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1.
Cell Mol Life Sci ; 71(2): 331-48, 2014 Jan.
Article in English | MEDLINE | ID: mdl-23760205

ABSTRACT

Programmed necrosis is important in many (patho)physiological settings. For specific therapeutic intervention, however, a better knowledge is required whether necrosis occurs through one single "core program" or through several independent pathways. Previously, the poly(ADP-ribose) polymerase (PARP) pathway has been suggested as a crucial element of tumor necrosis factor (TNF)-mediated necroptosis. Here, we show that TNF-induced necroptosis and the PARP pathway represent distinct and independent routes to programmed necrosis. First, DNA-alkylating agents such as 1-methyl-3-nitro-1-nitrosoguanidine (MNNG) or methyl methanesulfonate rapidly activate the PARP pathway, whereas this is a late and secondary event in TNF-induced necroptosis. Second, inhibition of the PARP pathway does not protect against TNF-induced necroptosis, e.g., the PARP-1 inhibitor 3-AB prevented MNNG- but not TNF-induced adenosine-5'-triposphate depletion, translocation of apoptosis-inducing factor, and necrosis. Likewise, olaparib, a more potent and selective PARP-1 inhibitor failed to block TNF-induced necroptosis, identical to knockdown/knockout of PARP-1, pharmacologic and genetic interference with c-Jun N-terminal kinases and calpain/cathepsin proteases as further components of the PARP pathway. Third, interruption of TNF-induced necroptosis by interference with ceramide generation, RIP1 or RIP3 function or by the radical scavenger butylated hydroxyanisole did not prevent programmed necrosis through the PARP pathway. In summary, our results suggest that the currently established role of the PARP pathway in TNF-induced necroptosis needs to be revised, with consequences for the design of future therapeutic strategies.


Subject(s)
Apoptosis/drug effects , Poly(ADP-ribose) Polymerases/metabolism , Tumor Necrosis Factor-alpha/pharmacology , Animals , Antineoplastic Agents, Alkylating/pharmacology , Benzamides/pharmacology , Calpain/metabolism , Cathepsins/metabolism , Cell Line , Ceramides/metabolism , Free Radical Scavengers/pharmacology , Guanidines/pharmacology , HEK293 Cells , HT29 Cells , HeLa Cells , Humans , JNK Mitogen-Activated Protein Kinases/metabolism , Jurkat Cells , MCF-7 Cells , Methyl Methanesulfonate/pharmacology , Mice , Necrosis , Nuclear Pore Complex Proteins/metabolism , Phthalazines/pharmacology , Piperazines/pharmacology , Poly(ADP-ribose) Polymerase Inhibitors , Poly(ADP-ribose) Polymerases/genetics , RNA Interference , RNA, Small Interfering/metabolism , RNA-Binding Proteins/metabolism , Receptor-Interacting Protein Serine-Threonine Kinases/metabolism
3.
FASEB J ; 19(14): 1945-56, 2005 Dec.
Article in English | MEDLINE | ID: mdl-16319138

ABSTRACT

Although numerous studies have implicated the sphingolipid ceramide in the induction of cell death, a causative function of ceramide in caspase-dependent apoptosis remains a highly debated issue. Here, we show that ceramide is a key mediator of a distinct route to programmed cell death (PCD), i.e., caspase-independent PCD. Under conditions where apoptosis is either not initiated or actively inhibited, TNF induces caspase-independent PCD in L929 fibrosarcoma cells, NIH3T3 fibroblasts, human leukemic Jurkat T cells, and lung fibroblasts by increasing intracellular ceramide levels prior to the onset of cell death. Survival is significantly enhanced when ceramide accumulation is prevented, as demonstrated in fibroblasts genetically deficient for acid sphingomyelinase, in L929 cells overexpressing acid ceramidase, by pharmacological intervention, or by RNA interference. Jurkat cells deficient for receptor-interacting protein 1 (RIP1) do not accumulate ceramide and therefore are fully resistant to caspase-independent PCD whereas Jurkat cells overexpressing the mitochondrial protein Bcl-2 are partially protected, implicating RIP1 and mitochondria as components of the ceramide death pathway. Our data point to a role of caspases (but not cathepsins) in suppressing the ceramide death pathway under physiological conditions. Moreover, clonogenic survival of tumor cells is clearly reduced by induction of the ceramide death pathway, promising additional options for the development of novel tumor therapies.


Subject(s)
Apoptosis , Caspases/metabolism , Ceramides/pharmacology , Animals , Benzoquinones , Cell Line , Ceramides/metabolism , Dose-Response Relationship, Drug , Fibroblasts/metabolism , Flow Cytometry , Humans , Immunoblotting , Jurkat Cells , Lactams, Macrocyclic , Lung/metabolism , Membrane Potentials , Mice , Mitochondria/metabolism , NIH 3T3 Cells , Protein Serine-Threonine Kinases/metabolism , Quinones/pharmacology , RNA Interference , Reactive Oxygen Species , Receptor-Interacting Protein Serine-Threonine Kinases , Sphingomyelin Phosphodiesterase/metabolism , Tumor Necrosis Factor Receptor-Associated Peptides and Proteins/metabolism , Tumor Necrosis Factor-alpha/metabolism
4.
Radiat Oncol ; 4: 41, 2009 Oct 09.
Article in English | MEDLINE | ID: mdl-19818125

ABSTRACT

BACKGROUND: Programmed cell death (PCD) is essential for development and homeostasis of multicellular organisms and can occur by caspase-dependent apoptosis or alternatively, by caspase-independent PCD (ciPCD). Bcl-2, a central regulator of apoptosis, localizes to both mitochondria and the endoplasmic reticulum (ER). Whereas a function of mitochondrial and ER-specific Bcl-2 in apoptosis has been established in multiple studies, corresponding data for ciPCD do not exist. METHODS: We utilized Bcl-2 constructs specifically localizing to mitochondria (Bcl-2 ActA), the ER (Bcl-2 cb5), both (Bcl-2 WT) or the cytosol/nucleus (Bcl-2 DeltaTM) and determined their protective effect on ceramide-mediated ciPCD in transiently and stably transfected Jurkat cells. Expression of the constructs was verified by immunoblots. Ceramide-mediated ciPCD was induced by treatment with human recombinant tumor necrosis factor and determined by flow cytometric measurement of propidium iodide uptake as well as by optical analysis of cell morphology. RESULTS: Only wildtype Bcl-2 had the ability to efficiently protect from ceramide-mediated ciPCD, whereas expression of Bcl-2 solely at mitochondria, the ER, or the cytosol/nucleus did not prevent ceramide-mediated ciPCD. CONCLUSION: Our data suggest a combined requirement for both mitochondria and the ER in the induction and the signaling pathways of ciPCD mediated by ceramide.


Subject(s)
Apoptosis/physiology , Endoplasmic Reticulum/metabolism , Mitochondria/metabolism , Proto-Oncogene Proteins c-bcl-2/genetics , Proto-Oncogene Proteins c-bcl-2/metabolism , Blotting, Western , Caspases/metabolism , Ceramides/toxicity , Flow Cytometry , Humans , Jurkat Cells , Signal Transduction/physiology , Transfection
5.
Cancer Res ; 68(5): 1443-50, 2008 Mar 01.
Article in English | MEDLINE | ID: mdl-18316608

ABSTRACT

Chronic inflammation has been implicated in the pathogenesis of many severe autoimmune disorders, as well as in diabetes, pulmonary diseases, and cancer. Inflammation accompanies most solid cancers including pancreatic ductal adenocarcinoma (PDAC), one of the most fatal cancers with surgery being the only curative therapeutic approach currently available. In the present work, we investigated the role of the major proinflammatory cytokine tumor necrosis factor alpha (TNFalpha) in the malignancy of PDAC cells in vitro and in vivo. In vitro, TNFalpha strongly increased invasiveness of Colo357, BxPc3, and PancTuI cells and showed only moderate antiproliferative effect. TNFalpha treatment of mice bearing orthotopically growing PDAC tumors led to dramatically enhanced tumor growth and metastasis. Notably, we found that PDAC cells themselves secrete TNFalpha. Although inhibition of TNFalpha with infliximab or etanercept only marginally affected proliferation and invasiveness of PDAC cells in vitro, both reagents exerted strong antitumoral effects in vivo. In severe combined immunodeficient mice with orthotopically growing Colo357, BxPc3, or PancTuI tumors, human-specific anti-TNF antibody infliximab reduced tumor growth and metastasis by about 30% and 50%, respectively. Importantly, in a PDAC resection model performed with PancTuI cells, we found an even stronger therapeutic effect for both anti-TNF compounds. Infliximab and etanercept reduced the number of liver metastases by 69% and 42%, respectively, as well as volumes of recurrent tumors by 73% and 51%. Thus, tumor cell-derived TNFalpha plays a profound role in malignancy of PDAC, and inhibition of TNFalpha represents a promising therapeutic option particularly in adjuvant therapy after subtotal pancreatectomy.


Subject(s)
Antineoplastic Agents/pharmacology , Carcinoma, Pancreatic Ductal/pathology , Gene Expression Regulation, Neoplastic , Pancreatic Neoplasms/pathology , Tumor Necrosis Factor-alpha/metabolism , Animals , Cell Line, Tumor , Cell Proliferation , Female , Humans , Interleukin-8/metabolism , Mice , Mice, SCID , Neoplasm Invasiveness , Neoplasm Metastasis
6.
Exp Cell Res ; 313(12): 2703-18, 2007 Jul 15.
Article in English | MEDLINE | ID: mdl-17512928

ABSTRACT

FAN (factor associated with neutral sphingomyelinase [N-SMase] activation) exhibits striking structural homologies to Lyst (lysosomal trafficking regulator), a BEACH protein whose inactivation causes formation of giant lysosomes/Chediak-Higashi syndrome. Here, we show that cells lacking FAN show a statistically significant increase in lysosome size (although less pronounced as Lyst), pointing to previously unrecognized functions of FAN in regulation of the lysosomal compartment. Since FAN regulates activation of N-SMase in complex with receptor for activated C-kinase (RACK)1, a scaffolding protein that recruits and stabilizes activated protein kinase C (PKC) isotypes at cellular membranes, and since an abnormal (calpain-mediated) downregulation/membrane recruitment of PKC has been linked to the defects observed in Lyst-deficient cells, we assessed whether PKC is also of relevance in FAN signaling. Our results demonstrate that activation of PKC is not required for regulation of N-SMase by FAN/RACK1. Conversely, activation of PKC and recruitment/stabilization by RACK1 occurs uniformly in the presence or absence of FAN (and equally, Lyst). Furthermore, regulation of lysosome size by FAN is not coupled to an abnormal downregulation/membrane recruitment of PKC by calpain. Identical results were obtained for Lyst, questioning the previously reported relevance of PKC for formation of giant lysosomes and in Chediak-Higashi syndrome. In summary, FAN mediates activation of N-SMase as well as regulation of lysosome size by signaling pathways that operate independent from activation/membrane recruitment of PKC.


Subject(s)
Cell Membrane/enzymology , Down-Regulation/genetics , Intracellular Signaling Peptides and Proteins/chemistry , Intracellular Signaling Peptides and Proteins/metabolism , Lysosomes/enzymology , Protein Kinase C/metabolism , Repetitive Sequences, Amino Acid , Animals , Cell Membrane/drug effects , Down-Regulation/drug effects , Enzyme Activation/drug effects , Fibroblasts/cytology , Fibroblasts/drug effects , Fibroblasts/enzymology , Humans , Isoenzymes/metabolism , Lysosomes/drug effects , Mice , Neuropeptides/deficiency , Neuropeptides/metabolism , Protein Processing, Post-Translational/drug effects , Protein Transport/drug effects , Proteins/metabolism , Receptors for Activated C Kinase , Tumor Necrosis Factor-alpha/pharmacology , Vesicular Transport Proteins
7.
Exp Cell Res ; 312(19): 3808-21, 2006 Nov 15.
Article in English | MEDLINE | ID: mdl-17026999

ABSTRACT

Death receptors such as the 55 kDa tumor necrosis factor (TNF) receptor (TNF-R55) or Fas can initiate both apoptotic (caspase-dependent) and caspase-independent routes to programmed cell death (PCD). Here, we demonstrate for the first time that the single murine receptor for (TNF)-related apoptosis-inducing ligand (mTRAIL-R2) can induce a caspase-independent form of PCD with necrosis-like features in addition to apoptosis. Analysis of morphological and cellular features of caspase-independent PCD in response to TRAIL and TNF suggests that mTRAIL-R2 and TNF-R55 elicit caspase-independent PCD through similar pathways, although without participation of cathepsins. Cells overexpressing acid ceramidase (AC), an enzyme that metabolizes the sphingolipid ceramide, show enhanced survival from TRAIL-induced caspase-independent PCD but not from apoptosis, implicating a function of ceramide as a key mediator in caspase-independent PCD (but not apoptosis) induced by mTRAIL-R2. In concert with the enhanced resistance of AC-overexpressing cells against caspase-independent PCD induced by TNF, our results suggest that ceramide acts as a common mediator of caspase-independent PCD caused by death receptors such as mTRAIL-R2 and TNF-R55.


Subject(s)
Apoptosis/physiology , Caspases/metabolism , Ceramides/metabolism , Receptors, TNF-Related Apoptosis-Inducing Ligand/metabolism , Animals , Cell Line , Galactosylgalactosylglucosylceramidase/metabolism , Humans , Mice , Receptors, Tumor Necrosis Factor, Type I/metabolism , Recombinant Proteins/metabolism , Signal Transduction , Tumor Necrosis Factor Decoy Receptors/metabolism
8.
J Immunol ; 177(6): 4086-93, 2006 Sep 15.
Article in English | MEDLINE | ID: mdl-16951373

ABSTRACT

Macrophages have a pivotal function in innate immunity against bacterial infections. They are present in all body compartments and able to detect invading microorganisms with high sensitivity. LPS (endotoxin) of Gram-negative bacteria is among the most potent stimuli for macrophages and initiates a wide panel of cellular activation responses. The release of mediators such as TNF-alpha and ILs is essential for the initiation of a proinflammatory antibacterial response. Here, we show that blockade of the large-conductance Ca2+ -activated potassium channel MaxiK (BK) inhibited cytokine production from LPS-stimulated macrophages at the transcriptional level. This inhibitory effect of channel blockade was specific to stimulation with LPS and affected neither stimulation of macrophages with the cytokine TNF-alpha nor LPS-induced activation of cells that do not express MaxiK. Investigation of the upstream intracellular signaling pathways induced by LPS revealed that the blockade of MaxiK selectively inhibited signaling pathways leading to the activation of the transcription factor NF-kappaB and the MAPK p38, whereas activation of ERK was unaffected. We present data supporting that proximal regulation of the inhibitory factor IkappaB-alpha is critically involved in the observed inhibition of NF-kappaB translocation. Using alveolar macrophages from rats, we could show that the necessity of MaxiK function in activation of NF-kappaB and subsequent cytokine production is not restricted to in vitro-generated monocyte-derived macrophages but also can be observed in primary cells. Thus, MaxiK appears to be a central molecule in the NF-kappaB-dependent inflammatory response of macrophages to bacterial LPS.


Subject(s)
I-kappa B Proteins/antagonists & inhibitors , Indoles/pharmacology , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/antagonists & inhibitors , Lipopolysaccharides/immunology , Macrophages, Alveolar/immunology , NF-kappa B/antagonists & inhibitors , Potassium Channel Blockers/pharmacology , Signal Transduction/physiology , Animals , Humans , I-kappa B Proteins/biosynthesis , I-kappa B Proteins/physiology , Inflammation/immunology , Inflammation/metabolism , Large-Conductance Calcium-Activated Potassium Channel alpha Subunits/physiology , Macrophages, Alveolar/drug effects , NF-KappaB Inhibitor alpha , NF-kappa B/physiology , Rats , Rats, Sprague-Dawley , Signal Transduction/drug effects
9.
Exp Cell Res ; 312(11): 2142-53, 2006 Jul 01.
Article in English | MEDLINE | ID: mdl-16631736

ABSTRACT

The cytoplasmic tail of the tumor necrosis factor receptor I (TNF-RI) contains several functionally distinct domains involved in apoptotic signaling. Mutants of TNF-RI carrying deletions of the death domain (DD), internalization domain (TRID), and neutral sphingomyelinase domain (NSD), respectively, retransfected in cells devoid of TNF-RI and TNF-RII, constituted distinct tools to evaluate the specific role of each domain in downstream apoptotic signaling events. Deletion of DD abolishes activation of caspase-3 and -9 and apoptosis following treatment with TNF because of blocked assembly of the DISC. Nevertheless, TNF-RI DeltaTRID, though lacking a DISC, still allows for residual activation of caspase-3 followed by cell death, although caspase-9 activation was not detected. This activity of caspase-3 is probably due to activation of neutral sphingomyelinase (N-SMase). Increased activity of this enzyme was detected in cells expressing TNF-RI DeltaTRID following treatment with TNF, but not in any other cell line investigated. N-SMase is activated by factor associated with N-SMase (FAN). Because TNF-RI DeltaTRID is retained at the cell surface, FAN may interact with the mutated receptor for a prolonged amount of time, thereby overactivating N-SMase. Double deletion of TRID and NSD abolished caspase-3 activation and apoptosis, confirming this hypothesis.


Subject(s)
Cell Death , Receptors, Tumor Necrosis Factor, Type I/physiology , Sphingomyelin Phosphodiesterase/metabolism , Amino Acid Motifs , Animals , Caspase 3 , Caspase 9 , Caspases/drug effects , Caspases/metabolism , Cell Death/genetics , Cells, Cultured , Ceramides/metabolism , Ceramides/pharmacology , DNA-Binding Proteins/genetics , Death Domain Receptor Signaling Adaptor Proteins , Enzyme Activation , Fibroblasts , Gene Deletion , Intracellular Signaling Peptides and Proteins/metabolism , Mice , Mice, Knockout , Models, Biological , Protein Structure, Tertiary/genetics , Protein Transport/physiology , RNA, Small Interfering/pharmacology , Receptors, Tumor Necrosis Factor, Type I/chemistry , Receptors, Tumor Necrosis Factor, Type I/genetics , Receptors, Tumor Necrosis Factor, Type II/genetics , Tumor Necrosis Factor-alpha/pharmacology
10.
Biochem Biophys Res Commun ; 335(2): 575-83, 2005 Sep 23.
Article in English | MEDLINE | ID: mdl-16083851

ABSTRACT

Tumor necrosis factor (TNF) can induce caspase-dependent (apoptotic) and caspase-independent pathways to programmed cell death (PCD). Here, we demonstrate that stable transfection of a cDNA encompassing the C-terminal apoptosis inhibitory domain (AID) of FE65-like protein 1 into mouse L929 fibrosarcoma cells protects from caspase-independent as well as from apoptotic PCD induced by TNF. We show that the AID does not protect from caspase-independent PCD elicited by 1-methyl-3-nitro-1-nitrosoguanidine, suggesting that the AID might prevent cell death by affecting assembly of the death inducing signaling complex of the 55 kDa TNF receptor or clustering of the receptor itself. Interference with caspase-independent PCD mediated by the sphingolipid ceramide further increases protection conferred by the AID, as does the antioxidant butylated hydroxyanisole, implicating ceramide and reactive oxygen species as potential factors interacting with caspase-independent PCD regulated by the AID.


Subject(s)
Adaptor Proteins, Signal Transducing/chemistry , Apoptosis , Caspases/metabolism , Tumor Necrosis Factor-alpha/metabolism , Adaptor Proteins, Signal Transducing/metabolism , Amino Acid Sequence , Animals , Antioxidants/pharmacology , Base Sequence , Cell Death , Cell Line , Cell Line, Tumor , Cell Survival , Ceramides/metabolism , Cloning, Molecular , Dose-Response Relationship, Drug , Genetic Vectors , Humans , Immunoblotting , Mice , Molecular Sequence Data , Nitrosoguanidines/pharmacology , Polymerase Chain Reaction , Protein Structure, Tertiary , Reactive Oxygen Species , Sequence Homology, Amino Acid , Sphingolipids/chemistry , Time Factors , Transfection
11.
J Immunol ; 174(7): 3880-90, 2005 Apr 01.
Article in English | MEDLINE | ID: mdl-15778342

ABSTRACT

Extracellular ATP and other nucleotides act through specific cell surface receptors and regulate a wide variety of cellular responses in many cell types and tissues. In this study, we demonstrate that murine mast cells express several P2Y and P2X receptor subtypes including P2X(7), and describe functional responses of these cells to extracellular ATP. Stimulation of bone marrow-derived mast cells (BMMC), as well as MC/9 and P815 mast cell lines with millimolar concentrations of ATP, resulted in Ca(2+) influx across the cellular membrane and cell permeabilization. Moreover, brief exposures to ATP were sufficient to induce apoptosis in BMMCs, MC/9, and P815 cells which involved activation of caspase-3 and -8. However, in the time period between commitment to apoptosis and actual cell death, ATP triggered rapid but transient phosphorylation of multiple signaling molecules in BMMCs and MC/9 cells, including ERK, Jak2, and STAT6. In addition, ATP stimulation enhanced the expression of several proinflammatory cytokines, such as IL-4, IL-6, IL-13, and TNF-alpha. The effects of ATP were mimicked by submillimolar concentrations of 3-O-(4'-benzoyl)-benzoyl-benzoyl-ATP, and were inhibited by pretreatment of mast cells with a selective blocker of human and mouse P2X(7) receptor, 1[N,O-bis(5-isoquinolinesulphonyl)-N-methyl-l-tyrosyl]-4-phenylpiperazine, as well as oxidized ATP. The nucleotide selectivity and pharmacological profile data support the role for P2X(7) receptor as the mediator of the ATP-induced responses. Given the importance of mast cells in diverse pathological conditions, the ability of extracellular ATP to induce the P2X(7)-mediated apoptosis in these cells may facilitate the development of new strategies to modulate mast cell activities.


Subject(s)
Adenosine Triphosphate/pharmacology , Apoptosis/drug effects , Cytokines/genetics , Mast Cells/drug effects , Receptors, Purinergic P2/physiology , Animals , Bone Marrow Cells , Calcium Signaling , Cells, Cultured , Gene Expression Regulation/drug effects , Mice , Mice, Inbred C57BL , Phosphorylation , Receptors, Purinergic P2X7 , Signal Transduction
12.
EMBO J ; 24(24): 4260-70, 2005 Dec 21.
Article in English | MEDLINE | ID: mdl-16308569

ABSTRACT

Discrimination between cytokine receptor and receptor tyrosine kinase (RTK) signaling pathways is a central paradigm in signal transduction research. Here, we report a 'promiscuous liaison' between both receptors that enables interleukin (IL)-15 to transactivate the signaling pathway of a tyrosine kinase. IL-15 protects murine L929 fibroblasts from tumor necrosis factor alpha (TNFalpha)-induced cell death, but fails to rescue them upon targeted depletion of the RTK, Axl; however, Axl-overexpressing fibroblasts are TNFalpha-resistant. IL-15Ralpha and Axl colocalize on the cell membrane and co-immunoprecipitate even in the absence of IL-15, whereby the extracellular part of Axl proved to be essential for Axl/IL-15Ralpha interaction. Most strikingly, IL-15 treatment mimics stimulation by the Axl ligand, Gas6, resulting in a rapid tyrosine phosphorylation of both Axl and IL-15Ralpha, and activation of the phosphatidylinositol 3-kinase/Akt pathway. This is also seen in mouse embryonic fibroblasts from wild-type but not Axl-/- or IL-15Ralpha-/- mice. Thus, IL-15-induced protection from TNFalpha-mediated cell death involves a hitherto unknown IL-15 receptor complex, consisting of IL-15Ralpha and Axl RTK, and requires their reciprocal activation initiated by ligand-induced IL-15Ralpha.


Subject(s)
Interleukin-15/physiology , Receptors, Interleukin-2/metabolism , Animals , Cell Death , Cell Differentiation , Cell Line , Cell Separation , Ceramides/metabolism , Cytokines/metabolism , Dendritic Cells/cytology , Enzyme Activation , Enzyme-Linked Immunosorbent Assay , Fibroblasts/metabolism , Flow Cytometry , Humans , Intercellular Signaling Peptides and Proteins/metabolism , Interleukin-15/metabolism , Ligands , Mice , Mice, Transgenic , Microscopy, Confocal , Models, Biological , Oncogene Proteins/metabolism , Phosphatidylinositol 3-Kinases/metabolism , Protein Binding , Proto-Oncogene Proteins , RNA, Small Interfering/metabolism , Receptor Protein-Tyrosine Kinases/metabolism , Receptors, Interleukin-15 , Receptors, Interleukin-2/chemistry , Recombinant Fusion Proteins/chemistry , Reverse Transcriptase Polymerase Chain Reaction , Signal Transduction , Transcriptional Activation , Transfection , Tumor Necrosis Factor-alpha/metabolism , Axl Receptor Tyrosine Kinase
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