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Methods in molecular biology

Methods in molecular biology

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Methods in molecular biology

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  1. Turkish Thoracic Society 10th Annual Congress Methods in molecular biology Ian Adcock National Heart & Lung Institute, Imperial College, London Imperial College London

  2. CoordinateGeneExpression T -Cells (CD4+) ––– Eosinophils ––– Macrophages ––– Epithelial Cells ––– Smooth Muscle Cells Pathology AcuteInflammation ––– ProgressiveChronicInflammation ––– AirwayRemodeling GeneProducts Cytokines ––– Chemokines ––– CellAdhesionMolecules ––– Enzymes GeneticFactors EnvironmentalFactors (Smoke, allergens) Corticosteroids Asthma: A Chronic Inflammatory Disease

  3. x x x x Inhibitor Induction of inflammatory mediators Cytokines Cigarette Smoke Infectious agents Airway Inflammation Epithelial cell Neutrophil Macrophage Lymphocyte IL-6 IL-8 Proteinases Adhesion Chemotaxis Proteinases TNFa IL-6 Eicosanoids Interferon g IL-2 IL-4 IL-5 IgE

  4. Detection of abnormal gene expression using genome-wide screens and microarrays Allen et al., 2003; Kerr et al., 2005

  5. Functional genomics/Proof of concept • Does an asthma/COPD gene have a relevant function? • What is the effect of selective overexpression? • Does loss of function have functional significance?

  6. Asthma COPD Normal Normal Asthma COPD Localisation of DPP10 in biopsies of asthma and COPD patients. Results were confirmed using Western blot analysis

  7. DPP10 expression is increased in bronchial biopsies of asthmatic subjects DPP10 β-actin Normal Moderate asthma Severe asthma

  8. DPP10 GAPDH * * Increase from control 4hr 24hr Non-stim 1hr Effect of CSE on DPP10 mRNA expression in Beas2B cells

  9. CSE- and H2O2-induced DPP10 is attenuated by NAC * * # Non-stim CSE H2O2 NAC (1mM) CSE + NAC H2O2 + NAC

  10. Overexpression of target genes • Transfection using naked DNA • Transfection aided by Liposomes • Adenoviral infection of cells

  11. Transfection reagent Cell line specific. Need to optimise individually. Manufacturer Transfection reagent Cell lines tested A549, C6, CHO-K1, COS-7, NIH3T3, HEK293, HeLa, Hepa1cLc7, HepG2, MCF-7, Neuro-2a, SK-N-MC (Transfection of siRNA) Primary cells work best with ds oligos and viral infection Amaxa Mirus TransIT-TKO HeLa, CHO (Transfection of RNA) Qiagen Transmessenger COS-7, NIH3T3, HEK293, HeLa, CHO, S2 (Transfection of RNA and siRNA) Invitrogen Oligofectamine

  12. Use of adenoviral vector to inhibit Protein Kinase A Meja KK et al.,JPET 2004

  13. Use of adenoviral vector to inhibit PKA: Effect on 3H-AA release Meja KK et al.,JPET 2004

  14. HDAC2 RNAi RT-PCR 1 2 3 4 5 HDAC2 HDAC1 Western Blotting 2 3 4 5 HDAC2 siRNA attenuates HDAC2 expression 1. Non treatment 2. control duplex siRNA 3. HDAC2 duplex siRNA-1 (612-) 4. HDAC2 duplex siRNA-2 (813-) 5. HDAC2 siRNA-1 & siRNA-2 72-96 hrs to KD HDAC2 protein/ 24-48h for mRNA

  15. HDAC2 RNAi ** 150 100 HDAC2 expression (% of control) -14 -10 50 0 0.1 1 10 100 Dex EC50 (10-10M) HDAC2 knockdown attenuates steroid sensitivity sc 100 % of control 50 0 Dex (log, M) -14 -10

  16. non-smoker COPD 5.0 NT Em H2 NT Em H2 GM-CSF (ng/ml) 2.5 * * * 0 smoker COPD Non-smoker Control HDAC1 overexpression IL-1b (1ng/ml) HDAC2 overexpression IL-1b + Dex (10-6M) HDAC2 overexpression restores steroid sensitivity in COPD BAL macrophages

  17. Acetylation of K494/495 is important in GR-mediated suppression of NF-kB activity Ito et al., JEM 2006

  18. DPP10 knockdown in BEAS-2B cells DPP10 120 100 80 % of control 60 40 20 0 50 n g siRNA 10 0

  19. Dex induced apoptosis following DPP10 knock down in the presence of IL-1β/H2O2 IL-1β (10ng/ml) H2O2 (50µM) Caspase 3 activity assay - - - - + + + + + + + + + + + + - - - - Dex (-log [M]) 8 6 0 0 8 6 0 8 6 0 8 6 Scrambled siRNA DPP10 siRNA Scrambled siRNA DPP10 siRNA

  20. GFP Increased transcription Co-activator complex Activated GR GRE Basal transcriptional machinery Upstream promoter elements The glucocorticoid receptor DBD NT (AF1) LBD (AF2) Classic nuclear receptor structure Held in cytosol until ligand binds

  21. YFP CFP 0.18 0.16 0.14 0.12 BRET Siganl 0.1 0.08 0.06 0.01 0.1 1 10 100 1000 Dexamethasone nM Mechanisms of GR transrepression Activated GR Transrepression depends on a physical interaction between GR and NF-kB Similar mechanism has been described for AP-1 Decreased transcription NF-kB p65 Basal transcriptional machinery Upstream promoter elements We can measure this association in cells using bioluminescent resonance energy transfer technology (BRET)

  22. 2000 100 1500 80 60 MMTV luciferase 1000 NF-kB activity (% TNF) 40 500 20 0 0 0.001 0.01 0.1 1 10 100 0.001 0.01 0.1 1 10 100 1000 [steroid] nM [Steroid] nM Investigating GR function using reporter genes in vitro Foetal rat lung epithelial cells cotransfected with an NF-kB-dependent reporter to measure transrepression and an MMTV -driven reporter to measure transactivation

  23. Reporter gene assays can indicate potential regulatory sequences Newton et al., 1997; Morello et al., 2006

  24. IL-10 gene polymorphism and asthma severity -1082 -819 -592 promoter IL-10 gene G/A C/T G/A Normal Mild Severe n=241 n=82 n=113 FEV1 (% pred) 96% 90% 64% GCC  IL-10 49.1 28.8 21.8 ** ACC IL-10 43.3 30.5 26.2 ** ATA  IL-10 38.5 30.5 30.5

  25. Measures of transcription factor activation Newton et al., 1997, Morello et al., 2006, Lee et al., 2006

  26. mild asthma severe asthma 200 Mr (kDa) Identification of affected Pathways 10 3.5 10 3.5 10 pI pI Systems analysis approach to determining protein function + +