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Tensin Homolog (tensin + homolog)
Selected AbstractsClinically reported heterozygous mutations in the PINK1 kinase domain exert a gene dosage effect,HUMAN MUTATION, Issue 11 2009Eng-King Tan Abstract Mutations in the gene encoding phosphatase and tensin homolog (PTEN)-induced kinase 1 (PINK1) have been associated with the loss of dopaminergic neurons characteristic of familial and sporadic Parkinson disease. We developed an in vitro system of stable human dopaminergic neuronal cell lines coexpressing an equivalent copy of normal and mutant PINK1 to simulate "heterozygous" and "homozygous" states in patients. Mutants in the N-terminus, C-terminus, and kinase domain were generated and cloned into a two-gene mammalian expression vector to generate stable mammalian expression cell lines producing an equivalent copy number of wild-type/mutant PINK1. The cell lines were subjected to oxidative stress and the rate of apoptosis and change in mitochondrial membrane potential (,,m) were assessed. Cell lines expressing kinase and C-terminus mutants exhibited a greater rate of apoptosis and decrease in ,,m, and increased time-dependent cell loss when subjected to oxidative stress compared to the wild-type. Cell lines expressing two copies of kinase mutants exhibited a greater apoptosis rate and ,,m decrease than those expressing one copy of the mutant. In time-dependent experiments, there was a significant difference between "homozygous," "heterozygous," and wild-type cell lines, with decreasing cell survival in cell lines expressing mutant copies of PINK1 compared to the wild-type. We provided the first experimental evidence that clinically reported PINK1 heterozygous mutations exert a gene dosage effect, suggesting that haploinsufficiency of PINK1 is the most likely mechanism that increased the susceptibility to dopaminergic cellular loss. Hum Mutat 30:1551,1557, 2009. © 2009 Wiley-Liss, Inc. [source] Relevance of insulin-like growth factor 2 in the etiopathophysiology of diabetic nephropathy: Possible roles of phosphatase and tensin homolog on chromosome 10 and secreted protein acidic and rich in cysteine as regulators of repairJOURNAL OF DIABETES, Issue 2 2009Movva SIREESHA Abstract Background: Diabetic nephropathy (DN) is a devastating complication of diabetes, the exact molecular pathophysiology of which is not well established. Hyperglycemia increases insulin-like growth factors (IGFs), especially IGF2, which acts via the IGF1 receptor present on renal cells. Elevated glucose levels damage the kidney, which is repaired by modulators such as secreted protein acidic and rich in cysteine (SPARC). Hence, it was hypothesized that IGF2 and SPARC may have an important role in the etiology of DN. Methods: Human renal biopsies, histopathologically categorized as normal, early Type 2 diabetes mellitus (T2DM), or established DN, were analyzed for the localization and expression of IGF2, its negative regulator phosphatase and tensin homolog on chromosome 10 (PTEN), and SPARC. Results: Expression of IGF2, PTEN, and SPARC was increased in renal biopsies from T2DM patients compared with normal samples. Although IGF2 protein was increased in biopsies from DN patients, PTEN and SPARC levels were decreased. Real-time reverse transcription,polymerase chain reaction indicated that transcript levels of IGF2 and PTEN were greater than those of ,-actin in all human renal biopsy samples. Conclusion: The results suggest the following molecular etiopathophysiology of DN: (i) hyperglycemia upregulates IGF2, which initiates PTEN, a regulator of IGF2 signaling; (ii) loss of this IGF2,PTEN feedback loop causes changes that are characteristic of DN; and (iii) lowered expression of the repair modulator SPARC results in the development and/or progression of DN. Hence, targeting relevant modulators, such as like IGF2, PTEN, and SPARC, may be important in the management of DN. [source] Microarray Analysis of Ethanol-Treated Cortical Neurons Reveals Disruption of Genes Related to the Ubiquitin-Proteasome Pathway and Protein SynthesisALCOHOLISM, Issue 12 2004Ramana Gutala Background: Chronic ethanol abuse results in deleterious behavioral responses such as tolerance, dependence, reinforcement, sensitization, and craving. The objective of this research was to identify transcripts that are differentially regulated in ethanol-treated cortical neurons compared with controls by using a pathway-focused complementary DNA microarray. Methods: Cortical neurons were isolated from postconception day 14 C57BL/6 mouse fetuses and cultured according to a standard protocol. The cortical neuronal cells were treated with 100 mM ethanol for five consecutive days with a change of media every day. A homeostatic pathway-focused microarray consisting of 638 sequence-verified genes was used to measure transcripts differentially regulated in four ethanol-treated cortical neuron samples and four control samples. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis was used to verify the mRNA expression levels of genes of interest detected from the microarray experiments. Results: We identified 56 down-regulated and 10 up-regulated genes in ethanol-treated cortical neurons relative to untreated controls at a 5% false-discovery rate. The expression of many genes involved in ubiquitin-proteasome and protein synthesis was decreased by ethanol, including ubiquitin B, ubiquitin-like 3, ubiquitin-conjugating enzyme E3A, 20S proteasome ,- and ,-subunits, and members of the ribosomal proteins. Furthermore, the mRNA expression of heat shock proteins, myristoylated alanine-rich protein kinase C substrate, phosphatase and tensin homolog deleted on chromosome 10, and FK506 binding protein rapamycin-associated protein (FKBP) (mTOR) was also decreased in ethanol-treated cortical neurons. Quantitative real-time reverse transcriptase-polymerase chain reaction analysis of genes involved in the ubiquitin-proteasome cascade revealed a down-regulation of these genes, thereby corroborating our microarray results. Conclusions: Our results indicate that chronic ethanol treatment of cortical neurons resulted in decreased mRNA expression of genes involving the ubiquitin-proteasome pathway and ribosomal proteins together with mTOR expression leading to disruption of protein degradation mechanism and impairment of protein synthesis machinery. [source] Oxidative stress may enhance the malignant potential of human hepatocellular carcinoma by telomerase activationLIVER INTERNATIONAL, Issue 6 2009Taichiro Nishikawa Abstract Background/Aims: Continuous oxidative stress (OS) plays an important role in the progression of chronic liver diseases and hepatocarcinogenesis through telomere shortening in hepatocytes. However, it has not been established how the OS influences the progression of human hepatocellular carcinomas (HCCs). We examined the correlations of OS with telomere length of cancer cells, telomerase activity and other clinicopathological factors in 68 HCCs. Methods: The level of 8-hydroxy-2,-deoxyguanosine (8-OHdG) as a marker of OS was examined immunohistochemically and OS was scored in four grades (0,3). The telomere length of cancer cells was measured by quantitative fluorescence in situ hybridization. Telomerase activity was measured by (i) immunodetection of human telomerase reverse transcriptase (hTERT) and (ii) telomere repeat amplification protocol (TRAP) assay. Telomerase related proteins, phosphatase and tensin homolog deleted on chromosome 10 (PTEN) and Akt, and other clinicopathological factors were also evaluated. Results: As the OS grade increased, the average telomere length became significantly shorter in HCCs, especially in the hTERT-negative group. In the state of high-grade OS, hTERT-positive HCC cells showed more proliferative and less apoptotic features compared with hTERT-negative HCC cells. Telomerase activity, as measured by the TRAP assay, was strongly correlated with OS grade in HCCs. Furthermore, a high OS grade was correlated with the downexpression of PTEN and the activation of Akt. Conclusions: Oxidative stress enhanced the malignant potential of HCCs through the activation of telomerase, which raises the possibility of using OS as a marker for assessing the clinical state of HCCs. [source] Analysis of phosphatase and tensin homolog tumor suppressor interacting proteins by in vitro and in silico proteomicsPROTEINS: STRUCTURE, FUNCTION AND BIOINFORMATICS, Issue 5 2005David K. Crockett Abstract The phosphatase and tensin homolog (PTEN) tumor suppressor is a multifunctional protein deregulated in many types of cancer. To date, a comprehensive documentation of PTEN interacting proteins has not been performed. The goal of our study was to characterize the PTEN interactome using affinity pull-down and tandem mass spectrometry (MS/MS). Wild-type PTEN cDNA was inserted into pTRC-His2 vector to create a 6-His tagged protein, which was expressed in Escherichia coli. Lysate from a human lymphoma cell line was used in pull-down assays, utilizing affinity for nickel-agarose beads. Bound proteins were eluted with imidazole, digested and analyzed on an LCQ DecaXP ion trap mass spectrometer. The nickel affinity pull-down efficiency was evaluated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot analysis. Acquired data were searched against the NCBI nr.fasta nonredundant protein database using the SEQUEST algorithm and screened using INTERACT and ProteinProphet. All experiments were performed in duplicate with 6-His- lacZ serving as control. A total of 79 proteins were identified in the wild-type 6-His-PTEN pull-down by MS/MS. We further validated a subset of the proteins present in the PTEN interactome by performing immunoprecipitation using an anti-PTEN antibody and establishing the presence of the proteins in the immunocomplex by Western blot analysis. A search of published PTEN interactions was also performed using Online Mendelian Inheritance in Man, Human Protein Reference Database, the IntAct Project database, and PubMed. This in silico analysis confirmed 42 out of 79 (53%) of the proteins identified by MS/MS. The remaining 37 proteins represent probable PTEN interactions not previously documented in public databases or reported in the literature. These results highlight the value of combining both in vitro biochemical approaches with in silico analyses for a comprehensive study of protein-protein interactions. [source] Conditional expression of PTEN alters the androgen responsiveness of prostate cancer cells,THE PROSTATE, Issue 10 2006Z. Wu Abstract BACKGROUND Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is lost as a function of prostate tumor androgen dependence. While the transcriptional activity of the androgen receptor (AR) is inhibited by PTEN in androgen sensitive prostate cancer (CaP), the role of PTEN in androgen disease is unclear. METHODS We developed a system where PTEN can be conditionally re-expressed at physiologic levels into a PTEN null metastatic human CaP cell line, C4-2, and androgen responsiveness examined. RESULTS PTEN induction reduces cell growth and blocks the growth effect of synthetic androgen R1881. The anti-androgen Casodex enhances the growth-inhibitory action of PTEN and this effect is independent of Akt phosphorylation. Combined PTEN induction and Casodex, result in a further decrease in prostate specific antigen promoter activity compared to PTEN but not Casodex alone. CONCLUSIONS PTEN induction confers androgen independent CaP cells enhanced responsiveness to the anti-proliferative effects of anti-androgens and this action may involve non-AR mediated effects. Prostate 66: 1114-1123, 2006. © 2006 Wiley-Liss, Inc. [source] PTEN expression is down-regulated in liver tissues of rats with hepatic fibrosis induced by biliary stenosisAPMIS, Issue 9 2009LI SEN HAO The gene phosphatase and tensin homolog deleted on chromosome 10 (PTEN) codes for a tumor-suppressor phospholipid phosphatase. Deletion, mutation or abnormal expression of PTEN is commonly found in many kinds of malignant tumors. At the time of this study, though, the role of PTEN expression in the pathology of hepatic fibrosis remains unclear. In this study, we investigate the dynamic expression of PTEN in a rat model of hepatic fibrosis, with special emphasis on the activation and proliferation of hepatic stellate cells (HSC) in vivo. The rat model of hepatic fibrosis used in this study employed common bile duct ligation. At four time points, the expression of PTEN in hepatic tissues and activated HSC in rat liver tissues was measured by immunohistochemical staining, Western blotting, real-time fluorescent quantitative PCR and immunofluorescence confocal laser scanning microscopy, respectively. Further, ,-smooth muscle actin (,-SMA), an activated HSC marker in rat liver tissues, was detected by immunohistochemical staining. This study showed that aggravation of hepatic fibrosis led to gradually decreasing expression of PTEN in the hepatic tissues. Further, as hepatic fibrosis worsens, PTEN-expressing activated HSC accounts for an increasingly smaller percentage of all activated HSC. In contrast, the percentage of ,-SMA-expressing HSC cells increases significantly. In conclusion, expression of PTEN mRNA and protein is down-regulated in fibrogenic rat liver tissue, and its expression in HSC in vivo also decreases with progression of fibrosis. Thus, these results show that the dynamic expression of PTEN in hepatic tissues negatively correlates with activation and proliferation of HSC. [source] Dihydrosphingosine 1-phosphate has a potent antifibrotic effect in scleroderma fibroblasts via normalization of phosphatase and tensin homolog levelsARTHRITIS & RHEUMATISM, Issue 7 2010Shizhong Bu Objective Previous studies have revealed a phosphatase and tensin homolog (PTEN),dependent interaction between the sphingolipid agonist dihydrosphingosine 1-phosphate (dhS1P) and the transforming growth factor ,/Smad3 signaling pathway. This study was undertaken to examine responses of systemic sclerosis (SSc) fibroblasts to sphingosine 1-phosphate (S1P) and dhS1P and to gain further insight into the regulation of the S1P/dhS1P/PTEN pathway in SSc fibrosis. Methods Fibroblast cultures were established from skin biopsy samples obtained from patients with SSc and matched healthy controls. Western blotting and quantitative polymerase chain reaction were used to measure protein and messenger RNA levels, respectively. PTEN protein was examined in skin biopsy samples by immunohistochemistry. Results PTEN protein levels were low in SSc fibroblasts and correlated with elevated levels of collagen and phospho-Smad3 and reduced levels of matrix metalloproteinase 1 (MMP-1). Treatment with dhS1P restored PTEN levels and normalized collagen and MMP-1 expression, as well as Smad3 phosphorylation status in SSc fibroblasts. S1P was strongly profibrotic in SSc and control fibroblasts. Distribution of S1P receptor isoforms was altered in SSc fibroblasts, which had reduced levels of S1P receptor 1 and S1P receptor 2 and elevated levels of S1P receptor 3. Only depletion of S1P receptor 1 abrogated the effects of dhS1P and S1P in control dermal fibroblasts. In contrast, depletion of either S1P receptor 1 or S1P receptor 2 prevented the effects of S1P and dhS1P in SSc fibroblasts. Conclusion Our findings demonstrate that PTEN deficiency is a critical determinant of the profibrotic phenotype of SSc fibroblasts. The antifibrotic effect of dhS1P is mediated through normalization of PTEN expression, suggesting that dhS1P or its derivatives may be effective as therapeutic antifibrotic agents. The distribution and function of S1P receptors differ in SSc and healthy fibroblasts, suggesting that alteration in the sphingolipid signaling pathway may contribute to SSc fibrosis. [source] Copy number gains in EGFR and copy number losses in PTEN are common events in osteosarcoma tumors,CANCER, Issue 6 2008Serena S. Freeman Abstract BACKGROUND. Osteosarcoma cell lines and tumors have been shown to express epidermal growth factor receptor (EGFR) and harbor amplifications at the EGFR locus. In this study, the authors further analyzed the genomic features of EGFR in osteosarcoma tumors and investigated whether they correlate with phosphatase and tensin homolog (PTEN) expression and copy number status. METHODS. EGFR and PTEN expression was assessed by immunohistochemistry (n = 28), and copy number alterations at the EGFR and PTEN loci were surveyed using Affymetrix (Santa Clara, Calif) 50K single nucleotide polymorphism (SNP) arrays (n = 31) and fluorescence in situ hybridization (FISH) (n = 27). The EGFR tyrosine kinase domain was sequenced to survey for activating mutations (n = 34). In addition, EGFRvIII expression was assessed using reverse transcriptase polymerase chain reaction (n = 24). Results were correlated with available clinical information on 59 patients, with a median age of 14.1 years (range, 5-23years) and median follow-up of 4.4 years. RESULTS. EGFR expression was detected in the majority of osteosarcoma tumors surveyed (23 of 28; 82%). SNP arrays revealed evidence of frequent copy number gains at 7p11.2 and losses at 10q23.21. A sizeable subset (16 of 27 cases; 59%) showed gains at the EGFR locus using FISH (amplification in 4 of 27 [15%] and balanced chromosome 7 polysomy in 12 of 27 [44%]), and 12 cases showed deletions at the PTEN locus (biallelic deletions in 4 of 27 [15%] and monoallelic deletion in 9 of 27 [33%]). No activating mutations in the EGFR tyrosine kinase domain, EGFRvIII expression, or association with clinical findings were detected. CONCLUSIONS. EGFR expression and genomic gains at the EGFR locus are prevalent in osteosarcoma tumors, which also commonly harbor deletions at the PTEN locus. Cancer 2008. © 2008 American Cancer Society. [source] | ||||||||||