BIO 5068 Fundamentals of Molecular and Cell Biology

1. BIO 5068Fundamentals of Molecular and Cell Biology Apoptosis December 1/6, 2005 Dr. Robert H. Arch arch@wustl.edu phone 747-4681

2. The emergent integrated circuit of the cell Hanahan and Weinberg (2000). Cell 100:57-70 2003 03230

3. The emergent integrated circuit of the cell Hanahan and Weinberg (2000). Cell 100:57-70 2003 0330

4. disorders of cell accumulation disorders of cell loss Cell proliferation - apoptosis = homeostasis proliferation apoptosis homeostasis time 2004 0481

5. “The term apoptosis is proposed for a hitherto little recognized mechanism of controlled cell deletion, which appears to play a complementary but opposite role to mitosis in the regulation of animal cell populations. Its morphological features suggest that it is an active, inherently programmed phenomenon, and it has been shown that it can be initiated or inhibited by a variety of environmental stimuli, both physiological and pathological…” Kerr, Wyllie, and Currie (1972). Br. J. Cancer 26:239-57 Apoptosis: A basic biological phenomenon with wide-ranging implications… 2003 0331

6. Cell death-related publications 2003 0286

7. Apoptosis (programmed cell death) – part II first described by Kerr, Wyllie and Currie(Br. J. Cancer, 1972, 26:239) Greek for “falling off” or “dropping off” describes the molecular and morphological processes leading to controlled cellular self-destruction stereotypical morphological changes: shrinkage, vacuoloation, loss of contact to ECM, chromatin condensation, fragmentation into apoptotic bodies active and defined process that plays a crucial role in regulation of tissue homeostasis 2003 0329

8. Morphologic features ofnormal, apoptotic and necrotic cells 1 2 3 4 5 6 Vitale et al. Purdue Cytometry CD-ROM Series, Vol. 4 2004 0477

9. cell swelling nuclear disintegration membrane dissolution annexin V+/PI+ apoptosis cell shrinkage nuclear condensation, apoptotic bodies cytoplasmic blebbing annexin V+/PIˉ single or few cells no inflammation Apoptosis vs. necrosis necrosis 2004 0478

10. PI annexV GITR crosslinking does not inhibitactivation-induced cell death -- aGITR +aGITR III I II 0.00 mg/ml anti-CD3 Esparza and Arch (2005). J. Immunol. 174:7869-74 0.02 mg/ml anti-CD3 2004 0459

11. cell swelling nuclear disintegration membrane dissolution annexin V+/PI+ whole organs inflammation apoptosis cell shrinkage nuclear condensation, apoptotic bodies cytoplasmic blebbing annexin V+/PIˉ single or few cells no inflammation Apoptosis vs. necrosis necrosis 2004 0478

12. Apoptosis (programmed cell death) – part II stimuli include signaling triggered by:cell surface receptors, growth factor withdrawal, hypoxia, heat shock, DNA damage, viral infection, chemotherapeutic agents involved in many physiologic events:embryogenesis, differentiation, homeostasis, aging, removal of defect and/or harmful cells cause for a variety of pathologic disorders:neurodegenerative disease, immunodeficiency,auto-immune disease, and cancer stimuli include signaling triggered by:cell surface receptors, growth factor withdrawal, hypoxia, heat shock, DNA damage, viral infection, chemotherapeutic agents involved in many physiologic events:embryogenesis, differentiation, homeostasis, aging, removal of defect and/or harmful cells cause for a variety of pathologic disorders:neurodegenerative disease, immunodeficiency,auto-immune disease, and cancer stimuli include signaling triggered by:cell surface receptors, growth factor withdrawal, hypoxia, heat shock, DNA damage, viral infection, chemotherapeutic agents involved in many physiologic events:embryogenesis, differentiation, homeostasis, aging, removal of defect and/or harmful cells cause for a variety of pathologic disorders:neurodegenerative disease, immunodeficiency,auto-immune disease, and cancer stimuli include signaling triggered by:cell surface receptors, growth factor withdrawal, hypoxia, heat shock, DNA damage, viral infection, chemotherapeutic agents involved in many physiologic events:embryogenesis, differentiation, homeostasis, aging, removal of defect and/or harmful cells cause for a variety of pathologic disorders:neurodegenerative disease, immunodeficiency,auto-immune disease, and cancer 2003 0329

13. Apoptosis vs. ‘programmed cell death’ • ‘programmed cell death’ and apoptosis are often used as synonyms, but the terms are not identical • ‘programmed cell death’ is a genetically defined process during development of multicellular organisms, e.g., Caenorhabditis elegans:1090 cells are generated during development,exactly 131 of these cells undergo apoptosis • ‘programmed cell death’ is mediated by apoptosis 2004 0479

14. Caenorhabditis elegans:a model for apoptosis research EGL-1 CED-9 CED-4 CED-3 apoptosis 2004 0480

15. The Nobel Prizein Physiology and Medicine 2002 "for their discoveries concerning 'genetic regulation of organ development and programmed cell death'" http://nobelprize.org/medicine/laureates/2002/ 2004 0491

16. Regulation of caspase activity:C. elegans vs. higher eukaryotes Ced-9 Bcl-2 Bcl-xL Bax Bad Ced-4 Apaf-1 cyto c Ced-3 caspases c-IAPs modified from Arch and Thompson (1999). Ann. Rev. Cell Dev. Biol. 15:113-40 2001 0221

17. Biochemical features of apoptosis proteolytic cleavage of cytoskeleton proteolytic cleavage of nuclear proteins endonucleolytic cleavage of genomic DNA crosslinking of proteins by transglutaminase exposure of phoshpatidyl serine on the cell surface 2004 0482

18. Proposed functions of biochemical changes during apoptosis changes of cytoskeleton and cell volume→ separation of cells from their environment degradation of nuclear proteins and genomic DNA→ destruction of genetic material and interference with DNA repair mechanisms Transglutaminase activity→ packaging cell content Plasma membrane changes→ activation of phagocytes changes of cytoskeleton and cell volume→ separation of cells from their environment degradation of nuclear proteins and genomic DNA→ destruction of genetic material and interference with DNA repair mechanisms Transglutaminase activity→ packaging cell content Plasma membrane changes→ activation of phagocytes changes of cytoskeleton and cell volume→ separation of cells from their environment degradation of nuclear proteins and genomic DNA→ destruction of genetic material and interference with DNA repair mechanisms Transglutaminase activity→ packaging cell content Plasma membrane changes→ activation of phagocytes changes of cytoskeleton and cell volume→ separation of cells from their environment degradation of nuclear proteins and genomic DNA→ destruction of genetic material and interference with DNA repair mechanisms Transglutaminase activity→ packaging cell content Plasma membrane changes→ activation of phagocytes changes of cytoskeleton and cell volume→ separation of cells from their environment degradation of nuclear proteins and genomic DNA→ destruction of genetic material and interference with DNA repair mechanisms Transglutaminase activity→ packaging cell content Plasma membrane changes→ activation of phagocytes 2004 0483

19. Cell-extrinsic and cell-intrinsicapoptotic signaling pathways Ashkenazi (2002). Nature Reviews Cancer Vol 2:420-30 2003 0280

20. TNFR signaling and apoptosis “death receptors” induce p53-independent apoptosis TNF was identified as a tumoricidal serum compound (Carswell et al. 1975). TNFR was identified years later(Rubin et al. 1985, Kull et al. 1985). TNF and TNFR belong to superfamilies(Aggarwal et al. 1985). TNFR superfamily can be divided based on the presence of a death domain in some of the receptors. 2003 0295

21. TNFR and TNF superfamilies Ashkenazi (2002). Nature Reviews Cancer Vol 2:420-30 2003 0344

22. Signaling triggered by TNFR family members modified from Arch and Thompson (1999). Ann. Rev. Cell Dev. Biol. 15:113-40 2005 0529

23. Therapeutic strategies utilizing death receptors Ashkenazi (2002). Nature Reviews Cancer Vol 2:420-30 2003 0278

24. Death receptor-induced signaling Ashkenazi (2002). Nature Reviews Cancer Vol 2:420-30 2003 0276

25. Targeting of the FADD gene Yeh et al. (1998). Science 279:1954-8 2003 0268

26. Defective apoptotic pathwaysin FADD-/- MEFs Yeh et al. (1998). Science 279:1954-8 2003 0296

27. ● FADD+/+ ■FADD-/- Apoptotic pathways in FADD-/- fibroblasts c-myc adenovirus 10 ng TNF-a + chx c-myc adenovirus E1B-neg adenovirus adriamycin Yeh et al. (1998). Science 279:1954-8 2003 0297

28. Death receptor-induced signaling Ashkenazi (2002). Nature Reviews Cancer Vol 2:420-30 2003 0276

29. Caspases (part I) cysteine proteases with specificity for aspartic acid. related to Caenorhabditis elegans gene ced-3. constitutively and ubiquitously expressed as catalytically inactive pro-enzymes (zymogens). N-terminal pro-domain, large and small subunit. activation requires proteolysis of the zymogen at specific Asp residues resulting in removal of thepro-domain and formation of heterodimers active caspases are tetramers composed of two heterodimers 2003 0301

30. Mechanism of caspase activation Asp Asp zymogen large small prodomain autoproteolysis, other caspases prodomain active caspase catalytic domain requires amino acids from large and small subunits 2004 0484

31. Caspases (part II) differences in length and sequence of pro-domain:long pro-domain [casp 1, 2, 4, 5, 8, 9, 10, 11, 12, 13]short pro-domain [casp 3, 6, 7, 14] long pro-domains mediate protein-protein interaction:death effector domain (DED) mediates interactionwith death receptors [casp 8, 10]casp recruitment domain (CARD) also found in Ced-4, Apaf-1 and RAIDD [casp 1, 2, 4, 5, 9, 11, 12, 13] arranged in proteolytic cascades that amplify signals. initiator caspases (instigators) cleaveeffector caspases (terminators). 2003 0301

32. Caspase-3, -8, and -9 knockouts caspase-8: embryonically lethal (>E11.5)death receptor (Fas, TNFR, DR3) pathway caspase-9: perinatally lethal<2% survive and develop normallyneuroepithelial progenitors; mitochondrial pathways (dexamethasone, staurosporin, etoposide, g-irradiation) caspase-3: perinatally lethaldepends on genetic backgroundneuroepithelial progenitors; lack of or delayed morphological changes and DNA fragmentation 2003 0320

33. Apoptotic signaling pathways Ashkenazi (2002). Nature Reviews Cancer Vol 2:420-30 2003 0280

34. Apoptotic pathways in caspase-3-/- ES cells Woo et al. (1998). Genes Dev. 12:806-19 2003 0317

35. Some examples of caspase substrates

36. DNA fragmentation in apoptosis agarose gel electrophoresis endonucleolytic DNA fragmentation nucleosome core histone DNA 180bp chromatin 2004 0485

37. Abnormal chromatin condensation andlack of DNA degradation in caspase-3-/- cells Woo et al. (1998). Genes Dev. 12:806-19 2003 0318

38. Abnormal chromatin condensationin caspase-3-/- MEFs Woo et al. (1998). Genes Dev. 12:806-19 2003 0319

39. Lethal effect of anti-Fas mAbin caspase-deficient animals Zheng et al. (2000). Nature Med. 6:1241-7 2003 0321

40. Apoptotic signaling pathways Ashkenazi (2002). Nature Reviews Cancer Vol 2:420-30 2003 0280

41. Bcl-2 Bcl-xL Bax BakBad tBid I + * cytochrome c Smac/DIABLO Cytochrome c release, Bid translocation and caspase activation triggered by anti-Fas mAb Zheng et al. (2000). Nature Med. 6:1241-7 2003 0322

42. Bcl-2 Bcl-xL Bax BakBad tBid I + * cytochrome c Smac/DIABLO Interactions betweenprocaspase-9, Apaf-1, and cytochrome c Zou et al. (1999). J. Biol. Chem. 274:11549-56 2003 0325

43. The apoptosome Zou et al. (1999). J. Biol. Chem. 274:11549-56 2003 0324

44. Phenotypes of Apaf1-/- embryos Apaf-1-/- Apaf-1+/+ Yoshida et al. (1998). Cell 94:739-50 2003 0302

45. Apoptotic pathways in Apaf1-/- ES and EF cells ES cells EF cells Yoshida et al. (1998). Cell 94:739-50 2003 0303

46. Regulators of apoptosis cellular IAPs– family of cytoplasmic proteins that interfere with caspase activation but are also involved in receptor-induced signal transduction pathways. Bcl-2/Bcl-xL– family of pro- and anti-apoptotic proteins that act as sensor for cellular damage or stress. p53 – transcription factor that senses DNA damage and regulates cell proliferation and apoptosis. PI-3K and Akt/PKB– lipid and protein kinases that integrate signals from growth factor receptor to regulate multiple pathways of cell survival and apoptosis. cellular IAPs – family of cytoplasmic proteins that interfere with caspase activation but are also involved in receptor-induced signal transduction pathways. Bcl-2/Bcl-xL– family of pro- and anti-apoptotic proteins that act as sensor for cellular damage or stress. p53 – transcription factor that senses DNA damage and regulates cell proliferation and apoptosis. PI-3K and Akt/PKB– lipid and protein kinases that integrate signals from growth factor receptor to regulate multiple pathways of cell survival and apoptosis. cellular IAPs – family of cytoplasmic proteins that interfere with caspase activation but are also involved in receptor-induced signal transduction pathways. Bcl-2/Bcl-xL – family of pro- and anti-apoptotic proteins that act as sensor for cellular damage or stress. p53 – transcription factor that senses DNA damage and regulates cell proliferation and apoptosis. PI-3K and Akt/PKB– lipid and protein kinases that integrate signals from growth factor receptor to regulate multiple pathways of cell survival and apoptosis. cellular IAPs – family of cytoplasmic proteins that interfere with caspase activation but are also involved in receptor-induced signal transduction pathways. Bcl-2/Bcl-xL – family of pro- and anti-apoptotic proteins that act as sensor for cellular damage or stress. p53 – transcription factor that senses DNA damage and regulates cell proliferation and apoptosis. PI-3K and Akt/PKB– lipid and protein kinases that integrate signals from growth factor receptor to regulate multiple pathways of cell survival and apoptosis. cellular IAPs – family of cytoplasmic proteins that interfere with caspase activation but are also involved in receptor-induced signal transduction pathways. Bcl-2/Bcl-xL – family of pro- and anti-apoptotic proteins that act as sensor for cellular damage or stress. p53 – transcription factor that senses DNA damage and regulates cell proliferation and apoptosis. PI-3K and Akt/PKB – lipid and protein kinases that integrate signals from growth factor receptor to regulate multiple pathways of cell survival and apoptosis. 2004 0494

47. Bcl-2 proteins as regulators of apoptosis inner membrane intermembrane space outer membrane Bcl-2 Bcl-xL Bax Bak Bad tBid I + * Cytochrome c release caspase activation Smac/DIABLO release X-IAP inhibition 2004 0486

48. Bcl-2 Bcl-xL Bax BakBad tBid I + * cytochrome c Smac/DIABLO Smac/DIABLO deficiencyimpairs caspase 3 cleavage in vitro Okada et al. (2002). Mol. Cell. Biol. 22:3509-17 2003 0311

49. Role of X-IAP in exogenous and endogenous cell death pathways Liston et al. (2003). Oncogene 22:8568-80 2004 0500

50. Bcl-2 family members Chan and Yu (2004). Clin. Exp. Pharm. Phys. 31:119-28 2004 0491