Explore the vast range of titles available.

Sweat glands play an important role in thermoregulation via sweating, and protect human vitals. The reduction in sweating may increase the incidence of hyperthermia. Myoepithelial cells in sweat glands exhibit stemness characteristics and play a major role in sweat gland homeostasis and sweating processes. Previously, we successfully passaged primary myoepithelial cells in spheroid culture systems; however, they could not be maintained for long under in vitro conditions. No myoepithelial cell line has been established to date. In this study, we transduced two immortalizing genes into primary myoepithelial cells and developed a myoepithelial cell line. When compared with primary sweat gland cells, the immortalized myoepithelial cells (designated \"iEM\") continued to form spheroids after the 4th passage and expressed α-smooth muscle actin and other proteins that characterize myoepithelial cells. Furthermore, treatment with small compounds targeting the Wnt signaling pathways induced differentiation of iEM cells into luminal cells. Thus, we successfully developed an immortalized myoepithelial cell line having differentiation potential. As animal models are not useful for studying human sweat glands, our cell line will be helpful for studying the mechanisms underlying the pathophysiology of sweating disorders.

Metformin, a first-line diabetes drug linked to cancer prevention in retrospective clinical analyses, inhibits cellular transformation and selectively kills breast cancer stem cells (CSCs). Although a few metabolic effects of metformin and the related biguanide phenformin have been investigated in established cancer cell lines, the global metabolic impact of biguanides during the process of neoplastic transformation and in CSCs is unknown. Here, we use LC/MS/MS metabolomics (>200 metabolites) to assess metabolic changes induced by metformin and phenformin in an Src-inducible model of cellular transformation and in mammosphere-derived breast CSCs. Although phenformin is the more potent biguanide in both systems, the metabolic profiles of these drugs are remarkably similar, although not identical. During the process of cellular transformation, biguanide treatment prevents the boost in glycolytic intermediates at a specific stage of the pathway and coordinately decreases tricarboxylic acid (TCA) cycle intermediates. In contrast, in breast CSCs, biguanides have a modest effect on glycolytic and TCA cycle intermediates, but they strongly deplete nucleotide triphosphates and may impede nucleotide synthesis. These metabolic profiles are consistent with the idea that biguanides inhibit mitochondrial complex 1, but they indicate that their metabolic effects differ depending on the stage of cellular transformation.

Diabetic patients exhibit a reduction in β cells, which secrete insulin to help regulate glucose homeostasis; however, little is known about the factors that regulate proliferation of these cells in human pancreas. Access to primary human β cells is limited and a challenge for both functional studies and drug discovery progress. We previously reported the generation of a human β cell line (EndoC-βH1) that was generated from human fetal pancreas by targeted oncogenesis followed by in vivo cell differentiation in mice. EndoC-βH1 cells display many functional properties of adult β cells, including expression of β cell markers and insulin secretion following glucose stimulation; however, unlike primary β cells, EndoC-βH1 cells continuously proliferate. Here, we devised a strategy to generate conditionally immortalized human β cell lines based on Cre-mediated excision of the immortalizing transgenes. The resulting cell line (EndoC-βH2) could be massively amplified in vitro. After expansion, transgenes were efficiently excised upon Cre expression, leading to an arrest of cell proliferation and pronounced enhancement of β cell-specific features such as insulin expression, content, and secretion. Our data indicate that excised EndoC-βH2 cells are highly representative of human β cells and should be a valuable tool for further analysis of human β cells.

Stromal cells with a myofibroblast phenotype present in the normal human esophagus are increased in individuals with gastro-esophageal reflux disease (GERD). We have previously demonstrated that myofibroblasts stimulated with acid and TLR4 agonists increase IL-6 and IL-8 secretion using primary cultures of myofibroblasts established from normal human esophagus. While primary cultures have the advantage of reflecting the in vivo environment, a short life span and unavoidable heterogeneity limits the usefulness of this model in larger scale in vitro cellular signaling studies. The major aim of this paper therefore was to generate a human esophageal myofibroblast line with an extended lifespan. In the work presented here we have generated and characterized an immortalized human esophageal myofibroblast line by transfection with a commercially available GFP-hTERT lentivirus. Immortalized human esophageal myofibroblasts demonstrate phenotypic, genotypic and functional similarity to primary cultures of esophageal myofibroblasts we have previously described. We found that immortalized esophageal myofibroblasts retain myofibroblast spindle-shaped morphology at low and high confluence beyond passage 80, and express α-SMA, vimentin, and CD90 myofibroblast markers. Immortalized human esophageal myofibroblasts also express the putative acid receptor TRPV1 and TLR4 and retain the functional capacity to respond to stimuli encountered in GERD with secretion of IL-6. Finally, immortalized human esophageal myofibroblasts also support the stratified growth of squamous esophageal epithelial cells in 3D organotypic cultures. This newly characterized immortalized human esophageal myofibroblast cell line can be used in future cellular signaling and co-culture studies.

The recepteur d'origine nantais (RON) is a receptor tyrosine kinase (RTK) in the scatter factor family, which includes the c-Met receptor. RON exhibits increased expression in a significant number of human breast cancer tissues as well as in many established breast cancer cell lines. Recent studies have indicated that in addition to ligand-dependent signaling events, RON also promotes signals in the absence of its only known ligand, MSP, when expressed in epithelial cells. In this study, we found that when expressed in MCF-10A breast epithelial cells, RON exhibits both MSP-dependent and MSP-independent signaling, which lead to distinct biological outcomes. In the absence of MSP, RON signaling promotes cell survival, increased cell spreading and enhanced migration in response to other growth factors. However, both RON-mediated proliferation and migration require the addition of MSP in MCF-10A cells. Both MSP-dependent and MSP-independent signaling by RON are mediated in part by Src family kinases. These data suggest that RON has two alternative modes of signaling that can contribute to oncogenic behavior in normal breast epithelial cells.

T cells transformed by Herpesvirus saimiri express seven viral U-rich noncoding RNAs of unknown function called HSURs. We noted that conserved sequences in HSURs 1 and 2 constitute potential binding sites for three host-cell microRNAs (miRNAs). Coimmunoprecipitation experiments confirmed that HSURs 1 and 2 interact with the predicted miRNAs in virally transformed T cells. The abundance of one of these miRNAs, miR-27, is dramatically lowered in transformed cells, with consequent effects on the expression of miR-27 target genes. Transient knockdown and ectopic expression of HSUR 1 demonstrate that it directs degradation of mature miR-27 in a sequence-specific and binding-dependent manner. This viral strategy illustrates use of a ncRNA to manipulate host-cell gene expression via the miRNA pathway.

In vitro human cell line models have been widely used for cancer pharmacogenomic studies to predict clinical response, to help generate pharmacogenomic hypothesis for further testing, and to help identify novel mechanisms associated with variation in drug response. Among cell line model systems, immortalized cell lines such as Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines (LCLs) have been used most often to test the effect of germline genetic variation on drug efficacy and toxicity. Another model, especially in cancer research, uses cancer cell lines such as the NCI-60 panel. These models have been used mainly to determine the effect of somatic alterations on response to anticancer therapy. Even though these cell line model systems are very useful for initial screening, results from integrated analyses of multiple omics data and drug response phenotypes using cell line model systems still need to be confirmed by functional validation and mechanistic studies, as well as validation studies using clinical samples. Future models might include the use of patient-specific inducible pluripotent stem cells and the incorporation of 3D culture which could further optimize in vitro cell line models to improve their predictive validity.

The periodontal ligament (PDL) is a highly specialized tissue connecting the cementum with the tooth socket bone and affects the life span of the tooth. However, little is known about the precise characteristics and regenerative mechanism of PDL cells because of the absence of specific markers and cell lines. Therefore, we aimed to establish three immortalized human PDL fibroblast cell lines by using simian virus40 T-antigen (SV40T-Ag) and human telomerase reverse transcriptase (hTERT) transfection, expecting these cells to have the characteristics of primary cells. The transfected cells were named STPLF. The expression of SV40T-Ag and hTERT in all STPLF lines was verified by using the semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) method, stretch PCR analysis, or Western blotting analysis. All STPLF showed stable proliferation at more than 120 population doublings (PD), whereas primary human PDL fibroblasts (HPLF) stopped at 10-20 PD. Characterization by RT-PCR analysis revealed that all STPLF genes mimicked the expression of their respective original HPLF genes. STPLF expressed runt-related transcription factor-2, osterix, alkaline phosphatase, osteopontin, osteocalcin, periostin, receptor activator of NF-kappa B ligand, osteoprotegerin, epidermal growth factor receptor, alpha-smooth muscle actin, and type XII collagen. STPLF stimulated with 50 micro g/ml ascorbic acid and 2 mM beta-glycerophosphate for 4 weeks produced more calcified deposits than did HPLF cultured with the same reagents. These results suggest that each STPLF line retained the characteristics of the respective original HPLF, that STPLF gained increased calcification activity, and that STPLF are helpful tools for studying the biology and regenerative mechanisms of human PDL.

Mammalian target of rapamycin (mTOR) complex 1 (mTORC1) is an important, highly conserved, regulator of cell growth. Ancient among the signals that regulate mTORC1 are nutrients. Amino acids direct mTORC1 to the surface of the late endosome/lysosome, where mTORC1 becomes receptive to other inputs. However, the interplay between endosomes and mTORC1 is poorly understood. Here, we report the discovery of a network that links mTORC1 to a critical component of the late endosome/lysosome, the V‐ATPase. In an unbiased screen, we found that mTORC1 regulated the expression of, among other lysosomal genes, the V‐ATPases. mTORC1 regulates V‐ATPase expression both in cells and in mice. V‐ATPase regulation by mTORC1 involves a transcription factor translocated in renal cancer, TFEB. TFEB is required for the expression of a large subset of mTORC1 responsive genes. mTORC1 coordinately regulates TFEB phosphorylation and nuclear localization and in a manner dependent on both TFEB and V‐ATPases, mTORC1 promotes endocytosis. These data uncover a regulatory network linking an oncogenic transcription factor that is a master regulator of lysosomal biogenesis, TFEB, to mTORC1 and endocytosis. TORC1 is a key regulator of cell growth in response to nutrients and acts at the surface of the late endosome. This study identifies V‐ATPase genes as transcriptional targets of TORC1 and implicates the transcription factor TFEB as an important mediator of TORC1‐dependent gene expression and TORC1‐regulated endocytosis.

Purpose To evaluate the inflammatory response and barrier formation of a new alveolar type 1-like (transformed type I; TT1) cell line to establish its suitability for toxicity and drug transport studies. Methods TT1 and A549 cells were challenged with lipopolysaccharide (LPS). Secretion of inflammatory mediators was quantified by ELISA. The barrier properties of TT1 cells were evaluated by transepithelial electrical resistance (TEER), fluorescein sodium (flu-Na) apparent permeability (P app) and staining of zona occludens-1 (ZO-1). Results LPS stimulated similar levels of secretion of IL-6 and IL-8 in TT1 and A549 cells. TNF-α was not produced by either cell line. In contrast to A549 cells, TT1 cells did not secrete SLPI or elafin. TT1 cells produced maximal TEER of ~55 Ω cm² and flu-Na P app of ~6.0 x 10⁻⁶ cm/s. ZO-1 staining was weak and discontinuous. Attempts to optimise culture conditions did not increase the barrier properties of the TT1 cell layers. Conclusions The TT1 cell line models the alveolar inflammatory response to LPS challenge and provides a valuable complement to cell lines currently used in toxicity assays. However, under the experimental conditions used the TT1 cell line did not form the highly restrictive tight junctions which exist in vivo.