Programmed Cell Death

• Programmed cell death is also known as APOPTOSIS
• An orderly disposal of cells

  • Sustained too many injury
  • Infected with a virus
  • Old cells
  • Needs to be removed for body parts

  Programmed cell death (PCD)
• Morphology of Apoptosis:

  1. Cell shrinks (pyknosis)
  2. Chromosomes condense and fragment
  3. Nuclear membrane breaks down
  4. Apoptotic body formation
  5. Engulfment of the cell corpse
• Apoptosis: dying cells shrink, are engulfed by other cells, leaving no trace and dont result in harmful outcomes
• Necrosis

  • Pathological cell death
  • dying cells swell and lyse; toxic contents leak out and result in inflammatory response. Cell death after an injury
• Caspases

  • Cysteine aspartyl-specific protease (degrading enzyme) involved in apoptosis
  • Effector proteins, cleave key proteins including cytosolic protein and nuclear lamin leading to cell death
• Two pathway of activation in apoptosis:

  1. Intrinsic
  2. Extrinsic
• Intrinsic pathway

  • Begins at the mitochondria
  • Mitochondria normally display Bcl-2 (inhibits apoptosis) on their surface
• Damage to cell alerts p53 which causes protein Bad (pro-apoptotic protein) to migrate to the mitochondrial membrane, inhibiting Bcl-2 and allowing the membrane channel (MAC) to open
• Opening of MAC causes cytochrome C (electron carriers) to leak out
• Extrinsic pathway

  • These cells are chosen for death by other cells, usually cytotoxic T cells (immune cells)
  • All cells have receptors for Fas and TNF (pro- apoptotic ligands)
• Extrinsic Pathway

  • When TNF or FasL binds the cell at these receptors, the cell activates caspase 8, which initiates a cascade of caspases
  • This lead to phagocytosis of the cell and its contents
• TUNEL (Terminal deoxynucleotidyl transferase dUTP nick end labeling) assay

  • Been designed to detect apoptotic cells that undergo extensive DNA degradation during the late stages of apoptosis.
  • Detecting DNA fragmentation by labelling the terminal end of nucleic acids
  • Relies on the presence of nicks in the DNA which can be identified by terminal deoxynucleotidyl transferase (TdT) thatwill catalyze the addition of dUTPs that are secondary labeled with a marker
• Alexa-fluor: bright green fluorescent dye label at the terminal end of the nucleic acid
• Cell viability

  1. Neutral red assay
  2. MTT
  3. WST
  4. Resazurin or alamar blue assay
• Membrane integrity

  1. Colony formation assay tryphan blue
  2. Calcein acetoxymethyl
  3. Lactate dehydrogenase
  4. Hemolytic assays
• Apoptosis

  1. Propidium iodide
  2. LDH assay
  3. DNA laddering
  4. Acridine orange/EtBr
  5. TUNEL assay
  6. Caspase-3 activity
• Flow cytometry: technique used to detect and measure the physical and chemical characteristics of a population of cells or particles