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By admin, on February 21st, 2012
Prostate Cancer Prostatic Dis. 2012 Feb 14;
Price RS, Cavazos DA, De Angel RE, Hursting SD, Degraffenried LA
Background:Obesity is associated with larger tumors, shorter time to PSA failure, and higher Gleason scores. However, the mechanism(s) by which obesity promotes aggressive prostate cancer remains unknown. We hypothesize that circulating factors related to obesity promote prostate cancer progression by modulating components of the metastatic cascade.Methods:Male C57BL/6 mice (6 weeks) were fed an ad libitum diet-induced obesity (60% fat) or control diet (10% fat) for 12 weeks. Serum was collected, metabolic and inflammatory proteins were measured by an antibody array. Sera were used to measure, in vitro, characteristics of a metastatic phenotype.Results:Comparable to obese men, obese sera contained higher levels or leptin, vascular endothelial growth factor, PAI-1, interleukin-6 (IL-6) and lower levels of testosterone. In prostate cells, serum was used to assess: proliferation, invasion, migration, epithelial-mesenchymal-transition (EMT) and matrix metalloproteinase (MMP) activity. LNCaP and PacMetUT1 cells exposed to obese sera increased proliferation, whereas PrEC and DU145 were unaffected. LNCaP, PacMetUT1 and DU145 cancer cells exposed to obese sera resulted in increased invasion, migration and MMP-9 activity. Prostate cancer cells exposed to obese sera showed increased vimentin, dispersion of e-cadherin and β-catenin from the plasma membrane.Conclusion:We report, prostate cancer cells exposed to sera from obese mice increases proliferation, invasion, migration, MMP activity and induces changes in proteins critical for EMT.Prostate Cancer and Prostatic Diseases advance online publication, 14 February 2012; doi:10.1038/pcan.2011.54.
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Obesity-related systemic factors promote an invasive phenotype in prostate cancer cells.
By admin, on February 21st, 2012
Prostate Cancer Prostatic Dis. 2012 Feb 14;
Price RS, Cavazos DA, De Angel RE, Hursting SD, Degraffenried LA
Background:Obesity is associated with larger tumors, shorter time to PSA failure, and higher Gleason scores. However, the mechanism(s) by which obesity promotes aggressive prostate cancer remains unknown. We hypothesize that circulating factors related to obesity promote prostate cancer progression by modulating components of the metastatic cascade.Methods:Male C57BL/6 mice (6 weeks) were fed an ad libitum diet-induced obesity (60% fat) or control diet (10% fat) for 12 weeks. Serum was collected, metabolic and inflammatory proteins were measured by an antibody array. Sera were used to measure, in vitro, characteristics of a metastatic phenotype.Results:Comparable to obese men, obese sera contained higher levels or leptin, vascular endothelial growth factor, PAI-1, interleukin-6 (IL-6) and lower levels of testosterone. In prostate cells, serum was used to assess: proliferation, invasion, migration, epithelial-mesenchymal-transition (EMT) and matrix metalloproteinase (MMP) activity. LNCaP and PacMetUT1 cells exposed to obese sera increased proliferation, whereas PrEC and DU145 were unaffected. LNCaP, PacMetUT1 and DU145 cancer cells exposed to obese sera resulted in increased invasion, migration and MMP-9 activity. Prostate cancer cells exposed to obese sera showed increased vimentin, dispersion of e-cadherin and β-catenin from the plasma membrane.Conclusion:We report, prostate cancer cells exposed to sera from obese mice increases proliferation, invasion, migration, MMP activity and induces changes in proteins critical for EMT.Prostate Cancer and Prostatic Diseases advance online publication, 14 February 2012; doi:10.1038/pcan.2011.54.
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Obesity-related systemic factors promote an invasive phenotype in prostate cancer cells.
By admin, on February 19th, 2012
Biochim Biophys Acta. 2012 Feb 7;
Weisová P, Anilkumar U, Ryan C, Concannon CG, Prehn JH, Ward MW
The preconditioning response conferred by a mild uncoupling of the mitochondrial membrane potential (Δψ(m)) has been attributed to altered reactive oxygen species (ROS) production and mitochondrial Ca(2+) uptake within the cells. Here we have explored if altered cellular energetics in response to a mild mitochondrial uncoupling stimulus may also contribute to the protection. The addition of 100nM FCCP for 30min to cerebellar granule neurons (CGNs) induced a transient depolarization of the Δψ(m), that was sufficient to significantly reduce CGN vulnerability to the excitotoxic stimulus, glutamate. On investigation, the mild mitochondrial 'uncoupling' stimulus resulted in a significant increase in the plasma membrane levels of the glucose transporter isoform 3, with a hyperpolarisation of Δψ(m) and increased cellular ATP levels also evident following the washout of FCCP. Furthermore, the phosphorylation state of AMP-activated protein kinase (AMPK) (Thr 172) was increased within 5min of the uncoupling stimulus and elevated up to 1h after washout. Significantly, the physiological changes and protection evident after the mild uncoupling stimulus were lost in CGNs when AMPK activity was inhibited. This study identifies an additional mechanism through which protection is mediated upon mild mitochondrial uncoupling: it implicates increased AMPK signalling and an adaptive shift in energy metabolism as mediators of the preconditioning response associated with FCCP-induced mild mitochondrial uncoupling.
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‘Mild mitochondrial uncoupling’ induced protection against glutamate excitotoxicity in primary neurons requires AMPK activity.
By admin, on February 18th, 2012
Exp Cell Res . 2012 Feb 3; Robinson AJ, Kunji ER, Gross A Recent studies report mitochondrial carrier homolog 2 (MTCH2) as a novel and uncharacterized protein that acts as a receptor-like protein for the truncated BH3-interacting domain death agonist (tBID) protein in the outer membrane of mitochondria. These studies using mouse embryonic stem cells and fibroblasts, as well as mice with a conditional knockout of MTCH2 in the liver showed that deletion of MTCH2 hindered recruitment of tBID to the mitochondria with subsequent reductions in the activation of pro-apoptotic proteins, mitochondrial outer membrane permeabilization and apoptosis.
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Mitochondrial carrier homolog 2 (MTCH2): The recruitment and evolution of a mitochondrial carrier protein to a critical player in apoptosis.
By admin, on February 18th, 2012
Exp Cell Res . 2012 Feb 3; Robinson AJ, Kunji ER, Gross A Recent studies report mitochondrial carrier homolog 2 (MTCH2) as a novel and uncharacterized protein that acts as a receptor-like protein for the truncated BH3-interacting domain death agonist (tBID) protein in the outer membrane of mitochondria. These studies using mouse embryonic stem cells and fibroblasts, as well as mice with a conditional knockout of MTCH2 in the liver showed that deletion of MTCH2 hindered recruitment of tBID to the mitochondria with subsequent reductions in the activation of pro-apoptotic proteins, mitochondrial outer membrane permeabilization and apoptosis
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Mitochondrial carrier homolog 2 (MTCH2): The recruitment and evolution of a mitochondrial carrier protein to a critical player in apoptosis.
By admin, on February 16th, 2012
Mol Cell Biol. 2012 Feb 13;
Abe M, Makino A, Hullin-Matsuda F, Kamijo K, Ohno-Iwashita Y, Hanada K, Mizuno H, Miyawaki A, Kobayashi T
Cytokinesis is a crucial step towards the creation of two daughter cells by the formation and ingression of the cleavage furrow. Here we show that sphingomyelin (SM), one of the major sphingolipids in mammalian cells, is required for the localization of PIP(2) to the cleavage furrow during cytokinesis. Real time observation with a labeled SM-specific protein, lysenin, revealed that SM is concentrated in the outer leaflet of the furrow at the time of cytokinesis. Super-resolution fluorescence microscopy analysis indicates a transbilayer co-localization between the SM-rich domains in the outer leaflet and PIP(2)-rich domains in the inner leaflet of the plasma membrane. Depletion of SM disperses PIP(2) and inhibits the recruitment of the small GTPase RhoA to the cleavage furrow, leading to abnormal cytokinesis. These results suggest that the formation of SM-rich domains is required for the accumulation of PIP(2) to the cleavage furrow, which is a prerequisite for the proper translocation of RhoA and the progression of cytokinesis.
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A Role for Sphingomyelin-rich Lipid Domains in the Accumulation of PIP2 to the Cleavage Furrow during Cytokinesis.
By admin, on February 11th, 2012
J Immunol. 2012 Feb 8;
Lin Q, Liu Y, Moore DJ, Elizer SK, Veach RA, Hawiger J, Ruley HE
The expression of proinflammatory cytokines and chemokines in response to TCR agonists is regulated by the caspase-recruitment domain membrane-associated guanylate kinase 1 (CARMA1) signalosome through the coordinated assembly of complexes containing the BCL10 adaptor protein. We describe a novel mechanism to negatively regulate the CARMA1 signalosome by the “death” adaptor protein caspase and receptor interacting protein adaptor with death domain (CRADD)/receptor interacting protein-associated ICH-1/CED-3 homologous protein with a death domain. We show that CRADD interacts with BCL10 through its caspase recruitment domain and suppresses interactions between BCL10 and CARMA1. TCR agonist-induced interaction between CRADD and BCL10 coincides with reduction of its complex formation with CARMA1 in wild-type, as compared with Cradd-deficient, primary cells. Finally, Cradd-deficient spleen cells, CD4(+) T cells, and mice respond to T cell agonists with strikingly higher production of proinflammatory mediators, including IFN-γ, IL-2, TNF-α, and IL-17. These results define a novel role for CRADD as a negative regulator of the CARMA1 signalosome and suppressor of Th1- and Th17-mediated inflammatory responses.
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Cutting Edge: The "Death" Adaptor CRADD/RAIDD Targets BCL10 and Suppresses Agonist-Induced Cytokine Expression in T Lymphocytes.
By admin, on February 11th, 2012
J Immunol. 2012 Feb 8;
Lin Q, Liu Y, Moore DJ, Elizer SK, Veach RA, Hawiger J, Ruley HE
The expression of proinflammatory cytokines and chemokines in response to TCR agonists is regulated by the caspase-recruitment domain membrane-associated guanylate kinase 1 (CARMA1) signalosome through the coordinated assembly of complexes containing the BCL10 adaptor protein. We describe a novel mechanism to negatively regulate the CARMA1 signalosome by the “death” adaptor protein caspase and receptor interacting protein adaptor with death domain (CRADD)/receptor interacting protein-associated ICH-1/CED-3 homologous protein with a death domain. We show that CRADD interacts with BCL10 through its caspase recruitment domain and suppresses interactions between BCL10 and CARMA1. TCR agonist-induced interaction between CRADD and BCL10 coincides with reduction of its complex formation with CARMA1 in wild-type, as compared with Cradd-deficient, primary cells. Finally, Cradd-deficient spleen cells, CD4(+) T cells, and mice respond to T cell agonists with strikingly higher production of proinflammatory mediators, including IFN-γ, IL-2, TNF-α, and IL-17. These results define a novel role for CRADD as a negative regulator of the CARMA1 signalosome and suppressor of Th1- and Th17-mediated inflammatory responses.
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Cutting Edge: The "Death" Adaptor CRADD/RAIDD Targets BCL10 and Suppresses Agonist-Induced Cytokine Expression in T Lymphocytes.
By admin, on February 10th, 2012
FEBS J. 2012 Feb 6;
Schmidt EM, Gu S, Anagnostopoulou V, Alevizopoulos K, Föller M, Lang F, Stournaras C
The serum- and glucocorticoid-dependent kinases-1-3 (SGK1-3) are downstream effectors of PI3 kinases, implicated in various cell responses including colon cancer tumorigenesis in mice. Here we investigated the role of SGK1 in the regulation of cell motility. Using Caco-2 colon tumor and Hek 293 embryonic kidney cells we report that transfection with the constitutively active SGK1 mutant (SGK1-SD) significantly enhanced cell motility. The cell adhesion protein vinculin was effectively de-phosphorylated in SGK1-SD-transfected cells. Treatment of the cells with phosphatase inhibitors restored vinculin phosphorylation and inhibited cell migration, indicating a significant role of vinculin phosphorylation in SGK1-induced motility. SGK1-SD-enhanced cell motility was inhibited by activation of membrane androgen binding sites (mAR) via testosterone-conjugates in both cell lines, while iAR-silencing and flutamide treatment revealed that these effects were clearly independent of the interaction of SGK1 with the classical androgen receptors (iAR). More importantly, mAR activation restored vinculin phosphorylation in SGK1-SD-transfected cells, while silencing of vinculin fully reversed the mAR-induced inhibition of the migratory capacity, implying that this protein is directly involved in cell motility regulation by SGK1 and mAR. The present report indicates for the first time that SGK1 regulates cell migration via vinculin de-phosphorylation, a mechanism that is controlled by mAR function.
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Serum- and Glucocorticoid-dependent Kinase-1-induced cell migration is dependent on vinculin and regulated by membrane Androgen Receptor.
By admin, on February 10th, 2012
Proc Natl Acad Sci U S A. 2012 Feb 6;
Nabhan JF, Hu R, Oh RS, Cohen SN, Lu Q
Mammalian cells are capable of delivering multiple types of membrane capsules extracellularly. The limiting membrane of late endosomes can fuse with the plasma membrane, leading to the extracellular release of multivesicular bodies (MVBs), initially contained within the endosomes, as exosomes. Budding viruses exploit the TSG101 protein and endosomal sorting complex required for transport (ESCRT) machinery used for MVB formation to mediate the egress of viral particles from host cells. Here we report the discovery of a virus-independent cellular process that generates microvesicles that are distinct from exosomes and which, like budding viruses, are produced by direct plasma membrane budding. Such budding is driven by a specific interaction of TSG101 with a tetrapeptide PSAP motif of an accessory protein, arrestin domain-containing protein 1 (ARRDC1), which we show is localized to the plasma membrane through its arrestin domain. This interaction results in relocation of TSG101 from endosomes to the plasma membrane and mediates the release of microvesicles that contain TSG101, ARRDC1, and other cellular proteins. Unlike exosomes, which are derived from MVBs, ARRDC1-mediated microvesicles (ARMMs) lack known late endosomal markers. ARMMs formation requires VPS4 ATPase and is enhanced by the E3 ligase WWP2, which interacts with and ubiquitinates ARRDC1. ARRDC1 protein discharged into ARMMs was observed in co-cultured cells, suggesting a role for ARMMs in intercellular communication. Our findings reveal an intrinsic cellular mechanism that results in direct budding of microvesicles from the plasma membrane, providing a formal paradigm for the evolutionary recruitment of ESCRT proteins in the release of budding viruses.
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Formation and release of arrestin domain-containing protein 1-mediated microvesicles (ARMMs) at plasma membrane by recruitment of TSG101 protein.
By admin, on February 9th, 2012
By admin, on February 9th, 2012
By admin, on February 9th, 2012
By admin, on February 9th, 2012
By admin, on February 8th, 2012
Anal Chim Acta. 2012 Mar 2; 717: 67-76
Ji C, Tschantz WR, Pfeifer ND, Ullah M, Sadagopan N
OATP1B1, OATP1B3 and OATP2B1 are important members of the organic anion transporting polypeptides (OATP) family and are implicated in the hepatic disposition of endobiotics and xenobiotics. Quantitating the expression levels of human OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissue samples could significantly improve attempts to scale up in vitro data and result in more effective in vitro-in vivo correlation of transporter-mediated effects on drug disposition, such as hepatic clearance. In the present study, a quantification method was developed, characterized, and implemented for simultaneous determination of human OATP1B1, OATP1B3 and OATP2B1 in HEK cells transfected with OATP-expressing plasmid vectors (SLCO1B1, SLCO1B3, and SLCO2B1, respectively), human hepatocytes, human brain capillary endothelial cells, and humanized mouse liver tissue using UPLC-MRM MS. Purified membrane protein standards prepared and characterized as previously reported (Protein Expr. Purif. 2008, 57, 163-71) were first used as standards for absolute quantification of the expression levels of the three human OATP membrane proteins. The specificity of the optimized MRM transitions were characterized by analyzing the tryptic digests of the membrane protein fraction of wild type HEK cells and control mouse liver tissue using the herein reported UPLC-MRM MS method. The linearity of the calibration curve spanned from 0.2μgmL(-1) (0.040μgmg(-1)) to 20μgmL(-1) (4.0μgmg(-1)), with accuracy (% RE) within 15% at all concentrations examined for all three OATPs of interest in this study. The intra- and inter-day assay accuracy (% RE) and coefficient of variations (% CV) of triplicates are all within 15% for all levels of quality control samples prepared by mixing the membrane fraction of control mouse liver tissue with the required amount of purified human OATP1B1, OATP1B3 and OATP2B1.
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Development of a multiplex UPLC-MRM MS method for quantification of human membrane transport proteins OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissues.
By admin, on February 8th, 2012
Anal Chim Acta. 2012 Mar 2; 717: 67-76
Ji C, Tschantz WR, Pfeifer ND, Ullah M, Sadagopan N
OATP1B1, OATP1B3 and OATP2B1 are important members of the organic anion transporting polypeptides (OATP) family and are implicated in the hepatic disposition of endobiotics and xenobiotics. Quantitating the expression levels of human OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissue samples could significantly improve attempts to scale up in vitro data and result in more effective in vitro-in vivo correlation of transporter-mediated effects on drug disposition, such as hepatic clearance. In the present study, a quantification method was developed, characterized, and implemented for simultaneous determination of human OATP1B1, OATP1B3 and OATP2B1 in HEK cells transfected with OATP-expressing plasmid vectors (SLCO1B1, SLCO1B3, and SLCO2B1, respectively), human hepatocytes, human brain capillary endothelial cells, and humanized mouse liver tissue using UPLC-MRM MS. Purified membrane protein standards prepared and characterized as previously reported (Protein Expr. Purif. 2008, 57, 163-71) were first used as standards for absolute quantification of the expression levels of the three human OATP membrane proteins. The specificity of the optimized MRM transitions were characterized by analyzing the tryptic digests of the membrane protein fraction of wild type HEK cells and control mouse liver tissue using the herein reported UPLC-MRM MS method. The linearity of the calibration curve spanned from 0.2μgmL(-1) (0.040μgmg(-1)) to 20μgmL(-1) (4.0μgmg(-1)), with accuracy (% RE) within 15% at all concentrations examined for all three OATPs of interest in this study. The intra- and inter-day assay accuracy (% RE) and coefficient of variations (% CV) of triplicates are all within 15% for all levels of quality control samples prepared by mixing the membrane fraction of control mouse liver tissue with the required amount of purified human OATP1B1, OATP1B3 and OATP2B1.
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Development of a multiplex UPLC-MRM MS method for quantification of human membrane transport proteins OATP1B1, OATP1B3 and OATP2B1 in in vitro systems and tissues.
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