Sample & Assay Technologies Cell death: New research solutions for apoptosis, autophagy, and necrosis Jennifer Gibbons, Ph.D. R&D Scientist
Sample & Assay Technologies
Cell death: New research solutions for apoptosis, autophagy, and necrosis
Jennifer Gibbons, Ph.D.R&D Scientist
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• SABiosciences is now a Company
Biology-focused solutions for pathway analysis
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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 recognized2002: Nobel Prize in Medicine for apoptosis
� Brenner, Sulston, Horvitz
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Introduction: Cell death
Definition: Cell death is a controlled mechanism ne cessary for development, immune regulation, and homeostasis.
Commonly accepted cell death mechanisms� Apoptosis� Autophagy� Necrosis� Cornification
Recognition of cell death� Cell morphology� Enzymology� Immunological/nonimmunological� Programmed/accidental� Physiological/pathological
Other potential cell death mechanisms� Pyroptosis/pyronecrosis� Mitotic catastrophe� Excitotoxicity� Wallerian degeneration� Paraptosis� Entosis
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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
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Apoptosis pathway
Duprez L (2009) Microbes & Infect. 11: 1050.
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Autophagy
Levine B (2005) J. Clin. Invest. 115: 2679.
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
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Autophagy (molecular mechanism and processes)
Duprez L (2009) Microbes & Infect. 11: 1050.
http://en.wikipedia.org/wiki/File:Macro-micro-autophagy.gif
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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?
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Necrosis pathway
Duprez L (2009) Microbes & Infect. 11: 1050.
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Cell death crosstalk
Christofferson DE (2010) Current Opinion in Cell Bio. 22: 263.
Zhivotovsky B (2010) Exp. Cell Res. 316: 1374.
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Cell death experimental design & QIAGEN
Gene expression� RT-PCR
Epigenetics� miRNA� DNA methylation� Histone modifications
Functional studies� Reporter assays� siRNA/shRNA
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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
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Experimental overview: Gene expression analysis
Stimulate cells Isolate RNA RT-PCR
Data analysis
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Apoptosis studies: Gene expression
Why is breast cancer resistant to apoptosis?
Experiment: TRAIL-induced apoptotic gene expression in normal breast cells vs. MDA-MB-231 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.
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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.
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Cell death experimental design & QIAGEN
Gene expression� RT-PCR
Epigenetics� miRNA� DNA methylation� Histone modifications
Functional studies� Reporter assays� siRNA/shRNA
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Epigenetics: Overview
Structural gene NFκB BS
Activated transcription factors
p53 BS
p53
NFκB
Protein “A”
mRNA ”A”
MeMeMe
Histones
Histone-DNA interactions
DNA methylation
Me AcMe Me MeMe
DNA methylation
miRNAshRNAsiRNA
Transcription initiation complex
+
–
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miRNA reagents from QIAGEN
3’ UTR reporters
Target identification Expression
Isolation
Function
miRNA mimics & inhibitors
miScript miRNA PCR Arrays
miRNeasy
miRNAstudies
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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.
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Epigenetics: Overview
Structural gene NFκB BS
Activated transcription factors
p53 BS
p53
NFκB
Protein “A”
mRNA ”A”
MeMeMe
Histones
Histone-DNA interactions
DNA methylation
Me AcMe Me MeMe
DNA methylation
miRNAshRNAsiRNA
Transcription initiation complex
+
–
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Apoptosis: DNA methylation and histone modification
Prolactinomas lacking dopamine receptors are resistant to chemotherapeutic treatment-induced 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.
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Cell death experimental design & QIAGEN
Gene expression� RT-PCR
Epigenetics� miRNA� DNA methylation� Histone modifications
Functional studies� Reporter assays� siRNA/shRNA
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Cignal Reporter Assays: Complete SolutionReporter assays: Overview
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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.
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shRNA/siRNA Function
Structural gene NFκB BS
Activated transcription factors
p53 BS
p53
NFκB
Protein “A”
mRNA ”A”
MeMeMe
Histones
Histone-DNA interactions
DNA methylation
Me AcMe Me MeMe
DNA methylation
miRNAshRNAsiRNA
Transcription initiation complex
+
–
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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.
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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
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Thank you for attending!Would you like to try a Cell Death PathwayFinder, Apoptosis, Autophagy, or Necrosis PCR Array?
Call 1-888-503-3187 to order
Email: [email protected]@QIAGEN.COM
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