Activation Of P70 S6 Kinase Research Articles

Apolipoprotein A-I Mimetic Peptides Inhibit Expression and Activity of Hypoxia-Inducible Factor-1α in Human Ovarian Cancer Cell Lines and a Mouse Ovarian Cancer Model

Our previous results demonstrated that the apolipoprotein A-I (apoA-I) mimetic peptides L-4F and L-5F inhibit vascular endothelial growth factor production and tumor angiogenesis. The present study was designed to test whether apoA-I mimetic peptides inhibit the expression and activity of hypoxia-inducible factor-1α (HIF-1α), which plays a critical role in the production of angiogenic factors and angiogenesis. Immunohistochemistry staining was used to examine the expression of HIF-1α in tumor tissues. Immunoblotting, real-time polymerase chain reaction, immunofluorescence, and luciferase activity assays were used to determine the expression and activity of HIF-1α in human ovarian cancer cell lines. Immunohistochemistry staining demonstrated that L-4F treatment dramatically decreased HIF-1α expression in mouse ovarian tumor tissues. L-4F inhibited the expression and activity of HIF-1α induced by low oxygen concentration, cobalt chloride (CoCl(2), a hypoxia-mimic compound), lysophosphatidic acid, and insulin in two human ovarian cancer cell lines, OV2008 and CAOV-3. L-4F had no effect on the insulin-induced phosphorylation of Akt, but inhibited the activation of extracellular signal-regulated kinase and p70s6 kinase, leading to the inhibition of HIF-1α synthesis. Pretreatment with L-4F dramatically accelerated the proteasome-dependent protein degradation of HIF-1α in both insulin- and CoCl(2)-treated cells. The inhibitory effect of L-4F on HIF-1α expression is in part mediated by the reactive oxygen species-scavenging effect of L-4F. ApoA-I mimetic peptides inhibit the expression and activity of HIF-1α in both in vivo and in vitro models, suggesting the inhibition of HIF-1α may be a critical mechanism responsible for the suppression of tumor progression by apoA-I mimetic peptides.

Abstract 2246: Combined grape polyphenols regulate mammalian target of rapamycin (mTOR) signaling via a dual mechanism of Akt inhibition and AMPK activation

Abstract Breast Cancer (BC) is the major prevalent cancer in women in the United States and Puerto Rico with metastatic breast cancer (MBC), often resulting in death. Recent studies have shown that the Akt/mammalian target of rapamycin (mTOR) pathway is a critical signaling cascade that drives MBC. We recently reported that a combined diet of grape polyphenols (resveratrol, quercetin and catechin: RQC) at equimolar concentration is effective at inhibition of mammary tumor growth and metastasis to lung and bones in nude mice. The objective of this study is to investigate a role for RQC in prevention of MBC via inhibition of the Akt/AMPK/mTOR pathway. We found that RQC at 5 μM each inhibits Akt and p70S6 kinase (downstream effector of mTOR) activities in a metastatic variant of the MDA-MB-435 cell line and MDA-MB-231 human BC cells. RQC at 5 μM demonstrated a 2-fold activation of adenosine monophophate-activated protein kinase (AMPK), an endogenous inhibitor of mTOR that acts opposite to Akt. To determine the contribution of individual resveratrol, quercetin, and catechin to the effect of combined RQC on mTOR signaling, MDA-MB-231 cells were treated with RQC at 1, 3 and 9 μM individually and in combination. Results show that at 1 μM, individual or combined RQC did not increase AMPK activity, while at 3 μM, only Quercetin increased AMPK activity. Individual compounds at 1 μM did not affect Akt activity, while combined RQC decreased Akt activity by 20%. At 3 μM, Akt activity is downregulated by all compounds individually and in combination with combined RQC exerting a higher inhibitory effect on Akt activity than individual compounds. In general, the combination of RQC was more efficient at inhibition of the Akt/mTOR pathway than individual compounds. Thus, RQC has the potential to be used alone or in combination with other cancer therapeutics to impede progressive BC. This study was sponsored by the Department of Defense/Breast Cancer Research Program award W81XWH-07-1-0330 to SD and the U54 grant No. CA-096297 to UPR Comprehensive Cancer Center. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2246. doi:1538-7445.AM2012-2246

Correction: TBC1D3, a Hominoid-Specific Gene, Delays IRS-1 Degradation and Promotes Insulin Signaling by Modulating p70 S6 Kinase Activity

Correction: TBC1D3, a Hominoid-Specific Gene, Delays IRS-1 Degradation and Promotes Insulin Signaling by Modulating p70 S6 Kinase Activity

Ischemic postconditioning regulates cardiomyocyte autophagic activity following ischemia/reperfusion injury.

Ischemic postconditioning (IPostC) is a promising protective mechanism for combating reperfusion injury. However, the role of autophagy in the protective effects of IPostC and the associated signaling pathways have remained to be elucidated. Male Sprague Dawley rats were subjected to 30 min ischemia and 1, 2, 3, 6, 12 and 24 h of reperfusion, with or without IPostC treatment. Autophagic flux was evaluated by detecting mRNA and protein expression levels of microtubule associated protein 1 light chain 3 and p62. Phosphorylated (p)-P70S6 kinase (P70S6K), p-adenosine monophosphate-activated kinase (AMPK) and Beclin 1 protein levels were measured by western blot analysis. Myocardial infarct size was measured using staining with Evans blue dye and myocardial apoptosis was evaluated by terminal deoxynucleotidyl transferase mediated dUTP nick end labeling staining. Autophagic activity was observed to be inhibited within the first hour of reperfusion, increased during 2-6 h and reduced from 12-24 h following IPostC compared with reperfusion without IPostC. Inhibition of autophagy by chloroquine significantly reversed the effects of IPostC on myocardial infarct size and the levels of apoptosis. IPostC significantly increased Beclin 1 levels, inhibited AMPK activation and increased P70S6K activation within the first hour compared with reperfusion without IPostC. IPostC attenuated the upregulation of Beclin 1 levels, increased AMPK activation and reduced P70S6K activation between 2 and 12 h, and subsequently exerted the opposite effects on these molecules between 12 and 24 h. IPostC was demonstrated to regulate autophagic activity in a time‑dependent manner. The Beclin 1 and AMPK-mammalian target of rapamycin signaling pathways are suggested to be involved in the regulation of IPostC in autophagy.

TBC1D3, a Hominoid-Specific Gene, Delays IRS-1 Degradation and Promotes Insulin Signaling by Modulating p70 S6 Kinase Activity

Insulin/IGF-1 signaling plays a pivotal role in the regulation of cellular homeostasis through its control of glucose metabolism as well as due to its effects on cell proliferation. Aberrant regulation of insulin signaling has been repeatedly implicated in uncontrolled cell growth and malignant transformations. TBC1D3 is a hominoid specific gene previously identified as an oncogene in breast and prostate cancers. Our efforts to identify the molecular mechanisms of TBC1D3-induced oncogenesis revealed the role of TBC1D3 in insulin/IGF-1 signaling pathway. We document here that TBC1D3 intensifies insulin/IGF-1-induced signal transduction through intricate, yet elegant fine-tuning of signaling mechanisms. We show that TBC1D3 expression substantially delayed ubiquitination and degradation of insulin receptor substrate-1 (IRS-1). This effect is achieved through suppression of serine phosphorylation at S636/639, S307 and S312 of IRS-1, which are key phosphorylation sites required for IRS-1 degradation. Furthermore, we report that the effect of TBC1D3 on IRS-1:S636/639 phosphorylation is mediated through TBC1D3-induced activation of protein phosphatase 2A (PP2A), followed by suppression of T389 phosphorylation on p70 S6 kinase (S6K). TBC1D3 specifically interacts with PP2A regulatory subunit B56γ, indicating that TBC1D3 and PP2A B56γ operate jointly to promote S6K:T389 dephosphorylation. These findings suggest that TBC1D3 plays an unanticipated and potentially unique role in the fine-tuning of insulin/IGF-1 signaling, while providing novel insights into the regulation of tumorigenesis by a hominoid-specific protein.

A Hexane Fraction of Guava Leaves (Psidium guajavaL.) Induces Anticancer Activity by Suppressing AKT/Mammalian Target of Rapamycin/Ribosomal p70 S6 Kinase in Human Prostate Cancer Cells

This study was carried out to evaluate the anticancer effects of guava leaf extracts and its fractions. The chemical compositions of the active extracts were also determined. In the present study, we set out to determine whether the anticancer effects of guava leaves are linked with their ability to suppress constitutive AKT/mammalian target of rapamycin (mTOR)/ribosomal p70 S6 kinase (S6K1) and mitogen-activated protein kinase (MAPK) activation pathways in human prostate cancer cells. We found that guava leaf hexane fraction (GHF) was the most potent inducer of cytotoxic and apoptotic effects in PC-3 cells. The molecular mechanism or mechanisms of GHF apoptotic potential were correlated with the suppression of AKT/mTOR/S6K1 and MAPK signaling pathways. This effect of GHF correlated with down-regulation of various proteins that mediate cell proliferation, cell survival, metastasis, and angiogenesis. Analysis of GHF by gas chromatography and gas chromatography-mass spectrometry tentatively identified 60 compounds, including β-eudesmol (11.98%), α-copaene (7.97%), phytol (7.95%), α-patchoulene (3.76%), β-caryophyllene oxide (CPO) (3.63%), caryophylla-3(15),7(14)-dien-6-ol (2.68%), (E)-methyl isoeugenol (1.90%), α-terpineol (1.76%), and octadecane (1.23%). Besides GHF, CPO, but not phytol, also inhibited the AKT/mTOR/S6K1 signaling pathway and induced apoptosis in prostate cancer cells. Overall, these findings suggest that guava leaves can interfere with multiple signaling cascades linked with tumorigenesis and provide a source of potential therapeutic compounds for both the prevention and treatment of cancer.

Cellular signals underlying β-adrenergic receptor mediated salivary gland enlargement

We examined the cellular signaling pathways involved in parotid gland enlargement induced by repeated isoproterenol administration in rats. Immunoblot analysis revealed early (1 h) activation of the mitogen activated protein kinase (MAPK) ERK1/2, and progressive activation of epidermal growth factor receptor (EGFR), p38MAPK and p70S6 kinase (p70S6K) during 72 h of isoproterenol treatment. Expression of β-adrenergic receptors (ARs) of the β2, but not β1, subtype increased over time in parallel with increases in the proliferation marker PCNA and parotid gland weight. Levels of β2-AR mRNA, assessed by quantitative RT-PCR and Northern blot analysis, were upregulated in parotid glands of isoproterenol treated rats. cAMP response element binding protein (CREB), a positive regulator of β2-AR transcription, was activated at 1 h after isoproterenol administration, as evidenced by increased nuclear translocation and DNA binding using immunohistochemical staining and electrophoretic mobility shift assay. ELISA of NF-κB, also a β2-AR transcriptional regulator, revealed an increase in p65 and p50 subunits in nuclear protein extracts from parotid glands of isoproterenol treated rats. Together, these results demonstrate that β-adrenergic stimulation activates diverse cell survival and progrowth signaling pathways, including cAMP and EGFR linked activation of ERK1/2, p38MAPK, and p70S6K, and also induction of β2-ARs, possibly mediated by CREB and NF-κB, resulting in salivary gland enlargement. We propose that during isoproterenol treatment activation of the β1-AR, the predominant β-AR subtype in unstimulated salivary glands, initiates proliferative signaling cascades, and that upregulation of the β2-AR plays an essential role in later stages of salivary gland growth.

Akt Stimulates Hepatic SREBP1c and Lipogenesis through Parallel mTORC1-Dependent and Independent Pathways

Through unknown mechanisms, insulin activates the sterol regulatory element-binding protein (SREBP1c) transcription factor to promote hepatic lipogenesis. We find that this induction is dependent on the mammalian target of rapamycin (mTOR) complex 1 (mTORC1). To further define the role of mTORC1 in the regulation of SREBP1c in the liver, we generated mice with liver-specific deletion of TSC1 (LTsc1KO), which results in insulin-independent activation of mTORC1. Surprisingly, the LTsc1KO mice are protected from age- and diet-induced hepatic steatosis and display hepatocyte-intrinsic defects in SREBP1c activation and de novo lipogenesis. These phenotypes result from attenuation of Akt signaling driven by mTORC1-dependent insulin resistance. Therefore, mTORC1 activation is not sufficient to stimulate hepatic SREBP1c in the absence of Akt signaling, revealing the existence of an additional downstream pathway also required for this induction. We provide evidence that this mTORC1-independent pathway involves Akt-mediated suppression of Insig2a, a liver-specific transcript encoding the SREBP1c inhibitor INSIG2.

The alpha 2A adrenergic receptor evokes activation of p70S6 kinase through G protein and transactivation of EGFR

The function of α2A adrenergic receptor (α2AAR) has been implicated in a wide range of physiological activities. The molecular mechanisms behind these activities are topics under intensive investigation. At the cellular level, stimulation of the α2AAR leads to propagation of a number of signaling cascades, such as MAPK, Akt and p70S6 kinase (p70S6K) pathways. In the current study, we characterized α2AAR‐mediated p70S6K activation. Stimulation of the α2AAR evokes activation of p70S6K in both embryonic fibroblasts (MEF) and neurons in a dose‐dependent manner. To delineate the molecular pathway of the α2AAR‐mediated p70S6K activation, pharmacological inhibitors for potential signaling molecules involved were applied on cells. Pertussis toxin treatment of cells blocked α2AAR‐mediated p70S6K activation, indicating the involvement of G proteins in this process. When cells were treated with an EGFR inhibitor or a PI3K inhibitor, α2AAR‐mediated p70S6K activation was also blocked. Additionally, rapamycin treatment diminished α2AAR‐mediated p70S6K activation. Taken together, these data suggests that α2AAR‐mediated activation of p70S6K is a G protein‐dependent process and achieved through transactivation of EGFR, which subsequently activates PI3K and mTOR to stimulate p70S6K. This research was supported by a NIH grant MH081917 (QW) and an AHA SDG grant 0630103N (QW).

Impact of combined HDAC and mTOR inhibition on adhesion, migration and invasion of prostate cancer cells

The concept of molecular tumor targeting might provide new hope in the treatment of advanced prostate cancer. We evaluated metastasis blocking properties of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell lines. RAD001 or VPA were applied to PC-3 or LNCaP cells, either separately or in combination. Adhesion to vascular endothelium or to immobilized collagen, fibronectin or laminin was quantified. Migration and invasion were explored by a modified Boyden chamber assay. Integrin α and β subtypes were analyzed by flow cytometry, western blotting and RT-PCR. Effects of drug treatment on integrin related signaling, Akt and p70S6kinase activation, histone H3 and H4 acetylation were also determined. Separate application of RAD001 or VPA distinctly reduced tumor cell adhesion, migration and invasion, accompanied by elevated Akt activation and p70S6kinase de-activation. Integrin subtype expression was altered significantly by both compounds (VPA > RAD001). When both drugs were used in concert additive effects were observed on the migratory and invasive behavior but not on tumor-endothelium and tumor-matrix interaction. Separate mTOR or HDAC inhibition slows processes related to tumor metastasis. The RAD001-VPA combination showed advantage over VPA monotreatment with particular respect to migration and invasion. Ongoing studies are required to assess the relevance of VPA monotherapy versus VPA-RAD001 combination on tumor cell motility.

C1q regulation of dendritic cell development from monocytes with distinct cytokine production and T cell stimulation

The causative association of complement C1q deficiency with systemic lupus erythematosus (SLE), which inevitably involves the breakdown of tolerance, remains poorly explained. Its non-hepatic, macrophage and dendritic cell (DC) origin may be highly relevant. In tissues, C1q is produced by DCs and macrophages which deposits around these cells and we ask whether this pericellular form of C1q regulates DC development from monocytes. DCs cultured on immobilized C1q (C1q-DCs) show similar MHC, CD40, CD80, CD86, CD83 and CCR7 expression as normal DCs, but these cells exhibit increased phagocytosis of apoptotic cells and elevated IL-10 but reduced IL-12 and IL-23 production. Intracellularly, C1q-DCs exhibit increased ERK, p38 and p70S6 kinase activity. By mixed leukocyte reaction, C1q-DCs show reduced Th1 and Th17 induction from allogeneic CD4+ T cells. LPS and IFNγ, which cause normal DCs to induce increased CD25 expression on CD4+ T cells, attenuate C1q-DC induction of CD25. These imply that the DC pericellular C1q may induce tolerogenic properties in developing DCs.

Mitotic lymphoma cells are characterized by high expression of phosphorylated ribosomal S6 protein

Growth factors and mitogens influence signaling pathways and often induce the activity of p70S6 kinase (p70S6K), which in turn phosphorylates the ribosomal S6 protein (S6). Although recent data are rather conflicting, the overall view suggests that phosphorylated S6 is a regulator of global protein synthesis, cell proliferation, cell size and glucose homeostasis. In the present work, emphasis was given to cell cycle-dependent activation of S6 focusing mainly on human lymphoid and lymphoma cells. Paraffin-embedded human tissue blocks from lymph node and different tumor biopsies as well as in vitro cell lines were investigated by immunohistochemistry, immunocytochemistry, flow cytometry and Western blotting using antibodies directed against phospho-S6, phospho-mTOR, phospho-p70S6K and phospho-Histone H3. To enrich the cell number in different phases of the cell cycle, nocodazole, staurosporine or rapamycin were used in cell cultures. We observed strong phospho-S6 positivity by immunostainings in the dividing lymphoid cells of reactive lymph nodes and in lymphoma cells cultured in vitro. Phospho-S6 protein levels were shown to be elevated throughout mitosis in lymphoma cells; however, the high expression of phospho-S6 in mitotic cells was not a general hallmark of tumor cell types studied so far: phospho-S6-negative mitotic cells were detected in several carcinoma and sarcoma biopsies. These observations may have practical implications as they raise the possibility to consider p70S6K and/or S6 as a potential therapeutic target-besides mTOR-in certain lymphomas and perhaps in clinical immunosuppression.

416 IMPACT OF COMBINED HDAC AND MTOR INHIBITION ON THE INVASIVE POTENTIAL OF PROSTATE CANCER CELLS

You have accessJournal of UrologyProstate Cancer: Basic Research1 Apr 2011416 IMPACT OF COMBINED HDAC AND MTOR INHIBITION ON THE INVASIVE POTENTIAL OF PROSTATE CANCER CELLS Steffen Wedel, Lucasz Hudak, Jasmina Makarevic, Jens-Michael Seibel, Eva Juengel, Axel Haferkamp, and Roman Blaheta Steffen WedelSteffen Wedel Frankfurt am Main, Germany More articles by this author , Lucasz HudakLucasz Hudak Frankfurt am Main, Germany More articles by this author , Jasmina MakarevicJasmina Makarevic Frankfurt am Main, Germany More articles by this author , Jens-Michael SeibelJens-Michael Seibel Frankfurt am Main, Germany More articles by this author , Eva JuengelEva Juengel Frankfurt am Main, Germany More articles by this author , Axel HaferkampAxel Haferkamp Frankfurt am Main, Germany More articles by this author , and Roman BlahetaRoman Blaheta Frankfurt am Main, Germany More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.505AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The concept of molecular tumor targeting has recently become standard in the therapy of renal cell carcinoma and might also provide new hope in the treatment of advanced prostate cancer. We evaluated metastasis blocking properties of the histone deacetylase (HDAC) inhibitor valproic acid (VPA) and the mammalian target of rapamycin (mTOR) inhibitor RAD001 on prostate cancer cell lines. METHODS RAD001 or VPA were applied to PC-3, DU-145 and LNCaP cells, either separately or in combination. Adhesion to vascular endothelium or to immobilized collagen, fibronectin or laminin was quantified. Migration and invasion were explored by a modified Boyden chamber assay. Integrin α and β subtypes were analyzed by flow cytometry, western blotting and RT-PCR. Effects of drug treatment on integrin related signaling, Akt and p70S6kinase activation, histone H3 and H4 acetylation were also determined. RESULTS Separate application of RAD001 or VPA distinctly reduced tumor cell adhesion, migration and invasion, accompanied by elevated Akt activation and p70S6kinase deactivation. Integrin subtype expression was altered significantly by both compounds (VPA > RAD001). Particularly,β3 coding mRNA was diminished drastically with the combination being superior then single drug treatment. Furthermore, when both drugs were used in concert additive effects were observed on the migratory and invasive behavior of the prostate cancer cells. CONCLUSIONS Separate mTOR or HDAC inhibition slows processes related to tumor metastasis. The RAD001-VPA combination showed advantage over VPA application alone with particular respect to migration and invasion. Ongoing studies are required to assess the relevance of VPA monotherapy versus VPA-RAD001 combination on tumor cell motility. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e167 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Steffen Wedel Frankfurt am Main, Germany More articles by this author Lucasz Hudak Frankfurt am Main, Germany More articles by this author Jasmina Makarevic Frankfurt am Main, Germany More articles by this author Jens-Michael Seibel Frankfurt am Main, Germany More articles by this author Eva Juengel Frankfurt am Main, Germany More articles by this author Axel Haferkamp Frankfurt am Main, Germany More articles by this author Roman Blaheta Frankfurt am Main, Germany More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Chronically Increased S6K1 Is Associated with Impaired IRS1 Signaling in Skeletal Muscle of GDM Women with Impaired Glucose Tolerance Postpartum

The rapidly increasing prevalence of gestational diabetes mellitus (GDM) globally places a growing population at risk for developing type 2 diabetes mellitus (T2DM), particularly those with persistent impaired glucose tolerance (IGT) postpartum. We sought to 1) identify dynamic insulin signaling abnormalities in vivo in a prospective, longitudinal study of GDM women compared to weight-matched pregnant controls both antepartum and postpartum; and 2) determine abnormalities that might distinguish GDM women who normalize their glucose tolerance postpartum from those with persistent IGT. Skeletal muscle biopsies were obtained before and after a 75-g glucose load in nine overweight to obese GDM women and 10 weight-matched pregnant controls antepartum and postpartum. Postpartum biopsies were collected in five weight-matched GDM women with IGT (GDM/IGT). GDM women had decreased skeletal muscle insulin-stimulated insulin receptor and insulin receptor substrate 1 (IRS1) tyrosine activation and reduced IRS1, concomitant with increased basal IRS1 serine phosphorylation and basal p70 S6-kinase (S6K1) activation, which resolved postpartum. However, GDM/IGT subjects had a persistent impairment in IRS1 activation and increased S6K1 phosphorylation compared to GDM subjects with normal glucose tolerance. This study reveals that women with GDM demonstrate impaired IRS1 signaling associated with increased S6K1 activation in skeletal muscle in vivo. This defect is maintained postpartum in GDM/IGT subjects, despite similar body weights and cytokine levels. Given that GDM women with persistent IGT are at a high risk of developing T2DM, understanding how the nutrient-sensitive mammalian target of rapamycin/S6K1 pathway is chronically activated in GDM may lead to important therapies that could prevent the progression to T2DM.

Oltipraz upregulates the nuclear respiratory factor 2 alpha subunit (NRF2) antioxidant system and prevents insulin resistance and obesity induced by a high-fat diet in C57BL/6J mice

We investigated whether oltipraz, a nuclear respiratory factor 2 alpha subunit (NRF2) activator, improves insulin sensitivity and prevents the development of obesity in mice. C57BL/6J mice were fed with a low-fat diet (10% of energy as fat), a high-fat diet (HFD) (45% of energy as fat) or a HFD with oltipraz for 28 weeks. The effects of oltipraz on body weight, fat content, glucose disposal, insulin signalling, metabolic profiles and endogenous NRF2 functional status in the three groups of mice were investigated. Oltipraz prevented or significantly attenuated the effect of HFD on glucose disposal, body weight and fat gain. Impairment of protein kinase B/Akt phosphorylation in this HFD-fed mouse model in response to intraperitoneal insulin injection was observed in adipose tissue, but not in the muscles, accompanied by inhibition of AMP-activated protein kinase signalling and activation of p70S6 kinase, as well as reduced GLUT4 content. These defects were attenuated by oltipraz administration. Nuclear content of NRF2 in adipose tissue was reduced by HFD feeding, associated with increased Keap1 mRNA expression and reduced production of haem oxygenase-1 and superoxide dismutase, increased protein oxidation, decreased plasma reduced:oxidised glutathione ratio and the appearance of macrophage marker F4/80. These defects were also restored by oltipraz. Finally, oltipraz attenuated HFD-induced inducible nitric oxide synthase overproduction. Impairment of the endogenous redox system is important in the development of obesity and insulin resistance in chronic HFD feeding. NRF2 activation represents a potential novel approach in the treatment and prevention of obesity and diabetes.

Increased expression of tuberin in human uterine leiomyoma

Female Eker rats harboring an insertional deletion in one copy of the tuberous sclerosis complex 2 (Tsc2) gene develop uterine leiomyoma, but the underlying mechanism of human uterine leiomyoma is not completely understood. To examine whether down-regulation of tuberin, a TSC2 gene product, is present in human uterine leiomyoma, we analyzed leiomyoma and matched myometrium tissues from 22 Chinese patients with Western blotting and real-time polymerase chain reaction analyses, and found that the expression of tuberin was significantly increased in leiomyoma tissues compared with matched myometrium tissues with inhibition of both the mammalian target of rapacmycin pathway and mitogen-activated protein kinase pathways.

Levels of Phosphorylated P70 S6 Kinase and Phosphorylated ERK1&2 Predict Overall Survival and Time to First Treatment In Chronic Lymphocytic Leukaemia

AbstractAbstract 1363Chronic lymphocytic leukaemia (CLL) is a clinically heterogeneous disease characterised by an accumulation of malignant B lymphocytes. Central to the survival of CLL-cells is the B-cell Receptor (BCR), a structurally complex moiety which contains an immunoglobulin molecule with antigenic specificity. Stimulation of the BCR by antigen binding may lead to the activation of multiple intracellular kinase cascades with a variety of intracellular functions. Kinases known to be activated include phosphorylated Extracellular Regulated Kinase (pERK) and P70 S6 Kinase (P70S6K).Increased levels of phosphorylated Extracellular Regulated Kinase (pERK) are found in CLL cells induced to proliferate and inhibition of the ERK pathway promotes chemotherapy induced apoptosis in B-cell lines. P70S6K regulates cell cycle behaviour in B cells and it has been shown that inhibition of P70S6K activation by phosphorylation with rapamycin leads to cell cycle arrest in CLL cells.We investigated the relationships between pERK isoforms 1&2 (pERK1&2) and phosphorylated-P70S6K concentrations (both baseline and after BCR-crosslinking) and overall survival (OS) as well as time to first treatment (TTFT) in a cohort of patients with CLL.Lymphocytes were isolated from patients with CLL attending Haematology Clinic at the Hull and East Yorkshire NHS Trust. Collections were made after taking informed consent according to the Declaration of Helsinki with Local Ethics Committee approval (05/Q1104/33). pERK1&2 concentrations and phospho-P70S6K were measured quantitatively using ELISA according to the manufacturer's protocol (#SUV1018 &#DYC896, RnD Systems) in 99 such cases. Kinase concentrations were assayed at baseline in unstimulated cells and also after stimulation by antibody mediated BCR-crosslinking using a goat anti-human IgM antibody (#109-006-129, Jackson ImmunoResearch). The mutational status of the variable region of the immunoglobulin gene (IgVH status) was determined in all cases. The pERK1&2 and phospho-P70S6K concentrations measured were correlated with clinical stage, IgVH, ZAP70 and CD38 status. The individual kinase concentration measurements were then ranked and those in the upper quartile were regarded as being elevated. Statistical analysis was performed to assess the relationships between elevated pERK1&2 and phospho-P70S6K concentrations and OS/TTFT in both unstimulated CLL cells as well as after BCR-crosslinking.An elevated baseline concentration of intracellular pERK1&2 was found to define a subset of IgVH mutated patients with a reduced mean overall survival (157 vs. 259 months, p=0.011) and, also, to identify patients with a shorter median time to first treatment (8 vs. 31 months, p=0.050). An elevated baseline concentration of P70S6K defined subsets of both IgVH mutated (168 vs. 254 months, p=0.046) and CD38 positive (57 vs. 234 months, p=0.000) patients with a reduced overall survival. An elevated concentration of pERK after BCR-crosslinking was shown to define a subset of ZAP70 negative patients with reduced overall survival (99 vs. 233 months, p=0.020), as well as identifying patients with a shorter median time to first treatment (8 vs. 37 months, p=0.011).Our results demonstrate that increased baseline intracellular pERK1&2 and phospho-P70S6K concentrations can be used in conjunction with established prognostic markers to identify patients with a reduced OS and, in the case of pERK1&2, TTFT. An unmutated IgVH status was associated with higher absolute pERK1&2 and phospho-P70S6K baseline concentrations. This suggests that increased intracellular signalling activity may contribute to the adverse prognosis seen in unmutated IgVH patients. The findings support the investigation of kinase inhibitors as novel agents in the management of bad risk CLL. Disclosures:No relevant conflicts of interest to declare.

Monitoring Molecular-Specific Pharmacodynamics of Rapamycin In vivo with Inducible Gal4→Fluc Transgenic Reporter Mice

Rapamycin (Rap), a small-molecule inhibitor of mTOR, is an immunosuppressant, and several Rap analogues are cancer chemotherapeutics. Further pharmacologic development will be significantly facilitated if in vivo reporter models are available to enable monitoring of molecular-specific pharmacodynamic actions of Rap and its analogues. Herein we present the use of a Gal4→Fluc reporter mouse for the study of Rap-induced mTOR/FKBP12 protein-protein interactions in vivo with the use of a mouse two-hybrid transactivation strategy, a derivative of the yeast two-hybrid system applied to live mice. Upon treatment with Rap, a bipartite transactivator was reconstituted, and transcription of a genomic firefly luciferase reporter was activated in a concentration-dependent (K(d) = 2.3 nmol/L) and FK506-competitive (K(i) = 17.1 nmol/L) manner in cellulo, as well as in a temporal and specific manner in vivo. In particular, after a single dose of Rap (4.5 mg/kg, i.p.), peak Rap-induced protein-protein interactions were observed in the liver at 24 hours post treatment, with photon flux signals 600-fold over baseline, which correlated temporally with suppression of p70S6 kinase activity, a downstream effector of mTOR. The Gal4→Fluc reporter mouse provides an intact physiologic system to interrogate protein-protein interactions and molecular-specific pharmacodynamics during drug discovery and lead characterization. Imaging protein interactions and functional proteomics in whole animals in vivo may serve as a basic tool for screening and mechanism-based analysis of small molecules targeting specific protein-protein interactions in human diseases.

Crosstalk between VEGFR2 and muscarinic receptors regulates the mTOR pathway in serum starved SK-N-SH human neuroblastoma cells

Muscarinic acetylcholine receptors (mAchRs) are guanosine nucleotide-binding protein (G protein) coupled receptors that crosstalk with receptor tyrosine kinases (RTKs) to signal mitogenic pathways. In particular, mAchRs are known to couple with RTKs for several growth factors to activate the mammalian target of rapamycin (mTOR)/Akt pathway, a regulator of protein synthesis. The RTK for the vascular endothelial growth factor (VEGF), VEGFR2, can signal protein synthesis but whether it cooperates with mAchRs to mediate mTOR activation has not been demonstrated. Using serum starved SK-N-SH neuroblastoma cells, we show that the muscarinic receptor agonists carbachol and pilocarpine enhance the activation of the mTOR substrate p70 S6 Kinase (S6K) and its target ribosomal protein S6 (S6) in a VEGFR2 dependent manner. Treatments with carbachol increased VEGFR2 phosphorylation, suggesting that mAchRs stimulate VEGFR2 transactivation to enhance mTOR signaling. Inhibitor studies revealed that phosphatidylinositol 3 kinase resides upstream from S6K, S6 and Akt phosphorylation while protein kinase C (PKC) functions in an opposing fashion by positively regulating S6K and S6 phosphorylation and suppressing Akt activation. Treatments with the phosphatase inhibitors sodium orthovanadate and okadaic acid increase S6, Akt and to a lesser extent S6K phosphorylation, indicating that tyrosine and serine/threonine dephosphorylation also regulates their activity. However, okadaic acid elicited a far greater increase in phosphorylation, implicating phosphatase 2A as a critical determinant of their function. Finally, pilocarpine but not carbachol induced a time and dose dependent cell death that was associated with caspase activation and oxidative stress but independent of S6K and S6 activation through VEGFR2. Accordingly, our findings suggest that mAchRs crosstalk with VEGFR2 to enhance mTOR activity but signal divergent effects on survival through alternate mechanisms.

Inactivation of p38 MAPK during liver regeneration

There is increasing evidence that p38 MAPK, which is classified as a stress-activated kinase, also participates in cell cycle regulation, functioning as a suppressor of cell proliferation and tumorigenesis. We conducted a study of p38 MAPK phosphorylation during liver regeneration in mice to determine whether p38 MAPK activation or inactivation may correlate with events that lead to DNA replication after partial hepatectomy (PH), and whether p38 MAPK activation may be required for hepatocyte DNA replication in vivo and in culture. We report that active p38 (Pi-p38 MAPK) is present in normal liver, is rapidly inactivated starting 30 min after PH, and is re-activated by 12 h. Although levels of Pi-MKK 3/6, the upstream kinases that activate p38 MAPK increase after PH, the expression of the dual protein phosphatase 1 is also elevated, and may be responsible for Pi-p38 MAPK dephosphorylation after PH. Inactivation and re-activation of p38 MAPK inversely correlates with the stimulation of protein synthesis and translation pathways, as indicated by activation of p70S6 kinase, increases in the phosphorylation of initiation factor elF-4E and translational repressor, 4E-BP. The activity of a p38 MAPK downstream substrate, MAPKAPK2 (MK2), did not reflect the changing levels of Pi-p38 MAPK during liver regeneration. Pi-p38 MAPK may be involved in TNF-stimulated DNA replication of murine hepatocytes in culture, but is not necessary for hepatocyte DNA replication after PH. Our results suggest that p38 MAPK inactivation plays a permissible role in DNA replication during liver regeneration and is consistent with a role for p38 MAPK in the maintenance of hepatocyte cell cycle arrest in adult liver.