Great overview of methods and data for detecting apoptosis in vivo and in vitro

1. Cytometry of Apoptosis Zbigniew Darzynkiewicz, M.D., Ph.D. Brander Cancer Research Institute New York Medical College Hawthorne, NY 10536 [email_address] Detection of Mitochondrial Changes and Activation of Caspases and Serine proteases

2. HeNe laser LSC Optical System Scan Lens CCD Camera Objective Lens Computer Controlled Stage Scatter Sensor Scanning Mirror Dichroic Mirrors and Optical Filters Photomultipliers Argon ion Laser Violet Diode Laser

3. Setting contours by LSC THRESHOLD CONTOUR INTEGRATION CONTOUR PERIPHERAL CONTOUR BACKGROUND CONTOUR nucleus cytoplasm

5. Apoptosis - Falling off petals of the flower. Connotation of natural death Apoptosis: Active cell death; “Cell suicide” Necrosis: Accidental cell death; “Cell murder”

7. Morphology of apoptotic and necrotic cells Apoptotic Reduced cell size Chromatin condensation Nuclear fragmentation Cell organelles unchanged Membrane “blebbing” Formation of apoptotic bodies Apoptotic bodies shed off Cell remnants phagocytized Cells detach from substrate Necrotic Cell and nuclear swelling Vacuolization of cytoplasm Patchy chromatin condensation Mitochondrial swelling Plasma membrane rupture Dissolution of chromatin Attraction of inflammatory cells

10. Confocal 3d images of nuclei from nonapoptotic (A) and apoptotic (B) cells stained with PI A B

11. HL-60 cells, Necrosis

12. Ca +2 Ca +2 Bad Raf-1 P Bad Bad Bad PT Pore Bax Conductance Pore Bag-1 AIF Bax Bax Bax Bax Bax Bax Bax CYTOCHROME C RELEASE CASPASE-9 APAF-1 (CED-4) OLIGOMERIZATION of APAF-1 ATP, dATP ACTIVATION OF CASPASE-9 “ Initiator caspase ” CARD ACTIVATED CASPASE-9 CASPASES-3,-6,-7 ACTIVATION OF CASPASES INTRACELLULAR DEATH SIGNALS nucleus PARP PARP p53 Smac/DIABLO IAP Smac/DIABLO IAP Bcl-2 Bcl-X L Bcl-X L Bcl-2 Bcl-2 Bcl-2 Bcl-2

13. Main pathways of apoptosis Execution Cleavage of Apoptosis Regulators Cleavage of Housekeeping Proteins DNA Fragmentation Signals Death ligands (TNF, TRAIL) Internal cell stress, DNA damage Death Receptors Cytochrome C Release Caspase a ctivator FADD Apaf-1 Bid Initiator c aspase Caspase-8, Caspase-10 Caspase-9 Effector c aspase Caspase-3, Caspase-6 or Caspase-7

14. Features of apoptosis measured by flow or laser scanning cytometry Plasma membrane Change in permeability Change in asymmetry of phosphatidylserine Nucleus Chromatin condensation Nuclear fragmentation DNA fragmentation Organelles Change in mitochondrial membrane potential (  m ) Other features Caspase activation PARP cleavage Ser -proteases activation Transglutaminase 2 activation

15. LINKER DNA Apoptosis-induced DNA fragmentation HISTONE OCTAMER (NUCLEOSOME CORE) DNA ELECTROPHORESIS Distance between cuts = multiplicity of 200 base pairs 200 bp 400 bp 600 bp 800 bp ICAD CAD DNA cleavage

16. Indirect labeling of DNA breaks dUTP -biotynylated TdT TdT avidin-FITC DNA cleavage ICAD CAD

17. Apoptotic in situ DNA strand breaks

18. Colocalization: DNA and DNA strand breaks

21. Early event of apoptosis: translocation of Bax to mitochondria Bcl-2 Bcl-X L Caspase 9 PARP PARP nucleus Bcl-X L - Bcl 2 Bcl-2 Ca +2 Ca +2 Bad Raf-1 P Bad Bad Bad PT Pore Bax Conductance Pore p53 Caspase 3 Bag-1 Bcl-2 Bcl-2 APAF-1 AIF Cytochrome C Bax Bax Bax Bax Bax Bax Bax Bax Bax

22. Bax immunofluorescence, MCF-7 cells, CPT-treated

23. Translocation of Bax to mitochondria N Bax in cytoplasm and in nucleus Bax in mitochondria C (diffuse) (punctate)

24. Translocation of Bax to mitochondria: Increase of maximal pixel of Bax immunofluorescence DNA Content DNA Content DNA Content CTRL CPT 24h CPT 48h 150 150 150 0 0 0 G 1 S G 2 M Bax Max Pixel G 1 S G 2 M Bax max pixel DNA content

25. Dissipation of the mitochondrial trans- membrane electrochemical potential Rhodamine 123 or carbo - cyanine dyes “ Aggregate” dyes (e.g. JC-1)

26. HL-60 CTRL HL-60 CPT 4h Rh 123 (rhodamine 123) Green Integral Green Integral Scatter Integral Scatter Integral

27. HL-60 CTRL HL-60 CPT 4h JC-1 (“aggregate” dye) (5,5’,6,6’-tetrachloro-1,1’,3,3’-tetraethylbenzimidazolcarbocyanine iodide) Green Integral Green Integral Orange Integral Orange Integral

29. Immunocytochemical detection of p-89 PARP

30. Immunocytochemical detection of PARP p89

31. PARP cleavage vs appearance of DNA strand breaks CPT-induced

32. PARP cleavage vs appearance of DNA strand breaks TNF-induced

33. PARP cleavage vs DNA strand break apperance

34. PARP cleavage vs appearance of DNA strand breaks

35. Is collapse of the mitochondrial potential a prerequisite for cytochrome c release ? Bcl-2 Bcl-X L Caspase 9 PARP PARP nucleus Bcl-X L - Bcl 2 Bcl-2 Ca +2 Ca +2 Bad Raf-1 P Bad Bad Bad PT Pore Bax Conductance Pore p53 Caspase 3 Bag-1 Bcl-2 Bcl-2 APAF-1 AIF Cytochrome C Bax Bax Bax Bax Bax Bax Bax Bax Bax

36. Multiparameter analysis; cause - effect relationship A B A B ~100% B+ will be A+ some B+ will be A-

37. Cell attributes studied supravitally Cell attributes that require cell fixation Integrity of plasma membrane Surface immuno-typing Phosphatidylserine on cell surface Transport through membrane Intracellular pH Oxidative stress (ROIs) Mitochondrial potential Level of glutathione Activation of intracellular enzymes Calcium flux and other ions Cell cycle position, DNA ploidy BrdU incorporation DNA strand breaks Chromatin condensation Activation of NF-kappa B Bax, cytochrome c translocations Translocation of other (AIF, APAF-1) molecules Caspase activation (e.g. PARP cleavage) Immunodetection of intracellular proteins Changes in cell morphology

38. Strategy to detect correlation between the paramaters measured on live (mitochondrial potential) vs fixed (PARP cleavage) cells by LSC Live cells Measure  m Fix cells Measure PARP File # 1 File # 2 Merge file # 1 and # 2 Analyze the merged file

39. Mitochondrial potential vs PARP cleavage

40. Fl uorochrome-labeled i nhibitors of ca spasses FLICA Affinity labeling probes of caspase enzymatic center FAM FMK VEID

41. INACTIVE CASPASE (ZYMOGEN) ACTIVATED CASPASE (HETERO-TETRAMER) BINDING OF FLICA prodomain A B C D FLICA Active center Active center FAM FMK VEID

42. FAM-DEVD-FMK

43. VAD CTRL CPT 3H CPT 4H VEID TNF+CHX 1h TNF+CHX 1.5h VAD CTRL

44. PI - fluorescence FAM-VAD-FMK 100 0 10 4 PI - fluorescence FAM-VAD-FMK 100 0 10 4 FAM-VAD-FMK FAM-VAD-FMK 100 100 Number of cells Number of cells 0 0 Control Control 3h CPT 3h CPT D C A B Caspases activation during apoptosis induced by CPT

45. Green Max Pixel Cell frequency 0 100 100 1 2 Protection of FAM-VAD-FMK binding by cell pre-exposure to z-VAD-FMK

46. A B Caspases activation detected by FAM-VAD-FMK binding

47. FAM-VAD-FMK binding; fixed cells

48. Concurrent cell staining with FAM-VAD-FMK and PI FAM-VAD-FMK fluorescence PI fluorescence

49. Time (h) control 2h 3h 4h 5h A B C D B A D C Cell number (%) P I f l u o r e s c e n c e 100 0 10 4 10 4 10 4 10 4 10 4 0 2 3 4 5 100 FAM-VAD-FMK fluorescence 0 0 0 0 0 100 100 100 100 Kinetics of FAM-VAD-FMK and PI binding

50. Caspase activation - cell cycle specificity Effect of TNF on HL-60 cells PI, INT FAM-VAD-FMK; MPX 0 a b 10 3 10 3 100 100 0  FAM-VAD-FMK; INT FAM-VAD-FMK; INT G 1 S G 2 /M

51. A B C D Propidium Iodide FAM-VAD-FMK Distinction between apoptotic and necrotic cells based on caspases activation (FAM-VAD-FMK binding) vs exclusion of PI Ctr Parthenolide -treated HL-60 cells

52. Correlation between caspase activation and Annexin V binding Annexin V, Max Pix FAM-VAD-FMK, Max Pix Annexin V, Max Pix

53. Stathmo-apoptosis; arresting progress of apoptotic process Not arrested Arrested FAM-VAD-FMK fluorescence P I f l u o r e s c e n c e P I f l u o r e s c e n c e FAM-VAD-FMK fluorescence FAM-VAD-FMK fluorescence FAM-VAD-FMK fluorescence FAM-VAD-FMK fluorescence 100 100 100 100 100 0 0 0 0 0 10 4 10 4 10 4 10 4 10 4 3 % 2 % 4 % 9 % 21 % 20 % 25 % 2 % 2 % 2 % 9 % 7 % 51% 10 % 2 % 6h 3h

54. Stathmo-apoptosis ARRESTED NOT ARRESTED A B C D FAM-VAD-FMK 10 4 10 4 0 Propidium iodide Control A C D P r o p i d i u m i o d i d e B FAM-VAD-FMK 10 4 10 4 0 10 4 10 4 0 10 4 10 4 0 FAM-VAD-FMK 10 4 10 4 0 TNF FLICA TNF + FLICA CPT + FLICA CPT FLICA

55. FAM-VAD-FMK fluorescence 10 0 Stathmo-apoptosis Apoptotic MCF 7 cells prevented from detachment 48h 72h Control Ap-1% Ap-13% Ap-23% Ap-39% Ap-24% P I f l u o r e s c e n c e 0 0 FAM-VAD-FMK fluorescence FAM-VAD-FMK fluorescence 0 0 10 2 0 FAM -VAD -FMK fluorescence FAM-VAD-FMK fluorescence 10 2 10 2 10 2 10 2 P I f l u o r e s c e n c e 10 0 10 0 10 0 10 0

56. Activation-induced apoptosis of lymphocytes Caspases activation detected by FLICA PI fluorescence FAM-VAD-FMK CTR PHA PHA+ONC A A D A B B B D D C C C A B C D

57. 0 24 48 72 0 0 Time (h) 40 40 0 0 60 60 0 60 Time (h) CAI (%) CAI (%) CAI (%) Patient 1 Patient 2 Patient 3 Patient 4 0 24 48 72 0 24 48 72 0 24 48 72 0 24 48 72 Patient 5 2-CdA Control Subtracted CAI

59. FLISP (Fluorochrome-labeled inhibitor of Ser -proteases) Labeling of active enzyme center of Ser -proteases with FLISP Asp-102 His-57 Ser-195 FAM F (Phe) CMK Imidazole Alkylated imidazole Active chymotrypsin Covalent binding of FLISP

60. CPT-induced apoptosis of T-24 cells - Texas Red-FCK vs DAPI

61. Propidium iodide 10 4 10 4 10 4 10 4 FFCK FFCK FLCK FLCK FFCK FLCK FFCK FLCK 100 100 100 100 100 100 100 100 0 0 0 0 Control 3h CPT Control Control Control 3h CPT 3h CPT 3h CPT Number of cells 0 0 0 0 Activation of Ser -proteases reactive with FFCK and FLCK during apoptosis of HL-60 cells induced by CPT

62. SR-VAD-FMK 100 0 FFCK 0 100 r = 0.72 Activation of caspases and Ser -protease(s) ( FFCK -reactive) in HL- 60 cells 3 h after addition of CPT

63. Activation of caspases and Ser-proteases SR-VAD-FMK / FFCK

64. FLCK SR-VAD-FMK 100 0 100 r = 0.82 Activation of caspases and Ser -protease(s) ( FLCK -reactive) in HL- 60 cells 3 h after addition of CPT

65. FAM-VAD-FMK FFCK r = 0.98 FLCK FAM-VAD-FMK r = 0.982 Correlation between activation of caspases (FAM-VAD-FMK) and Ser -proteases during apoptosis of HL-60 cells induced by CPT r = 0.986

66. SR-VAD-FMK (Int) FFCK (Int) 0 100 SR-VAD-FMK (Int) FFCK (Int) 0 100 100 100 100 0 Apoptosis of HL-60 cells induced by Onconase FFCK vs SR-VAD-FMK binding Ctrl Onc

67. 100 SR-VAD-FMK (Int) FLCK (Int) 0 100 100 SR-VAD-FMK (Int) FLCK (Int) 0 100 100 Apoptosis of HL-60 cells induced by Onconase FLCK vs FAM-VAD-FMK binding

68. Activation of caspases and Ser-proteases during apoptosis of HL-60 cells induced by Onconase PI fluorescence FAM-VAD-FMK FFCK FLCK Ctrl Ctrl Ctrl Onc Onc Onc

69. Proteins covalently reactive with FFCK and FLCK (anti-fluorescein Ab) 205 112 70 52.4 Control FFCK FFCK+TNF Control FLCK FLCK+TNF

70. Brander Cancer Research Institute at NYMC, Valhalla, NY E. Bedner W. Gorczyca J. Grabarek G. Juan X. Li P. Pozarowski P. Smolewski F. Traganos Z. Darzynkiewicz Immunochemistry Technologies, Bloomington NM B.W. Lee G. Johnson

72. A B C D * * * * * * * * TGase 2 Resistance to detergent (A,B) Fluorescein-cadaverine binding (C,D)

73. SULFORHODAMINE (protein) 100 100 0 A D 100 100 0 C 100 100 0 B G 1 G 2 M S DAPI; DNA content TGase 2, cell resistance to detergent

74. FL - cadaverine DNA content 100 0 100 FL - cadaverine DNA content 100 0 100 G 1 G 2 M S TGase 2; binding of fluoresceinated cadaverine

75. FAM-VAD-FCK vs PI FFCK vs PI FLCK vs PI