Cell Proliferation and Death

Transcription

1 Cell Proliferation and Death Derek Davies, Cancer Research UK

2 Proliferation A cell Apoptosis Cell death Proliferation signals Senescence

3 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

4 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

5 Dead cell discrimination Dead Dead Propidium Iodide Note log scale! Forward Scatter

6 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

7 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

8 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

9 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

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

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

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

13 DNA stained with propidium iodide Cell count Propidium Iodide DNA Content We can quantitate the percentage of cells in each phase of the cell cycle and monitor the effect of treatments

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

15 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= Cell count Cell count Aneuploid G2 Cell count DNA Index = Propidium Iodide - Area Propidium Iodide - Area Propidium Iodide - Area

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

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

18 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

19 Typical dual parameter plot Anti-BrdU FITC BrdU-FITC S Phase 10 G1 G2/M Propidium Iodide Propidium Iodide

20 Compare comparative growth rates MCF10A Breast cancer cell line Control Drug-treated BrdU FITC % 33% BrdU FITC Propidium iodide Propidium iodide

21 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

22 Measuring Proliferation by dye dilution Divisions: # Cells /40 Violet-A

23 Measuring Proliferation by dye dilution Serum free + Serum Overlay # Cells # Cells % of Max /40 Violet-A /40 Violet-A /40 Violet-A

24 Measuring Proliferation by dye dilution B-Cell Marker CFSE

25 The other side of the coin

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

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

28 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

29 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

30 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 kb cleavage Intra-nucleosomal cleavage Protein cross-linking

31 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 Cell. 100:57.

32 Put simply. reviews/poster/apoptosis

33 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

34 The road to commitment

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

36 Apoptosis detection by Flow Cytometry Light scattering/cell permeability (PI, DAPI, To-Pro-3) Untreated Treated Dead 10 3 Dead Propidium Iodide 10 2 Propidium Iodide Live 10 1 Live Forward Scatter Forward Scatter

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

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

39 Apoptosis detection by Flow Cytometry Changes in enzyme expression (Caspases 3, 8 and 9) Untreated Treated Cell count 400 Cell count 400 Cell count Cleaved Caspase-3 FITC Cleaved Caspase-3 FITC Cleaved Caspase-3 FITC

40 Apoptosis detection by Flow Cytometry Changes in cellular DNA (Fragmentation and strand breaks) Untreated Treated Counts 400 Counts Propidium Iodide Propidium Iodide

41 Integration of apoptosis methods TMRE, Annexin, 7-AAD, Hoechst AAD 10 2 TMRE Annexin V-FITC Hoechst TMRE 10 2 TMRE Hoechst Hoechst 33342

42 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