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1
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Liu X, Shi X, Zhao H, Wang C. Exploring the molecular mechanisms of comorbidity of myocardial infarction and anxiety disorders by combining multiple data sets with in vivo experimental validation. Int Immunopharmacol 2025; 146:113852. [PMID: 39733641 DOI: 10.1016/j.intimp.2024.113852] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/24/2024] [Revised: 11/25/2024] [Accepted: 12/10/2024] [Indexed: 12/31/2024]
Abstract
BACKGROUND The incidence of comorbidity between myocardial infarction (MI) and anxiety disorders is increasing. However, the biological association between them has not been fully understood. OBJECTIVE This study aims to investigate the molecular mechanisms of comorbidity between MI and anxiety disorders and to predict their key genes and potential therapeutic drugs. METHODS We searched Gene Expression Omnibus databases and performed differential analyses using the limma package to identify the functional enrichment of differential genes. Next, we constructed regulatory networks to investigate the relationship between hub genes and autophagy, ferroptosis, and immunity. Furthermore, we predicted transcription factors by R package, constructed a miRNA network, performed the single-cell analysis of key gene expression, and predicted drug targeting of differential genes using the Connectivity Map database. RESULTS The datasets for MI and anxiety disorders were analyzed for up and down-regulated differential genes, resulting in 35 intersecting differential genes. The top 10 feature genes from each dataset were intersected using Random Forest, resulting in the identification of three intersecting genes: STK17B, AKIRIN2, and WDR77. Validation of the above key genes was carried out by in vitro experiments. We examined the gene expression of STK17B, WDR77 and AKIRIN2 in the hippocampus and myocardial infarction border zone respectively by qPCR and WB, and the results confirmed that the above are the key genes for myocardial infarction and anxiety. There is a significant correlation between the comorbidity mechanism of myocardial infarction and anxiety disorders with ferroptosis and immunity. The construction of the miRNA network revealed that miR-205 and let-7 had higher average connectivity among the three hub genes. The single-cell analysis revealed significant expression of key genes in Endothelial cells, Cardiomyocytes, Macrophages, and Fibroblasts datasets. Cd274 showed a higher correlation with key genes in myocardial infarction and anxiety disorders. CONCLUSION Validation by multiple datasets and in vitro experiments showed that STK17B, AKIRIN2, and WDR77 are the key genes in the comorbidity of myocardial infarction and anxiety disorders, and ferroptosis and immunity are the key links in the comorbidity mechanism of myocardial infarction and anxiety disorders.
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Affiliation(s)
- Xiang Liu
- Beijing University of Chinese Medicine, Beijing, China.
| | - Xiaojun Shi
- Beijing University of Chinese Medicine, Beijing, China
| | - Haibin Zhao
- Dongfang Hospital of Beijing University of Chinese Medicine, Beijing, China
| | - Chao Wang
- Dongfang Hospital of Beijing University of Chinese Medicine, Beijing, China.
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2
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Wilander BA, Harris TL, Mandarano AH, Guy CS, Prater MS, Pruett-Miller SM, Ogden SK, McGargill MA. DRAK2 regulates myosin light chain phosphorylation in T cells. J Cell Sci 2024; 137:jcs261813. [PMID: 39421891 DOI: 10.1242/jcs.261813] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/16/2023] [Accepted: 10/09/2024] [Indexed: 10/19/2024] Open
Abstract
Death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2; also known as STK17B) is a serine/threonine kinase expressed in T cells. Drak2-deficient (Drak2-/-) mice respond effectively to tumors and pathogens while displaying resistance to T cell-mediated autoimmune disease. However, the molecular mechanisms by which DRAK2 impacts T cell function remain unclear. Gaining further insight into the function of DRAK2 in T cells will shed light on differentially regulated pathways in autoreactive and pathogen-specific T cells, which is crucial for improving autoimmune therapies. Here, we demonstrate that DRAK2 contributes to activation of myosin light chain (MLC2, encoded by Myl2) in both murine and human T cells. In the absence of Drak2, the amount of polymerized actin was decreased, suggesting that DRAK2 modulates actomyosin dynamics. We further show that myosin-dependent T cell functions, such as migration, T cell receptor microcluster accumulation, and conjugation to antigen presenting cells are decreased in the absence of Drak2. These findings reveal that DRAK2 plays an important role in regulating MLC activation within T cells.
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Affiliation(s)
- Benjamin A Wilander
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
- Graduate School of Biomedical Sciences, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Tarsha L Harris
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Alexandra H Mandarano
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Cliff S Guy
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Mollie S Prater
- The Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
- Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Shondra M Pruett-Miller
- The Center for Advanced Genome Engineering, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
- Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Stacey K Ogden
- Department of Cell and Molecular Biology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Maureen A McGargill
- Department of Immunology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
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3
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Wang D, Yan B, Wang A, Sun Q, Pang J, Cui Y, Tian G. Tu-Xian Decoction ameliorates diabetic cognitive impairment by inhibiting DAPK-1. Chin J Nat Med 2023; 21:950-960. [PMID: 38143108 DOI: 10.1016/s1875-5364(23)60428-5] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2023] [Indexed: 12/26/2023]
Abstract
Tu-Xian decoction (TXD), a traditional Chinese medicine (TCM) formula, has been frequently administered to manage diabetic cognitive impairment (DCI). Despite its widespread use, the mechanisms underlying TXD's protective effects on DCI have yet to be fully elucidated. As a significant regulator in neurodegenerative conditions, death-associated protein kinase-1 (DAPK-1) serves as a focus for understanding the action of TXD. This study was designed to whether TXD mediates its beneficial outcomes by inhibiting DAPK-1. To this end, a diabetic model was established using Sprague-Dawley (SD) rats through a high-fat, high-sugar (HFHS) diet regimen, followed by streptozotocin (STZ) injection. The experimental cohort was stratified into six groups: Control, Diabetic, TC-DAPK6, high-dose TXD, medium-dose TXD, and low-dose TXD groups. Following a 12-week treatment period, various assessments-including blood glucose levels, body weight measurements, Morris water maze (MWM) testing for cognitive function, brain magnetic resonance imaging (MRI), and histological analyses using hematoxylin-eosin (H&E), and Nissl staining-were conducted. Protein expression in the hippocampus was quantified through Western blotting analysis. The results revealed that TXD significantly improved spatial learning and memory abilities, and preserved hippocampal structure in diabetic rats. Importantly, TXD administration led to a down-regulation of proteins indicative of neurological damage and suppressed DAPK-1 activity within the hippocampal region. These results underscore TXD's potential in mitigating DCIvia DAPK-1 inhibition, positioning it as a viable therapeutic candidate for addressing this condition. Further investigation into TXD's molecular mechanisms may elucidate new pathways for the treatment of DCI.
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Affiliation(s)
- Danyang Wang
- Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Beijing 100730, China; Chinese Academy of Mediucal Sciences & Peking Union Medical College, Beijing 100730, China
| | - Bin Yan
- Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Beijing 100730, China
| | - An Wang
- Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Beijing 100730, China; Chinese Academy of Mediucal Sciences & Peking Union Medical College, Beijing 100730, China
| | - Qing Sun
- Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Beijing 100730, China
| | - Junyi Pang
- Department of Pathology, Peking Union Medical College Hospital, State Key Laboratory of Complex Severe and Rare Disease, Molecular Pathology Research Center, Beijing 100730, China
| | - Yangming Cui
- Animal Research Laboratory Platform, Peking Union Medical College Hospital, the National Science and Technology Key Infrastructure on Translational Medicine, Beijing 100730, China
| | - Guoqing Tian
- Department of Traditional Chinese Medicine, Peking Union Medical College Hospital, Beijing 100730, China.
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4
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Park S, Kye S, Jung ME, Chae CH, Yang K, Kim S, Choi G, Lee K. Discovery of TRD‐93 as a novel
DRAK2
inhibitor. B KOREAN CHEM SOC 2023. [DOI: 10.1002/bkcs.12680] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/28/2023]
Affiliation(s)
- Sangjun Park
- Bio & Drug Discovery Division Korea Research Institute of Chemical Technology Daejeon South Korea
- Medicinal Chemistry & Pharmacology University of Science & Technology Daejeon South Korea
| | - Seungmin Kye
- Bio & Drug Discovery Division Korea Research Institute of Chemical Technology Daejeon South Korea
- Medicinal Chemistry & Pharmacology University of Science & Technology Daejeon South Korea
| | - Myoung Eun Jung
- Bio & Drug Discovery Division Korea Research Institute of Chemical Technology Daejeon South Korea
| | - Chong Hak Chae
- Bio & Drug Discovery Division Korea Research Institute of Chemical Technology Daejeon South Korea
| | | | | | - Gildon Choi
- Bio & Drug Discovery Division Korea Research Institute of Chemical Technology Daejeon South Korea
- Medicinal Chemistry & Pharmacology University of Science & Technology Daejeon South Korea
| | - Kwangho Lee
- Bio & Drug Discovery Division Korea Research Institute of Chemical Technology Daejeon South Korea
- Medicinal Chemistry & Pharmacology University of Science & Technology Daejeon South Korea
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5
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Shi X, Zhou Q, Huang B, Xia S, Jiang Y, Fang S, Lin J. Prognostic and immune-related value of STK17B in skin cutaneous melanoma. PLoS One 2022; 17:e0263311. [PMID: 35171924 PMCID: PMC8849620 DOI: 10.1371/journal.pone.0263311] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/17/2021] [Accepted: 01/16/2022] [Indexed: 11/26/2022] Open
Abstract
Skin cutaneous melanoma (SKCM) is a common cancer of which mortality is increasing continuously. Our study conducted a series of analyses on the clinical significance of Serine/threonine kinase 17B (STK17B) in SKCM to provide a new biomarker for diagnosis and treatment. The RNA-sequence data were obtained from The Cancer Genome Atlas and Genotype-Tissue Expression databases. The data of 468 SKCM patients were divided into STK17B high- and low-expression groups and analyzed by Bioconductor package to identify the differential expressed genes. The R package of "clusterProfiler" was used for Gene Ontology, Kyoto Encyclopedia of Genes and Genomes, and Gene-Set Enrichment Analysis analyses. A protein-protein interaction network and immune infiltration landscape were respectively constructed via STRING database and ssGSEA. STK17B had lower expression in SKCM than normal tissues. Besides, STK17B expression was significantly related to some clinicopathological characteristics in SKCM patients including T stage, Breslow depth, radiation therapy, melanoma Clark level, and pathologic stage. The Kaplan-Meier curve analyses revealed that the low expression of STK17B was correlated with poor overall survival and disease-specific survival. We constructed nomograms to predict the 1-, 3-, and 5-year survival of SKCM patients. The function enrichment analyses showed STK17B-related differential expressed genes were enriched in cellular differentiation and immune-related progress. STK17B expression level were positively correlated with infiltrating level of immune cells. In this study, we found that STK17B, which played an important role in immune infiltration, could be a new biomarker for diagnosis and prognosis in SKCM patients.
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Affiliation(s)
- Xueying Shi
- Clinical Laboratory of Integrative Medicine, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China
- Institute (College) of Integrative Medicine, Dalian Medical University, Dalian, Liaoning, China
| | - Qi Zhou
- Clinical Laboratory of Integrative Medicine, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China
- Institute (College) of Integrative Medicine, Dalian Medical University, Dalian, Liaoning, China
| | - Bingqian Huang
- Clinical Laboratory of Integrative Medicine, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China
- Institute (College) of Integrative Medicine, Dalian Medical University, Dalian, Liaoning, China
| | - Shilin Xia
- Clinical Laboratory of Integrative Medicine, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China
| | - Yuankuan Jiang
- Department of Dermatology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China
| | - Shifeng Fang
- Department of Ophthalmology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China
| | - Jingrong Lin
- Department of Dermatology, The First Affiliated Hospital of Dalian Medical University, Dalian, Liaoning, China
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6
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Horvath M, Petrvalska O, Herman P, Obsilova V, Obsil T. 14-3-3 proteins inactivate DAPK2 by promoting its dimerization and protecting key regulatory phosphosites. Commun Biol 2021; 4:986. [PMID: 34413451 PMCID: PMC8376927 DOI: 10.1038/s42003-021-02518-y] [Citation(s) in RCA: 19] [Impact Index Per Article: 4.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/11/2021] [Accepted: 08/03/2021] [Indexed: 01/05/2023] Open
Abstract
Death-associated protein kinase 2 (DAPK2) is a CaM-regulated Ser/Thr protein kinase, involved in apoptosis, autophagy, granulocyte differentiation and motility regulation, whose activity is controlled by autoinhibition, autophosphorylation, dimerization and interaction with scaffolding proteins 14-3-3. However, the structural basis of 14-3-3-mediated DAPK2 regulation remains unclear. Here, we structurally and biochemically characterize the full-length human DAPK2:14-3-3 complex by combining several biophysical techniques. The results from our X-ray crystallographic analysis revealed that Thr369 phosphorylation at the DAPK2 C terminus creates a high-affinity canonical mode III 14-3-3-binding motif, further enhanced by the diterpene glycoside Fusicoccin A. Moreover, concentration-dependent DAPK2 dimerization is disrupted by Ca2+/CaM binding and stabilized by 14-3-3 binding in solution, thereby protecting the DAPK2 inhibitory autophosphorylation site Ser318 against dephosphorylation and preventing Ca2+/CaM binding. Overall, our findings provide mechanistic insights into 14-3-3-mediated DAPK2 inhibition and highlight the potential of the DAPK2:14-3-3 complex as a target for anti‐inflammatory therapies. Horvath et al. structurally and biochemically characterize the full-length human DAPK2-14-3-3 complex to investigate the effects of binding to DAPK2 on its dimerization, activation by dephosphorylation of Ser318, and Ca2+/calmodulin binding. Their results provide mechanistic insights into 14- 3-3-mediated DAPK2 inhibition and highlight the potential of the DAPK2:14-3-3 complex as a target for anti-inflammatory therapies.
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Affiliation(s)
- Matej Horvath
- Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic.,Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Vestec, Czech Republic
| | - Olivia Petrvalska
- Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic.,Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Vestec, Czech Republic
| | - Petr Herman
- Institute of Physics, Faculty of Mathematics and Physics, Charles University, Prague, Czech Republic
| | - Veronika Obsilova
- Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Vestec, Czech Republic.
| | - Tomas Obsil
- Department of Physical and Macromolecular Chemistry, Faculty of Science, Charles University, Prague, Czech Republic. .,Department of Structural Biology of Signaling Proteins, Division BIOCEV, Institute of Physiology of the Czech Academy of Sciences, Vestec, Czech Republic.
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7
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Dwivedi R, Pandey R, Chandra S, Mehrotra D. Apoptosis and genes involved in oral cancer - a comprehensive review. Oncol Rev 2020; 14:472. [PMID: 32685111 PMCID: PMC7365992 DOI: 10.4081/oncol.2020.472] [Citation(s) in RCA: 22] [Impact Index Per Article: 4.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/24/2019] [Accepted: 03/20/2020] [Indexed: 12/24/2022] Open
Abstract
Oral cancers needs relentless research due to high mortality and morbidity associated with it. Despite of the comparable ease in accessibility to these sites, more than 2/3rd cases are diagnosed in advanced stages. Molecular/genetic studies augment clinical assessment, classification and prediction of malignant potential of oral lesions, thereby reducing its incidence and increasing the scope for early diagnosis and treatment of oral cancers. Herein we aim to review the role of apoptosis and genes associated with it in oral cancer development in order to aid in early diagnosis, prediction of malignant potential and evaluation of possible treatment targets in oral cancer. An internet-based search was done with key words apoptosis, genes, mutations, targets and analysis to extract 72 articles after considering inclusion and exclusion criteria. The knowledge of genetics and genomics of oral cancer is of utmost need in order to stop the rising prevalence of oral cancer. Translational approach and interventions at the early stage of oral cancer, targeted destruction of cancerous cells by silencing or promoting involved genes should be the ideal intervention.
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Affiliation(s)
- Ruby Dwivedi
- DHR-MRU & Department of Oral and Maxillofacial Surgery, Faculty of Dental Sciences, King George's Medical University, Lucknow, Uttar Pradesh, India
| | - Rahul Pandey
- DHR-MRU & Department of Oral and Maxillofacial Surgery, Faculty of Dental Sciences, King George's Medical University, Lucknow, Uttar Pradesh, India
| | - Shaleen Chandra
- DHR-MRU & Department of Oral and Maxillofacial Surgery, Faculty of Dental Sciences, King George's Medical University, Lucknow, Uttar Pradesh, India
| | - Divya Mehrotra
- DHR-MRU & Department of Oral and Maxillofacial Surgery, Faculty of Dental Sciences, King George's Medical University, Lucknow, Uttar Pradesh, India
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8
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Thomas F, Holmes KB, Kreuz S, Hillmen P, Lefevre PF. DAPK3 participates in the mRNA processing of immediate early genes in chronic lymphocytic leukaemia. Mol Oncol 2020; 14:1268-1281. [PMID: 32306542 PMCID: PMC7266284 DOI: 10.1002/1878-0261.12692] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/13/2019] [Revised: 03/24/2020] [Accepted: 03/29/2020] [Indexed: 11/29/2022] Open
Abstract
Cross‐linking of the B‐cell receptor (BCR) induces transcriptional activation of immediate early genes (IEGs) including EGR1 and DUSP2 in chronic lymphocytic leukaemia (CLL). Here, we have shown that this transcriptional activation correlated with histone H3 threonine 6 and 11 phosphorylation. Both transcription and histone post‐translational modifications are repressed by ibrutinib, a small molecule inhibitor used in CLL treatment. Moreover, we have identified the death‐associated protein kinase 3 (DAPK3), as the kinase mediating these histone phosphorylation marks in response to activation of the BCR signalling pathway with this kinase being recruited to RNA polymerase II in an anti‐IgM‐dependent manner. DAPK inhibition mimics ibrutinib‐induced repression of both IEG mRNA and histone H3 phosphorylation and has anti‐proliferative effect comparable to ibrutinib in CLL in vitro. DAPK inhibitor does not repress transcription itself but impacts on mRNA processing and has a broader anti‐tumour effect than ibrutinib, by repressing both anti‐IgM‐ and CD40L‐dependent activation.
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Affiliation(s)
- Fraser Thomas
- Division of Haematology and Immunology, Leeds Institute of Medical Research at St. James's, University of Leeds, UK
| | - Katie B Holmes
- Division of Haematology and Immunology, Leeds Institute of Medical Research at St. James's, University of Leeds, UK
| | - Sarah Kreuz
- Division of Haematology and Immunology, Leeds Institute of Medical Research at St. James's, University of Leeds, UK
| | - Peter Hillmen
- Division of Haematology and Immunology, Leeds Institute of Medical Research at St. James's, University of Leeds, UK
| | - Pascal F Lefevre
- Division of Haematology and Immunology, Leeds Institute of Medical Research at St. James's, University of Leeds, UK
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9
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10
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Tur MK, Daramola AK, Gattenlöhner S, Herling M, Chetty S, Barth S. Restoration of DAP Kinase Tumor Suppressor Function: A Therapeutic Strategy to Selectively Induce Apoptosis in Cancer Cells Using Immunokinase Fusion Proteins. Biomedicines 2017; 5:biomedicines5040059. [PMID: 28976934 PMCID: PMC5744083 DOI: 10.3390/biomedicines5040059] [Citation(s) in RCA: 10] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/11/2017] [Revised: 09/26/2017] [Accepted: 09/27/2017] [Indexed: 12/16/2022] Open
Abstract
Targeted cancer immunotherapy is designed to selectively eliminate tumor cells without harming the surrounding healthy tissues. The death-associated protein kinases (DAPk) are a family of proapoptotic proteins that play a vital role in the regulation of cellular process and have been identified as positive mediators of apoptosis via extrinsic and intrinsic death-regulating signaling pathways. Tumor suppressor activities have been shown for DAPk1 and DAPk2 and they are downregulated in e.g., Hodgkin's (HL) and B cell lymphoma (CLL), respectively. Here, we review a targeted therapeutic approach which involves reconstitution of DAPks by the generation of immunokinase fusion proteins. These recombinant proteins consist of a disease-specific ligand fused to a modified version of DAPk1 or DAPk2. HL was targeted via CD30 and B-CLL via CD22 cell surface antigens.
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Affiliation(s)
- Mehmet Kemal Tur
- Institute of Pathology, University Hospital, Justus Liebig University Giessen, 35390 Giessen, Germany.
| | - Adebukola K Daramola
- South African Research Chair in Cancer Biotechnology, Institute of Infectious Disease and Molecular Medicine, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town 7700, South Africa.
| | - Stefan Gattenlöhner
- Institute of Pathology, University Hospital, Justus Liebig University Giessen, 35390 Giessen, Germany.
| | - Marco Herling
- Laboratory of Lymphocyte Signaling and Oncoproteome, Excellence Cluster for Cellular Stress Response and Aging-Associated Diseases, University of Cologne, 50923 Köln, Germany.
- Department I of Internal Medicine, Center for Integrated Oncology Köln-Bonn, and CECAD, University of Cologne, 50923 Köln, Germany.
| | - Shivan Chetty
- South African Research Chair in Cancer Biotechnology, Institute of Infectious Disease and Molecular Medicine, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town 7700, South Africa.
| | - Stefan Barth
- South African Research Chair in Cancer Biotechnology, Institute of Infectious Disease and Molecular Medicine, Department of Integrative Biomedical Sciences, Faculty of Health Sciences, University of Cape Town, Cape Town 7700, South Africa.
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11
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Wang S, Xu L, Lu YT, Liu YF, Han B, Liu T, Tang J, Li J, Wu J, Li JY, Yu LF, Yang F. Discovery of benzofuran-3(2H)-one derivatives as novel DRAK2 inhibitors that protect islet β-cells from apoptosis. Eur J Med Chem 2017; 130:195-208. [PMID: 28249207 DOI: 10.1016/j.ejmech.2017.02.048] [Citation(s) in RCA: 29] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/11/2017] [Revised: 02/17/2017] [Accepted: 02/18/2017] [Indexed: 12/11/2022]
Abstract
Death-associated protein kinase-related apoptosis-inducing kinase-2 (DRAK2) is a serine/threonine kinase that plays a key role in a wide variety of cell death signaling pathways. Inhibition of DRAK2 was found to efficiently protect islet β-cells from apoptosis and hence DRAK2 inhibitors represent a promising therapeutic strategy for the treatment of diabetes. Only very few chemical entities targeting DRAK2 are currently known. We carried out a high throughput screening and identified compound 4 as a moderate DRAK2 inhibitor with an IC50 value of 3.15 μM. Subsequent SAR studies of hit compound 4 led to the development of novel benzofuran-3(2H)-one series of DRAK2 inhibitors with improved potency and favorable selectivity profiles against 26 selected kinases. Importantly, most potent compounds 40 (IC50 = 0.33 μM) and 41 (IC50 = 0.25 μM) were found to protect islet β-cells from apoptosis in dose-dependent manners. These data support the notion that small molecule inhibitors of DRAK2 represents a promising strategy for the treatment of diabetes.
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Affiliation(s)
- Sheng Wang
- Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
| | - Lei Xu
- Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guoshoujing Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China
| | - Yu-Ting Lu
- Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guoshoujing Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China
| | - Yu-Fei Liu
- Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
| | - Bing Han
- Laboratory of Immunology and Cardiovascular Research, Centre Hospitalier de l'Université de Montréal, 900 rue St-Denis, Montréal, Québec, Canada
| | - Ting Liu
- Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
| | - Jie Tang
- Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China; Shanghai Key Laboratory of Green Chemistry and Chemical Process, School of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China
| | - Jia Li
- Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guoshoujing Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China
| | - Jiangping Wu
- Laboratory of Immunology and Cardiovascular Research, Centre Hospitalier de l'Université de Montréal, 900 rue St-Denis, Montréal, Québec, Canada.
| | - Jing-Ya Li
- Chinese National Center for Drug Screening, State Key Laboratory of Drug Research, Shanghai Institute of Materia Medica, Chinese Academy of Sciences, 189 Guoshoujing Road, Zhangjiang Hi-Tech Park, Shanghai 201203, China.
| | - Li-Fang Yu
- Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China.
| | - Fan Yang
- Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development, School of Chemistry and Molecular Engineering, East China Normal University, 3663 North Zhongshan Road, Shanghai 200062, China.
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Abstract
The identification of genetic variants responsible for behavioral variation is an enduring goal in biology, with wide-scale ramifications, ranging from medical research to evolutionary theory on personality syndromes. Here, we use for the first time a large-scale genetical genomics analysis in the brains of chickens to identify genes affecting anxiety as measured by an open field test. We combine quantitative trait locus (QTL) analysis in 572 individuals and expression QTL (eQTL) analysis in 129 individuals from an advanced intercross between domestic chickens and Red Junglefowl. We identify 10 putative quantitative trait genes affecting anxiety behavior. These genes were tested for an association in the mouse Heterogeneous Stock anxiety (open field) data set and human GWAS data sets for bipolar disorder, major depressive disorder, and schizophrenia. Although comparisons between species are complex, associations were observed for four of the candidate genes in mice and three of the candidate genes in humans. Using a multimodel approach we have therefore identified a number of putative quantitative trait genes affecting anxiety behavior, principally in chickens but also with some potentially translational effects as well. This study demonstrates that chickens are an excellent model organism for the genetic dissection of behavior.
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13
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Discovery of indirubin derivatives as new class of DRAK2 inhibitors from high throughput screening. Bioorg Med Chem Lett 2016; 26:2719-23. [PMID: 27106709 DOI: 10.1016/j.bmcl.2016.03.111] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/23/2015] [Revised: 03/27/2016] [Accepted: 03/31/2016] [Indexed: 01/14/2023]
Abstract
DRAK2 is a serine/threonine kinase belonging to the death-associated protein kinase (DAPK) family and has emerged as a promising drug target for the treatment of autoimmune diseases and cancers. To identify small molecule inhibitors for DRAK2, we performed a high throughput screening campaign using in-house chemical library and identified indirubin-3'-monoximes as novel class of DRAK2 inhibitors. Among the compounds tested, compound 16 exhibited the most potent inhibitory activity against DRAK2 (IC50=0.003μM). We also propose that compound 16 may bind to the ATP-binding site of the enzyme based on enzyme kinetics and molecular docking studies.
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Matsuda T, Muromoto R, Sekine Y, Togi S, Kitai Y, Kon S, Oritani K. Signal transducer and activator of transcription 3 regulation by novel binding partners. World J Biol Chem 2015; 6:324-332. [PMID: 26629315 PMCID: PMC4657126 DOI: 10.4331/wjbc.v6.i4.324] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/20/2015] [Revised: 05/02/2015] [Accepted: 09/02/2015] [Indexed: 02/05/2023] Open
Abstract
Signal transducers and activators of transcription (STATs) mediate essential signals for various biological processes, including immune responses, hematopoiesis, and neurogenesis. STAT3, for example, is involved in the pathogenesis of various human diseases, including cancers, autoimmune and inflammatory disorders. STAT3 activation is therefore tightly regulated at multiple levels to prevent these pathological conditions. A number of proteins have been reported to associate with STAT3 and regulate its activity. These STAT3-interacting proteins function to modulate STAT3-mediated signaling at various steps and mediate the crosstalk of STAT3 with other cellular signaling pathways. This article reviews the roles of novel STAT3 binding partners such as DAXX, zipper-interacting protein kinase, Krüppel-associated box-associated protein 1, Y14, PDZ and LIM domain 2 and signal transducing adaptor protein-2, in the regulation of STAT3-mediated signaling.
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Edwards BA, Harris TL, Floersh H, Lukens JR, Zaki MH, Vogel P, Kanneganti TD, Bui JD, McGargill MA. Drak2 is not required for tumor surveillance and suppression. Int Immunol 2015; 27:161-6. [PMID: 25568303 DOI: 10.1093/intimm/dxu146] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/09/2023] Open
Abstract
Drak2 is a promising therapeutic target to treat organ-specific autoimmune diseases such as type 1 diabetes and multiple sclerosis without causing generalized immune suppression. Inhibition of Drak2 may also prevent graft rejection following organ transplantation. However, Drak2 may function as a critical tumor suppressor, which would challenge the prospect of targeting Drak2 for therapeutic treatment. Thus, we examined the susceptibility of Drak2 (-/-) mice in several tumor models. We show that Drak2 is not required to prevent tumor formation in a variety of settings. Therefore, Drak2 does not function as an essential tumor suppressor in in vivo tumor models. These data further validate Drak2 as a viable therapeutic target to treat autoimmune disease and graft rejection. Importantly, these data also indicate that while Drak2 may induce apoptosis when overexpressed in cell lines, it is not an essential tumor suppressor.
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Affiliation(s)
- Benjamin A Edwards
- Department of Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, MS 351, Memphis, TN 38105, USA
| | - Tarsha L Harris
- Department of Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, MS 351, Memphis, TN 38105, USA
| | - Helen Floersh
- Department of Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, MS 351, Memphis, TN 38105, USA
| | - John R Lukens
- Department of Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, MS 351, Memphis, TN 38105, USA
| | - Md Hasan Zaki
- Department of Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, MS 351, Memphis, TN 38105, USA
| | - Peter Vogel
- Department of Veterinary Pathology, St. Jude Children's Research Hospital, Memphis, TN 38105, USA
| | - Thirumala-Devi Kanneganti
- Department of Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, MS 351, Memphis, TN 38105, USA
| | - Jack D Bui
- Department of Pathology, University of California, San Diego, La Jolla, CA 92093, USA
| | - Maureen A McGargill
- Department of Immunology, St. Jude Children's Research Hospital, 262 Danny Thomas Place, MS 351, Memphis, TN 38105, USA
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16
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Cytoplasmic DRAK1 overexpressed in head and neck cancers inhibits TGF-β1 tumor suppressor activity by binding to Smad3 to interrupt its complex formation with Smad4. Oncogene 2014; 34:5037-45. [PMID: 25531329 DOI: 10.1038/onc.2014.423] [Citation(s) in RCA: 24] [Impact Index Per Article: 2.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/25/2014] [Revised: 11/11/2014] [Accepted: 11/15/2014] [Indexed: 12/11/2022]
Abstract
Head and neck squamous cell carcinoma (HNSCC) is an extremely aggressive cancer with a poor prognosis and low patient survival. Because chemotherapy for advanced HNSCC is often ineffective, discovering new therapeutic targets that are important for HNSCC development and progression and elucidating their molecular mechanisms are required. In the present study, we describe the role of DRAK1 (death-associated protein kinase-related apoptosis-inducing kinase 1) as a novel negative regulator of the transforming growth factor-β (TGF-β) tumor suppressor signaling pathway for the first time in human HNSCC cells. DRAK1 was significantly overexpressed in primary human HNSCCs and in HNSCC cell lines. Through gain- and loss-of-function experiments, we demonstrated that the DRAK1 expression level regulated TGF-β1-induced transcriptional activity and expression of the tumor suppressor gene p21(Waf1/Cip1). DRAK1 depletion enhanced TGF-β1-induced growth inhibition in vitro and suppressed tumorigenicity in xenograft models in vivo. Mechanistically, DRAK1 was predominantly localized in the cytoplasm and bound to Smad3, thereby interrupting Smad3/Smad4 complex formation, which is the core process for the induction of tumor suppressor genes by TGF-β1. Thus, our findings suggest that cytoplasmic DRAK1 increases tumorigenic potential through inhibition of TGF-β1-mediated tumor suppressor activity in HNSCC cells and may be a potential therapeutic target for HNSCCs.
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Huang L, Lv X, Huang Y, Hu Y, Yan H, Zheng M, Zeng H, Li X, Liang C, Wu Z, Yu X. Identification, sequence analysis, and characterization of serine/threonine protein kinase 17A from Clonorchis sinensis. Parasitol Res 2014; 113:1713-23. [PMID: 24578258 DOI: 10.1007/s00436-014-3816-x] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/12/2014] [Accepted: 02/07/2014] [Indexed: 11/24/2022]
Abstract
This is the first report of a novel protein from Clonorchis sinensis (C. sinensis), serine/threonine protein kinase 17A (CsSTK17A), which belongs to a member of the death-associated protein kinase (DAPK) family known to regulate diverse biological processes. The full-length sequence encoding CsSTK17A was isolated from C. sinensis adult cDNA plasmid library. Two transcribed isoforms of the gene were identified from the genome of C. sinensis. CsSTK17A contains a kinase domain at the N-terminus that shares a degree of conservation with the DAPK families. Besides, the catalytic domain contains 11 subdomains conserved among STKs and shares the highest identity with STK from Schistosoma mansoni (55.9%). Three-dimensional structure of CsSTK17A displays the canonical STK fold, including the helix C, P-loop, and the activation loop. We obtained recombinant CsSTK17A (rCsSTK17A) and anti-rCsSTK17A IgG. The rCsSTK17A could be probed by anti-rCsSTK17A rat serum, C. sinensis-infected rat serum and the sera from rats immunized with C. sinensis excretory-secretory products, indicating that it is a circulating antigen possessing a strong immunocompetence. Moreover, quantitative RT-PCR and western blotting analyses revealed that CsSTK17A exhibited the highest mRNA and protein expression level in eggs, followed by metacercariae and adult worms. Intriguingly, in the immunolocalization assay, CsSTK17A was intensively localized to the operculum region of eggs in uterus, as well as the vitelline gland of both adult worm and metacercaria, implying that the protein was associated with the reproduction and development of C. sinensis. Overall, these fundamental studies might contribute to further researches on signaling systems of the parasite.
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Affiliation(s)
- Lisi Huang
- Department of Clinical Laboratory, Sun Yat-sen Memorial Hospital of Sun Yat-sen University, Guangzhou, 510120, People's Republic of China
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Gallagher PJ, Blue EK. Post-translational regulation of the cellular levels of DAPK. Apoptosis 2013; 19:306-15. [DOI: 10.1007/s10495-013-0936-1] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
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Ye P, Zhao L, Gonda TJ. The MYB oncogene can suppress apoptosis in acute myeloid leukemia cells by transcriptional repression of DRAK2 expression. Leuk Res 2013; 37:595-601. [PMID: 23398943 DOI: 10.1016/j.leukres.2013.01.012] [Citation(s) in RCA: 18] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2012] [Revised: 01/14/2013] [Accepted: 01/16/2013] [Indexed: 11/19/2022]
Abstract
RNA interference-mediated suppression of MYB expression promoted apoptosis in the AML cell line U937, without affecting expression of the anti-apoptotic MYB target BCL2. This was accompanied by up-regulation of the pro-apoptotic gene DRAK2 and stimulation of caspase-9 activity. Moreover, RNA interference-mediated suppression of DRAK2 in U937 cells alleviated apoptosis induced by MYB down-regulation. Finally ChIP assays showed that in U937 cells MYB binds to a conserved element upstream of the DRAK2 transcription start site. Together, these findings identify a novel mechanism by which MYB suppresses apoptosis in an AML model cell line.
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Affiliation(s)
- Ping Ye
- School of Pharmacy, The University of Queensland, Brisbane, Queensland, Australia
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Yashiro M, Qiu H, Hasegawa T, Zhang X, Matsuzaki T, Hirakawa K. An EGFR inhibitor enhances the efficacy of SN38, an active metabolite of irinotecan, in SN38-refractory gastric carcinoma cells. Br J Cancer 2011; 105:1522-32. [PMID: 21997136 PMCID: PMC3242520 DOI: 10.1038/bjc.2011.397] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
BACKGROUND Acquired drug resistance to irinotecan is one of the significant obstacles in the treatment of advanced gastric cancer. This study was performed to clarify the effect of epidermal growth factor receptor (EGFR) inhibitors in combination with SN38, an active metabolite of irinotecan, on the proliferation of irinotecan-refractory gastric cancer. METHODS Two irinotecan-resistant gastric cancer cell lines, OCUM-2M/SN38 and OCUM-8/SN38 were, respectively, established by stepwise exposure to SN38 from the parent gastric cancer cell lines OCUM-2M and OCUM-8. The combination effects of two EGFR inhibitors, gefitinib and lapatinib, with SN38 on proliferation, apoptosis, and cell cycle on gastric cancer cells were examined. RESULTS Gefitinib or lapatinib showed synergistic anti-tumour effects against OCUM-2M/SN38 and OCUM-8/SN38 cells when used in combination with SN38, but not against OCUM-2M or OCUM-8 cells. SN38 increased the expression of EGFR and HER2 in OCUM-2M/SN38 and OCUM-8/SN38 cells. The combination of an EGFR inhibitor and SN38 significantly increased the levels of apoptosis-related molecules, caspase-6, p53, and DAPK-2, and resulted in the induction of apoptosis of irinotecan-resistant cells. The EGFR inhibitors increased the S-phase and decreased the UGT1A1 and ABCG expression in irinotecan-resistant cells. The SN38 plus Lapatinib group more effectively suppressed in vivo tumour growth by OCUM-2M/SN38 cells than either alone group. CONCLUSION The combination treatment with an EGFR inhibitor and irinotecan might produce synergistic anti-tumour effects for irinotecan-refractory gastric cancer cells. The regulation of SN38 metabolism-related genes and cell cycle by EGFR inhibitors might be responsible for the synergism.
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Affiliation(s)
- M Yashiro
- Oncology Institute of Geriatrics and Medical Science, Osaka City University Graduate School of Medicine, 1-4-3 Asahi-machi, Abeno-ku, Osaka 545-8585, Japan.
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Newton RH, Leverrier S, Srikanth S, Gwack Y, Cahalan MD, Walsh CM. Protein kinase D orchestrates the activation of DRAK2 in response to TCR-induced Ca2+ influx and mitochondrial reactive oxygen generation. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2011; 186:940-50. [PMID: 21148796 PMCID: PMC3133617 DOI: 10.4049/jimmunol.1000942] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
DRAK2 is a serine/threonine kinase highly enriched in lymphocytes that raises the threshold for T cell activation and maintains T cell survival following productive activation. T cells lacking DRAK2 are prone to activation under suboptimal conditions and exhibit enhanced calcium responses to AgR stimulation. Despite this, mice lacking DRAK2 are resistant to organ-specific autoimmune diseases due to defective autoreactive T cell survival. DRAK2 kinase activity is induced by AgR signaling, and in this study we show that the induction of DRAK2 activity requires Ca(2+) influx through the Ca(2+) release-activated Ca(2+) channel formed from Orai1 subunits. Blockade of DRAK2 activity with the protein kinase D (PKD) inhibitor Gö6976 or expression of a kinase-dead PKD mutant prevented activation of DRAK2, whereas a constitutively active PKD mutant promoted DRAK2 function. Knockdown of PKD in T cells strongly blocked endogenous DRAK2 activation following TCR ligation, implicating PKD as an essential intermediate in the activation of DRAK2 by Ca(2+) influx. Furthermore, we identify DRAK2 as a novel substrate of PKD, and demonstrate that DRAK2 and PKD physically interact under conditions that activate PKD. Mitochondrial generation of reactive oxygen intermediates was necessary and sufficient for DRAK2 activation in response to Ca(2+) influx. Taken together, DRAK2 and PKD form a novel signaling module that controls calcium homeostasis following T cell activation.
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Affiliation(s)
- Ryan H. Newton
- Institute for Immunology, University of California, Irvine, Irvine, CA 92697
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697
| | - Sabrina Leverrier
- Institute for Immunology, University of California, Irvine, Irvine, CA 92697
- Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697
| | - Sonal Srikanth
- Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095
| | - Yousang Gwack
- Department of Physiology, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA 90095
| | - Michael D. Cahalan
- Institute for Immunology, University of California, Irvine, Irvine, CA 92697
- Department of Physiology and Biophysics, University of California, Irvine, Irvine, CA 92697
| | - Craig M. Walsh
- Institute for Immunology, University of California, Irvine, Irvine, CA 92697
- Department of Molecular Biology and Biochemistry, University of California, Irvine, Irvine, CA 92697
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Ghorbel MT, Cherif M, Jenkins E, Mokhtari A, Kenny D, Angelini GD, Caputo M. Transcriptomic analysis of patients with tetralogy of Fallot reveals the effect of chronic hypoxia on myocardial gene expression. J Thorac Cardiovasc Surg 2010; 140:337-345.e26. [PMID: 20416888 PMCID: PMC2951593 DOI: 10.1016/j.jtcvs.2009.12.055] [Citation(s) in RCA: 46] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/01/2009] [Revised: 11/19/2009] [Accepted: 12/19/2009] [Indexed: 11/20/2022]
Abstract
OBJECTIVES In cyanotic patients undergoing repair of heart defects, chronic hypoxia is thought to lead to greater susceptibility to ischemia and reoxygenation injury. We sought to find an explanation to such a hypothesis by investigating the cardiac gene expression in patients with tetralogy of Fallot undergoing cardiac surgery. METHODS The myocardial gene profile was investigated in right ventricular biopsy specimens obtained from 20 patients with a diagnosis of cyanotic (n = 11) or acyanotic (n = 9) tetralogy of Fallot undergoing surgical repair. Oligonucleotide microarray analyses were performed on the samples, and the array results were validated with Western blotting and enzyme-linked immunosorbent assay. RESULTS Data revealed 795 differentially expressed genes in cyanotic versus acyanotic hearts, with 198 upregulated and 597 downregulated. Growth/morphogenesis, remodeling, and apoptosis emerged as dominant functional themes for the upregulated genes and included the apoptotic gene TRAIL (tumor necrosis factor-related apoptosis-inducing ligand), the remodeling factor OPN (osteopontin), and the mitochondrial function gene COX11 (cytochrome-c oxidase 11). In contrast, transcription, mitogen-activated protein kinase signaling, and contractile machinery were the dominant functional classes for the downregulated genes, which included the calcium-handling gene NCX1 (sodium-calcium exchanger). Protein levels of COX11, NCX1, OPN, and LYZ (lysozyme) in the myocardium followed the same pattern obtained by means of transcriptomics. The TRAIL level did not change in myocardium but increased in circulating blood of cyanotic patients, suggesting the myocardium as a possible source. Additionally, our data showed increased protein expression of apoptosis markers in cyanotic myocardium. CONCLUSIONS Chronic hypoxia in cyanotic children with tetralogy of Fallot induced the expression of genes associated with apoptosis and remodeling and reduced the expression of genes associated with myocardium contractility and function.
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Affiliation(s)
- Mohamed T Ghorbel
- Bristol Heart Institute, University of Bristol, Bristol Royal Infirmary, Bristol, United Kingdom
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Anamika K, Garnier N, Srinivasan N. Functional diversity of human protein kinase splice variants marks significant expansion of human kinome. BMC Genomics 2009; 10:622. [PMID: 20028505 PMCID: PMC2805699 DOI: 10.1186/1471-2164-10-622] [Citation(s) in RCA: 14] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/22/2009] [Accepted: 12/22/2009] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Protein kinases are involved in diverse spectrum of cellular processes. Availability of draft version of the human genomic data in the year 2001 enabled recognition of repertoire of protein kinases. However, over the years the human genomic data is being refined and the current release of human genomic data has helped us to recognize a larger repertoire of over 900 human protein kinases represented mainly by splice variants. RESULTS Many of these identified protein kinases are alternatively spliced products. Interestingly, some of the human kinase splice variants appear to be significantly diverged in terms of their functional properties as represented by incorporation or absence of one or more domains. Many sets of protein kinase splice variants have substantially different domain organization and in a few sets of splice variants kinase domains belong to different subfamilies of kinases suggesting potential participation in different signal transduction pathways. CONCLUSIONS Addition or deletion of a domain between splice variants of multi-domain kinases appears to be a means of generating differences in the functional features of otherwise similar kinases. It is intriguing that marked sequence diversity within the catalytic regions of some of the splice variant kinases result in kinases belonging to different subfamilies. These human kinase splice variants with different functions might contribute to diversity of eukaryotic cellular signaling.
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Hertle ML, Popp C, Petermann S, Maier S, Kremmer E, Lang R, Mages J, Kempkes B. Differential gene expression patterns of EBV infected EBNA-3A positive and negative human B lymphocytes. PLoS Pathog 2009; 5:e1000506. [PMID: 19578441 PMCID: PMC2700271 DOI: 10.1371/journal.ppat.1000506] [Citation(s) in RCA: 64] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/23/2009] [Accepted: 06/05/2009] [Indexed: 01/05/2023] Open
Abstract
The genome of Epstein-Barr virus (EBV) encodes 86 proteins, but only a limited set is expressed in EBV–growth transformed B cells, termed lymphoblastoid cell lines (LCLs). These cells proliferate via the concerted action of EBV nuclear antigens (EBNAs) and latent membrane proteins (LMPs), some of which are rate limiting to establish a stable homeostasis of growth promoting and anti-apoptotic activities. We show here that EBV mutants, which lack the EBNA-3A gene, are impaired but can still initiate cell cycle entry and proliferation of primary human B cells in contrast to an EBNA-2 deficient mutant virus. Surprisingly, and in contrast to previous reports, these viral mutants are attenuated in growth transformation assays but give rise to permanently growing EBNA-3A negative B cell lines which exhibit reduced proliferation rates and elevated levels of apoptosis. Expression profiles of EBNA-3A deficient LCLs are characterized by 129 down-regulated and 167 up-regulated genes, which are significantly enriched for genes involved in apoptotic processes or cell cycle progression like the tumor suppressor gene p16/INK4A, or might contribute to essential steps of the viral life cycle in the infected host. In addition, EBNA-3A cellular target genes remarkably overlap with previously identified targets of EBNA-2. This study comprises the first genome wide expression profiles of EBNA-3A target genes generated within the complex network of viral proteins of the growth transformed B cell and permits a more detailed understanding of EBNA-3A's function and contribution to viral pathogenesis. Epstein-Barr virus (EBV) infects primary human B cells and establishes a latent infection, which leads to permanently growing B cell cultures. These growth transformed B cells express a well defined set of latent viral genes, which are also expressed in post-transplant lymphomas of immunosuppressed patients. In a concerted action these latent viral proteins drive cellular proliferation and prevent apoptosis. For this study, recombinant Epstein-Barr virus mutants that lack the gene for the Epstein-Barr virus nuclear antigen-3A (EBNA-3A) were generated. EBNA-3A is a transcriptional modulator of gene expression. We show here that EBNA-3A deficient growth transformed B cells can be established in vitro. Our results suggest that EBNA-3A supports viability but is not absolutely essential for proliferation of the infected B cell. By virtue of the established EBNA-3A deficient cell lines, we could for the first time identify a broad array of cellular target genes controlled by EBNA-3A in EBV infected B cells. These EBNA-3A target genes will permit a more detailed understanding of EBNA-3A's function and contribution to viral pathogenesis.
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Affiliation(s)
- Marie L. Hertle
- Department of Gene Vectors, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany
| | - Claudia Popp
- Department of Gene Vectors, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany
| | - Sabine Petermann
- Department of Gene Vectors, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany
| | - Sabine Maier
- Department of Gene Vectors, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany
| | - Elisabeth Kremmer
- Institute of Molecular Immunology, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany
| | - Roland Lang
- Institute of Clinical Microbiology, Immunology and Hygiene, Friedrich-Alexander-University Erlangen-Nürnberg, Erlangen, Germany
- Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany
| | - Jörg Mages
- Institute of Medical Microbiology, Immunology and Hygiene, Technical University Munich, Munich, Germany
- Biotools B&M Labs, S.A., Madrid, Spain
| | - Bettina Kempkes
- Department of Gene Vectors, Helmholtz Center Munich, German Research Center for Environmental Health, Munich, Germany
- * E-mail:
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Qiu H, Ding FY, Xiong HH, Zhang MS, Li RC, Chen Y. Establishment of VP16-resistant gastric cell line and its multi-drug resistance mechanism. Shijie Huaren Xiaohua Zazhi 2009; 17:1809-1814. [DOI: 10.11569/wcjd.v17.i18.1809] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To establish the VP16 resistant gastric cell line and to explore its potential multi-drug resistant mechanism.
METHODS: We used the drug concentration step-elevation method to establish VP16 resistant sub-line of gastric cell line OCUM-2M. Growth curves of cells were delineated and cell doubling times of cells were calculated using cell-counting methods. IC50 of chemotherapy drugs in two cell lines were determined by MTT methods. Cell cycle distributions were tested by FCM analysis. mRNA expression levels of caspase-3, P53, DAPK-1, DAPK-2, DAPK-3, Bcl-2, ERCC-1, MDR-1, and MRP were determined by RT-PCR.
RESULTS: OCUM-2M was a successful drug-resistant cell line, and the resistance index to VP16 was 40.53. The cell doubling time of OCUM-2M/VP16 was 30.29 ± 2.55 h, while that of parental cell line OCUM-2M was 22.96 ± 0.96 h (P < 0.01). The cross-drug-resistance of Sn38, oxaliplatin, and gemcitabine in OCUM-2M/VP16 was observed, while chemo-sensitivity of 5-FU and paclitaxol in OCUM-2M/VP16 remained the same with OCUM-2M. In OCUM-2M/VP16, the mean expression levels of apoptosis related genes, DAPK-2, DAPK-3 and Bcl-2 were respectively 0.24, 0.45, and 0.44, which were lower than OCUM-2M (0.61, 0.79 and 0.81). The expression levels of drug-resistance related genes ERCC-1 and MDR-1 in OCUM-2M/VP16 were respectively 0.84 and 0.41, which were higher than OCUM-2M (0.53 and 0.20, P < 0.01). The expression levels of caspase-3, P53, DAPK-1 and MRP had no significant change in the two cell lines.
CONCLUSION: OCUM-2M/VP16 is a successful VP16-resistant gastric cell line with cross-resistance ability, and the expression level changes of apoptosis and drug resistance related genes might contribute to drug-resistance in OCUM-2M/VP16.
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Targeted Restoration of Down-regulated DAPK2 Tumor Suppressor Activity Induces Apoptosis in Hodgkin Lymphoma Cells. J Immunother 2009; 32:431-41. [DOI: 10.1097/cji.0b013e31819f1cb6] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/26/2022]
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Ramos SJ, Hernandez JB, Gatzka M, Walsh CM. Enhanced T cell apoptosis within Drak2-deficient mice promotes resistance to autoimmunity. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2008; 181:7606-16. [PMID: 19017949 PMCID: PMC2709975 DOI: 10.4049/jimmunol.181.11.7606] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 12/22/2022]
Abstract
Clonal expansion of T cells is vital to adaptive immunity, yet this process must be tightly controlled to prevent autoimmune disease. The serine/threonine kinase death-associated protein kinase-related apoptosis-inducing kinase 2 (DRAK2) is a negative regulator of TCR signaling and sets the threshold for the activation of naive and memory T cells and selected thymocytes. Despite enhanced T cell activation, Drak2(-/-) mice are resistant to experimental autoimmune encephalomyelitis, an autoimmune demyelinating disease that resembles multiple sclerosis. However, the basis for this autoimmune resistance is currently unknown. In this study, we show that, in the absence of DRAK2 signaling, T cells require greater tonic signaling for maintenance during clonal expansion. Following stimulation, Drak2(-/-) T cells were more sensitive to an intrinsic form of apoptosis that was prevented by CD28 ligation, homeostatic cytokines, or enforced Bcl-x(L) expression. T cell-specific Bcl-x(L) expression also restored the susceptibility of Drak2(-/-) mice to experimental autoimmune encephalomyelitis and enhanced thymic positive selection. These findings demonstrate that DRAK2 is selectively important for T cell survival and highlight the potential that DRAK2 blockade may lead to permanent autoimmune T cell destruction via intrinsic apoptosis pathways.
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MESH Headings
- Animals
- Apoptosis/genetics
- Apoptosis/immunology
- Autoimmunity/genetics
- Autoimmunity/immunology
- CD28 Antigens/genetics
- CD28 Antigens/immunology
- Cell Survival/genetics
- Cell Survival/immunology
- Cytokines/genetics
- Cytokines/immunology
- Encephalomyelitis, Autoimmune, Experimental/enzymology
- Encephalomyelitis, Autoimmune, Experimental/genetics
- Encephalomyelitis, Autoimmune, Experimental/immunology
- Immunologic Memory/genetics
- Immunologic Memory/immunology
- Lymphocyte Activation/genetics
- Lymphocyte Activation/immunology
- Mice
- Mice, Knockout
- Multiple Sclerosis/enzymology
- Multiple Sclerosis/genetics
- Multiple Sclerosis/immunology
- Protein Serine-Threonine Kinases/genetics
- Protein Serine-Threonine Kinases/immunology
- Receptors, Antigen, T-Cell/genetics
- Receptors, Antigen, T-Cell/immunology
- Signal Transduction/genetics
- Signal Transduction/immunology
- Thymus Gland/immunology
- bcl-X Protein/genetics
- bcl-X Protein/immunology
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Affiliation(s)
| | | | - Martina Gatzka
- Center for Immunology and the Department of Molecular Biology and Biochemistry, University of California, Irvine. Irvine, CA 92697-3900
| | - Craig M. Walsh
- Center for Immunology and the Department of Molecular Biology and Biochemistry, University of California, Irvine. Irvine, CA 92697-3900
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McGargill MA, Choy C, Wen BG, Hedrick SM. Drak2 regulates the survival of activated T cells and is required for organ-specific autoimmune disease. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2008; 181:7593-605. [PMID: 19017948 PMCID: PMC2792703 DOI: 10.4049/jimmunol.181.11.7593] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 01/22/2023]
Abstract
Drak2 is a serine/threonine kinase expressed in T and B cells. The absence of Drak2 renders T cells hypersensitive to suboptimal stimulation, yet Drak2(-/-) mice are enigmatically resistant to experimental autoimmune encephalomyelitis, an animal model of multiple sclerosis. We show in this study that Drak2(-/-) mice were also completely resistant to type 1 diabetes when bred to the NOD strain of mice that spontaneously develop autoimmune diabetes. However, there was not a generalized suppression of the immune system, because Drak2(-/-) mice remained susceptible to other models of autoimmunity. Adoptive transfer experiments revealed that resistance to disease was intrinsic to the T cells and was due to a loss of T cell survival under conditions of chronic autoimmune stimulation. Importantly, the absence of Drak2 did not alter the survival of naive T cells, memory T cells, or T cells responding to an acute viral infection. These experiments reveal a distinction between the immune response to persistent self-encoded molecules and transiently present infectious agents. We present a model whereby T cell survival depends on a balance of TCR and costimulatory signals to explain how the absence of Drak2 affects autoimmune disease without generalized suppression of the immune system.
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Affiliation(s)
- Maureen A. McGargill
- Molecular Biology Section, Division of Biological Sciences, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093
| | - Carmen Choy
- Molecular Biology Section, Division of Biological Sciences, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093
| | - Ben G. Wen
- Department of Pharmacology, Genomics Institute of Novartis Research Foundation, San Diego, CA 92121
| | - Stephen M. Hedrick
- Molecular Biology Section, Division of Biological Sciences, Department of Cellular and Molecular Medicine, University of California, San Diego, La Jolla, CA 92093
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30
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Al-Qahtani A, Xu Z, Zan H, Walsh CM, Casali P. A role for DRAK2 in the germinal center reaction and the antibody response. Autoimmunity 2008; 41:341-52. [PMID: 18568639 PMCID: PMC3140869 DOI: 10.1080/08916930802170633] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/07/2023]
Abstract
DAP-related apoptotic kinase-2 (DRAK2), a death-associated protein kinase family member, is highly expressed in B and T lymphocytes in the human and the mouse. To determine whether DRAK2 plays a role in B-cell activation and differentiation, we analyzed germinal centers (GCs) and the specific antibody response to NP in drak2-/- mice immunized with the thymus-dependent (TD) conjugated hapten NP16-CGG. In drak2-/- mice, spleen GCs were normal in size and morphology, but their number was reduced by as much as 5-fold, as compared to their wild-type littermates. This was not due to a defect in B-cell proliferation, as the BrdU uptake was comparable in DRAK2-deficient and wild-type B cells. Rather, the proportion of apoptotic GC B and T cells in drak2-/- mice was significantly higher than that in wild-type control mice, as shown by 7-AAD and terminal deoxynucleotide transferase dUTP nick end labeling (TUNEL) staining. In drak2-/- mice, the generation high affinity IgG antibodies was impaired in spite of the seemingly normal somatic hypermutation and class switch DNA recombination machineries in drak2-/- B cells. In NP16-CGG-immunized drak2-/- mice, T-cell-intrinsic Bcl-xL transgene expression increased the number of GCs and rescued the high affinity IgG response to NP. These findings suggest a novel role for DRAK2 in regulating the GC reaction and the response to TD antigens, perhaps through increased survival of T cells and enhanced B-cell positive selection. They also suggest that DRAK2-deficiency is not involved in regulating intrinsic B-cell apoptosis.
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Affiliation(s)
- Ahmed Al-Qahtani
- Center for Immunology, University of California, Irvine, CA, USA
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31
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Wang S, Welte T, McGargill M, Town T, Thompson J, Anderson JF, Flavell RA, Fikrig E, Hedrick SM, Wang T. Drak2 contributes to West Nile virus entry into the brain and lethal encephalitis. JOURNAL OF IMMUNOLOGY (BALTIMORE, MD. : 1950) 2008; 181:2084-91. [PMID: 18641347 PMCID: PMC2494872 DOI: 10.4049/jimmunol.181.3.2084] [Citation(s) in RCA: 55] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
Death-associated protein kinase-related apoptosis-inducing kinase-2 (Drak2), a member of the death-associated protein family of serine/threonine kinases, is specifically expressed in T and B cells. In the absence of Drak2, mice are resistant to experimental autoimmune encephalomyelitis due to a decrease in the number of cells infiltrating the CNS. In the present study, we investigated the role of Drak2 in West Nile virus (WNV)-induced encephalitis and found that Drak2(-/-) mice were also more resistant to lethal WNV infection than wild-type mice. Although Drak2(-/-) mice had an increase in the number of IFN-gamma-producing T cells in the spleen after infection, viral levels in the peripheral tissues were not significantly different between these two groups of mice. In contrast, there was a reduced viral load in the brains of Drak2(-/-) mice, which was accompanied by a decrease in the number of Drak2(-/-) CD4(+) and CD8(+) T cells in the brain following WNV infection. Moreover, we detected viral Ags in T cells isolated from the spleen or brain of WNV-infected mice. These results suggest that following a systemic infection, WNV might cross the blood brain barrier and enter the CNS by being carried by infected infiltrating T cells.
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Affiliation(s)
- Shuhui Wang
- Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine & Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
| | - Thomas Welte
- Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine & Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
| | - Maureen McGargill
- Department of Biology and Cancer Center, University of California-San Diego, La Jolla, CA 92093
| | - Terrence Town
- Section of Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520
| | - Jesse Thompson
- Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine & Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
| | - John F Anderson
- Department of Entomology, Connecticut Agricultural Experiment Station, P. O. Box 1106, New Haven, CT 06504
| | - Richard A Flavell
- Section of Immunobiology, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520
- The Howard Hughes Medical Institute, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520
| | - Erol Fikrig
- The Howard Hughes Medical Institute, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520
- Section of Infectious Diseases, Department of Internal Medicine, Yale University School of Medicine, 300 Cedar Street, New Haven, CT 06520
| | - Stephen M. Hedrick
- Department of Biology and Cancer Center, University of California-San Diego, La Jolla, CA 92093
| | - Tian Wang
- Department of Microbiology, Immunology and Pathology, College of Veterinary Medicine & Biomedical Sciences, Colorado State University, Fort Collins, CO 80523
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32
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Physical and functional interactions between ZIP kinase and UbcH5. Biochem Biophys Res Commun 2008; 372:708-12. [PMID: 18515077 DOI: 10.1016/j.bbrc.2008.05.113] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/14/2008] [Accepted: 05/18/2008] [Indexed: 01/14/2023]
Abstract
Zipper-interacting protein kinase (ZIPK) is a widely expressed serine/threonine kinase that has been implicated in cell death and transcriptional regulation, but its mechanism of regulation remains unknown. In our previous study, we showed that leukemia inhibitory factor stimulated threonine-265 phosphorylation of ZIPK, thereby leading to phosphorylation and activation of signal transducer and activator of transcription 3. Here, we identified UbcH5c as a novel ZIPK-binding partner by yeast two-hybrid screening. Importantly, we found that UbcH5c induced ubiquitination of ZIPK. Small-interfering RNA-mediated reduction of endogenous UbcH5 expression decreased ZIPK ubiquitination. Furthermore, coexpression of UbcH5c with ZIPK influenced promyelocytic leukemia protein nuclear body (PML-NB) formation. These results suggest that UbcH5 regulates ZIPK accumulation in PML-NBs by interacting with ZIPK and stimulating its ubiquitination.
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33
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Schaumburg CS, Gatzka M, Walsh CM, Lane TE. DRAK2 regulates memory T cell responses following murine coronavirus infection. Autoimmunity 2008; 40:483-8. [PMID: 17966037 DOI: 10.1080/08916930701651139] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/05/2023]
Abstract
The contribution of DRAK2 [death-associated protein kinase (DAPK)-related apoptosis-inducing kinase 2] to anti-viral memory T cell responses following infection with mouse hepatitis virus (MHV) was examined. DRAK2 is a lymphoid-enriched serine/threonine kinase that is an important regulatory molecule involved in modulating T cell responses. Memory T cells derived from MHV-immunized Drak2(-/-) mice exhibited amplified proliferation and IFN-gamma secretion following stimulation with viral epitopes. Transfer of Drak2(-/-) memory T cells into Rag1(-/-) mice infected intracerebrally with MHV resulted in accelerated clearance of virus from the brain. Thus, DRAK2 may be a novel target for stimulating protective immunity to viral pathogens.
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Affiliation(s)
- Chris S Schaumburg
- Department of Molecular Biology and Biochemistry, University of California, Irvine, CA 92697-3900, USA
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34
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Leister P, Felten A, Chasan AI, Scheidtmann KH. ZIP kinase plays a crucial role in androgen receptor-mediated transcription. Oncogene 2007; 27:3292-300. [PMID: 18084323 DOI: 10.1038/sj.onc.1210995] [Citation(s) in RCA: 20] [Impact Index Per Article: 1.1] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
The androgen receptor (AR) is a ligand-dependent transcription factor that plays a crucial role in the development and homeostasis of the prostate and in prostate cancer. The transcriptional activity of AR is mediated by interaction with multiple co-activators, which serve in chromatin modification or remodeling, or provide a link between specific and general transcription factors. We have identified zipper interacting protein (ZIP) kinase as a novel transcriptional co-activator of the AR. ZIP kinase enhanced expression of AR-responsive promotor/luciferase reporter constructs in a hormone- and kinase-dependent manner. Similar results were obtained for glucocorticoid receptor but not for progesterone receptor and estrogen receptor. Following hormone treatment, AR and ZIP kinase formed physical complexes and associated with the promoter and enhancer of the prostate-specific antigen gene, as revealed by chromatin immunoprecipitation. Strikingly, depletion of ZIP kinase by siRNA led to significant reduction of AR-mediated transactivation. The interaction of ZIP kinase with AR seems to be mediated in part by apoptosis antagonizing transcription factor and in part by direct binding. Interestingly, AR was not phosphorylated by ZIP kinase in vitro, suggesting that it phosphorylates other co-activators or chromatin proteins.
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Affiliation(s)
- P Leister
- Institute of Genetics, University of Bonn, Bonn, Germany
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35
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Shivapurkar N, Sherman ME, Stastny V, Echebiri C, Rader JS, Nayar R, Bonfiglio TA, Gazdar AF, Wang SS. Evaluation of candidate methylation markers to detect cervical neoplasia. Gynecol Oncol 2007; 107:549-53. [PMID: 17894941 PMCID: PMC2718832 DOI: 10.1016/j.ygyno.2007.08.057] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/25/2007] [Revised: 08/14/2007] [Accepted: 08/15/2007] [Indexed: 11/18/2022]
Abstract
OBJECTIVE Studies of cervical cancer and its immediate precursor, cervical intraepithelial neoplasia 3 (CIN3), have identified genes that often show aberrant DNA methylation and therefore represent candidate early detection markers. We used quantitative PCR assays to evaluate methylation in five candidate genes (TNFRSF10C, DAPK1, SOCS3, HS3ST2 and CDH1) previously demonstrated as methylated in cervical cancer. METHODS In this analysis, we performed methylation assays for the five candidate genes in 45 invasive cervical cancers, 12 histologically normal cervical specimens, and 23 liquid-based cervical cytology specimens confirmed by expert review as unequivocal demonstrating cytologic high-grade squamous intraepithelial lesions, thus representing the counterparts of histologic CIN3. RESULTS We found hypermethylation of HS3ST2 in 93% of cancer tissues and 70% of cytology specimens interpreted as CIN3; hypermethylation of CDH1 was found in 89% of cancers and 26% of CIN3 cytology specimens. Methylation of either HS3ST2 or CDH1 was observed in 100% of cervical cancer tissues and 83% of CIN3 cytology specimens. None of the five genes showed detectable methylation in normal cervical tissues. CONCLUSION Our data support further evaluation of HS3ST2 and CDH1 methylation as potential markers of cervical cancer and its precursor lesions.
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Affiliation(s)
- Narayan Shivapurkar
- Hamon Center for Therapeutic Oncology Research, Department of Pathology, UT Southwestern Medical Center, Dallas, Texas 75930
| | - Mark E. Sherman
- Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, MD 20852-7234
| | - Victor Stastny
- Hamon Center for Therapeutic Oncology Research, Department of Pathology, UT Southwestern Medical Center, Dallas, Texas 75930
| | - Chinyere Echebiri
- Hamon Center for Therapeutic Oncology Research, Department of Pathology, UT Southwestern Medical Center, Dallas, Texas 75930
| | - Janet S. Rader
- Department of Obstetrics and Gynecology, Washington University School of Medicine, St. Louis, MO 63110
| | - Ritu Nayar
- Northwestern University Feinberg School of Medicine, Chicago, IL 60208
| | | | - Adi F. Gazdar
- Hamon Center for Therapeutic Oncology Research, Department of Pathology, UT Southwestern Medical Center, Dallas, Texas 75930
| | - Sophia S. Wang
- Division of Cancer Epidemiology and Genetics, National Cancer Institute, Rockville, MD 20852-7234
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36
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Lee JW, Lee KF, Hsu HY, Hsu LP, Shih WL, Chu YC, Hsiao WT, Liu PF. Protein expression and intracellular localization of prostate apoptosis response-4 (Par-4) are associated with apoptosis induction in nasopharyngeal carcinoma cell lines. Cancer Lett 2007; 257:252-62. [PMID: 17881119 DOI: 10.1016/j.canlet.2007.08.004] [Citation(s) in RCA: 17] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/16/2007] [Revised: 07/03/2007] [Accepted: 08/01/2007] [Indexed: 10/22/2022]
Abstract
Prostate apoptosis response-4 (Par-4) is a proapoptotic gene that selectively induces cell death in most cancer cells. In addition to the increased percentage of apoptotic cells, caspase-3 activity, and poly (ADP-ribose) polymerase (PARP) cleavage, we demonstrate that elevated expression of Par-4 and nuclear entry resulted in apoptosis of nasopharyngeal carcinoma (NPC) cell lines either in serum deprivation or by ectopic overexpression of Par-4. Moreover, disassociation from the Par-4/Akt complex was correlated with the induced proapoptotic ability of Par-4. Therefore, our data suggest that the cytoplasmic localization and expression level of endogenous Par-4 in NPC cells are not sufficient to augment apoptosis.
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Affiliation(s)
- Jeng-Woei Lee
- Department and Institute of Life Science, College of Life Science, Tzu-Chi University, 701, Sec. 3, Chung-Yang Road, Hualien 97071, Taiwan.
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37
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Scheidtmann KH. Dlk/ZIP kinase, a novel Ser/Thr-specific protein kinase with multiple functions. ACTA ACUST UNITED AC 2007. [DOI: 10.1002/sita.200600112] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/26/2023]
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38
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Shivapurkar N, Stastny V, Suzuki M, Wistuba II, Li L, Zheng Y, Feng Z, Hol B, Prinsen C, Thunnissen FB, Gazdar AF. Application of a methylation gene panel by quantitative PCR for lung cancers. Cancer Lett 2007; 247:56-71. [PMID: 16644104 PMCID: PMC3379713 DOI: 10.1016/j.canlet.2006.03.020] [Citation(s) in RCA: 69] [Impact Index Per Article: 3.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/31/2006] [Revised: 03/19/2006] [Accepted: 03/24/2006] [Indexed: 01/29/2023]
Abstract
Detection of lung cancer at early stages could potentially increase survival rates. One promising approach is the application of suitable lung cancer-specific biomarkers to specimens obtained by non-invasive methods. Thus far, clinically useful biomarkers that have high sensitivity have proven elusive. Certain genes, which are involved in cellular pathways such as signal transduction, apoptosis, cell to cell communication, cell cycles and cytokine signaling are down-regulated in cancers and may be considered as potential tumor suppressor genes. Aberrant promoter hypermethylation is a major mechanism for silencing tumor suppressor genes in many kinds of human cancers. Using quantitative real time PCR, we tested 11 genes (3-OST-2, RASSF1A, DcR1, DcR2, P16, DAPK, APC, ECAD, HCAD, SOCS1, SOCS3) for levels of methylation within their promoter sequences in non-small cell lung cancers (NSCLC), adjacent non-malignant lung tissues, in peripheral blood mononuclear cells (PBMC) from cancer free patients, in sputum of cancer patients and controls. Of all the 11 genes tested 3-OST-2 showed the highest levels of promoter methylation in tumors combined with lowest levels of promoter methylation in control tissues. 3-OST-2 followed by, RASSF1A showed increased levels of methylation with advanced tumor stage (P<0.05). Thus, quantitative analysis of 3-OST-2 and RASSF1A methylation appears to be a promising biomarker assay for NSCLC and should be further explored in a clinical study. Our preliminary data on the analysis of sputum DNA specimens from cancer patients further support these observations.
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Affiliation(s)
- Narayan Shivapurkar
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA
- Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
| | - Victor Stastny
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA
| | - Makoto Suzuki
- Department of Thoracic Surgery, Graduate School of Medicine, Chiba University, Chiba, Japan
| | - Ignacio I. Wistuba
- Department of Pathology, MD Anderson Cancer Center Houston, Houston, TX 77030, USA
| | - Lin Li
- Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Yingye Zheng
- Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Ziding Feng
- Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA 98109, USA
| | - Bernard Hol
- Department of Pulmonology, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands
| | - Clemens Prinsen
- Department of Pathology, Canisius Wilhelmina Hospital, Nijmegen, The Netherlands
| | | | - Adi F. Gazdar
- Hamon Center for Therapeutic Oncology Research, University of Texas Southwestern Medical Center, 6000 Harry Hines Boulevard, Dallas, TX 75390, USA
- Department of Pathology, University of Texas Southwestern Medical Center, Dallas, TX 75390, USA
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39
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Abstract
Transcription factor AP-1 is a dimer complex composed by DNA-binding proteins of Jun, Fos, and ATF families. AP-1 mediates cell response on growth factors, cytokines, neurotransmitters and other intercellular signaling molecules. AP-1 activity is mediated by G-proteins, adapter proteins, MAP kinases and other elements of cellular signaling systems. AP-1 dependent genes play a pivotal role in regulation of cell proliferation, morphogenesis, apoptosis, and differentiation.
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40
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Houben A, Demidov D, Caperta AD, Karimi R, Agueci F, Vlasenko L. Phosphorylation of histone H3 in plants--a dynamic affair. ACTA ACUST UNITED AC 2007; 1769:308-15. [PMID: 17320987 DOI: 10.1016/j.bbaexp.2007.01.002] [Citation(s) in RCA: 85] [Impact Index Per Article: 4.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2006] [Revised: 01/08/2007] [Accepted: 01/11/2007] [Indexed: 01/15/2023]
Abstract
Histones are the main protein components of chromatin: they undergo extensive post-translational modifications, particularly acetylation, methylation, phosphorylation, ubiquitination and ADP-ribosylation which modify the structural/functional properties of chromatin. Post-translational modifications of the N-terminal tails of the core histones within the nucleosome particle are thought to act as signals from the chromatin to the cell, for various processes. Thus, in many ways histone tails can be viewed as complex protein-protein interaction surfaces that are regulated by numerous post-translational modifications. Histone phosphorylation has been linked to chromosome condensation/segregation, activation of transcription, apoptosis and DNA damage repair. In plants, the cell cycle dependent phosphorylation of histone H3 has been described; it is hyperphosphorylated at serines 10/28 and at threonines 3/11 during both mitosis and meiosis in patterns that are specifically coordinated in both space and time. Although this post-translational modification is highly conserved, data show that the chromosomal distribution of individual modifications can differ between groups of eukaryotes. Initial results indicate that members of the plant Aurora kinase family have the capacity to control cell cycle regulated histone H3 phosphorylation, and in addition we describe other potential H3 kinases and discuss their functions.
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Affiliation(s)
- Andreas Houben
- Leibniz-Institute of Plant Genetics and Crop Plant Research, Chromosome Structure and Function Group, Corrensstrasse 3, D-06466 Gatersleben, Germany.
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41
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Mehta SL, Manhas N, Raghubir R. Molecular targets in cerebral ischemia for developing novel therapeutics. ACTA ACUST UNITED AC 2007; 54:34-66. [PMID: 17222914 DOI: 10.1016/j.brainresrev.2006.11.003] [Citation(s) in RCA: 541] [Impact Index Per Article: 30.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/11/2006] [Revised: 11/09/2006] [Accepted: 11/10/2006] [Indexed: 11/20/2022]
Abstract
Cerebral ischemia (stroke) triggers a complex series of biochemical and molecular mechanisms that impairs the neurologic functions through breakdown of cellular integrity mediated by excitotoxic glutamatergic signalling, ionic imbalance, free-radical reactions, etc. These intricate processes lead to activation of signalling mechanisms involving calcium/calmodulin-dependent kinases (CaMKs) and mitogen-activated protein kinases (MAPKs) such as extracellular signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). The distribution of these transducers bring them in contact with appropriate molecular targets leading to altered gene expression, e.g. ERK and JNK mediated early gene induction, responsible for activation of cell survival/damaging mechanisms. Moreover, inflammatory reactions initiated at the neurovascular interface and alterations in the dynamic communication between the endothelial cells, astrocytes and neurons are thought to substantially contribute to the pathogenesis of the disease. The damaging mechanisms may proceed through rapid nonspecific cell lysis (necrosis) or by active form of cell demise (apoptosis or necroptosis), depending upon the severity and duration of the ischemic insult. A systematic understanding of these molecular mechanisms with prospect of modulating the chain of events leading to cellular survival/damage may help to generate the potential strategies for neuroprotection. This review briefly covers the current status on the molecular mechanisms of stroke pathophysiology with an endeavour to identify potential molecular targets such as targeting postsynaptic density-95 (PSD-95)/N-methyl-d-aspartate (NMDA) receptor interaction, certain key proteins involved in oxidative stress, CaMKs and MAPKs (ERK, p38 and JNK) signalling, inflammation (cytokines, adhesion molecules, etc.) and cell death pathways (caspases, Bcl-2 family proteins, poly (ADP-ribose) polymerase-1 (PARP-1), apoptosis-inducing factor (AIF), inhibitors of apoptosis proteins (IAPs), heat shock protein 70 (HSP70), receptor interacting protein (RIP), etc., besides targeting directly the genes itself. However, selecting promising targets from various signalling cascades, for drug discovery and development is very challenging, nevertheless such novel approaches may lead to the emergence of new avenues for therapeutic intervention in cerebral ischemia.
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Affiliation(s)
- Suresh L Mehta
- Division of Pharmacology, Central Drug Research Institute, Chatter Manzil Palace, POB-173, Lucknow-226001, India
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42
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Felten A, Leister P, Burgdorf S, Uhlmann L, Scheidtmann KH. Characterization of rat BLOS2/Ceap, a putative yeast She3 homolog, as interaction partner of apoptosis antagonizing transcription factor/Che-1. Biol Chem 2007; 388:569-82. [PMID: 17552904 DOI: 10.1515/bc.2007.073] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/13/2023]
Abstract
AATF/Che-1 is a coactivator of several transcription factors, including steroid hormone receptors. In search of novel interaction partners of AATF, we identified BLOS2 (BLOC1S2, also termed Ceap) from a rat cDNA library. BLOS2 is extremely conserved with a high degree of homology to yeast She3p. The clone isolated represents a splice variant encoding a polypeptide of 168 residues. Rat BLOS2 mRNA is highly expressed in brain and testis and at lower levels in other tissues, but not in skeletal or smooth muscle. Expression as a tagged fusion protein revealed predominant cytoplasmic, but also nuclear localization. In the cytoplasm, BLOS2 fusion proteins exhibit diffuse, filamentous, or dotted distribution, with the latter partially co-localizing with recycling endosomes. In addition, BLOS2 localizes to centrosomes or the pericentrosomal region. Moreover, BLOS2 co-localizes with myosin V globular tail domains in vesicle-like structures. However, a direct interaction could not be demonstrated. In transactivation assays, BLOS2 enhanced transcription from androgen receptor and p53-responsive promoters. However, this enhancement correlated with accumulation of both androgen receptor and p53, suggesting that BLOS2 has a stabilizing effect on these transcription factors. We propose that BLOS2 functions as an adapter in processes such as protein and vesicle processing and transport, and perhaps transcription.
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Affiliation(s)
- Andrea Felten
- Institute of Genetics, University of Bonn, Roemerstr. 164, D-53117 Bonn, Germany
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43
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Friedrich ML, Cui M, Hernandez JB, Weist BM, Andersen HM, Zhang X, Huang L, Walsh CM. Modulation of DRAK2 autophosphorylation by antigen receptor signaling in primary lymphocytes. J Biol Chem 2006; 282:4573-4584. [PMID: 17182616 DOI: 10.1074/jbc.m606675200] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/06/2022] Open
Abstract
Death-associated protein-related apoptotic kinase-2 (DRAK2), a member of the death-associated protein-like family of serine/threonine kinases, is highly expressed in lymphoid organs and is a negative regulator of T cell activation. To investigate the regulation of DRAK2 activity in primary lymphocytes, we employed mass spectrometry to identify sites of autophosphorylation on DRAK2. These studies have revealed a key site of autophosphorylation on serine 12. Using a phospho-specific antibody to detect Ser(12) phosphorylation, we found that autophosphorylation is induced by antigen receptor stimulation in T and B cells. In Jurkat T cells, resting B cells and thymocytes, DRAK2 was hypophosphorylated on Ser(12) but rapidly phosphorylated with antigen receptor ligation. This increase in phosphorylation was dependent on intracellular calcium mobilization, because BAPTA-AM blocked DRAK2 kinase activity, whereas the SERCA inhibitor thapsigargin promoted Ser(12) phosphorylation. Our results show that DRAK2 kinase activity is regulated in a calcium-dependent manner and that Ser(12) phosphorylation is necessary for optimal suppression of T cell activation by this kinase, suggesting a potential feedback loop may act to modulate the activity of this kinase following antigen receptor signaling.
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Affiliation(s)
- Monica L Friedrich
- Center for Immunology and Department of Molecular Biology & Biochemistry, University of California, Irvine, Irvine, California 92697
| | - Meng Cui
- Departments of Physiology & Biophysics and Developmental & Cell Biology, University of California, Irvine, Irvine, California 92697
| | - Jeniffer B Hernandez
- Center for Immunology and Department of Molecular Biology & Biochemistry, University of California, Irvine, Irvine, California 92697
| | - Brian M Weist
- Center for Immunology and Department of Molecular Biology & Biochemistry, University of California, Irvine, Irvine, California 92697
| | - Hilde-Marie Andersen
- Center for Immunology and Department of Molecular Biology & Biochemistry, University of California, Irvine, Irvine, California 92697
| | - Xiaowu Zhang
- Cell Signaling Technology, Inc., Danvers, Massachusetts 01923
| | - Lan Huang
- Departments of Physiology & Biophysics and Developmental & Cell Biology, University of California, Irvine, Irvine, California 92697; Cancer Research Institute, University of California, Irvine, Irvine, California 92697 and.
| | - Craig M Walsh
- Center for Immunology and Department of Molecular Biology & Biochemistry, University of California, Irvine, Irvine, California 92697; Cancer Research Institute, University of California, Irvine, Irvine, California 92697 and.
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Zhang X, Yashiro M, Ohira M, Ren J, Hirakawa K. Synergic antiproliferative effect of DNA methyltransferase inhibitor in combination with anticancer drugs in gastric carcinoma. Cancer Sci 2006; 97:938-44. [PMID: 16805821 PMCID: PMC11158058 DOI: 10.1111/j.1349-7006.2006.00253.x] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Epigenetic alterations of DNA methylation play an important role in the regulation of gene expression associated with chemosensitivity of gastric carcinomas. With the aim of improving the chemotherapeutic efficacy of gastric carcinoma, the effect of DNA methyltransferase inhibitor, 5-aza-CdR, on the chemosensitivity of five anticancer drugs was investigated. Human gastric cancer cell lines, OCUM-2M and MKN-74, and five anticancer drugs, 5-FU, PTX, OXA, SN38, and GEM, were used. In both gastric cancer cell lines, a synergistic antiproliferative effect by a combination of 5-aza-CdR at 5 microM was found in SN38 and GEM. 5-Aza-CdR at 5 microM increased apoptosis induced by SN38 and GEM in both cell lines. 5-Aza-CdR increases the expression of DAPK-2 and DAPK-3, RASSF1, and THBS1 genes in both OCUM-2M and MKN-74 cells, but not that of hMLH1, p16, MGMT, E-cadherin, and p53 genes. These findings suggest that 5-aza-CdR is a promising chemotherapeutical agent for gastric carcinomas, in combination with the anticancer drugs SN38 and GEM, in apoptosis signaling. The upregulation of DAPK-2 and DAPK-3, RASSF1, and THBS1 genes by 5-aza-CdR might be associated with the synergistic effect.
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Affiliation(s)
- Xiaotian Zhang
- Department of Medical Oncology, Beijing Cancer Hospital, School of Oncology, Peking University, Beijing, China
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Kohya N, Koga Y, Kitajima Y, Miyazaki K. Aberrant promoter hypermethylation in biliary tract carcinoma. ACTA ACUST UNITED AC 2006; 13:296-305. [PMID: 16858540 DOI: 10.1007/s00534-005-1058-0] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/01/2005] [Accepted: 09/01/2005] [Indexed: 10/24/2022]
Abstract
Biliary tract carcinoma is a relatively rare tumor with a poor survival rate. The molecular biological mechanisms underlying the development of biliary tract carcinomas are not well understood. Promoter methylation is an important epigenetic mechanism for suppressing tumor-suppressor gene activity. There is limited information regarding the abnormal methylation of cancer-related genes in biliary tract carcinoma; however, a few insights have been obtained into the role of epigenetic silencing in the progression of biliary tract carcinoma. In this review, we summarize recent data on gene silencing by promoter hypermethylation, and we discuss the implications for biliary tract carcinomas.
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Affiliation(s)
- Naohiko Kohya
- Department of Surgery, Saga University Faculty of Medicine, 5-1-1 Nabeshima, Saga, 849-8501, Japan
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Chen RH, Wang WJ, Kuo JC. The tumor suppressor DAP-kinase links cell adhesion and cytoskeleton reorganization to cell death regulation. J Biomed Sci 2006; 13:193-9. [PMID: 16456710 DOI: 10.1007/s11373-005-9063-5] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2005] [Accepted: 12/19/2005] [Indexed: 01/01/2023] Open
Abstract
Death-associated protein (DAP)-kinase, an actin-cytoskeleton localized serine/threonine kinase, functions as a novel tumor suppressor and participates in a wide variety of cell death systems. Recent studies indicate that DAP-kinase elicits a potent cytoskeletal reorganization effect and is capable of modulating integrin inside-out signaling. Using this understanding of DAP-kinase protein function as a framework, we discuss the functional mechanisms of this kinase in regulating death-associated morphological and signaling events. Furthermore, a potential role of DAP-kinase to be a drug target is also discussed.
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Affiliation(s)
- Ruey-Hwa Chen
- Institute of Molecular Medicine, College of Medicine, National Taiwan University, Taipei, Taiwan,
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Abstract
The recognition that cardiac myocytes die by multiple mechanisms and thus substantially affect ventricular remodeling in diseased human hearts supports the concept of ongoing myocyte death in the progression of heart failure and constitutes the basis of this review. In addition, based on the pathophysiology of myocardial cell deaths, the present study emphasizes that currently methodologies, although with some inherent limitations, are available to recognize and measure quantitatively the contribution of myocyte cell death to the progression of the pathologic state of the heart. Our own studies show that application of such methodologies including modern microscopy techniques and the use of different molecular and immunohistochemical markers may generate the consensus that myocyte cell death is a quantifiable parameter in the normal and pathological human heart. The present study also demonstrates that myocyte cell death, apoptotic, oncotic or autophagic in nature, has to be regarded as an additional critical variable of the multifactorial events implicated in the alterations of cardiac anatomy and myocardial structure of the diseased human heart.
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Affiliation(s)
- Sawa Kostin
- Department of Experimental Cardiology, Max-Planck Institute, Bad Nauheim, Germany
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Kuwahara H, Nakamura N, Kanazawa H. Nuclear Localization of the Serine/Threonine Kinase DRAK2 Is Involved in UV-Induced Apoptosis. Biol Pharm Bull 2006; 29:225-33. [PMID: 16462023 DOI: 10.1248/bpb.29.225] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
Abstract
DAP kinase-related apoptosis-inducing kinase 2 (DRAK2), a member of the DAP kinase family, is a serine/threonine kinase capable of inducing apoptosis. Here we studied the relationship between DRAK2 intracellular localization and apoptosis, and found that UV light acts as a stimulus for apoptosis induced by DRAK2. The intracellular location of DRAK2 depended on the cell line: DRAK2 was found primarily in the nuclei of NRK, NIH3T3, and Caco-2 cells while it was present primarily in the cytoplasm of ACL-15, HeLa, and WI-38 cells. Overexpression of Myc-tagged DRAK2 led to apoptosis-like cell death in NRK cells, but not in ACL-15 cells. A GFP fusion protein of DRAK2 was spontaneously localized to the nucleus of ACL-15 cells and resulted in cell death. Nuclear localization and cell death were also observed with DRAK2(1-293) fused to the NLS of SV40 but not with DRAK2(1-293) alone. These results suggested that nuclear accumulation of DRAK2 and the resulting increase in the endogenous level of its kinase activity are required for cell death. UV irradiation caused nuclear accumulation of endogenous DRAK2 in ACL-15 cells, which was followed by apoptosis-like cell death. Knockdown of DRAK2 expression by siRNA partially suppressed UV-induced apoptosis. These results suggest that DRAK2 plays a role in UV induced apoptosis.
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Affiliation(s)
- Hiroshi Kuwahara
- Department of Biological Sciences, Graduate School of Science, Osaka University, 1-1 Machikaneyama-cho, Toyonaka, Osaka 560-0043, Japan
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Schneider-Stock R, Kuester D, Ullrich O, Mittag F, Habold C, Boltze C, Peters B, Krueger S, Hintze C, Meyer F, Hartig R, Roessner A. Close localization of DAP-kinase positive tumour-associated macrophages and apoptotic colorectal cancer cells. J Pathol 2006; 209:95-105. [PMID: 16575786 DOI: 10.1002/path.1951] [Citation(s) in RCA: 19] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/08/2022]
Abstract
The death-associated protein kinase (DAP-kinase) is a cytoskeleton-associated protein crucially involved in the induction of early apoptotic pathways. Aberrant hypermethylation of the DAP-kinase promoter plays a major role in tumorigenesis. We aimed to investigate the inactivation of DAP-kinase and its association with apoptotic cell death in 94 colorectal carcinomas. DAP-kinase promoter hypermethylation and mRNA expression were investigated using methylation-specific PCR and real-time RT-PCR, respectively. The expression of DAP-kinase, Fas, and Fas-ligand (FasL) proteins was studied by immunohistochemistry and immunofluorescence. Apoptosis of tumour cells was investigated using the TUNEL assay. DAP-kinase was expressed in tumour cells and tumour-invading macrophages and was closely associated with high numbers of apoptotic tumour cells. DAP-kinase expression co-localized with FasL overexpression in tumour-associated macrophages, and aberrant promoter hypermethylation was verified in more than 50% of carcinomas. There was a tendency for proximal tumours to show DAP-kinase promoter methylation more frequently (p = 0.07). Promoter methylation resulted in a decrease or loss of DAP-kinase protein expression in tumour cells and tumour-associated macrophages. Simultaneously, a decreased apoptotic count and loss of Fas/FasL expression was observed in tumour cells. Our study is the first to demonstrate DAP-kinase expression in invading tumour-associated macrophages in colorectal cancer. The presence of similar expression levels of DAP-kinase in tumour cells and associated macrophages, and their dependence on the promoter methylation status of the tumour cells, suggests cross talk between these cell types during apoptotic cell death.
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Affiliation(s)
- R Schneider-Stock
- Department of Pathology, Otto-von-Guericke University Magdeburg, Germany
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Vanharanta S, Pollard PJ, Lehtonen HJ, Laiho P, Sjöberg J, Leminen A, Aittomäki K, Arola J, Kruhoffer M, Orntoft TF, Tomlinson IP, Kiuru M, Arango D, Aaltonen LA. Distinct expression profile in fumarate-hydratase-deficient uterine fibroids. Hum Mol Genet 2005; 15:97-103. [PMID: 16319128 DOI: 10.1093/hmg/ddi431] [Citation(s) in RCA: 49] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022] Open
Abstract
Defects in mitochondrial enzymes predispose to severe developmental defects as well as tumorigenesis. Heterozygous germline mutations in the nuclear gene encoding fumarate hydratase (FH), an enzyme catalyzing the hydration of fumarate in the Krebs tricarboxylic acid cycle, cause hereditary leiomyomatosis and renal cell cancer; yet the connection between disruption of mitochondrial metabolic pathways and neoplasia remains to be discovered. We have used an expression microarray approach for studying differences in global gene expression pattern caused by mutations in FH. Seven uterine fibroids carrying FH mutations were compared with 15 fibroids with wild-type FH. The two groups showed markedly different expression profiles, and multiple differentially expressed genes were detected. The most significant increase in FH mutants was seen in the expression of carbohydrate metabolism- and glycolysis-related genes. Other significantly up-regulated gene categories in FH mutants were, for example, iron ion homeostasis and oxidoreduction. Genes with lower expression in FH-mutant fibroids belonged to groups such as extracellular matrix, cell adhesion, muscle development and cell contraction. We show that FH mutations alter significantly the expression profiles of fibroids, most strikingly increasing the expression of genes involved in glycolysis.
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