Lecturer : Khalil Abou-El-Ardat and Michaël Beck

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1 Module n : 406 Title : Apoptosis and DNA Repair Lecturer : Khalil Abou-El-Ardat and Michaël Beck Molecular Biology and Cytometry Course, May , Mol

2 Workshop Session C: 406 Apoptosis and DNA repair Khalil Abou-el-Ardat Michaël Beck 2

Consequences of DNA damage DAMAGE DNA turnover Endogenous agents ROS Exogenous agents SSB: 55000 events/cell.day DSB: 8 Events/cell.

3 Consequences of DNA damage DAMAGE DNA turnover Endogenous agents ROS Exogenous agents SSB: events/cell.day DSB: 8 Events/cell.day Cell death -Apoptosis -Necrosis? Cell survival ATM/ATR Cellular p53 checkpoint (proliferation) 3 DNA repair -HEJ -NER -NHEJ -BER -Mismatch

4 Consequences of DNA damage DAMAGE DNA turnover Endogenous agents ROS Exogenous agents SSB: events/cell.day DSB: 8 Events/cell.day Cell death -Apoptosis -Necrosis? Cell survival ATM/ATR Cellular p53 checkpoint (proliferation) 4 DNA repair -HEJ -NER -NHEJ -BER -Mismatch

5 Consequences of DNA damage DAMAGE DNA turnover Endogenous agents ROS Exogenous agents SSB: events/cell.day DSB: 8 Events/cell.day Cell death -Apoptosis -Necrosis? Cell survival ATM/ATR Cellular p53 checkpoint (proliferation) 5 DNA repair -HEJ -NER -NHEJ -BER -Mismatch

6 Apoptosis An active process of cell death. Proceeds via two main pathways: Intrinsic (mitochondria-mediated) Extrinsic (receptor-mediated) Hallmarks of apoptosis: DNA fragmentation, chromatin condensation, membrane blebbing, activation of caspases 6

7 Extrinsic Pathway Mediated by death receptors. Activation of death receptor causes trimerization of receptor. Activation of caspases which are cytosine aspartatases that cleave each other and other cellular targets. Are two types: initiators and effectors. 7

8 Extrinsic Pathway 8

9 Intrinsic Pathway Starts with the mitochondria. Pores in mitochondria due to Bax and Bax-like proteins causes release of cytochrome c. Formation of apoptosome which activates caspase 9. 9

10 10

11 Assessing apoptosis Morphology -Blebs - Chromatin condensation - DNA fragmentation Indicators of apoptosis -Proteins (caspases, PARP, BCL-2, Bax, etc.) - Membrane alteration DNA based assays -Sub G1 peak - Ladder -TUNEL -H2A.X 11

cells ( polynuclei or DNA fragmentation, in blue) and a general increase in cell size.

12 Morphology: cytospin 0 Gy 4 Gy X-rays cause a dose-dependent but not linear increase of apoptotic cells ( blebs, examples in yellow), in cells with vacuolized cytoplasms ( in red), in DNA damaged cells ( polynuclei or DNA fragmentation, in blue) and a general increase in cell size. At 1 Gy or more, less than 50% of the cells remain normal. One-way ANOVA, all three p values <0.001, Bonferroni post-hoc. 12

13 Morphology: cytospin X-rays cause a dose-dependent but not linear increase of apoptotic cells ( blebs, examples in yellow), in cells with vacuolized cytoplasms ( in red), in DNA damaged cells ( polynuclei or DNA fragmentation, in blue) and a general increase in cell size. At 1 Gy or more, less than 50% of the cells remain normal. One-way ANOVA, all three p values <0.001, Bonferroni post-hoc. 13

14 Indicators of apoptosis Caspase-3: effector proteinase PARP: substrate of caspase-3 (DNA repair enzyme). Bax: creating channels in the mitochondrion, leading to cytochrome C release and apoptosis BCL-2: direct or indirect inhibition of bax Any other protein acting in the apoptosis pathway Flow cytometry, western blotting, microscopy (immunostaining) 14

15 Indicators of apoptosis Caspase-3 activity Gy Gy 15

16 Membrane alteration Annexin V / Propidium iodide Principle: - Loss of phosphatidylserine (PS) assymetry - PS externalized in apoptotic cells Protocol: - Staining with Annexin V shows available PS - Staining with PI shows membrane integrity - Flow cytometry analysis: apoptotic cells are positive for Annexin V and negative for PI since PS is outside an intact membrane 16

17 Phospholipid membrane assymetry 17

18 Membrane alteration Annexin V / Propidium iodide 0 Gy 4 Gy 18

19 DNA based assays Sub G1 peak Principle: - DNA fragmentation leads to less DNA content than 2N cells in G1 phase of the cell cycle Protocol: - Staining the DNA with PI after membrane permeabilisation with EtOH allows to count the number of cells with less DNA than the ones in G1 phase - Flow cytometry analysis 19

20 DNA based assays Sub G1 peak Number of cells G1/2 DNA content 0 Gy 4 Gy 20

21 DNA based assays DNA ladder Principle: - PARP makes DNA available for repair, releasing the histones - PARP is a target of caspase 3 - DNA fragmentation during apoptosis leads to fragments multiple of ~180bp Protocol: - Electrophoresis gel of DNA has a ladder pattern in apoptotic cells 21

22 22

23 DNA based assays DNA ladder M = Size marker = Control cells without camptothecin + = Cells treated with camptothecin C = Positive control from the kit 23 Image from Roche

24 DNA based assays Ladder: Histone-complexed DNA Principle: - Immunostaining of histone-complexed DNA fragments (mono- and oligonucleosomes) Protocol: Lysate with CAM -ELISA Lysate without CAM Supernatant 24 Image from Roche

25 DNA based assays TUNEL Principle: - Stains free DNA ends - Not only apoptotic breaks are measured Protocol: - TdT-mediated X dutp Nick End Labeling - Flow cytometry, fluorescence microscopy 25

26 DNA based assays TUNEL 26

27 DNA based assays H2A.X Principle: - Histone H2A.X is phosphorylated directly after DSB - Used as early apoptosis assay, but stains DNA breaks! Protocol: - Flow cytometry, fluorescence microscopy 27

28 DNA based assays H2A.X 28

29 Consequences of DNA damage DAMAGE DNA turnover Endogenous agents ROS Exogenous agents SSB: events/cell.day DSB: 8 Events/cell.day Cell death -Apoptosis -Necrosis? Cell survival ATM/ATR Cellular p53 checkpoint (proliferation) 29 DNA repair -HEJ -NER -NHEJ -BER -Mismatch

30 Cytotoxicity assay: LDH Principle: - Lactate dehydrogenase (LDH) is a cytosolic enzyme responsible for the reduction of pyruvate into lactate - Loss of membrane permeability indicates cell death Protocol: - Measurement of LDH content in the supernatant (colorimetric assay) 30

31 31

32 Cell proliferation Kinetic assay Integration of dnucleotides Time point Cell cycle & mitotic index Count of the absolute number of cells Metabolic activity assays (MTT, XTT) Cell cycle & cellular checkpoints 32

33 Cell proliferation Kinetic assay Principle: - Cells in S phase are copying their DNA in order to enter into mitosis - Measuring the integration of marked dnucleotides in the DNA Protocol: - Culture with 5-bromo-2 -deoxy-uridine (BrdU) - Immunostaining of BrdY, measured by flow cytometry, fluorescence microscopy, ELISA 33

34 Cell proliferation Kinetic assay Fluorescein Legend: - Total DNA content stained by PI - BrdU integration stained by fluorescein Observations: - Cells in S phase incorporate more BrdU PI 34 Image from Roche

35 Cell proliferation Cell cycle & mitotic index Principle: - The cell cycle gives indications about the number of proliferating cells - MI = [(1xG1) + (1.5xS) + (2xG2)] / 100 Protocol: - Cell cycle analysis by flow cytometry (PI staining after membrane permeabilisation with EtOH) 35

36 Cell proliferation Mitotic index Principle: - Counting the number of cells with visible chromosomes are in mitosis - MI = number of cells with visible chrom / total Protocol: - DNA staining and count by microscopy 36

37 Cell proliferation Absolute count of cells Principle: - The number of cells remaining a certain time after a stress is an indicator of cell death AND cell proliferation Protocol: - Count the cells by flow cytometry (using beads for volume reference) or microscopy (trypan blue) 37

38 Cell proliferation - MTT Principle: - Active (living and proliferating) cells reduce the tetrazolium salt MTT into formazan salt Protocol: - Incubation of cells with MTT, measure by colorimetry 38

39 Cell cycle & cellular checkpoints G1 G2 (2xG1) Number of cells Cell cycle arrest in G2 at 4Gy DNA content 0 Gy 4 Gy 39

40 Assessing DNA Damage 40

41 DNA Damage DNA damage is induced by several factors as chemicals, energetic radiation, and oxidative stress. The cell is equipped with a battery of responses against DNA damage involving halting proliferation and repair of damage. If all else fails, the cell resorts to cell death. 41

42 ssdna vs. dsdna Two types of DNA damage: ssdna dsdna ssdna can be fixed using: NER, BER, and MR dsdna employ NHEJ or HR 42

43 Players in DNA repair BER: Glycosylase, DNA polymerase, Ligase NER: Nuclease, helicase, DNA polymerase, Ligase MR: MutH, MutT NHEJ: Ku70/80, DNA-PK, XRCC, Ligase IV HR: Rad51, MRN complex, RPA 43

44 Assessing DNA Damage 1. Detecting DNA damage directly 1. Detecting DNA modifications 2. Detecting DNA breaks 2. Detecting DNA repair mechanism activation 44

45 DNA Modifications Reactive oxygen species (ROS) cause DNA damage to DNA One modification is 8-hydroxylation to guanine 8- oxo-7,8-dihydro-2 -deoxyguanosine (8-oxodG) Could be mutagenic if not repaired 45

46 Detection 1. HPLC: detect number of molecules of 8- oxodg. Report as mol./10 5 mol 2. Gas chromatography Mass spectrometry: used for tissue DNA analysis. High sensitivity and requirment for only µg DNA 1. Problem: presence of artefacts 3. Liquid chromatography Mass spectrometry: e.g: in urine. But: expensive and requires experienced technicians 46

47 Detection 4. Flow Cytometry: Utilizing antibodies against 8-oxodG conjugated to FITC Kits produced by companies: binding of FITC-labeled protein conjugate emitting green/yellow fluorescence Argustus Medical OxyDNA Test, Kamiya Biomedical Company 47

48 8-oxoguanine staining 48

49 8-oxodG by flow cytometry Gy 4 Gy 49

50 Detection 5. ELISA Kits: Using 8-oxodG antibody and based on a colorimetric method to quantify damage. High sensitivity Possibility of doing it in a 96-well microtiter plate Quick and required little starting material 50

51 51

52 Histone H2AX modification Upon DNA damage, ATM, ATR, and DNA- PK phosphorylate histone H2AX on Serine 139 and is therefore called γ-h2ax 52

53 53

54 Detection Targeting γ-h2ax with FITC-conjugated γ- H2AX antibody and measuring fluorescence: Flow Cytometry (kits available: Fluorescence microscopy. 54

55 55

56 DNA based assays H2A.X 0 Gy 4 Gy 56

57 DNA based assays H2A.X Gy Gy 57

58 Comet Assay Called the single cell gel electrophoresis (SCGE) Cells are placed in a thin layer of agarose gel, lysed, and placed in an electric field The relaxed DNA loops, indicative of damage and breaks, migrates into the gel forming a tail while the rest remains in the head 58

59 Comet Assay The DNA can be visualized using fluorescent dyes: DAPI, ethidium bromide, YOYO1, and acridine orange Acridine orange can also distinguish ssdna and dsdna by giving different colors: red for ssdna and yellow/green for dsdna 59

60 Comet Assay Extent of damage can also be assessed either visually by naked eye or by using specialized software Parameters: tail length, relative fluorescence intensity of head and tail, tail moment Assigning a score (0-4) for each comet 60

62 Comet Assay Pros: Simplicity, sensitivity, versatility, speed and economy Applications: genotoxicity testing, ecological monitoring Human studies: biomonitoring, nutritional studies, assessing background levels of damage, diagnosis 62

63 Aldehyde Reactive Probe Aldehyde Reactive Probe (ARP) is a reagent that reacts specifically with an aldehyde group which is the open ring form of apurine/apyrimidine sites After treating DNA containing AP sites with ARP reagents, AP sites are tagged with biotin residues, which can be quantified using avidin-biotin assay followed by colorimetric detection 63

64 Cont d AP sites are 1 major type of DNA lesions formed during BER of oxidized, deaminated or alkylated bases Can be done in a 96-well plate 64

65 65

66 Micronucleus Test A mutagenic test for detection of chemicals which induce formation of small membrane-bound DNA fragments (micronuclei) Micronuclei may originate from acentric fragments or whole chromosomes which are unable to migrate with the rest of chromosomes during the anaphase 66

67 Micronucleus 67

68 Flow Cytometry DNA damage causes a relaxation of DNA supercoiling and thus into longer loops DNA is stained using ethidium bromide and measured by flow cytometry (forward scatter) The nucleoid supercoiling is thought to measure both ssdna breaks and changed DNA-protein conformation 68

69 TdT Terminal deoxynucleotidyl Transferase is a DNA polymerase that catalyzes addition of dntps to the 3 -OH end of DNA strands without need for template or primer First assay was Terminal Uridine Nucleotide End Labeling (TUNEL) Use of digoxigenin-conjugated dutp or biotin-conjugated dutp 69

70 TdT TUNEL staining can also be used to detect active DNA repair 70

71 Visualization Flow cytometry Electron microscopy Laser scanning cytometer 71

72 Flow Cytometry 72

73 Electron Microscopy Visualization of DNA damage Establish definitely whether the labeling affects an apoptotic or necrotic cells 73

74 Laser Scanning Cytometer Microscope-based cytofluorimetric method allowing rapid measurement of fluorescence High sensitivity and accuracy Combines advantages of both flow and image cytometry 74

75 75 TUNEL

76 DNA Polymerase I A mixture of labeled nucleotides and enzymes are added to permeabilized cells These nucleotides and enzymes are able to be incorporated into gaps and nicks Can be visualized by flow cytometry or fluorescence microscopy 76

77 Labeling DNA Breaks in Situ By use of the Klenow Enzyme 75 kda proteolytic fragment of the Kornberg polymerase of E. coli Requires a ssdna template and a DNA or RNA primer with a 3 -OH terminus 77

78 Cont d The incorporated biotinylated nucleotides are bound by a streptavidin-horseradish peroxidase conjugate Detected by conversion of diaminobenzamide tetrahydrochloride (DAB) to an insoluble brown precipitate Visualized by light microscopy 78

79 79

80 T4 DNA Ligase Ligation of ds oligoprobes with specific ends to ends of DNA breaks by T4 DNA ligase Allows visualization of exact areas of damage in chromosomes 80

81 DBD-FISH DNA Breakage Detection FISH Allows both intragenomic and intercellular heterogeneity in DNA breakage induction and repair to be assigned Cells in a thin insert of agarose on a glass slide are incubated in an alkaline unwinding solution transforming DNA breaks into ssdna 81

82 Cont d These motifs serve as targets for hybridization of DNA probes As DNA breaks increase = more ssdna produced = more FISH probes 82

83 DBD-FISH Comet 83

84 Western blotting Of course levels of various players in the DNA repair and apoptotic pathway can be assessed by Western blotting. 84

85 85

86 Immunostaining Using immunostaining allows the visualization of repair foci. Use antibodies against players in the DNA repair pathway (eg: Rad51). Limited by resolution of microscope. 86

Technical Bulletin. Multiple Methods for Detecting Apoptosis on the BD Accuri C6 Flow Cytometer. Introduction

Technical Bulletin. Multiple Methods for Detecting Apoptosis on the BD Accuri C6 Flow Cytometer. Introduction March 212 Multiple Methods for Detecting Apoptosis on the BD Accuri C6 Flow Cytometer Contents 1 Introduction 2 Annexin V 4 JC-1 5 Caspase-3 6 APO-BrdU and APO-Direct Introduction Apoptosis (programmed