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
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Sample & Assay Technologies
4. Introduction: Cell death
Definition: Cell death is a controlled mechanism necessary for development, immune regulation,
and homeostasis.
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Other potential cell death mechanisms
Pyroptosis/pyronecrosis
Mitotic catastrophe
Excitotoxicity
Wallerian degeneration
Paraptosis
Entosis
Commonly accepted cell death mechanisms
Apoptosis
Autophagy
Necrosis
Cornification
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Recognition of cell death
Cell morphology
Enzymology
Immunological/nonimmunological
Programmed/accidental
Physiological/pathological
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Sample & Assay Technologies
15. Apoptosis studies: Gene expression
Why is breast cancer resistant to apoptosis?
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Experiment: TRAIL-induced apoptotic gene expression in normal breast cells vs. MDA-MB231 cells. (Human Apoptosis PCR Array [384 well]; PAHS-3012)
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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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Sample & Assay Technologies
16. Apoptosis & autophagy: Which pathway to choose?
Gene expression analysis
When does glucocorticoid stimulation induce autophagy or apoptosis in osteocytes?
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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)
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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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Sample & Assay Technologies
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?
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1) Measure methylation of the CpG island within the D2R gene.
Result: Tumors are hypermethylated, reducing gene expression
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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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Sample & Assay Technologies
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?
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Experiment: NOD2-dependent NFκB signaling leads to autophagy. Are typical Crohn’s NOD2
mutants pathological?
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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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Sample & Assay Technologies
27. Necrosis studies: siRNA knockdown
How does 24S-OHC (common brain-derived cholesterol metabolite) induce neuronal cell
death?
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Experiment: Morphological studies of SH-SY5Y cells suggest necrosis as the primary form of
cell death. siRNA-mediated knockdown of RIPK1 tested this hypothesis.
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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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Sample & Assay Technologies
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
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Sample & Assay Technologies
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
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Sample & Assay Technologies
