87 research outputs found
Identification and validation of genes with expression patterns inverse to multiple metastasis suppressor genes in breast cancer cell lines
Metastasis suppressor genes (MSGs) have contributed to an understanding of regulatory pathways unique to the lethal metastatic process. When re-expressed in experimental models, MSGs block cancer spread to, and colonization of distant sites without affecting primary tumor formation. Genes have been identified with expression patterns inverse to a single MSG, and found to encode functional, druggable signaling pathways. We now hypothesize that common signaling pathways mediate the effects of multiple MSGs. By gene expression profiling of human MCF7 breast carcinoma cells expressing a scrambled siRNA, or siRNAs to each of 19 validated MSGs (NME1, BRMS1, CD82, CDH1, CDH2, CDH11, CASP8, MAP2K4, MAP2K6, MAP2K7, MAPK14, GSN, ARHGDIB, AKAP12, DRG1, CD44, PEBP1, RRM1, KISS1), we identified genes whose expression was significantly opposite to at least five MSGs. Five genes were selected for further analysis: PDE5A, UGT1A, IL11RA, DNM3 and OAS1. After stable downregulation of each candidate gene in the aggressive human breast cancer cell line MDA-MB-231T, in vitro motility was significantly inhibited. Two stable clones downregulating PDE5A (phosphodiesterase 5A), an enzyme involved in the regulation of cGMP-specific signaling, exhibited no difference in cell proliferation, but reduced motility by 47 and 66 % compared to the empty vector-expressing cells (p = 0.01 and p = 0.005). In an experimental metastasis assay, two shPDE5A-MDA-MB-231T clones produced 47-62 % fewer lung metastases than shRNA-scramble expressing cells (p = 0.045 and p = 0.009 respectively). This study demonstrates that previously unrecognized genes are inversely related to the expression of multiple MSGs, contribute to aspects of metastasis, and may stand as novel therapeutic targets
Diet, physical activity, and adiposity in children in poor and rich neighbourhoods: a cross-sectional comparison
BACKGROUND: Obesity in Canadian children increased three-fold in twenty years. Children living in low-income neighborhoods exercise less and are more overweight than those living in more affluent neighborhoods after accounting for family socio-economic status. Strategies to prevent obesity in children have focused on personal habits, ignoring neighborhood characteristics. It is essential to evaluate diet and physical activity patterns in relation to socio-economic conditions to understand the determinants of obesity. The objective of this pilot study was to compare diet, physical activity, and the built environment in two Hamilton area elementary schools serving socio-economically different communities. METHODS: We conducted a cross-sectional study (November 2005-March 2006) in two public elementary schools in Hamilton, Ontario, School A and School B, located in low and high socioeconomic areas respectively. We assessed dietary intake, physical activity, dietary restraint, and anthropometric measures in consenting children in grades 1 and higher. From their parents we assessed family characteristics and walkability of the built environment. RESULTS: 160 children (n = 48, School A and n = 112, School B), and 156 parents (n = 43, School A and n = 113, School B) participated in this study. The parents with children at School A were less educated and had lower incomes than those at School B. The School A neighborhood was perceived to be less walkable than the School B neighborhood. Children at School A consumed more baked foods, chips, sodas, gelatin desserts, and candies and less low fat dairy, and dark bread than those at School B. Children at School A watched more television and spent more time in front of the computer than children studying at School B, but reported spending less time sitting on weekdays and weekends. Children at both schools were overweight but there was no difference in their mean BMI z-scores (School A = 0.65 versus School B = 0.81, p-value = 0.38). CONCLUSION: The determinants of overweight in children may be more complex than imagined. In future intervention programs researchers may consider addressing environmental factors, and customizing lifestyle interventions so that they are closer to community needs
Aboriginal birth cohort (ABC): a prospective cohort study of early life determinants of adiposity and associated risk factors among Aboriginal people in Canada
Nutraceutical therapies for atherosclerosis
Atherosclerosis is a chronic inflammatory disease affecting large and medium arteries and is considered to be a major underlying cause of cardiovascular disease (CVD). Although the development of pharmacotherapies to treat CVD has contributed to a decline in cardiac mortality in the past few decades, CVD is estimated to be the cause of one-third of deaths globally. Nutraceuticals are natural nutritional compounds that are beneficial for the prevention or treatment of disease and, therefore, are a possible therapeutic avenue for the treatment of atherosclerosis. The purpose of this Review is to highlight potential nutraceuticals for use as antiatherogenic therapies with evidence from in vitro and in vivo studies. Furthermore, the current evidence from observational and randomized clinical studies into the role of nutraceuticals in preventing atherosclerosis in humans will also be discussed
The Performance Implications of Timing of Entry and Involvement in Multipartner Alliances
Enigma prevents Cbl-c-mediated ubiquitination and degradation of RETMEN2A.
The Cbl proteins (Cbl, Cbl-b, and Cbl-c) are a highly conserved family of RING finger ubiquitin ligases (E3s) that function as negative regulators of tyrosine kinases in a wide variety of signal transduction pathways. In this study, we identify a new Cbl-c interacting protein, Enigma (PDLIM7). This interaction is specific to Cbl-c as Enigma fails to bind either of its closely related homologues, Cbl and Cbl-b. The binding between Enigma and Cbl-c is mediated through the LIM domains of Enigma as removal of all three LIM domains abrogates this interaction, while only LIM1 is sufficient for binding. Here we show that Cbl-c binds wild-type and MEN2A isoforms of the receptor tyrosine kinase, RET, and that Cbl-c enhances ubiquitination and degradation of activated RET. Enigma blocks Cbl-c-mediated RETMEN2A ubiquitination and degradation. Cbl-c decreased downstream ERK activation by RETMEN2A and co-expression of Enigma blocked the Cbl-c-mediated decrease in ERK activation. Enigma showed no detectable effect on Cbl-c-mediated ubiquitination of activated EGFR suggesting that this effect is specific to RET. Through mapping studies, we show that Cbl-c and Enigma bind RETMEN2A at different residues. However, binding of Enigma to RETMENA prevents Cbl-c recruitment to RETMEN2A. Consistent with these biochemical data, exploratory analyses of breast cancer patients with high expression of RET suggest that high expression of Cbl-c correlates with a good outcome, and high expression of Enigma correlates with a poor outcome. Together, these data demonstrate that Cbl-c can ubiquitinate and downregulate RETMEN2A and implicate Enigma as a positive regulator of RETMEN2A through blocking of Cbl-mediated ubiquitination and degradation
Enigma abrogates RETMEN2A ubiquitination.
<p>(<b>A</b>) RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates described above and immunoblotted for HA-ubiquitin and phosphotyrosine (pY) as indicated on the right. (<b>B</b>) HEK293T cells were transfected with GST-Cbl-c with and without FLAG-Enigma along with either RET9MEN2A or RET51MEN2A. Cells were collected 48h post-transfection, and whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates and immunoblotted for RET and HA-ubiquitin as indicated on the right. Molecular weight standards are indicated on the left and Ponceau S staining serves as a measure of protein loading. (<b>C</b>) HEK293T cells were transfected with RET9MEN2A with GST-Cbl-c and FLAG-Enigma both alone and in combination as indicated above the blots. All transfections were performed in triplicate. At 24h post-transfection, triplicate plates were pooled and replated. At 40h post-transfection, replicate plates were treated with 100ng/mL cycloheximide (CHX) for either 3 or 5h as indicated. Control plates received an equivalent volume of DMSO vehicle control for 5h prior to cell collection. A total of 20 µg of each of the whole cell lysates was immunoblotted as indicated. RET steady state levels were then assessed using β-actin as a loading control. (<b>D</b>) Densitometric analysis of RET steady state levels in the presence of GST-Cbl-c and Enigma, either alone or in combination. Levels were all compared to Vector transfected cells in the absence of cycloheximide. Error bars denote mean ± SE (n  = 3). (<b>E</b>) HEK293T cells were transfected with RET9MEN2A with GST-Cbl-c and FLAG-Enigma both alone and in combination as indicated above each blot. At 24h post-transfection, all cells were starved of FBS for 24h prior to cell harvesting. A total of 20 µgs of each of the whole cell lysates were immunoblotted as indicated to the right of each panel. Phospho-MAPK steady state levels were assessed using MAPK and Hsc70 as loading controls. Molecular weight standards are indicated on the left of each panel. (<b>F</b>) HEK293T cells were transfected with wild-type RET and the RET co-receptor, GFRα1 along with GST-Cbl-c and Enigma both alone and in combination. Vector controls included empty GST vector and all transfections were performed in duplicate and included HA-tagged ubiquitin. At 24h post-transfection, duplicate plates were pooled and re-plated to allow reattachment overnight. At 40h post-transfection, plates were rinsed and starved in media lacking FBS for 24h, then stimulated as described previously <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0087116#pone.0087116-Boulay1" target="_blank">[43]</a>, using 30ng/mL GDNF and 100ng/mL exogenous GFRα1 (+) or water control (-) for 15m as indicated prior to collection and lysis. Whole cell lysates were immunoblotted as indicated. To assess RET ubiquitination, RET immunoprecipitations were performed on 300ugs of whole cell lysate and immunoblotted for HA-ubiquitin as indicated on the right. (<b>G</b>) HEK293T cells were transfected with EGFR along with GST-Cbl-c and Enigma both alone and in combination. All transfections were performed in duplicate. At 24h post-transfection, duplicate plates were pooled and replated to allow reattachment overnight. At 40h post-transfection, plates were rinsed and starved in media lacking FBS for 8h, then stimulated with 100ng/mL EGF (+) or water control (-) for 10m as indicated prior to collection and lysis. Whole cell lysates were immunoblotted as indicated. To assess EGFR ubiquitination, EGFR immunoprecipitations were performed on 300 µg of whole cell lysate and immunoblotted for EGFR and HA-ubiquitin as indicated on the right. Molecular weight standards are shown to the left of each panel and tubulin serves as loading control.</p
Enigma blocks Cbl-c-mediated RET9MEN2A ubiquitination.
<p>(<b>A</b>) HEK293T cells were transfected with RET9MEN2A along with GST-Cbl-c, FLAG-Enigma or FLAG-EnigmaΔLIM2-3 alone and in combination as indicated above the blots. All transfections included an equal amount of HA-ubiquitin. At 40h post-transfection, all cells were treated with 20nM MG-132 for 5h prior to cell collection. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of whole cell lysate and immunoblotted for RET and HA-ubiquitin. Co-immunoprecipitation of Cbl-c and Enigma were evaluated as indicated on the right. Molecular weight standards are indicated to the left of each panel and Hsc70 serves as a loading control. (<b>B</b>) RET truncation constructs used for mapping Cbl-c and Enigma interaction sites. Structural domains include the extracellular cadherin-like repeats (C1-4), a cysteine rich region (CR), a hydrophobic transmembrane region (TM), a split cytoplasmic tyrosine kinase domain (TK1 and TK2), and intracellular tyrosines subsequently mutated to an F or stop (*) to create each construct used in this study. (<b>C</b>) HEK293T cells were transfected with GFP-tagged Enigma both alone and in combination with RET9MEN2A or one of a series of RET9MEN2A mutant constructs as indicated above each blot. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. RET immunoprecipitations were performed on 300 µg of each of the whole cell lysates and immunoblotted for GFP-Enigma. Molecular weight standards are indicated on the left of each panel and Hsc70 serves as loading control. (<b>D</b>) HEK293T cells were transfected with each of the RET9MEN2A constructs both alone and in combination with GST-Cbl-c as indicated above each blot. All transfections were balanced with empty vector controls, and cells were collected 48 h post-transfection. A total of 20 µg of each of the whole cell lysates were immunoblotted as indicated on the right. GST pull-downs were performed on 300 µg of each of the whole cell lysates and immunoblotted for Cbl-c, phosphotyrosine (pY), and RET as indicated on the right. Molecular weight standards are indicated to the left of each panel and Hsc70 serves as a loading control.</p
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