CELL DEATH an overview

1. Intercalated BSc 2007-08 CELL DEATHan overview Dr Cathy Baker 22nd October 2007

2. How do cells die? • Killed by injurious agents • Induced to commit suicide NECROSIS APOPTOSIS

3. LEARNING OBJECTIVES Understand, describe and illustrate … • Differences: necrosis vs. apoptosis • Morphological changes of apoptosis • Function of apoptosis • Principal biochemical mechanisms • Role of apoptosis in pathologies

4. Necrosis Apoptosis Lecture overview Function Morphological changes Biochemistry Pathology

5. Necrosis • Mechanical injury & toxic agents • Cell groups • Membrane integrity destroyed • Cells and organelles swell, burst and leak contents • Inflammatory response • Other cells and tissues damaged

6. Cell death by necrosis

7. John Kerr et al Br.J.Cancer 26: 239-257, 1972

8. Apoptosis • Essential biological process • Cells have role in own death - told or decide to commit suicide • Programmed cell death (PCD)

9. Apoptosis • Distinct form of single cell death • Tightly regulated • Very localised • Energy consuming process • Membranes intact (early stages) • Safe disposal of cell corpse • No inflammation

10. Necrosis Apoptosis Morphological changes

11. Changes in cell morphology • Cells shrink and become detached from adjoining cells • Cytoskeleton collapses • Mitochondria remain intact • Plasma membrane develops bubbles (blebs) on surface

12. Membrane blebs during apoptosis

13. Nucleus and chromatin condense • Aggregates at periphery of nucleus • Nuclear envelope disintegrates • DNA fragmentation • Budding off and breakage into small membrane wrapped fragments - apoptotic bodies

14. Formation of apoptotic bodies

15. What happens to apoptotic cells and apoptotic bodies? • Ingested & degraded by phagocytes • Macrophages and dendritic cells • Adjacent cells in tissue • High speed and efficiency • Histologically inconspicuous • No inflammation

16. Phagocytosis of apoptotic cells and bodies

17. Necrosis Function Apoptosis Morphological changes

18. Function of apoptosis? • Deliberate removal of specific, unwanted cells • Organised and controlled manner • Without damaging other cells or tissues Circumstances?

19. Homeostasis • Constancy of internal environment • Tissue turnover • Cell numbers have to be maintained Homeodynamics

20. Embryonic development Removal of unwanted cells • Damage • Organ and tissue differentiation • Vestigial structures • Alteration of tissue form

21. 5 weeks 8 weeks

22. Neurologicaldevelopment • Deletion of excess immature neurons that have failed to establish synaptic connections • Occurs in CNS and PNS • Prevents redundant cell in mature nervous system

23. Involution of tissue • Endometrial breakdown prior to menstruation • Regression of lactating breast tissue after weaning

24. Cell damage • Internal cell damage • Inappropriate 3o protein structure • Cell Infection • Viral • Stress • Starvation • DNA damage • Ionizing radiation, ROS

25. Necrosis Function Apoptosis Morphological changes Biochemistry

26. Biochemistry of apoptosis • Intense area of research • Complicated integrated mechanisms • Much more to be revealed! • Common core process • Underpins morphological changes • Four stage process

27. Stage 1 - The Death Signal

28. Stage 2 - Integration and Transduction

29. Stage 3 - Execution

30. Stage 4 - Cell Removal

31. Stage 1- The Death Signal • Absence or withdrawal of positive survival factors • Presence of negative pro-apoptotic factors

32. Survival or positive signals • Cell survival relies receiving positive stimuli • Neuronal growth factor • Interleukin 2 for lymphocytes • Hormones • Withdrawal is a death signal • Default pathway for many cells

33. Death or negative signals • Signals to induce apoptosis • Damaged DNA • UV light and X rays • Chemotherapeutic drugs • Oxidants/free radicals • Oxidative stress • Death activators or receptor ligands

34. What are Death Activators? • Molecules that bind to specific receptors on cell surface • Tumour necrosis factor alpha • Lymphotoxin TNF beta • Fas ligand (CD95) • Binding of death activator to its specific receptor is a pro-apoptotic signal

35. Stage 2 - Integration and Transduction • Signals linked to execution phase through an integration stage • Uses positive and negative regulatory molecules • Inhibit, stimulate or forestall apoptosis

36. To die or not to die? Integrated balance between positive survival factors and negative death signals decides fate of cell

37. Common intracellular machinery for apoptosis The three main players • Family of enzymes - Caspases • Protein family - Bcl-2 proteins • Regulating gene - p53 gene

38. Caspases • Family of protease enzymes • 14 isoforms identified • Have Cysteine at active site • Synthesised as inactive precursors - procaspase • Not all involved in apoptosis

39. cleavage sites prodomain large subunits small subunits Procaspase structure

40. Procaspase are activated through cleavage

42. Re-association of large and small subunits

43. Initiator caspases Effector caspases Activated caspase has proteolytic activity

44. Activate other caspases • Amplify caspase activity Initiator caspases Apoptosis execution Effector caspases

46. Bcl-2 proteins • Large family of proteins • Named from B cell lymphoma • Some are pro-apoptotic some are anti-apoptotic

48. Bcl-2 proteins and apoptosis • Main mechanism is regulation of mitochondrial permeability • Cell survival stimuli induce the expression of anti-apoptotic Bcl proteins • Death signals induce pro-apoptotic Bcl proteins

49. p53 gene and p53 protein • p53 is tumour suppressor gene • Active gene product p53 produced in response to DNA and cell damage • Prevents cell completing cell cycle • If damage is minor - allows repair • If major - induces apoptosis • Complex mechanisms

50. Apoptotic transduction pathways • Mitochondrial or intrinsic pathway • Death activator or extrinsic pathway