Overexpression of Ras(G12V) in primary cells induces a permanent growth arrest called oncogene-induced senescence (OIS) that serves as a fail-safe mechanism against malignant transformation. We have ...performed a genome-wide small interfering RNA (siRNA) screen and a microRNA (miRNA) screen to identify mediators of OIS and show that siRNA-mediated knockdown of p21(Waf1/Cip1) rescues from Ras(G12V)-induced senescence in human mammary epithelial cells (HMECs). Moreover, we isolated a total of 28 miRNAs that prevented Ras(G12V)-induced growth arrest, among which all of the miR-106b family members were present. In addition, we obtained a number of hits, miR-130b, miR-302a, miR-302b, miR302c, miR-302d, miR-512-3p and miR-515-3p with seed sequences very similar to miR-106b family members. We show that overexpression of all these miRNAs rescues HMECs from Ras(G12V)-induced senescence by prevention of Ras(G12V)-induced upregulation of p21(Waf1/Cip1). Our results establish an important role for the cell cycle inhibitor p21(Waf1/Cip1) in growth control of HMECs and extend the repertoire of miRNAs that modulate the activity of this tumour suppressor.
Abstract Background In the current practice of lung transplantation, donor and recipient genders are neither directly considered nor matched. However, some data have suggested a possible effect of ...gender combinations on survival following lung transplantation. Methods A total of 249 adult lung transplant recipients at a single center between February 1988 and December 2008, were analyzed retrospectively for donor-recipient gender matching. We compared the mortality by calculating one-term survival rates after transplantation using the Kaplan-Meier method with comparisons using the log-rank (Mantel-Cox) test. Statistical significance of the mean effects of size matching was assessed by paired Student t tests and Wilcoxon signed rank tests. Results Kaplan-Meier survival analysis shown that male compared to female recipients did not have an effect on outcomes after lung transplantation at 5 years ( P = .5379), 10 years ( P = .107), 15 years ( P = .0841), 20 years ( P = .0711). No effect of gender on lung transplantation outcomes was observed with donor-recipient gender mismatches at 5 years ( P = .1804), 10 years ( P = .1457), 15 years ( P = .0731), or 20 years ( P = .0629). Similarly, no differences were observed for each gender combination. The degree of size matching was defined as the ratio of donor-to-recipient predicted total lung capacity. The ratios were similar for the donor-recipient gender match and significantly different for the donor-recipient gender mismatch. Conclusions These analyses suggested that gender was not a significant independent risk factor affecting survival after lung transplantation. Size mismatch caused by gender mismatch did not increase mortality.
Abstract Background The aim of this study was to investigate the relationship between donor-to-recipient weight ratio and post-transplantation survival. Methods From February 1988 to November 2006, ...255 adult bilateral lung transplantation patients from 2 different centers were retrospectively analyzed. The cohort was divided into 4 groups depending on the quartile ranges of the donor-to-recipient weight ratio. A time-to-event analysis was performed for risk of death after transplantation conditional on 5-year survival using Kaplan-Meier and Cox proportional hazards models. Results The mean weight ratio for the study cohort was 1.23 ± 0.39. For all lung transplant recipients during the study period, survival rate at 5 years was 58%. Median survival was 6.3 years in the cohort subgroup with weight ratio <1.23, whereas the median survival was 7.7 years for the cohort subgroup with weight ratio >1.23. Weight ratio >1.23 recipients had a significant survival advantage out to 5 years compared with weight ratio <1.23 recipients (66.1% vs 51.1%, P = .0126). With the aim to assess underweight and overweight donors vs recipients, we have divided all patients into 4 groups, from quartile 1 to 4, based on donor-to-recipient weight ratio. Weight ratio strata affected overall survival, with quartile 1 (lower weight ratio recipients) experiencing the lowest 5-year survival (39.1%), followed by quartile 2 (57.8%), quartile 4 (68.2%), and quartile 3 (70.3%) recipients. The effect of weight ratio strata on survival was statistically significant for the quartile 1 recipients (lower quartile) as compared with the 3 other quartiles. Conclusions Our findings show a statistically significant effect of donor-to-recipient weight ratios on bilateral lung transplantation survival. A higher donor-to-recipient weight ratio was associated with improved survival after bilateral lung transplantation and likely reflects a mismatch between a relatively overweight donor vs recipient. In contrast, a lower donor-to-recipient ratio was associated with increased mortality after bilateral lung transplantation.
The extracellular matrix (ECM) plays an instrumental role in determining the spatial orientation of epithelial polarity and the formation of lumens in glandular tissues during morphogenesis. Here, we ...show that the Endoplasmic Reticulum (ER)-resident protein anterior gradient-2 (AGR2), a soluble protein-disulfide isomerase involved in ER protein folding and quality control, is secreted and interacts with the ECM. Extracellular AGR2 (eAGR2) is a microenvironmental regulator of epithelial tissue architecture, which plays a role in the preneoplastic phenotype and contributes to epithelial tumorigenicity. Indeed, eAGR2, is secreted as a functionally active protein independently of its thioredoxin-like domain (CXXS) and of its ER-retention domain (KTEL), and is sufficient, by itself, to promote the acquisition of invasive and metastatic features. Therefore, we conclude that eAGR2 plays an extracellular role independent of its ER function and we elucidate this gain-of-function as a novel and unexpected critical ECM microenvironmental pro-oncogenic regulator of epithelial morphogenesis and tumorigenesis.
β-Arrestins are multifunctional adapters involved in the internalization and signaling of G protein-coupled receptors (GPCRs). They target receptors to clathrin-coated pits (CCPs) through binding ...with clathrin and clathrin adapter 2 (AP-2) complex. They also act as transducers of signaling by recruiting c-Src kinase to certain GPCRs. Here we sought to determine whether c-Src regulates the recruitment of AP-2 to β-arrestin and the angiotensin II (Ang II) type 1 receptor (AT1R) during internalization. We show that the agonist stimulation of native AT1R in vascular smooth muscle cells (VSMCs) induces the formation of an endogenous complex containing c-Src, β-arrestins and AP-2. In vitro studies using coimmunoprecipitation experiments and a yeast three-hybrid assay reveal that c-Src stabilizes the agonist-independent association between β-arrestin2 and the β-subunit of AP-2 independently of the kinase activity of c-Src. However, although c-Src expression promoted the rapid dissociation of AP-2 from both β-arrestin and AT1R after receptor stimulation, a kinase-inactive mutant of c-Src failed to induce the dissociation of AP-2 from the agonist-occupied receptor. Thus, the consequence of c-Src in regulating the dissociation of AP-2 from the receptor was also examined on the internalization of AT1R by depleting c-Src in human embryonic kidney (HEK) 293 cells using a small interfering RNA strategy. Experiments in c-Src depleted cells reveal that AT1R remained mostly colocalized with AP-2 at the plasma membrane after Ang II stimulation, consistent with the observed delay in receptor internalization. Moreover, coimmunoprecipitation experiments in c-Src depleted HEK 293 cells and VSMCs showed an increased association of AP-2 to the agonist-occupied AT1R and β-arrestin, respectively. Together, our results support a role for c-Src in regulating the dissociation of AP-2 from agonist-occupied AT1R and β-arrestin during the clathrin-mediated internalization of receptors and suggest a novel function for c-Src kinase in the internalization of AT1R.
Overexpression of Ras.sup.G12V in primary cells induces a permanent growth arrest called oncogene-induced senescence (OIS) that serves as a fail-safe mechanism against malignant transformation. We ...have performed a genome-wide small interfering RNA (siRNA) screen and a microRNA (miRNA) screen to identify mediators of OIS and show that siRNA-mediated knockdown of p21.sup.Waf1/Cip1 rescues from Ras.sup.G12V-induced senescence in human mammary epithelial cells (HMECs). Moreover, we isolated a total of 28 miRNAs that prevented Ras.sup.G12V-induced growth arrest, among which all of the miR-106b family members were present. In addition, we obtained a number of hits, miR-130b, miR-302a, miR-302b, miR302c, miR-302d, miR-512-3p and miR-515-3p with seed sequences very similar to miR-106b family members. We show that overexpression of all these miRNAs rescues HMECs from Ras.sup.G12V-induced senescence by prevention of Ras.sup.G12V-induced upregulation of p21.sup.Waf1/Cip1. Our results establish an important role for the cell cycle inhibitor p21.sup.Waf1/Cip1 in growth control of HMECs and extend the repertoire of miRNAs that modulate the activity of this tumour suppressor. Oncogene (2010) 29, 2262-2271; doi:10.1038/onc.2009.497; published online 25 January 2010 Keywords: oncogene-induced senescence; Ras; siRNA; miRNA; p21.sup.Waf1/Cip1
Beta-arrestins are multifunctional adaptors that bind agonist-activated G protein-coupled receptors (GPCRs), mediate their desensitization and internalization, and control the rate at which receptors ...recycle back at the plasma membrane ready for subsequent stimulation. The activation of the bradykinin (BK) type 2 receptor (B2R) results in the rapid desensitization and internalization of the receptor. Little is known, however, about the role of β-arrestin in regulating the intracellular trafficking and the resensitization of the B2R. Using confocal microscopy, we show that BK stimulation of COS-7 cells expressing B2R induces the colocalization of the agonist-activated receptor with β-arrestin into endosomes. Fluorescent imaging and ligand binding experiments also reveal that upon agonist removal, β-arrestin rapidly dissociates from B2R into endosomes, and that receptors return back to the plasma membrane, fully competent for reactivating B2R signaling as measured by NO production upon a second BK challenge. However, when the receptor is mutated in its C-terminal domain to increase its avidity for β-arrestin, B2R remains associated with β-arrestin into endosomes, and receptors fail to recycle to the plasma membrane postagonist wash. Similarly, the recycling of receptors is prevented when a β-arrestin mutant exhibiting increased avidity for agonist-bound GPCRs is expressed with B2R. Stabilizing receptor/β-arrestin complexes into endosomes results in the dampening of the BK-mediated NO production. These results provide evidence for the involvement of β-arrestin in the intracellular trafficking of B2R, and highlight the importance of receptor recycling in reestablishing B2R signaling.
Overexpression of Ras super(G12V) in primary cells induces a permanent growth arrest called oncogene-induced senescence (OIS) that serves as a fail-safe mechanism against malignant transformation. We ...have performed a genome-wide small interfering RNA (siRNA) screen and a microRNA (miRNA) screen to identify mediators of OIS and show that siRNA-mediated knockdown of p21 super(Waf1/Cip1) rescues from Ras super(G12V)-induced senescence in human mammary epithelial cells (HMECs). Moreover, we isolated a total of 28 miRNAs that prevented Ras super(G12V)-induced growth arrest, among which all of the miR-106b family members were present. In addition, we obtained a number of hits, miR-130b, miR-302a, miR-302b, miR302c, miR-302d, miR-512-3p and miR-515-3p with seed sequences very similar to miR-106b family members. We show that overexpression of all these miRNAs rescues HMECs from Ras super(G12V)-induced senescence by prevention of Ras super(G12V)-induced upregulation of p21 super(Waf1/Cip1). Our results establish an important role for the cell cycle inhibitor p21 super(Waf1/Cip1) in growth control of HMECs and extend the repertoire of miRNAs that modulate the activity of this tumour suppressor.
The Tat protein from HIV-1 fused with heterologous proteins traverses biological membranes in a transcellular process called: protein transduction. This has already been successfully exploited in ...various biological models, but never in the nematode worm
Caenorhabditis elegans. TAT-eGFP or GST-eGFP proteins were fed to
C. elegans worms, which resulted in the specific localization of Tat-eGFP to epithelial intestinal cells. This system represents an efficient tool for transcellular transduction in
C. elegans intestinal cells. Indeed, this approach avoids the use of tedious purification steps to purify the TAT fusion proteins and allows for rapid analyses of the transduced proteins. In addition, it may represent an efficient tool to functionally analyze the mechanisms of protein transduction as well as to complement RNAi/KO in the epithelial intestinal system. To sum up, the advantage of this technology is to combine the potential of bacterial expression system and the Tat-mediated transduction technique in living worm.
Overexpression of Ras(G12V) in primary cells induces a permanent growth arrest called oncogene-induced senescence (OIS) that serves as a fail-safe mechanism against malignant transformation. We have ...performed a genome-wide small interfering RNA (siRNA) screen and a microRNA (miRNA) screen to identify mediators of OIS and show that siRNA-mediated knockdown of p21(Waf1/Cip1) rescues from Ras(G12V)-induced senescence in human mammary epithelial cells (HMECs). Moreover, we isolated a total of 28 miRNAs that prevented Ras(G12V)-induced growth arrest, among which all of the miR-106b family members were present. In addition, we obtained a number of hits, miR-130b, miR-302a, miR-302b, miR302c, miR-302d, miR-512-3p and miR-515-3p with seed sequences very similar to miR-106b family members. We show that overexpression of all these miRNAs rescues HMECs from Ras(G12V)-induced senescence by prevention of Ras(G12V)-induced upregulation of p21(Waf1/Cip1). Our results establish an important role for the cell cycle inhibitor p21(Waf1/Cip1) in growth control of HMECs and extend the repertoire of miRNAs that modulate the activity of this tumour suppressor. PUBLICATION ABSTRACT
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