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Primary Human Fibroblasts (primary + human_fibroblast)
Selected AbstractsNicotine inhibits human gingival fibroblast migration via modulation of Rac signalling pathwaysJOURNAL OF CLINICAL PERIODONTOLOGY, Issue 12 2005Yiyu Fang Abstract Aim: Cigarette smoking is a risk factor in the development of periodontal diseases. In addition, a delayed healing process has been shown in smokers compared with non-smokers after periodontal treatment. Cell migration is a key process of wound healing and it is highly regulated by a variety of signalling pathways. The small G protein, Rac, is necessary for cell migration. Our aim was to determine if nicotine disrupted Rac and its downstream signalling proteins, p21-activated kinase 1/2 (PAK1/2), and p44/42 mitogen-activated protein kinase (MAPK) (extracellular regulated kinase 1/2). Material and Methods: Primary human fibroblasts from healthy gingival tissues were cultured and grown to confluence. Cells were serum starved for 24 h, and then treated with nicotine (0 or 0.5 ,M) prior to in vitro wounding. Cell migration was analysed in live cell assays following in vitro wounds. Rac activity, phosphorylation levels of PAK1/2, and p44/42 MAPK were assessed in cultures treated with or without nicotine after multiple wounds. Results: Nicotine decreased cell migration rates by 50% compared with controls. In addition, nicotine altered the activation patterns of Rac and PAK 1/2 and up-regulated p44/42 MAPK. Conclusion: Decreased cell migration in periodontal wounds exposed to nicotine may be mediated through the Rac and PAK1/2 signalling pathways. [source] Cell cycle-related variation in subcellular localization of eIF3e/INT6 in human fibroblastsCELL PROLIFERATION, Issue 2 2004S. J. Watkins In addition, the protein can interact with two other multi-subunit complexes: the COP9 signalosome (CSN) and the proteasome. The role of INT6 in tumourigenesis is nonetheless currently unclear. Here, using immunofluorescence microscopy, we show that eIF3e/INT6 is localized in part to the nucleus, while other eIF3 components are cytoplasmic. Primary human fibroblasts, but not their transformed counterparts, showed reduced nuclear INT6 staining in some cells, and this reduction was maximal in early S phase. This variation in eIF3e/INT6 may indicate regulated shuttling between cellular compartments and would be consistent with the presence of a nuclear export signal as well as a nuclear localization signal in the protein sequence. Loss of regulation of eIF3e/INT6 redistribution may therefore be a significant feature of malignancy in human cells. [source] Cdt1 and geminin are down-regulated upon cell cycle exit and are over-expressed in cancer-derived cell linesFEBS JOURNAL, Issue 16 2004Georgia Xouri Licensing origins for replication upon completion of mitosis ensures genomic stability in cycling cells. Cdt1 was recently discovered as an essential licensing factor, which is inhibited by geminin. Over-expression of Cdt1 was shown to predispose cells for malignant transformation. We show here that Cdt1 is down-regulated at both the protein and RNA level when primary human fibroblasts exit the cell cycle into G0, and its expression is induced as cells re-enter the cell cycle, prior to S phase onset. Cdt1's inhibitor, geminin, is similarly down-regulated upon cell cycle exit at both the protein and RNA level, and geminin protein accumulates with a 3,6 h delay over Cdt1, following serum re-addition. Similarly, mouse NIH3T3 cells down-regulate Cdt1 and geminin mRNA and protein when serum starved. Our data suggest a transcriptional control over Cdt1 and geminin at the transition from quiescence to proliferation. In situ hybridization and immunohistochemistry localize Cdt1 as well as geminin to the proliferative compartment of the developing mouse gut epithelium. Cdt1 and geminin levels were compared in primary cells vs. cancer-derived human cell lines. We show that Cdt1 is consistently over-expressed in cancer cell lines at both the protein and RNA level, and that the Cdt1 protein accumulates to higher levels in individual cancer cells. Geminin is similarly over-expressed in the majority of cancer cell lines tested. The relative ratios of Cdt1 and geminin differ significantly amongst cell lines. Our data establish that Cdt1 and geminin are regulated at cell cycle exit, and suggest that the mechanisms controlling Cdt1 and geminin levels may be altered in cancer cells. [source] Capturing Complex Protein Gradients on Biomimetic Hydrogels for Cell-Based AssaysADVANCED FUNCTIONAL MATERIALS, Issue 21 2009Steffen Cosson Abstract A versatile strategy to rapidly immobilize complex gradients of virtually any desired protein on soft poly(ethylene glycol) (PEG) hydrogel surfaces that are reminiscent of natural extracellular matrices (ECM) is reported. A microfluidic chip is used to generate steady-state gradients of biotinylated or Fc-tagged fusion proteins that are captured and bound to the surface in less than 5,min by NeutrAvidin or ProteinA, displayed on the surface. The selectivity and orthogonality of the binding schemes enables the formation of parallel and orthogonal overlapping gradients of multiple proteins, which is not possible on conventional cell culture substrates. After patterning, the hydrogels are released from the microfluidic chip and used for cell culture. This novel platform is validated by conducting single-cell migration experiments using time-lapse microscopy. The orientation of cell migration, as well as the migration rate of primary human fibroblasts, depends on the concentration of an immobilized fibronectin fragment. This technique can be readily applied to other proteins to address a wealth of biological questions with different cell types. [source] Two new lipoaminoacids with complementary modes of action: new prospects to fight out against skin agingINTERNATIONAL JOURNAL OF COSMETIC SCIENCE, Issue 1 2010S. Dumont Synopsis The mode of action of two cosmetic active ingredients (AIs), palmitoyl glycine (PG) and cocoyl alanine (CA) was studied with cDNA array experiments and quantitative PCR confirmations, which were performed on experimentally aged human fibroblasts. These preliminary studies revealed complementary profiles. Thus, specific supplementary investigations were then carried out for each AI. Protocols used were based either on in vitro models: (i) biochemical assays, (ii) monolayer cell culture (primary human fibroblasts and keratinocytes) and (iii) the model of capillary-like tube formation by human endothelial cells or on ex vivo models, i.e. topically treated skin explants and both immunohistochemical and ChromameterTM investigations. New prospects are proposed to fight out against skin aging. Indeed, PG and CA showed complementary properties and thus enabled a regulation or a restoration effect on main aging-associated disorders. Thus, they can not only act on tissue architecture, cell,cell interactions and extracellular matrix protection but also on inflammation, cell longevity, skin immune system protection, skin radiance and stem cell survey. Finally, a clinical trial performed on Caucasian women confirmed AI anti-wrinkle efficacy, which was superior to that of a market reference ingredient. In the future, complementary experiments enabling a better understanding of the aging-induced decline of epidermal stem cells would be of a great interest. Résumé Le mode d'action de deux actifs cosmétiques, Palmitoyl glycine (PG) et Cocoyl Alanine (CA), a été déterminéà l'aide d'expériences de cDNA arrays et de confirmations par qPCR, réalisées sur des fibroblastes humains vieillis expérimentalement. Ces études préliminaires ont révélé des modes d'action complémentaires. Des expériences supplémentaires spécifiques ont donc ensuite été réalisées pour chaque actif. Les protocoles utilisés étaient basés sur des modèles in vitro: i) études biochimiques, ii) cultures cellulaires en monocouches (cultures primaires de fibroblastes et de kératinocytes humains) et iii) modèle de formation de pseudo-tubules par des cellules endothéliales humaines; ou sur des modèles ex-vivo, i.e. des explants de peau traités de manière topique et analysés à l'aide d'études immuno-histochimiques et d'un ChromamètreTM. De nouvelles perspectives s'ouvrent pour combattre le vieillissement cutané. En effet, PG et CA montrent des propriétés complémentaires et permettent ainsi une régulation ou une restauration des principaux dysfonctionnements liés à l'âge. Ainsi, ils peuvent agir non-seulement sur l'architecture des tissus, l'interaction entre les cellules et la protection de la matrice extracellulaire mais aussi sur l'inflammation, la longévité cellulaire, la survie des cellules souches, le système de protection immunitaire et l'éclat de la peau. Finalement, des essais cliniques réalisés sur des femmes de type Caucasien ont confirmé l'efficacité antirides des actifs, laquelle était supérieure à celle d'une référence anti-âge du marché. Dans un futur proche, des tests complémentaires pourraient permettre une meilleure compréhension de la dégradation des cellules souches épidermiques au cours du vieillissement. [source] Nicotinamide enhances mitochondria quality through autophagy activation in human cellsAGING CELL, Issue 4 2009Hyun Tae Kang Summary Nicotinamide (NAM) treatment causes a decrease in mitochondrial respiration and reactive oxygen species production in primary human fibroblasts and extends their replicative lifespan. In the current study, it is reported that NAM treatment induces a decrease in mitochondrial mass and an increase in membrane potential (,,m) by accelerating autophagic degradation of mitochondria. In the NAM-treated cells, the level of LC3-II as well as the number of LC3 puncta and lysosomes co-localizing with mitochondria substantially increased. Furthermore, in the NAM-treated cells, the levels of Fis1, Drp1, and Mfn1, proteins that regulate mitochondrial fission and fusion, increased and mitochondria experienced dramatic changes in structure from filaments to dots or rings. This structural change is required for the decrease of mitochondrial mass indicating that NAM accelerates mitochondrial autophagy, at least in part, by inducing mitochondrial fragmentation. The decrease in mitochondria mass was attenuated by treatment with cyclosporine A, which prevents the loss of mitochondrial membrane potential by blocking the mitochondrial permeability transition, suggesting autophagic degradation selective for mitochondria with low ,,m. All these changes were accompanied by and dependent on an increase in the levels of GAPDH, and are blocked by inhibition of the cellular conversion of NAM to NAD+. Taken together with our previous findings, these results suggest that up-regulation of GAPDH activity may prolong healthy lifespan of human cells through autophagy-mediated mitochondria quality maintenance. [source] Tumorigenic study on hepatocytes coexpressing SV40 with RasMOLECULAR CARCINOGENESIS, Issue 4 2006Beicheng Sun Abstract A model of neoplastic transformation by the combination of SV40 large T antigen (LT), SV40 small T antigen (ST), oncogenic Ras, and human telomerase reverse trasncriptase subunit (hTERT) has become established and replicated in primary human fibroblasts, however, there is no report on human hepatocytes. Here we use cell transplantation model, and show that transplantation of human hepatocytes of HL-7702 and HL-7703 expressing Ha-RasV12 and SV40 LT into subrenal capsule of immunodeficient mice results in fully malignant tumors, in contrast to conventional subcutaneous injections where tumors fail to develop. In GM-847 cell study, we have found that hTERT is not required for tumorigenic growth in subrenal capsule transplantation, however, it is required in subcutaneous injection assay. These results demonstrate that Human hepatocytes can be transformed under kidney capsule by coexpressing SV40 LT and Ha-RasV12, neither hTERT nor protein phosphatase 2A (PP2A) inhibition are required for malignant transformation, a gene which increases cell survival in the subcutaneous injection model is not required for tumorigenic growth in subrenal capsule. © 2005 Wiley-Liss, Inc. [source] Induction of CCL13 expression in synovial fibroblasts highlights a significant role of oncostatin M in rheumatoid arthritisARTHRITIS & RHEUMATISM, Issue 7 2009Christoph Hintzen Objective To investigate the molecular mechanisms of CCL13/monocyte chemoattractant protein 4 (MCP-4) chemokine expression through proinflammatory cytokines in different primary human fibroblasts and the contribution of CCL13 to monocyte migration. Methods Using RNase protection assays and enzyme-linked immunosorbent assays, we quantified the expression of CCL13 compared with that of CCL2/MCP-1 in primary human fibroblasts. Boyden chamber assays were performed to determine the importance of CCL13 for migration of primary monocytes. Pharmacologic inhibitors as well as small interfering RNA knockdown approaches were used to investigate the signaling pathways regulating CCL13 expression. Results The interleukin-6 (IL-6),type cytokine oncostatin M (OSM) was a powerful inducer of CCL13 expression in primary synovial fibroblasts from patients with rheumatoid arthritis (RA) as well as those from healthy control subjects but not in other types of fibroblasts. Neither IL-6 nor tumor necrosis factor , could stimulate the expression of CCL13 in synovial fibroblasts; IL-1, was a very weak inducer. Synovial fibroblasts from patients with RA constitutively produced low amounts of CCL13, which was partially dependent on constitutive production of OSM. By investigating the underlying molecular mechanism, we identified STAT-5, ERK-1/2, and p38 as critical factors involved in OSM-dependent transcription and messenger RNA stabilization of CCL13. Conclusion In contrast to other prominent cytokines involved in the pathogenesis of RA, OSM can strongly up-regulate the expression of CCL13, a chemokine recently identified in the synovial fluid of patients with RA. Despite potent OSM-induced signal transduction in all types of fibroblasts analyzed, only synovial fibroblasts secreted CCL13, which might be indicative of tissue-specific imprinting of different fibroblasts during development. [source] | |||||||||||||