DOCSLIB.ORG

Pyruvate Dehydrogenase Kinase 4 Deficiency Results in Expedited Cellular Proliferation Through E2F1-Mediated Increase of Cyclins S

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Pyruvate Dehydrogenase Kinase 4 Deficiency Results in Expedited Cellular Proliferation Through E2F1-Mediated Increase of Cyclins S Supplemental material to this article can be found at: http://molpharm.aspetjournals.org/content/suppl/2016/12/21/mol.116.106757.DC1 1521-0111/91/3/189–196$25.00 http://dx.doi.org/10.1124/mol.116.106757 MOLECULAR PHARMACOLOGY Mol Pharmacol 91:189–196, March 2017 Copyright ª 2017 by The American Society for Pharmacology and Experimental Therapeutics Pyruvate Dehydrogenase Kinase 4 Deficiency Results in Expedited Cellular Proliferation through E2F1-Mediated Increase of Cyclins s Jonathan Choiniere, Jianguo Wu, and Li Wang Department of Physiology and Neurobiology, Institute for Systems Genomics, University of Connecticut, Storrs, Connecticut (J.C., J.W., L.W.); Veterans Affairs Connecticut Healthcare System, West Haven, Connecticut (L.W.); Section of Digestive Diseases, Department of Internal Medicine, Yale University, New Haven, Connecticut (L.W.); and School of Pharmaceutical Sciences, Wenzhou Medical University, Wenzhou, Zhejiang, China (L.W.) Downloaded from Received August 31, 2016; accepted December 9, 2016 ABSTRACT Hepatocellular carcinoma (HCC) is a common form of cancer PDK4-knockdown HCC cells also progressed faster through with prevalence worldwide. There are many factors that lead to the cell cycle, which concurrently expressed high levels of molpharm.aspetjournals.org the development and progression of HCC. This study aimed to cyclins and E2F1 as seen in the Pdk42/2 mice. Interestingly, identify potential new tumor suppressors, examine their function the induced cyclin E1 and cyclin A2 caused by Pdk4 deficiency as cell cycle modulators, and investigate their impact on the was repressed by arsenic treatment in mouse liver and in HCC cyclin family of proteins and cyclin-dependent kinases (CDKs). cells. E2f1 deficiency in E2f12/2 mouse liver or knockdown E2F1 In this study, the pyruvate dehydrogenase kinase (PDK)4 gene using small hairpin RNAs in HCC cells significantly decreased was shown to have potential tumor suppressor characteristics. cyclin E1, cyclin A2, and E2F1 proteins. In contrast, inhibition of PDK4 expression was significantly downregulated in human PDK4 activity in HCC cells increased cyclin E1, cyclin A2, and HCC. Pdk42/2 mouse liver exhibited a consistent increase in E2F1 proteins. These findings demonstrate that PDK4 is a critical cell cycle regulator proteins, including cyclin D1, cyclin E1, cyclin regulator of hepatocyte proliferation via E2F1-mediated regula- A2, some associated CDKs, and transcription factor E2F1. tion of cyclins. at ASPET Journals on September 25, 2021 Introduction 2003). Identification of cyclin modulators will aid in the under- standing of HCC development and progression. Liver cancer is the second leading cause of cancer-related Pyruvate dehydrogenase kinase PDK4 is a mitochondrial death worldwide, with an estimated 745,500 deaths yearly. protein with a histidine kinase domain that inhibits the The majority of liver cancer deaths are identified as hepato- pyruvate dehydrogenase complex (PDC). Inhibition of the cellular carcinoma (HCC). The mechanisms behind HCC PDC results in reduced conversion of pyruvate to acetyl- progression are continually studied. Many factors that in- CoA. Acetyl-CoA is used in the citric acid cycle to carry out crease the risk of HCC have been identified, including cellular respiration (Sugden and Holness, 2003). PDK4 is a environmental factors, viral infection, alcohol consumption, major factor in cellular respiration, since it works to inhibit and smoking (Kanda et al., 2015). Cyclins are vital cell cycle the progression from glycolysis to the citric acid cycle. Cellular regulators that normally function to ensure the control of respiration is a major physiologic factor that was shown to be cellular proliferation. The cyclin family of proteins and the altered in cancer cells (Scatena, 2012); therefore, PDK4 is a associated cyclin-dependent kinases (CDKs) have been shown prime molecular suspect in crosstalk between cellular respi- to be significantly elevated in HCC tissues (Masaki et al., ration and cell cycle progression. Because liver cells are highly aerobic and metabolically active, liver tissue and associated cells serve as excellent test subjects. This research was supported by the National Institutes of Health National Institute of Environmental Health Sciences [Grant R01-ES025909], the Arsenic is identified as a group 1 carcinogen and is the 20th National Institutes of Health National Institute of Diabetes and Digestive most common element in the earth’s crust (International and Kidney Diseases [R01-DK080440, R01-DK104656, and P30-DK34989 (to Agency for Research on Cancer, 2012). Over 100 million people Yale Liver Center)], the National Institutes of Health National Institute on Alcohol Abuse and Alcoholism [Grants R21-AA022482 and R21-AA024935], worldwide are exposed to arsenic-contaminated drinking and the U.S. Department of Veterans Affairs [Merit Award 1I01BX002634]. water, making it a global health concern (Polya and Charlet, dx.doi.org/10.1124/mol.116.106757. s This article has supplemental material available at molpharm. 2009). Arsenic has been reported to induce epigenetic alter- aspetjournals.org. ations related to HCC progression (Liu et al., 2014) and to ABBREVIATIONS: Aza, 59-aza-29-deoxycytidine; CDK, cyclin-dependent kinase; DNMT, DNA methyltransferase; HCC, hepatocellular carcinoma; MeDIP, methylated DNA immunoprecipitation on chromatin immunoprecipitation; PCNA, proliferating cell nuclear antigen; PDC, pyruvate dehydrogenase complex; PDK, pyruvate dehydrogenase kinase; qPCR, quantitative polymerase chain reaction; RNA-seq, RNA sequencing; SHP, small heterodimer partner; shRNA, small hairpin RNA; WT, wild type. 189 190 Choiniere et al. silence hepatic PDK4 expression through activation of histone Liver Specimens. The coded human liver specimens were H3K9 methyltransferase G9a (Zhang et al., 2016). obtained through the Liver Tissue Procurement and Distribution Nuclear receptors are a class of proteins that mediate the System (Minneapolis, MN) and were described previously (He et al., activity of hormones and have been implicated in various 2008). Because we did not ascertain individual identities associated diseases, including HCC (Rudraiah et al., 2016). The small with the samples, the Institutional Review Board for Human Research at the University of Connecticut determined that the project was not heterodimer partner (SHP) is a unique transcriptional re- research involving human subjects. pressor (Zhou et al., 2010) and has been implicated as a critical Cell Culture Experiments, Fluorescence-Activated Cell inhibitor of HCC progression (Zhang et al., 2011b) by regulat- Sorting Analysis, and Luciferase Promoter Assay. Huh7 ing multiple pathways involved in tumor growth, including (Zhang and Wang, 2011) and 293T (Yang and Wang, 2012) cells cell proliferation (Zhang et al., 2008), apoptosis (Farhana were described previously. Cultured cells were maintained in et al., 2007; Zhang et al., 2010), and migration and invasion Dulbecco’s modified Eagle’s medium (Gibco, Grand Island, NY) with (Yang et al., 2016). Intriguingly, SHP represses DNA meth- 10% fetal bovine serum (Gibco) and 100 U/ml penicillin-streptomycin ylation via suppressing DNA methyltransferase (DNMT) (Mediatech, Manassas, VA). High- and low-efficiency (shPDK4 H expression (Zhang and Wang, 2011; Zhang et al., 2012), and shPDK4 L, respectively) PDK4 knockdown cell lines were suggesting that modulating SHP function by its agonist may generated by a lentiviral vector containing the small hairpin RNA (shRNA) along with packing vector and envelope vector. These be useful to develop epigenetic-based therapeutic treatment of components were cotransfected into 293T cells using X-tremeGENE Downloaded from HCC. HP DNA transfection reagent (Sigma-Aldrich) according to the Using an unbiased approach that included a combination manufacturer’s protocol. The supernatant containing virus particles of methylated DNA immunoprecipitation on chromatin was collected and concentrated. Suspended virus was applied to immunoprecipitation (MeDIP) and RNA sequencing (RNA- target cells with 6 mg/ml polybrene. The shPDK4H cell line was seq) methods in Shp2/2 mice, this study identified PDK4 as generated using shRNA (TRCN0000006264, clone ID NM_002612.2- a potential tumor suppressor gene. The expression of PDK4 2954s1c) from Sigma-Aldrich, and the shPDK4 L was generated was markedly reduced in human HCC specimens and in using shRNA (TRCN0000194917, clone ID NM_002612.2-1297s1c1) molpharm.aspetjournals.org 2 2 m mouse liver tumors. The Pdk4 / liver had a significant also from Sigma-Aldrich. shPDK4L cells were treated with 15 M induction in multiple cyclin proteins, with the most striking arsenic for 24 hours. E2F1 shRNAs were from Sigma-Aldrich (shE2F1 1: TRCN0000039659, clone ID NM_005225.1-502s1c1; and shE2F1 2: elevation of cyclin E1, cyclin A2, and E2F1, whereas Pdk4- TRCN0000010328, clone ID NM_005225.x-1171s1c1). The PDK4 knockdown HCC cells exhibited a similar activation of cyclin inhibitor diisopropylamine dichloroacetate was from Fisher Scien- proteins and progressed faster through the G2/M phase of the tific (cat. no. AAH6134103, Hampton, NH). cell cycle compared with control cells. Interestingly, arsenic Western Blotting, Quantitative Polymerase Chain Reaction, decreased the levels of cyclin proteins in both mouse livers and Transient Transfection and Luciferase Assay. Western and cultured HCC cells that were induced by Pdk4 de- blotting and quantitative polymerase chain reaction
Load more

Recommended publications
  • Gene Symbol Gene Description ACVR1B Activin a Receptor, Type IB
    Table S1. Kinase clones included in human kinase cDNA library for yeast two-hybrid screening Gene Symbol Gene Description ACVR1B activin A receptor, type IB ADCK2 aarF domain containing kinase 2 ADCK4 aarF domain containing kinase 4 AGK multiple substrate lipid kinase;MULK AK1 adenylate kinase 1 AK3 adenylate kinase 3 like 1 AK3L1 adenylate kinase 3 ALDH18A1 aldehyde dehydrogenase 18 family, member A1;ALDH18A1 ALK anaplastic lymphoma kinase (Ki-1) ALPK1 alpha-kinase 1 ALPK2 alpha-kinase 2 AMHR2 anti-Mullerian hormone receptor, type II ARAF v-raf murine sarcoma 3611 viral oncogene homolog 1 ARSG arylsulfatase G;ARSG AURKB aurora kinase B AURKC aurora kinase C BCKDK branched chain alpha-ketoacid dehydrogenase kinase BMPR1A bone morphogenetic protein receptor, type IA BMPR2 bone morphogenetic protein receptor, type II (serine/threonine kinase) BRAF v-raf murine sarcoma viral oncogene homolog B1 BRD3 bromodomain containing 3 BRD4 bromodomain containing 4 BTK Bruton agammaglobulinemia tyrosine kinase BUB1 BUB1 budding uninhibited by benzimidazoles 1 homolog (yeast) BUB1B BUB1 budding uninhibited by benzimidazoles 1 homolog beta (yeast) C9orf98 chromosome 9 open reading frame 98;C9orf98 CABC1 chaperone, ABC1 activity of bc1 complex like (S. pombe) CALM1 calmodulin 1 (phosphorylase kinase, delta) CALM2 calmodulin 2 (phosphorylase kinase, delta) CALM3 calmodulin 3 (phosphorylase kinase, delta) CAMK1 calcium/calmodulin-dependent protein kinase I CAMK2A calcium/calmodulin-dependent protein kinase (CaM kinase) II alpha CAMK2B calcium/calmodulin-dependent
    [Show full text]
  • ROS Production Induced by BRAF Inhibitor Treatment Rewires
    Cesi et al. Molecular Cancer (2017) 16:102 DOI 10.1186/s12943-017-0667-y RESEARCH Open Access ROS production induced by BRAF inhibitor treatment rewires metabolic processes affecting cell growth of melanoma cells Giulia Cesi, Geoffroy Walbrecq, Andreas Zimmer, Stephanie Kreis*† and Claude Haan† Abstract Background: Most melanoma patients with BRAFV600E positive tumors respond well to a combination of BRAF kinase and MEK inhibitors. However, some patients are intrinsically resistant while the majority of patients eventually develop drug resistance to the treatment. For patients insufficiently responding to BRAF and MEK inhibitors, there is an ongoing need for new treatment targets. Cellular metabolism is such a promising new target line: mutant BRAFV600E has been shown to affect the metabolism. Methods: Time course experiments and a series of western blots were performed in a panel of BRAFV600E and BRAFWT/ NRASmut human melanoma cells, which were incubated with BRAF and MEK1 kinase inhibitors. siRNA approaches were used to investigate the metabolic players involved. Reactive oxygen species (ROS) were measured by confocal microscopy and AZD7545, an inhibitor targeting PDKs (pyruvate dehydrogenase kinase) was tested. Results: We show that inhibition of the RAS/RAF/MEK/ERK pathway induces phosphorylation of the pyruvate dehydrogenase PDH-E1α subunit in BRAFV600E and in BRAFWT/NRASmut harboring cells. Inhibition of BRAF, MEK1 and siRNA knock-down of ERK1/2 mediated phosphorylation of PDH. siRNA-mediated knock-down of all PDKs or the use of DCA (a pan-PDK inhibitor) abolished PDH-E1α phosphorylation. BRAF inhibitor treatment also induced the upregulation of ROS, concomitantly with the induction of PDH phosphorylation.
    [Show full text]
  • A Computational Approach for Defining a Signature of Β-Cell Golgi Stress in Diabetes Mellitus
    Page 1 of 781 Diabetes A Computational Approach for Defining a Signature of β-Cell Golgi Stress in Diabetes Mellitus Robert N. Bone1,6,7, Olufunmilola Oyebamiji2, Sayali Talware2, Sharmila Selvaraj2, Preethi Krishnan3,6, Farooq Syed1,6,7, Huanmei Wu2, Carmella Evans-Molina 1,3,4,5,6,7,8* Departments of 1Pediatrics, 3Medicine, 4Anatomy, Cell Biology & Physiology, 5Biochemistry & Molecular Biology, the 6Center for Diabetes & Metabolic Diseases, and the 7Herman B. Wells Center for Pediatric Research, Indiana University School of Medicine, Indianapolis, IN 46202; 2Department of BioHealth Informatics, Indiana University-Purdue University Indianapolis, Indianapolis, IN, 46202; 8Roudebush VA Medical Center, Indianapolis, IN 46202. *Corresponding Author(s): Carmella Evans-Molina, MD, PhD ([email protected]) Indiana University School of Medicine, 635 Barnhill Drive, MS 2031A, Indianapolis, IN 46202, Telephone: (317) 274-4145, Fax (317) 274-4107 Running Title: Golgi Stress Response in Diabetes Word Count: 4358 Number of Figures: 6 Keywords: Golgi apparatus stress, Islets, β cell, Type 1 diabetes, Type 2 diabetes 1 Diabetes Publish Ahead of Print, published online August 20, 2020 Diabetes Page 2 of 781 ABSTRACT The Golgi apparatus (GA) is an important site of insulin processing and granule maturation, but whether GA organelle dysfunction and GA stress are present in the diabetic β-cell has not been tested. We utilized an informatics-based approach to develop a transcriptional signature of β-cell GA stress using existing RNA sequencing and microarray datasets generated using human islets from donors with diabetes and islets where type 1(T1D) and type 2 diabetes (T2D) had been modeled ex vivo. To narrow our results to GA-specific genes, we applied a filter set of 1,030 genes accepted as GA associated.
    [Show full text]
  • Tricarboxylic Acid (TCA) Cycle Intermediates: Regulators of Immune Responses
    life Review Tricarboxylic Acid (TCA) Cycle Intermediates: Regulators of Immune Responses Inseok Choi , Hyewon Son and Jea-Hyun Baek * School of Life Science, Handong Global University, Pohang, Gyeongbuk 37554, Korea; [email protected] (I.C.); [email protected] (H.S.) * Correspondence: [email protected]; Tel.: +82-54-260-1347 Abstract: The tricarboxylic acid cycle (TCA) is a series of chemical reactions used in aerobic organisms to generate energy via the oxidation of acetylcoenzyme A (CoA) derived from carbohydrates, fatty acids and proteins. In the eukaryotic system, the TCA cycle occurs completely in mitochondria, while the intermediates of the TCA cycle are retained inside mitochondria due to their polarity and hydrophilicity. Under cell stress conditions, mitochondria can become disrupted and release their contents, which act as danger signals in the cytosol. Of note, the TCA cycle intermediates may also leak from dysfunctioning mitochondria and regulate cellular processes. Increasing evidence shows that the metabolites of the TCA cycle are substantially involved in the regulation of immune responses. In this review, we aimed to provide a comprehensive systematic overview of the molecular mechanisms of each TCA cycle intermediate that may play key roles in regulating cellular immunity in cell stress and discuss its implication for immune activation and suppression. Keywords: Krebs cycle; tricarboxylic acid cycle; cellular immunity; immunometabolism 1. Introduction The tricarboxylic acid cycle (TCA, also known as the Krebs cycle or the citric acid Citation: Choi, I.; Son, H.; Baek, J.-H. Tricarboxylic Acid (TCA) Cycle cycle) is a series of chemical reactions used in aerobic organisms (pro- and eukaryotes) to Intermediates: Regulators of Immune generate energy via the oxidation of acetyl-coenzyme A (CoA) derived from carbohydrates, Responses.
    [Show full text]
  • Microrna‑186‑5P Downregulation Inhibits Osteoarthritis Development by Targeting MAPK1
    MOLECULAR MEDICINE REPORTS 23: 253, 2021 MicroRNA‑186‑5p downregulation inhibits osteoarthritis development by targeting MAPK1 QING LI1, MINGJIE WU1, GUOFANG FANG1, KUANGWEN LI1, WENGANG CUI1, LIANG LI1, XIA LI2, JUNSHENG WANG2 and YANHONG CANG2 1Department of Orthopedics, Shenzhen Hospital of Southern Medical University, Shenzhen, Guangdong 518101; 2Department of Orthopedics, The Second People's Hospital of Huai'an, Huai'an, Jiangsu 223002, P.R. China Received February 26, 2020; Accepted September 11, 2020 DOI: 10.3892/mmr.2021.11892 Abstract. As a chronic degenerative joint disease, the char‑ expression, suggesting that miR‑186‑5p may be used as a acteristics of osteoarthritis (OA) are degeneration of articular potential therapeutic target for OA. cartilage, subchondral bone sclerosis and bone hyperplasia. It has been reported that microRNA (miR)‑186‑5p serves a key Introduction role in the development of various tumors, such as osteosar‑ coma, non‑small‑cell lung cancer cells, glioma and colorectal As a chronic degenerative joint disease, the characteristics cancer. The present study aimed to investigate the effect of of osteoarthritis (OA) are degeneration of articular cartilage, miR‑186‑5p in OA. Different concentrations of IL‑1β were subchondral bone sclerosis and bone hyperplasia (1). OA used to treat the human chondrocyte cell line CHON‑001 affects an estimated 10% of men and 18% of women >60 years to simulate inflammation, and CHON‑001 cell injury was of age, worldwide (2). OA is affected by multiple factors, such assessed by detecting cell viability, apoptosis, caspase‑3 as age, sex, trauma history, obesity, heredity and joint defor‑ activity and the levels of TNF‑α, IL‑8 and IL‑6.
    [Show full text]
  • Upregulation of SLC2A3 Gene and Prognosis in Colorectal Carcinoma
    Kim et al. BMC Cancer (2019) 19:302 https://doi.org/10.1186/s12885-019-5475-x RESEARCH ARTICLE Open Access Upregulation of SLC2A3 gene and prognosis in colorectal carcinoma: analysis of TCGA data Eunyoung Kim1†, Sohee Jung2†, Won Seo Park3, Joon-Hyop Lee4, Rumi Shin5, Seung Chul Heo5, Eun Kyung Choe6, Jae Hyun Lee7, Kwangsoo Kim2* and Young Jun Chai5* Abstract Background: Upregulation of SLC2A genes that encode glucose transporter (GLUT) protein is associated with poor prognosis in many cancers. In colorectal cancer, studies reporting the association between overexpression of GLUT and poor clinical outcomes were flawed by small sample sizes or subjective interpretation of immunohistochemical staining. Here, we analyzed mRNA expressions in all 14 SLC2A genes and evaluated the association with prognosis in colorectal cancer using data from the Cancer Genome Atlas (TCGA) database. Methods: In the present study, we analyzed the expression of SLC2A genes in colorectal cancer and their association with prognosis using data obtained from the TCGA for the discovery sample, and a dataset from the Gene Expression Omnibus for the validation sample. Results: SLC2A3 was significantly associated with overall survival (OS) and disease-free survival (DFS) in both the discovery sample (345 patients) and validation sample (501 patients). High SLC2A3 expression resulted in shorter OS and DFS. In multivariate analyses, high SLC2A3 levels predicted unfavorable OS (adjusted HR 1.95, 95% CI 1.22–3.11; P = 0.005) and were associated with poor DFS (adjusted HR 1.85, 95% CI 1.10–3.12; P = 0.02). Similar results were found in the discovery set.
    [Show full text]
  • PKD3 Promotes Metastasis and Growth of Oral Squamous Cell Carcinoma Through Positive Feedback Regulation with PD-L1 and Activation of ERK-STAT1/3-EMT Signalling
    International Journal of Oral Science www.nature.com/ijos ARTICLE OPEN PKD3 promotes metastasis and growth of oral squamous cell carcinoma through positive feedback regulation with PD-L1 and activation of ERK-STAT1/3-EMT signalling Bomiao Cui1, Jiao Chen1, Min Luo 1, Yiying Liu1, Hongli Chen1, Die Lü1, Liwei Wang1, Yingzhu Kang1, Yun Feng1, Libin Huang2 and Ping Zhang1 Oral squamous cell carcinoma (OSCC) has a high incidence of metastasis. Tumour immunotherapy targeting PD-L1 or PD-1 has been revolutionary; however, only a few patients with OSCC respond to this treatment. Therefore, it is essential to gain insights into the molecular mechanisms underlying the growth and metastasis of OSCC. In this study, we analysed the expression levels of protein kinase D3 (PKD3) and PD-L1 and their correlation with the expression of mesenchymal and epithelial markers. We found that the expression of PKD3 and PD-L1 in OSCC cells and tissues was significantly increased, which correlated positively with that of mesenchymal markers but negatively with that of epithelial markers. Silencing PKD3 significantly inhibited the growth, metastasis and invasion of OSCC cells, while its overexpression promoted these processes. Our further analyses revealed that there was positive feedback regulation between PKD3 and PD-L1, which could drive EMT of OSCC cells via the ERK/STAT1/3 pathway, thereby promoting tumour growth and metastasis. Furthermore, silencing PKD3 significantly inhibited the expression of PD-L1, and lymph node metastasis of OSCC was investigated with a mouse footpad xenograft model. Thus, our findings provide a 1234567890();,: theoretical basis for targeting PKD3 as an alternative method to block EMT for regulating PD-L1 expression and inhibiting OSCC growth and metastasis.
    [Show full text]
  • Human Kinases Info Page
    Human Kinase Open Reading Frame Collecon Description: The Center for Cancer Systems Biology (Dana Farber Cancer Institute)- Broad Institute of Harvard and MIT Human Kinase ORF collection from Addgene consists of 559 distinct human kinases and kinase-related protein ORFs in pDONR-223 Gateway® Entry vectors. All clones are clonal isolates and have been end-read sequenced to confirm identity. Kinase ORFs were assembled from a number of sources; 56% were isolated as single cloned isolates from the ORFeome 5.1 collection (horfdb.dfci.harvard.edu); 31% were cloned from normal human tissue RNA (Ambion) by reverse transcription and subsequent PCR amplification adding Gateway® sequences; 11% were cloned into Entry vectors from templates provided by the Harvard Institute of Proteomics (HIP); 2% additional kinases were cloned into Entry vectors from templates obtained from collaborating laboratories. All ORFs are open (stop codons removed) except for 5 (MST1R, PTK7, JAK3, AXL, TIE1) which are closed (have stop codons). Detailed information can be found at: www.addgene.org/human_kinases Handling and Storage: Store glycerol stocks at -80oC and minimize freeze-thaw cycles. To access a plasmid, keep the plate on dry ice to prevent thawing. Using a sterile pipette tip, puncture the seal above an individual well and spread a portion of the glycerol stock onto an agar plate. To patch the hole, use sterile tape or a portion of a fresh aluminum seal. Note: These plasmid constructs are being distributed to non-profit institutions for the purpose of basic
    [Show full text]
  • PRODUCTS and SERVICES Target List
    PRODUCTS AND SERVICES Target list Kinase Products P.1-11 Kinase Products Biochemical Assays P.12 "QuickScout Screening Assist™ Kits" Kinase Protein Assay Kits P.13 "QuickScout Custom Profiling & Panel Profiling Series" Targets P.14 "QuickScout Custom Profiling Series" Preincubation Targets Cell-Based Assays P.15 NanoBRET™ TE Intracellular Kinase Cell-Based Assay Service Targets P.16 Tyrosine Kinase Ba/F3 Cell-Based Assay Service Targets P.17 Kinase HEK293 Cell-Based Assay Service ～ClariCELL™ ～ Targets P.18 Detection of Protein-Protein Interactions ～ProbeX™～ Stable Cell Lines Crystallization Services P.19 FastLane™ Structures ～Premium～ P.20-21 FastLane™ Structures ～Standard～ Kinase Products For details of products, please see "PRODUCTS AND SERVICES" on page 1～3. Tyrosine Kinases Note: Please contact us for availability or further information. Information may be changed without notice. Expression Protein Kinase Tag Carna Product Name Catalog No. Construct Sequence Accession Number Tag Location System HIS ABL(ABL1) 08-001 Full-length 2-1130 NP_005148.2 N-terminal His Insect (sf21) ABL(ABL1) BTN BTN-ABL(ABL1) 08-401-20N Full-length 2-1130 NP_005148.2 N-terminal DYKDDDDK Insect (sf21) ABL(ABL1) [E255K] HIS ABL(ABL1)[E255K] 08-094 Full-length 2-1130 NP_005148.2 N-terminal His Insect (sf21) HIS ABL(ABL1)[T315I] 08-093 Full-length 2-1130 NP_005148.2 N-terminal His Insect (sf21) ABL(ABL1) [T315I] BTN BTN-ABL(ABL1)[T315I] 08-493-20N Full-length 2-1130 NP_005148.2 N-terminal DYKDDDDK Insect (sf21) ACK(TNK2) GST ACK(TNK2) 08-196 Catalytic domain
    [Show full text]
  • Supplementary Information
    Immune differentiation regulator p100 tunes NF-κB responses to TNF Budhaditya Chatterjee,1, 2* Payel Roy,1, # * Uday Aditya Sarkar,1 Yashika Ratra,1 Meenakshi Chawla,1 James Gomes,2 Soumen Basak1§ 1Systems Immunology Laboratory, National Institute of Immunology, Aruna Asaf Ali Marg, New Delhi-110067, India. 2Kusuma School of Biological Sciences, Indian Institute of Technology Delhi, India. #Current address: La Jolla Institute for Allergy and Immunology, USA *These authors contributed equally to this work. §Corresponding author. Email: [email protected] Supplementary Information I. Supplementary Fig. S1 – Fig. S4 and corresponding figure legends II. Supplementary Tables III. Detailed description of global gene expression analyses IV. A description of the mathematical model and related parameterization V. Supplementary References 1 I. Supplementary Fig. S1 – Fig. S4 and corresponding figure legends a Theoretical IKK2 activity inputs b Theoretical IKK2 activity inputs of varying peak amplitude of varying durations 60 100 50 30 NEMO-IKK2(nM) 0 NEMO-IKK2 (nM) 0 simulated NF-κB activities simulated NF-κB activities 100 100 (nM) (nM) 50 50 Bn Bn κ κ NF NF 0 0 0 2 4 6 8 0 2 4 6 8 time (hr) time (hr) c experimentally-derived IKK2 activity inputs 100 TNFc TNFp 50 NEMO-IKK2 activity(nM) 0 0 2 4 6 8 0 2 4 6 8 time (hr) Figure S1: In silico studies of the NF-κB system: a) A library of twelve theoretical IKK2 activity profiles with an invariant signal duration of 8 hr and peak amplitude uniformly varying from 10 nM to 100 nM was used as model inputs (top) for simulating NF-κBn responses in a time course (bottom).
    [Show full text]
  • Inhaltsverzeichnis
    Inhaltsverzeichnis 1 Inhaltsverzeichnis 47 2 Einführung 49 2.1 Die Pathologie . 49 2.2 Geschichte . 49 2.3 Berufsbild des Pathologen . 50 2.4 Grundbegriffe . 52 2.5 Krankheitsverlauf . 53 2.6 Krankheitsausgang . 53 2.7 Der Tod . 54 2.7.1 Todesart . 55 2.7.2 Todesursache . 56 2.7.3 Sterbetypen . 56 2.8 Krankheitsstatistik . 56 2.9 Klassifikation von Krankheiten . 56 3 Technik und Methoden 59 3.1 Untersuchungsmaterial und Materialgewinnung . 59 3.2 Materialtransport . 59 3.2.1 Nativmaterial (NM) . 59 3.2.2 Formalin-fixiertes Material (FFM) . 60 3.2.3 Zytologie . 60 3.2.4 Begleitformular . 61 3.3 Materialaufbereitung . 61 3.3.1 Zuschnitt und makroskopischer Befund . 62 3.3.2 Einbettung . 62 1 2 3.3.3 Schnittanfertigung . 62 3.4 Histochemie (Färbungen) . 66 3.5 Immunhistochemie (IHC) . 68 3.5.1 Antigen-Tabelle geordnet nach Gewebe . 68 3.5.2 Antigen-Tabelle geordnet nach Marker . 74 3.5.3 Antigene mit therapeutischer Relevanz . 76 3.6 Elektronenmikroskopie (EM) . 78 3.7 Immunfluoreszenz-Techniken (IF) . 78 3.7.1 Direkte Immunfluoreszenz (DIF) . 80 3.8 Molekularbiologische und genetische Methoden . 80 4 Anpassungsreaktionen 81 5 Zell- und Gewebsschäden 85 6 Exogene Noxen 87 6.1 Physikalische Noxen . 87 6.2 Chemische Noxen . 88 6.3 Biologische Noxen . 92 6.3.1 Biologische Gifte . 92 6.3.2 Prionen . 94 6.3.3 Viren . 94 6.3.4 Bakterien . 99 6.3.5 Protozoen . 103 6.3.6 Pilze . 104 6.3.7 Helminthen (Würmer) . 105 6.3.8 Arthropoden . 107 7 Kardiovaskuläres System 109 8 Herz 111 8.1 Herzinsuffizienz .
    [Show full text]
  • Inactivation of the HIF-1A/PDK3 Signaling Axis Drives Melanoma Toward Mitochondrial Oxidative Metabolism and Potentiates the Therapeutic Activity of Pro-Oxidants
    Published OnlineFirst August 3, 2012; DOI: 10.1158/0008-5472.CAN-12-0979 Cancer Therapeutics, Targets, and Chemical Biology Research Inactivation of the HIF-1a/PDK3 Signaling Axis Drives Melanoma toward Mitochondrial Oxidative Metabolism and Potentiates the Therapeutic Activity of Pro-Oxidants Jerome Kluza1, Paola Corazao-Rozas1, Yasmine Touil1, Manel Jendoubi1, Cyril Maire1, Pierre Guerreschi1, Aurelie Jonneaux1, Caroline Ballot1,Stephane Balayssac4, Samuel Valable2,3, Aurelien Corroyer-Dulmont2,3, Myriam Bernaudin2,3, Myriam Malet-Martino4, Elisabeth Martin de Lassalle1, Patrice Maboudou5, Pierre Formstecher1, Renata Polakowska1, Laurent Mortier1, and Philippe Marchetti1,5 Abstract Cancer cells can undergo a metabolic reprogramming from oxidative phosphorylation to glycolysis that allows them to adapt to nutrient-poor microenvironments, thereby imposing a selection for aggressive variants. However, the mechanisms underlying this reprogramming are not fully understood. Using complementary approaches in validated cell lines and freshly obtained human specimens, we report here that mitochondrial respiration and oxidative phosphorylation are slowed in metastatic melanomas, even under normoxic conditions due to the persistence of a high nuclear expression of hypoxia-inducible factor-1a (HIF-1a). Pharmacologic or genetic blockades of the HIF-1a pathway decreased glycolysis and promoted mitochondrial respiration via specific reduction in the expression of pyruvate dehydrogenase kinase-3 (PDK3). Inhibiting PDK3 activity by dichloroacetate (DCA) or siRNA-mediated attenuation was sufficient to increase pyruvate dehydrogenase activity, oxidative phosphorylation, and mitochondrial reactive oxygen species generation. Notably, DCA potentiated the antitumor effects of elesclomol, a pro-oxidative drug currently in clinical development, both by limiting cell proliferation and promoting cell death. Interestingly, this combination was also effective against BRAF V600E-mutant melanoma cells that were resistant to the BRAF inhibitor vemurafenib.
    [Show full text]