Regulation of the activity of the tumor suppressor PTEN by thioredoxin in Drosophila melanogaster

International Nuclear Information System (INIS)

Song, Zuohe; Saghafi, Negin; Gokhale, Vijay; Brabant, Marc; Meuillet, Emmanuelle J.

2007-01-01

Human Thioredoxin-1 (hTrx-1) is a small redox protein with a molecular weight of 12 kDa that contains two cysteine residues found in its catalytic site. HTrx-1 plays an important role in cell growth, apoptosis, and cancer patient prognosis. Recently, we have demonstrated that hTrx-1 binds to the C2 domain of the human tumor suppressor, PTEN, in a redox dependent manner. This binding leads to the inhibition of PTEN lipid phosphatase activity in mammalian tissue culture systems. In this study, we show that over-expression of hTrx-1 in Drosophila melanogaster promotes cell growth and proliferation during eye development as measured by eye size and ommatidia size. Furthermore, hTrx-1 rescues the small eye phenotype induced by the over-expression of PTEN. We demonstrate that this rescue of the PTEN-induced eye size phenotype requires cysteine-218 in the C2 domain of PTEN. We also show that hTrx-1 over-expression results in increased Akt phosphorylation in fly head extracts supporting our observations that the hTrx-1-induced eye size increase results from the inhibition of PTEN activity. Our study confirms the redox regulation of PTEN through disulfide bond formation with the hTrx-1 in Drosophila and suggests conserved mechanisms for thioredoxins and their interactions with the phosphatidylinositol-3-kinase signaling pathway in humans and fruit flies

RAS/ERK modulates TGFbeta-regulated PTEN expression in human pancreatic adenocarcinoma cells.

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Chow, Jimmy Y C; Quach, Khai T; Cabrera, Betty L; Cabral, Jennifer A; Beck, Stayce E; Carethers, John M

2007-11-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-beta might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFbeta and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFbeta surface receptors. Cells were treated with TGFbeta1 and separated into cytosolic/nuclear fractions for western blotting with phospho-SMAD2, SMAD 2, 4 phospho-ATP-dependent tyrosine kinases (Akt), Akt and PTEN antibodies. PTEN mRNA levels were assessed by reverse transcriptase-polymerase chain reaction. The MEK1 inhibitor, PD98059, was used to block the downstream action of oncogenic K-RAS/ERK, as was a dominant-negative (DN) K-RAS construct. TGFbeta increased phospho-SMAD2 in both cytosolic and nuclear fractions. PD98059 treatment further increased phospho-SMAD2 in the nucleus of both pancreatic cell lines, and DN-K-RAS further improved SMAD translocation in K-RAS mutant CAPAN cells. TGFbeta treatment significantly suppressed PTEN protein levels concomitant with activation of Akt by 48 h through transcriptional reduction of PTEN mRNA that was evident by 6 h. TGFbeta-induced PTEN suppression was reversed by PD98059 and DN-K-RAS compared with treatments without TGFbeta. TGFbeta-induced PTEN expression was inversely related to cellular proliferation. Thus, oncogenic K-RAS/ERK in pancreatic adenocarcinoma facilitates TGFbeta-induced transcriptional down-regulation of the tumor suppressor PTEN in a SMAD4-independent manner and could constitute a signaling switch mechanism from growth suppression to growth promotion in pancreatic cancers.

Deletion of the N-terminus of IKKγ induces apoptosis in keratinocytes and impairs the AKT/PTEN signaling pathway

International Nuclear Information System (INIS)

Leis, Hugo; Sanchis, Ana; Perez, Paloma

2007-01-01

The regulatory subunit IKKγ/NEMO is crucial for skin development and function and although devoid of kinase activity, loss of IKKγ function completely abolishes the activation of NF-κB by all pro-inflammatory cytokines. To inhibit the IκB kinase (IKK) complex in keratinocytes, we have used a dominant negative approach by generating stable transfectants of an N-terminal deletion of IKKγ (IKKγ-DN97) that uncouples formation of the IKK complex. Expression of this mutant in PB keratinocytes (PB-IKKγ-DN97) delayed growth kinetics, caused morphological changes and dramatically augmented apoptosis even in the absence of pro-apoptotic stimuli, as determined by cell morphology, TUNEL and caspase-3 cleavage. Moreover, in PB-IKKγ-DN97 cells, TNF-α and IL-1 treatment failed to induce degradation of IκBα, phosphorylation of p65 on Ser 536 and nuclear translocation which, consequently, reduced κB-binding activity. In PB-IKKγ-DN97 cells, accumulation of IκBα correlated with a downregulation of AKT activity and an increase of PTEN protein levels whereas pro-apoptotic p53 target genes Bax and Puma were upregulated. These effects were most likely mediated through IKK since coexpression of the wild-type form of IKKγ in keratinocytes partially reversed apoptosis and reduced PTEN expression. Thus, our data suggest a negative cross-talk mechanism involving PTEN and NF-κB, critical for the anti-apoptotic role of NF-κB in keratinocytes

Kaempferol Promotes Apoptosis in Human Bladder Cancer Cells by Inducing the Tumor Suppressor, PTEN

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Liqun Zhou

2013-10-01

Full Text Available Kaempferol (Kae, a natural flavonoid, is widely distributed in fruits and vegetables. Previous studies have identified Kae as a possible cancer preventive and therapeutic agent. We found Kae to exhibit potent antiproliferation and anti-migration effects in human bladder cancer EJ cells. Kaempferol robustly induced apoptosis in EJ cells in a dose-dependent manner, as evidenced by increased cleavage of caspase-3. Furthermore, we found Kae-induced apoptosis in EJ cells to be associated with phosphatase and the tensin homolog deleted on the chromosome 10 (PTEN/PI3K/Akt pathway. Kae significantly increased PTEN and decreased Akt phosphorylation. Kae-induced apoptosis was partially attenuated in PTEN-knockdown cells. Our findings indicate that Kae could be an alternative medicine for bladder cancer, based on a PTEN activation mechanism.

Voltage-sensing phosphatase: its molecular relationship with PTEN.

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Okamura, Yasushi; Dixon, Jack E

2011-02-01

Voltage-sensing phosphoinositide phosphatase (VSP) contains voltage sensor and cytoplasmic phosphatase domains. A unique feature of this protein is that depolarization-induced motions of the voltage sensor activate PtdIns(3,4,5)P(3) and PtdIns(4,5)P(2) phosphatase activities. VSP exhibits remarkable structural similarities with PTEN, the phosphatase and tensin homolog deleted on chromosome 10. These similarities include the cytoplasmic phosphatase region, the phosphoinositide binding region, and the putative membrane interacting C2 domain.

PTEN gene and phosphorylation of Akt protein expression in the LPS-induced lung fibroblast

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Mao-lin HUANG

2014-09-01

Full Text Available Objective: To investigate PTEN gene expression and the Akt phosphorylation of protein expression in the LPS-induced lung fibroblast, to initially reveal the relation between PTEN gene and the Akt phosphorylated proteins to LPS-induced lung fibroblast proliferation mechanism. Methods: BrdU experiments was performed to evaluate the LPS-induced lung fibroblast proliferation,  RT-PCR and Western Blot analysis were used to analyze the PTEN gene expression and Western blot was performed to analyze Akt phosphorylated protein expression. Results: PTEN mRNA level of the experimental group were significantly lower than the control group (P<0.05 with LPS simulation for 24h and 72h , and there were no significant difference between the experimental group and control group the experimental group and control group (P>0.05 . PTEN protein expression levels of the experimental group were significantly lower than the control group (P<0.05 , at 72h, and PTEN mRNA levels had no significant differences between these of the experimental and control group at 6h,12h and 24h(p>0.05. Phosphorylation Akt protein level (relative to total Akt protein was significantly higer than the control group (P<0.05 at 24h and 72h, and phosphorylation Akt protein levels had no significant differences between these of the experimental and control group at 6h and 12h (P>0.05 .Conclusion: PTEN gene and phosphorylation Akt protein involve in LPS-induced lung fibroblast proliferation signal transduction pathway.

Superoxide anion radicals induce IGF-1 resistance through concomitant activation of PTP1B and PTEN

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Singh, Karmveer; Maity, Pallab; Krug, Linda; Meyer, Patrick; Treiber, Nicolai; Lucas, Tanja; Basu, Abhijit; Kochanek, Stefan; Wlaschek, Meinhard; Geiger, Hartmut; Scharffetter-Kochanek, Karin

2015-01-01

The evolutionarily conserved IGF-1 signalling pathway is associated with longevity, metabolism, tissue homeostasis, and cancer progression. Its regulation relies on the delicate balance between activating kinases and suppressing phosphatases and is still not very well understood. We report here that IGF-1 signalling in vitro and in a murine ageing model in vivo is suppressed in response to accumulation of superoxide anions () in mitochondria, either by chemical inhibition of complex I or by genetic silencing of -dismutating mitochondrial Sod2. The -dependent suppression of IGF-1 signalling resulted in decreased proliferation of murine dermal fibroblasts, affected translation initiation factors and suppressed the expression of α1(I), α1(III), and α2(I) collagen, the hallmarks of skin ageing. Enhanced led to activation of the phosphatases PTP1B and PTEN, which via dephosphorylation of the IGF-1 receptor and phosphatidylinositol 3,4,5-triphosphate dampened IGF-1 signalling. Genetic and pharmacologic inhibition of PTP1B and PTEN abrogated -induced IGF-1 resistance and rescued the ageing skin phenotype. We thus identify previously unreported signature events with , PTP1B, and PTEN as promising targets for drug development to prevent IGF-1 resistance-related pathologies. PMID:25520316

An inducible knockout mouse to model the cell-autonomous role of PTEN in initiating endometrial, prostate and thyroid neoplasias

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Mirantes, Cristina; Eritja, Núria; Dosil, Maria Alba; Santacana, Maria; Pallares, Judit; Gatius, Sónia; Bergadà, Laura; Maiques, Oscar; Matias-Guiu, Xavier; Dolcet, Xavier

2013-01-01

SUMMARY PTEN is one of the most frequently mutated tumor suppressor genes in human cancers. The role of PTEN in carcinogenesis has been validated by knockout mouse models. PTEN heterozygous mice develop neoplasms in multiple organs. Unfortunately, the embryonic lethality of biallelic excision of PTEN has inhibited the study of complete PTEN deletion in the development and progression of cancer. By crossing PTEN conditional knockout mice with transgenic mice expressing a tamoxifen-inducible Cre-ERT under the control of a chicken actin promoter, we have generated a tamoxifen-inducible mouse model that allows temporal control of PTEN deletion. Interestingly, administration of a single dose of tamoxifen resulted in PTEN deletion mainly in epithelial cells, but not in stromal, mesenchymal or hematopoietic cells. Using the mT/mG double-fluorescent Cre reporter mice, we demonstrate that epithelial-specific PTEN excision was caused by differential Cre activity among tissues and cells types. Tamoxifen-induced deletion of PTEN resulted in extremely rapid and consistent formation of endometrial in situ adenocarcinoma, prostate intraepithelial neoplasia and thyroid hyperplasia. We also analyzed the role of PTEN ablation in other epithelial cells, such as the tubular cells of the kidney, hepatocytes, colonic epithelial cells or bronchiolar epithelium, but those tissues did not exhibit neoplastic growth. Finally, to validate this model as a tool to assay the efficacy of anti-tumor drugs in PTEN deficiency, we administered the mTOR inhibitor everolimus to mice with induced PTEN deletion. Everolimus dramatically reduced the progression of endometrial proliferations and significantly reduced thyroid hyperplasia. This model could be a valuable tool to study the cell-autonomous mechanisms involved in PTEN-loss-induced carcinogenesis and provides a good platform to study the effect of anti-neoplastic drugs on PTEN-negative tumors. PMID:23471917

PTEN-induction in U251 glioma cells decreases the expression of insulin-like growth factor binding protein-2

International Nuclear Information System (INIS)

Levitt, Randy J.; Georgescu, Maria-Magdalena; Pollak, Michael

2005-01-01

PTEN is a tumor suppressor gene whose loss of function is observed in ∼40-50% of human cancers. Although insulin-like growth factor binding protein-2 (IGFBP-2) was classically described as a growth inhibitor, multiple recent reports have shown an association of overexpression and/or high serum levels of IGFBP-2 with poor prognosis of several malignancies, including gliomas. Using an inducible PTEN expression system in the PTEN-null glioma cell line U251, we demonstrate that PTEN-induction is associated with reduced proliferation, increased apoptosis, and a substantial reduction of the high levels of IGFBP-2 expression. The PTEN-induced decrease in IGFBP-2 expression could be mimicked with the PI3-kinase inhibitor LY294002, indicating that the lipid phosphatase activity of PTEN is responsible for the observed effect. However, the rapamycin analog CCI-779 did not affect IGFBP-2 expression, suggesting that the PTEN-induced decrease in IGFBP-2 expression is not attributable to decreased mTOR signalling. Recombinant human IGFBP-2 was unable to rescue U251-PTEN cells from the antiproliferative effects of PTEN, and IGFBP-2 siRNA did not affect the IGF-dependent or -independent growth of this cell line. These results suggest that the clinical data linking IGFBP-2 expression to poor prognosis may arise, at least in part, because high levels of IGFBP-2 expression correlate with loss of function of PTEN, which is well known to lead to aggressive behavior of gliomas. Our results motivate translational research regarding the relationship between IGFBP-2 expression and loss of function of PTEN

Functional genomics identifies specific vulnerabilities in PTEN-deficient breast cancer.

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Tang, Yew Chung; Ho, Szu-Chi; Tan, Elisabeth; Ng, Alvin Wei Tian; McPherson, John R; Goh, Germaine Yen Lin; Teh, Bin Tean; Bard, Frederic; Rozen, Steven G

2018-03-22

Phosphatase and tensin homolog (PTEN) is one of the most frequently inactivated tumor suppressors in breast cancer. While PTEN itself is not considered a druggable target, PTEN synthetic-sick or synthetic-lethal (PTEN-SSL) genes are potential drug targets in PTEN-deficient breast cancers. Therefore, with the aim of identifying potential targets for precision breast cancer therapy, we sought to discover PTEN-SSL genes present in a broad spectrum of breast cancers. To discover broad-spectrum PTEN-SSL genes in breast cancer, we used a multi-step approach that started with (1) a genome-wide short interfering RNA (siRNA) screen of ~ 21,000 genes in a pair of isogenic human mammary epithelial cell lines, followed by (2) a short hairpin RNA (shRNA) screen of ~ 1200 genes focused on hits from the first screen in a panel of 11 breast cancer cell lines; we then determined reproducibility of hits by (3) identification of overlaps between our results and reanalyzed data from 3 independent gene-essentiality screens, and finally, for selected candidate PTEN-SSL genes we (4) confirmed PTEN-SSL activity using either drug sensitivity experiments in a panel of 19 cell lines or mutual exclusivity analysis of publicly available pan-cancer somatic mutation data. The screens (steps 1 and 2) and the reproducibility analysis (step 3) identified six candidate broad-spectrum PTEN-SSL genes (PIK3CB, ADAMTS20, AP1M2, HMMR, STK11, and NUAK1). PIK3CB was previously identified as PTEN-SSL, while the other five genes represent novel PTEN-SSL candidates. Confirmation studies (step 4) provided additional evidence that NUAK1 and STK11 have PTEN-SSL patterns of activity. Consistent with PTEN-SSL status, inhibition of the NUAK1 protein kinase by the small molecule drug HTH-01-015 selectively impaired viability in multiple PTEN-deficient breast cancer cell lines, while mutations affecting STK11 and PTEN were largely mutually exclusive across large pan-cancer data sets. Six genes showed PTEN

Suppression of PTEN transcription by UVA

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Zhao, Baozhong; Ming, Mei; He, Yu-Ying

2012-01-01

Although UVA has different physical and biological targets than UVB, the contribution of UVA to skin cancer susceptibility and its molecular basis remain largely unknown. Here we show that chronic UVA radiation suppresses PTEN expression at the mRNA level. Subchronic and acute UVA radiation also down-regulated PTEN in normal human epidermal keratinocytes, skin culture and mouse skin. At the molecular level, chronic UVA radiation decreased the transcriptional activity of the PTEN promoter in a methylation-independent manner, while it had no effect on the protein stability or mRNA stability of PTEN. In contrast, we found that UVA-induced activation of the Ras/ERK/AKT and NF-κB pathways plays an important role in UV-induced PTEN down-regulation. Inhibiting ERK or AKT increases PTEN expression. Our findings may provide unique insights into PTEN down-regulation as a critical component of UVA’s molecular impact during keratinocyte transformation. PMID:23129115

A unified nomenclature and amino acid numbering for human PTEN

NARCIS (Netherlands)

Pulido, Rafael; Baker, Suzanne J; Barata, Joao T; Carracedo, Arkaitz; Cid, Victor J; Chin-Sang, Ian D; Davé, Vrushank; den Hertog, Jeroen; Devreotes, Peter; Eickholt, Britta J; Eng, Charis; Furnari, Frank B; Georgescu, Maria-Magdalena; Gericke, Arne; Hopkins, Benjamin; Jiang, Xeujun; Lee, Seung-Rock; Lösche, Mathias; Malaney, Prerna; Matias-Guiu, Xavier; Molina, María; Pandolfi, Pier Paolo; Parsons, Ramon; Pinton, Paolo; Rivas, Carmen; Rocha, Rafael M; Rodríguez, Manuel S; Ross, Alonzo H; Serrano, Manuel; Stambolic, Vuk; Stiles, Bangyan; Suzuki, Akira; Tan, Seong-Seng; Tonks, Nicholas K; Trotman, Lloyd C; Wolff, Nicolas; Woscholski, Rudiger; Wu, Hong; Leslie, Nicholas R

2014-01-01

The tumor suppressor PTEN is a major brake for cell transformation, mainly due to its phosphatidylinositol 3,4,5-trisphosphate [PI(3,4,5)P3] phosphatase activity that directly counteracts the oncogenicity of phosphoinositide 3-kinase (PI3K). PTEN mutations are frequent in tumors and in the germ line

Effects of PTEN inhibition on the regulation of Tau phosphorylation in rat cortical neuronal injury after oxygen and glucose deprivation.

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Zhao, Jing; Chen, Yurong; Xu, Yuxia; Pi, Guanghuan

2016-01-01

This report investigated the involvement of the PTEN pathway in the regulation of Tau phosphorylation using an oxygen and glucose deprivation (OGD) model with rat cortical neurons. Primary cortical neurons were used to establish the oxygen and glucose deprivation (OGD) model in vitro. These were randomly divided into control, OGD, bpV+OGD, As+OGD, Se+OGD and Mock treatment groups. The neuron viability was assessed by MTT, the cell apoptosis was detected using TUNEL staining. The expression of Phospho-PTEN/PTEN, Phospho-Tau/Tau, Phospho-Akt/Akt and Phospho-GSK-3β/GSK-3β were detected by Western blotting. OGD induced Tau phosphorylation through PTEN and glycogen synthase kinase-3β (GSK-3β) activation, together with a decrease in AKT activity. Pre-treatment with bpv, a potent PTEN inhibitor, and PTEN antisense nucleotides decreased PTEN and GSK-3β activity and caused alterations in Tau phosphorylation. Neuronal apoptosis was also reduced. The PTEN/Akt/GSK-3β/Tau pathway is involved in the regulation of neuronal injury, providing a novel route for protecting neurons following neonatal HI.

Molecular Analysis of AFP and HSA Interactions with PTEN Protein

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Mingyue Zhu

2015-01-01

Full Text Available Human cytoplasmic alpha-fetoprotein (AFP has been classified as a member of the albuminoid gene family. The protein sequence of AFP has significant homology to that of human serum albumin (HSA, but its biological characteristics are vastly different from HSA. The AFP functions as a regulator in the phosphatidylinositol 3-kinase (PI3K/protein kinase B (AKT pathway, but HSA plays a key role as a transport protein. To probe their molecular mechanisms, we have applied colocalization, coimmunoprecipitation (co-IP, and molecular docking approaches to analyze the differences between AFP and HSA. The data from colocalization and co-IP displayed a strong interaction between AFP and PTEN (phosphatase and tensin homolog, demonstrating that AFP did bind to PTEN, but HSA did not. The molecular docking study further showed that the AFP domains I and III could contact with PTEN. In silicon substitutions of AFP binding site residues at position 490M/K and 105L/R corresponding to residues K490 and R105 in HSA resulted in steric clashes with PTEN residues R150 and K46, respectively. These steric clashes may explain the reason why HSA cannot bind to PTEN. Ultimately, the experimental results and the molecular modeling data from the interactions of AFP and HSA with PTEN will help us to identify targets for designing drugs and vaccines against human hepatocellular carcinoma.

Variation in the PTEN-induced putative kinase 1 gene associated ...

Indian Academy of Sciences (India)

with the increase risk of type 2 diabetes in northern Chinese ... 2National Institute of Geriatric Medicines, Beijing Hospital, Ministry of Health, Beijing 100730, People's Republic of ... Two hundred and ninety two unrelated T2DM patients were.

Variation in the PTEN-induced putative kinase 1 gene associated ...

Indian Academy of Sciences (India)

with the increase risk of type 2 diabetes in northern Chinese. YANCHUN QU1†∗ ... and genetic variation analysis have indicated the involvement of PINK1 gene in the ... Qualitative variables were analysed by a chi-squared test. The level of ...

MDM2 regulates hypoxic hypoxia-inducible factor 1α stability in an E3 ligase, proteasome, and PTEN-phosphatidylinositol 3-kinase-AKT-dependent manner.

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Joshi, Shweta; Singh, Alok R; Durden, Donald L

2014-08-15

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1β subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α-HIF1β heterodimers undergo post-translational modifications that promote transactivation. Previous reports suggest that amplified signaling through PI3K enhances HIF1-dependent gene expression; however, its role is controversial, and the mechanism is unclear. Using genetically engineered PTEN-deficient cell lines, we demonstrate that PTEN specifically inhibited the accumulation of HIF1α in response to hypoxia. Furthermore, we report that in glioblastoma cell lines, inhibition of PI3K pathway, using pan as well as isoform-specific PI3K inhibitors SF1126, PF4691502, BEZ-235, GDC0941, and TGX221 blocked the induction of HIF1α protein and its targets vascular endothelial growth factor, HK1, and GLUT1 mRNA in response to hypoxia. Herein, we describe the first evidence that HIF1α can be degraded under hypoxic conditions via the 26 S proteasome and that MDM2 is the E3 ligase that induces the hypoxic degradation of HIF1α. Moreover, the action of MDM2 on HIF1α under hypoxia occurs in the cytoplasm and is controlled by the PTEN-PI3K-AKT signaling axis. These data strongly suggest a new role for PTEN in the regulation of HIF1α and importantly that PI3K-AKT activation is required for the hypoxic stabilization of HIF1α and that hypoxia alone is not sufficient to render HIF1α resistant to proteasomal cleavage and degradation. Moreover, these findings suggest new therapeutic considerations for PI3K and/or AKT inhibitors for cancer therapeutics. © 2014 by The American Society for Biochemistry and Molecular Biology, Inc.

MDM2 Regulates Hypoxic Hypoxia-inducible Factor 1α Stability in an E3 Ligase, Proteasome, and PTEN-Phosphatidylinositol 3-Kinase-AKT-dependent Manner*

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Joshi, Shweta; Singh, Alok R.; Durden, Donald L.

2014-01-01

Hypoxia-inducible factor 1 (HIF1) is a heterodimeric transcription factor containing an inducibly expressed HIF1α subunit and a constitutively expressed HIF1β subunit. Under hypoxic conditions, the HIF1α subunit accumulates because of a decrease in the rate of proteolytic degradation, and the resulting HIF1α–HIF1β heterodimers undergo post-translational modifications that promote transactivation. Previous reports suggest that amplified signaling through PI3K enhances HIF1-dependent gene expression; however, its role is controversial, and the mechanism is unclear. Using genetically engineered PTEN-deficient cell lines, we demonstrate that PTEN specifically inhibited the accumulation of HIF1α in response to hypoxia. Furthermore, we report that in glioblastoma cell lines, inhibition of PI3K pathway, using pan as well as isoform-specific PI3K inhibitors SF1126, PF4691502, BEZ-235, GDC0941, and TGX221 blocked the induction of HIF1α protein and its targets vascular endothelial growth factor, HK1, and GLUT1 mRNA in response to hypoxia. Herein, we describe the first evidence that HIF1α can be degraded under hypoxic conditions via the 26 S proteasome and that MDM2 is the E3 ligase that induces the hypoxic degradation of HIF1α. Moreover, the action of MDM2 on HIF1α under hypoxia occurs in the cytoplasm and is controlled by the PTEN-PI3K-AKT signaling axis. These data strongly suggest a new role for PTEN in the regulation of HIF1α and importantly that PI3K-AKT activation is required for the hypoxic stabilization of HIF1α and that hypoxia alone is not sufficient to render HIF1α resistant to proteasomal cleavage and degradation. Moreover, these findings suggest new therapeutic considerations for PI3K and/or AKT inhibitors for cancer therapeutics. PMID:24982421

Establishment of pten knockout medaka with transcription activator-like effector nucleases (TALENs as a model of PTEN deficiency disease.

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Yuriko Matsuzaki

Full Text Available Phosphatase and tensin homolog (PTEN is a lipid and protein phosphatase that antagonizes signaling by the phosphatidylinositol 3-kinase (PI3K-AKT signaling pathway. The PTEN gene is a major tumor suppressor, with mutations of this gene occurring frequently in tumors of humans and mice. We have now developed mutant medaka deficient in PTEN with the use of transcription activator-like effector nuclease (TALEN technology. Medaka possesses two pten genes, ptena and ptenb, similar to zebrafish. We established 16 ptena mutant lines and two ptenb mutant lines. Homozygous single pten mutants were found to be viable and fertile. In contrast, pten double-knockout (dko embryos manifested severe abnormalities in vasculogenesis, eye size, and tail development at 72 hours post fertilization(hpf and died before hatching. Immunoblot analysis revealed that the ratio of phosphorylated to total forms of AKT (pAKT/AKT in pten dko embryos was four times that in wild-type embryos, indicative of up-regulation of signaling by the PI3K-AKT pathway. Treatment of pten dko embryos with the PI3K inhibitor LY294002 reduced the pAKT/AKT ratio by about one-half and partially rescued the defect in vasculogenesis. Additional inhibitors of the PI3K-AKT pathway, including rapamycin and N-α-tosyl-L-phenylalanyl chloromethyl ketone, also partially restored vasculogenesis in the dko embryos. Our model system thus allows pten dko embryos to be readily distinguished from wild-type embryos at an early stage of development and is suitable for the screening of drugs able to compensate for PTEN deficiency.

Cisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer cells: a potential mechanism of chemoresistance

International Nuclear Information System (INIS)

Singh, Mohan; Chaudhry, Parvesh; Fabi, Francois; Asselin, Eric

2013-01-01

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines. Cells were treated with 10μM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation. PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells. The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells

Cisplatin-induced caspase activation mediates PTEN cleavage in ovarian cancer cells: a potential mechanism of chemoresistance.

Science.gov (United States)

Singh, Mohan; Chaudhry, Parvesh; Fabi, Francois; Asselin, Eric

2013-05-10

The phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor protein is a central negative regulator of the PI3K/AKT signaling cascade and suppresses cell survival as well as cell proliferation. PTEN is found to be either inactivated or mutated in various human malignancies. In the present study, we have investigated the regulation of PTEN during cisplatin induced apoptosis in A2780, A270-CP (cisplatin resistant), OVCAR-3 and SKOV3 ovarian cancer cell lines. Cells were treated with 10μM of cisplatin for 24h. Transcript and protein levels were analysed by quantitative reverse transcriptase-polymerase chain reaction (qRT-PCR) and western blotting, respectively. Immunofluorescence microscopy was used to assess the intracellular localization of PTEN. Proteasome inhibitor and various caspases inhibitors were used to find the mechanism of PTEN degradation. PTEN protein levels were found to be decreased significantly in A2780 cells; however, there was no change in PTEN protein levels in A2780-CP, OVCAR-3 and SKOV3 cells with cisplatin treatment. The decrease in PTEN protein was accompanied with an increase in the levels of AKT phosphorylation (pAKT) in A2780 cells and a decrease of BCL-2. Cisplatin treatment induced the activation/cleavage of caspase-3, -6, -7, -8, -9 in all cell lines tested in this study except the resistant variant A2780-CP cells. In A2780 cells, restoration of PTEN levels was achieved upon pre-treatment with Z-DEVD-FMK (broad range caspases inhibitor) and not with MG132 (proteasome inhibitor) and by overexpression of BCL-2, suggesting that caspases and BCL-2 are involved in the decrease of PTEN protein levels in A2780 cells. The decrease in pro-apoptotic PTEN protein levels and increase in survival factor pAKT in A2780 ovarian cancer cells suggest that cisplatin treatment could further exacerbate drug resistance in A2780 ovarian cancer cells.

MicroRNA-22 promotes cell survival upon UV radiation by repressing PTEN

International Nuclear Information System (INIS)

Tan, Guangyun; Shi, Yuling; Wu, Zhao-Hui

2012-01-01

Highlights: ► miR-22 is induced in cells treated with UV radiation. ► ATM is required for miR-22 induction in response to UV. ► miR-22 targets 3′-UTR of PTEN to repress its expression in UV-treated cells. ► Upregulated miR-22 inhibits apoptosis in cells exposed to UV. -- Abstract: DNA damage response upon UV radiation involves a complex network of cellular events required for maintaining the homeostasis and restoring genomic stability of the cells. As a new class of players involved in DNA damage response, the regulation and function of microRNAs in response to UV remain poorly understood. Here we show that UV radiation induces a significant increase of miR-22 expression, which appears to be dependent on the activation of DNA damage responding kinase ATM (ataxia telangiectasia mutated). Increased miR-22 expression may result from enhanced miR-22 maturation in cells exposed to UV. We further found that tumor suppressor gene phosphatase and tensin homolog (PTEN) expression was inversely correlated with miR-22 induction and UV-induced PTEN repression was attenuated by overexpression of a miR-22 inhibitor. Moreover, increased miR-22 expression significantly inhibited the activation of caspase signaling cascade, leading to enhanced cell survival upon UV radiation. Collectively, these results indicate that miR-22 is an important player in the cellular stress response upon UV radiation, which may promote cell survival via the repression of PTEN expression.

Differential role of PTEN in transforming growth factor β (TGF-β) effects on proliferation and migration in prostate cancer cells.

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Kimbrough-Allah, Mawiyah N; Millena, Ana C; Khan, Shafiq A

2018-04-01

Transforming growth factor-β (TGF-β) acts as a tumor suppressor in normal epithelial cells but as a tumor promoter in advanced prostate cancer cells. PI3-kinase pathway mediates TGF-β effects on prostate cancer cell migration and invasion. PTEN inhibits PI3-kinase pathway and is frequently mutated in prostate cancers. We investigated possible role(s) of PTEN in TGF-β effects on proliferation and migration in prostate cancer cells. Expression of PTEN mRNA and proteins were determined using RT-PCR and Western blotting in RWPE1 and DU145 cells. We also studied the role of PTEN in TGF-β effects on cell proliferation and migration in DU145 cells after transient silencing of endogenous PTEN. Conversely, we determined the role of PTEN in cell proliferation and migration after over-expression of PTEN in PC3 cells which lack endogenous PTEN. TGF-β1 and TGF-β3 had no effect on PTEN mRNA levels but both isoforms increased PTEN protein levels in DU145 and RWPE1 cells indicating that PTEN may mediate TGF-β effects on cell proliferation. Knockdown of PTEN in DU145 cells resulted in significant increase in cell proliferation which was not affected by TGF-β isoforms. PTEN overexpression in PC3 cells inhibited cell proliferation. Knockdown of endogenous PTEN enhanced cell migration in DU145 cells, whereas PTEN overexpression reduced migration in PC3 cells and reduced phosphorylation of AKT in response to TGF-β. We conclude that PTEN plays a role in inhibitory effects of TGF-β on cell proliferation whereas its absence may enhance TGF-β effects on activation of PI3-kinase pathway and cell migration. © 2018 Wiley Periodicals, Inc.

The human PINK1 locus is regulated in vivo by a non-coding natural antisense RNA during modulation of mitochondrial function

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Scheele, Camilla; Petrovic, Natasa; Faghihi, Mohammad A

2007-01-01

BACKGROUND: Mutations in the PTEN induced putative kinase 1 (PINK1) are implicated in early-onset Parkinson's disease. PINK1 is expressed abundantly in mitochondria rich tissues, such as skeletal muscle, where it plays a critical role determining mitochondrial structural integrity in Drosophila. ...

Tumor suppressor PTEN affects tau phosphorylation: deficiency in the phosphatase activity of PTEN increases aggregation of an FTDP-17 mutant Tau

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Zhang Xue

2006-07-01

Full Text Available Abstract Background Aberrant hyperphosphorylation of tau protein has been implicated in a variety of neurodegenerative disorders. Although a number of protein kinases have been shown to phosphorylate tau in vitro and in vivo, the molecular mechanisms by which tau phosphorylation is regulated pathophysiologically are largely unknown. Recently, a growing body of evidence suggests a link between tau phosphorylation and PI3K signaling. In this study, phosphorylation, aggregation and binding to the microtubule of a mutant frontal temporal dementia and parkinsonism linked to chromosome 17 (FTDP-17 tau in the presence of tumor suppressor PTEN, a major regulatory component in PI3K signaling, were investigated. Results Phosphorylation of the human mutant FTDP-17 tau, T40RW, was evaluated using different phospho-tau specific antibodies in the presence of human wild-type or phosphatase activity null mutant PTEN. Among the evaluated phosphorylation sites, the levels of Ser214 and Thr212 phospho-tau proteins were significantly decreased in the presence of wild-type PTEN, and significantly increased when the phosphatase activity null mutant PTEN was ectopically expressed. Fractionation of the mutant tau transfected cells revealed a significantly increased level of soluble tau in cytosol when wild-type PTEN was expressed, and an elevated level of SDS-soluble tau aggregates in the presence of the mutant PTEN. In addition, the filter/trap assays detected more SDS-insoluble mutant tau aggregates in the cells overexpressing the mutant PTEN compared to those in the cells overexpressing wild-type PTEN and control DNA. This notion was confirmed by the immunocytochemical experiment which demonstrated that the overexpression of the phosphatase activity null mutant PTEN caused the mutant tau to form aggregates in the COS-7 cells. Conclusion Tumor suppressor PTEN can alleviate the phosporylation of the mutant FTDP-17 tau at specific sites, and the phosphatase activity

The 5-lipoxygenase inhibitor tepoxalin induces oxidative damage and altered PTEN status prior to apoptosis in canine osteosarcoma cell lines.

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Loftus, J P; Cavatorta, D; Bushey, J J; Levine, C B; Sevier, C S; Wakshlag, J J

2016-06-01

The 5-lipoxygenase (5-LOX) inhibitor tepoxalin has been shown to slow canine osteosarcoma (OSA) tumour xenografts growth, yet the mechanisms are poorly elucidated. Further examination of tepoxalin in canine OSA cell lines shows that tepoxalin treated cells undergo apoptosis through caspase-3 activation and annexin staining. Interestingly, apoptosis is superseded by an increase in reactive oxygen species (ROS), as measured by activation of dihydrorhodamine 123 and mitosox. This increase in ROS appears to be related to the 5-LOX inhibitor regardless of cellular 5-LOX status, and was not observed after treatment with the tepoxalin metabolite RWJ20142. Additionally, 5-LOX inhibition by tepoxalin appears to increase phosphatase and tensin (PTEN) homolog activity by preventing its alkylation or oxidation. PTEN modification or inhibition allows phosphoinositide-3 (PI3) kinase activity thereby heightening activation of protein kinase B (AKT) phosphorylation. Our data suggest that off target oxidation and LOX inhibition play roles in the apoptotic response. © 2014 John Wiley & Sons Ltd.

Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

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Qiao, Lan [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Paul, Pritha; Lee, Sora [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Qiao, Jingbo [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Wang, Yongsheng [Department of Pharmaceutical Sciences, Jilin University, Changchun 130021 (China); Chung, Dai H., E-mail: dai.chung@vanderbilt.edu [Department of Pediatric Surgery, Vanderbilt University Medical Center, Nashville, TN 37232 (United States); Department of Cancer Biology, Vanderbilt University Medical Center, Nashville, TN 37232 (United States)

2013-05-31

Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma.

Differential regulation of cyclin-dependent kinase inhibitors in neuroblastoma cells

International Nuclear Information System (INIS)

Qiao, Lan; Paul, Pritha; Lee, Sora; Qiao, Jingbo; Wang, Yongsheng; Chung, Dai H.

2013-01-01

Highlights: •GRP-R signaling differentially regulated the expression of p21 and p27. •Silencing GRP/GRP-R downregulated p21, while p27 expression was upregulated. •Inhibition of GRP/GRP-R signaling enhanced PTEN expression, correlative to the increased expression of p27. •PTEN and p27 co-localized in cytoplasm and silencing PTEN decreased p27 expression. -- Abstract: Gastrin-releasing peptide (GRP) and its receptor (GRP-R) are highly expressed in undifferentiated neuroblastoma, and they play critical roles in oncogenesis. We previously reported that GRP activates the PI3K/AKT signaling pathway to promote DNA synthesis and cell cycle progression in neuroblastoma cells. Conversely, GRP-R silencing induces cell cycle arrest. Here, we speculated that GRP/GRP-R signaling induces neuroblastoma cell proliferation via regulation of cyclin-dependent kinase (CDK) inhibitors. Surprisingly, we found that GRP/GRP-R differentially induced expressions of p21 and p27. Silencing GRP/GRP-R decreased p21, but it increased p27 expressions in neuroblastoma cells. Furthermore, we found that the intracellular localization of p21 and p27 in the nuclear and cytoplasmic compartments, respectively. In addition, we found that GRP/GRP-R silencing increased the expression and accumulation of PTEN in the cytoplasm of neuroblastoma cells where it co-localized with p27, thus suggesting that p27 promotes the function of PTEN as a tumor suppressor by stabilizing PTEN in the cytoplasm. GRP/GRP-R regulation of CDK inhibitors and tumor suppressor PTEN may be critical for tumoriogenesis of neuroblastoma

PTEN Loss in E-Cadherin-Deficient Mouse Mammary Epithelial Cells Rescues Apoptosis and Results in Development of Classical Invasive Lobular Carcinoma

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Mirjam C. Boelens

2016-08-01

Full Text Available Invasive lobular carcinoma (ILC is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC, the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K signaling as a potential therapeutic strategy for targeting CLC.

Study on construction of pEgr-hPTEN expression vector induced by irradiation and its anti-tumor effect in vitro

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Tian Mei; Jin Guanghui; Piao Chunji; Li Xiuyi; Liu Linlin

2003-01-01

Objective: To clone the cDNA of human tumor suppressor gene-PTEN, construct pEgr-hPTEN expression vector induced by irradiation and study its inhibitory effect on proliferation of malignant glioma cell line SHG-44 transfected steadily with pEgr-hPTEN after different doses of X-ray irradiation. Methods: A DNA fragment about 1200 bp, PTEN, was amplified from human placenta tissues by using RT-nested PCR and was cloned into pUCm-T vector after automatic sequencing, then the fragment was inserted into a vector pcD-NA3.1-Egr to construct an expression vector pEgr-hPTEN. pEgr-hPTEN was transfected into SHG-44 cells in vitro. Stably transfected cell line SHG-44-sPTEN was selected through G418. The inhibitor effect on SHG-44-sPTEN was observed after different doses of X-ray irradiation in vitro. Results: The PTEN cDNA has been cloned correctly and its expression vector pEgr-hPTEN was also constructed. Growth of SHG-44 cells was inhibited significantly by stable pEgr-hPTEN transfection combined with X-ray irradiation. With the increase of dose, the inhibitory effect was enhanced within 5 Gy. Conclusion: Human tumor suppressor gene-PTEN cDNA has been cloned and its expression vector has been constructed. The tumor was inhibited significantly by gene-radiotherapy in vitro. The result provides the theoretical and experimental basis for improvement of clinical radiotherapeutic effect on tumors

The PTEN protein: cellular localization and post-translational regulation.

Science.gov (United States)

Leslie, Nick R; Kriplani, Nisha; Hermida, Miguel A; Alvarez-Garcia, Virginia; Wise, Helen M

2016-02-01

The phosphatase and tensin homologue deleted on chromosome 10 (PTEN) phosphatase dephosphorylates PIP3, the lipid product of the class I PI 3-kinases, and suppresses the growth and proliferation of many cell types. It has been heavily studied, in large part due to its status as a tumour suppressor, the loss of function of which is observed through diverse mechanisms in many tumour types. Here we present a concise review of our understanding of the PTEN protein and highlight recent advances, particularly in our understanding of its localization and regulation by ubiquitination and SUMOylation. © 2016 Authors; published by Portland Press Limited.

PTEN differentially regulates expressions of ICAM-1 and VCAM-1 through PI3K/Akt/GSK-3β/GATA-6 signaling pathways in TNF-α-activated human endothelial cells.

Science.gov (United States)

Tsoyi, Konstantin; Jang, Hwa Jin; Nizamutdinova, Irina Tsoy; Park, Kyungok; Kim, Young Min; Kim, Hye Jung; Seo, Han Geuk; Lee, Jae Heun; Chang, Ki Churl

2010-11-01

Phosphotase and tensin homolog deleted on chromosome 10 (PTEN) is a potent negative regulator of PI3K/Akt pathway. Here, we tried to elucidate the role of PTEN in the regulation of endothelial adhesion molecules, vascular cell adhesion molecule (VCAM)-1 and intracellular adhesion molecule (ICAM)-1, induced by TNF-α in human endothelial cells (ECs). Transfection with PTEN overexpressing vector resulted in the significant decrease in phosphorylation of Akt in TNF-α-treated ECs. PTEN strongly inhibited VCAM-1 but not ICAM-1, however this inhibitory effect was reversed by co-transfection with constitutively active-Akt (CA-Akt-HA) in TNF-α-stimulated ECs. Additionally, silencing of PTEN with specific siRNA showed significant increase of phosphor-Akt compared with TNF-α alone treated ECs. siPTEN significantly upregulated VCAM-1 but was indifferent to ICAM-1 in TNF-α-treated cells. Further, chromatin immunoprecipitation (ChIP) assay showed that PTEN targets GATA-6 but not IRF-1 binding to VCAM-1 promoter. In addition, GATA-6 is associated with glycogen synthesis kinase-3beta (GSK-3β) which is in turn regulated by PTEN-dependent Akt activity. Finally, PTEN significantly prevented monocyte adhesion to TNF-α-induced ECs probably through VCAM-1 regulation. It is concluded that PTEN selectively inhibits expression of VCAM-1 but not ICAM-1 through modulation of PI3K/Akt/GSK-3β/GATA-6 signaling cascade in TNF-α-treated ECs. Copyright © 2010 Elsevier Ireland Ltd. All rights reserved.

A common variation of the PTEN gene is associated with peripheral insulin resistance

DEFF Research Database (Denmark)

Grinder-Hansen, L; Ribel-Madsen, R; Wojtaszewski, Jørgen

2016-01-01

. RESULTS: The minor G allele of PTEN rs11202614 was associated with elevated fasting plasma insulin levels and a decreased peripheral glucose disposal rate, but not with the hepatic insulin resistance index or insulin secretion measured as the first-phase insulin response and disposition index. The single...... nucleotide polymorphism was not associated with either PI3K or Akt activities. CONCLUSION: A common PTEN variation is associated with peripheral insulin resistance and subsequent risk of developing T2D. However, the association with insulin resistance is not explained by decreased proximal insulin signalling......AIM: Phosphatase and tensin homologue (PTEN) reduces insulin sensitivity by inhibiting the phosphatidylinositol 3-kinase (PI3K)/v-akt murine thymoma viral oncogene homologue (Akt) pathway. This study investigated how a common single nucleotide polymorphism near PTEN, previously associated...

PTEN Loss in E-Cadherin-Deficient Mouse Mammary Epithelial Cells Rescues Apoptosis and Results in Development of Classical Invasive Lobular Carcinoma.

Science.gov (United States)

Boelens, Mirjam C; Nethe, Micha; Klarenbeek, Sjoerd; de Ruiter, Julian R; Schut, Eva; Bonzanni, Nicola; Zeeman, Amber L; Wientjens, Ellen; van der Burg, Eline; Wessels, Lodewyk; van Amerongen, Renée; Jonkers, Jos

2016-08-23

Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary epithelium, we generated a mouse model of classical ILC (CLC), the main histological ILC subtype. While loss of E-cadherin induced cell dissemination and apoptosis, additional PTEN inactivation promoted cell survival and rapid formation of invasive mammary tumors that recapitulate the histological and molecular features, estrogen receptor (ER) status, growth kinetics, metastatic behavior, and tumor microenvironment of human CLC. Combined inactivation of E-cadherin and PTEN is sufficient to cause CLC development. These CLCs showed significant tumor regression upon BEZ235-mediated inhibition of PI3K signaling. In summary, this mouse model provides important insights into CLC development and suggests inhibition of phosphatidylinositol 3-kinase (PI3K) signaling as a potential therapeutic strategy for targeting CLC. Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.

Dermal Delivery of Constructs Encoding Cre Recombinase to Induce Skin Tumors in PtenLoxP/LoxP;BrafCA/+ Mice

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Marcel A. Deken

2016-12-01

Full Text Available Current genetically-engineered mouse melanoma models are often based on Tyr::CreERT2-controlled MAPK pathway activation by the BRAFV600E mutation and PI3K pathway activation by loss of PTEN. The major drawback of these models is the occurrence of spontaneous tumors caused by leakiness of the Tyr::CreERT2 system, hampering long-term experiments. To address this problem, we investigated several approaches to optimally provide local delivery of Cre recombinase, including injection of lentiviral particles, DNA tattoo administration and particle-mediated gene transfer, to induce melanomas in PtenLoxP/LoxP;BrafCA/+ mice lacking the Tyr::CreERT2 allele. We found that dermal delivery of the Cre recombinase gene under the control of a non-specific CAG promoter induced the formation of melanomas, but also keratoacanthoma and squamous cell carcinomas. Delivery of Cre recombinase DNA under the control of melanocyte-specific promoters in PtenLoxP/LoxP;BrafCA/+ mice resulted in sole melanoma induction. The growth rate and histological features of the induced tumors were similar to 4-hydroxytamoxifen-induced tumors in Tyr::CreERT2;PtenLoxP/LoxP;BrafCA/+ mice, while the onset of spontaneous tumors was prevented completely. These novel induction methods will allow long-term experiments in mouse models of skin malignancies.

Studies of variability in the PTEN gene among Danish caucasian patients with Type II diabetes mellitus

DEFF Research Database (Denmark)

Hansen, L; Jensen, J N; Ekstrøm, C T

2001-01-01

Phosphatase and tensin homologue deleted from chromosome ten (PTEN) has recently been characterized as a novel member in the expanding network of proteins regulating the intracellular effects of insulin. By dephosphorylation of phosphatidyl-inositol-(3, 4, 5)-trisphosphate (PIP3) the PTEN protein...... regulates the insulin-dependent phosphoinositide 3-kinase (PI3K) signalling cassette and accordingly might function as a regulator of insulin sensitivity in skeletal muscle and adipose tissue. In this study we tested PTEN as a candidate gene for insulin resistance and late-onset Type II (non...

PTEN C-Terminal Deletion Causes Genomic Instability and Tumor Development

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Zhuo Sun

2014-03-01

Full Text Available Tumor suppressor PTEN controls genomic stability and inhibits tumorigenesis. The N-terminal phosphatase domain of PTEN antagonizes the PI3K/AKT pathway, but its C-terminal function is less defined. Here, we describe a knockin mouse model of a nonsense mutation that results in the deletion of the entire Pten C-terminal region, referred to as PtenΔC. Mice heterozygous for PtenΔC develop multiple spontaneous tumors, including cancers and B cell lymphoma. Heterozygous deletion of the Pten C-terminal domain also causes genomic instability and common fragile site rearrangement. We found that Pten C-terminal disruption induces p53 and its downstream targets. Simultaneous depletion of p53 promotes metastasis without influencing the initiation of tumors, suggesting that p53 mainly suppresses tumor progression. Our data highlight the essential role of the PTEN C terminus in the maintenance of genomic stability and suppression of tumorigenesis.

DMA mitigates ionizing radiation induced damage in Balb/c mice through Akt/NFκB/PTEN pathway

International Nuclear Information System (INIS)

Tiwari, Vinod; Ranjan, Atul; Tandon, Vibha

2014-01-01

Ionizing radiation is associated with massive apoptosis in tumor as well as in radiosensitive organs. DMA, (5-(4-methylpiperazin-1-yl)-2-(2'-(3,4-dimethoxy-phenyl)-5'-benzimidazolyl) a cytoprotective radiomodulator, work in dual mode of action as free radical quencher and modifier of genomic instability caused by radiation. We observed 34% radioprotection with 50 mg/Kg bw intravenous dose of DMA in Balb/c mice at 8 Gy. DMA treatment before irradiation restored the normal crypts and villi architecture in Balb/c mice. The villi height was restored equivalent to control group in DMA treated animals, whereas, it was degenerated in irradiated animals. IR-induced apoptosis was reduced in spleen in presence of DMA as a result of preservation of splenic lymphocytes from radiation. This clearly exhibits the radioprotective ability of DMA to mitigate radiation induced tissue damage. IR-induced S phase check point was overcome by DMA. DMA promoted activation and phosphorylation of GSK3β through the activation of Akt in Balb/c mice. There was reduction in PTEN level in DMA pretreated mice where as it was upregulated in irradiated mice. Relative enhanced kinase activity of Akt was observed in DMA treated Balb/c mice and irradiated A549, MRC5 cell lines. There was no significant radioprotection in DMA treated Akt siRNA transfected cells in comparison to only Akt siRNA transfected cells with increasing dose of radiation. Akt activation was found in a dose-dependent manner by DMA through Luciferase reporter assay. We observed that DMA treated HEK cells transfected with control siRNA, resulted in less early apoptotic cells within 24h, but radiation (5 Gy) treated cells showed 20% early apoptotic cells within 3 h which were reduced to 12% at 3 h, 9% at 6 h and 8% at 24 h in DMA+radiation treated cells determined by Annexin V binding assay. Further molecular mRNA expression analysis of key regulatory genes unveil that DMA inhibited p21 and augmented Akt and Gadd45 in

BMP suppresses PTEN expression via RAS/ERK signaling.

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Beck, Stayce E; Carethers, John M

2007-08-01

Bone morphogenetic protein (BMP), a member of the transforming growth factor beta family, classically utilizes the SMAD signaling pathway for its growth suppressive effects,and loss of this signaling cascade may accelerate cell growth. In the colon cancer predisposition syndrome Juvenile Polyposis, as well as in the late progression stages of nonsyndromic colorectal cancers, SMAD4 function is typically abrogated. Here, we utilized the SMAD4-null SW480 colon cancer cell line to examine BMPs effect on a potential target gene, PTEN, and how its expression might be regulated. Initial treatment of the SMAD4-null cells with BMP resulted in mild growth suppression, but with prolonged exposure to BMP, the cells become growth stimulatory, which coincided with observed decreases in transcription and translation of PTEN, and with corresponding increases in phospho-AKT protein levels. BMP-induced PTEN suppression was mediated via the RAS/ERK pathway, as pharmacologic inhibition of RAS/ERK, or interference with protein function in the cytosol by DN-RAS prevented BMP-induced growth promotion and changes in PTEN levels, as did treatment with noggin, a BMP ligand inhibitor. Thus, BMP downregulates PTEN via RAS/ERK in a SMAD4-null environment that contributes to cell growth, and constitutes a SMAD4-independent but BMP-responsive signaling pathway.

PPAR, PTEN, and the Fight against Cancer

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Rosemary E. Teresi

2008-01-01

Full Text Available Peroxisome proliferator-activated receptor gamma (PPAR is a ligand-activated transcription factor, which belongs to the family of nuclear hormone receptors. Recent in vitro studies have shown that PPAR can regulate the transcription of phosphatase and tensin homolog on chromosome ten (PTEN, a known tumor suppressor. PTEN is a susceptibility gene for a number of disorders, including breast and thyroid cancer. Activation of PPAR through agonists increases functional PTEN protein levels that subsequently induces apoptosis and inhibits cellular growth, which suggests that PPAR may be a tumor suppressor. Indeed, several in vivo studies have demonstrated that genetic alterations of PPAR can promote tumor progression. These results are supported by observations of the beneficial effects of PPAR agonists in the in vivo cancer setting. These studies signify the importance of PPAR and PTEN's interaction in cancer prevention.

Characterization of novel non-clonal intrachromosomal rearrangements between the H4 and PTEN genes (H4/PTEN) in human thyroid cell lines and papillary thyroid cancer specimens

International Nuclear Information System (INIS)

Puxeddu, Efisio; Zhao Guisheng; Stringer, James R.; Medvedovic, Mario; Moretti, Sonia; Fagin, James A.

2005-01-01

The two main forms of RET rearrangement in papillary thyroid carcinomas (PTC) arise from intrachromosomal inversions fusing the tyrosine kinase domain of RET with either the H4 (RET/PTC1) or the ELE1/RFG genes (RET/PTC3). PTEN codes for a dual-specificity phosphatase and maps to chromosome 10q22-23. Germline mutations confer susceptibility to Cowden syndrome whereas somatic mutations or deletions are common in several sporadic human tumors. Decreased PTEN expression has been implicated in thyroid cancer development. We report the characterization of a new chromosome 10 rearrangement involving H4 and PTEN. The initial H4/PTEN rearrangement was discovered as a non-specific product of RT-PCR for RET/PTC1 in irradiated thyroid cell lines. Sequencing revealed a transcript consisting of exon 1 and 2 of H4 fused with exons 3-6 of PTEN. Nested RT-PCR with specific primers bracketing the breakpoints confirmed the H4/PTEN rearrangements in irradiated KAT-1 and KAT-50 cells. Additional H4/PTEN variants, generated by recombination of either exon 1 or exon 2 of H4 with exon 6 of PTEN, were found in non-irradiated KAK-1, KAT-50, ARO and NPA cells. Their origin through chromosomal recombination was confirmed by detection of the reciprocal PTEN/H4 product. H4/PTEN recombination was not a clonal event in any of the cell lines, as Southern blots with appropriate probes failed to demonstrate aberrant bands, and multicolor FISH of KAK1 cells with BAC probes for H4 and PTEN did not show a signal overlap in all cells. Based on PCR of serially diluted samples, the minimal frequency of spontaneous recombination between these loci was estimated to be approximately 1/10 6 cells. H4/PTEN products were found by nested RT-PCR in 4/14 normal thyroid tissues (28%) and 14/18 PTC (78%) (P < 0.01). H4/PTEN is another example of recombination involving the H4 locus, and points to the high susceptibility of thyroid cells to intrachromosomal gene rearrangements. As this also represents a plausible

Characterization of novel non-clonal intrachromosomal rearrangements between the H4 and PTEN genes (H4/PTEN) in human thyroid cell lines and papillary thyroid cancer specimens

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Puxeddu, Efisio [Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Zhao Guisheng [Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Stringer, James R. [Department of Molecular Genetics, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Medvedovic, Mario [Center for Biostatistic Service, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States); Moretti, Sonia [Dipartimento di Medicina Interna, Universita degli Studi di Perugia, Via E. dal Pozzo, Perugia 06126, (Italy); Fagin, James A. [Division of Endocrinology and Metabolism, University of Cincinnati College of Medicine, PO Box 670547, Cincinnati, OH 45267-0547 (United States)]. E-mail: james.fagin@uc.edu

2005-02-15

The two main forms of RET rearrangement in papillary thyroid carcinomas (PTC) arise from intrachromosomal inversions fusing the tyrosine kinase domain of RET with either the H4 (RET/PTC1) or the ELE1/RFG genes (RET/PTC3). PTEN codes for a dual-specificity phosphatase and maps to chromosome 10q22-23. Germline mutations confer susceptibility to Cowden syndrome whereas somatic mutations or deletions are common in several sporadic human tumors. Decreased PTEN expression has been implicated in thyroid cancer development. We report the characterization of a new chromosome 10 rearrangement involving H4 and PTEN. The initial H4/PTEN rearrangement was discovered as a non-specific product of RT-PCR for RET/PTC1 in irradiated thyroid cell lines. Sequencing revealed a transcript consisting of exon 1 and 2 of H4 fused with exons 3-6 of PTEN. Nested RT-PCR with specific primers bracketing the breakpoints confirmed the H4/PTEN rearrangements in irradiated KAT-1 and KAT-50 cells. Additional H4/PTEN variants, generated by recombination of either exon 1 or exon 2 of H4 with exon 6 of PTEN, were found in non-irradiated KAK-1, KAT-50, ARO and NPA cells. Their origin through chromosomal recombination was confirmed by detection of the reciprocal PTEN/H4 product. H4/PTEN recombination was not a clonal event in any of the cell lines, as Southern blots with appropriate probes failed to demonstrate aberrant bands, and multicolor FISH of KAK1 cells with BAC probes for H4 and PTEN did not show a signal overlap in all cells. Based on PCR of serially diluted samples, the minimal frequency of spontaneous recombination between these loci was estimated to be approximately 1/10{sup 6} cells. H4/PTEN products were found by nested RT-PCR in 4/14 normal thyroid tissues (28%) and 14/18 PTC (78%) (P < 0.01). H4/PTEN is another example of recombination involving the H4 locus, and points to the high susceptibility of thyroid cells to intrachromosomal gene rearrangements. As this also represents a

PTEN expression is consistent in colorectal cancer primaries and metastases and associates with patient survival

International Nuclear Information System (INIS)

Atreya, Chloe E; Sangale, Zaina; Xu, Nafei; Matli, Mary R; Tikishvili, Eliso; Welbourn, William; Stone, Steven; Shokat, Kevan M; Warren, Robert S

2013-01-01

Phosphatase and tensin homologue deleted on chromosome 10 (PTEN) negatively regulates the phosphoinositide-3-kinase (PI3K) signaling pathway. In colorectal cancer (CRC), observed frequencies of loss of PTEN expression, concordant expression in primary tumors and metastases, and the association of PTEN status with outcome vary markedly by detection method. We determined the degree to which PTEN expression is consistent in 70 matched human CRC primaries and liver metastases using a validated immunohistochemistry assay. We found loss of PTEN expression in 12.3% of assessable CRC primaries and 10.3% of assessable liver metastases. PTEN expression (positive or negative) was concordant in 98% of matched colorectal primaries and liver metastases. Next we related PTEN status to mutations in RAS and PI3K pathway genes (KRAS, NRAS, BRAF, and PIK3CA) and to overall survival (OS). PTEN expression was not significantly associated with the presence or absence of mutations in RAS or PI3K pathway genes. The median OS of patients whose tumors did not express PTEN was 9 months, compared to 49 months for patients whose tumors did express PTEN (HR = 6.25, 95% confidence intervals (CI) (1.98, 15.42), P = 0.0017). The association of absent PTEN expression with increased risk of death remained significant in multivariate analysis (HR = 6.31, 95% CI (2.03, 17.93), P = 0.0023). In summary, PTEN expression was consistent in matched CRC primaries and in liver metastases. Therefore, future investigations of PTEN in metastatic CRC can use primary tumor tissue. In patients with liver-only metastases, loss of PTEN expression predicted poor OS. We observed concordant PTEN expression in 98% of colorectal cancer (CRC) primary and liver metastasis pairs using a validated immunohistochemistry assay. Consistent PTEN expression at both disease sites is significant because tumor tissue is usually available from CRC primaries but not metastases. Loss of PTEN expression associated with poor survival of

PLZF mediates the PTEN/AKT/FOXO3a signaling in suppression of prostate tumorigenesis.

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JingPing Cao

Full Text Available Promyelocytic leukemia zinc finger (PLZF protein expression is closely related to the progression of human cancers, including prostate cancer (PCa. However, the according context of a signaling pathway for PLZF to suppress prostate tumorigenesis remains greatly unknown. Here we report that PLZF is a downstream mediator of the PTEN signaling pathway in PCa. We found that PLZF expression is closely correlated with PTEN expression in a cohort of prostate cancer specimens. Interestingly, both PTEN rescue and phosphoinositide 3-kinase (PI3K inhibitor LY294002 treatment increase the PLZF expression in prostate cancer cell lines. Further, luciferase reporter assay and chromatin immunoprecipitation assay demonstrate that FOXO3a, a transcriptional factor phosphorylated by PI3K/AKT, could directly bind to the promoter of PLZF gene. These results indicate that PTEN regulates PLZF expression by AKT/FOXO3a. Moreover, our animal experiments also demonstrate that PLZF is capable of inhibiting prostate tumorigenesis in vivo. Taken together, our study defines a PTEN/PLZF pathway and would shed new lights for developing therapeutic strategy of prostate cancer.

Reduced expression levels of PTEN are associated with decreased sensitivity of HCC827 cells to icotinib.

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Zhai, Yang; Zhang, Yanjun; Nan, Kejun; Liang, Xuan

2017-05-01

The clinical resistance of non-small cell lung cancer (NSCLC) to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) has been linked to EGFR T790M resistance mutations or MET amplifications. Additional mechanisms underlying EGFR-TKI drug resistance remain unclear. The present study demonstrated that icotinib significantly inhibited the proliferation and increased the apoptosis rate of HCC827 cells; the cellular mRNA and protein expression levels of phosphatase and tensin homolog (PTEN) were also significantly downregulated. To investigate the effect of PTEN expression levels on the sensitivity of HCC827 cells to icotinib, PTEN expression was silenced using a PTEN-specific small interfering RNA. The current study identified that the downregulation of PTEN expression levels may promote cellular proliferation in addition to decreasing the apoptosis of HCC827 cells, and may reduce the sensitivity of HCC827 cells to icotinib. These results suggested that reduced PTEN expression levels were associated with the decreased sensitivity of HCC827 cells to icotinib. Furthermore, PTEN expression levels may be a useful marker for predicting icotinib resistance and elucidating the resistance mechanisms underlying EGFR-mutated NSCLC.

Apoptosis and inactivation of the PI3-kinase pathway by tetrocarcin A in breast cancers

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Nakajima, Hiroo; Sakaguchi, Koichi; Fujiwara, Ikuya; Mizuta, Mitsuhiko; Tsuruga, Mie; Magae, Junji; Mizuta, Naruhiko

2007-01-01

A survival kinase, Akt, is a downstream factor in the phosphatidylinositide-3'-kinase-dependent pathway, which mediates many biological responses including glucose uptake, protein synthesis and the regulation of proliferation and apoptosis, which is assumed to contribute to acquisition of malignant properties of human cancers. Here we find that an anti-tumor antibiotic, tetrocarcin A, directly induces apoptosis of human breast cancer cells. The apoptosis is accompanied by the activation of a proteolytic cascade of caspases including caspase-3 and -9, and concomitantly decreases phosphorylation of Akt, PDK1, and PTEN, a tumor suppressor that regulates the activity of Akt through the dephosphorylation of polyphosphoinositides. Tetrocarcin A affected neither expression of Akt, PDK1, or PTEN, nor did it affect the expression of Bcl family members including Bcl-2, Bcl-X L , and Bax. These results suggest that tetrocarcin A could be a potent chemotherapeutic agent for human breast cancer targeting the phosphatidylinositide-3'-kinase/Akt signaling pathway

PTEN overexpression improves cisplatin-resistance of human ovarian cancer cells through upregulating KRT10 expression

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Wu, Huijuan; Wang, Ke; Liu, Wenxin; Hao, Quan

2014-01-01

Highlights: • Overexpression of PTEN enhanced the sensitivity of C13K cells to cisplatin. • KRT10 is a downstream molecule of PTEN involved in the resistance-reversing effect. • Overexpression of KRT10 enhanced the chemosensitivity of C13K cells to cisplatin. - Abstract: Multi-drug resistance (MDR) is a common cause of the failure of chemotherapy in ovarian cancer. PTEN, a tumor suppressor gene, has been demonstrated to be able to reverse cisplatin-resistance in ovarian cancer cell line C13K. However, the downstream molecules of PTEN involved in the resistance-reversing effect have not been completely clarified. Therefore, we screened the downstream molecules of PTEN and studied their interactions in C13K ovarian cancer cells using a 3D culture model. Firstly, we constructed an ovarian cancer cell line stably expressing PTEN, C13K/PTEN. MTT assay showed that overexpression of PTEN enhanced the sensitivity of C13K cells to cisplatin, but not to paclitaxel. Then we examined the differently expressed proteins that interacted with PTEN in C13K/PTEN cells with or without cisplatin treatment by co-immunoprecipitation. KRT10 was identified as a differently expressed protein in cisplatin-treated C13K/PTEN cells. Further study confirmed that cisplatin could induce upregulation of KRT10 mRNA and protein in C13K/PTEN cells and there was a directly interaction between KRT10 and PTEN. Forced expression of KRT10 in C13K cells also enhanced cisplatin-induced proliferation inhibition and apoptosis of C13K cells. In addition, KRT10 siRNA blocked cisplatin-induced proliferation inhibition of C13K/PTEN cells. In conclusion, our data demonstrate that KRT10 is a downstream molecule of PTEN which improves cisplatin-resistance of ovarian cancer and forced KRT10 overexpression may also act as a therapeutic method for overcoming MDR in ovarian cancer

MicroRNA-486–dependent modulation of DOCK3/PTEN/AKT signaling pathways improves muscular dystrophy–associated symptoms

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Alexander, Matthew S.; Casar, Juan Carlos; Motohashi, Norio; Vieira, Natássia M.; Eisenberg, Iris; Marshall, Jamie L.; Gasperini, Molly J.; Lek, Angela; Myers, Jennifer A.; Estrella, Elicia A.; Kang, Peter B.; Shapiro, Frederic; Rahimov, Fedik; Kawahara, Genri; Widrick, Jeffrey J.; Kunkel, Louis M.

2014-01-01

Duchenne muscular dystrophy (DMD) is caused by mutations in the gene encoding dystrophin, which results in dysfunctional signaling pathways within muscle. Previously, we identified microRNA-486 (miR-486) as a muscle-enriched microRNA that is markedly reduced in the muscles of dystrophin-deficient mice (Dmdmdx-5Cv mice) and in DMD patient muscles. Here, we determined that muscle-specific transgenic overexpression of miR-486 in muscle of Dmdmdx-5Cv mice results in reduced serum creatine kinase levels, improved sarcolemmal integrity, fewer centralized myonuclei, increased myofiber size, and improved muscle physiology and performance. Additionally, we identified dedicator of cytokinesis 3 (DOCK3) as a miR-486 target in skeletal muscle and determined that DOCK3 expression is induced in dystrophic muscles. DOCK3 overexpression in human myotubes modulated PTEN/AKT signaling, which regulates muscle hypertrophy and growth, and induced apoptosis. Furthermore, several components of the PTEN/AKT pathway were markedly modulated by miR-486 in dystrophin-deficient muscle. Skeletal muscle–specific miR-486 overexpression in Dmdmdx-5Cv animals decreased levels of DOCK3, reduced PTEN expression, and subsequently increased levels of phosphorylated AKT, which resulted in an overall beneficial effect. Together, these studies demonstrate that stable overexpression of miR-486 ameliorates the disease progression of dystrophin-deficient skeletal muscle. PMID:24789910

Cystic Fibrosis Transmembrane Conductance Regulator Attaches Tumor Suppressor PTEN to the Membrane and Promotes Anti Pseudomonas aeruginosa Immunity.

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Riquelme, Sebastián A; Hopkins, Benjamin D; Wolfe, Andrew L; DiMango, Emily; Kitur, Kipyegon; Parsons, Ramon; Prince, Alice

2017-12-19

The tumor suppressor PTEN controls cell proliferation by regulating phosphatidylinositol-3-kinase (PI3K) activity, but the participation of PTEN in host defense against bacterial infection is less well understood. Anti-inflammatory PI3K-Akt signaling is suppressed in patients with cystic fibrosis (CF), a disease characterized by hyper-inflammatory responses to airway infection. We found that Ptenl -/- mice, which lack the NH 2 -amino terminal splice variant of PTEN, were unable to eradicate Pseudomonas aeruginosa from the airways and could not generate sufficient anti-inflammatory PI3K activity, similar to what is observed in CF. PTEN and the CF transmembrane conductance regulator (CFTR) interacted directly and this interaction was necessary to position PTEN at the membrane. CF patients under corrector-potentiator therapy, which enhances CFTR transport to the membrane, have increased PTEN amounts. These findings suggest that improved CFTR trafficking could enhance P. aeruginosa clearance from the CF airway by activating PTEN-mediated anti-bacterial responses and might represent a therapeutic strategy. Published by Elsevier Inc.

Skin Aging-Dependent Activation of the PI3K Signaling Pathway via Downregulation of PTEN Increases Intracellular ROS in Human Dermal Fibroblasts

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Eun-Mi Noh

2016-01-01

Full Text Available Reactive oxygen species (ROS play a major role in both chronological aging and photoaging. ROS induce skin aging through their damaging effect on cellular constituents. However, the origins of ROS have not been fully elucidated. We investigated that ROS generation of replicative senescent fibroblasts is generated by the modulation of phosphatidylinositol 3,4,5-triphosphate (PIP3 metabolism. Reduction of the PTEN protein, which dephosphorylates PIP3, was responsible for maintaining a high level of PIP3 in replicative cells and consequently mediated the activation of the phosphatidylinositol-3-OH kinase (PI3K/Akt pathway. Increased ROS production was blocked by inhibition of PI3K or protein kinase C (PKC or by NADPH oxidase activating in replicative senescent cells. These data indicate that the signal pathway to ROS generation in replicative aged skin cells can be stimulated by reduced PTEN level. Our results provide new insights into skin aging-associated modification of the PI3K/NADPH oxidase signaling pathway and its relationship with a skin aging-dependent increase of ROS in human dermal fibroblasts.

Loss of PTEN causes SHP2 activation, making lung cancer cells unresponsive to IFN-γ

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Chen, Chia-Ling [Translational Research Center, Taipei Medical University, Taipei 110, Taiwan (China); Chiang, Tzu-Hui; Tseng, Po-Chun [Institute of Clinical Medicine, College of Medicine, National Cheng Kung University, Tainan 701, Taiwan (China); Wang, Yu-Chih [Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan (China); Lin, Chiou-Feng, E-mail: cflin2014@tmu.edu.tw [Department of Microbiology and Immunology, School of Medicine, College of Medicine, Taipei Medical University, Taipei 110, Taiwan (China); Graduate Institute of Medical Sciences, College of Medicine, Taipei Medical University, Taipei 110, Taiwan (China)

2015-10-23

Src homology-2 domain-containing phosphatase (SHP) 2, an oncogenic phosphatase, inhibits type II immune interferon (IFN)-γ signaling by subverting signal transducers and activators of transcription 1 tyrosine phosphorylation and activation. For cancer immunoediting, this study aimed to investigate the decrease of phosphatase and tensin homolog deleted on chromosome 10 (PTEN), a tumor suppressor protein, leading to cellular impairment of IFN-γ signaling. In comparison with human lung adenocarcinoma A549 cells, the natural PTEN loss in another human lung adenocarcinoma line, PC14PE6/AS2 cells, presents reduced responsiveness in IFN-γ-induced IFN regulatory factor 1 activation and CD54 expression. Artificially silencing PTEN expression in A549 cells also caused cells to be unresponsive to IFN-γ without affecting IFN-γ receptor expression. IFN-γ-induced inhibition of cell proliferation and cytotoxicity were demonstrated in A549 cells but were defective in PC14PE6/AS2 cells and in PTEN-deficient A549 cells. Aberrant activation of SHP2 by ROS was specifically shown in PC14PE6/AS2 cells and PTEN-deficient A549 cells. Inhibiting ROS and SHP2 rescued cellular responses to IFN-γ-induced cytotoxicity and inhibition of cell proliferation in PC14PE6/AS2 cells. These results demonstrate that a decrease in PTEN facilitates ROS/SHP2 signaling, causing lung cancer cells to become unresponsive to IFN-γ. - Highlights: • This study demonstrates that PTEN decrease causes cellular unresponsive to IFN-γ. • Lung cancer cells with PTEN deficiency show unresponsive to IFN-γ signaling. • PTEN decrease inhibits IFN-γ-induced CD54, cell proliferation inhibition, and cytotoxicity. • ROS-mediated SHP2 activation makes PTEN-deficient cells unresponsive to IFN-γ.

Novel receptor-like protein kinases induced by Erwinia carotovora and short oligogalacturonides in potato.

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Montesano, M; Kõiv, V; Mäe, A; Palva, E T

2001-11-01

summary Identification of potato genes responsive to cell wall-degrading enzymes of Erwinia carotovora resulted in the isolation of cDNA clones for four related receptor-like protein kinases. One of the putative serine-threonine protein kinases might have arisen through alternative splicing. These potato receptor-like kinases (PRK1-4) were highly equivalent (91-99%), most likely constituting a family of related receptors. All PRKs and four other plant RLKs share in their extracellular domain a conserved bi-modular pattern of cysteine repeats distinct from that in previously characterized plant RLKs, suggesting that they represent a new class of receptors. The corresponding genes were rapidly induced by E. carotovora culture filtrate (CF), both in the leaves and tubers of potato. Furthermore, the genes were transiently induced by short oligogalacturonides. The structural identity of PRKs and their induction pattern suggested that they constitute part of the early response of potato to E. carotovora infection.

Resistance to EGF receptor inhibitors in glioblastoma mediated by phosphorylation of the PTEN tumor suppressor at tyrosine 240.

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Fenton, Tim R; Nathanson, David; Ponte de Albuquerque, Claudio; Kuga, Daisuke; Iwanami, Akio; Dang, Julie; Yang, Huijun; Tanaka, Kazuhiro; Oba-Shinjo, Sueli Mieko; Uno, Miyuki; Inda, Maria del Mar; Wykosky, Jill; Bachoo, Robert M; James, C David; DePinho, Ronald A; Vandenberg, Scott R; Zhou, Huilin; Marie, Suely K N; Mischel, Paul S; Cavenee, Webster K; Furnari, Frank B

2012-08-28

Glioblastoma multiforme (GBM) is the most aggressive of the astrocytic malignancies and the most common intracranial tumor in adults. Although the epidermal growth factor receptor (EGFR) is overexpressed and/or mutated in at least 50% of GBM cases and is required for tumor maintenance in animal models, EGFR inhibitors have thus far failed to deliver significant responses in GBM patients. One inherent resistance mechanism in GBM is the coactivation of multiple receptor tyrosine kinases, which generates redundancy in activation of phosphoinositide-3'-kinase (PI3K) signaling. Here we demonstrate that the phosphatase and tensin homolog deleted on chromosome 10 (PTEN) tumor suppressor is frequently phosphorylated at a conserved tyrosine residue, Y240, in GBM clinical samples. Phosphorylation of Y240 is associated with shortened overall survival and resistance to EGFR inhibitor therapy in GBM patients and plays an active role in mediating resistance to EGFR inhibition in vitro. Y240 phosphorylation can be mediated by both fibroblast growth factor receptors and SRC family kinases (SFKs) but does not affect the ability of PTEN to antagonize PI3K signaling. These findings show that, in addition to genetic loss and mutation of PTEN, its modulation by tyrosine phosphorylation has important implications for the development and treatment of GBM.

MicroRNA-21 induces 5-fluorouracil resistance in human pancreatic cancer cells by regulating PTEN and PDCD4

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Wei, Xueju; Wang, Weibin; Wang, Lanlan; Zhang, Yuanyuan; Zhang, Xian; Chen, Mingtai; Wang, Fang; Yu, Jia; Ma, Yanni; Sun, Guotao

2016-01-01

Pancreatic cancer patients are often resistant to chemotherapy treatment, which results in poor prognosis. The objective of this study was to delineate the mechanism by which miR-21 induces drug resistance to 5-fluorouracil (5-FU) in human pancreatic cancer cells (PATU8988 and PANC-1). We report that PATU8988 cells resistant to 5-FU express high levels of miR-21 in comparison to sensitive primary PATU8988 cells. Suppression of miR-21 expression in 5-Fu-resistant PATU8988 cells can alleviate its 5-FU resistance. Meanwhile, lentiviral vector-mediated overexpression of miR-21 not only conferred resistance to 5-FU but also promoted proliferation, migration, and invasion of PATU8988 and PANC-1 cells. The proresistance effects of miR-21 were attributed to the attenuated expression of tumor suppressor genes, including PTEN and PDCD4. Overexpression of PTEN and PDCD4 antagonized miR-21-induced resistance to 5-FU and migration activity. Our work demonstrates that miR-21 can confer drug resistance to 5-FU in pancreatic cancer cells by regulating the expression of tumor suppressor genes, as the target genes of miR-21, PTEN and PDCD4 can rescue 5-FU sensitivity and the phenotypic characteristics disrupted by miR-21

E3 Ligase Subunit Fbxo15 and PINK1 Kinase Regulate Cardiolipin Synthase 1 Stability and Mitochondrial Function in Pneumonia

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Bill B. Chen

2014-04-01

Full Text Available Acute lung injury (ALI is linked to mitochondrial injury, resulting in impaired cellular oxygen utilization; however, it is unknown how these events are linked on the molecular level. Cardiolipin, a mitochondrial-specific lipid, is generated by cardiolipin synthase (CLS1. Here, we show that S. aureus activates a ubiquitin E3 ligase component, Fbxo15, that is sufficient to mediate proteasomal degradation of CLS1 in epithelia, resulting in decreased cardiolipin availability and disrupted mitochondrial function. CLS1 is destabilized by the phosphatase and tensin homolog (PTEN-induced putative kinase 1 (PINK1, which binds CLS1 to phosphorylate and regulates CLS1 disposal. Like Fbxo15, PINK1 interacts with and regulates levels of CLS1 through a mechanism dependent upon Thr219. S. aureus infection upregulates this Fbxo15-PINK1 pathway to impair mitochondrial integrity, and Pink1 knockout mice are less prone to S. aureus-induced ALI. Thus, ALI-associated disruption of cellular bioenergetics involves bioeffectors that utilize a phosphodegron to elicit ubiquitin-mediated disposal of a key mitochondrial enzyme.

PTEN genomic deletion predicts prostate cancer recurrence and is associated with low AR expression and transcriptional activity

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Choucair Khalil

2012-11-01

Full Text Available Abstract Background Prostate cancer (PCa, a leading cause of cancer death in North American men, displays a broad range of clinical outcome from relatively indolent to lethal metastatic disease. Several genomic alterations have been identified in PCa which may serve as predictors of progression. PTEN, (10q23.3, is a negative regulator of the phosphatidylinositol 3-kinase (PIK3/AKT survival pathway and a tumor suppressor frequently deleted in PCa. The androgen receptor (AR signalling pathway is known to play an important role in PCa and its blockade constitutes a commonly used treatment modality. In this study, we assessed the deletion status of PTEN along with AR expression levels in 43 primary PCa specimens with clinical follow-up. Methods Fluorescence In Situ Hybridization (FISH was done on formalin fixed paraffin embedded (FFPE PCa samples to examine the deletion status of PTEN. AR expression levels were determined using immunohistochemistry (IHC. Results Using FISH, we found 18 cases of PTEN deletion. Kaplan-Meier analysis showed an association with disease recurrence (P=0.03. Concurrently, IHC staining for AR found significantly lower levels of AR expression within those tumors deleted for PTEN (PPTEN deleted. We confirmed the predictive value of PTEN deletion in disease recurrence (P=0.03. PTEN deletion was also linked to diminished expression of PTEN (PP=0.02. Furthermore, gene set enrichment analysis revealed a diminished expression of genes downstream of AR signalling in PTEN deleted tumors. Conclusions Altogether, our data suggest that PTEN deleted tumors expressing low levels of AR may represent a worse prognostic subset of PCa establishing a challenge for therapeutic management.

PTEN phosphatase-independent maintenance of glandular morphology in a predictive colorectal cancer model system.

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Jagan, Ishaan C; Deevi, Ravi K; Fatehullah, Aliya; Topley, Rebecca; Eves, Joshua; Stevenson, Michael; Loughrey, Maurice; Arthur, Kenneth; Campbell, Frederick Charles

2013-11-01

Organotypic models may provide mechanistic insight into colorectal cancer (CRC) morphology. Three-dimensional (3D) colorectal gland formation is regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN) coupling of cell division cycle 42 (cdc42) to atypical protein kinase C (aPKC). This study investigated PTEN phosphatase-dependent and phosphatase-independent morphogenic functions in 3D models and assessed translational relevance in human studies. Isogenic PTEN-expressing or PTEN-deficient 3D colorectal cultures were used. In translational studies, apical aPKC activity readout was assessed against apical membrane (AM) orientation and gland morphology in 3D models and human CRC. We found that catalytically active or inactive PTEN constructs containing an intact C2 domain enhanced cdc42 activity, whereas mutants of the C2 domain calcium binding region 3 membrane-binding loop (M-CBR3) were ineffective. The isolated PTEN C2 domain (C2) accumulated in membrane fractions, but C2 M-CBR3 remained in cytosol. Transfection of C2 but not C2 M-CBR3 rescued defective AM orientation and 3D morphogenesis of PTEN-deficient Caco-2 cultures. The signal intensity of apical phospho-aPKC correlated with that of Na(+)/H(+) exchanger regulatory factor-1 (NHERF-1) in the 3D model. Apical NHERF-1 intensity thus provided readout of apical aPKC activity and associated with glandular morphology in the model system and human colon. Low apical NHERF-1 intensity in CRC associated with disruption of glandular architecture, high cancer grade, and metastatic dissemination. We conclude that the membrane-binding function of the catalytically inert PTEN C2 domain influences cdc42/aPKC-dependent AM dynamics and gland formation in a highly relevant 3D CRC morphogenesis model system.

PTEN Phosphatase-Independent Maintenance of Glandular Morphology in a Predictive Colorectal Cancer Model System

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Ishaan C. Jagan

2013-11-01

Full Text Available Organotypic models may provide mechanistic insight into colorectal cancer (CRC morphology. Three-dimensional (3D colorectal gland formation is regulated by phosphatase and tensin homologue deleted on chromosome 10 (PTEN coupling of cell division cycle 42 (cdc42 to atypical protein kinase C (aPKC. This study investigated PTEN phosphatase-dependent and phosphatase-independent morphogenic functions in 3D models and assessed translational relevance in human studies. Isogenic PTEN-expressing or PTEN-deficient 3D colorectal cultures were used. In translational studies, apical aPKC activity readout was assessed against apical membrane (AM orientation and gland morphology in 3D models and human CRC. We found that catalytically active or inactive PTEN constructs containing an intact C2 domain enhanced cdc42 activity, whereas mutants of the C2 domain calcium binding region 3 membrane-binding loop (M-CBR3 were ineffective. The isolated PTEN C2 domain (C2 accumulated in membrane fractions, but C2 M-CBR3 remained in cytosol. Transfection of C2 but not C2 M-CBR3 rescued defective AM orientation and 3D morphogenesis of PTEN-deficient Caco-2 cultures. The signal intensity of apical phospho-aPKC correlated with that of Na+/H+ exchanger regulatory factor-1 (NHERF-1 in the 3D model. Apical NHERF-1 intensity thus provided readout of apical aPKC activity and associated with glandular morphology in the model system and human colon. Low apical NHERF-1 intensity in CRC associated with disruption of glandular architecture, high cancer grade, and metastatic dissemination. We conclude that the membrane-binding function of the catalytically inert PTEN C2 domain influences cdc42/aPKC-dependent AM dynamics and gland formation in a highly relevant 3D CRC morphogenesis model system.

Putative tyrosine kinases expressed in K-562 human leukemia cells

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Partanen, J.; Maekelae, T.P.; Lehvaeslaiho, H.; Alitalo, K.; Alitalo, R.

1990-01-01

Tyrosine phosphorylation is important in the transmission of growth and differentiation signals; known tyrosine kinases include several oncoproteins and growth factor receptors. Interestingly, some differentiated cell types, such as erythrocytes and platelets contain high amounts of phosphotyrosine. The authors analyzed tyrosine kinases expressed in the K-562 chronic myelogenous leukemia cell line, which has a bipotential erythroid and megakaryoblastoid differentiation capacity. Analysis of 359 polymerase chain reaction-amplified cDNA clones led to the identification of 14 different tyrosine kinase-related sequences (JTK1-14). Two of the clones (JTK2 and JTK4) represent unusual members of the fibroblast growth factor receptor gene family, and the clones JTK5, JTK11, and JTK14 may also belong to the family of receptor tyrosine kinases but lack a close relationship to any known tyrosine kinase. Each of these different genes has its own characteristic expression pattern in K-562 cells and several other human tumor cell lines. In addition, the JTK11 and JTK14 mRNAs are induced during the megakaryoblastoid differentiation of K-562 cells. These tyrosine kinases may have a role in the differentiation of megakaryoblasts or in the physiology of platelets

Pten Regulates Retinal Amacrine Cell Number by Modulating Akt, Tgfβ, and Erk Signaling.

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Tachibana, Nobuhiko; Cantrup, Robert; Dixit, Rajiv; Touahri, Yacine; Kaushik, Gaurav; Zinyk, Dawn; Daftarian, Narsis; Biernaskie, Jeff; McFarlane, Sarah; Schuurmans, Carol

2016-09-07

All tissues are genetically programmed to acquire an optimal size that is defined by total cell number and individual cellular dimensions. The retina contains stereotyped proportions of one glial and six neuronal cell types that are generated in overlapping waves. How multipotent retinal progenitors know when to switch from making one cell type to the next so that appropriate numbers of each cell type are generated is poorly understood. Pten is a phosphatase that controls progenitor cell proliferation and differentiation in several lineages. Here, using a conditional loss-of-function strategy, we found that Pten regulates retinal cell division and is required to produce the full complement of rod photoreceptors and amacrine cells in mouse. We focused on amacrine cell number control, identifying three downstream Pten effector pathways. First, phosphoinositide 3-kinase/Akt signaling is hyperactivated in Pten conditional knock-out (cKO) retinas, and misexpression of constitutively active Akt (Akt-CA) in retinal explants phenocopies the reduction in amacrine cell production observed in Pten cKOs. Second, Akt-CA activates Tgfβ signaling in retinal explants, which is a negative feedback pathway for amacrine cell production. Accordingly, Tgfβ signaling is elevated in Pten cKO retinas, and epistatic analyses placed Pten downstream of TgfβRII in amacrine cell number control. Finally, Pten regulates Raf/Mek/Erk signaling levels to promote the differentiation of all amacrine cell subtypes, which are each reduced in number in Pten cKOs. Pten is thus a positive regulator of amacrine cell production, acting via multiple downstream pathways, highlighting its diverse actions as a mediator of cell number control. Despite the importance of size for optimal organ function, how individual cell types are generated in correct proportions is poorly understood. There are several ways to control cell number, including readouts of organ function (e.g., secreted hormones reach functional

δ-Tocopherol inhibits receptor tyrosine kinase-induced AKT activation in prostate cancer cells.

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Wang, Hong; Hong, Jungil; Yang, Chung S

2016-11-01

The cancer preventive activity of vitamin E is suggested by epidemiological studies and supported by animal studies with vitamin E forms, γ-tocopherol and δ-tocopherol (δ-T). Several recent large-scale cancer prevention trials with high dose of α-tocopherol, however, yielded disappointing results. Whether vitamin E prevents or promotes cancer is a serious concern. A better understanding of the molecular mechanisms of action of the different forms of tocopherols would enhance our understanding of this topic. In this study, we demonstrated that δ-T was the most effective tocopherol form in inhibiting prostate cancer cell growth, by inducing cell cycle arrest and apoptosis. By profiling the effects of δ-T on the cell signaling using the phospho-kinase array, we found that the most inhibited target was the phosphorylation of AKT on T308. Further study on the activation of AKT by EGFR and IGFR revealed that δ-T attenuated the EGF/IGF-induced activation of AKT (via the phosphorylation of AKT on T308 induced by the activation of PIK3). Expression of dominant active PIK3 and AKT in prostate cancer cell line DU145 in which PIK3, AKT, and PTEN are wild type caused the cells to be reflectory to the inhibition of δ-T, supporting that δ-T inhibits the PIK3-mediated activation of AKT. Our data also suggest that δ-T interferes with the EGF-induced EGFR internalization, which leads to the inhibition of the receptor tyrosine kinase-dependent activation of AKT. In summary, our results revealed a novel mechanism of δ-T in inhibiting prostate cancer cell growth, supporting the cancer preventive activity δ-T. © 2015 Wiley Periodicals, Inc. © 2015 Wiley Periodicals, Inc.

Hesperitin derivative-11 suppress hepatic stellate cell activation and proliferation by targeting PTEN/AKT pathway

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Li, Wan-xia; Chen, Xin; Yang, Yang; Huang, Hui-min; Li, Hai-di; Huang, Cheng; Meng, Xiao-ming; Li, Jun

2017-01-01

Hesperitin derivative (HD-11) is a monomeric compound derived from Hesperidin, which is a naturally occurring flavanone glycoside that exerts extensive clinical effects such as anti-inflammatory, anti-oxidant and anti-angiogenic. However, the role and fundamental mechanism of HD-11 in hepatic fibrosis are still unrevealed. In this study, HD-11 not only alleviates ECM deposition in rats with liver fibrosis, but also reduces the expression of α-SMA and col1a1 in TGF-β1-induced HSC-T6 cells. Moreover, it was demonstrated that HD-11 significantly promoted the expression of PTEN in vivo and in vitro. In order to evaluate the involvement of HD-11 in TGF-β1-induced HSC-T6 activation, a specific blocking agent of PTEN (bpv) and PTEN small interfering (si)-RNA-mediated silencing were used. Interestingly, HD-11 treatment couldn’t inhibit α-SMA and col1a1 expression on the basis of PTEN knockdown. On the contrary, over-expression of PTEN had an opposite effect on the expression of α-SMA and col1a1 in TGF-β1-induced HSC-T6 cells after treatment of HD-11. In addition, HD-11 remarkably inhibited the expression of p-AKT in vivo and in vitro. Taken together, all the above results indicate that HD-11 may play the part of an effective modulator of PTEN/AKT signaling pathway.

Phenotype selection reveals coevolution of muscle glycogen and protein and PTEN as a gate keeper for the accretion of muscle mass in adult female mice.

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Mandy Sawitzky

Full Text Available We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK, were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice.

Exosomes promote cetuximab resistance via the PTEN/Akt pathway in colon cancer cells.

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Zhang, S; Zhang, Y; Qu, J; Che, X; Fan, Y; Hou, K; Guo, T; Deng, G; Song, N; Li, C; Wan, X; Qu, X; Liu, Y

2017-11-13

Cetuximab is widely used in patients with metastatic colon cancer expressing wildtype KRAS. However, acquired drug resistance limits its clinical efficacy. Exosomes are nanosized vesicles secreted by various cell types. Tumor cell-derived exosomes participate in many biological processes, including tumor invasion, metastasis, and drug resistance. In this study, exosomes derived from cetuximab-resistant RKO colon cancer cells induced cetuximab resistance in cetuximab-sensitive Caco-2 cells. Meanwhile, exosomes from RKO and Caco-2 cells showed different levels of phosphatase and tensin homolog (PTEN) and phosphor-Akt. Furthermore, reduced PTEN and increased phosphorylated Akt levels were found in Caco-2 cells after exposure to RKO cell-derived exosomes. Moreover, an Akt inhibitor prevented RKO cell-derived exosome-induced drug resistance in Caco-2 cells. These findings provide novel evidence that exosomes derived from cetuximab-resistant cells could induce cetuximab resistance in cetuximab-sensitive cells, by downregulating PTEN and increasing phosphorylated Akt levels.

Genomic rearrangements of PTEN in prostate cancer

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Sopheap ePhin

2013-09-01

Full Text Available The phosphatase and tensin homolog gene on chromosome 10q23.3 (PTEN is a negative regulator of the PIK3/Akt survival pathway and is the most frequently deleted tumor suppressor gene in prostate cancer. Monoallelic loss of PTEN is present in up to 60% of localized prostate cancers and complete loss of PTEN in prostate cancer is linked to metastasis and androgen independent progression. Studies on the genomic status of PTEN in prostate cancer initially used a two-color fluorescence in-situ hybridization (FISH assay for PTEN copy number detection in formalin fixed paraffin embedded tissue preparations. More recently, a four-color FISH assay containing two additional control probes flanking the PTEN locus with a lower false-positive rate was reported. Combined with the detection of other critical genomic biomarkers for prostate cancer such as ERG, AR, and MYC, the evaluation of PTEN genomic status has proven to be invaluable for patient stratification and management. Although less frequent than allelic deletions, point mutations in the gene and epigenetic silencing are also known to contribute to loss of PTEN function, and ultimately to prostate cancer initiation. Overall, it is clear that PTEN is a powerful biomarker for prostate cancer. Used as a companion diagnostic for emerging therapeutic drugs, FISH analysis of PTEN is promisingly moving human prostate cancer closer to more effective cancer management and therapies.

A Critical Role of the PTEN/PDGF Signaling Network for the Regulation of Radiosensitivity in Adenocarcinoma of the Prostate

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Christensen, Michael, E-mail: mechristense@uwalumni.com [Department of Radiation Oncology, Wayne State University School of Medicine, Barbara Ann Karmanos Cancer Center, Detroit, Michigan (United States); Najy, Abdo J. [Department of Pathology, Wayne State University School of Medicine, Barbara Ann Karmanos Cancer Center, Detroit, Michigan (United States); Snyder, Michael; Movilla, Lisa S. [Department of Radiation Oncology, Wayne State University School of Medicine, Barbara Ann Karmanos Cancer Center, Detroit, Michigan (United States); Kim, Hyeong-Reh Choi [Department of Pathology, Wayne State University School of Medicine, Barbara Ann Karmanos Cancer Center, Detroit, Michigan (United States)

2014-01-01

Purpose: Loss or mutation of the phosphate and tensin homologue (PTEN) is a common genetic abnormality in prostate cancer (PCa) and induces platelet-derived growth factor D (PDGF D) signaling. We examined the role of the PTEN/PDGF axis on radioresponse using a murine PTEN null prostate epithelial cell model. Methods and Materials: PTEN wild-type (PTEN{sup +/+}) and PTEN knockout (PTEN{sup −/−}) murine prostate epithelial cell lines were used to examine the relationship between the PTEN status and radiosensitivity and also to modulate the PDGF D expression levels. PTEN{sup −/−} cells were transduced with a small hairpin RNA (shRNA) lentiviral vector containing either scrambled nucleotides (SCRM) or sequences targeted to PDGF D (shPDGF D). Tumorigenesis and morphogenesis of these cell lines were evaluated in vivo via subcutaneous injection of male nude mice and in vitro using Matrigel 3-dimensional (3D) culture. Effects of irradiation on clonogenic survival, cell migration, and invasion were measured with respect to the PTEN status and the PDGF D expression level. In addition, apoptosis and cell cycle redistribution were examined as potential mechanisms for differences seen. Results: PTEN{sup −/−} cells were highly tumorigenic in animals and effectively formed foci in 3D culture. Importantly, loss of PDGF D in these cell lines drastically diminished these phenotypes. Furthermore, PTEN{sup −/−} cells demonstrated increased clonogenic survival in vitro compared to PTEN{sup +/+}, and attenuation of PDGF D significantly reversed this radioresistant phenotype. PTEN{sup −/−} cells displayed greater migratory and invasive potential at baseline as well as after irradiation. Both the basal and radiation-induced migratory and invasive phenotypes in PTEN{sup −/−} cells required PDGF D expression. Interestingly, these differences were independent of apoptosis and cell cycle redistribution, as they showed no significant difference. Conclusions: We propose

PTEN loss promotes intratumoral androgen synthesis and tumor microenvironment remodeling via aberrant activation of RUNX2 in castration-resistant prostate cancer

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Yang, Yinhui; Bai, Yang; He, Yundong; Zhao, Yu; Chen, Jiaxiang; Ma, Linlin; Pan, Yunqian; Hinten, Michael; Zhang, Jun; Karnes, R. Jeffrey; Kohli, Manish; Westendorf, Jennifer J.; Li, Benyi; Zhu, Runzhi; Huang, Haojie; Xu, Wanhai

2018-01-01

Purpose Intratumoral androgen synthesis (IAS) is a key mechanism promoting androgen receptor (AR)reactivation and anti-androgen resistance in castration-resistant prostate cancer (CRPC). However, signaling pathways driving aberrant IAS remain poorly understood. Experimental Design The effect of components of the AKT-RUNX2-osteocalcin (OCN)-GPRC6A-CREB signaling axis on expression of steroidogenesis genes CYP11A1 and CYP17A1 and testosterone level were examined in PTEN-null human PCa cell lines. Pten knockout mice were employed to examine the effect of Runx2 heterozygous deletion or abiraterone acetate (ABA), a prodrug of the CYP17A1 inhibitor abiraterone on Cyp11a1 and Cyp17a1 expression, testosterone level and tumor microenvironment (TME) remodeling in vivo. Results We uncovered that activation of the AKT-RUNX2-OCN-GPRC6A-CREB signaling axis induced expression of CYP11A1 and CYP17A1 and testosterone production in PTEN-null PCa cell lines in culture. Deletion of Runx2 in Pten homozygous knockout prostate tumors decreased Cyp11a1 and Cyp17a1 expression, testosterone level and tumor growth in castrated mice. ABA treatment also inhibited testosterone synthesis and alleviated Pten loss-induced tumorigenesis in vivo. Pten deletion induced TME remodeling, but Runx2 heterozygous deletion or ABA treatment reversed the effect of Pten loss by decreasing expression of the collagenase Mmp9. Conclusions Abnormal RUNX2 activation plays a pivotal role in PTEN loss-induced IAS and TME remodeling, suggesting that the identified signaling cascade represents a viable target for effective treatment of PTEN-null PCa including CRPC. PMID:29167276

The Involvement of Phosphatase and Tensin Homolog Deleted on Chromosome Ten (PTEN in the Regulation of Inflammation Following Coronary Microembolization

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Jiangyou Wang

2014-06-01

Full Text Available Background/Aims: Growing evidence shows that phosphatase and tensin homolog deleted on chromosome ten (PTEN is involved in regulating inflammation in different pathological conditions. Therefore, we hypothesized that the upregulation of PTEN correlates with the impairment of cardiac function in swine following coronary microembolization (CME. Methods: To possibly disclose an anti-inflammatory effect of PTEN, we induced swine CME by injecting inertia plastic microspheres (42 μm in diameter into the left anterior descending coronary artery and analyzed the myocardial tissue by immunochemistry, qRT-PCR and western blot analyses. In addition, we downregulated PTEN using siRNA. Results: Following CME, PTEN mRNA and protein levels were elevated as early as 3 h, peaked at 12 h, and then continuously decreased at 24 h and 48 h but remained elevated. Through linear correlation analysis, the PTEN protein level positively correlated with cTnI and TNF-α but was negatively correlated with LVEF. Furthermore, PTEN siRNA reduced the microinfarct volume, improved cardiac function (LVEF, reduced the release of cTnI, and suppressed PTEN and TNF-α protein expression. Conclusion: This study demonstrated, for the first time, that PTEN is involved in CME-induced inflammatory injury. The data generated from this study provide a rationale for the development of PTEN-based anti-inflammatory strategies.

Four MicroRNAs Promote Prostate Cell Proliferation with Regulation of PTEN and Its Downstream Signals In Vitro

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Xue, Jing-lun; Chen, Jin-zhong

2013-01-01

Background Phosphatase and tensin homologue (PTEN), as a tumor suppressor, plays vital roles in tumorigenesis and progression of prostate cancer. However, the mechanisms of PTEN regulation still need further investigation. We here report that a combination of four microRNAs (miR-19b, miR-23b, miR-26a and miR-92a) promotes prostate cell proliferation by regulating PTEN and its downstream signals in vitro. Methodology/Principal Findings We found that the four microRNAs (miRNAs) could effectively suppress PTEN expression by directly interacting with its 3’ UTR in prostate epithelial and cancer cells. Under-expression of the four miRNAs by antisense neutralization up-regulates PTEN expression, while overexpression of the four miRNAs accelerates epithelial and prostate cancer cell proliferation. Furthermore, the expression of the four miRNAs could, singly or jointly, alter the expression of the key components in the phosphoinositide 3-kinase (PI3K)/Akt pathway, including PIK3CA, PIK3CD, PIK3R1 and Akt, along with their downstream signal, cyclin D1. Conclusions These results suggested that the four miRNAs could promote prostate cancer cell proliferation by co-regulating the expression of PTEN, PI3K/Akt pathway and cyclin D1 in vitro. These findings increase understanding of the molecular mechanisms of prostate carcinogenesis and progression, even provide valuable insights into the diagnosis, prognosis, and rational design of novel therapeutics for prostate cancer. PMID:24098737

Assessing PIK3CA and PTEN in Early-Phase Trials with PI3K/AKT/mTOR Inhibitors

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Filip Janku

2014-01-01

Full Text Available Despite a wealth of preclinical studies, it is unclear whether PIK3CA or phosphatase and tensin homolog (PTEN gene aberrations are actionable in the clinical setting. Of 1,656 patients with advanced, refractory cancers tested for PIK3CA or PTEN abnormalities, PIK3CA mutations were found in 9% (146/1,589, and PTEN loss and/or mutation was found in 13% (149/1,157. In multicovariable analysis, treatment with a phosphatidylinositol 3-kinase (PI3K/AKT/mammalian target of rapamycin (mTOR inhibitor was the only independent factor predicting response to therapy in individuals harboring a PIK3CA or PTEN aberration. The rate of stable disease ≥6 months/partial response reached 45% in a subgroup of individuals with H1047R PIK3CA mutations. Aberrations in the PI3K/AKT/mTOR pathway are common and potentially actionable in patients with diverse advanced cancers. This work provides further important clinical validation for continued and accelerated use of biomarker-driven trials incorporating rational drug combinations.

PTENα, a PTEN isoform translated through alternative initiation, regulates mitochondrial function and energy metabolism.

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Liang, Hui; He, Shiming; Yang, Jingyi; Jia, Xinying; Wang, Pan; Chen, Xi; Zhang, Zhong; Zou, Xiajuan; McNutt, Michael A; Shen, Wen Hong; Yin, Yuxin

2014-05-06

PTEN is one of the most frequently mutated genes in human cancer. It is known that PTEN has a wide range of biological functions beyond tumor suppression. Here, we report that PTENα, an N-terminally extended form of PTEN, functions in mitochondrial metabolism. Translation of PTENα is initiated from a CUG codon upstream of and in-frame with the coding region of canonical PTEN. Eukaryotic translation initiation factor 2A (eIF2A) controls PTENα translation, which requires a CUG-centered palindromic motif. We show that PTENα induces cytochrome c oxidase activity and ATP production in mitochondria. TALEN-mediated somatic deletion of PTENα impairs mitochondrial respiratory chain function. PTENα interacts with canonical PTEN to increase PINK1 protein levels and promote energy production. Our studies demonstrate the importance of eIF2A-mediated alternative translation for generation of protein diversity in eukaryotic systems and provide insights into the mechanism by which the PTEN family is involved in multiple cellular processes. Copyright © 2014 Elsevier Inc. All rights reserved.

Deletion of PTEN Produces Deficits in Conditioned Fear and Increases Fragile X Mental Retardation Protein

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Lugo, Joaquin N.; Smith, Gregory D.; Morrison, Jessica B.; White, Jessika

2013-01-01

The phosphatase and tensin homolog detected on chromosome 10 (PTEN) gene product modulates activation of the phosphatidylinositol 3-kinase (PI3K)/AKT pathway. The PI3K pathway has been found to be involved in the regulation of the fragile X mental retardation protein, which is important for long-term depression and in the formation of new…

PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70-KILODALTON HEAT SHOCK PROTEIN HSPA2

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THE PUTATIVE CREATINE KINASE M-ISOFORM IN HUMAN SPERM IS IDENTIFIED AS THE 70 kDa HEAT SHOCK PROTEIN HSPA2* Gabor Huszar1, Kathryn Stone2, David Dix3 and Lynne Vigue11The Sperm Physiology Laboratory, Department of Obstetrics and Gynecology, 2 W.M. Keck Foundatio...

The role of PTEN in regulation of hepatic macrophages activation and function in progression and reversal of liver fibrosis

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Cheng, Yahui; Tian, Yuanyao; Xia, Jialu; Wu, Xiaoqin; Yang, Yang; Li, Xiaofeng; Huang, Cheng; Meng, Xiaoming; Ma, Taotao; Li, Jun, E-mail: lj@ahmu.edu.cn

2017-02-15

Activation of Kupffer cells (KCs) plays a pivotal role in the pathogenesis of liver fibrosis. The progression and reversal of CCl{sub 4}-induced mouse liver fibrosis showed a mixed induction of hepatic classical (M1) and alternative (M2) macrophage markers. Although the role of phosphatase and tensin homolog deleted on chromosome 10 (PTEN) in modulating myeloid cell activation has recently been identified, its function in macrophage activation during hepatic fibrosis remains to be fully appreciated. In our study, PTEN expression of KCs was remarkably decreased in CCl{sub 4}-induced mice but increased to a near-normal level in reversed mice. Moreover, PTEN was significantly decreased in IL4-induced RAW 264.7 cells in vitro and lower expression of PTEN was observed in M2 macrophages in vivo. In addition, loss- and gain-of-function studies suggested that PTEN regulates M2 macrophages polarization via activation of PI3K/Akt/STAT6 signaling, but had a limited effect on M1 macrophages polarization in vitro. Additionally, Ly294002, a chemical inhibitor of PI3K/Akt, could dramatically down-regulate the hallmarks of M2 macrophages. In conclusion, PTEN mediates macrophages activation by PI3K/Akt/STAT6 signaling pathway, which provides novel compelling evidences on the potential of PTEN in liver injury and opens new cellular target for the pharmacological therapy of liver fibrosis. - Highlights: • CCl{sub 4} treatment triggered a mixed M1/M2 macrophage phenotype in fibrosis. • Lower expression of PTEN in murine M2 macrophages in vivo and vitro. • PTEN modulates M2 macrophages activation via PI3K/Akt/STAT6 signaling. • Provide a new cellular target modulate macrophage mediated hepatic fibrosis.

Protein Kinase C Epsilon Cooperates with PTEN Loss for Prostate Tumorigenesis through the CXCL13-CXCR5 Pathway

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Rachana Garg

2017-04-01

Full Text Available Summary: PKCε, an oncogenic member of the PKC family, is aberrantly overexpressed in epithelial cancers. To date, little is known about functional interactions of PKCε with other genetic alterations, as well as the effectors contributing to its tumorigenic and metastatic phenotype. Here, we demonstrate that PKCε cooperates with the loss of the tumor suppressor Pten for the development of prostate cancer in a mouse model. Mechanistic analysis revealed that PKCε overexpression and Pten loss individually and synergistically upregulate the production of the chemokine CXCL13, which involves the transcriptional activation of the CXCL13 gene via the non-canonical nuclear factor κB (NF-κB pathway. Notably, targeted disruption of CXCL13 or its receptor, CXCR5, in prostate cancer cells impaired their migratory and tumorigenic properties. In addition to providing evidence for an autonomous vicious cycle driven by PKCε, our studies identified a compelling rationale for targeting the CXCL13-CXCR5 axis for prostate cancer treatment. : Garg et al. find that PKCε overexpression cooperates with Pten loss to promote prostate cancer in mice. These two alterations together confer enhanced growth, tumorigenic, migratory and invasive capabilities to prostate epithelial cells, and promote the release of CXCL13, an effect that is mediated by the non-canonical NF-κB pathway. Keywords: PKCε, PTEN, CXCL13, NF-κB, prostate cancer, CXCR5, migration, proliferation, transgenic mice

Curcumin prevents cisplatin-induced renal alterations in mitochondrial bioenergetics and dynamic.

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Ortega-Domínguez, Bibiana; Aparicio-Trejo, Omar Emiliano; García-Arroyo, Fernando E; León-Contreras, Juan Carlos; Tapia, Edilia; Molina-Jijón, Eduardo; Hernández-Pando, Rogelio; Sánchez-Lozada, Laura Gabriela; Barrera-Oviedo, Diana; Pedraza-Chaverri, José

2017-09-01

Cisplatin is widely used as chemotherapeutic agent for treatment of diverse types of cancer, however, acute kidney injury (AKI) is an important side effect of this treatment. Diverse mechanisms have been involved in cisplatin-induced AKI, such as oxidative stress, apoptosis and mitochondrial damage. On the other hand, curcumin is a polyphenol extracted from the rhizome of Curcuma longa L. Previous studies have shown that curcumin protects against the cisplatin-induced AKI; however, it is unknown whether curcumin can reduce alterations in mitochondrial bioenergetics and dynamic in this model. It was found that curcumin prevents cisplatin-induced: (a) AKI and (b) alterations in the following mitochondrial parameters: bioenergetics, ultrastructure, hydrogen peroxide production and dynamic. In fact, curcumin prevented the increase of mitochondrial fission 1 protein (FIS1), the decrease of optic atrophy 1 protein (OPA1) and the decrease of NAD + -dependent deacetylase sirtuin-3 (SIRT3), a mitochondrial dynamic regulator as well as the increase in the mitophagy associated proteins parkin and phosphatase and tensin homologue (PTEN)-induced putative kinase protein 1 (PINK1). In conclusion, the protective effect of curcumin in cisplatin-induced AKI was associated with the prevention of the alterations in mitochondrial bioenergetics, ultrastructure, redox balance, dynamic, and SIRT3 levels. Copyright © 2017 Elsevier Ltd. All rights reserved.

The Putative HORMA Domain Protein Atg101 Dimerizes and Is Required for Starvation-Induced and Selective Autophagy in Drosophila

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Krisztina Hegedűs

2014-01-01

Full Text Available The large-scale turnover of intracellular material including organelles is achieved by autophagy-mediated degradation in lysosomes. Initiation of autophagy is controlled by a protein kinase complex consisting of an Atg1-family kinase, Atg13, FIP200/Atg17, and the metazoan-specific subunit Atg101. Here we show that loss of Atg101 impairs both starvation-induced and basal autophagy in Drosophila. This leads to accumulation of protein aggregates containing the selective autophagy cargo ref(2P/p62. Mapping experiments suggest that Atg101 binds to the N-terminal HORMA domain of Atg13 and may also interact with two unstructured regions of Atg1. Another HORMA domain-containing protein, Mad2, forms a conformational homodimer. We show that Drosophila Atg101 also dimerizes, and it is predicted to fold into a HORMA domain. Atg101 interacts with ref(2P as well, similar to Atg13, Atg8a, Atg16, Atg18, Keap1, and RagC, a known regulator of Tor kinase which coordinates cell growth and autophagy. These results raise the possibility that the interactions and dimerization of the putative HORMA domain protein Atg101 play critical roles in starvation-induced autophagy and proteostasis, by promoting the formation of protein aggregate-containing autophagosomes.

C-reactive protein inhibits survivin expression via Akt/mTOR pathway downregulation by PTEN expression in cardiac myocytes.

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Beom Seob Lee

Full Text Available C-reactive protein (CRP is one of the most important biomarkers for arteriosclerosis and cardiovascular disease. Recent studies have shown that CRP affects cell cycle and inflammatory process in cardiac myocytes. Survivin is also involved in cardiac myocytes replication and apoptosis. Reduction of survivin expression is associated with less favorable cardiac remodeling in animal models. However, the effect of CRP on survivin expression and its cellular mechanism has not yet been studied. We demonstrated that treatment of CRP resulted in a significant decrease of survivin protein expression in a concentration-dependent manner in cardiac myocytes. The upstream signaling proteins of survivin, such as Akt, mTOR and p70S6K, were also downregulated by CRP treatment. In addition, CRP increased the protein and mRNA levels of PTEN. The siRNA transfection or specific inhibitor treatment for PTEN restored the CRP-induced downregulation of Akt/mTOR/p70S6K pathway and survivin protein expression. Moreover, pretreatment with a specific p53 inhibitor decreased the CRP-induced PTEN expression. ERK-specific inhibitor also blocked the p53 phosphorylation and PTEN expression induced by CRP. Our study provides a novel insight into CRP-induced downregulation of survivin protein expression in cardiac myocytes through mechanisms that involved in downregulation of Akt/mTOR/p70S6K pathway by expression of PTEN.

Overexpression of miR-21 in stem cells improves ovarian structure and function in rats with chemotherapy-induced ovarian damage by targeting PDCD4 and PTEN to inhibit granulosa cell apoptosis.

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Fu, Xiafei; He, Yuanli; Wang, Xuefeng; Peng, Dongxian; Chen, Xiaoying; Li, Xinran; Wang, Qing

2017-08-14

Chemotherapy-induced premature ovarian failure (POF) is a severe complication affecting tumor patients at a childbearing age. Mesenchymal stem cells (MSCs) can partially restore the ovarian structure and function damaged by chemotherapy. miR-21 is a microRNA that can regulate cell apoptosis. This study discusses the repair effect and mechanism of MSCs overexpressing miR-21 on chemotherapy-induced POF. Rat MSCs and granulosa cells (GCs) were isolated in vitro. MSCs were transfected with miR-21 lentiviral vector (LV-miR-21) to obtain MSCs stably expressing miR-21 (miR-21-MSCs). The microenvironment of an ovary receiving chemotherapy was mimicked by adding phosphamide mustard (PM) into the cellular culture medium. The apoptosis rate and the mRNA and protein expression of target genes PTEN and PDCD4 were detected in MSCs. Apoptosis was induced by adding PM into the culture medium for GCs, which were cocultured with miR-21-MSCs. The apoptosis rate and the mRNA and protein expression of PTEN and PDCD4 were detected. The chemotherapy-induced POF model was built into rats by intraperitoneal cyclophosphamide injection. miR-21-MSCs were transplanted into the bilateral ovary. The rats were sacrificed at 15, 30, 45, and 60 days after the last injection. The ovarian weights, follicle count, estrous cycle, and sex hormone levels (estradiol (E2) and follicle-stimulating hormone (FSH)) were detected. Apoptosis of GCs was determined by TUNEL assay. The miR-21 and mRNA and protein expression of PTEN and PDCD4 were determined. The apoptosis decreased in MSCs transfected with miR-21. The mRNA and protein expression of target genes PTEN and PDCD4 was downregulated. GCs cocultured with miR-21-MSCs showed a decreased apoptosis, an upregulation of miR-21, and a downregulation of PTEN and PDCD4. Following the injection of miR-21-MSCs, the ovarian weight and follicle counts increased; E 2 levels increased while FSH levels decreased, with less severe apoptosis of GCs. The miR-21 expression

Region-specific role for Pten in maintenance of epithelial phenotype and integrity

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Flodby, Per; Sunohara, Mitsuhiro; Castillo, Dan R.; McConnell, Alicia M.; Krishnaveni, Manda S.; Banfalvi, Agnes; Li, Min; Stripp, Barry; Zhou, Beiyun; Crandall, Edward D.; Minoo, Parviz

2017-01-01

Previous studies have demonstrated resistance to naphthalene-induced injury in proximal airways of mice with lung epithelial-specific deletion of the tumor-suppressor gene Pten, attributed to increased proliferation of airway progenitors. We tested effects of Pten loss following bleomycin injury, a model typically used to study distal lung epithelial injury, in conditional PtenSFTPC-cre knockout mice. Pten-deficient airway epithelium exhibited marked hyperplasia, particularly in small bronchioles and at bronchoalveolar duct junctions, with reduced E-cadherin and β-catenin expression between cells toward the luminal aspect of the hyperplastic epithelium. Bronchiolar epithelial and alveolar epithelial type II (AT2) cells in PtenSFTPC-cre mice showed decreased expression of epithelial markers and increased expression of mesenchymal markers, suggesting at least partial epithelial-mesenchymal transition at baseline. Surprisingly, and in contrast to previous studies, mutant mice were exquisitely sensitive to bleomycin, manifesting rapid weight loss, respiratory distress, increased early mortality (by day 5), and reduced dynamic lung compliance. This was accompanied by sloughing of the hyperplastic airway epithelium with occlusion of small bronchioles by cellular debris, without evidence of increased parenchymal lung injury. Increased airway epithelial cell apoptosis due to loss of antioxidant defenses, reflected by decreased expression of superoxide dismutase 3, in combination with deficient intercellular adhesion, likely predisposed to airway sloughing in knockout mice. These findings demonstrate an important role for Pten in maintenance of airway epithelial phenotype integrity and indicate that responses to Pten deletion in respiratory epithelium following acute lung injury are highly context-dependent and region-specific. PMID:27864284

Role played by Disabled-2 in albumin induced MAP Kinase signalling

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Diwakar, Ramaswamy; Pearson, Alexander L.; Colville-Nash, Paul; Baines, Deborah L.; Dockrell, Mark E.C.

2008-01-01

Albumin has been shown to activate the mitogen activated protein kinase (MAPK) pathway in proximal tubular cells (PTECs) of the kidney. Megalin, the putative receptor for albumin has potential signalling properties. However, the mechanisms by which megalin signals are unclear. The adaptor phosphoprotein Disabled-2 (Dab2) is known to interact with the cytoplasmic tail of megalin and may be involved in albumin-mediated MAPK signalling. In this study, we investigated the role of Dab2 in albumin-mediated MAPK signalling and further studied the role of Dab2 in albumin-induced TGFβ-1 secretion, a MAPK dependent event. We used RNA interference to knockdown Dab2 protein abundance in HKC-8 cells a model of human PTECs. Albumin activated ERK1,2 and Elk-1 in a MEK-1 dependent manner and resulted in secretion of TGFβ-1. In the absence of albumin, knockdown of Dab2 resulted in a trend towards increase in pERK1,2 consistent with its putative role as an inhibitor of cell proliferation. However albumin-induced ERK1,2 activation was completely abolished by Dab2 knockdown. Dab2 knockdown did not however result in inhibition of albumin-induced TGFβ-1 secretion. These results suggest that Dab2 is a ligand dependent bi-directional regulator of ERK1,2 activity by demonstrating that in addition to its more traditional role as an inhibitor of ERK1,2 it may also activate ERK1,2

Recombinant adenovirus-mediated overexpression of PTEN and KRT10 improves cisplatin resistance of ovarian cancer in vitro and in vivo.

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Wu, H; Wang, K; Liu, W; Hao, Q

2015-06-18

Drug resistance is a major cause of treatment failure in ovarian cancer patients, and novel therapeutic strategies are urgently needed. Overexpression of phosphatase and tensin homolog (PTEN) has been shown to preserve the cisplatin-resistance of ovarian cancer cells, while cisplatin-induced keratin 10 (KRT10) overexpression mediates the resistance-reversing effect of PTEN. However, whether overexpression of PTEN or KRT10 can improve the cisplatin resistance of ovarian cancer in vivo has not been investigated. Therefore, we investigated the effects of adenovirus-mediated PTEN or KRT10 overexpression on the cisplatin resistance of ovarian cancer in vivo. Recombinant adenoviruses carrying the gene for PTEN or KRT10 were constructed. The effects of overexpression of PTEN and KRT10 on cisplatin resistance of ovarian cancer cells were examined using the 3(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide (MTT) and TdT-mediated dUTP nick-end labeling (TUNEL) assays in vitro. Subcutaneously transplanted nude mice, as a model of human ovarian cancer, were used to test the effects of PTEN and KRT10 on cisplatin resistance of ovarian cancer in vivo. The MTT assay showed that recombinant adenovirus-mediated overexpression of KRT10 and PTEN enhanced the proliferation inhibition effect of cisplatin on C13K cells. Recombinant adenovirus-mediated overexpression of KRT10 and PTEN also increased the cisplatin-induced apoptosis rate of C13K cells. Furthermore, recombinant adenovirus-mediated overexpression of KRT10 and PTEN enhanced the inhibitory effect of cisplatin on C13K xenograft tumor growth. Thus, recombinant adenovirus-mediated overexpression of KRT10 and PTEN may improve the cisplatin resistance of ovarian cancer in vitro and in vivo.

Cytoplasm-predominant Pten associates with increased region-specific brain tyrosine hydroxylase and dopamine D2 receptors in mouse model with autistic traits.

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He, Xin; Thacker, Stetson; Romigh, Todd; Yu, Qi; Frazier, Thomas W; Eng, Charis

2015-01-01

Autism spectrum disorder (ASD) is a group of neurodevelopmental disorders characterized by impairment in social communication/interaction and inflexible/repetitive behavior. Several lines of evidence support genetic factors as a predominant cause of ASD. Among those autism susceptibility genes that have been identified, the PTEN tumor suppressor gene, initially identified as predisposing to Cowden heritable cancer syndrome, was found to be mutated in a subset of ASD patients with extreme macrocephaly. However, the ASD-relevant molecular mechanism mediating the effect of PTEN mutations remains elusive. We developed a Pten knock-in murine model to study the effects of Pten germline mutations, specifically altering subcellular localization, in ASD. Proteins were isolated from the hemispheres of the male littermates, and Western blots were performed to determine protein expression levels of tyrosine hydroxylase (TH). Immunohistochemical stains were carried out to validate the localization of TH and dopamine D2 receptors (D2R). PC12 cells ectopically expressing either wild-type or missense mutant PTEN were then compared for the differences in TH expression. Mice carrying Pten mutations have high TH and D2R in the striatum and prefrontal cortex. They also have increased phosphorylation of cAMP response element-binding protein (CREB) and TH. Mechanistically, PTEN downregulates TH production in PC12 cells via inhibiting the phosphoinositide 3-kinase (PI3K)/CREB signaling pathway, while PTEN reduces TH phosphorylation via suppressing MAPK pathway. Unlike wild-type PTEN but similar to the mouse knock-in mutant Pten, three naturally occurring missense mutations of PTEN that we previously identified in ASD patients, H93R, F241S, and D252G, were not able to suppress TH when overexpressed in PC12 cells. In addition, two other PTEN missense mutations, C124S (pan phosphatase dead) and G129E (lipid phosphatase dead), failed to suppress TH when ectopically expressed in PC12 cells

An integrative genomic and proteomic analysis of PIK3CA, PTEN and AKT mutations in breast cancer

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Stemke-Hale, Katherine; Gonzalez-Angulo, Ana Maria; Lluch, Ana; Neve, Richard M.; Kuo, Wen-Lin; Davies, Michael; Carey, Mark; Hu, Zhi; Guan, Yinghui; Sahin, Aysegul; Symmans, W. Fraser; Pusztai, Lajos; Nolden, Laura K.; Horlings, Hugo; Berns, Katrien; Hung, Mien-Chie; van de Vijver, Marc J.; Valero, Vicente; Gray, Joe W.; Bernards, Rene; Mills, Gordon B.; Hennessy, Bryan T.

2008-05-06

Phosphatidylinositol-3-kinase (PI3K)/AKT pathway aberrations are common in cancer. By applying mass spectroscopy-based sequencing and reverse phase protein arrays to 547 human breast cancers and 41 cell lines, we determined the subtype specificity and signaling effects of PIK3CA, AKT and PTEN mutations, and the effects of PIK3CA mutations on responsiveness to PI3K inhibition in-vitro and on outcome after adjuvant tamoxifen. PIK3CA mutations were more common in hormone receptor positive (33.8%) and HER2-positive (24.6%) than in basal-like tumors (8.3%). AKT1 (1.4%) and PTEN (2.3%) mutations were restricted to hormone receptor-positive cancers with PTEN protein levels also being significantly lower in hormone receptor-positive cancers. Unlike AKT1 mutations, PIK3CA (39%) and PTEN (20%) mutations were more common in cell lines than tumors, suggesting a selection for these but not AKT1 mutations during adaptation to culture. PIK3CA mutations did not have a significant impact on outcome in 166 hormone receptor-positive breast cancer patients after adjuvant tamoxifen. PIK3CA mutations, in comparison with PTEN loss and AKT1 mutations, were associated with significantly less and indeed inconsistent activation of AKT and of downstream PI3K/AKT signaling in tumors and cell lines, and PTEN loss and PIK3CA mutation were frequently concordant, suggesting different contributions to pathophysiology. PTEN loss but not PIK3CA mutations rendered cells sensitive to growth inhibition by the PI3K inhibitor LY294002. Thus, PI3K pathway aberrations likely play a distinct role in the pathogenesis of different breast cancer subtypes. The specific aberration may have implications for the selection of PI3K-targeted therapies in hormone receptor-positive breast cancer.

Downregulation of PI3-K/Akt/PTEN pathway and activation of mitochondrial intrinsic apoptosis by Diclofenac and Curcumin in colon cancer.

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Rana, Chandan; Piplani, Honit; Vaish, Vivek; Nehru, Bimla; Sanyal, S N

2015-04-01

Phosphatidylinositol 3-kinase (PI3-K)/PTEN/Akt signaling is over activated in various tumors including colon cancer. Activation of this pathway regulates multiple biological processes such as apoptosis, metabolism, cell proliferation, and cell growth that underlie the biology of a cancer cell. In the present study, the chemopreventive effects have been observed of Diclofenac, a preferential COX-2 inhibitory non-steroidal anti-inflammatory drugs, and Curcumin, a natural anti-inflammatory agent, in the early stage of colorectal carcinogenesis induced by 1,2-dimethylhydrazine dihydrochloride in rats. The tumor-promoting role of PI3-K/Akt/PTEN signal transduction pathway and its association with anti-apoptotic family of proteins are also observed. Both Diclofenac and Curcumin downregulated the PI3-K and Akt expression while promoting the apoptotic mechanism. Diclofenac and Curcumin administration significantly increased the expression of pro-apoptotic Bcl-2 family members (Bad and Bax) while decreasing the anti-apoptotic Bcl-2 protein. An up-regulation of cysteine protease family apoptosis executioner, such as caspase-3 and -9, is seen. Diclofenac and Curcumin inhibited the Bcl-2 protein by directly interacting at the active site by multiple hydrogen bonding, as also evident by negative glide score of Bcl-2. These drugs stimulated apoptosis by increasing reactive oxygen species (ROS) generation and simultaneously decreasing the mitochondrial membrane potential (ΔΨ M). Diclofenac and Curcumin showed anti-neoplastic effects by downregulating PI3-K/Akt/PTEN pathway, inducing apoptosis, increasing ROS generation, and decreasing ΔΨ M. The anti-neoplastic and apoptotic effects were found enhanced when both Diclofenac and Curcumin were administered together, rather than individually.

Loss of CDH1 and Pten accelerates cellular invasiveness and angiogenesis in the mouse uterus.

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Lindberg, Mallory E; Stodden, Genna R; King, Mandy L; MacLean, James A; Mann, Jordan L; DeMayo, Francesco J; Lydon, John P; Hayashi, Kanako

2013-07-01

E-cadherin (CDH1) is a cell adhesion molecule that coordinates key morphogenetic processes regulating cell growth, cell proliferation, and apoptosis. Loss of CDH1 is a trademark of the cellular event epithelial to mesenchymal transition, which increases the metastatic potential of malignant cells. PTEN is a tumor-suppressor gene commonly mutated in many human cancers, including endometrial cancer. In the mouse uterus, ablation of Pten induces epithelial hyperplasia, leading to endometrial carcinomas. However, loss of Pten alone does not affect longevity until around 5 mo. Similarly, conditional ablation of Cdh1 alone does not predispose mice to cancer. In this study, we characterized the impact of dual Cdh1 and Pten ablation (Cdh1(d/d) Pten(d/d)) in the mouse uterus. We observed that Cdh1(d/d) Pten(d/d) mice died at Postnatal Days 15-19 with massive blood loss. Their uteri were abnormally structured with curly horns, disorganized epithelial structure, and increased cell proliferation. Co-immunostaining of KRT8 and ACTA2 showed invasion of epithelial cells into the myometrium. Further, the uteri of Cdh1(d/d) Pten(d/d) mice had prevalent vascularization in both the endometrium and myometrium. We also observed reduced expression of estrogen and progesterone receptors, loss of cell adherens, and tight junction molecules (CTNNB1 and claudin), as well as activation of AKT in the uteri of Cdh1(d/d) Pten(d/d) mice. However, complex hyperplasia was not found in the uteri of Cdh1(d/d) Pten(d/d) mice. Collectively, these findings suggest that ablation of Pten with Cdh1 in the uterus accelerates cellular invasiveness and angiogenesis and causes early death.

MicroRNA-221 and -222 Regulate Radiation Sensitivity by Targeting the PTEN Pathway

International Nuclear Information System (INIS)

Zhang Chunzhi; Kang Chunsheng; Wang Ping; Cao Yongzhen; Lv Zhonghong; Yu Shizhu; Wang Guangxiu; Zhang Anling; Jia Zhifan; Han Lei; Yang Chunying; Ishiyama, Hiromichi; Teh, Bin S.; Xu Bo; Pu Peiyu

2011-01-01

Purpose: MicroRNAs (miRNAs) are noncoding RNAs inhibiting expression of numerous target genes by posttranscriptional regulation. miRNA-221 and miRNA-222 (miRNA-221/-222) expression is elevated in radioresistant tumor cell lines; however, it is not known whether and how miRNAs control cellular responses to ionizing irradiation. Methods and Materials: We used bioinformatic analyses, luciferase reporter assay, and genetic knockdown and biochemical assays to characterize the regulation pathways of miRNA-221/-222 in response to radiation treatment. Results: We identified the PTEN gene as a target of miRNA-221/-222. Furthermore, we found that knocking down miRNA-221/-222 by antisense oligonucleotides upregulated PTEN expression. Upregulated PTEN expression suppressed AKT activity and increased radiation-induced apoptosis, resulting in enhancement of radiosensitivity in tumor cells. Conclusions: miRNA-221/-222 control radiation sensitivity by regulating the PTEN/AKT pathway and can be explored as novel targets for radiosensitization.

Alkylation of the tumor suppressor PTEN activates Akt and β-catenin signaling: a mechanism linking inflammation and oxidative stress with cancer.

Directory of Open Access Journals (Sweden)

Tracy M Covey

2010-10-01

Full Text Available PTEN, a phosphoinositide-3-phosphatase, serves dual roles as a tumor suppressor and regulator of cellular anabolic/catabolic metabolism. Adaptation of a redox-sensitive cysteinyl thiol in PTEN for signal transduction by hydrogen peroxide may have superimposed a vulnerability to other mediators of oxidative stress and inflammation, especially reactive carbonyl species, which are commonly occurring by-products of arachidonic acid peroxidation. Using MCF7 and HEK-293 cells, we report that several reactive aldehydes and ketones, e.g. electrophilic α,β-enals (acrolein, 4-hydroxy-2-nonenal and α,β-enones (prostaglandin A(2, Δ12-prostaglandin J(2 and 15-deoxy-Δ-12,14-prostaglandin J(2 covalently modify and inactivate cellular PTEN, with ensuing activation of PKB/Akt kinase; phosphorylation of Akt substrates; increased cell proliferation; and increased nuclear β-catenin signaling. Alkylation of PTEN by α,β-enals/enones and interference with its restraint of cellular PKB/Akt signaling may accentuate hyperplastic and neoplastic disorders associated with chronic inflammation, oxidative stress, or aging.

TGF-β1 stimulates migration of type II endometrial cancer cells by down-regulating PTEN via activation of SMAD and ERK1/2 signaling pathways.

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Xiong, Siyuan; Cheng, Jung-Chien; Klausen, Christian; Zhao, Jianfang; Leung, Peter C K

2016-09-20

PTEN acts as a tumor suppressor primarily by antagonizing the PI3K/AKT signaling pathway. PTEN is frequently mutated in human cancers; however, in type II endometrial cancers its mutation rate is very low. Overexpression of TGF-β1 and its receptors has been reported to correlate with metastasis of human cancers and reduced survival rates. Although TGF-β1 has been shown to regulate PTEN expression through various mechanisms, it is not yet known if the same is true in type II endometrial cancer. In the present study, we show that treatment with TGF-β1 stimulates the migration of two type II endometrial cancer cell lines, KLE and HEC-50. In addition, TGF-β1 treatment down-regulates both mRNA and protein levels of PTEN. Overexpression of PTEN or inhibition of PI3K abolishes TGF-β1-stimulated cell migration. TGF-β1 induces SMAD2/3 phosphorylation and knockdown of common SMAD4 inhibits the suppressive effects of TGF-β1 on PTEN mRNA and protein. Interestingly, TGF-β1 induces ERK1/2 phosphorylation and pre-treatment with a MEK inhibitor attenuates the suppression of PTEN protein, but not mRNA, by TGF-β1. This study provides important insights into the molecular mechanisms mediating TGF-β1-induced down-regulation of PTEN and demonstrates an important role of PTEN in the regulation of type II endometrial cancer cell migration.

Aspergillus nidulans Natural Product Biosynthesis Is Regulated by MpkB, a Putative Pheromone Response Mitogen-Activated Protein Kinase

International Nuclear Information System (INIS)

Atoui, A.; Bao, D.; Kaur, N.; Grayburn, W.S.; Calvo, A.M.

2008-01-01

The Aspergillus nidulans putative mitogen-activated protein kinase encoded by mpkB has a role in natural product biosynthesis. An mpkB mutant exhibited a decrease in sterigmatocystin gene expression and low mycotoxin levels. The mutation also affected the expression of genes involved in penicillin and terrequinone A synthesis. mpkB was necessary for normal expression of laeA, which has been found to regulate secondary metabolism gene clusters. (author)

The AKT inhibitor AZD5363 is selectively active in PI3KCA mutant gastric cancer, and sensitizes a patient-derived gastric cancer xenograft model with PTEN loss to Taxotere.

Science.gov (United States)

Li, Jing; Davies, Barry R; Han, Sufang; Zhou, Minhua; Bai, Yu; Zhang, Jingchuan; Xu, Yan; Tang, Lily; Wang, Huiying; Liu, Yuan Jie; Yin, Xiaolu; Ji, Qunsheng; Yu, De-Hua

2013-10-02

Activation of the PI3K/AKT pathway is a common phenomenon in cancer due to multiple mechanisms, including mutation of PI3KCA, loss or mutation of PTEN, or over-expression of receptor tyrosine kinases. We recently developed a novel AKT kinase inhibitor, AZD5363, and demonstrated that HGC27, a cell line harboring both PI3KCA mutation and PTEN loss, displayed the greatest sensitivity to this AKT inhibitor in vitro and in vivo. To further elucidate the correlation between AZD5363 response and genetic alterations in gastric cancer (GC) and identify GC patients with both PI3KCA mutations and PTEN loss, we investigated the effects of pharmacological inhibition of AKT on a panel of 20 GC cell lines and genetic aberrations in tumor samples from a cohort of Chinese GC patients. We demonstrated that GC cells with PI3KCA mutations were selectively sensitive to AZD5363. Disease linkage studies showed that PI3KCA activating mutations or PTEN loss were found in 2.7% (4/150) and 23% (14/61) of Chinese GC patients respectively. To further dissect the role of PI3KCA mutation and PTEN loss in response to AKT inhibition, we tested the antitumor activity of AZD5363 in two patient-derived GC xenograft (PDGCX) models harboring either PI3KCA mutation or PTEN loss. Our data indicated that AZD5363 monotherapy treatment led to a moderate response in the PI3KCA mutant PDGCX model. Whilst monotherapy AZD5363 or Taxotere were ineffective in the PTEN negative PDGCX model, significant anti-tumor activity was observed when AZD5363 was combined with Taxotere. Our results indicated that PI3KCA mutation is an important determinant of response to AKT inhibition in GC and combination with AZD5363 can overcome innate resistance to Taxotere in a PTEN loss PDGCX model. It is suggested that AKT inhibitor is an attractive option for treatment of a new segment of GC patients with aberrant PI3K/AKT signaling.

Reciprocal feedback regulation of PI3K and androgen receptor signaling in PTEN-deficient prostate cancer.

Science.gov (United States)

Carver, Brett S; Chapinski, Caren; Wongvipat, John; Hieronymus, Haley; Chen, Yu; Chandarlapaty, Sarat; Arora, Vivek K; Le, Carl; Koutcher, Jason; Scher, Howard; Scardino, Peter T; Rosen, Neal; Sawyers, Charles L

2011-05-17

Prostate cancer is characterized by its dependence on androgen receptor (AR) and frequent activation of PI3K signaling. We find that AR transcriptional output is decreased in human and murine tumors with PTEN deletion and that PI3K pathway inhibition activates AR signaling by relieving feedback inhibition of HER kinases. Similarly, AR inhibition activates AKT signaling by reducing levels of the AKT phosphatase PHLPP. Thus, these two oncogenic pathways cross-regulate each other by reciprocal feedback. Inhibition of one activates the other, thereby maintaining tumor cell survival. However, combined pharmacologic inhibition of PI3K and AR signaling caused near-complete prostate cancer regressions in a Pten-deficient murine prostate cancer model and in human prostate cancer xenografts, indicating that both pathways coordinately support survival. Copyright © 2011 Elsevier Inc. All rights reserved.

Cell Cycle Control by PTEN.

Science.gov (United States)

Brandmaier, Andrew; Hou, Sheng-Qi; Shen, Wen H

2017-07-21

Continuous and error-free chromosome inheritance through the cell cycle is essential for genomic stability and tumor suppression. However, accumulation of aberrant genetic materials often causes the cell cycle to go awry, leading to malignant transformation. In response to genotoxic stress, cells employ diverse adaptive mechanisms to halt or exit the cell cycle temporarily or permanently. The intrinsic machinery of cycling, resting, and exiting shapes the cellular response to extrinsic stimuli, whereas prevalent disruption of the cell cycle machinery in tumor cells often confers resistance to anticancer therapy. Phosphatase and tensin homolog (PTEN) is a tumor suppressor and a guardian of the genome that is frequently mutated or deleted in human cancer. Moreover, it is increasingly evident that PTEN deficiency disrupts the fundamental processes of genetic transmission. Cells lacking PTEN exhibit cell cycle deregulation and cell fate reprogramming. Here, we review the role of PTEN in regulating the key processes in and out of cell cycle to optimize genomic integrity. Copyright © 2017 Elsevier Ltd. All rights reserved.

A phosphatase-independent gain-of-function mutation in PTEN triggers aberrant cell growth in astrocytes through an autocrine IGF-1 loop.

Science.gov (United States)

Fernández, S; Genis, L; Torres-Alemán, I

2014-08-07

Loss-of-function mutations in the phosphatase PTEN (phosphatase and tensin homolog deleted on chromosome10) contribute to aberrant cell growth in part through upregulation of the mitogenic IGF-1/PI3K/Akt pathway. In turn, this pathway exerts a homeostatic feedback over PTEN. Using mutagenesis analysis to explore a possible impact of this mutual control on astrocyte growth, we found that truncation of the C-terminal region of PTEN (Δ51) associates with a marked increase in NFκB activity, a transcription factor overactivated in astrocyte tumors. Whereas mutations of PTEN are considered to lead to a loss-of-function, PTENΔ51, a truncation that comprises a region frequently mutated in human gliomas, displayed a neomorphic (gain-of-function) activity that was independent of its phosphatase activity. This gain-of-function of PTENΔ51 includes stimulation of IGF-1 synthesis through protein kinase A activation of the IGF-1 promoter. Increased IGF-1 originates an autocrine loop that activates Akt and NFκB. Constitutive activation of NFκB in PTENΔ51-expressing astrocytes leads to aberrant cell growth; astrocytes expressing this mutant PTEN generate colonies in vitro and tumors in vivo. Mutations converting a tumor suppressor such as PTEN into a tumor promoter through a gain-of-function involving IGF-1 production may further our understanding of the role played by this growth factor in glioma growth and help us define druggable targets for personalized therapy.

PKA Phosphorylation of NCLX Reverses Mitochondrial Calcium Overload and Depolarization, Promoting Survival of PINK1-Deficient Dopaminergic Neurons

Directory of Open Access Journals (Sweden)

Marko Kostic

2015-10-01

Full Text Available Mitochondrial Ca2+ overload is a critical, preceding event in neuronal damage encountered during neurodegenerative and ischemic insults. We found that loss of PTEN-induced putative kinase 1 (PINK1 function, implicated in Parkinson disease, inhibits the mitochondrial Na+/Ca2+ exchanger (NCLX, leading to impaired mitochondrial Ca2+ extrusion. NCLX activity was, however, fully rescued by activation of the protein kinase A (PKA pathway. We further show that PKA rescues NCLX activity by phosphorylating serine 258, a putative regulatory NCLX site. Remarkably, a constitutively active phosphomimetic mutant of NCLX (NCLXS258D prevents mitochondrial Ca2+ overload and mitochondrial depolarization in PINK1 knockout neurons, thereby enhancing neuronal survival. Our results identify an mitochondrial Ca2+ transport regulatory pathway that protects against mitochondrial Ca2+ overload. Because mitochondrial Ca2+ dyshomeostasis is a prominent feature of multiple disorders, the link between NCLX and PKA may offer a therapeutic target.

Conditional Deletion of Pten Causes Bronchiolar Hyperplasia

OpenAIRE

Davé, Vrushank; Wert, Susan E.; Tanner, Tiffany; Thitoff, Angela R.; Loudy, Dave E.; Whitsett, Jeffrey A.

2007-01-01

Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (PtenΔ/Δ) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as in...

Diacylglycerol kinase regulation of protein kinase D during oxidative stress-induced intestinal cell injury

International Nuclear Information System (INIS)

Song Jun; Li Jing; Mourot, Joshua M.; Mark Evers, B.; Chung, Dai H.

2008-01-01

We recently demonstrated that protein kinase D (PKD) exerts a protective function during oxidative stress-induced intestinal epithelial cell injury; however, the exact role of DAG kinase (DGK)ζ, an isoform expressed in intestine, during this process is unknown. We sought to determine the role of DGK during oxidative stress-induced intestinal cell injury and whether DGK acts as an upstream regulator of PKD. Inhibition of DGK with R59022 compound or DGKζ siRNA transfection decreased H 2 O 2 -induced RIE-1 cell apoptosis as measured by DNA fragmentation and increased PKD phosphorylation. Overexpression of kinase-dead DGKζ also significantly increased PKD phosphorylation. Additionally, endogenous nuclear DGKζ rapidly translocated to the cytoplasm following H 2 O 2 treatment. Our findings demonstrate that DGK is involved in the regulation of oxidative stress-induced intestinal cell injury. PKD activation is induced by DGKζ, suggesting DGK is an upstream regulator of oxidative stress-induced activation of the PKD signaling pathway in intestinal epithelial cells

PIK3CA, HRAS and PTEN in human papillomavirus positive oropharyngeal squamous cell carcinoma

International Nuclear Information System (INIS)

Chiosea, Simion I; Nikiforova, Marina N; Grandis, Jennifer R; Lui, Vivian W Y; Diergaarde, Brenda; Maxwell, Jessica H; Ferris, Robert L; Kim, Seungwon W; Luvison, Alyssa; Miller, Megan

2013-01-01

Recent genomic evidence suggests frequent phosphatidylinositide 3-kinase (PI3K) pathway activation in human papillomavirus (HPV) positive oropharyngeal squamous cell carcinoma. Mutations/amplification of the gene encoding p110α catalytic subunit of phosphoinositide 3-kinase (PIK3CA), loss of phosphatase and tensin homolog (PTEN) and HRAS mutations are known to activate PI3K pathway. PIK3CA mutations were identified by Sanger sequencing in 23 of 75 (31%) HPV-positive oropharyngeal carcinomas, including exon 9 (p.E545K [n = 10] and p.E542K [n = 5]) or exon 20 (p.H1047Y, n = 2) mutations. Five rare and one novel (p.R537Q) PIK3CA mutations were identified. HRAS mutation (p.Q61L) was detected in 1 of 62 tested cases. PIK3CA amplification by fluorescence in situ hybridization (FISH) was identified in 4 cases (4/21, 20%), while PTEN loss was seen in 7 (7/21, 33%) cases (chromosome 10 monosomy [n = 4], homozygous deletion [n = 3]). Overall, genetic alterations that likely lead to PI3K pathway activation were identified in 34 of 75 cases (45%) and did not correlate with disease specific survival. These findings offer a molecular rationale for therapeutic targeting of PI3K pathway in patients with HPV-positive oropharyngeal carcinoma

PTEN status in advanced colorectal cancer treated with cetuximab

Science.gov (United States)

Negri, F V; Bozzetti, C; Lagrasta, C A; Crafa, P; Bonasoni, M P; Camisa, R; Pedrazzi, G; Ardizzoni, A

2009-01-01

Background: Loss of phosphatase and tensin homologue deleted in chromosome 10 (PTEN) function in advanced colorectal cancer (CRC) may represent one of the resistance mechanisms to cetuximab by interfering with the epidermal growth factor receptor signal transduction pathway. Methods: PTEN expression tested by indirect immunofluorescence was evaluated both on primary (n=43) and on metastatic (n=24) sites in CRC patients treated with cetuximab. Results: The loss of PTEN expression tested on metastatic sites was negatively associated with response (100% progressive disease (PD) in PTEN-negative cases vs 30% PD in PTEN-positive cases; P<0.05), PFS (0.8 vs 8.2 months; P<0.001) and OS (2.9 vs 14.2 months; P<0.001). Conclusion: A potential role of PTEN in the anti-tumour activity of cetuximab could be hypothesised. PMID:19953097

Lead (Pb) induced ATM-dependent mitophagy via PINK1/Parkin pathway.

Science.gov (United States)

Gu, Xueyan; Qi, Yongmei; Feng, Zengxiu; Ma, Lin; Gao, Ke; Zhang, Yingmei

2018-07-01

Lead (Pb), a widely distributed environmental pollutant, is known to induce mitochondrial damage as well as autophagy in vitro and in vivo. In this study, we found that Pb could trigger mitophagy in both HEK293 cells and the kidney cortex of male Kunming mice. However, whether ataxia telangiectasis mutated (ATM) which is reported to be linked with PTEN-induced putative kinase 1 (PINK1)/Parkin pathway (a well-characterized mitophagic pathway) participates in the regulation of Pb-induced mitophagy and its exact role remains enigmatic. Our results indicated that Pb activated ATM in vitro and in vivo, and further in vitro studies showed that ATM could co-localize with PINK1 and Parkin in cytosol and interact with PINK1. Knockdown of ATM by siRNA blocked Pb-induced mitophagy even under the circumstance of enhanced accumulation of PINK1 and mitochondrial Parkin. Intriguingly, elevation instead of reduction in phosphorylation level of PINK1 and Parkin was observed in response to ATM knockdown and Pb did not contribute to the further increase of their phosphorylation level, implying that ATM indirectly regulated PINK1/Parkin pathway. These findings reveal a novel mechanism for Pb toxicity and suggest the regulatory importance of ATM in PINK1/Parkin-mediated mitophagy. Copyright © 2018 Elsevier B.V. All rights reserved.

MicroRNA-103 Promotes Colorectal Cancer by Targeting Tumor Suppressor DICER and PTEN

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Li Geng

2014-05-01

Full Text Available MicroRNAs (miRNAs are a class of small, noncoding RNAs that act as key regulators in various physiological and pathological processes. However, the regulatory mechanisms for miRNAs in colorectal cancer remain largely unknown. Here, we found that miR-103 is up-regulated in colorectal cancer and its overexpression is closely associated with tumor proliferation and migration. In addition, repressing the expression of miR-103 apparently inhibits colorectal cancer cell proliferation and migration in vitro and HCT-116 xenograft tumor growth in vivo. Subsequent software analysis and dual-luciferase reporter assay identified two tumor suppressor genes DICER and PTEN as direct targets of miR-103, and up-regulation of DICER and PTEN obtained similar results to that occurred in the silencing of miR-103. In addition, restoration of DICER and PTEN can inhibit miR-103-induced colorectal cancer cell proliferation and migration. Our data collectively demonstrate that miR-103 is an oncogene miRNA that promotes colorectal cancer proliferation and migration through down-regulation of the tumor suppressor genes DICER and PTEN. Thus, miR-103 may represent a new potential diagnostic and therapeutic target for colorectal cancer treatment.

Hydrogen sulfide prevents hydrogen peroxide-induced activation of epithelial sodium channel through a PTEN/PI(3,4,5P3 dependent pathway.

Directory of Open Access Journals (Sweden)

Jianing Zhang

Full Text Available Sodium reabsorption through the epithelial sodium channel (ENaC at the distal segment of the kidney plays an important role in salt-sensitive hypertension. We reported previously that hydrogen peroxide (H2O2 stimulates ENaC in A6 distal nephron cells via elevation of phosphatidylinositol 3,4,5-trisphosphate (PI(3,4,5P3 in the apical membrane. Here we report that H2S can antagonize H2O2-induced activation of ENaC in A6 cells. Our cell-attached patch-clamp data show that ENaC open probability (PO was significantly increased by exogenous H2O2, which is consistent with our previous finding. The aberrant activation of ENaC induced by exogenous H2O2 was completely abolished by H2S (0.1 mM NaHS. Pre-treatment of A6 cells with H2S slightly decreased ENaC P(O; however, in these cells H2O2 failed to elevate ENaC PO . Confocal microscopy data show that application of exogenous H2O2 to A6 cells significantly increased intracellular reactive oxygen species (ROS level and induced accumulation of PI(3,4,5P3 in the apical compartment of the cell membrane. These effects of exogenous H2O2 on intracellular ROS levels and on apical PI(3,4,5P3 levels were almost completely abolished by treatment of A6 cells with H2S. In addition, H2S significantly inhibited H2O2-induced oxidative inactivation of the tumor suppressor phosphatase and tensin homolog (PTEN which is a negative regulator of PI(3,4,5P3. Moreover, BPV(pic, a specific inhibitor of PTEN, elevated PI(3,4,5P3 and ENaC activity in a manner similar to that of H2O2 in A6 cells. Our data show, for the first time, that H2S prevents H2O2-induced activation of ENaC through a PTEN-PI(3,4,5P3 dependent pathway.

Conditional deletion of Pten causes bronchiolar hyperplasia.

Science.gov (United States)

Davé, Vrushank; Wert, Susan E; Tanner, Tiffany; Thitoff, Angela R; Loudy, Dave E; Whitsett, Jeffrey A

2008-03-01

Tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a lipid phosphatase that regulates multiple cellular processes including cell polarity, migration, proliferation, and carcinogenesis. In this work, we demonstrate that conditional deletion of Pten (Pten(Delta/Delta)) in the respiratory epithelial cells of the developing mouse lung caused epithelial cell proliferation and hyperplasia as early as 4 to 6 weeks of age. While bronchiolar cell differentiation was normal, as indicated by beta-tubulin and FOXJ1 expression in ciliated cells and by CCSP expression in nonciliated cells, cell proliferation (detected by expression of Ki-67, phospho-histone-H3, and cyclin D1) was increased and associated with activation of the AKT/mTOR survival pathway. Deletion of Pten caused papillary epithelial hyperplasia characterized by a hypercellular epithelium lining papillae with fibrovascular cores that protruded into the airway lumens. Cell polarity, as assessed by subcellular localization of cadherin, beta-catenin, and zonula occludens-1, was unaltered. PTEN is required for regulation of epithelial cell proliferation in the lung and for the maintenance of the normal simple columnar epithelium characteristics of bronchi and bronchioles.

Selective deletion of Pten in theca-interstitial cells leads to androgen excess and ovarian dysfunction in mice.

Science.gov (United States)

Lan, Zi-Jian; Krause, M S; Redding, S D; Li, X; Wu, G Z; Zhou, H X; Bohler, H C; Ko, C; Cooney, A J; Zhou, Junmei; Lei, Z M

2017-03-15

Theca cell-selective Pten mutation (tPtenMT) in mice resulted in increases in PDK1 and Akt phosphorylation, indicating an over-activation of PI3K signaling in the ovaries. These mice displayed elevated androgen levels, ovary enlargement, antral follicle accumulation, early fertility loss and increased expression of Lhcgr and genes that are crucial to androgenesis. These abnormalities were partially reversed by treatments of PI3K or Akt inhibitor. LH actions in Pten deficient theca cells were potentiated. The phosphorylation of Foxo1 was increased, while the binding of Foxo1 to forkhead response elements in the Lhcgr promoter was reduced in tPtenMT theca cells, implying a mechanism by which PI3K/Akt-induced upregulation of Lhcgr in theca cells might be mediated by reducing the inhibitory effect of Foxo1 on the Lhcgr promoter. The phenotype of tPtenMT females is reminiscent of human PCOS and suggests that dysregulated PI3K cascade in theca cells may be involved in certain types of PCOS pathogenesis. Copyright © 2017 Elsevier B.V. All rights reserved.

Pten Regulates Epithelial Cytodifferentiation during Prostate Development

DEFF Research Database (Denmark)

Lokody, Isabel B; Francis, Jeffrey C; Gardiner, Jennifer R

2015-01-01

that are shared with Pten mutant prostate cancer models, including a decrease in androgen receptor regulated genes. In depth analysis of the phenotype of these mice during development revealed that loss of Pten leads to the precocious differentiation of epithelial cells towards a luminal cell fate. This study......Gene expression and functional studies have indicated that the molecular programmes involved in prostate development are also active in prostate cancer. PTEN has been implicated in human prostate cancer and is frequently mutated in this disease. Here, using the Nkx3.1:Cre mouse strain and a genetic...... deletion approach, we investigate the role of Pten specifically in the developing mouse prostate epithelia. In contrast to its role in other developing organs, this gene is dispensable for the initial developmental processes such as budding and branching. However, as cytodifferentiation progresses...

Ischemic preconditioning negatively regulates plenty of SH3s-mixed lineage kinase 3-Rac1 complex and c-Jun N-terminal kinase 3 signaling via activation of Akt.

Science.gov (United States)

Zhang, Q-G; Han, D; Xu, J; Lv, Q; Wang, R; Yin, X-H; Xu, T-L; Zhang, G-Y

2006-12-01

Activation of Akt/protein kinase B has been recently reported to play an important role in ischemic tolerance. We here demonstrate that the decreased protein expression and phosphorylation of phosphatase and tensin homolog deleted from chromosome 10 (PTEN) underlie the increased Akt-Ser-473 phosphorylation in the hippocampal CA1 subfield in ischemic preconditioning (IPC). Co-immunoprecipitation analysis reveals that Akt physically interacts with Rac1, a small Rho family GTPase required for mixed lineage kinase 3 (MLK3) autophosphorylation, and both this interaction and Rac1-Ser-71 phosphorylation induced by Akt are promoted in preconditioned rats. In addition, we show that Akt activation results in the disassembly of the plenty of SH3s (POSH)-MLK3-Rac1 signaling complex and down-regulation of the activation of MLK3/c-Jun N-terminal kinase (JNK) pathway. Akt activation results in decreased serine phosphorylation of 14-3-3, a cytoplasmic anchor of Bax, and prevents ischemia-induced mitochondrial translocation of Bax, release of cytochrome c, and activation of caspase-3. The expression of Fas ligand is also decreased in the CA1 region. Akt activation protects against apoptotic neuronal death as shown in TUNEL staining following IPC. Intracerebral infusion of LY294002 before IPC reverses the increase in Akt phosphorylation and the decrease in JNK signaling activation, as well as the neuroprotective action of IPC. Our results suggest that activation of pro-apoptotic MLK3/JNK3 cascade can be suppressed through activating anti-apoptotic phosphoinositide 3-kinase/Akt pathway induced by a sublethal ischemic insult, which provides a functional link between Akt and the JNK family of stress-activated kinases in ischemic tolerance.

Molecular and phenotypic abnormalities in individuals with germline heterozygous PTEN mutations and autism.

Science.gov (United States)

Frazier, T W; Embacher, R; Tilot, A K; Koenig, K; Mester, J; Eng, C

2015-09-01

PTEN is a tumor suppressor associated with an inherited cancer syndrome and an important regulator of ongoing neural connectivity and plasticity. The present study examined molecular and phenotypic characteristics of individuals with germline heterozygous PTEN mutations and autism spectrum disorder (ASD) (PTEN-ASD), with the aim of identifying pathophysiologic markers that specifically associate with PTEN-ASD and that may serve as targets for future treatment trials. PTEN-ASD patients (n=17) were compared with idiopathic (non-PTEN) ASD patients with (macro-ASD, n=16) and without macrocephaly (normo-ASD, n=38) and healthy controls (n=14). Group differences were evaluated for PTEN pathway protein expression levels, global and regional structural brain volumes and cortical thickness measures, neurocognition and adaptive behavior. RNA expression patterns and brain characteristics of a murine model of Pten mislocalization were used to further evaluate abnormalities observed in human PTEN-ASD patients. PTEN-ASD had a high proportion of missense mutations and showed reduced PTEN protein levels. Compared with the other groups, prominent white-matter and cognitive abnormalities were specifically associated with PTEN-ASD patients, with strong reductions in processing speed and working memory. White-matter abnormalities mediated the relationship between PTEN protein reductions and reduced cognitive ability. The Pten(m3m4) murine model had differential expression of genes related to myelination and increased corpus callosum. Processing speed and working memory deficits and white-matter abnormalities may serve as useful features that signal clinicians that PTEN is etiologic and prompting referral to genetic professionals for gene testing, genetic counseling and cancer risk management; and could reveal treatment targets in trials of treatments for PTEN-ASD.

A role for Pten in paediatric intestinal dysmotility disorders.

LENUS (Irish Health Repository)

O'Donnell, Anne-Marie

2012-02-01

PURPOSE: The enteric nervous system (ENS) is a network of neurons and glia that lies within the gut wall. It is responsible for the normal regulation of gut motility and secretory activities. Hirschsprung\\'s disease (HD) is a congenital defect of the ENS, characterised by an absence of ganglia in the distal colon. Intestinal neuronal dysplasia (IND) is a condition that clinically resembles HD, characterised by hyperganglionosis, giant and ectopic ganglia, resulting in intestinal dysmotility. Intestinal ganglioneuromatosis is characterised by hyperplasia and hypertrophy of enteric neuronal cells and causes chronic intestinal pseudo-obstruction (CIPO). Phosphatase and tensin homolog deleted on chromosome 10 (Pten) is a phosphatase that is critical for controlling cell growth, proliferation and cell death. A recent study of Pten knockout mice showed evidence of ganglioneuromatosis in the ENS suggesting a role for this protein in ENS development. Ganglioneuromatosis patients have also been shown to have a decreased level of Pten expression in the colon. The aim of our study was to investigate Pten expression in the ENS of HD and IND patients compared to normal controls. METHODS: Resected tissue from 10 HD and 10 IND type B patients was fixed and embedded in paraffin wax. Normal control colon tissue was obtained from ten patients who underwent a colostomy closure for imperforate anus. Sections were cut and immunohistochemistry was carried out using a Pten antibody. Results were analysed by light microscopy. RESULTS: Staining showed that Pten was strongly expressed in ganglia of both the submucosal and myenteric plexus of normal and HD specimens from the ganglionic colon. Pten expression was significantly reduced in the giant ganglia in IND patients in both the myenteric and submucosal plexuses compared to the normal controls. Specimens from the aganglionic region of HD did not show Pten expression. CONCLUSION: To the best of our knowledge, this is the first study

Effect of Jianpi Jiedu Recipe on angiogenesis and the PTEN/PI3K/AKT signaling pathway in the course of Helicobacter pylori-induced gastric cancer in C57BL/6 mice

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Ning-Ning Liu

2018-01-01

Full Text Available Objective: To reveal the effect of Jianpi Jiedu recipe (JPJDR on angiogenesis and the PTEN (Phosphatase and tensin homolog deleted on chromosome ten/PI3K/AKT signaling pathway in the course of H. pylori infection-induced carcinogenesis of gastric mucosa in C57BL/6 mice. Methods: Two-hundred C57BL/6 mice were randomly divided into five groups (control group, model group, JPJDR low-dose group, JPJDR medium-dose group, and JPJDR high-dose group, 40 in each group. A mouse model of gastric cancer, induced by H. pylori standard strain infection, was established. The mice of JPJDR low-dose, middle-dose, and high-dose groups were intragastrically administered 250, 500, and 1000 mg/kg JPJDR per day, respectively. After 72 weeks, the H. pylori infection in gastric mucosa of the mice was analyzed by rapid urease test; the pathological changes in the gastric mucosa of mice were assessed by histopathological examination, and micro-vessel density (MVD, vascular endothelial growth factor (VEGF, and PTEN/PI3K/AKT levels were determined. Results: The incidence of gastric cancer in each group (control group, model group, JPJDR low-dose, medium-dose, high-dose group was 0%, 26.3%, 13.2%, 10%, and 7.5% respectively. The incidence of gastric cancer in the Chinese medicine group was significantly lower than that of the model group (P = 0.020, P = 0.023, P = 0.007. The expression of MVD and VEGF in the model group was significantly higher than that in the control group (P = 0.002, P < 0.001, while the expression of MVD and VEGF decreased in the Chinese medicine group. The expression of p-PTEN and p-AKT in the model group was significantly higher than that in the control group (All P < 0.001, while Chinese medicine could reduce the expression of p-PTEN and p-AKT to varying extents. Conclusion: Long-term infection of C57BL/6 mice with H. pylori induces gastric carcinogenesis, by increasing gastric mucosal MVD, promoting the expression of VEGF, inhibiting the activity of

Thymidine kinases in archaea

DEFF Research Database (Denmark)

Clausen, A.R.; Matakos, A.; Sandrini, Michael

2006-01-01

Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarcha...

Adenovirus mediated homozygous endometrial epithelial Pten deletion results in aggressive endometrial carcinoma

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Joshi, Ayesha; Ellenson, Lora Hedrick, E-mail: lora.ellenson@med.cornell.edu

2011-07-01

Pten is the most frequently mutated gene in uterine endometriod carcinoma (UEC) and its precursor complex atypical hyperplasia (CAH). Because the mutation frequency is similar in CAH and UEC, Pten mutations are thought to occur relatively early in endometrial tumorigenesis. Previous work from our laboratory using the Pten{sup +/-} mouse model has demonstrated somatic inactivation of the wild type allele of Pten in both CAH and UEC. In the present study, we injected adenoviruses expressing Cre into the uterine lumen of adult Pten floxed mice in an attempt to somatically delete both alleles of Pten specifically in the endometrium. Our results demonstrate that biallelic inactivation of Pten results in an increased incidence of carcinoma as compared to the Pten{sup +/-} mouse model. In addition, the carcinomas were more aggressive with extension beyond the uterus into adjacent tissues and were associated with decreased expression of nuclear ER{alpha} as compared to associated CAH. Primary cultures of epithelial and stromal cells were prepared from uteri of Pten floxed mice and Pten was deleted in vitro using Cre expressing adenovirus. Pten deletion was evident in both the epithelial and stromal cells and the treatment of the primary cultures with estrogen had different effects on Akt activation as well as Cyclin D3 expression in the two purified components. This study demonstrates that somatic biallelic inactivation of Pten in endometrial epithelium in vivo results in an increased incidence and aggressiveness of endometrial carcinoma compared to mice carrying a germline deletion of one allele and provides an important in vivo and in vitro model system for understanding the genetic underpinnings of endometrial carcinoma.

Adenovirus mediated homozygous endometrial epithelial Pten deletion results in aggressive endometrial carcinoma

International Nuclear Information System (INIS)

Joshi, Ayesha; Ellenson, Lora Hedrick

2011-01-01

Pten is the most frequently mutated gene in uterine endometriod carcinoma (UEC) and its precursor complex atypical hyperplasia (CAH). Because the mutation frequency is similar in CAH and UEC, Pten mutations are thought to occur relatively early in endometrial tumorigenesis. Previous work from our laboratory using the Pten +/- mouse model has demonstrated somatic inactivation of the wild type allele of Pten in both CAH and UEC. In the present study, we injected adenoviruses expressing Cre into the uterine lumen of adult Pten floxed mice in an attempt to somatically delete both alleles of Pten specifically in the endometrium. Our results demonstrate that biallelic inactivation of Pten results in an increased incidence of carcinoma as compared to the Pten +/- mouse model. In addition, the carcinomas were more aggressive with extension beyond the uterus into adjacent tissues and were associated with decreased expression of nuclear ERα as compared to associated CAH. Primary cultures of epithelial and stromal cells were prepared from uteri of Pten floxed mice and Pten was deleted in vitro using Cre expressing adenovirus. Pten deletion was evident in both the epithelial and stromal cells and the treatment of the primary cultures with estrogen had different effects on Akt activation as well as Cyclin D3 expression in the two purified components. This study demonstrates that somatic biallelic inactivation of Pten in endometrial epithelium in vivo results in an increased incidence and aggressiveness of endometrial carcinoma compared to mice carrying a germline deletion of one allele and provides an important in vivo and in vitro model system for understanding the genetic underpinnings of endometrial carcinoma.

Facile construction of fused benzimidazole-isoquinolinones that induce cell-cycle arrest and apoptosis in colorectal cancer cells.

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He, Liu-Jun; Yang, Dong-Lin; Li, Shi-Qiang; Zhang, Ya-Jun; Tang, Yan; Lei, Jie; Frett, Brendan; Lin, Hui-Kuan; Li, Hong-Yu; Chen, Zhong-Zhu; Xu, Zhi-Gang

2018-06-12

Colorectal cancer (CRC) is one of the most frequent, malignant gastrointestinal tumors, and strategies and effectiveness of current therapy are limited. A series of benzimidazole-isoquinolinone derivatives (BIDs) was synthesized and screened to identify novel scaffolds for CRC. Of the compounds evaluated, 7g exhibited the most promising anti-cancer properties. Employing two CRC cell lines, SW620 and HT29, 7g was found to suppress growth and proliferation of the cell lines at a concentration of ∼20 µM. Treatment followed an increase in G 2 /M cell cycle arrest, which was attributed to cyclin B1 and cyclin-dependent kinase 1 (CDK1) signaling deficiencies with simultaneous enhancement in p21 and p53 activity. In addition, mitochondrial-mediated apoptosis was induced in CRC cells. Interestingly, 7g decreased phosphorylated AKT, mTOR and 4E-BP1 levels, while promoting the expression/stability of PTEN. Since PTEN controls input into the PI3K/AKT/mTOR pathway, antiproliferative effects can be attributed to PTEN-mediated tumor suppression. Collectively, these results suggest that BIDs exert antitumor activity in CRC by impairing PI3K/AKT/mTOR signaling. Against a small kinase panel, 7g exhibited low affinity at 5 µM suggesting anticancer properties likely stem through a non-kinase mechanism. Because of the novelty of BIDs, the structure can serve as a lead scaffold to design new CRC therapies. Copyright © 2018. Published by Elsevier Ltd.

Quercetogetin protects against cigarette smoke extract-induced apoptosis in epithelial cells by inhibiting mitophagy.

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Son, Eun Suk; Kim, Se-Hee; Ryter, Stefan W; Yeo, Eui-Ju; Kyung, Sun Young; Kim, Yu Jin; Jeong, Sung Hwan; Lee, Chang Soo; Park, Jeong-Woong

2018-04-01

Recent studies demonstrate that the autophagy-dependent turnover of mitochondria (mitophagy) mediates pulmonary epithelial cell death in response to cigarette smoke extract (CSE) exposure, and contributes to emphysema development in vivo during chronic cigarette smoke (CS)-exposure, although the underlying mechanisms remain unclear. Here, we investigated the role of mitophagy in regulating apoptosis in CSE-exposed human lung bronchial epithelial cells. Furthermore, we investigated the potential of the polymethoxylated flavone antioxidant quercetogetin (QUE) to inhibit CSE-induced mitophagy-dependent apoptosis. Our results demonstrate that CSE induces mitophagy in epithelial cells via mitochondrial dysfunction, and causes increased expression levels of the mitophagy-regulator protein PTEN-induced putative kinase-1 (PINK1) and the mitochondrial fission protein dynamin-1-like protein (DRP-1). CSE induced epithelial cell death and increased the expression of the apoptosis-related proteins cleaved caspase-3, -8 and -9. Caspase-3 activity was significantly increased in Beas-2B cells exposed to CSE, and decreased by siRNA-dependent knockdown of DRP-1. Treatment of epithelial cells with QUE inhibited CSE-induced mitochondrial dysfunction and mitophagy by inhibiting phospho (p)-DRP-1 and PINK1 expression. QUE suppressed mitophagy-dependent apoptosis by inhibiting the expression of cleaved caspase-3, -8 and -9 and downregulating caspase activity in human bronchial epithelial cells. These findings suggest that QUE may serve as a potential therapeutic in CS-induced pulmonary diseases. Copyright © 2018 Elsevier Ltd. All rights reserved.

Negative Regulation of Receptor Tyrosine Kinase (RTK Signaling: A Developing Field

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Fernanda Ledda

2007-01-01

Full Text Available ophic factors control cellular physiology by activating specific receptor tyrosine kinases (RTKs. While the over activation of RTK signaling pathways is associated with cell growth and cancer, recent findings support the concept that impaired down-regulation or deactivation of RTKs may also be a mechanism involved in tumor formation. Under this perspective, the molecular determinants of RTK signaling inhibition may act as tumor-suppressor genes and have a potential role as tumor markers to monitor and predict disease progression. Here, we review the current understanding of the physiological mechanisms that attenuate RTK signaling and discuss evidence that implicates deregulation of these events in cancer.Abbreviations: BDP1: Brain-derived phosphatase 1; Cbl: Casitas B-lineage lymphoma; CIN-85: Cbl-interacting protein of 85 kDa; DER: Drosophila EGFR; EGFR: Epidermal growth factor receptor; ERK 1/2: Extracellular signal-regulated kinase 1/2; Grb2: Growth factor receptor-bound protein 2; HER2: Human epidermal growth factor receptor 2; LRIG: Leucine-rich repeats and immunoglobulin-like domain 1; MAPK: Mitogen-activated protein kinase; Mig 6: Mitogen-inducible gene 6; PTEN: Phosphatase and tensin homologue; RET: Rearranged in transformation; RTK: Receptor tyrosine kinase. SH2 domain: Src-homology 2 domain; SH3 domain: Src-homology 3 domain; Spry: Sprouty.

DNA methylation of PTEN gene promoter region is not correlated ...

African Journals Online (AJOL)

Tumor suppressor gene PTEN plays an important role in cell cycle. Disorder of PTEN protein can cause cell growth and division in an uncontrolled way, which can lead to the formation of tumors. It has been proven that epigenetic mechanisms, such as promoter hypermethylation, may account for inactivation of PTEN in a ...

Atorvastatin Inhibits Myocardial Apoptosis in a Swine Model of Coronary Microembolization by Regulating PTEN/PI3K/Akt Signaling Pathway

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Jiangyou Wang

2016-01-01

Full Text Available Background/Aims: Phosphatase and tensin homolog deleted on chromosome ten (PTEN has been recognized as a promoter of apoptosis in various tissues, and revealed to be up-regulated in circumstances of coronary microembolization (CME. However, whether this functional protein could be modified by pretreatment of atorvastatin in models of CME has not been disclosed yet. Methods: Swine CME was induced by intra-coronary injection of inertia plastic microspheres (diameter 42 μm into left anterior descending coronary, with or without pretreatment of atorvastatin or PTEN siRNA. Echocardiologic measurements, pathologic examination, TUNEL staining and western blotting were applied to assess their functional, morphological and molecular effects in CME. Results: PTEN were aberrantly up-regulated in cardiomyocytes following CME, with both the mRNA and protein levels increased after CME modeling. Pretreatment with atorvastatin could attenuate the induction of PTEN. Furthermore, down-regulation of PTEN in vivo via siRNA was associated with an improved cardiac function, attenuated myocardial apoptosis, and concomitantly inhibited expressions of key proapoptotic proteins such as Bax, cleaved-caspase-3. Interestingly, atorvastatin could markedly attenuate PTEN expression and therefore partially reverse cardiac dysfunction and attenuate the apoptosis of the myocardium following CME. Conclusion: Modulation of PTEN was probably as a potential mechanism involved in the beneficial effects of pretreatment of atorvastatin to cardiac function and apoptosis in large animal models of CME.

The PTEN/NRF2 Axis Promotes Human Carcinogenesis

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Rojo, Ana I; Rada, Patricia; Mendiola, Marta

2014-01-01

and tumorigenic advantage. Tissue microarrays from endometrioid carcinomas showed that 80% of PTEN-negative tumors expressed high levels of NRF2 or its target heme oxygenase-1 (HO-1). INNOVATION: These results uncover a new mechanism of oncogenic activation of NRF2 by loss of its negative regulation by PTEN/GSK-3....../β-TrCP that may be relevant to a large number of tumors, including endometrioid carcinomas. CONCLUSION: Increased activity of NRF2 due to loss of PTEN is instrumental in human carcinogenesis and represents a novel therapeutic target. Antioxid. Redox Signal. 21, 2498-2514....

Exploring the Hypersensitivity of PTEN Deleted Prostate Cancer Stem Cells to WEE1 Tyrosine Kinase Inhibitors

Science.gov (United States)

2015-12-01

performed the clonogenic survival assay using LAPC4 as a model as these cell form well defined colonies compared to C42B cells. This assay revealed that...cells. Future studies with other PTEN deficient models are important to validate the effectiveness of WEE1 inhibitors to tackle recurrent PC...Amplifications and missense mutations Cixutumumab (IMC-A12), Dalotuzumab (MK-0646; h7C10), Linsitinib ( OSI -906) EPHA5 12% LAC, 10% SCLC, 9% SAC, Lung

Hydrocephalus caused by conditional ablation of the Pten or beta-catenin gene

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Ohtoshi Akihira

2008-10-01

Full Text Available Abstract To investigate the roles of Pten and β-Catenin in the midbrain, either the Pten gene or the β-catenin gene was conditionally ablated, using Dmbx1 (diencephalon/mesencephalon-expressed brain homeobox gene 1-Cre mice. Homozygous disruption of the Pten or β-catenin gene in Dmbx1-expressing cells caused severe hydrocephalus and mortality during the postnatal period. Conditional deletion of Pten resulted in enlargement of midbrain structures. β-catenin conditional mutant mice showed malformation of the superior and inferior colliculi and stenosis of the midbrain aqueduct. These results demonstrate that both Pten and β-Catenin are essential for proper midbrain development, and provide the direct evidence that mutations of both Pten and β-catenin lead to hydrocephalus.

PTEN drives Th17 cell differentiation by preventing IL-2 production.

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Kim, Hyeong Su; Jang, Sung Woong; Lee, Wonyong; Kim, Kiwan; Sohn, Hyogon; Hwang, Soo Seok; Lee, Gap Ryol

2017-11-06

T helper 17 (Th17) cells are a CD4 + T cell subset that produces IL-17A to mediate inflammation and autoimmunity. IL-2 inhibits Th17 cell differentiation. However, the mechanism by which IL-2 is suppressed during Th17 cell differentiation remains unclear. Here, we show that phosphatase and tensin homologue (PTEN) is a key factor that regulates Th17 cell differentiation by suppressing IL-2 production. Th17-specific Pten deletion ( Pten fl/fl Il17a cre ) impairs Th17 cell differentiation in vitro and ameliorated symptoms of experimental autoimmune encephalomyelitis (EAE), a model of Th17-mediated autoimmune disease. Mechanistically, Pten deficiency up-regulates IL-2 and phosphorylation of STAT5, but reduces STAT3 phosphorylation, thereby inhibiting Th17 cell differentiation. PTEN inhibitors block Th17 cell differentiation in vitro and in the EAE model. Thus, PTEN plays a key role in Th17 cell differentiation by blocking IL-2 expression. © 2017 Kim et al.

Expression patterns and role of PTEN in rat peripheral nerve development and injury.

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Chen, Hui; Xiang, Jianping; Wu, Junxia; He, Bo; Lin, Tao; Zhu, Qingtang; Liu, Xiaolin; Zheng, Canbin

2018-05-29

Studies have suggested that phosphatase and tensin homolog (PTEN) plays an important role in neuroprotection and neuronal regeneration. To better understand the potential role of PTEN with respect to peripheral nerve development and injury, we investigated the expression pattern of PTEN at different stages of rat peripheral nerve development and injury and subsequently assessed the effect of pharmacological inhibition of PTEN using bpV(pic) on axonal regeneration in a rat sciatic nerve crush injury model. During the early stages of development, PTEN exhibits low expression in neuronal cell bodies and axons. From embryonic day (E) 18.5 and postnatal day (P)5 to adult, PTEN protein becomes more detectable, with high expression in the dorsal root ganglia (DRG) and axons. PTEN expression is inhibited in peripheral nerves, preceding myelination during neuronal development and remyelination after acute nerve injury. Low PTEN expression after nerve injury promotes Akt/mammalian target of rapamycin (mTOR) signaling pathway activity. In vivo pharmacological inhibition of PTEN using bpV(pic) promoted axonal regrowth, increased the number of myelinated nerve fibers, improved locomotive recovery and enhanced the amplitude response and nerve conduction velocity following stimulation in a rat sciatic nerve crush injury model. Thus, we suggest that PTEN may play potential roles in peripheral nerve development and regeneration and that inhibition of PTEN expression is beneficial for nerve regeneration and functional recovery after peripheral nerve injury. Copyright © 2018 Elsevier B.V. All rights reserved.

Cell-type specific roles for PTEN in establishing a functional retinal architecture.

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Robert Cantrup

Full Text Available The retina has a unique three-dimensional architecture, the precise organization of which allows for complete sampling of the visual field. Along the radial or apicobasal axis, retinal neurons and their dendritic and axonal arbors are segregated into layers, while perpendicular to this axis, in the tangential plane, four of the six neuronal types form patterned cellular arrays, or mosaics. Currently, the molecular cues that control retinal cell positioning are not well-understood, especially those that operate in the tangential plane. Here we investigated the role of the PTEN phosphatase in establishing a functional retinal architecture.In the developing retina, PTEN was localized preferentially to ganglion, amacrine and horizontal cells, whose somata are distributed in mosaic patterns in the tangential plane. Generation of a retina-specific Pten knock-out resulted in retinal ganglion, amacrine and horizontal cell hypertrophy, and expansion of the inner plexiform layer. The spacing of Pten mutant mosaic populations was also aberrant, as were the arborization and fasciculation patterns of their processes, displaying cell type-specific defects in the radial and tangential dimensions. Irregular oscillatory potentials were also observed in Pten mutant electroretinograms, indicative of asynchronous amacrine cell firing. Furthermore, while Pten mutant RGC axons targeted appropriate brain regions, optokinetic spatial acuity was reduced in Pten mutant animals. Finally, while some features of the Pten mutant retina appeared similar to those reported in Dscam-mutant mice, PTEN expression and activity were normal in the absence of Dscam.We conclude that Pten regulates somal positioning and neurite arborization patterns of a subset of retinal cells that form mosaics, likely functioning independently of Dscam, at least during the embryonic period. Our findings thus reveal an unexpected level of cellular specificity for the multi-purpose phosphatase, and

A Potent HER3 Monoclonal Antibody That Blocks Both Ligand-Dependent and -Independent Activities: Differential Impacts of PTEN Status on Tumor Response.

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Xiao, Zhan; Carrasco, Rosa A; Schifferli, Kevin; Kinneer, Krista; Tammali, Ravinder; Chen, Hong; Rothstein, Ray; Wetzel, Leslie; Yang, Chunning; Chowdhury, Partha; Tsui, Ping; Steiner, Philipp; Jallal, Bahija; Herbst, Ronald; Hollingsworth, Robert E; Tice, David A

2016-04-01

HER3/ERBB3 is a kinase-deficient member of the EGFR family receptor tyrosine kinases (RTK) that is broadly expressed and activated in human cancers. HER3 is a compelling cancer target due to its important role in activation of the oncogenic PI3K/AKT pathway. It has also been demonstrated to confer tumor resistance to a variety of cancer therapies, especially targeted drugs against EGFR and HER2. HER3 can be activated by its ligand (heregulin/HRG), which induces HER3 heterodimerization with EGFR, HER2, or other RTKs. Alternatively, HER3 can be activated in a ligand-independent manner through heterodimerization with HER2 in HER2-amplified cells. We developed a fully human mAb against HER3 (KTN3379) that efficiently suppressed HER3 activity in both ligand-dependent and independent settings. Correspondingly, KTN3379 inhibited tumor growth in divergent tumor models driven by either ligand-dependent or independent mechanisms in vitro and in vivo Most intriguingly, while investigating the mechanistic underpinnings of tumor response to KTN3379, we discovered an interesting dichotomy in that PTEN loss, a frequently occurring oncogenic lesion in a broad range of cancer types, substantially blunted the tumor response in HER2-amplified cancer, but not in the ligand-driven cancer. To our knowledge, this represents the first study ascertaining the impact of PTEN loss on the antitumor efficacy of a HER3 mAb. KTN3379 is currently undergoing a phase Ib clinical trial in patients with advanced solid tumors. Our current study may help us optimize patient selection schemes for KTN3379 to maximize its clinical benefits. Mol Cancer Ther; 15(4); 689-701. ©2016 AACR. ©2016 American Association for Cancer Research.

RNA interference mediated pten knock-down inhibit the formation of polycystic ovary.

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Ouyang, Jie-Xiu; Luo, Tao; Sun, Hui-Yun; Huang, Jian; Tang, Dan-Feng; Wu, Lei; Zheng, Yue-Hui; Zheng, Li-Ping

2013-08-01

Pten (phosphatase and tensin homolog deleted on chromosome 10), a kind of tumor suppressor gene, plays important roles in female reproductive system. But its expression and roles in the formation of polycystic ovaries are yet to be known. In this study, we constructed a rat model of PCOS using norethindrone and HCG injections and found the expressions of pten mRNA and PTEN protein increased significantly in the polycystic ovary tissue by immunohistochemistry, RT-PCR, and western blot. Furthermore, the results showed that in vivo ovaries could be effectively transfected by lentiviral vectors through the ovarian microinjection method and indicated that pten shRNA may inhibit the formation of polycystic ovaries by pten down-regulation. Our study provides new information regarding the role of PTEN in female reproductive disorders, such as polycystic ovary syndrome.

miR-18a promotes cell proliferation of esophageal squamous cell carcinoma cells by increasing cylin D1 via regulating PTEN-PI3K-AKT-mTOR signaling axis

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Zhang, Weiguo, E-mail: weiguozhangHU@gmail.com; Lei, Caipeng; Fan, Junli; Wang, Jing

2016-08-12

Esophageal squamous cell carcinoma (ESCC) is one of the lethal cancers with a high incidence rate in Asia. Cyclin D1 is overexpressed and plays an important role in the carcinogenesis of ESCC; however the mechanism of the deregulation of Cyclin D1 in ESCC remains to be determined. In the study, we found that miR-18a promotes the expression Cyclin D1 by targeting PTEN in eophageal squamous cell carcinoma TE13 and Eca109 cells. Transfection of miR-18a mimetics increased cyclin D1, while transfection of miR-18a antagomir decreased D1. Moreover, miR-18a-mediated upregulation of cyclin D1 was accompanied with downregulation of PTEN, which is a direct target of miR-18a, and increase of the phosphorylation of AKT and S6K1. In addition, pharmacologic inhibition of AKT or mTOR kinases abolished the increase of cyclinD1 by miR-18a, which was accompanied with decreased phosphorylation of Rb−S780 and inhibition of cell proliferation. Our results demonstrated the upregulation of miR-18a promoted cell proliferation by increasing cylin D1 via regulating PTEN-PI3K-AKT-mTOR signaling axis, suggesting that small molecule inhibitors of AKT-mTOR signaling are potential agents for the treatment of ESCC patients with upregulation of miR-17-92 cluster. - Highlights: • miR-18a promotes the proliferation of ESCC cells. • miR-18a increase cyclin D1 expression in ESCC cells. • miR-18a directly targets PTEN in ESCC cells. • Inhibition of AKT-mTOR prevents miR-18a-induced cyclin D1 in ESCC cells. • miR-18a antagomir sensitizes ESCC cells to cisplatin.

Protein Kinase Mitogen-activated Protein Kinase Kinase Kinase Kinase 4 (MAP4K4) Promotes Obesity-induced Hyperinsulinemia.

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Roth Flach, Rachel J; Danai, Laura V; DiStefano, Marina T; Kelly, Mark; Menendez, Lorena Garcia; Jurczyk, Agata; Sharma, Rohit B; Jung, Dae Young; Kim, Jong Hun; Kim, Jason K; Bortell, Rita; Alonso, Laura C; Czech, Michael P

2016-07-29

Previous studies revealed a paradox whereby mitogen-activated protein kinase kinase kinase kinase 4 (Map4k4) acted as a negative regulator of insulin sensitivity in chronically obese mice, yet systemic deletion of Map4k4 did not improve glucose tolerance. Here, we report markedly reduced glucose-responsive plasma insulin and C-peptide levels in whole body Map4k4-depleted mice (M4K4 iKO) as well as an impaired first phase of insulin secretion from islets derived from M4K4 iKO mice ex vivo After long-term high fat diet (HFD), M4K4 iKO mice pancreata also displayed reduced β cell mass, fewer proliferating β cells and reduced islet-specific gene mRNA expression compared with controls, although insulin content was normal. Interestingly, the reduced plasma insulin in M4K4 iKO mice exposed to chronic (16 weeks) HFD was not observed in response to acute HFD challenge or short term treatment with the insulin receptor antagonist S961. Furthermore, the improved insulin sensitivity in obese M4K4 iKO mice was abrogated by high exogenous insulin over the course of a euglycemic clamp study, indicating that hypoinsulinemia promotes insulin sensitivity in chronically obese M4K4 iKO mice. These results demonstrate that protein kinase Map4k4 drives obesity-induced hyperinsulinemia and insulin resistance in part by promoting insulin secretion from β cells in mice. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

α(V)β(6) integrin expression is induced in the POET and Pten(pc-/-) mouse models of prostatic inflammation and prostatic adenocarcinoma.

Science.gov (United States)

Garlick, David S; Li, Jing; Sansoucy, Brian; Wang, Tao; Griffith, Leeanne; Fitzgerald, Tj; Butterfield, Julie; Charbonneau, Bridget; Violette, Shelia M; Weinreb, Paul H; Ratliff, Timothy L; Liao, Chun-Peng; Roy-Burman, Pradip; Vietri, Michele; Lian, Jane B; Stein, Gary S; Altieri, Dario C; Languino, Lucia R

2012-01-01

Chronic inflammation is proposed to prime the development of prostate cancer. However, the mechanisms of prostate cancer initiation and development are not completely understood. The α(v)β(6) integrin has been shown to play a role in epithelial development, wound healing and some epithelial cancers [1, 2]. Here, we investigate the expression of α(v)β(6) in mouse models of prostatic inflammation and prostate cancer to establish a possible relationship between inflammation of the prostate, α(v)β(6) expression and the progression of prostate cancer. Using immunohistochemical techniques, we show expression of α(v)β(6) in two in vivo mouse models; the Pten(pc)-/- model containing a prostate- specific Pten tumor suppressor deletion that causes cancer, and the prostate ovalbumin-expressing transgenic (POET) inflammation mouse model. We show that the α(v)β(6) integrin is induced in prostate cancer and inflammation in vivo in these two mouse models. α(v)β(6) is expressed in all the mice with cancer in the Pten(pc-/-) model but not in age-matched wild-type mice. In the POET inflammation model, α(v)β(6) is expressed in mice injected with activated T-cells, but in none of the control mice. In the POET model, we also used real time PCR to assess the expression of Transforming Growth Factor Beta 1 (TGFβ1), a factor in inflammation that is activated by α(v)β(6). In conclusion, through in vivo evidence, we conclude that α(v)β(6) integrin may be a crucial link between prostatic inflammation and prostatic adenocarcinoma.

The Phosphodiesterase 4 Inhibitor Roflumilast Protects against Cigarette Smoke Extract-Induced Mitophagy-Dependent Cell Death in Epithelial Cells.

Science.gov (United States)

Kyung, Sun Young; Kim, Yu Jin; Son, Eun Suk; Jeong, Sung Hwan; Park, Jeong Woong

2018-04-01

Recent studies show that mitophagy, the autophagy-dependent turnover of mitochondria, mediates pulmonary epithelial cell death in response to cigarette smoke extract (CSE) exposure and contributes to the development of emphysema in vivo during chronic cigarette smoke (CS) exposure, although the underlying mechanisms remain unclear. In this study, we investigated the role of mitophagy in the regulation of CSE-exposed lung bronchial epithelial cell (Beas-2B) death. We also investigated the role of a phosphodiesterase 4 inhibitor, roflumilast, in CSE-induced mitophagy-dependent cell death. Our results demonstrated that CSE induces mitophagy in Beas-2B cells through mitochondrial dysfunction and increased the expression levels of the mitophagy regulator protein, PTEN-induced putative kinase-1 (PINK1), and the mitochondrial fission protein, dynamin-1-like protein (DRP1). CSE-induced epithelial cell death was significantly increased in Beas-2B cells exposed to CSE but was decreased by small interfering RNA-dependent knockdown of DRP1. Treatment with roflumilast in Beas-2B cells inhibited CSE-induced mitochondrial dysfunction and mitophagy by inhibiting the expression of phospho-DRP1 and -PINK1. Roflumilast protected against cell death and increased cell viability, as determined by the lactate dehydrogenase release test and the MTT assay, respectively, in Beas-2B cells exposed to CSE. These findings suggest that roflumilast plays a protective role in CS-induced mitophagy-dependent cell death. Copyright©2018. The Korean Academy of Tuberculosis and Respiratory Diseases.

Creatine kinase and creatine kinase subunit-B in coronary sinus blood in pacing-induced angina pectoris

DEFF Research Database (Denmark)

Bagger, J P; Ingerslev, J; Heinsvig, E M

1982-01-01

In nine out of 10 patients with angiographic documented coronary artery disease, pacing-induced angina pectoris provoked myocardial production of lactate, whereas no significant release of either creatine kinase or creatine kinase subunit-B to coronary sinus and peripheral venous blood could...

Hyperactivity of newborn Pten knock-out neurons results from increased excitatory synaptic drive.

Science.gov (United States)

Williams, Michael R; DeSpenza, Tyrone; Li, Meijie; Gulledge, Allan T; Luikart, Bryan W

2015-01-21

Developing neurons must regulate morphology, intrinsic excitability, and synaptogenesis to form neural circuits. When these processes go awry, disorders, including autism spectrum disorder (ASD) or epilepsy, may result. The phosphatase Pten is mutated in some patients having ASD and seizures, suggesting that its mutation disrupts neurological function in part through increasing neuronal activity. Supporting this idea, neuronal knock-out of Pten in mice can cause macrocephaly, behavioral changes similar to ASD, and seizures. However, the mechanisms through which excitability is enhanced following Pten depletion are unclear. Previous studies have separately shown that Pten-depleted neurons can drive seizures, receive elevated excitatory synaptic input, and have abnormal dendrites. We therefore tested the hypothesis that developing Pten-depleted neurons are hyperactive due to increased excitatory synaptogenesis using electrophysiology, calcium imaging, morphological analyses, and modeling. This was accomplished by coinjecting retroviruses to either "birthdate" or birthdate and knock-out Pten in granule neurons of the murine neonatal dentate gyrus. We found that Pten knock-out neurons, despite a rapid onset of hypertrophy, were more active in vivo. Pten knock-out neurons fired at more hyperpolarized membrane potentials, displayed greater peak spike rates, and were more sensitive to depolarizing synaptic input. The increased sensitivity of Pten knock-out neurons was due, in part, to a higher density of synapses located more proximal to the soma. We determined that increased synaptic drive was sufficient to drive hypertrophic Pten knock-out neurons beyond their altered action potential threshold. Thus, our work contributes a developmental mechanism for the increased activity of Pten-depleted neurons. Copyright © 2015 the authors 0270-6474/15/350943-17$15.00/0.

Endogenous S-sulfhydration of PTEN helps protect against modification by nitric oxide

International Nuclear Information System (INIS)

Ohno, Kazuki; Okuda, Kosaku; Uehara, Takashi

2015-01-01

Highlights: • PTEN is S-sulfhydrated endogenously in SH-SY5Y human neuroblastoma cells. • Preventing this modification by knocking down CBS renders PTEN sensitive to NO. • pAkt levels are increased significantly in CBS siRNA-transfected cells. • H 2 S functions as an endogenous regulator of PTEN in neuronal cells. - Abstract: Hydrogen sulfide (H 2 S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H 2 S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H 2 S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine β-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H 2 S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H 2 S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions

Endogenous S-sulfhydration of PTEN helps protect against modification by nitric oxide

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Ohno, Kazuki; Okuda, Kosaku; Uehara, Takashi, E-mail: uehara@pharm.okayama-u.ac.jp

2015-01-02

Highlights: • PTEN is S-sulfhydrated endogenously in SH-SY5Y human neuroblastoma cells. • Preventing this modification by knocking down CBS renders PTEN sensitive to NO. • pAkt levels are increased significantly in CBS siRNA-transfected cells. • H{sub 2}S functions as an endogenous regulator of PTEN in neuronal cells. - Abstract: Hydrogen sulfide (H{sub 2}S) is a gaseous regulatory factor produced by several enzymes, and plays a pivotal role in processes such as proliferation or vasodilation. Recent reports demonstrated the physiological and pathophysiological functions of H{sub 2}S in neurons. PTEN is a target of nitric oxide (NO) or hydrogen peroxide, and the oxidative modification of cysteine (Cys) residue(s) attenuates its enzymatic activity. In the present study, we assessed the effect of H{sub 2}S on the direct modification of PTEN and the resulting downstream signaling. A modified biotin switch assay in SH-SY5Y human neuroblastoma cells revealed that PTEN is S-sulfhydrated endogenously. Subsequently, site-directed mutagenesis demonstrated that both Cys71 and Cys124 in PTEN are targets for S-sulfhydration. Further, the knockdown of cystathionine β-synthetase (CBS) using siRNA decreased this modification in a manner that was correlated to amount of H{sub 2}S. PTEN was more sensitive to NO under these conditions. These results suggest that the endogenous S-sulfhydration of PTEN via CBS/H{sub 2}S plays a role in preventing the S-nitrosylation that would inhibition its enzymatic activity under physiological conditions.

Hydrogen peroxide induces activation of insulin signaling pathway via AMP-dependent kinase in podocytes

International Nuclear Information System (INIS)

Piwkowska, Agnieszka; Rogacka, Dorota; Angielski, Stefan; Jankowski, Maciej

2012-01-01

Highlights: ► H 2 O 2 activates the insulin signaling pathway and glucose uptake in podocytes. ► H 2 O 2 induces time-dependent changes in AMPK phosphorylation. ► H 2 O 2 enhances insulin signaling pathways via AMPK activation. ► H 2 O 2 stimulation of glucose uptake is AMPK-dependent. -- Abstract: Podocytes are cells that form the glomerular filtration barrier in the kidney. Insulin signaling in podocytes is critical for normal kidney function. Insulin signaling is regulated by oxidative stress and intracellular energy levels. We cultured rat podocytes to investigate the effects of hydrogen peroxide (H 2 O 2 ) on the phosphorylation of proximal and distal elements of insulin signaling. We also investigated H 2 O 2 -induced intracellular changes in the distribution of protein kinase B (Akt). Western blots showed that H 2 O 2 (100 μM) induced rapid, transient phosphorylation of the insulin receptor (IR), the IR substrate-1 (IRS1), and Akt with peak activities at 5 min (Δ 183%, P 2 O 2 >. Furthermore, H 2 O 2 inhibited phosphorylation of the phosphatase and tensin homologue (PTEN; peak activity at 10 min; Δ −32%, P 2 O 2 on IR phosphorylation by about 40% (from 2.07 ± 0.28 to 1.28 ± 0.12, P 2 O 2 increased glucose uptake in podocytes (from 0.88 ± 0.04 to 1.29 ± 0.12 nmol/min/mg protein, P 2 O 2 activated the insulin signaling pathway and glucose uptake via AMPK in cultured rat podocytes. This signaling may play a potential role in the prevention of insulin resistance under conditions associated with oxidative stress.

A Chimeric Protein PTEN-L-p53 Enters U251 Cells to Repress Proliferation and Invasion.

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Xiao, Man; An, Yang; Wang, Fengling; Yao, Chao; Zhang, Chu; Xin, Junfang; Duan, Yongjian; Zhao, Xiaofang; Fang, Na; Ji, Shaoping

2018-05-23

PTEN, a well-known tumor suppressor, dephosphorylates PIP3 and inhibits AKT activity. A translational variant of PTEN has been identified and termed PTEN-Long (PTEN-L). The additional 173 amino acids (PTEN-L leader) at the N-terminal constitute a potential signal peptide. Differing from canonical PTEN, PTEN-L is secreted into the extracellular fluid and re-enters recipient cells, playing the similar roles as PTEN in vivo and in vitro. This character confers the PTEN-L a therapeutic ability via directly protein delivering instead of traditional DNA and RNA vector options. In the present study, we employed PTEN-L leader to assemble a fusion protein, PTEN-L-p53, inosculated with the transcriptional regulator TP53, which is another powerful tumor suppressor. We overexpressed PTEN-L-p53 in HEK293T cells and detected it in both the cytoplasm and nucleus. Subsequently, we found that PTEN-L-p53 was secreted outside of the cells and detected in the culture media by immunoblotting. Furthermore, we demonstrated that PTEN-L-p53 freely entered the cells and suppressed the viability of U251cells (p53 R273H , a cell line with p53 R273H-mutation). PTEN-L-p53 is composed of endogenous protein/peptide bearing low immunogenicity, and only the junction region between PTEN-L leader and p53 can act as a new immune epitope. Accordingly, this fusion protein can potentially be used as a therapeutic option for TP53-abnormality cancers. Copyright © 2018. Published by Elsevier Inc.

Intermittent fasting uncovers and rescues cognitive phenotypes in PTEN neuronal haploinsufficient mice.

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Cabral-Costa, J V; Andreotti, D Z; Mello, N P; Scavone, C; Camandola, S; Kawamoto, E M

2018-06-05

Phosphatase and tensin homolog (PTEN) is an important protein with key modulatory functions in cell growth and survival. PTEN is crucial during embryogenesis and plays a key role in the central nervous system (CNS), where it directly modulates neuronal development and synaptic plasticity. Loss of PTEN signaling function is associated with cognitive deficits and synaptic plasticity impairment. Accordingly, Pten mutations have a strong link with autism spectrum disorder. In this study, neuronal Pten haploinsufficient male mice were subjected to a long-term environmental intervention - intermittent fasting (IF) - and then evaluated for alterations in exploratory, anxiety and learning and memory behaviors. Although no significant effects on spatial memory were observed, mutant mice showed impaired contextual fear memory in the passive avoidance test - an outcome that was effectively rescued by IF. In this study, we demonstrated that IF modulation, in addition to its rescue of the memory deficit, was also required to uncover behavioral phenotypes otherwise hidden in this neuronal Pten haploinsufficiency model.

Combinatorial therapy with adenoviral-mediated PTEN and a PI3K inhibitor suppresses malignant glioma cell growth in vitro and in vivo by regulating the PI3K/AKT signaling pathway.

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Nan, Yang; Guo, Liyun; Song, Yunpeng; Wang, Le; Yu, Kai; Huang, Qiang; Zhong, Yue

2017-08-01

Glioblastoma is a highly invasive and challenging tumor of the central nervous system. The mutation/deletion of the tumor suppressor phosphatase and tensin homolog (PTEN) gene is the main genetic change identified in glioblastomas. PTEN plays a critical role in tumorigenesis and has been shown to be an important therapeutic target. The phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002 is commonly used to inhibit glioma cell growth via regulation of the PI3K/AKT signaling pathway. In this study, we examined the growth inhibitory effects of a combinatorial therapy of adenoviral-mediated PTEN (Ad-PTEN) and LY294002 on LN229 and U251 glioma cells in vitro and on tumor xenografts in vivo. In vitro, LN229 and U251 glioma cells were treated by combinatorial therapy with Ad-PTEN and LY294002. The growth ability was determined by MTT assay. The cell cycle distribution was analyzed by flow cytometry. Cell invasive ability was analyzed by transwell invasion assay and cell apoptosis analysis via FITC-Annexin V analysis. In vivo, U251 subcutaneous glioblastoma xenograft was used to assay anti-tumor effect of combinatorial therapy with Ad-PTEN and LY294002 by mean volume of tumors, immunohistochemistry and TUNEL method. The combinatorial treatment clearly suppressed cell proliferation, arrested the cell cycle, reduced cell invasion and promoted cell apoptosis compared with the Ad-PTEN or LY294002 treatment alone. The treatment worked by inhibiting the PI3K/AKT pathway. In addition, the growth of U251 glioma xenografts treated with the combination of Ad-PTEN and LY294002 was significantly inhibited compared with those treated with Ad-PTEN or LY294002 alone. Our data indicated that the combination of Ad-PTEN and LY294002 effectively suppressed the malignant growth of human glioma cells in vitro and in tumor xenografts, suggesting a promising new approach for glioma gene therapy that warrants further investigation.

PTEN Signaling in the Postnatal Perivascular Progenitor Niche Drives Medulloblastoma Formation.

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Zhu, Guo; Rankin, Sherri L; Larson, Jon D; Zhu, Xiaoyan; Chow, Lionel M L; Qu, Chunxu; Zhang, Jinghui; Ellison, David W; Baker, Suzanne J

2017-01-01

Loss of the tumor suppressor gene PTEN exerts diverse outcomes on cancer in different developmental contexts. To gain insight into the effect of its loss on outcomes in the brain, we conditionally inactivated the murine Pten gene in neonatal neural stem/progenitor cells. Pten inactivation created an abnormal perivascular proliferative niche in the cerebellum that persisted in adult animals but did not progress to malignancy. Proliferating cells showed undifferentiated morphology and expressed the progenitor marker Nestin but not Math1, a marker of committed granule neuron progenitors. Codeletion of Pten and Trp53 resulted in fully penetrant medulloblastoma originating from the perivascular niche, which exhibited abnormal blood vessel networks and advanced neuronal differentiation of tumor cells. EdU pulse-chase experiments demonstrated a perivascular cancer stem cell population in Pten/Trp53 double mutant medulloblastomas. Genetic analyses revealed recurrent somatic inactivations of the tumor suppressor gene Ptch1 and a recapitulation of the sonic hedgehog subgroup of human medulloblastomas. Overall, our results showed that PTEN acts to prevent the proliferation of a progenitor niche in postnatal cerebellum predisposed to oncogenic induction of medulloblastoma. Cancer Res; 77(1); 123-33. ©2016 AACR. ©2016 American Association for Cancer Research.

Interaction of IGF2 and PTEN in ( M alignant Breast T issues

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Preetha J Shetty

2012-07-01

Full Text Available Background: Breast Cancer (BC is one of the leading malignancies affecting women worldwide. Epigenetic mechanisms regulate gene expression playing an important role in the pathophysiology of cancer. In the present study IGF2 and PTEN genes in AKT pathway were selected for evaluation. Objective: To investigate the role of methylation and interaction of IGF2 and PTEN and in the pathoetiology of BC. Methods: Paraffin embedded archival breast tumor and adjacent normal tissue samples were used for carrying out PCR based methylation assay, genomic PCR, immunohistochemistry and qRT PCR. Results: In-Silico study indicated the absence of hormone responsive elements in the promoters of the selected genes. Methylation results indicated significant loss of methylation in IGF2 exon 9 CpG cluster and significant gain of PTEN promoter methylation in tumors. Immunohistochemistry revealed enhanced cytoplasmic expression o f IGF2 protein (p< 0.0001 and decreased nuclear localization of PTEN protein (p=0.0069 in the breast tumors. RT-PCR results indicated an increased IGF2 (p=0.024 and decreased PTEN transcripts (p<0.0001 in the tumors. Conclusion: Increased IGF2 in normal tissues increases PTEN which acts as a negative regulator of AKT pathway in the cytoplasm controlling excessive proliferation while in tumors this regulation is lost. PTEN acts as a negative regulator of MAPK pathway in the nucleus, plays an important role in cell cycle arrest in normal breast tissue. Reduction of PTEN in tumor tissue affects this pathway leading to cell survival. IGF2 and PTEN have a role in breast cancer and these molecular factors can be used for targeting therapy in future.

Inhibition of PTEN and activation of Akt by menadione.

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Yoshikawa, Kyoko; Nigorikawa, Kiyomi; Tsukamoto, Mariko; Tamura, Namiko; Hazeki, Kaoru; Hazeki, Osamu

2007-04-01

Menadione (vitamin K(3)) has been shown to activate Erk in several cell lines. This effect has been shown to be due to the activation of EGF receptors (EGFR) as a result of inhibition of some protein tyrosine phosphatases. In the present study, we examined the effects of menadione on Akt in Chinese hamster ovary cells. The phosphorylation of Akt by menadione was not inhibited by AG1478, an inhibitor of EGFR. Menadione inhibited the lipid phosphatase activity of PTEN in a cell-free system. In an intact cell system, menadione inhibited the effect of transfected PTEN on Akt. Thus, one mechanism of its action was considered the accelerated activation of Akt through inhibition of PTEN. This was not the sole mechanism responsible for the EGFR-independent activation of Akt, because menadione attenuated the rate of Akt dephosphorylation even in PTEN-null PC3 cells. The decelerated inactivation of Akt, probably through inhibition of some tyrosine phosphatases, was considered another mechanism of its action.

Hepatitis B virus induces cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma by targeting programmed cell death protein4 (PDCD4 and phosphatase and tensin homologue (PTEN.

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Preeti Damania

Full Text Available Hepatitis B viral infection-induced hepatocellular carcinoma is one of the major problems in the developing countries. One of the HBV proteins, HBx, modulates the host cell machinery via several mechanisms. In this study we hypothesized that HBV enhances cell proliferation via HBx-induced microRNA-21 in hepatocellular carcinoma. HBx gene was over-expressed, and miRNA-21 expression and cell proliferation were measured in Huh 7 and Hep G2 cells. miRNA-21 was over-expressed in these cells, cell proliferation and the target proteins were analyzed. To confirm the role of miRNA-21 in HBx-induced proliferation, Hep G 2.2.1.5 cells (a cell line that expresses HBV stably were used for miRNA-21 inhibition studies. HBx over-expression enhanced proliferation (3.7- and 4.5-fold increase; n = 3; p<0.01 and miRNA-21 expression (24- and 36-fold increase, normalized with 5S rRNA; p<0.001 in Huh 7 and Hep G2 cells respectively. HBx also resulted in the inhibition of miRNA-21 target proteins, PDCD4 and PTEN. miRNA-21 resulted in a significant increase in proliferation (2- and 2.3-fold increase over control cells; p<0.05 in Huh 7 and Hep G2 cells respectively and decreased target proteins, PDCD4 and PTEN expression. Anti-miR-21 resulted in a significant decrease in proliferation (p<0.05 and increased miRNA-21 target protein expression. We conclude that HBV infection enhances cell proliferation, at least in part, via HBx-induced miRNA-21 expression during hepatocellular carcinoma progression.

Imaging the impact of genes on Parkinson's disease

DEFF Research Database (Denmark)

van der Vegt, J P M; van Nuenen, B F L; Bloem, B R

2009-01-01

by the discovery of mutations in single genes that can cause autosomal dominant (alpha-synuclein (SNCA)) and leucine rich repeat kinase 2 (LRRK2) gene) or recessive (Parkin, PTEN-induced putative kinase 1 (PINK1), DJ-1, and ATP13A2 gene) forms of PD. Here, we review how structural and functional neuroimaging...... of individuals carrying a mutation in one of the PD genes has offered a unique avenue of research into the pathogenesis of PD. In symptomatic mutation carriers (i.e. those with overt disease), brain mapping can help to link the molecular pathogenesis of PD more directly with functional and structural changes...... monogenic forms of PD, common polymorphisms in genes that influence mono-aminergic signaling or synaptic plasticity may have modifying effects on distinct aspects of PD. We also discuss how functional and structural neuroimaging can be used to better characterize these genotype-phenotype correlations....

Regulation of T Cell Homeostasis and Responses by Pten

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Ryan H. Newton

2012-06-01

Full Text Available The generation of lipid products catalyzed by PI3K is critical for normal T cell homeostasis and a productive immune response. PI3K can be activated in response to antigen receptor, costimulatory, cytokine and chemokine signals. Moreover, dysregulation of this pathway frequently occurs in T cell lymphomas and is implicated in lymphoproliferative autoimmune disease. Akt acts as a central mediator of PI3K signals, downstream of which is the mTOR pathway, controlling cell growth and metabolism. Members of the Foxo family of transcription factors are also regulated by Akt, thus linking control over homing and migration of T cells, as well cell cycle entry, apoptosis, and DNA damage and oxidative stress responses, to PI3K signaling. PTEN, first identified as a tumor suppressor gene, encodes a lipid phosphatase that, by catalyzing the reverse of the PI3K reaction, directly opposes PI3K signaling. However, PTEN may have other functions as well, and recent reports have suggested roles for PTEN as a tumor suppressor independent of its effects on PI3K signaling. Through the use of models in which Pten is deleted specifically in T cells, it is becoming increasingly clear that control over autoimmunity and lymphomagenesis by PTEN involves multi-faceted functions of this molecule at multiple stages of T cell development.

Targeting of the tumor suppressor GRHL3 by a miR-21-dependent proto-oncogenic network results in PTEN loss and tumorigenesis.

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Darido, Charbel; Georgy, Smitha R; Wilanowski, Tomasz; Dworkin, Sebastian; Auden, Alana; Zhao, Quan; Rank, Gerhard; Srivastava, Seema; Finlay, Moira J; Papenfuss, Anthony T; Pandolfi, Pier Paolo; Pearson, Richard B; Jane, Stephen M

2011-11-15

Despite its prevalence, the molecular basis of squamous cell carcinoma (SCC) remains poorly understood. Here, we identify the developmental transcription factor Grhl3 as a potent tumor suppressor of SCC in mice, and demonstrate that targeting of Grhl3 by a miR-21-dependent proto-oncogenic network underpins SCC in humans. Deletion of Grhl3 in adult epidermis evokes loss of expression of PTEN, a direct GRHL3 target, resulting in aggressive SCC induced by activation of PI3K/AKT/mTOR signaling. Restoration of Pten expression completely abrogates SCC formation. Reduced levels of GRHL3 and PTEN are evident in human skin, and head and neck SCC, associated with increased expression of miR-21, which targets both tumor suppressors. Our data define the GRHL3-PTEN axis as a critical tumor suppressor pathway in SCC. 2011 Elsevier Inc. All rights reserved.

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

International Nuclear Information System (INIS)

Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

1988-01-01

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn 2+ , while Fe 2+ and Mn 2+ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca 2+ , phorbol ester, or antigen

Interleukin-1 beta induced synthesis of protein kinase C-delta and protein kinase C-epsilon in EL4 thymoma cells: possible involvement of phosphatidylinositol 3-kinase.

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Varley, C L; Royds, J A; Brown, B L; Dobson, P R

2001-01-01

We present evidence here that the proinflammatory cytokine, interleukin-1 beta (IL-1 beta) stimulates a significant increase in protein kinase C (PKC)-epsilon and PKC-delta protein levels and increases PKC-epsilon, but not PKC-delta, transcripts in EL4 thymoma cells. Incubation of EL4 cells with IL-1 beta induced protein synthesis of PKC-epsilon (6-fold increase) by 7 h and had a biphasic effect on PKC-delta levels with peaks at 4 h (2-fold increase) and 24 h (4-fold increase). At the level of mRNA, PKC-epsilon, but not PKC-delta levels, were induced after incubation of EL4 cells with IL-1 beta. The signalling mechanisms utilized by IL-1 beta to induce the synthesis of these PKC isoforms were investigated. Two phosphatidylinositol (PI) 3-kinase-specific inhibitors, wortmannin and LY294002, inhibited IL-1 beta-induced synthesis of PKC-epsilon. However, the PI 3-kinase inhibitors had little effect on the IL-1 beta-induced synthesis of PKC-delta in these cells. Our results indicate that IL-1 beta induced both PKC-delta and PKC-epsilon expression over different time periods. Furthermore, our evidence suggests that IL-1 beta induction of PKC-epsilon, but not PKC-delta, may occur via the PI 3-kinase pathway. Copyright 2001 S. Karger AG, Basel

Germline disruption of Pten localization causes enhanced sex-dependent social motivation and increased glial production.

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Tilot, Amanda K; Gaugler, Mary K; Yu, Qi; Romigh, Todd; Yu, Wanfeng; Miller, Robert H; Frazier, Thomas W; Eng, Charis

2014-06-15

PTEN Hamartoma Tumor Syndrome (PHTS) is an autosomal-dominant genetic condition underlying a subset of autism spectrum disorder (ASD) with macrocephaly. Caused by germline mutations in PTEN, PHTS also causes increased risks of multiple cancers via dysregulation of the PI3K and MAPK signaling pathways. Conditional knockout models have shown that neural Pten regulates social behavior, proliferation and cell size. Although much is known about how the intracellular localization of PTEN regulates signaling in cancer cell lines, we know little of how PTEN localization influences normal brain physiology and behavior. To address this, we generated a germline knock-in mouse model of cytoplasm-predominant Pten and characterized its behavioral and cellular phenotypes. The homozygous Pten(m3m4) mice have decreased total Pten levels including a specific drop in nuclear Pten and exhibit region-specific increases in brain weight. The Pten(m3m4) model displays sex-specific increases in social motivation, poor balance and normal recognition memory-a profile reminiscent of some individuals with high functioning ASD. The cytoplasm-predominant protein caused cellular hypertrophy limited to the soma and led to increased NG2 cell proliferation and accumulation of glia. The animals also exhibit significant astrogliosis and microglial activation, indicating a neuroinflammatory phenotype. At the signaling level, Pten(m3m4) mice show brain region-specific differences in Akt activation. These results demonstrate that differing alterations to the same autism-linked gene can cause distinct behavioral profiles. The Pten(m3m4) model is the first murine model of inappropriately elevated social motivation in the context of normal cognition and may expand the range of autism-related behaviors replicated in animal models. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Genomic Deletion at 10q23 in Prostate Cancer: More Than PTEN Loss?

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Raghavendra Tejo Karthik Poluri

2018-06-01

Full Text Available The PTEN gene encodes for the phosphatase and tensin homolog; it is a tumor suppressor gene that is among the most frequently inactivated genes throughout the human cancer spectrum. The most recent sequencing approaches have allowed the identification of PTEN genomic alterations, including deletion, mutation, or rearrangement in about 50% of prostate cancer (PCa cases. It appears that mechanisms leading to PTEN inactivation are cancer-specific, comprising gene mutations, small insertions/deletions, copy number alterations (CNAs, promoter hypermethylation, and RNA interference. The examination of publicly available results from deep-sequencing studies of various cancers showed that PCa appears to be the only cancer in which PTEN is lost mostly through CNA. Instead of inactivating mutations, which are seen in other cancers, deletion of the 10q23 locus is the most common form of PTEN inactivation in PCa. By investigating the minimal deleted region at 10q23, several other genes appear to be lost simultaneously with PTEN. Expression data indicate that, like PTEN, these genes are also downregulated upon loss of 10q23. These analyses raise the possibility that 10q23 is lost upon selective pressure not only to inactivate PTEN but also to impair the expression of surrounding genes. As such, several genes from this deleted region, which represents about 500 kb, may also act as tumor suppressors in PCa, requiring further studies on their respective functions in that context.

Loss of Cdh1 and Pten Accelerates Cellular Invasiveness and Angiogenesis in the Mouse Uterus1

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Lindberg, Mallory E.; Stodden, Genna R.; King, Mandy L.; MacLean, James A.; Mann, Jordan L.; DeMayo, Francesco J.; Lydon, John P.; Hayashi, Kanako

2013-01-01

ABSTRACT E-cadherin (CDH1) is a cell adhesion molecule that coordinates key morphogenetic processes regulating cell growth, cell proliferation, and apoptosis. Loss of CDH1 is a trademark of the cellular event epithelial to mesenchymal transition, which increases the metastatic potential of malignant cells. PTEN is a tumor-suppressor gene commonly mutated in many human cancers, including endometrial cancer. In the mouse uterus, ablation of Pten induces epithelial hyperplasia, leading to endometrial carcinomas. However, loss of Pten alone does not affect longevity until around 5 mo. Similarly, conditional ablation of Cdh1 alone does not predispose mice to cancer. In this study, we characterized the impact of dual Cdh1 and Pten ablation (Cdh1d/d Ptend/d) in the mouse uterus. We observed that Cdh1d/d Ptend/d mice died at Postnatal Days 15–19 with massive blood loss. Their uteri were abnormally structured with curly horns, disorganized epithelial structure, and increased cell proliferation. Co-immunostaining of KRT8 and ACTA2 showed invasion of epithelial cells into the myometrium. Further, the uteri of Cdh1d/d Ptend/d mice had prevalent vascularization in both the endometrium and myometrium. We also observed reduced expression of estrogen and progesterone receptors, loss of cell adherens, and tight junction molecules (CTNNB1 and claudin), as well as activation of AKT in the uteri of Cdh1d/d Ptend/d mice. However, complex hyperplasia was not found in the uteri of Cdh1d/d Ptend/d mice. Collectively, these findings suggest that ablation of Pten with Cdh1 in the uterus accelerates cellular invasiveness and angiogenesis and causes early death. PMID:23740945

c-Jun N-terminal kinase mediates AML1-ETO protein-induced connexin-43 expression

International Nuclear Information System (INIS)

Gao Fenghou; Wang Qiong; Wu Yingli; Li Xi; Zhao Kewen; Chen Guoqiang

2007-01-01

AML1-ETO fusion protein, a product of leukemia-related chromosomal translocation t(8;21), was reported to upregulate expression of connexin-43 (Cx43), a member of gap junction-constituted connexin family. However, its mechanism(s) remains unclear. By bioinformatic analysis, here we showed that there are two putative AML1-binding consensus sequences followed by two activated protein (AP)1 sites in the 5'-flanking region upstream to Cx43 gene. AML1-ETO could directly bind to these two AML1-binding sites in electrophoretic mobility shift assay, but luciferase reporter assay revealed that the AML1 binding sites were not indispensable for Cx43 induction by AML1-ETO protein. Conversely, AP1 sites exerted an important role in this event. In agreement, AML1-ETO overexpression in leukemic U937 cells activated c-Jun N-terminal kinase (JNK), while its specific inhibitor SP600125 effectively abrogated AML1-ETO-induced Cx43 expression, indicating that JNK signaling pathway contributes to AML1-ETO induced Cx43 expression. These results would shed new insights for understanding mechanisms of AML1-ETO-associated leukemogenesis

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes

Energy Technology Data Exchange (ETDEWEB)

Csermely, P.; Szamel, M.; Resch, K.; Somogyi, J.

1988-05-15

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested. In the present report, the authors demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes,and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn/sup 2 +/, while Fe/sup 2 +/ and Mn/sup 2 +/ are only partially counteractive. The results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca/sup 2 +/, phorbol ester, or antigen.

Alcohol Dehydrogenase Protects against Endoplasmic Reticulum Stress-Induced Myocardial Contractile Dysfunction via Attenuation of Oxidative Stress and Autophagy: Role of PTEN-Akt-mTOR Signaling.

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Jiaojiao Pang

Full Text Available The endoplasmic reticulum (ER plays an essential role in ensuring proper folding of the newly synthesized proteins. Aberrant ER homeostasis triggers ER stress and development of cardiovascular diseases. ADH is involved in catalyzing ethanol to acetaldehyde although its role in cardiovascular diseases other than ethanol metabolism still remains elusive. This study was designed to examine the impact of ADH on ER stress-induced cardiac anomalies and underlying mechanisms involved using cardiac-specific overexpression of alcohol dehydrogenase (ADH.ADH and wild-type FVB mice were subjected to the ER stress inducer tunicamycin (1 mg/kg, i.p., for 48 hrs. Myocardial mechanical and intracellular Ca(2+ properties, ER stress, autophagy and associated cell signaling molecules were evaluated.ER stress compromised cardiac contractile function (evidenced as reduced fractional shortening, peak shortening, maximal velocity of shortening/relengthening, prolonged relengthening duration and impaired intracellular Ca(2+ homeostasis, oxidative stress and upregulated autophagy (increased LC3B, Atg5, Atg7 and p62, along with dephosphorylation of PTEN, Akt and mTOR, all of which were attenuated by ADH. In vitro study revealed that ER stress-induced cardiomyocyte anomaly was abrogated by ADH overexpression or autophagy inhibition using 3-MA. Interestingly, the beneficial effect of ADH was obliterated by autophagy induction, inhibition of Akt and mTOR. ER stress also promoted phosphorylation of the stress signaling ERK and JNK, the effect of which was unaffected by ADH transgene.Taken together, these findings suggested that ADH protects against ER stress-induced cardiac anomalies possibly via attenuation of oxidative stress and PTEN/Akt/mTOR pathway-regulated autophagy.

Cytokine-induced loss of glucocorticoid function: effect of kinase inhibitors, long-acting β(2-adrenoceptor [corrected] agonist and glucocorticoid receptor ligands.

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Christopher F Rider

Full Text Available Acting on the glucocorticoid receptor (NR3C1, glucocorticoids are widely used to treat inflammatory diseases. However, glucocorticoid resistance often leads to suboptimal asthma control. Since glucocorticoid-induced gene expression contributes to glucocorticoid activity, the aim of this study was to use a 2 × glucocorticoid response element (GRE reporter and glucocorticoid-induced gene expression to investigate approaches to combat cytokine-induced glucocorticoid resistance. Pre-treatment with tumor necrosis factor-α (TNF or interleukin-1β inhibited dexamethasone-induced mRNA expression of the putative anti-inflammatory genes RGS2 and TSC22D3, or just TSC22D3, in primary human airway epithelial and smooth muscle cells, respectively. Dexamethasone-induced DUSP1 mRNA was unaffected. In human bronchial epithelial BEAS-2B cells, dexamethasone-induced TSC22D3 and CDKN1C expression (at 6 h was reduced by TNF pre-treatment, whereas DUSP1 and RGS2 mRNAs were unaffected. TNF pre-treatment also reduced dexamethasone-dependent 2×GRE reporter activation. This was partially reversed by PS-1145 and c-jun N-terminal kinase (JNK inhibitor VIII, inhibitors of IKK2 and JNK, respectively. However, neither inhibitor affected TNF-dependent loss of dexamethasone-induced CDKN1C or TSC22D3 mRNA. Similarly, inhibitors of the extracellular signal-regulated kinase, p38, phosphoinositide 3-kinase or protein kinase C pathways failed to attenuate TNF-dependent repression of the 2×GRE reporter. Fluticasone furoate, fluticasone propionate and budesonide were full agonists relative to dexamethasone, while GSK9027, RU24858, des-ciclesonide and GW870086X were partial agonists on the 2×GRE reporter. TNF reduced reporter activity in proportion with agonist efficacy. Full and partial agonists showed various degrees of agonism on RGS2 and TSC22D3 expression, but were equally effective at inducing CDKN1C and DUSP1, and did not affect the repression of CDKN1C or TSC22D3

Sensor histidine kinase is a β-lactam receptor and induces resistance to β-lactam antibiotics.

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Li, Lu; Wang, Qiyao; Zhang, Hui; Yang, Minjun; Khan, Mazhar I; Zhou, Xiaohui

2016-02-09

β-Lactams disrupt bacterial cell wall synthesis, and these agents are the most widely used antibiotics. One of the principle mechanisms by which bacteria resist the action of β-lactams is by producing β-lactamases, enzymes that degrade β-lactams. In Gram-negative bacteria, production of β-lactamases is often induced in response to the antibiotic-associated damage to the cell wall. Here, we have identified a previously unidentified mechanism that governs β-lactamase production. In the Gram-negative enteric pathogen Vibrio parahaemolyticus, we found a histidine kinase/response regulator pair (VbrK/VbrR) that controls expression of a β-lactamase. Mutants lacking either VbrK or VbrR do not produce the β-lactamase and are no longer resistant to β-lactam antibiotics. Notably, VbrK autophosphorylation is activated by β-lactam antibiotics, but not by other lactams. However, single amino acid substitutions in the putative periplasmic binding pocket of VbrK leads its phosphorylation in response to both β-lactam and other lactams, suggesting that this kinase is a β-lactam receptor that can directly detect β-lactam antibiotics instead of detecting the damage to cell wall resulting from β-lactams. In strong support of this idea, we found that purified periplasmic sensor domain of VbrK binds penicillin, and that such binding is critical for VbrK autophosphorylation and β-lactamase production. Direct recognition of β-lactam antibiotics by a histidine kinase receptor may represent an evolutionarily favorable mechanism to defend against β-lactam antibiotics.

Constitutive Activation of the Fission Yeast Pheromone-Responsive Pathway Induces Ectopic Meiosis and Reveals Ste11 as a Mitogen-Activated Protein Kinase Target

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Kjærulff, Søren; Lautrup-Larsen, I.; Truelsen, S.

2005-01-01

In the fission yeast Schizosaccharomyces pombe, meiosis normally takes place in diploid zygotes resulting from conjugation of haploid cells. In the present study, we report that the expression of a constitutively activated version of the pheromone-responsive mitogen-activated protein kinase kinase...... found that haploid meiosis was dramatically reduced when Ste11 was mutated to mimic phosphorylation by Pat1. The mutation of two putative MAPK sites in Ste11 also dramatically reduced the level of haploid meiosis, suggesting that Ste11 is a direct target of Spk1. Supporting this, we show that Spk1 can...... interact physically with Ste11 and also phosphorylate the transcription factor in vitro. Finally, we demonstrate that ste11 is required for pheromone-induced G1 arrest. Interestingly, when we mutated Ste11 in the sites for Pat1 and Spk1 phosphorylation simultaneously, the cells could still arrest in G1...

The significance of PTEN and AKT aberrations in pediatric T-cell acute lymphoblastic leukemia

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Zuurbier, Linda; Petricoin, Emanuel F.; Vuerhard, Maartje J.; Calvert, Valerie; Kooi, Clarissa; Buijs-Gladdines, Jessica G.C.A.M.; Smits, Willem K.; Sonneveld, Edwin; Veerman, Anjo J.P.; Kamps, Willem A.; Horstmann, Martin; Pieters, Rob; Meijerink, Jules P.P.

2012-01-01

Background PI3K/AKT pathway mutations are found in T-cell acute lymphoblastic leukemia, but their overall impact and associations with other genetic aberrations is unknown. PTEN mutations have been proposed as secondary mutations that follow NOTCH1-activating mutations and cause cellular resistance to γ-secretase inhibitors. Design and Methods The impact of PTEN, PI3K and AKT aberrations was studied in a genetically well-characterized pediatric T-cell leukemia patient cohort (n=146) treated on DCOG or COALL protocols. Results PTEN and AKT E17K aberrations were detected in 13% and 2% of patients, respectively. Defective PTEN-splicing was identified in incidental cases. Patients without PTEN protein but lacking exon-, splice-, promoter mutations or promoter hypermethylation were present. PTEN/AKT mutations were especially abundant in TAL- or LMO-rearranged leukemia but nearly absent in TLX3-rearranged patients (P=0.03), the opposite to that observed for NOTCH1-activating mutations. Most PTEN/AKT mutant patients either lacked NOTCH1-activating mutations (P=0.006) or had weak NOTCH1-activating mutations (P=0.011), and consequently expressed low intracellular NOTCH1, cMYC and MUSASHI levels. T-cell leukemia patients without PTEN/AKT and NOTCH1-activating mutations fared well, with a cumulative incidence of relapse of only 8% versus 35% for PTEN/AKT and/or NOTCH1-activated patients (P=0.005). Conclusions PI3K/AKT pathway aberrations are present in 18% of pediatric T-cell acute lymphoblastic leukemia patients. Absence of strong NOTCH1-activating mutations in these cases may explain cellular insensitivity to γ-secretase inhibitors. PMID:22491738

The GAS5/miR-222 Axis Regulates Proliferation of Gastric Cancer Cells Through the PTEN/Akt/mTOR Pathway.

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Li, Yanhua; Gu, Junjiao; Lu, Hong

2017-12-01

Several lines of evidence have indicated that growth arrest-specific transcript 5 (GAS5) functions as a tumor suppressor and is aberrantly expressed in multiple cancers. GAS5 was found to be downregulated in gastric cancer (GC) tissues, and ectopic expression of GAS5 inhibited GC cell proliferation. The present study aimed to explore the underlying mechanisms of GAS5 involved in GC cell proliferation. GAS5 and miR-222 expressions in GC cell lines were estimated by quantitative real-time polymerase chain reaction. The effects of GAS5 and miR-222 on GC cell proliferation were assessed by MTT assay and 5-bromo-2-deoxyuridine (BrdU) incorporation assays. The interaction between GAS5 and miR-222 was confirmed by luciferase reporter assay and RNA immunoprecipitation assay. The protein levels of the phosphatase and tensin homolog (PTEN), phosphorylated protein kinase B (Akt) (p-Akt), Akt, phosphorylated mammalian target of rapamycin (mTOR) (p-mTOR), and mTOR were determined by western blot. GAS5 was downregulated and miR-222 was upregulated in GC cells. GAS5 directly targeted and suppressed miR-222 expression. GAS5 overexpression and miR-222 inhibition suppressed cell proliferation, increased PTEN protein level and decreased p-Akt and p-mTOR protein levels in GC cells while GAS5 knockdown and miR-222 overexpression exhibited the opposite effects. Moreover, mechanistic analyses revealed that GAS5 regulated GC cell proliferation through the PTEN/Akt/mTOR pathway by negatively regulating miR-222. GAS5/miR-222 axis regulated proliferation of GC cells through the PTEN/Akt/mTOR pathway, which facilitated the development of lncRNA-directed therapy against this deadly disease.

Selective neuronal PTEN deletion: can we take the brakes off of growth without losing control?

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Erin A Gutilla

2016-01-01

Full Text Available The limited ability for injured adult axons to regenerate is a major cause for limited functional recovery after injury to the nervous system, motivating numerous efforts to uncover mechanisms capable of enhancing regeneration potential. One promising strategy involves deletion or knockdown of the phosphatase and tensin (PTEN gene. Conditional genetic deletion of PTEN before, immediately following, or several months after spinal cord injury enables neurons of the corticospinal tract (CST to regenerate their axons across the lesion, which is accompanied by enhanced recovery of skilled voluntary motor functions mediated by the CST. Although conditional genetic deletion or knockdown ofPTEN in neurons enables axon regeneration, PTEN is a well-known tumor suppressor and mutations of the PTEN gene disrupt brain development leading to neurological abnormalities including macrocephaly, seizures, and early mortality. The long-term consequences of manipulating PTEN in the adult nervous system, as would be done for therapeutic intervention after injury, are only now being explored. Here, we summarize evidence indicating that long-term deletion of PTEN in mature neurons does not cause evident pathology; indeed, cortical neurons that have lived without PTEN for over 1 year appear robust and healthy. Studies to date provide only a first look at potential negative consequences of PTEN deletion or knockdown, but the absence of any detectable neuropathology supports guarded optimism that interventions to enable axon regeneration after injury are achievable.

AMP-activated protein kinase induces actin cytoskeleton reorganization in epithelial cells

International Nuclear Information System (INIS)

Miranda, Lisa; Carpentier, Sarah; Platek, Anna; Hussain, Nusrat; Gueuning, Marie-Agnes; Vertommen, Didier; Ozkan, Yurda; Sid, Brice; Hue, Louis; Courtoy, Pierre J.; Rider, Mark H.; Horman, Sandrine

2010-01-01

AMP-activated protein kinase (AMPK), a known regulator of cellular and systemic energy balance, is now recognized to control cell division, cell polarity and cell migration, all of which depend on the actin cytoskeleton. Here we report the effects of A769662, a pharmacological activator of AMPK, on cytoskeletal organization and signalling in epithelial Madin-Darby canine kidney (MDCK) cells. We show that AMPK activation induced shortening or radiation of stress fibers, uncoupling from paxillin and predominance of cortical F-actin. In parallel, Rho-kinase downstream targets, namely myosin regulatory light chain and cofilin, were phosphorylated. These effects resembled the morphological changes in MDCK cells exposed to hyperosmotic shock, which led to Ca 2+ -dependent AMPK activation via calmodulin-dependent protein kinase kinase-β(CaMKKβ), a known upstream kinase of AMPK. Indeed, hypertonicity-induced AMPK activation was markedly reduced by the STO-609 CaMKKβ inhibitor, as was the increase in MLC and cofilin phosphorylation. We suggest that AMPK links osmotic stress to the reorganization of the actin cytoskeleton.

Zinc can increase the activity of protein kinase C and contributes to its binding to plasma membranes in T lymphocytes.

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Csermely, P; Szamel, M; Resch, K; Somogyi, J

1988-05-15

In the primary structure of protein kinase C, the presence of a putative metal-binding site has been suggested (Parker, P.J., Coussens, L., Totty, N., Rhee, L., Young, S., Chen, E., Stabel, S., Waterfield, M.D., and Ullrich, A. (1986) Science 233, 853-859). In the present report, we demonstrate that the most abundant intracellular heavy metal, zinc, can increase the activity of cytosolic protein kinase C. Zinc reversibly binds the enzyme to plasma membranes, and it may contribute to the calcium-induced binding as well. The intracellular heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl) ethylenediamine prevents the phorbol ester- and antigen-induced translocation of protein kinase C. This effect can be totally reversed by the concomitant addition of Zn2+, while Fe2+ and Mn2+ are only partially counteractive. Our results suggest that zinc can activate protein kinase C and contributes to its binding to plasma membranes in T lymphocytes induced by Ca2+, phorbol ester, or antigen.

Hydrogen peroxide induces activation of insulin signaling pathway via AMP-dependent kinase in podocytes

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Piwkowska, Agnieszka, E-mail: apiwkowska@cmdik.pan.pl [Mossakowski Medical Research Centre, Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdansk (Poland); Rogacka, Dorota; Angielski, Stefan [Mossakowski Medical Research Centre, Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdansk (Poland); Jankowski, Maciej [Mossakowski Medical Research Centre, Polish Academy of Sciences, Laboratory of Molecular and Cellular Nephrology, Gdansk (Poland); Medical University of Gdansk, Department of Therapy Monitoring and Pharmacogenetics (Poland)

2012-11-09

Highlights: Black-Right-Pointing-Pointer H{sub 2}O{sub 2} activates the insulin signaling pathway and glucose uptake in podocytes. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} induces time-dependent changes in AMPK phosphorylation. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} enhances insulin signaling pathways via AMPK activation. Black-Right-Pointing-Pointer H{sub 2}O{sub 2} stimulation of glucose uptake is AMPK-dependent. -- Abstract: Podocytes are cells that form the glomerular filtration barrier in the kidney. Insulin signaling in podocytes is critical for normal kidney function. Insulin signaling is regulated by oxidative stress and intracellular energy levels. We cultured rat podocytes to investigate the effects of hydrogen peroxide (H{sub 2}O{sub 2}) on the phosphorylation of proximal and distal elements of insulin signaling. We also investigated H{sub 2}O{sub 2}-induced intracellular changes in the distribution of protein kinase B (Akt). Western blots showed that H{sub 2}O{sub 2} (100 {mu}M) induced rapid, transient phosphorylation of the insulin receptor (IR), the IR substrate-1 (IRS1), and Akt with peak activities at 5 min ({Delta} 183%, P < 0.05), 3 min ({Delta} 414%, P < 0.05), and 10 min ({Delta} 35%, P < 0.05), respectively. Immunostaining cells with an Akt-specific antibody showed increased intensity at the plasma membrane after treatment with H{sub 2}O{sub 2}>. Furthermore, H{sub 2}O{sub 2} inhibited phosphorylation of the phosphatase and tensin homologue (PTEN; peak activity at 10 min; {Delta} -32%, P < 0.05) and stimulated phosphorylation of the AMP-dependent kinase alpha subunit (AMPK{alpha}; 78% at 3 min and 244% at 10 min). The stimulation of AMPK was abolished with an AMPK inhibitor, Compound C (100 {mu}M, 2 h). Moreover, Compound C significantly reduced the effect of H{sub 2}O{sub 2} on IR phosphorylation by about 40% (from 2.07 {+-} 0.28 to 1.28 {+-} 0.12, P < 0.05). In addition, H{sub 2}O{sub 2} increased glucose uptake in podocytes

Bosutinib induced pleural effusions: Case report and review of tyrosine kinase inhibitors induced pulmonary toxicity

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Natalia I. Moguillansky, MD

2017-01-01

Full Text Available Tyrosine kinase inhibitors are known to cause pulmonary complications. We report a case of bosutinib related bilateral pleural effusions in a patient with chronic myeloid leukemia. Characteristics of the pleural fluid are presented. We also discuss other tyrosine kinase inhibitors induced pulmonary toxicities, including pulmonary hypertension and interstitial lung disease.

Role of contractile prostaglandins and Rho-kinase in growth factor-induced airway smooth muscle contraction

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Zaagsma Johan

2005-07-01

Full Text Available Abstract Background In addition to their proliferative and differentiating effects, several growth factors are capable of inducing a sustained airway smooth muscle (ASM contraction. These contractile effects were previously found to be dependent on Rho-kinase and have also been associated with the production of eicosanoids. However, the precise mechanisms underlying growth factor-induced contraction are still unknown. In this study we investigated the role of contractile prostaglandins and Rho-kinase in growth factor-induced ASM contraction. Methods Growth factor-induced contractions of guinea pig open-ring tracheal preparations were studied by isometric tension measurements. The contribution of Rho-kinase, mitogen-activated protein kinase (MAPK and cyclooxygenase (COX to these reponses was established, using the inhibitors Y-27632 (1 μM, U-0126 (3 μM and indomethacin (3 μM, respectively. The Rho-kinase dependency of contractions induced by exogenously applied prostaglandin F2α (PGF2α and prostaglandin E2 (PGE2 was also studied. In addition, the effects of the selective FP-receptor antagonist AL-8810 (10 μM and the selective EP1-antagonist AH-6809 (10 μM on growth factor-induced contractions were investigated, both in intact and epithelium-denuded preparations. Growth factor-induced PGF2α-and PGE2-release in the absence and presence of Y-27632, U-0126 and indomethacin, was assessed by an ELISA-assay. Results Epidermal growth factor (EGF-and platelet-derived growth factor (PDGF-induced contractions of guinea pig tracheal smooth muscle preparations were dependent on Rho-kinase, MAPK and COX. Interestingly, growth factor-induced PGF2α-and PGE2-release from tracheal rings was significantly reduced by U-0126 and indomethacin, but not by Y-27632. Also, PGF2α-and PGE2-induced ASM contractions were largely dependent on Rho-kinase, in contrast to other contractile agonists like histamine. The FP-receptor antagonist AL-8810 (10 μM significantly

Developing a PTEN-ERG Signature to Improve Molecular Risk Stratification in Prostate Cancer

Science.gov (United States)

2017-10-01

AWARD NUMBER: W81XWH-16-1-0737 TITLE: Developing a PTEN-ERG Signature to Improve Molecular Risk Stratification in Prostate Cancer PRINCIPAL...AND SUBTITLE 5a. CONTRACT NUMBER Developing a PTEN-ERG Signature to Improve Molecular Risk Stratification in Prostate Cancer 5b. GRANT NUMBER W81XWH...that there exist distinctive molecular correlates of PTEN loss in the context of ETS-negative versus ETS-positive human prostate cancers and that

A chimeric cyclic interferon-α2b peptide induces apoptosis by sequential activation of phosphatidylinositol 3-kinase, protein kinase Cδ and p38 MAP kinase.

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Blank, V C; Bertucci, L; Furmento, V A; Peña, C; Marino, V J; Roguin, L P

2013-06-10

We have previously demonstrated that tyrosine phosphorylation of STAT1/3 and p38 mitogen-activated protein kinase (p38 MAPK) activation are involved in the apoptotic response triggered by a chimeric cyclic peptide of the interferon-α2b (IFN-α2b) in WISH cells. Since the peptide also induced serine phosphorylation of STAT proteins, in the present study we examined the kinase involved in serine STAT1 phosphorylation and the signaling effectors acting upstream such activation. We first found that p38 MAPK is involved in serine STAT1 phosphorylation, since a reduction of phophoserine-STAT1 levels was evident after incubating WISH cells with cyclic peptide in the presence of a p38 pharmacological inhibitor or a dominant-negative p38 mutant. Next, we demonstrated that the peptide induced activation of protein kinase Cδ (PKCδ). Based on this finding, the role of this kinase was then evaluated. After incubating WISH cells with a PKCδ inhibitor or after decreasing PKCδ expression levels by RNA interference, both peptide-induced serine STAT1 and p38 phosphorylation levels were significantly decreased, indicating that PKCδ functions as an upstream regulator of p38. We also showed that PKCδ and p38 activation stimulated by the peptide was inhibited by a specific pharmacological inhibitor of phosphatidylinositol 3-kinase (PI3K) or by a dominant-negative p85 PI3K-regulatory subunit, suggesting that PI3K is upstream in the signaling cascade. In addition, the role of PI3K and PKCδ in cyclic peptide-induced apoptosis was examined. Both signaling effectors were found to regulate the antiproliferative activity and the apoptotic response triggered by the cyclic peptide in WISH cells. In conclusion, we herein demonstrated that STAT1 serine phosphorylation is mediated by the sequential activation of PI3K, PKCδ and p38 MAPK. This signaling cascade contributes to the antitumor effect induced by the chimeric IFN-α2b cyclic peptide in WISH cells. Copyright © 2013 Elsevier Inc

A study of the dynamics of PTEN proteins in living cells using in vivo fluorescence correlation spectroscopy

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Du, Zhixue; Dong, Chaoqing; Ren, Jicun

2017-06-01

PTEN (phosphatase and tensin homolog on chromosome 10) is one of the most important tumor-suppressor proteins, which plays a key role in negative regulation of the PI3K/AKT pathway, and governs many cellular processes including growth, proliferation, survival and migration. The dynamics of PTEN proteins in single living cells is as yet unclear owing to a shortage of suitable in vivo approaches. Here, we report a single-molecule method for in vivo study of the dynamics of PTEN proteins in living cells using fluorescence correlation spectroscopy (FCS). First, we established a monoclonal H1299 stable cell line expressing enhanced green fluorescent protein (EGFP) and PTEN (EGFP-PTEN) fusion proteins; we then developed an in vivo FCS method to study the dynamics of EGFP-PTEN both in the nucleus and the cytoplasm. We investigated the diffusion behaviors of EGFP and EGFP-PTEN in solution, nucleus and cytosol, and observed that the motion of PTEN in living cells was restricted compared with EGFP. Finally, we investigated the protein dynamics in living cells under oxidative stress stimulation and a cellular ATP depletion treatment. Under oxidative stress stimulation, the EGFP-PTEN concentration increased in the nucleus, but slightly decreased in the cytoplasm. The diffusion coefficient and alpha value of EGFP-PTEN reduced significantly both in the nucleus and cytoplasm; the significantly decreased alpha parameter indicates a more restricted Brownian diffusion behavior. Under the cellular ATP depletion treatment, the concentration of EGFP-PTEN remained unchanged in the nucleus and decreased significantly in cytosol. The diffusion coefficient of EGFP-PTEN decreased significantly in cytosol, but showed no significant change in the nucleus; the alpha value decreased significantly in both the nucleus and cytoplasm. These results suggest that the concentration and mobility of PTEN in the nucleus and cytoplasm can be regulated by stimulation methods. Our approach provides a unique

Posttranslational regulation of phosphatase and tensin homolog (PTEN and its functional impact on cancer behaviors

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Xu WT

2014-10-01

Full Text Available Wenting Xu,1 Zhen Yang,1 Shu-Feng Zhou,2 Nonghua Lu1 1Department of Gastroenterology, The First Affiliated Hospital of Nanchang University, Nanchang, Jiangxi, People’s Republic of China; 2Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA Abstract: The incidence of cancer is increasing worldwide, but the biochemical mechanisms for the occurrence of cancer is not fully understood, and there is no cure for advanced tumors. Defects of posttranslational modifications of proteins are linked to a number of important diseases, such as cancer. This review will update our knowledge on the critical role of posttranscriptional regulation of phosphatase and tensin homolog (PTEN and its activities and the functional impact on cancer behaviors. PTEN is a tumor suppressor gene that occupies a key position in regulating cell growth, proliferation, apoptosis, mobility, signal transduction, and other crucial cellular processes. The activity and function of PTEN are regulated by coordinated epigenetic, transcriptional, posttranscriptional, and posttranslational modifications. In particular, PTEN is subject to phosphorylation, ubiquitylation, somoylation, acetylation, and active site oxidation. Posttranslational modifications of PTEN can dynamically change its activity and function. Deficiency in the posttranslational regulation of PTEN leads to abnormal cell proliferation, apoptosis, migration, and adhesion, which are associated with cancer initiation, progression, and metastasis. With increasing information on how PTEN is regulated by multiple mechanisms and networked proteins, its exact role in cancer initiation, growth, and metastasis will be revealed. PTEN and its functionally related proteins may represent useful targets for the discovery of new anticancer drugs, and gene therapy and the therapeutic potentials should be fully explored. Keywords: phosphorylation, ubiquitination, acetylation, oxidation

Lithium blocks ethanol-induced modulation of protein kinases in the developing brain

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Chakraborty, Goutam; Saito, Mitsuo; Mao, Rui-Fen; Wang, Ray; Vadasz, Csaba; Saito, Mariko

2008-01-01

Lithium has been shown to be neuroprotective against various insults including ethanol exposure. We previously reported that ethanol-induced apoptotic neurodegeneration in the postnatal day 7 (P7) mice is associated with decreases in phosphorylation levels of Akt, glycogen synthase kinase-3β (GSK-3β), and AMP-activated protein kinase (AMPK), and alteration in lipid profiles in the brain. Here, P7 mice were injected with ethanol and lithium, and the effects of lithium on ethanol-induced alterations in phosphorylation levels of protein kinases and lipid profiles in the brain were examined. Immunoblot and immunohistochemical analyses showed that lithium significantly blocked ethanol-induced caspase-3 activation and reduction in phosphorylation levels of Akt, GSK-3β, and AMPK. Further, lithium inhibited accumulation of cholesterol ester (ChE) and N-acylphosphatidylethanolamine (NAPE) triggered by ethanol in the brain. These results suggest that Akt, GSK-3β, and AMPK are involved in ethanol-induced neurodegeneration and the neuroprotective effects of lithium by modulating both apoptotic and survival pathways

Sphingosine kinase-1 mediates androgen-induced osteoblast cell growth

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Martin, Claire [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); Lafosse, Jean-Michel [CHU Toulouse, Hopital Rangueil, Service d' orthopedie et Traumatologie, Toulouse F-31000 (France); Malavaud, Bernard [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France); CHU Toulouse, Hopital Rangueil, Service d' Urologie et de Transplantation Renale, Toulouse F-31000 (France); Cuvillier, Olivier, E-mail: olivier.cuvillier@ipbs.fr [CNRS, Institut de Pharmacologie et de Biologie Structurale, Toulouse F-31000 (France); Universite de Toulouse, UPS, IPBS, Toulouse F-31000 (France)

2010-01-01

Herein we report that the lipid kinase sphingosine kinase-1 (SphK1) is instrumental in mediating androgen-induced cell proliferation in osteoblasts. Dihydrotestosterone (DHT) triggered cell growth in steroid-deprived MC3T3 cells, which was associated with a rapid stimulation of SphK1 and activation of both Akt and ERK signaling pathways. This mechanism relied on functional androgen receptor/PI3K/Akt nongenotropic signaling as pharmacological antagonists could block SphK1 stimulation by DHT and its consequences. Finally, SphK1 inhibition not only abrogated DHT-induced ERK activation but also blocked cell proliferation, while ERK inhibition had no impact, suggesting that SphK1 was critical for DHT signaling yet independently of the ERK.

Creatine kinase activity in dogs with experimentally induced acute inflammation

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Dimitrinka Zapryanova

2013-01-01

Full Text Available The main purpose of this study was to investigate the effect of acute inflammation on total creatine kinase (CK activity in dogs. In these animals, CK is an enzyme found predominantly in skeletal muscle and significantly elevated serum activity is largely associated with muscle damage. Plasma increases in dogs are associated with cell membrane leakage and will therefore be seen in any condition associated with muscular inflammation. The study was induced in 15 mongrel male dogs (n=9 in experimental group and n=6 in control group at the age of two years and body weight 12-15 kg. The inflammation was reproduced by inoculation of 2 ml turpentine oil subcutaneously in lumbar region. The plasma activity of creatine kinase was evaluated at 0, 6, 24, 48, 72 hours after inoculation and on days 7, 14 and 21 by a kit from Hospitex Diagnostics. In the experimental group, the plasma concentrations of the CK-activity were increased at the 48th hour (97.48±6.92 U/L and remained significantly higher (p<0.05 at the 72 hour (97.43±2.93 U/L compared to the control group (77.08±5.27 U/L. The results of this study suggest that the evaluation of creatine kinase in dogs with experimentally induced acute inflammation has a limited diagnostic value. It was observed that the creatine kinase activity is slightly affected by the experimentally induced acute inflammation in dogs.

Synergistic action of Smad4 and Pten in suppressing pancreatic ductal adenocarcinoma formation in mice.

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Xu, X; Ehdaie, B; Ohara, N; Yoshino, T; Deng, C-X

2010-02-04

Mutations of SMAD4/DPC4 are found in about 60% of human invasive pancreatic ductal adenocarcinomas (PDACs); yet, the manner in which SMAD4 deficiency enhances tumorigenesis remains elusive. Using a Cre-LoxP approach, we generated a mutant mouse carrying a targeted deletion of Smad4 in the pancreas. We showed that the absence of Smad4 alone did not trigger pancreas tumor formation; however, it increased the expression of an inactivated form of Pten, suggesting a role of Pten in preventing Smad4-/- cells from undergoing malignancy. To investigate this, we disrupted both Pten and Smad4. We showed that Pten deficiency initiated widespread premalignant lesions, and a low tumor incidence that was significantly accelerated by Smad4-deficiency. The absence of Smad4 in a Pten-mutant background enhanced cell proliferation and triggered transdifferentiation from acinar, centroacinar and islet cells, accompanied by activation of Notch1 signaling. We showed that all tumors developed in the Smad4/Pten-mutant pancreas exhibited high levels of pAKT and mTOR, and that about 50 and 83% of human pancreatic cancers examined showed increased pAKT and pmTOR, respectively. Besides the similarity in gene expression, the pAKT and/or pmTOR-positive human PDACs and mouse pancreatic tumors also shared some histopathological similarities. These observations indicate that Smad4/Pten-mutant mice mimic the tumor progression of human pancreatic cancers that are driven by activation of the AKT-mTOR pathway, and uncovered a synergistic action of Smad4 and Pten in repressing pancreatic tumorigenesis.

Avian Reovirus Protein p17 Functions as a Nucleoporin Tpr Suppressor Leading to Activation of p53, p21 and PTEN and Inactivation of PI3K/AKT/mTOR and ERK Signaling Pathways.

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Wei-Ru Huang

Full Text Available Avian reovirus (ARV protein p17 has been shown to regulate cell cycle and autophagy by activation of p53/PTEN pathway; nevertheless, it is still unclear how p53 and PTEN are activated by p17. Here, we report for the first time that p17 functions as a nucleoporin Tpr suppressor that leads to p53 nuclear accumulation and consequently activates p53, p21, and PTEN. The nuclear localization signal (119IAAKRGRQLD128 of p17 has been identified for Tpr binding. This study has shown that Tpr suppression occurs by p17 interacting with Tpr and by reducing the transcription level of Tpr, which together inhibit Tpr function. In addition to upregulation of PTEN by activation of p53 pathway, this study also suggests that ARV protein p17 acts as a positive regulator of PTEN. ARV p17 stabilizes PTEN by stimulating phosphorylation of cytoplasmic PTEN and by elevating Rak-PTEN association to prevent it from E3 ligase NEDD4-1 targeting. To activate PTEN, p17 is able to promote β-arrestin-mediated PTEN translocation from the cytoplasm to the plasma membrane via a Rock-1-dependent manner. The accumulation of p53 in the nucleus induces the PTEN- and p21-mediated downregulation of cyclin D1 and CDK4. Furthermore, Tpr and CDK4 knockdown increased virus production in contrast to depletion of p53, PTEN, and LC3 reducing virus yield. Taken together, our data suggest that p17-mediated Tpr suppression positively regulates p53, PTEN, and p21 and negatively regulates PI3K/AKT/mTOR and ERK signaling pathways, both of which are beneficial for virus replication.

Damage-induced DNA replication stalling relies on MAPK-activated protein kinase 2 activity

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Köpper, Frederik; Bierwirth, Cathrin; Schön, Margarete

2013-01-01

knockdown of the MAP kinase-activated protein kinase 2 (MK2), a kinase currently implicated in p38 stress signaling and G2 arrest. Depletion or inhibition of MK2 also protected cells from DNA damage-induced cell death, and mice deficient for MK2 displayed decreased apoptosis in the skin upon UV irradiation...

Rho-Kinase Inhibition Ameliorates Dasatinib-Induced Endothelial Dysfunction and Pulmonary Hypertension

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Csilla Fazakas

2018-05-01

Full Text Available The multi-kinase inhibitor dasatinib is used for treatment of imatinib-resistant chronic myeloid leukemia, but is prone to induce microvascular dysfunction. In lung this can manifest as capillary leakage with pleural effusion, pulmonary edema or even pulmonary arterial hypertension. To understand how dasatinib causes endothelial dysfunction we examined the effects of clinically relevant concentrations of dasatinib on both human pulmonary arterial macro- and microvascular endothelial cells (ECs. The effects of dasatinib was compared to imatinib and nilotinib, two other clinically used BCR/Abl kinase inhibitors that do not inhibit Src. Real three-dimensional morphology and high resolution stiffness mapping revealed softening of both macro- and microvascular ECs upon dasatinib treatment, which was not observed in response to imatinib. In a dose-dependent manner, dasatinib decreased transendothelial electrical resistance/impedance and caused a permeability increase as well as disruption of tight adherens junctions in both cell types. In isolated perfused and ventilated rat lungs, dasatinib increased mean pulmonary arterial pressure, which was accompanied by a gain in lung weight. The Rho-kinase inhibitor Y27632 partly reversed the dasatinib-induced changes in vitro and ex vivo, presumably by acting downstream of Src. Co-administration of the Rho-kinase inhibitor Y27632 completely blunted the increased pulmonary pressure in response to dasatinib. In conclusion, a dasatinib-induced permeability increase in human pulmonary arterial macro- and microvascular ECs might explain many of the adverse effects of dasatinib in patients. Rho-kinase inhibition might be suitable to ameliorate these effects.

Von Hippel-Lindau status influences phenotype of liver cancers arising from PTEN loss

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Sendor AB

2015-02-01

Full Text Available Adam B Sendor,1 Kathryn E Hacker,1 Shufen Chen,1 Armando L Corona,1 Oishee Sen,1 Derek Y Chiang,1 Anna Snavely,1 Arlin B Rogers,2 Stephanie A Montgomery,1 W Kimryn Rathmell,1 Autumn J McRee11Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, USA; 2Section of Pathology, Department of Biomedical Sciences, Cummings School of Veterinary Medicine, Tufts University, Boston, MA, USABackground: PTEN loss contributes to the development of liver diseases including hepatic steatosis and both hepatocellular carcinoma (HCC and cholangiocarcinoma (CC. The factors that influence the penetrance of these conditions are unclear. We explored the influence of sustained hypoxia signaling through co-deletion of Pten and Vhl in a murine model.Methods: We used a CreER-linked Keratin 18 mouse model to conditionally delete Pten, Vhl or both in somatic cells of adult mice, evaluating the resultant tumors by histology and gene expression microarray. Existing sets of gene expression data for human HCC and CC were examined for pathways related to those observed in the murine tumors, and a cohort of human CC samples was evaluated for relationships between HIF-1α expression and clinical outcomes.Results: Both Pten deletion genotypes developed liver tumors, but with differing phenotypes. Pten deletion alone led to large hepatic tumors with widespread hepatosteatosis. Co-deletion of Pten and Vhl with the Keratin 18 promoter resulted in reduced steatosis and a reduced tumor burden that was characterized by a trabecular architecture similar to CC. Genes associated with hepatic steatosis were coordinately expressed in the human HCC dataset, while genes involved in hypoxia response were upregulated in tumors from the human CC dataset. HIF-1α expression and overall survival were examined in an independent cohort of human CC tumors with no statistical differences uncovered.Conclusion: Pten deletion in Keratin 18 expressing cells leads to

Multifocal demyelinating motor neuropathy and hamartoma syndrome associated with a de novo PTEN mutation.

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Bansagi, Boglarka; Phan, Vietxuan; Baker, Mark R; O'Sullivan, Julia; Jennings, Matthew J; Whittaker, Roger G; Müller, Juliane S; Duff, Jennifer; Griffin, Helen; Miller, James A L; Gorman, Grainne S; Lochmüller, Hanns; Chinnery, Patrick F; Roos, Andreas; Swan, Laura E; Horvath, Rita

2018-05-22

To describe a patient with a multifocal demyelinating motor neuropathy with onset in childhood and a mutation in phosphatase and tensin homolog ( PTEN ), a tumor suppressor gene associated with inherited tumor susceptibility conditions, macrocephaly, autism, ataxia, tremor, and epilepsy. Functional implications of this protein have been investigated in Parkinson and Alzheimer diseases. We performed whole-exome sequencing in the patient's genomic DNA validated by Sanger sequencing. Immunoblotting, in vitro enzymatic assay, and label-free shotgun proteomic profiling were performed in the patient's fibroblasts. The predominant clinical presentation of the patient was a childhood onset, asymmetric progressive multifocal motor neuropathy. In addition, he presented with macrocephaly, autism spectrum disorder, and skin hamartomas, considered as clinical criteria for PTEN-related hamartoma tumor syndrome. Extensive tumor screening did not detect any malignancies. We detected a novel de novo heterozygous c.269T>C, p.(Phe90Ser) PTEN variant, which was absent in both parents. The pathogenicity of the variant is supported by altered expression of several PTEN-associated proteins involved in tumorigenesis. Moreover, fibroblasts showed a defect in catalytic activity of PTEN against the secondary substrate, phosphatidylinositol 3,4-trisphosphate. In support of our findings, focal hypermyelination leading to peripheral neuropathy has been reported in PTEN-deficient mice. We describe a novel phenotype, PTEN-associated multifocal demyelinating motor neuropathy with a skin hamartoma syndrome. A similar mechanism may potentially underlie other forms of Charcot-Marie-Tooth disease with involvement of the phosphatidylinositol pathway. Copyright © 2018 The Author(s). Published by Wolters Kluwer Health, Inc. on behalf of the American Academy of Neurology.

Cellular reprogramming through mitogen-activated protein kinases

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Justin eLee

2015-10-01

Full Text Available Mitogen-activated protein kinase (MAPK cascades are conserved eukaryote signaling modules where MAPKs, as the final kinases in the cascade, phosphorylate protein substrates to regulate cellular processes. While some progress in the identification of MAPK substrates has been made in plants, the knowledge on the spectrum of substrates and their mechanistic action is still fragmentary. In this focused review, we discuss the biological implications of the data in our original paper (Sustained mitogen-activated protein kinase activation reprograms defense metabolism and phosphoprotein profile in Arabidopsis thaliana; Frontiers in Plant Science 5: 554 in the context of related research. In our work, we mimicked in vivo activation of two stress-activated MAPKs, MPK3 and MPK6, through transgenic manipulation of Arabidopsis thaliana and used phosphoproteomics analysis to identify potential novel MAPK substrates. Here, we plotted the identified putative MAPK substrates (and downstream phosphoproteins as a global protein clustering network. Based on a highly stringent selection confidence level, the core networks highlighted a MAPK-induced cellular reprogramming at multiple levels of gene and protein expression – including transcriptional, post-transcriptional, translational, post-translational (such as protein modification, folding and degradation steps, and also protein re-compartmentalization. Additionally, the increase in putative substrates/phosphoproteins of energy metabolism and various secondary metabolite biosynthesis pathways coincides with the observed accumulation of defense antimicrobial substances as detected by metabolome analysis. Furthermore, detection of protein networks in phospholipid or redox elements suggests activation of downstream signaling events. Taken in context with other studies, MAPKs are key regulators that reprogram cellular events to orchestrate defense signaling in eukaryotes.

The amoebal MAP kinase response to Legionella pneumophila is regulated by DupA.

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Li, Zhiru; Dugan, Aisling S; Bloomfield, Gareth; Skelton, Jason; Ivens, Alasdair; Losick, Vicki; Isberg, Ralph R

2009-09-17

The amoeba Dictyostelium discoideum can support replication of Legionella pneumophila. Here we identify the dupA gene, encoding a putative tyrosine kinase/dual-specificity phosphatase, in a screen for D. discoideum mutants altered in allowing L. pneumophila intracellular replication. Inactivation of dupA resulted in depressed L. pneumophila growth and sustained hyperphosphorylation of the amoebal MAP kinase ERK1, consistent with loss of a phosphatase activity. Bacterial challenge of wild-type amoebae induced dupA expression and resulted in transiently increased ERK1 phosphorylation, suggesting that dupA and ERK1 are part of a response to bacteria. Indeed, over 500 of the genes misregulated in the dupA(-) mutant were regulated in response to L. pneumophila infection, including some thought to have immune-like functions. MAP kinase phosphatases are known to be highly upregulated in macrophages challenged with L. pneumophila. Thus, DupA may regulate a MAP kinase response to bacteria that is conserved from amoebae to mammals.

Interaction of E-cadherin and PTEN regulates morphogenesis and growth arrest in human mammary epithelial cells

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Fournier, Marcia V.; Fata, Jimmie E.; Martin, Katherine J.; Yaswen, Paul; Bissell, Mina J.

2009-06-03

PTEN is a dual function phosphatase with tumor suppressor function compromised in a wide spectrum of cancers. Because tissue polarity and architecture are crucial modulators of normal and malignant behavior, we postulated that PTEN may play a role in maintenance of tissue integrity. We used two non-malignant human mammary epithelial cell lines (HMECs) that form polarized, growth-arrested structures (acini) when cultured in 3-dimensional laminin-rich extracellular matrix gels (3D lrECM). As acini begin to form, PTEN accumulates in both the cytoplasm, and at cell-cell contacts where it colocalizes with E-cadherin/{beta}-catenin complex. Reduction of PTEN levels by shRNA in lrECM prevents formation of organized breast acini and disrupts growth arrest. Importantly, disruption of acinar polarity and cell-cell contact by E-cadherin function-blocking antibodies reduces endogenous PTEN protein levels and inhibits its accumulation at cell-cell contacts. Conversely, in SKBR3 breast cancer cells lacking endogenous E-cadherin expression, exogenous introduction of E-cadherin gene causes induction of PTEN expression and its accumulation at sites of cell interactions. These studies provide evidence that E-cadherin regulates both the PTEN protein levels and its recruitment to cell-cell junctions in 3D lrECM indicating a dynamic reciprocity between architectural integrity and the levels and localization of PTEN. This interaction thus appears to be a critical integrator of proliferative and morphogenetic signaling in breast epithelial cells.

miR-367 promotes proliferation and invasion of hepatocellular carcinoma cells by negatively regulating PTEN

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Meng, Xiangrui, E-mail: mengxiangruibb2008@163.com [Oncology Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou (China); Lu, Peng [Gastrointestinal Surgery Department, People' s Hospital of Zhengzhou, Zhengzhou (China); Fan, Qingxia [Oncology Department, The First Affiliated Hospital of Zhengzhou University, Zhengzhou (China)

2016-01-29

MicroRNAs play important roles in the carcinogenesis of many types of cancers by inhibiting gene expression at posttranscriptional level. However, the roles of microRNAs in hepatocellular carcinoma, are still unclear. Here, we identified that miR-367 promotes hepatocellular carcinoma (HCC) cell proliferation by negatively regulates its target gene PTEN. The expression of miR-367 and PTEN are significantly inverse correlated in 35 HCC patients. In HCC cell line, CCK-8 proliferation assay indicated that the cell proliferation was promoted by miR-367, while miR-367 inhibitor significantly inhibited the cell proliferation. Transwell assay showed that miR-367 mimics significantly promoted the migration and invasion of HCC cells, whereas miR-367 inhibitors significantly reduced cell migration and invasion. Luciferase assays confirmed that miR-367 directly bound to the 3'untranslated region of PTEN, and western blotting showed that miR-367 suppressed the expression of PTEN at the protein levels. This study indicated that miR-367 negatively regulates PTEN and promotes proliferation and invasion of HCC cells. Thus, miR-367 may represent a potential therapeutic target for HCC intervention. - Highlights: • miR-367 mimics promote the proliferation and invasion of HCC cells. • miR-367 inhibitors inhibit the proliferation and invasion of HCC cells. • miR-367 targets 3′UTR of PTEN in HCC cells. • miR-367 negatively regulates PTEN in HCC cells.

A novel deleterious PTEN mutation in a patient with early-onset bilateral breast cancer

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Pradella, Laura Maria; Gasparre, Giuseppe; Turchetti, Daniela; Evangelisti, Cecilia; Ligorio, Claudia; Ceccarelli, Claudio; Neri, Iria; Zuntini, Roberta; Amato, Laura Benedetta; Ferrari, Simona; Martelli, Alberto Maria

2014-01-01

An early age at Breast Cancer (BC) onset may be a hallmark of inherited predisposition, but BRCA1/2 mutations are only found in a minority of younger BC patients. Among the others, a fraction may carry mutations in rarer BC genes, such as TP53, STK11, CDH1 and PTEN. As the identification of women harboring such mutations allows for targeted risk-management, the knowledge of associated manifestations and an accurate clinical and family history evaluation are warranted. We describe the case of a woman who developed an infiltrating ductal carcinoma of the right breast at the age of 32, a contralateral BC at age 36 and another BC of the right breast at 40. When she was 39 years-old, during a dermatological examination, mucocutaneous features suggestive of Cowden Syndrome, a disorder associated to germ-line PTEN mutations, were noticed. PTEN genetic testing revealed the novel c.71A > T (p.Asp24Val) mutation, whose deleterious effect, suggested by conservation data and in silico tools, was definitely demonstrated by the incapacity of mutant PTEN to inhibit Akt phosphorylation when used to complement PTEN-null cells. In BC tissue, despite the absence of LOH or somatic mutations of PTEN, Akt phosphorylation was markedly increased in comparison to normal tissue, thus implying additional somatic events into the deregulation of the PI3K/Akt/mTOR pathway and, presumably, into carcinogenesis. Hence, known oncogenic mutations in PIK3CA (exons 10 and 21) and AKT1 (exon 2) were screened in tumor DNA with negative results, which suggests that the responsible somatic event(s) is a different, uncommon one. This case stresses the importance of clinical/genetic assessment of early-onset BC patients in order to identify mutation carriers, who are at high risk of new events, so requiring tailored management. Moreover, it revealed a novel PTEN mutation with pathogenic effect, pointing out, however, the need for further efforts to elucidate the molecular steps of PTEN

Aurora A kinase RNAi and small molecule inhibition of Aurora kinases with VE-465 induce apoptotic death in multiple myeloma cells.

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Evans, Robert; Naber, Claudia; Steffler, Tara; Checkland, Tamara; Keats, Jonathan; Maxwell, Christopher; Perry, Troy; Chau, Heidi; Belch, Andrew; Pilarski, Linda; Reiman, Tony

2008-03-01

The expression of RHAMM and other centrosome-associated genes are known to correlate with the extent of centrosome amplification in multiple myeloma, and with poor prognosis. RHAMM has a significant interaction with TPX2, a protein which regulates the localization and action of Aurora A kinase (AURKA) at the spindle poles. AURKA is known to be a central determinant of centrosome and spindle function and is a target for cancer therapy. Given these observations, we investigated the role of Aurora kinases as therapeutic targets in myeloma. Here we report that AURKA is expressed ubiquitously in myeloma, to varying degrees, in both cell lines and patients' bone marrow plasma cells. siRNA targeting AURKA induces apoptotic cell death in myeloma cell lines. The Aurora kinase inhibitor VE-465 also induces apoptosis and death in myeloma cell lines and primary myeloma plasma cells. The combination of VE-465 and dexamethasone improves cell killing compared with the use of either agent alone, even in cells resistant to the single agents. The phenotype of myeloma cells treated with VE-465 is consistent with published reports on the effects of Aurora kinase inhibition. Aurora kinase inhibitors should be pursued as potential treatments for myeloma.

Inhibition of phosphatidylinositol-3-kinase causes increased sensitivity to radiation through a PKB-dependent mechanism

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Gottschalk, Alexander R.; Doan, Albert; Nakamura, Jean L.; Stokoe, David; Haas-Kogan, Daphne A.

2005-01-01

Purpose: To identify whether inhibition of phosphatidylinositol-3-kinase (PI3K) causes increased radiosensitivity through inhibition of protein kinase B (PKB), implicating PKB as an important therapeutic target in prostate cancer. Methods and Materials: The prostate cancer cell line LNCaP was treated with the PI3K inhibitor LY294002, radiation, and combinations of the two therapies. Apoptosis and survival were measured by cell cycle analysis, Western blot analysis for cleaved poly (ADP-ribose) polymerase, and clonogenic survival. To test the hypothesis that inhibition of PKB is responsible for LY294002-induced radiosensitivity, LNCaP cells expressing a constitutively active form of PKB were used. Results: The combination of PI3K inhibition and radiation caused an increase in apoptosis and a decrease in clonogenic survival when compared to either modality alone. The expression of constitutively activated PKB blocked apoptosis induced by combination of PI3K inhibition and radiation and prevented radiosensitization by LY294002. Conclusion: These data indicate that PI3K inhibition increases sensitivity of prostate cancer cell lines to ionizing radiation through inactivation of PKB. Therefore, PTEN mutations, which lead to PKB activation, may play an important role in the resistance of prostate cancer to radiation therapy. Targeted therapy against PKB could be beneficial in the management of prostate cancer patients

A recessive form of extreme macrocephaly and mild intellectual disability complements the spectrum of PTEN hamartoma tumour syndrome.

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Schwerd, Tobias; Khaled, Andrea V; Schürmann, Manfred; Chen, Hannah; Händel, Norman; Reis, André; Gillessen-Kaesbach, Gabriele; Uhlig, Holm H; Abou Jamra, Rami

2016-06-01

PTEN hamartoma tumour syndrome (PHTS) is caused by heterozygous variants in PTEN and is characterised by tumour predisposition, macrocephaly, and cognition impairment. Bi-allelic loss of PTEN activity has not been reported so far and animal models suggest that bi-allelic loss of PTEN activity is embryonically lethal. Here, we report the identification of a novel homozygous variant in PTEN, NM_000314.4; c.545T>C; p.Leu182Ser, in two adolescent siblings with severe macrocephaly and mild intellectual disability. The variant is predicted to be damaging and is associated with significantly increased phospho-S6 downstream of PTEN. The absence of tumours in the two homozygous siblings as well as lack of symptoms of PHTS in the heterozygous carriers of the family suggest that this particular variant is functionally hypomorphic rather than deleterious.

The microbiome in PTEN hamartoma tumor syndrome.

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Byrd, Victoria; Getz, Ted; Padmanabhan, Roshan; Arora, Hans; Eng, Charis

2018-03-01

Germline PTEN mutations defining PTEN hamartoma tumor syndrome (PHTS) confer heritable predisposition to breast, endometrial, thyroid and other cancers with known age-related risks, but it remains impossible to predict if any individual will develop cancer. In the general population, gut microbial dysbiosis has been linked to cancer, yet is unclear whether these are associated in PHTS patients. In this pilot study, we aimed to characterize microbial composition of stool, urine, and oral wash from 32 PTEN mutation-positive individuals using 16S rRNA gene sequencing. PCoA revealed clustering of the fecal microbiome by cancer history ( P  = 0.03, R 2  = 0.04). Fecal samples from PHTS cancer patients had relatively more abundant operational taxonomic units (OTUs) from family Rikenellaceae and unclassified members of Clostridia compared to those from non-cancer patients, whereas families Peptostreptococcaceae, Enterobacteriaceae, and Bifidobacteriaceae represented relatively more abundant OTUs among fecal samples from PHTS non-cancer patients. Functional metagenomic prediction revealed enrichment of the folate biosynthesis, genetic information processing and cell growth and death pathways among fecal samples from PHTS cancer patients compared to non-cancer patients. We found no major shifts in overall diversity and no clustering by cancer history among oral wash or urine samples. Our observations suggest the utility of an expanded study to interrogate gut dysbiosis as a potential cancer risk modifier in PHTS patients. © 2018 The authors.

Association of promoter methylation and 32-bp deletion of the PTEN gene with susceptibility to metabolic syndrome.

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Hashemi, Mohammad; Rezaei, Hamzeh; Eskandari-Nasab, Ebrahim; Kaykhaei, Mahmoud-Ali; Taheri, Mohsen

2013-01-01

Metabolic syndrome (MeS), a cluster of several metabolic disorders, is increasingly being recognized as a risk factor for type II diabetes (T2D) and cardiovascular disease. Genetic and epigenetic alteration of the phosphatase and tensin homolog deleted on chromosome ten (PTEN) has been associated with components of MeS. The aim of the present study was to investigate the possible association of a 32-bp deletion polymorphism and promoter methylation of the PTEN gene with MeS. DNA was extracted from the peripheral blood of 151 subjects with and 149 subjects without MeS. The 32-bp deletion variant of PTEN was detected by polymerase chain reaction (PCR) and PTEN promoter methylation was defined by a nested methylation‑specific PCR (MSP) method. No significant differences were found in the allelic and genotypic frequencies of the 32-bp deletion variant of PTEN between the groups [odds ratio (OR), 0.77; 95% confidence interval (CI), 0.41-1.45; P=0.431]. However, patients with MeS were identified to have lower levels of PTEN promoter hypermethylation than subjects without MeS. Promoter methylation may be a protective factor against susceptibility to MeS (OR, 0.52; 95% CI, 0.29-0.92; P=0.029). Our findings suggest that PTEN promoter methylation may be a mechanism for PTEN downregulation or silencing in MeS, which remains to be fully clarified.

Alterations in PTEN and PIK3CA in colorectal cancers in the EPIC Norfolk study: associations with clinicopathological and dietary factors

International Nuclear Information System (INIS)

Naguib, Adam; Arends, Mark J; Cooke, James C; Happerfield, Lisa; Kerr, Lucy; Gay, Laura J; Luben, Robert N; Ball, Richard Y; Mitrou, Panagiota N; McTaggart, Alison

2011-01-01

The PTEN tumour suppressor gene and PIK3CA proto-oncogene encode proteins which contribute to regulation and propagation of signal transduction through the PI3K/AKT signalling pathway. This study investigates the prevalence of loss of PTEN expression and mutations in both PTEN and PIK3CA in colorectal cancers (CRC) and their associations with tumour clinicopathological features, lifestyle factors and dietary consumptions. 186 adenocarcinomas and 16 adenomas from the EPIC Norfolk study were tested for PTEN and PIK3CA mutations by DNA sequencing and PTEN expression changes by immunohistochemistry. Dietary and lifestyle data were collected prospectively using seven day food diaries and lifestyle questionnaires. Mutations in exons 7 and 8 of PTEN were observed in 2.2% of CRC and PTEN loss of expression was identified in 34.9% CRC. Negative PTEN expression was associated with lower blood low-density lipoprotein concentrations (p = 0.05). PIK3CA mutations were observed in 7% of cancers and were more frequent in CRCs in females (p = 0.04). Analysis of dietary intakes demonstrated no link between PTEN expression status and any specific dietary factor. PTEN expression negative, proximal CRC were of more advanced Dukes' stage (p = 0.02) and poor differentiation (p < 0.01). Testing of the prevalence of PIK3CA mutations and loss of PTEN expression demonstrated that these two events were independent (p = 0.55). These data demonstrated the frequent occurrence (34.9%) of PTEN loss of expression in colorectal cancers, for which gene mutations do not appear to be the main cause. Furthermore, dietary factors are not associated with loss of PTEN expression. PTEN expression negative CRC were not homogenous, as proximal cancers were associated with a more advanced Dukes' stage and poor differentiation, whereas distal cancers were associated with earlier Dukes' stage

CSK negatively regulates nerve growth factor induced neural differentiation and augments AKT kinase activity

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Dey, Nandini; Howell, Brian W.; De, Pradip K.; Durden, Donald L.

2005-01-01

Src family kinases are involved in transducing growth factor signals for cellular differentiation and proliferation in a variety of cell types. The activity of all Src family kinases (SFKs) is controlled by phosphorylation at their C-terminal 527-tyrosine residue by C-terminal SRC kinase, CSK. There is a paucity of information regarding the role of CSK and/or specific Src family kinases in neuronal differentiation. Pretreatment of PC12 cells with the Src family kinase inhibitor, PP1, blocked NGF-induced activation of SFKs and obliterated neurite outgrowth. To confirm a role for CSK and specific isoforms of SFKs in neuronal differentiation, we overexpressed active and catalytically dead CSK in the rat pheochromocytoma cell line, PC12. CSK overexpression caused a profound inhibition of NGF-induced activation of FYN, YES, RAS, and ERK and inhibited neurite outgrowth, NGF-stimulated integrin-directed migration and blocked the NGF-induced conversion of GDP-RAC to its GTP-bound active state. CSK overexpression markedly augmented the activation state of AKT following NGF stimulation. In contrast, kinase-dead CSK augmented the activation of FYN, RAS, and ERK and increased neurite outgrowth. These data suggest a distinct requirement for CSK in the regulation of NGF/TrkA activation of RAS, RAC, ERK, and AKT via the differential control of SFKs in the orchestration of neuronal differentiation

Lindersin B from Lindernia crustacea induces neuritogenesis by activation of tyrosine kinase A/phosphatidylinositol 3 kinase/extracellular signal-regulated kinase signaling pathway.

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Cheng, Lihong; Ye, Ying; Xiang, Lan; Osada, Hiroyuki; Qi, Jianhua

2017-01-15

Neurotrophic factors such as nerve growth factor (NGF) play important roles in nervous system. NGF is a potential therapeutic drug for treatment of neurodegenerative diseases. However, because of physicochemical property, NGF cannot pass through the blood-brain barrier (BBB). Hence, small molecules which exhibit NGF-mimic activity and can pass through the BBB are considered to be promising drug candidates for treatment of such diseases. The present study was designed to isolate NGF-mimic substance from extract of natural products, determine their structures and investigate mechanism of action of the active substance. Extract of Lindernia crustacean was partitioned between water and ethyl acetate to obtain water layer and ethyl acetate layer samples, respectively, and then evaluated their neuritogenic activity in PC12 cells. The active sample was separated by open columns, followed by HPLC purification to obtain active compound. Then, specific inhibitors were used to investigate signaling pathway of neurite outgrowth induced by the active compound. Finally, western blot analysis was performed to confirm the pathway proposed by inhibitor experiments. The ethyl acetate layer sample of extract of Lindernia crustacea exhibited significant neuritogenic activity. Two new compounds, named as linderside A and lindersin B, were isolated; their structures were elucidated by spectroscopic and chemical derivatization methods. Linderside A is a cucurbitane glycoside, whereas lindersin B is a cucurbitane triterpenoid. Each compound has an unusual isopentene unit, namely, a double bond bound to an unmodified isopropyl group at the end of cucurbitane triterpenoid side chain. Among them, lindersin B induced significant neurite outgrowth in PC12 cells, while linderside A was inactive against PC12 cells. Western blotting analysis results showed that lindersin B-induced neuritogenic activity depended on the activation of the mitogen-activated protein kinase (MAPK)/extracellular signal

VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

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Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica; Gonzalez Espinosa, Claudia

2010-01-01

Research highlights: → Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. → CoCl 2 -induced VEGF secretion in mast cells occurs by a Ca 2+ -insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. → Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits FcεRI-dependent anaphylactic degranulation in mast cells. → Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl 2 ) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl 2 promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl 2 -induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl 2 -induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl 2 in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals-dependent Fyn kinase activation.

VEGF secretion during hypoxia depends on free radicals-induced Fyn kinase activity in mast cells

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Garcia-Roman, Jonathan; Ibarra-Sanchez, Alfredo; Lamas, Monica [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico); Gonzalez Espinosa, Claudia, E-mail: cgonzal@cinvestav.mx [Departamento de Farmacobiologia, Centro de Investigacion y de Estudios Avanzados del IPN (Cinvestav, IPN) (Mexico)

2010-10-15

Research highlights: {yields} Bone marrow-derived mast cells (BMMCs) secrete functional VEGF but do not degranulate after Cobalt chloride-induced hypoxia. {yields} CoCl{sub 2}-induced VEGF secretion in mast cells occurs by a Ca{sup 2+}-insensitive but brefeldin A and Tetanus toxin-sensitive mechanism. {yields} Trolox and N-acetylcysteine inhibit hypoxia-induced VEGF secretion but only Trolox inhibits Fc{epsilon}RI-dependent anaphylactic degranulation in mast cells. {yields} Src family kinase Fyn activation after free radical production is necessary for hypoxia-induced VEGF secretion in mast cells. -- Abstract: Mast cells (MC) have an important role in pathologic conditions such as asthma and chronic obstructive pulmonary disease (COPD), where hypoxia conduce to deleterious inflammatory response. MC contribute to hypoxia-induced angiogenesis producing factors such as vascular endothelial growth factor (VEGF), but the mechanisms behind the control of hypoxia-induced VEGF secretion in this cell type is poorly understood. We used the hypoxia-mimicking agent cobalt chloride (CoCl{sub 2}) to analyze VEGF secretion in murine bone marrow-derived mast cells (BMMCs). We found that CoCl{sub 2} promotes a sustained production of functional VEGF, able to induce proliferation of endothelial cells in vitro. CoCl{sub 2}-induced VEGF secretion was independent of calcium rise but dependent on tetanus toxin-sensitive vesicle-associated membrane proteins (VAMPs). VEGF exocytosis required free radicals formation and the activation of Src family kinases. Interestingly, an important deficiency on CoCl{sub 2}-induced VEGF secretion was observed in Fyn kinase-deficient BMMCs. Moreover, Fyn kinase was activated by CoCl{sub 2} in WT cells and this activation was prevented by treatment with antioxidants such as Trolox and N-acetylcysteine. Our results show that BMMCs are able to release VEGF under hypoxic conditions through a tetanus toxin-sensitive mechanism, promoted by free radicals

Signaling via class IA Phosphoinositide 3-kinases (PI3K in human, breast-derived cell lines.

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Veronique Juvin

Full Text Available We have addressed the differential roles of class I Phosphoinositide 3-kinases (PI3K in human breast-derived MCF10a (and iso-genetic derivatives and MDA-MB 231 and 468 cells. Class I PI3Ks are heterodimers of p110 catalytic (α, β, δ and γ and p50-101 regulatory subunits and make the signaling lipid, phosphatidylinositol (3,4,5-trisphosphate (PtdIns(3,4,5P3 that can activate effectors, eg protein kinase B (PKB, and responses, eg migration. The PtdIns(3,4,5P3-3-phosphatase and tumour-suppressor, PTEN inhibits this pathway. p110α, but not other p110s, has a number of onco-mutant variants that are commonly found in cancers. mRNA-seq data shows that MCF10a cells express p110β>>α>δ with undetectable p110γ. Despite this, EGF-stimulated phosphorylation of PKB depended upon p110α-, but not β- or δ- activity. EGF-stimulated chemokinesis, but not chemotaxis, was also dependent upon p110α, but not β- or δ- activity. In the presence of single, endogenous alleles of onco-mutant p110α (H1047R or E545K, basal, but not EGF-stimulated, phosphorylation of PKB was increased and the effect of EGF was fully reversed by p110α inhibitors. Cells expressing either onco-mutant displayed higher basal motility and EGF-stimulated chemokinesis.This latter effect was, however, only partially-sensitive to PI3K inhibitors. In PTEN(-/- cells, basal and EGF-stimulated phosphorylation of PKB was substantially increased, but the p110-dependency was variable between cell types. In MDA-MB 468s phosphorylation of PKB was significantly dependent on p110β, but not α- or δ- activity; in PTEN(-/- MCF10a it remained, like the parental cells, p110α-dependent. Surprisingly, loss of PTEN suppressed basal motility and EGF-stimulated chemokinesis. These results indicate that; p110α is required for EGF signaling to PKB and chemokinesis, but not chemotaxis; onco-mutant alleles of p110α augment signaling in the absence of EGF and may increase motility, in part, via acutely

PTEN deletion from adult-generated dentate granule cells disrupts granule cell mossy fiber axon structure.

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LaSarge, Candi L; Santos, Victor R; Danzer, Steve C

2015-03-01

Dysregulation of the mTOR-signaling pathway is implicated in the development of temporal lobe epilepsy. In mice, deletion of PTEN from hippocampal dentate granule cells leads to mTOR hyperactivation and promotes the rapid onset of spontaneous seizures. The mechanism by which these abnormal cells initiate epileptogenesis, however, is unclear. PTEN-knockout granule cells develop abnormally, exhibiting morphological features indicative of increased excitatory input. If these cells are directly responsible for seizure genesis, it follows that they should also possess increased output. To test this prediction, dentate granule cell axon morphology was quantified in control and PTEN-knockout mice. Unexpectedly, PTEN deletion increased giant mossy fiber bouton spacing along the axon length, suggesting reduced innervation of CA3. Increased width of the mossy fiber axon pathway in stratum lucidum, however, which likely reflects an unusual increase in mossy fiber axon collateralization in this region, offsets the reduction in boutons per axon length. These morphological changes predict a net increase in granule cell innervation of CA3. Increased diameter of axons from PTEN-knockout cells would further enhance granule cell communication with CA3. Altogether, these findings suggest that amplified information flow through the hippocampal circuit contributes to seizure occurrence in the PTEN-knockout mouse model of temporal lobe epilepsy. Copyright © 2015 Elsevier Inc. All rights reserved.

Aging-associated dysfunction of Akt/protein kinase B: S-nitrosylation and acetaminophen intervention.

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Miaozong Wu

Full Text Available BACKGROUND: Aged skeletal muscle is characterized by an increased incidence of metabolic and functional disorders, which if allowed to proceed unchecked can lead to increased morbidity and mortality. The mechanism(s underlying the development of these disorders in aging skeletal muscle are not well understood. Protein kinase B (Akt/PKB is an important regulator of cellular metabolism and survival, but it is unclear if aged muscle exhibits alterations in Akt function. Here we report a novel dysfunction of Akt in aging muscle, which may relate to S-nitrosylation and can be prevented by acetaminophen intervention. PRINCIPAL FINDINGS: Compared to 6- and 27-month rats, the phosphorylation of Akt (Ser473 and Thr308 was higher in soleus muscles of very aged rats (33-months. Paradoxically, these increases in Akt phosphorylation were associated with diminished mammalian target of rapamycin (mTOR phosphorylation, along with decreased levels of insulin receptor beta (IR-beta, phosphoinositide 3-kinase (PI3K, phosphatase and tensin homolog deleted on chromosome 10 (PTEN and phosphorylation of phosphoinositide-dependent kinase-1 (PDK1 (Ser241. In vitro Akt kinase measurements and ex vivo muscle incubation experiments demonstrated age-related impairments of Akt kinase activity, which were associated with increases in Akt S-nitrosylation and inducible nitric oxide synthase (iNOS. Impairments in Akt function occurred parallel to increases in myocyte apoptosis and decreases in myocyte size and the expression of myosin and actin. These age-related disorders were attenuated by treating aged (27-month animals with acetaminophen (30 mg/kg body weight/day for 6-months. CONCLUSIONS: These data demonstrate that Akt dysfunction and increased S-nitrosylation of Akt may contribute to age-associated disorders in skeletal muscle and that acetaminophen may be efficacious for the treatment of age-related muscle dysfunction.

Protein kinase D stabilizes aldosterone-induced ERK1/2 MAP kinase activation in M1 renal cortical collecting duct cells to promote cell proliferation.

LENUS (Irish Health Repository)

McEneaney, Victoria

2010-01-01

Aldosterone elicits transcriptional responses in target tissues and also rapidly stimulates the activation of protein kinase signalling cascades independently of de novo protein synthesis. Here we investigated aldosterone-induced cell proliferation and extra-cellular regulated kinase 1 and 2 (ERK1\\/2) mitogen activated protein (MAP) kinase signalling in the M1 cortical collecting duct cell line (M1-CCD). Aldosterone promoted the proliferative growth of M1-CCD cells, an effect that was protein kinase D1 (PKD1), PKCdelta and ERK1\\/2-dependent. Aldosterone induced the rapid activation of ERK1\\/2 with peaks of activation at 2 and 10 to 30 min after hormone treatment followed by sustained activation lasting beyond 120 min. M1-CCD cells suppressed in PKD1 expression exhibited only the early, transient peaks in ERK1\\/2 activation without the sustained phase. Aldosterone stimulated the physical association of PKD1 with ERK1\\/2 within 2 min of treatment. The mineralocorticoid receptor (MR) antagonist RU28318 inhibited the early and late phases of aldosterone-induced ERK1\\/2 activation, and also aldosterone-induced proliferative cell growth. Aldosterone induced the sub-cellular redistribution of ERK1\\/2 to the nuclei at 2 min and to cytoplasmic sites, proximal to the nuclei after 30 min. This sub-cellular distribution of ERK1\\/2 was inhibited in cells suppressed in the expression of PKD1.

Loss of PTEN expression is associated with aggressive behavior and poor prognosis in Middle Eastern triple-negative breast cancer.

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Beg, Shaham; Siraj, Abdul K; Prabhakaran, Sarita; Jehan, Zeenath; Ajarim, Dahish; Al-Dayel, Fouad; Tulbah, Asma; Al-Kuraya, Khawla S

2015-06-01

PTEN is a tumor suppressor that negatively regulates the PI3 K-AKT signaling pathway which is involved in the pathogenesis of many different tumor types and serves as a prognostic marker in breast cancer. However, the significance of the role of PTEN in Middle Eastern ethnic breast cancer has not been explored especially with the fact that breast cancer originating from this ethnic population tend to behave more aggressively than breast cancer in the west. In this study, we analyzed PTEN alteration in a tissue microarray format containing more than 1000 primary breast cancers with clinical follow-up data. Tissue Microarray sections were analyzed for protein expression and copy number change using immunohistochemistry and fluorescence in situ hybridization. Loss of PTEN immunostaining was observed in 77 % of the cases. PTEN loss was significantly associated with large tumor size (p = 0.0030), high grade (p = 0.0281), tumor recurrence (p = 0.0333), and triple-negative breast cancers (p = 0.0086). PTEN loss in triple-negative breast cancers was significantly associated with rapid tumor cell proliferation (p = 0.0396) and poor prognosis (p = 0.0408). PTEN deletion was found only in 60 cases (6.4 %). Loss of PTEN protein expression occurs at high frequency in Middle Eastern breast cancer. PTEN inactivation may potentially lead to an aggressive behavior of tumor cells through stimulation of tumor cell proliferation. Furthermore, PTEN signaling pathway might be used as potential therapeutic target in triple-negative breast cancers since loss of its expression is shown to be significantly associated with this aggressive subtype of breast cancer.

MHC-I-induced apoptosis in human B-lymphoma cells is dependent on protein tyrosine and serine/threonine kinases

DEFF Research Database (Denmark)

Pedersen, Anders Elm; Bregenholt, S; Johansen, B

1999-01-01

B lymphoma cells, is dependent on protein tyrosine kinases and the phosphatidylinositol 3 (PI-3) kinase. Functional studies showed that MHC-I crosslinking induced almost complete inhibition of the spontaneous proliferation of the B lymphoma cells as early as 6 h post-crosslinking and apoptosis 24 h...... post-crosslinking. Preincubation with either protein tyrosine kinase or protein serine/threonine kinase inhibitors reduced the MHC-I-induced apoptosis to background levels, whereas inhibition of PI-3 kinase had no effect. These data demonstrate a pivotal role for protein tyrosine and serine...

Kinase-dead ATM protein is highly oncogenic and can be preferentially targeted by Topo-isomerase I inhibitors.

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Yamamoto, Kenta; Wang, Jiguang; Sprinzen, Lisa; Xu, Jun; Haddock, Christopher J; Li, Chen; Lee, Brian J; Loredan, Denis G; Jiang, Wenxia; Vindigni, Alessandro; Wang, Dong; Rabadan, Raul; Zha, Shan

2016-06-15

Missense mutations in ATM kinase, a master regulator of DNA damage responses, are found in many cancers, but their impact on ATM function and implications for cancer therapy are largely unknown. Here we report that 72% of cancer-associated ATM mutations are missense mutations that are enriched around the kinase domain. Expression of kinase-dead ATM (Atm(KD/-)) is more oncogenic than loss of ATM (Atm(-/-)) in mouse models, leading to earlier and more frequent lymphomas with Pten deletions. Kinase-dead ATM protein (Atm-KD), but not loss of ATM (Atm-null), prevents replication-dependent removal of Topo-isomerase I-DNA adducts at the step of strand cleavage, leading to severe genomic instability and hypersensitivity to Topo-isomerase I inhibitors. Correspondingly, Topo-isomerase I inhibitors effectively and preferentially eliminate Atm(KD/-), but not Atm-proficientor Atm(-/-) leukemia in animal models. These findings identify ATM kinase-domain missense mutations as a potent oncogenic event and a biomarker for Topo-isomerase I inhibitor based therapy.

PTEN and DMBT1 homozygous deletion and expression in medulloblastomas and supratentorial primitive neuroectodermal tumors.

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Inda, María Mar; Mercapide, Javier; Muñoz, Jorge; Coullin, Philippe; Danglot, Giséle; Tuñon, Teresa; Martínez-Peñuela, José María; Rivera, José María; Burgos, Juan J; Bernheim, Alain; Castresana, Javier S

2004-12-01

Medulloblastoma, which accounts for 20-25% of all childhood brain tumors, is defined as a primitive neuroectodermal tumor (PNET) located in the cerebellum. Supratentorial PNET are less frequent than medulloblastoma. But their clinical outcome is worse than in medulloblastomas. Chromosome 10q contains at least 2 tumor suppressor genes that might play a role in brain tumor development: PTEN and DMBT1. The aim of this study was to compare the status of homozygous deletion and expression of PTEN and DMBT1 genes in PNET primary tumor samples and cell lines. Homozygous deletions of PTEN and DMBT1 were studied in 32 paraffin-embedded PNET samples (23 medulloblastomas and 9 supratentorial PNET) and in 7 PNET cell lines, by differential PCR and by FISH. PTEN homozygous losses were demonstrated in 7 medulloblastomas (32%) and in no supratentorial PNET, while homozygous deletions of DMBT1 appeared in 1 supratentorial PNET (20%) and in 7 medulloblastomas (33%). No homozygous deletion of PTEN or DMBT1 was detected in any of the PNET cell lines either by differential PCR or by FISH. Expression study of the 2 genes was performed in the 7 PNET cell lines by RT-PCR. One PNET cell line lacked PTEN and DMBT1 expression, while 2 medulloblastoma cell lines did not express DMBT1. Our results add some positive data to the hypothesis that supratentorial PNETs and medulloblastomas might be genetically different.

PTEN expression as a predictor for the response to trastuzumab-based therapy in Her-2 overexpressing metastatic breast cancer.

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Daphne Gschwantler-Kaulich

Full Text Available Even though trastuzumab is an effective therapy in early stage Her-2+ breast cancer, 40-50% of advanced Her-2+ breast cancer patients develop trastuzumab resistance. A potential resistance mechanism is aberrant downstream signal transmission due to loss of phosphatase and tensin homologue (PTEN. This study investigated the relationship between the expression of PTEN and trastuzumab response in Her-2 overexpressing metastatic breast cancer patients.Between 2000 and 2007, 164 patients with Her-2+ metastatic breast cancer received trastuzumab-based therapy in our institution. We analyzed PTEN status by immunohistochemistry of 115 available tumor tissues and analyzed associations with other histopathological parameters, response rate, progression free survival (PFS and overall survival (OS with a median follow-up of 60 months.Eighty patients were PTEN positive (69.6% and 35 patients PTEN negative (30.4%. We found a significant association of the expression of PTEN and p53 (p = 0.041, while there was no association with grading, hormone receptor status, IGFR or MIB. We found significantly more cases with progressive disease under trastuzumab-based therapy in patients with PTEN positive breast cancers (p = 0.018, while there was no significant correlation with PFS or OS.In Her-2-positive metastatic breast cancers, PTEN positivity was significantly associated with progressive disease, but not with PFS or OS.

PTEN Sequence Analysis in Endometrial Hyperplasia and Endometrial Carcinoma in Slovak Women

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H. Gbelcová

2015-01-01

Full Text Available Phosphatase and tensin homolog (PTEN is a protein that acts as a tumor suppressor by dephosphorylating the lipid second messenger phosphatidylinositol 3,4,5-trisphosphate. Loss of PTEN function has been implicated in the pathogenesis of a number of different tumors, particularly endometrial carcinoma (ECa. ECa is the most common neoplasia of the female genital tract. Our study evaluates an association between the morphological appearance of endometrial hyperplasia and endometrial carcinoma and the degree of PTEN alterations. A total of 45 endometrial biopsies from Slovak women were included in present study. Formalin-fixed and paraffin-embedded tissue samples with simple hyperplasia (3, complex hyperplasia (5, atypical complex hyperplasia (7, endometrioid carcinomas G1 (20 and G3 (5, and serous carcinoma (5 were evaluated for the presence of mutations in coding regions of PTEN gene, the most frequently mutated tumor suppressor gene in endometrial carcinoma. 75% of the detected mutations were clustered in exons 5 and 8. Out of the 39 mutations detected in 24 cases, 20 were frameshifts and 19 were nonsense, missense, or silent mutations. Some specimens harboured more than one mutation. The results of current study on Slovak women were compared to a previous study performed on Polish population. The two sets of results were similar.

Pten regulates spindle pole movement through Dlg1-mediated recruitment of Eg5 to centrosomes

NARCIS (Netherlands)

Ree, J.H. van; Nam, H.J.; Jeganathan, K.B.; Kanakkanthara, A.; Deursen, J.M.A. van

2016-01-01

Phosphatase and tensin homologue (Pten) suppresses neoplastic growth by negatively regulating PI(3)K signalling through its phosphatase activity. To gain insight into the actions of non-catalytic Pten domains in normal physiological processes and tumorigenesis, we engineered mice lacking the

[Overexpression of liver kinase B1 inhibits the proliferation of lung cancer cells].

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Li, Yang; Zhang, Libin; Wang, Ping

2017-01-01

Objective To explore the effect of overexpressed liver kinase B1(LKB1) on the proliferation of lung cancer cell lines. Methods The expression levels of LKB1 and PTEN in A549, NCI-H23, NCI-H157, XWLC-05, NCI-H446 lung cancer cells were detected by immunocytochemistry (ICC) and Western blotting. Plasmid pcDNA3.1 + -LKB1 and empty vector pcDNA3.1 + -null were separately transfected into the above five cell lines, and then the expression of LKB1 mRNA and protein were determined by quantitative real-time PCR and Western blotting, respectively. Finally, CCK-8 assay was used to analyze the proliferation ability of the transfected cells. Results LKB1 and PTEN were positive in NCI-H23 cells; LKB1 was negative while PTEN was positive in A549 and NCI-H446 cells; both LKB1 and PTEN were negative in NCI-H157 and XWLC-05 cells. Quantitative real-time PCR and Western blotting showed that the expression level of LKB1 significantly increased in the above cell lines transfected with plasmid pcDNA3.1 + -LKB1 compared with the ones with empty vector pcDNA3.1 + -null. Besides, CCK-8 assay showed that the overexpression of LKB1 in the lung cancer cells transfected with pcDNA3.1 + -LKB1 had an obvious inhibitory effect on cell proliferation. Conclusion The expression of LKB1 is down-regulated in most of the lung cell lines to different extent and the over-expression of LKB1 can remarkably inhibit the proliferation ability of lung cancer cell lines.

Rac1-mediated membrane raft localization of PI3K/p110β is required for its activation by GPCRs or PTEN loss

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Cizmecioglu, Onur; Ni, Jing; Xie, Shaozhen; Zhao, Jean J; Roberts, Thomas M

2016-01-01

We aimed to understand how spatial compartmentalization in the plasma membrane might contribute to the functions of the ubiquitous class IA phosphoinositide 3-kinase (PI3K) isoforms, p110α and p110β. We found that p110β localizes to membrane rafts in a Rac1-dependent manner. This localization potentiates Akt activation by G-protein-coupled receptors (GPCRs). Thus genetic targeting of a Rac1 binding-deficient allele of p110β to rafts alleviated the requirement for p110β-Rac1 association for GPCR signaling, cell growth and migration. In contrast, p110α, which does not play a physiological role in GPCR signaling, is found to reside in nonraft regions of the plasma membrane. Raft targeting of p110α allowed its EGFR-mediated activation by GPCRs. Notably, p110β dependent, PTEN null tumor cells critically rely upon raft-associated PI3K activity. Collectively, our findings provide a mechanistic account of how membrane raft localization regulates differential activation of distinct PI3K isoforms and offer insight into why PTEN-deficient cancers depend on p110β. DOI: http://dx.doi.org/10.7554/eLife.17635.001 PMID:27700986

High-incidence of PTEN mutations in Chinese patients with primary small cell carcinoma of the esophagus

International Nuclear Information System (INIS)

Zhang, Zhimin; Wang, Ge; Xiao, Hualiang; Xie, Fei; Zhang, Hui; Chen, Chuan; Xiao, He; Yang, Zhenzhou; Wang, Dong; Li, Zengpeng

2014-01-01

Primary small cell carcinoma of the esophagus (PSCCE) is a rare and aggressive tumor with poor prognosis. The aim of this study was to investigate the existence of EGFR, KRAS, PIK3CA and PTEN mutations in PSCCE. Clinical–pathological data and paraffin-embedded specimens were collected from 38 patients. Exons 18 to 21 of EGFR, KRAS and PIK3CA status were analyzed by real-time PCR based on ARMS and Scorpion technology in all patients, and the PTEN gene was also screened using real-time PCR and high-resolution melting curve analysis (HRMA). Only 1 (2.63%) out of 38 patients had EGFR mutations in L858R missense, and KRAS and PIK3CA were not found in the mutational spot in all patients. However, PTEN mutations presented in 14 (36.84%) out of 38 patients, including exon 5 coding for PTEN missense mutation (n =4, 10.53%), exon 6 (n =7, 18.42%), concurrent exon 5 and exon 6 (n =2, 5.26%), and exon 8 (n =1, 2.63%). Concurrent mutations of these genes were not detected in all samples. No statistically significant associations were found between the clinicopathological features and the mutation status of PTEN. The incidence of PTEN mutations in Chinese patients with PSCCE was higher than that of previous reports in other histological subtypes of esophageal cancer

Evidence from bioinformatics, expression and inhibition studies of phosphoinositide-3 kinase signalling in Giardia intestinalis

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Crompton Mark R

2006-05-01

Full Text Available Abstract Background Giardia intestinalis is a parasitic protozoan and major cause of diarrhoeal disease. Disease transmission is dependent on the ability of the parasite to differentiate back and forth between an intestine-colonising trophozoite and an environmentally-resistant infective cyst. Our current understanding of the intracellular signalling mechanisms that regulate parasite replication and differentiation is limited, yet such information could suggest new methods of disease control. Phosphoinositide-3 kinase (PI3K signalling pathways have a central involvement in many vital eukaryotic processes, such as regulation of cell growth, intracellular membrane trafficking and cell motility. Here we present evidence for the existence of functional PI3K intracellular signalling pathways in G. intestinalis. Results We have identified and characterised two genes, Gipi3k1 and Gipi3k2, which encode putative PI3Ks. Both genes are expressed in trophozoites and encysting cells, suggesting a possible role of GiPI3K1 and GiPI3K2 in regulating giardial growth and differentiation. Extensive nucleotide and amino acid sequence characterisation predicts that both encoded PI3Ks are functional as indicated by the presence of highly conserved structural domains and essential catalytic residues. The inhibitory effect of the PI3K inhibitor LY294002 on trophozoite proliferation also supports their functionality. Phylogenetic analysis supports the identity of GiPI3K1 as a Class I isoform and GiPI3K2 as a Class III isoform. In addition, giardial genes encoding putative homologues of phosphoinositide-metabolising enzymes such as PTEN, MTM, PIPkin and PI 5-phosphatase as well as downstream effectors with phosphoinositide-binding domains have been identified, placing GiPI3K1 and GiPI3K2 in a broader signalling context. Compared with twenty-six PI3Ks from other organisms, GiPI3K1 and GiPI3K2 are unique in that they contain large insertions within their highly conserved

miR-1297 mediates PTEN expression and contributes to cell progression in LSCC

International Nuclear Information System (INIS)

Li, Xin; Wang, Hong-liang; Peng, Xin; Zhou, Hui-fang; Wang, Xin

2012-01-01

Highlights: ► miR-1297 was found to be overexpressed in LSCC and contribute to the cell progression. ► PTEN was confirmed to be a target gene of miR-1297. ► Downregulation of PTEN can rescue the proliferation and invasion ability of miR-1297 downregulated Hep-2 cells. ► Downregulation of miR-1297 inhibits tumor growth in vivo. -- Abstract: MicroRNAs (miRNAs) are small noncoding RNAs that regulate gene expression after transcription, and are involved in cancer development. Laryngeal squamous cell carcinoma (LSCC) is one of the most common malignant neoplasms with increasing incidence in recent years. In this paper, we report the overexpression of miR-1297 in LSCC and Hep-2 cells. In addition, PTEN was identified to be directly regulated by miR-1297 through western blot and luciferase activity assay. Furthermore, downregulation of miR-1297 in Hep-2 cells was shown to inhibit cancer cell proliferation, migration, and tumor genesis. Our results document a new epigenetic mechanism for PTEN regulation in LSCC, which is crucial for the development of these tumors.

The melatonin-MT1 receptor axis modulates tumor growth in PTEN-mutated gliomas.

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Ma, Huihui; Wang, Zhen; Hu, Lei; Zhang, Shangrong; Zhao, Chenggang; Yang, Haoran; Wang, Hongzhi; Fang, Zhiyou; Wu, Lijun; Chen, Xueran

2018-02-19

More than 40% of glioma patients have tumors that harbor PTEN (phosphatase and tensin homologue deleted on chromosome ten) mutations; this disease is associated with poor therapeutic resistance and outcome. Such mutations are linked to increased cell survival and growth, decreased apoptosis, and drug resistance; thus, new therapeutic strategies focusing on inhibiting glioma tumorigenesis and progression are urgently needed. Melatonin, an indolamine produced and secreted predominantly by the pineal gland, mediates a variety of physiological functions and possesses antioxidant and antitumor properties. Here, we analyzed the relationship between PTEN and the inhibitory effect of melatonin in primary human glioma cells and cultured glioma cell lines. The results showed that melatonin can inhibit glioma cell growth both in culture and in vivo. This inhibition was associated with PTEN levels, which significantly correlated with the expression level of MT1 in patients. In fact, c-fos-mediated MT1 was shown to be a key modulator of the effect of melatonin on gliomas that harbor wild type PTEN. Taken together, these data suggest that melatonin-MT1 receptor complexes represent a potential target for the treatment of glioma. Copyright © 2018 Elsevier Inc. All rights reserved.

The Role of PAS Kinase in PASsing the Glucose Signal

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Julianne H. Grose

2010-06-01

Full Text Available PAS kinase is an evolutionarily conserved nutrient responsive protein kinase that regulates glucose homeostasis. Mammalian PAS kinase is activated by glucose in pancreatic beta cells, and knockout mice are protected from obesity, liver triglyceride accumulation, and insulin resistance when fed a high-fat diet. Yeast PAS kinase is regulated by both carbon source and cell integrity stress and stimulates the partitioning of glucose toward structural carbohydrate biosynthesis. In our current model for PAS kinase regulation, a small molecule metabolite binds the sensory PAS domain and activates the enzyme. Although bona fide PAS kinase substrates are scarce, in vitro substrate searches provide putative targets for exploration.

Sensitization of human colon cancer cells to sodium butyrate-induced apoptosis by modulation of sphingosine kinase 2 and protein kinase D

International Nuclear Information System (INIS)

Xiao, Min; Liu, Yungang; Zou, Fei

2012-01-01

Sphingosine kinases (SphKs) have been recognized as important proteins regulating cell proliferation and apoptosis. Of the two isoforms of SphK (SphK1 and SphK2), little is known about the functions of SphK2. Sodium butyrate (NaBT) has been established as a promising chemotherapeutic agent, but the precise mechanism for its effects is unknown. In this study, we investigated the role of SphK2 in NaBT-induced apoptosis of HCT116 colon cancer cells. The results indicated that following NaBT treatment SphK2 was translocated from the nucleus to the cytoplasm, leading to its accumulation in the cytoplasm; in the meantime, only mild apoptosis occurred. However, downregulation of SphK2 resulted in sensitized apoptosis, and overexpression of SphK2 led to even lighter apoptosis; these strongly indicate an inhibitory role of SphK2 in cell apoptosis induced by NaBT. After knocking down protein kinase D (PKD), another protein reported to be critical in cell proliferation/apoptosis process, by using siRNA, blockage of cytoplasmic accumulation of SphK2 and sensitized apoptosis following NaBT treatment were observed. The present study suggests that PKD and SphK2 may form a mechanism for the resistance of cancer cells to tumor chemotherapies, such as HCT116 colon cancer cells to NaBT, and these two proteins may become molecular targets for designation of new tumor-therapeutic drugs. -- Highlights: ► In the present study sodium butyrate (10 mM) induced mild apoptosis of cancer cells. ► The apoptosis was negatively regulated by cytoplasmic Sphingosine Kinase 2 (SphK2). ► Translocation of SphK2 from nucleus to cytoplasm was mediated by protein kinase D. ► Downregulation of SphK2 or protein kinase D leads to sensitized cell apoptosis.

Doxycyclin ameliorates a starvation-induced germline tumor in C. elegans daf-18/PTEN mutant background.

Science.gov (United States)

Wolf, Tim; Qi, Wenjing; Schindler, Verena; Runkel, Eva Diana; Baumeister, Ralf

2014-08-01

Managing available resources is a key necessity of each organism to cope with the environment. The nematode C. elegans responds to nutritional deprivation or harsh environmental conditions with a multitude of developmental adaptations, among them a starvation-induced quiescence at early larval development (L1). daf-18, the C. elegans homolog of the human tumor suppressor gene PTEN, is essential for the maintenance of survival and germline stem cell arrest during the L1 diapause. We show here that daf-18 mutants, independently to their failure to maintain G2 arrest of the primordial germ cells, develop a gonad phenotype after refeeding. This highly penetrant gonadal phenotype is further enhanced by a mutation in shc-1, encoding a protein homologous to the human adaptor ShcA. Features of this phenotype are a tumor-like phenotype encompassing hyper-proliferation of germ cell nuclei and disruption/invasion of the basement membrane surrounding the gonad. The penetrance of this phenotype is reduced by decreasing starvation temperature. In addition, it is also ameliorated in a dose-dependent way by exposure to the antibiotic doxycyclin either during starvation or during subsequent refeeding. Since, in eukaryotic cells, doxycyclin specifically blocks mitochondrial translation, our results suggest that daf-18 and shc-1;daf-18 mutants fail to adapt mitochondrial activity to reduced nutritional availability during early larval developing. Copyright © 2014 Elsevier Inc. All rights reserved.

Age-related clinical and biological features of PTEN abnormalities in T-cell acute lymphoblastic leukaemia.

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Tesio, M; Trinquand, A; Ballerini, P; Hypolite, G; Lhermitte, L; Petit, A; Ifrah, N; Baruchel, A; Dombret, H; Macintyre, E; Asnafi, V

2017-12-01

The tumour suppressor gene PTEN is commonly altered in T-cell acute lymphoblastic leukaemia but its prognostic impact is still debated. We screened a cohort of 573 fully characterised adult and paediatric T-cell acute lymphoblastic leukaemia (T-ALL) patients for genomic PTEN abnormalities. PTEN-inactivating mutations and/or deletions were identified in 91 cases (16%), including 18% of paediatric (49/277) and 14% of adult cases (42/296). Thirty-four patients harboured only mutations, 12 cases demonstrated only large deletions and 9 only microdeletions. About 36 patients had combined alterations. Different mechanisms of PTEN inactivation predicted differences in the clinical outcome for both adult and paediatric patients treated according to the GRAALL03/05 and FRALLE2000 protocols. Whereas large deletions predicted lower 5-year overall survival (P=0.0053 in adults, P=0.001 in children) and disease-free survival (P=0.0009 in adults, P=0.0002 in children), mutations were not associated with a worse prognosis. The prognostic impact of PTEN loss is therefore linked to the underlying type of genomic abnormality, both in adult and paediatric T-ALLs, demonstrating that detailed analysis of the type of abnormality type would be useful to refine risk stratification.

Negative regulatory role of PI3-kinase in TNF-induced tumor necrosis.

Science.gov (United States)

Matschurat, Susanne; Blum, Sabine; Mitnacht-Kraus, Rita; Dijkman, Henry B P M; Kanal, Levent; De Waal, Robert M W; Clauss, Matthias

2003-10-20

Tissue factor is the prime initiator of blood coagulation. Expression of tissue factor in tumor endothelial cells leads to thrombus formation, occlusion of vessels and development of hemorrhagic infarctions in the tumor tissue, often followed by regression of the tumor. Tumor cells produce endogenous vascular endothelial growth factor (VEGF), which sensitizes endothelial cells for systemically administered tumor necrosis factor alpha (TNF alpha) and synergistically enhances the TNF-induced expression of tissue factor. We have analyzed the pathways involved in the induction of tissue factor in human umbilical cord vein endothelial cells (HUVECs) after combined stimulation with TNF and VEGF. By using specific low molecular weight inhibitors, we demonstrated that protein kinase C (PKC), p44/42 and p38 mitogen-activated protein (MAP) kinases, and stress-activated protein kinase (JNK) are essentially involved in the induction of tissue factor. In contrast, the application of wortmannin, an inhibitor of phosphatidylinositol 3 (PI3)-kinase, led to strongly enhanced expression of tissue factor in TNF- and VEGF-treated cells, implicating a negative regulatory role for PI3-kinase. In vivo, the application of wortmannin promoted the formation of TNF-induced hemorrhages and intratumoral necroses in murine meth A tumors. The co-injection of wortmannin lowered the effective dose of applied TNF. Therefore, it is conceivable that the treatment of TNF-sensitive tumors with a combination of TNF and wortmannin will ensure the selective damage of the tumor endothelium and minimize the risk of systemic toxicity of TNF. TNF-treatment in combination with specific inhibition of PI3-kinase is a novel concept in anti-cancer therapy. Copyright 2003 Wiley-Liss, Inc.

Cancer/testis Antigen-Plac1 Promotes Invasion and Metastasis of Breast Cancer through Furin/NICD/PTEN Signaling Pathway.

Science.gov (United States)

Li, Yongfei; Chu, Jiahui; Li, Jun; Feng, Wanting; Yang, Fan; Wang, Yifan; Zhang, Yanhong; Sun, Chunxiao; Yang, Mengzhu; Vasilatos, Shauna N; Huang, Yi; Fu, Ziyi; Yin, Yongmei

2018-04-28

Plac1 is a cancer-testis antigen that plays a critical role in promoting cancer initiation and progression. However, the clinical significance and mechanism of Plac1 in cancer progression remains elusive. Here we report that Plac1 is an important oncogenic and prognostic factor which physically interacts with Furin to drive breast cancer invasion and metastasis. We have shown that Plac1 expression positively correlates with clinical stage, lymph node metastasis, HR status and overall patient survival. Overexpression of Plac1 promoted invasion and metastasis of breast cancer cells in vitro and in vivo. Co-immunoprecipitation and immunofluorescence cell staining assays revealed that interaction of Plac1 and Furin degraded Notch1 and generated Notch1 intracellular domain (NICD) that could inhibit PTEN activity. These findings are consistent with the results of microarray study in MDA-MB-231 cells overexpressing Plac1. A rescue study showed that inhibition of Furin and overexpression of PTEN in Plac1 overexpression cells blocked Plac1-induced tumor cell progression. Taken together, our findings suggest that functional interaction between Plac1 and Furin enhances breast cancer invasion and metastasis and the Furin/NICD/PTEN axis may act as an important therapeutic target for breast cancer treatment. Molecular Oncology (2018) © 2018 The Authors. Published by FEBS Press and John Wiley & Sons Ltd.

Differential Requirements for c-Myc in Chronic Hematopoietic Hyperplasia and Acute Hematopoietic Malignancies in Pten-null Mice

Science.gov (United States)

Zhang, Jun; Xiao, Yechen; Guo, Yinshi; Breslin, Peter; Zhang, Shubin; Wei, Wei; Zhang, Zhou; Zhang, Jiwang

2011-01-01

Myeloproliferative disorders (MPDs), lymphoproliferative disorders (LPDs), acute T-lymphocytic or myeloid leukemia and T-lymphocytic lymphoma were developed in inducible Pten-knockout (Pten−/−) mice. The appearance of these multiple diseases in one animal model provides an opportunity to study the pathogenesis of multiple diseases simultaneously. To study whether Myc function is required for the development of these hematopoietic disorders in Pten−/− mice, we generated inducible Pten/Myc double-knockout mice (Pten−/−/Myc−/−). By comparing the hematopoietic phenotypes of these double-knockout mice with those of Pten−/− mice, we found that both sets of animals developed MPDs and LPDs. However, none of the compound-mutant mice developed acute leukemia or lymphoma. Interestingly, in contrast to the MPDs which developed in Pten−/− mice which are dominated by granulocytes, megakaryocytes predominate in the MPDs of Pten−/−/Myc−/− mice. Our study suggests that the deregulation of PI3K/Akt signaling in Pten−/− hematopoietic cells protects these cells from apoptotic cell death, resulting in chronic proliferative disorders. But due to the differential requirement for Myc in granulocyte as compared to megakaryocyte proliferation, Myc deletion converts Pten−/− MPDs from granulocyte-dominated to megakaryocyte-dominated conditions. Myc is absolutely required for the development of acute hematopoietic malignancies. PMID:21926961

Broad spectrum of neuropsychiatric phenotypes associated with white matter disease in PTEN hamartoma tumor syndrome.

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Balci, Tugce B; Davila, Jorge; Lewis, Denice; Boafo, Addo; Sell, Erick; Richer, Julie; Nikkel, Sarah M; Armour, Christine M; Tomiak, Eva; Lines, Matthew A; Sawyer, Sarah L

2018-01-01

White matter lesions have been described in patients with PTEN hamartoma tumor syndrome (PHTS). How these lesions correlate with the neurocognitive features associated with PTEN mutations, such as autism spectrum disorder (ASD) or developmental delay, has not been well established. We report nine patients with PTEN mutations and white matter changes on brain magnetic resonance imaging (MRI), eight of whom were referred for reasons other than developmental delay or ASD. Their clinical presentations ranged from asymptomatic macrocephaly with normal development/intellect, to obsessive compulsive disorder, and debilitating neurological disease. To our knowledge, this report constitutes the first detailed description of PTEN-related white matter changes in adult patients and in children with normal development and intelligence. We present a detailed assessment of the neuropsychological phenotype of our patients and discuss the relationship between the wide array of neuropsychiatric features and observed white matter findings in the context of these individuals. © 2017 Wiley Periodicals, Inc.

PINK1-Parkin alleviates metabolic stress induced by obesity in adipose tissue and in 3T3-L1 preadipocytes.

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Cui, Chen; Chen, Shihong; Qiao, Jingting; Qing, Li; Wang, Lingshu; He, Tianyi; Wang, Chuan; Liu, Fuqiang; Gong, Lei; Chen, Li; Hou, Xinguo

2018-04-06

Mitochondria play an important role in cellular metabolism and are closely related with metabolic stress. Recently, several studies have shown that mitophagy mediated by PTEN-induced putative kinase 1 (PINK1) and Parkin may play a critical role in clearing the damaged mitochondria and maintaining the overall balance of intracellular mitochondria in quality and quantity. A previous study showed that PINK1 and Parkin were overexpressed in adipose tissue in obese subjects. However, it is still unclear whether a direct relationship exists between obesity and mitophagy. In this study, we created a high-fat-diet (HFD)-induced obese mouse model and examined the expression of PINK1 and Parkin in adipose tissue using western blot and real-time quantitative PCR. After we confirmed that there is an interesting difference between regular-chow-fed mice and HFD-induced obese mice in the expression of PINK1 and Parkin in vivo, we further tested the expression of PINK1 and Parkin in 3T3-L1 preadipocytes in vitro by treating cells with palmitic acid (PA) to induce metabolic stress. To better understand the role of PINK1 and Parkin in metabolic stress, 3T3-L1 preadipocytes were transfected with small interfering RNA (siRNA) of PINK1 and Parkin followed by PA treatment. Our results showed that under lower concentrations of PA, PINK1 and Parkin can be activated and play a protective role in resisting the harmful effects of PA, including protecting the mitochondrial function and resisting cellular death, while under higher concentrations of PA, the expression of PINK1 and Parkin can be inhibited. These results suggest that PINK1-Parkin can protect mitochondrial function against metabolic stress induced by obesity or PA to a certain degree. Copyright © 2018 Elsevier Inc. All rights reserved.

Reciprocal positive regulation between TRPV6 and NUMB in PTEN-deficient prostate cancer cells

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Kim, Sung-Young; Hong, Chansik; Wie, Jinhong [Department of Physiology, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Kim, Euiyong [Department of Physiology, College of Medicine, Inje University, Busan 614-735 (Korea, Republic of); Kim, Byung Joo [Division of Longevity and Biofunctional Medicine, Pusan National University School of Korean Medicine, Yangsan 626-870 (Korea, Republic of); Ha, Kotdaji [Department of Physiology, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Cho, Nam-Hyuk; Kim, In-Gyu [Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Jeon, Ju-Hong [Department of Physiology, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); So, Insuk, E-mail: insuk@snu.ac.kr [Department of Physiology, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of); Department of Biomedical Sciences, Seoul National University College of Medicine, Seoul 110-799 (Korea, Republic of)

2014-04-25

Highlights: • TRPV6 interacts with tumor suppressor proteins. • Numb has a selective effect on TRPV6, depending on the prostate cancer cell line. • PTEN is a novel regulator of TRPV6–Numb complex. - Abstract: Calcium acts as a second messenger and plays a crucial role in signaling pathways involved in cell proliferation. Recently, calcium channels related to calcium influx into the cytosol of epithelial cells have attracted attention as a cancer therapy target. Of these calcium channels, TRPV6 is overexpressed in prostate cancer and is considered an important molecule in the process of metastasis. However, its exact role and mechanism is unclear. NUMB, well-known tumor suppressor gene, is a novel interacting partner of TRPV6. We show that NUMB and TRPV6 have a reciprocal positive regulatory relationship in PC-3 cells. We repeated this experiment in two other prostate cancer cell lines, DU145 and LNCaP. Interestingly, there were no significant changes in TRPV6 expression following NUMB knockdown in DU145. We revealed that the presence or absence of PTEN was the cause of NUMB–TRPV6 function. Loss of PTEN caused a positive correlation of TRPV6–NUMB expression. Collectively, we determined that PTEN is a novel interacting partner of TRPV6 and NUMB. These results demonstrated a novel relationship of NUMB–TRPV6 in prostate cancer cells, and show that PTEN is a novel regulator of this complex.

Mitochondrial Stress Signalling: HTRA2 and Parkinson's Disease

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Enrico Desideri

2012-01-01

Full Text Available Mitochondria are cellular energy generators whose activity requires a continuous supply of oxygen. Recent genetic analysis has suggested that defects in mitochondrial quality control may be key factors in the development of Parkinson’s disease (PD. Mitochondria have a crucial role in supplying energy to the brain, and their deterioration can affect the function and viability of neurons, contributing to neurodegeneration. These organelles can sow the seeds of their own demise because they generate damaging oxygen-free radicals as a byproduct of their intrinsic physiological functions. Mitochondria have therefore evolved specific molecular quality control mechanisms to compensate for the action of damaging agents such as oxygen-free radicals. PTEN-induced putative kinase 1 (PINK1 and high-temperature-regulated A2 (HTRA2, a mitochondrial protease, have recently been proposed to be key modulators of mitochondrial molecular quality control. Here, we review some of the most recent advances in our understanding of mitochondria stress-control pathways, focusing on how signalling by the p38 stress kinase pathway may regulate mitochondrial stress by modulating the activity of HTRA2 via PINK1 and cyclin-dependent kinase 5 (CDK5. We also propose how defects in this pathway may contribute to PD.

Heterogeneity of PTEN and ERG expression in prostate cancer on core needle biopsies: implications for cancer risk stratification and biomarker sampling.

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Shah, Rajal B; Bentley, James; Jeffery, Zach; DeMarzo, Angelo M

2015-05-01

ERG and PTEN biomarkers are increasingly being analyzed on prostate core biopsies (NBXs); ERG as a marker of clonality and number of separately arising tumor foci and PTEN for prognostic information. Yet, in patients with multiple biopsy cores positive for cancer (PCa), there is no standardized approach for interrogation of these biomarkers in terms of the number of positive cores to evaluate. A total of 194 NBX cases containing more than one positive core with cancer were evaluated for ERG overexpression and PTEN loss by immunostaining (immunohistochemistry) of all positive cores. ERG overexpression or PTEN loss in at least one cancer core was present in 111 (57%) and 69 (36%) cases respectively. ERG overexpression was significantly associated with PTEN loss (P < .0001), and PTEN loss was associated with a high Gleason score (P < .0001). Inter- and intra-tumor core staining heterogeneity for ERG overexpression occurred in 42% and 5% cases and for PTEN loss both intra- and inter-tumor core heterogeneity was 68%. PTEN staining was highly discordant between PCa sites regardless of laterality. When the Gleason score was non-uniform across PCa sites, the combination of cores showing the highest Gleason score and largest tumor volume provided the best representation of ERG overexpression (92%) and PTEN loss (98%). When grades were uniform across cancer sites, the highest tumor volume core was generally representative of ERG overexpression (90%) but was less representative for PTEN loss (76%). Our results suggest that knowledge of this heterogeneity is critical for developing optimal yet cost-effective strategies to identify these underlying molecular abnormalities. Copyright © 2015 Elsevier Inc. All rights reserved.

Escitalopram Ameliorates Tau Hyperphosphorylation and Spatial Memory Deficits Induced by Protein Kinase A Activation in Sprague Dawley Rats.

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Ren, Qing-Guo; Wang, Yan-Juan; Gong, Wei-Gang; Xu, Lin; Zhang, Zhi-Jun

2015-01-01

Here, we investigated the effect of escitalopram pretreatment on protein kinase A (PKA)-induced tau hyperphosphorylation and spatial memory deficits in rats using western blot and behavioral tests, respectively. We demonstrated that escitalopram effectively ameliorated tau hyperphosphorylation and the spatial memory deficits induced by PKA activation. We measured the total and activity-dependent Ser9-phosphorylated levels of glycogen synthase kinase (GSK)-3β in hippocampal extracts. No significant change in the total level of GSK-3β was observed between the different groups. However, compared with forskolin injection alone, pretreatment with escitalopram increased the level of Ser9-phosphorylated GSK-3β. We also demonstrated that escitalopram increased Akt phosphorylation at Ser473 (the active form of Akt). Furthermore, we identified other important kinases and phosphatases, such as protein phosphatase 2A, extracellular signal-regulated kinases 1 and 2, and MAP kinase kinase-1/2, that have previously been reported to play a crucial role in tau phosphorylation; however, we did not detect any significant change in the activation of these kinases or phosphatases in our study. We unexpectedly demonstrated that forskolin caused anxiety-like behavior in rats, and pretreatment with escitalopram did not significantly ameliorate the anxiety-like behavior induced by forskolin. These data provide the first evidence that escitalopram ameliorates forskolin-induced tau hyperphosphorylation and spatial memory impairment in rats; these effects do not occur via the anti-anxiety activity of escitalopram but may involve the Akt/GSK-3β signaling pathway.

RAS/ERK modulates TGFβ-regulated PTEN expression in human pancreatic adenocarcinoma cells

OpenAIRE

Chow, Jimmy Y.C.; Quach, Khai T.; Cabrera, Betty L.; Cabral, Jennifer A.; Beck, Stayce E.; Carethers, John M.

2007-01-01

Phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is rarely mutated in pancreatic cancers, but its regulation by transforming growth factor (TGF)-β might mediate growth suppression and other oncogenic actions. Here, we examined the role of TGFβ and the effects of oncogenic K-RAS/ERK upon PTEN expression in the absence of SMAD4. We utilized two SMAD4-null pancreatic cell lines, CAPAN-1 (K-RAS mutant) and BxPc-3 (WT-K-RAS), both of which express TGFβ surface receptors. Cells were t...

Crystal structure of the cytoplasmic phosphatase and tensin homolog (PTEN)-like region of Ciona intestinalis voltage-sensing phosphatase provides insight into substrate specificity and redox regulation of the phosphoinositide phosphatase activity.

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Matsuda, Makoto; Takeshita, Kohei; Kurokawa, Tatsuki; Sakata, Souhei; Suzuki, Mamoru; Yamashita, Eiki; Okamura, Yasushi; Nakagawa, Atsushi

2011-07-01

Ciona intestinalis voltage-sensing phosphatase (Ci-VSP) has a transmembrane voltage sensor domain and a cytoplasmic region sharing similarity to the phosphatase and tensin homolog (PTEN). It dephosphorylates phosphatidylinositol 4,5-bisphosphate and phosphatidylinositol 3,4,5-trisphosphate upon membrane depolarization. The cytoplasmic region is composed of a phosphatase domain and a putative membrane interaction domain, C2. Here we determined the crystal structures of the Ci-VSP cytoplasmic region in three distinct constructs, wild-type (248-576), wild-type (236-576), and G365A mutant (248-576). The crystal structure of WT-236 and G365A-248 had the disulfide bond between the catalytic residue Cys-363 and the adjacent residue Cys-310. On the other hand, the disulfide bond was not present in the crystal structure of WT-248. These suggest the possibility that Ci-VSP is regulated by reactive oxygen species as found in PTEN. These structures also revealed that the conformation of the TI loop in the active site of the Ci-VSP cytoplasmic region was distinct from the corresponding region of PTEN; Ci-VSP has glutamic acid (Glu-411) in the TI loop, orienting toward the center of active site pocket. Mutation of Glu-411 led to acquirement of increased activity toward phosphatidylinositol 3,5-bisphosphate, suggesting that this site is required for determining substrate specificity. Our results provide the basic information of the enzymatic mechanism of Ci-VSP.

Antibody-induced dimerization activates the epidermal growth factor receptor tyrosine kinase

NARCIS (Netherlands)

Spaargaren, M.; Defize, L. H.; Boonstra, J.; de Laat, S. W.

1991-01-01

The relationship between epidermal growth factor receptor (EGF-R) protein tyrosine kinase activation and ligand-induced receptor dimerization was investigated using several bivalent anti-EGF-R antibodies directed against various receptor epitopes. In A431 membrane preparations and permeabilized

Role of PTEN in the Tumor Microenvironment

Science.gov (United States)

2009-06-01

themselves, as well as the matrix that holds these cells together. Because the entire system is so complex, we plan to study how the Pten gene behaves as...Rev. 17, 501-519 8. Gould HJ, Sutton BJ. (2008). IgE in allergy and asthma today. Nat. Rev. Immunol. 8, 205-217. 9. Granucci F, Petralia F, Urbano M

Induction of intrahepatic cholangiocellular carcinoma by liver-specific disruption of Smad4 and Pten in mice.

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Xu, Xiaoling; Kobayashi, Shogo; Qiao, Wenhui; Li, Cuiling; Xiao, Cuiying; Radaeva, Svetlana; Stiles, Bangyan; Wang, Rui-Hong; Ohara, Nobuya; Yoshino, Tadashi; LeRoith, Derek; Torbenson, Michael S; Gores, Gregory J; Wu, Hong; Gao, Bin; Deng, Chu-Xia

2006-07-01

Cholangiocellular carcinoma (CC), the second most common primary liver cancer, is associated with a poor prognosis. It has been shown that CCs harbor alterations of a number of tumor-suppressor genes and oncogenes, yet key regulators for tumorigenesis remain unknown. Here we have generated a mouse model that develops CC with high penetrance using liver-specific targeted disruption of tumor suppressors SMAD4 and PTEN. In the absence of SMAD4 and PTEN, hyperplastic foci emerge exclusively from bile ducts of mutant mice at 2 months of age and continue to grow, leading to tumor formation in all animals at 4-7 months of age. We show that CC formation follows a multistep progression of histopathological changes that are associated with significant alterations, including increased levels of phosphorylated AKT, FOXO1, GSK-3beta, mTOR, and ERK and increased nuclear levels of cyclin D1. We further demonstrate that SMAD4 and PTEN regulate each other through a novel feedback mechanism to maintain an expression balance and synergistically repress CC formation. Finally, our analysis of human CC detected PTEN inactivation in a majority of p-AKT-positive CCs, while about half also lost SMAD4 expression. These findings elucidate the relationship between SMAD4 and PTEN and extend our understanding of CC formation.

Failure of the PTEN/aPKC/Lgl Axis Primes Formation of Adult Brain Tumours in Drosophila

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Simona Paglia

2017-01-01

Full Text Available Different regions in the mammalian adult brain contain immature precursors, reinforcing the concept that brain cancers, such as glioblastoma multiforme (GBM, may originate from cells endowed with stem-like properties. Alterations of the tumour suppressor gene PTEN are very common in primary GBMs. Very recently, PTEN loss was shown to undermine a specific molecular axis, whose failure is associated with the maintenance of the GBM stem cells in mammals. This axis is composed of PTEN, aPKC, and the polarity determinant Lethal giant larvae (Lgl: PTEN loss promotes aPKC activation through the PI3K pathway, which in turn leads to Lgl inhibition, ultimately preventing stem cell differentiation. To find the neural precursors responding to perturbations of this molecular axis, we targeted different neurogenic regions of the Drosophila brain. Here we show that PTEN mutation impacts aPKC and Lgl protein levels also in Drosophila. Moreover, we demonstrate that PI3K activation is not sufficient to trigger tumourigenesis, while aPKC promotes hyperplastic growth of the neuroepithelium and a noticeable expansion of the type II neuroblasts. Finally, we show that these neuroblasts form invasive tumours that persist and keep growing in the adult, leading the affected animals to untimely death, thus displaying frankly malignant behaviours.

[Protein kinase A inhibitor H-89 blocks polyploidization of SP600125-induced CMK cells by regulating phosphorylation of ribosomal protein S6 kinase 1].

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Zhao, Song; Yang, Jingang; Li, Changling; Xing, Sining; Yu, Ying; Liu, Shuo; Pu, Feifei; Ma, Dongchu

2016-10-01

Objective To investigate the regulatory effect of post-translation modification of ribosomal protein S6 kinase 1 (S6K1) on the polyploidization of megakaryocytes. Methods SP600125, a c-Jun N-terminal kinase (JNK) inhibitor, and H-89, a cAMP-dependent protein kinase (PKA) inhibitor, were used to treat CMK cells separately or in combination. With propidium iodide (PI) to dye DNA in the treated cells, the relative DNA content was detected by flow cytometry, and then the DNA polyploidy was analyzed. The change of expression and phosphorylation of ribosomal protein S6 kinase 1 (S6K1), an important mammalian target of rapamycin (mTOR) downstream target molecule, was analyzed by Western blotting. Molecular docking study and kinase activity assay were performed to analyze the combination of H-89 with S6K1 and the effect of H-89 on the activity of S6K1 kinase. Results SP600125 induced CMK cell polyploidization in a time-dependent and dose-dependent manner. At the same time, it increased the phosphorylation of S6K1 at Thr421/Ser424 and decreased the phosphorylation of S6K1 at Thr389. H-89 not only blocked polyploidization, but also decreased the phosphorylation of S6K1 at Thr421/Ser424 and increased the phosphorylation of S6K1 at Thr389. Molecular docking and kinase activity assay showed that H-89 occupied the ATP binding sites of S6K1 and inhibited its activity. Noticeably, both H-89 and SP600125 inhibited the activity of PKA. Moreover, the two drugs further inhibited the activity of PKA when used together. Therefore, these data indicated that H-89 blocked the SP600125-induced polyploidization of CMK cells mainly by changing S6K1 phosphorylation state, rather than its inhibitory effect on PKA. Conclusion H-89 can block the polyploidization of SP600125-induced CMK cells by regulating S6K1 phosphorylation state.

Chitin and stress induced protein kinase activation

DEFF Research Database (Denmark)

Kenchappa, Chandra Shekar; Azevedo da Silva, Raquel; Bressendorff, Simon

2017-01-01

The assays described here are pertinent to protein kinase studies in any plant. They include an immunoblot phosphorylation/activation assay and an in-gel activity assay for MAP kinases (MPKs) using the general protein kinase substrate myelin basic protein. They also include a novel in-gel peptide...... substrate assay for Snf1-related kinase family 2 members (SnRK2s). This kinase family-specific assay overcomes some limitations of in-gel assays and permits the identification of different types of kinase activities in total protein extracts....

Epidermal growth factor receptor inhibition reduces angiogenesis via hypoxia-inducible factor-1α and Notch1 in head neck squamous cell carcinoma.

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Wei-Ming Wang

Full Text Available Angiogenesis, a marker of cancer development, affects response to radiotherapy sensibility. This preclinical study aims to understand the receptor tyrosine kinase-mediated angiogenesis in head neck squamous cell carcinoma (HNSCC. The receptor tyrosine kinase activity in a transgenic mouse model of HNSCC was assessed. The anti-tumorigenetic and anti-angiogenetic effects of cetuximab-induced epidermal growth factor receptor (EGFR inhibition were investigated in xenograft and transgenic mouse models of HNSCC. The signaling transduction of Notch1 and hypoxia-inducible factor-1α (HIF-1α was also analyzed. EGFR was overexpressed and activated in the Tgfbr1/Pten deletion (2cKO mouse model of HNSCC. Cetuximab significantly delayed tumor onset by reducing tumor angiogenesis. This drug exerted similar effects on heterotopic xenograft tumors. In the human HNSCC tissue array, increased EGFR expression correlated with increased HIF-1α and micro vessel density. Cetuximab inhibited tumor-induced angiogenesis in vitro and in vivo by significantly downregulating HIF-1α and Notch1. EGFR is involved in the tumor angiogenesis of HNSCC via the HIF-1α and Notch1 pathways. Therefore, targeting EGFR by suppressing hypoxia- and Notch-induced angiogenesis may benefit HNSCC therapy.

Cocoa Procyanidins Suppress Transformation by Inhibiting Mitogen-activated Protein Kinase Kinase*S⃞

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Kang, Nam Joo; Lee, Ki Won; Lee, Dong Eun; Rogozin, Evgeny A.; Bode, Ann M.; Lee, Hyong Joo; Dong, Zigang

2008-01-01

Cocoa was shown to inhibit chemically induced carcinogenesis in animals and exert antioxidant activity in humans. However, the molecular mechanisms of the chemopreventive potential of cocoa and its active ingredient(s) remain unknown. Here we report that cocoa procyanidins inhibit neoplastic cell transformation by suppressing the kinase activity of mitogen-activated protein kinase kinase (MEK). A cocoa procyanidin fraction (CPF) and procyanidin B2 at 5 μg/ml and 40 μm, respectively, inhibited 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced neoplastic transformation of JB6 P+ mouse epidermal (JB6 P+) cells by 47 and 93%, respectively. The TPA-induced promoter activity and expression of cyclooxygenase-2, which is involved in tumor promotion and inflammation, were dose-dependently inhibited by CPF or procyanidin B2. The activation of activator protein-1 and nuclear factor-κB induced by TPA was also attenuated by CPF or procyanidin B2. The TPA-induced phosphorylation of MEK, extracellular signal-regulated kinase, and p90 ribosomal s6 kinase was suppressed by CPF or procyanidin B2. In vitro and ex vivo kinase assay data demonstrated that CPF or procyanidin B2 inhibited the kinase activity of MEK1 and directly bound with MEK1. CPF or procyanidin B2 suppressed JB6 P+ cell transformation induced by epidermal growth factor or H-Ras, both of which are known to be involved in MEK/ERK signal activation. In contrast, theobromine (up to 80 μm) had no effect on TPA-induced transformation, cyclooxygenase-2 expression, the transactivation of activator protein-1 or nuclear factor-κB, or MEK. Notably, procyanidin B2 exerted stronger inhibitory effects compared with PD098059 (a well known pharmacological inhibitor of MEK) on MEK1 activity and neoplastic cell transformation. PMID:18519570

Activation of PI3K/AKT and ERK MAPK signal pathways is required for the induction of lytic cycle replication of Kaposi's Sarcoma-associated herpesvirus by herpes simplex virus type 1

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Lv Zhigang

2011-10-01

Full Text Available Abstract Background Kaposi's sarcoma-associated herpesvirus (KSHV is causally linked to several acquired immunodeficiency syndrome-related malignancies, including Kaposi's sarcoma (KS, primary effusion lymphoma (PEL and a subset of multicentric Castleman's disease. Regulation of viral lytic replication is critical to the initiation and progression of KS. Recently, we reported that herpes simplex virus type 1 (HSV-1 was an important cofactor that activated lytic cycle replication of KSHV. Here, we further investigated the possible signal pathways involved in HSV-1-induced reactivation of KSHV. Results By transfecting a series of dominant negative mutants and protein expressing constructs and using pharmacologic inhibitors, we found that either Janus kinase 1 (JAK1/signal transducer and activator of transcription 3 (STAT3 or JAK1/STAT6 signaling failed to regulate HSV-1-induced KSHV replication. However, HSV-1 infection of BCBL-1 cells activated phosphatidylinositol 3-kinase (PI3K/protein kinase B (PKB, also called AKT pathway and inactivated phosphatase and tensin homologue deleted on chromosome ten (PTEN and glycogen synthase kinase-3β (GSK-3β. PTEN/PI3K/AKT/GSK-3β pathway was found to be involved in HSV-1-induced KSHV reactivation. Additionally, extracellular signal-regulated protein kinase (ERK mitogen-activated protein kinase (MAPK pathway also partially contributed to HSV-1-induced KSHV replication. Conclusions HSV-1 infection stimulated PI3K/AKT and ERK MAPK signaling pathways that in turn contributed to KSHV reactivation, which provided further insights into the molecular mechanism controlling KSHV lytic replication, particularly in the context of HSV-1 and KSHV co-infection.

"PINK1"-Linked Parkinsonism Is Associated with Lewy Body Pathology

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Samaranch, Lluis; Lorenzo-Betancor, Oswaldo; Arbelo, Jose M.; Ferrer, Isidre; Lorenzo, Elena; Irigoyen, Jaione; Pastor, Maria A.; Marrero, Carmen; Isla, Concepcion; Herrera-Henriquez, Joanna; Pastor, Pau

2010-01-01

Phosphatase and tensin homolog-induced putative kinase 1 gene mutations have been associated with autosomal recessive early-onset Parkinson's disease. To date, no neuropathological reports have been published from patients with Parkinson's disease with both phosphatase and tensin homolog-induced putative kinase 1 gene copies mutated. We analysed…

Involvement of the N-terminal unique domain of Chk tyrosine kinase in Chk-induced tyrosine phosphorylation in the nucleus

International Nuclear Information System (INIS)

Nakayama, Yuji; Kawana, Akiko; Igarashi, Asae; Yamaguchi, Naoto

2006-01-01

Chk tyrosine kinase phosphorylates Src-family kinases and suppresses their kinase activity. We recently showed that Chk localizes to the nucleus as well as the cytoplasm and inhibits cell proliferation. In this study, we explored the role of the N-terminal unique domain of Chk in nuclear localization and Chk-induced tyrosine phosphorylation in the nucleus. In situ binding experiments showed that the N-terminal domain of Chk was associated with the nucleus and the nuclear matrix. The presence of the N-terminal domain of Chk led to a fourfold increase in cell population exhibiting Chk-induced tyrosine phosphorylation in the nucleus. Expression of Chk but not kinase-deficient Chk induced tyrosine phosphorylation of a variety of proteins ranging from 23 kDa to ∼200 kDa, especially in Triton X-100-insoluble fraction that included chromatin and the nuclear matrix. Intriguingly, in situ subnuclear fractionations revealed that Chk induced tyrosine phosphorylation of proteins that were associated with the nuclear matrix. These results suggest that various unidentified substrates of Chk, besides Src-family kinases, may be present in the nucleus. Thus, our findings indicate that the importance of the N-terminal domain to Chk-induced tyrosine phosphorylation in the nucleus, implicating that these nuclear tyrosine-phosphorylated proteins may contribute to inhibition of cell proliferation

Saw palmetto extract suppresses insulin-like growth factor-I signaling and induces stress-activated protein kinase/c-Jun N-terminal kinase phosphorylation in human prostate epithelial cells.

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Wadsworth, Teri L; Carroll, Julie M; Mallinson, Rebecca A; Roberts, Charles T; Roselli, Charles E

2004-07-01

A common alternative therapy for benign prostatic hyperplasia (BPH) is the extract from the fruit of saw palmetto (SPE). BPH is caused by nonmalignant growth of epithelial and stromal elements of the prostate. IGF action is important for prostate growth and development, and changes in the IGF system have been documented in BPH tissues. The main signaling pathways activated by the binding of IGF-I to the IGF-I receptor (IGF-IR) are the ERK arm of the MAPK cascade and the phosphoinositol-3-kinase (PI3K)/protein kinase B (PKB/Akt) cascade. We tested the hypothesis that SPE suppresses growth and induces apoptosis in the P69 prostate epithelial cell line by inhibiting IGF-I signaling. Treatment with 150 microg/ml SPE for 24 h decreased IGF-I-induced proliferation of P69 cells and induced cleavage of the enzyme poly(ADP-ribose)polymerase (PARP), an index of apoptosis. Treatment of serum-starved P69 cells with 150 microg/ml SPE for 6 h reduced IGF-I-induced phosphorylation of Akt (assessed by Western blot) and Akt activity (assessed by an Akt kinase assay). Western blot analysis showed that SPE reduced IGF-I-induced phosphorylation of the adapter protein insulin receptor substrate-1 and decreased downstream effects of Akt activation, including increased cyclin D1 levels and phosphorylation of glycogen synthase kinase-3 and p70(s6k). There was no effect on IGF-I-induced phosphorylation of MAPK, IGF-IR, or Shc. Treatment of starved cells with SPE alone induced phosphorylation the proapoptotic protein JNK. SPE treatment may relieve symptoms of BPH, in part, by inhibiting specific components of the IGF-I signaling pathway and inducing JNK activation, thus mediating antiproliferative and proapoptotic effects on prostate epithelia.

Bioinformatics prediction of miR-30a targets and its inhibition of cell proliferation of osteosarcoma by up-regulating the expression of PTEN

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Biao Zhong

2017-11-01

Full Text Available Abstract Background MiRNAs are frequently abnormally expressed in the progression of human osteosarcoma. Phosphatase and tensin homologue deleted on chromosome 10 (PTEN is one of the tumor suppressors in various types of human cancer. In the present study, we detected how hsa-miR-30a-3p regulated PTEN and further tested the role of hsa-miR-30a-3p in the cell proliferation of osteosarcoma cells. Methods The levels of miR-30a were determined by real time PCR. The expression of PTEN was tested by western blotting analysis. Cell distribution of PTEN was observed with confocal laser scanning microscope. Cell viability was determined by MTT assay. Results The expression of miR-30a and PTEN was obviously decreased in MG-63, 143B and Saos-2 cells compared with primary osteoblasts. TargetScan analysis data showed miR-30a might bind with position 30-57 of 3’UTR of PTEN. Transfection with miR-30a-3p increased the level of PTEN in MG-63 cells, while transfection with miR-30a-3p inhibitor significantly decreased the expression of PTEN in osteosarcoma cells. Transfection with miR-30a-3p significantly inhibited cell proliferation of osteosarcoma cells, while miR-30a inhibitor obviously promoted cell viability of MG63 cells and Saos-2 cells. Inhibition of PTEN eliminated the proliferation inhibitory effect of miR-30a-3p. Conclusion Thus, all these findings revealed the anti-tumor effects of miR-30a in human osteosarcoma cells, which could be mediated by regulating the level of PTEN.

PTENp1, a natural sponge of miR-21, mediates PTEN expression to inhibit the proliferation of oral squamous cell carcinoma.

Science.gov (United States)

Gao, Ling; Ren, Wenhao; Zhang, Linmei; Li, Shaoming; Kong, Xinjuan; Zhang, Hao; Dong, Jianwei; Cai, Guangfeng; Jin, Changxiong; Zheng, Danqing; Zhi, Keqian

2017-04-01

PTENp1, non-coding RNA (ncRNA) pseudogene, is involved in oral squamous cell carcinoma (OSCC). The precise effects mediated by PTENp1 transcripts within intricate regulatory networks involving molecular interactions with ancestral gene PTEN and tumorigenicity in OSCC remain unclear. Here, we found that PTENp1 was aberrantly expressed in OSCC. There was a positive correlation between the expression levels of PTENp1 and PTEN. Further, we showed that PTENp1 acted as a competing endogenous RNA that protects PTEN transcripts from being inhibited by miR-21, and consequently inhibited proliferation and colony formation and triggered S-G2/M cell cycle arrest through the AKT pathway. Also, the homogeneous relationship between expression of PTENp1 and PTEN was confirmed in OSCC tumor xenografts. Finally, low expression of PTENp1 and PTEN was negatively associated with histological differentiation and OSCC prognosis. The present work provided the first evidence for the extraordinary crosstalk among PTENp1, PTEN, and miR-21, and rendered a new light on the treatment of OSCC. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Inhibition of protein kinase C induces differentiation in Neuro-2a cells

International Nuclear Information System (INIS)

Minana, M.D.; Felipo, V.; Grisolia, S.

1990-01-01

1-(5-Isoquinolinylsulfonyl)-2-methylpiperazine (H7), a potent inhibitor of protein kinase C, induced neuritogenesis in Neuro-2a cells, whereas N-(2-guanidinoethyl)-5-isoquinolinesulfonamide (HA 1004), which inhibits more efficiently cAMP- and cGMP-dependent protein kinases, did not. The effect, noticeable after 3 hr, was maximum (13-fold increase at 500 μM H7) between 1 and 3 days and was maintained over 2 months. In controls, 90% of the cells were undifferentiated, whereas after 3 hr with 500 μM H7 only 25% of the cells remained undifferentiated. DNA synthesis decreased as the number of differentiated cells increased. Differentiation is also functional since acetylcholinesterase activity increased ∼7-fold after 48 hr with 500 μM H7. Phorbol 12-myristate 13-acetate, a specific activator of protein kinase C, prevented or reversed the induction of neuritogenesis and the inhibition of DNA synthesis by H7. There is a good correlation between the level of protein kinase C and the percentage of differentiated cells. The results indicate that protein kinase C may play a key role in the control of differentiation of neural cells. Some possible clinical implications are briefly discussed

Molecular Mechanism of Nkx3.1 Deregulation and its Function in Murine Pten Prostate Cancer Model

Science.gov (United States)

2006-09-01

One possible explanation is that Pten null cells have become ‘‘ addicted ’’ to high levels of PI3K/AKT activity and, consequently, are hypersensitive...H.H., Kertesz, N., Lesche, R., Whale , A.D., Martinez-Diaz, H., Rozengurt, N., Cardiff, R.D., et al. (2003). PTEN tumor suppressor regulates p53 protein

Phorbol-ester-induced down-regulation of protein kinase C in mouse pancreatic islets. Potentiation of phase 1 and inhibition of phase 2 of glucose-induced insulin secretion

DEFF Research Database (Denmark)

Thams, P; Capito, K; Hedeskov, C J

1990-01-01

and potentiated phase 1 of glucose-induced secretion. Furthermore, perifusion of islets in the presence of staurosporine (1 microM), an inhibitor of protein kinase C, potentiated phase 1 and inhibited phase 2 of glucose-induced secretion. In addition, down-regulation of protein kinase C potentiated phase 1...

Perfluorooctanoic acid exposure for 28 days affects glucose homeostasis and induces insulin hypersensitivity in mice

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Yan, Shengmin; Zhang, Hongxia; Zheng, Fei; Sheng, Nan; Guo, Xuejiang; Dai, Jiayin

2015-06-01

Perfluoroalkyl acids (PFAAs) are widely used in many applications due to their unique physical and chemical characteristics. Because of the increasing prevalence of metabolic syndromes, including obesity, dyslipidemia and insulin resistance, concern has arisen about the roles of environmental pollutants in such diseases. Earlier epidemiologic studies showed a potential association between perfluorooctanoic acid (PFOA) and glucose metabolism, but how PFOA influences glucose homeostasis is still unknown. Here, we report on the modulation of the phosphatidylinositol 3-kinase-serine/threonine protein kinase (PI3K-AKT) signaling pathway in the livers of mice after 28 d of exposure to PFOA. Compared with normal mice, PFOA exposure significantly decreased the expression of the phosphatase and tensin homologue (PTEN) protein and affected the PI3K-AKT signaling pathway in the liver. Tolerance tests further indicated that PFOA exposure induced higher insulin sensitivity and glucose tolerance in mice. Biochemical analysis revealed that PFOA exposure reduced hepatic glycogen synthesis, which might be attributed to gluconeogenesis inhibition. The levels of several circulating proteins were altered after PFOA exposure, including proteins potentially related to diabetes and liver disease. Our results suggest that PFOA affected glucose metabolism and induced insulin hypersensitivity in mice.

RPS6KA2, a putative tumour suppressor gene at 6q27 in sporadic epithelial ovarian cancer

DEFF Research Database (Denmark)

Bignone, P A; Lee, K Y; Liu, Y

2007-01-01

We had previously defined by allele loss studies a minimal region at 6q27 (between D6S264 and D6S297) to contain a putative tumour suppressor gene. The p90 ribosomal S6 kinase-3 gene (p90 Rsk-3, RPS6KA2) maps in this interval. It is a serine-threonine kinase that signals downstream of the mitogen...

Enhancement of high glucose-induced PINK1 expression by melatonin stimulates neuronal cell survival: Involvement of MT2 /Akt/NF-κB pathway.

Science.gov (United States)

Onphachanh, Xaykham; Lee, Hyun Jik; Lim, Jae Ryong; Jung, Young Hyun; Kim, Jun Sung; Chae, Chang Woo; Lee, Sei-Jung; Gabr, Amr Ahmed; Han, Ho Jae

2017-09-01

Hyperglycemia is a representative hallmark and risk factor for diabetes mellitus (DM) and is closely linked to DM-associated neuronal cell death. Previous investigators reported on a genome-wide association study and showed relationships between DM and melatonin receptor (MT), highlighting the role of MT signaling by assessing melatonin in DM. However, the role of MT signaling in DM pathogenesis is unclear. Therefore, we investigated the role of mitophagy regulators in high glucose-induced neuronal cell death and the effect of melatonin against high glucose-induced mitophagy regulators in neuronal cells. In our results, high glucose significantly increased PTEN-induced putative kinase 1 (PINK1) and LC-3B expressions; as well it decreased cytochrome c oxidase subunit 4 expression and Mitotracker™ fluorescence intensity. Silencing of PINK1 induced mitochondrial reactive oxygen species (ROS) accumulation and mitochondrial membrane potential impairment, increased expressions of cleaved caspases, and increased the number of annexin V-positive cells. In addition, high glucose-stimulated melatonin receptor 1B (MTNR1B) mRNA and PINK1 expressions were reversed by ROS scavenger N-acetyl cysteine pretreatment. Upregulation of PINK1 expression in neuronal cells is suppressed by pretreatment with MT 2 receptor-specific inhibitor 4-P-PDOT. We further showed melatonin stimulated Akt phosphorylation, which was followed by nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) phosphorylation and nuclear translocation. Silencing of PINK1 expression abolished melatonin-regulated mitochondrial ROS production, cleaved caspase-3 and caspase-9 expressions, and the number of annexin V-positive cells. In conclusion, we have demonstrated the melatonin stimulates PINK1 expression via an MT 2 /Akt/NF-κB pathway, and such stimulation is important for the prevention of neuronal cell apoptosis under high glucose conditions. © 2017 The Authors. Journal of Pineal Research

Klf5 deletion promotes Pten deletion-initiated luminal-type mouse prostate tumors through multiple oncogenic signaling pathways.

Science.gov (United States)

Xing, Changsheng; Ci, Xinpei; Sun, Xiaodong; Fu, Xiaoying; Zhang, Zhiqian; Dong, Eric N; Hao, Zhao-Zhe; Dong, Jin-Tang

2014-11-01

Krüppel-like factor 5 (KLF5) regulates multiple biologic processes. Its function in tumorigenesis appears contradictory though, showing both tumor suppressor and tumor promoting activities. In this study, we examined whether and how Klf5 functions in prostatic tumorigenesis using mice with prostate-specific deletion of Klf5 and phosphatase and tensin homolog (Pten), both of which are frequently inactivated in human prostate cancer. Histologic analysis demonstrated that when one Pten allele was deleted, which causes mouse prostatic intraepithelial neoplasia (mPIN), Klf5 deletion accelerated the emergence and progression of mPIN. When both Pten alleles were deleted, which causes prostate cancer, Klf5 deletion promoted tumor growth, increased cell proliferation, and caused more severe morphologic and molecular alterations. Homozygous deletion of Klf5 was more effective than hemizygous deletion. Unexpectedly, while Pten deletion alone expanded basal cell population in a tumor as reported, Klf5 deletion in the Pten-null background clearly reduced basal cell population while expanding luminal cell population. Global gene expression profiling, pathway analysis, and experimental validation indicate that multiple mechanisms could mediate the tumor-promoting effect of Klf5 deletion, including the up-regulation of epidermal growth factor and its downstream signaling molecules AKT and ERK and the inactivation of the p15 cell cycle inhibitor. KLF5 also appears to cooperate with several transcription factors, including CREB1, Sp1, Myc, ER and AR, to regulate gene expression. These findings validate the tumor suppressor function of KLF5. They also yield a mouse model that shares two common genetic alterations with human prostate cancer-mutation/deletion of Pten and deletion of Klf5.

HSP90 inhibitors potentiate PGF2α-induced IL-6 synthesis via p38 MAP kinase in osteoblasts.

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Kazuhiko Fujita

Full Text Available Heat shock protein 90 (HSP90 that is ubiquitously expressed in various tissues, is recognized to be a major molecular chaperone. We have previously reported that prostaglandin F2α (PGF2α, a potent bone remodeling mediator, stimulates the synthesis of interleukin-6 (IL-6 through p44/p42 mitogen-activated protein (MAP kinase and p38 MAP kinase in osteoblast-like MC3T3-E1 cells, and that Rho-kinase acts at a point upstream of p38 MAP kinase. In the present study, we investigated the involvement of HSP90 in the PGF2α-stimulated IL-6 synthesis and the underlying mechanism in MC3T3-E1 cells. Geldanamycin, an inhibitor of HSP90, significantly amplified both the PGF2α-stimulated IL-6 release and the mRNA expression levels. In addition, other HSP90 inhibitors, 17-allylamino-17demethoxy-geldanamycin (17-AAG and 17-dimethylamino-ethylamino-17-demethoxy-geldanamycin (17-DMAG and onalespib, enhanced the PGF2α-stimulated IL-6 release. Geldanamycin, 17-AAG and onalespib markedly strengthened the PGF2α-induced phosphorylation of p38 MAP kinase. Geldanamycin and 17-AAG did not affect the PGF2α-induced phosphorylation of p44/p42 MAP kinase and myosin phosphatase targeting subunit (MYPT-1, a substrate of Rho-kinase, and the protein levels of RhoA and Rho-kinase. In addition, HSP90-siRNA enhanced the PGF2α-induced phosphorylation of p38 MAP kinase. Furthermore, SB203580, an inhibitor of p38 MAP kinase, significantly suppressed the amplification by geldanamycin, 17-AAG or 17-DMAG of the PGF2α-stimulated IL-6 release. Our results strongly suggest that HSP90 negatively regulates the PGF2α-stimulated IL-6 synthesis in osteoblasts, and that the effect of HSP90 is exerted through regulating p38 MAP kinase activation.

Expression of putative expansin genes in phylloxera (Daktulosphaira vitifoliae Fitch) induced root galls of Vitis spp.

Science.gov (United States)

Lawo, N C; Griesser, M; Forneck, A

Grape phylloxera ( Daktulosphaira vitifoliae Fitch) is a serious global pest in viticulture. The insects are sedentary feeders and require a gall to feed and reproduce. The insects induce their feeding site within the meristematic zone of the root tip, where they stay attached, feeding both intra- and intercellularly, and causing damage by reducing plant vigour. Several changes in cell structure and composition, including increased cell division and tissue swelling close to the feeding site, cause an organoid gall called a nodosity to develop. Because alpha expansin genes are involved in cell enlargement and cell wall loosening in many plant tissues it may be anticipated that they are also involved in nodosity formation. To identify expansin genes in Vitis vinifera cv. Pinot noir , we mined for orthologues genes in a comparative analysis. Eleven putative expansin genes were identified and shown to be present in the rootstock Teleki 5C ( V. berlandieri Planch. x V. riparia Michx.) using specific PCR followed by DNA sequencing. Expression analysis of young and mature nodosities and uninfested root tips were conducted via quantitative real time PCR (qRT-PCR). Up-regulation was measured for three putative expansin genes (VvEXPA15, -A17 and partly -A20) or down-regulation for three other putative genes (VvEXPA7, -A12, -A20) in nodosities. The present study clearly shows the involvement of putative expansin genes in the phylloxera-root interaction.

Rho Kinase ROCK2 Mediates Acid-Induced NADPH Oxidase NOX5-S Expression in Human Esophageal Adenocarcinoma Cells.

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Jie Hong

Full Text Available Mechanisms of the progression from Barrett's esophagus (BE to esophageal adenocarcinoma (EA are not fully understood. We have shown that NOX5-S may be involved in this progression. However, how acid upregulates NOX5-S is not well known. We found that acid-induced increase in NOX5-S expression was significantly decreased by the Rho kinase (ROCK inhibitor Y27632 in BE mucosal biopsies and FLO-1 EA cells. In addition, acid treatment significantly increased the Rho kinase activity in FLO-1 cells. The acid-induced increase in NOX5-S expression and H2O2 production was significantly decreased by knockdown of Rho kinase ROCK2, but not by knockdown of ROCK1. Conversely, the overexpression of the constitutively active ROCK2, but not the constitutively active ROCK1, significantly enhanced the NOX5-S expression and H2O2 production. Moreover, the acid-induced increase in Rho kinase activity and in NOX5-S mRNA expression was blocked by the removal of calcium in both FLO-1 and OE33 cells. The calcium ionophore A23187 significantly increased the Rho kinase activity and NOX5-S mRNA expression. We conclude that acid-induced increase in NOX5-S expression and H2O2 production may depend on the activation of ROCK2, but not ROCK1, in EA cells. The acid-induced activation of Rho kinase may be mediated by the intracellular calcium increase. It is possible that persistent acid reflux present in BE patients may increase the intracellular calcium, activate ROCK2 and thereby upregulate NOX5-S. High levels of reactive oxygen species derived from NOX5-S may cause DNA damage and thereby contribute to the progression from BE to EA.

Variable laterality of corticospinal tract axons that regenerate after spinal cord injury as a result of PTEN deletion or knock-down

Science.gov (United States)

Willenberg, Rafer; Zukor, Katherine; Liu, Kai; He, Zhigang; Steward, Oswald

2016-01-01

Corticospinal tract (CST) axons from one hemisphere normally extend and terminate predominantly in the contralateral spinal cord. We previously showed that deleting PTEN in the sensorimotor cortex enables CST axons to regenerate after spinal cord injury and that some regenerating axons extend along the “wrong” side. Here, we characterize the degree of specificity of regrowth in terms of laterality. PTEN was selectively deleted via cortical AAV-Cre injections in neonatal PTEN-floxed mice. As adults, mice received dorsal hemisection injuries at T12 or complete crush injuries at T9. CST axons from one hemisphere were traced by unilateral BDA injections in PTEN-deleted mice with spinal cord injury and in non-injured PTEN-floxed mice that had not received AAV-Cre. In non-injured mice, 97.9 ± 0.7% of BDA-labeled axons in white matter and 88.5 ± 1.0% of BDA-labeled axons in grey matter were contralateral to the cortex of origin. In contrast, laterality of CST axons that extended past a lesion due to PTEN deletion varied across animals. In some cases, regenerated axons extended predominantly on the ipsilateral side, in other cases, axons extended predominantly contralaterally, and in others, axons were similar in numbers on both sides. Similar results were seen in analyses of cases from previous studies using shRNA-mediated PTEN knock-down. These results indicate that CST axons that extend past a lesion due to PTEN deletion or knock-down do not maintain the contralateral rule of the non-injured CST, highlighting one aspect for how resultant circuitry from regenerating axons may differ from that of the uninjured CST. PMID:26878190

Role of PTEN in the Tumor Microenvironment

Science.gov (United States)

2010-06-01

fibroblasts (Fig. 2f, g), presumably owing to the constitutive Pten-independent activation of Erk1/2 by serum -stimulation23. Among the many expression...and provide rigorous validation proof. Using man- ual ground- truth , we compare the performance of the tensor classification framework with the k...tive hues in the standard H&E histological staining protocol. In an earlier effort, Pan and Huang (2005) devised a Bayesian supervised segmentation

Protein kinase Cη activates NF-κB in response to camptothecin-induced DNA damage

International Nuclear Information System (INIS)

Raveh-Amit, Hadas; Hai, Naama; Rotem-Dai, Noa; Shahaf, Galit; Gopas, Jacob; Livneh, Etta

2011-01-01

Highlights: → Protein kinase C-eta (PKCη) is an upstream regulator of the NF-κB signaling pathway. → PKCη activates NF-κB in non-stressed conditions and in response to DNA damage. → PKCη regulates NF-κB by activating IκB kinase (IKK) and inducing IκB degradation. -- Abstract: The nuclear factor κB (NF-κB) family of transcription factors participates in the regulation of genes involved in innate- and adaptive-immune responses, cell death and inflammation. The involvement of the Protein kinase C (PKC) family in the regulation of NF-κB in inflammation and immune-related signaling has been extensively studied. However, not much is known on the role of PKC in NF-κB regulation in response to DNA damage. Here we demonstrate for the first time that PKC-eta (PKCη) regulates NF-κB upstream signaling by activating the IκB kinase (IKK) and the degradation of IκB. Furthermore, PKCη enhances the nuclear translocation and transactivation of NF-κB under non-stressed conditions and in response to the anticancer drug camptothecin. We and others have previously shown that PKCη confers protection against DNA damage-induced apoptosis. Our present study suggests that PKCη is involved in NF-κB signaling leading to drug resistance.

Impact of the putative cancer stem cell markers and growth factor receptor expression on the sensitivity of ovarian cancer cells to treatment with various forms of small molecule tyrosine kinase inhibitors and cytotoxic drugs

OpenAIRE

Puvanenthiran, Soozana; Essapen, Sharadah; Seddon, Alan M.; Modjtahedi, Helmout

2016-01-01

Increased expression and activation of human epidermal growth factor receptor (EGFR) and HER-2 have been reported in numerous cancers. The aim of this study was to determine the sensitivity of a large panel of human ovarian cancer cell lines (OCCLs) to treatment with various forms of small molecule tyrosine kinase inhibitors (TKIs) and cytotoxic drugs. The aim was to see if there was any association between the protein expression of various biomarkers including three putative ovarian cancer s...

The inducible CAM plants in putative lunar lander experiments

Science.gov (United States)

Burlak, Olexii; Zaetz, Iryna; Soldatkin, Olexii; Rogutskyy, Ivan; Danilchenko, Boris; Mikheev, Olexander; de Vera, Jean-Pierre; Vidmachenko, Anatolii; Foing, Bernard H.; Kozyrovska, Natalia

Precursory lunar lander experiments on growing plants in locker-based chambers will increase our understanding of effect of lunar conditions on plant physiology. The inducible CAM (Cras-sulacean Acid Metabolism)-plants are reasonable model for a study of relationships between environmental challenges and changes in plant/bacteria gene expression. In inducible CAM-plants the enzymatic machinery for the environmentally activated CAM switches on from a C3-to a full-CAM mode of photosynthesis in response to any stresses (Winter et al., 2008). In our study, Kalanchoe spp. are shown to be promising candidates for putative lunar experiments as resistant to irradiation and desiccation, especially after inoculation with a bacterial consortium (Boorlak et al., 2010). Within frames of the experiment we expect to get information about the functional activity of CAM-plants, in particular, its organogenesis, photosystem, the circadian regulation of plant metabolism on the base of data gaining with instrumental indications from expression of the reporter genes fused to any genes involved in vital functions of the plant (Kozyrovska et al., 2009). References 1. Winter K., Garcia M., Holtum J. (2008) J. Exp. Bot. 59(7):1829-1840 2. Bourlak O., Lar O., Rogutskyy I., Mikheev A., Zaets I., Chervatyuk N., de Vera J.-P., Danilchenko A.B. Foing B.H., zyrovska N. (2010) Space Sci. Technol. 3. Kozyrovska N.O., Vidmachenko A.P., Foing B.H. et al. Exploration/call/estec/ESA. 2009.

Tauroursodeoxycholate Protects Rat Hepatocytes from Bile Acid-Induced Apoptosis via β1-Integrin- and Protein Kinase A-Dependent Mechanisms

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Annika Sommerfeld

2015-05-01

Full Text Available Background/Aims: Ursodeoxycholic acid, which in vivo is rapidly converted into its taurine conjugate, is frequently used for the treatment of cholestatic liver disease. Apart from its choleretic effects, tauroursodeoxycholate (TUDC can protect hepatocytes from bile acid-induced apoptosis, but the mechanisms underlying its anti-apoptotic effects are poorly understood. Methods: These mechanisms were investigated in perfused rat liver and isolated rat hepatocytes. Results: It was found that TUDC inhibited the glycochenodeoxycholate (GCDC-induced activation of the CD95 death receptor at the level of association between CD95 and the epidermal growth factor receptor. This was due to a rapid TUDC-induced β1-integrin-dependent cyclic AMP (cAMP signal with induction of the dual specificity mitogen-activated protein (MAP kinase phosphatase 1 (MKP-1, which prevented GCDC-induced phosphorylation of mitogen-activated protein kinase kinase 4 (MKK4 and c-jun-NH2-terminal kinase (JNK activation. Furthermore, TUDC induced a protein kinase A (PKA-mediated serine/threonine phosphorylation of the CD95, which was recently identified as an internalization signal for CD95. Furthermore, TUDC inhibited GCDC-induced CD95 targeting to the plasma membrane in a β1-integrin-and PKA-dependent manner. In line with this, the β1-integrin siRNA knockdown in sodium taurocholate cotransporting polypeptide (Ntcp-transfected HepG2 cells abolished the protective effect of TUDC against GCDC-induced apoptosis. Conclusion: TUDC exerts its anti-apoptotic effect via a β1-integrin-mediated formation of cAMP, which prevents CD95 activation by hydrophobic bile acids at the levels of JNK activation and CD95 serine/threonine phosphorylation.

Membrane depolarization-induced RhoA/Rho-associated kinase activation and sustained contraction of rat caudal arterial smooth muscle involves genistein-sensitive tyrosine phosphorylation

Science.gov (United States)

Mita, Mitsuo; Tanaka, Hitoshi; Yanagihara, Hayato; Nakagawa, Jun-ichi; Hishinuma, Shigeru; Sutherland, Cindy; Walsh, Michael P.; Shoji, Masaru

2013-01-01

Rho-associated kinase (ROK) activation plays an important role in K+-induced contraction of rat caudal arterial smooth muscle (Mita et al., Biochem J. 2002; 364: 431–40). The present study investigated a potential role for tyrosine kinase activity in K+-induced RhoA activation and contraction. The non-selective tyrosine kinase inhibitor genistein, but not the src family tyrosine kinase inhibitor PP2, inhibited K+-induced sustained contraction (IC50 = 11.3 ± 2.4 µM). Genistein (10 µM) inhibited the K+-induced increase in myosin light chain (LC20) phosphorylation without affecting the Ca2+ transient. The tyrosine phosphatase inhibitor vanadate induced contraction that was reversed by genistein (IC50 = 6.5 ± 2.3 µM) and the ROK inhibitor Y-27632 (IC50 = 0.27 ± 0.04 µM). Vanadate also increased LC20 phosphorylation in a genistein- and Y-27632-dependent manner. K+ stimulation induced translocation of RhoA to the membrane, which was inhibited by genistein. Phosphorylation of MYPT1 (myosin-targeting subunit of myosin light chain phosphatase) was significantly increased at Thr855 and Thr697 by K+ stimulation in a genistein- and Y-27632-sensitive manner. Finally, K+ stimulation induced genistein-sensitive tyrosine phosphorylation of proteins of ∼55, 70 and 113 kDa. We conclude that a genistein-sensitive tyrosine kinase, activated by the membrane depolarization-induced increase in [Ca2+]i, is involved in the RhoA/ROK activation and sustained contraction induced by K+. Ca2+ sensitization, myosin light chain phosphatase, RhoA, Rho-associated kinase, tyrosine kinase PMID:24133693

Induced overexpression of protein kinase D1 stimulates mitogenic signaling in human pancreatic carcinoma PANC-1 cells.

Science.gov (United States)

Kisfalvi, Krisztina; Hurd, Cliff; Guha, Sushovan; Rozengurt, Enrique

2010-05-01

Neurotensin (NT) stimulates protein kinase D1 (PKD1), extracellular signal regulated kinase (ERK), c-Jun N-terminal Kinase (JNK), and DNA synthesis in the human pancreatic adenocarcinoma cell line PANC-1. To determine the effect of PKD1 overexpression on these biological responses, we generated inducible stable PANC-1 clones that express wild-type (WT) or kinase-dead (K618N) forms of PKD1 in response to the ecdysone analog ponasterone-A (PonA). NT potently stimulated c-Jun Ser(63) phosphorylation in both wild type and clonal derivatives of PANC-1 cells. PonA-induced expression of WT, but not K618N PKD1, rapidly blocked NT-mediated c-Jun Ser(63) phosphorylation either at the level of or upstream of MKK4, a dual-specificity kinase that leads to JNK activation. This is the first demonstration that PKD1 suppresses NT-induced JNK/cJun activation in PANC-1 cells. In contrast, PKD1 overexpression markedly increased the duration of NT-induced ERK activation in these cells. The reciprocal influence of PKD1 signaling on pro-mitogenicERK and pro-apopotic JNK/c-Jun pathways prompted us to examine whether PKD1 overexpression promotes DNA synthesis and proliferation of PANC-1 cells. Our results show that PKD1 overexpression increased DNA synthesis and cell numbers of PANC-1 cells cultured in regular dishes or in polyhydroxyethylmethacrylate [Poly-(HEMA)]-coated dishes to eliminate cell adhesion (anchorage-independent growth). Furthermore, PKD1 overexpression markedly enhanced DNA synthesis induced by NT (1-10 nM). These results indicate that PKD1 mediates mitogenic signaling in PANC-1 and suggests that this enzyme could be a novel target for the development of therapeutic drugs that restrict the proliferation of these cells.

Apc inactivation, but not obesity, synergizes with Pten deficiency to drive intestinal stem cell-derived tumorigenesis.

Science.gov (United States)

Tabrizian, Tahmineh; Wang, Donghai; Guan, Fangxia; Hu, Zunju; Beck, Amanda P; Delahaye, Fabien; Huffman, Derek M

2017-06-01

Obesity is a major risk factor for colorectal cancer and can accelerate Lgr5+ intestinal stem cell (ISC)-derived tumorigenesis after the inactivation of Apc However, whether non-canonical pathways involving PI3K-Akt signaling in ISCs can lead to tumor formation, and if this can be further exacerbated by obesity is unknown. Despite the synergy between Pten and Apc inactivation in epithelial cells on intestinal tumor formation, their combined role in Lgr5+-ISCs, which are the most rapidly dividing ISC population in the intestine, is unknown. Lgr5+-GFP mice were provided low-fat diet (LFD) or high-fat diet (HFD) for 8 months, and the transcriptome was evaluated in Lgr5+-ISCs. For tumor studies, Lgr5+-GFP and Lgr5+-GFP- Pten flox/flox mice were tamoxifen treated to inactivate Pten in ISCs and provided LFD or HFD until 14-15 months of age. Finally, various combinations of Lgr5+-ISC-specific, Apc- and Pten -deleted mice were generated and evaluated for histopathology and survival. HFD did not overtly alter Akt signaling in ISCs, but did increase other metabolic pathways. Pten deficiency, but not HFD, increased BrdU-positive cells in the small intestine ( P  Apc deficiency synergistically increased proliferative markers, tumor pathology and mortality, in a dose-dependent fashion ( P  Apc deficiency in ISCs synergistically increases proliferation, tumor formation and mortality. Thus, aberrant Wnt/β-catenin, rather than PI3K-Akt signaling, is requisite for obesity to drive Lgr5+ ISC-derived tumorigenesis. © 2017 Society for Endocrinology.

A Comparative Study of the Aneugenic and Polyploidy-inducing Effects of Fisetin and Two Model Aurora Kinase Inhibitors

Science.gov (United States)

Gollapudi, P.; Hasegawa, L.S.; Eastmond, D.A.

2014-01-01

Fisetin, a plant flavonol commonly found in fruits, nuts and vegetables, is frequently added to nutritional supplements due to its reported cardioprotective, anti-carcinogenic and antioxidant properties. Earlier reports from our laboratory and others have indicated that fisetin has both aneugenic and clastogenic properties in cultured cells. More recently, fisetin has also been reported to target Aurora B kinase, a Ser/Thr kinase involved in ensuring proper microtubule attachment at the spindle assembly checkpoint, and an enzyme that is overexpressed in several types of cancer. Here we have further characterized the chromosome damage caused by fisetin and compared it with that induced by two known Aurora kinase inhibitors, VX-680 and ZM-447439, in cultured TK6 cells using the micronucleus assay with CREST staining as well as a flow cytometry-based assay that measures multiple types of numerical chromosomal aberrations. The three compounds were highly effective in inducing aneuploidy and polyploidy as evidenced by increases in kinetochore-positive micronuclei, hyperdiploidy, and polyploidy. With fisetin, however, the latter two effects were most significantly observed only after cells were allowed to overcome a cell cycle delay, and occurred at higher concentrations than those induced by the other Aurora kinase inhibitors. Modest increases in kinetochore-negative micronuclei were also seen with the model Aurora kinase inhibitors. These results indicate that fisetin induces multiple types of chromosome abnormalities in human cells, and indicate a need for a thorough investigation of fisetin-augmented dietary supplements. PMID:24680981

LY294002 inhibits glucocorticoid-induced COX-2 gene expression in cardiomyocytes through a phosphatidylinositol 3 kinase-independent mechanism

International Nuclear Information System (INIS)

Sun Haipeng; Xu Beibei; Sheveleva, Elena; Chen, Qin M.

2008-01-01

Glucocorticoids induce COX-2 expression in rat cardiomyocytes. While investigating whether phosphatidylinositol 3 kinase (PI3K) plays a role in corticosterone (CT)-induced COX-2, we found that LY294002 (LY29) but not wortmannin (WM) attenuates CT from inducing COX-2 gene expression. Expression of a dominant-negative mutant of p85 subunit of PI3K failed to inhibit CT from inducing COX-2 expression. CT did not activate PI3K/AKT signaling pathway whereas LY29 and WM decreased the activity of PI3K. LY303511 (LY30), a structural analogue and a negative control for PI3K inhibitory activity of LY29, also suppressed COX-2 induction. These data suggest PI3K-independent mechanisms in regulating CT-induced COX-2 expression. LY29 and LY30 do not inhibit glucocorticoid receptor transactivity. Both compounds have been reported to inhibit Casein Kinase 2 activity and modulate potassium and calcium levels independent of PI3K, while LY29 has been reported to inhibit mammalian Target of Rapamycin (mTOR), and DNA-dependent Protein Kinase (DNA-PK). Inhibitor of Casein Kinase 2 (CK2), mTOR or DNA-PK failed to prevent CT from inducing COX-2 expression. Tetraethylammonium (TEA), a potassium channel blocker, and nimodipine, a calcium channel blocker, both attenuated CT from inducing COX-2 gene expression. CT was found to increase intracellular Ca 2+ concentration, which can be inhibited by LY29, TEA or nimodipine. These data suggest a possible role of calcium instead of PI3K in CT-induced COX-2 expression in cardiomyocytes

Activation of AMP-activated protein kinase by tributyltin induces neuronal cell death

International Nuclear Information System (INIS)

Nakatsu, Yusuke; Kotake, Yaichiro; Hino, Atsuko; Ohta, Shigeru

2008-01-01

AMP-activated protein kinase (AMPK), a member of the metabolite-sensing protein kinase family, is activated by energy deficiency and is abundantly expressed in neurons. The environmental pollutant, tributyltin chloride (TBT), is a neurotoxin, and has been reported to decrease cellular ATP in some types of cells. Therefore, we investigated whether TBT activates AMPK, and whether its activation contributes to neuronal cell death, using primary cultures of cortical neurons. Cellular ATP levels were decreased 0.5 h after exposure to 500 nM TBT, and the reduction was time-dependent. It was confirmed that most neurons in our culture system express AMPK, and that TBT induced phosphorylation of AMPK. Compound C, an AMPK inhibitor, reduced the neurotoxicity of TBT, suggesting that AMPK is involved in TBT-induced cell death. Next, the downstream target of AMPK activation was investigated. Nitric oxide synthase, p38 phosphorylation and Akt dephosphorylation were not downstream of TBT-induced AMPK activation because these factors were not affected by compound C, but glutamate release was suggested to be controlled by AMPK. Our results suggest that activation of AMPK by TBT causes neuronal death through mediating glutamate release

Pervanadate induces Mammalian Ste20 Kinase 3 (MST3) tyrosine phosphorylation but not activation.

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Kan, Wei-Chih; Lu, Te-Ling; Ling, Pin; Lee, Te-Hsiu; Cho, Chien-Yu; Huang, Chi-Ying F; Jeng, Wen-Yih; Weng, Yui-Ping; Chiang, Chun-Yen; Wu, Jin Bin; Lu, Te-Jung

2016-07-01

The yeast Ste20 (sterile) protein kinase, which is a serine/threonine kinase, responds to the stimulation of the G proteincoupled receptor (GPCR) pheromone receptor. Ste20 protein kinase serves as the critical component that links signaling from the GPCR/G proteins to the mitogen-activated protein kinase (MAPK) cascade in yeast. The yeast Ste20p functions as a MAP kinase kinase kinase kinase (MAP4K) in the pheromone response. Ste20-like kinases are structurally conserved from yeast to mammals. The mechanism by which MAP4K links GPCR to the MAPK pathway is less clearly defined in vertebrates. In addition to MAP4K, the tyrosine kinase cascade bridges G proteins and the MAPK pathway in vertebrate cells. Mammalian Ste20 Kinase 3 (MST3) has been categorized into the Ste20 family and has been reported to function in the regulation of cell polarity and migration. However, whether MST3 tyrosine phosphorylation regulates diverse signaling pathways is unknown. In this study, the tyrosine phosphatase inhibitor pervanadate was found to induce MST3 tyrosine phosphorylation in intact cells, and the activity of tyrosine-phosphorylated MST3 was measured. This tyrosine-directed phosphorylation was independent of MST3 activity. Parameters including protein conformation, Triton concentration and ionic concentration influenced the sensitivity of MST3 activity. Taken together, our data suggests that the serine/threonine kinase MST3 undergoes tyrosinedirected phosphorylation. The tyrosine-phosphorylated MST3 may create a docking site for the structurally conserved SH2/SH3 (Src Homology 2 and 3) domains within the Src oncoprotein. The unusual tyrosinephosphorylated MST3 may recruit MST3 to various signaling components. Copyright © 2016. Published by Elsevier Inc.

Loss of mTOR repressors Tsc1 or Pten has divergent effects on excitatory and inhibitory synaptic transmission in single hippocampal neuron cultures.

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Weston, Matthew C; Chen, Hongmei; Swann, John W

2014-01-01

The Pten and Tsc1 genes both encode proteins that repress mechanistic target of rapamycin (mTOR) signaling. Disruption of either gene in the brain results in epilepsy and autism-like symptoms in humans and mouse models, therefore it is important to understand the molecular and physiological events that lead from gene disruption to disease phenotypes. Given the similar roles these two molecules play in the regulation of cellular growth and the overlap in the phenotypes that result from their loss, we predicted that the deletion of either the Pten or Tsc1 gene from autaptic hippocampal neurons would have similar effects on neuronal morphology and synaptic transmission. Accordingly, we found that loss of either Pten or Tsc1 caused comparable increases in soma size, dendrite length and action potential properties. However, the effects of Pten and Tsc1 loss on synaptic transmission were different. Loss of Pten lead to an increase in both excitatory and inhibitory neurotransmission, while loss of Tsc1 did not affect excitatory neurotransmission and reduced inhibitory transmission by decreasing mIPSC amplitude. Although the loss of Pten or Tsc1 both increased downstream mTORC1 signaling, phosphorylation of Akt was increased in Pten-ko and decreased in Tsc1-ko neurons, potentially accounting for the different effects on synaptic transmission. Despite the different effects at the synaptic level, our data suggest that loss of Pten or Tsc1 may both lead to an increase in the ratio of excitation to inhibition at the network level, an effect that has been proposed to underlie both epilepsy and autism.

HRR25, a putative protein kinase from budding yeast: Association with repair of damaged DNA

International Nuclear Information System (INIS)

Hoekstra, M.F.; Ou, A.C.; DeMaggio, A.J.; Burbee, D.G.; Liskay, R.M.; Heffron, F.

1991-01-01

In simple eukaryotes, protein kinases regulate mitotic and meiotic cell cycles, the response to polypeptide pheromones, and the initiation of nuclear DNA synthesis. The protein HRR25 from the budding yeast Saccharomyces cerevisiae was defined by the mutation hrr25-1. This mutation resulted in sensitivity to continuous expression of the HO double-strand endonuclease, to methyl methanesulfonate, and to x-irradiation. Homozygotes of hrr25-1 were unable to sporulate and disruption and deletion of HRR25 interfered with mitotic and meiotic cell division. Sequence analysis revealed two distinctive regions in the protein. The NH 2 -terminus of HRR25 contains the hallmark features of protein kinases, whereas the COOH-terminus is rich in proline and glutamine. Mutations in HRR25 at conserved residues found in all protein kinases inactivated the gene, and these mutants exhibited the hrr25 null phenotypes. Taken together, the hrr25 mutant phenotypes and the features of the gene product indicate that HRR25 is a distinctive member of the protein kinase superfamily

The Src family kinase inhibitor dasatinib delays pain-related behaviour and conserves bone in a rat model of cancer-induced bone pain

DEFF Research Database (Denmark)

Appel, Camilla Kristine; Gallego-Pedersen, Simone; Andersen, Line

2017-01-01

Pain is a severe and debilitating complication of metastatic bone cancer. Current analgesics do not provide sufficient pain relief for all patients, creating a great need for new treatment options. The Src kinase, a non-receptor protein tyrosine kinase, is implicated in processes involved in cancer......-induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p...

Premature Senescence Induced by Ionizing Radiation Requires AKT Activity and Reactive Oxygen Species in Glioma

International Nuclear Information System (INIS)

Lee, Je Jung; Kim, Bong Cho; Yoo, Hee Jung; Lee, Jae Seon

2010-01-01

Loss of PTEN, a tumor suppressor gene has frequently observed in human gliomas, which conferred AKT activation and resistance to ionizing radiation (IR) and anti-cancer drugs. Recent reports have shown that AKT activation induces premature senescence through increase of oxygen consumption and inhibition of expression of ROS scavenging enzymes. In this study, we compared cellular response to IR in the PTEN-deficient U87, U251, U373 or PTEN-proficient LN18, LN428 glioma cells

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

OpenAIRE

Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2012-01-01

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substra...

A multicenter study shows PTEN deletion is strongly associated with seminal vesicle involvement and extracapsular extension in localized prostate cancer.

Science.gov (United States)

Troyer, Dean A; Jamaspishvili, Tamara; Wei, Wei; Feng, Ziding; Good, Jennifer; Hawley, Sarah; Fazli, Ladan; McKenney, Jesse K; Simko, Jeff; Hurtado-Coll, Antonio; Carroll, Peter R; Gleave, Martin; Lance, Raymond; Lin, Daniel W; Nelson, Peter S; Thompson, Ian M; True, Lawrence D; Brooks, James D; Squire, Jeremy A

2015-08-01

Loss of the phosphatase and tensin homolog (PTEN) tumor suppressor gene is a promising marker of aggressive prostate cancer. Active surveillance and watchful waiting are increasingly recommended to patients with small tumors felt to be low risk, highlighting the difficulties of Gleason scoring in this setting. There is an urgent need for predictive biomarkers that can be rapidly deployed to aid in clinical decision-making. Our objectives were to assess the incidence and ability of PTEN alterations to predict aggressive disease in a multicenter study. We used recently developed probes optimized for sensitivity and specificity in a four-color FISH deletion assay to study the Canary Retrospective multicenter Prostate Cancer Tissue Microarray (TMA). This TMA was constructed specifically for biomarker validation from radical prostatectomy specimens, and is accompanied by detailed clinical information with long-term follow-up. In 612 prostate cancers, the overall rate of PTEN deletion was 112 (18.3%). Hemizygous PTEN losses were present in 55/612 (9.0%) of cancers, whereas homozygous PTEN deletion was observed in 57/612 (9.3%) of tumors. Significant associations were found between PTEN status and pathologic stage (P free survival (number of events = 189), pre-operative prostate specific antigen (PSA) (P free survival in multivariate models, as were seminal vesicle invasion, extracapsular extension, and Gleason score, and preoperative PSA. Furthermore, these data demonstrate that the assay can be readily introduced at first diagnosis in a cost effective manner analogous to the use of FISH for analysis of HER2/neu status in breast cancer. Combined with published research beginning 17 years ago, both the data and tools now exist to implement a PTEN assay in the clinic. © 2015 The Authors. The Prostate, published by Wiley Periodicals, Inc.

DNA-dependent protein kinase inhibits AID-induced antibody gene conversion.

Directory of Open Access Journals (Sweden)

Adam J L Cook

2007-04-01

Full Text Available Affinity maturation and class switching of antibodies requires activation-induced cytidine deaminase (AID-dependent hypermutation of Ig V(DJ rearrangements and Ig S regions, respectively, in activated B cells. AID deaminates deoxycytidine bases in Ig genes, converting them into deoxyuridines. In V(DJ regions, subsequent excision of the deaminated bases by uracil-DNA glycosylase, or by mismatch repair, leads to further point mutation or gene conversion, depending on the species. In Ig S regions, nicking at the abasic sites produced by AID and uracil-DNA glycosylases results in staggered double-strand breaks, whose repair by nonhomologous end joining mediates Ig class switching. We have tested whether nonhomologous end joining also plays a role in V(DJ hypermutation using chicken DT40 cells deficient for Ku70 or the DNA-dependent protein kinase catalytic subunit (DNA-PKcs. Inactivation of the Ku70 or DNA-PKcs genes in DT40 cells elevated the rate of AID-induced gene conversion as much as 5-fold. Furthermore, DNA-PKcs-deficiency appeared to reduce point mutation. The data provide strong evidence that double-strand DNA ends capable of recruiting the DNA-dependent protein kinase complex are important intermediates in Ig V gene conversion.

Oryza sativa (Rice) Hull Extract Inhibits Lipopolysaccharide-Induced Inflammatory Response in RAW264.7 Macrophages by Suppressing Extracellular Signal-regulated Kinase, c-Jun N-terminal Kinase, and Nuclear Factor-κB Activation.

Science.gov (United States)

Ha, Sang Keun; Sung, Jeehye; Choi, Inwook; Kim, Yoonsook

2016-01-01

Rice ( Oryza sativa ) is a major cereal crop in many Asian countries and an important staple food source. Rice hulls have been reported to possess antioxidant activities. In this study, we evaluated the antiinflammatory effects of rice hull extract and associated signal transduction mechanisms in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages. We found that rice hull extract inhibited nitric oxide (NO) and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively. The release of interleukin-1β and tumor necrosis factor-α was also reduced in a dose-dependent manner. Furthermore, rice hull extract attenuated the activation of nuclear factor-kappa B (NF-κB), as well as the phosphorylation of mitogen-activated protein kinases, extracellular signal-regulated kinase (ERK), and c-Jun N-terminal kinase (JNK), in LPS-stimulated RAW264.7 cells. This suggests that rice hull extract decreases the production of inflammatory mediators by downregulating ERK and JNK and the NF-κB signal pathway in RAW 264.7 cells. Rice hull extract inhibits the lipopolysaccharide-induced inflammatory response in RAW264.7 macrophages.Rice hull extract inhibited nitric oxide and prostaglandin E 2 by suppressing the expression of inducible NO synthase and cyclooxygenase-2, respectively.Rice hull extract exerted anti-inflammatory effect through inhibition of nuclear factor-kappa B, extracellular signal-regulated kinase and c-Jun N-terminal kinase signaling pathways.Rice hull extract may provide a potential therapeutic approach for inflammatory diseases. Abbreviations used: COX-2: cyclooxygenase-2, ERK: extracellular signal-regulated kinase, IκB: inhibitory kappa B, IL-1β: interleukin-1β, iNOS: inducible NO synthase, JNK: c-Jun N-terminal kinase, LPS: lipopolysaccharide, MAPKs: mitogen-activated protein kinases, NF-κB: nuclear factor-κB, NO: nitric oxide, PGE2: prostaglandin E2, RHE: rice hull extract, ROS: reactive oxygen species

Overexpression of a novel endogenous NADH kinase in Aspergillus nidulans enhances growth

DEFF Research Database (Denmark)

Panagiotou, Gianni; Grotkjær, Thomas; Hofmann, Gerald

2009-01-01

.7.1.86) has been identified. The enzyme has a predicted molecular weight of 49 kDa. We characterised the role of this NADH kinase by genomic integration of the putative gene AN8837.2 under a strong constitutive promoter. The physiological effects of overexpressed NADH kinase in combination with different...... yield on glucose and the maximum specific growth rate increased from 0.47 g/g and 0.22 h(-1) (wild type) to 0.54 g/g and 0.26 h(-1) (NADH kinase overexpressed), respectively. The results suggest that overexpression of NADH kinase improves the growth efficiency of the cell by increasing the access...

Molecular mechanism of 17-allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL receptor tyrosine kinase degradation.

Science.gov (United States)

Krishnamoorthy, Gnana Prakasam; Guida, Teresa; Alfano, Luigi; Avilla, Elvira; Santoro, Massimo; Carlomagno, Francesca; Melillo, Rosa Marina

2013-06-14

The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [(35)S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL.

Molecular Mechanism of 17-Allylamino-17-demethoxygeldanamycin (17-AAG)-induced AXL Receptor Tyrosine Kinase Degradation*

Science.gov (United States)

Krishnamoorthy, Gnana Prakasam; Guida, Teresa; Alfano, Luigi; Avilla, Elvira; Santoro, Massimo; Carlomagno, Francesca; Melillo, Rosa Marina

2013-01-01

The receptor tyrosine kinase AXL is overexpressed in many cancer types including thyroid carcinomas and has well established roles in tumor formation and progression. Proper folding, maturation, and activity of several oncogenic receptor tyrosine kinases require HSP90 chaperoning. HSP90 inhibition by the antibiotic geldanamycin or its derivative 17-allylamino-17-demethoxygeldanamycin (17-AAG) causes destabilization of its client proteins. Here we show that AXL is a novel client protein of HSP90. 17-AAG induced a time- and dose-dependent down-regulation of endogenous or ectopically expressed AXL protein, thereby inhibiting AXL-mediated signaling and biological activity. 17-AAG-induced AXL down-regulation specifically affected fully glycosylated mature receptor present on cell membrane. By using biotin and [35S]methionine labeling, we showed that 17-AAG caused depletion of membrane-localized AXL by mediating its degradation in the intracellular compartment, thus restricting its exposure on the cell surface. 17-AAG induced AXL polyubiquitination and subsequent proteasomal degradation; under basal conditions, AXL co-immunoprecipitated with HSP90. Upon 17-AAG treatment, AXL associated with the co-chaperone HSP70 and the ubiquitin E3 ligase carboxyl terminus of HSC70-interacting protein (CHIP). Overexpression of CHIP, but not of the inactive mutant CHIP K30A, induced accumulation of AXL polyubiquitinated species upon 17-AAG treatment. The sensitivity of AXL to 17-AAG required its intracellular domain because an AXL intracellular domain-deleted mutant was insensitive to the compound. Active AXL and kinase-dead AXL were similarly sensitive to 17-AAG, implying that 17-AAG sensitivity does not require receptor phosphorylation. Overall our data elucidate the molecular basis of AXL down-regulation by HSP90 inhibitors and suggest that HSP90 inhibition in anticancer therapy can exert its effect through inhibition of multiple kinases including AXL. PMID:23629654

Diffuse Gastric Ganglioneuromatosis: Novel Presentation of PTEN Hamartoma Syndrome—Case Report and Review of Gastric Ganglioneuromatous Proliferations and a Novel PTEN Gene Mutation

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Alexander J. Williams

2018-01-01

Full Text Available Gastrointestinal ganglioneuromatous proliferations are rare, most often found in the colon, and are three types: polypoid ganglioneuromas, ganglioneuromatous polyposis, and diffuse ganglioneuromatosis. We present a case of diffuse ganglioneuromatosis in the posterior gastric wall in a nine-year-old female. To our knowledge, this is the first reported case of diffuse ganglioneuromatosis located in the stomach. Only six cases of gastric ganglioneuromatous proliferations have previously been reported, two in English and none were diffuse ganglioneuromatosis. A diagnosis of diffuse ganglioneuromatosis is relevant for patient care because, unlike sporadic polypoid ganglioneuromas or ganglioneuromatous polyposis, most are syndromic. Diffuse ganglioneuromatosis is commonly associated with neurofibromatosis type 1, multiple endocrine neoplasia type 2b, and Cowden Syndrome, one of the phenotypes of PTEN hamartoma tumor syndrome. The patient had the noted gastric diffuse ganglioneuromatosis, as well as other major and minor criteria for Cowden syndrome. Genetic testing revealed a novel frameshift mutation in the PTEN gene in the patient, her father, paternal aunt, and the aunt’s son who is a paternal first cousin of the patient.

Insulin-induced decrease in protein phosphorylation in rat adipocytes not explained by decreased A-kinase activity

International Nuclear Information System (INIS)

Egan, J.J.; Greenberg, A.S.; Chang, M.K.; Londos, C.

1987-01-01

In isolated rat adipocytes, insulin inhibits lipolysis to a greater extent than would be predicted by the decrease in (-/+)cAMP activity ratio of cAMP-dependent protein kinase [A-kinase], from which it was speculated that insulin promotes the dephosphorylation of hormone-sensitive lipase. They have examined the phosphorylation state of cellular proteins under conditions of varying A-kinase activities in the presence and absence of insulin. Protein phosphorylation was determined by SDS-PAGE electrophoresis of extracts from 32 P-loaded cells; glycerol and A-kinase activity ratios were measured in the cytosolic extracts from control, non-radioactive cells. Increased protein phosphorylation in general occurred over the same range of A-kinase activity ratios, 0.1-0.3, associated with increased glycerol release. The insulin-induced decrease in lipolysis was associated with a decrease in the 32 P content of several proteins, an effect not explained by the modest reduction in A-kinase activity by insulin. This effect of insulin on protein phosphorylation was lost as the A-kinase activity ratios exceeded 0.5. The results suggest that insulin promotes the dephosphorylation of those adipocyte proteins which are subject to phosphorylation by A-kinase

Role of phosphoinositide 3-kinase in ischemic postconditioning-induced attenuation of cerebral ischemia-evoked behavioral deficits in mice.

Science.gov (United States)

Rehni, Ashish K; Singh, Nirmal

2007-01-01

The present study has been designed to pharmacologically investigate the role of phosphoinositide 3-kinase in ischemic postconditioning-induced reversal of global cerebral ischemia and reperfusion-induced behavioral dysfunction in mice. Bilateral carotid artery occlusion for 10 min followed by reperfusion for 24 h was employed in the present study to produce ischemia and reperfusion-induced cerebral injury in mice. Short-term memory was evaluated using the elevated plus maze test. The inclined beam walking test was employed to assess motor incoordination. Bilateral carotid artery occlusion followed by reperfusion produced impaired short-term memory, motor co-ordination and lateral push response. Three episodes of carotid artery occlusion for a period of 10 s and reperfusion of 10 s (ischemic postconditioning) significantly prevented ischemia-reperfusion-induced behavioral deficit measured in terms of loss of short-term memory, motor coordination and lateral push response. Wortmannin (2 mg/kg, iv), a phosphoinositide 3-kinase inhibitor given 10 min before ischemia attenuated the beneficial effects of ischemic postconditioning. It may be concluded that beneficial effects of ischemic postconditioning on global cerebral ischemia and reperfusion-induced behavioral deficits may involve activation of phosphoinositide 3-kinase-linked pathway.

Interactions between TGF-β1, canonical WNT/β-catenin pathway and PPAR γ in radiation-induced fibrosis.

Science.gov (United States)

Vallée, Alexandre; Lecarpentier, Yves; Guillevin, Rémy; Vallée, Jean-Noël

2017-10-27

Radiation therapy induces DNA damage and inflammation leading to fibrosis. Fibrosis can occur 4 to 12 months after radiation therapy. This process worsens with time and years. Radiation-induced fibrosis is characterized by fibroblasts proliferation, myofibroblast differentiation, and synthesis of collagen, proteoglycans and extracellular matrix. Myofibroblasts are non-muscle cells that can contract and relax. Myofibroblasts evolve towards irreversible retraction during fibrosis process. In this review, we discussed the interplays between transforming growth factor-β1 (TGF-β1), canonical WNT/β-catenin pathway and peroxisome proliferator-activated receptor gamma (PPAR γ) in regulating the molecular mechanisms underlying the radiation-induced fibrosis, and the potential role of PPAR γ agonists. Overexpression of TGF-β and canonical WNT/β-catenin pathway stimulate fibroblasts accumulation and myofibroblast differentiation whereas PPAR γ expression decreases due to the opposite interplay of canonical WNT/β-catenin pathway. Both TGF-β1 and canonical WNT/β-catenin pathway stimulate each other through the Smad pathway and non-Smad pathways such as phosphatidylinositol 3-kinase/serine/threonine kinase (PI3K/Akt) signaling. WNT/β-catenin pathway and PPAR γ interact in an opposite manner. PPAR γ agonists decrease β-catenin levels through activation of inhibitors of the WNT pathway such as Smad7, glycogen synthase kinase-3 (GSK-3 β) and dickkopf-related protein 1 (DKK1). PPAR γ agonists also stimulate phosphatase and tensin homolog (PTEN) expression, which decreases both TGF-β1 and PI3K/Akt pathways. PPAR γ agonists by activating Smad7 decrease Smads pathway and then TGF-β signaling leading to decrease radiation-induced fibrosis. TGF-β1 and canonical WNT/β-catenin pathway promote radiation-induced fibrosis whereas PPAR γ agonists can prevent radiation-induced fibrosis.

Effects of AdR-siPTEN on learning capability, memory and ...

African Journals Online (AJOL)

USER

2010-07-12

Jul 12, 2010 ... protein expression of a phosphatase and tensin homolog deleted on chromosome 10 (PTEN), ... disease (AD) and refers to a step-wise deterioration in cognitive ..... Pappas BA, de la Torre JC, Davidson CM (1996).Chronic ...

Potential value of PTEN in predicting cetuximab response in colorectal cancer: An exploratory study

International Nuclear Information System (INIS)

Razis, Evangelia; Galanidi, Eleni; Bai, Maria; Gikonti, Ioanna; Koukouma, Alona; Kafiri, Georgia; Papakostas, Pavlos; Kalogeras, Konstantine T; Kosmidis, Paris; Fountzilas, George; Briasoulis, Evangelos; Vrettou, Eleni; Skarlos, Dimosthenis V; Papamichael, Dimitrios; Kostopoulos, Ioannis; Samantas, Epaminontas; Xanthakis, Ioannis; Bobos, Mattheos

2008-01-01

The epidermal growth factor receptor (EGFR) is over-expressed in 70–75% of colorectal adenocarcinomas (CRC). The anti-EGFR monoclonal antibody cetuximab has been approved for the treatment of metastatic CRC, however tumor response to cetuximab has not been found to be associated with EGFR over-expression by immunohistochemistry (IHC). The aim of this study was to explore EGFR and the downstream effector phosphatase and tensin homologue deleted on chromosome 10 (PTEN) as potential predictors of response to cetuximab. CRC patients treated with cetuximab by the Hellenic Cooperative Oncology group, whose formalin-fixed paraffin-embedded tumor tissue was available, were included. Tissue was tested for EGFR and PTEN by IHC and fluorescence in situ hybridization (FISH). Eighty-eight patients were identified and 72 were included based on the availability of tissue blocks with adequate material for analysis on them. All patients, except one, received cetuximab in combination with chemotherapy. Median follow-up was 53 months from diagnosis and 17 months from cetuximab initiation. At the time of the analysis 53% of the patients had died. Best response was complete response in one and partial response in 23 patients. In 16 patients disease stabilized. Lack of PTEN gene amplification was associated with more responses to cetuximab and longer time to progression (p = 0.042). PTEN could be one of the molecular determinants of cetuximab response. Due to the heterogeneity of the population and the retrospective nature of the study, our results are hypothesis generating and should be approached with caution. Further prospective studies are needed to validate this finding

Maternal high-fat diet induces metabolic stress response disorders in offspring hypothalamus.

Science.gov (United States)

Nguyen, Long The; Saad, Sonia; Tan, Yi; Pollock, Carol; Chen, Hui

2017-07-01

Maternal obesity has been shown to increase the risk of obesity and related disorders in the offspring, which has been partially attributed to changes of appetite regulators in the offspring hypothalamus. On the other hand, endoplasmic reticulum (ER) stress and autophagy have been implicated in hypothalamic neuropeptide dysregulation, thus may also play important roles in such transgenerational effect. In this study, we show that offspring born to high-fat diet-fed dams showed significantly increased body weight and glucose intolerance, adiposity and plasma triglyceride level at weaning. Hypothalamic mRNA level of the orexigenic neuropeptide Y (NPY) was increased, while the levels of the anorexigenic pro-opiomelanocortin (POMC), NPY1 receptor (NPY1R) and melanocortin-4 receptor (MC4R) were significantly downregulated. In association, the expression of unfolded protein response (UPR) markers including glucose-regulated protein (GRP)94 and endoplasmic reticulum DNA J domain-containing protein (Erdj)4 was reduced. By contrast, protein levels of autophagy-related genes Atg5 and Atg7, as well as mitophagy marker Parkin, were slightly increased. The administration of 4-phenyl butyrate (PBA), a chemical chaperone of protein folding and UPR activator, in the offspring from postnatal day 4 significantly reduced their body weight, fat deposition, which were in association with increased activating transcription factor (ATF)4, immunoglobulin-binding protein (BiP) and Erdj4 mRNA as well as reduced Parkin, PTEN-induced putative kinase (PINK)1 and dynamin-related protein (Drp)1 protein expression levels. These results suggest that hypothalamic ER stress and mitophagy are among the regulatory factors of offspring metabolic changes due to maternal obesity. © 2017 Society for Endocrinology.

A Transition Zone Showing Highly Discontinuous or Alternating Levels of Stem Cell and Proliferation Markers Characterizes the Development of PTEN-Haploinsufficient Colorectal Cancer.

Science.gov (United States)

Arvai, Kevin J; Hsu, Ya-Hsuan; Lee, Lobin A; Jones, Dan

2015-01-01

Stepwise acquisition of oncogene mutations and deletion/inactivation of tumor suppressor genes characterize the development of colorectal cancer (CRC). These genetic events interact with discrete morphologic transitions from hyperplastic mucosa to adenomatous areas, followed by in situ malignant transformation and finally invasive carcinoma. The goal of this study was to identify tissue markers of the adenoma-carcinoma morphogenetic transitions in CRC. We analyzed the patterns of expression of growth regulatory and stem cell markers across these distinct morphologic transition zones in 735 primary CRC tumors. In 202 cases with preserved adenoma-adenocarcinoma transition, we identified, in 37.1% of cases, a zone of adenomatous epithelium, located immediately adjacent to the invasive component, that showed rapidly alternating intraglandular stretches of PTEN+ and PTEN- epithelium. This zone exactly overlapped with similar alternating expression of Ki-67 and inversely with the transforming growth factor-beta (TGF-β) growth regulator SMAD4. These zones also show parallel alternating levels and/or subcellular localization of multiple cancer stem/progenitor cell (CSC) markers, including β-catenin/CTNNB1, ALDH1, and CD44. PTEN was always re-expressed in the invasive tumor in these cases, unlike those with complete loss of PTEN expression. Genomic microarray analysis of CRC with prominent CSC-like expansions demonstrated a high frequency of PTEN genomic deletion/haploinsufficiency in tumors with CSC-like transition zones (62.5%) but not in tumors with downregulated but non-alternating PTEN expression (14.3%). There were no significant differences in the levels of KRAS mutation or CTNNB1 mutation in CSC-like tumors as compared to unselected CRC cases. In conclusion, we have identified a distinctive CSC-like pre-invasive transition zone in PTEN-haploinsufficient CRC that shows convergent on-off regulation of the PTEN/AKT, TGF-β/SMAD and Wnt/β-catenin pathways. This

A Transition Zone Showing Highly Discontinuous or Alternating Levels of Stem Cell and Proliferation Markers Characterizes the Development of PTEN-Haploinsufficient Colorectal Cancer.

Directory of Open Access Journals (Sweden)

Kevin J Arvai

Full Text Available Stepwise acquisition of oncogene mutations and deletion/inactivation of tumor suppressor genes characterize the development of colorectal cancer (CRC. These genetic events interact with discrete morphologic transitions from hyperplastic mucosa to adenomatous areas, followed by in situ malignant transformation and finally invasive carcinoma. The goal of this study was to identify tissue markers of the adenoma-carcinoma morphogenetic transitions in CRC.We analyzed the patterns of expression of growth regulatory and stem cell markers across these distinct morphologic transition zones in 735 primary CRC tumors. In 202 cases with preserved adenoma-adenocarcinoma transition, we identified, in 37.1% of cases, a zone of adenomatous epithelium, located immediately adjacent to the invasive component, that showed rapidly alternating intraglandular stretches of PTEN+ and PTEN- epithelium. This zone exactly overlapped with similar alternating expression of Ki-67 and inversely with the transforming growth factor-beta (TGF-β growth regulator SMAD4. These zones also show parallel alternating levels and/or subcellular localization of multiple cancer stem/progenitor cell (CSC markers, including β-catenin/CTNNB1, ALDH1, and CD44. PTEN was always re-expressed in the invasive tumor in these cases, unlike those with complete loss of PTEN expression. Genomic microarray analysis of CRC with prominent CSC-like expansions demonstrated a high frequency of PTEN genomic deletion/haploinsufficiency in tumors with CSC-like transition zones (62.5% but not in tumors with downregulated but non-alternating PTEN expression (14.3%. There were no significant differences in the levels of KRAS mutation or CTNNB1 mutation in CSC-like tumors as compared to unselected CRC cases.In conclusion, we have identified a distinctive CSC-like pre-invasive transition zone in PTEN-haploinsufficient CRC that shows convergent on-off regulation of the PTEN/AKT, TGF-β/SMAD and Wnt

Calcium Hydroxide-induced Proliferation, Migration, Osteogenic Differentiation, and Mineralization via the Mitogen-activated Protein Kinase Pathway in Human Dental Pulp Stem Cells.

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Chen, Luoping; Zheng, Lisha; Jiang, Jingyi; Gui, Jinpeng; Zhang, Lingyu; Huang, Yan; Chen, Xiaofang; Ji, Jing; Fan, Yubo

2016-09-01

Calcium hydroxide has been extensively used as the gold standard for direct pulp capping in clinical dentistry. It induces proliferation, migration, and mineralization in dental pulp stem cells (DPSCs), but the underlying mechanisms are still unclear. The aim of this study was to investigate the role of the mitogen-activated protein (MAP) kinase pathway in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Human DPSCs between passages 3 and 6 were used. DPSCs were preincubated with inhibitors of MAP kinases and cultured with calcium hydroxide. The phosphorylated MAP kinases were detected by Western blot analysis. Cell viability was analyzed via the methylthiazol tetrazolium assay. Cell migration was estimated using the wound healing assay. Alkaline phosphatase (ALP) expression was analyzed using the ALP staining assay. Mineralization was studied by alizarin red staining analysis. Calcium hydroxide significantly promoted the phosphorylation of the c-Jun N-terminal kinase (JNK), p38, and extracellular signal-regulated kinase. The inhibition of JNK and p38 signaling abolished calcium hydroxide-induced proliferation of DPSCs. The inhibition of JNK, p38, and extracellular signal-regulated kinase signaling suppressed the migration, ALP expression, and mineralization of DPSCs. Our study showed that the MAP kinase pathway was involved in calcium hydroxide-induced proliferation, migration, osteogenic differentiation, and mineralization in human DPSCs. Copyright © 2016 American Association of Endodontists. Published by Elsevier Inc. All rights reserved.

Role of Glycogen Synthase Kinase-3β in APP Hyperphosphorylation Induced by NMDA Stimulation in Cortical Neurons

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Xanthi Antoniou

2010-01-01

Full Text Available The phosphorylation of Amyloid Precursor Protein (APP at Thr668 plays a key role in APP metabolism that is highly relevant to AD. The c-Jun-N-terminal kinase (JNK, glycogen synthase kinase-3β (GSK-3β and cyclin-dependent kinase 5 (Cdk5 can all be responsible for this phosphorylation. These kinases are activated by excitotoxic stimuli fundamental hallmarks of AD. The exposure of cortical neurons to a high dose of NMDA (100 μM for 30’-45’ led to an increase of P-APP Thr668. During NMDA stimulation APP hyperphosphorylation has to be assigned to GSK-3β activity, since addition of L803-mts, a substrate competitive inhibitor of GSK-3β reduced APP phosphorylation induced by NMDA. On the contrary, inhibition of JNK and Cdk5 with D-JNKI1 and Roscovitine respectively did not prevent NMDA-induced P-APP increase. These data show a tight connection, in excitotoxic conditions, between APP metabolism and the GSK-3β signaling pathway.

Myristoylation of Src kinase mediates Src-induced and high-fat diet-accelerated prostate tumor progression in mice.

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Kim, Sungjin; Yang, Xiangkun; Li, Qianjin; Wu, Meng; Costyn, Leah; Beharry, Zanna; Bartlett, Michael G; Cai, Houjian

2017-11-10

Exogenous fatty acids provide substrates for energy production and biogenesis of the cytoplasmic membrane, but they also enhance cellular signaling during cancer cell proliferation. However, it remains controversial whether dietary fatty acids are correlated with tumor progression. In this study, we demonstrate that increased Src kinase activity is associated with high-fat diet-accelerated progression of prostate tumors and that Src kinases mediate this pathological process. Moreover, in the in vivo prostate regeneration assay, host SCID mice carrying Src(Y529F)-transduced regeneration tissues were fed a low-fat diet or a high-fat diet and treated with vehicle or dasatinib. The high-fat diet not only accelerated Src-induced prostate tumorigenesis in mice but also compromised the inhibitory effect of the anticancer drug dasatinib on Src kinase oncogenic potential in vivo We further show that myristoylation of Src kinase is essential to facilitate Src-induced and high-fat diet-accelerated tumor progression. Mechanistically, metabolism of exogenous myristic acid increased the biosynthesis of myristoyl CoA and myristoylated Src and promoted Src kinase-mediated oncogenic signaling in human cells. Of the fatty acids tested, only exogenous myristic acid contributed to increased intracellular myristoyl CoA levels. Our results suggest that targeting Src kinase myristoylation, which is required for Src kinase association at the cellular membrane, blocks dietary fat-accelerated tumorigenesis in vivo Our findings uncover the molecular basis of how the metabolism of myristic acid stimulates high-fat diet-mediated prostate tumor progression. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

Gallic Acid Induces a Reactive Oxygen Species-Provoked c-Jun NH2-Terminal Kinase-Dependent Apoptosis in Lung Fibroblasts

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Chen, Chiu-Yuan; Chen, Kun-Chieh; Yang, Tsung-Ying; Liu, Hsiang-Chun; Hsu, Shih-Lan

2013-01-01

Idiopathic pulmonary fibrosis is a chronic lung disorder characterized by fibroblasts proliferation and extracellular matrix accumulation. Induction of fibroblast apoptosis therefore plays a crucial role in the resolution of this disease. Gallic acid (3,4,5-trihydroxybenzoic acid), a common botanic phenolic compound, has been reported to induce apoptosis in tumor cell lines and renal fibroblasts. The present study was undertaken to examine the role of mitogen-activated protein kinases (MAPKs) in lung fibroblasts apoptosis induced by gallic acid. We found that treatment with gallic acid resulted in activation of c-Jun NH2-terminal kinase (JNK), extracellular signal-regulated kinase (ERK), and protein kinase B (PKB, Akt), but not p38MAPK, in mouse lung fibroblasts. Inhibition of JNK using pharmacologic inhibitor (SP600125) and genetic knockdown (JNK specific siRNA) significantly inhibited p53 accumulation, reduced PUMA and Fas expression, and abolished apoptosis induced by gallic acid. Moreover, treatment with antioxidants (vitamin C, N-acetyl cysteine, and catalase) effectively diminished gallic acid-induced hydrogen peroxide production, JNK and p53 activation, and cell death. These observations imply that gallic acid-mediated hydrogen peroxide formation acts as an initiator of JNK signaling pathways, leading to p53 activation and apoptosis in mouse lung fibroblasts. PMID:23533505

Cisplatin Induces Cytotoxicity through the Mitogen-Activated Protein Kinase Pathways ana Activating Transcription Factor 3

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Carly St. Germain

2010-07-01

Full Text Available The mechanisms underlying the proapoptotic effect of the chemotherapeutic agent, cisplatin, are largely undefined. Understanding the mechanisms regulating cisplatin cytotoxicity may uncover strategies to enhance the efficacy of this important therapeutic agent. This study evaluates the role of activating transcription factor 3 (ATF3 as a mediator of cisplatin-induced cytotoxicity. Cytotoxic doses of cisplatin and carboplatin treatments consistently induced ATF3 expression in five tumor-derived cell lines. Characterization of this induction revealed a p53, BRCA1, and integrated stress response-independent mechanism, all previously implicated in stress-mediated ATF3 induction. Analysis of mitogenactivated protein kinase (MAPK pathway involvement in ATF3 induction by cisplatin revealed a MAPK-dependent mechanism. Cisplatin treatment combined with specific inhibitors to each MAPK pathway (c-Jun N-terminal kinase, extracellularsignal-regulated kinase, and p38 resulted in decreasedATF3 induction at the protein level. MAPK pathway inhibition led to decreased ATF3 messenger RNA expression and reduced cytotoxic effects of cisplatin as measured by the 3-(4,5-dimethylthiazol-2-ylF2,5-diphenyltetrazolium bromide cell viability assay. In A549 lung carcinoma cells, targeting ATF3 with specific small hairpin RNA also attenuated the cytotoxic effects of cisplatin. Similarly, ATF3-/murine embryonic fibroblasts (MEFs were shown to be less sensitive to cisplatin-induced cytotoxicity compared with ATF3+/+ MEFs. This study identifies cisplatin as a MAPK pathway-dependent inducer of ATF3, whose expression influences cisplatin’s cytotoxic effects.

Matriptase is required for the active form of hepatocyte growth factor induced Met, focal adhesion kinase and protein kinase B activation on neural stem/progenitor cell motility.

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Fang, Jung-Da; Lee, Sheau-Ling

2014-07-01

Hepatocyte growth factor (HGF) is a chemoattractant and inducer for neural stem/progenitor (NS/P) cell migration. Although the type II transmembrane serine protease, matriptase (MTP) is an activator of the latent HGF, MTP is indispensable on NS/P cell motility induced by the active form of HGF. This suggests that MTP's action on NS/P cell motility involves mechanisms other than proteolytic activation of HGF. In the present study, we investigate the role of MTP in HGF-stimulated signaling events. Using specific inhibitors of phosphatidylinositol-3-kinase (PI3K), protein kinase B (Akt) or focal adhesion kinase (FAK), we demonstrated that in NS/P cells HGF-activated c-Met induces PI3k-Akt signaling which then leads to FAK activation. This signaling pathway ultimately induces MMP2 expression and NS/P cell motility. Knocking down of MTP in NS/P cells with specific siRNA impaired HGF-stimulation of c-Met, Akt and FAK activation, blocked HGF-induced production of MMP2 and inhibited HGF-stimulated NS/P cell motility. MTP-knockdown NS/P cells cultured in the presence of recombinant protein of MTP protease domain or transfected with the full-length wild-type but not the protease-defected MTP restored HGF-responsive events in NS/P cells. In addition to functioning as HGF activator, our data revealed novel function of MTP on HGF-stimulated c-Met signaling activation. Copyright © 2014. Published by Elsevier B.V.

Induction of intrahepatic cholangiocellular carcinoma by liver-specific disruption of Smad4 and Pten in mice

OpenAIRE

Xu, Xiaoling; Kobayashi, Shogo; Qiao, Wenhui; Li, Cuiling; Xiao, Cuiying; Radaeva, Svetlana; Stiles, Bangyan; Wang, Rui-Hong; Ohara, Nobuya; Yoshino, Tadashi; LeRoith, Derek; Torbenson, Michael S.; Gores, Gregory J.; Wu, Hong; Gao, Bin

2006-01-01

Cholangiocellular carcinoma (CC), the second most common primary liver cancer, is associated with a poor prognosis. It has been shown that CCs harbor alterations of a number of tumor-suppressor genes and oncogenes, yet key regulators for tumorigenesis remain unknown. Here we have generated a mouse model that develops CC with high penetrance using liver-specific targeted disruption of tumor suppressors SMAD4 and PTEN. In the absence of SMAD4 and PTEN, hyperplastic foci emerge exclusively from ...

ROS and CDPK-like kinase-mediated activation of MAP kinase in rice roots exposed to lead.

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Huang, Tsai-Lien; Huang, Hao-Jen

2008-04-01

Lead (Pb2+) is a cytotoxic metal ion in plants, the mechanism of which is not yet established. The aim of this study is to investigate the signalling pathways that are activated by elevated concentrations of Pb2+ in rice roots. Root growth was stunted and cell death was accelerated when exposed to different dosages of Pb2+ during extended time periods. Using ROS-sensitive dye and Ca2+ indicator, we demonstrated that Pb2+ induced ROS production and Ca2+ accumulation, respectively. In addition, Pb2+ elicited a remarkable increase in myelin basic protein (MBP) kinase activities. By immunoblot and immunoprecipitation analysis, 40- and 42-kDa MBP kinases that were activated by Pb2+ were identified to be mitogen-activated protein (MAP) kinases. Pre-treatment of rice roots with an antioxidant and a NADPH oxidase inhibitor, glutathione (GSH) and diphenylene iodonium (DPI), effectively reduced Pb2+-induced cell death and MAP kinase activation. Moreover, calcium-dependent protein kinase (CDPK) antagonist, W7, attenuated Pb2+-induced cell death and MAP kinase activation. These results suggested that the ROS and CDPK may function in the Pb2+-triggered cell death and MAP kinase signalling pathway in rice roots.

Angiogenin-induced protein kinase B/Akt activation is necessary for angiogenesis but is independent of nuclear translocation of angiogenin in HUVE cells

International Nuclear Information System (INIS)

Kim, Hye-Mi; Kang, Dong-Ku; Kim, Hak Yong; Kang, Sang Sun; Chang, Soo-Ik

2007-01-01

Angiogenin, a potent angiogenic factor, binds to endothelial cells and is endocytosed and rapidly translocated to and concentrated in the nucleolus where it binds to DNA. In this study, we report that angiogenin induces transient phosphorylation of protein kinase B/Akt in cultured human umbilical vein endothelial (HUVE) cells. LY294002 inhibits the angiogenin-induced protein kinase B/Akt activation and also angiogenin-induced cell migration in vitro as well as angiogenesis in chick embryo chorioallantoic membrane in vivo without affecting nuclear translocation of angiogenin in HUVE cells. These results suggest that cross-talk between angiogenin and protein kinase B/Akt signaling pathways is essential for angiogenin-induced angiogenesis in vitro and in vivo, and that angiogenin-induced PKB/Akt activation is independent of nuclear translocation of angiogenin in HUVE cells

MiR-21/PTEN Axis Promotes Skin Wound Healing by Dendritic Cells Enhancement.

Science.gov (United States)

Han, Zhaofeng; Chen, Ya; Zhang, Yile; Wei, Aizhou; Zhou, Jian; Li, Qian; Guo, Lili

2017-10-01

A number of miRNAs associated with wound repair have been identified and characterized, but the mechanism has not been fully clarified. MiR-21 is one of wound-related lncRNAs, and the study aimed to explore the functional involvement of miR-21 and its concrete mechanism in wound healing. In this study, the rat model of skin wounds was established. The expression of miR-21, PTEN and related molecules of wound tissues or cells was determined by quantitative real-time PCR and Western blot, respectively. The regulatory role of miR-21 on PTEN was examined by luciferase reporter gene assay. Flow cytometry assay was applied to measure cell number changes. MiR-21 was upregulated at 6, 24, 48, 72 h after model establishment, and the increase reached a maximum at 24 h in wound tissues. MMP-9 expression presented the same tread as miR-21 and was significantly enhanced within 6 h of wound formation, and then remained to be increased to the maximum at 24 h. The increase of miR-21 was accompanied by the increase of cell total number and DCs ratio in wound fluids. MiR-21 overexpression significantly improved the healing of skin wounds and increased the ratio of DCs in rats. The results of using FL confirmed that miR-21 overexpression obviously promoted DCs differentiation. Additionally, miR-21 could activate AKT/PI3K signaling pathway via inhibition of PTEN. MiR-21 contributes to wound healing via inhibition of PTEN that activated AKT/PI3K signaling pathway to increase DCs. J. Cell. Biochem. 118: 3511-3519, 2017. © 2017 Wiley Periodicals, Inc. © 2017 Wiley Periodicals, Inc.

The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

KAUST Repository

Tewari, Rita; Straschil, Ursula; Bateman, Alex; Bö hme, Ulrike; Cherevach, Inna; Gong, Peng; Pain, Arnab; Billker, Oliver

2010-01-01

Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

The systematic functional analysis of plasmodium protein kinases identifies essential regulators of mosquito transmission

KAUST Repository

Tewari, Rita

2010-10-21

Although eukaryotic protein kinases (ePKs) contribute to many cellular processes, only three Plasmodium falciparum ePKs have thus far been identified as essential for parasite asexual blood stage development. To identify pathways essential for parasite transmission between their mammalian host and mosquito vector, we undertook a systematic functional analysis of ePKs in the genetically tractable rodent parasite Plasmodium berghei. Modeling domain signatures of conventional ePKs identified 66 putative Plasmodium ePKs. Kinomes are highly conserved between Plasmodium species. Using reverse genetics, we show that 23 ePKs are redundant for asexual erythrocytic parasite development in mice. Phenotyping mutants at four life cycle stages in Anopheles stephensi mosquitoes revealed functional clusters of kinases required for sexual development and sporogony. Roles for a putative SR protein kinase (SRPK) in microgamete formation, a conserved regulator of clathrin uncoating (GAK) in ookinete formation, and a likely regulator of energy metabolism (SNF1/KIN) in sporozoite development were identified. 2010 Elsevier Inc.

Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

International Nuclear Information System (INIS)

Souza, C.F.; Carneiro, A.B.; Silveira, A.B.; Laranja, G.A.T.; Silva-Neto, M.A.C.; Costa, S.C. Goncalves da; Paes, M.C.

2009-01-01

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

Heme-induced Trypanosoma cruzi proliferation is mediated by CaM kinase II

Energy Technology Data Exchange (ETDEWEB)

Souza, C.F. [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Carneiro, A.B.; Silveira, A.B. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); Laranja, G.A.T. [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); Silva-Neto, M.A.C. [Laboratorio de Sinalizacao Celular, Instituto de Bioquimica Medica, UFRJ (Brazil); INCT, Entomologia Molecular (Brazil); Costa, S.C. Goncalves da [Laboratorio de Imunomodulacao e Protozoologia, Instituto Oswaldo Cruz, Fiocruz (Brazil); Paes, M.C., E-mail: mcpaes@uerj.br [Laboratorio de Interacao Tripanosomatideos e Vetores, Departamento de Bioquimica, IBRAG, UERJ, 20551-030 Rio de Janeiro (Brazil); INCT, Entomologia Molecular (Brazil)

2009-12-18

Trypanosoma cruzi, the etiologic agent of Chagas disease, is transmitted through triatomine vectors during their blood-meal on vertebrate hosts. These hematophagous insects usually ingest approximately 10 mM of heme bound to hemoglobin in a single meal. Blood forms of the parasite are transformed into epimastigotes in the crop which initiates a few hours after parasite ingestion. In a previous work, we investigated the role of heme in parasite cell proliferation and showed that the addition of heme significantly increased parasite proliferation in a dose-dependent manner . To investigate whether the heme effect is mediated by protein kinase signalling pathways, parasite proliferation was evaluated in the presence of several protein kinase (PK) inhibitors. We found that only KN-93, a classical inhibitor of calcium-calmodulin-dependent kinases (CaMKs), blocked heme-induced cell proliferation. KN-92, an inactive analogue of KN-93, was not able to block this effect. A T. cruzi CaMKII homologue is most likely the main enzyme involved in this process since parasite proliferation was also blocked when Myr-AIP, an inhibitory peptide for mammalian CaMKII, was included in the cell proliferation assay. Moreover, CaMK activity increased in parasite cells with the addition of heme as shown by immunological and biochemical assays. In conclusion, the present results are the first strong indications that CaMKII is involved in the heme-induced cell signalling pathway that mediates parasite proliferation.

A G-protein-coupled chemokine receptor: A putative insertion site for a multi-pathogen recombinant capripoxvirus vaccine strategy.

Science.gov (United States)

Cêtre-Sossah, Catherine; Dickmu, Simon; Kwiatek, Olivier; Albina, Emmanuel

2017-09-01

Capripoxviruses (CaPVs) have been shown to be ideal viral vectors for the development of recombinant multivalent vaccines to enable delivery of immunogenic genes from ruminant pathogens. So far, the viral thymidine kinase (TK) gene is the only gene used to generate recombinants. A putative non-essential gene encoding a G-protein-coupled chemokine receptor subfamily homologue (GPCR) was targeted as an additional insertion site. Peste des petits ruminants (PPR) was chosen as a disease model. A new recombinant CaPV expressing the viral attachment hemagglutinin (H) of the PPR virus (PPRV) in the GPCR insertion site (rKS1-HPPR-GPCR) was generated in the backbone North African isolate KS1 strain of lumpy skin disease virus (LSDV). Comparison with the recombinant CaPV expressing the H of PPRV in the TK gene (rKS1-HPPR-TK) shown to induce protection against both PPR and LSD in both sheep and goats was assessed. The suitability of the GPCR gene to be a putative additional insertion site in the CaPV genome is evaluated and discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Increased plasma levels of FABP4 and PTEN is associated with more severe insulin resistance in women with gestational diabetes mellitus.

Science.gov (United States)

Li, Yuan-yuan; Xiao, Rui; Li, Cai-ping; Huangfu, Jian; Mao, Jiang-feng

2015-02-08

The aim of this study was to investigate the relationship between plasma fatty acid binding protein 4 (FABP4), phosphatase and tensin homolog (PTEN), and insulin resistance in patients with gestational diabetes mellitus (GDM). Plasma FABP4 and PTEN were determined by ELISA in GDM patients (GDM group, n=30) and in euglycemic pregnant women (control group, n=30). The clinical features, body mass index (BMI), homeostasis model assessment of insulin resistance (HOMA-IR), and lipid profiles were compared between the 2 groups. The influence of risk factors on insulin resistance, including BMI, lipid profiles, FABP4, and PTEN, were further investigated by multiple-factor stepwise regression analysis. Higher levels of BMI, ΔBMI, triglyceride (TG), fasting plasma glucose (FPG), 2-hour plasma glucose (2hPG), fasting insulin, HOMA-IR, FABP4, PTEN, and lower level of high-density lipoprotein cholesterol (HDL-C) were found in the GDM patients than in the controls (all Pinsulin resistance. GDM patients have more severe insulin resistance compared to euglycemic pregnant women. Higher levels of plasma FABP4 and PTEN are associated with increased insulin resistance and may participate in the pathogenesis of insulin resistance during gestation.

Disruption of IGF-1R signaling increases TRAIL-induced apoptosis: A new potential therapy for the treatment of melanoma

Energy Technology Data Exchange (ETDEWEB)

Karasic, Thomas B.; Hei, Tom K. [Center for Radiological Research, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States); Ivanov, Vladimir N., E-mail: vni3@columbia.edu [Center for Radiological Research, Department of Radiation Oncology, College of Physicians and Surgeons, Columbia University, New York, NY 10032 (United States)

2010-07-15

Resistance of cancer cells to apoptosis is dependent on a balance of multiple genetic and epigenetic mechanisms, which up-regulate efficacy of the surviving growth factor-receptor signaling pathways and suppress death-receptor signaling pathways. The Insulin-like Growth Factor-1 Receptor (IGF-1R) signaling pathway is highly active in metastatic melanoma cells by mediating downstream activation of PI3K-AKT and MAPK pathways and controlling general cell survival and proliferation. In the present study, we used human melanoma lines with established genotypes that represented different phases of cancer development: radial-growth-phase WM35, vertical-growth-phase WM793, metastatic LU1205 and WM9 [1]. All these lines have normal NRAS. WM35, WM793, LU1205 and WM9 cells have mutated BRAF (V600E). WM35 and WM9 cells express normal PTEN, while in WM793 cells PTEN expression is down-regulated; finally, in LU1205 cells PTEN is inactivated by mutation. Cyclolignan picropodophyllin (PPP), a specific inhibitor of IGF-1R kinase activity, strongly down-regulated the basal levels of AKT activity in WM9 and in WM793 cells, modestly does so in LU1205, but has no effect on AKT activity in the early stage WM35 cells that are deficient in IGF-1R. In addition, PPP partially down-regulated the basal levels of active ERK1/2 in all lines used, highlighting the role of an alternative, non-BRAF pathway in MAPK activation. The final result of PPP treatment was an induction of apoptosis in WM793, WM9 and LU1205 melanoma cells. On the other hand, dose-dependent inhibition of IGF-1R kinase activity by PPP at a relatively narrow dose range (near 500 nM) has different effects on melanoma cells versus normal cells, inducing apoptosis in cancer cells and G2/M arrest of fibroblasts. To further enhance the pro-apoptotic effects of PPP on melanoma cells, we used a combined treatment of TNF-Related Apoptosis-Inducing Ligand (TRAIL) and PPP. This combination substantially increased death by apoptosis for

Concurrent deletion of 16q23 and PTEN is an independent prognostic feature in prostate cancer.

Science.gov (United States)

Kluth, Martina; Runte, Frederic; Barow, Philipp; Omari, Jazan; Abdelaziz, Zaid M; Paustian, Lisa; Steurer, Stefan; Christina Tsourlakis, Maria; Fisch, Margit; Graefen, Markus; Tennstedt, Pierre; Huland, Hartwig; Michl, Uwe; Minner, Sarah; Sauter, Guido; Simon, Ronald; Adam, Meike; Schlomm, Thorsten

2015-11-15

The deletion of 16q23-q24 belongs to the most frequent chromosomal changes in prostate cancer, but the clinical consequences of this alteration have not been studied in detail. We performed fluorescence in situ hybridization analysis using a 16q23 probe in more than 7,400 prostate cancers with clinical follow-up data assembled in a tissue microarray format. Chromosome 16q deletion was found in 21% of cancers, and was linked to advanced tumor stage, high Gleason grade, accelerated cell proliferation, the presence of lymph node metastases (p Deletion was more frequent in ERG fusion-positive (27%) as compared to ERG fusion-negative cancers (16%, p deletions including phosphatase and tensin homolog (PTEN) (p deletion of 16q was linked to early biochemical recurrence independently from the ERG status (p deletion of 16q alone. Multivariate modeling revealed that the prognostic value of 16q/PTEN deletion patterns was independent from the established prognostic factors. In summary, the results of our study demonstrate that the deletion of 16q and PTEN cooperatively drives prostate cancer progression, and suggests that deletion analysis of 16q and PTEN could be of important clinical value particularly for preoperative risk assessment of the clinically most challenging group of low- and intermediated grade prostate cancers. © 2015 UICC.

The Src family kinase inhibitor dasatinib delays pain-related behaviour and conserves bone in a rat model of cancer-induced bone pain

DEFF Research Database (Denmark)

Appel, Camilla Kristine; Gallego-Pedersen, Simone; Andersen, Line

2017-01-01

-induced bone pain, including cancer growth, osteoclastic bone degradation and nociceptive signalling. Here we investigate the role of dasatinib, an oral Src kinase family and Bcr-Abl tyrosine kinase inhibitor, in an animal model of cancer-induced bone pain. Daily administration of dasatinib (15 mg/kg, p...

Cytotoxic activities of amentoflavone against human breast and cervical cancers are mediated by increasing of PTEN expression levels due to peroxisomes proliferate-activated receptor {gamma} activation

Energy Technology Data Exchange (ETDEWEB)

Lee, Eunjung; Shin, Soyoung; Lee, Jeeyoung; Lee, So Jung; Kim, Jinkyoung; Yoon, Doyoung; Kim, Yangmee [Konkuk Univ., Seoul (Korea, Republic of); Woo, Eunrhan [Chosun Univ., Gwangju (Korea, Republic of)

2012-07-15

Human peroxisomes proliferate-activated receptor gamma (hPPAR{gamma}) has been implicated in numerous pathologies, including obesity, diabetes, and cancer. Previously, we verified that amentoflavone is an activator of hPPAR{gamma} and probed the molecular basis of its action. In this study, we investigated the mechanism of action of amentoflavone in cancer cells and demonstrated that amentoflavone showed strong cytotoxicity against MCF-7 and HeLa cancer cell lines. We showed that hPPAR{gamma} expression in MCF-7 and HeLa cells is specifically stimulated by amentoflavone, and suggested that amentoflavone-induced cytotoxic activities are mediated by activation of hPPAR{gamma} in these two cancer cell lines. Moreover, amentoflavone increased PTEN levels in these two cancer cell lines, indicating that the cytotoxic activities of amentoflavone are mediated by increasing of PTEN expression levels due to hPPAR{gamma} activation.

Melittin restores PTEN expression by down-regulating HDAC2 in human hepatocelluar carcinoma HepG2 cells.

Directory of Open Access Journals (Sweden)

Hui Zhang

Full Text Available Melittin is a water-soluble toxic peptide derived from the venom of the bee. Although many studies show the anti-tumor activity of melittin in human cancer including glioma cells, the underlying mechanisms remain elusive. Here the effect of melittin on human hepatocelluar carcinoma HepG2 cell proliferation in vitro and further mechanisms was investigated. We found melittin could inhibit cell proliferation in vitro using Flow cytometry and MTT method. Besides, we discovered that melittin significantly downregulated the expressions of CyclinD1 and CDK4. Results of western Blot and Real-time PCR analysis indicated that melittin was capable to upregulate the expression of PTEN and attenuate histone deacetylase 2 (HDAC2 expression. Further studies demonstrated that knockdown of HDAC2 completely mimicked the effects of melittin on PTEN gene expression. Conversely, it was that the potential utility of melittin on PTEN expression was reversed in cells treated with a recombinant pEGFP-C2-HDAC2 plasmid. In addition, treatment with melittin caused a downregulation of Akt phosphorylation, while overexpression of HDAC2 promoted Akt phosphorylation. These findings suggested that the inhibitory of cell growth by melittin might be led by HDAC2-mediated PTEN upregulation, Akt inactivation, and inhibition of the PI3K/Akt signaling pathways.

Calcium-mediated signaling and calmodulin-dependent kinase regulate hepatocyte-inducible nitric oxide synthase expression.

Science.gov (United States)

Zhang, Baochun; Crankshaw, Will; Nesemeier, Ryan; Patel, Jay; Nweze, Ikenna; Lakshmanan, Jaganathan; Harbrecht, Brian G

2015-02-01

Induced nitric oxide synthase (iNOS) is induced in hepatocytes by shock and inflammatory stimuli. Excessive NO from iNOS mediates shock-induced hepatic injury and death, so understanding the regulation of iNOS will help elucidate the pathophysiology of septic shock. In vitro, cytokines induce iNOS expression through activation of signaling pathways including mitogen-activated protein kinases and nuclear factor κB. Cytokines also induce calcium (Ca(2+)) mobilization and activate calcium-mediated intracellular signaling pathways, typically through activation of calmodulin-dependent kinases (CaMK). Calcium regulates NO production in macrophages but the role of calcium and calcium-mediated signaling in hepatocyte iNOS expression has not been defined. Primary rat hepatocytes were isolated, cultured, and induced to produce NO with proinflammatory cytokines. Calcium mobilization and Ca(2+)-mediated signaling were altered with ionophore, Ca(2+) channel blockers, and inhibitors of CaMK. The Ca(2+) ionophore A23187 suppressed cytokine-stimulated NO production, whereas Ethylene glycol tetraacetic acid and nifedipine increased NO production, iNOS messenger RNA, and iNOS protein expression. Inhibition of CaMK with KN93 and CBD increased NO production but the calcineurin inhibitor FK 506 decreased iNOS expression. These data demonstrate that calcium-mediated signaling regulates hepatocyte iNOS expression and does so through a mechanism independent of calcineurin. Changes in intracellular calcium levels may regulate iNOS expression during hepatic inflammation induced by proinflammatory cytokines. Copyright © 2015 Elsevier Inc. All rights reserved.

Triptolide, a diterpenoid triepoxide, induces antitumor proliferation via activation of c-Jun NH2-terminal kinase 1 by decreasing phosphatidylinositol 3-kinase activity in human tumor cells

International Nuclear Information System (INIS)

Miyata, Yoshiki; Sato, Takashi; Ito, Akira

2005-01-01

Triptolide, a diterpenoid triepoxide extracted from the Chinese herb Tripterygium wilfordii Hook f., exerts antitumorigenic actions against several tumor cells, but the intracellular target signal molecule(s) for this antitumorigenesis activity of triptolide remains to be identified. In the present study, we demonstrated that triptolide, in a dose-dependent manner, inhibited the proliferation of human fibrosarcoma HT-1080, human squamous carcinoma SAS, and human uterine cervical carcinoma SKG-II cells. In addition, triptolide was found to decrease phosphatidylinositol 3-kinase (PI3K) activity. A PI3K inhibitor, LY-294002, mimicked the triptolide-induced antiproliferative activity in HT-1080, SAS, and SKG-II cells. There was no change in the activity of Akt or protein kinase C (PKC), both of which are downstream effectors in the PI3K pathway. Furthermore, the phosphorylation of Ras, Raf, and mitogen-activated protein/extracellular signal-regulated kinase 1/2 was not modified in HT-1080 cells treated with triptolide. However, the phosphorylation of c-Jun NH 2 -terminal kinase 1 (JNK1) was found to increase in both triptolide- and LY-294002-treated cells. Furthermore, the triptolide-induced inhibition of HT-1080 cell proliferation was not observed by JNK1 siRNA-treatment. These results provide novel evidence that PI3K is a crucial target molecule in the antitumorigenic action of triptolide. They further suggest a possible triptolide-induced inhibitory signal for tumor cell proliferation that is initiated by the decrease in PI3K activity, which in turn leads to the augmentation of JNK1 phosphorylation via the Akt and/or PKC-independent pathway(s). Moreover, it is likely that the activation of JNK1 is required for the triptolide-induced inhibition of tumor proliferation

The Amoebal MAP Kinase Response to Legionella pneumophila Is Regulated by DupA

OpenAIRE

Li, Zhiru; Dugan, Aisling S.; Bloomfield, Gareth; Skelton, Jason; Ivens, Alasdair; Losick, Vicki; Isberg, Ralph R.

2009-01-01

The amoeba Dictyostelium discoideum can support replication of Legionella pneumophila. Here we identify the dupA gene, encoding a putative tyrosine kinase/dual-specificity phosphatase, in a screen for D. discoideum mutants altered in allowing L. pneumophila intracellular replication. Inactivation of dupA resulted in depressed L. pneumophila growth and sustained hyperphosphorylation of the amoebal MAP kinase ERK1, consistent with loss of a phosphatase activity. Bacterial challenge of wild-type...

The safe use of a PTEN inhibitor for the activation of dormant mouse primordial follicles and generation of fertilizable eggs.

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Deepak Adhikari

Full Text Available Primordial ovarian follicles, which are often present in the ovaries of premature ovarian failure (POF patients or are cryopreserved from the ovaries of young cancer patients who are undergoing gonadotoxic anticancer therapies, cannot be used to generate mature oocytes for in vitro fertilization (IVF. There has been very little success in triggering growth of primordial follicles to obtain fertilizable oocytes due to the poor understanding of the biology of primordial follicle activation.We have recently reported that PTEN (phosphatase and tensin homolog deleted on chromosome ten prevents primordial follicle activation in mice, and deletion of Pten from the oocytes of primordial follicles leads to follicular activation. Consequently, the PTEN inhibitor has been successfully used in vitro to activate primordial follicles in both mouse and human ovaries. These results suggest that PTEN inhibitors could be used in ovarian culture medium to trigger the activation of primordial follicle. To study the safety and efficacy of the use of such inhibitors, we activated primordial follicles from neonatal mouse ovaries by transient treatment with a PTEN inhibitor bpV(HOpic. These ovaries were then transplanted under the kidney capsules of recipient mice to generate mature oocytes. The mature oocytes were fertilized in vitro and progeny mice were obtained after embryo transfer.Long-term monitoring up to the second generation of progeny mice showed that the mice were reproductively active and were free from any overt signs or symptoms of chronic illnesses. Our results indicate that the use of PTEN inhibitors could be a safe and effective way of generating mature human oocytes for use in novel IVF techniques.

Resveratrol alleviates diabetes-induced testicular dysfunction by inhibiting oxidative stress and c-Jun N-terminal kinase signaling in rats

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Faid, Iman; Al-Hussaini, Heba; Kilarkaje, Narayana, E-mail: knarayana@hsc.edu.kw

2015-12-15

Diabetes adversely affects reproductive functions in humans and animals. The present study investigated the effects of Resveratrol on diabetes-induced alterations in oxidative stress, c-Jun N-terminal kinase (JNK) signaling and apoptosis in the testis. Adult male Wistar rats (13–15 weeks; n = 6/group) were segregated into 1) normal control, 2) Resveratrol-treated (5 mg/kg; ip; given during last 3 weeks), 3) Streptozotocin-induced diabetic and, 4) Resveratrol-treated diabetic groups, and euthanized on day 42 after the confirmation of diabetes. Resveratrol did not normalize blood glucose levels in diabetic rats. Resveratrol supplementation recovered diabetes-induced decreases in reproductive organ weights, sperm count and motility, intra-testicular levels of superoxide dismutase, catalase, and glutathione peroxidase and an increase in 4-hydroxynonenal activities (P < 0.05). Resveratrol also recovered diabetes-induced increases in JNK signaling pathway proteins, namely, ASK1 (apoptosis signal-regulating kinase 1), JNKs (46 and 54 kDa isoforms) and p-JNK to normal control levels (P < 0.05). Interestingly, the expression of a down-stream target of ASK1, MKK4 (mitogen-activated protein kinase kinase 4) and its phosphorylated form (p-MKK4) did not change in experimental groups. Resveratrol inhibited diabetes-induced increases in AP-1 (activator protein-1) components, c-Jun and ATF2 (activating transcription factor 2), but not their phosphorylated forms, to normal control levels (P < 0.05). Further, Resveratrol inhibited diabetes-induced increase in cleaved-caspase-3 to normal control levels. In conclusion, Resveratrol alleviates diabetes-induced apoptosis in testis by modulating oxidative stress, JNK signaling pathway and caspase-3 activities, but not by inhibiting hyperglycemia, in rats. These results suggest that Resveratrol supplementation may be a useful strategy to treat diabetes-induced testicular dysfunction. - Highlights: • Resveratrol up-regulates glutathione

Resveratrol alleviates diabetes-induced testicular dysfunction by inhibiting oxidative stress and c-Jun N-terminal kinase signaling in rats

International Nuclear Information System (INIS)

Faid, Iman; Al-Hussaini, Heba; Kilarkaje, Narayana

2015-01-01

Diabetes adversely affects reproductive functions in humans and animals. The present study investigated the effects of Resveratrol on diabetes-induced alterations in oxidative stress, c-Jun N-terminal kinase (JNK) signaling and apoptosis in the testis. Adult male Wistar rats (13–15 weeks; n = 6/group) were segregated into 1) normal control, 2) Resveratrol-treated (5 mg/kg; ip; given during last 3 weeks), 3) Streptozotocin-induced diabetic and, 4) Resveratrol-treated diabetic groups, and euthanized on day 42 after the confirmation of diabetes. Resveratrol did not normalize blood glucose levels in diabetic rats. Resveratrol supplementation recovered diabetes-induced decreases in reproductive organ weights, sperm count and motility, intra-testicular levels of superoxide dismutase, catalase, and glutathione peroxidase and an increase in 4-hydroxynonenal activities (P < 0.05). Resveratrol also recovered diabetes-induced increases in JNK signaling pathway proteins, namely, ASK1 (apoptosis signal-regulating kinase 1), JNKs (46 and 54 kDa isoforms) and p-JNK to normal control levels (P < 0.05). Interestingly, the expression of a down-stream target of ASK1, MKK4 (mitogen-activated protein kinase kinase 4) and its phosphorylated form (p-MKK4) did not change in experimental groups. Resveratrol inhibited diabetes-induced increases in AP-1 (activator protein-1) components, c-Jun and ATF2 (activating transcription factor 2), but not their phosphorylated forms, to normal control levels (P < 0.05). Further, Resveratrol inhibited diabetes-induced increase in cleaved-caspase-3 to normal control levels. In conclusion, Resveratrol alleviates diabetes-induced apoptosis in testis by modulating oxidative stress, JNK signaling pathway and caspase-3 activities, but not by inhibiting hyperglycemia, in rats. These results suggest that Resveratrol supplementation may be a useful strategy to treat diabetes-induced testicular dysfunction. - Highlights: • Resveratrol up-regulates glutathione

Cell-type-specific activation of mitogen-activated protein kinases in PAN-induced progressive renal disease in rats

International Nuclear Information System (INIS)

Park, Sang-Joon; Jeong, Kyu-Shik

2004-01-01

We examined the time-course activation and the cell-type specific role of MAP kinases in puromycin aminonucleoside (PAN)-induced renal disease. The maximal activation of c-Jun-NH 2 -terminal kinase (JNK), extracellular signal regulated kinase (ERK), and p38 MAP kinase was detected on Days 52, 38, and 38 after PAN-treatment, respectively. p-JNK was localized in mesangial and proximal tubular cells at the early renal injury. It was expressed, therefore, in the inflammatory cells of tubulointerstitial lesions. While, p-ERK was markedly increased in the glomerular regions and macrophages p-p38 was observed in glomerular endothelial cells, tubular cells, and some inflammatory cells. The results show that the activation of MAP kinases in the early renal injury by PAN-treatment involves cellular changes such as cell proliferation or apoptosis in renal native cells. The activation of MAP kinases in infiltrated inflammatory cells and fibrotic cells plays an important role in destructive events such as glomerulosclerosis and tubulointerstitial fibrosis

Simvastatin and metformin inhibit cell growth in hepatitis C virus infected cells via mTOR increasing PTEN and autophagy.

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José A Del Campo

Full Text Available Hepatitis C virus (HCV infection has been related to increased risk of development of hepatocellular carcinoma (HCC while metformin (M and statins treatment seemed to protect against HCC development. In this work, we aim to identify the mechanisms by which metformin and simvastatin (S could protect from liver cancer. Huh7.5 cells were infected with HCV particles and treated with M+S. Human primary hepatocytes were treated with M+S. Treatment with both drugs inhibited Huh7.5 cell growth and HCV infection. In non-infected cells S increased translational controlled tumor protein (TCTP and phosphatase and tensin homolog (PTEN proteins while M inhibited mammalian target of rapamycin (mTOR and TCTP. Simvastatin and metformin co-administered down-regulated mTOR and TCTP, while PTEN was increased. In cells infected by HCV, mTOR, TCTP, p62 and light chain 3B II (LC3BII were increased and PTEN was decreased. S+M treatment increased PTEN, p62 and LC3BII in Huh7.5 cells. In human primary hepatocytes, metformin treatment inhibited mTOR and PTEN, but up-regulated p62, LC3BII and Caspase 3. In conclusion, simvastatin and metformin inhibited cell growth and HCV infection in vitro. In human hepatocytes, metformin increased cell-death markers. These findings suggest that M+S treatment could be useful in therapeutic prevention of HCV-related hepatocellular carcinoma.

Characterization of Heterogeneous Prostate Tumors in Targeted Pten Knockout Mice.

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Hanneke Korsten

Full Text Available Previously, we generated a preclinical mouse prostate tumor model based on PSA-Cre driven inactivation of Pten. In this model homogeneous hyperplastic prostates (4-5m developed at older age (>10m into tumors. Here, we describe the molecular and histological characterization of the tumors in order to better understand the processes that are associated with prostate tumorigenesis in this targeted mouse Pten knockout model. The morphologies of the tumors that developed were very heterogeneous. Different histopathological growth patterns could be identified, including intraductal carcinoma (IDC, adenocarcinoma and undifferentiated carcinoma, all strongly positive for the epithelial cell marker Cytokeratin (CK, and carcinosarcomas, which were negative for CK. IDC pattern was already detected in prostates of 7-8 month old mice, indicating that it could be a precursor stage. At more than 10 months IDC and carcinosarcoma were most frequently observed. Gene expression profiling discriminated essentially two molecular subtypes, denoted tumor class 1 (TC1 and tumor class 2 (TC2. TC1 tumors were characterized by high expression of epithelial markers like Cytokeratin 8 and E-Cadherin whereas TC2 tumors showed high expression of mesenchyme/stroma markers such as Snail and Fibronectin. These molecular subtypes corresponded with histological growth patterns: where TC1 tumors mainly represented adenocarcinoma/intraductal carcinoma, in TC2 tumors carcinosarcoma was the dominant growth pattern. Further molecular characterization of the prostate tumors revealed an increased expression of genes associated with the inflammatory response. Moreover, functional markers for senescence, proliferation, angiogenesis and apoptosis were higher expressed in tumors compared to hyperplasia. The highest expression of proliferation and angiogenesis markers was detected in TC2 tumors. Our data clearly showed that in the genetically well-defined PSA-Cre;Pten-loxP/loxP prostate tumor

PTEN Loss in E-Cadherin-Deficient Mouse Mammary Epithelial Cells Rescues Apoptosis and Results in Development of Classical Invasive Lobular Carcinoma

NARCIS (Netherlands)

Boelens, M.C.; Nethe, M.; Klarenbeek, S.; de Ruiter, J.R.; Schut, E.; Bonzanni, N.; Zeeman, A.L.; Wientjens, E.; van der Burg, E.; Wessels, L.; van Amerongen, R.; Jonkers, J.

2016-01-01

Invasive lobular carcinoma (ILC) is an aggressive breast cancer subtype with poor response to chemotherapy. Besides loss of E-cadherin, a hallmark of ILC, genetic inactivation of PTEN is frequently observed in patients. Through concomitant Cre-mediated inactivation of E-cadherin and PTEN in mammary

Phosphoinositide 3–kinase γ participates in T cell receptor–induced T cell activation

Science.gov (United States)

Alcázar, Isabela; Marqués, Miriam; Kumar, Amit; Hirsch, Emilio; Wymann, Matthias; Carrera, Ana C.; Barber, Domingo F.

2007-01-01

Class I phosphoinositide 3–kinases (PI3Ks) constitute a family of enzymes that generates 3-phosphorylated polyphosphoinositides at the cell membrane after stimulation of protein tyrosine (Tyr) kinase–associated receptors or G protein–coupled receptors (GPCRs). The class I PI3Ks are divided into two types: class IA p85/p110 heterodimers, which are activated by Tyr kinases, and the class IB p110γ isoform, which is activated by GPCR. Although the T cell receptor (TCR) is a protein Tyr kinase–associated receptor, p110γ deletion affects TCR-induced T cell stimulation. We examined whether the TCR activates p110γ, as well as the consequences of interfering with p110γ expression or function for T cell activation. We found that after TCR ligation, p110γ interacts with Gαq/11, lymphocyte-specific Tyr kinase, and ζ-associated protein. TCR stimulation activates p110γ, which affects 3-phosphorylated polyphosphoinositide levels at the immunological synapse. We show that TCR-stimulated p110γ controls RAS-related C3 botulinum substrate 1 activity, F-actin polarization, and the interaction between T cells and antigen-presenting cells, illustrating a crucial role for p110γ in TCR-induced T cell activation. PMID:17998387

Nuclear localization of Src-family tyrosine kinases is required for growth factor-induced euchromatinization

International Nuclear Information System (INIS)

Takahashi, Akinori; Obata, Yuuki; Fukumoto, Yasunori; Nakayama, Yuji; Kasahara, Kousuke; Kuga, Takahisa; Higashiyama, Yukihiro; Saito, Takashi; Yokoyama, Kazunari K.; Yamaguchi, Naoto

2009-01-01

Src-family kinases (SFKs), which participate in various signaling events, are found at not only the plasma membrane but also several subcellular compartments, including the nucleus. Nuclear structural changes are frequently observed during transcription, cell differentiation, senescence, tumorigenesis, and cell cycle. However, little is known about signal transduction in the alteration of chromatin texture. Here, we develop a pixel imaging method for quantitatively evaluating chromatin structural changes. Growth factor stimulation increases euchromatic hypocondensation and concomitant heterochromatic hypercondensation in G 1 phase, and the levels reach a plateau by 30 min, sustain for at least 5 h and return to the basal levels after 24 h. Serum-activated SFKs in the nucleus were more frequently detected in the euchromatin areas than the heterochromatin areas. Nuclear expression of kinase-active SFKs, but not unrelated Syk kinase, drastically increases both euchromatinization and heterochromatinization in a manner dependent on the levels of nuclear tyrosine phosphorylation. However, growth factor stimulation does not induce chromatin structural changes in SYF cells lacking SFKs, and reintroduction of one SFK member into SYF cells can, albeit insufficiently, induce chromatin structural changes. These results suggest that nuclear tyrosine phosphorylation by SFKs plays an important role in chromatin structural changes upon growth factor stimulation.

Homozygous deletion and expression of PTEN and DMBT1 in human primary neuroblastoma and cell lines.

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Muñoz, Jorge; Lázcoz, Paula; Inda, María Mar; Nistal, Manuel; Pestaña, Angel; Encío, Ignacio J; Castresana, Javier S

2004-05-01

Neuroblastoma is the most common pediatric solid tumor. Although many allelic imbalances have been described, a bona fide tumor suppressor gene for this disease has not been found yet. In our study, we analyzed 2 genes, PTEN and DMBT1, mapping 10q23.31 and 10q25.3-26.1, respectively, which have been found frequently altered in other kinds of neoplasms. We screened both genes for homozygous deletions in 45 primary neuroblastic tumors and 12 neuroblastoma cell lines. Expression of these genes in cell lines was assessed by RT-PCR analysis. We could detect 2 of 41 (5%) primary tumors harboring PTEN homozygous deletions. Three of 41 (7%) primary tumors and 2 of 12 cell lines presented homozygous losses at the g14 STS on the DMBT1 locus. All cell lines analyzed expressed PTEN, but lack of DMBT1 mRNA expression was detected in 2 of them. We tried to see whether epigenetic mechanisms, such as aberrant promoter hypermethylation, had any role in DMBT1 silencing. The 2 cell lines lacking DMBT1 expression were treated with 5-aza-2'-deoxycytidine; DMBT1 expression was restored in only one of them (MC-IXC). From our work, we can conclude that PTEN and DMBT1 seem to contribute to the development of a small fraction of neuroblastomas, and that promoter hypermethylation might have a role in DMBT1 gene silencing. Copyright 2004 Wiley-Liss, Inc.

Apoptosis signal-regulating kinase 1 mediates denbinobin-induced apoptosis in human lung adenocarcinoma cells

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Pan Shiow-Lin

2009-05-01

Full Text Available Abstract In the present study, we explore the role of apoptosis signal-regulating kinase 1 (ASK1 in denbinobin-induced apoptosis in human lung adenocarcinoma (A549 cells. Denbinobin-induced cell apoptosis was attenuated by an ASK1 dominant-negative mutant (ASK1DN, two antioxidants (N-acetyl-L-cysteine (NAC and glutathione (GSH, a c-Jun N-terminal kinase (JNK inhibitor (SP600125, and an activator protein-1 (AP-1 inhibitor (curcumin. Treatment of A549 cells with denbinobin caused increases in ASK1 activity and reactive oxygen species (ROS production, and these effects were inhibited by NAC and GSH. Stimulation of A549 cells with denbinobin caused JNK activation; this effect was markedly inhibited by NAC, GSH, and ASK1DN. Denbinobin induced c-Jun phosphorylation, the formation of an AP-1-specific DNA-protein complex, and Bim expression. Bim knockdown using a bim short interfering RNA strategy also reduced denbinobin-induced A549 cell apoptosis. The denbinobin-mediated increases in c-Jun phosphorylation and Bim expression were inhibited by NAC, GSH, SP600125, ASK1DN, JNK1DN, and JNK2DN. These results suggest that denbinobin might activate ASK1 through ROS production to cause JNK/AP-1 activation, which in turn induces Bim expression, and ultimately results in A549 cell apoptosis.

Src Kinase Dependent Rapid Non-genomic Modulation of Hippocampal Spinogenesis Induced by Androgen and Estrogen

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Mika Soma

2018-05-01

Full Text Available Dendritic spine is a small membranous protrusion from a neuron's dendrite that typically receives input from an axon terminal at the synapse. Memories are stored in synapses which consist of spines and presynapses. Rapid modulations of dendritic spines induced by hippocampal sex steroids, including dihydrotestosterone (DHT, testosterone (T, and estradiol (E2, are essential for synaptic plasticity. Molecular mechanisms underlying the rapid non-genomic modulation through synaptic receptors of androgen (AR and estrogen (ER as well as its downstream kinase signaling, however, have not been well understood. We investigated the possible involvement of Src tyrosine kinase in rapid changes of dendritic spines in response to androgen and estrogen, including DHT, T, and E2, using hippocampal slices from adult male rats. We found that the treatments with DHT (10 nM, T (10 nM, and E2 (1 nM increased the total density of spines by ~1.22 to 1.26-fold within 2 h using super resolution confocal imaging of Lucifer Yellow-injected CA1 pyramidal neurons. We examined also morphological changes of spines in order to clarify differences between three sex steroids. From spine head diameter analysis, DHT increased middle- and large-head spines, whereas T increased small- and middle-head spines, and E2 increased small-head spines. Upon application of Src tyrosine kinase inhibitor, the spine increases induced through DHT, T, and E2 treatments were completely blocked. These results imply that Src kinase is essentially involved in sex steroid-induced non-genomic modulation of the spine density and morphology. These results also suggest that rapid effects of exogenously applied androgen and estrogen can occur in steroid-depleted conditions, including “acute” hippocampal slices and the hippocampus of gonadectomized animals.

Helicobacter pylori induces cell migration and invasion through casein kinase 2 in gastric epithelial cells.

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Lee, Yeo Song; Lee, Do Yeon; Yu, Da Yeon; Kim, Shin; Lee, Yong Chan

2014-12-01

Chronic infection with Helicobacter pylori (H. pylori) is causally linked with gastric carcinogenesis. Virulent H. pylori strains deliver bacterial CagA into gastric epithelial cells. Induction of high motility and an elongated phenotype is considered to be CagA-dependent process. Casein kinase 2 plays a critical role in carcinogenesis through signaling pathways related to the epithelial mesenchymal transition. This study was aimed to investigate the effect of H. pylori infection on the casein kinase 2-mediated migration and invasion in gastric epithelial cells. AGS or MKN28 cells as human gastric epithelial cells and H. pylori strains Hp60190 (ATCC 49503, CagA(+)) and Hp8822 (CagA(-)) were used. Cells were infected with H. pylori at multiplicity of infection of 100 : 1 for various times. We measured in vitro kinase assay to examine casein kinase 2 activity and performed immunofluorescent staining to observe E-cadherin complex. We also examined β-catenin transactivation through promoter assay and MMP7 expression by real-time PCR and ELISA. H. pylori upregulates casein kinase 2 activity and inhibition of casein kinase 2 in H. pylori-infected cells profoundly suppressed cell invasiveness and motility. We confirmed that casein kinase 2 mediates membranous α-catenin depletion through dissociation of the α-/β-catenin complex in H. pylori-infected cells. We also found that H. pylori induces β-catenin nuclear translocation and increases MMP7 expressions mediated through casein kinase 2. We show for the first time that CagA(+) H. pylori upregulates cellular invasiveness and motility through casein kinase 2. The demonstration of a mechanistic interplay between H. pylori and casein kinase 2 provides important insights into the role of CagA(+) H. pylori in the gastric cancer invasion and metastasis. © 2014 John Wiley & Sons Ltd.

Tyrosine Kinase Inhibitors Induced Thyroid Dysfunction: A Review of Its Incidence, Pathophysiology, Clinical Relevance, and Treatment

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Hala Ahmadieh

2013-01-01

Full Text Available Tyrosine kinase inhibitors (TKI belong to a new class of molecular multitargeted anticancer therapy which targets different growth factor receptors and hence attenuates cancer cell survival and growth. Since their introduction as adjunct treatment for renal cell carcinoma and gastrointestinal stromal tumors (GIST, a number of reports have demonstrated that TKI can induce thyroid dysfunction which was especially more common with sunitinib maleate. Many mechanisms with respect to this adverse effect of tyrosine kinase inhibitors have been proposed including their induction of thyroiditis, capillary regression in the thyroid gland, antithyroid peroxidase antibody production, and their ability to decrease iodine uptake by the thyroid gland. Of interest is the observation that TKI-induced thyroid dysfunction may actually be protective as it was shown to improve overall survival, and it was suggested that it may have a prognostic value. Followup on thyroid function tests while patients are maintained on tyrosine kinase inhibitor is strongly recommended. When thyroid dysfunction occurs, appropriate treatment should be individualized depending on patients symptoms and thyroid stimulating hormone level.

Integrin-linked kinase is involved in matrix-induced hepatocyte differentiation

International Nuclear Information System (INIS)

Gkretsi, Vasiliki; Bowen, William C.; Yang, Yu; Wu, Chuanyue; Michalopoulos, George K.

2007-01-01

Hepatocytes have restricted proliferative capacity in culture and when cultured without matrix, lose the hepatocyte-specific gene expression and characteristic cellular micro-architecture. Overlay of matrix-preparations on de-differentiated hepatocytes restores differentiation. Integrin-linked kinase (ILK) is a cell-matrix-adhesion protein crucial in fundamental processes such as differentiation and survival. In this study, we investigated the role of ILK, and its binding partners PINCH, α-parvin, and Mig-2 in matrix-induced hepatocyte differentiation. We report here that ILK is present in the liver and localizes at cell-matrix adhesions of cultured hepatocytes. We also show that ILK, PINCH, α-parvin, and Mig-2 expression level is dramatically reduced in the re-differentiated hepatocytes. Interestingly, hepatocytes lacking ILK undergo matrix-induced differentiation but their differentiation is incomplete, as judged by monitoring cell morphology and production of albumin. Our results show that ILK and cell-matrix adhesion proteins play an important role in the process of matrix-induced hepatocyte differentiation

Strigolactone-Induced Putative Secreted Protein 1 Is Required for the Establishment of Symbiosis by the Arbuscular Mycorrhizal Fungus Rhizophagus irregularis.

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Tsuzuki, Syusaku; Handa, Yoshihiro; Takeda, Naoya; Kawaguchi, Masayoshi

2016-04-01

Arbuscular mycorrhizal (AM) symbiosis is the most widespread association between plants and fungi. To provide novel insights into the molecular mechanisms of AM symbiosis, we screened and investigated genes of the AM fungus Rhizophagus irregularis that contribute to the infection of host plants. R. irregularis genes involved in the infection were explored by RNA-sequencing (RNA-seq) analysis. One of the identified genes was then characterized by a reverse genetic approach using host-induced gene silencing (HIGS), which causes RNA interference in the fungus via the host plant. The RNA-seq analysis revealed that 19 genes are up-regulated by both treatment with strigolactone (SL) (a plant symbiotic signal) and symbiosis. Eleven of the 19 genes were predicted to encode secreted proteins and, of these, SL-induced putative secreted protein 1 (SIS1) showed the largest induction under both conditions. In hairy roots of Medicago truncatula, SIS1 expression is knocked down by HIGS, resulting in significant suppression of colonization and formation of stunted arbuscules. These results suggest that SIS1 is a putative secreted protein that is induced in a wide spatiotemporal range including both the presymbiotic and symbiotic stages and that SIS1 positively regulates colonization of host plants by R. irregularis.

Mitochondrial dynamics in type 2 diabetes: Pathophysiological implications

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Susana Rovira-Llopis

2017-04-01

Full Text Available Mitochondria play a key role in maintaining cellular metabolic homeostasis. These organelles have a high plasticity and are involved in dynamic processes such as mitochondrial fusion and fission, mitophagy and mitochondrial biogenesis. Type 2 diabetes is characterised by mitochondrial dysfunction, high production of reactive oxygen species (ROS and low levels of ATP. Mitochondrial fusion is modulated by different proteins, including mitofusin-1 (MFN1, mitofusin-2 (MFN2 and optic atrophy (OPA-1, while fission is controlled by mitochondrial fission 1 (FIS1, dynamin-related protein 1 (DRP1 and mitochondrial fission factor (MFF. PARKIN and (PTEN-induced putative kinase 1 (PINK1 participate in the process of mitophagy, for which mitochondrial fission is necessary. In this review, we discuss the molecular pathways of mitochondrial dynamics, their impairment under type 2 diabetes, and pharmaceutical approaches for targeting mitochondrial dynamics, such as mitochondrial division inhibitor-1 (mdivi-1, dynasore, P110 and 15-oxospiramilactone. Furthermore, we discuss the pathophysiological implications of impaired mitochondrial dynamics, especially in type 2 diabetes.

PINK1 Primes Parkin-Mediated Ubiquitination of PARIS in Dopaminergic Neuronal Survival

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Yunjong Lee

2017-01-01

Full Text Available Mutations in PTEN-induced putative kinase 1 (PINK1 and parkin cause autosomal-recessive Parkinson’s disease through a common pathway involving mitochondrial quality control. Parkin inactivation leads to accumulation of the parkin interacting substrate (PARIS, ZNF746 that plays an important role in dopamine cell loss through repression of proliferator-activated receptor gamma coactivator-1-alpha (PGC-1α promoter activity. Here, we show that PARIS links PINK1 and parkin in a common pathway that regulates dopaminergic neuron survival. PINK1 interacts with and phosphorylates serines 322 and 613 of PARIS to control its ubiquitination and clearance by parkin. PINK1 phosphorylation of PARIS alleviates PARIS toxicity, as well as repression of PGC-1α promoter activity. Conditional knockdown of PINK1 in adult mouse brains leads to a progressive loss of dopaminergic neurons in the substantia nigra that is dependent on PARIS. Altogether, these results uncover a function of PINK1 to direct parkin-PARIS-regulated PGC-1α expression and dopaminergic neuronal survival.

Crystallization and preliminary crystallographic analysis of a putative glucokinase/hexokinase from Thermus thermophilus

International Nuclear Information System (INIS)

Nakamura, Tsutomu; Kashima, Yasuhiro; Mine, Shouhei; Oku, Takashi; Uegaki, Koichi

2011-01-01

In this study, a putative glucokinase/hexokinase from T. thermophilus was purified and crystallized. Diffraction data were collected and processed to 2.02 Å resolution. Glucokinase/hexokinase catalyzes the phosphorylation of glucose to glucose 6-phosphate, which is the first step of glycolysis. The open reading frame TTHA0299 of the extreme thermophile Thermus thermophilus encodes a putative glucokinase/hexokinase which contains the consensus sequence for proteins from the repressors, open reading frames and sugar kinases family. In this study, the glucokinase/hexokinase from T. thermophilus was purified and crystallized using polyethylene glycol 8000 as a precipitant. Diffraction data were collected and processed to 2.02 Å resolution. The crystal belonged to space group P2 1 , with unit-cell parameters a = 70.93, b = 138.14, c = 75.16 Å, β = 95.41°

Ibuprofen abates cypermethrin-induced expression of pro-inflammatory mediators and mitogen-activated protein kinases and averts the nigrostriatal dopaminergic neurodegeneration.

Science.gov (United States)

Singh, Ashish; Tripathi, Pratibha; Prakash, Om; Singh, Mahendra Pratap

2016-12-01

Cypermethrin induces oxidative stress, microglial activation, inflammation and apoptosis leading to Parkinsonism in rats. While ibuprofen, a non-steroidal anti-inflammatory drug, relieves from inflammation, its efficacy against cypermethrin-induced Parkinsonism has not yet been investigated. The study aimed to explore the protective role of ibuprofen in cypermethrin-induced Parkinsonism, an environmentally relevant model of Parkinson's disease (PD), along with its underlying mechanism. Animals were treated with/without cypermethrin in the presence/absence of ibuprofen. Behavioural, immunohistochemical and biochemical parameters of Parkinsonism and expression of pro-inflammatory and pro-apoptotic proteins along with mitogen-activated protein kinases (MAPKs) were determined. Ibuprofen resisted cypermethrin-induced behavioural impairments, striatal dopamine depletion, oxidative stress in the nigrostriatal tissues and loss of the nigral dopamine producing cells and increase in microglial activation along with atypical expression of pro-inflammatory and apoptotic proteins that include cyclooxygenase-2, tumour necrosis factor-α, MAPKs (c-Jun N-terminal kinase, p38 and extracellular signal-regulated kinase), B cell lymphoma 2-associated protein X, tumour suppressor protein p53, cytochrome c and caspase-3 in the nigrostriatal tissue. The results obtained thus demonstrate that ibuprofen lessens inflammation and regulates MAPKs expression thereby averts cypermethrin-induced Parkinsonism.

Fisetin Attenuates AKT Associated Growth Promoting Events in AflatoxinB1 Induced Hepatocellular Carcinoma.

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Maurya, Brajesh Kumar; Trigun, Surendra Kumar

2017-12-29

Recently we have reported that Fisetin, a natural flavonol, is able to regress Aflatoxin-B1 (AFB1) induced hepatocellular carcinoma (HCC) by suppressing reactive oxygen species (ROS) led pro-inflammatory factors in rats. In the current study, we aimed to delineate whether Fisetin does so by modulating the cell growth promoting signaling cascade in HCC. The reciprocal interplay of 3-phosphoinositol kinase (PI3K) vs phosphatase and tensin homologue deleted on chromosome 10 (PTEN) displays Akt, a protein kinase B, to get phosphorylated at Thr308 by a 3-phosphoinositol dependent kinase 1 (PDK1). This commits cells of neoplastic niche to undergo rapid proliferation by p-Akt thr308 dependent phosphorylation of glycogen synthase kinase 3β (GSK3β) at Ser 9 position. In this study, the effect of in vivo treatment of 20 mg/kg b.w. Fisetin on relative profile of all these factors were studied in the liver from the HCC rats induced by two doses of 1mg/kg b.w. AFB1 i.p. As compared to the untreated HCC liver, liver from Fisetin treated HCC group rats showed a significant decline in the activity and level of p-Aktthr308 which was consistent with a similar decline in PDK1 level. Concordantly, the level of p-GSK3βSer 9 was also found to be declined significantly in those Fisetin-treated HCC livers. A concomitant decline in immunohistochemically detected number of the proliferating cell nuclear antigen (PCNA), a cell proliferation marker, in the HCC liver, further confirmed anti-cell proliferative role of Fisetin during HCC growth in vivo. This findings suggest that Fisetin is able to suppress Akt dependent cell growth signaling mechanisms in HCC mainly by down regulating PDK1 dependent Akt phosphorylation. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

Norathyriol Suppresses Skin Cancers Induced by Solar Ultraviolet Radiation by Targeting ERK Kinases

Energy Technology Data Exchange (ETDEWEB)

Li, Jixia; Malakhova, Margarita; Mottamal, Madhusoodanan; Reddy, Kanamata; Kurinov, Igor; Carper, Andria; Langfald, Alyssa; Oi, Naomi; Kim, Myoung Ok; Zhu, Feng; Sosa, Carlos P.; Zhou, Keyuan; Bode, Ann M.; Dong, Zigang (Cornell); (Guangdong); (UMM)

2012-06-27

Ultraviolet (UV) irradiation is the leading factor in the development of skin cancer, prompting great interest in chemopreventive agents for this disease. In this study, we report the discovery of norathyriol, a plant-derived chemopreventive compound identified through an in silico virtual screening of the Chinese Medicine Library. Norathyriol is a metabolite of mangiferin found in mango, Hypericum elegans, and Tripterospermum lanceolatum and is known to have anticancer activity. Mechanistic investigations determined that norathyriol acted as an inhibitor of extracellular signal-regulated kinase (ERK)1/2 activity to attenuate UVB-induced phosphorylation in mitogen-activated protein kinases signaling cascades. We confirmed the direct and specific binding of norathyriol with ERK2 through a cocrystal structural analysis. The xanthone moiety in norathyriol acted as an adenine mimetic to anchor the compound by hydrogen bonds to the hinge region of the protein ATP-binding site on ERK2. Norathyriol inhibited in vitro cell growth in mouse skin epidermal JB6 P+ cells at the level of G{sub 2}-M phase arrest. In mouse skin tumorigenesis assays, norathyriol significantly suppressed solar UV-induced skin carcinogenesis. Further analysis indicated that norathyriol mediates its chemopreventive activity by inhibiting the ERK-dependent activity of transcriptional factors AP-1 and NF-{kappa}B during UV-induced skin carcinogenesis. Taken together, our results identify norathyriol as a safe new chemopreventive agent that is highly effective against development of UV-induced skin cancer.

Targeting Glutamatergic Signaling and the PI3 Kinase Pathway to Halt Melanoma Progression

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Stephen A. Rosenberg

2015-02-01

Full Text Available Our group has previously reported that the majority of human melanomas (>60% express the metabotropic glutamate receptor 1 (GRM1 and that the glutamate release inhibitor riluzole, a drug currently used to treat amyotrophic lateral sclerosis, can induce apoptosis in GRM1-expressing melanoma cells. Our group previously reported that in vitro riluzole treatment reduces cell growth in three-dimensional (3D soft agar colony assays by 80% in cells with wildtype phosphoinositide 3-kinase (PI3K pathway activation. However, melanoma cell lines harboring constitutive activating mutations of the PI3K pathway (PTEN and NRAS mutations showed only a 35% to 40% decrease in colony formation in soft agar in the presence of riluzole. In this study, we have continued our preclinical studies of riluzole and its effect on melanoma cells alone and in combination with inhibitors of the PI3 kinase pathway: the AKT inhibitor, API-2, and the mammalian target of rapamycin (mTOR inhibitor, rapamycin. We modeled these combinatorial therapies on various melanoma cell lines in 3D and 2D systems and in vivo. Riluzole combined with mTOR inhibition is more effective at halting melanoma anchorage-independent growth and xenograft tumor progression than either agent alone. PI3K signaling changes associated with this combinatorial treatment shows that 3D (nanoculture modeling of cell signaling more closely resembles in vivo signaling than monolayer models. Riluzole combined with mTOR inhibition is effective at halting tumor cell progression independent of BRAF mutational status. This makes this combinatorial therapy a potentially viable alternative for metastatic melanoma patients who are BRAF WT and are therefore ineligible for vemurafenib therapy.

The selective and inducible activation of endogenous PI 3-kinase in PC12 cells results in efficient NGF-mediated survival but defective neurite outgrowth.

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Ashcroft, M; Stephens, R M; Hallberg, B; Downward, J; Kaplan, D R

1999-08-12

The Trk/Nerve Growth Factor receptor mediates the rapid activation of a number of intracellular signaling proteins, including phosphatidylinositol 3-kinase (PI 3-kinase). Here, we describe a novel, NGF-inducible system that we used to specifically address the signaling potential of endogenous PI 3-kinase in NGF-mediated neuronal survival and differentiation processes. This system utilizes a Trk receptor mutant (Trk(def)) lacking sequences Y490, Y785 and KFG important for the activation of the major Trk targets; SHC, PLC-gammal, Ras, PI 3-kinase and SNT. Trk(def) was kinase active but defective for NGF-induced responses when stably expressed in PC12nnr5 cells (which lack detectable levels of TrkA and are non-responsive to NGF). The PI 3-kinase consensus binding site, YxxM (YVPM), was introduced into the insert region within the kinase domain of Trk(def). NGF-stimulated tyrosine phosphorylation of the Trk(def)+PI 3-kinase addback receptor, resulted in the direct association and selective activation of PI 3-kinase in vitro and the production of PI(3,4)P2 and PI(3,4,5)P3 in vivo (comparable to wild-type). PC12nnr5 cells stably expressing Trk(def) + PI 3-kinase, initiated neurite outgrowth but failed to stably extend and maintain these neurites in response to NGF as compared to PC12 parental cells, or PC12nnr5 cells overexpressing wild-type Trk. However, Trk(def) + PI 3-kinase was fully competent in mediating NGF-induced survival processes. We propose that while endogenous PI 3-kinase can contribute in part to neurite initiation processes, its selective activation and subsequent signaling to downstream effectors such as Akt, functions mainly to promote cell survival in the PC12 system.

Protein kinase CK2 phosphorylates the Fas-associated factor FAF1 in vivo and influences its transport into the nucleus

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Olsen, Birgitte B; Jessen, Vibeke; Højrup, Peter

2003-01-01

We previously identified the Fas-associated factor FAF1 as an in vitro substrate of protein kinase CK2 and determined Ser289 and Ser291 as phosphorylation sites. Here we demonstrate that these two serine residues are the only sites phosphorylated by CK2 in vitro, and that at least one site...... is phosphorylated in vivo. Furthermore, we analyzed putative physiological functions of FAF1 phosphorylation. The ability of FAF1 to potentiate Fas-induced apoptosis is not influenced by the FAF1 phosphorylation status; however, the nuclear import of a phosphorylation-deficient FAF1 mutant was delayed in comparison...

Deceleration of arginine kinase refolding by induced helical structures.

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Li, Hai-Long; Zhou, Sheng-Mei; Park, Daeui; Jeong, Hyoung Oh; Chung, Hae Young; Yang, Jun-Mo; Meng, Fan-Guo; Hu, Wei-Jiang

2012-04-01

Arginine kinase (AK) is a key metabolic enzyme for keeping energy balance in invertebrates. Therefore, regulation of the enzymatic activity and the folding studies of AK from the various invertebrates have been the focus of investigation. We studied the effects of helical structures by using hexafluoroisopropanol (HFIP) on AK folding. Folding kinetic studies showed that the folding rates of the urea-denatured AKs were significantly decelerated after being induced in various concentrations of HFIP. AK lost its activity completely at concentrations greater than 60%. The results indicated that the HFIP-induced helical structures in the denatured state play a negative role in protein folding, and the helical structures induced in 5% (v/v) HFIP act as the most effective barrier against AK taking its native structure. The computational docking simulations (binding energies for -2.19 kcal/mol for AutoDock4.2 and -20.47 kcal/mol for Dock6.3) suggested that HFIP interacts with the several important residues that are predicted by both programs. The excessively pre-organized helical structures not only hampered the folding process, but also ultimately brought about changes in the three-dimensional conformation and biological function of AK.

The two putative comS homologs of the biotechnologically important Bacillus licheniformis do not contribute to competence development.

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Jakobs, Mareike; Hoffmann, Kerstin; Liesegang, Heiko; Volland, Sonja; Meinhardt, Friedhelm

2015-03-01

In Bacillus subtilis, natural genetic competence is subject to complex genetic regulation and quorum sensing dependent. Upon extracellular accumulation of the peptide-pheromone ComX, the membrane-bound sensor histidine kinase ComP initiates diverse signaling pathways by activating-among others-DegQ and ComS. While DegQ favors the expression of extracellular enzymes rather than competence development, ComS is crucial for competence development as it prevents proteolytic degradation of ComK, the key transcriptional activator of all genes required for the uptake and integration of DNA. In Bacillus licheniformis, ComX/ComP sensed cell density negatively influences competence development, suggesting differences from the quorum-sensing-dependent control mechanism in Bacillus subtilis. Here, we show that each of six investigated strains possesses both of two different, recently identified putative comS genes. When expressed from an inducible promoter, none of the comS candidate genes displayed an impact on competence development neither in B. subtilis nor in B. licheniformis. Moreover, disruption of the genes did not reduce transformation efficiency. While the putative comS homologs do not contribute to competence development, we provide evidence that the degQ gene as for B. subtilis negatively influences genetic competency in B. licheniformis.

Role of nongenomic activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase kinase/extracellular signal-regulated kinase 1/2 pathways in 1,25D3-mediated apoptosis in squamous cell carcinoma cells.

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Ma, Yingyu; Yu, Wei-Dong; Kong, Rui-Xian; Trump, Donald L; Johnson, Candace S

2006-08-15

Vitamin D is a steroid hormone that regulates calcium homeostasis and bone metabolism. The active form of vitamin D [1 alpha,25-dihydroxyvitamin D(3) (1,25D3)] acts through both genomic and nongenomic pathways. 1,25D3 has antitumor effects in a variety of cancers, including colorectal, prostate, breast, ovarian, and skin cancers. 1,25D3 exerts growth-inhibitory effects in cancer cells through the induction of apoptosis, cell cycle arrest, and differentiation. The mechanisms regulating 1,25D3-induced apoptosis remain unclear. We investigated the role of nongenomic signaling in 1,25D3-mediated apoptosis in squamous cell carcinoma (SCC) cells. 1,25D3 induced rapid and sustained activation of phosphatidylinositol 3-kinase/Akt and mitogen-activated protein kinase/extracellular signal-regulated kinase (ERK) 1/2 pathways in SCC cells. These effects were nongenomic: they occurred rapidly and were not inhibited by cycloheximide or actinomycin D. To examine whether the nongenomic activation of Akt and ERK1/2 plays a role in 1,25D3-mediated apoptosis, the expression of Akt or ERK1/2 was reduced by small interfering RNA (siRNA). siRNA-Akt significantly enhanced 1,25D3-induced apoptosis as indicated by increased levels of Annexin V-positive cells and increased sub-G(1) population and DNA fragmentation. In contrast, siRNA-ERK1/2 had no effects on 1,25D3-induced apoptosis. In addition, siRNA-Akt transfection followed by 1,25D3 treatment induced apoptosis much sooner than 1,25D3 alone. siRNA-Akt and 1,25D3 induced caspase-10 activation, suppressed the expression of c-IAP1 and XIAP, and promoted 1,25D3-induced caspase-3 activation. These results support a link between 1,25D3-induced nongenomic signaling and apoptosis. 1,25D3 induces the activation of phosphatidylinositol 3-kinase/Akt, which suppresses 1,25D3-mediated apoptosis and prolongs the survival of SCC cells.

SPINK1 Overexpression in Localized Prostate Cancer: a Rare Event Inversely Associated with ERG Expression and Exclusive of Homozygous PTEN Deletion.

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Huang, Kuo-Cheng; Evans, Andrew; Donnelly, Bryan; Bismar, Tarek A

2017-04-01

SPINK1 is proposed as potential prognostic marker in prostate cancer (PCA). However, its relation to PTEN and ERG in localized PCA remains unclear. The study population consisted of two independent cohorts of men treated by radical prostatectomy for localized PCA (discovery n = 218 and validation n = 129). Patterns of association between SPINK1 and each of ERG and PTEN were evaluated by immunohistochemistry and fluorescence in situ hybridization. Associations between SPINK1 expression and various pathologic parameters and clinical outcome were also investigated. SPINK1 was expressed in 15.3 % and 10.9 % of cases in the discovery and validation cohort, respectively. SPINK expression was observed in 5.56 % of high-grade prostatic intraepithelial neoplasia and 1.1 % of adjacent morphologically benign prostatic glands. SPINK1 and ERG expression were almost exclusive, with only 1.0 % of the cases co-expressing both in the same core sample. SPINK1 interfocal and within-core heterogeneity was noted in 29.2 % and 64.6 % of cases, respectively. SPINK1 expression was not significantly associated with PTEN deletion in the two cohorts (p = 0.871 for discovery cohort and p = 0.293 for validation cohort). While SPINK1 expression did occur with hemizygous PTEN deletion, there was a complete absence of SPINK1 expression in PCA showing homozygous PTEN deletion, which was confirmed in the validation cohort (p = 0.02). Despite SPINK1's association with higher Gleason score (>7) (p = 0.02), it was not associated with other pathological parameters or biochemical recurrence post-radical prostatectomy. We documented absolute exclusivity between SPINK1 overexpression and homozygous PTEN deletion in localized PCA. SPINK1 and ERG expressions are exclusive events in PCA. SPINK1 is not of added prognostic value in localized PCA.

Downregulation of catalase by reactive oxygen species via PI 3 kinase/Akt signaling in mesangial cells.

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Venkatesan, Balachandar; Mahimainathan, Lenin; Das, Falguni; Ghosh-Choudhury, Nandini; Ghosh Choudhury, Goutam

2007-05-01

Reactive oxygen species (ROS) contribute to many glomerular diseases by targeting mesangial cells. ROS have been shown to regulate expression of many antioxidant enzymes including catalase. The mechanism by which the expression of catalase protein is regulated by ROS is not precisely known. Here we report that increased intracellular ROS level by hydrogen peroxide (H(2)O(2)) reduced the expression of catalase. H(2)O(2) increased phosphorylation of Akt kinase in a dose-dependent and sustained manner with a concomitant increase in the phosphorylation of FoxO1 transcription factor. Further analysis revealed that H(2)O(2) promoted rapid activation of phosphatidylinositol (PI) 3 kinase. The PI 3 kinase inhibitor Ly294002 and expression of tumor suppressor protein PTEN inhibited Akt kinase activity, resulting in the attenuation of FoxO1 phosphorylation and preventing the downregulating effect of H(2)O(2) on catalase protein level. Dominant negative Akt attenuated the inhibitory effect of H(2)O(2) on expression of catalase. Constitutively active FoxO1 increased the expression of catalase. However, dominant negative FoxO1 inhibited catalase protein level. Catalase transcription was reduced by H(2)O(2) treatment. Furthermore, expression of dominant negative Akt and constitutively active FoxO1 increased catalase transcription, respectively. These results demonstrate that ROS downregulate the expression of catalase in mesangial cells by PI 3 kinase/Akt signaling via FoxO1 as a target. (c) 2007 Wiley-Liss, Inc.

Impairment of Akt activity by CYP2E1 mediated oxidative stress is involved in chronic ethanol-induced fatty liver

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Tao Zeng

2018-04-01

Full Text Available Protein kinase B (PKB/Akt plays important roles in the regulation of lipid homeostasis, and impairment of Akt activity has been demonstrated to be involved in the development of non-alcoholic fatty liver disease (NAFLD. Previous studies suggest that cytochrome P4502E1 (CYP2E1 plays causal roles in the pathogenesis of alcoholic fatty liver (AFL. We hypothesized that Akt activity might be impaired due to CYP2E1-induced oxidative stress in chronic ethanol-induced hepatic steatosis. In this study, we found that chronic ethanol-induced hepatic steatosis was accompanied with reduced phosphorylation of Akt at Thr308 in mice liver. Chronic ethanol exposure had no effects on the protein levels of phosphatidylinositol 3 kinase (PI3K and phosphatase and tensin homologue deleted on chromosome ten (PTEN, and led to a slight decrease of phosphoinositide-dependent protein kinase 1 (PDK-1 protein level. Ethanol exposure resulted in increased levels of malondialdehyde (MDA and 4-hydroxynonenal (4-HNE-Akt adducts, which was significantly inhibited by chlormethiazole (CMZ, an efficient CYP2E1 inhibitor. Interestingly, N-acetyl-L-cysteine (NAC significantly attenuated chronic ethanol-induced hepatic fat accumulation and the decline of Akt phosphorylation at Thr308. In the in vitro studies, Akt phosphorylation was suppressed in CYP2E1-expressing HepG2 (CYP2E1-HepG2 cells compared with the negative control HepG2 (NC-HepG2 cells, and 4-HNE treatment led to significant decrease of Akt phosphorylation at Thr308 in wild type HepG2 cells. Lastly, pharmacological activation of Akt by insulin-like growth factor-1 (IGF-1 significantly alleviated chronic ethanol-induced fatty liver in mice. Collectively, these results indicate that CYP2E1-induced oxidative stress may be responsible for ethanol-induced suppression of Akt phosphorylation and pharmacological modulation of Akt in liver may be an effective strategy for the treatment of ethanol-induced fatty liver. Keywords

Crucial role of rho-kinase in pressure overload-induced right ventricular hypertrophy and dysfunction in mice.

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Ikeda, Shohei; Satoh, Kimio; Kikuchi, Nobuhiro; Miyata, Satoshi; Suzuki, Kota; Omura, Junichi; Shimizu, Toru; Kobayashi, Kenta; Kobayashi, Kazuto; Fukumoto, Yoshihiro; Sakata, Yasuhiko; Shimokawa, Hiroaki

2014-06-01

Right ventricular (RV) failure is the leading cause of death in various cardiopulmonary diseases, including pulmonary hypertension. It is generally considered that the RV is vulnerable to pressure overload as compared with the left ventricle (LV). However, as compared with LV failure, the molecular mechanisms of RV failure are poorly understood, and hence therapeutic targets of the disorder remain to be elucidated. Thus, we aimed to identify molecular therapeutic targets for RV failure in a mouse model of pressure overload. To induce pressure overload to respective ventricles, we performed pulmonary artery constriction or transverse aortic constriction in mice. We first performed microarray analysis and found that the molecules related to RhoA/Rho-kinase and integrin pathways were significantly upregulated in the RV with pulmonary artery constriction compared with the LV with transverse aortic constriction. Then, we examined the responses of both ventricles to chronic pressure overload in vivo. We demonstrated that compared with transverse aortic constriction, pulmonary artery constriction caused greater extents of mortality, Rho-kinase expression (especially ROCK2 isoform), and oxidative stress in pressure-overloaded RV, reflecting the weakness of the RV in response to pressure overload. Furthermore, mice with myocardial-specific overexpression of dominant-negative Rho-kinase showed resistance to pressure overload-induced hypertrophy and dysfunction associated with reduced oxidative stress. Finally, dominant-negative Rho-kinase mice showed a significantly improved long-term survival in both pulmonary artery constriction and transverse aortic constriction as compared with littermate controls. These results indicate that the Rho-kinase pathway plays a crucial role in RV hypertrophy and dysfunction, suggesting that the pathway is a novel therapeutic target of RV failure in humans. © 2014 American Heart Association, Inc.

Growth Inhibition by Bupivacaine Is Associated with Inactivation of Ribosomal Protein S6 Kinase 1

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Beigh, Mushtaq Ahmad; Showkat, Mehvish; Bashir, Basharat; Bashir, Asma; Hussain, Mahboob ul; Andrabi, Khurshid Iqbal

2014-01-01

Bupivacaine is an amide type long acting local anesthetic used for epidural anesthesia and nerve blockade in patients. Use of bupivacaine is associated with severe cytotoxicity and apoptosis along with inhibition of cell growth and proliferation. Although inhibition of Erk, Akt, and AMPK seemingly appears to mediate some of the bupivacaine effects, potential downstream targets that mediate its effect remain unknown. S6 kinase 1 is a common downstream effector of several growth regulatory pathways involved in cell growth and proliferation known to be affected by bupivacaine. We have accordingly attempted to relate the growth inhibitory effects of bupivacaine with the status of S6K1 activity and we present evidence that decrease in cell growth and proliferation by bupivacaine is mediated through inactivation of S6 kinase 1 in a concentration and time dependent manner. We also show that ectopic expression of constitutively active S6 kinase 1 imparts substantial protection from bupivacaine induced cytotoxicity. Inactivation of S6K1 though associated with loss of putative mTOR mediated phosphorylation did not correspond with loss of similar phosphorylations in 4EBP1 indicating that S6K1 inhibition was not mediated through inactivation of mTORC1 signaling pathway or its down regulation. PMID:24605337

Protein Kinase G Induces an Immune Response in Cows Exposed to Mycobacterium avium Subsp. paratuberculosis

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Horacio Bach

2018-01-01

Full Text Available To establish infection, pathogens secrete virulence factors, such as protein kinases and phosphatases, to modulate the signal transduction pathways used by host cells to initiate immune response. The protein MAP3893c is annotated in the genome sequence of Mycobacterium avium subspecies paratuberculosis (MAP, the causative agent of Johne’s disease, as the serine/threonine protein kinase G (PknG. In this work, we report that PknG is a functional kinase that is secreted within macrophages at early stages of infection. The antigen is able to induce an immune response from cattle exposed to MAP in the form of interferon gamma production after stimulation of whole blood with PknG. These findings suggest that PknG may contribute to the pathogenesis of MAP by phosphorylating macrophage signalling and/or adaptor molecules as observed with other pathogenic mycobacterial species.

Characterization of cyclin-dependent kinases and Cdc2/Cdc28 kinase subunits in Trichomonas vaginalis.

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Amador, Erick; López-Pacheco, Karla; Morales, Nataly; Coria, Roberto; López-Villaseñor, Imelda

2017-04-01

Cyclin-dependent kinases (CDKs) have important roles in regulating key checkpoints between stages of the cell cycle. Their activity is tightly regulated through a variety of mechanisms, including through binding with cyclin proteins and the Cdc2/Cdc28 kinase subunit (CKS), and their phosphorylation at specific amino acids. Studies of the components involved in cell cycle control in parasitic protozoa are limited. Trichomonas vaginalis is the causative agent of trichomoniasis in humans and is therefore important in public health; however, some of the basic biological processes used by this organism have not been defined. Here, we characterized proteins potentially involved in cell cycle regulation in T. vaginalis. Three genes encoding protein kinases were identified in the T. vaginalis genome, and the corresponding recombinant proteins (TvCRK1, TvCRK2, TvCRK5) were studied. These proteins displayed similar sequence features to CDKs. Two genes encoding CKSs were also identified, and the corresponding recombinant proteins were found to interact with TvCRK1 and TvCRK2 by a yeast two-hybrid system. One putative cyclin B protein from T. vaginalis was found to bind to and activate the kinase activities of TvCRK1 and TvCRK5, but not TvCRK2. This work is the first characterization of proteins involved in cell cycle control in T. vaginalis.

A Calcium-Dependent Protein Kinase Is Systemically Induced upon Wounding in Tomato Plants1

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Chico, José Manuel; Raíces, Marcela; Téllez-Iñón, María Teresa; Ulloa, Rita María

2002-01-01

A full-length cDNA clone (LeCDPK1) from tomato (Lycopersicon esculentum) encoding a calcium-dependent protein kinase (CDPK) was isolated by screening a cDNA library from tomato cell cultures exposed to Cladosporium fulvum elicitor preparations. The predicted amino acid sequence of the cDNA reveals a high degree of similarity with other members of the CDPK family. LeCDPK1 has a putative N-terminal myristoylation sequence and presents a possible palmitoylation site. The in vitro translated protein conserves the biochemical properties of a member of the CDPK family. In addition, CDPK activity was detected in soluble and particulate extracts of tomato leaves. Basal levels of LeCDPK1 mRNA were detected by northern-blot analysis in roots, stems, leaves, and flowers of tomato plants. The expression of LeCDPK1 was rapidly and transiently enhanced in detached tomato leaves treated with pathogen elicitors and H2O2. Moreover, when tomato greenhouse plants were subjected to mechanical wounding, a transient increase of LeCDPK1 steady-state mRNA levels was detected locally at the site of the injury and systemically in distant non-wounded leaves. The increase observed in LeCDPK1 mRNA upon wounding correlates with an increase in the amount and in the activity of a soluble CDPK detected in extracts of tomato leaves, suggesting that this kinase is part of physiological plant defense mechanisms against biotic or abiotic attacks. PMID:11788771

Targeting Src family kinases inhibits bevacizumab-induced glioma cell invasion.

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Deborah Huveldt

Full Text Available Anti-VEGF antibody therapy with bevacizumab provides significant clinical benefit in patients with recurrent glioblastoma multiforme (GBM. Unfortunately, progression on bevacizumab therapy is often associated with a diffuse disease recurrence pattern, which limits subsequent therapeutic options. Therefore, there is an urgent need to understand bevacizumab's influence on glioma biology and block it's actions towards cell invasion. To explore the mechanism(s of GBM cell invasion we have examined a panel of serially transplanted human GBM lines grown either in short-term culture, as xenografts in mouse flank, or injected orthotopically in mouse brain. Using an orthotopic xenograft model that exhibits increased invasiveness upon bevacizumab treatment, we also tested the effect of dasatinib, a broad spectrum SFK inhibitor, on bevacizumab-induced invasion.We show that 1 activation of Src family kinases (SFKs is common in GBM, 2 the relative invasiveness of 17 serially transplanted GBM xenografts correlates strongly with p120 catenin phosphorylation at Y228, a Src kinase site, and 3 SFK activation assessed immunohistochemically in orthotopic xenografts, as well as the phosphorylation of downstream substrates occurs specifically at the invasive tumor edge. Further, we show that SFK signaling is markedly elevated at the invasive tumor front upon bevacizumab administration, and that dasatinib treatment effectively blocked the increased invasion induced by bevacizumab.Our data are consistent with the hypothesis that the increased invasiveness associated with anti-VEGF therapy is due to increased SFK signaling, and support testing the combination of dasatinib with bevacizumab in the clinic.

Identifying Inhibitors of Inflammation: A Novel High-Throughput MALDI-TOF Screening Assay for Salt-Inducible Kinases (SIKs).

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Heap, Rachel E; Hope, Anthony G; Pearson, Lesley-Anne; Reyskens, Kathleen M S E; McElroy, Stuart P; Hastie, C James; Porter, David W; Arthur, J Simon C; Gray, David W; Trost, Matthias

2017-12-01

Matrix-assisted laser desorption/ionization time-of-flight (MALDI TOF) mass spectrometry has become a promising alternative for high-throughput drug discovery as new instruments offer high speed, flexibility and sensitivity, and the ability to measure physiological substrates label free. Here we developed and applied high-throughput MALDI TOF mass spectrometry to identify inhibitors of the salt-inducible kinase (SIK) family, which are interesting drug targets in the field of inflammatory disease as they control production of the anti-inflammatory cytokine interleukin-10 (IL-10) in macrophages. Using peptide substrates in in vitro kinase assays, we can show that hit identification of the MALDI TOF kinase assay correlates with indirect ADP-Hunter kinase assays. Moreover, we can show that both techniques generate comparable IC 50 data for a number of hit compounds and known inhibitors of SIK kinases. We further take these inhibitors to a fluorescence-based cellular assay using the SIK activity-dependent translocation of CRTC3 into the nucleus, thereby providing a complete assay pipeline for the identification of SIK kinase inhibitors in vitro and in cells. Our data demonstrate that MALDI TOF mass spectrometry is fully applicable to high-throughput kinase screening, providing label-free data comparable to that of current high-throughput fluorescence assays.

miR-494 up-regulates the PI3K/Akt pathway via targetting PTEN and attenuates hepatic ischemia/reperfusion injury in a rat model.

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Su, Song; Luo, De; Liu, Xiangdong; Liu, Jiang; Peng, Fangyi; Fang, Cheng; Li, Bo

2017-10-31

A rat HIRI model was constructed and treated with an intraperitoneal injection of agomir- miR-494 or agomir-NC (negative control) for 7 days after the surgery. The pathophysiological changes in sham-operated rats, HIRI, HIRI + agomir- miR-494 , and HIRI + agomir-NC were compared. The effect of miR-494 was also assessed in an H 2 O 2 -induced apoptosis model. Hepatic AML12 cells were transfected with mimics NC or miR-494 mimics, followed by 6-h H 2 O 2 treatment. Cell proliferation and apoptosis were detected by CCK8 assay and flow cytometry, respectively. Further, the miR-494 target gene was identified by luciferase reporter assay, and verified both in vitro and in vivo experiments. The activity of AKT pathway was further analyzed in vivo by Western blot. HIRI + agomir- miR-494 rats exhibited significantly higher miR-494 expression, lower serum alanine aminotransferase (ALT), aspartate aminotransferase (AST), lactate dehydrogenase (LDH), and glutamate dehydrogenase (GLDH) level, lower hepatic MDA, TOA, and OSI, alleviated hepatic necrosis, reduced hepatocyte apoptosis, and decreased expression of apoptosis-related proteins, when compared with HIRI + agomir-NC rats ( P <0.05 or 0.01). After H 2 O 2 treatment, AML-12 cells transfected with miR-494 mimics had significantly higher proliferation and lower apoptosis rate compared with mimics NC group ( P <0.01). PTEN was identified as an miR-494 target gene. PTEN expression was significantly down-regulated in AML12 cells transfected with miR-494 mimics, and was up-regulated by treatment of miR-494 inhibitor ( P <0.01). Moreover, HIRI + agomir- miR-494 rats exhibited significantly lower PTEN expression, and higher p-AKT, p-mTOR, and p-p70S6K levels compared with HIRI + agomir-NC rats. Therefore, miR-494 protected rats against hepatic ischemia/reperfusion (I/R) injury through down-regulating its downstream target gene PTEN , leading to the activation of PI3K/AKT signaling pathway. © 2017 The Author(s).

Cell surface area and membrane folding in glioblastoma cell lines differing in PTEN and p53 status.

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Simon Memmel

Full Text Available Glioblastoma multiforme (GBM is characterized by rapid growth, invasion and resistance to chemo-/radiotherapy. The complex cell surface morphology with abundant membrane folds, microvilli, filopodia and other membrane extensions is believed to contribute to the highly invasive behavior and therapy resistance of GBM cells. The present study addresses the mechanisms leading to the excessive cell membrane area in five GBM lines differing in mutational status for PTEN and p53. In addition to scanning electron microscopy (SEM, the membrane area and folding were quantified by dielectric measurements of membrane capacitance using the single-cell electrorotation (ROT technique. The osmotic stability and volume regulation of GBM cells were analyzed by video microscopy. The expression of PTEN, p53, mTOR and several other marker proteins involved in cell growth and membrane synthesis were examined by Western blotting. The combined SEM, ROT and osmotic data provided independent lines of evidence for a large variability in membrane area and folding among tested GBM lines. Thus, DK-MG cells (wild type p53 and wild type PTEN exhibited the lowest degree of membrane folding, probed by the area-specific capacitance C m = 1.9 µF/cm(2. In contrast, cell lines carrying mutations in both p53 and PTEN (U373-MG and SNB19 showed the highest C m values of 3.7-4.0 µF/cm(2, which corroborate well with their heavily villated cell surface revealed by SEM. Since PTEN and p53 are well-known inhibitors of mTOR, the increased membrane area/folding in mutant GBM lines may be related to the enhanced protein and lipid synthesis due to a deregulation of the mTOR-dependent downstream signaling pathway. Given that membrane folds and extensions are implicated in tumor cell motility and metastasis, the dielectric approach presented here provides a rapid and simple tool for screening the biophysical cell properties in studies on targeting chemo- or radiotherapeutically the

Corticosterone induces rapid spinogenesis via synaptic glucocorticoid receptors and kinase networks in hippocampus.

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Yoshimasa Komatsuzaki

Full Text Available BACKGROUND: Modulation of dendritic spines under acute stress is attracting much attention. Exposure to acute stress induces corticosterone (CORT secretion from the adrenal cortex, resulting in rapid increase of CORT levels in plasma and the hippocampus. METHODOLOGY/PRINCIPAL FINDINGS: Here we demonstrated the mechanisms of rapid effect (∼1 h of CORT on the density and morphology of spines by imaging neurons in adult male rat hippocampal slices. The application of CORT at 100-1000 nM induced a rapid increase in the density of spines of CA1 pyramidal neurons. The density of small-head spines (0.2-0.4 µm was increased even at low CORT levels (100-200 nM. The density of middle-head spines (0.4-0.5 µm was increased at high CORT levels between 400-1000 nM. The density of large-head spines (0.5-1.0 µm was increased only at 1000 nM CORT. Co-administration of RU486, an antagonist of glucocorticoid receptor (GR, abolished the effect of CORT. Blocking a single kinase, such as MAPK, PKA, PKC or PI3K, suppressed CORT-induced enhancement of spinogenesis. Blocking NMDA receptors suppressed the CORT effect. CONCLUSIONS/SIGNIFICANCE: These results imply that stress levels of CORT (100-1000 nM drive the spinogenesis via synaptic GR and multiple kinase pathways.

Glycogen synthase kinase-3β ablation limits pancreatitis-induced acinar-to-ductal metaplasia.

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Ding, Li; Liou, Geou-Yarh; Schmitt, Daniel M; Storz, Peter; Zhang, Jin-San; Billadeau, Daniel D

2017-09-01

Acinar-to-ductal metaplasia (ADM) is a reversible epithelial transdifferentiation process that occurs in the pancreas in response to acute inflammation. ADM can rapidly progress towards pre-malignant pancreatic intraepithelial neoplasia (PanIN) lesions in the presence of mutant KRas and ultimately pancreatic adenocarcinoma (PDAC). In the present work, we elucidate the role and related mechanism of glycogen synthase kinase-3beta (GSK-3β) in ADM development using in vitro 3D cultures and genetically engineered mouse models. We show that GSK-3β promotes TGF-α-induced ADM in 3D cultured primary acinar cells, whereas deletion of GSK-3β attenuates caerulein-induced ADM formation and PanIN progression in Kras G12D transgenic mice. Furthermore, we demonstrate that GSK-3β ablation influences ADM formation and PanIN progression by suppressing oncogenic KRas-driven cell proliferation. Mechanistically, we show that GSK-3β regulates proliferation by increasing the activation of S6 kinase. Taken together, these results indicate that GSK-3β participates in early pancreatitis-induced ADM and thus could be a target for the treatment of chronic pancreatitis and the prevention of PDAC progression. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. Copyright © 2017 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Alteration of major vault protein in human glioblastoma and its relation with EGFR and PTEN status.

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Navarro, L; Gil-Benso, R; Megías, J; Muñoz-Hidalgo, L; San-Miguel, T; Callaghan, R C; González-Darder, J M; López-Ginés, C; Cerdá-Nicolás, M J

2015-06-25

Glioblastoma (GBM) is the most frequent and malignant primary brain tumor. Conventional therapy of surgical removal, radiation and chemotherapy is largely palliative. Major vault protein (MVP), the main component of the vault organelle has been associated with multidrug resistance by reducing cellular accumulation of chemotherapeutic agents. With regard to cancer, MVP has been shown to be overexpressed in drug resistance development and malignant progression. The aim of the present study was to evaluate the MVP gene dosage levels in 113 archival samples from GBM and its correlation with patients' survival and epidermal growth factor receptor (EGFR) and phosphatase and tensin homolog (PTEN) gene dosages. Fluorescent in situ hybridization revealed polysomy of chromosome 7 in 76.1% of the GBMs and EGFR amplification in a 64.6% of the tumors. Genetic status of EGFR, PTEN and MVP copies was determined by multiplex ligation-dependent probe amplification (MLPA) technique. 31% of the tumors showed the EGFR is variant III mutation (EGFRvIII) mutation and 74.3% of them presented amplification of MVP gene. Amplification of EGFR and MVP was found in a 63.7% and 56.6% of the GBM, respectively. An inverse correlation between MVP and PTEN dosage values was observed. Besides, an inverse relationship between the survival of the patients treated with chemotherapy and the levels of MVP copies was determined. In conclusion, our study reveals an important role of MVP, together with EGFRvIII and PTEN, in the progression of GBM and proposes it as a novel and interesting target for new treatment approaches. Copyright © 2015 IBRO. Published by Elsevier Ltd. All rights reserved.

Protein kinase C activation induces conductance changes in Hermissenda photoreceptors like those seen in associative learning.

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Farley, J; Auerbach, S

Phosphorylation of ion channels has been suggested as one molecular mechanism responsible for learning-produced long-term changes in neuronal excitability. Persistent training-produced changes in two distinct K+ currents (IA (ref. 2), IK-Ca (refs 3,4)) and a voltage-dependent calcium current (ICa; refs 3,4) have previously been shown to occur in type B photoreceptors of Hermissenda, as a result of associative learning. But the identity of the phosphorylation pathway(s) responsible for these changes has not as yet been determined. Injections of cyclic AMP-dependent protein kinase reduce a K+ current (IK) in B cells which is different from those changed by training, but fails to reduce IA and IK-Ca. Phosphorylase b kinase (an exogenous calcium/calmodulin-dependent kinase) reduces IA, but whether IK-Ca and ICa are changed in the manner of associative training is not yet known. Another protein kinase present in high concentrations in both mammalian brain and molluscan nervous systems is protein kinase C, which is both calcium- and phospholipid-sensitive. We now present evidence that activation of protein kinase C by the tumour promoter phorbol ester (PDB) and intracellular injection of the enzyme induce conductance changes similar to those caused by associative training in Hermissenda B cells (that is a reduction of IA and IK-Ca, and enhancement of ICa). These results represent the first direct demonstration that protein kinase C affects membrane K+ ion conductance mechanisms.

Eotaxin induces degranulation and chemotaxis of eosinophils through the activation of ERK2 and p38 mitogen-activated protein kinases

DEFF Research Database (Denmark)

Kampen, G T; Stafford, S; Adachi, T

2000-01-01

Eotaxin and other CC chemokines acting via CC chemokine receptor-3 (CCR3) are believed to play an integral role in the development of eosinophilic inflammation in asthma and allergic inflammatory diseases. However, little is known about the intracellular events following agonist binding to CCR3...... and the relationship of these events to the functional response of the cell. The objectives of this study were to investigate CCR3-mediated activation of the mitogen-activated protein (MAP) kinases extracellular signal-regulated kinase-2 (ERK2), p38, and c-jun N-terminal kinase (JNK) in eosinophils and to assess...... the requirement for MAP kinases in eotaxin-induced eosinophil cationic protein (ECP) release and chemotaxis. MAP kinase activation was studied in eotaxin-stimulated eosinophils (more than 97% purity) by Western blotting and immune-complex kinase assays. ECP release was measured by radioimmunoassay. Chemotaxis...

Fish oil suppresses cell growth and metastatic potential by regulating PTEN and NF-κB signaling in colorectal cancer.

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Shevali Kansal

Full Text Available Homeostasis in eukaryotic tissues is tightly regulated by an intricate balance of the prosurvival and antisurvival signals. The tumor suppressor PTEN (phosphatase and tensin homolog deleted on chromosome 10, a dual-specificity phosphatase, plays a functional role in cell cycle arrest and apoptosis. NF-κB and its downstream regulators (such as VEGF play a central role in prevention of apoptosis, promotion of inflammation and tumor growth. Therefore, we thought to estimate the expression of PTEN, Poly-ADP-ribose polymerase (PARP, NF-κBp50, NF-κBp65 and VEGF to evaluate the effect of supplementation of fish oil on apoptotic and inflammatory signaling in colon carcinoma. Male wistar rats in Group I received purified diet while Group II and III received modified diet supplemented with FO∶CO(1∶1&FO∶CO(2.5∶1 respectively. These were further subdivided into controls receiving ethylenediamine-tetra acetic-acid and treated groups received dimethylhydrazine-dihydrochloride (DMH/week for 4 weeks. Animals sacrificed 48 hours after last injection constituted initiation phase and that sacrificed after 16 weeks constituted post-initiation phase. We have analysed expression of PTEN, NF-κBp50, NF-κBp65 by flowcytometer and nuclear localization of NF-κB by immunofluorescence. PARP and VEGF were assessed by immunohistochemistry. In the initiation phase, animals receiving DMH have shown increased % of apoptotic cells, PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF however in post-initiation phase no significant alteration in apoptosis with decreased PTEN and increased PARP, NF-κBp50, NF-κBp65 and VEGF were observed as compared to control animals. On treatment with both ratios of fish oil in both the phases, augmentation in % of apoptotic cells, decreased PTEN, PARP, NF-κBp50, NF-κBp65 and VEGF were documented with respect to DMH treated animals with effect being more exerted with higher ration in post-initiation phase. Hence, fish oil activates

Growth- and Stress-Induced PASTA Kinase Phosphorylation in Enterococcus faecalis.

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Labbe, Benjamin D; Kristich, Christopher J

2017-11-01

Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes Such PASTA kinases regulate critical processes, including antibiotic resistance, cell division, toxin production, and virulence, and are essential for viability in certain organisms. Based on in vitro studies with purified extracellular and intracellular fragments of PASTA kinases, a model for signaling has been proposed, in which the extracellular PASTA domains bind currently undefined ligands (typically thought to be peptidoglycan, or fragments thereof) to drive kinase dimerization, which leads to enhanced kinase autophosphorylation and enhanced phosphorylation of substrates. However, this model has not been rigorously tested in vivo Enterococcus faecalis is a Gram-positive intestinal commensal and major antibiotic-resistant opportunistic pathogen. In E. faecalis , the PASTA kinase IreK drives intrinsic resistance to cell wall-active antimicrobials, suggesting that such antimicrobials may trigger IreK signaling. Here we show that IreK responds to cell wall stress in vivo by enhancing its phosphorylation and that of a downstream substrate. This response requires both the extracellular PASTA domains and specific phosphorylatable residues in the kinase domain. Thus, our results provide in vivo evidence, with an intact full-length PASTA kinase in its native physiological environment, that supports the prevailing model of PASTA kinase signaling. In addition, we show that IreK responds to a signal associated with growth and/or cell division, in the absence of cell wall-active antimicrobials. Surprisingly, the ability of IreK to respond to growth and/or division does not require the extracellular PASTA domains, suggesting that IreK monitors multiple parameters for sensory input in vivo IMPORTANCE Transmembrane Ser/Thr kinases containing extracellular PASTA domains are ubiquitous among Actinobacteria and Firmicutes and regulate critical processes. The

A Global Analysis of Kinase Function in Candida albicans Hyphal Morphogenesis Reveals a Role for the Endocytosis Regulator Akl1.

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Bar-Yosef, Hagit; Gildor, Tsvia; Ramírez-Zavala, Bernardo; Schmauch, Christian; Weissman, Ziva; Pinsky, Mariel; Naddaf, Rawi; Morschhäuser, Joachim; Arkowitz, Robert A; Kornitzer, Daniel

2018-01-01

The human pathogenic fungus Candida albicans can switch between yeast and hyphal morphologies as a function of environmental conditions and cellular physiology. The yeast-to-hyphae morphogenetic switch is activated by well-established, kinase-based signal transduction pathways that are induced by extracellular stimuli. In order to identify possible inhibitory pathways of the yeast-to-hyphae transition, we interrogated a collection of C. albicans protein kinases and phosphatases ectopically expressed under the regulation of the TETon promoter. Proportionately more phosphatases than kinases were identified that inhibited hyphal morphogenesis, consistent with the known role of protein phosphorylation in hyphal induction. Among the kinases, we identified AKL1 as a gene that significantly suppressed hyphal morphogenesis in serum. Akl1 specifically affected hyphal elongation rather than initiation: overexpression of AKL1 repressed hyphal growth, and deletion of AKL1 resulted in acceleration of the rate of hyphal elongation. Akl1 suppressed fluid-phase endocytosis, probably via Pan1, a putative clathrin-mediated endocytosis scaffolding protein. In the absence of Akl1, the Pan1 patches were delocalized from the sub-apical region, and fluid-phase endocytosis was intensified. These results underscore the requirement of an active endocytic pathway for hyphal morphogenesis. Furthermore, these results suggest that under standard conditions, endocytosis is rate-limiting for hyphal elongation.

Cardiac hyporesponsiveness in severe sepsis is associated with nitric oxide-dependent activation of G protein receptor kinase.

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Dal-Secco, Daniela; DalBó, Silvia; Lautherbach, Natalia E S; Gava, Fábio N; Celes, Mara R N; Benedet, Patricia O; Souza, Adriana H; Akinaga, Juliana; Lima, Vanessa; Silva, Katiussia P; Kiguti, Luiz Ricardo A; Rossi, Marcos A; Kettelhut, Isis C; Pupo, André S; Cunha, Fernando Q; Assreuy, Jamil

2017-07-01

G protein-coupled receptor kinase isoform 2 (GRK2) has a critical role in physiological and pharmacological responses to endogenous and exogenous substances. Sepsis causes an important cardiovascular dysfunction in which nitric oxide (NO) has a relevant role. The present study aimed to assess the putative effect of inducible NO synthase (NOS2)-derived NO on the activity of GRK2 in the context of septic cardiac dysfunction. C57BL/6 mice were submitted to severe septic injury by cecal ligation and puncture (CLP). Heart function was assessed by isolated and perfused heart, echocardiography, and β-adrenergic receptor binding. GRK2 was determined by immunofluorescence and Western blot analysis in the heart and isolated cardiac myocytes. Sepsis increased NOS2 expression in the heart, increased plasma nitrite + nitrate levels, and reduced isoproterenol-induced isolated ventricle contraction, whole heart tension development, and β-adrenergic receptor density. Treatment with 1400W or with GRK2 inhibitor prevented CLP-induced cardiac hyporesponsiveness 12 and 24 h after CLP. Increased labeling of total and phosphorylated GRK2 was detected in hearts after CLP. With treatment of 1400W or in hearts taken from septic NOS2 knockout mice, the activation of GRK2 was reduced. 1400W or GRK2 inhibitor reduced mortality, improved echocardiographic cardiac parameters, and prevented organ damage. Therefore, during sepsis, NOS2-derived NO increases GRK2, which leads to a reduction in β-adrenergic receptor density, contributing to the heart dysfunction. Isolated cardiac myocyte data indicate that NO acts through the soluble guanylyl cyclase/cGMP/PKG pathway. GRK2 inhibition may be a potential therapeutic target in sepsis-induced cardiac dysfunction. NEW & NOTEWORTHY The main novelty presented here is to show that septic shock induces cardiac hyporesponsiveness to isoproterenol by a mechanism dependent on nitric oxide and mediated by G protein-coupled receptor kinase isoform 2. Therefore

Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK) Complex.

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Frey, Stefan; Reschka, Eva J; Pöggeler, Stefanie

2015-01-01

The striatin-interacting phosphatase and kinase (STRIPAK) complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs) MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H) interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP). In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

Germinal Center Kinases SmKIN3 and SmKIN24 Are Associated with the Sordaria macrospora Striatin-Interacting Phosphatase and Kinase (STRIPAK Complex.

Directory of Open Access Journals (Sweden)

Stefan Frey

Full Text Available The striatin-interacting phosphatase and kinase (STRIPAK complex is composed of striatin, protein phosphatase PP2A and protein kinases that regulate development in animals and fungi. In the filamentous ascomycete Sordaria macrospora, it is required for fruiting-body development and cell fusion. Here, we report on the presence and function of STRIPAK-associated kinases in ascomycetes. Using the mammalian germinal center kinases (GCKs MST4, STK24, STK25 and MINK1 as query, we identified the two putative homologs SmKIN3 and SmKIN24 in S. macrospora. A BLASTP search revealed that both kinases are conserved among filamentous ascomycetes. The physical interaction of the striatin homolog PRO11 with SmKIN3 and SmKIN24 were verified by yeast two-hybrid (Y2H interaction studies and for SmKIN3 by co-Immunoprecipitation (co-IP. In vivo localization found that both kinases were present at the septa and deletion of both Smkin3 and Smkin24 led to abnormal septum distribution. While deletion of Smkin3 caused larger distances between adjacent septa and increased aerial hyphae, deletion of Smkin24 led to closer spacing of septa and to sterility. Although phenotypically distinct, both kinases appear to function independently because the double-knockout strain ΔSmkin3/ΔSmkin24 displayed the combined phenotypes of each single-deletion strain.

Odontogenic ameloblast-associated protein (ODAM) inhibits growth and migration of human melanoma cells and elicits PTEN elevation and inactivation of PI3K/AKT signaling

International Nuclear Information System (INIS)

Foster, James S; Fish, Lindsay M; Phipps, Jonathan E; Bruker, Charles T; Lewis, James M; Bell, John L; Solomon, Alan; Kestler, Daniel P

2013-01-01

The Odontogenic Ameloblast-associated Protein (ODAM) is expressed in a wide range of normal epithelial, and neoplastic tissues, and we have posited that ODAM serves as a novel prognostic biomarker for breast cancer and melanoma. Transfection of ODAM into breast cancer cells yields suppression of cellular growth, motility, and in vivo tumorigenicity. Herein we have extended these studies to the effects of ODAM on cultured melanoma cell lines. The A375 and C8161 melanoma cell lines were stably transfected with ODAM and assayed for properties associated with tumorigenicity including cell growth, motility, and extracellular matrix adhesion. In addition, ODAM–transfected cells were assayed for signal transduction via AKT which promotes cell proliferation and survival in many neoplasms. ODAM expression in A375 and C8161 cells strongly inhibited cell growth and motility in vitro, increased cell adhesion to extracellular matrix, and yielded significant cytoskeletal/morphologic rearrangement. Furthermore, AKT activity was downregulated by ODAM expression while an increase was noted in expression of the PTEN (phosphatase and tensin homolog on chromosome 10) tumor suppressor gene, an antagonist of AKT activation. Increased PTEN in ODAM-expressing cells was associated with increases in PTEN mRNA levels and de novo protein synthesis. Silencing of PTEN expression yielded recovery of AKT activity in ODAM-expressing melanoma cells. Similar PTEN elevation and inhibition of AKT by ODAM was observed in MDA-MB-231 breast cancer cells while ODAM expression had no effect in PTEN-deficient BT-549 breast cancer cells. The apparent anti-neoplastic effects of ODAM in cultured melanoma and breast cancer cells are associated with increased PTEN expression, and suppression of AKT activity. This association should serve to clarify the clinical import of ODAM expression and any role it may serve as an indicator of tumor behavior

Platelet-activating factor stimulation of tyrosine kinase and its relationship to phospholipase C in rabbit platelets: Studies with genistein and monoclonal antibody to phosphotyrosine

International Nuclear Information System (INIS)

Dhar, A.; Paul, A.K.; Shukla, S.D.

1990-01-01

Platelet-activating factor (PAF) is a proinflammatory lipid that has platelet-stimulating property. PAF receptor-coupled activation of phosphoinositide-specific phospholipase C (PLC) and phosphorylation of several proteins has already been established in our laboratory. To investigate further the molecular mechanism and relationship between activation of PLC and protein phosphorylation, we have used Genistein (a putative inhibitor of tyrosine-specific protein kinases), phosphotyrosine antibody, and phosphoamino acid analysis to probe the involvement of tyrosine kinase in this process. Washed rabbit platelets were loaded with myo-[2-3H]inositol and challenged with PAF (100 nM) after pretreatment with Genistein. PLC-mediated production of radioactive inositol monophosphate, inositol diphosphate, and inositol triphosphate was monitored. PAF alone caused stimulation of PLC activity [( 3H]inositol triphosphate production), whereas pretreatment with Genistein (0.5 mM) diminished PAF-stimulated PLC activity to basal level. Genistein also blocked PAF-stimulated platelet aggregation at this dose. In contrast to Genistein, staurosporine which inhibits protein kinase C, potentiated PAF-stimulated [3H]inositol triphosphate production. Genistein substantially inhibited the combined effects of staurosporine and PAF on inositol triphosphate production. Genistein also reduced PAF-induced phosphorylation of Mr 20,000 and 50,000 proteins. Phorbol 12-myristate 13-acetate-induced Mr 40,000 protein phosphorylation was also affected by Genistein. The above results suggested that Genistein inhibited tyrosine kinase at an early stage of signal transduction by inhibiting PLC. This, in turn, decreased the activation of protein kinase C and, therefore, caused a reduction in Mr 40,000 protein phosphorylation

KRAS and BRAF Mutations and PTEN Expression Do Not Predict Efficacy of Cetuximab-Based Chemoradiotherapy in Locally Advanced Rectal Cancer

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Erben, Philipp, E-mail: philipp.erben@medma.uni-heidelberg.de [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Stroebel, Philipp [Pathologisches Institut, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Horisberger, Karoline [Chirurgische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Popa, Juliana; Bohn, Beatrice; Hanfstein, Benjamin [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Kaehler, Georg; Kienle, Peter; Post, Stefan [Chirurgische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Wenz, Frederik [Klinik fuer Strahlentherapie und Radioonkologie, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Hochhaus, Andreas [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany); Klinik fuer Innere Medizin II, Abteilung Haematologie/Onkologie, Universitaetsklinikum Jena, Jena (Germany); Hofheinz, Ralf-Dieter [III. Medizinische Klinik, Universitaetsmedizin Mannheim, Universitaet Heidelberg, Mannheim (Germany)

2011-11-15

Purpose: Mutations in KRAS and BRAF genes as well as the loss of expression of phosphatase and tensin homolog (PTEN) (deleted on chromosome 10) are associated with impaired activity of antibodies directed against epidermal growth factor receptor in patients with metastatic colorectal cancer. The predictive and prognostic value of the KRAS and BRAF point mutations as well as PTEN expression in patients with locally advanced rectal cancer (LARC) treated with cetuximab-based neoadjuvant chemoradiotherapy is unknown. Methods and Materials: We have conducted phase I and II trials of the combination of weekly administration of cetuximab and irinotecan and daily doses of capecitabine in conjunction with radiotherapy (45 Gy plus 5.4 Gy) in patients with LARC (stage uT3/4 or uN+). The status of KRAS and BRAF mutations was determined with direct sequencing, and PTEN expression status was determined with immunohistochemistry testing of diagnostic tumor biopsies. Tumor regression was evaluated by using standardized regression grading, and disease-free survival (DFS) was calculated according to the Kaplan-Meier method. Results: A total of 57 patients were available for analyses. A total of 31.6% of patients carried mutations in the KRAS genes. No BRAF mutations were found, while the loss of PTEN expression was observed in 9.6% of patients. Six patients achieved complete remission, and the 3-year DFS rate was 73%. No correlation was seen between tumor regression or DFS rate and a single marker or a combination of all markers. Conclusions: In the present series, no BRAF mutation was detected. The presence of KRAS mutations and loss of PTEN expression were not associated with impaired response to cetuximab-based chemoradiotherapy and 3-year DFS.

KRAS and BRAF Mutations and PTEN Expression Do Not Predict Efficacy of Cetuximab-Based Chemoradiotherapy in Locally Advanced Rectal Cancer

International Nuclear Information System (INIS)

Erben, Philipp; Ströbel, Philipp; Horisberger, Karoline; Popa, Juliana; Bohn, Beatrice; Hanfstein, Benjamin; Kähler, Georg; Kienle, Peter; Post, Stefan; Wenz, Frederik; Hochhaus, Andreas; Hofheinz, Ralf-Dieter

2011-01-01

Purpose: Mutations in KRAS and BRAF genes as well as the loss of expression of phosphatase and tensin homolog (PTEN) (deleted on chromosome 10) are associated with impaired activity of antibodies directed against epidermal growth factor receptor in patients with metastatic colorectal cancer. The predictive and prognostic value of the KRAS and BRAF point mutations as well as PTEN expression in patients with locally advanced rectal cancer (LARC) treated with cetuximab-based neoadjuvant chemoradiotherapy is unknown. Methods and Materials: We have conducted phase I and II trials of the combination of weekly administration of cetuximab and irinotecan and daily doses of capecitabine in conjunction with radiotherapy (45 Gy plus 5.4 Gy) in patients with LARC (stage uT3/4 or uN+). The status of KRAS and BRAF mutations was determined with direct sequencing, and PTEN expression status was determined with immunohistochemistry testing of diagnostic tumor biopsies. Tumor regression was evaluated by using standardized regression grading, and disease-free survival (DFS) was calculated according to the Kaplan–Meier method. Results: A total of 57 patients were available for analyses. A total of 31.6% of patients carried mutations in the KRAS genes. No BRAF mutations were found, while the loss of PTEN expression was observed in 9.6% of patients. Six patients achieved complete remission, and the 3-year DFS rate was 73%. No correlation was seen between tumor regression or DFS rate and a single marker or a combination of all markers. Conclusions: In the present series, no BRAF mutation was detected. The presence of KRAS mutations and loss of PTEN expression were not associated with impaired response to cetuximab-based chemoradiotherapy and 3-year DFS.

Spontaneous nisin-resistant Listeria monocytogenes mutants with increased expression of a putative penicillin-binding protein and their sensitivity to various antibiotics

DEFF Research Database (Denmark)

Gravesen, Anne; Sorensen, K.; Aarestrup, Frank Møller

2001-01-01

-molecular-weight penicillin-binding proteins (PBPs), a histidine protein kinase, a protein of unknown function, and ClpB (putative functions from homology), The three former proteins had increased expression in a total of six out of 10 independent mutants originating from five different wildtype strains, indicating...

Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status

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Djuzenova, Cholpon S.; Fiedler, Vanessa; Memmel, Simon; Katzer, Astrid; Hartmann, Susanne; Krohne, Georg; Zimmermann, Heiko; Scholz, Claus-Jürgen; Polat, Bülent; Flentje, Michael

2015-01-01

Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion. - Highlights: • We examine 5 glioblastoma lines on the invasion capacity and actin cytoskeleton. • Glioblastoma cell lines mutated in both p53 and PTEN were the most invasive. • Less invasive cells showed much less lamellipodia, but more actin stress fibers. • A mechanism for the differences in tumor cell invasion is proposed

Actin cytoskeleton organization, cell surface modification and invasion rate of 5 glioblastoma cell lines differing in PTEN and p53 status

Energy Technology Data Exchange (ETDEWEB)

Djuzenova, Cholpon S., E-mail: djuzenova_t@ukw.de [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); Fiedler, Vanessa [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); Memmel, Simon [Lehrstuhl für Biotechnologie und Biophysik, Universität Würzburg, Biozentrum Am Hubland, 97070 Würzburg (Germany); Katzer, Astrid; Hartmann, Susanne [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); Krohne, Georg [Elektronenmikroskopie, Biozentrum, Universität Würzburg, Am Hubland, 97070 Würzburg (Germany); Zimmermann, Heiko [Hauptabteilung Biophysik and Kryotechnologie, Fraunhofer-Institut für Biomedizinische Technik, Lehrstuhl für Molekulare und Zelluläre Biotechnologie/Nanotechnologie, Universität des Saarlandes, Ensheimer Strasse 48, 66386 St. Ingbert (Germany); Scholz, Claus-Jürgen [Interdisciplinary Center for Clinical Research, University Hospital, Versbacher Strasse 7, 97078 Würzburg (Germany); Polat, Bülent; Flentje, Michael [Department of Radiation Oncology, University Hospital, Josef-Schneider-Strasse 11, D-97080 Würzburg (Germany); and others

2015-01-15

Glioblastoma cells exhibit highly invasive behavior whose mechanisms are not yet fully understood. The present study explores the relationship between the invasion capacity of 5 glioblastoma cell lines differing in p53 and PTEN status, expression of mTOR and several other marker proteins involved in cell invasion, actin cytoskeleton organization and cell morphology. We found that two glioblastoma lines mutated in both p53 and PTEN genes (U373-MG and SNB19) exhibited the highest invasion rates through the Matrigel or collagen matrix. In DK-MG (p53wt/PTENwt) and GaMG (p53mut/PTENwt) cells, F-actin mainly occurred in the numerous stress fibers spanning the cytoplasm, whereas U87-MG (p53wt/PTENmut), U373-MG and SNB19 (both p53mut/PTENmut) cells preferentially expressed F-actin in filopodia and lamellipodia. Scanning electron microscopy confirmed the abundant filopodia and lamellipodia in the PTEN mutated cell lines. Interestingly, the gene profiling analysis revealed two clusters of cell lines, corresponding to the most (U373-MG and SNB19, i.e. p53 and PTEN mutated cells) and less invasive phenotypes. The results of this study might shed new light on the mechanisms of glioblastoma invasion. - Highlights: • We examine 5 glioblastoma lines on the invasion capacity and actin cytoskeleton. • Glioblastoma cell lines mutated in both p53 and PTEN were the most invasive. • Less invasive cells showed much less lamellipodia, but more actin stress fibers. • A mechanism for the differences in tumor cell invasion is proposed.

Activation of the Cph1-dependent MAP kinase signaling pathway induces white-opaque switching in Candida albicans.

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Bernardo Ramírez-Zavala

Full Text Available Depending on the environmental conditions, the pathogenic yeast Candida albicans can undergo different developmental programs, which are controlled by dedicated transcription factors and upstream signaling pathways. C. albicans strains that are homozygous at the mating type locus can switch from the normal yeast form (white to an elongated cell type (opaque, which is the mating-competent form of this fungus. Both white and opaque cells use the Ste11-Hst7-Cek1/Cek2 MAP kinase signaling pathway to react to the presence of mating pheromone. However, while opaque cells employ the transcription factor Cph1 to induce the mating response, white cells recruit a different downstream transcription factor, Tec1, to promote the formation of a biofilm that facilitates mating of opaque cells in the population. The switch from the white to the opaque cell form is itself induced by environmental signals that result in the upregulation of the transcription factor Wor1, the master regulator of white-opaque switching. To get insight into the upstream signaling pathways controlling the switch, we expressed all C. albicans protein kinases from a tetracycline-inducible promoter in a switching-competent strain. Screening of this library of strains showed that a hyperactive form of Ste11 lacking its N-terminal domain (Ste11(ΔN467 efficiently stimulated white cells to switch to the opaque phase, a behavior that did not occur in response to pheromone. Ste11(ΔN467-induced switching specifically required the downstream MAP kinase Cek1 and its target transcription factor Cph1, but not Cek2 and Tec1, and forced expression of Cph1 also promoted white-opaque switching in a Wor1-dependent manner. Therefore, depending on the activation mechanism, components of the pheromone-responsive MAP kinase pathway can be reconnected to stimulate an alternative developmental program, switching of white cells to the mating-competent opaque phase.

Resveratrol induces growth arrest and apoptosis through activation of FOXO transcription factors in prostate cancer cells.

Directory of Open Access Journals (Sweden)

Qinghe Chen

2010-12-01

Full Text Available Resveratrol, a naturally occurring phytopolyphenol compound, has attracted extensive interest in recent years because of its diverse pharmacological characteristics. Although resveratrol possesses chemopreventive properties against several cancers, the molecular mechanisms by which it inhibits cell growth and induces apoptosis have not been clearly understood. The present study was carried out to examine whether PI3K/AKT/FOXO pathway mediates the biological effects of resveratrol.Resveratrol inhibited the phosphorylation of PI3K, AKT and mTOR. Resveratrol, PI3K inhibitors (LY294002 and Wortmannin and AKT inhibitor alone slightly induced apoptosis in LNCaP cells. These inhibitors further enhanced the apoptosis-inducing potential of resveratrol. Overexpression of wild-type PTEN slightly induced apoptosis. Wild type PTEN and PTEN-G129E enhanced resveratrol-induced apoptosis, whereas PTEN-G129R had no effect on proapoptotic effects of resveratrol. Furthermore, apoptosis-inducing potential of resveratrol was enhanced by dominant negative AKT, and inhibited by wild-type AKT and constitutively active AKT. Resveratrol has no effect on the expression of FKHR, FKHRL1 and AFX genes. The inhibition of FOXO phosphorylation by resveratrol resulted in its nuclear translocation, DNA binding and transcriptional activity. The inhibition of PI3K/AKT pathway induced FOXO transcriptional activity resulting in induction of Bim, TRAIL, p27/KIP1, DR4 and DR5, and inhibition of cyclin D1. Similarly, resveratrol-induced FOXO transcriptional activity was further enhanced when activation of PI3K/AKT pathway was blocked. Over-expression of phosphorylation deficient mutants of FOXO proteins (FOXO1-TM, FOXO3A-TM and FOXO4-TM induced FOXO transcriptional activity, which was further enhanced by resveratrol. Inhibition of FOXO transcription factors by shRNA blocked resveratrol-induced upregulation of Bim, TRAIL, DR4, DR5, p27/KIP1 and apoptosis, and inhibition of cyclin D1 by

Rho kinase inhibitor fasudil mitigates high-cholesterol diet-induced hypercholesterolemia and vascular damage.

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Abdali, Nibrass Taher; Yaseen, Awny H; Said, Eman; Ibrahim, Tarek M

2017-04-01

The current study was designed to investigate the potential beneficial therapeutic outcome of Rho kinase inhibitor (fasudil) against hypercholesterolemia-induced myocardial and vascular injury in rabbits together with diet modification. Sixteen male rabbits were randomly divided into four groups: normal control group which received standard rabbit chow, hypercholesterolemic control group, and treated groups which received cholesterol-rich rabbit chow (1.5% cholesterol) for 8 weeks. Treated groups received either fasudil (100 mg/kg/day) or rosuvastatin (2.5 mg/kg/day) starting from the ninth week for further 4 weeks with interruption of the cholesterol-rich chow. Biochemical assessment of serum cholesterol, triglyceride, high-density lipoprotein (HDL), low-density lipoprotein (LDL), and myocardial oxidative/antioxidant biomarkers malondialdehyde (MDA), superoxide dismutase (SOD), and reduced glutathione (GSH), besides biochemical assessment of serum nitric oxide (NO), creatine kinase (CK), and lactate dehydrogenase (LDH) activities and serum total antioxidant capacity (TAC), was conducted. Serum vascular cell adhesion molecule 1 (VCAM-1) and serum Rho-associated protein kinase 1 (ROCK-1) were also evaluated along with histopathological examination of aorta specimens. Fasudil administration significantly decreased serum cholesterol, triglyceride (TG), and LDL and significantly increased serum HDL, with concomitant decrease in serum CK and LDH activities, NO, and restoration of serum TAC. Myocardial MDA significantly declined; SOD activity and GSH contents were restored. Serum ROCK-1 and VCAM-1 levels significantly declined as well. Vascular improvement was confirmed with histopathological examination, which revealed normal aortic intema with the absence of atheromas. Fasudil has promising anti-atherogenic activity mediated primarily via alleviation of hypercholesterolemia-induced oxidative stress and modulation of inflammatory response.

JAK kinases are required for the bacterial RNA and poly I:C induced tyrosine phosphorylation of PKR

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Bleiblo, Farag; Michael, Paul; Brabant, Danielle; Ramana, Chilakamarti V; Tai, TC; Saleh, Mazen; Parrillo, Joseph E; Kumar, Anand; Kumar, Aseem

2013-01-01

Discriminating the molecular patterns associated with RNA is central to innate immunity. The protein kinase PKR is a cytosolic sensor involved in the recognition of viral dsRNA and triggering interferon-induced signaling. Here, we identified bacterial RNA as a novel distinct pattern recognized by PKR. We show that the tyrosine phosphorylation of PKR induced by either bacterial RNA or poly I:C is impaired in mutant cells lacking TYK2, JAK1, or JAK2 kinases. PKR was found to be a direct substrate for the activated JAKs. Our results indicated that the double-stranded structures of bacterial RNA are required to fully activate PKR. These results suggest that bacterial RNA signaling is analogous in some respects to that of viral RNA and interferons and may have implications in bacterial immunity. PMID:23236554

Phenotypic analyses of Arabidopsis T-DNA insertion lines and expression profiling reveal that multiple L-type lectin receptor kinases are involved in plant immunity

NARCIS (Netherlands)

Wang, Y.; Bouwmeester, K.; Beseh, P.; Shan, W.; Govers, F.

2014-01-01

L-type lectin receptor kinases (LecRKs) are membrane-spanning receptor-like kinases with putative roles in biotic and abiotic stress responses and in plant development. In Arabidopsis, 45 LecRKs were identified but their functions are largely unknown. Here, a systematic functional analysis was

Fps/Fes and Fer non-receptor protein-tyrosine kinases regulate collagen- and ADP-induced platelet aggregation.

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Senis, Y A; Sangrar, W; Zirngibl, R A; Craig, A W B; Lee, D H; Greer, P A

2003-05-01

Fps/Fes and Fer proto-oncoproteins are structurally related non-receptor protein-tyrosine kinases implicated in signaling downstream from cytokines, growth factors and immune receptors. We show that Fps/Fes and Fer are expressed in human and mouse platelets, and are activated following stimulation with collagen and collagen-related peptide (CRP), suggesting a role in GPVI receptor signaling. Fer was also activated following stimulation with thrombin and a protease-activated receptor4 (PAR4)-activating peptide, suggesting a role in signaling downstream from the G protein-coupled PAR4. There were no detectable perturbations in CRP-induced activation of Syk, PLCgamma2, cortactin, Erk, Jnk, Akt or p38 in platelets from mice lacking Fps/Fes, Fer, or both kinases. Platelets lacking Fps/Fes, from a targeted fps/fes null strain of mice, showed increased rates and amplitudes of collagen-induced aggregation, relative to wild-type platelets. P-Selectin expression was also elevated on the surface of Fps/Fes-null platelets in response to CRP. Fer-deficient platelets, from mice targeted with a kinase-inactivating mutation, disaggregated more rapidly than wild-type platelets in response to ADP. This report provides the first evidence that Fps/Fes and Fer are expressed in platelets and become activated downstream from the GPVI collagen receptor, and that Fer is activated downstream from a G-protein coupled receptor. Furthermore, using targeted mouse models we show that deficiency in Fps/Fes or Fer resulted in disregulated platelet aggregation and disaggregation, demonstrating a role for these kinases in regulating platelet functions.

Ghrelin augments murine T-cell proliferation by activation of the phosphatidylinositol-3-kinase, extracellular signal-regulated kinase and protein kinase C signaling pathways

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Lee, Jun Ho; Patel, Kalpesh; Tae, Hyun Jin; Lustig, Ana; Kim, Jie Wan; Mattson, Mark P.; Taub, Dennis D.

2014-01-01

Thymic atrophy occurs during normal aging, and is accelerated by exposure to chronic stressors that elevate glucocorticoid levelsand impair the naïve T cell output. The orexigenic hormone ghrelin was recently shown to attenuate age-associated thymic atrophy. Here, we report that ghrelin enhances the proliferation of murine CD4+ primary T cells and a CD4+ T-cell line. Ghrelin induced activation of the ERK1/2 and Akt signaling pathways, via upstream activation of phosphatidylinositol-3-kinase and protein kinase C, to enhance T-cell proliferation. Moreover, ghrelin induced expression of the cell cycle proteins cyclin D1, cyclin E, cyclin-dependent kinase 2 (CDK2) and retinoblastoma phosphorylation. Finally, ghrelin activated the above-mentioned signaling pathways and stimulated thymocyte proliferation in young and older mice in vivo. PMID:25447526

CIKS, a connection to Ikappa B kinase and stress-activated protein kinase.

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Leonardi, A; Chariot, A; Claudio, E; Cunningham, K; Siebenlist, U

2000-09-12

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-kappaB and AP-1/ATF families. Activation of NF-kappaB factors is thought to be mediated primarily via IkappaB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKalpha and IKKbeta are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-kappaB essential modulator)/IKKgamma. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKgamma in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-kappaB-dependent reporter. Activation of NF-kappaB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins.

CIKS, a connection to IκB kinase and stress-activated protein kinase

Science.gov (United States)

Leonardi, Antonio; Chariot, Alain; Claudio, Estefania; Cunningham, Kirk; Siebenlist, Ulrich

2000-01-01

Pathogens, inflammatory signals, and stress cause acute transcriptional responses in cells. The induced expression of genes in response to these signals invariably involves transcription factors of the NF-κB and AP-1/ATF families. Activation of NF-κB factors is thought to be mediated primarily via IκB kinases (IKK), whereas that of AP-1/ATF can be mediated by stress-activated protein kinases (SAPKs; also named Jun kinases or JNKs). IKKα and IKKβ are two catalytic subunits of a core IKK complex that also contains the regulatory subunit NEMO (NF-κB essential modulator)/IKKγ. The latter protein is essential for activation of the IKKs, but its mechanism of action is not known. Here we describe the molecular cloning of CIKS (connection to IKK and SAPK/JNK), a previously unknown protein that directly interacts with NEMO/IKKγ in cells. When ectopically expressed, CIKS stimulates IKK and SAPK/JNK kinases and it transactivates an NF-κB-dependent reporter. Activation of NF-κB is prevented in the presence of kinase-deficient, interfering mutants of the IKKs. CIKS may help to connect upstream signaling events to IKK and SAPK/JNK modules. CIKS could coordinate the activation of two stress-induced signaling pathways, functions reminiscent of those noted for tumor necrosis factor receptor-associated factor adaptor proteins. PMID:10962033

Modeling invasive lobular breast carcinoma by CRISPR/Cas9-mediated somatic genome editing of the mammary gland.

Science.gov (United States)

Annunziato, Stefano; Kas, Sjors M; Nethe, Micha; Yücel, Hatice; Del Bravo, Jessica; Pritchard, Colin; Bin Ali, Rahmen; van Gerwen, Bas; Siteur, Bjørn; Drenth, Anne Paulien; Schut, Eva; van de Ven, Marieke; Boelens, Mirjam C; Klarenbeek, Sjoerd; Huijbers, Ivo J; van Miltenburg, Martine H; Jonkers, Jos

2016-06-15

Large-scale sequencing studies are rapidly identifying putative oncogenic mutations in human tumors. However, discrimination between passenger and driver events in tumorigenesis remains challenging and requires in vivo validation studies in reliable animal models of human cancer. In this study, we describe a novel strategy for in vivo validation of candidate tumor suppressors implicated in invasive lobular breast carcinoma (ILC), which is hallmarked by loss of the cell-cell adhesion molecule E-cadherin. We describe an approach to model ILC by intraductal injection of lentiviral vectors encoding Cre recombinase, the CRISPR/Cas9 system, or both in female mice carrying conditional alleles of the Cdh1 gene, encoding for E-cadherin. Using this approach, we were able to target ILC-initiating cells and induce specific gene disruption of Pten by CRISPR/Cas9-mediated somatic gene editing. Whereas intraductal injection of Cas9-encoding lentiviruses induced Cas9-specific immune responses and development of tumors that did not resemble ILC, lentiviral delivery of a Pten targeting single-guide RNA (sgRNA) in mice with mammary gland-specific loss of E-cadherin and expression of Cas9 efficiently induced ILC development. This versatile platform can be used for rapid in vivo testing of putative tumor suppressor genes implicated in ILC, providing new opportunities for modeling invasive lobular breast carcinoma in mice. © 2016 Annunziato et al.; Published by Cold Spring Harbor Laboratory Press.

The Putative Son's Attractiveness Alters the Perceived Attractiveness of the Putative Father.

Science.gov (United States)

Prokop, Pavol

2015-08-01

A body of literature has investigated female mate choice in the pre-mating context (pre-mating sexual selection). Humans, however, are long-living mammals forming pair-bonds which sequentially produce offspring. Post-mating evaluations of a partner's attractiveness may thus significantly influence the reproductive success of men and women. I tested herein the theory that the attractiveness of putative sons provides extra information about the genetic quality of fathers, thereby influencing fathers' attractiveness across three studies. As predicted, facially attractive boys were more frequently attributed to attractive putative fathers and vice versa (Study 1). Furthermore, priming with an attractive putative son increased the attractiveness of the putative father with the reverse being true for unattractive putative sons. When putative fathers were presented as stepfathers, the effect of the boy's attractiveness on the stepfather's attractiveness was lower and less consistent (Study 2). This suggests that the presence of an attractive boy has the strongest effect on the perceived attractiveness of putative fathers rather than on non-fathers. The generalized effect of priming with beautiful non-human objects also exists, but its effect is much weaker compared with the effects of putative biological sons (Study 3). Overall, this study highlighted the importance of post-mating sexual selection in humans and suggests that the heritable attractive traits of men are also evaluated by females after mating and/or may be used by females in mate poaching.

Calcium Channels, Rho-Kinase, Protein Kinase-C, and Phospholipase-C Pathways Mediate Mercury Chloride-Induced Myometrial Contractions in Rats.

Science.gov (United States)

Koli, Swati; Prakash, Atul; Choudhury, Soumen; Mandil, Rajesh; Garg, Satish K

2018-05-21

Adverse effects of mercury on female reproduction are reported; however, its effect on myogenic activity of uterus and mechanism thereof is obscure. Present study was undertaken to unravel the mechanistic pathways of mercuric chloride (HgCl 2 )-induced myometrial contraction in rats. Isometric tension in myometrial strips of rats following in vitro exposure to HgCl 2 was recorded using data acquisition system-based physiograph. HgCl 2 produced concentration-dependent (10 nM-100 μM) uterotonic effect which was significantly (p Graphical Abstract Graphical abstract depicting the mechanism of mercury-induced myometrial contraction in rats. M receptor: Muscarinic receptor; PIP2: phospho-inositol bisphosphate; PLC: phospholipase-C; DAG: diacyl glycerol; IP3: inositol triphosphate; IP3R: inositol triphosphate receptor; PKC; protein kinase-C; MLCP: myosin light chain phosphatise; MYPT: myosin phosphatase; SR: sarco-endoplasmic reticulum.

Inhibition of PTEN and activation of Akt by menadione

OpenAIRE

Yoshikawa, Kyoko; Nigorikawa, Kiyomi; Tsukamoto, Mariko; Tamura, Namiko; Hazeki, Kaoru; Hazeki, Osamu

2007-01-01

Menadione (vitamin K3) has been shown to activate Erk in several cell lines. This effect has been shown to be due to the activation of EGF receptors (EGFR) as a result of inhibition of some protein tyrosine phosphatases. In the present study, we examined the effects of menadione on Akt in Chinese hamster ovary cells. The phosphorylation of Akt by menadione was not inhibited by AG1478, an inhibitor of EGFR. Menadione inhibited the lipid phosphatase activity of PTEN in a cell-free system. In an...

Trivalent dimethylarsenic compound induces histone H3 phosphorylation and abnormal localization of Aurora B kinase in HepG2 cells

International Nuclear Information System (INIS)

Suzuki, Toshihide; Miyazaki, Koichi; Kita, Kayoko; Ochi, Takafumi

2009-01-01

Trivalent dimethylarsinous acid [DMA(III)] has been shown to induce mitotic abnormalities, such as centrosome abnormality, multipolar spindles, multipolar division, and aneuploidy, in several cell lines. In order to elucidate the mechanisms underlying these mitotic abnormalities, we investigated DMA(III)-mediated changes in histone H3 phosphorylation and localization of Aurora B kinase, which is a key molecule in cell mitosis. DMA(III) caused the phosphorylation of histone H3 (ser10) and was distributed predominantly in mitotic cells, especially in prometaphase cells. By contrast, most of the phospho-histone H3 was found to be localized in interphase cells after treatment with inorganic arsenite [iAs(III)], suggesting the involvement of a different pathway in phosphorylation. DMA(III) activated Aurora B kinase and slightly activated ERK MAP kinase. Phosphorylation of histone H3 by DMA(III) was effectively reduced by ZM447439 (Aurora kinase inhibitor) and slightly reduced by U0126 (MEK inhibitor). By contrast, iAs(III)-dependent histone H3 phosphorylation was markedly reduced by U0126. Aurora B kinase is generally localized in the midbody during telophase and plays an important role in cytokinesis. However, in some cells treated with DMA(III), Aurora B was not localized in the midbody of telophase cells. These findings suggested that DMA(III) induced a spindle abnormality, thereby activating the spindle assembly checkpoint (SAC) through the Aurora B kinase pathway. In addition, cytokinesis was not completed because of the abnormal localization of Aurora B kinase by DMA(III), thereby resulting in the generation of multinucleated cells. These results provide insight into the mechanism of arsenic tumorigenesis.

Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

Science.gov (United States)

Ali, Shawkat; Magne, Maxime; Chen, Shiyan; Côté, Olivier; Stare, Barbara Gerič; Obradovic, Natasa; Jamshaid, Lubna; Wang, Xiaohong; Bélair, Guy; Moffett, Peter

2015-01-01

The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12) and an expansin-like protein (GrEXPB2), suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.

Analysis of putative apoplastic effectors from the nematode, Globodera rostochiensis, and identification of an expansin-like protein that can induce and suppress host defenses.

Directory of Open Access Journals (Sweden)

Shawkat Ali

Full Text Available The potato cyst nematode, Globodera rostochiensis, is an important pest of potato. Like other pathogens, plant parasitic nematodes are presumed to employ effector proteins, secreted into the apoplast as well as the host cytoplasm, to alter plant cellular functions and successfully infect their hosts. We have generated a library of ORFs encoding putative G. rostochiensis putative apoplastic effectors in vectors for expression in planta. These clones were assessed for morphological and developmental effects on plants as well as their ability to induce or suppress plant defenses. Several CLAVATA3/ESR-like proteins induced developmental phenotypes, whereas predicted cell wall-modifying proteins induced necrosis and chlorosis, consistent with roles in cell fate alteration and tissue invasion, respectively. When directed to the apoplast with a signal peptide, two effectors, an ubiquitin extension protein (GrUBCEP12 and an expansin-like protein (GrEXPB2, suppressed defense responses including NB-LRR signaling induced in the cytoplasm. GrEXPB2 also elicited defense response in species- and sequence-specific manner. Our results are consistent with the scenario whereby potato cyst nematodes secrete effectors that modulate host cell fate and metabolism as well as modifying host cell walls. Furthermore, we show a novel role for an apoplastic expansin-like protein in suppressing intra-cellular defense responses.

Characterization and response of newly developed high-grade glioma cultures to the tyrosine kinase inhibitors, erlotinib, gefitinib and imatinib

International Nuclear Information System (INIS)

Kinsella, Paula; Howley, Rachel; Doolan, Padraig; Clarke, Colin; Madden, Stephen F.; Clynes, Martin; Farrell, Michael; Amberger-Murphy, Verena

2012-01-01

High-grade gliomas (HGG), are the most common aggressive brain tumours in adults. Inhibitors targeting growth factor signalling pathways in glioma have shown a low clinical response rate. To accurately evaluate response to targeted therapies further in vitro studies are necessary. Growth factor pathway expression using epidermal growth factor receptor (EGFR), mutant EGFR (EGFRvIII), platelet derived growth factor receptor (PDGFR), C-Kit and C-Abl together with phosphatase and tensin homolog (PTEN) expression and downstream activation of AKT and phosphorylated ribosomal protein S6 (P70S6K) was analysed in 26 primary glioma cultures treated with the tyrosine kinase inhibitors (TKIs) erlotinib, gefitinib and imatinib. Response to TKIs was assessed using 50% inhibitory concentrations (IC 50 ). Response for each culture was compared with the EGFR/PDGFR immunocytochemical pathway profile using hierarchical cluster analysis (HCA) and principal component analysis (PCA). Erlotinib response was not strongly associated with high expression of the growth factor pathway components. PTEN expression did not correlate with response to any of the three TKIs. Increased EGFR expression was associated with gefitinib response; increased PDGFR-α expression was associated with imatinib response. The results of this in vitro study suggest gefitinib and imatinib may have therapeutic potential in HGG tumours with a corresponding growth factor receptor expression profile. -- Highlights: ► Non-responders had low EGFR expression, high PDGFR-β, and a low proliferation rate. ► PTEN is not indicative of response to a TKI. ► Erlotinib response was not associated with expression of the proteins examined. ► Imatinib-response correlated with expression of PDGFR-α. ► Gefitinib response correlated with increased expression of EGFR.

Platelet-derived-growth-factor-induced signalling in human platelets: phosphoinositide-3-kinase-dependent inhibition of platelet activation.

Science.gov (United States)

Selheim, F; Fukami, M H; Holmsen, H; Vassbotn, F S

2000-09-01

Human platelets release platelet-derived growth factor (PDGF) from alpha-granules during platelet activation. We have previously shown that platelets have PDGF alpha-receptors, a transmembrane tyrosine kinase that takes part in negative feedback regulation during platelet activation. Here we have described a study of PDGF-induced tyrosine phosphorylation of platelet substrates and phosphoinositide 3-kinase (PI-3K) activity in collagen-stimulated platelets. By immunoblotting with phosphotyrosine antibodies of collagen-activated platelets we found that PDGF increased the phosphorylation of several platelet substrates, e.g. pp140, pp120 and pp85. PDGF inhibited collagen-induced platelet activation in the presence of inhibitors of autocrine stimulation, thus blocking the pure collagen-induced signal transduction. PDGF enhanced the collagen-induced formation of PtdIns(3,4)P(2) and PtdIns(3,4,5)P(3) as measured by HPLC. Wortmannin and LY294002, two unrelated inhibitors of PI-3K, were used to investigate the role of PI-3K in PDGF-induced platelet signalling. Incubation of platelets with wortmannin and LY294002 blocked the formation of three phosphorylated inositides as well as the inhibitory effect of PDGF on collagen-induced platelet activation. We conclude that the inhibitory effect of PDGF on platelet activation is PI-3K dependent. This is the first demonstration of a negative regulatory function of 3-phosphorylated inositides in platelets.

Genome-Wide Identification and Evolutionary Analysis of Sarcocystis neurona Protein Kinases.

Science.gov (United States)

Murungi, Edwin K; Kariithi, Henry M

2017-03-21

The apicomplexan parasite Sarcocystis neurona causes equine protozoal myeloencephalitis (EPM), a degenerative neurological disease of horses. Due to its host range expansion, S. neurona is an emerging threat that requires close monitoring. In apicomplexans, protein kinases (PKs) have been implicated in a myriad of critical functions, such as host cell invasion, cell cycle progression and host immune response evasion. Here, we used various bioinformatics methods to define the kinome of S. neurona and phylogenetic relatedness of its PKs to other apicomplexans. We identified 97 putative PKs clustering within the various eukaryotic kinase groups. Although containing the universally-conserved PKA (AGC group), S. neurona kinome was devoid of PKB and PKC. Moreover, the kinome contains the six-conserved apicomplexan CDPKs (CAMK group). Several OPK atypical kinases, including ROPKs 19A, 27, 30, 33, 35 and 37 were identified. Notably, S. neurona is devoid of the virulence-associated ROPKs 5, 6, 18 and 38, as well as the Alpha and RIO kinases. Two out of the three S. neurona CK1 enzymes had high sequence similarities to Toxoplasma gondii TgCK1-α and TgCK1-β and the Plasmodium PfCK1. Further experimental studies on the S. neurona putative PKs identified in this study are required to validate the functional roles of the PKs and to understand their involvement in mechanisms that regulate various cellular processes and host-parasite interactions. Given the essentiality of apicomplexan PKs in the survival of apicomplexans, the current study offers a platform for future development of novel therapeutics for EPM, for instance via application of PK inhibitors to block parasite invasion and development in their host.

Genome-Wide Identification and Evolutionary Analysis of Sarcocystis neurona Protein Kinases

Directory of Open Access Journals (Sweden)

Edwin K. Murungi

2017-03-01

Full Text Available The apicomplexan parasite Sarcocystis neurona causes equine protozoal myeloencephalitis (EPM, a degenerative neurological disease of horses. Due to its host range expansion, S. neurona is an emerging threat that requires close monitoring. In apicomplexans, protein kinases (PKs have been implicated in a myriad of critical functions, such as host cell invasion, cell cycle progression and host immune response evasion. Here, we used various bioinformatics methods to define the kinome of S. neurona and phylogenetic relatedness of its PKs to other apicomplexans. We identified 97 putative PKs clustering within the various eukaryotic kinase groups. Although containing the universally-conserved PKA (AGC group, S. neurona kinome was devoid of PKB and PKC. Moreover, the kinome contains the six-conserved apicomplexan CDPKs (CAMK group. Several OPK atypical kinases, including ROPKs 19A, 27, 30, 33, 35 and 37 were identified. Notably, S. neurona is devoid of the virulence-associated ROPKs 5, 6, 18 and 38, as well as the Alpha and RIO kinases. Two out of the three S. neurona CK1 enzymes had high sequence similarities to Toxoplasma gondii TgCK1-α and TgCK1-β and the Plasmodium PfCK1. Further experimental studies on the S. neurona putative PKs identified in this study are required to validate the functional roles of the PKs and to understand their involvement in mechanisms that regulate various cellular processes and host-parasite interactions. Given the essentiality of apicomplexan PKs in the survival of apicomplexans, the current study offers a platform for future development of novel therapeutics for EPM, for instance via application of PK inhibitors to block parasite invasion and development in their host.

The double-stranded RNA-activated protein kinase mediates viral-induced encephalitis

International Nuclear Information System (INIS)

Scheuner, Donalyn; Gromeier, Matthias; Davies, Monique V.; Dorner, Andrew J.; Song Benbo; Patel, Rupali V.; Wimmer, Eckard J.; McLendon, Roger E.; Kaufman, Randal J.

2003-01-01

The double-stranded (ds) RNA-activated protein kinase (PKR) plays an important role in control of viral infections and cell growth. We have studied the role of PKR in viral infection in mice that are defective in the PKR signaling pathway. Transgenic mice were derived that constitutively express a trans-dominant-negative kinase-defective mutant PKR under control of the β-actin promoter. The trans-dominant-negative PKR mutant expressing transgenic mice do not have a detectable phenotype, similar to observations with PKR knock-out mice. The requirement for PKR in viral pathogenesis was studied by intracerebral infection of mice with a mouse-adapted poliovirus. Histopathological analysis revealed diffuse encephalomyelitis with severe inflammatory lesions throughout the central nervous system (CNS) in infected wild-type mice. In contrast, histopathological evaluation of virus-injected trans-dominant-negative PKR transgenic mice as well as PKR knock-out mice yielded no signs of tissue damage associated with inflammatory host responses. However, the virus did replicate in both models of PKR-deficient mice at a level equal to that observed in wild-type infected mice. Although the results indicate a clear difference in susceptibility to poliovirus-induced encephalitis, this difference manifests clinically as a slight delay in fatal neuropathy in trans-dominant-negative PKR transgenic and PKR knock-out animals. Our observations support the finding that viral-induced PKR activation may play a significant role in pathogenesis by mediating the host response to viral CNS infection. They support PKR to be an effective target to control tissue damage due to deleterious host responses to viral infection

Mutation of serine 1333 in the ATR HEAT repeats creates a hyperactive kinase.

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Jessica W Luzwick

Full Text Available Subcellular localization, protein interactions, and post-translational modifications regulate the DNA damage response kinases ATR, ATM, and DNA-PK. During an analysis of putative ATR phosphorylation sites, we found that a single mutation at S1333 creates a hyperactive kinase. In vitro and in cells, mutation of S1333 to alanine (S1333A-ATR causes elevated levels of kinase activity with and without the addition of the protein activator TOPBP1. S1333 mutations to glycine, arginine, or lysine also create a hyperactive kinase, while mutation to aspartic acid decreases ATR activity. S1333A-ATR maintains the G2 checkpoint and promotes completion of DNA replication after transient exposure to replication stress but the less active kinase, S1333D-ATR, has modest defects in both of these functions. While we find no evidence that S1333 is phosphorylated in cultured cells, our data indicate that small changes in the HEAT repeats can have large effects on kinase activity. These mutants may serve as useful tools for future studies of the ATR pathway.

Analysis of the complexity of protein kinases within the phloem sieve tube system. Characterization of Cucurbita maxima calmodulin-like domain protein kinase 1.

Science.gov (United States)

Yoo, Byung-Chun; Lee, Jung-Youn; Lucas, William J

2002-05-03

In angiosperms, functional, mature sieve elements lack nuclei, vacuoles, ribosomes, and most of the endomembrane network. In this study, the complexity, number, and nature of protein kinases within the phloem sap of Cucurbita maxima were investigated to test the hypothesis that the enucleate sieve tube system utilizes a simplified signal transduction network. Supporting evidence was obtained in that only five putative protein kinases (three calcium-independent and two calcium-dependent protein kinases) were detected within the phloem sap extracted from stem tissues. Biochemical methods were used to purify one such calcium-dependent protein kinase. The gene for this C. maxima calmodulin-like domain protein kinase 1 (CmCPK1), was cloned using peptide microsequences. A combination of mass spectrometry, peptide fingerprinting, and amino-terminal sequencing established that, in the phloem sap, CmCPK1 exists as an amino-terminally cleaved protein. A second highly homologous isoform, CmCPK2, was identified, but although transcripts could be detected in the companion cells, peptide fingerprint analysis suggested that CmCPK2 does not enter the phloem sap. Potential substrates for CmCPK1, within the phloem sap, were also detected using an on-membrane phosphorylation assay. Entry of CmCPK1 into sieve elements via plasmodesmata and the potential roles played by these phloem protein kinases are discussed.

Requirement of Sequences outside the Conserved Kinase Domain of Fission Yeast Rad3p for Checkpoint Control

Science.gov (United States)

Chapman, Carolyn Riley; Evans, Sarah Tyler; Carr, Antony M.; Enoch, Tamar

1999-01-01

The fission yeast Rad3p checkpoint protein is a member of the phosphatidylinositol 3-kinase-related family of protein kinases, which includes human ATMp. Mutation of the ATM gene is responsible for the disease ataxia-telangiectasia. The kinase domain of Rad3p has previously been shown to be essential for function. Here, we show that although this domain is necessary, it is not sufficient, because the isolated kinase domain does not have kinase activity in vitro and cannot complement a rad3 deletion strain. Using dominant negative alleles of rad3, we have identified two sites N-terminal to the conserved kinase domain that are essential for Rad3p function. One of these sites is the putative leucine zipper, which is conserved in other phosphatidylinositol 3-kinase-related family members. The other is a novel motif, which may also mediate Rad3p protein–protein interactions. PMID:10512862