MOLECULAR BIOLOGY OF APOPTOSIS

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MOLECULAR BIOLOGY OF APOPTOSIS

• Definition
• Apo apart
• Ptosis fallen
• Shedding of leaves from tress
• During embriogenesis ------? occurs as PCD
• Post-embrional life-------? as apoptosis

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apoptosis

• Apoptosis is used as a synonymous for PCD but PCD
  is physiological death, occurs only during
  embriogenesis.
• It is a functional death and it is a good
  mechanism to eliminate wasted, useless, unwanted,
  or crippled cells!

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• Thymus
• Prostate
• Endometrium
• Adrenal cortex
• Lymphoid cells
• Neurons are all subject to apoptosis

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Why have we developed such a self-destructive
system?

• A. PCD allows a constant selection for the
  fittest cell in a colony
• Every cell carries the molecular machinery to do
  PCD!
• Cells that are sensitive to extracellular signals
  will survive, cell that cannot compete with their
  more vital sisters will undergo apoptosis.

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• Ischemia
• XRT
• Toxins
• Chemicals

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• PCD machinery is silent until signals arrive to
  start PCD
• Signals
• damage to DNA
• Activation of membrane receptors. Ligands are
  peptides, cytokines, ATP, ROS etc
• Deprivation of specific signals of GFs, hormones
  or survival signals for apoptosis

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PHYSIOLOGICAL VS PATHOLOGICAL CELL DEATH

• Necrosis
• Apoptosis
• MOLECULAR PATHWAY OF APOPTOSIS
• The initiating phase by signals
• External that trigger receptors on the plasma
  membrane.
• Intracellular alterations

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• Fas receptor?
• Receptors for growth factors, cytokines and
  hormones
• Membrane alterations cause apoptosis.
• Q. What kind of membrane alterations ??
• A. Phospholipid redistributions, changes in
  membrane charge, carbohydrate and surface
  markers.

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Decision phase

• Final decision " to live or die"
• The final decision depends on expression of
  several proto-oncogenes, called as Bcll-2 gene
  family (B-Cell Leukemia Lymphoma)
• Bcl-2 gene product protects B lymphocytes, T
  cells against apoptosis induced by
• Drugs
• XRT
• Heat shock
• Oxidative stress
• What are nuclear changes?

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MEASUREMENT OF APOPTOSIS

• TECHNIQUES BASED ON MORPHOLOGICAL CHANGES
• Light microscopy
• Electron microscopy
• TECHNIQUES BASED ON DNA FRAGMENTATION
• Measurement of endonuclease activity

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MEASUREMENT OF APOPTOSIS

• TECHNIQUES BASED ON MEMBRANE ALTERATIONS
• Measurement of dye exclusion
• TECHNIQUES BASED ON CYTOPLASMIC CHANGES
• Changes in intracellular enzyme activity
• Measurement of calcium influx

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APOPTOSIS AND ILLNESS

• APOPTOSIS AND OXIDATIVE STRESS
• Background and introduction
• Promotion of apoptosis by oxidative stress
• Modulation of apoptosis by oxidative stress

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Background and Intro

• Ox-stress can cause apoptotic or necrotic cell
  death.
• This section well talk about ways in which
  Ox-Stress can intersect apoptotic pathways.
• ROS may accumulate due to toxic insults or normal
  metabolic processes

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• Cell shrinkage, chromatin condensation,
  internucleosomal DNA fragmentation and formation
  of apoptotic bodies are all characteristic
  features of apoptosis.
• Several protease families have been implicated in
  apoptosis the most prominent being CASPASES
• Caspase is aspartic acid-specific cysteine
  protease which is found in zymogens almost in all
  cells!

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• 3 models of caspase action is proposed.
• TNF receptor mediated
• No receptor involvement
• cytotoxic cell activation of caspase
• Regardless of the mechanism, upon activation
  caspases cleave many proteins and finally DNA.

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• A role for Ox-Stress in apoptosis has been
  clarified by many scientists.
• Promotion of apoptosis by OX-Stress
• Proposed mechanisms
• Fig
• Modulation of apoptosis by oxidative stress

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• Apoptotic cell death can be switched to necrosis
  during oxidative stress by 2 mechanisms
• Inactivation of caspases due to oxidation of
  their active site thiol group by oxidants or
  S-nitrosylation.
• Decrease in ATP due to failure of mitochondrial
  energy production by oxidants (Table 30.1).

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• NO can also have dual effects on apoptosis
• NO is reactive, unstable free radical gas that
  can easily cross cell membranes.
• L-Arg------? NO
• Low NO Neurotransmitter, regulator in
  vasodilation and platelet aggregation.
• High NO Cytotoxicity

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• NO may also mediate apoptosis
• Macrophages
• Beta cell line
• Thymocytes

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• How???
• Formation of iron-nitrosyl complexes with
  FeS-containing enzymes This leads to impairment
  of mitochondrial function
• -?ATP depletion.
• NO may directly damage DNA-?mutagenesis
• Generation of OONO-? Apoptosis
• NO may inactivate several antioxidant enzymes
  (CAT, GPx, SOD etc)

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• NO exposure or iNOS activation may inhibit
  apoptosis in
• Lymphocytes
• Endothelial cells
• Neurons
• Hepatocytes
• Kidney cels

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• How??
• Direct inhibition of caspase (S-nitrosylation of
  the active site Cys)
• R-S-NO is important component of signal
  transduction cascades.
• S-nitrosylation can regulate many proteins
• Enzymes
• Ion channels
• G-proteins
• Transcription factors
• NO may act as a modular switch to control protein
  function via SH groups.

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• For example, S-nitrosylation was shown to occur
  in
• Papain protease
• Calpain
• NF-KB
• AP-1
• These are all implicated in the regulation of
  apoptosis.
• NO inhibition of caspase is reversible.
• Pro-caspase-3 was recently shown to be
  S-nitrosylated on its catalytic site Cys (Cys
  163).
• Nitrosylation/denitrosylation-? may serve asa
  regulatory mechanism just like.?