermal growth factor receptor (EGFR) gene, on the radiosensitivity of esophageal squamous cell carcinoma cells. EGFR gene siRNAs (EGFR-siRNA) were introduced into esophageal cancer Eca109 cells using Lipofectamine® 2000. The EGFR messenger (m)RNA expression levels, EGFR protein expression and cell growth were assessed using reverse transcription-polymerase chain reaction analysis, western blot analysis and a Cell Counting Kit-8 (CCK-8), respectively. In addition, colony assays were used to determine the inhibitory effects of X-ray radiation on EGFR-silenced cells. EGFR mRNA and protein levels were reduced in the Eca109 cells transfected with EGFR-siRNA. The relative EGFR mRNA expression levels were reduced to 26.74, 9.52 and 4.61% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These mRNA levels were significantly reduced compared with the those of the control group (42.44%; P<0.0001). Transfection with siRNA3 resulted in the greatest reduction in EGFR mRNA expression, with an inhibition rate of 85%. The relative EGFR protein expression levels were reduced to 24.05, 34.91 and 34.14% in Eca109 cells transfected with EGFR-siRNA1, 2 and 3, respectively. These protein levels were significantly reduced compared with those of the control group (78.57%; P<0.0001). Transfection with siRNA1 resulted in the greatest reduction in EGFR protein expression, with an inhibition rate of 72.84%. This reduction in EGFR expression inhibited the proliferation of Eca109 cells, which was identified using the CCK-8 assay. The proliferation inhibition ratio was 28.2%. The cells treated with irradiation in addition to EGFR-siRNA, demonstrated reduced radiobiological parameters (D0, Dq and SF2) compared with those of cells treated with irradiation only, with a sensitization enhancing ratio of 1.5. In conclusion, suppression of EGFR expression may enhance the radiosensitivity

  4. Characterization of a Putative Receptor Binding Surface on Skint-1, a Critical Determinant of Dendritic Epidermal T Cell Selection*

    PubMed Central

    Salim, Mahboob; Knowles, Timothy J.; Hart, Rosie; Mohammed, Fiyaz; Woodward, Martin J.; Willcox, Carrie R.; Overduin, Michael; Hayday, Adrian C.; Willcox, Benjamin E.

    2016-01-01

    Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection. PMID:26917727

  5. Characterization of a Putative Receptor Binding Surface on Skint-1, a Critical Determinant of Dendritic Epidermal T Cell Selection.

    PubMed

    Salim, Mahboob; Knowles, Timothy J; Hart, Rosie; Mohammed, Fiyaz; Woodward, Martin J; Willcox, Carrie R; Overduin, Michael; Hayday, Adrian C; Willcox, Benjamin E

    2016-04-22

    Dendritic epidermal T cells (DETC) form a skin-resident γδ T cell population that makes key contributions to cutaneous immune stress surveillance, including non-redundant contributions to protection from cutaneous carcinogens. How DETC become uniquely associated with the epidermis was in large part solved by the identification of Skint-1, the prototypic member of a novel B7-related multigene family. Expressed only by thymic epithelial cells and epidermal keratinocytes, Skint-1 drives specifically the development of DETC progenitors, making it the first clear candidate for a selecting ligand for non-MHC/CD1-restricted T cells. However, the molecular mechanisms underpinning Skint-1 activity are unresolved. Here, we provide evidence that DETC selection requires Skint-1 expression on the surface of thymic epithelial cells, and depends upon specific residues on the CDR3-like loop within the membrane-distal variable domain of Skint-1 (Skint-1 DV). Nuclear magnetic resonance of Skint-1 DV revealed a core tertiary structure conserved across the Skint family, but a highly distinct surface charge distribution, possibly explaining its unique function. Crucially, the CDR3-like loop formed an electrostatically distinct surface, featuring key charged and hydrophobic solvent-exposed residues, at the membrane-distal tip of DV. These results provide the first structural insights into the Skint family, identifying a putative receptor binding surface that directly implicates Skint-1 in receptor-ligand interactions crucial for DETC selection. PMID:26917727

  6. A COMPARISON BETWEEN DIFFERENT EXISTING METHODS USED TO SEPARATE EPIDERMAL CELLS FROM SKIN BIOPSIES FOR AUTOLOGOUS TRANSPLANTATION

    PubMed Central

    Maharlooei, Mohsen Khosravi; Mohammadi, Ali Akbar; Farsi, Ali; Ahrari, Iman; Attar, Armin; Monabati, Ahmad

    2011-01-01

    Background: Burn surgeons use autologous skin graft technique for patients, but a challenge remains for large surface wounds. Recently, a method was described which used a small piece of skin to cover a 70 times greater surface by spraying epidermal cells on injured skin. We designed a comparative study to find the best method to make an epidermal cell suspension. Materials and Methods: Eleven discarded skin samples were sent to our laboratory from Ghotboddin Burn Hospital, Shiraz. Each sample was sliced into four small pieces (1 cm2) and each piece was treated with a different chemical including sodium bromide (2N) and (4N), ammonium hydroxide (2N), and trypsin (0.05%) for 20 minutes. The epidermis and dermis were separated using forceps. Trypsin was added to all samples (except the trypsinized sample) to begin the intercellular detachment. Afterward, epidermis was sliced into small pieces followed by filtration and centrifugation. Cells were counted using hemocytometer. Identification of keratinocytes and melanocytes was made through immunocytochemical staining for cytokeratin and melanosome antigens, respectively. Results: There was a significant difference in alive cell counts comparing cells obtained from NaBr (4N) method to other methods. Considering total cell count and alive cell count, NaBr (4N) yielded the most cells. Immunocytochemical staining showed that in all methods, some cells are stained positively for cytokeratin antibody and some for melanosome antibody. Conclusion: Although recent papers had advised trypsin method to make a cell suspension to use for burn patients, we found that NaBr (4N) method yields more alive cells and less toxicity. PMID:22345767

  7. Induction of suppression of delayed type hypersensitivity to herpes simplex virus by epidermal cells exposed to UV-irradiated urocanic acid in vivo

    SciTech Connect

    Ross, J.A.; Howie, S.E.; Norval, M.; Maingay, J.P. )

    1987-01-01

    Urocanic acid (UCA), the putative photoreceptor for ultraviolet radiation (UV)-induced suppression, undergoes a UV-dependent trans to cis isomerisation. Epidermal cells from mice painted with UCA, containing a known proportion of the cis-isomer, generate suppression of the delayed type hypersensitivity response to herpes simplex virus type 1 (HSV-1) when transferred to naive syngeneic recipients at the same time and site as infection with HSV-1. One T suppressor cell subset, of phenotype (Thy1+, L3T4+, Ly2-), is induced by the cis-UCA modified epidermal cell transfer. Flow cytometric analysis of the epidermal cells from skin treated with UV or cis-UCA indicates an overall reduction from normal in the number of cells expressing MHC Class II antigens, but no alteration in the number expressing I-J antigens.

  8. Distribution of apoptosis-mediating Fas antigen in human skin and effects of anti-Fas monoclonal antibody on human epidermal keratinocyte and squamous cell carcinoma cell lines.

    PubMed

    Oishi, M; Maeda, K; Sugiyama, S

    1994-01-01

    Fast antigen is a cell surface protein that mediates apoptosis. Using immunohistological, flow cytometry and electron microscopic analyses, we investigated the expression of Fas antigen on various skin tissues, and on cultured SV40-transformed human epidermal keratinocyte cell line KJD and human skin squamous cell carcinoma cell line HSC. The Fas antigen was widely distributed in skin components such as the keratinocytes in the lower portion of the epidermis, epidermal dendritic cells, endothelial cells, fibroblasts, apocrine glands, eccrine sweat glands, sebaceous glands, some normal melanocytes and infiltrating lymphoid cells. It was also strongly expressed on the keratinocytes of lichenoid eruptions seen in lupus erythematosus and lichen planus, and on the spongiotic or acanthotic epidermis seen in chronic eczema, adult T-cell leukaemia/lymphoma (ATLL) and atopic dermatitis. Its expression was closely correlated with lymphoid infiltrating cells and it was strongly expressed in lymphoid neoplastic cells, particularly ATLL cells, and fibroblasts seen in dermatofibroma. However, the antigen was not detected on basal cell epithelioma cells, some malignant melanomas or any junctional naevi. The cell lines KJD and HSC strongly expressed the Fas antigen, and crosslinking of the Fas antigen by an anti-Fas monoclonal antibody induced apoptosis of these cell lines. These results indicate that the apoptosis-mediating Fas antigen may play an important role in normal skin turnover and cell differentiation, in immune regulation of skin tumours, and in the pathogenesis of various skin diseases. PMID:7529480

  9. Immunolocalization of FGF8/10 in the Apical Epidermal Peg and Blastema of the regenerating tail in lizard marks this apical growing area.

    PubMed

    Alibardi, Lorenzo

    2016-07-01

    Previous studies have shown that Fibroblast Growth Factors are present in the regenerating tail tissues of lizards where they may stimulate the process of regeneration. The present study is focused on the immunolocalization of FGF8 and FGF10 in the regenerating lizard tail, two signaling proteins of the apical epidermal cup/ridge and mesenchymal blastema sustaining tail and limb regeneration in amphibians and the development of the tail and limbs in vertebrate embryos. Main immunoreactive protein bands at 15-18kDa for FGF8/10 are detected in the regenerating epidermis and only a band at 30 or 35kDa in the underlying connective tissues. FGF8 appears particularly localized in cells and nuclei of the apical epidermal peg and of the ependymal ampulla present at the tip of the regenerating tail. FGF10 is also immuno-localized in the apical epidermis but is particularly intensely localized in the mesenchyme of the apical blastema. In accordance with previous studies, the present observations supports the hypothesis that the apical epidermal peg and the ependymal tube with the few regenerated neurons present within it, release FGF8/10 that may contribute to maintenance of cell proliferation in the apical front of the mesenchyme for the growth of the regenerating tail. PMID:27113329

  10. Epidermal growth factor receptor (EGFR) mutations in small cell lung cancers: Two cases and a review of the literature.

    PubMed

    Siegele, Bradford J; Shilo, Konstantin; Chao, Bo H; Carbone, David P; Zhao, Weiqiang; Ioffe, Olga; Franklin, Wilbur A; Edelman, Martin J; Aisner, Dara L

    2016-05-01

    Activating mutations in the epidermal growth factor receptor (EGFR) gene are exceedingly rare in small cell lung cancer (SCLC). We present two cases of SCLC harboring EGFR mutations, one in an 82 year-old male smoker with a combined SCLC and adenocarcinoma with a novel D855H point mutation in exon 21, and the second in a 68 year-old female never smoker with the L858R point mutation in exon 21. The cases, accompanied by a review of the literature, highlight the importance of integration of clinicopathologic considerations and adherence to recently promulgated Guideline recommendations for molecular testing in lung cancer. PMID:27040854

  11. Starting to gel: how Arabidopsis seed coat epidermal cells produce specialized secondary cell walls.

    PubMed

    Voiniciuc, Cătălin; Yang, Bo; Schmidt, Maximilian Heinrich-Wilhelm; Günl, Markus; Usadel, Björn

    2015-01-01

    For more than a decade, the Arabidopsis seed coat epidermis (SCE) has been used as a model system to study the synthesis, secretion and modification of cell wall polysaccharides, particularly pectin. Our detailed re-evaluation of available biochemical data highlights that Arabidopsis seed mucilage is more than just pectin. Typical secondary wall polymers such as xylans and heteromannans are also present in mucilage. Despite their low abundance, these components appear to play essential roles in controlling mucilage properties, and should be further investigated. We also provide a comprehensive community resource by re-assessing the mucilage phenotypes of almost 20 mutants using the same conditions. We conduct an in-depth functional evaluation of all the SCE genes described in the literature and propose a revised model for mucilage production. Further investigation of SCE cells will improve our understanding of plant cell walls. PMID:25658798

  12. Starting to Gel: How Arabidopsis Seed Coat Epidermal Cells Produce Specialized Secondary Cell Walls

    PubMed Central

    Voiniciuc, Cătălin; Yang, Bo; Schmidt, Maximilian Heinrich-Wilhelm; Günl, Markus; Usadel, Björn

    2015-01-01

    For more than a decade, the Arabidopsis seed coat epidermis (SCE) has been used as a model system to study the synthesis, secretion and modification of cell wall polysaccharides, particularly pectin. Our detailed re-evaluation of available biochemical data highlights that Arabidopsis seed mucilage is more than just pectin. Typical secondary wall polymers such as xylans and heteromannans are also present in mucilage. Despite their low abundance, these components appear to play essential roles in controlling mucilage properties, and should be further investigated. We also provide a comprehensive community resource by re-assessing the mucilage phenotypes of almost 20 mutants using the same conditions. We conduct an in-depth functional evaluation of all the SCE genes described in the literature and propose a revised model for mucilage production. Further investigation of SCE cells will improve our understanding of plant cell walls. PMID:25658798

  13. Increased epidermal cell proliferation in normal human skin in vivo following local administration of interferon-gamma.

    PubMed Central

    Barker, J. N.; Goodlad, J. R.; Ross, E. L.; Yu, C. C.; Groves, R. W.; MacDonald, D. M.

    1993-01-01

    Recombinant human interferon-gamma was administered intradermally (10 micrograms in 0.1 ml) to healthy adult human volunteers from day 1 to day 3, and epidermal cell proliferation was measured on whole skin biopsies at day 6. Three independent parameters were assessed, namely, a) epidermal keratin-16 expression, b) keratinocyte proliferating cell nuclear antigen expression, and c) keratinocyte silver nucleolar organizer region counts. Significantly increased scores for each parameter were observed after interferon-gamma injection (P < 0.01 in each case) compared to site-matched controls. Keratin-16 expression was confined to suprabasal epidermis, whereas proliferating cell nuclear antigen and silver nucleolar organizer region counts were particularly elevated in the basal epidermis. Taken together with previous findings, these studies indicate both proinflammatory and growth regulatory roles for interferon-gamma in human skin. These data are likely to be of particular importance to pathophysiological mechanisms of psoriasis and related cutaneous inflammatory diseases. Images Figure 1 Figure 2 Figure 3 PMID:7682760

  14. Arabidopsis IRT2 cooperates with the high-affinity iron uptake system to maintain iron homeostasis in root epidermal cells.

    PubMed

    Vert, Grégory; Barberon, Marie; Zelazny, Enric; Séguéla, Mathilde; Briat, Jean-François; Curie, Catherine

    2009-05-01

    Iron is an essential nutrient for all organisms but toxic when present in excess. Consequently, plants carefully regulate their iron uptake, dependent on the FRO2 ferric reductase and the IRT1 transporter, to control its homeostasis. Arabidopsis IRT2 gene, whose expression is induced in root epidermis upon iron deprivation, was shown to encode a functional iron/zinc transporter in yeast, and proposed to function in iron acquisition from the soil. In this study, we demonstrate that, unlike its close homolog IRT1, IRT2 is not involved in iron absorption from the soil since overexpression of IRT2 does not rescue the iron uptake defect of irt1-1 mutant and since a null irt2 mutant shows no chlorosis in low iron. Consistently, an IRT2-green fluorescent fusion protein, transiently expressed in culture cells, localizes to intracellular vesicles. However, IRT2 appears strictly co-regulated with FRO2 and IRT1, supporting the view that IRT2 is an integral component of the root response to iron deficiency in root epidermal cells. We propose a model where IRT2 likely prevents toxicity from IRT1-dependent iron fluxes in epidermal cells, through compartmentalization. PMID:19252923

  15. Cadmium induces autophagy through ROS-dependent activation of the LKB1-AMPK signaling in skin epidermal cells

    SciTech Connect

    Son, Young-Ok; Wang Xin; Hitron, John Andrew; Zhang Zhuo; Cheng Senping; Budhraja, Amit; Ding Songze; Lee, Jeong-Chae; Shi Xianglin

    2011-09-15

    Cadmium is a toxic heavy metal which is environmentally and occupationally relevant. The mechanisms underlying cadmium-induced autophagy are not yet completely understood. The present study shows that cadmium induces autophagy, as demonstrated by the increase of LC3-II formation and the GFP-LC3 puncta cells. The induction of autophagosomes was directly visualized by electron microscopy in cadmium-exposed skin epidermal cells. Blockage of LKB1 or AMPK by siRNA transfection suppressed cadmium-induced autophagy. Cadmium-induced autophagy was inhibited in dominant-negative AMPK-transfected cells, whereas it was accelerated in cells transfected with the constitutively active form of AMPK. mTOR signaling, a negative regulator of autophagy, was downregulated in cadmium-exposed cells. In addition, cadmium generated reactive oxygen species (ROS) at relatively low levels, and caused poly(ADP-ribose) polymerase-1 (PARP) activation and ATP depletion. Inhibition of PARP by pharmacological inhibitors or its siRNA transfection suppressed ATP reduction and autophagy in cadmium-exposed cells. Furthermore, cadmium-induced autophagy signaling was attenuated by either exogenous addition of catalase and superoxide dismutase, or by overexpression of these enzymes. Consequently, these results suggest that cadmium-mediated ROS generation causes PARP activation and energy depletion, and eventually induces autophagy through the activation of LKB1-AMPK signaling and the down-regulation of mTOR in skin epidermal cells. - Highlights: > Cadmium, a toxic heavy metal, induces autophagic cell death through ROS-dependent activation of the LKB1-AMPK signaling. > Cadmium generates intracellular ROS at low levels and this leads to severe DNA damage and PARP activation, resulting in ATP depletion, which are the upstream events of LKB1-AMPK-mediated autophagy. > This novel finding may contribute to further understanding of cadmium-mediated diseases.

  16. Mechanism of interleukin-1α transcriptional regulation of S100A9 in a human epidermal keratinocyte cell line

    PubMed Central

    Bando, Mika; Zou, Xianqiong; Hiroshima, Yuka; Kataoka, Masatoshi; Ross, Karen F; Shinohara, Yasuo; Nagata, Toshihiko; Herzberg, Mark C; Kido, Jun-ichi

    2013-01-01

    S100A9 is a calcium-binding protein and subunit of antimicrobial calprotectin complex (S100A8/A9). Produced by neutrophils, monocytes/ macrophages and keratinocytes, S100A9 expression increases in response to inflammation. For example, IL-1α produced by epithelial cells acts autonomously on the same cells to induce expression of S100A8/A9 and cellular differentiation. Whereas it is well known that IL-1α and members of the IL-10 family of cytokines upregulate S100A8 and S100A9 in several cell lineages, the pathway and mechanism of IL-1α-dependent transcriptional control of S100A9 in epithelial cells is not established. Modeled using human epidermal keratinocytes (HaCaT cells), IL-1α stimulated phosphorylation of p38 MAPK and induced S100A9 expression, which was blocked by IL-1 receptor antagonist, RNAi suppression of p38, or a p38 MAPK inhibitor. Transcription of S100A9 in HaCaT cells depended on nucleotides -94 to -53 in the upstream promoter region, based upon use of deletion constructs and luciferase reporter activity. Within the responsive promoter region, IL-1α increased the binding activity of CCAAT/enhancer binding protein β (C/EBPβ). Mutated C/EBPβ binding sequences or C/EBPβ-specific siRNA inhibited the S100A9 transcriptional response. Hence, IL-1α is strongly suggested to increase S100A9 expression in a human epidermal keratinocyte cell line by signaling through the IL-1 receptor and p38 MAPK, increasing C/EBPβ-dependent transcriptional activity. PMID:23563247

  17. Investigation of macromolecule orientation in dry and hydrated walls of single onion epidermal cells by FTIR microspectroscopy

    NASA Astrophysics Data System (ADS)

    Chen, Limei; Wilson, Reginald H.; McCann, Maureen C.

    1997-06-01

    Polarised infrared spectra from the wall of a single epidermal onion cell were obtained using a Fourier transform infrared (FTIR) microscope. The use of a newly constructed hydration cell allowed studies of both composition and architecture of intact walls of single hydrated plant cells. By comparing spectra taken with infrared light polarised perpendicular, or parallel, to the long axis of the cell, orientations of macromolecules in dry and hydrated cell walls were investigated. It was observed that bands associated with pectin were stronger with polarisation perpendicular to the direction of the cell elongation. On the other hand, bands associated with cellulose were more intense with polarisation parallel to the direction of cell elongation. These results show that in dry and hydrated cell walls, not only was there a net orientation of cellulose, but also of pectin. The implication of this is that pectin, which was previously thought to play no structural role in cell walls may, in fact, contribute to the mechanical and structural properties of the cell network. Such results are likely to have a tremendous impact on the formulation of definitive models for the static and growing cell wall.

  18. Protein kinase C is differentially regulated by thrombin, insulin, and epidermal growth factor in human mammary tumor cells

    SciTech Connect

    Gomez, M.L.; Tellez-Inon, M.T. ); Medrano, E.E.; Cafferatta, E.G.A. )

    1988-03-01

    The exposure of serum-deprived mammary tumor cells MCF-7 and T-47D to insulin, thrombin, and epidermal growth factor (EGF) resulted in dramatic modifications in the activity and in the translocation capacity of protein kinase C from cytosol to membrane fractions. Insulin induces a 600% activation of the enzyme after 5 h of exposure to the hormone in MCF-7 cells; thrombin either activates (200% in MCF-7) or down-regulates (in T-47D), and EGF exerts only a moderate effect. Thus, the growth factors studied modulate differentially the protein kinase C activity in human mammary tumor cells. The physiological significance of the results obtained are discussed in terms of the growth response elicited by insulin, thrombin, and EGF.

  19. DNA vaccination strategy targets epidermal dendritic cells, initiating their migration and induction of a host immune response.

    PubMed

    Smith, Trevor Rf; Schultheis, Katherine; Kiosses, William B; Amante, Dinah H; Mendoza, Janess M; Stone, John C; McCoy, Jay R; Sardesai, Niranjan Y; Broderick, Kate E

    2014-01-01

    The immunocompetence and clinical accessibility of dermal tissue offers an appropriate and attractive target for vaccination. We previously demonstrated that pDNA injection into the skin in combination with surface electroporation (SEP), results in rapid and robust expression of the encoded antigen in the epidermis. Here, we demonstrate that intradermally EP-enhanced pDNA vaccination results in the rapid induction of a host humoral immune response. In the dermally relevant guinea pig model, we used high-resolution laser scanning confocal microscopy to observe direct dendritic cell (DC) transfections in the epidermis, to determine the migration kinetics of these cells from the epidermal layer into the dermis, and to follow them sequentially to the immediate draining lymph nodes. Furthermore, we delineate the relationship between the migration of directly transfected epidermal DCs and the generation of the host immune response. In summary, these data indicate that direct presentation of antigen to the immune system by DCs through SEP-based in vivo transfection in the epidermis, is related to the generation of a humoral immune response. PMID:26052522

  20. The Effect of MCP-1/CCR2 on the Proliferation and Senescence of Epidermal Constituent Cells in Solar Lentigo.

    PubMed

    Lee, Woo Jin; Jo, Soo Youn; Lee, Mi Hye; Won, Chong Hyun; Lee, Mi Woo; Choi, Jee Ho; Chang, Sung Eun

    2016-01-01

    Solar lentigo (SL) is a representative photoaging skin disorder. Alteration of the main epidermal constituent cells-keratinocytes and melanocytes-in relation to the photoaged dermal environment or chemokine/cytokine network is suggested as its pathogenesis. Among these, we focused on monocyte chemoattractant protein-1 (MCP-1), as it is known to be associated with tissue aging. For the first time, we report that the MCP-1 receptor, CCR2, is expressed in normal human melanocytes. In SL tissue, there was an increase of CCR2+Melan A+ melanocytes with positivity to Rb protein compared to peri-lesional normal skin. MCP-1 induced the proliferation of normal human melanocytes without a significant change in the melanin content. MCP-1 treatment in normal human keratinocytes showed an increase in senescence-associated β-galactosidase staining and p53 and p21 protein expressions. In summary, MCP-1 may participate in the development of SL by affecting epidermal constituent cells, for example, by inducing melanocyte proliferation and keratinocyte senescence. PMID:27314341

  1. DNA vaccination strategy targets epidermal dendritic cells, initiating their migration and induction of a host immune response

    PubMed Central

    Smith, Trevor RF; Schultheis, Katherine; Kiosses, William B; Amante, Dinah H; Mendoza, Janess M; Stone, John C; McCoy, Jay R; Sardesai, Niranjan Y; Broderick, Kate E

    2014-01-01

    The immunocompetence and clinical accessibility of dermal tissue offers an appropriate and attractive target for vaccination. We previously demonstrated that pDNA injection into the skin in combination with surface electroporation (SEP), results in rapid and robust expression of the encoded antigen in the epidermis. Here, we demonstrate that intradermally EP-enhanced pDNA vaccination results in the rapid induction of a host humoral immune response. In the dermally relevant guinea pig model, we used high-resolution laser scanning confocal microscopy to observe direct dendritic cell (DC) transfections in the epidermis, to determine the migration kinetics of these cells from the epidermal layer into the dermis, and to follow them sequentially to the immediate draining lymph nodes. Furthermore, we delineate the relationship between the migration of directly transfected epidermal DCs and the generation of the host immune response. In summary, these data indicate that direct presentation of antigen to the immune system by DCs through SEP-based in vivo transfection in the epidermis, is related to the generation of a humoral immune response. PMID:26052522

  2. Mechanical properties of epidermal cells of whole living roots of Arabidopsis thaliana: An atomic force microscopy study

    NASA Astrophysics Data System (ADS)

    Fernandes, Anwesha N.; Chen, Xinyong; Scotchford, Colin A.; Walker, James; Wells, Darren M.; Roberts, Clive J.; Everitt, Nicola M.

    2012-02-01

    The knowledge of mechanical properties of root cell walls is vital to understand how these properties interact with relevant genetic and physiological processes to bring about growth. Expansion of cell walls is an essential component of growth, and the regulation of cell wall expansion is one of the ways in which the mechanics of growth is controlled, managed and directed. In this study, the inherent surface mechanical properties of living Arabidopsis thaliana whole-root epidermal cells were studied at the nanoscale using the technique of atomic force microscopy (AFM). A novel methodology was successfully developed to adapt AFM to live plant roots. Force-Indentation (F-I) experiments were conducted to investigate the mechanical properties along the length of the root. F-I curves for epidermal cells of roots were also generated by varying turgor pressure. The F-I curves displayed a variety of features due to the heterogeneity of the surface. Hysteresis is observed. Application of conventional models to living biological systems such as cell walls in nanometer regimes tends to increase error margins to a large extent. Hence information from the F-I curves were used in a preliminary semiquantitative analysis to infer material properties and calculate two parameters. The work done in the loading and unloading phases (hysteresis) of the force measurements were determined separately and were expressed in terms of “Index of Plasticity” (η), which characterized the elasticity properties of roots as a viscoelastic response. Scaling approaches were used to find the ratio of hardness to reduced modulus ((H)/(E*)).

  3. Surface tethered epidermal growth factor protects proliferating and differentiating multipotential stromal cells from FasL induced apoptosis

    PubMed Central

    Rodrigues, Melanie; Blair, Harry; Stockdale, Linda; Griffith, Linda; Wells, Alan

    2012-01-01

    Multipotential stromal cells, or mesenchymal stem cells, (MSC) have ben proposed as aids in regenerating bone and adipose tissues, as these cells form osteoblasts and adipocytes. A major obstacle to this use of MSC is the initial loss of cells post-implantation. This cell death in part, is due to ubiquitous non-specific inflammatory cytokines such as FasL generated in the implant site. Our group previously found that soluble epidermal growth factor (sEGF) promotes MSC expansion. Further, tethering EGF onto a two-dimensional surface (tEGF) altered MSC responses, by restricting epidermal growth factor receptor (EGFR) to the cell surface, causing sustained activation of EGFR, and promoting survival from FasL-induced death. sEGF by causing internalization of EGFR does not support MSC survival. However, for tEGF to be useful in bone regeneration, it needs to allow for MSC differentiation into osteoblasts while also protecting emerging osteoblasts from apoptosis. tEGF did not block induced differentiation of MSCs into osteoblasts, or adipocytes, a common default MSC-differentiation pathway. MSC-derived pre-osteoblasts showed increased Fas levels and became more susceptible to FasL induced death, which tEGF prevented. Differentiating adipocytes underwent a reduction in Fas expression and became resistant to FasL-induced death, with tEGF having no further survival effect. tEGF protected undifferentiated MSC from combined insults of FasL, serum deprivation and physiologic hypoxia. Additionally, tEGF was dominant in the face of sEGF to protect MSC from FasL-induced death. Our results suggest that MSCs and differentiating osteoblasts need protective signals to survive in the inflammatory wound milieu and that tEGF can serve this function. PMID:22948863

  4. Gastrointestinal cell proliferation and crypt fission are separate but complementary means of increasing tissue mass following infusion of epidermal growth factor in rats

    PubMed Central

    Berlanga-Acosta, J; Playford, R; Mandir, N; Goodlad, R

    2001-01-01

    BACKGROUND AND AIMS—Epidermal growth factor (EGF) is a potent mitogen for the gastrointestinal tract and also influences the number of new crypts formed by crypt fission. The time course of these events and possible linkage between these two complementary mechanisms is however poorly understood. We therefore examined the temporal relationship of proliferation and fission in rats treated with EGF.
METHODS—Osmotic minipumps were implanted subcutaneously into male Wistar rats to infuse EGF continuously (60 µg/rat/day) for periods of 1-14 days. Proliferation and crypt branching were quantified following vincristine induced metaphase arrest and morphometric assessment of microdissected tissue.
RESULTS—In the small intestine, EGF significantly increased epithelial cell proliferation and crypt and villus area after 24 hours of EGF, although maximal effects were only reached following six days of infusion. EGF also resulted in an approximate 30% reduction in crypt fission in the small bowel. In the colon, EGF caused a twofold increase in epithelial cell proliferation one day after infusion, from 15.3 (2.3) to 29.6 (3.5) metaphases per crypt (p<0.01). Maximal effects were seen in rats receiving EGF for seven days. For all time points, colonic crypt size increased in response to EGF. The amount of branching increased following one day of infusion with EGF (from 15.3 (1.9) to 32.4 (5.5)%; p<0.001) but was significantly lower (approximately 25% of control values) following longer periods of infusion. Crypt fission did not correlate with crypt area.
CONCLUSION—EGF has profound effects on cell proliferation and also altered crypt fission, with its actions on crypt fission most pronounced in the colon where it first increased and then decreased fission. EGF can thus be a potent stimulus for crypt fission during short term infusion and may reduce the number of branched crypts present in a resting or quiescent stage. Growth factors can alter cell mass by two

  5. CELLULOSE SYNTHASE9 Serves a Nonredundant Role in Secondary Cell Wall Synthesis in Arabidopsis Epidermal Testa Cells1[C][W][OA

    PubMed Central

    Stork, Jozsef; Harris, Darby; Griffiths, Jonathan; Williams, Brian; Beisson, Fred; Li-Beisson, Yonghua; Mendu, Venugopal; Haughn, George; DeBolt, Seth

    2010-01-01

    Herein, we sought to explore the contribution of cellulose biosynthesis to the shape and morphogenesis of hexagonal seed coat cells in Arabidopsis (Arabidopsis thaliana). Consistent with seed preferential expression of CELLULOSE SYNTHASE9 (CESA9), null mutations in CESA9 caused no change in cellulose content in leaves or stems, but caused a 25% reduction in seeds. Compositional studies of cesa9 seeds uncovered substantial proportional increases in cell wall neutral sugars and in several monomers of cell wall-associated polyesters. Despite these metabolic compensations, cesa9 seeds were permeable to tetrazolium salt, implying that cellulose biosynthesis, via CESA9, is required for correct barrier function of the seed coat. A syndrome of depleted radial wall, altered seed coat cell size, shape, and internal angle uniformity was quantified using scanning electron micrographs in cesa9 epidermal cells. By contrast, morphological defects were absent in cesa9 embryos, visually inspected from torpedo to bent cotyledon, consistent with no reduction in postgermination radical or hypocotyl elongation. These data implied that CESA9 was seed coat specific or functionally redundant in other tissues. Assessment of sections from glutaraldehyde fixed wild-type and cesa9 mature seeds supported results of scanning electron micrographs and quantitatively showed depletion of secondary cell wall synthesis in the radial cell wall. Herein, we show a nonredundant role for CESA9 in secondary cell wall biosynthesis in radial cell walls of epidermal seed coats and document its importance for cell morphogenesis and barrier function of the seed coat. PMID:20335403

  6. Toxic Epidermal Necrolysis.

    PubMed

    Castelain, Florence; Humbert, Phillip

    2013-02-01

    Toxic epidermal necrolysis (TEN) is a severe mucocutaneous drug-induced syndrome that causes massive keratinocyte apoptosis and therefore hydro-electrolytic disorders and systemic infection. TEN approximately affects one to two cases per million per year. Mortality rate may reach thirty percent of cases. Thus, TEN constitute a therapeutic emergency at diagnosis. Typically, clinical examination shows a mucocutaneous detachment involving more than thirty percent of body area. Definitive diagnosis is made on cutaneous biopsy with histological exam that shows the blister of necrotic keratinocytes. Main differential diagnosis are acute staphylococcus epidermis, acute generalized exanthematous pustulosis, linear IgA bullous dermatosis, paraneoplastic pemphigus, bullous fixed pigmented erythema, acute lupus erythematosus. In the early days, SCORTEN gives a good estimation and is now widely used as prognostic score. Drugs are generally considered as the main etiology of TEN but in some cases bacterial or viral infections could be involved. Physiopathology remains unclear even if recent advances have reported the possible implication of immune pathways based on activation of T and NK cells. Treatment of TEN requires to be instituted as soon as the diagnosis is made and the patient is preferentially referred to a specialized unit. Supportive care consist in covering areas of cutaneous detachment. No other therapy have demonstrated its efficiency, but high-dose intravenous immunoglobulin might improve the prognosis. PMID:23373551

  7. Toxic epidermal necrolysis.

    PubMed

    Castelain, Florence; Humbert, Philippe

    2012-11-01

    Toxic epidermal necrolysis (TEN) is a severe mucocutaneous drug-induced syndrome that causes massive keratinocyte apoptosis and therefore hydro-electrolytic disorders and systemic infection. TEN approximately affects one to two cases per million per year. Mortality rate may reach thirty percent of cases. Thus, TEN constitutes a therapeutic emergency at diagnosis. Typically, clinical examination shows a mucocutaneous detachment involving more than thirty percent of body area. Definitive diagnosis is made on cutaneous biopsy with histological exam that shows the blister of necrotic keratinocytes. Main differential diagnosis are acute staphylococcus epidermis, acute generalized exanthematous pustulosis, linear IgA bullous dermatosis, paraneoplastic pemphigus, bullous fixed pigmented erythema, acute lupus erythematosus. In the early days, SCORTEN gives a good estimation and is now widely used as prognostic score. Drugs are generally considered as the main etiology of TEN but in some cases bacterial or viral infections could be involved. Physiopathology remains unclear even if recent advances have reported the possible implication of immune pathways based on activation of T and NK cells. Treatment of TEN requires to be instituted as soon as the diagnosis is made and the patient is preferentially referred to a specialized unit. Supportive care consist of covering areas of cutaneous detachment. No other therapy has demonstrated its efficiency, but high-dose intravenous immunoglobulin might improve the prognosis. PMID:23441982

  8. Parametrial fat tissue from high fat diet-treated SKH-1 mice stimulates transformation of mouse epidermal JB6 cells

    PubMed Central

    Bernard, Jamie J.; Lou, You-Rong; Peng, Qing-Yun; Li, Tao; Vakil, Priyal R.; Ding, Ning; Laskin, Jeffrey D.; Dong, Zigang; Conney, Allan H.; Lu, Yao-Ping

    2015-01-01

    Our previous studies indicated that decreasing visceral adipose tissue by surgical removal of the parametrial fat pads inhibited UVB-induced carcinogenesis in SKH-1 mice fed a high fat diet (HFD), but not a low fat diet (LFD) indicating that the parametrial fat tissue from mice fed a HFD played a role in skin carcinogenesis. Objective In the present study, we sought to investigate how a HFD may influence the intrinsic properties of the parametrial fat tissue to influence UVB-induced skin tumor formation. Methods and results Immunohistochemical staining, adipokine array, and flow cytometry showed that parametrial fat tissue from mice fed a HFD had a higher density of macrophage-fused dead adipocytes (crown-like structures), more adipokines, and stimulated the production of more reactive oxygen species compared with parametrial fat tissue from mice fed a LFD. These differences between parametrial fat tissue from mice fed a HFD and LFD were associated with their effect on the in vitro transformation of mouse epidermal JB6 cells. Our results indicated that fat tissue filtrate (an aqueous filtrate made from the parametrial fat pad) from mice fed a HFD enhanced the conversion of JB6 cells from an epithelial-like morphology to cells with a fibroblast-like morphology to a greater extent than fat tissue filtrate from mice fed a LFD. Studies indicated that the fibroblast-like cells had decreased levels of E-cadherin, increased levels of Twist as assayed by western blot. Fat tissue filtrate made from the parametrial fat tissue of mice fed a HFD had 160% more transforming activity than that from mice fed a LFD and formed malignant mesenchymal tumors in vivo. Conclusion These studies provide the first in vitro demonstration of a parametrial fat tissue-induced transformation of an epidermal cell. PMID:25821644

  9. Potent endogenous allelopathic compounds in Lepidium sativum seed exudate: effects on epidermal cell growth in Amaranthus caudatus seedlings.

    PubMed

    Iqbal, Amjad; Fry, Stephen C

    2012-04-01

    Many plants exude allelochemicals--compounds that affect the growth of neighbouring plants. This study reports further studies of the reported effect of cress (Lepidium sativum) seed(ling) exudates on seedling growth in Amaranthus caudatus and Lactuca sativa. In the presence of live cress seedlings, both species grew longer hypocotyls and shorter roots than cress-free controls. The effects of cress seedlings were allelopathic and not due to competition for resources. Amaranthus seedlings grown in the presence of cress allelochemical(s) had longer, thinner hypocotyls and shorter, thicker roots--effects previously attributed to lepidimoide. The active principle was more abundant in cress seed exudate than in seedling (root) exudates. It was present in non-imbibed seeds and releasable from heat-killed seeds. Release from live seeds was biphasic, starting rapidly but then continuing gradually for 24 h. The active principle was generated by aseptic cress tissue and was not a microbial digestion product or seed-treatment chemical. Crude seed exudate affected hypocotyl and root growth at ~25 and ~450 μg ml(-1) respectively. The exudate slightly (28%) increased epidermal cell number along the length of the Amaranthus hypocotyl but increased total hypocotyl elongation by 129%; it resulted in a 26% smaller hypocotyl circumference but a 55% greater epidermal cell number counted round the circumference. Therefore, the effect of the allelochemical(s) on organ morphology was imposed primarily by regulation of cell expansion, not cell division. It is concluded that cress seeds exude endogenous substances, probably including lepidimoide, that principally regulate cell expansion in receiver plants. PMID:22268144

  10. PECTIN METHYLESTERASE INHIBITOR6 Promotes Arabidopsis Mucilage Release by Limiting Methylesterification of Homogalacturonan in Seed Coat Epidermal Cells[C][W

    PubMed Central

    Saez-Aguayo, Susana; Ralet, Marie-Christine; Berger, Adeline; Botran, Lucy; Ropartz, David; Marion-Poll, Annie; North, Helen M.

    2013-01-01

    Imbibed seeds of the Arabidopsis thaliana accession Djarly are affected in mucilage release from seed coat epidermal cells. The impaired locus was identified as a pectin methylesterase inhibitor gene, PECTIN METHYLESTERASE INHIBITOR6 (PMEI6), specifically expressed in seed coat epidermal cells at the time when mucilage polysaccharides are accumulated. This spatio-temporal regulation appears to be modulated by GLABRA2 and LEUNIG HOMOLOG/MUCILAGE MODIFIED1, as expression of PMEI6 is reduced in mutants of these transcription regulators. In pmei6, mucilage release was delayed and outer cell walls of epidermal cells did not fragment. Pectin methylesterases (PMEs) demethylate homogalacturonan (HG), and the majority of HG found in wild-type mucilage was in fact derived from outer cell wall fragments. This correlated with the absence of methylesterified HG labeling in pmei6, whereas transgenic plants expressing the PMEI6 coding sequence under the control of the 35S promoter had increased labeling of cell wall fragments. Activity tests on seeds from pmei6 and 35S:PMEI6 transgenic plants showed that PMEI6 inhibits endogenous PME activities, in agreement with reduced overall methylesterification of mucilage fractions and demucilaged seeds. Another regulator of PME activity in seed coat epidermal cells, the subtilisin-like Ser protease SBT1.7, acts on different PMEs, as a pmei6 sbt1.7 mutant showed an additive phenotype. PMID:23362209

  11. Study of lung-metastasized prostate cancer cell line chemotaxis to epidermal growth factor with a BIOMEMS device

    NASA Astrophysics Data System (ADS)

    Tata, Uday; Rao, Smitha M. N.; Sharma, Akash; Pabba, Krishna; Pokhrel, Kushal; Adhikari, Bandita; Lin, Victor K.; Chiao, J.-C.

    2012-09-01

    Understanding the effects of different growth factors on cancer metastasis will enable researchers to develop effective post-surgery therapeutic strategies to stop the spread of cancer. Conventional Boyden chamber assays to evaluate cell motility in metastasis studies require high volumes of reagents and are impractical for high-throughput analysis. A microfluidic device was designed for arrayed assaying of prostate cancer cell migration towards different growth factors. The device was created with polydimethylsiloxane (PDMS) and featured two wells connected by 10 micro channels. One well was for cell seeding and the other well for specific growth factors. Each channel has a width of 20 μm, a length of 1 mm and a depth of 10 μm. The device was placed on a culture dish and primed with growth media. Lung-metastasized cells in suspension of RPMI 1640 media1 supplemented with 2% of fetal bovine serum (FBS) were seeded in the cell wells. Cell culture media with epidermal growth factor (EGF) of 25, 50, 75, 100 and 125 ng ml‑1 concentrations were individually added in the respective growth factor wells. A 5-day time-lapsed study of cell migration towards the chemoattractant was performed. The average numbers of cells per device in the microchannels were obtained for each attractant condition. The results indicated migration of cells increased from 50 to 100 ng ml‑1 of EGF and significantly decreased at 125 ng ml‑1 of EGF, as compared to control.

  12. miR-143 suppresses the proliferation of NSCLC cells by inhibiting the epidermal growth factor receptor

    PubMed Central

    Zhang, Hong-Bo; Sun, Li-Chao; Ling, Lan; Cong, Lu-Hong; Lian, Rui

    2016-01-01

    MicroRNAs (miRs) regulate the proliferation and metastasis of numerous cancer cell types. It was previously reported that miR-143 levels were downregulated in non-small cell lung cancer (NSCLC) tissues and cell lines, and that the migration and invasion of NSCLC cells was inhibited upon suppression of cell proliferation and colony formation by the upregulation of miR-143. Epidermal growth factor receptor (EGFR), which is a vital factor in the promotion of cancer cell proliferation and has been investigated as a potential focus in cancer therapy, has been reported to be a possible target of miR-143. The present study aimed to investigate the role of miR-143 in NSCLC using NSCLC cell lines and primary cells from NSCLC patients. NSCLC cells were co-transfected with EGFR and miR-143, and the mRNA and protein expression of EGFR were analyzed. Furthermore, the activity of the transfected cancer cells with regard to colony formation, migration, invasion and apoptosis were evaluated. The levels of miR-143 were decreased in the NSCLC cell lines and primary cells from patients with NSCLC compared with the controls. Following transfection with miR-143, the ability of NSCLC cells to proliferate, form colonies, migrate and invade was inhibited. Similarly, knockdown of EGFR led to the suppression of NSCLC cell proliferation. The mRNA and protein expression levels of EGFR were significantly reduced following miR-143 overexpression, and the level of miR-143 was inversely correlated with that of EGFR in NSCLC cells. The results of the present study demonstrated that miR-143 was able to suppress NSCLC cell proliferation and invasion by inhibiting the effects of EGFR, suggesting that EGFR may be considered a potential target for NSCLC therapy. PMID:27602093

  13. The Effect of MCP-1/CCR2 on the Proliferation and Senescence of Epidermal Constituent Cells in Solar Lentigo

    PubMed Central

    Lee, Woo Jin; Jo, Soo Youn; Lee, Mi Hye; Won, Chong Hyun; Lee, Mi Woo; Choi, Jee Ho; Chang, Sung Eun

    2016-01-01

    Solar lentigo (SL) is a representative photoaging skin disorder. Alteration of the main epidermal constituent cells—keratinocytes and melanocytes—in relation to the photoaged dermal environment or chemokine/cytokine network is suggested as its pathogenesis. Among these, we focused on monocyte chemoattractant protein-1 (MCP-1), as it is known to be associated with tissue aging. For the first time, we report that the MCP-1 receptor, CCR2, is expressed in normal human melanocytes. In SL tissue, there was an increase of CCR2+Melan A+ melanocytes with positivity to Rb protein compared to peri-lesional normal skin. MCP-1 induced the proliferation of normal human melanocytes without a significant change in the melanin content. MCP-1 treatment in normal human keratinocytes showed an increase in senescence-associated β-galactosidase staining and p53 and p21 protein expressions. In summary, MCP-1 may participate in the development of SL by affecting epidermal constituent cells, for example, by inducing melanocyte proliferation and keratinocyte senescence. PMID:27314341

  14. Increased epidermal growth factor-receptor protein in a human mesothelial cell line in response to long asbestos fibers.

    PubMed Central

    Pache, J. C.; Janssen, Y. M.; Walsh, E. S.; Quinlan, T. R.; Zanella, C. L.; Low, R. B.; Taatjes, D. J.; Mossman, B. T.

    1998-01-01

    Epidermal growth factor (EGF) is a potent mitogen for human mesothelial cells, and autophosphorylation of the EGF receptor (EGF-R) occurs in these cell types after exposure to asbestos, a carcinogen associated with the development of mesothelioma. Here, the intensity and distribution of EGF-R protein was documented by immunocytochemistry in a human mesothelial cell line (MET5A) exposed to various concentrations of crocidolite asbestos and man-made vitreous fibers (MMVF-10). Whereas cells in contact with or phagocytizing shorter asbestos fibers (<60 microm length) or MMVF-10 at a range of concentrations showed no increase in EGF-R protein as determined by immunofluorescence, elongated cells phagocytizing and surrounding longer fibers (> or =60 microm) showed intense staining for EGF-R. In contrast, human A549 lung carcinoma cells showed neither elongation nor increased accumulation of EGF-R protein in response to long fibers. Patterns of aggregation and increases in EGF-R protein in mesothelial cells phagocytizing long asbestos fibers were distinct from diffuse staining of phosphotyrosine residues observed in asbestos-exposed cultures. These studies indicate that aggregation of EGF-R by long fibers may initiate cell signaling cascades important in asbestos-induced mitogenesis and carcinogenesis. Images Figure 1 Figure 2 Figure 3 PMID:9466557

  15. Epidermal Growth Factor-Induced Tumor Cell Invasion and Metastasis Initiated by Dephosphorylation and Downregulation of Focal Adhesion Kinase

    PubMed Central

    Lu, Zhimin; Jiang, Guoqiang; Blume-Jensen, Peter; Hunter, Tony

    2001-01-01

    Upregulated epidermal growth factor (EGF) receptor (EGFR) expression and EGFR-induced signaling have been correlated with progression to invasion and metastasis in a wide variety of carcinomas, but the mechanism behind this is not well understood. We show here that, in various human carcinoma cells that overexpress EGFR, EGF treatment induced rapid tyrosine dephosphorylation of focal adhesion kinase (FAK) associated with downregulation of its kinase activity. The downregulation of FAK activity was both required and sufficient for EGF-induced refractile morphological changes, detachment of cells from the extracellular matrix, and increased tumor cell motility, invasion, and metastasis. Tumor cells with downregulated FAK activity became less adherent to the extracellular matrix. However, once cells started reattaching, FAK activity was restored by activated integrin signaling. Moreover, this process of readhesion and spreading could not be abrogated by further EGF stimulation. Interruption of transforming growth factor alpha-EGFR autocrine regulation with an EGFR tyrosine kinase inhibitor led to a substantial increase in FAK tyrosine phosphorylation and inhibition of tumor cell invasion in vitro. Consistent with this, FAK tyrosine phosphorylation was reduced in cells from tumors growing in transplanted, athymic, nude mice, which have an intact autocrine regulation of the EGFR. We suggest that the dynamic regulation of FAK activity, initiated by EGF-induced downregulation of FAK leading to cell detachment and increased motility and invasion, followed by integrin-dependent reactivation during readhesion, plays a role in EGF-associated tumor invasion and metastasis. PMID:11359909

  16. Epidermal growth factor-induced cyclooxygenase-2 enhances head and neck squamous cell carcinoma metastasis through fibronectin up-regulation

    PubMed Central

    Hsu, Jinn-Yuan; Chang, Kwang-Yu; Chen, Shang-Hung; Lee, Chung-Ta; Chang, Sheng-Tsung; Cheng, Hung-Chi; Chang, Wen-Chang; Chen, Ben-Kuen

    2015-01-01

    Epidermal growth factor receptor (EGFR) activation is a major cause of metastasis in many cancers, such as head and neck squamous cell carcinoma (HNSCC). However, whether the induction of cyclooxygenase-2 (COX-2) mediates EGF-enhanced HNSCC metastasis remains unclear. Interestingly, we found that EGF induced COX-2 expression mainly in HNSCC. The tumor cell transformation induced by EGF was repressed by COX-2 knockdown, and this repression was reversed by simultaneously treating the cells with EGF and prostaglandin E2 (PGE2). The down-regulation of COX-2 expression or inhibition of COX-2 activity significantly blocked EGF enhancement of cell migration and invasion, but the addition of PGE2 compensated for this inhibitory effect in COX-2-knockdown cells. COX-2 depletion inhibited EGF-induced matrix metalloproteinase (MMP)-1, MMP-2, MMP-3, MMP-9, and fibronectin expression and Rac1/cdc42 activation. The inhibitory effect of COX-2 depletion on MMPs and the fibronectin/Rac1/cdc42 axis were reversed by co-treatment with PGE2. Furthermore, depletion of fibronectin impeded the COX-2-enhanced binding of HNSCC cells to endothelial cells and tumor cells metastatic seeding of the lungs. These results demonstrate that EGF-induced COX-2 expression enhances HNSCC metastasis via activation of the fibronectin signaling pathway. The inhibition of COX-2 expression and activation may be a potential strategy for the treatment of EGFR-mediated HNSCC metastasis. PMID:25595899

  17. Raman Spectroscopy of Human Neuronal and Epidermal Cells Exposed to an Insecticide Mixture of Chlorpyrifos and Deltamethrin.

    PubMed

    2014-10-01

    Many pesticides are increasingly used in combinations for crop protection. Their chemical stability ensures the presence of such mixtures, both in the workspaces of the operators involved in agricultural activities and in foodstuffs, thus making probable human exposure to such chemicals in the environment. We report an investigation, performed by means of Raman microspectroscopy and principal component analysis, concerning the effects of in vitro cellular exposure to a commercial insecticide based on a chlorpyrifos and deltamethrin mixture. The investigated cells belong to the SHSY-5Y and human keratinocyte (HUKE) cell lines, which can be considered representative of neuronal and epidermal cells, respectively. After 24 h exposure at a concentration one-tenth of that usually used by operators, about 50% of the investigated cells were dead and the relative content of the biochemical components of both types of cells that were still alive had been affected by the exposure. A statistically significant decrease in the protein and nucleic acid content occurred in the SHSY-5Y cells, and a lowering of the lipid and carbohydrate content was observed in the HUKE cells. This study shows the utility of Raman microspectroscopy and principal component analysis for the investigation of the effects on human cells of environmental exposure to any chemicals. PMID:25199150

  18. 3D In Vitro Model of a Functional Epidermal Permeability Barrier from Human Embryonic Stem Cells and Induced Pluripotent Stem Cells

    PubMed Central

    Petrova, Anastasia; Celli, Anna; Jacquet, Laureen; Dafou, Dimitra; Crumrine, Debra; Hupe, Melanie; Arno, Matthew; Hobbs, Carl; Cvoro, Aleksandra; Karagiannis, Panagiotis; Devito, Liani; Sun, Richard; Adame, Lillian C.; Vaughan, Robert; McGrath, John A.; Mauro, Theodora M.; Ilic, Dusko

    2014-01-01

    Summary Cornification and epidermal barrier defects are associated with a number of clinically diverse skin disorders. However, a suitable in vitro model for studying normal barrier function and barrier defects is still lacking. Here, we demonstrate the generation of human epidermal equivalents (HEEs) from human embryonic stem cells (hESCs) and induced pluripotent stem cells (iPSCs). HEEs are structurally similar to native epidermis, with a functional permeability barrier. We exposed a pure population of hESC/iPSC-derived keratinocytes, whose transcriptome corresponds to the gene signature of normal primary human keratinocytes (NHKs), to a sequential high-to-low humidity environment in an air/liquid interface culture. The resulting HEEs had all of the cellular strata of the human epidermis, with skin barrier properties similar to those of normal skin. Such HEEs generated from disease-specific iPSCs will be an invaluable tool not only for dissecting molecular mechanisms that lead to epidermal barrier defects but also for drug development and screening. PMID:24936454

  19. [The pathogenesis of psoriasis. Autoradiographic in vitro studies on cell proliferation in psoriasis vulgaris and other normal and hyperproliferative states of epidermal and dermal human cells].

    PubMed

    Pullman, H

    1978-07-01

    In the epidermal cells of patients suffering from psoriasis we found a significant prolongation of DNA-synthesis time (ts) in uninvolved skin, very early lesions, and fully developed plaques. In uninvolved psoriatic skin ts in addition increased significantly within 6 hours after stripping of the horny layer. In normal epidermis and in other states of epidermal inflammation and hyperproliferation (akanthosis by petrolatum, toxic dermatitis, chronic allergic ekzema, neurodermitis, allergic patch test reaction) a comparable prolongation of ts was not ascertainable. This prolongation is most distinct in the early lesions and proceeds the development of hyperproliferation and akanthosis. A dermal infiltrate with increased proliferative activity seems to be a stimulus, in the sense of a Koebner-phenomenon. The abnormal psoriatic epidermis, with disturbed DNA-synthesis, reacts to this infiltrate as well as to other irritants not with a limited hyperproliferation but with the development of psoriatic plaque. PMID:149749

  20. The Effects of Adenoviral Transfection of the Keratinocyte Growth Factor Gene on Epidermal Stem Cells: an In Vitro Study

    PubMed Central

    Li, Xinping; Liang, Ling; Zhao, Pin; Uchida, Kenzo; Baba, Hisatoshi; Huang, Hong; Bai, Wenfang; Bai, Liming; Zhang, Mingsheng

    2013-01-01

    Epidermal stem cells (ESCs) are characterized as slow-cycling, multi-potent, and self-renewing cells that not only maintain somatic homeostasis but also participate in tissue regeneration and repair. To examine the feasibility of adenoviral vector-mediated keratinocyte growth factor (KGF) gene transfer into in vitro-expanded ESCs, ESCs were isolated from samples of human skin, cultured in vitro, and then transfected with recombinant adenovirus (Ad) carrying the human KGF gene (AdKGF) or green fluorescent protein gene (AdGFP). The effects of KGF gene transfer on cell proliferation, cell cycle arrest, cell surface antigen phenotype, and β-catenin expression were investigated. Compared to ESCs transfected with AdGFP, AdKGF-transfected ESCs grew well, maintained a high proliferative capacity in keratinocyte serum-free medium, and expressed high levels of β-catenin. AdKGF infection increased the number of ESCs in the G0/G1 phase and promoted ESCs entry into the G2/M phase, but had no effect on cell surface antigen phenotype (CD49f+/CD71−). The results suggest that KGF gene transfer can stimulate ESCs to grow and undergo cell division, which can be applied to enhance cutaneous wound healing. PMID:24170090

  1. Epidermal growth factor receptor-dependent stimulation of amphiregulin expression in androgen-stimulated human prostate cancer cells.

    PubMed Central

    Sehgal, I; Bailey, J; Hitzemann, K; Pittelkow, M R; Maihle, N J

    1994-01-01

    Amphiregulin is a heparin-binding epidermal growth factor (EGF)-related peptide that binds to the EGF receptor (EGF-R) with high affinity. In this study, we report a role for amphiregulin in androgen-stimulated regulation of prostate cancer cell growth. Androgen is known to enhance EGF-R expression in the androgen-sensitive LNCaP human prostate carcinoma cell line, and it has been suggested that androgenic stimuli may regulate proliferation, in part, through autocrine mechanisms involving the EGF-R. In this study, we demonstrate that LNCaP cells express amphiregulin mRNA and peptide and that this expression is elevated by androgenic stimulation. We also show that ligand-dependent EGF-R stimulation induces amphiregulin expression and that androgenic effects on amphiregulin synthesis are mediated through this EGF-R pathway. Parallel studies using the estrogen-responsive breast carcinoma cell line, MCF-7, suggest that regulation of amphiregulin by estrogen may also be mediated via an EGF-R pathway. In addition, heparin treatment of LNCaP cells inhibits androgen-stimulated cell growth further suggesting that amphiregulin can mediate androgen-stimulated LNCaP proliferation. Together, these results implicate an androgen-regulated autocrine loop composed of amphiregulin and its receptor in prostate cancer cell growth and suggest that the mechanism of steroid hormone regulation of amphiregulin synthesis may occur through androgen upregulation of the EGF-R and subsequent receptor-dependent pathways. Images PMID:8049525

  2. Imaging of epidermal growth factor receptor on single breast cancer cells using surface-enhanced Raman spectroscopy

    PubMed Central

    Xiao, Lifu; Harihar, Sitaram; Welch, Danny R.; Zhou, Anhong

    2014-01-01

    Epidermal growth factor receptor (EGFR) is widely used as a biomarker for pathological grading and therapeutic targeting of human cancers. This study investigates expression, spatial distribution as well as the endocytosis of EGFR in single breast cancer cells using surface-enhanced Raman spectroscopy (SERS). By incubating anti-EGFR antibody conjugated SERS nanoprobes with an EGFR-over-expressing cancer cell line, A431, EGFR localization was measured over time and found to be located primarily at the cell surface. To further validate the constructed SERS probes, we applied this SERS probes to detect the EGFR expression on breast cancer cells (MDA-MB-435, MDA-MB-231) and their counterpart cell lines in which EGFR expression was down-regulated by breast cancer metastasis suppressor 1 (BRMS1). The results showed that SERS method not only confirms immunoblot data measuring EGFR levels, but also adds new insights regarding EGFR localization and internalization in living cells which is impossible in immunoblot method. Thus, SERS provides a powerful new tool to measure biomarkers in living cancer cells. PMID:25150698

  3. Divergent effects of epidermal growth factor and transforming growth factors on a human endometrial carcinoma cell line.

    PubMed

    Korc, M; Haussler, C A; Trookman, N S

    1987-09-15

    Epidermal growth factor (EGF), at concentrations ranging from 0.83 to 4.98 nM, markedly inhibited the proliferation of RL95-2 cells that were seeded at low plating densities (4.7 X 10(3) cells/cm2). Under the same incubation conditions, 16.6 pM EGF enhanced cell proliferation. At high plating densities (2.5 X 10(4) cells/cm2) 0.83 nM EGF also stimulated cell proliferation. Both the inhibitory and stimulatory effects of EGF were mimicked by transforming growth factor-alpha (TGF-alpha). However, the inhibitory action of TGF-alpha was always greater that of EGF. Binding studies with 125I-labeled TGF-alpha indicated that maximal cell surface binding of TGF-alpha occurred at 15 min, whereas maximal internalization occurred at 45 min. Both cell surface and internalized radioactivity declined sharply thereafter. Analysis of radioactivity released into the incubation medium during pulse-chase experiments indicated that RL95-2 cells extensively degraded both TGF-alpha and EGF. The lysosomotropic compound methylamine arrested the generation of low-molecular-weight degradation products of EGF, but not of TGF-alpha. In contrast to EGF and TGF-alpha, transforming growth factor-beta (TGF-beta) inhibited the proliferation of RL95-2 cells that were seeded at either low or high plating densities. Further, transforming growth factor-beta induced the appearance of large cuboidal cells that were readily distinguished from cells treated with either EGF or TGF-alpha. These findings point to complex regulatory actions of growth factors on the proliferation of RL95-2 cells and suggest that the processing of TGF-alpha following EGF receptor activation is distinct from the processing of EGF. PMID:3497713

  4. ACE inhibitors can induce circulating antibodies directed to antigens of the superficial epidermal cells.

    PubMed

    Cozzani, Emanuele; Rosa, Gian Marco; Drosera, Massimo; Intra, Chiara; Barsotti, Antonio; Parodi, Aurora

    2011-07-01

    Drug-induced pemphigus has been reported in patients receiving angiotensin-converting enzyme inhibitors. The aim of this work was to study a group of hypertensive patients without skin diseases treated with angiotensin-converting enzyme (ACE) Inhibitors (I), to verify the presence of serum circulating anti-antibodies. The indirect immunofluorescence showed that 33 sera (52.38%) presented autoantibodies directed to an antigen of the cytoplasm of the superficial epidermal keratinocytes. Two of the 33 positive sera had antibodies to Dsg1 and/or 3 in ELISA. Immunoblot analyses were negative. All the 48 control sera were found to have no circulating antibodies using the three assays. Our results would confirm that ACEI drugs may trigger the production of circulating autoantibodies also in patients without clinical manifestations of pemphigus. PMID:20563876

  5. Epidermal growth factor receptor variant III mediates head and neck cancer cell invasion via STAT3 activation

    PubMed Central

    Suzuki, Shinsuke; Morgan, Sarah E.; Thomas, Sufi M.; Sen, Malabika; Leeman-Neill, Rebecca J.; Kuan, Chien-Tsun; Bigner, Darrell; Gooding, William E.; Lai, Stephen Y.; Grandis, Jennifer R.

    2009-01-01

    Epidermal Growth Factor Receptor (EGFR) is frequently over-expressed in head and neck squamous cell carcinoma (HNSCC) where aberrant signaling downstream of this receptor contributes to tumor growth. EGFR variant III (EGFRvIII) is the most commonly altered form of EGFR and contains a truncated ligand-binding domain. We previously reported that EGFRvIII is expressed in up to 40% of HNSCC tumors where it is associated with increased proliferation, tumor growth and chemoresistance to anti-tumor drugs including the EGFR targeting monoclonal antibody cetuximab. Cetuximab was FDA-approved in 2006 for HNSCC but has not been shown to prevent invasion or metastasis. The present study was undertaken to evaluate the mechanisms of EGFRvIII-mediated cell motility and invasion in HNSCC. We found that EGFRvIII induced HNSCC cell migration and invasion in conjunction with increased STAT3 activation, which was not abrogated by cetuximab treatment. Further investigation demonstrated that EGF-induced expression of the STAT3 target gene HIF1-α, was abolished by cetuximab in HNSCC cells expressing wild-type EGFR under hypoxic conditions, but not in EGFRvIII-expressing HNSCC cells. These results suggest that EGFRvIII mediates HNSCC cell migration and invasion via increased STAT3 activation and induction of HIF1-α, which contribute to cetuximab resistance in EGFRvIII-expressing HNSCC tumors. PMID:20622897

  6. Differential regulation of human Eag1 channel expression by serum and epidermal growth factor in lung and breast cancer cells

    PubMed Central

    Acuña-Macías, Isabel; Vera, Eunice; Vázquez-Sánchez, Alma Yolanda; Mendoza-Garrido, María Eugenia; Camacho, Javier

    2015-01-01

    Oncogenic ether à-go-go-1 (Eag1) potassium channels are overexpressed in most primary human solid tumors. Low oxygen and nutrient/growth factor concentrations play critical roles in tumorigenesis. However, the mechanisms by which tumor cells survive and proliferate under growth factor-depleted conditions remain elusive. Here, we investigated whether serum-deprived conditions and epidermal growth factor (EGF) regulate Eag1 expression in human lung and breast cancer cells. The human cancer cell lines A549 and MCF-7 (from the lungs and breast, respectively) were obtained from the American Type Culture Collection and cultured following the manufacturer’s recommendations. Eag1 gene and protein expression were studied by real-time PCR and immunocytochemistry, respectively. Cell proliferation was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay, and ERK1/2 phosphorylation was investigated by Western blot. Serum-deprived conditions increased Eag1 mRNA and protein expression in both cell lines. This Eag1 upregulation was prevented by EGF and the ERK1/2 inhibitor U0126 in only lung cancer cells; vascular endothelial growth factor did not prevent Eag1 upregulation. Our results suggest that Eag1 may act as a survival and mitogenic factor under low-serum and nutrient conditions and may be a clinical target during the early stages of tumor development. PMID:26527881

  7. Activation of AMP-activated kinase modulates sensitivity of glioma cells against epidermal growth factor receptor inhibition.

    PubMed

    Hartel, Ines; Ronellenfitsch, Michael; Wanka, Christina; Wolking, Stefan; Steinbach, Joachim P; Rieger, Johannes

    2016-07-01

    The epidermal growth factor (EGFR) pathway is frequently activated in glioblastoma but the clinical efficacy of EGFR inhibitors in malignant glioma has been disappointing. The reasons for the failure of the mechanisms of resistance of these inhibitors are unclear, but may involve factors of the tumor microenvironment such as limited glucose availability and hypoxia. It was therefore examined whether glucose and oxygen influenced the response of glioma cells to EGFR inhibition. Decreased levels of glucose and oxygen led to resistance against the EGFR inhibitor PD153035, whereas high glucose amounts and normoxia sensitised glioma cells towards the inhibitor. Low levels of glucose and oxygen stimulated AMP-activated kinase (AMPK) in glioma cells. 2DG, an inhibitor of glycolysis, and the AMPK activator A769662 reduced glucose consumption, induced phosphorylation of AMPK and mimicked the effects of low glucose availability on the toxicity of PD153035. Similarly, 2DG reduced toxicity of imatinib in K562 leukemia cells. In contrast, inhibition of AMPK by compound C or by short-hairpin (sh)-mediated gene suppression increased cell death induced by the EGFR inhibitor and reverted the protective effects of 2DG and A769662. In conclusion, cytotoxicity of EGFR inhibition can be diminished by AMPK activation in glioma cells. These results may provide one explanation for the low activity of EGFR inhibitors in clinical trials and suggest antagonism of AMPK or of AMPK-regulated metabolic alterations as a promising approach to enhance their therapeutic efficacy. PMID:27121290

  8. The effect of all-trans-retinoic acid on the synthesis of epidermal cell-surface-associated carbohydrates

    PubMed Central

    King, Ian A.; Tabiowo, Anne

    1981-01-01

    1. all-trans-Retinoic acid at concentrations greater than 10−7m stimulated the incorporation of d-[3H]glucosamine into 8m-urea/5% (w/v) sodium dodecyl sulphate extracts of 1m-CaCl2-separated epidermis from pig ear skin slices cultured for 18h. The incorporation of 35SO42−, l-[14C]fucose and U-14C-labelled l-amino acids was not significantly affected. 2. Electrophoresis of the solubilized epidermis showed increased incorporation of d-[3H]glucosamine into a high-molecular-weight glycosaminoglycan-containing peak when skin slices were cultured in the presence of 10−5m-all-trans-retinoic acid. The labelling of other epidermal components with d-[3H]glucosamine, 35SO42−, l-[14C]fucose and U-14C-labelled l-amino acids was not significantly affected by 10−5m-all-trans-retinoic acid. 3. Trypsinization dispersed the epidermal cells and released 75–85% of the total d-[3H]glucosamine-labelled material in the glycosaminoglycan peak. Thus most of this material was extracellular in both control and 10−5m-all-trans-retinoic acid-treated epidermis. 4. Increased labelling of extracellular epidermal glycosaminoglycans was also observed when human skin slices were treated with all-trans-retinoic acid, indicating a similar mechanism in both tissues. Increased labelling was also found when the epidermis was cultured in the absence of the dermis, suggesting a direct effect of all-trans-retinoic acid on the epidermis. 5. Increased incorporation of d-[3H]-glucosamine into extracellular epidermal glycosaminoglycans in 10−5m-all-trans-retinoic acid-treated skin slices was apparent after 4–8h in culture and continued up to 48h. all-trans-Retinoic acid (10−5m) did not affect the rate of degradation of this material in cultures `chased' with 5mm-unlabelled glucosamine after 4 or 18h. 6. Cellulose acetate electrophoresis at pH7.2 revealed that hyaluronic acid was the major labelled glycosaminoglycan (80–90%) in both control and 10−5m-all-trans-retinoic acid

  9. MATRIX METALLOPROTEINS (MMP)-MEDIATED PHOSPHORYLATION OF THE EPIDERMAL GROWTH FACTOR RECEPTOR (EGFR) IN HUMAN AIRWAY EPITHELIAL CELLS (HAEC) EXPOSED TO ZINC (ZN)

    EPA Science Inventory

    Matrix Metalloproteinase (MMP)-Mediated Phosphorylation of The Epidermal Growth Factor Receptor (EGFR) in Human Airway Epithelial Cells (HAEC) Exposed to Zinc (Zn)
    Weidong Wu, James M. Samet, Robert Silbajoris, Lisa A. Dailey, Lee M. Graves, and Philip A. Bromberg
    Center fo...

  10. Increase in epidermal planar cell density accompanies decreased russeting of “Golden Delicious” apples treated with gibberellin A4+7

    Technology Transfer Automated Retrieval System (TEKTRAN)

    A two-year study was conducted in a “Golden Delicious” (Malus Xdomestica Borkh.) orchard having a high historical incidence of physiological fruit russeting, to examine the effect of gibberellin A4+7 (GA4+7) on apple epidermal cell size. Beginning at petal fall, four sequential applications of GA4+7...

  11. Increased epidermal growth factor receptor gene expression by gamma-interferon in a human breast carcinoma cell line.

    PubMed Central

    Hamburger, A. W.; Pinnamaneni, G. D.

    1991-01-01

    The interferons are a group of naturally occurring proteins that inhibit the growth of tumours in vivo and many transformed cell lines in vitro. The mechanisms of action of interferon, however, remain unclear. The IFN induced inhibition of growth of many epithelial cancer cell lines is associated with changes in Epidermal Growth Factor Receptor (EGFR) binding or expression. Therefore, we examined the effect of IFN treatment on the expression of EGFR in a human breast carcinoma cell line, MDA 468. We have found the IFN-gamma inhibited, in a dose dependent fashion, the growth of MDA 468 cells. IFN decreased cell surface binding of 125I-EGF to EGFR by changing receptor number rather than affinity. However, total cellular receptor protein, as measured by immunoprecipitation with monoclonal antibodies, was increased in IFN-treated cells. The half-life of the metabolically labelled receptor was unchanged by treatment with IFN. Increased amounts of EGFR mRNA were observed in MDA 468 cells treated with IFN-gamma for 3 days. The levels of mRNA increased with time in culture, reaching a peak of four times control values after 5 days of treatment. This effect was observable with as little as 10 U ml-1 of IFN-gamma. Treatment of the cells with Actinomycin D to inhibit new RNA synthesis suggested that the stability of EGFR mRNA was not enhanced in IFN-gamma treated cells. The increase in receptor mRNA induced by IFN was not inhibited by cycloheximide. These data suggest IFN-gamma can increase expression of EGFR mRNA and protein in MDA 468 cells. Increased expression of EGFR mRNA and protein by IFN-gamma is associated with inhibition of cell growth. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 PMID:1906727

  12. Benzyladenine and gibberellin treatment of developing "Pink Lady" apples results in mature fruits with a thicker cuticle comprising clusters of epidermal cells.

    PubMed

    Fogelman, Edna; Stern, Raphael A; Ginzberg, Idit

    2015-07-01

    A mixture of 6-benzyladenine (BA) and gibberellins GA4 plus GA7 applied to "Pink Lady" apple at early phenological stages was previously shown to result in an immediate increase in epidermal cell density and associated reduction in calyx-end cracking disorder in the mature fruit, implying a long-term effect of the BA + GA4+7 mixture. Here, we analyzed the anatomical changes in the mature peel at the calyx end 210 days after full bloom (DAFB), following application of the plant growth regulators (PGRs) at the cell-division phase of fruit development, 21-50 DAFB. Experiments were conducted in northern Israel, and the PGRs were applied as the commercial formulation Superlon™ (Fine Agrochemicals Ltd.), composed of 19 g l(-1) BA and 19 g l(-1) GA4+7. Trees were sprayed with 0.025, 0.1, or 0.2 % (v/v) Superlon™. The most obvious phenomenon was the presence of epidermal cell clusters within the cuticular matrix that were detached from the native epidermal layer located at the bottom of the cuticle and which could not be detected in the untreated control fruits. Treatment with 20 mg l(-1) BA + GA4+7 (0.1 % Superlon™) resulted in a markedly thicker cuticle, a higher percentage of detached epidermal cells within the cuticular membrane and a significant reduction in calyx-end cracking at harvest. The presence of cuticle-embedded epidermal cell clusters may have contributed to strengthening the peel by adding more cell-wall components, thickening the cuticle layer and possibly enhancing crack repair. PMID:25433445

  13. Extracellular hydrogen peroxide, produced through a respiratory burst oxidase/superoxide dismutase pathway, directs ingrowth wall formation in epidermal transfer cells of Vicia faba cotyledons.

    PubMed

    Xia, Xue; Zhang, Hui-Ming; Andriunas, Felicity A; Offler, Christina E; Patrick, John W

    2012-09-01

    The intricate, and often polarized, ingrowth walls of transfer cells (TCs) amplify their plasma membrane surface areas to confer a transport function of supporting high rates of nutrient exchange across apo-/symplasmic interfaces. The TC ingrowth wall comprises a uniform wall layer on which wall ingrowths are deposited. Signals and signal cascades inducing trans-differentiation events leading to formation of TC ingrowth walls are poorly understood. Vicia faba cotyledons offer a robust experimental model to examine TC induction as, when placed into culture, their adaxial epidermal cells rapidly (h) and synchronously form polarized ingrowth walls accessible for experimental observations. Using this model, we recently reported findings consistent with extracellular hydrogen peroxide, produced through a respiratory burst oxidase homolog/superoxide dismutase pathway, initiating cell wall biosynthetic activity and providing directional information guiding deposition of the polarized uniform wall. Our conclusions rested on observations derived from pharmacological manipulations of hydrogen peroxide production and correlative gene expression data sets. A series of additional studies were undertaken, the results of which verify that extracellular hydrogen peroxide contributes to regulating ingrowth wall formation and is generated by a respiratory burst oxidase homolog/superoxide dismutase pathway. PMID:22899058

  14. MIIP accelerates epidermal growth factor receptor protein turnover and attenuates proliferation in non-small cell lung cancer

    PubMed Central

    Wen, Jing; Fu, Jianhua; Ling, Yihong; Zhang, Wei

    2016-01-01

    The migration and invasion inhibitory protein (MIIP) has been discovered recently to have inhibitory functions in cell proliferation and migration. Overexpression of MIIP reduced the intracellular steady-state level of epidermal growth factor receptor (EGFR) protein in lung cancer cells with no effect on EGFR mRNA expression compared to that in the control cells. This MIIP-promoted EGFR protein degradation was reversed by proteasome and lysosome inhibitors, suggesting the involvement of both proteasomal and lysosomal pathways in this degradation. This finding was further validated by pulse-chase experiments using 35S-methionine metabolic labeling. We found that MIIP accelerates EGFR protein turnover via proteasomal degradation in the endoplasmic reticulum and then via the lysosomal pathway after its entry into endocytic trafficking. MIIP-stimulated downregulation of EGFR inhibits downstream activation of Ras and blocks the MEK signal transduction pathway, resulting in inhibition of cell proliferation. The negative correlation between MIIP and EGFR protein expression was validated in lung adenocarcinoma samples. Furthermore, the higher MIIP protein expression predicts a better overall survival of Stage IA-IIIA lung adenocarcinoma patients who underwent radical surgery. These findings reveal a new mechanism by which MIIP inhibits cell proliferation. PMID:26824318

  15. Axl mediates acquired resistance of head and neck cancer cells to the epidermal growth factor receptor inhibitor erlotinib.

    PubMed

    Giles, Keith M; Kalinowski, Felicity C; Candy, Patrick A; Epis, Michael R; Zhang, Priscilla M; Redfern, Andrew D; Stuart, Lisa M; Goodall, Gregory J; Leedman, Peter J

    2013-11-01

    Elevated expression and activity of the epidermal growth factor receptor (EGFR) is associated with development and progression of head and neck cancer (HNC) and a poor prognosis. Clinical trials with EGFR tyrosine kinase inhibitors (e.g., erlotinib) have been disappointing in HNC. To investigate the mechanisms mediating resistance to these agents, we developed an HNC cell line (HN5-ER) with acquired erlotinib resistance. In contrast to parental HN5 HNC cells, HN5-ER cells exhibited an epithelial-mesenchymal (EMT) phenotype with increased migratory potential, reduced E-cadherin and epithelial-associated microRNAs (miRNA), and elevated vimentin expression. Phosphorylated receptor tyrosine kinase profiling identified Axl activation in HN5-ER cells. Growth and migration of HN5-ER cells were blocked with a specific Axl inhibitor, R428, and R428 resensitized HN5-ER cells to erlotinib. Microarray analysis of HN5-ER cells confirmed the EMT phenotype associated with acquired erlotinib resistance, and identified activation of gene expression associated with cell migration and inflammation pathways. Moreover, increased expression and secretion of interleukin (IL)-6 and IL-8 in HN5-ER cells suggested a role for inflammatory cytokine signaling in EMT and erlotinib resistance. Expression of the tumor suppressor miR-34a was reduced in HN5-ER cells and increasing its expression abrogated Axl expression and reversed erlotinib resistance. Finally, analysis of 302 HNC patients revealed that high tumor Axl mRNA expression was associated with poorer survival (HR = 1.66, P = 0.007). In summary, our results identify Axl as a key mediator of acquired erlotinib resistance in HNC and suggest that therapeutic inhibition of Axl by small molecule drugs or specific miRNAs might overcome anti-EGFR therapy resistance. PMID:24026012

  16. The trifunctional antibody ertumaxomab destroys tumor cells that express low levels of human epidermal growth factor receptor 2.

    PubMed

    Jäger, Michael; Schoberth, Alexandra; Ruf, Peter; Hess, Jürgen; Lindhofer, Horst

    2009-05-15

    Human epidermal growth factor receptor 2 (HER2/neu) is an important target for the treatment of the breast cancers in which it is overexpressed. However, no approved anti-HER2/neu therapy is available for the majority of breast cancer patients, who express HER2/neu at low levels (with scores of 1+ or 2+/fluorescence in situ hybridization-negative). The trifunctional antibody ertumaxomab targets HER2/neu, CD3, and activating Fcgamma receptors. In presence of ertumaxomab, tri-cell complexes consisting of tumor cells, T cells, and accessory cells form to cause tumor cell lysis. In a phase I trial with metastatic breast cancer patients, ertumaxomab could be applied safely and resulted in radiographically confirmed clinical responses. In this study, we compare ertumaxomab- and trastuzumab-mediated killing of cancer cell lines that express HER2/neu at low and high levels. Under optimal conditions for trastuzumab-mediated destruction of HER2/neu-overexpressing cells, only ertumaxomab was able to mediate the elimination of tumor cell lines that express HER2/neu at low levels (1+). Ertumaxomab-mediated activity was accompanied by a Th1-based cytokine release, a unique mode of action of trifunctional antibodies. Competitive binding studies with trastuzumab and 520C9 mapped the binding site of ertumaxomab to the extracellular regions II and III of the HER2/neu ectodomain. This site is distinct from the binding site of trastuzumab, so that HER2/neu-expressing tumor cells can be eliminated by ertumaxomab in the presence of high amounts of trastuzumab. The ability of ertumaxomab to induce cytotoxicity against various tumor cell lines, including those with low HER2/neu antigen density, may provide a novel therapeutic option for breast cancer patients who are not eligible for trastuzumab treatment. PMID:19435924

  17. MicroRNA-27a functions as a tumor suppressor in renal cell carcinoma by targeting epidermal growth factor receptor

    PubMed Central

    LI, YUEYAN; LI, JIE; SUN, XIAOLEI; CHEN, JIACUN; SUN, XIAOQING; ZHENG, JUNNIAN; CHEN, RENFU

    2016-01-01

    Numerous studies have suggested that microRNAs (miRNAs) are vital in the development of various types of human cancers, including renal cell carcinoma (RCC), and the regulation of tumor progression and invasion. However, the effect of miRNA-27a (miR-27a) on the tumorigenesis of RCC is unclear. The aim of the present study was to investigate the function of miR-27a and identify its possible target genes in RCC cells. In the present study, cell proliferation, migration and invasion and the percentage of apoptotic cells were detected by methylthiazol tetrazolium assays, Annexin V analysis, wound-healing assays and Transwell invasion assays. Western blot analysis was performed to validate the protein expression level and assess whether the epidermal growth factor receptor (EGFR) was a target gene of miR-27a. A tumor xenograft animal model was used to detect the role of miR-27a on RCC cell growth in vivo. The present study demonstrated that miR-27a significantly suppressed human RCC 786-O cell proliferation and induced cell apoptosis. Restoration of miR-27 also resulted in 786-O cell migration and invasion inhibition. Furthermore, upregulated miR-27a attenuated RCC tumor growth in the tumor xenograft animal model. The present results suggested that miR-27a functions as a tumor suppressor in RCC. The western blot analysis assay revealed that EGFR was a novel target of miR-27a. The growth suppression of RCC cells was attributed partly to the downregulation of the cell cycle by ERFR inhibition. The present findings may aid in the understanding of the molecular mechanism of miR-27a in the tumorigenesis of RCC, and may provide novel diagnostic and therapeutic options for RCC. PMID:27313769

  18. Forward genetics identifies Kdf1/1810019J16Rik as an essential regulator of the proliferation-differentiation decision in epidermal progenitor cells

    PubMed Central

    Lee, Sunjin; Kong, Yong; Weatherbee, Scott D.

    2013-01-01

    Cell fate decisions during embryogenesis and adult life govern tissue formation, homeostasis and repair. Two key decisions that must be tightly coordinated are proliferation and differentiation. Overproliferation can lead to hyperplasia or tumor formation while premature differentiation can result in a depletion of proliferating cells and organ failure. Maintaining this balance is especially important in tissues that undergo rapid turnover like skin however, despite recent advances, the genetic mechanisms that balance cell differentiation and proliferation are still unclear. In an unbiased genetic screen to identify genes affecting early development, we identified an essential regulator of the proliferation-differentiation balance in epidermal progenitor cells, the Keratinocyte differentiation factor 1 (Kdf1; 1810019J16Rik) gene. Kdf1 is expressed in epidermal cells from early stages of epidermis formation through adulthood. Specifically, Kdf1 is expressed both in epidermal progenitor cells where it acts to curb the rate of proliferation as well as in their progeny where it is required to block proliferation and promote differentiation. Consequently, Kdf1 mutants display both uncontrolled cell proliferation in the epidermis and failure to develop terminal fates. Our findings reveal a dual role for the novel gene Kdf1 both as a repressive signal for progenitor cell proliferation through its inhibition of p63 and a strong inductive signal for terminal differentiation through its interaction with the cell cycle regulator Stratifin. PMID:24075906

  19. The effects of folate-conjugated gold nanorods in combination with plasmonic photothermal therapy on mouth epidermal carcinoma cells.

    PubMed

    Mehdizadeh, Alireza; Pandesh, Sajjad; Shakeri-Zadeh, Ali; Kamrava, Seyed Kamran; Habib-Agahi, Mojtaba; Farhadi, Mohammad; Pishghadam, Morteza; Ahmadi, Amirhossein; Arami, Sanam; Fedutik, Yuri

    2014-05-01

    The use of lasers has emerged to be highly promising for cancer therapy modalities, most commonly, the photothermal therapy method. Unfortunately, the most common disadvantage of laser therapy is its nonselectivity and requirement of high power density. The use of plasmonic nanoparticles as highly enhanced photoabsorbing agents has thus introduced a much more selective and efficient cancer therapy strategy. In this study, we aimed to demonstrate the selective targeting and destruction of mouth epidermal carcinoma cells (KB cells) using the photothermal therapy of folate-conjugated gold nanorods (F-GNRs). Considering the beneficial characteristics of GNRs and overexpression of the folate receptor by KB cells, we selected F-GNRs as a targeted photothermal therapy agent. Cell viability was evaluated using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Apoptosis was determined by flow cytometry using an annexin V-fluorescein isothiocyanate/propidium iodide apoptosis detection kit. No cell damage or cytotoxicity from the individual treatment of laser light or F-GNRs was observed. However, a 56% cell lethality was achieved for KB cells using combined plasmonic photothermal therapy of 20 μM F-GNRs with seven pulses of laser light and 6-h incubation periods. Cell lethality strongly depends on the concentration of F-GNRs and the incubation period that is mainly due to the induction of apoptosis. This targeted damage is due to the F-GNRs present in the cancer cells strongly absorbing near-infrared laser light and rapidly converting it to heat. This new therapeutic avenue for cancer therapy merits further investigation using in vivo models for application in humans. PMID:24013622

  20. Stimulation of prostaglandin E/sub 2/ production by phorbol esters and epidermal growth factor in porcine thyroid cells

    SciTech Connect

    Kasai, K.; Hiraiwa, M.; Emoto, T.; Akimoto, K.; Takaoka, T.; Shimoda, S.I.

    1987-07-13

    Effects of phorbol esters and epidermal growth factor (EGF) on prostaglandin E/sub 2/ production by cultured porcine thyroid cells were examined. Both phorbol 12-myristate 13-acetate (PMA) and EGF stimulated prostaglandin E/sub 2/ production by the cells in dose related fashion. PMA stimulated prostaglandin E/sub 2/ production over fifty-fold with the dose of 10/sup -7/ M compared with control. EGF (10/sup -7/ M) also stimulated it about ten-fold. The ED/sub 50/ values of PMA and EGF were respectively around 1 x 10/sup -9/ M and 5 x 10/sup -10/ M. Thyroid stimulating hormone (TSH), however, did not stimulate prostaglandin E/sub 2/ production from 1 to 24-h incubation. The release of radioactivity from (/sup 3/H)-arachidonic acid prelabeled cells was also stimulated by PMA and EGF, but not by TSH. These results indicate that both PMA and EGF are potent stimulators of prostaglandin E/sub 2/ production, associated with the activity to stimulate arachidonic acid release in porcine thyroid cells. 36 references, 2 figures, 1 table.

  1. Shedding of epidermal growth factor receptor is a regulated process that occurs with overexpression in malignant cells.

    PubMed

    Perez-Torres, Marianela; Valle, Blanca L; Maihle, Nita J; Negron-Vega, Lisandra; Nieves-Alicea, Rene; Cora, Elsa M

    2008-10-01

    Soluble isoforms of the epidermal growth factor receptor (sEGFR) previously have been identified in the conditioned culture media (CCM) of the vulvar adenocarcinoma cell line, A431 and within exosomes of the keratinocyte cell line HaCaT. Here, we report that the extracellular domain (ECD) of EGFR is shed from the cell surface of human carcinoma cell lines that express 7x10(5) receptors/cell or more. We purified this proteolytic isoform of EGFR (PI-sEGFR) from the CCM of MDA-MB-468 breast cancer cells. The amino acid sequence of PI-sEGFR was determined by reverse-phase HPLC nano-electrospray tandem mass spectrometry of peptides generated by trypsin, chymotrypsin or GluC digestion. The PI-sEGFR protein is identical in amino acid sequence to the EGFR ECD. The release of PI-sEGFR from MDA-MB-468 cells is enhanced by phorbol 12-myristate 13-acetate, heat-inactivated fetal bovine serum, pervanadate, and EGFR ligands (i.e., EGF and TGF-alpha). In addition, 4-aminophenylmercuric acetate, an activator of metalloproteases, increased PI-sEGFR levels in the CCM of MDA-MB-468 cells. Inhibitors of metalloproteases decreased the constitutive shedding of EGFR while the PMA-induced shedding was inhibited by metalloprotease inhibitors, by the two serine protease inhibitors leupeptin and 3,4-dichloroisocoumarin (DCI), and by the aspartyl inhibitor pepstatin. These results suggest that PI-sEGFR arises by proteolytic cleavage of EGFR via a mechanism that is regulated by both PKC- and phosphorylation-dependent pathways. Our results further suggest that when proteolytic shedding of EGFR does occur, it is correlated with a highly malignant phenotype. PMID:18687326

  2. Zebrafish grainyhead-like1 is a common marker of different non-keratinocyte epidermal cell lineages, which segregate from each other in a Foxi3-dependent manner

    PubMed Central

    JÄNICKE, MARTINA; RENISCH, BJÖRN; HAMMERSCHMIDT, MATTHIAS

    2012-01-01

    Grainyhead/CP2 transcription factor family members are widely conserved among the animal kingdom and have been implicated in different developmental processes. Thus far, nothing has been known about their roles in zebrafish. Here we identify seven zebrafish grainyhead-like (grhl) / cp2 genes, with focus on grhl1, which is expressed in the periderm and in epidermal ionocyte progenitors, but downregulated when ionocytes differentiate. In addition, expression was detected in other “non-keratinocyte” cell types of the epidermis, such as pvalb8-expressing cells, which according to our lineage tracing experiments are derived from the same pool of progenitor cells like keratinocytes and ionocytes. Antisense morpholino oligonucleotide-based loss-of-function analysis revealed that grhl1 is dispensable for the development and function of all investigated epidermal cell types, but required as a negative regulator of its own transcription during ionocyte differentiation. Knockdown of the transcription factor Foxi3a, which is expressed in a subset of the grhl1 population, caused a loss of ionocytes and a corresponding increase in the number of pvalb8-expressing cells, while leaving the number of grhl1-positive cells unaltered. We propose that grhl1 is a novel common marker of all or most “non-keratinocyte” epidermal progenitors, and that the sub-functionalisation of these cells is regulated by differential positive and negative effects of Foxi3 factors. PMID:19757382

  3. Role of epidermal γδ T-cell-derived interleukin 13 in the skin-whitening effect of Ginsenoside F1.

    PubMed

    Han, Jiyeon; Lee, Eunkyung; Kim, EunJoo; Yeom, Myung Hun; Kwon, Ohsang; Yoon, Tae Hong; Lee, Tae Ryong; Kim, Kwangmi

    2014-11-01

    Ginsenoside F1 (GF1) is a metabolite of ginsenoside Rg1. Although GF1 has several benefits for skin physiology, the effect of GF1 on skin pigmentation has not been reported. We found that a cream containing 0.1% GF1 showed a significant whitening effect on artificially tanned human skin after 8 weeks of application. However, GF1 did not inhibit mRNA expression of tyrosinase or dopachrome tautomerase (DCT) in normal human epidermal melanocytes (NHEMs) or cocultured NHEMs/normal human epidermal keratinocytes. Interestingly, GF1 enhanced production of interleukin 13 (IL-13) from human epidermal γδ T cells. IL-13 significantly reduced the mRNA expression and protein amount of both tyrosinase and DCT and reduced melanin synthesis activities in NHEMs, resulting in visible brightening of NHEM pellet. These results suggest that enhancement of IL-13 production by GF1 from epidermal γδ T cells might play a role in the skin-whitening effect of GF1 via the suppression of tyrosinase and DCT. PMID:25091975

  4. Inhibitory effects of tetrandrine on epidermal growth factor-induced invasion and migration in HT29 human colorectal adenocarcinoma cells.

    PubMed

    Horng, Chi-Ting; Yang, Jai-Sing; Chiang, Jo-Hua; Lu, Chi-Cheng; Lee, Chiu-Fang; Chiang, Ni-Na; Chen, Fu-An

    2016-01-01

    Tetrandrine has been shown to reduce cancer cell proliferation and to inhibit metastatic effects in multiple cancer models in vitro and in vivo. However, the effects of tetrandrine on the underlying mechanism of HT29 human colorectal adenocarcinoma cell metastasis remain to be fully elucidated. The aim of the present study was focused on tetrandrine‑treated HT29 cells following epidermal growth factor (EGF) treatment, and Transwell, gelatin zymography, gene expression and immunoblotting assays were performed to investigate metastatic effects in vitro. Tetrandrine was observed to dose‑dependently inhibit EGF‑induced HT29 cell invasion and migration, however, no effect on cell viability occurred following exposure to tetradrine between 0.5 and 2 µM. Tetrandrine treatment inhibited the enzymatic activity of matrix metalloprotease (MMP)‑2 and MMP‑9 in a concentration‑dependent manner. The present study also found a reduction in the mRNA expression levels of MMP‑2 and MMP‑9 in the tetrandrine‑treated HT29 cells. Tetrandrine also suppressed the phosphorylation of EGF receptor (EGFR) and its downstream pathway, including phosphoinositide‑dependent kinase 1, phosphatidylinositol 3‑kinase and phosphorylated AKT, suppressing the gene expression of MMP‑2 and MMP‑9. Furthermore, tetrandrine triggered mitogen‑activated protein kinase signaling through the suppressing the activation of phosphorylated extracellular signal‑regulated protein kinase. These data suggested that targeting EGFR signaling and its downstream molecules contributed to the inhibition of EGF‑induced HT29 cell metastasis caused by tetrandrine, eventually leading to a reduction in the mRNA and gelatinase activities of MMP-2 and MMP-9, respectively. PMID:26648313

  5. Downregulation of Epidermal Growth Factor Receptor Expression Contributes to α-TEA's Proapoptotic Effects in Human Ovarian Cancer Cell Lines

    PubMed Central

    Shun, Ming-Chieh; Yu, Weiping; Park, Sook-Kyung; Sanders, Bob G.; Kline, Kimberly

    2010-01-01

    RRR-α-tocopherol derivative α-TEA (RRR-α-tocopherol ether-linked acetic acid analog) has been shown to be a potent antitumor agent both in vivo and in vitro. In this study, we investigated the effects of α-TEA on the expression of epidermal growth factor receptor (EGFR) family members, ErbB1, 2 and 3, and the role of ErbB 2 and 3 in α-TEA-induced apoptosis and suppression of Akt, FLIP and survivin in the cisplatin-sensitive (A2780S) and -resistant (A2780/CP70R) human ovarian cancer cell lines. Data show that α-TEA's ability to induced apoptosis was associated with reduced expression of ErbB1 (cisplatin-resistant cells), 2 and 3 (both cell types) and reduced levels of the phosphorylated (active) form of Akt; as well as, reduced levels of FLIP and survivin proteins in both cell types. Ectopic overexpression and siRNA knockdown studies showed that ErbB2, ErbB3, Akt, FLIP and survivin are involved in α-TEA-induce apoptosis and that α-TEA downregulates FLIP and survivin via suppression of pAkt, which is mediated by ErbB2 and ErB3. Thus, α-TEA is a potent pro-apoptotic agent for both cisplatin-sensitive and -resistant ovarian cancer cell lines in cell culture and it produces cell death, at least in part, by downregulation of members of the EGFR family. PMID:20224651

  6. Epidermal Growth Factor Removal or Tyrphostin AG1478 Treatment Reduces Goblet Cells & Mucus Secretion of Epithelial Cells from Asthmatic Children Using the Air-Liquid Interface Model

    PubMed Central

    Parker, Jeremy C.; Douglas, Isobel; Bell, Jennifer; Comer, David; Bailie, Keith; Skibinski, Grzegorz; Heaney, Liam G.; Shields, Michael D.

    2015-01-01

    Rationale Epithelial remodelling in asthma is characterised by goblet cell hyperplasia and mucus hypersecretion for which no therapies exist. Differentiated bronchial air-liquid interface cultures from asthmatic children display high goblet cell numbers. Epidermal growth factor and its receptor have been implicated in goblet cell hyperplasia. Objectives We hypothesised that EGF removal or tyrphostin AG1478 treatment of differentiating air-liquid interface cultures from asthmatic children would result in a reduction of epithelial goblet cells and mucus secretion. Methods In Aim 1 primary bronchial epithelial cells from non-asthmatic (n = 5) and asthmatic (n = 5) children were differentiated under EGF-positive (10ng/ml EGF) and EGF-negative culture conditions for 28 days. In Aim 2, cultures from a further group of asthmatic children (n = 5) were grown under tyrphostin AG1478, a tyrosine kinase inhibitor, conditions. All cultures were analysed for epithelial resistance, markers of differentiation using immunocytochemistry, ELISA for MUC5AC mucin secretion and qPCR for MUC5AC mRNA. Results In cultures from asthmatic children the goblet cell number was reduced in the EGF negative group (p = 0.01). Tyrphostin AG1478 treatment of cultures from asthmatic children had significant reductions in goblet cells at 0.2μg/ml (p = 0.03) and 2μg/ml (p = 0.003) as well as mucus secretion at 2μg/ml (p = 0.04). Conclusions We have shown in this preliminary study that through EGF removal and tyrphostin AG1478 treatment the goblet cell number and mucus hypersecretion in differentiating air-liquid interface cultures from asthmatic children is significantly reduced. This further highlights the epidermal growth factor receptor as a potential therapeutic target to inhibit goblet cell hyperplasia and mucus hypersecretion in asthma. PMID:26057128

  7. Blister fluid T lymphocytes during toxic epidermal necrolysis are functional cytotoxic cells which express human natural killer (NK) inhibitory receptors

    PubMed Central

    Le Cleach, L; Delaire, S; Boumsell, L; Bagot, M; Bourgault-Villada, I; Bensussan, A; Roujeau, J C

    2000-01-01

    Toxic epidermal necrolysis (TEN) is a rare life-threatening adverse drug reaction characterized by a massive destruction of the epidermis. Immunohistological studies of skin biopsies of TEN showed infiltrates of predominantly CD8+ T lymphocytes even though other authors reported a prominent involvement of cells of the monocyte-macrophage lineage. The aim of this study was to characterize phenotypically and functionally the cells present in the cutaneous blister fluid of four patients with TEN. We first determined that lymphocytes were predominant in blister fluid obtained early, while monocytes/macrophages later became the most important population. We then showed that this lymphocyte population, mainly CD3+CD8+, corresponded to a peculiar cell subset as they expressed cutaneous leucocyte antigen, killer inhibitory receptors KIR/KAR and failed to express CD28 molecule. Functionally, we determined that blister T lymphocytes had a cytotoxic T lymphocyte (CTL)- and NK-like cytotoxicity. The role of this cytotoxic lymphocyte population present at the site of lesions during TEN remains to be understood. PMID:10606987

  8. Targeting of epidermal growth factor receptor (EGFR)-expressing tumor cells with sterically stabilized affibody liposomes (SAL).

    PubMed

    Beuttler, Julia; Rothdiener, Miriam; Müller, Dafne; Frejd, Fredrik Y; Kontermann, Roland E

    2009-06-01

    Affibody molecules are small and stable antigen-binding molecules derived from the B domain of protein A. We applied a bivalent, high-affinity epidermal growth factor receptor (EGFR)-specific affibody molecule for the generation of targeted PEGylated liposomes. These sterically stabilized affibody liposomes (SAL) were produced by chemical coupling of the cysteine-modified affibody molecule to maleimide-PEG(2000)-DSPE and subsequent insertion into PEGylated liposomes. These SAL showed strong and selective binding to EGFR-expressing tumor cell lines. Binding was dependent on the amount of inserted affibody molecule-lipid conjugates and could be blocked by soluble EGF. Approximately 30% of binding activity was still retained after 6 days of incubation in human plasma at 37 degrees C. Binding of SAL to cells led to efficient internalization of the liposomes. Using mitoxantrone-loaded liposomes, we observed for SAL, compared to untargeted liposomes, an enhanced cytotoxicity toward EGFR-expressing cells. In summary, we show that SAL can be easily prepared from affibody molecules and thus may be suitable for the development of carrier systems for targeted delivery of drugs. PMID:19435362

  9. Progressive alopecia reveals decreasing stem cell activation probability during aging of mice with epidermal deletion of DNA methyltransferase 1.

    PubMed

    Li, Ji; Jiang, Ting-Xin; Hughes, Michael W; Wu, Ping; Yu, Juehua; Widelitz, Randall B; Fan, Guoping; Chuong, Cheng-Ming

    2012-12-01

    To examine the roles of epigenetic modulation on hair follicle regeneration, we generated mice with a K14-Cre-mediated loss of DNA methyltransferase 1 (DNMT1). The mutant shows an uneven epidermal thickness and alterations in hair follicle size. When formed, hair follicle architecture and differentiation appear normal. Hair subtypes exist but hair fibers are shorter and thinner. Hair numbers appear normal at birth but gradually decrease to <50% of control in 1-year-old mice. Sections of old mutant skin show follicles in prolonged telogen with hyperplastic sebaceous glands. Anagen follicles in mutants exhibit decreased proliferation and increased apoptosis in matrix transient-amplifying cells. Although K15-positive stem cells in the mutant bulge are comparable in number to the control, their ability to proliferate and become activated to form a hair germ is reduced. As mice age, residual DNMT activity declines further, and the probability of successful anagen reentry decreases, leading to progressive alopecia. Paradoxically, there is increased proliferation in the epidermis, which also shows aberrant differentiation. These results highlight the importance of DNA methylation in maintaining stem cell homeostasis during the development and regeneration of ectodermal organs. PMID:22763785

  10. Epidermal growth factor receptor gene-amplified MDA-468 breast cancer cell line and its nonamplified variants.

    PubMed Central

    Filmus, J; Trent, J M; Pollak, M N; Buick, R N

    1987-01-01

    We have recently reported (J. Filmus, M. N. Pollak, R. Cailleau, and R. N. Buick, Biochem. Biophys. Res. Commun. 128:898-905, 1985) that MDA-468, a human breast cancer cell line with a high number of epidermal growth factor (EGF) receptors, has an amplified EGF receptor gene and is growth inhibited in vitro pharmacological doses of EGF. We have derived several MDA-468 clonal variants which are resistant to EGF-induced growth inhibition. These clones had a number of EGF receptors, similar to normal human fibroblasts, and had lost the EGF receptor gene amplification. Karyotype analysis showed that MDA-468 cells had an abnormally banded region (ABR) in chromosome 7p which was not present in the variants. It was shown by in situ hybridization that the amplified EGF receptor sequences were located in that chromosome, 7pABR. Five of the six variants studied were able to generate tumors in nude mice, but their growth rate was significantly lower than that of tumors derived from the parental cell line. The variant that was unable to produce tumors was found to be uniquely dependent on EGF for growth in soft agar. Images PMID:3494191

  11. Screening and discovery of nitro-benzoxadiazole compounds activating epidermal growth factor receptor (EGFR) in cancer cells.

    PubMed

    Sakanyan, Vehary; Angelini, Marie; Le Béchec, Mickael; Lecocq, Michèle Françoise; Benaiteau, Florence; Rousseau, Bénédicte; Gyulkhandanyan, Aram; Gyulkhandanyan, Lusine; Logé, Cédric; Reiter, Eric; Roussakis, Christos; Fleury, Fabrice

    2014-01-01

    Peptide ligand-induced dimerization of the extracellular region of the epidermal growth factor receptor (sEGFR) is central to the signal transduction of many cellular processes. A small molecule microarray screen has been developed to search for non-peptide compounds able to bind to sEGFR. We describe the discovery of nitro-benzoxadiazole (NBD) compounds that enhance tyrosine phosphorylation of EGFR and thereby trigger downstream signaling pathways and other receptor tyrosine kinases in cancer cells. The protein phosphorylation profile in cells exposed to NBD compounds is to some extent reminiscent of the profile induced by the cognate ligand. Experimental studies indicate that the small compounds bind to the dimerization domain of sEGFR, and generate stable dimers providing allosteric activation of the receptor. Moreover, receptor phosphorylation is associated with inhibition of PTP-1B phosphatase. Our data offer a promising paradigm for investigating new aspects of signal transduction mediated by EGFR in cancer cells exposed to electrophilic NBD compounds. PMID:24496106

  12. Screening and discovery of nitro-benzoxadiazole compounds activating epidermal growth factor receptor (EGFR) in cancer cells

    PubMed Central

    Sakanyan, Vehary; Angelini, Marie; Le Béchec, Mickael; Lecocq, Michèle Françoise; Benaiteau, Florence; Rousseau, Bénédicte; Gyulkhandanyan, Aram; Gyulkhandanyan, Lusine; Logé, Cédric; Reiter, Eric; Roussakis, Christos; Fleury, Fabrice

    2014-01-01

    Peptide ligand-induced dimerization of the extracellular region of the epidermal growth factor receptor (sEGFR) is central to the signal transduction of many cellular processes. A small molecule microarray screen has been developed to search for non-peptide compounds able to bind to sEGFR. We describe the discovery of nitro-benzoxadiazole (NBD) compounds that enhance tyrosine phosphorylation of EGFR and thereby trigger downstream signaling pathways and other receptor tyrosine kinases in cancer cells. The protein phosphorylation profile in cells exposed to NBD compounds is to some extent reminiscent of the profile induced by the cognate ligand. Experimental studies indicate that the small compounds bind to the dimerization domain of sEGFR, and generate stable dimers providing allosteric activation of the receptor. Moreover, receptor phosphorylation is associated with inhibition of PTP-1B phosphatase. Our data offer a promising paradigm for investigating new aspects of signal transduction mediated by EGFR in cancer cells exposed to electrophilic NBD compounds. PMID:24496106

  13. Role of 3'-5'-cyclic adenosine monophosphate on the epidermal growth factor dependent survival in mammary epithelial cells.

    PubMed

    Grinman, Diego Y; Romorini, Leonardo; Presman, Diego M; Rocha-Viegas, Luciana; Coso, Omar A; Davio, Carlos; Pecci, Adali

    2016-01-01

    Epidermal growth factor (EGF) has been suggested to play a key role in the maintenance of epithelial cell survival during lactation. Previously, we demonstrated that EGF dependent activation of PI3K pathway prevents apoptosis in confluent murine HC11 cells cultured under low nutrient conditions. The EGF protective effect is associated with increased levels of the antiapoptotic protein Bcl-XL. Here, we identify the EGF-dependent mechanism involved in cell survival that converges in the regulation of bcl-X expression by activated CREB. EGF induces Bcl-XL expression through activation of a unique bcl-X promoter, the P1; being not only the PI3K/AKT signaling pathway but also the increase in cAMP levels and the concomitant PKA/CREB activation necessary for both bcl-XL upregulation and apoptosis avoidance. Results presented in this work suggest the existence of a novel connection between the EGF receptor and the adenylate cyclase that would have an impact in preventing apoptosis under low nutrient conditions. PMID:26522133

  14. Lactoperoxidase-catalyzed iodination of membrane proteins in normal and neoplastic epidermal cells

    SciTech Connect

    Brysk, M.M.; Snider, J.M.

    1982-01-01

    Cell surface proteins of normal human, mouse, and rat cells in primary culture, of human basal cell carcinoma, and of carcinogen-transformed cell lines were examined by lactoperoxidase-catalyzed iodination. Autoradiography was used to record the distribution of label in the polypeptide subunits separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis. There was no significant difference in the results for normal cells of human, mouse, and rat. On the other hand, carcinogen-transformed mouse cells had many more labeled polypeptide bands of widely distributed molecular weights. The iodination profiles from human basal cell carcinoma cells were much more akin to those from normal cells than to those from carcinogen-transformed cells. Treatment of iodinated cells with proteolytic enzymes visibly altered the polypeptide bands.

  15. Cross-talk Between Estrogen Receptor and Epidermal Growth Factor Receptor in Head and Neck Squamous Cell Carcinoma

    PubMed Central

    Egloff, Ann Marie; Rothstein, Mary E.; Seethala, Raja; Siegfried, Jill M.; Grandis, Jennifer Rubin.; Stabile, Laura P.

    2009-01-01

    Purpose To characterize estrogen receptor (ER) expression and signaling in head and neck squamous cell carcinoma (HNSCC) cell lines and patient tissues and evaluate ER and epidermal growth factor (EGF) receptor (EGFR) cross-activation in HNSCC. Experimental Design ER expression and signaling in HNSCC cell lines were assessed by immunoblotting. In vitro proliferation and invasion were evaluated in HNSCC cell lines in response to ER and EGFR ligands or inhibitors. ER and EGFR protein expression in patient tissues was assessed by immunohistochemical (IHC) staining. Results Phospho-MAP kinase (P-MAPK) levels were significantly increased following combined estrogen (E2) and EGF treatment. Treatment of HNSCC cells with E2 and EGF significantly increased cell invasion compared to either treatment alone while inhibiting these two pathways resulted in reduced invasion compared to inhibiting either pathway alone. EGFR (P=0.008) and nuclear ERα (ERαnuc) (P<0.001) levels were significantly increased in HNSCC tumors (n=56) compared to adjacent mucosa (n=30) while ERβnuc levels did not differ (P=0.67). Patients with high ERαnuc and EGFR tumor levels had significantly reduced PFS compared to patients low tumor ERαnuc and EGFR levels (H.R. = 4.09, P = 0.01; Cox proportional hazards). In contrast, high ERβnuc tumor levels were not associated with reduced PFS alone or when combined with EGFR. Conclusions ERα and ERβ were expressed in HNSCC and stimulation with ER ligands resulted in both cytoplasmic signal transduction and transcriptional activation. ER and EGFR cross-talk was observed. Collectively, these studies indicate ER and EGFR together may contribute to HNSCC development and disease progression. PMID:19825947

  16. 7,3',4'-Trihydroxyisoflavone inhibits epidermal growth factor-induced proliferation and transformation of JB6 P+ mouse epidermal cells by suppressing cyclin-dependent kinases and phosphatidylinositol 3-kinase.

    PubMed

    Lee, Dong Eun; Lee, Ki Won; Song, Nu Ry; Seo, Sang Kwon; Heo, Yong-Seok; Kang, Nam Joo; Bode, Ann M; Lee, Hyong Joo; Dong, Zigang

    2010-07-01

    Numerous in vitro and in vivo studies have shown that isoflavones exhibit anti-proliferative activity against epidermal growth factor (EGF) receptor-positive malignancies of the breast, colon, skin, and prostate. 7,3',4'-Trihydroxyisoflavone (7,3',4'-THIF) is one of the metabolites of daidzein, a well known soy isoflavone, but its chemopreventive activity and the underlying molecular mechanisms are poorly understood. In this study, 7,3',4'-THIF prevented EGF-induced neoplastic transformation and proliferation of JB6 P+ mouse epidermal cells. It significantly blocked cell cycle progression of EGF-stimulated cells at the G(1) phase. As shown by Western blot, 7,3',4'-THIF suppressed the phosphorylation of retinoblastoma protein at Ser-795 and Ser-807/Ser-811, which are the specific sites of phosphorylation by cyclin-dependent kinase (CDK) 4. It also inhibited the expression of G(1) phase-regulatory proteins, including cyclin D1, CDK4, cyclin E, and CDK2. In addition to regulating the expression of cell cycle-regulatory proteins, 7,3',4'-THIF bound to CDK4 and CDK2 and strongly inhibited their kinase activities. It also bound to phosphatidylinositol 3-kinase (PI3K), strongly inhibiting its kinase activity and thereby suppressing the Akt/GSK-3beta/AP-1 pathway and subsequently attenuating the expression of cyclin D1. Collectively, these results suggest that CDKs and PI3K are the primary molecular targets of 7,3',4'-THIF in the suppression of EGF-induced cell proliferation. These insights into the biological actions of 7,3',4'-THIF provide a molecular basis for the possible development of new chemoprotective agents. PMID:20444693

  17. Transient Silencing of CHALCONE SYNTHASE during Fruit Ripening Modifies Tomato Epidermal Cells and Cuticle Properties1[C][W

    PubMed Central

    España, Laura; Heredia-Guerrero, José A.; Reina-Pinto, José J.; Fernández-Muñoz, Rafael; Heredia, Antonio; Domínguez, Eva

    2014-01-01

    Tomato (Solanum lycopersicum) fruit ripening is accompanied by an increase in CHALCONE SYNTHASE (CHS) activity and flavonoid biosynthesis. Flavonoids accumulate in the cuticle, giving its characteristic orange color that contributes to the eventual red color of the ripe fruit. Using virus-induced gene silencing in fruits, we have down-regulated the expression of SlCHS during ripening and compared the cuticles derived from silenced and nonsilenced regions. Silenced regions showed a pink color due to the lack of flavonoids incorporated to the cuticle. This change in color was accompanied by several other changes in the cuticle and epidermis. The epidermal cells displayed a decreased tangential cell width; a decrease in the amount of cuticle and its main components, cutin and polysaccharides, was also observed. Flavonoids dramatically altered the cuticle biomechanical properties by stiffening the elastic and viscoelastic phase and by reducing the ability of the cuticle to deform. There seemed to be a negative relation between SlCHS expression and wax accumulation during ripening that could be related to the decreased cuticle permeability to water observed in the regions silencing SlCHS. A reduction in the overall number of ester linkages present in the cutin matrix was also dependent on the presence of flavonoids. PMID:25277718

  18. The relationship of quantitative epidermal growth factor receptor expression in non-small cell lung cancer to long term survival.

    PubMed Central

    Veale, D.; Kerr, N.; Gibson, G. J.; Kelly, P. J.; Harris, A. L.

    1993-01-01

    Increased expression of epidermal growth factor receptor (EGFr) has been reported in non small cell lung cancers (NSCLC) when compared to normal lung. We have examined post-operative survival in 19 surgically treated patients with NSCLC who had full characterisation of EGFr on primary tumour membrane preparations from resection specimens. There were ten squamous, seven adeno and two large cell carcinomas. The median concentration of high affinity sites was 31 fmol per mg of protein (4-1532) and the median dissociation constant (Kd) of these high affinity sites was 2.3 x 10(-10) per mol (1.2-30 x 10(-10)). Seven patients survived over 5 years. Twelve patients died between 8.5 and 55 months from the time of surgery. When > 5 year survivors were compared to non-survivors there was no difference as regards tumour size or stage, or as regards age or sex. The survivors had a median concentration of high affinity EGFr sites of 16.1 fmol mg-1 protein compared to a median concentration of 68.6 fmol mg-1 protein in the non-survivors (P = 0.01 Wilcoxon test). No long term survivor had > 35 fmol mg-1 protein of receptor. Thus EGFr quantitation may give independent prognostic information in NSCLC and help to select patients for adjuvant therapy after surgery. These results need confirmation in a larger prospective study. PMID:8391303

  19. Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells

    PubMed Central

    Takeuchi, Miyuki; Karahara, Ichirou; Kajimura, Naoko; Takaoka, Akio; Murata, Kazuyoshi; Misaki, Kazuyo; Yonemura, Shigenobu; Staehelin, L. Andrew; Mineyuki, Yoshinobu

    2016-01-01

    The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs. PMID:27053663

  20. Single microfilaments mediate the early steps of microtubule bundling during preprophase band formation in onion cotyledon epidermal cells.

    PubMed

    Takeuchi, Miyuki; Karahara, Ichirou; Kajimura, Naoko; Takaoka, Akio; Murata, Kazuyoshi; Misaki, Kazuyo; Yonemura, Shigenobu; Staehelin, L Andrew; Mineyuki, Yoshinobu

    2016-06-01

    The preprophase band (PPB) is a cytokinetic apparatus that determines the site of cell division in plants. It originates as a broad band of microtubules (MTs) in G2 and narrows to demarcate the future division site during late prophase. Studies with fluorescent probes have shown that PPBs contain F-actin during early stages of their development but become actin depleted in late prophase. Although this suggests that actins contribute to the early stages of PPB formation, how actins contribute to PPB-MT organization remains unsolved. To address this question, we used electron tomography to investigate the spatial relationship between microfilaments (MFs) and MTs at different stages of PPB assembly in onion cotyledon epidermal cells. We demonstrate that the PPB actins observed by fluorescence microscopy correspond to short, single MFs. A majority of the MFs are bound to MTs, with a subset forming MT-MF-MT bridging structures. During the later stages of PPB assembly, the MF-mediated links between MTs are displaced by MT-MT linkers as the PPB MT arrays mature into tightly packed MT bundles. On the basis of these observations, we propose that the primary function of actins during PPB formation is to mediate the initial bundling of the PPB MTs. PMID:27053663

  1. Loss of integrin α3 prevents skin tumor formation by promoting epidermal turnover and depletion of slow-cycling cells

    PubMed Central

    Sachs, Norman; Secades, Pablo; van Hulst, Laura; Kreft, Maaike; Song, Ji-Ying; Sonnenberg, Arnoud

    2012-01-01

    Progression through the various stages of skin tumorigenesis is correlated with an altered expression of the integrin α3β1, suggesting that it plays an important role in the tumorigenic process. Using epidermis-specific Itga3 KO mice subjected to the 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate two-stage skin carcinogenesis protocol, we demonstrate that efficient tumor development is critically dependent on the presence of α3β1. In the absence of α3β1, tumor initiation is dramatically decreased because of increased epidermal turnover, leading to a loss of DMBA-initiated label-retaining keratinocytes. Lineage tracing revealed emigration of α3-deficient keratinocytes residing in the bulge of the hair follicle toward the interfollicular epidermis. Furthermore, tumor growth and cell proliferation were strongly reduced in mice with an epidermis-specific deletion of Itga3. However, the rate of progression of α3β1-null squamous cell carcinomas to undifferentiated, invasive carcinomas was increased. Therefore, α3β1 critically affects skin carcinogenesis with opposing effects early and late in tumorigenesis. PMID:23236172

  2. Loss of integrin α3 prevents skin tumor formation by promoting epidermal turnover and depletion of slow-cycling cells.

    PubMed

    Sachs, Norman; Secades, Pablo; van Hulst, Laura; Kreft, Maaike; Song, Ji-Ying; Sonnenberg, Arnoud

    2012-12-26

    Progression through the various stages of skin tumorigenesis is correlated with an altered expression of the integrin α3β1, suggesting that it plays an important role in the tumorigenic process. Using epidermis-specific Itga3 KO mice subjected to the 7,12-dimethylbenzanthracene (DMBA)/12-O-tetradecanoylphorbol-13-acetate two-stage skin carcinogenesis protocol, we demonstrate that efficient tumor development is critically dependent on the presence of α3β1. In the absence of α3β1, tumor initiation is dramatically decreased because of increased epidermal turnover, leading to a loss of DMBA-initiated label-retaining keratinocytes. Lineage tracing revealed emigration of α3-deficient keratinocytes residing in the bulge of the hair follicle toward the interfollicular epidermis. Furthermore, tumor growth and cell proliferation were strongly reduced in mice with an epidermis-specific deletion of Itga3. However, the rate of progression of α3β1-null squamous cell carcinomas to undifferentiated, invasive carcinomas was increased. Therefore, α3β1 critically affects skin carcinogenesis with opposing effects early and late in tumorigenesis. PMID:23236172

  3. Effects of retinoids on differentiation, lipid metabolism, epidermal growth factor, and low-density lipoprotein binding in squamous carcinoma cells

    SciTech Connect

    Ponec, M.; Weerheim, A. ); Havekes, L. ); Boonstra, J. )

    1987-08-01

    The relationship among keratinocyte differentiation capacity, lipid synthesis, low-density lipoprotein (LDL) metabolism, plasma membrane composition, and epidermal growth factor (EGF) binding has been studied in SCC-12F2 cells. The differentiation capacity of the cells, i.e., ionophore-induced cornified envelope formation, was inhibited by various retinoids and stimulated by hydrocortisone. Retinoids that caused a significant reduction of cornified envelope formation, i.e., retinoic acid and 13-cis-retinoic acid, caused only minor changes in lipid synthesis and plasma membrane composition. Arotinoid ethylsulfone, having a minor effect on cornified envelope formation, caused a drastic inhibition of cholesterol synthesis resulting in changes in the plasma membrane composition. Hydrocortisone stimulated cornified envelope formation but had only minor effects on lipid synthesis and plasma membrane composition. Of all retinoids tested, only arotinoid ethylsulfone caused a drastic increase in EGF binding, while hydrocortisone had no effect. These results clearly demonstrate that the plasma membrane composition is not related to keratinocyte differentiation capacity, but most likely does determine EGF binding. Furthermore, EGF binding does not determine keratinocyte differentiation capacity.

  4. Heparin-Binding Epidermal Growth Factor and Its Receptors Mediate Decidualization and Potentiate Survival of Human Endometrial Stromal Cells

    PubMed Central

    Chobotova, Katya; Karpovich, Natalia; Carver, Janet; Manek, Sanjiv; Gullick, William J.; Barlow, David H.; Mardon, Helen J.

    2006-01-01

    Heparin-binding epidermal growth factor (HB-EGF) has pleiotropic biological functions in many tissues, including those of the female reproductive tract. It facilitates embryo development and mediates implantation and is thought to have a function in endometrial receptivity and maturation. The mature HB-EGF molecule manifests its activity as either a soluble factor (sol-HB-EGF) or a transmembrane precursor (tm-HB-EGF) and can bind two receptors, EGFR and ErbB4/HER4. In this study, we identify factors that modulate expression of HB-EGF, EGFR, and ErbB4 in endometrial stromal cells in vitro. We demonstrate that levels of sol- and tm-HB-EGF, EGFR, and ErbB4 are increased by cAMP, a potent inducer of decidualization of the endometrial stroma. We also show that production of sol- and tm-HB-EGF is differentially modulated by TNFα and TGFβ. Our data suggest that HB-EGF has a function in endometrial maturation in mediating decidualization and attenuating TNFα- and TGFβ-induced apoptosis of endometrial stromal cells. PMID:15562026

  5. Phorbol ester and interferon-gamma modulation of epidermal growth factor receptors on human amniotic (WISH) cells.

    PubMed

    Karasaki, Y; Jaken, S; Komoriya, A; Zoon, K C

    1989-04-15

    In this study we report that pretreatment of human amniotic (WISH) cells with interferon gamma (IFN-gamma) in the presence of 12-O-tetradecanoylphorbol 13-acetate (TPA) resulted in the down-modulation of epidermal growth factor (EGF) receptors with respect to both receptor number and affinity. Scatchard analysis of EGF binding in the absence of both IFN-gamma and TPA indicated biphasic binding whereas addition of TPA resulted in the loss of the higher affinity class of sites. Pretreatment with IFN-gamma for 24 h enhanced the TPA-induced inhibition of EGF binding whereas IFN-gamma alone had no effect on binding. Protein kinase C (Ca2+/phospholipid-dependent enzyme) was examined as a possible mediator of IFN-induced EGF-receptor modulation; pretreatment of cells with IFN-gamma affected neither the binding of [3H]phorbol 12,13-dibutyrate to membrane or cytosolic fractions nor the protein kinase C activity, suggesting that IFN-gamma pretreatment did not result in translocation or activation of protein kinase C. PMID:2495278

  6. Transactivation of the epidermal growth factor receptor mediates muscarinic stimulation of focal adhesion kinase in intestinal epithelial cells.

    PubMed

    Calandrella, Sean O; Barrett, Kim E; Keely, Stephen J

    2005-04-01

    We have previously shown that the Gq protein coupled receptor (GqPCR) agonist, carbachol (CCh), transactivates and recruits epidermal growth factor receptor (EGFr)-dependent signaling mechanisms in intestinal epithelial cells. Increasing evidence suggests that GqPCR agonists can also recruit focal adhesion-dependent signaling pathways in some cell types. Therefore, the aim of the present study was to investigate if CCh stimulates activation of the focal adhesion-associated protein, focal adhesion kinase (FAK), in intestinal epithelia and, if so, to examine the signaling mechanisms involved. Experiments were carried out on monolayers of T84 cells grown on permeable supports. CCh rapidly induced tyrosine phosphorylation of FAK in T84 cells. This effect was accompanied by phosphorylation of another focal adhesion-associated protein, paxillin, and association of FAK with paxillin. CCh-stimulated FAK phosphorylation was inhibited by a chelator of intracellular Ca2+, BAPTA/AM (20 microM), and was mimicked by thapsigargin (2 microM), which mobilizes intracellular Ca2+ in a receptor-independent fashion. CCh also induced association of FAK with the EGFr and FAK phosphorylation was attenuated by an EGFr inhibitor, tyrphostin AG1478, and an inhibitor of Src family kinases, PP2. The actin cytoskeleton disruptor, cytochalasin D (20 microM), abolished FAK phosphorylation in response to CCh but did not alter CCh-induced EGFr or ERK MAPK activation. In summary, these data demonstrate that agonists of GqPCRs have the ability to induce FAK activation in intestinal epithelial cells. GqPCR-induced FAK activation is mediated by via a pathway involving transactivation of the EGFr and alterations in the actin cytoskeleton. PMID:15389641

  7. The Mediator Kinase Module Restrains Epidermal Growth Factor Receptor Signaling and Represses Vulval Cell Fate Specification in Caenorhabditis elegans.

    PubMed

    Grants, Jennifer M; Ying, Lisa T L; Yoda, Akinori; You, Charlotte C; Okano, Hideyuki; Sawa, Hitoshi; Taubert, Stefan

    2016-02-01

    Cell signaling pathways that control proliferation and determine cell fates are tightly regulated to prevent developmental anomalies and cancer. Transcription factors and coregulators are important effectors of signaling pathway output, as they regulate downstream gene programs. In Caenorhabditis elegans, several subunits of the Mediator transcriptional coregulator complex promote or inhibit vulva development, but pertinent mechanisms are poorly defined. Here, we show that Mediator's dissociable cyclin dependent kinase 8 (CDK8) module (CKM), consisting of cdk-8, cic-1/Cyclin C, mdt-12/dpy-22, and mdt-13/let-19, is required to inhibit ectopic vulval cell fates downstream of the epidermal growth factor receptor (EGFR)-Ras-extracellular signal-regulated kinase (ERK) pathway. cdk-8 inhibits ectopic vulva formation by acting downstream of mpk-1/ERK, cell autonomously in vulval cells, and in a kinase-dependent manner. We also provide evidence that the CKM acts as a corepressor for the Ets-family transcription factor LIN-1, as cdk-8 promotes transcriptional repression by LIN-1. In addition, we find that CKM mutation alters Mediator subunit requirements in vulva development: the mdt-23/sur-2 subunit, which is required for vulva development in wild-type worms, is dispensable for ectopic vulva formation in CKM mutants, which instead display hallmarks of unrestrained Mediator tail module activity. We propose a model whereby the CKM controls EGFR-Ras-ERK transcriptional output by corepressing LIN-1 and by fine tuning Mediator specificity, thus balancing transcriptional repression vs. activation in a critical developmental signaling pathway. Collectively, these data offer an explanation for CKM repression of EGFR signaling output and ectopic vulva formation and provide the first evidence of Mediator CKM-tail module subunit crosstalk in animals. PMID:26715664

  8. Partial protection against epidermal IL-10 transcription and Langerhans cell depletion by sunscreens after exposure of human skin to UVB.

    PubMed

    Hochberg, M; Enk, C D

    1999-11-01

    Sunscreens capable of inhibiting erythema are assumed to protect against UV-induced carcinogenesis as well. However, the correlation between inflammation and carcinogenesis is uncertain, and the prevention of UV-induced erythema might in fact be biologically irrelevant as an indicator of protection against UV-induced skin cancer. Ultraviolet-B radiation promotes cutaneous immunosuppression by the release of immunoregulatory cytokines and by depletion of Langerhans cells. We investigated the ability of two different sunscreens to inhibit UVB-induced expression of epidermal interleukin (IL)-10 and depletion of Langerhans cells. Chemical and physical sunscreens were applied to the forearms of volunteers 15 min prior to 4 minimal erythemal doses of UVB exposure. Suction blisters were induced 24 h after irradiation, and RNA was extracted from the blister roofs. Reverse transcription polymerase chain reaction was performed using primers for IL-10 and CD1a. A chemical sunscreen containing octyl methoxycinnamate (12 sun protection factor [SPF]) and a physical sunscreen containing zinc oxide (16 SPF) were assayed: UVB-induced IL-10 mRNA expression was nearly totally inhibited by both sunscreens (median protection for chemical and physical sunscreens was 95% and 78%, respectively), whereas UVB-induced Langerhans cell depletion was partially prevented (47% and 50% for chemical and physical sunscreens, respectively). Langerhans cell protection by sunscreens was confirmed by estimation of cell density after ATPase staining. In contrast, both sunscreens effectively prevented the induction of UVB-induced erythema. We believe this to be the first demonstration that sunscreens can prevent the induction of cutaneous mediators of immunosuppression, and that the results indicate that the immunoprotection offered by the sunscreens is significantly lower than their ability to prevent erythema. PMID:10568168

  9. In vivo demonstration of cell types in bone that harbor epidermal growth factor receptors

    SciTech Connect

    Martineau-Doize, B.; Lai, W.H.; Warshawsky, H.; Bergeron, J.J.

    1988-08-01

    The binding and internalization of (/sup 125/I)iodoepidermal growth factor (EGF) by bone cells of the rat was demonstrated in situ by quantitative radioautography. Specific binding sites were observed on a cell profile enriched in endocytic components, including lysosome-like structures, a rough endoplasmic reticulum-rich cell profile, and a cell profile that histologically resembles an undifferentiated precursor cell. By the criteria of gel filtration and precipitability by trichloroacetic acid, most of the bound (/sup 125/I)iodo-EGF was considered intact. By morphological criteria none of the cell profiles that bound (/sup 125/I)iodo-EGF corresponded to fully formed osteoclasts or osteoblasts. The endocytic cell was found in the epiphyseal plate between the invading capillary and the transverse and longitudinal cartilage septa as well as near osteoclasts in the zone of mixed spicules. The rough endoplasmic reticulum-rich cell was present in vacated chondrocyte lacunae of the epiphyseal plate close to the metaphysis, and the poorly differentiated cell was observed between the mixed spicules of the metaphysis. Similar cell types were also found in the alveolar bone surrounding the incisors. These cells may be the origin of established bone cell lines that harbor high concentrations of EGF receptors and may also be responsible for the humoral hypercalcemia in response to the reported actions of injected EGF or transforming growth factor-alpha as well as that of malignancy.

  10. Enediyne lidamycin enhances the effect of epidermal growth factor receptor tyrosine kinase inhibitor, gefitinib, in epidermoid carcinoma A431 cells and lung carcinoma H460 cells.

    PubMed

    Liu, Hong; Li, Liang; Li, Xing-Qi; Liu, Xiu-Jun; Zhen, Yong-Su

    2009-01-01

    Gefitinib, a low-molecular-weight epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, is effective in a wide variety of tumor types. Preclinical studies have shown potentiated antitumor efficacies of this agent in combination with chemotherapy or radiotherapy. The antitumor antibiotic lidamycin (LDM) showed extremely potent cytotoxicity in vitro and marked therapeutic effect in vivo. In this report, the cytotoxic and biochemical activity of LDM and gefitinib on human epidermoid carcinoma A431 cells and human large cell lung cancer H460 cells as a single agent or in combination has been evaluated. In the MTT assay, LDM showed much more potent cytotoxicity than gefitinib to both cell lines. A431 cells with a highly EGFR-expressing level were more sensitive to gefitinib than H460 cells, which expressed EGFR at an intermediate level. LDM plus gefitinib showed potentiation of antiproliferative activity and apoptosis induction, which were associated with downregulation of EGFR signaling pathway and nuclear factor-kappa B expression, and the increase of cleaved poly (adenosine diphosphate-ribose) polymerase in the two cell lines, although to a lesser degree in H460 cells. Combined treatment induced G1 phase arrest similar to that of gefitinib alone in A431 cells and intensified G2/M phase accumulation in H460 cells. The above results indicate that LDM potentiates the effects of gefitinib in both gefitinib sensitive and less sensitive cells in association with enhanced inhibition of EGFR-dependent signaling. PMID:19342999

  11. Targeting of epidermal Langerhans cells with antigenic proteins: attempts to harness their properties for immunotherapy.

    PubMed

    Flacher, Vincent; Sparber, Florian; Tripp, Christoph H; Romani, Nikolaus; Stoitzner, Patrizia

    2009-07-01

    Langerhans cells, a subset of skin dendritic cells in the epidermis, survey peripheral tissue for invading pathogens. In recent functional studies it was proven that Langerhans cells can present exogenous antigen not merely on major histocompatibility complexes (MHC)-class II molecules to CD4+ T cells, but also on MHC-class I molecules to CD8+ T cells. Immune responses against topically applied antigen could be measured in skin-draining lymph nodes. Skin barrier disruption or co-application of adjuvants was required for maximal induction of T cell responses. Cytotoxic T cells induced by topically applied antigen inhibited tumor growth in vivo, thus underlining the potential of Langerhans cells for immunotherapy. Here we review recent work and report novel observations relating to the potential use of Langerhans cells for immunotherapy. We investigated the potential of epicutaneous immunization strategies in which resident skin dendritic cells are loaded with tumor antigen in situ. This contrasts with current clinical approaches, where dendritic cells generated from progenitors in blood are loaded with tumor antigen ex vivo before injection into cancer patients. In the current study, we applied either fluorescently labeled protein antigen or targeting antibodies against DEC-205/CD205 and langerin/CD207 topically onto barrier-disrupted skin and examined antigen capture and transport by Langerhans cells. Protein antigen could be detected in Langerhans cells in situ, and they were the main skin dendritic cell subset transporting antigen during emigration from skin explants. Potent in vivo proliferative responses of CD4+ and CD8+ T cells were measured after epicutaneous immunization with low amounts of protein antigen. Targeting antibodies were mainly transported by langerin+ migratory dendritic cells of which the majority represented migratory Langerhans cells and a smaller subset the new langerin+ dermal dendritic cell population located in the upper dermis. The

  12. Basal cell carcinoma develops in contact with the epidermal basal cell layer - a three-dimensional morphological study.

    PubMed

    Pirici, Ionica; Ciurea, Marius Eugen; Mîndrilă, Ion; Avrămoiu, Ioan; Pirici, Alexandru; Nicola, Monica Georgiana; Rogoveanu, Otilia Constantina

    2016-01-01

    Basal cell carcinoma is the most common malignant tumor of the skin, and it develops most frequently on the areas of the body that make its treatment and care extremely difficult, especially in cases of neglecting or aggressive growth and invasion. Both typical mild cases as well as locally aggressive tumor types do not tend to metastasize, and it has been postulated that they should share some common biological and morphological features that might explain this behavior. In this study, we have utilized a high-resolution three-dimensional reconstruction technique on pathological samples from 15 cases of common aggressive (fibrosing and adenoid types) and mild (superficial type) basal cell carcinomas, and showed that all these types shared contact points and bridges with the underlying basal cell layer of the epidermis or with the outmost layer of the hair follicle. The connections found had in fact the highest number for fibrosing type (100%), compared to the superficial (85.71%) and adenoid (55%) types. The morphology of the connection bridges was also different, adjacent moderate to abundant inflammatory infiltrate seeming to lead to a loss of basaloid features in these areas. For the adenoid type, tumor islands seemed to be connected also to each other more strongly, forming a common "tumor lace", and while it has been showed that superficial and fibrosing types have higher recurrence risks, all together these data might iterate a connection between the number of bridging points and the biological and clinical manifestation of this skin tumor. PMID:27151694

  13. Dendritic cell and macrophage staining by monoclonal antibodies in tissue sections and epidermal sheets.

    PubMed Central

    Flotte, T. J.; Springer, T. A.; Thorbecke, G. J.

    1983-01-01

    Mouse tissue sections were stained by monoclonal antibodies to macrophage antigens (Mac-1 (M1/70), Mac-2 (M3/38), Mac-3 (M3/84) with the use of immunoperoxidase. Mac-1 was located diffusely in the cytoplasm of round cells in a high percentage of alveolar macrophages, resident peritoneal and bone marrow cells, in splenic red pulp, and in rare perivascular cells in the thymus. Mac-1 was absent in epithelial cells and Langerhans cells. Mac-2 was strongly positive in many dendritic cells in the thymic medulla, more than the cortex, in paracortex and medulla of lymph nodes, sparing the follicles, and in the marginal zone of spleen. There were a few positive cells in germinal centers. Mac-2 was located in a low percentage of bone marrow and a high percentage of resident peritoneal cells. When positive in sections Mac-3 always showed granular cytoplasmic staining. Bone marrow showed a high percentage of cytoplasmic staining (greater than 50%), as compared with low surface staining (less than 1%). It was found in hematopoietic cells, and in all endothelium, including postcapillary venules and lining of sinuses. It was probable that the resulting dendritic staining pattern for Mac-3 in paracortex of lymph node, white and red pulp, thymic cortex, and medulla included dendritic cells other than endothelial cells. Alveolar macrophages and Kupffer cells were positive for Mac-2 and Mac-3. Mac-3 also stained bile canaliculi. Clearly different staining patterns were found in epithelial cells for Mac-2 and Mac-3 in kidney tubules, intestinal mucosal lining, bronchi, choroid plexus, and epidermis. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 PMID:6340516

  14. The heat shock protein 90-binding geldanamycin inhibits cancer cell proliferation, down-regulates oncoproteins, and inhibits epidermal growth factor-induced invasion in thyroid cancer cell lines.

    PubMed

    Park, Jin-Woo; Yeh, Michael W; Wong, Mariwil G; Lobo, Margaret; Hyun, William C; Duh, Quan-Yang; Clark, Orlo H

    2003-07-01

    Heat shock protein 90 (HSP90) serves as a chaperone protein and plays a critical role in tumor cell growth and/or survival. Geldanamycin, a specific inhibitor of HSP90, is cytotoxic to several human cancer cell lines, but its effect in thyroid cancer is unknown. We, therefore, investigated the effect of geldanamycin on cell proliferation, oncoprotein expression, and invasion in human thyroid cancer cell lines. We used six thyroid cancer cell lines: TPC-1 (papillary), FTC-133, FTC-236, FTC-238 (follicular), XTC-1 (Hürthle cell), and ARO (anaplastic). We used the dimethyl-thiazol-diphenyltetrazolium bromide assay, a clonogenic assay, an apoptotic assay, and a Matrigel invasion assay. We evaluated oncoprotein expression using Western blots and flow cytometry. After 6 d of treatment with 50 nM geldanamycin, the percent inhibition of growth was 29.4% in TPC-1, 97.5% in FTC-133, 96.7% in FTC-236, 10.8% in FTC-238, 70.9% in XTC-1, and 45.5% in ARO cell lines. In the FTC-133 cell line, geldanamycin treatment decreased clonogenicity by 21% at a concentration of 50 nM; geldanamycin induced apoptosis and down-regulated c-Raf-1, mutant p53, and epidermal growth factor (EGF) receptor expression; geldanamycin inhibited EGF-stimulated invasion. In conclusion, geldanamycin inhibited cancer cell proliferation, down-regulated oncoproteins, and inhibited EGF-induced invasion in thyroid cancer cell lines. PMID:12843186

  15. Ultrastructural localization of photosynthetic and photorespiratory enzymes in epidermal, mesophyll, bundle sheath, and vascular bundle cells of the C4 dicot Amaranthus viridis.

    PubMed

    Ueno, O

    2001-05-01

    In the leaves of the NAD-malic enzyme (NAD-ME)-type C4 dicot Amaranthus viridis L., there are chloroplasts in the vascular parenchyma cells (VPC), companion cells (CC), ordinary epidermal cells (EC), and guard cells (GC), as well as in the mesophyll cells (MC) and the bundle sheath cells (BSC). However, the chloroplasts of the VPC, CC, EC, and GC are smaller than those of the MC and BSC. In this study, the accumulation of photosynthetic and photorespiratory enzymes in these leaf cell types was investigated by immunogold labelling and electron microscopy. Strong labelling for phosphoenolpyruvate carboxylase was found in the MC cytosol. Weak labelling was observed in the CC and GC cytosol. Labelling for pyruvate, Pi dikinase occurred to varying degrees in the chloroplasts of all cell types except CC. Labelling for the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase was detected in the chloroplasts of all cell types except MC. For both NAD-ME and the P-protein of glycine decarboxylase, intense labelling was found in the BSC mitochondria; weaker labelling was recognized in the VPC mitochondria. These data indicate that when not only the MC and BSC but also the other leaf cell types are included, the cell-specific expression of the enzymes in C4 leaves becomes more complex than has been known previously. These findings are discussed in relation to the metabolic function of epidermal and vascular bundle cells. PMID:11432917

  16. Epidermal growth factor attenuates tubular necrosis following mercuric chloride damage by regeneration of indigenous, not bone marrow-derived cells

    PubMed Central

    Yen, Tzung-Hai; Alison, Malcolm R; Goodlad, Robert A; Otto, William R; Jeffery, Rosemary; Cook, H Terence; Wright, Nicholas A; Poulsom, Richard

    2015-01-01

    To assess effects of epidermal growth factor (EGF) and pegylated granulocyte colony-stimulating factor (P-GCSF; pegfilgrastim) administration on the cellular origin of renal tubular epithelium regenerating after acute kidney injury initiated by mercuric chloride (HgCl2). Female mice were irradiated and male whole bone marrow (BM) was transplanted into them. Six weeks later recipient mice were assigned to one of eight groups: control, P-GCSF+, EGF+, P-GCSF+EGF+, HgCl2, HgCl2+P-GCSF+, HgCl2+EGF+ and HgCl2+P-GCSF+EGF+. Following HgCl2, injection tubular injury scores increased and serum urea nitrogen levels reached uraemia after 3 days, but EGF-treated groups were resistant to this acute kidney injury. A four-in-one analytical technique for identification of cellular origin, tubular phenotype, basement membrane and S-phase status revealed that BM contributed 1% of proximal tubular epithelium in undamaged kidneys and 3% after HgCl2 damage, with no effects of exogenous EGF or P-GCSF. Only 0.5% proximal tubular cells were seen in S-phase in the undamaged group kidneys; this increased to 7–8% after HgCl2 damage and to 15% after addition of EGF. Most of the regenerating tubular epithelium originated from the indigenous pool. BM contributed up to 6.6% of the proximal tubular cells in S-phase after HgCl2 damage, but only to 3.3% after additional EGF. EGF administration attenuated tubular necrosis following HgCl2 damage, and the major cause of this protective effect was division of indigenous cells, whereas BM-derived cells were less responsive. P-GCSF did not influence damage or regeneration. PMID:25389045

  17. Shikonin and its derivatives inhibit the epidermal growth factor receptor signaling and synergistically kill glioblastoma cells in combination with erlotinib.

    PubMed

    Zhao, Qiaoli; Kretschmer, Nadine; Bauer, Rudolf; Efferth, Thomas

    2015-09-15

    Overexpression and mutation of the epidermal growth factor receptor (EGFR) gene play a causal role in tumorigenesis and resistance to treatment of glioblastoma (GBM). EGFR inhibitors such as erlotinib are currently used for the treatment of GBM; however, their efficacy has been limited due to drug resistance. New treatment strategies are therefore urgently needed. Shikonin, a natural naphthoquinone, induces both apoptosis and necroptosis in human glioma cells, but the effectiveness of erlotinib-shikonin combination treatment as well as the underlying molecular mechanisms is unknown yet. In this study, we investigated erlotinib in combination with shikonin and 14 shikonin derivatives in parental U87MG and transfected U87MG.ΔEGFR GBM cells. Most of the shikonin derivatives revealed strong cytotoxicity. Shikonin together with five other derivatives, namely deoxyshikonin, isobutyrylshikonin, acetylshikonin, β,β-dimethylacrylshikonin and acetylalkannin showed synergistic cytotoxicity toward U87MG.ΔEGFR in combination with erlotinib. Moreover, the combined cytotoxic effect of shikonin and erlotinib was further confirmed with another three EGFR-expressing cell lines, BS153, A431 and DK-MG. Shikonin not only dose-dependently inhibited EGFR phosphorylation and decreased phosphorylation of EGFR downstream molecules, including AKT, P44/42MAPK and PLCγ1, but also together with erlotinib synergistically inhibited ΔEGFR phosphorylation in U87MG.ΔEGFR cells as determined by Loewe additivity and Bliss independence drug interaction models. These results suggest that the combination of erlotinib with shikonin or its derivatives might be a potential strategy to overcome drug resistance to erlotinib. PMID:25688715

  18. Anticancer activity of pristimerin in epidermal growth factor receptor 2-positive SKBR3 human breast cancer cells.

    PubMed

    Lee, Jin Sun; Yoon, In Sang; Lee, Myung Sun; Cha, Eun Young; Thuong, Phuong Thien; Diep, Trinh Thi; Kim, Je Ryong

    2013-01-01

    Pristimerin is a naturally occurring triterpenoid that causes cytotoxicity in several cancer cell lines. However, the mechanism of action for the cytotoxic effect of pristimerin has not been unexplored. The purpose of this study was to investigate the effect of pristimerin on cytotoxicity using the epidermal growth factor receptor 2 (HER2)-positive SKBR3 human breast cancer cell line. Pristimerin inhibited proliferation in dose- and time-dependent manners in cells. We found it to be effective for suppressing HER2 protein and mRNA expression. Fatty acid synthase (FASN) expression and FASN activity were downregulated by pristimerin. Adding of exogenous palmitate, the end product of de novo fatty acid synthesis, reduced the proliferation activity of pristimerin. The changes in HER2 and FASN expression induced by pristimerin altered the levels of Akt and mitogen-activated protein kinase (MAPK) phosphorylation (Erk1/2, p38, and c-Jun N-terminal kinase (JNK)). Pristimerin lowered the levels of phosphorylated mammalian target of rapamycin (mTOR) and its downstream targets such as phosphoprotein 70 ribosomal protein S6 kinase and 4E binding protein1. Pristimerin inhibited migration and invasion of cells, and co-treatment with the mTOR inhibitor rapamycin additionally suppressed these activities. Pristimerin-induced apoptosis was evaluated using Western blotting for caspase-3, -8, -9, and poly (ADP-ribose) polymerase expression and flow cytometric analysis for propidium iodide labeling. These results suggest that pristimerin is a novel HER2-downregulated compound that is able to decrease fatty acid synthase and modulate the Akt, MAPK, and mTOR signaling pathways to influence metastasis and apoptosis. Pristimerin may be further evaluated as a chemotherapeutic agent for HER2-positive breast cancers. PMID:23370361

  19. Differential regulation of epidermal growth factor receptor by hydrogen peroxide and flagellin in cultured lung alveolar epithelial cells.

    PubMed

    Nishi, Hiroyuki; Maeda, Noriko; Izumi, Shunsuke; Higa-Nakamine, Sayomi; Toku, Seikichi; Kakinohana, Manabu; Sugahara, Kazuhiro; Yamamoto, Hideyuki

    2015-02-01

    In previous studies, we found that stimulation of Toll-like receptor 5 (TLR5) by flagellin induced the activation of mitogen-activated protein kinase (MAPK)-activated protein kinase-2 (MAPKAPK-2) through activation of the p38 MAPK pathway in cultured alveolar epithelial A549 cells. Our studies strongly suggested that MAPKAPK-2 phosphorylated epidermal growth factor receptor (EGFR) at Ser1047. It has been reported that phosphorylation of Ser1047 after treatment with tumor necrosis factor α (TNFα) induced the internalization of EGFR. In the present study, we first found that treatment of A549 cells with hydrogen peroxide induced the activation of MAPKAPK-2 and phosphorylation of EGFR at Ser1047 within 30 min. This was different from flagellin treatment because hydrogen peroxide treatment induced the phosphorylation of EGFR at Tyr1173 as well as Ser1047, indicating the activation of EGFR. We also found that KN93, an inhibitor of CaM kinase II, inhibited the hydrogen peroxide-induced phosphorylation of EGFR at Ser1047 through inhibition of the activation of the p38 MAPK pathway. Furthermore, we examined the internalization of EGFR by three different methods. Flow cytometry with an antibody against the extracellular domain of EGFR and biotinylation of cell surface proteins revealed that flagellin, but not hydrogen peroxide, decreased the amount of cell-surface EGFR. In addition, activation of extracellular signal-regulated kinase by EGF treatment was reduced by flagellin pre-treatment. These results strongly suggested that hydrogen peroxide activated the p38 MAPK pathway via activation of CaM kinase II and that flagellin and hydrogen peroxide regulate the functions of EGFR by different mechanisms. PMID:25542757

  20. Intradermal injection of an anti-Langerin-HIVGag fusion vaccine targets epidermal Langerhans cells in nonhuman primates and can be tracked in vivo.

    PubMed

    Salabert, Nina; Todorova, Biliana; Martinon, Frédéric; Boisgard, Raphaël; Zurawski, Gerard; Zurawski, Sandra; Dereuddre-Bosquet, Nathalie; Cosma, Antonio; Kortulewski, Thierry; Banchereau, Jacques; Levy, Yves; Le Grand, Roger; Chapon, Catherine

    2016-03-01

    The development of new immunization strategies requires a better understanding of early molecular and cellular events occurring at the site of injection. The skin is particularly rich in immune cells and represents an attractive site for vaccine administration. Here, we specifically targeted vaccine antigens to epidermal Langerhans cells (LCs) using a fusion protein composed of HIV antigens and a monoclonal antibody targeting Langerin. We developed a fluorescence imaging approach to visualize, in vivo, the vaccine-targeted cells. Studies were performed in nonhuman primates (NHPs) because of their relevance as a model to assess human vaccines. We directly demonstrated that in NHPs, intradermally injected anti-Langerin-HIVGag specifically targets epidermal LCs and induces rapid changes in the LC network, including LC activation and migration out of the epidermis. Vaccine targeting of LCs significantly improved anti-HIV immune response without requirement of an adjuvant. Although the co-injection of the TLR-7/8 synthetic ligand, R-848 (resiquimod), with the vaccine, did not enhance significantly the antibody response, it stimulated recruitment of HLA-DR+ inflammatory cells to the site of immunization. This study allowed us to characterize the dynamics of early local events following the injection of a vaccine-targeted epidermal LCs and R-848. PMID:26678013

  1. Integrin-linked kinase regulates the niche of quiescent epidermal stem cells

    PubMed Central

    Morgner, Jessica; Ghatak, Sushmita; Jakobi, Tobias; Dieterich, Christoph; Aumailley, Monique; Wickström, Sara A.

    2015-01-01

    Stem cells reside in specialized niches that are critical for their function. Quiescent hair follicle stem cells (HFSCs) are confined within the bulge niche, but how the molecular composition of the niche regulates stem cell behaviour is poorly understood. Here we show that integrin-linked kinase (ILK) is a key regulator of the bulge extracellular matrix microenvironment, thereby governing the activation and maintenance of HFSCs. ILK mediates deposition of inverse laminin (LN)-332 and LN-511 gradients within the basement membrane (BM) wrapping the hair follicles. The precise BM composition tunes activities of Wnt and transforming growth factor-β pathways and subsequently regulates HFSC activation. Notably, reconstituting an optimal LN microenvironment restores the altered signalling in ILK-deficient cells. Aberrant stem cell activation in ILK-deficient epidermis leads to increased replicative stress, predisposing the tissue to carcinogenesis. Overall, our findings uncover a critical role for the BM niche in regulating stem cell activation and thereby skin homeostasis. PMID:26349061

  2. Changes of epidermal cell morphology and keratin expression induced by inhibitors of protein kinase C.

    PubMed

    Hegemann, L; Wevers, A; Bonnekoh, B; Mahrle, G

    1992-03-01

    Several lines of evidence show protein kinase C as being involved in various regulatory processes in keratinocyte biology, e.g. proliferation and differentiation. In the present study, we investigated the effects of three different inhibitors of protein kinase C, staurosporine, CP 46'665-1, and tiflucarbine, on cell morphology and keratin expression in a non-tumorigenic human keratinocyte cell line (HaCaT cells). Staurosporine, being the most potent inhibitor of protein kinase C activity in vitro, and CP 46'665-1 induced morphological transformation to a fibroblast-like cell shape. In contrast, no changes in cell morphology were observed after exposure to tiflucarbine. The investigation of keratin expression in HaCaT cells grown in the presence of the different compounds revealed the following changes: After 72 h of cultivation, keratins 8 and 18 were still expressed in treated cells, whereas expression of keratin 13 was decreased as compared to control cells. Immunoblotting to detect vimentin demonstrated its absence in treated and control cells. Since tiflucarbine is known as a dual protein kinase C/calmodulin inhibitor whereas staurosporine and CP 46'665-1 do not antagonize calmodulin function, it might be possible that not only protein kinase C but also calmodulin is involved in the process leading to the morphological changes. PMID:1376142

  3. The Evolving Role of Maintenance Therapy Using Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors (EGFR TKIs) in the Management of Advanced Non-Small-Cell Lung Cancer

    PubMed Central

    Huang, Chao H.; Powers, Benjamin C.

    2012-01-01

    The epidermal growth factor receptor (EGFR) plays an important role in the development of many cancers, including non-small cell lung cancer. Epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) are a class of novel biologically-targeted agents widely used in the management of recurrent non-small cell lung cancer. Erlotinib, one of the EGFR TKIs, is currently FDA approved in second and third line therapy. However, recent studies showed that erlotinib is also effective as maintenance therapy after initial chemotherapy, improving disease free survival and possibly overall survival. Our current understanding of erlotinib’s mechanism of action, with the discovery that EGFR mutation confers higher response rate, has propelled this agent into the first line setting. Advances in molecular testing and clinical research of this agent and other agents in this class will eventually change the way we utilize EGFR TKIs in the near future. PMID:22550402

  4. Regulators of floral fragrance production and their target genes in petunia are not exclusively active in the epidermal cells of petals.

    PubMed

    Van Moerkercke, Alex; Galván-Ampudia, Carlos S; Verdonk, Julian C; Haring, Michel A; Schuurink, Robert C

    2012-05-01

    In which cells of the flower volatile biosynthesis takes place is unclear. In rose and snapdragon, some enzymes of the volatile phenylpropanoid/benzenoid pathway have been shown to be present in the epidermal cells of petals. It is therefore generally believed that the production of these compounds occurs in these cells. However, whether the entire pathway is active in these cells and whether it is exclusively active in these cells remains to be proven. Cell-specific transcription factors activating these genes will determine in which cells they are expressed. In petunia, the transcription factor EMISSION OF BENZENOIDS II (EOBII) activates the ODORANT1 (ODO1) promoter and the promoter of the biosynthetic gene isoeugenol synthase (IGS). The regulator ODO1 in turn activates the promoter of the shikimate gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS). Here the identification of a new target gene of ODO1, encoding an ABC transporter localized on the plasma membrane, PhABCG1, which is co-expressed with ODO1, is described. PhABCG1 expression is up-regulated in petals overexpressing ODO1 through activation of the PhABCG1 promoter. Interestingly, the ODO1, PhABCG1, and IGS promoters were active in petunia protoplasts originating from both epidermal and mesophyll cell layers of the petal, suggesting that the volatile phenylpropanoid/benzenoid pathway in petunia is active in these different cell types. Since volatile release occurs from epidermal cells, trafficking of (volatile) compounds between cell layers must be involved, but the exact function of PhABCG1 remains to be resolved. PMID:22345641

  5. Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus

    PubMed Central

    Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

    2016-01-01

    RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh. RACB is required for positioning of the nucleus near the site of attack from Bgh. We therefore suggest that Bgh profits from RACB’s function in cell polarity rather than from immunity-regulating functions of RACB. PMID:27056842

  6. Barley disease susceptibility factor RACB acts in epidermal cell polarity and positioning of the nucleus.

    PubMed

    Scheler, Björn; Schnepf, Vera; Galgenmüller, Carolina; Ranf, Stefanie; Hückelhoven, Ralph

    2016-05-01

    RHO GTPases are regulators of cell polarity and immunity in eukaryotes. In plants, RHO-like RAC/ROP GTPases are regulators of cell shaping, hormone responses, and responses to microbial pathogens. The barley (Hordeum vulgare L.) RAC/ROP protein RACB is required for full susceptibility to penetration by Blumeria graminis f.sp. hordei (Bgh), the barley powdery mildew fungus. Disease susceptibility factors often control host immune responses. Here we show that RACB does not interfere with early microbe-associated molecular pattern-triggered immune responses such as the oxidative burst or activation of mitogen-activated protein kinases. RACB also supports rather than restricts expression of defence-related genes in barley. Instead, silencing of RACB expression by RNAi leads to defects in cell polarity. In particular, initiation and maintenance of root hair growth and development of stomatal subsidiary cells by asymmetric cell division is affected by silencing expression of RACB. Nucleus migration is a common factor of developmental cell polarity and cell-autonomous interaction with Bgh RACB is required for positioning of the nucleus near the site of attack from Bgh We therefore suggest that Bgh profits from RACB's function in cell polarity rather than from immunity-regulating functions of RACB. PMID:27056842

  7. Turgor Regulation in Osmotically Stressed Arabidopsis Epidermal Root Cells. Direct Support for the Role of Inorganic Ion Uptake as Revealed by Concurrent Flux and Cell Turgor Measurements1

    PubMed Central

    Shabala, Sergey N.; Lew, Roger R.

    2002-01-01

    Hyperosmotic stress is known to significantly enhance net uptake of inorganic ions into plant cells. Direct evidence for cell turgor recovery via such a mechanism, however, is still lacking. In the present study, we performed concurrent measurements of net ion fluxes (with the noninvasive microelectrode ion flux estimation technique) and cell turgor changes (with the pressure-probe technique) to provide direct evidence that inorganic ion uptake regulates turgor in osmotically stressed Arabidopsis epidermal root cells. Immediately after onset of hyperosmotic stress (100/100 mm mannitol/sorbitol treatment), the cell turgor dropped from 0.65 to about 0.25 MPa. Turgor recovery started within 2 to 10 min after the treatment and was accompanied by a significant (30–80 nmol m−2 s−1) increase in uptake of K+, Cl−, and Na+ by root cells. In most cells, almost complete (>90% of initial values) recovery of the cell turgor was observed within 40 to 50 min after stress onset. In another set of experiments, we combined the voltage-clamp and the microelectrode ion flux estimation techniques to show that this process is, in part, mediated by voltage-gated K+ transporters at the cell plasma membrane. The possible physiological significance of these findings is discussed. PMID:12011359

  8. Autocrine epidermal growth factor signaling stimulates directionally persistent mammary epithelial cell migration

    SciTech Connect

    Maheshwari, Gargi; Wiley, H Steven ); Lauffenburger, Douglas A.

    2001-12-24

    Autocrine receptor/ligand signaling loops were first identified in tumor cells, where it was found that transformation of cells resulted in overexpression of certain growth factors leading to unregulated proliferation of the tumor cells (Sporn and Todaro, 1980). However, in the ensuing decades autocrine signaling has been found to operate in numerous physiological situations (Sporn and Roberts, 1992), including wound healing (Tokumaru et al., 2000), angiogenesis (Seghezzi et al., 1998), and tissue organization during development (Wasserman and Freeman, 1998) and reproductive cycles (Xie et al., 1997). Although it is becoming evident that autocrine loops play crucial roles in regulation of cell function within tissue contexts, it is unclear whether their effects on cell responses are different from the effects of the same ligand presented in exogenous or paracrine manner.

  9. Epidermal growth factor receptor tyrosine kinase inhibitors with conventional chemotherapy for the treatment of non-small cell lung cancer

    PubMed Central

    Gao, Yuan; Song, PingPing; Li, Hui; Guo, HongBo; Jia, Hui; Zhang, BaiJiang

    2016-01-01

    We report a Chinese male patient with advanced stage lung squamous cell carcinoma who developed brain metastases after responding to treatment comprising six cycles of conventional chemotherapy with docetaxel and cisplatin. The patient was then treated with oral erlotinib (150 mg/day) and whole-brain radiation therapy followed by four cycles of docetaxel and carboplatin chemotherapy. The patient then received gefitinib (250 mg/day) as a maintenance therapy until the end of the follow-up period. In this patient, progression-free survival, defined as the interval from the initiation of first-line chemotherapy to the cessation of erlotinib due to progressive disease or death from any cause, was 3 months. Overall survival, defined as the interval from the initiation of first-line chemotherapy to death from any cause, was 75 months. Erlotinib was well tolerated in combination with whole-brain radiation therapy and a favorable objective response rate was observed. Furthermore, targeted drug treatment warrants consideration in patients with a negative epidermal growth factor receptor mutation status and male patients with a history of smoking. PMID:26719713

  10. Amlexanox Blocks the Interaction between S100A4 and Epidermal Growth Factor and Inhibits Cell Proliferation.

    PubMed

    Cho, Ching Chang; Chou, Ruey-Hwang; Yu, Chin

    2016-01-01

    The human S100A4 protein binds calcium, resulting in a change in its conformation to promote the interaction with its target protein. Human epidermal growth factor (EGF) is the target protein of S100A4 and a critical ligand of the receptor EGFR. The EGF/EGFR system promotes cell survival, differentiation, and growth by activating several signaling pathways. Amlexanox is an anti-inflammatory and anti-allergic drug that is used to treat recurrent aphthous ulcers. In the present study, we determined that amlexanox interacts with S100A4 using heteronuclear single quantum correlation titration. We elucidated the interactions of S100A4 with EGF and amlexanox using fluorescence and nuclear magnetic resonance spectroscopy. We generated two binary models (for the S100A4-EGF and S100A4-amlexanox complexes) and observed that amlexanox and EGF share a similar binding region in mS100A4. We also used a WST-1 assay to investigate the bioactivity of S100A4, EGF, and amlexanox, and found that amlexanox blocks the binding between S100A4 and EGF, and is therefore useful for the development of new anti-proliferation drugs. PMID:27559743

  11. Amlexanox Blocks the Interaction between S100A4 and Epidermal Growth Factor and Inhibits Cell Proliferation

    PubMed Central

    Cho, Ching Chang; Chou, Ruey-Hwang; Yu, Chin

    2016-01-01

    The human S100A4 protein binds calcium, resulting in a change in its conformation to promote the interaction with its target protein. Human epidermal growth factor (EGF) is the target protein of S100A4 and a critical ligand of the receptor EGFR. The EGF/EGFR system promotes cell survival, differentiation, and growth by activating several signaling pathways. Amlexanox is an anti-inflammatory and anti-allergic drug that is used to treat recurrent aphthous ulcers. In the present study, we determined that amlexanox interacts with S100A4 using heteronuclear single quantum correlation titration. We elucidated the interactions of S100A4 with EGF and amlexanox using fluorescence and nuclear magnetic resonance spectroscopy. We generated two binary models (for the S100A4-EGF and S100A4-amlexanox complexes) and observed that amlexanox and EGF share a similar binding region in mS100A4. We also used a WST-1 assay to investigate the bioactivity of S100A4, EGF, and amlexanox, and found that amlexanox blocks the binding between S100A4 and EGF, and is therefore useful for the development of new anti-proliferation drugs. PMID:27559743

  12. Golgi- and trans-Golgi network-mediated vesicle trafficking is required for wax secretion from epidermal cells.

    PubMed

    McFarlane, Heather E; Watanabe, Yoichiro; Yang, Weili; Huang, Yan; Ohlrogge, John; Samuels, A Lacey

    2014-03-01

    Lipid secretion from epidermal cells to the plant surface is essential to create the protective plant cuticle. Cuticular waxes are unusual secretory products, consisting of a variety of highly hydrophobic compounds including saturated very-long-chain alkanes, ketones, and alcohols. These compounds are synthesized in the endoplasmic reticulum (ER) but must be trafficked to the plasma membrane for export by ATP-binding cassette transporters. To test the hypothesis that wax components are trafficked via the endomembrane system and packaged in Golgi-derived secretory vesicles, Arabidopsis (Arabidopsis thaliana) stem wax secretion was assayed in a series of vesicle-trafficking mutants, including gnom like1-1 (gnl1-1), transport particle protein subunit120-4, and echidna (ech). Wax secretion was dependent upon GNL1 and ECH. Independent of secretion phenotypes, mutants with altered ER morphology also had decreased wax biosynthesis phenotypes, implying that the biosynthetic capacity of the ER is closely related to its structure. These results provide genetic evidence that wax export requires GNL1- and ECH-dependent endomembrane vesicle trafficking to deliver cargo to plasma membrane-localized ATP-binding cassette transporters. PMID:24468625

  13. Epidermal Growth Factor Receptor Mutation Status in the Treatment of Non-small Cell Lung Cancer: Lessons Learned

    PubMed Central

    Lee, Dae Ho; Srimuninnimit, Vichien; Cheng, Rebecca; Wang, Xin; Orlando, Mauro

    2015-01-01

    Advances in oncology research have led to identification of tumor-specific biomarkers, some of which are important predictive indicators and ideal targets for novel therapeutics. One such biomarker in non-small cell lung cancer (NSCLC) is the epidermal growth factor receptor (EGFR). Patients with NSCLC who harbor an activating EGFR mutation show a more favorable response to treatment with an EGFR inhibitor, such as gefitinib, erlotinib, or afatinib, than to chemotherapy. The prevalence of EGFR mutations in East Asian patients is higher than that in other populations, and in some clinical settings, patients have been treated with EGFR inhibitors based on clinicopathologic characteristics with no information on EGFR status. However, based on results from a series of studies in which East Asian patients with advanced non-squamous NSCLC were treated with EGFR inhibitors alone or in combination with standard chemotherapy, this may not be the best practice because EGFR mutation status was found to be a key predictor of outcome. Data from these studies highlight the necessity of EGFR testing in determining the most suitable treatment for patients with advanced or metastatic NSCLC. PMID:25943319

  14. Deep mutational scanning of an antibody against epidermal growth factor receptor using mammalian cell display and massively parallel pyrosequencing

    PubMed Central

    Forsyth, Charles M.; Juan, Veronica; Akamatsu, Yoshiko; DuBridge, Robert B.; Doan, Minhtam; Ivanov, Alexander V.; Ma, Zhiyuan; Polakoff, Dixie; Razo, Jennifer; Wilson, Keith; Powers, David B.

    2013-01-01

    We developed a method for deep mutational scanning of antibody complementarity-determining regions (CDRs) that can determine in parallel the effect of every possible single amino acid CDR substitution on antigen binding. The method uses libraries of full length IgGs containing more than 1000 CDR point mutations displayed on mammalian cells, sorted by flow cytometry into subpopulations based on antigen affinity and analyzed by massively parallel pyrosequencing. Higher, lower and neutral affinity mutations are identified by their enrichment or depletion in the FACS subpopulations. We applied this method to a humanized version of the anti-epidermal growth factor receptor antibody cetuximab, generated a near comprehensive data set for 1060 point mutations that recapitulates previously determined structural and mutational data for these CDRs and identified 67 point mutations that increase affinity. The large-scale, comprehensive sequence-function data sets generated by this method should have broad utility for engineering properties such as antibody affinity and specificity and may advance theoretical understanding of antibody-antigen recognition. PMID:23765106

  15. Strain differences in the expression of an H-2K/sup k/ gene product by epidermal and spleen cells

    SciTech Connect

    Hadley, G.A.; Steinmuller, D.

    1986-03-01

    Cytotoxic T lymphocytes (CTL) directed against Epa-1, a non-H-2 alloantigen expressed by epidermal cells (EC) but no lymphoid cells, lyse EC of different H-2/sup k/, Epa-1/sup +/ strains at different levels. For example, the mean percent lysis values for EC of strains CBA, AKR, C58, and RF are 60, 46, 41, and 35 respectively. Since the CTL used to obtain these values recognize Epa-1 only in the context of H-2K/sup k/, the different levels of lysis could reflect differences in either Epa-1 or K/sup k/ antigens. The goal of this investigation was to test the second alternative. For this purpose, the authors obtained hybridoma 16-1-11N that secretes a K/sup k/-specific MoAb. They first demonstrated the capacity of MoAb 16-1-11N to block the lysis of CBA EC by Epa-1-specific CTL. They then utilized it as the probe in a cellar RIA, with /sup 125/I-protein A as the second reagent, to quantitate the expression of K/sup k/ antigens on EC of strains CBA, AKR, C58, and RF. They found that C58 and RF EC bound significantly less of the K/sup k/ MoAb than CBA EC. Although AKR EC also bound less of the MoAb than CBA EC, the difference was not significant. Nonetheless, these data support the hypothesis that the differential susceptibility of the strains to lysis by Epa-1-specific CTL is due to differences in the expression of the H-2 restricting element. The authors also tested spleen cells (SC) of the four strains in the RIA described above and found that SC of RF, but not of C58 or AKR, express reduced levels of K/sup k/ antigens compared to CBA SC.

  16. Two-photon fluorescence imaging and bimodal phototherapy of epidermal cancer cells with biocompatible self-assembled polymer nanoparticles.

    PubMed

    Kandoth, Noufal; Kirejev, Vladimir; Monti, Sandra; Gref, Ruxandra; Ericson, Marica B; Sortino, Salvatore

    2014-05-12

    We have developed herein an engineered polymer-based nanoplatform showing the convergence of two-photon fluorescence imaging and bimodal phototherapeutic activity in a single nanostructure. It was achieved through the appropriate choice of three different components: a β-cyclodextrin-based polymer acting as a suitable carrier, a zinc phthalocyanine emitting red fluorescence simultaneously as being a singlet oxygen ((1)O2) photosensitizer, and a tailored nitroaniline derivative, functioning as a nitric oxide (NO) photodonor. The self-assembly of these components results in photoactivable nanoparticles, approximately 35 nm in diameter, coencapsulating a multifunctional cargo, which can be delivered to carcinoma cells. The combination of steady-state and time-resolved spectroscopic and photochemical techniques shows that the two photoresponsive guests do not interfere with each other while being enclosed in their supramolecular container and can thus be operated in parallel under control of light stimuli. Specifically, two-photon fluorescence microscopy allows mapping of the nanoassembly, here applied to epidermal cancer cells. By detecting the red emission from the phthalocyanine fluorophore it was also possible to investigate the tissue distribution after topical delivery onto human skin ex vivo. Irradiation of the nanoassembly with visible light triggers the simultaneous delivery of cytotoxic (1)O2 and NO, resulting in an amplified cell photomortality due to a combinatory effect of the two cytotoxic agents. The potential of dual therapeutic photodynamic action and two-photon fluorescence imaging capability in a single nanostructure make this system an appealing candidate for further studies in biomedical research. PMID:24673610

  17. Mn-doped Zinc Sulphide nanocrystals for immunofluorescent labeling of epidermal growth factor receptors on cells and clinical tumor tissues

    NASA Astrophysics Data System (ADS)

    J, Aswathy; V, Seethalekshmy N.; R, Hiran K.; R, Bindhu M.; K, Manzoor; Nair, Shantikumar V.; Menon, Deepthy

    2014-11-01

    The field of molecular detection and targeted imaging has evolved considerably with the introduction of fluorescent semiconductor nanocrystals. Manganese-doped zinc sulphide nanocrystals (ZnS:Mn NCs), which are widely used in electroluminescent displays, have been explored for the first time for direct immunofluorescent (IF) labeling of clinical tumor tissues. ZnS:Mn NCs developed through a facile wet chemistry route were capped using amino acid cysteine, conjugated to streptavidin and thereafter coupled to biotinylated epidermal growth factor receptor (EGFR) antibody utilizing the streptavidin-biotin linkage. The overall conjugation yielded stable EGFR antibody conjugated ZnS:Mn NCs (EGFR ZnS:Mn NCs) with a hydrodynamic diameter of 65 ± 15 nm, and having an intense orange-red fluorescence emission at 598 nm. Specific labeling of EGF receptors on EGFR+ve A431 cells in a co-culture with EGFR-ve NIH3T3 cells was demonstrated using these nanoprobes. The primary antibody conjugated fluorescent NCs could also clearly delineate EGFR over-expressing cells on clinical tumor tissues processed by formalin fixation as well as cryopreservation with a specificity of 86% and accuracy of 88%, in comparison to immunohistochemistry. Tumor tissues labeled with EGFR ZnS:Mn NCs showed good fluorescence emission when imaged after storage even at 15 months. Thus, ZnS nanobioconjugates with dopant-dependent and stable fluorescence emission show promise as an efficient, target-specific fluorophore that would enable long term IF labeling of any antigen of interest on clinical tissues.

  18. Antiestrogen fulvestrant enhances the antiproliferative effects of epidermal growth factor receptor inhibitors in human non-small cell lung cancer

    PubMed Central

    Garon, Edward B.; Pietras, Richard J.; Finn, Richard S.; Kamranpour, Naeimeh; Pitts, Sharon; Márquez-Garbán, Diana C.; Desai, Amrita J.; Dering, Judy; Hosmer, Wylie; von Euw, Erika M.; Dubinett, Steven M.; Slamon, Dennis J.

    2012-01-01

    Introduction Estrogen receptor (ER) signaling and its interaction with epidermal growth factor receptor (EGFR) is a potential therapeutic target in non-small cell lung cancer (NSCLC). To explore cross-communication between ER and EGFR, we have correlated ER pathway gene and protein expression profiles and examined effects of antiestrogens with or without EGFR inhibitors in preclinical models of human NSCLC. Methods We evaluated 54 NSCLC cell lines for growth inhibition with EGFR inhibitors, antiestrogen treatment or the combination. Each line was evaluated for baseline ER pathway protein expression. The majority were also evaluated for baseline ER pathway gene expression. Human NSCLC xenografts were evaluated for effects of inhibition of each pathway either individually or in combination. Results The specific antiestrogen fulvestrant has modest single agent activity in vitro, but in many lines fulvestrant adds to effects of EGFR inhibitors, including synergy in the EGFR mutant, erlotinib-resistant H1975 line. ERα, ERβ, progesterone receptor (PR)-A, PR-B and aromatase proteins are expressed in all lines to varying degrees, with trends towards lower aromatase in more sensitive cell lines. Sensitivity to fulvestrant correlates with greater baseline ERα gene expression. Tumor stability is achieved in human tumor xenografts with either fulvestrant or EGFR inhibitors, but tumors regress significantly when both pathways are inhibited. Conclusions These data provide a rationale for further investigation of the antitumor activity of combined therapy with antiestrogen and anti-EGFR agents in the clinic. Future work should also evaluate dual ER and EGFR inhibition in the setting of secondary resistance to EGFR inhibition. PMID:23399957

  19. Blockade of Hedgehog Signaling Synergistically Increases Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Cell Lines

    PubMed Central

    Bai, Xiao-Yan; Zhang, Xu-Chao; Yang, Su-Qing; An, She-Juan; Chen, Zhi-Hong; Su, Jian; Xie, Zhi; Gou, Lan-Ying; Wu, Yi-Long

    2016-01-01

    Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs. PMID:26943330

  20. Blockade of Hedgehog Signaling Synergistically Increases Sensitivity to Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors in Non-Small-Cell Lung Cancer Cell Lines.

    PubMed

    Bai, Xiao-Yan; Zhang, Xu-Chao; Yang, Su-Qing; An, She-Juan; Chen, Zhi-Hong; Su, Jian; Xie, Zhi; Gou, Lan-Ying; Wu, Yi-Long

    2016-01-01

    Aberrant activation of the hedgehog (Hh) signaling pathway has been implicated in the epithelial-to-mesenchymal transition (EMT) and cancer stem-like cell (CSC) maintenance; both processes can result in tumor progression and treatment resistance in several types of human cancer. Hh cooperates with the epidermal growth factor receptor (EGFR) signaling pathway in embryogenesis. We found that the Hh signaling pathway was silenced in EGFR-TKI-sensitive non-small-cell lung cancer (NSCLC) cells, while it was inappropriately activated in EGFR-TKI-resistant NSCLC cells, accompanied by EMT induction and ABCG2 overexpression. Upregulation of Hh signaling through extrinsic SHH exposure downregulated E-cadherin expression and elevated Snail and ABCG2 expression, resulting in gefitinib tolerance (P < 0.001) in EGFR-TKI-sensitive cells. Blockade of the Hh signaling pathway using the SMO antagonist SANT-1 restored E-cadherin expression and downregulate Snail and ABCG2 in EGFR-TKI-resistant cells. A combination of SANT-1 and gefitinib markedly inhibited tumorigenesis and proliferation in EGFR-TKI-resistant cells (P < 0.001). These findings indicate that hyperactivity of Hh signaling resulted in EGFR-TKI resistance, by EMT introduction and ABCG2 upregulation, and blockade of Hh signaling synergistically increased sensitivity to EGFR-TKIs in primary and secondary resistant NSCLC cells. E-cadherin expression may be a potential biomarker of the suitability of the combined application of an Hh inhibitor and EGFR-TKIs in EGFR-TKI-resistant NSCLCs. PMID:26943330

  1. Epidermal stem cells and skin tissue engineering in hair follicle regeneration

    PubMed Central

    Balañá, María Eugenia; Charreau, Hernán Eduardo; Leirós, Gustavo José

    2015-01-01

    The reconstitution of a fully organized and functional hair follicle from dissociated cells propagated under defined tissue culture conditions is a challenge still pending in tissue engineering. The loss of hair follicles caused by injuries or pathologies such as alopecia not only affects the patients’ psychological well-being, but also endangers certain inherent functions of the skin. It is then of great interest to find different strategies aiming to regenerate or neogenerate the hair follicle under conditions proper of an adult individual. Based upon current knowledge on the epithelial and dermal cells and their interactions during the embryonic hair generation and adult hair cycling, many researchers have tried to obtain mature hair follicles using different strategies and approaches depending on the causes of hair loss. This review summarizes current advances in the different experimental strategies to regenerate or neogenerate hair follicles, with emphasis on those involving neogenesis of hair follicles in adult individuals using isolated cells and tissue engineering. Most of these experiments were performed using rodent cells, particularly from embryonic or newborn origin. However, no successful strategy to generate human hair follicles from adult cells has yet been reported. This review identifies several issues that should be considered to achieve this objective. Perhaps the most important challenge is to provide three-dimensional culture conditions mimicking the structure of living tissue. Improving culture conditions that allow the expansion of specific cells while protecting their inductive properties, as well as methods for selecting populations of epithelial stem cells, should give us the necessary tools to overcome the difficulties that constrain human hair follicle neogenesis. An analysis of patent trends shows that the number of patent applications aimed at hair follicle regeneration and neogenesis has been increasing during the last decade. This

  2. Epidermal stem cells and skin tissue engineering in hair follicle regeneration.

    PubMed

    Balañá, María Eugenia; Charreau, Hernán Eduardo; Leirós, Gustavo José

    2015-05-26

    The reconstitution of a fully organized and functional hair follicle from dissociated cells propagated under defined tissue culture conditions is a challenge still pending in tissue engineering. The loss of hair follicles caused by injuries or pathologies such as alopecia not only affects the patients' psychological well-being, but also endangers certain inherent functions of the skin. It is then of great interest to find different strategies aiming to regenerate or neogenerate the hair follicle under conditions proper of an adult individual. Based upon current knowledge on the epithelial and dermal cells and their interactions during the embryonic hair generation and adult hair cycling, many researchers have tried to obtain mature hair follicles using different strategies and approaches depending on the causes of hair loss. This review summarizes current advances in the different experimental strategies to regenerate or neogenerate hair follicles, with emphasis on those involving neogenesis of hair follicles in adult individuals using isolated cells and tissue engineering. Most of these experiments were performed using rodent cells, particularly from embryonic or newborn origin. However, no successful strategy to generate human hair follicles from adult cells has yet been reported. This review identifies several issues that should be considered to achieve this objective. Perhaps the most important challenge is to provide three-dimensional culture conditions mimicking the structure of living tissue. Improving culture conditions that allow the expansion of specific cells while protecting their inductive properties, as well as methods for selecting populations of epithelial stem cells, should give us the necessary tools to overcome the difficulties that constrain human hair follicle neogenesis. An analysis of patent trends shows that the number of patent applications aimed at hair follicle regeneration and neogenesis has been increasing during the last decade. This

  3. Cord Blood Stem Cells Inhibit Epidermal Growth Factor Receptor Translocation to Mitochondria in Glioblastoma

    PubMed Central

    Dasari, Venkata Ramesh; Velpula, Kiran Kumar; Alapati, Kiranmai; Gujrati, Meena; Tsung, Andrew J.

    2012-01-01

    Background Overexpression of EGFR is one of the most frequently diagnosed genetic aberrations of glioblastoma multiforme (GBM). EGFR signaling is involved in diverse cellular functions and is dependent on the type of preferred receptor complexes. EGFR translocation to mitochondria has been reported recently in different cancer types. However, mechanistic aspects of EGFR translocation to mitochondria in GBM have not been evaluated to date. Methodology/Principle Findings In the present study, we analyzed the expression of EGFR in GBM-patient derived specimens using immunohistochemistry, reverse-transcription based PCR and Western blotting techniques. In clinical samples, EGFR co-localizes with FAK in mitochondria. We evaluated this previous observation in standard glioma cell lines and in vivo mice xenografts. We further analyzed the effect of human umbilical cord blood stem cells (hUCBSC) on the inhibition of EGFR expression and EGFR signaling in glioma cells and xenografts. Treatment with hUCBSC inhibited the expression of EGFR and its co-localization with FAK in glioma cells. Also, hUCBSC inhibited the co-localization of activated forms of EGFR, FAK and c-Src in mitochondria of glioma cells and xenografts. In addition, hUCBSC also inhibited EGFR signaling proteins in glioma cells both in vitro and in vivo. Conclusions/Significance We have shown that hUCBSC treatments inhibit phosphorylation of EGFR, FAK and c-Src forms. Our findings associate EGFR expression and its localization to mitochondria with specific biological functions in GBM cells and provide relevant preclinical information that can be used for the development of effective hUCBSC-based therapies. PMID:22348136

  4. Mospd1, a new player in mesenchymal versus epidermal cell differentiation.

    PubMed

    Thaler, R; Rumpler, M; Spitzer, S; Klaushofer, K; Varga, F

    2011-10-01

    Mospd1 codes for a small protein with unknown physiological function, which is part of a family of genes, including Mospd2 and Mospd3, defined by the presence of the major sperm protein domain and two transmembrane domains. This work characterizes the Mospd1 gene, the intracellular location of the protein and its expression in different mouse tissues and mesenchymal cell lines during differentiation. The role of Mospd1 in mesenchymal cellular differentiation was studied by siRNA knockdown experiments in mouse osteoblastic MC3T3-E1 cells. Transfection experiments of the targeted cDNA show MOSPD1 located in the endoplasmatic reticulum and in the Golgi apparatus. Removal of the last exon of the gene resulted in localization of the protein in the nucleus, which was attributed to a nuclear export sequence in the N-terminal part. In mouse tissues the gene was generally strongly expressed while mesenchymal tissues showed the highest expression. In mesenchymal cell lines Mospd1 mRNA was higher expressed in cells with advanced differentiation status. In osteoblastic, myoblastic, and adipocytic cell lines Mospd1 was up-regulated during differentiation. Genome-wide gene expression analysis after knockdown of Mospd1 by siRNA in MC3T3-E1 cells revealed a shift in the gene expression pattern from mesenchymal to epithelial genes featuring up-regulation of the epithelial cadherin Cdh1 and down-regulation of its inhibitors Snail1 and 2 and the mesenchymal cadherin Cdh11, suggesting a mesenchymal to epithelial transition. From these data we conclude that Mospd1 plays a pivotal role in the developmental regulation at the switch between mesenchymal and epithelial cells. PMID:21792907

  5. Non-small-cell lung cancer cells combat epidermal growth factor receptor tyrosine kinase inhibition through immediate adhesion-related responses

    PubMed Central

    Wang, Hsian-Yu; Hsu, Min-Kung; Wang, Kai-Hsuan; Tseng, Ching-Ping; Chen, Feng-Chi; Hsu, John T-A

    2016-01-01

    Background Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs), such as gefitinib, erlotinib, and afatinib, have greatly improved treatment efficacy in non-small cell lung cancer (NSCLC) patients with drug-sensitive EGFR mutations. However, in some TKI responders, the benefits of such targeted therapies are limited by the rapid development of resistance, and strategies to overcome this resistance are urgently needed. Studies of drug resistance in cancer cells typically involve long term in vitro induction to obtain stably acquired drug-resistant cells followed by elucidation of resistance mechanisms, but the immediate responses of cancer cells upon drug treatment have been ignored. The aim of this study was to investigate the immediate responses of NSCLC cells upon treatment with EGFR TKIs. Results Both NSCLC cells, ie, PC9 and H1975, showed immediate enhanced adhesion-related responses as an apoptosis-countering mechanism upon first-time TKI treatment. By gene expression and pathway analysis, adhesion-related pathways were enriched in gefitinib-treated PC9 cells. Pathway inhibition by small-hairpin RNAs or small-molecule drugs revealed that within hours of EGFR TKI treatment, NSCLC cells used adhesion-related responses to combat the drugs. Importantly, we show here that the Src family inhibitor, dasatinib, dramatically inhibits cell adhesion-related response and greatly enhances the cell-killing effects of EGFR TKI (gefitinib for the PC9 cells; afatinib for the H1975 cells) in NSCLC cells, which would otherwise escape the TKI-induced apoptosis. Conclusion Results from this study indicate that NSCLC cells can employ the adhesion response as a survival pathway to survive under EGFR-targeted therapy. Simultaneous targeting of EGFR signaling and adhesion pathways would further boost the efficacy of EGFR-targeted therapy in NSCLC. PMID:27284246

  6. Tumor cells can follow distinct evolutionary paths to become resistant to epidermal growth factor receptor inhibition

    PubMed Central

    Hata, Aaron N; Niederst, Matthew J; Archibald, Hannah L; Gomez-Caraballo, Maria; Siddiqui, Faria M; Mulvey, Hillary E; Maruvka, Yosef E; Ji, Fei; Bhang, Hyo-eun C; Radhakrishna, Viveksagar Krishnamurthy; Siravegna, Giulia; Hu, Haichuan; Raoof, Sana; Lockerman, Elizabeth; Kalsy, Anuj; Lee, Dana; Keating, Celina L; Ruddy, David A; Damon, Leah J; Crystal, Adam S; Costa, Carlotta; Piotrowska, Zofia; Bardelli, Alberto; Iafrate, Anthony J; Sadreyev, Ruslan I; Stegmeier, Frank; Getz, Gad; Sequist, Lecia V; Faber, Anthony C; Engelman, Jeffrey A

    2016-01-01

    Although mechanisms of acquired resistance of EGFR mutant non-small cell lung cancers to EGFR inhibitors have been identified, little is known about how resistant clones evolve during drug therapy. Here, we observe that acquired resistance caused by the T790M gatekeeper mutation can occur either by selection of pre-existing T790M clones or via genetic evolution of initially T790M-negative drug tolerant cells. The path to resistance impacts the biology of the resistant clone, as those that evolved from drug tolerant cells had a diminished apoptotic response to third generation EGFR inhibitors that target T790M EGFR; treatment with navitoclax, an inhibitor of BCL-XL and BCL-2 restored sensitivity. We corroborated these findings using cultures derived directly from EGFR inhibitor-resistant patient tumors. These findings provide evidence that clinically relevant drug resistant cancer cells can both pre-exist and evolve from drug tolerant cells, and point to therapeutic opportunities to prevent or overcome resistance in the clinic. PMID:26828195

  7. Epidermal ‘alarm substance’ cells of fishes maintained by non-alarm functions: possible defence against pathogens, parasites and UVB radiation

    PubMed Central

    Chivers, Douglas P; Wisenden, Brian D; Hindman, Carrie J; Michalak, Tracy A; Kusch, Robin C; Kaminskyj, Susan G.W; Jack, Kristin L; Ferrari, Maud C.O; Pollock, Robyn J; Halbgewachs, Colin F; Pollock, Michael S; Alemadi, Shireen; James, Clayton T; Savaloja, Rachel K; Goater, Cameron P; Corwin, Amber; Mirza, Reehan S; Kiesecker, Joseph M; Brown, Grant E; Adrian, James C; Krone, Patrick H; Blaustein, Andrew R; Mathis, Alicia

    2007-01-01

    Many fishes possess specialized epidermal cells that are ruptured by the teeth of predators, thus reliably indicating the presence of an actively foraging predator. Understanding the evolution of these cells has intrigued evolutionary ecologists because the release of these alarm chemicals is not voluntary. Here, we show that predation pressure does not influence alarm cell production in fishes. Alarm cell production is stimulated by exposure to skin-penetrating pathogens (water moulds: Saprolegnia ferax and Saprolegnia parasitica), skin-penetrating parasites (larval trematodes: Teleorchis sp. and Uvulifer sp.) and correlated with exposure to UV radiation. Suppression of the immune system with environmentally relevant levels of Cd inhibits alarm cell production of fishes challenged with Saprolegnia. These data are the first evidence that alarm substance cells have an immune function against ubiquitous environmental challenges to epidermal integrity. Our results indicate that these specialized cells arose and are maintained by natural selection owing to selfish benefits unrelated to predator–prey interactions. Cell contents released when these cells are damaged in predator attacks have secondarily acquired an ecological role as alarm cues because selection favours receivers to detect and respond adaptively to public information about predation. PMID:17686729

  8. Forces generated by cell intercalation tow epidermal sheets in mammalian tissue morphogenesis.

    PubMed

    Heller, Evan; Kumar, K Vijay; Grill, Stephan W; Fuchs, Elaine

    2014-03-31

    While gastrulation movements offer mechanistic paradigms for how collective cellular movements shape developing embryos, far less is known about coordinated cellular movements that occur later in development. Studying eyelid closure, we explore a case where an epithelium locally reshapes, expands, and moves over another epithelium. Live imaging, gene targeting, and cell-cycle inhibitors reveal that closure does not require overlying periderm, proliferation, or supracellular actin cable assembly. Laser ablation and quantitative analyses of tissue deformations further distinguish the mechanism from wound repair and dorsal closure. Rather, cell intercalations parallel to the tissue front locally compress it perpendicularly, pulling the surrounding epidermis along the closure axis. Functional analyses in vivo show that the mechanism requires localized myosin-IIA- and α5β1 integrin/fibronectin-mediated migration and E-cadherin downregulation likely stimulated by Wnt signaling. These studies uncover a mode of epithelial closure in which forces generated by cell intercalation are leveraged to tow the surrounding tissue. PMID:24697897

  9. Visualization of the melanosome transfer-inhibition in a mouse epidermal cell co-culture model.

    PubMed

    Kim, Hae Jong; Kazi, Julhash U; Lee, You-Ree; Nguyen, Dung H; Lee, Hyang-Bok; Shin, Jeong-Hyun; Soh, Jae-Won; Kim, Eun-Ki

    2010-02-01

    Transfer of melanin-containing melanosomes from melanocytes to neighboring keratinocytes results in skin pigmentation. To provide a more practical method of visualizing melanosomes in melanocytes as well as in keratinocytes, we attempted to use murine cell lines instead of human primary cells. We generated various fluorescent fusion proteins of tyrosinase, a melanin synthesis enzyme located in the melanosome, by using green fluorescent protein and red fluorescent protein. The intracellular localization of tyrosinase was then examined by fluorescence and confocal microscopy. Co-culture of murine melanocytes and keratinocytes was optimized and melanosome transfer was either stimulated with alphaMSH or partially inhibited by niacinamide. To the best of our knowledge, this is the first study showing that a murine co-culture model, in addition to human primary cell co-culture, can be a good tool for depigmenting agent screening by monitoring melanosome transfer. PMID:20043134

  10. Forces Generated by Cell Intercalation Tow Epidermal Sheets in Mammalian Tissue Morphogenesis

    PubMed Central

    Heller, Evan; Kumar, K. Vijay; Grill, Stephan W.; Fuchs, Elaine

    2014-01-01

    Summary While gastrulation movements offer mechanistic paradigms for how collective cellular movements shape developing embryos, far less is known about coordinated cellular movements that occur later in development. Studying eyelid closure, we explore a case where an epithelium locally reshapes, expands, and moves over another epithelium. Live imaging, gene targeting and cell cycle inhibitors reveal that closure does not require overlying periderm, proliferation or supracellular actin cable assembly. Laser ablation and quantitative analyses of tissue deformations further distinguish the mechanism from wound-repair and dorsal closure. Rather, cell intercalations parallel to the tissue front locally compress it perpendicularly, pulling the surrounding epidermis along the closure axis. Functional analyses in vivo show that the mechanism requires localized myosin-IIA and α5β1-fibronectin-mediated migration, and E-cadherin downregulation likely stimulated by Wnt signaling. These studies uncover a mode of epithelial closure in which forces generated by cell intercalation are leveraged to tow the surrounding tissue. PMID:24697897

  11. Cellular and Tumor Radiosensitivity is Correlated to Epidermal Growth Factor Receptor Protein Expression Level in Tumors Without EGFR Amplification;Epidermal growth factor receptor; Radiotherapy; Squamous cell carcinoma; Biomarker; Local tumor control

    SciTech Connect

    Kasten-Pisula, Ulla; Saker, Jarob; Eicheler, Wolfgang; Krause, Mechthild; Yaromina, Ala; Meyer-Staeckling, Soenke; Scherkl, Benjamin; Kriegs, Malte; Brandt, Burkhard; Grenman, Reidar; Petersen, Cordula; Baumann, Michael; Dikomey, Ekkehard

    2011-07-15

    Purpose: There is conflicting evidence for whether the expression of epidermal growth factor receptor in human tumors can be used as a marker of radioresponse. Therefore, this association was studied in a systematic manner using squamous cell carcinoma (SCC) cell lines grown as cell cultures and xenografts. Methods and Materials: The study was performed with 24 tumor cell lines of different tumor types, including 10 SCC lines, which were also investigated as xenografts on nude mice. Egfr gene dose and the length of CA-repeats in intron 1 were determined by polymerase chain reaction, protein expression in vitro by Western blot and in vivo by enzyme-linked immunosorbent assay, and radiosensitivity in vitro by colony formation. Data were correlated with previously published tumor control dose 50% data after fractionated irradiation of xenografts of the 10 SCC. Results: EGFR protein expression varies considerably, with most tumor cell lines showing moderate and only few showing pronounced upregulation. EGFR upregulation could only be attributed to massive gene amplification in the latter. In the case of little or no amplification, in vitro EGFR expression correlated with both cellular and tumor radioresponse. In vivo EGFR expression did not show this correlation. Conclusions: Local tumor control after the fractionated irradiation of tumors with little or no gene amplification seems to be dependent on in vitro EGFR via its effect on cellular radiosensitivity.

  12. Epidermal growth factor: Porcine uterine luminal epithelial cell migratory signal during the peri-implantation period of pregnancy.

    PubMed

    Jeong, Wooyoung; Jung, Seoungo; Bazer, Fuller W; Song, Gwonhwa; Kim, Jinyoung

    2016-01-15

    The majority of early conceptus mortality in pregnancy occurs during the peri-implantation period, suggesting that this period is important for conceptus viability and the establishment of pregnancy. Successful establishment of pregnancy in all mammalian species depends on the orchestrated molecular events that transpire at the conceptus-uterine interface during the peri-implantation period of pregnancy. This maternal-conceptus interaction is especially crucial in pigs because they have a non-invasive epitheliochorial placentation during a protracted peri-implantation period. During the pre-implantation period of pregnancy, conceptus survival and the establishment of pregnancy depend on the developing conceptus receiving an adequate supply of histotroph which contains a wide range of nutrients and growth factors. Evidence links epidermal growth factor (EGF) to embryogenesis or implantation in various mammalian species. EGF exhibits potential growth-promoting activities on the conceptus and endometrium; however, in the case of pigs, little is known its functions, especially their regulatory mechanisms at the maternal-conceptus interface. EGF receptor (EGFR) mRNA and protein are abundant in endometrial luminal (LE) and glandular (GE) epithelia and conceptus trophectoderm on Days 13-14 of pregnancy, suggesting that EGF provides an autocrine signal to uterine LE and GE just prior to implantation. Therefore, the objectives of this study were to determine: 1) the potential intracellular signaling pathways responsible for the activities of EGF in porcine uterine LE (pLE) cells; and 2) the changes in cellular activities induced by EGF. EGF treatment of pLE cells increased the abundance of phosphorylated (p)-ERK1/2, p-P70RSK and p-RPS6 compared to that for control cells. Furthermore, EGF-stimulated phosphorylation of ERK1/2 MAPK was inhibited in pLE cells transfected with an EGFR siRNA compared with control siRNA-transfected pLE cells. Moreover, EGF stimulated migration of

  13. Combined effects of lapatinib and bortezomib in human epidermal receptor 2 (HER2)-overexpressing breast cancer cells and activity of bortezomib against lapatinib-resistant breast cancer cells.

    PubMed

    Ma, Chuandong; Niu, Xiuqing; Luo, Jianmin; Shao, Zhimin; Shen, Kunwei

    2010-10-01

    Lapatinib and bortezomib are highly active against breast cancer cells. Breast cancer patients who initially respond to lapatinib may eventually manifest acquired resistance to this treatment. Thus, the identification of novel agents that may prevent or delay the development of acquired resistance to lapatinib is critical. In the current study, we show that the combination of lapatinib and bortezomib results in a synergistic growth inhibition in human epidermal receptor 2 (HER2)-overexpressing breast cancer cells and that the combination enhances apoptosis of SK-BR-3 cells. Importantly, we found that the combination of lapatinib plus bortezomib more effectively blocked activation of the HER2 pathway in SK-BR-3 cells, compared with monotherapy. In addition, we established a model of acquired resistance to lapatinib by chronically challenging SK-BR-3 breast cancer cells with increasing concentrations of lapatinib. Here, we showed that bortezomib notably induced apoptosis of lapatinib-resistant SK-BR-3 pools and further inhibited HER2 signaling in the resistant cells. Taken together, the current data indicate a synergistic interaction between lapatinib and bortezomib in HER2-overexpressing breast cancer cells and provide the rationale for the clinical evaluation of these two noncross-resistant targeted therapies. The combination of lapatinib and bortezomib may be a potentially novel approach to prevent or delay the onset of acquired resistance to lapatinib in HER2-overxpressing/estrogen receptor (ER)-negative breast cancers. PMID:20701607

  14. Epidermal growth factor upregulates Skp2/Cks1 and p27kip1 in human extrahepatic cholangiocarcinoma cells

    PubMed Central

    Kim, Ja-yeon; Kim, Hong Joo; Park, Jung Ho; Park, Dong Il; Cho, Yong Kyun; Sohn, Chong Il; Jeon, Woo Kyu; Kim, Byung Ik; Kim, Dong Hoon; Chae, Seoung Wan; Sohn, Jin Hee

    2014-01-01

    AIM: To evaluate the expression status of S-phase kinase-associated protein 2 (Skp2)/cyclin-dependent kinases regulatory subunit 1 (Cks1) and p27kip1, and assess the prognostic significance of Skp2/Cks1 expression with p27kip1 in patients with extrahepatic cholangiocarcinoma. METHODS: Seventy-six patients who underwent curative resection for histologically confirmed extrahepatic cholangiocarcinoma at our institution from December 1994 to March 2008 were enrolled. Immunohistochemical staining for Skp2, Cks1, p27kip1, and Ki67, along with other relevant molecular biologic experiments, were performed. RESULTS: By Cox regression analyses, advanced age (> 65 years), advanced AJCC tumor stage, poorly differentiated histology, and higher immunostaining intensity of Skp2 were identified as independent prognostic factors in patients with extrahepatic cholangiocarcinoma. Exogenous epidermal growth factor (EGF, especially 0.1-10 ng/mL) significantly increased the proliferation indices by MTT assay and the mRNA levels of Skp2/Cks1 and p27kip1 in SNU-1196, SNU-1079, and SNU-245 cells. The protein levels of Skp2/Cks1 (from nuclear lysates) and p27kip1 (from cytosolic lysate) were also significantly increased in these cells. There were significant reductions in the protein levels of Skp2/Cks1 and p27kip1 (from nuclear lysate) after the treatment of LY294002. By chromatin immunoprecipitation assay, we found that E2F1 transcription factor directly binds to the promoter site of Skp2. CONCLUSION: Higher immunostaining intensity of Skp2/Cks1 was an independent prognostic factor for patients with extrahepatic cholangiocarcinoma. EGF upregulates the mRNA and protein levels of Skp2/Cks1 and p27kip1 via the PI3K/Akt pathway and direct binding of E2F1 transcription factor with the Skp2 promoter. PMID:24574749

  15. Dermabrasion and Thin Epidermal Grafting for Treatment of Large and Small Areas of Postburn Leukoderma: A Case Series and Review of the Literature.

    PubMed

    Driscoll, Daniel N; Levy, Alexander N; Gama, Amon-Ra

    2016-01-01

    Deep burn injuries can have serious aesthetic consequences as it often results in scar tissue and pigmentary changes of the skin. The focus of this article is to report our experience and results using dermabrasion and thin split-thickness skin grafting as a technique for restoring skin pigmentation after burn injuries. Patient records were obtained from a pediatric burn hospital medical record database from 1990 to 2007. Both charts and photographs were retrospectively reviewed. The treatment was evaluated for body region treated, surface area involved, effectiveness of treatment, and number of treatments required. Indications for the procedure included longstanding depigmentation, defined as greater than 1 year, and a patient wiling to have a donor site. The areas of vitiligo were marked and dermabraded with a mechanical dermabrader. Thin epidermal grafts with a thickness of 6 thousands of an inch were harvested with an air-powered dermatome. The grafts were affixed to the dermabraded bed and dressed open or with nonstick gauze for areas of the face and wrapped for areas in the extremities. Eleven patients underwent 16 procedures. The average size of the graft per procedure was 87 cm (4-500 cm). All results were consistent and long-lasting at follow-up. Postburn leukoderma of long duration is well treated by dermabrasion and thin split-thickness skin grafting. This study is unique in describing grafting on multiple occasions and for larger areas than previously described, with two patients undergoing grafting more than 200 cm. PMID:26135526

  16. Arabidopsis Actin Depolymerizing Factor4 Modulates the Stochastic Dynamic Behavior of Actin Filaments in the Cortical Array of Epidermal Cells[C][W

    PubMed Central

    Henty, Jessica L.; Bledsoe, Samuel W.; Khurana, Parul; Meagher, Richard B.; Day, Brad; Blanchoin, Laurent; Staiger, Christopher J.

    2011-01-01

    Actin filament arrays are constantly remodeled as the needs of cells change as well as during responses to biotic and abiotic stimuli. Previous studies demonstrate that many single actin filaments in the cortical array of living Arabidopsis thaliana epidermal cells undergo stochastic dynamics, a combination of rapid growth balanced by disassembly from prolific severing activity. Filament turnover and dynamics are well understood from in vitro biochemical analyses and simple reconstituted systems. However, the identification in living cells of the molecular players involved in controlling actin dynamics awaits the use of model systems, especially ones where the power of genetics can be combined with imaging of individual actin filaments at high spatial and temporal resolution. Here, we test the hypothesis that actin depolymerizing factor (ADF)/cofilin contributes to stochastic filament severing and facilitates actin turnover. A knockout mutant for Arabidopsis ADF4 has longer hypocotyls and epidermal cells when compared with wild-type seedlings. This correlates with a change in actin filament architecture; cytoskeletal arrays in adf4 cells are significantly more bundled and less dense than in wild-type cells. Several parameters of single actin filament turnover are also altered. Notably, adf4 mutant cells have a 2.5-fold reduced severing frequency as well as significantly increased actin filament lengths and lifetimes. Thus, we provide evidence that ADF4 contributes to the stochastic dynamic turnover of actin filaments in plant cells. PMID:22010035

  17. Selective Killing Effects of Cold Atmospheric Pressure Plasma with NO Induced Dysfunction of Epidermal Growth Factor Receptor in Oral Squamous Cell Carcinoma

    PubMed Central

    Lee, Jung-Hwan; Om, Ji-Yeon; Kim, Yong-Hee; Kim, Kwang-Mahn; Choi, Eun-Ha; Kim, Kyoung-Nam

    2016-01-01

    The aim of this study is to investigate the effects of cold atmospheric pressure plasma (CAP)-induced radicals on the epidermal growth factor receptor (EGFR), which is overexpressed by oral squamous cell carcinoma, to determine the underlying mechanism of selective killing. CAP-induced highly reactive radicals were observed in both plasma plume and cell culture media. The selective killing effect was observed in oral squamous cell carcinoma compared with normal human gingival fibroblast. Degradation and dysfunction of EGFRs were observed only in the EGFR-overexpressing oral squamous cell carcinoma and not in the normal cell. Nitric oxide scavenger pretreatment in cell culture media before CAP treatment rescued above degradation and dysfunction of the EGFR as well as the killing effect in oral squamous cell carcinoma. CAP may be a promising cancer treatment method by inducing EGFR dysfunction in EGFR-overexpressing oral squamous cell carcinoma via nitric oxide radicals. PMID:26919318

  18. In Vivo Quantification of Peroxisome Tethering to Chloroplasts in Tobacco Epidermal Cells Using Optical Tweezers.

    PubMed

    Gao, Hongbo; Metz, Jeremy; Teanby, Nick A; Ward, Andy D; Botchway, Stanley W; Coles, Benjamin; Pollard, Mark R; Sparkes, Imogen

    2016-01-01

    Peroxisomes are highly motile organelles that display a range of motions within a short time frame. In static snapshots, they can be juxtaposed to chloroplasts, which has led to the hypothesis that they are physically interacting. Here, using optical tweezers, we tested the dynamic physical interaction in vivo. Using near-infrared optical tweezers combined with TIRF microscopy, we were able to trap peroxisomes and approximate the forces involved in chloroplast association in vivo in tobacco (Nicotiana tabacum) and observed weaker tethering to additional unknown structures within the cell. We show that chloroplasts and peroxisomes are physically tethered through peroxules, a poorly described structure in plant cells. We suggest that peroxules have a novel role in maintaining peroxisome-organelle interactions in the dynamic environment. This could be important for fatty acid mobilization and photorespiration through the interaction with oil bodies and chloroplasts, highlighting a fundamentally important role for organelle interactions for essential biochemistry and physiological processes. PMID:26518344

  19. Characterization of A Three-Dimensional Organotypic Co-Culture Skin Model for Epidermal Differentiation of Rat Adipose-Derived Stem Cells

    PubMed Central

    Ghanavati, Zeinab; Orazizadeh, Mahmoud; Bayati, Vahid; Abbaspour, Mohammad Reza; Khorsandi, Layasadat; Mansouri, Esrafil; Neisi, Niloofar

    2016-01-01

    Objective The organotypic co-culture is a well-known technique to examine cellular interactions and their roles in stem cell proliferation and differentiation. This study aims to evaluate the effects of dermal fibroblasts (DFs) on epidermal differentiation of adipose-derived stem cells (ASCs) using a three-dimensional (3D) organotypic co- culture technique. Materials and Methods In this experimental research study, rat DFs and ASCs were isolated and cultured separately on electrospun polycaprolactone (PCL) matrices. The PCL matrices seeded by ASCs were superimposed on to the matrices seeded by DFs in order to create a 3D organotypic co-culture. In the control groups, PCL matrices seeded by ASCs were placed on matrices devoid of DFs. After 10 days, we assessed the expressions of keratinocyte-related genes by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and expression of pan-cytokeratin protein by immunofluorescence in the differentiated keratinocyte-like cells from co- culture and control groups. Keratinocyte-like cell morphologies were also observed by scanning electron microscopy (SEM). Results The early, intermediate, and terminal differentiation keratinocyte markers-Cytokeratin14, Filaggrin, and Involucrin significantly expressed in the co-culture groups com- pared to the control ones (P<0.05). We observed pan-cytokeratin in keratinocyte-like cells of both groups by immunofluorescence. SEM observation of the co-culture groups showed that the differentiated keratinocyte-like cells developed a polygonal cobblestone shape, considered characteristic of keratinocytes. Conclusion The 3D organotypic co-culture bilayered construct that consisted of DFs and ASCs was an effective technique for epidermal differentiation of ASCs. This co-culture might be useful for epidermal differentiation of stem cells for future applications in skin regeneration. PMID:27602310

  20. PDK1 Is a Regulator of Epidermal Differentiation that Activates and Organizes Asymmetric Cell Division.

    PubMed

    Dainichi, Teruki; Hayden, Matthew S; Park, Sung-Gyoo; Oh, Hyunju; Seeley, John J; Grinberg-Bleyer, Yenkel; Beck, Kristen M; Miyachi, Yoshiki; Kabashima, Kenji; Hashimoto, Takashi; Ghosh, Sankar

    2016-05-24

    Asymmetric cell division (ACD) in a perpendicular orientation promotes cell differentiation and organizes the stratified epithelium. However, the upstream cues regulating ACD have not been identified. Here, we report that phosphoinositide-dependent kinase 1 (PDK1) plays a critical role in establishing ACD in the epithelium. Production of phosphatidyl inositol triphosphate (PIP3) is localized to the apical side of basal cells. Asymmetric recruitment of atypical protein kinase C (aPKC) and partitioning defective (PAR) 3 is impaired in PDK1 conditional knockout (CKO) epidermis. PDK1(CKO) keratinocytes do not undergo calcium-induced activation of aPKC or IGF1-induced activation of AKT and fail to differentiate. PDK1(CKO) epidermis shows decreased expression of Notch, a downstream effector of ACD, and restoration of Notch rescues defective expression of differentiation-induced Notch targets in vitro. We therefore propose that PDK1 signaling regulates the basal-to-suprabasal switch in developing epidermis by acting as both an activator and organizer of ACD and the Notch-dependent differentiation program. PMID:27184845

  1. Modulation of estrogen and epidermal growth factor receptors by rosemary extract in breast cancer cells.

    PubMed

    González-Vallinas, Margarita; Molina, Susana; Vicente, Gonzalo; Sánchez-Martínez, Ruth; Vargas, Teodoro; García-Risco, Mónica R; Fornari, Tiziana; Reglero, Guillermo; Ramírez de Molina, Ana

    2014-06-01

    Breast cancer is the leading cause of cancer-related mortality among females worldwide, and therefore the development of new therapeutic approaches is still needed. Rosemary (Rosmarinus officinalis L.) extract possesses antitumor properties against tumor cells from several organs, including breast. However, in order to apply it as a complementary therapeutic agent in breast cancer, more information is needed regarding the sensitivity of the different breast tumor subtypes and its effect in combination with the currently used chemotherapy. Here, we analyzed the antitumor activities of a supercritical fluid rosemary extract (SFRE) in different breast cancer cells, and used a genomic approach to explore its effect on the modulation of ER-α and HER2 signaling pathways, the most important mitogen pathways related to breast cancer progression. We found that SFRE exerts antitumor activity against breast cancer cells from different tumor subtypes and the downregulation of ER-α and HER2 receptors by SFRE might be involved in its antitumor effect against estrogen-dependent (ER+) and HER2 overexpressing (HER2+) breast cancer subtypes. Moreover, SFRE significantly enhanced the effect of breast cancer chemotherapy (tamoxifen, trastuzumab, and paclitaxel). Overall, our results support the potential utility of SFRE as a complementary approach in breast cancer therapy. PMID:24615943

  2. Tyrosine kinase inhibitors for epidermal growth factor receptor gene mutation-positive non-small cell lung cancers: an update for recent advances in therapeutics.

    PubMed

    Chung, Clement

    2016-06-01

    The presence of activating gene mutations in the epidermal growth factor receptor of non-small cell lung cancer patients is predictive (improved progression-free survival and improved response rate) when treated with small molecule tyrosine kinase inhibitors such as gefitinib, erlotinib and afatinib. The two most common mutations that account for greater than 85% of all EGFR gene mutations are in-frame deletions in exon 19 (LREA deletions) and substitution in exon 21 (L858R). Exon 18 mutations occur much less frequently at about 4% of all EGFR gene mutations. Together, exon 19 deletion and exon 21 L858R gene substitution are present in about 10% of Caucasian patients and 20-40% of Asian patients with non-small cell lung cancer. T790M gene mutation at exon 20 is associated with acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors. Early studies showed that activating EGFR gene mutations are most common in patients with adenocarcinoma histology, women, never smokers and those of Asian ethnicity. A recent multi-center phase III trial suggested that frontline epidermal growth factor receptor tyrosine kinase inhibitor therapy with afatinib is associated with improved progression-free survival compared to chemotherapy regardless of race. Moreover, guidelines now suggest EGFR gene mutation testing should be conducted in all patients with lung adenocarcinoma or mixed lung cancers with an adenocarcinoma component, regardless of characteristics such as smoking status, gender or race. The success of targeted therapies in non-small cell lung cancer patients has changed the treatment paradigm in metastatic non-small cell lung cancer. However, despite a durable response of greater than a year, resistance to epidermal growth factor receptor tyrosine kinase inhibitors inevitably occurs. This mini-review describes the clinically relevant EGFR gene mutations and the efficacy/toxicity of small molecule epidermal growth factor receptor tyrosine kinase

  3. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR)

    PubMed Central

    Trussoni, Christy E.; Tabibian, James H.; Splinter, Patrick L.; O’Hara, Steven P.

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes. PMID:25915403

  4. Lipopolysaccharide (LPS)-Induced Biliary Epithelial Cell NRas Activation Requires Epidermal Growth Factor Receptor (EGFR).

    PubMed

    Trussoni, Christy E; Tabibian, James H; Splinter, Patrick L; O'Hara, Steven P

    2015-01-01

    Cholangiocytes (biliary epithelial cells) actively participate in microbe-induced proinflammatory responses in the liver and contribute to inflammatory and infectious cholangiopathies. We previously demonstrated that cholangiocyte TLR-dependent NRas activation contributes to proinflammatory/ proliferative responses. We test the hypothesis that LPS-induced activation of NRas requires the EGFR. SV40-transformed human cholangiocytes (H69 cells), or low passage normal human cholangiocytes (NHC), were treated with LPS in the presence or absence of EGFR or ADAM metallopeptidase domain 17 (TACE) inhibitors. Ras activation assays, quantitative RT-PCR, and proliferation assays were performed in cells cultured with or without inhibitors or an siRNA to Grb2. Immunofluorescence for phospho-EGFR was performed on LPS-treated mouse samples and specimens from patients with primary sclerosing cholangitis, primary biliary cirrhosis, hepatitis C, and normal livers. LPS-treatment induced an association between the TLR/MyD88 and EGFR/Grb2 signaling apparatus, NRas activation, and EGFR phosphorylation. NRas activation was sensitive to EGFR and TACE inhibitors and correlated with EGFR phosphorylation. The TACE inhibitor and Grb2 depletion prevented LPS-induced IL6 expression (p<0.05) and proliferation (p<0.01). Additionally, cholangiocytes from LPS-treated mouse livers and human primary sclerosing cholangitis (PSC) livers exhibited increased phospho-EGFR (p<0.01). Moreover, LPS-induced mouse cholangiocyte proliferation was inhibited by concurrent treatment with the EGFR inhibitor, Erlotinib. Our results suggest that EGFR is essential for LPS-induced, TLR4/MyD88-mediated NRas activation and induction of a robust proinflammatory cholangiocyte response. These findings have implications not only for revealing the signaling potential of TLRs, but also implicate EGFR as an integral component of cholangiocyte TLR-induced proinflammatory processes. PMID:25915403

  5. Rapid and simple isolation of vascular, epidermal and mesophyll cells from plant leaf tissue.

    PubMed

    Endo, Motomu; Shimizu, Hanako; Araki, Takashi

    2016-08-01

    To understand physiological phenomena at the tissue level, elucidation of tissue-specific molecular functions in vivo is required. As an example of the current state of affairs, many genes in plants have been reported to have discordant levels of expression between bulk tissues and the specific tissues in which the respective gene product is principally functional. The principal challenge in deciphering such tissue-specific functions lies in separating tissues with high spatiotemporal resolution to evaluate accurate gene expression profiles. Here, we provide a simple and rapid tissue isolation protocol to isolate all three major leaf tissues (mesophyll, vasculature and epidermis) from Arabidopsis within 30 min with high purity. On the basis of the different cell-to-cell connectivities of tissues, the mesophyll isolation is achieved by making protoplasts, and the vasculature and epidermis isolation is achieved through sonication and enzymatic digestion of leaves. We have successfully tested the protocol on several other plant species, including crop plants such as soybean, tomato and wheat. Furthermore, isolated tissues can be used not only for tissue-specific transcriptome assays but also potentially for tissue-specific proteome and methylome assays. PMID:27388555

  6. Establishment of 3D organotypic cultures using human neonatal epidermal cells.

    PubMed

    Gangatirkar, Pradnya; Paquet-Fifield, Sophie; Li, Amy; Rossi, Ralph; Kaur, Pritinder

    2007-01-01

    This protocol describes an ex vivo three-dimensional coculture system optimized to study the skin regenerative ability of primary human keratinocytes grown at the air-liquid interface on collagen matrices embedded with human dermal fibroblasts. An option for enrichment of keratinocyte stem cells and their progeny using fluorescence-activated cell sorting is also provided. Initially, dermal equivalents, comprising human passaged fibroblasts seeded in a collagen matrix, are grown on porous filters (3 mum) placed in transwells. After 1 week, primary human keratinocytes are seeded on this base. One week later, an air-lift transition is performed, leading to the differentiation of the keratinocytes, which are macroscopically visible as artificial skin after a couple of days. The cultures can be harvested 1 week after the air-lift and processed for immunohistochemistry or gene expression analysis. The overall procedure can be completed in 3 weeks, including the preparation of the dermal equivalent and the seeding of the primary keratinocytes. PMID:17401352

  7. Intercalated chemotherapy and erlotinib for non-small cell lung cancer (NSCLC) with activating epidermal growth factor receptor (EGFR) mutations.

    PubMed

    Zwitter, Matjaz; Rajer, Mirjana; Stanic, Karmen; Vrankar, Martina; Doma, Andrej; Cuderman, Anka; Grmek, Marko; Kern, Izidor; Kovac, Viljem

    2016-08-01

    Among attempts to delay development of resistance to tyrosine kinase inhibitors (TKIs) in patients with advanced non-small cell lung cancer (NSCLC) with activating mutations of epidermal growth factor receptor (EGFR), intercalated therapy has not been properly evaluated. In a phase II trial, 38 patients with EGFR mutated NSCLC in advanced stage were treated with 4 to 6 3-weekly cycles of intercalated schedule with gemcitabine (1250 mg/m2, days 1 and 4), cisplatin (75 mg/m2, day 2) and erlotinib (150 mg, days 5 - 15), followed by continuous erlotinib as maintenance. In addition to standard radiologic evaluation according to RECIST, PET/CT was done prior to treatment and at 6 months, using PERCIST as a method for assessment of response. The primary endpoint was progression-free survival (PFS). In general, tolerance to treatment was good, even among 8 patients with performance status 2-3 and 13 patients with brain metastases; grade 4 toxicity included 2 cases of neutropenia and 4 thrombo-embolic events. Complete response (CR) or partial response (PR) were seen in 15 (39.5%) and 17 (44.7%) cases, respectively. All cases of CR were confirmed also by PET/CT. Median PFS was 23.4 months and median overall survival (OS) was 38.3  months. After a median follow-up of 35 months, 8 patients are still in CR and on maintenance erlotinib. In conclusion, intercalated treatment for treatment-naive patients with EGFR activating mutations leads to excellent response rate and prolonged PFS and survival. Comparison of the intercalated schedule to monotherapy with TKIs in a randomized trial is warranted. PMID:27261103

  8. Epigenetic modifications of triterpenoid ursolic acid in activating Nrf2 and blocking cellular transformation of mouse epidermal cells.

    PubMed

    Kim, Hyuck; Ramirez, Christina N; Su, Zheng-Yuan; Kong, Ah-Ng Tony

    2016-07-01

    Ursolic acid (UA), a well-known natural triterpenoid found in abundance in blueberries, cranberries and apple peels, has been reported to possess many beneficial health effects. These effects include anticancer activity in various cancers, such as skin cancer. Skin cancer is the most common cancer in the world. Nuclear factor E2-related factor 2 (Nrf2) is a master regulator of antioxidative stress response with anticarcinogenic activity against UV- and chemical-induced tumor formation in the skin. Recent studies show that epigenetic modifications of Nrf2 play an important role in cancer prevention. However, the epigenetic impact of UA on Nrf2 signaling remains poorly understood in skin cancer. In this study, we investigated the epigenetic effects of UA on mouse epidermal JB6 P+ cells. UA inhibited cellular transformation by 12-O-tetradecanoylphorbol-13-acetate at a concentration at which the cytotoxicity was no more than 25%. Under this condition, UA induced the expression of the Nrf2-mediated detoxifying/antioxidant enzymes heme oxygenase-1, NAD(P)H:quinone oxidoreductase 1 and UDP-glucuronosyltransferase 1A1. DNA methylation analysis revealed that UA demethylated the first 15 CpG sites of the Nrf2 promoter region, which correlated with the reexpression of Nrf2. Furthermore, UA reduced the expression of epigenetic modifying enzymes, including the DNA methyltransferases DNMT1 and DNMT3a and the histone deacetylases (HDACs) HDAC1, HDAC2, HDAC3 and HDAC8 (Class I) and HDAC6 and HDAC7 (Class II), and HDAC activity. Taken together, these results suggest that the epigenetic effects of the triterpenoid UA could potentially contribute to its beneficial effects, including the prevention of skin cancer. PMID:27260468

  9. Expressed miRNAs target feather related mRNAs involved in cell signaling, cell adhesion and structure during chicken epidermal development.

    PubMed

    Bao, Weier; Greenwold, Matthew J; Sawyer, Roger H

    2016-10-15

    MicroRNAs (miRNAs) are small non-coding RNAs that regulate gene expression at the post-transcriptional level. Previous studies have shown that miRNA regulation contributes to a diverse set of processes including cellular differentiation and morphogenesis which leads to the creation of different cell types in multicellular organisms and is thus key to animal development. Feathers are one of the most distinctive features of extant birds and are important for multiple functions including flight, thermal regulation, and sexual selection. However, the role of miRNAs in feather development has been woefully understudied despite the identification of cell signaling pathways, cell adhesion molecules and structural genes involved in feather development. In this study, we performed a microarray experiment comparing the expression of miRNAs and mRNAs among three embryonic stages of development and two tissues (scutate scale and feather) of the chicken. We combined this expression data with miRNA target prediction tools and a curated list of feather related genes to produce a set of 19 miRNA-mRNA duplexes. These targeted mRNAs have been previously identified as important cell signaling and cell adhesion genes as well as structural genes involved in feather and scale morphogenesis. Interestingly, the miRNA target site of the cell signaling pathway gene, Aldehyde Dehydrogenase 1 Family, Member A3 (ALDH1A3), is unique to birds indicating a novel role in Aves. The identified miRNA target site of the cell adhesion gene, Tenascin C (TNC), is only found in specific chicken TNC splice variants that are differentially expressed in developing scutate scale and feather tissue indicating an important role of miRNA regulation in epidermal differentiation. Additionally, we found that β-keratins, a major structural component of avian and reptilian epidermal appendages, are targeted by multiple miRNA genes. In conclusion, our work provides quantitative expression data on miRNAs and m

  10. Quantitative differences in host cell reactivation of ultraviolet-damaged virus in human skin fibroblasts and epidermal keratinocytes cultured from the same foreskin biopsy

    SciTech Connect

    Tyrrell, R.M.; Pidoux, M.

    1986-06-01

    Repair efficiency of cultured cells may be estimated by measuring the ability of a particular cell type to support virus damaged by an appropriate agent. In this study we have compared the inactivation of ultraviolet (254 nm)-damaged herpes simplex virus in human fibroblast and epidermal keratinocyte cell lines derived from the same foreskin biopsy and found the epithelial cells to be a factor of 3 times less efficient in supporting the damaged virus. The two different cell types show comparable ultraviolet inactivation of clone-forming ability, indicating that the difference is specific to viral host cell reactivation. This study required the development of a quantitative infectious centers assay for the measurement of viral titer in human epithelial cells, a system which may be of more general application in studies of potential human carcinogens.

  11. Lactobacillus reuteri protects epidermal keratinocytes from Staphylococcus aureus-induced cell death by competitive exclusion.

    PubMed

    Prince, Tessa; McBain, Andrew J; O'Neill, Catherine A

    2012-08-01

    Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit Staphylococcus aureus infection. Evaluation of the candidate probiotics Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus AC413, and Lactobacillus salivarius UCC118 demonstrated that both L. reuteri and L. rhamnosus, but not L. salivarius, reduced S. aureus-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (P < 0.01) or simultaneously with (P < 0.01) infection with S. aureus but not when added after infection had commenced (P > 0.05). The protective effect of L. reuteri was not dependent on the elaboration of inhibitory substances such as lactic acid. L. reuteri inhibited adherence of S. aureus to keratinocytes by competitive exclusion (P = 0.026). L. salivarius UCC118, however, did not inhibit S. aureus from adhering to keratinocytes (P > 0.05) and did not protect keratinocyte viability. S. aureus utilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (P = 0.03). This suggests that the protective mechanism for L. reuteri-mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin by S. aureus and thus aid in the prevention of infection. PMID:22582077

  12. Transcriptional and Secretomic Profiling of Epidermal Cells Exposed to Alpha Particle Radiation

    PubMed Central

    Chauhan, Vinita; Howland, Matthew; Greene, Hillary Boulay; Wilkins, Ruth C

    2012-01-01

    Alpha (α)-particle emitters are probable isotopes to be used in a terrorist attack. The development of biological assessment tools to identify those who have handled these difficult to detect materials would be an asset to our current forensic capacity. In this study, for the purposes of biomarker discovery, human keratinocytes were exposed to α-particle and X-radiation (0.98 Gy/h at 0, 0.5, 1.0, 1.5 Gy) and assessed for differential gene and protein expression using microarray and Bio-Plex technology, respectively. Secretomic analysis of supernatants showed expression of two pro-inflammatory cytokines (IL-13 and PDGF-bb) to be exclusively affected in α-particle exposed cells. The highest dose of α-particle radiation modulated a total of 67 transcripts (fold change>|1.5|, (False discovery rate) FDR<0.05) in exposed cells. Several genes which responded with high expression levels (>2 fold) included KIF20A, NEFM, C7orf10, HIST1H2BD, BMP6, and HIST1H2AC. Among the high expressing genes, five (CCNB2, BUB1, NEK2, CDC20, AURKA) were also differentially expressed at the medium (1.0 Gy) dose however, these genes were unmodulated following exposure to X-irradiation. Networks of these genes clustered around tumor protein-53 and transforming growth factor-beta signaling. This study has identified some potential gene /protein responses and networks that may be validated further to confirm their specificity and potential to be signature biomarkers of α-particle exposure. PMID:23002402

  13. Lactobacillus reuteri Protects Epidermal Keratinocytes from Staphylococcus aureus-Induced Cell Death by Competitive Exclusion

    PubMed Central

    Prince, Tessa; McBain, Andrew J.

    2012-01-01

    Recent studies have suggested that the topical application of probiotic bacteria can improve skin health or combat disease. We have utilized a primary human keratinocyte culture model to investigate whether probiotic bacteria can inhibit Staphylococcus aureus infection. Evaluation of the candidate probiotics Lactobacillus reuteri ATCC 55730, Lactobacillus rhamnosus AC413, and Lactobacillus salivarius UCC118 demonstrated that both L. reuteri and L. rhamnosus, but not L. salivarius, reduced S. aureus-induced keratinocyte cell death in both undifferentiated and differentiated keratinocytes. Keratinocyte survival was significantly higher if the probiotic was applied prior to (P < 0.01) or simultaneously with (P < 0.01) infection with S. aureus but not when added after infection had commenced (P > 0.05). The protective effect of L. reuteri was not dependent on the elaboration of inhibitory substances such as lactic acid. L. reuteri inhibited adherence of S. aureus to keratinocytes by competitive exclusion (P = 0.026). L. salivarius UCC118, however, did not inhibit S. aureus from adhering to keratinocytes (P > 0.05) and did not protect keratinocyte viability. S. aureus utilizes the α5β1 integrin to adhere to keratinocytes, and blocking of this integrin resulted in a protective effect similar to that observed with probiotics (P = 0.03). This suggests that the protective mechanism for L. reuteri-mediated protection of keratinocytes was by competitive exclusion of the pathogen from its binding sites on the cells. Our results suggest that use of a topical probiotic prophylactically could inhibit the colonization of skin by S. aureus and thus aid in the prevention of infection. PMID:22582077

  14. Crosstalk with cancer-associated fibroblasts induces resistance of non-small cell lung cancer cells to epidermal growth factor receptor tyrosine kinase inhibition

    PubMed Central

    Choe, Chungyoul; Shin, Yong-Sung; Kim, Changhoon; Choi, So-Jung; Lee, Jinseon; Kim, So Young; Cho, Yong Beom; Kim, Jhingook

    2015-01-01

    Although lung cancers with activating mutations in the epidermal growth factor receptor (EGFR) are highly sensitive to selective EGFR tyrosine kinase inhibitors (TKIs), these tumors invariably develop acquired drug resistance. Host stromal cells have been found to have a considerable effect on the sensitivity of cancer cells to EGFR TKIs. Little is known, however, about the signaling mechanisms through which stromal cells contribute to the response to EGFR TKI in non-small cell lung cancer. This work examined the role of hedgehog signaling in cancer-associated fibroblast (CAF)-mediated resistance of lung cancer cells to the EGFR TKI erlotinib. PC9 cells, non-small cell lung cancer cells with EGFR-activating mutations, became resistant to the EGFR TKI erlotinib when cocultured in vitro with CAFs. Polymerase chain reaction and immunocytochemical assays showed that CAFs induced epithelial to mesenchymal transition phenotype in PC9 cells, with an associated change in the expression of epithelial to mesenchymal transition marker proteins including vimentin. Importantly, CAFs induce upregulation of the 7-transmembrane protein smoothened, the central signal transducer of hedgehog, suggesting that the hedgehog signaling pathway is active in CAF-mediated drug resistance. Indeed, downregulation of smoothened activity with the smoothened antagonist cyclopamine induces remodeling of the actin cytoskeleton independently of Gli-mediated transcriptional activity in PC9 cells. These findings indicate that crosstalk with CAFs plays a critical role in resistance of lung cancer to EGFR TKIs through induction of the epithelial to mesenchymal transition and may be an ideal therapeutic target in lung cancer. PMID:26676152

  15. Epidermal growth factor receptor expression affects the efficacy of the combined application of saponin and a targeted toxin on human cervical carcinoma cells.

    PubMed

    Bachran, Diana; Schneider, Stefanie; Bachran, Christopher; Urban, Romy; Weng, Alexander; Melzig, Matthias F; Hoffmann, Corinna; Kaufmann, Andreas M; Fuchs, Hendrik

    2010-09-01

    Cervical cancer is the second most common cancer in women worldwide. Targeting the epidermal growth factor receptor (EGFR) is a very promising approach since it is overexpressed in about 90% of cervical tumors. Here, we quantified the toxic effect of SE, a targeted toxin consisting of epidermal growth factor (EGF) as targeting moiety and the plant toxin saporin-3, on 3 common human cervical carcinoma cell lines (HeLa, CaSki and SiHa) and recently established lines (PHCC1 and PHCC2) from 2 different individuals. A human melanocytic and a mouse cell line served as negative control. Additionally, we combined SE with saponinum album, a saponin composite from Gypsophila paniculata, which exhibited synergistic properties in previous studies. The cell lines, except for SiHa cells, revealed high sensitivity to SE with 50% cell survival in the range of 5-24.5 nM. The combination with saponin resulted in a remarkable enhancement of cytotoxicity with enhancement factors ranging from 9,000-fold to 2,500,000-fold. The cytotoxicity of SE was clearly target receptor specific since free EGF blocks the effect and saporin-3 alone was considerably less toxic. For all cervical carcinoma cell lines, we evinced a clear correlation between EGFR expression and SE sensitivity. Our data indicate a potential use of targeted toxins for the treatment of cervical cancer. In particular, the combination with saponins is a promising approach since efficacy is drastically improved. PMID:20020492

  16. Role of oxygen intermediates in UV-induced epidermal cell injury

    SciTech Connect

    Danno, K.; Horio, T.; Takigawa, M.; Imamura, S.

    1984-09-01

    To investigate the role of oxygen intermediates (OIs) in sunburn cell (SC) formation and development of UV-inflammation in vivo, groups of mice were injected intravenously with OI scavengers, including bovine blood superoxide dismutase (SOD), bovine liver catalase, L-histidine, D-mannitol, and saline (controls) before and/or after UV irradiation with sunlamp tubes (mainly 280-320 nm; 300 mJ/cm2; UVR). Ear thickness was measured before and 6 and 24 h after UVR. Ears were removed 24 h after UVR and the number of SCs per unit length of ear epidermis was counted using hematoxylineosin stained sections. The number of SCs was significantly decreased (p less than 0.02) by a single injection of SOD (10-30 units/g body weight) given either just before or immediately after (less than 15 min) UVR, while SC formation was no longer suppressed by injections given more than 2 h before or after UVR. Four repeated injections of SOD (10 units/g) also reduced SC counts but did not significantly alter ear-swelling responses (ESR). Neither SC counts nor ESR were remarkably suppressed by 4 injections of any of the other active OI scavengers, inactivated SOD, or bovine serum albumin. A single injection of diethyldithiocarbamate, an SOD inactivator, significantly augmented SC formation (p less than 0.05), but did not change ESR. These findings suggest that OIs generated by UVR participate in SC formation but are not apparently involved in UV-edema.

  17. Altered growth, differentiation, and responsiveness to epidermal growth factor of human embryonic mesenchymal cells of palate by persistent rubella virus infection

    SciTech Connect

    Yoneda, T.; Urade, M.; Sakuda, M.; Miyazaki, T.

    1986-05-01

    We previously demonstrated that human embryonic mesenchymal cells derived from the palate (HEMP cells) retain alkaline phosphatase (ALP) content and capacity for collagen synthesis after long-term culture, and their growth is markedly stimulated by epidermal growth factor (EGF). There was a dramatic decrease in ALP content and capacity to synthesize collagen in HEMP cells (HEMP-RV cells) persistently infected with rubella virus (RV). EGF increased ALP activity and decreased collagen synthesis in HEMP cells, whereas EGF showed no effect on these activities in HEMP-RV cells. Growth of HEMP-RV cells was slightly reduced compared with that of HEMP cells. EGF stimulated growth of HEMP cells and to a lesser extent of HEMP-RV cells. Binding of /sup 125/I-EGF to cell-surface receptors in HEMP-RV cells was, to our surprise, twice as much as that in HEMP cells. However, internalization of bound /sup 125/I-EGF in HEMP-RV cells was profoundly diminished. Thus, persistent RV infection causes not only changes in HEMP cell growth and differentiation but a decrease in or loss of HEMP cell responsiveness to EGF. The effects of persistent RV infection on palatal cell differentiation as well as growth may be responsible for the pathogenesis of congenital rubella. Furthermore, since HEMP cells appear to be closely related to osteoblasts, these results suggest a mechanism for RV-induced osseous abnormalities manifested in congenital rubella patients.

  18. Loss of BRCA1 leads to an increase in epidermal growth factor receptor expression in mammary epithelial cells, and epidermal growth factor receptor inhibition prevents estrogen receptor-negative cancers in BRCA1-mutant mice

    PubMed Central

    2011-01-01

    Introduction Women who carry a BRCA1 mutation typically develop "triple-negative" breast cancers (TNBC), defined by the absence of estrogen receptor (ER), progesterone receptor and Her2/neu. In contrast to ER-positive tumors, TNBCs frequently express high levels of epidermal growth factor receptor (EGFR). Previously, we found a disproportionate fraction of progenitor cells in BRCA1 mutation carriers with EGFR overexpression. Here we examine the role of EGFR in mammary epithelial cells (MECs) in the emergence of BRCA1-related tumors and as a potential target for the prevention of TNBC. Methods Cultures of MECs were used to examine EGFR protein levels and promoter activity in response to BRCA1 suppression with inhibitory RNA. EGFR was assessed by immunoblot and immunofluorescence analysis, real-time reverse transcriptase-polymerase chain reaction assay (RT-PCR) and flow cytometry. Binding of epidermal growth factor (EGF) to subpopulations of MECs was examined by Scatchard analysis. The responsiveness of MECs to the EGFR inhibitor erlotinib was assessed in vitro in three-dimensional cultures and in vivo. Mouse mammary tumor virus-Cre recombinase (MMTV-Cre) BRCA1flox/flox p53+/- mice were treated daily with erlotinib or vehicle control, and breast cancer-free survival was analyzed using the Kaplan-Meier method. Results Inhibition of BRCA1 in MECs led to upregulation of EGFR with an inverse correlation of BRCA1 with cellular EGFR protein levels (r2 = 0.87) and to an increase in cell surface-expressed EGFR. EGFR upregulation in response to BRCA1 suppression was mediated by transcriptional and posttranslational mechanisms. Aldehyde dehydrogenase 1 (ALDH1)-positive MECs expressed higher levels of EGFR than ALDH1-negative MECs and were expanded two- to threefold in the BRCA1-inhibited MEC population. All MECs were exquisitely sensitive to EGFR inhibition with erlotinib in vitro. EGFR inhibition in MMTV-Cre BRCA1flox/flox p53+/- female mice starting at age 3 months increased

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

    PubMed

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

    2015-12-01

    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. PMID:26367466

  20. Control of proliferation of human vascular endothelial cells. Characterization of the response of human umbilical vein endothelial cells to fibroblast growth factor, epidermal growth factor, and thrombin.

    PubMed

    Gospodarowicz, D; Brown, K D; Birdwell, C R; Zetter, B R

    1978-06-01

    Because the response of human endothelial cells to growth factors and conditioning agents has broad implications for our understanding of wound healing angiogenesis, and human atherogenesis, we have investigated the responses of these cells to the fibroblast (FGF) and epidermal growth factors (EGF), as well as to the protease thrombin, which has been previously shown to potentiate the growth response of other cell types of FGF and EGF. Because the vascular endothelial cells that form the inner lining of blood vessels may be expected to be exposed to high thrombin concentrations after trauma or in pathological states associated with thrombosis, they are of particular interest with respect to the physiological role of this protease in potentiating cell proliferation. Our results indicate that human vascular endothelial cells respond poorly to either FGF or thrombin alone. In contrast, when cells are maintained in the presence of thrombin, their proliferative response to FGF is greatly increased even in cultures seeded at a density as low as 3 cells/mm2. Human vascular endothelial cells also respond to EGF and thrombin, although their rate of proliferation is much slower than when maintained with FGF and thrombin. In contrast, bovine vascular endothelial cells derived from vascular territories as diverse as the bovine heart, aortic arch, and umbilical vein respond maximally to FGF alone and neither respond to nor bind EGF. Furthermore, the response of bovine vascular endothelial cells to FGF was not potentiated by thrombin, indicating that the set of factors controlling the proliferation of vascular endothelial cells could be species-dependent. The requirement of cultured human vascular endothelial cells for thrombin could explain why the human cells, in contrast to bovine endothelial cells, are so difficult to maintain in tissue culture. Our results demonstrate that by using FGF and thrombin one can develop cultures of human vascular endothelial cells capable of

  1. Responses of epidermal cell turgor pressure and photosynthetic activity of leaves of the atmospheric epiphyte Tillandsia usneoides (Bromeliaceae) after exposure to high humidity.

    PubMed

    Martin, Craig E; Rux, Guido; Herppich, Werner B

    2013-01-01

    It has been well-established that many epiphytic bromeliads of the atmospheric-type morphology, i.e., with leaf surfaces completely covered by large, overlapping, multicellular trichomes, are capable of absorbing water vapor from the atmosphere when air humidity increases. It is much less clear, however, whether this absorption of water vapor can hydrate the living cells of the leaves and, as a consequence, enhance physiological processes in such cells. The goal of this research was to determine if the absorption of atmospheric water vapor by the atmospheric epiphyte Tillandsia usneoides results in an increase in turgor pressure in leaf epidermal cells that subtend the large trichomes, and, by using chlorophyll fluorescence techniques, to determine if the absorption of atmospheric water vapor by leaves of this epiphyte results in increased photosynthetic activity. Results of measurements on living cells of attached leaves of this epiphytic bromeliad, using a pressure probe and of whole-shoot fluorescence imaging analyses clearly illustrated that the turgor pressure of leaf epidermal cells did not increase, and the photosynthetic activity of leaves did not increase, following exposure of the leaves to high humidity air. These results experimentally demonstrate, for the first time, that the absorption of water vapor following increases in atmospheric humidity in atmospheric epiphytic bromeliads is mostly likely a physical phenomenon resulting from hydration of non-living leaf structures, e.g., trichomes, and has no physiological significance for the plant's living tissues. PMID:23000465

  2. Epidermal growth factor and Ras regulate gene expression in GH4 pituitary cells by separate, antagonistic signal transduction pathways.

    PubMed Central

    Pickett, C A; Gutierrez-Hartmann, A

    1995-01-01

    We have previously demonstrated that epidermal growth factor (EGF) produces activation of the rat prolactin (rPRL) promoter in GH4 neuroendocrine cells via a Ras-independent mechanism. This Ras independence of the EGF response appears to be cell rather than promoter specific. Oncogenic Ras also produces activation of the rPRL promoter when transfected into GH4 cells and requires the sequential activation of Raf kinase, mitogen-activated protein (MAP) kinase, and c-Ets-1/GHF-1 to mediate this response. In these studies, we have investigated the interaction between EGF and Ras in stimulating rPRL promoter activity and the role of Raf and MAP kinases in mediating the EGF response. We have also examined the role of several transcription factors and used various promoter mutants of the rPRL gene in order to better define the trans- and cis-acting components of the EGF response. EGF treatment of GH4 cells inhibits activation of the rPRL promoter produced by transfection of V12Ras from 24- to 4-fold in an EGF dose-dependent manner. This antagonistic effect of EGF and Ras is mutual in that transfection of V12Ras also blocks EGF-induced activation of the rPRL promoter in a Ras dose-dependent manner, from 5.5- to 1.6-fold. Transfection of a plasmid encoding the dominant-negative Raf C4 blocks Ras-induced activation by 66% but fails to inhibit EGF-mediated activation of the rPRL promoter. Similarly, transfection of a construct encoding an inhibitory form of MAP kinase decreases the Ras response by 50% but does not inhibit the EGF response. Previous studies have demonstrated that c-Ets-1 is necessary and that GHF-1 acts synergistically with c-Ets-1 in the Ras response of the rPRL promoter. In contrast, overexpression of neither c-Ets-1 nor GHF-1 enhanced EGF-mediated activation of the rPRL promoter, and dominant-negative forms of these transcription factors failed to inhibit the EGF response. Using 5' deletion and site-specific mutations, we have mapped the EGF response to two

  3. Subfunctionalization of Cellulose Synthases in Seed Coat Epidermal Cells Mediates Secondary Radial Wall Synthesis and Mucilage Attachment1[C][W][OA

    PubMed Central

    Mendu, Venugopal; Griffiths, Jonathan S.; Persson, Staffan; Stork, Jozsef; Downie, A. Bruce; Voiniciuc, Cătălin; Haughn, George W.; DeBolt, Seth

    2011-01-01

    Arabidopsis (Arabidopsis thaliana) epidermal seed coat cells follow a complex developmental program where, following fertilization, cells of the ovule outer integument differentiate into a unique cell type. Two hallmarks of these cells are the production of a doughnut-shaped apoplastic pocket filled with pectinaceous mucilage and the columella, a thick secondary cell wall. Cellulose is thought to be a key component of both these secondary cell wall processes. Here, we investigated the role of cellulose synthase (CESA) subunits CESA2, CESA5, and CESA9 in the seed coat epidermis. We characterized the roles of these CESA proteins in the seed coat by analyzing cell wall composition and morphology in cesa mutant lines. Mutations in any one of these three genes resulted in lower cellulose content, a loss of cell shape uniformity, and reduced radial wall integrity. In addition, we found that attachment of the mucilage halo to the parent seed following extrusion is maintained by cellulose-based connections requiring CESA5. Hence, we show that cellulose fulfills an adhesion role between the extracellular mucilage matrix and the parent cell in seed coat epidermal cells. We propose that mucilage remains attached to the seed coat through interactions between components in the seed mucilage and cellulose. Our data suggest that CESA2 and CESA9 serve in radial wall reinforcement, as does CESA5, but CESA5 also functions in mucilage biosynthesis. These data suggest unique roles for different CESA subunits in one cell type and illustrate a complex role for cellulose biosynthesis in plant developmental biology. PMID:21750228

  4. Effects of transforming growth factor beta and epidermal growth factor on cell proliferation and the formation of bone nodules in isolated fetal rat calvaria cells.

    PubMed

    Antosz, M E; Bellows, C G; Aubin, J E

    1989-08-01

    When cells enzymatically isolated from fetal rat calvaria (RC cells) are cultured in vitro in the presence of ascorbic acid and Na beta-glycerophosphate, discrete three-dimensional nodules form with the histologic, immunohistochemical, and ultrastructural characteristics of bone (Bellows et al; Calcified Tissue International 38:143-154, 1986; Bhargava et al., Bone, 9:155-163, 1988). Quantitation of the number of bone nodules that forms provides a colony assay for osteoprogenitor cells present in the RC population (Bellows and Aubin, Develop. Biol., 133:8-13, 1989). Continuous culture with either epidermal growth factor (EGF) or transforming growth factor beta (TGF-beta) results in dose-dependent inhibition of bone nodule formation; however, the former causes increased proliferation and saturation density, while the latter reduces both parameters. Addition of EGF (48 h pulse, 2-200 ng/ml) to RC cells at day 1 after plating results in increased proliferation and population saturation density and an increased number of bone nodules formed. Similar pulses at confluence and in postconfluent multilayered cultures when nodules first begin forming (approx. day 11) inhibited bone nodule formation and resulted in a smaller stimulation of cell proliferation. Forty-eight hour pulses of TGF-beta (0.01-1 ng/ml) reduced bone nodule formation and proliferation at all times examined, with pulses on day 1 causing maximum inhibition. The effects of pulses with TGF-beta and EGF on inhibition of nodule formation are independent of the presence of serum in the culture medium during the pulse. The data suggest that whereas EGF can either stimulate or inhibit the formation of bone nodules depending upon the time and duration of exposure, TGF-B inhibits bone nodule formation under all conditions tested. Moreover, these effects on osteoprogenitor cell differentiation do not always correlate with the effects of the growth factors on RC cell proliferation. PMID:2787326

  5. Microsurgical removal of epidermal and cortical cells: evidence that the gravitropic signal moves through the outer cell layers in primary roots of maize

    NASA Technical Reports Server (NTRS)

    Yang, R. L.; Evans, M. L.; Moore, R.

    1990-01-01

    There is general agreement that during root gravitropism some sort of growth-modifying signal moves from the cap to the elongation zone and that this signal ultimately induces the curvature that leads to reorientation of the root. However, there is disagreement regarding both the nature of the signal and the pathway of its movement from the root cap to the elongation zone. We examined the pathway of movement by testing gravitropism in primary roots of maize (Zea mays L.) from which narrow (0.5 mm) rings of epidermal and cortical tissue were surgically removed from various positions within the elongation zone. When roots were girdled in the apical part of the elongation zone gravitropic curvature occurred apical to the girdle but not basal to the girdle. Filling the girdle with agar allowed curvature basal to the girdle to occur. Shallow girdles, in which only two or three cell layers (epidermis plus one or two cortical cell layers) were removed, prevented or greatly delayed gravitropic curvature basal to the girdle. The results indicate that the gravitropic signal moves basipetally through the outermost cell layers, perhaps through the epidermis itself.

  6. Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells.

    PubMed

    Zhang, Hui-ming; Talbot, Mark J; McCurdy, David W; Patrick, John W; Offler, Christina E

    2015-09-01

    Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca(2+) levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane-microtubule inter-relationship is discussed. PMID:26136268

  7. A barley ROP GTPase ACTIVATING PROTEIN associates with microtubules and regulates entry of the barley powdery mildew fungus into leaf epidermal cells.

    PubMed

    Hoefle, Caroline; Huesmann, Christina; Schultheiss, Holger; Börnke, Frederik; Hensel, Götz; Kumlehn, Jochen; Hückelhoven, Ralph

    2011-06-01

    Little is known about the function of host factors involved in disease susceptibility. The barley (Hordeum vulgare) ROP (RHO of plants) G-protein RACB is required for full susceptibility of the leaf epidermis to invasion by the biotrophic fungus Blumeria graminis f. sp hordei. Stable transgenic knockdown of RACB reduced the ability of barley to accommodate haustoria of B. graminis in intact epidermal leaf cells and to form hairs on the root epidermis, suggesting that RACB is a common element of root hair outgrowth and ingrowth of haustoria in leaf epidermal cells. We further identified a barley MICROTUBULE-ASSOCIATED ROP-GTPASE ACTIVATING PROTEIN (MAGAP1) interacting with RACB in yeast and in planta. Fluorescent MAGAP1 decorated cortical microtubules and was recruited by activated RACB to the cell periphery. Under fungal attack, MAGAP1-labeled microtubules built a polarized network at sites of successful defense. By contrast, microtubules loosened where the fungus succeeded in penetration. Genetic evidence suggests a function of MAGAP1 in limiting susceptibility to penetration by B. graminis. Additionally, MAGAP1 influenced the polar organization of cortical microtubules. These results add to our understanding of how intact plant cells accommodate fungal infection structures and suggest that RACB and MAGAP1 might be antagonistic players in cytoskeleton organization for fungal entry. PMID:21685259

  8. Calcium-dependent depletion zones in the cortical microtubule array coincide with sites of, but do not regulate, wall ingrowth papillae deposition in epidermal transfer cells

    PubMed Central

    Zhang, Hui-ming; Talbot, Mark J.; McCurdy, David W.; Patrick, John W.; Offler, Christina E.

    2015-01-01

    Trans-differentiation to a transfer-cell morphology is characterized by the localized deposition of wall ingrowth papillae that protrude into the cytosol. Whether the cortical microtubule array directs wall ingrowth papillae formation was investigated using a Vicia faba cotyledon culture system in which their adaxial epidermal cells were spontaneously induced to trans-differentiate to transfer cells. During deposition of wall ingrowth papillae, the aligned cortical microtubule arrays in precursor epidermal cells were reorganized into a randomized array characterized by circular depletion zones. Concurrence of the temporal appearance, spatial pattern, and size of depletion zones and wall ingrowth papillae was consistent with each papilla occupying a depletion zone. Surprisingly, microtubules appeared not to regulate construction of wall ingrowth papillae, as neither depolymerization nor stabilization of cortical microtubules changed their deposition pattern or morphology. Moreover, the size and spatial pattern of depletion zones was unaltered when the formation of wall ingrowth papillae was blocked by inhibiting cellulose biosynthesis. In contrast, the depletion zones were absent when the cytosolic calcium plumes, responsible for directing wall ingrowth papillae formation, were blocked or dissipated. Thus, we conclude that the depletion zones within the cortical microtubule array result from localized depolymerization of microtubules initiated by elevated cytosolic Ca2+ levels at loci where wall ingrowth papillae are deposited. The physiological significance of the depletion zones as a mechanism to accommodate the construction of wall ingrowth papillae without compromising maintenance of the plasma membrane–microtubule inter-relationship is discussed. PMID:26136268

  9. Clinical approaches to treat patients with non-small cell lung cancer and epidermal growth factor receptor tyrosine kinase inhibitor acquired resistance.

    PubMed

    Tartarone, Alfredo; Lerose, Rosa

    2015-10-01

    The discovery of epidermal growth factor receptor activating mutations (EGFR Mut+) has determined a paradigm shift in the treatment of non-small cell lung cancer (NSCLC). In several phase III studies, patients with NSCLC EGFR Mut+ achieved a significantly better progression-free survival when treated with a first- (gefitinib, erlotinib) or second-generation (afatinib) EGFR tyrosine kinase inhibitor (TKI) compared with standard chemotherapy. However, despite these impressive results, most patients with NSCLC EGFR Mut+ develop acquired resistance to TKIs. This review will discuss both the mechanisms of resistance to TKIs and the therapeutic strategies to overcome resistance, including emerging data on third-generation TKIs. PMID:26016841

  10. Epidermal growth factor promotes a mesenchymal over an amoeboid motility of MDA-MB-231 cells embedded within a 3D collagen matrix

    NASA Astrophysics Data System (ADS)

    Geum, Dongil T.; Kim, Beum Jun; Chang, Audrey E.; Hall, Matthew S.; Wu, Mingming

    2016-01-01

    The receptor of epidermal growth factor (EGFR) critically regulates tumor cell invasion and is a potent therapeutic target for treatment of many types of cancers, including carcinomas and glioblastomas. It is known that EGF regulates cell motility when tumor cells are embedded within a 3D biomatrix. However, roles of EGF in modulating tumor cell motility phenotype are largely unknown. In this article, we report that EGF promotes a mesenchymal over an amoeboid motility phenotype using a malignant breast tumor cell line, MDA-MB-231, embedded within a 3D collagen matrix. Amoeboid cells are rounded in shape, while mesenchymal cells are elongated, and their migrations are governed by a distinctly different set of biomolecules. Using single cell tracking analysis, we also show that EGF promotes cell dissemination through a significant increase in cell persistence along with a moderate increase of speed. The increase of persistence is correlated with the increase of the percentage of the mesenchymal cells within the population. Our work reveals a novel role of microenvironmental cue, EGF, in modulating heterogeneity and plasticity of tumor cell motility phenotype. In addition, it suggests a potential visual cue for diagnosing invasive states of breast cancer cells. This work can be easily extended beyond breast cancer cells.

  11. Phosphorylated Epidermal Growth Factor Receptor on Tumor-Associated Endothelial Cells Is a Primary Target for Therapy with Tyrosine Kinase Inhibitors1

    PubMed Central

    Kuwai, Toshio; Nakamura, Toru; Sasaki, Takamitsu; Kim, Sun-Jin; Fan, Dominic; Villares, Gabriel J; Zigler, Maya; Wang, Hua; Bar-Eli, Menashe; Kerbel, Robert S; Fidler, Isaiah J

    2008-01-01

    We determined whether phosphorylated epidermal growth factor receptor (EGFR) expressed on tumor-associated endothelial cells is a primary target for therapy with EGFR tyrosine kinase inhibitors (TKIs). Human colon cancer cells SW620CE2 (parental) that do not express EGFR or human epidermal growth factor receptor 2 (HER2) but express transforming growth factor α (TGF-α) were transduced with a lentivirus carrying nontargeting small hairpin RNA (shRNA) or TGF-α shRNA. The cell lines were implanted into the cecum of nude mice. Two weeks later, treatment began with saline, 4-[R]-phenethylamino-6-[hydroxyl] phenyl-7H-pyrrolo [2,3-d]-pyrimidine (PKI166), or irinotecan. Endothelial cells in parental and nontargeting shRNA tumors expressed phosphorylated EGFR. Therapy with PKI166 alone or with irinotecan produced apoptosis of these endothelial cells and necrosis of the EGFR-negative tumors. Endothelial cells in tumors that did not express TGF-α did not express EGFR, and these tumors were resistant to treatment with PKI166. The response of neoplasms to EGFR antagonists has been correlated with EGFR mutations, HER2 expression, Akt activation, and EGFR gene copy number. Our present data using colon cancer cells that do not express EGFR or HER2 suggest that the expression of TGF-α by tumor cells leading to the activation of EGFR in tumor-associated endothelial cells is a major determinant for the susceptibility of neoplasms to therapy by specific EGFR-TKI. PMID:18472966

  12. Phosphorylated epidermal growth factor receptor on tumor-associated endothelial cells is a primary target for therapy with tyrosine kinase inhibitors.

    PubMed

    Kuwai, Toshio; Nakamura, Toru; Sasaki, Takamitsu; Kim, Sun-Jin; Fan, Dominic; Villares, Gabriel J; Zigler, Maya; Wang, Hua; Bar-Eli, Menashe; Kerbel, Robert S; Fidler, Isaiah J

    2008-05-01

    We determined whether phosphorylated epidermal growth factor receptor (EGFR) expressed on tumor-associated endothelial cells is a primary target for therapy with EGFR tyrosine kinase inhibitors (TKIs). Human colon cancer cells SW620CE2 (parental) that do not express EGFR or human epidermal growth factor receptor 2 (HER2) but express transforming growth factor alpha (TGF-alpha) were transduced with a lentivirus carrying nontargeting small hairpin RNA (shRNA) or TGF-alpha shRNA. The cell lines were implanted into the cecum of nude mice. Two weeks later, treatment began with saline, 4-[R]-phenethylamino-6-[hydroxyl] phenyl-7H-pyrrolo [2,3-D]-pyrimidine (PKI166), or irinotecan. Endothelial cells in parental and nontargeting shRNA tumors expressed phosphorylated EGFR. Therapy with PKI166 alone or with irinotecan produced apoptosis of these endothelial cells and necrosis of the EGFR-negative tumors. Endothelial cells in tumors that did not express TGF-alpha did not express EGFR, and these tumors were resistant to treatment with PKI166. The response of neoplasms to EGFR antagonists has been correlated with EGFR mutations, HER2 expression, Akt activation, and EGFR gene copy number. Our present data using colon cancer cells that do not express EGFR or HER2 suggest that the expression of TGF-alpha by tumor cells leading to the activation of EGFR in tumor-associated endothelial cells is a major determinant for the susceptibility of neoplasms to therapy by specific EGFR-TKI. PMID:18472966

  13. Toxic epidermal necrolysis

    PubMed Central

    Hoetzenecker, Wolfram; Mehra, Tarun; Saulite, Ieva; Glatz, Martin; Schmid-Grendelmeier, Peter; Guenova, Emmanuella; Cozzio, Antonio; French, Lars E.

    2016-01-01

    Toxic epidermal necrolysis (TEN) is a rare, life-threatening drug-induced skin disease with a mortality rate of approximately 30%. The clinical hallmark of TEN is a marked skin detachment caused by extensive keratinocyte cell death associated with mucosal involvement. The exact pathogenic mechanism of TEN is still uncertain. Recent advances in this field have led to the identification of several factors that might contribute to the induction of excessive apoptosis of keratinocytes. In addition, specific human leukocyte antigen types seem to be associated with certain drugs and the development of TEN. As well-controlled studies are lacking, patients are treated with various immunomodulators (e.g. intravenous immunoglobulin) in addition to the best supportive care. PMID:27239294

  14. Release of infectious cells from epidermal ulcers in Ichthyophonus sp.–infected Pacific herring (Clupea pallasii): Evidence for multiple mechanisms of transmission

    USGS Publications Warehouse

    Hershberger, Paul K.; Gregg, Jacob L.; Kocan, R.M.

    2010-01-01

    A common clinical sign of ichthyophoniasis in herring and trout is “sandpaper” skin, a roughening of the epidermis characterized by the appearance of small papules, followed by ulceration and sloughing of the epithelium; early investigators hypothesized that these ulcers might be a means of transmitting the parasite, Ichthyophonus sp., without the necessity of ingesting an infected host. We examined the cells associated with the epidermal lesions and confirmed that they were viable Ichthyophonus sp. cells that were readily released from the skin into the mucous layer and ultimately into the aquatic environment. The released cells were infectious when injected into the body cavity of specific-pathogen-free herring. Our hypothesis is that different mechanisms of transmission occur in carnivorous and planktivorous hosts: Planktonic feeders become infected by ingestion of ulcer-derived cells, while carnivores become infected by ingestion of whole infected fish.

  15. Simultaneous suppression of epidermal growth factor receptor and c-erbB-2 reverses aneuploidy and malignant phenotype of a human ovarian carcinoma cell line.

    PubMed

    Pack, Svetlana D; Alper, Ozgül M; Stromberg, Kurt; Augustus, Meena; Ozdemirli, Metin; Miermont, Anne M; Klus, Greg; Rusin, Marek; Slack, Rebecca; Hacker, Neville F; Ried, Thomas; Szallasi, Zoltan; Alper, Ozge

    2004-02-01

    Coexpression of epidermal growth factor receptor (EGFR) and c-erbB-2 in 47-68% of ovarian cancer cells indicate their strong association with tumor formation. We examined the effects of simultaneous antisense- or immunosuppression of EGFR and c-erbB-2 expression on the invasive phenotype, aneuploidy, and genotype of cultured human ovarian carcinoma cells (NIH:OVCAR-8). We report here that suppression of both EGFR and c-erbB-2 results in regression of aneuploidy and genomic imbalances in NIH:OVCAR-8 cells, restores a more normal phenotype, and results in a more normal gene expression profile. Combined with cytogenetic analysis, our data demonstrate that the regression of aneuploidy is due to the selective apoptosis of double antisense transfected cells with highly abnormal karyotype. PMID:14871800

  16. Synergistic effect of targeting the epidermal growth factor receptor and hyaluronan synthesis in oesophageal squamous cell carcinoma cells

    PubMed Central

    Kretschmer, I; Freudenberger, T; Twarock, S; Fischer, J W

    2015-01-01

    Background and Purpose Worldwide, oesophageal cancer is the eighth most common cancer and has a very poor survival rate. In order to identify new tolerable treatment options for oesophageal squamous cell carcinoma (ESCC), erlotinib was tested with moderate efficacy in phase I and II studies. As 4-methylumbelliferone (4-MU), an hyaluronan (HA) synthesis inhibitor showed anti-cancer effects in vitro, and in ESCC xenograft tumours, we investigated whether the anti-cancer effects of erlotinib could be augmented by combining it with 4-MU. Experimental Approach ESCC cell lines were treated with erlotinib or gefitinib (1 μmol·L−1) and 4-MU (300 μmol·L−1), and the cell count, cell cycle progression and migration were determined as compared to the single agents and the solvent-control. Key Results The combination of erlotinib and 4-MU synergistically inhibited the proliferation of ESCC cell lines. Furthermore, the migration speed of ESCC cell line KYSE-410 in gap closure assays was significantly reduced by the combination of erlotinib and 4-MU. Decreased ERK phosphorylation could explain the anti-proliferative and anti-migratory effects in the combined treatment group. Finally, the combination was additionally able to decrease the growth of multicellular tumour spheroids, a three-dimensional cell culture model that was associated with sustained inhibition of ERK1/2 phosphorylation. Conclusions and Implications The combination of 4-MU and erlotinib showed promising anti-cancer efficacies in the ESCC cell lines. PMID:26140525

  17. Size-dependent effects of tungsten carbide-cobalt particles on oxygen radical production and activation of cell signaling pathways in murine epidermal cells

    SciTech Connect

    Ding, M.; Kisin, E.R.; Zhao, J.; Bowman, L.; Lu, Y.; Jiang, B.; Leonard, S.; Vallyathan, V.; Castranova, V.; Murray, A.R.; Fadeel, B.; Shvedova, A.A.

    2009-12-15

    Hard metal or cemented carbide consists of a mixture of tungsten carbide (WC) (85%) and metallic cobalt (Co) (5-15%). WC-Co is considered to be potentially carcinogenic to humans. However, no comparison of the adverse effects of nano-sized WC-Co particles is available to date. In the present study, we compared the ability of nano- and fine-sized WC-Co particles to form free radicals and propensity to activate the transcription factors, AP-1 and NF-kappaB, along with stimulation of mitogen-activated protein kinase (MAPK) signaling pathways in a mouse epidermal cell line (JB6 P{sup +}). Our results demonstrated that nano-WC-Co generated a higher level of hydroxyl radicals, induced greater oxidative stress, as evidenced by a decrease of GSH levels, and caused faster JB6 P{sup +} cell growth/proliferation than observed after exposure of cells to fine WC-Co. In addition, nano-WC-Co activated AP-1 and NF-kappaB more efficiently in JB6{sup +/+} cells as compared to fine WC-Co. Experiments using AP-1-luciferase reporter transgenic mice confirmed the activation of AP-1 by nano-WC-Co. Nano- and fine-sized WC-Co particles also stimulated MAPKs, including ERKs, p38, and JNKs with significantly higher potency of nano-WC-Co. Finally, co-incubation of the JB6{sup +/+} cells with N-acetyl-cysteine decreased AP-1 activation and phosphorylation of ERKs, p38 kinase, and JNKs, thus suggesting that oxidative stress is involved in WC-Co-induced toxicity and AP-1 activation.

  18. The role of peroxisome proliferator-activated receptor-{beta}/{delta} in epidermal growth factor-induced HaCaT cell proliferation

    SciTech Connect

    Liang Pengfei; Jiang Bimei; Yang Xinghua; Xiao Xianzhong Huang Xu; Long Jianhong; Zhang Pihong; Zhang Minghua; Xiao Muzhang; Xie Tinghong; Huang Xiaoyuan

    2008-10-15

    Epidermal growth factor (EGF) has been shown to be a potent mitogen for epidermal cells both in vitro and in vivo, thus contributing to the development of an organism. It has recently become clear that peroxisome proliferator-activated receptor-{beta}/{delta} (PPAR{beta}/{delta}) expression and activation is involved in the cell proliferation. However, little is known about the role of PPAR{beta}/{delta} in EGF-induced proliferation of HaCaT keratinocytes. In this study, HaCaT cells were cultured in the presence and absence of EGF and we identified that EGF induced an increase of PPAR{beta}/{delta} mRNA and protein level expression in time-dependent and dose-dependent manner, and AG1487, an EGF receptor (EGFR) special inhibitor, caused attenuation of PPAR{beta}/{delta} protein expression. Electrophoretic mobility shift assay (EMSA) revealed that EGF significantly increased PPAR{beta}/{delta} binding activity in HaCaT keratinocytes. Antisense phosphorothioate oligonucleotides (asODNs) against PPAR{beta}/{delta} caused selectively inhibition of PPAR{beta}/{delta} protein content induced by EGF and significantly attenuated EGF-mediated cell proliferation. Treatment of the cells with L165041, a specific synthetic ligand for PPAR{beta}/{delta}, significantly enhanced EGF-mediated cell proliferation. Finally, c-Jun ablation inhibited PPAR{beta}/{delta} up-regulation induced by EGF, and chromatin immunoprecipitation (ChIP) showed that c-Jun bound to the PPAR{beta}/{delta} promoter and the binding increased in EGF-stimulated cells. These results demonstrate that EGF induces PPAR{beta}/{delta} expression in a c-Jun-dependent manner and PPAR{beta}/{delta} plays a vital role in EGF-stimulated proliferation of HaCaT cells.

  19. Faster DNA Repair of Ultraviolet-Induced Cyclobutane Pyrimidine Dimers and Lower Sensitivity to Apoptosis in Human Corneal Epithelial Cells than in Epidermal Keratinocytes.

    PubMed

    Mallet, Justin D; Dorr, Marie M; Drigeard Desgarnier, Marie-Catherine; Bastien, Nathalie; Gendron, Sébastien P; Rochette, Patrick J

    2016-01-01

    Absorption of UV rays by DNA generates the formation of mutagenic cyclobutane pyrimidine dimers (CPD) and pyrimidine (6-4) pyrimidone photoproducts (6-4PP). These damages are the major cause of skin cancer because in turn, they can lead to signature UV mutations. The eye is exposed to UV light, but the cornea is orders of magnitude less prone to UV-induced cancer. In an attempt to shed light on this paradox, we compared cells of the corneal epithelium and the epidermis for UVB-induced DNA damage frequency, repair and cell death sensitivity. We found similar CPD levels but a 4-time faster UVB-induced CPD, but not 6-4PP, repair and lower UV-induced apoptosis sensitivity in corneal epithelial cells than epidermal. We then investigated levels of DDB2, a UV-induced DNA damage recognition protein mostly impacting CPD repair, XPC, essential for the repair of both CPD and 6-4PP and p53 a protein upstream of the genotoxic stress response. We found more DDB2, XPC and p53 in corneal epithelial cells than in epidermal cells. According to our results analyzing the protein stability of DDB2 and XPC, the higher level of DDB2 and XPC in corneal epithelial cells is most likely due to an increased stability of the protein. Taken together, our results show that corneal epithelial cells have a better efficiency to repair UV-induced mutagenic CPD. On the other hand, they are less prone to UV-induced apoptosis, which could be related to the fact that since the repair is more efficient in the HCEC, the need to eliminate highly damaged cells by apoptosis is reduced. PMID:27611318

  20. Epidermal Fatty Acid Binding Protein (E-FABP) Is Not Required for the Generation or Maintenance of Effector and Memory T Cells following Infection with Listeria monocytogenes.

    PubMed

    Li, Bing; Schmidt, Nathan W

    2016-01-01

    Following activation of naïve T cells there are dynamic changes in the metabolic pathways used by T cells to support both the energetic needs of the cell and the macromolecules required for growth and proliferation. Among other changes, lipid metabolism undergoes dynamic transitions between fatty acid oxidation and fatty acid synthesis as cells progress from naïve to effector and effector to memory T cells. The hydrophobic nature of lipids requires that they be bound to protein chaperones within a cell. Fatty acid binding proteins (FABPs) represent a large class of lipid chaperones, with epidermal FABP (E-FABP) expressed in T cells. The objective of this study was to determine the contribution of E-FABP in antigen-specific T cell responses. Following infection with Listeria monocytogenes, we observed similar clonal expansion, contraction and formation of memory CD8 T cells in WT and E-FABP-/- mice, which also exhibited similar phenotypic and functional characteristics. Analysis of Listeria-specific CD4 T cells also revealed no defect in the expansion, contraction, and formation of memory CD4 T cells in E-FABP-/- mice. These data demonstrate that E-FABP is dispensable for antigen-specific T cell responses following a bacterial infection. PMID:27588422

  1. The Epidermal Cell Structure of the Secondary Pollen Presenter in Vangueria infausta (Rubiaceae: Vanguerieae) Suggests a Functional Association with Protruding Onci in Pollen Grains

    PubMed Central

    Tilney, Patricia M.; van Wyk, Abraham E.; van der Merwe, Chris F.

    2014-01-01

    Secondary pollen presentation is a well-known phenomenon in the Rubiaceae with particularly conspicuous pollen presenters occurring in the tribe Vanguerieae. These knob-like structures are formed by a modification of the upper portion of the style and stigma, together known as the stylar head complex. In the flower bud and shortly before anthesis, the anthers surrounding the stylar head complex dehisce and release pollen grains which adhere to the pollen presenter. The epidermal cells of the pollen presenter facing the anthers are radially elongated with a characteristic wall thickening encircling the anticlinal walls of each cell towards the distal end. These cells were studied in the pollen presenter of Vangueria infausta using electron and light microscopy in conjunction with histochemical tests and immunohistochemical methods. Other prominent thickenings of the cell wall were also observed on the distal and proximal walls. All these thickenings were found to be rich in pectin and possibly xyloglucan. The terms “thickenings of Igersheim” and “bands of Igersheim” are proposed to refer, respectively, to these wall structures in general and those encircling the anticlinal walls of each cell near the distal end. The epidermal cells have an intricate ultrastructure with an abundance of organelles, including smooth and rough endoplasmic reticulum, Golgi apparatus, mitochondria and secretory vesicles. This indicates that these cells are likely to have an active physiological role. The pollen grains possess prominent protruding onci and observations were made on their structure and development. Walls of the protruding onci are also rich in pectin. Pectins are hydrophilic and known to be involved in the dehydration and rehydration of pollen grains. We hypothesise that the thickenings of Igersheim, as well as the protruding onci of the pollen grains, are functionally associated and part of the adaptive syndrome of secondary pollen presentation, at least in the

  2. The epidermal cell structure of the secondary pollen presenter in Vangueria infausta (Rubiaceae: Vanguerieae) suggests a functional association with protruding onci in pollen grains.

    PubMed

    Tilney, Patricia M; van Wyk, Abraham E; van der Merwe, Chris F

    2014-01-01

    Secondary pollen presentation is a well-known phenomenon in the Rubiaceae with particularly conspicuous pollen presenters occurring in the tribe Vanguerieae. These knob-like structures are formed by a modification of the upper portion of the style and stigma, together known as the stylar head complex. In the flower bud and shortly before anthesis, the anthers surrounding the stylar head complex dehisce and release pollen grains which adhere to the pollen presenter. The epidermal cells of the pollen presenter facing the anthers are radially elongated with a characteristic wall thickening encircling the anticlinal walls of each cell towards the distal end. These cells were studied in the pollen presenter of Vangueria infausta using electron and light microscopy in conjunction with histochemical tests and immunohistochemical methods. Other prominent thickenings of the cell wall were also observed on the distal and proximal walls. All these thickenings were found to be rich in pectin and possibly xyloglucan. The terms "thickenings of Igersheim" and "bands of Igersheim" are proposed to refer, respectively, to these wall structures in general and those encircling the anticlinal walls of each cell near the distal end. The epidermal cells have an intricate ultrastructure with an abundance of organelles, including smooth and rough endoplasmic reticulum, Golgi apparatus, mitochondria and secretory vesicles. This indicates that these cells are likely to have an active physiological role. The pollen grains possess prominent protruding onci and observations were made on their structure and development. Walls of the protruding onci are also rich in pectin. Pectins are hydrophilic and known to be involved in the dehydration and rehydration of pollen grains. We hypothesise that the thickenings of Igersheim, as well as the protruding onci of the pollen grains, are functionally associated and part of the adaptive syndrome of secondary pollen presentation, at least in the Vanguerieae

  3. Rapid auxin-mediated changes in the proteome of the epidermal cells in rye coleoptiles: Implications for the initiation of growth

    PubMed Central

    Deng, Z.; Xu, S.; Chalkley, R. J.; Oses-Prieto, J. A.; Burlingame, A. L.; Wang, Z.-Y.; Kutschera, U.

    2011-01-01

    In axial organs of juvenile plants, the phytohormone auxin (indole-3-acetic acid, IAA) rapidly allows cell wall loosening and hence promotes turgor-driven elongation. In this study, we used rye (Secale cereale) coleoptile sections to investigate possible effects of IAA on the proteome of cells. In a first set of experiments, we document that IAA causes organ elongation via promotion of expansion of the rigid outer wall of the outer epidermis. A quantitative comparison of the proteome (membrane-associated proteins), using two-dimensional difference gel electrophoresis (2-D DIGE), revealed that, within 2 h of auxin treatment, at least 16 protein spots were up- or down-regulated by IAA. These proteins were identified using reverse-phase liquid chromatography electrospray tandem mass spectrometry. Four of these proteins were detected in the growth-controlling outer epidermis and were further analysed. One epidermal polypeptide, a small Ras-related GTP-binding protein, was rapidly down-regulated by IAA (after 0.5 h of incubation) by −35% compared to the control. Concomitantly, a subunit of the 26S proteasome was up-regulated by IAA (+30% within 1 h). In addition, this protein displayed IAA-mediated post-translational modification. The implications of these rapid auxin effects with respect to signal transduction and IAA-mediated secretion of glycoproteins (osmiophilic nano-particles) into the growth-controlling outer epidermal wall are discussed. PMID:22117532

  4. Templated green synthesis of plasmonic silver nanoparticles in onion epidermal cells suitable for surface-enhanced Raman and hyper-Raman scattering.

    PubMed

    Espina Palanco, Marta; Bo Mogensen, Klaus; Gühlke, Marina; Heiner, Zsuzsanna; Kneipp, Janina; Kneipp, Katrin

    2016-01-01

    We report fast and simple green synthesis of plasmonic silver nanoparticles in the epidermal cells of onions after incubation with AgNO3 solution. The biological environment supports the generation of silver nanostructures in two ways. The plant tissue delivers reducing chemicals for the initial formation of small silver clusters and their following conversion to plasmonic particles. Additionally, the natural morphological structures of the onion layers, in particular the extracellular matrix provides a biological template for the growth of plasmonic nanostructures. This is indicated by red glowing images of extracellular spaces in dark field microscopy of onion layers a few hours after AgNO3 exposure due to the formation of silver nanoparticles. Silver nanostructures generated in the extracellular space of onion layers and within the epidermal cell walls can serve as enhancing plasmonic structures for one- and two-photon-excited spectroscopy such as surface enhanced Raman scattering (SERS) and surface enhanced hyper-Raman scattering (SEHRS). Our studies demonstrate a templated green preparation of enhancing plasmonic nanoparticles and suggest a new route to deliver silver nanoparticles as basic building blocks of plasmonic nanosensors to plants by the uptake of solutions of metal salts. PMID:27547600

  5. Templated green synthesis of plasmonic silver nanoparticles in onion epidermal cells suitable for surface-enhanced Raman and hyper-Raman scattering

    PubMed Central

    Espina Palanco, Marta; Bo Mogensen, Klaus; Gühlke, Marina; Heiner, Zsuzsanna; Kneipp, Janina

    2016-01-01

    Summary We report fast and simple green synthesis of plasmonic silver nanoparticles in the epidermal cells of onions after incubation with AgNO3 solution. The biological environment supports the generation of silver nanostructures in two ways. The plant tissue delivers reducing chemicals for the initial formation of small silver clusters and their following conversion to plasmonic particles. Additionally, the natural morphological structures of the onion layers, in particular the extracellular matrix provides a biological template for the growth of plasmonic nanostructures. This is indicated by red glowing images of extracellular spaces in dark field microscopy of onion layers a few hours after AgNO3 exposure due to the formation of silver nanoparticles. Silver nanostructures generated in the extracellular space of onion layers and within the epidermal cell walls can serve as enhancing plasmonic structures for one- and two-photon-excited spectroscopy such as surface enhanced Raman scattering (SERS) and surface enhanced hyper-Raman scattering (SEHRS). Our studies demonstrate a templated green preparation of enhancing plasmonic nanoparticles and suggest a new route to deliver silver nanoparticles as basic building blocks of plasmonic nanosensors to plants by the uptake of solutions of metal salts. PMID:27547600

  6. Large area space solar cell assemblies

    NASA Technical Reports Server (NTRS)

    Spitzer, M. B.; Nowlan, M. J.

    1982-01-01

    Development of a large area space solar cell assembly is presented. The assembly consists of an ion implanted silicon cell and glass cover. The important attributes of fabrication are (1) use of a back surface field which is compatible with a back surface reflector, and (2) integration of coverglass application and call fabrication.

  7. Requirement for neurogenesis to proceed through the division of neuronal progenitors following differentiation of epidermal growth factor and fibroblast growth factor-2-responsive human neural stem cells.

    PubMed

    Ostenfeld, Thor; Svendsen, Clive N

    2004-01-01

    Epidermal growth factor (EGF)- and fibroblast growth factor-2 (FGF-2)-responsive human neural stem cells may provide insight into mechanisms of neural development and have applications in cell-based therapeutics for neurological disease. However, their biology after expansion in vitro is currently poorly understood. Cells grown in either EGF or FGF-2 or a combination of both mitogens displayed characteristically similar levels of transcriptional activation and comparable proliferative profiles with linear cell-cycle kinetics and possessed similar neuronal differentiation capabilities. These data support the view that human neurospheres at later stages of expansion (>10 weeks) are comprised overwhelmingly of a single type of stem cell responsive to both EGF and FGF-2. After mitogen withdrawal and neurosphere plating, bromodeoxyuridine pulse-chase experiments revealed that the stem cells did not undergo differentiation directly into neurons. Instead, most immature neurons arose via the division of emerging progenitor cells in the absence of exogenous EGF or FGF-2. Neurogenesis was abolished by application of high concentrations of either EGF/FGF-2 or the mitotic inhibitor cytosine-b-arabinofuranoside, suggesting that there is an obligatory requirement for at least one round of cell division in the absence of mitogens as a prelude to terminal neuronal differentiation. The differentiation of human neurospheres provides a useful model of human neurogenesis, and the data presented indicate that it proceeds through the division of committed neuronal progenitor cells rather than directly from the neural stem cell. PMID:15342944

  8. Calcium, Orai1 and Epidermal Proliferation

    PubMed Central

    Bikle, DD; Mauro, T

    2014-01-01

    Ca2+ influx controls essential epidermal functions, including proliferation, differentiation, cell migration, itch, and barrier homeostasis. The Orai1 ion channel allows capacitive Ca2+ influx after Ca2+ release from the endoplasmic reticulum, and it has now been shown to modulate epidermal atrophy. These findings reveal new interactions among various Ca2+ signaling pathways and uncover novel functions for Ca2+ signaling via the Orai1 channel. PMID:24825060

  9. Epidermal growth factor and perlecan fragments produced by apoptotic endothelial cells co-ordinately activate ERK1/2-dependent antiapoptotic pathways in mesenchymal stem cells.

    PubMed

    Soulez, Mathilde; Sirois, Isabelle; Brassard, Nathalie; Raymond, Marc-André; Nicodème, Frédéric; Noiseux, Nicolas; Durocher, Yves; Pshezhetsky, Alexei V; Hébert, Marie-Josée

    2010-04-01

    Mounting evidence indicates that mesenchymal stem cells (MSC) are pivotal to vascular repair and neointima formation in various forms of vascular disease. Yet, the mechanisms that allow MSC to resist apoptosis at sites where other cell types, such as endothelial cells (EC), are dying are not well defined. In the present work, we demonstrate that apoptotic EC actively release paracrine mediators which, in turn, inhibit apoptosis of MSC. Serum-free medium conditioned by apoptotic EC increases extracellular signal-regulated kinases 1 and 2 (ERK1/2) activation and inhibits apoptosis (evaluated by Bcl-xL protein levels and poly (ADP-ribose) polymerase cleavage) of human MSC. A C-terminal fragment of perlecan (LG3) released by apoptotic EC is one of the mediators activating this antiapoptotic response in MSC. LG3 interacts with beta1-integrins, which triggers downstream ERK1/2 activation in MSC, albeit to a lesser degree than medium conditioned by apoptotic EC. Hence, other mediators released by apoptotic EC are probably required for induction of the full antiapoptotic phenotype in MSC. Adopting a comparative proteomic strategy, we identified epidermal growth factor (EGF) as a novel mediator of the paracrine component of the endothelial apoptotic program. LG3 and EGF cooperate in triggering beta1-integrin and EGF receptor-dependent antiapoptotic signals in MSC centering on ERK1/2 activation. The present work, providing novel insights into the mechanisms facilitating the survival of MSC in a hostile environment, identifies EGF and LG3 released by apoptotic EC as central antiapoptotic mediators involved in this paracrine response. PMID:20201065

  10. Dermo-epidermal interactions in reptilian scales: speculations on the evolution of scales, feathers, and hairs.

    PubMed

    Alibardi, Lorenzo

    2004-07-15

    The dermal influence on the epidermis during scale formation in reptiles is poorly known. Cells of the superficial dermis are not homogeneously distributed beneath the epidermis, but are instead connected to specific areas of the epidermis. Dermal cells are joined temporarily or cyclically through the basement membrane, with the reactive region of the epidermis forming specific regions of dermo-epidermal interactions. In these regions morphoregulatory molecules may be exchanged between the dermis and the connected epidermis. Possible changes in the localization of these regions in the skin may result in the production of different appendages, in accordance with the genetic makeup of the epidermis in each species. Regions of dermo-epidermal interactions seem to move their position during development. A hypothesis on the development and evolution of scales, hairs, and feathers from sarcopterigian fish to amniotes is presented, based on the different localization and extension of regions of dermo-epidermal interactions in the skin. It is hypothesized that, during phylogenesis, possible variations in the localization and extension of these regions, from the large scales of basic amniotes to those of sauropsid amniotes, may have originated scales with hard (beta)-keratin. In extant reptiles, extended regions of dermo-epidermal interaction form most of the expanse of outer scale surface. It is hypothesized that the reduction of large regions of dermo-epidermal interactions into small areas in the skin were the origin of dermal condensations. In mammals, small regions of dermo-epidermal interactions have invaginated, forming the dermal papilla with the associated hair matrix epidermis. In birds, small regions of dermo-epidermal interactions have reduced the original scale surface of archosaurian scales, forming the dermal papilla. The latter has invaginated in association with the collar epidermis from which feathers were produced. PMID:15287101

  11. Combination of basic fibroblast growth factor and epidermal growth factor enhances proliferation and neuronal/glial differential of postnatal human enteric neurosphere cells in vitro.

    PubMed

    Pan, Wei-Kang; Yu, Hui; Wu, A-Li; Gao, Ya; Zheng, Bai-Jun; Li, Peng; Yang, Wei-Li; Huang, Qiang; Wang, Huai-Jie; Ge, Xin

    2016-08-01

    Human enteric neural stem cells (hENSCs) proliferate and differentiate into neurons and glial cells in response to a complex network of neurotrophic factors to form the enteric nervous system. The primary aim of this study was to determine the effect of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF) on in-vitro expansion and differentiation of postnatal hENSCs-containing enteric neurosphere cells. Enteric neurosphere cells were isolated from rectal polyp specimens of 75 children (age, 1-13 years) and conditioned with bFGF, EGF, bFGF+EGF, or plain culture media. Proliferation of enteric neurosphere cells was examined using the methyl thiazolyl tetrazolium colorimetric assay over 7 days of culture. Fetal bovine serum (10%) was added to induce the differentiation of parental enteric neurosphere cells, and differentiated offspring cells were immunophenotyped against p75 neutrophin receptor (neural stem cells), peripherin (neuronal cells), and glial fibrillary acidic protein (glial cells). Combining bFGF and EGF significantly improved the proliferation of enteric neurosphere cells compared with bFGF or EGF alone (both P<0.01) throughout 7 days of culture. The addition of bFGF drove a significantly greater proportion of enteric neurosphere cells to differentiate into neuronal cells than that of EGF (P<0.01), whereas addition of EGF resulted in significantly more glial differentiation compared with addition of bFGF (P<0.01). Combining bFGF and EGF drove enteric neurosphere cells to differentiate into neuronal cells in a proportion similar to glial cells. Our results showed that the combination of bFGF and EGF significantly enhanced the proliferation and differentiation of postnatal hENSCs-containing enteric neurosphere cells in vitro. PMID:27306591

  12. EPR measurements showing that plasma membrane viscosity can vary from 30 to 100 cP in human epidermal cell strains

    NASA Astrophysics Data System (ADS)

    Dunham, W. R.; Sands, R. H.; Klein, S. B.; Duell, E. A.; Rhodes, L. M.; Marcelo, C. L.

    1996-09-01

    A rigorous technique for the measurement of human membrane viscosity by electron paramagnetic resonance (EPR) spectroscopy has been developed by designing a sample preparation procedure to optimize the spin labeling process and using a special (grown in essential fatty acid free medium) epidermal cell strain. The essential fatty acid deficient cell strains (keratinocytes) were also grown in fatty acid supplemented media formulated to alter the fatty acid composition of the phospholipids that form the cell membrane. Fatty acid free bovine serum albumin was used as a carrier for the spin label (16-doxyl stearate methyl ester) at an approximately equimolar ratio. Monolayers grown in T-75 flasks were labeled for 15 min at 4°C with 12 μM bovine serum albumin plus 20 μM spin label. The cells were then washed and transferred (at 4°C) to a flatcell for EPR studies at 37°C. The spectra were computer simulated and the results were interpreted by comparison with a "standard curve" obtained from the EPR spectra of the spin label in oil at multiple temperatures. Arguments are presented for preferring this measurement technique over the more conventional use of order parameters and over the use of some other spin labels. The EPR spectra were completely insensitive to the effects of molecular dioxygen in the growth medium and cytoplasm, but remarkabley sensitive to the fatty acid composition of the cellular phospholipids. Fatty acid modified epidermal cells showed a very strong correlation between membrane fluidity (a three-fold change in the membrane viscosity) and a fatty acid double bond index.

  13. c-Jun/AP-1 pathway-mediated cyclin D1 expression participates in low dose arsenite-induced transformation in mouse epidermal JB6 Cl41 cells

    SciTech Connect

    Zhang Dongyun; Li Jingxia; Gao Jimin; Huang Chuanshu

    2009-02-15

    Arsenic is a well-documented human carcinogen associated with skin carcinogenesis. Our previous work reveals that arsenite exposure is able to induce cell transformation in mouse epidermal cell JB6 Cl41 through the activation of ERK, rather than JNK pathway. Our current studies further evaluate downstream pathway in low dose arsenite-induced cell transformation in JB6 Cl41 cells. Our results showed that treatment of cells with low dose arsenite induced activation of c-Jun/AP-1 pathway, and ectopic expression of dominant negative mutant of c-Jun (TAM67) blocked arsenite-induced transformation. Furthermore, our data indicated that cyclin D1 was an important downstream molecule involved in c-Jun/AP-1-mediated cell transformation upon low dose arsenite exposure, because inhibition of cyclin D1 expression by its specific siRNA in the JB6 Cl41 cells resulted in impairment of anchorage-independent growth of cells induced by low dose arsenite. Collectively, our results demonstrate that c-Jun/AP-1-mediated cyclin D1 expression is at least one of the key events implicated in cell transformation upon low dose arsenite exposure.

  14. Epidermal Neural Crest Stem Cell (EPI-NCSC)—Mediated Recovery of Sensory Function in a Mouse Model of Spinal Cord Injury

    PubMed Central

    Hu, Yao Fei; Gourab, Krishnaj; Wells, Clive; Clewes, Oliver; Schmit, Brian D.

    2010-01-01

    Here we show that epidermal neural crest stem cell (EPI-NCSC) transplants in the contused spinal cord caused a 24% improvement in sensory connectivity and a substantial recovery of touch perception. Furthermore we present a novel method for the ex vivo expansion of EPI-NCSC into millions of stem cells that takes advantage of the migratory ability of neural crest stem cells and is based on a new culture medium and the use of microcarriers. Functional improvement was shown by two independent methods, spinal somatosensory evoked potentials (SpSEP) and the Semmes-Weinstein touch test. Subsets of transplanted cells differentiated into myelinating oligodendrocytes. Unilateral injections of EPI-NCSC into the lesion of midline contused mouse spinal cords elicited bilateral improvements. Intraspinal EPI-NCSC did not migrate laterally in the spinal cord or invade the spinal roots and dorsal root ganglia, thus implicating diffusible factors. EPI-NCSC expressed neurotrophic factors, angiogenic factors, and metalloproteases. The strength of EPI-NCSC thus is that they can exert a combination of pertinent functions in the contused spinal cord, including cell replacement, neuroprotection, angiogenesis and modulation of scar formation. EPI-NCSC are uniquely qualified for cell-based therapy in spinal cord injury, as neural crest cells and neural tube stem cells share a higher order stem cell and are thus ontologically closely related. PMID:20414748

  15. A Functional Role of RB-Dependent Pathway in the Control of Quiescence in Adult Epidermal Stem Cells Revealed by Genomic Profiling

    PubMed Central

    Lorz, Corina; García-Escudero, Ramón; Segrelles, Carmen; Garín, Marina I.; Ariza, José M.; Santos, Mirentxu; Ruiz, Sergio; Lara, María F.; Martínez-Cruz, Ana B.; Costa, Clotilde; Buitrago-Pérez, Águeda; Saiz-Ladera, Cristina; Dueñas, Marta

    2010-01-01

    Continuous cell renewal in mouse epidermis is at the expense of a pool of pluripotent cells that lie in a well defined niche in the hair follicle known as the bulge. To identify mechanisms controlling hair follicle stem cell homeostasis, we developed a strategy to isolate adult bulge stem cells in mice and to define their transcriptional profile. We observed that a large number of transcripts are underexpressed in hair follicle stem cells when compared to non-stem cells. Importantly, the majority of these downregulated genes are involved in cell cycle. Using bioinformatics tools, we identified the E2F transcription factor family as a potential element involved in the regulation of these transcripts. To determine their functional role, we used engineered mice lacking Rb gene in epidermis, which showed increased expression of most E2F family members and increased E2F transcriptional activity. Experiments designed to analyze epidermal stem cell functionality (i.e.: hair regrowth and wound healing) imply a role of the Rb-E2F axis in the control of stem cell quiescence in epidermis. Electronic supplementary material The online version of this article (doi:10.1007/s12015-010-9139-0) contains supplementary material, which is available to authorized users. PMID:20376578

  16. VEGF-A acts via neuropilin-1 to enhance epidermal cancer stem cell survival and formation of aggressive and highly vascularized tumors.

    PubMed

    Grun, D; Adhikary, G; Eckert, R L

    2016-08-18

    We identify a limited subpopulation of epidermal cancer stem cells (ECS cells), in squamous cell carcinoma, that form rapidly growing, invasive and highly vascularized tumors, as compared with non-stem cancer cells. These ECS cells grow as non-attached spheroids, and display enhanced migration and invasion. We show that ECS cell-produced vascular endothelial growth factor (VEGF)-A is required for the maintenance of this phenotype, as knockdown of VEGF-A gene expression or treatment with VEGF-A-inactivating antibody reduces these responses. In addition, treatment with bevacizumab reduces tumor vascularity and growth. Surprisingly, the classical mechanism of VEGF-A action via interaction with VEGF receptors does not mediate these events, as these cells lack VEGFR1 and VEGFR2. Instead, VEGF-A acts via the neuropilin-1 (NRP-1) co-receptor. Knockdown of NRP-1 inhibits ECS cell spheroid formation, invasion and migration, and attenuates tumor formation. These studies suggest that VEGF-A acts via interaction with NRP-1 to trigger intracellular events leading to ECS cell survival and formation of aggressive, invasive and highly vascularized tumors. PMID:26804163

  17. Combinatorial-Designed Epidermal Growth Factor Receptor-Targeted Chitosan Nanoparticles for Encapsulation and Delivery of Lipid-Modified Platinum Derivatives in Wild-Type and Resistant Non-Small-Cell Lung Cancer Cells.

    PubMed

    Nascimento, Ana Vanessa; Singh, Amit; Bousbaa, Hassan; Ferreira, Domingos; Sarmento, Bruno; Amiji, Mansoor M

    2015-12-01

    Development of efficient and versatile drug delivery platforms to overcome the physical and biological challenges in cancer therapeutics is an area of great interest, and novel materials are actively sought for such applications. Recent strides in polymer science have led to a combinatorial approach for generating a library of materials with different functional identities that can be "mixed and matched" to attain desired characteristics of a delivery vector. We have applied the combinatorial design to chitosan (CS), where the polymer backbone has been modified with polyethylene glycol, epidermal growth factor receptor-binding peptide, and lipid derivatives of varying chain length to encapsulate hydrophobic drugs. Cisplatin, cis-([PtCl2(NH3)2]), is one of the most potent chemotherapy drugs broadly administered for cancer treatment. Cisplatin is a hydrophilic drug, and in order for it to be encapsulated in the developed nanosystems, it was modified with lipids of varying chain length. The library of four CS derivatives and six platinum derivatives was self-assembled in aqueous medium and evaluated for physicochemical characteristics and cytotoxic effects in platinum-sensitive and -resistant lung cancer cells. The results show that the lipid-modified platinate encapsulation into CS nanoparticles significantly improved cellular cytotoxicity of the drug. In this work, we have also reinforced the idea that CS is a multifaceted system that can be as successful in delivering small molecules as it has been as a nucleic acids carrier. PMID:26523837

  18. Antacid Use and De Novo Brain Metastases in Patients with Epidermal Growth Factor Receptor-Mutant Non-Small Cell Lung Cancer Who Were Treated Using First-Line First-Generation Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors

    PubMed Central

    Chen, Yu-Mu; Lai, Chien-Hao; Chang, Huang-Chih; Chao, Tung-Ying; Tseng, Chia-Cheng; Fang, Wen-Feng; Wang, Chin-Chou; Chung, Yu-Hsiu; Wang, Yi-Hsi; Su, Mao-Chang; Liu, Shih-Feng; Huang, Kuo-Tung; Chen, Hung-Chen; Chang, Ya-Chun; Lin, Meng-Chih

    2016-01-01

    Background Antacid treatments decrease the serum concentrations of first-generation epidermal growth factor receptor (EGFR)-tyrosine kinase inhibitors (TKIs), although it is unknown whether antacids affect clinical outcomes. As cerebrospinal fluid concentrations of TKIs are much lower than serum concentrations, we hypothesized that this drug-drug interaction might affect the prognosis of patients with de novo brain metastases. Materials and Methods This retrospective study evaluated 269 patients with EGFR-mutant non-small cell lung cancer (NSCLC) who had been diagnosed between December 2010 and December 2013, and had been treated using first-line first-generation EGFR-TKIs. Among these patients, we identified patients who concurrently used H2 receptor antagonists (H2RAs) and proton pump inhibitors (PPIs) as antacids. Patients who exhibited >30% overlap between the use of TKIs and antacids were considered antacid users. Results Fifty-seven patients (57/269, 21.2%) were antacid users, and antacid use did not significantly affect progression-free survival (PFS; no antacids: 11.2 months, H2RAs: 9.4 months, PPIs: 6.7 months; p = 0.234). However, antacid use significantly reduced overall survival (OS; no antacids: 25.0 months, H2RAs: 15.5 months, PPIs: 11.3 months; p = 0.002). Antacid use did not affect PFS for various metastasis sites, although antacid users with de novo brain metastases exhibited significantly shorter OS, compared to non-users (11.8 vs. 16.3 months, respectively; p = 0.041). Antacid use did not significantly affect OS in patients with bone, liver, or pleural metastases. Conclusion Antacid use reduced OS among patients with EGFR-mutant NSCLC who were treated using first-line first-generation EGFR-TKIs, and especially among patients with de novo brain metastases. PMID:26894507

  19. Detection of epidermal growth factor receptor mutations in formalin fixed paraffin embedded biopsies in Malaysian non-small cell lung cancer patients

    PubMed Central

    2013-01-01

    Background Somatic mutations of the epidermal growth factor receptor (EGFR) are reportedly associated with various responses in non-small cell lung cancer (NSCLC) patients receiving the anti-EGFR agents. Detection of the mutation therefore plays an important role in therapeutic decision making. The aim of this study was to detect EGFR mutations in formalin fixed paraffin embedded (FFPE) samples using both Scorpion ARMS and high resolution melt (HRM) assay, and to compare the sensitivity of these methods. Results All of the mutations were found in adenocarcinoma, except one that was in squamous cell carcinoma. The mutation rate was 45.7% (221/484). Complex mutations were also observed, wherein 8 tumours carried 2 mutations and 1 tumour carried 3 mutations. Conclusions Both methods detected EGFR mutations in FFPE samples. HRM assays gave more EGFR positive results compared to Scorpion ARMS. PMID:23590575

  20. A cyclic peptide derived from alpha-fetoprotein inhibits the proliferative effects of the epidermal growth factor and estradiol in MCF7 cells.

    PubMed

    Torres, Cristian; Antileo, Elmer; Epuñán, Maráa José; Pino, Ana María; Valladares, Luis Emilio; Sierralta, Walter Daniel

    2008-06-01

    A cyclic peptide derived from the active domain of alpha-fetoprotein (AFP) significantly inhibited the proliferation of MCF7 cells stimulated with the epidermal growth factor (EGF) or estradiol (E2). The action of these three agents on cell growth was independent of the presence of calf serum in the culture medium. Our results demonstrated that the cyclic peptide interfered markedly with the regulation of MAPK by activated c-erbB2. The cyclic peptide showed no effect on the E2-stimulated release of matrix metalloproteinases 2 and 9 nor on the shedding of heparin-binding EGF into the culture medium. We propose that the AFP-derived cyclic peptide represents a valuable novel antiproliferative agent for treating breast cancer. PMID:18497971

  1. Multiphoton high-resolution 3D imaging of Langerhans cells and keratinocytes in the mouse skin model adopted for epidermal powdered immunization.

    PubMed

    Mulholland, William J; Arbuthnott, Edward A H; Bellhouse, Brian J; Cornhill, J Frederick; Austyn, Jonathan M; Kendall, Mark A F; Cui, Zhanfeng; Tirlapur, Uday K

    2006-07-01

    Langerhans cells (LCs) can be targeted with DNA-coated gold micro-projectiles ("Gene Gun") to induce potent cellular and humoral immune responses. It is likely that the relative volumetric distribution of LCs and keratinocytes within the epidermis impacts on the efficacy of Gene Gun immunization protocols. This study quantified the three-dimensional (3D) distribution of LCs and keratinocytes in the mouse skin model with a near-infrared multiphoton laser-scanning microscope (NIR-MPLSM). Stratum corneum (SC) and viable epidermal thickness measured with MPLSM was found in close agreement with conventional histology. LCs were located in the vertical plane at a mean depth of 14.9 microm, less than 3 mum above the dermo-epidermal boundary and with a normal histogram distribution. This likely corresponds to the fact that LCs reside in the suprabasal layer (stratum germinativum). The nuclear volume of keratinocytes was found to be approximately 1.4 times larger than that of resident LCs (88.6 microm3). Importantly, the ratio of LCs to keratinocytes in mouse ear skin (1:15) is more than three times higher than that reported for human breast skin (1:53). Accordingly, cross-presentation may be more significant in clinical Gene Gun applications than in pre-clinical mouse studies. These interspecies differences should be considered in pre-clinical trials using mouse models. PMID:16645596

  2. Hemangiosarcoma and its cancer stem cell subpopulation are effectively killed by a toxin targeted through epidermal growth factor and urokinase receptors.

    PubMed

    Schappa, Jill T; Frantz, Aric M; Gorden, Brandi H; Dickerson, Erin B; Vallera, Daniel A; Modiano, Jaime F

    2013-10-15

    Targeted toxins have the potential to overcome intrinsic or acquired resistance of cancer cells to conventional cytotoxic agents. Here, we hypothesized that EGFuPA-toxin, a bispecific ligand-targeted toxin (BLT) consisting of a deimmunized Pseudomonas exotoxin (PE) conjugated to epidermal growth factor and urokinase, would efficiently target and kill cells derived from canine hemangiosarcoma (HSA), a highly chemotherapy resistant tumor, as well as cultured hemangiospheres, used as a surrogate for cancer stem cells (CSC). EGFuPA-toxin showed cytotoxicity in four HSA cell lines (Emma, Frog, DD-1 and SB) at a concentration of ≤100 nM, and the cytotoxicity was dependent on specific ligand-receptor interactions. Monospecific targeted toxins also killed these chemoresistant cells; in this case, a "threshold" level of EGFR expression appeared to be required to make cells sensitive to the monospecific EGF-toxin, but not to the monospecific uPA-toxin. The IC₅₀ of CSCs was higher by approximately two orders of magnitude as compared to non-CSCs, but these cells were still sensitive to EGFuPA-toxin at nanomolar (i.e., pharmacologically relevant) concentrations, and when targeted by EGFuPA-toxin, resulted in death of the entire cell population. Taken together, our results support the use of these toxins to treat chemoresistant tumors such as sarcomas, including those that conform to the CSC model. Our results also support the use of companion animals with cancer for further translational development of these cytotoxic molecules. PMID:23553371

  3. Down-Regulation of ClC-3 Expression Reduces Epidermal Stem Cell Migration by Inhibiting Volume-Activated Chloride Currents.

    PubMed

    Guo, Rui; Pan, Fuqiang; Tian, Yanping; Li, Hongli; Li, Shirong; Cao, Chuan

    2016-06-01

    ClC-3, a member of the ClC chloride (Cl(-)) channel family, has recently been proposed as the primary Cl(-) channel involved in cell volume regulation. Changes in cell volume influence excitability, contraction, migration, pathogen-host interactions, cell proliferation, and cell death processes. In this study, expression and function of ClC-3 channels were investigated during epidermal stem cell (ESC) migration. We observed differential expression of CLC-3 regulates migration of ESCs. Further, whole-cell patch-clamp recordings and image analysis demonstrated ClC-3 expression affected volume-activated Cl(-) current (I Cl,Vol) within ESCs. Live cell imaging systems, designed to observe cellular responses to overexpression and suppression of ClC-3 in real time, indicated ClC-3 may regulate ESC migratory dynamics. We employed IMARIS software to analyze the velocity and distance of ESC migration in vitro to demonstrate the function of ClC-3 channel in ESCs. As our data suggest volume-activated Cl(-) channels play a vital role in migration of ESCs, which contribute to skin repair by migrating from neighboring unwounded epidermis infundibulum, hair follicle or sebaceous glands, ClC-3 may represent a new and valuable target for stem cell therapies. PMID:26769712

  4. Squamosamide derivative FLZ protects retinal pigment epithelium cells from oxidative stress through activation of epidermal growth factor receptor (EGFR)-AKT signaling.

    PubMed

    Cheng, Li-Bo; Chen, Chun-Ming; Zhong, Hong; Zhu, Li-Juan

    2014-01-01

    Reactive oxygen species (ROS)-mediated retinal pigment epithelium (RPE) cell apoptosis is attributed to age-related macular degeneration (AMD) pathogenesis. FLZ, a novel synthetic squamosamide derivative from a Chinese herb, Annona glabra, has displayed significant cyto-protective activity. In the current study, we explored the pro-survival effect of FLZ in oxidative stressed-RPE cells and studied the underlying signaling mechanisms. Our results showed that FLZ attenuated hydrogen peroxide (H2O2)-induced viability decrease and apoptosis in the RPE cell line (ARPE-19 cells) and in primary mouse RPE cells. Western blotting results showed that FLZ activated AKT signaling in RPE cells. The AKT-specific inhibitor, MK-2206, the phosphoinositide 3-kinase (PI3K)/AKT pan inhibitor, wortmannin, and AKT1-shRNA (short hairpin RNA) depletion almost abolished FLZ-mediated pro-survival/anti-apoptosis activity. We discovered that epidermal growth factor receptor (EGFR) trans-activation mediated FLZ-induced AKT activation and the pro-survival effect in RPE cells, and the anti-apoptosis effect of FLZ against H2O2 was inhibited by the EGFR inhibitor, PD153035, or by EGFR shRNA-knockdown. In conclusion, FLZ protects RPE cells from oxidative stress through activation of EGFR-AKT signaling, and our results suggest that FLZ might have therapeutic values for AMD. PMID:25329617

  5. Gefitinib and Erlotinib Lead to Phosphorylation of Eukaryotic Initiation Factor 2 Alpha Independent of Epidermal Growth Factor Receptor in A549 Cells

    PubMed Central

    Koyama, Satoshi; Omura, Tomohiro; Yonezawa, Atsushi; Imai, Satoshi; Nakagawa, Shunsaku; Nakagawa, Takayuki; Yano, Ikuko; Matsubara, Kazuo

    2015-01-01

    Gefitinib and erlotinib are anticancer agents, which inhibit epidermal growth factor receptor (EGFR) tyrosine kinase. Interstitial lung disease (ILD) occurs in patients with non-small cell lung cancer receiving EGFR inhibitors. In the present study, we examined whether gefitinib- and erlotinib-induced lung injury related to ILD through endoplasmic reticulum (ER) stress, which is a causative intracellular mechanism in cytotoxicity caused by various chemicals in adenocarcinomic human alveolar basal epithelial cells. These two EGFR inhibitors increased Parkinson juvenile disease protein 2 and C/EBP homologous protein mRNA expressions, and activated the eukaryotic initiation factor (eIF) 2α/activating transcription factor 4 pathway without protein kinase R-like ER kinase activation in A549 cells. Gefitinib and erlotinib caused neither ER stress nor cell death; however, these agents inhibited cell growth via the reduction of cyclin-D1 expression. Tauroursodeoxycholic acid, which is known to suppress eIF2α phosphorylation, cancelled the effects of EGFR inhibitors on cyclin-D1 expression and cell proliferation in a concentration-dependent manner. The results of an EGFR-silencing study using siRNA showed that gefitinib and erlotinib affected eIF2α phosphorylation and cyclin-D1 expression independent of EGFR inhibition. Therefore, the inhibition of cell growth by these EGFR inhibitors might equate to impairment of the alveolar epithelial cell repair system via eIF2α phosphorylation and reduced cyclin-D1 expression. PMID:26288223

  6. Gefitinib and Erlotinib Lead to Phosphorylation of Eukaryotic Initiation Factor 2 Alpha Independent of Epidermal Growth Factor Receptor in A549 Cells.

    PubMed

    Koyama, Satoshi; Omura, Tomohiro; Yonezawa, Atsushi; Imai, Satoshi; Nakagawa, Shunsaku; Nakagawa, Takayuki; Yano, Ikuko; Matsubara, Kazuo

    2015-01-01

    Gefitinib and erlotinib are anticancer agents, which inhibit epidermal growth factor receptor (EGFR) tyrosine kinase. Interstitial lung disease (ILD) occurs in patients with non-small cell lung cancer receiving EGFR inhibitors. In the present study, we examined whether gefitinib- and erlotinib-induced lung injury related to ILD through endoplasmic reticulum (ER) stress, which is a causative intracellular mechanism in cytotoxicity caused by various chemicals in adenocarcinomic human alveolar basal epithelial cells. These two EGFR inhibitors increased Parkinson juvenile disease protein 2 and C/EBP homologous protein mRNA expressions, and activated the eukaryotic initiation factor (eIF) 2α/activating transcription factor 4 pathway without protein kinase R-like ER kinase activation in A549 cells. Gefitinib and erlotinib caused neither ER stress nor cell death; however, these agents inhibited cell growth via the reduction of cyclin-D1 expression. Tauroursodeoxycholic acid, which is known to suppress eIF2α phosphorylation, cancelled the effects of EGFR inhibitors on cyclin-D1 expression and cell proliferation in a concentration-dependent manner. The results of an EGFR-silencing study using siRNA showed that gefitinib and erlotinib affected eIF2α phosphorylation and cyclin-D1 expression independent of EGFR inhibition. Therefore, the inhibition of cell growth by these EGFR inhibitors might equate to impairment of the alveolar epithelial cell repair system via eIF2α phosphorylation and reduced cyclin-D1 expression. PMID:26288223

  7. Epidermal growth factor receptor pathway substrate 8 (Eps8) is a novel regulator of cell adhesion and the blood-testis barrier integrity in the seminiferous epithelium

    PubMed Central

    Lie, Pearl P. Y.; Mruk, Dolores D.; Lee, Will M.; Cheng, C. Yan

    2009-01-01

    In the seminiferous epithelium, Eps8 is localized to actin-based cell junctions at the blood-testis barrier (BTB) and the apical ectoplasmic specialization (ES) in stage V–VI tubules but is considerably diminished in stage VIII tubules. Eps8 down-regulation coincides with the time of BTB restructuring and apical ES disassembly, implicating the role of Eps8 in cell adhesion. Its involvement in Sertoli-germ cell adhesion was substantiated in studies using an in vivo animal model by treating rats with 1-(2,4-dichlorobenzy)-1H-indazole-3-carbohydrazide (adjudin) to induce anchoring junction restructuring, during which Eps8 disappeared at the apical ES before germ cell departure. In Sertoli cell cultures with established permeability barrier mimicking the BTB in vivo, the knockdown of Eps8 by RNAi led to F-actin disorganization and the mislocalization of the tight junction proteins occludin and ZO-1, suggesting the function of Eps8 in maintaining BTB integrity. In vivo knockdown of Eps8 in the testis caused germ cell sloughing and BTB damage, concomitant with occludin mislocalization, further validating that Eps8 is a novel regulator of cell adhesion and BTB integrity in the seminiferous epithelium.—Lie, P. P. Y., Mruk, D. D., Lee, W. M., Cheng, C. Y. Epidermal growth factor receptor pathway substrate 8 (Eps8) is a novel regulator of cell adhesion and the blood-testis barrier integrity in the seminiferous epithelium. PMID:19293393

  8. Establishment and characterization of a skin epidermal cell line from mud loach, Misgurnus anguillicaudatus, (MASE) and its interaction with three bacterial pathogens.

    PubMed

    Xu, Xiaohui; Sivaramasamy, Elayaraja; Jin, Songjun; Li, Fuhua; Xiang, Jianhai

    2016-08-01

    A continuous skin epidermal cell line from mud Loach (Misgurnus anguillicaudatus) (MASE cell line) was established with its application in bacteria infection demonstrated in this study. Primary MASE cell culture was initiated at 26 °C in Dulbecco's modified Eagle medium/F12 medium (1:1; pH7.2) supplemented with 20% fetal bovine serum (FBS). The primary MASE cells in spindle morphology proliferated into a confluent monolayer within 2 weeks, and were continuously subcultured even in 10% FBS- DMEM/F12 after 10 passages. Impacts of medium and temperature on the growth of the cells were examined. The optimum growth was found in DMEM/F12 with 20% FBS and at 26 °C. The MASE cells have been subcultured steadily over Passage 90 with a population doubling time of 53.3 h at Passage 60. Chromosome analysis revealed that 60.5% of MASE cells at Passage 60 maintained the normal diploid chromosome number (50) with a normal karyotype of 10m+4sm + 36t. Bacteria from the three species (Aeromonas veronii, Vibrio parahaemolyticus and Escherichia coli) were used to investigate the interactions between bacteria and cellular hosts. The three strains could be attached to the MASE cells and replicate at different levels. A. veronii could induce apoptosis in the MASE cells, with highest adherence rate among the three strains, whereas V. parahaemolyticus could cause highest cell death rate through a non-apoptotic cell death pathway, with high level of replication. The results revealed that different bacteria could interact with the MASE cells in different manners, and divergent pathways might lie in mediating cell death when cellular hosts confronted with pathogen infection. Therefore, the MASE cell line may serve as a useful tool for studying the interaction between skin bacteria and fish cells. PMID:27288257

  9. EGF–FGF{sub 2} stimulates the proliferation and improves the neuronal commitment of mouse epidermal neural crest stem cells (EPI-NCSCs)

    SciTech Connect

    Bressan, Raul Bardini; Melo, Fernanda Rosene; Almeida, Patricia Alves; Bittencourt, Denise Avani; Visoni, Silvia; Jeremias, Talita Silva; Costa, Ana Paula; Leal, Rodrigo Bainy; Trentin, Andrea Gonçalves

    2014-09-10

    Epidermal neural crest stem cells (EPI-NCSCs), which reside in the bulge of hair follicles, are attractive candidates for several applications in cell therapy, drug screening and tissue engineering. As suggested remnants of the embryonic neural crest (NC) in an adult location, EPI-NCSCs are able to generate a wide variety of cell types and are readily accessible by a minimally invasive procedure. Since the combination of epidermal growth factor (EGF) and fibroblast growth factor type 2 (FGF{sub 2}) is mitogenic and promotes the neuronal commitment of various stem cell populations, we examined its effects in the proliferation and neuronal potential of mouse EPI-NCSCs. By using a recognized culture protocol of bulge whiskers follicles, we were able to isolate a population of EPI-NCSCs, characterized by the migratory potential, cell morphology and expression of phenotypic markers of NC cells. EPI-NCSCs expressed neuronal, glial and smooth muscle markers and exhibited the NC-like fibroblastic morphology. The treatment with the combination EGF and FGF{sub 2}, however, increased their proliferation rate and promoted the acquisition of a neuronal-like morphology accompanied by reorganization of neural cytoskeletal proteins βIII-tubulin and nestin, as well as upregulation of the pan neuronal marker βIII-tubulin and down regulation of the undifferentiated NC, glial and smooth muscle cell markers. Moreover, the treatment enhanced the response of EPI-NCSCs to neurogenic stimulation, as evidenced by induction of GAP43, and increased expression of Mash-1 in neuron-like cell, both neuronal-specific proteins. Together, the results suggest that the combination of EGF–FGF2 stimulates the proliferation and improves the neuronal potential of EPI-NCSCs similarly to embryonic NC cells, ES cells and neural progenitor/stem cells of the central nervous system and highlights the advantage of using EGF–FGF{sub 2} in neuronal differentiation protocols. - Highlights: • EPI

  10. Transient proliferation of proanthocyanidin-accumulating cells on the epidermal apex contributes to highly aluminum-resistant root elongation in camphor tree.

    PubMed

    Osawa, Hiroki; Endo, Izuki; Hara, Yukari; Matsushima, Yuki; Tange, Takeshi

    2011-01-01

    Aluminum (Al) is a harmful element that rapidly inhibits the elongation of plant roots in acidic soils. The release of organic anions explains Al resistance in annual crops, but the mechanisms that are responsible for superior Al resistance in some woody plants remain unclear. We examined cell properties at the surface layer of the root apex in the camphor tree (Cinnamomum camphora) to understand its high Al resistance mechanism. Exposure to 500 μm Al for 8 d, more than 20-fold higher concentration and longer duration than what soybean (Glycine max) can tolerate, only reduced root elongation in the camphor tree to 64% of the control despite the slight induction of citrate release. In addition, Al content in the root apices was maintained at low levels. Histochemical profiling revealed that proanthocyanidin (PA)-accumulating cells were present at the adjacent outer layer of epidermis cells at the root apex, having distinctive zones for cell division and the early phase of cell expansion. Then the PA cells were gradually detached off the root, leaving thin debris behind, and the root surface was replaced with the elongating epidermis cells at the 3- to 4-mm region behind the tip. Al did not affect the proliferation of PA cells or epidermis cells, except for the delay in the start of expansion and the accelerated detachment of the former. In soybean roots, the innermost lateral root cap cells were absent in both PA accumulation and active cell division and failed to protect the epidermal cell expansion at 25 μm Al. These results suggest that transient proliferation and detachment of PA cells may facilitate the expansion of epidermis cells away from Al during root elongation in camphor tree. PMID:21045123

  11. Tissue kallikrein induces SH-SY5Y cell proliferation via epidermal growth factor receptor and extracellular signal-regulated kinase1/2 pathway

    SciTech Connect

    Lu, Zhengyu; Yang, Qi; Cui, Mei; Liu, Yanping; Wang, Tao; Zhao, Hong; Dong, Qiang

    2014-03-28

    Highlights: • TK promotes EGFR phosphorylation in SH-SY5Y cells. • TK activates ERK1/2 and p38 phosphorylation in SH-SY5Y cells. • TK mediates SH-SY5Y cell proliferation via EGFR and ERK1/2 pathway. - Abstract: Tissue kallikrein (TK) is well known to take most of its biological functions through bradykinin receptors. In the present study, we found a novel signaling pathway mediated by TK through epidermal growth factor receptor (EGFR) in human SH-SY5Y cells. We discovered that TK facilitated the activation of EGFR, extracellular signal-regulated kinase (ERK) 1/2 and p38 cascade. Interestingly, not p38 but ERK1/2 phosphorylation was severely compromised in cells depleted of EGFR. Nevertheless, impairment of signaling of ERK1/2 seemed not to be restricted to EGFR phosphorylation. We also observed that TK stimulation could induce SH-SY5Y cell proliferation, which was reduced by EGFR down-regulation or ERK1/2 inhibitor. Overall, our findings provided convincing evidence that TK could mediate cell proliferation via EGFR and ERK1/2 pathway in vitro.

  12. Combined Inhibition of c-Src and Epidermal Growth Factor Receptor Abrogates Growth and Invasion of Head and Neck Squamous Cell Carcinoma

    PubMed Central

    Koppikar, Priya; Choi, Seung-Ho; Egloff, Ann Marie; Cai, Quan; Suzuki, Shinsuke; Freilino, Maria; Nozawa, Hiroshi; Thomas, Sufi M.; Gooding, William E.; Siegfried, Jill M.; Grandis, Jennifer R.

    2012-01-01

    Purpose Increased expression and/or activation of epidermal growth factor receptor (EGFR) is associated with tumor progression and poor prognosis in many cancers including head and neck squamous cell carcinoma (HNSCC). Src family kinases, including c-Src, mediate a variety of intra- or extracellular signals that contribute to tumor formation and progression. This study was undertaken to elucidate the role of c-Src in the growth and invasion of HNSCC and to determine the effects of combined targeting of EGFR and Src kinases in HNSCC cell lines. Experimental design HNSCC cells were engineered to stably express a dominant-active (DA) form of c-Src and investigated in cell growth and invasion assays. The biochemical effects of combined treatment with the Src inhibitor, AZD0530, a potent, orally active Src inhibitor with Bcr/Abl activity and the EGFR kinase inhibitor, gefitinib, were examined as well as the consequences of dual Src/EGFR targeting on the growth and invasion of a panel of HNSCC cell lines. Results HNSCC cells expressing DA c-Src demonstrated increased growth and invasion compared with vector-transfected controls. Combined treatment with AZD0530 and gefitinib resulted in greater inhibition of HNSCC cell growth and invasion compared with either agent alone. Conclusions These results suggest that increased expression and activation of c-Src promotes HNSCC progression where combined targeting of EGFR and c-Src may be an efficacious treatment approach. PMID:18594011

  13. Matrix metalloproteinase-1 is induced by epidermal growth factor in human bladder tumour cell lines and is detectable in urine of patients with bladder tumours.

    PubMed Central

    Nutt, J. E.; Mellon, J. K.; Qureshi, K.; Lunec, J.

    1998-01-01

    The matrix metalloproteinases are a family of enzymes that degrade the extracellular matrix and are considered to be important in tumour invasion and metastasis. The effect of epidermal growth factor (EGF) on matrix metalloproteinase-1 (MMP1) production in two human bladder tumour cell lines, RT112 and RT4, has been investigated. In the RT112 cell line, an increase in MMP1 mRNA levels was found after a 6-h incubation with EGF, and this further increased to 20-fold that of control levels at 24- and 48-h treatment with 50 ng ml(-1) of EGF. MMP2 mRNA levels remained constant over this time period, whereas in the RT4 cells no MMP2 transcripts were detectable, but MMP1 transcripts again increased with 24- and 48-h treatment with 50 ng ml(-1) of EGF. MMP1 protein concentration in the conditioned medium from both cell lines increased with 24- and 48-h treatment of the cells and the total MMP1 was higher in the medium than the cells, demonstrating that the bladder tumour cell lines synthesize and secrete MMP1 protein after continuous stimulation with EGF. MMP1 protein was detected in urine from patients with bladder tumours, with a significant increase in concentration with increased stage and grade of tumour. MMP1 urine concentrations may therefore be a useful prognostic indicator for bladder tumour progression. Images Figure 1 Figure 2 PMID:9683296

  14. Penile epidermal inclusion cyst.

    PubMed

    Saini, Pradeep; Mansoor, M N; Jalali, Sanjay; Sharma, Abhishek

    2010-07-01

    We report a case of epidermal inclusion cyst of penis in a five-year-old boy, who had presented to the outpatient department of our hospital. Epidermal inclusion cysts are benign lesions that can develop in any part of the body. However, the finding of an epidermal inclusion cyst in the penis is rare. The child was operated and discharged uneventfully. The objective of reporting this case is to highlight the rare possibility of an inclusion cyst arising from penis as a late complication of circumcision. PMID:20589475

  15. JAK2-related pathway induces acquired erlotinib resistance in lung cancer cells harboring an epidermal growth factor receptor-activating mutation.

    PubMed

    Harada, Daijiro; Takigawa, Nagio; Ochi, Nobuaki; Ninomiya, Takashi; Yasugi, Masayuki; Kubo, Toshio; Takeda, Hiromasa; Ichihara, Eiki; Ohashi, Kadoaki; Takata, Saburo; Tanimoto, Mitsune; Kiura, Katsuyuki

    2012-10-01

    Epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors, such as gefitinib and erlotinib, are effective for non-small cell lung cancer with activating EGFR mutations. However, even in patients with an initial dramatic response to such a drug, acquired resistance develops after 6-12 months. A secondary mutation of T790M in EGFR and amplification of the MET gene account for this resistance; however, the mechanism(s) of approximately 30% of acquired resistance cases remain unknown. We established an erlotinib-resistant lung cancer cell line named PC-9/ER3 that harbors an EGFR mutation after continuously exposing PC-9 cells to erlotinib. PC-9/ER3 cells were 136-fold more resistant to erlotinib than the parental cells. Although the PC-9/ER3 cells did not carry the T790M mutation or MET amplification and had similar levels of phosphorylated (p) STAT3, pJAK2 increased in the resistant cells. It was found in the present study that 3-12 h of exposure to erlotinib in both cell lines did not affect pJAK2 expression, but did result in increased pSTAT3 expression. pAkt in PC-9/ER3 cells was less suppressed than in PC-9 cells, although pEGFR and pMAPK were markedly suppressed in both cell lines. The combined treatment of erlotinib plus a JAK2 inhibitor (JSI-124) suppressed pAkt in PC-9/ER3 cells. Similarly, the combination of erlotinib plus JSI-124 or siRNA against JAK2 restored sensitivity to erlotinib in PC-9/ER3 cells. The combination of erlotinib plus JSI-124 was also effective for reducing PC-9/ER3 tumors in a murine xenograft model. Our results suggest that the activation of JAK2 partially accounts for acquired erlotinib resistance. PMID:22712764

  16. Epidermal nevi with aberrant epidermal structure in keratinocytes and melanocytes.

    PubMed

    Oiso, Naoki; Sugawara, Koji; Yonamine, Ayano; Tsuruta, Daisuke; Kawada, Akira

    2015-04-01

    Epidermal nevi are congenital cutaneous hamartomas caused by embryonic somatic mutations. Ultrastructural features of adult epidermal nevi have rarely been investigated. Herein, we report a case involving a Japanese adult who had epidermal nevi with right congenital blindness and a right accessory nipple. The histopathologic and ultrastructural studies showed divergent abnormal epidermal structures in both melanocytes and keratinocytes. Our case indicates the need to further investigate histopathologic, ultrastructural, and genetic associations in adult epidermal nevi. PMID:25657059

  17. Human Epidermal Growth Factor Receptor 2 (HER2) –Specific Chimeric Antigen Receptor–Modified T Cells for the Immunotherapy of HER2-Positive Sarcoma

    PubMed Central

    Ahmed, Nabil; Brawley, Vita S.; Hegde, Meenakshi; Robertson, Catherine; Ghazi, Alexia; Gerken, Claudia; Liu, Enli; Dakhova, Olga; Ashoori, Aidin; Corder, Amanda; Gray, Tara; Wu, Meng-Fen; Liu, Hao; Hicks, John; Rainusso, Nino; Dotti, Gianpietro; Mei, Zhuyong; Grilley, Bambi; Gee, Adrian; Rooney, Cliona M.; Brenner, Malcolm K.; Heslop, Helen E.; Wels, Winfried S.; Wang, Lisa L.; Anderson, Peter; Gottschalk, Stephen

    2015-01-01

    Purpose The outcome for patients with metastatic or recurrent sarcoma remains poor. Adoptive therapy with tumor-directed T cells is an attractive therapeutic option but has never been evaluated in sarcoma. Patients and Methods We conducted a phase I/II clinical study in which patients with recurrent/refractory human epidermal growth factor receptor 2 (HER2) –positive sarcoma received escalating doses (1 × 104/m2 to 1 × 108/m2) of T cells expressing an HER2-specific chimeric antigen receptor with a CD28.ζ signaling domain (HER2-CAR T cells). Results We enrolled 19 patients with HER2-positive tumors (16 osteosarcomas, one Ewing sarcoma, one primitive neuroectodermal tumor, and one desmoplastic small round cell tumor). HER2-CAR T-cell infusions were well tolerated with no dose-limiting toxicity. At dose level 3 (1 × 105/m2) and above, we detected HER2-CAR T cells 3 hours after infusion by quantitative polymerase chain reaction in 14 of 16 patients. HER2-CAR T cells persisted for at least 6 weeks in seven of the nine evaluable patients who received greater than 1 × 106/m2 HER2-CAR T cells (P = .005). HER2-CAR T cells were detected at tumor sites of two of two patients examined. Of 17 evaluable patients, four had stable disease for 12 weeks to 14 months. Three of these patients had their tumor removed, with one showing ≥ 90% necrosis. The median overall survival of all 19 infused patients was 10.3 months (range, 5.1 to 29.1 months). Conclusion This first evaluation of the safety and efficacy of HER2-CAR T cells in patients with cancer shows the cells can persist for 6 weeks without evident toxicities, setting the stage for studies that combine HER2-CAR T cells with other immunomodulatory approaches to enhance their expansion and persistence. PMID:25800760

  18. Dactylone inhibits epidermal growth factor-induced transformation and phenotype expression of human cancer cells and induces G1-S arrest and apoptosis.

    PubMed

    Fedorov, Sergey N; Shubina, Larisa K; Bode, Ann M; Stonik, Valentin A; Dong, Zigang

    2007-06-15

    The marine natural chamigrane-type sesquiterpenoid, dactylone, is closely related to secondary metabolites of some edible species of red algae. In the present study, the effect of dactylone was tested on the mouse skin epidermal JB6 P+ Cl41 cell line and its stable transfectants as well as on several human tumor cell lines, including lung (H460), colon (HCT-116), and skin melanomas (SK-MEL-5 and SK-MEL-28). This natural product was effective at nontoxic doses as a cancer-preventive agent, which exerted its actions, at least in part, through the inhibition of cyclin D3 and Cdk4 expression and retinoblastoma tumor suppressor protein (Rb) phosphorylation. The inhibition of these cell cycle components was followed by cell cycle arrest at the G1-S transition with subsequent p53-independent apoptosis. Therefore, these data showed that application of dactylone and related compounds may lead to decreased malignant cell transformation and/or decreased tumor cell proliferation. PMID:17575161