Cell Proliferation and Death - PDF Free Download

Cell Proliferation and Death Derek Davies, Cancer Research UK http://www.london-research-institute.org.uk/technologies/120

Proliferation A cell Apoptosis Cell death Proliferation signals Senescence

DNA analysis Propidium Iodide Ethidium Bromide Hoechst dyes Cyanine dyes eg TO-PRO-3, SYTO/SYTOX dyes Acridine Orange Pyronin Y Styryl Dyes eg LDS-751 Mithramycin, Chromomycin 7 Aminoactinomycin D (7AAD) Diamino-2-phenylindole (DAPI) DRAQ5, DRAQ7

DNA analysis We can use DNA dyes in two ways: As viability dyes Exclusion of dead cells during analysis and sorting Identification and quantification of apoptotic cells To measure DNA content and monitor cell cycle and its regulation

Dead cell discrimination Dead Dead 10000 1000 Propidium Iodide 100 10 Note log scale! 1 0 200 400 600 800 1000 Forward Scatter

The mammalian cell cycle G 1 G 0 S G1: Gap 1 S: Synthetic G2: Gap 2 M: Mitosis G0: cells that cease division M G 2

The mammalian cell cycle RB Phosphorylation Cdk4/Cyclin D Cdk6/Cyclin D Cdk2/cyclin E Cdk2/Cyclin A G 1 INK4, p21, p27, p57 P21, p27, p57 G 0 S Cdk2/Cyclin A M G 2 Cdc2/Cyclin A Cdc2/Cyclin B RB Dephosphorylation

Cell cycle analysis by flow cytometry Cells must be permeable - can use detergent or fixation (ethanol is best) DNA in cells can be stained with a fluorescent dye DNA probes like PI are stochiometric and increase fluorescence on binding Dyes either intercalate or bind specific base pairs So we can measure how much DNA is in a cell Basic protocol - fix, wash twice, remove RNA and stain with DNA-binding dye

In an ideal world. # of Events Increase in Fluorescence Intensity

In the real world. # of Events CV: SD/mean x 100. Increase in Fluorescence Intensity

DNA stained with propidium iodide 1000 S Phase 800 G1 Cell count 600 400 G2/M 200 0 0 200 400 600 800 1000 Propidium Iodide DNA Content Note linear scale!

DNA stained with propidium iodide 1000 800 Cell count 600 400 200 0 0 200 400 600 800 1000 Propidium Iodide DNA Content We can quantitate the percentage of cells in each phase of the cell cycle and monitor the effect of treatments

Example 1: Compare cycles Example 2: S phase block Example 3: M block G1 S G2 G1 S G2 G1 S G2

DNA analysis in a clinical situation Many tumours show altered DNA content Diploid index may have prognostic significance Many tumours show increased proliferation S phase fraction may have prognostic significance Diploid G1 Aneuploid G1 DI=1.36 1200 100 Cell count 600 400 Cell count 900 600 Aneuploid G2 Cell count 80 60 40 DNA Index = 1.32 200 300 20 0 0 0 0 200 400 600 800 1000 Propidium Iodide - Area 0 200 400 600 800 1000 Propidium Iodide - Area 0 200 400 600 800 1000 Propidium Iodide - Area

Analysis of DNA histograms - pitfalls and a better approach 1000 800 G1 54% S 27% G2 18% The use of markers gives a good indication but is only an estimate! # Cells 600 400 200 0 0 200 400 600 800 1000 Propidium Iodide # Cells 1200 900 600 G1 43% S 45% G2 10% Mathematical modeling is a better approach but still not ideal! 300 0 0 200 400 600 800 1000 Propidium Iodide

DNA analysis by a single fluorochrome can only take us so far! G 0 -G 1 Cell Number S G 2 -M Fluorescence Intensity

Cell cycle analysis - Bromodeoxyuridine (BrdU) method Thymidine analog Taken up by cycling cells Use for comparative growth rates, length of cell cycle, pulse labelling Staining procedure involves unwinding DNA Combine with Propidium iodide

Typical dual parameter plot 10000 Anti-BrdU FITC BrdU-FITC 1000 100 S Phase 10 G1 G2/M 1 0 200 400 600 800 1000 Propidium Iodide Propidium Iodide

Compare comparative growth rates MCF10A Breast cancer cell line Control Drug-treated 10 4 10 4 BrdU FITC 10 3 10 2 50% 33% BrdU FITC 10 3 10 2 10 1 10 1 10 0 0 200 400 600 800 1000 Propidium iodide 10 0 0 200 400 600 800 1000 Propidium iodide

Measuring Proliferation by dye dilution Dye must be taken up by live cells Dye must have low toxicity Dye must be compatible with flow cytometric set-up Dye must be equally apportioned between daughter cells Lipophilic dyes that label cell membrane Succinimidyl dyes that label intracellular proteins

Measuring Proliferation by dye dilution Divisions: 3 2 1 0 1200 900 # Cells 600 300 0 0 10 2 10 3 10 4 10 5 440/40 Violet-A

Measuring Proliferation by dye dilution Serum free + Serum Overlay 300 100 1200 80 # Cells 200 100 # Cells 900 600 % of Max 60 40 300 20 0 0 10 2 10 3 10 4 10 5 440/40 Violet-A 0 0 10 2 10 3 10 4 10 5 440/40 Violet-A 0 0 10 2 10 3 10 4 10 5 440/40 Violet-A

Measuring Proliferation by dye dilution B-Cell Marker CFSE

The other side of the coin

apoptosis Falling off Distinct from necrosis and oncosis Programmed cell death Kerr, Wyllie and Currie BJC (1972), 26:239

Normal development Normal tissue turnover Negative selection in immune system T cell killing Exposure to certain conditions Where is apoptosis seen?

Where is apoptosis seen? Normal development Normal tissue turnover Negative selection in immune system T cell killing Exposure to certain conditions Alzheimer s Disease Parkinson s Disease Autoimmune disorders Neurodegenerative disease Cancer

Necrosis Apoptosis Affects groups of cells Affects individual cells Non-physiological induction Physiological induction (viral, poison, ischemia) (lack of signals, changes) Phagocytosis by macrophages Phagocytosis by macrophages or other cells Inflammatory response No inflammatory response

Apoptosis Morphological Functional Cell Shrinkage Free Ca2+ rise Cell shape change bcl2/bax interaction Condensation of cytoplasm Cell dehydration Nuclear envelope changes Loss of mitochondrial membrane potential Nuclear fragmentation Enzyme activation (caspases) Loss of cell surface structures Phosphatidylserine externalisation Apoptotic bodies Lamin B proteolysis Cell detachment DNA denaturation Phagocytosis of remains 50-300kb cleavage Intra-nucleosomal cleavage Protein cross-linking

Why is apoptosis important? Self-sufficiency in growth signals Insensitivity to anti-growth signals Evading apoptosis Cancer Limitless replicative potential Sustained angiogenesis Tissue invasion and metastasis Hanahan, D. and Weinberg, R.A. 2000. Cell. 100:57.

Put simply. http://www.nature.com/ reviews/poster/apoptosis

Major Apoptotic Pathways in Mammalian Cells Death Receptor Pathway Mitochondrial Pathway DISC Fas/Apo1 /CD95 D D D D D Caspase 8 FasL FADD Procaspase 8 Cellular targets BID DNA damage Procaspase 3 Caspase 3 oxidants ceramide others Apaf-1 Procaspase 9 datp Apaf -1 Caspase 9 apoptosome Bcl-2 datp Cytochrome c

The road to commitment

How can apoptosis be detected? DNA Laddering Comet assay Electron microscopy Flow cytometry

Apoptosis detection by Flow Cytometry Light scattering/cell permeability (PI, DAPI, To-Pro-3) Untreated Treated 10 4 10 4 10 3 Dead 10 3 Dead Propidium Iodide 10 2 Propidium Iodide 10 2 10 1 Live 10 1 Live 10 0 0 200 400 600 800 1000 Forward Scatter 10 0 0 200 400 600 800 1000 Forward Scatter

Apoptosis detection by Flow Cytometry Changes to the mitochondria (TMRE, CMX dyes, JC-1) Untreated Treated 10 4 10 4 Dead Dead 10 3 10 3 TO-PRO-3 10 2 TO-PRO-3 10 2 10 1 Live 10 1 Live 10 0 Apoptotic 10 0 10 1 10 2 10 3 10 4 CMXRos 10 0 Apoptotic 10 0 10 1 10 2 10 3 10 4 CMXRos

Apoptosis detection by Flow Cytometry Changes to the cell membrane (Annexin binding) 10 4 Dead Untreated 10 4 Dead Treated 10 3 10 3 Propidium Iodide 10 2 Live Propidium Iodide 10 2 Live 10 1 Apop 10 1 Apop 10 0 10 0 10 1 10 2 10 3 10 4 Annexin V-FITC 10 0 10 0 10 1 10 2 10 3 10 4 Annexin V-FITC

Apoptosis detection by Flow Cytometry Changes in enzyme expression (Caspases 3, 8 and 9) Untreated Treated 800 800 100 600 600 80 Cell count 400 Cell count 400 Cell count 60 40 200 200 20 0 10 0 10 1 10 2 10 3 10 4 Cleaved Caspase-3 FITC 0 10 0 10 1 10 2 10 3 10 4 Cleaved Caspase-3 FITC 0 10 0 10 1 10 2 10 3 10 4 Cleaved Caspase-3 FITC

Apoptosis detection by Flow Cytometry Changes in cellular DNA (Fragmentation and strand breaks) Untreated Treated 300 600 Counts 400 Counts 200 200 100 0 0 200 400 600 800 1000 Propidium Iodide 0 0 200 400 600 800 1000 Propidium Iodide

Integration of apoptosis methods TMRE, Annexin, 7-AAD, Hoechst33342 10 4 10 4 10 3 10 3 7-AAD 10 2 TMRE 10 2 10 1 10 1 10 0 10 0 10 1 10 2 10 3 10 4 Annexin V-FITC 10 0 0 200 400 600 800 1000 Hoechst 33342 10 4 10 4 10 3 10 3 TMRE 10 2 TMRE 10 2 10 1 10 1 10 0 0 200 400 600 800 1000 Hoechst 33342 10 0 0 200 400 600 800 1000 Hoechst 33342

Which method should I use to assess proliferation and death? What is the question? Cell type? Cultured cells? Suspension or adherent? Primary cells?. What has happened to the cells? Treatment? Time course? What other information is being sought e.g. concurrent phenotyping. Are there any technical restrictions e.g. lasers. Cost, simplicity and number of samples. Expertise available