Programmed Cell Death A genetically controlled cell suicide pathway

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Programmed Cell Death A genetically controlled
cell suicide pathway
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The History of the Cell Death Study

• First observed in 1842 by a German scientist Carl
  Vogt in Jent und Gassman, Solothurn 1842 130
• He wrote The role of the core of the notochord
  in the formation of the vertebrae is quite simply
  that its cells are resolved , beginning when the
  proliferation of the surrounding cartilage exerts
  pressure on the notochore. The light
  vesicular nuclei of the embryonic cells have
  disappeared at least I could not detect any
  trace of them.
• 1951, Glucksmann, A (in Cambridge Philosophical
  Society of bilogical review 26, 59) reviewed and
  rediscovered developmental cell deaths in
  embryological tissues
• 1964, Lockshin, RA and Williams, CM first used
  the term of programmed cell death to describe
  the breakdown of the intersegmental muscles of
  silkworms
• 1972, three British scientists Kerr, Wyllie and
  Currie proposed the term of apoptosis for
  morphology of naturally occurring or
  physiological cell deaths. They recognized the
  importance of apoptosis and suggested that it is
  a basic biological phenomenon with wide-ranging
  implications in tissue kinetics.

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The Morphology of Apoptosis

• Cytoplasm shrinks

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video
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Difference Between Apoptosis and Necrosis

• Necrosis (pathological cell death) dying cells
  swell and lyse toxic contents leak out and
  result in inflammatory response.
• Apoptosis (physiological or programmed cell
  death) dying cells shrink, are engulfed by
  other cells, leave no trace, and dont result in
  harmful outcomes

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Functions of apoptosis

• Sculpt body structures, e.g. hand digit

Produced in excess, e.g. extra neurons are
removed by apoptosis during neurogenesis.
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The Nematode C. elegans As a Model Organism in
the Study of PCD

• A great genetic system
• Completely defined cell lineage
• Study of cell death at a single cell resolution
  in living animals

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The C. elegans Cell Lineage
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Cell Death Can Be Studied at a Single Cell
Resolution
P11
X
X
P11aap
Adapted from Sulston and Horvitz, Dev. Bology 56,
110-150, 1977
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The First Few Cell Death Mutants Identified in C.
elegans

• In 1976, J. Sulston first described programmed
  cell death in nematodes and reported the first
  cell death mutant (nuc-1), in which DNA in the
  death cells fail to be degraded.

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In 1980, E. Hedgecock isolated two cell death
mutants (ced-1 and ced-2) which are pivotal for
identification of the other cell death genes.
What went wrong with the ced-1 mutant?
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Phenotypic analysis of ced-1 and ced-2 mutants

1. More cell deaths?
2. Dying cells cannot be removed or engulfed

How to distinguish these two possibilities?
Follow the cell lineage in the mutant animals
What is next?
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Suppressor screens ced-3 and ced-4
What are the functions of ced-3 and ced-4?
H. Ellis and R.H. Horvitz
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What are the functions of ced-3 and ced-4?

• ced-3 and ced-4 promote cell corpse engulfment?
• Then the mutations must be increase-of-function
• 2) Inhibitors of cell corpse engulfment?
• Then the mutations should be loss-of-function
• 3) ced-3 and ced-4 could promote cell deaths
• Then the mutations should be loss-of-function

How to distinguish 2) and 3)?
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• Lineage analysis suggest
• Many cells that normally die now survive
• ced-3 and ced-4 are involved in cell killing

• How do ced-3 and ced-4 kill the cells?
• Cells die by murder?
• Cells die by suicide?
• cells die by aging?
• Cells die because of injuries?
• Cells die by sickness?

How to distinguish these possibilities?
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Mosaic Analysis

• Using specific techniques to create genetically
  mosaic worms, in which some cells carry a
  wild-type gene and others are homozygous mutant.
  Analysis of such animals can reveal where in the
  animal a gene is needed for its functions
• How to generate C. elegans genetic mosaics?
• Mosaic animals have been generated by the
  spontaneous mitotic loss of an extrachromosomal
  genetic element that carries the wild-type allele
  of a gene in an otherwise homozygous mutant
  background.
• free chromosome fragments
• extrachromosomal arrays

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Cells Die by Suicide Rather Than Murder

• Yuan and Horvitz demonstrated by mosaic analysis
  that ced-3 and ced-4 function in the dying cells
  to kill.
• ced-3 encodes a protein with homology with IL-1b
  converting enzyme (ICE), a cysteine protease.
• ced-4 encodes a protein similar to apoptotic
  protease-activating factor (Apaf-1).

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cps-6
ceh-30
CEM cells
psr-1
wah-1
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Caspases Are Cell Death Executors

• Yuans group using cell culture experiments
  showed ICE and CED-3 can both kill in mammalian
  cells
• CED-3/ICE define a family of cysteine proteases,
  named caspases (aspartate-specific proteases),
  which so far has 16 family members
• A caspase is first synthesized as an inactive
  protease precursor and later activated by
  specific proteolysis at specific aspartate
  residues.

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Caspase Family
caspase
Adapted from Thornberryand Lazebnik, Science 281,
1312-1216, 1998
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Stucture of Caspase-3 (CPP32)
Adapted from Thornberryand Lazebnik, Science 281,
1312-1216, 1998
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Activation of Caspases
1) By self-activation
2) By another cysteine protease or caspase
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Discovery of Bcl-2

• In 1986, three groups independently cloned bcl-2
  oncogene. bcl-2 oncogene causes follicular
  lymphoma and is a result of chromosome
  translocation t(1418 that has coupled the
  immunoglobulin heavy chain locus to a chromosome
  18 gene denoted bcl-2
• In 1988, Vaux, Cory, and Adams discovered bcl-2
  oncogene causes cancer by inhibiting lymphocyte
  cell deaths, providing the first evidence that
  cancer can result from inhibition of cell death
• 1990, Stanley Korsmyers group showed Bcl-2
  localized to mitochondria

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C. elegans ced-9 Gene Is a Functional Homologue
of Bcl-2

• A gain-of-function mutation in ced-9 protects
  against all cell deaths in nematodes, while
  loss-of-function mutations cause massive ectopic
  cell deaths
• ced-9 encodes a protein similar to Bcl-2
• Bcl-2 inhibits cell death in nematodes and can
  partially substitute for ced-9
• CED-9 is localized at mitochondria

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Bcl-2/ced-9 Define a Family of Cell Death
Regulators

• Korsmyers group purified a protein, Bax, that
  associates with and modulates the activity of
  Bcl-2. Bax by itself can also cause apoptosis in
  a Bcl-2-independent and caspase-independent
  pathways.
• Thompsons group identified a gene, named bcl-x,
  which can be alternatively spliced to generate
  two proteins that have opposite functions in
  apoptosis. The long form (Bcl-xL) inhibits
  apoptosis and the short form (Bcl-xs) cause cell
  death.
• Korsmyers group identified another
  Bcl-2-interacting and death inducing-protein,
  Bid, which only has one Bcl-2 homology domain
  (BH3).
• Subsequently, more Bid-like death-inducing
  proteins were identified, all of which has only
  one BH3 domain. This protein family was called
  BH3-only Bcl-2 subfamily.

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Anti-apoptotic Bcl-2 family members
Adapted from Adams and Cory, Science 281,
1322-1226, 1998
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Pro-apoptotic Bcl-2 family protein
Adapted from Adams and Cory, Science 281,
1322-1226, 1998
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Key Features of Bcl-2 Family Proteins

• They localize either inducibly or constitutively
  to outer membranes of mitochondria and nuclei,
  and membranes of ER
• They are capable of forming heterodimers with
  other family members, especially those with an
  amphipathic helical BH3 domain
• They are capable of forming ion-conducting
  channels on synthetic membranes

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Structures of Bcl-xL and Bid
BH3
BH3
Adapted from Chou et al., Cell, 1322-1226, 1999
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Structure of the Bcl-xl/Bak Complex
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Puzzles About Bcl-2

• Do they need to localize to mitochondria to
  function?
• Do they need to dimerize with other family
  members or apoptotic regulators to function?
• Do they function as channels to regulate cell
  death?

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Bcl-2 Family Proteins and Cancer

• Overexpression of Bcl-2 caused follicular
  lymphomas
• Mutations in Bax cause human gastrointestinal
  cancer and some leukemias.
• In many tumor cell lines, the expression levels
  of pro- and anti-apoptotic Bcl-2 family members
  are altered.