Cell death 2013

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Cell death 2013

  1. 1. Cell death: New research solutions for apoptosis, autophagy, and necrosis Jennifer Gibbons, Ph.D. R&D Scientist Sample & Assay Technologies
  2. 2. Biology-focused solutions for pathway analysis • SABiosciences is now a Company -2- Sample & Assay Technologies
  3. 3. Cell death: Key points in history 1842: Carl Vogt identifies regulated cell death during vertebrate development 1964: The term “programmed cell death” first used Publication on insect tissue development (Lockshin and Williams) 1972: The term “apoptosis” proposed (Kerr et al) 1976: Cell death recognized in Caenorhabditis elegans (Sulston) 1986: Bcl2 cloned (Cleary et al) First component of the cell death system recognized 2002: Nobel Prize in Medicine for apoptosis Brenner, Sulston, Horvitz ̣ ̣ ̣ ̣ ̣ ̣ -3- Sample & Assay Technologies
  4. 4. Introduction: Cell death Definition: Cell death is a controlled mechanism necessary for development, immune regulation, and homeostasis. ̣ Other potential cell death mechanisms Pyroptosis/pyronecrosis Mitotic catastrophe Excitotoxicity Wallerian degeneration Paraptosis Entosis Commonly accepted cell death mechanisms Apoptosis Autophagy Necrosis Cornification ̣ ̣ Recognition of cell death Cell morphology Enzymology Immunological/nonimmunological Programmed/accidental Physiological/pathological ̣ -4- Sample & Assay Technologies
  5. 5. Apoptosis Morphology Membrane blebbing Cell shrinkage Chromatin condensation and fragmentation PS exposure Phagocyte engulfment Physiological functions Embryonic Development Cellular differentiation Cellular damage Infection Pathophysiological functions Cancer Autoimmune diseases Chronic inflammation ̣ ̣ ̣ http://www.sciencephoto.com/media/253988/enlarge -5- Sample & Assay Technologies
  6. 6. Apoptosis pathway Duprez L (2009) Microbes & Infect. 11: 1050. -6- Sample & Assay Technologies
  7. 7. Autophagy Morphology NO Chromatin condensation and fragmentation NO phagocyte engulfment Cytoplasm vacuolization Physiological functions Antigen presentation Unfolded protein response Cellular survival Caloric restriction Pathophysiological functions Lysosomal glycogen storage diseases Cancer Neurodegenerative diseases ̣ ̣ ̣ Levine B (2005) J. Clin. Invest. 115: 2679. -7- Sample & Assay Technologies
  8. 8. Autophagy (molecular mechanism and processes) http://en.wikipedia.org/wiki/File:Macro-micro-autophagy.gif Duprez L (2009) Microbes & Infect. 11: 1050. -8- Sample & Assay Technologies
  9. 9. Necrosis (programmed necrosis) Morphology Gain in cell volume Organelle swelling Plasma membrane rupture and loss of contents Physiological functions Immune system stimulation (inflammation) Pathophysiological functions Ischemia/reperfusion injury? Neurodegenerative diseases? ̣ ̣ ̣ http://www.pathology.med.ohio-state.edu/ext/MedEd/ Med2Visuals/Scripts/descall.idc?FolderNumber=10534 -9- Sample & Assay Technologies
  10. 10. Necrosis pathway Duprez L (2009) Microbes & Infect. 11: 1050. - 10 - Sample & Assay Technologies
  11. 11. Cell death crosstalk Zhivotovsky B (2010) Exp. Cell Res. 316: 1374. Christofferson DE (2010) Current Opinion in Cell Bio. 22: 263. - 11 - Sample & Assay Technologies
  12. 12. Cell death experimental design & QIAGEN Gene expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional studies Reporter assays siRNA/shRNA ̣ ̣ ̣ - 12 - Sample & Assay Technologies
  13. 13. Principles of qRT-PCR: Overview • Real-time PCR • Amplify and simultaneously quantify target DNA • Reverse transcription real-time PCR • Amplify and simultaneously quantify mRNA • Ct values: Threshold cycle - 13 - Sample & Assay Technologies
  14. 14. Experimental overview: Gene expression analysis Stimulate cells Isolate RNA RT-PCR Data analysis - 14 - Sample & Assay Technologies
  15. 15. Apoptosis studies: Gene expression Why is breast cancer resistant to apoptosis? ̣ Experiment: TRAIL-induced apoptotic gene expression in normal breast cells vs. MDA-MB231 cells. (Human Apoptosis PCR Array [384 well]; PAHS-3012) ̣ Results: c-FLIP, STAT5A, and STAT5B are upregulated. STAT5 signaling is involved in the development of resistance to TRAIL-induced apoptosis. ̣ - 15 - Sample & Assay Technologies
  16. 16. Apoptosis & autophagy: Which pathway to choose? Gene expression analysis When does glucocorticoid stimulation induce autophagy or apoptosis in osteocytes? ̣ Experiment: Treat mice with varying concentrations of prednisolone for 28 days. Tibial RNA analyzed with Apoptosis and Autophagy RT2 Profiler PCR Arrays (PAMM-012 & PAMM-084) ̣ Conclusion: Lower doses induced autophagy, and higher doses induced apoptosis. The autophagic response may be induced during cellular stress to promote survival. ̣ - 16 - Sample & Assay Technologies
  17. 17. Cell death experimental design & QIAGEN Gene expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional studies Reporter assays siRNA/shRNA ̣ ̣ ̣ - 17 - Sample & Assay Technologies
  18. 18. Epigenetics: Overview Activated transcription factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription initiation complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA methylation Histone-DNA interactions Ac Structural gene Me Me Me DNA methylation - 18 - Sample & Assay Technologies
  19. 19. miRNA reagents from QIAGEN miRNeasy Isolation Target identification miRNA studies Expression miScript miRNA PCR Arrays 3’ UTR reporters Function miRNA mimics & inhibitors - 19 - Sample & Assay Technologies
  20. 20. Apoptosis studies: miRNA function 1) miRNA expression profiling in renal cancer cell lines (A498 and Caki2) shows significant downregulation of hsa-miR-708. 2) Overexpression of miR-708 suppresses tumorigenicity and induces apoptosis. 3) Bioinformatic predictions of miR-708 targets include Survivin (BIRC5, inhibitor of apoptosis family). 4) Survivin was confirmed as a miR-708 target via 3’ UTR reporter assay. ̣ ̣ ̣ ̣ Conclusion: miR-708 induces apoptosis via Survivin downregulation. ̣ - 20 - Sample & Assay Technologies
  21. 21. Epigenetics: Overview Activated transcription factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription initiation complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA methylation Histone-DNA interactions Ac Structural gene Me Me Me DNA methylation - 21 - Sample & Assay Technologies
  22. 22. Apoptosis: DNA methylation and histone modification Prolactinomas lacking dopamine receptors are resistant to chemotherapeutic treatmentinduced apoptosis. Why don’t these tumors express dopamine receptors? ̣ 1) Measure methylation of the CpG island within the D2R gene. Result: Tumors are hypermethylated, reducing gene expression ̣ ̣ 2) Measure D2R histone modifications. Result: Tumors have high H3K27me3, a marker for gene silencing, and low H3K9ac, a marker for active gene transcription. ̣ ̣ - 22 - Sample & Assay Technologies
  23. 23. Cell death experimental design & QIAGEN Gene expression RT-PCR Epigenetics miRNA DNA methylation Histone modifications Functional studies Reporter assays siRNA/shRNA ̣ ̣ ̣ - 23 - Sample & Assay Technologies
  24. 24. Cignal Reporter Assays: Complete Solution Reporter assays: Overview - 24 - Sample & Assay Technologies
  25. 25. Autophagy: Reporter assay NOD2 and ATG16L1 are susceptibility genes in Crohn’s disease. Could their dysregulation lead to autophagy disruption and the pathogenesis of Crohn’s? ̣ Experiment: NOD2-dependent NFκB signaling leads to autophagy. Are typical Crohn’s NOD2 mutants pathological? ̣ Conclusions: Crohn’s NOD2 mutants inhibit the autophagic response. NOD2 is also involved in the antibacterial response, identifying additional potential mechanisms for Crohn’s pathology. ̣ - 25 - Sample & Assay Technologies
  26. 26. shRNA/siRNA Function Activated transcription factors miRNA shRNA siRNA Protein “A” NFκB + p53 Transcription initiation complex mRNA ”A” – Histones p53 BS Me Me Me Me Me NFκB BS DNA methylation Histone-DNA interactions Ac Structural gene Me Me Me DNA methylation - 26 - Sample & Assay Technologies
  27. 27. Necrosis studies: siRNA knockdown How does 24S-OHC (common brain-derived cholesterol metabolite) induce neuronal cell death? ̣ Experiment: Morphological studies of SH-SY5Y cells suggest necrosis as the primary form of cell death. siRNA-mediated knockdown of RIPK1 tested this hypothesis. ̣ Conclusion: SH-SY5Y cells treated with RIPK1 siRNA and 24S-OHC did not undergo cell death, showing that 24S-OHC induces necrosis in neuronal cells. ̣ - 27 - Sample & Assay Technologies
  28. 28. Conclusions Three major forms of cell death Apoptosis Autophagy Necrosis QIAGEN offers many methods to study these cellular processes Gene expression RT2 Profiler PCR Arrays & Assays Epigenetics miScript miRNA PCR System EpiTect Methyl II PCR Arrays & Assays EpiTect ChIP qPCR Arrays & Assays Functional studies Cignal Reporter Assays SureSilencing shRNA Plasmid Flexitube siRNA, Flexitube GeneSolution, Flexitube Premix Flexiplate siRNA, AllStar Control ̣ ̣ - 28 - Sample & Assay Technologies
  29. 29. Thank you for attending! Would you like to try a Cell Death PathwayFinder, Apoptosis, Autophagy, or Necrosis PCR Array? PCR array starter pack- Promotion code: FDK-PAFASQ4 • PCR arrays of any pathway (FREE) • 2 96-well/100-well (2 samples) OR • 1 384-well (4 samples) • Required reagents (w/ purchase) • RT2 First-Strand cDNA Synthesis Kit • RT2 SYBR Green Mastermix (2-Pack) • US and Canada Only Call 1-888-503-3187 to order Email: support@SABiosciences.com QIAWEBINARS@QIAGEN.COM - 29 - Sample & Assay Technologies

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