Sample & Assay TechnologiesCell Death: New Research Solutions for Apoptosis, Autophagy, and NecrosisSample & Assay Technologies - 2 - SABiosciences is now aCompanyBiology-focused solutions for Pathway AnalysisSample & Assay Technologies - 3 -Cell Death: Key Points in History1842: 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 recognized2002: Nobel Prize in Medicine for apoptosis Brenner, Sulston, HorvitzSample & Assay Technologies - 4 -Introduction: Cell DeathDefinition: Cell death is a controlled mechanism necessary for development, immune regulation, and homeostasis. Commonly accepted cell death mechanisms Apoptosis Autophagy Necrosis CornificationRecognition of cell death Cell morphology Enzymology Immunological/nonimmunological Programmed/accidental Physiological/pathologicalOther potential cell death mechanisms Pyroptosis/pyronecrosis Mitotic catastrophe Excitotoxicity Wallerian degeneration Paraptosis EntosisSample & Assay Technologies - 5 -ApoptosisMorphology Membrane blebbing Cell shrinkage Chromatin condensation and fragmentation PS exposure Phagocyte engulfmentPhysiological functions Embryonic Development Cellular differentiation Cellular damage InfectionPathophysiological functions Cancer Autoimmune diseases Chronic inflammationSample & Assay Technologies - 6 -Apoptosis PathwayDuprez L (2009) Microbes & Infect. 11: 1050.Sample & Assay Technologies - 7 -AutophagyLevine B (2005) J. Clin. Invest. 115: 2679.Morphology NO Chromatin condensation and fragmentation NO phagocyte engulfment Cytoplasm vacuolizationPhysiological functions Antigen presentation Unfolded protein response Cellular survival Caloric restrictionPathophysiological functions Lysosomal glycogen storage diseases Cancer Neurodegenerative diseasesSample & Assay Technologies - 8 -Autophagy (Molecular Mechanism and Processes)Duprez L (2009) Microbes & Infect. 11: 1050.Sample & Assay Technologies - 9 -Necrosis (Programmed Necrosis)http://www.pathology.med.ohio-state.edu/ext/MedEd/ Med2Visuals/Scripts/descall.idc?FolderNumber=10534 Morphology Gain in cell volume Organelle swelling Plasma membrane rupture and loss of contentsPhysiological functions Immune system stimulation (inflammation)Pathophysiological functions Ischemia/reperfusion injury? Neurodegenerative diseases?Sample & Assay Technologies - 10 -Necrosis PathwayDuprez L (2009) Microbes & Infect. 11: 1050.Sample & Assay Technologies - 11 -Cell Death Pathway CrosstalkChristofferson DE (2010) Current Opinion in Cell Bio. 22: 263.Zhivotovsky B (2010) Exp. Cell Res. 316: 1374.Sample & Assay Technologies - 12 -Cell Death Experimental Design & QIAGENGene Expression RT-PCREpigenetics miRNA DNA methylation Histone modificationsFunctional Studies Reporter assays siRNA/shRNASample & Assay Technologies - 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 CycleSample & Assay Technologies - 14 -Experimental Overview: Gene Expression AnalysisStimulate Cells Isolate RNART-PCRData AnalysisSample & Assay Technologies - 15 -Apoptosis Studies: Gene ExpressionWhy is breast cancer resistant to apoptosis?Experiment: TRAIL-induced apoptotic gene expression in normal breast cells vs. MDA-MB- 231 cells. (Human Apoptosis 384HT PCR Array; PAHS-3012)Results: c-FLIP, STAT5A, and STAT5B are upregulated. STAT5 signaling is involved in the development of resistance to TRAIL-induced apoptosis.Sample & Assay Technologies - 16 -Apoptosis Studies: Gene ExpressionWhy is breast cancer resistant to apoptosis?Experiment: TRAIL-induced apoptotic gene expression in normal breast cells vs. MDA-MB- 231 cells. (Human Apoptosis 384HT PCR Array; PAHS-3012)Results: c-FLIP, STAT5A, and STAT5B are upregulated. STAT5 signaling is involved in the development of resistance to TRAIL-induced apoptosis.Sample & Assay Technologies - 17 -Epigenetics: OverviewStructural Gene NFB BSActivated Transcription Factorsp53 BSp53NFBProtein AmRNA AMeMe MeHistonesHistone-DNA InteractionsDNA MethylationMeAcMeMe Me MeDNA MethylationmiRNAshRNAsiRNATranscription Initiation Complex+Sample & Assay Technologies - 18 -miRNA Reagents from QIAGEN3 UTR ReportersTarget Identification ExpressionIsolationFunctionmiRNA mimics & inhibitorsmiScript miRNA PCR ArraysmiRNeasymiRNAStudiesSample & Assay Technologies - 19 -Apoptosis Studies: miRNA Function1) 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, a member of the 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.Sample & Assay Technologies - 20 -Apoptosis Studies: miRNA Function1) 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, a member of the 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.Sample & Assay Technologies - 21 -Apoptosis Studies: miRNA Function1) 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, a member of the 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.Sample & Assay Technologies - 22 -Epigenetics: OverviewStructural Gene NFB BSActivated Transcription Factorsp53 BSp53NFBProtein AmRNA AMeMe MeHistonesHistone-DNA InteractionsDNA MethylationMeAcMeMe Me MeDNA MethylationmiRNAshRNAsiRNATranscription Initiation Complex+Sample & Assay Technologies - 23 -Apoptosis: DNA Methylation and Histone ModificationProlactinomas lacking dopamine receptors are resistant to chemotherapeutic treatment- induced apoptosis. Why dont these tumors express dopamine receptors?1) Measure methylation of the CpG island within the D2R gene. Result: Tumors are hypermethylated, reducing gene expression2) Measure D2R histone modifications. Result: Tumors have high H3K27me3, a marker for gene silencing, and low H3K9ac, a marker for active gene transcription.Sample & Assay Technologies - 24 -Apoptosis: DNA Methylation and Histone ModificationProlactinomas lacking dopamine receptors are resistant to chemotherapeutic treatment- induced apoptosis. Why dont these tumors express dopamine receptors?1) Measure methylation of the CpG island within the D2R gene. Result: Tumors are hypermethylated, reducing gene expression2) Measure D2R histone modifications. Result: Tumors have high H3K27me3, a marker for gene silencing, and low H3K9ac, a marker for active gene transcription.Sample & Assay Technologies - 25 -Apoptosis: DNA Methylation and Histone ModificationProlactinomas lacking dopamine receptors are resistant to chemotherapeutic treatment- induced apoptosis. Why dont these tumors express dopamine receptors?1) Measure methylation of the CpG island within the D2R gene. Result: Tumors are hypermethylated, reducing gene expression2) Measure D2R histone modifications. Result: Tumors have high H3K27me3, a marker for gene silencing, and low H3K9ac, a marker for active gene transcription.Sample & Assay Technologies - 26 -Reporter ConstructGFP/firefly luciferaseTATAboxTandem repeats of TRETranscriptional Regulatory Elements (TRE), which establish thespecificity of each reporterCignal Reporter Assays: Complete SolutionReporter Assays: OverviewTFEGFPFLUpstream SignalingEventsSample & Assay Technologies - 27 -Autophagy: Reporter AssayNOD2 and ATG16L1 are susceptibility genes in Crohns disease. Could their dysregulation lead to autophagy disruption and the pathogenesis of Crohns?Experiment: Does NOD2-dependent NFB signaling lead to autophagy? Yes. Are typical Crohns NOD2 mutants pathological?Conclusions: Crohns NOD2 mutants inhibit the autophagic response. NOD2 is also involved in the antibacterial response, identifying additional potential mechanisms for Crohns pathology. Sample & Assay Technologies - 28 -Autophagy: Reporter AssayNOD2 and ATG16L1 are susceptibility genes in Crohns disease. Could their dysregulation lead to autophagy disruption and the pathogenesis of Crohns?Experiment: Does NOD2-dependent NFB signaling lead to autophagy? Yes. Are typical Crohns NOD2 mutants pathological?Conclusions: Crohns NOD2 mutants inhibit the autophagic response. NOD2 is also involved in the antibacterial response, identifying additional potential mechanisms for Crohns pathology. Sample & Assay Technologies - 29 -Autophagy: Reporter AssayNOD2 and ATG16L1 are susceptibility genes in Crohns disease. Could their dysregulation lead to autophagy disruption and the pathogenesis of Crohns?Experiment: Does NOD2-dependent NFB signaling lead to autophagy? Yes. Are typical Crohns NOD2 mutants pathological?Conclusions: Crohns NOD2 mutants inhibit the autophagic response. NOD2 is also involved in the antibacterial response, identifying additional potential mechanisms for Crohns pathology. Sample & Assay Technologies - 30 -Epigenetics: OverviewStructural Gene NFB BSActivated Transcription Factorsp53 BSp53NFBProtein AmRNA AMeMe MeHistonesHistone-DNA InteractionsDNA MethylationMeAcMeMe Me MeDNA MethylationmiRNAshRNAsiRNATranscription Initiation Complex+Sample & Assay Technologies - 31 -Necrosis Studies: siRNA KnockdownHow 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. Sample & Assay Technologies - 32 -Necrosis Studies: siRNA KnockdownHow 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. Sample & Assay Technologies - 33 -Necrosis Studies: siRNA KnockdownHow 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. Sample & Assay Technologies - 34 -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. Sample & Assay Technologies - 35 -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. Sample & Assay Technologies - 36 -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. Sample & Assay Technologies - 37 -ConclusionsThree major forms of cell death Apoptosis Autophagy NecrosisQIAGEN offers many methods to study these cellular processes Gene Expression RT2 Profiler PCR Arrays Epigenetics miScript miRNA PCR Arrays miScript miRNA System EpiTect Methyl qPCR Arrays Pyromark CpG Assays EpiTect ChIP qPCR Arrays Functional Studies Cignal Reporter System SureSilencing RNAiSample & Assay Technologies - 38 -Thank you!PCR Array Starter Pack FREE PCR Arrays of any Pathway 2 96-well/100-well (2 samples) OR 1 384-well (4 samples) With the purchase of: RT2 First-Strand cDNA Synthesis Kit RT2 SYBR Green Mastermix (2-Pack)Use Promo code: SAB-PAStarter2Details: Click HereFor more information, please Contact Us:1-888-503-3187 support@SABiosciences.comWould you like to try a Cell Death, Apoptosis, Autophagy or Necrosis PCR Array?