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1.
Hepatology ; 62(6): 1847-57, 2015 Dec.
Article in English | MEDLINE | ID: mdl-26077809

ABSTRACT

UNLABELLED: Although necrosis in the acetaminophen (APAP) model is known to be regulated by c-Jun NH2-terminal kinase (JNK) through interaction with mitochondria, the role of necroptosis through receptor-interacting proteins 1 and 3 (RIPK1 and RIPK3) has also been suggested. Our aim was to determine the relationship between these two mechanisms of cell death. To verify the participation of RIPK1, we used antisense knockdown and confirmed protection comparable to the RIPK1 inhibitor, necrostatin, in vivo and in vitro. However, we found no evidence that RIPK3 is expressed in primary mouse hepatocytes under basal conditions or after APAP and RIPK3(-/-) mice were not protected. RIPK3 was exclusively expressed in nonparenchymal cells. RIPK1 knockdown protected RIPK3(-/-) mice to the same extent as wild-type mice, underscoring the independent role of RIPK1. We confirmed that necroptosis is not involved in APAP toxicity by using mixed lineage kinase domain-like protein (MLKL) knockout mice, which were not protected from APAP. Next, we addressed whether there is interplay between RIPK1 and JNK. RIPK1 knockdown decreased the level of JNK activation and translocation to mitochondria and abrogated subsequent translocation of dynamin-related protein 1 (Drp1). Interestingly, APAP induced translocation of RIPK1 to mitochondria, which was unaffected by knockdown of the mitochondrial JNK docking protein, Sh3 homology 3 binding protein 5 (Sab). CONCLUSION: RIPK1 participates in APAP-induced necrosis upstream of JNK activation whereas RIPK3 and MLKL are dispensable, indicating that necroptosis does not contribute to APAP-induced necrosis and RIPK1 has a unique, independent role.


Subject(s)
Acetaminophen/toxicity , Protein Kinases/physiology , Receptor-Interacting Protein Serine-Threonine Kinases/physiology , Animals , Apoptosis , Male , Mice , Mice, Inbred C57BL , Necrosis , Organelles
2.
Hepatology ; 59(4): 1543-1554, 2014 Apr.
Article in English | MEDLINE | ID: mdl-23873604

ABSTRACT

UNLABELLED: This study examines the role of protein kinase C (PKC) and AMP-activated kinase (AMPK) in acetaminophen (APAP) hepatotoxicity. Treatment of primary mouse hepatocytes with broad-spectrum PKC inhibitors (Ro-31-8245, Go6983), protected against APAP cytotoxicity despite sustained c-jun-N-terminal kinase (JNK) activation. Broad-spectrum PKC inhibitor treatment enhanced p-AMPK levels and AMPK regulated survival-energy pathways including autophagy. AMPK inhibition by compound C or activation using an AMPK activator oppositely modulated APAP cytotoxicity, suggesting that p-AMPK and AMPK regulated energy survival pathways, particularly autophagy, play a critical role in APAP cytotoxicity. Ro-31-8245 treatment in mice up-regulated p-AMPK levels, increased autophagy (i.e., increased LC3-II formation, p62 degradation), and protected against APAP-induced liver injury, even in the presence of sustained JNK activation and translocation to mitochondria. In contrast, treatment of hepatocytes with a classical PKC inhibitor (Go6976) protected against APAP by inhibiting JNK activation. Knockdown of PKC-α using antisense (ASO) in mice also protected against APAP-induced liver injury by inhibiting JNK activation. APAP treatment resulted in PKC-α translocation to mitochondria and phosphorylation of mitochondrial PKC substrates. JNK 1 and 2 silencing in vivo decreased APAP-induced PKC-α translocation to mitochondria, suggesting PKC-α and JNK interplay in a feed-forward mechanism to mediate APAP-induced liver injury. CONCLUSION: PKC-α and other PKC(s) regulate death (JNK) and survival (AMPK) proteins, to modulate APAP-induced liver injury.


Subject(s)
AMP-Activated Protein Kinases/physiology , Acetaminophen/adverse effects , Chemical and Drug Induced Liver Injury/physiopathology , JNK Mitogen-Activated Protein Kinases/physiology , Protein Kinase C/physiology , Signal Transduction/physiology , AMP-Activated Protein Kinases/antagonists & inhibitors , AMP-Activated Protein Kinases/drug effects , Animals , Cells, Cultured , Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Disease Models, Animal , Hepatocytes/drug effects , Hepatocytes/metabolism , Hepatocytes/pathology , In Vitro Techniques , Indoles/pharmacology , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/drug effects , Male , Maleimides/pharmacology , Mice , Mice, Inbred C57BL , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism , Necrosis/metabolism , Necrosis/pathology , Protein Kinase C/antagonists & inhibitors , Protein Kinase C/drug effects , Protein Kinase Inhibitors/pharmacology
3.
Gastroenterology ; 143(6): 1555-1563.e2, 2012 Dec.
Article in English | MEDLINE | ID: mdl-22902870

ABSTRACT

BACKGROUND & AIMS: After liver injury, bone marrow-derived liver sinusoidal endothelial cell progenitor cells (BM SPCs) repopulate the sinusoid as liver sinusoidal endothelial cells (LSECs). After partial hepatectomy, BM SPCs provide hepatocyte growth factor, promote hepatocyte proliferation, and are necessary for normal liver regeneration. We examined how hepatic vascular endothelial growth factor (VEGF) regulates recruitment of BM SPCs and their effects on liver injury. METHODS: Rats were given injections of dimethylnitrosamine to induce liver injury, which was assessed by histology and transaminase assays. Recruitment of SPCs was analyzed by examining BM SPC proliferation, mobilization to the circulation, engraftment in liver, and development of fenestration (differentiation). RESULTS: Dimethylnitrosamine caused extensive denudation of LSECs at 24 hours, followed by centrilobular hemorrhagic necrosis at 48 hours. Proliferation of BM SPCs, the number of SPCs in the bone marrow, and mobilization of BM SPCs to the circulation increased 2- to 4-fold by 24 hours after injection of dimethylnitrosamine; within 5 days, 40% of all LSECs came from engrafted BM SPCs. Allogeneic resident SPCs, infused 24 hours after injection of dimethylnitrosamine, repopulated the sinusoid as LSECs and reduced liver injury. Expression of hepatic VEGF messenger RNA and protein increased 5-fold by 24 hours after dimethylnitrosamine injection. Knockdown of hepatic VEGF with antisense oligonucleotides completely prevented dimethylnitrosamine-induced proliferation of BM SPCs and their mobilization to the circulation, reduced their engraftment by 46%, completely prevented formation of fenestration after engraftment as LSECs, and exacerbated dimethylnitrosamine injury. CONCLUSIONS: BM SPC recruitment is a repair response to dimethylnitrosamine liver injury in rats. Hepatic VEGF regulates recruitment of BM SPCs to liver and reduces this form of liver injury.


Subject(s)
Chemical and Drug Induced Liver Injury/metabolism , Chemical and Drug Induced Liver Injury/pathology , Endothelial Cells/pathology , Liver/metabolism , Liver/pathology , Stem Cells/pathology , Vascular Endothelial Growth Factor A/metabolism , Animals , Bone Marrow Cells/pathology , Bone Marrow Transplantation , Cell Movement , Cell Proliferation , Chemical and Drug Induced Liver Injury/prevention & control , Dimethylnitrosamine/adverse effects , Hepatectomy , Models, Animal , Rats , Rats, Inbred Lew , Time Factors
4.
J Pharmacol Exp Ther ; 342(1): 150-62, 2012 Jul.
Article in English | MEDLINE | ID: mdl-22505629

ABSTRACT

Antisense oligonucleotides (ASO) containing 2'-O-methoxyethyl ribose (2'-MOE) modifications have been shown to possess both excellent pharmacokinetic properties and robust pharmacological activity in several animal models of human disease. 2'-MOE ASOs are generally well tolerated, displaying minimal to mild proinflammatory effect at doses far exceeding therapeutic doses. Although the vast majority of 2'-MOE ASOs are safe and well tolerated, a small subset of ASOs inducing acute inflammation in mice has been identified. The mechanism for these findings is not clear at this point, but the effects are clearly sequence-specific. One of those ASOs, ISIS 147420, causes a severe inflammatory response atypical of this class of oligonucleotides characterized by induction in interferon-ß (IFN-ß) at 48 h followed by acute transaminitis and extensive hepatocyte apoptosis and necrosis at 72 h. A large number of interferon-stimulated genes were significantly up-regulated in liver as early as 24 h. We speculated that a specific sequence motif might cause ISIS 147420 to be mistaken for viral RNA or DNA, thus triggering an acute innate immune response. ISIS 147420 toxicity was independent of Toll-like receptors, because there was no decrease in IFN-ß in Toll/interleukin-1 receptor-domain-containing adapter-inducing IFN-ß or Myd88-deficient mice. The involvement of cytosolic retinoic acid-inducible gene (RIG)-I-like pattern recognition receptors was also investigated. Pretreatment of mice with melanoma differentiation-associated gene 5 (MDA5) and IFN-ß promoter stimulator-1 ASOs, but not RIG-I or laboratory of genetics and physiology 2 (LGP2) ASOs, prevented the increase in IFN-ß and alanine aminotransferase induced by ISIS 147420. These results revealed a novel mechanism of oligonucleotide-mediated toxicity requiring both MDA5 and IPS-1 and resulting in the activation of the innate immune response.


Subject(s)
DEAD-box RNA Helicases/immunology , DNA/immunology , Immunity, Innate/immunology , Interferon Type I/immunology , Oligonucleotides, Antisense/immunology , Ribose/immunology , Adaptor Proteins, Signal Transducing/genetics , Adaptor Proteins, Signal Transducing/immunology , Adaptor Proteins, Signal Transducing/metabolism , Adaptor Proteins, Vesicular Transport/genetics , Adaptor Proteins, Vesicular Transport/immunology , Adaptor Proteins, Vesicular Transport/metabolism , Alanine Transaminase/genetics , Alanine Transaminase/immunology , Alanine Transaminase/metabolism , Animals , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , DNA/genetics , DNA/metabolism , Hepatocytes/immunology , Hepatocytes/metabolism , Immunity, Innate/genetics , Inflammation/genetics , Inflammation/immunology , Inflammation/metabolism , Interferon Type I/genetics , Interferon Type I/metabolism , Interferon-Induced Helicase, IFIH1 , Interferon-beta/genetics , Interferon-beta/immunology , Interferon-beta/metabolism , Male , Mice , Mice, Inbred BALB C , Mice, Inbred C57BL , Mice, Knockout , Myeloid Differentiation Factor 88/genetics , Myeloid Differentiation Factor 88/immunology , Myeloid Differentiation Factor 88/metabolism , Oligonucleotides, Antisense/genetics , Receptors, Interleukin-1/genetics , Receptors, Interleukin-1/immunology , Receptors, Interleukin-1/metabolism , Ribose/genetics , Ribose/metabolism , Signal Transduction/genetics , Signal Transduction/immunology , Toll-Like Receptors/genetics , Toll-Like Receptors/immunology , Toll-Like Receptors/metabolism
5.
Gastroenterology ; 142(4): 918-927.e6, 2012 Apr.
Article in English | MEDLINE | ID: mdl-22178212

ABSTRACT

BACKGROUND & AIMS: Capillarization, characterized by loss of differentiation of liver sinusoidal endothelial cells (LSECs), precedes the onset of hepatic fibrosis. We investigated whether restoration of LSEC differentiation would normalize crosstalk with activated hepatic stellate cells (HSC) and thereby promote quiescence of HSC and regression of fibrosis. METHODS: Rat LSECs were cultured with inhibitors and/or agonists and examined by scanning electron microscopy for fenestrae in sieve plates. Cirrhosis was induced in rats using thioacetamide, followed by administration of BAY 60-2770, an activator of soluble guanylate cyclase (sGC). Fibrosis was assessed by Sirius red staining; expression of α-smooth muscle actin was measured by immunoblot analysis. RESULTS: Maintenance of LSEC differentiation requires vascular endothelial growth factor-A stimulation of nitric oxide-dependent signaling (via sGC and cyclic guanosine monophosphate) and nitric oxide-independent signaling. In rats with thioacetamide-induced cirrhosis, BAY 60-2770 accelerated the complete reversal of capillarization (restored differentiation of LSECs) without directly affecting activation of HSCs or fibrosis. Restoration of differentiation to LSECs led to quiescence of HSCs and regression of fibrosis in the absence of further exposure to BAY 60-2770. Activation of sGC with BAY 60-2770 prevented progression of cirrhosis, despite continued administration of thioacetamide. CONCLUSIONS: The state of LSEC differentiation plays a pivotal role in HSC activation and the fibrotic process.


Subject(s)
Capillaries/pathology , Cell Differentiation , Endothelial Cells/pathology , Hepatic Stellate Cells/pathology , Liver Cirrhosis, Experimental/pathology , Liver/blood supply , Liver/pathology , Paracrine Communication , Actins/metabolism , Animals , Benzoates/pharmacology , Biphenyl Compounds , Blotting, Western , Capillaries/drug effects , Capillaries/metabolism , Cell Differentiation/drug effects , Cell Proliferation , Cells, Cultured , Cyclic GMP/metabolism , Disease Progression , Endothelial Cells/drug effects , Endothelial Cells/metabolism , Enzyme Activation , Enzyme Activators/pharmacology , Guanylate Cyclase/metabolism , Hepatic Stellate Cells/drug effects , Hepatic Stellate Cells/metabolism , Hydrocarbons, Fluorinated/pharmacology , Liver/drug effects , Liver/metabolism , Liver Cirrhosis, Experimental/chemically induced , Liver Cirrhosis, Experimental/metabolism , Liver Cirrhosis, Experimental/prevention & control , Male , Microscopy, Electron, Scanning , Nitric Oxide , Paracrine Communication/drug effects , Phenotype , Rats , Rats, Sprague-Dawley , Receptors, Cytoplasmic and Nuclear/metabolism , Signal Transduction , Soluble Guanylyl Cyclase , Thioacetamide , Vascular Endothelial Growth Factor A/metabolism
6.
J Biol Chem ; 285(11): 8244-55, 2010 Mar 12.
Article in English | MEDLINE | ID: mdl-20061376

ABSTRACT

Previously we demonstrated that c-Jun N-terminal kinase (JNK) plays a central role in acetaminophen (APAP)-induced liver injury. In the current work, we examined other possible signaling pathways that may also contribute to APAP hepatotoxicity. APAP treatment to mice caused glycogen synthase kinase-3beta (GSK-3beta) activation and translocation to mitochondria during the initial phase of APAP-induced liver injury ( approximately 1 h). The silencing of GSK-3beta, but not Akt-2 (protein kinase B) or glycogen synthase kinase-3alpha (GSK-3alpha), using antisense significantly protected mice from APAP-induced liver injury. The silencing of GSK-3beta affected several key pathways important in conferring protection against APAP-induced liver injury. APAP treatment was observed to promote the loss of glutamate cysteine ligase (GCL, rate-limiting enzyme in GSH synthesis) in liver. The silencing of GSK-3beta decreased the loss of hepatic GCL, and promoted greater GSH recovery in liver following APAP treatment. Silencing JNK1 and -2 also prevented the loss of GCL. APAP treatment also resulted in GSK-3beta translocation to mitochondria and the degradation of myeloid cell leukemia sequence 1 (Mcl-1) in mitochondrial membranes in liver. The silencing of GSK-3beta reduced Mcl-1 degradation caused by APAP treatment. The silencing of GSK-3beta also resulted in an inhibition of the early phase (0-2 h), and blunted the late phase (after 4 h) of JNK activation and translocation to mitochondria in liver following APAP treatment. Taken together our results suggest that activation of GSK-3beta is a key mediator of the initial phase of APAP-induced liver injury through modulating GCL and Mcl-1 degradation, as well as JNK activation in liver.


Subject(s)
Acetaminophen/toxicity , Chemical and Drug Induced Liver Injury/metabolism , Glutamate-Cysteine Ligase/metabolism , Glycogen Synthase Kinase 3/metabolism , JNK Mitogen-Activated Protein Kinases/metabolism , Proto-Oncogene Proteins c-bcl-2/metabolism , Analgesics, Non-Narcotic/toxicity , Animals , Buthionine Sulfoximine/pharmacology , Cells, Cultured , Chemical and Drug Induced Liver Injury/pathology , Cytoplasm/enzymology , Enzyme Inhibitors/pharmacology , Gene Expression Regulation, Enzymologic , Glutamate-Cysteine Ligase/genetics , Glycogen Synthase Kinase 3/antagonists & inhibitors , Glycogen Synthase Kinase 3 beta , Hepatocytes/cytology , Hepatocytes/enzymology , Male , Mice , Mice, Inbred C57BL , Mitochondria, Liver/enzymology , Mitochondria, Liver/pathology , Myeloid Cell Leukemia Sequence 1 Protein , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction/physiology , bcl-2-Associated X Protein/metabolism
7.
Hepatology ; 49(1): 87-96, 2009 Jan.
Article in English | MEDLINE | ID: mdl-19053047

ABSTRACT

UNLABELLED: Activation of c-Jun N-terminal kinase (JNK) has been implicated as a mechanism in the development of steatohepatitis. This finding, together with the reported role of JNK signaling in the development of obesity and insulin resistance, two components of the metabolic syndrome and predisposing factors for fatty liver disease, suggests that JNK may be a central mediator of the metabolic syndrome and an important therapeutic target in steatohepatitis. To define the isoform-specific functions of JNK in steatohepatitis associated with obesity and insulin resistance, the effects of JNK1 or JNK2 ablation were determined in developing and established steatohepatitis induced by a high-fat diet (HFD). HFD-fed jnk1 null mice failed to develop excessive weight gain, insulin resistance, or steatohepatitis. In contrast, jnk2(-/-) mice fed a HFD were obese and insulin-resistant, similar to wild-type mice, and had increased liver injury. In mice with established steatohepatitis, an antisense oligonucleotide knockdown of jnk1 decreased the amount of steatohepatitis in concert with a normalization of insulin sensitivity. Knockdown of jnk2 improved insulin sensitivity but had no effect on hepatic steatosis and markedly increased liver injury. A jnk2 knockdown increased hepatic expression of the proapoptotic Bcl-2 family members Bim and Bax and the increase in liver injury resulted in part from a Bim-dependent activation of the mitochondrial death pathway. CONCLUSION: JNK1 and JNK2 both mediate insulin resistance in HFD-fed mice, but the JNK isoforms have distinct effects on steatohepatitis, with JNK1 promoting steatosis and hepatitis and JNK2 inhibiting hepatocyte cell death by blocking the mitochondrial death pathway.


Subject(s)
Fatty Liver/drug therapy , Fatty Liver/etiology , Insulin Resistance , Mitogen-Activated Protein Kinase 8/antagonists & inhibitors , Mitogen-Activated Protein Kinase 9/antagonists & inhibitors , Animals , Apoptosis Regulatory Proteins/metabolism , Bcl-2-Like Protein 11 , Dietary Fats/administration & dosage , Fatty Liver/pathology , Liver/metabolism , Male , Membrane Proteins/metabolism , Mice , Mice, Inbred C57BL , Mice, Knockout , Mitogen-Activated Protein Kinase 8/physiology , Mitogen-Activated Protein Kinase 9/physiology , Oligonucleotides, Antisense/pharmacology , Proto-Oncogene Proteins/metabolism , bcl-2-Associated X Protein/metabolism
8.
Am J Physiol Cell Physiol ; 295(1): C50-63, 2008 Jul.
Article in English | MEDLINE | ID: mdl-18463227

ABSTRACT

Recent studies have suggested that, in certain cases, necrosis, like apoptosis, may be programmed, involving the activation and inhibition of many signaling pathways. In this study, we examined whether necrosis induced by H(2)O(2) is regulated by signaling pathways in primary hepatocytes. A detailed time course revealed that H(2)O(2) treated to hepatocytes is consumed within minutes, but hepatocytes undergo necrosis several hours later. Thus, H(2)O(2) treatment induces a "lag phase" where signaling changes occur, including PKC activation, Akt (PKB) downregulation, activation of JNK, and downregulation of AMP-activated kinase (AMPK). Investigation of various inhibitors demonstrated that PKC inhibitors were effective in reducing necrosis caused by H(2)O(2) (~80%). PKC inhibitor treatment decreased PKC activity but, surprisingly, also upregulated Akt and AMPK, suggesting that various PKC isoforms negatively regulate Akt and AMPK. Akt did not appear to play a significant role in H(2)O(2)-induced necrosis, since PKC inhibitor treatment protected hepatocytes from H(2)O(2) even when Akt was inhibited. On the other hand, compound C, a selective AMPK inhibitor, abrogated the protective effect of PKC inhibitors against necrosis induced by H(2)O(2). Furthermore, AMPK activators protected against H(2)O(2)-induced necrosis, suggesting that much of the protective effect of PKC inhibition was mediated through the upregulation of AMPK. Work with PKC inhibitors suggested that atypical PKC downregulates AMPK in response to H(2)O(2). Knockdown of PKC-alpha using antisense oligonucleotides also slightly protected (~22%) against H(2)O(2). Taken together, our data demonstrate that the modulation of signaling pathways involving PKC and AMPK can alter H(2)O(2)-induced necrosis, suggesting that a signaling "program" is important in mediating H(2)O(2)-induced necrosis in primary hepatocytes.


Subject(s)
Hepatocytes/cytology , Hydrogen Peroxide/metabolism , Multienzyme Complexes/physiology , Necrosis , Protein Kinase C/physiology , Protein Serine-Threonine Kinases/physiology , AMP-Activated Protein Kinases , Animals , Cells, Cultured , Enzyme Activation , Hepatocytes/drug effects , Hepatocytes/metabolism , Hydrogen Peroxide/pharmacology , Isoenzymes/antagonists & inhibitors , Isoenzymes/metabolism , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , JNK Mitogen-Activated Protein Kinases/metabolism , Mice , Mice, Inbred C57BL , Multienzyme Complexes/antagonists & inhibitors , Phosphorylation , Protein Kinase C/antagonists & inhibitors , Protein Serine-Threonine Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/antagonists & inhibitors , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction
9.
J Biol Chem ; 283(20): 13565-77, 2008 May 16.
Article in English | MEDLINE | ID: mdl-18337250

ABSTRACT

Previously, we demonstrated JNK plays a central role in acetaminophen (APAP)-induced liver injury (Gunawan, B. K., Liu, Z. X., Han, D., Hanawa, N., Gaarde, W. A., and Kaplowitz, N. (2006) Gastroenterology 131, 165-178). In this study, we examine the mechanism involved in activating JNK and explore the downstream targets of JNK important in promoting APAP-induced liver injury in vivo. JNK inhibitor (SP600125) was observed to significantly protect against APAP-induced liver injury. Increased mitochondria-derived reactive oxygen species were implicated in APAP-induced JNK activation based on the following: 1) mitochondrial GSH depletion (maximal at 2 h) caused increased H2O2 release from mitochondria, which preceded JNK activation (maximal at 4 h); 2) treatment of isolated hepatocytes with H2O2 or inhibitors (e.g. antimycin) that cause increased H2O2 release from mitochondria-activated JNK. An important downstream target of JNK following activation was mitochondria based on the following: 1) JNK translocated to mitochondria following activation; 2) JNK inhibitor treatment partially protected against a decline in mitochondria respiration caused by APAP treatment; and 3) addition of purified active JNK to mitochondria isolated from mice treated with APAP plus JNK inhibitor (mitochondria with severe GSH depletion, covalent binding) directly inhibited respiration. Cyclosporin A blocked the inhibitory effect of JNK on mitochondria respiration, suggesting JNK was directly inducing mitochondrial permeability transition in isolated mitochondria from mice treated with APAP plus JNK inhibitor. Addition of JNK to mitochondria isolated from control mice did not affect respiration. Our results suggests that APAP-induced liver injury involves JNK activation, due to increased reactive oxygen species generated by GSH-depleted mitochondria, and translocation of activated JNK to mitochondria where JNK induces mitochondrial permeability transition and inhibits mitochondria bioenergetics.


Subject(s)
Acetaminophen/adverse effects , Analgesics, Non-Narcotic/adverse effects , Energy Metabolism , Liver/injuries , MAP Kinase Kinase 4/metabolism , Mitochondria, Liver/metabolism , Mitochondria/metabolism , Animals , Anthracenes/pharmacology , Enzyme Inhibitors/pharmacology , Hydrogen Peroxide/chemistry , Hydrogen Peroxide/metabolism , Mice , Models, Biological , Oxygen Consumption , Protein Transport
10.
Am J Respir Cell Mol Biol ; 36(3): 276-85, 2007 Mar.
Article in English | MEDLINE | ID: mdl-16990616

ABSTRACT

The Th2 cytokines IL-4 and IL-13 mediate allergic pulmonary inflammation and airways hyperreactivity (AHR) in asthma models through signaling dependent upon the IL-4 receptor-alpha chain (IL-4Ralpha). IL-13 has been further implicated in the overproduction of mucus by the airway epithelium and in lung remodeling that commonly accompanies chronic inflammation. IL-4Ralpha-deficient mice are resistant to allergen-induced asthma, highlighting the therapeutic promise of selective molecular inhibitors of IL-4Ralpha. We designed a chemically modified IL-4Ralpha antisense oligonucleotide (IL-4Ralpha ASO) that specifically inhibits IL-4Ralpha protein expression in lung eosinophils, macrophages, dendritic cells, and airway epithelium after inhalation in allergen-challenged mice. Inhalation of IL-4Ralpha ASO attenuated allergen-induced AHR, suppressed airway eosinophilia and neutrophilia, and inhibited production of airway Th2 cytokines and chemokines in previously allergen-primed and -challenged mice. Histologic analysis of lungs from these animals demonstrated reduced goblet cell metaplasia and mucus staining that correlated with inhibition of Muc5AC gene expression in lung tissue. Therapeutic administration of inhaled IL-4Ralpha ASO in chronically allergen-challenged mice produced a spectrum of anti-inflammatory activity similar to that of systemically administered Dexamethasone with the added benefit of reduced airway neutrophilia. These data support the potential utility of a dual IL-4 and IL-13 oligonucleotide inhibitor in allergy/asthma, and suggest that local inhibition of IL-4Ralpha in the lung is sufficient to suppress allergen-induced pulmonary inflammation and AHR.


Subject(s)
Anti-Inflammatory Agents/metabolism , Oligonucleotides, Antisense/pharmacology , Receptors, Cell Surface/antagonists & inhibitors , Administration, Inhalation , Aerosols , Animals , Asthma/physiopathology , Bronchial Hyperreactivity/therapy , Bronchial Provocation Tests , Chemokines/biosynthesis , Dendritic Cells/drug effects , Dendritic Cells/metabolism , Disease Models, Animal , Eosinophils/drug effects , Eosinophils/metabolism , Gene Expression Regulation/drug effects , Goblet Cells/drug effects , Goblet Cells/pathology , Inflammation , Lung/drug effects , Lung/pathology , Macrophages, Alveolar/drug effects , Macrophages, Alveolar/metabolism , Male , Metaplasia , Mice , Mucins/genetics , Mucins/metabolism , Oligonucleotides, Antisense/administration & dosage , Ovalbumin , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Th2 Cells/drug effects , Th2 Cells/immunology , Treatment Outcome
11.
Gastroenterology ; 131(1): 165-78, 2006 Jul.
Article in English | MEDLINE | ID: mdl-16831600

ABSTRACT

BACKGROUND & AIMS: In searching for effects of acetaminophen (APAP) on hepatocytes downstream of its metabolism that may participate in hepatotoxicity, we examined the role of stress kinases. METHODS: Mouse hepatocytes and C57BL/6 mice were administered a toxic dose of APAP with or without SP600125, a chemical c-jun N-terminal kinase (JNK) inhibitor. JNK activity as reflected in phospho-c-jun levels, serum alanine transaminase (ALT), and liver histology were assessed. Similar experiments were repeated in JNK1 and JNK2 knockout mice and by using antisense oligonucleotide (ASO) to knockdown JNK. RESULTS: Sustained activation of JNK was observed in cultured mouse hepatocytes and in vivo in the liver after APAP treatment. The importance of this pathway was identified by the marked protective effect of SP600125 against APAP toxicity in vitro and in vivo. The specificity of this protective effect was confirmed in vivo by the knockdown of JNK1 and 2 using ASO pretreatment. JNK2 knockout mice and mice treated with JNK2 ASO exhibited partial protection against APAP. One potential target of JNK is Bax translocation, which was enhanced by APAP and blocked by the JNK inhibitor. Protection by the JNK inhibitor persisted in Kupffer cell-depleted mice, whereas there was no protection against CCl(4) or concanavalin A toxicity. CONCLUSIONS: This work suggests that JNK acts downstream of APAP metabolism to promote hepatotoxicity. The results suggest that JNK2 plays a predominant role, although maximum protection was seen with decrease in both forms of JNK.


Subject(s)
Acetaminophen/toxicity , Chemical and Drug Induced Liver Injury/enzymology , JNK Mitogen-Activated Protein Kinases/metabolism , Alanine Transaminase/blood , Analgesics, Non-Narcotic/toxicity , Animals , Anthracenes/therapeutic use , Apoptosis , Blotting, Western , Cells, Cultured , Chemical and Drug Induced Liver Injury/pathology , Chemical and Drug Induced Liver Injury/prevention & control , Disease Models, Animal , Hepatocytes/drug effects , Hepatocytes/enzymology , Hepatocytes/pathology , JNK Mitogen-Activated Protein Kinases/antagonists & inhibitors , Male , Mice , Mice, Inbred C3H , Mice, Inbred C57BL , Mitochondria, Liver/drug effects , Mitochondria, Liver/metabolism
12.
J Immunol ; 176(2): 1218-27, 2006 Jan 15.
Article in English | MEDLINE | ID: mdl-16394012

ABSTRACT

Endothelial cells (ECs) respond to TNF-alpha by altering their F-actin cytoskeleton and junctional permeability through mechanisms that include protein kinase C (PKC) and p38 MAPK. Ezrin, radixin, and moesin (ERM) regulate many cell processes that often require a conformational change of these proteins as a result of phosphorylation on a conserved threonine residue near the C terminus. This study tested the hypothesis that ERM proteins are phosphorylated on this critical threonine residue through TNF-alpha-induced activation of PKC and p38 and modulate permeability increases in pulmonary microvascular ECs. TNF-alpha induced ERM phosphorylation on the threonine residue that required activation of p38, PKC isoforms, and phosphatidylinositol-4-phosphate 5-kinase Ialpha, a major enzyme generating phosphatidylinositol 4,5-bisphosphate, and phosphorylated ERM were prominently localized at the EC periphery. TNF-alpha-induced ERM phosphorylation was accompanied by cytoskeletal changes, paracellular gap formation, and increased permeability to fluxes of dextran and albumin. These changes required activation of p38 and PKC and were completely prevented by inhibition of ERM protein expression using small interfering RNA. Thus, ERM proteins are phosphorylated through p38 and PKC-dependent mechanisms and modulate TNF-alpha-induced increases in endothelial permeability. Phosphorylation of ERM likely plays important roles in EC responses to TNF-alpha by modulating the F-actin cytoskeleton, adhesion molecules, and signaling events.


Subject(s)
Cytoskeletal Proteins/metabolism , Endothelium, Vascular/drug effects , Endothelium, Vascular/metabolism , Lung/blood supply , Membrane Proteins/metabolism , Microfilament Proteins/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Binding Sites , Cell Membrane Permeability/drug effects , Cells, Cultured , Cytoskeletal Proteins/chemistry , Endothelium, Vascular/cytology , Humans , MAP Kinase Signaling System , Membrane Proteins/chemistry , Microcirculation/cytology , Microcirculation/drug effects , Microcirculation/metabolism , Microfilament Proteins/chemistry , Phosphorylation , Protein Kinase C/metabolism , Recombinant Proteins/pharmacology , Signal Transduction , Threonine/chemistry , p38 Mitogen-Activated Protein Kinases/metabolism
13.
Am J Physiol Lung Cell Mol Physiol ; 288(2): L359-69, 2005 Feb.
Article in English | MEDLINE | ID: mdl-15516490

ABSTRACT

Previous studies demonstrated that neutrophil adherence induces ICAM-1-dependent cytoskeletal changes in TNF-alpha-treated pulmonary microvascular endothelial cells that are prevented by a pharmacological inhibitor of p38 MAP kinase. This study determined whether neutrophil adherence induces activation of p38 MAP kinase in endothelial cells, the subcellular localization of phosphorylated p38, which MAP kinase kinases lead to p38 activation, which p38 isoform is activated, and what the downstream targets may be. Confocal microscopy showed that neutrophil adhesion for 2 or 6 min induced an increase in phosphorylated p38 in endothelial cells that was punctate and concentrated in the central region of the endothelial cells. Studies using small interfering RNA (siRNA) to inhibit the protein expression of MAP kinase kinase 3 and 6, either singly or in combination, showed that both MAP kinase kinases were required for p38 phosphorylation. Studies using an antisense oligonucleotide to p38alpha demonstrated that inhibition of the protein expression of p38alpha 1) inhibited activation of p38 MAP kinase without affecting the protein expression of p38beta; 2) prevented phosphorylation of heat shock protein 27, an actin binding protein that may induce actin polymerization upon phosphorylation; 3) attenuated cytoskeletal changes; and 4) attenuated neutrophil migration to the EC borders. Thus MAP kinase kinase3- and 6-dependent activation of the alpha-isoform of p38 MAP kinase is required for the cytoskeletal changes induced by neutrophil adherence and influences subsequent neutrophil migration toward endothelial cell junctions.


Subject(s)
Cytoskeleton/ultrastructure , Endothelium, Vascular/metabolism , Endothelium, Vascular/ultrastructure , Intercellular Adhesion Molecule-1/metabolism , Lung/blood supply , MAP Kinase Kinase 3/metabolism , MAP Kinase Kinase 6/metabolism , p38 Mitogen-Activated Protein Kinases/metabolism , Actins/chemistry , Actins/drug effects , Cell Adhesion/drug effects , Cell Movement/drug effects , Cells, Cultured , Cross-Linking Reagents/pharmacology , Endothelial Cells/metabolism , HSP27 Heat-Shock Proteins , Heat-Shock Proteins/metabolism , Humans , Intercellular Adhesion Molecule-1/drug effects , Isoenzymes/genetics , Isoenzymes/metabolism , Molecular Chaperones , Neoplasm Proteins/metabolism , Neutrophils/physiology , Oligonucleotides, Antisense/pharmacology , Phosphorylation/drug effects , Signal Transduction , p38 Mitogen-Activated Protein Kinases/genetics
14.
J Biol Chem ; 279(12): 11957-66, 2004 Mar 19.
Article in English | MEDLINE | ID: mdl-14704147

ABSTRACT

The c-Jun NH(2)-terminal kinase (JNK) subgroup of mitogen-activated protein kinases has been implicated largely in stress responses, but an increasing body of evidence has suggested that JNK also plays a role in cell proliferation and survival. We examined the effect of JNK inhibition, using either SP600125 or specific antisense oligonucleotides, on cell proliferation and cell cycle progression. SP600125 was selective for JNK in vitro and in vivo versus other kinases tested including ERK, p38, cyclin-dependent protein kinase 1 (CDK1), and CDK2. SP600125 inhibited JNK activity and KB-3 cell proliferation with the same dose dependence, suggesting that inhibition of proliferation was a direct consequence of JNK inhibition. Inhibition of proliferation by SP600125 was associated with an increase in the G(2)-M and apoptotic fractions of cells but was not associated with p53 or p21 induction. Antisense oligonucleotides to JNK2 but not JNK1 caused highly significant inhibition of cell proliferation. Wild-type mouse fibroblasts responded similarly with proliferation inhibition and apoptosis induction, whereas c-jun(-/-) fibroblasts were refractory to the effects of SP600125, suggesting that JNK signaling to c-Jun is required for cell proliferation. Studies in synchronized KB-3 cells indicated that SP600125 delayed transit time through S and G(2)-M phases. Correspondingly, JNK activity increased in late S phase and peaked in late G(2) phase. During synchronous mitotic progression, cyclin B levels increased concomitant with phosphorylation of c-Jun, H1 histone, and Bcl-2. In the presence of SP600125, mitotic progression was prolonged, and c-Jun phosphorylation was inhibited, but neither H1 nor Bcl-2 phosphorylation was inhibited. However, the CDK inhibitor roscovitine inhibited mitotic Bcl-2 phosphorylation. These results indicate that JNK, and more specifically the JNK2 isoform, plays a key role in cell proliferation and cell cycle progression. In addition, conclusive evidence is presented that a kinase other than JNK, most likely CDK1 or a CDK1-regulated kinase, is responsible for mitotic Bcl-2 phosphorylation.


Subject(s)
Cell Cycle , Cell Division , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinase Kinases/antagonists & inhibitors , Proto-Oncogene Proteins c-bcl-2/metabolism , Anthracenes/pharmacology , Base Sequence , Cell Line, Tumor , DNA Primers , Enzyme Inhibitors/pharmacology , Humans , MAP Kinase Kinase 4 , Mitogen-Activated Protein Kinase Kinases/metabolism , Oligonucleotides, Antisense/pharmacology , Phosphorylation
15.
Diabetes ; 53(2): 410-7, 2004 Feb.
Article in English | MEDLINE | ID: mdl-14747292

ABSTRACT

Excess glucagon levels contribute to the hyperglycemia associated with type 2 diabetes. Reducing glucagon receptor expression may thus ameliorate the consequences of hyperglucagonemia and improve blood glucose control in diabetic patients. This study describes the antidiabetic effects of a specific glucagon receptor antisense oligonucleotide (GR-ASO) in db/db mice. The ability of GR-ASOs to inhibit glucagon receptor mRNA expression was demonstrated in primary mouse hepatocytes by quantitative real-time RT-PCR. Intraperitoneal administration of GR-ASO at a dosage of 25 mg/kg twice a week in db/db mice for 3 weeks resulted in 1) decreased glucagon receptor mRNA expression in liver; 2) decreased glucagon-stimulated cAMP production in hepatocytes isolated from GR-ASO-treated db/db mice; 3) significantly reduced blood levels of glucose, triglyceride, and free fatty acids; 4) improved glucose tolerance; and 5) a diminished hyperglycemic response to glucagon challenge. Neither lean nor db/db mice treated with GR-ASO exhibited hypoglycemia. Suppression of GR expression was also associated with increased ( approximately 10-fold) levels of plasma glucagon. No changes were observed in pancreatic islet cytoarchitecture, islet size, or alpha-cell number. However, alpha-cell glucagon levels were increased significantly. Our studies support the concept that antagonism of glucagon receptors could be an effective approach for controlling blood glucose in diabetes.


Subject(s)
Diabetes Mellitus/genetics , Diabetes Mellitus/prevention & control , Down-Regulation/drug effects , Hepatocytes/metabolism , Oligonucleotides, Antisense/pharmacology , Receptors, Glucagon/genetics , Animals , Blood Glucose/metabolism , Cyclic AMP/metabolism , Disease Models, Animal , Female , Gluconeogenesis , Mice , Mice, Inbred C57BL , Mice, Mutant Strains , Oligonucleotides, Antisense/therapeutic use , Transcription, Genetic
16.
J Biol Chem ; 278(48): 47731-43, 2003 Nov 28.
Article in English | MEDLINE | ID: mdl-14504278

ABSTRACT

Previous studies demonstrated that ICAM-1 ligation on human pulmonary microvascular endothelial cells (ECs) sequentially induces activation of xanthine oxidase and p38 MAPK. Inhibition of these signaling events reduces neutrophil migration to the EC borders. This study examined the role of SRC tyrosine kinases in ICAM-1-initiated signaling within these ECs. Cross-linking ICAM-1 on tumor necrosis factor-alpha-pretreated ECs induced an increase in the activity of SRC tyrosine kinases. This increase was inhibited by allopurinol (a xanthine oxidase inhibitor), Me2SO (a hydroxyl radical scavenger), or deferoxamine (an iron chelator). Phenylarsine oxide, a tyrosine phosphatase inhibitor, reduced the base-line activity of SRC as well as the increase in SRC activity induced by ICAM-1 cross-linking. Specific inhibition of the protein expression of the SRC homology 2-containing protein-tyrosine phosphatase-2 (SHP-2) by an antisense oligonucleotide prevented the induced SRC activation but had no effect on the basal SRC activity. Activation of SRC tyrosine kinases was accompanied by tyrosine phosphorylation of ezrin at Tyr-146, which was inhibited by PP2, an SRC tyrosine kinase inhibitor. Moreover, PP2 completely inhibited p38 activation, suggesting a role for SRC tyrosine kinases in p38 activation. These data demonstrate that ICAM-1 ligation activates SRC tyrosine kinases and that this activation requires SHP-2 as well as production of reactive oxygen species generated from xanthine oxidase. Activation of SRC tyrosine kinases in turn leads to tyrosine phosphorylation of ezrin, as well as activation of p38, a kinase previously identified to be required for cytoskeletal changes induced by ICAM-1 ligation and for neutrophil migration along the EC surface.


Subject(s)
Endothelium, Vascular/metabolism , Intercellular Adhesion Molecule-1/metabolism , Lung/blood supply , Microcirculation/metabolism , src-Family Kinases/metabolism , Allopurinol/pharmacology , Animals , Arsenicals/pharmacology , Chelating Agents/pharmacology , Cross-Linking Reagents/pharmacology , Cytoskeletal Proteins , Deferoxamine/pharmacology , Detergents/pharmacology , Dimethyl Sulfoxide/pharmacology , Enzyme Activation , Enzyme Inhibitors/pharmacology , Free Radical Scavengers/pharmacology , Humans , Intracellular Signaling Peptides and Proteins , Mice , Mitogen-Activated Protein Kinases/metabolism , Oligonucleotides, Antisense/pharmacology , Phosphoproteins/metabolism , Phosphorylation , Precipitin Tests , Protein Phosphatase 2 , Protein Structure, Tertiary , Protein Tyrosine Phosphatase, Non-Receptor Type 11 , Protein Tyrosine Phosphatases/metabolism , Reactive Oxygen Species , Recombinant Proteins/metabolism , Signal Transduction , Threonine/chemistry , Time Factors , Tumor Necrosis Factor-alpha/metabolism , Tyrosine/chemistry , Xanthine Oxidase/metabolism , p38 Mitogen-Activated Protein Kinases , src-Family Kinases/chemistry
17.
J Biol Chem ; 278(31): 28388-94, 2003 Aug 01.
Article in English | MEDLINE | ID: mdl-12743126

ABSTRACT

Using cDNA microarrays coupled with bioinformatics tools, we elucidated a signaling cascade regulating cyclooxygenase-2 (COX-2), a pivotal pro-inflammatory enzyme expressed in rheumatic and osteoarthritic, but not normal, cartilage. Exposure of T/C-28a2 chondrocytic cells to fluid shear results in co-regulation of c-Jun N-terminal kinase2 (JNK2), c-Jun, and COX-2 as well as concomitant downstream expression of prostaglandin receptors EP2 and EP3a1. JNK2 transcript inhibition abrogated shear-induced COX-2, EP2, and EP3a1 mRNA up-regulation as well as c-Jun phosphorylation. Functional knock-out experiments using an antisense c-Jun oligonucleotide revealed the abolition of shear-induced COX-2, EP2, and EP3a1, but not JNK2, transcripts. Moreover, inhibition of COX-2 activity eliminated mRNA upregulation of EP2 and EP3a1 induced by shear. Hence, a biochemical pathway exists wherein fluid shear activates COX-2, via a JNK2/c-Jun-dependent pathway, which in turn elicits downstream EP2 and EP3a1 mRNA synthesis.


Subject(s)
Chondrocytes/metabolism , Isoenzymes/biosynthesis , Mitogen-Activated Protein Kinases/metabolism , Prostaglandin-Endoperoxide Synthases/biosynthesis , Proto-Oncogene Proteins c-jun/metabolism , Receptors, Prostaglandin E/genetics , Cell Line , Chondrocytes/enzymology , Cyclooxygenase 2 , Enzyme Induction , Gene Expression Regulation , Humans , Isoenzymes/genetics , Membrane Proteins , Mitogen-Activated Protein Kinase 9 , Mitogen-Activated Protein Kinases/genetics , Oligonucleotide Array Sequence Analysis , Oligonucleotides, Antisense/genetics , Prostaglandin-Endoperoxide Synthases/genetics , Proto-Oncogene Proteins c-jun/genetics , RNA, Messenger/analysis , Receptors, Prostaglandin E, EP2 Subtype , Receptors, Prostaglandin E, EP3 Subtype , Rheology , Signal Transduction , Transfection
18.
Cancer Res ; 62(11): 3257-63, 2002 Jun 01.
Article in English | MEDLINE | ID: mdl-12036942

ABSTRACT

Although the c-Jun NH(2)-terminal kinase (JNK) pathway has been implicated in mediating cell growth and transformation, its downstream effectors remain to be identified. Using JNK2 antisense oligonucleotides (JNK2AS), we uncovered previously a role for JNK2 in regulating cell cycle progression and survival of human PC3 prostate carcinoma cells. Here, to identify genes involved in implementing JNK2-mediated effects, we have analyzed global gene expression changes in JNK2-deprived PC3 cells using Serial Analysis of Gene Expression. More than 40,000 tags each were generated from control and PC3-JNK2AS libraries, corresponding to 15,999 and 20,698 unique transcripts, respectively. Transcripts corresponding to transcription factors, stress-induced genes, and apoptosis-related genes were up-regulated in the PC3-JNK2AS library, revealing a significant stress response after the inhibition of JNK2 expression. Genes involved in DNA repair, mRNA turnover, and drug resistance were found to be down-regulated by inhibition of JNK2 expression, further highlighting the importance of JNK2 signaling in regulating cell homeostasis and tumor cell growth.


Subject(s)
Mitogen-Activated Protein Kinases/physiology , Prostatic Neoplasms/enzymology , Prostatic Neoplasms/genetics , Apoptosis/drug effects , Apoptosis/physiology , Blotting, Northern , Cell Cycle/drug effects , Cell Cycle/physiology , Cell Division/physiology , Gene Expression/drug effects , Gene Expression Profiling , Humans , JNK Mitogen-Activated Protein Kinases , Male , Mitogen-Activated Protein Kinases/antagonists & inhibitors , Mitogen-Activated Protein Kinases/genetics , Oligonucleotides, Antisense/genetics , Oligonucleotides, Antisense/pharmacology , Prostatic Neoplasms/pathology , Signal Transduction/physiology
19.
Diabetes ; 51(4): 1028-34, 2002 Apr.
Article in English | MEDLINE | ID: mdl-11916922

ABSTRACT

Signaling through the phosphatidylinositol 3'-kinase (PI3K) pathway is crucial for metabolic responses to insulin, and defects in PI3K signaling have been demonstrated in type 2 diabetes. PTEN (MMAC1) is a lipid/protein phosphatase that can negatively regulate the PI3K pathway by dephosphorylating phosphatidylinositol (3,4,5)-triphosphate, but it is unclear whether PTEN is physiologically relevant to insulin signaling in vivo. We employed an antisense oligonucleotide (ASO) strategy in an effort to specifically inhibit the expression of PTEN. Transfection of cells in culture with ASO targeting PTEN reduced PTEN mRNA and protein levels and increased insulin-stimulated Akt phosphorylation in alpha-mouse liver-12 (AML12) cells. Systemic administration of PTEN ASO once a week in mice suppressed PTEN mRNA and protein expression in liver and fat by up to 90 and 75%, respectively, and normalized blood glucose concentrations in db/db and ob/ob mice. Inhibition of PTEN expression also dramatically reduced insulin concentrations in ob/ob mice, improved the performance of db/db mice during insulin tolerance tests, and increased Akt phosphorylation in liver in response to insulin. These results suggest that PTEN plays a significant role in regulating glucose metabolism in vivo by negatively regulating insulin signaling.


Subject(s)
Phosphoric Monoester Hydrolases/genetics , Protein Serine-Threonine Kinases , Tumor Suppressor Proteins/genetics , 3T3 Cells , Adipocytes/physiology , Animals , Cell Line , Cells, Cultured , Gene Expression Regulation , Genes, Tumor Suppressor , Glucose/metabolism , Hepatocytes , Insulin/metabolism , Kinetics , Lipid Metabolism , Mice , Oligodeoxyribonucleotides, Antisense/pharmacology , PTEN Phosphohydrolase , Phosphatidylinositol 3-Kinases/metabolism , Phosphoric Monoester Hydrolases/antagonists & inhibitors , Protein-Tyrosine Kinases/genetics , Protein-Tyrosine Kinases/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Proto-Oncogene Proteins c-akt , RNA, Messenger/genetics , Recombinant Proteins/antagonists & inhibitors , Transcription, Genetic/drug effects , Transfection , Tumor Suppressor Proteins/antagonists & inhibitors
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