Cell death 2013

Sample & Assay Technologies

Cell death: New research solutions for apoptosis, autophagy, and necrosis

Jennifer Gibbons, Ph.D.R&D Scientist

Sample & Assay Technologies- 2 -

• SABiosciences is now a Company

Biology-focused solutions for pathway analysis


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


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


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


Apoptosis pathway

Duprez L (2009) Microbes & Infect. 11: 1050.


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


Autophagy (molecular mechanism and processes)


http://en.wikipedia.org/wiki/File:Macro-micro-autophagy.gif


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?


Necrosis pathway



Cell death crosstalk

Christofferson DE (2010) Current Opinion in Cell Bio. 22: 263.

Zhivotovsky B (2010) Exp. Cell Res. 316: 1374.


Cell death experimental design & QIAGEN

Gene expression� RT-PCR

Epigenetics� miRNA� DNA methylation� Histone modifications

Functional studies� Reporter assays� siRNA/shRNA


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


Experimental overview: Gene expression analysis

Stimulate cells Isolate RNA RT-PCR

Data analysis


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.


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.







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

+

–


miRNA reagents from QIAGEN

3’ UTR reporters

Target identification Expression

Isolation

Function

miRNA mimics & inhibitors

miScript miRNA PCR Arrays

miRNeasy

miRNAstudies


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.


Epigenetics: Overview



p53 BS

p53

NFκB

Protein “A”

mRNA ”A”

MeMeMe

Histones


DNA methylation

Me AcMe Me MeMe

DNA methylation

miRNAshRNAsiRNA


+

–


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.







Cignal Reporter Assays: Complete SolutionReporter assays: Overview


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.


shRNA/siRNA Function



p53 BS

p53

NFκB

Protein “A”

mRNA ”A”

MeMeMe

Histones


DNA methylation

Me AcMe Me MeMe

DNA methylation

miRNAshRNAsiRNA


+

–


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.


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


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

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• 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

Cell death 2013

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