WEEK 2.2

• Replicative senescence


  Telomere dysfunction – telomere erosion, Hayflick limit, p53-p21
• Stress-induced premature senescence


  DNA damage, Reactive oxidation species (ROS), p16
• Oncogene-induced senescence


  Oncogene activation, p53
• Replicative stress-induced senescence


  Haemopoietic stem cells
• Developmental senescence


  Embryogenesis, p21 (independent of p53)
• Cell-cell fusion


  Placental syncitiotrophoblast
• Cell senescence


  Antagonistic pleiotropy
• Chromosome capping function of telomeres

  • Protects against homologous recombination and non-homologous end joining (chromosomal instability)
  • Prevents ends of chromosomes from being "seen" as double stranded breaks
• Telomere structure


  • Terminal protein complex (shelterin complex)
  • 3'-single strand DNA overhang - T-loop and D-loop
• What happens if the ends of chromosomes become "visible" to the DNA checking enzymes?

  
  

  can trigger cellular responses aimed at preventing the proliferation of damaged cells and maintaining genomic stability.
• End replication problem


  • DNA polymerases cannot lengthen terminal section (approx 100 to 200bp) of lagging strand (primer site)
  • Telomere shortening
  • Replicative senescence – p21
  • CIP1/CDKI1
• Telomere length and ageing
• Telomerase


  • Special DNA replication enzyme which can lengthen telomeric DNA (discovered in 1984 by Elizabeth Blackburn)
  • Reverse transcriptase (TERT) synthesises (maintains) telomere length in germ cells, some stem cells (e.g. embryonic stem cells), some white blood cells
  • Lengthens lagging strand (5'-3'), DNA polymerase-alpha completes complementary strand
  • Induced telomerase can immortalise cells (Zou Y et al. Exp. Cell Research 2002)
  • Activated in cancers (about 90%) e.g. breast, lung, colon, prostate and pancreas
  • Telomerase expression may also resist apoptosis
  • Alternative Lengthening of Telomeres (ALT) pathway – recombination mediated lengthening of telomeres
• Telomere length, telomerase and ageing
• Cell death


  Apoptosis and oncosis/necrosis
• Apoptosis


  • Programmed cell death. Most somatic cells have a limited life span and are programmed to die (~300 million cells/min) after they have reached their maturity or when their functional properties are no longer required.
  • Programmed cell death is intrinsic to the cell
• Ways a cell can die

  • Killed by injurious agents (necrosis)
  • Induced to commit suicide (apoptosis)
• Differences between Necrosis and Apoptosis

  • Necrosis: Large swathes of cells dies, cells and nuclei swollen, plasma membranes disrupted
  • Apoptosis: Individual cells die, nuclei and whole cells shrunken, rounded and darkly stained, chromatin condensed
• Causes of Necrosis


  Usually the result of injury
• Causes of Apoptosis


  Can be physiogical, developmental or the result of injury
• Inflammatory effects


  • Necrosis: Wide range of cells attracted
  • Apoptosis: Only phagocytic macrophages attracted
• Cell necrosis


  • Cells damaged by injury, e.g. mechanical damage, exposure to toxic chemicals
  • Characteristic series of changes: ATP depletion, disruption of ion/water control, cells and organelles swell, cell contents leak out leading to inflammation
• Cell necrosis in liver

  • Irreversible damage - paracetamol
  • Cells pale, vacuolated. Dead cells pink, small basophilic nucleus "pyknosis"
• Cell necrosis in kidney

  • Nuclei fragment to form small basophilic structures "karyorrhesis" followed by karyolysis (loss)
• Coagulative necrosis


  Gelatinous (gel-like) substance, protein denaturation, typically seen in hypoxic environments like infarction, primarily in tissues like kidney, heart and adrenal glands
• Liquefactive necrosis


  Digestion of dead cells to form a viscous liquid mass, typical of bacterial or fungal infections, frequently creamy yellow due to dead leukocytes (pus), frequently occurs in the brain
• Gangrenous necrosis


  Mummified tissue (type of coagulative necrosis), characteristic of ischaemia of lower limb and GI tract, subsequent infection produces liquefactive necrosis (wet gangrene)
• Caseous necrosis


  Combination of coagulative and liquefactive necrosis, white and friable tissue appearance ("clumped cheese"), caused by mycobacteria (e.g. tuberculosis), fungi, dead cells not completely digested, leaving granular particles (e.g. granuloma)
• Fat necrosis


  Action of activated lipases on fatty tissues (e.g. pancreas), calcium, magnesium or sodium may bind to lesions to produce a chalky-white substance, appearance - gritty white flecks
• Fibrinoid necrosis


  Immune-mediated vascular damage, deposition of fibrin and immune complexes within arterial walls
• Apoptosis


  • Cells that are induced to commit suicide: shrink, develop bubble-like blebs, have chromatin degraded, break into small, membrane-wrapped, fragments
  • Phosphatidylserine exposed on surface, bound by receptors on phagocytic cells like macrophages and dendritic cells which then engulf the cell fragments
  • Phagocytic cells secrete cytokines that inhibit inflammation (e.g., IL-10 and TGF-β)
• Extrinsic pathway of apoptosis

  Instructed death (Death domain receptors) e.g. during viral infection
• Intrinsic pathways of apoptosis

  Default death (absence of growth factors) (Activation of bcl 2 Associated Death (BAD) promoter due to non-functioning of phosphate kinase B)
• Stress activated apoptosis


  • Direct activation of mitochondria
  • p53 which activates a bcl2 protein called BAX
  • Through protein kinase p38 (p38 MAPK)
• Cells activate apoptosis in response to 3 types of event
• Apoptosis and clearance of dead cells

  • Lymph node high rate of turn over. T is apoptic cell in macrophage. Cells pull away from adjacent cells.
  • Apoptosis in colonic columnar epithelial cells. Fragments of nuclear material in eosinic cytoplasm
• Morphological changes in apoptosis

  • Nuclear condensation, breakdown of chromatin, nuclei take on "horse-shoe" like appearance
  • Cells continue to shrink packaging themselves into a form that allows for easy clearance by macrophages
  • Membrane changes can often be observed morphologically through the appearance of membrane blebs or blisters which often appear towards the end of the apoptotic process
• Death receptor pathway (TNF)

  1. TNF (Tumour necrosis factor) TNFR (TNF receptor)

  2. TRADD (TNFR-associated death domain)

  3. FADD (Fas-associated death domain DD (death domain))

  4. Caspase 8

  5. Caspase cascade

  6. Apoptosis
• Mitochondrial apoptotic pathway


  1. Mitochondria: aspiration

  2. Pore: cytochrome c release

  3. Pore forming: Bax, Bak

  4. Pore facilitator: Bad, Bid

  5. Anti-pore: Bcl-2

  6. Apoptosome Cytochrome c Apaf-1

  7. Pro-caspase 9

  8. Caspase 9

  9. Apoptosis
• Death receptor and mitochondrial apoptotic pathways

  • Extracellular
  • Intracellular
  • endonucleases
  • apoptosis