CBIO320CANB320

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CBIO320/CANB320 Cancer Development Cell
Proliferation Survival in Development Laura
Lee Jan. 15, 2007
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Cell Proliferation Survival in Development
C. elegans Screen for regulators of apoptosis
D. melanogaster Screen for negative regulators of
cell proliferation
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• The of cells in a multicellular organism is
  tightly regulated
• By controlling the rate of cell division
• By controlling the rate of cell death

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Programmed Cell Death (PCD) Apoptosis
Apoptosis (Greek) the falling of leaves from a
tree
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Importance of Apoptosis

• Maintaining homeostasis
• Cell death is balanced with mitosis to regulate
• total cell number
• Embryonic development
• Errors in apoptosis can lead to birth defects
• Improper regulation contributes to human disease

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Each day in a human 100,000,000,000 cells are
born
Each day in a human 100,000,000,000 cells
commit suicide
(to maintain homeostasis, cell death balances
cell division)
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Cell division Cell death (in adult tissues)
Example adult rat liver Remove liver --gt Liver
cell proliferation to make up loss Treat rat
with phenobarbital --gt Stimulates liver cell
division -- gt Liver enlargement Withdraw
phenobarbital --gt Apoptosis in liver stimulated
--gt Liver returns to normal size (usually by 1
week)
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Apoptosis and Development
Skin cell
Embryonic cell
Blood Cell
Differentiated Cell Types
Apoptotic Cell
Undifferentiated Cell
During development, the terminal fate of certain
cells is to become apoptotic
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Apoptosis and Development Metamorphosis
Tadpole ------------gt Frog

• Cells in the tadpoles tail are induced to
  undergo apoptosis
• Stimulated by an increase in thyroid hormone in
  the blood

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Apoptosis and Development Removal of temporary
structures
Apoptosis sculpts the digits in the developing
mouse paw
Interdigital programmed cell death (marked by
bright green dots) eliminates tissue between
developing digits of the embryonic mouse paw
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Apoptosis and Development Neurogenesis
In the developing vertebrate nervous system, most
nerve cells normally die soon after they are
formed
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Apoptosis and Development Neurogenesis
Apoptosis adjusts the number of nerve cells to
match the number of target cells that require
innervation
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Apoptosis in the Immune System
Lymphocytes infected with Epstein-Barr virus do
not undergo apoptosis because a viral protein
induces bcl-2 activity (anti-apoptotic)
Lymphocytes that respond to self are removed by
apoptosis -gtDefects in apoptosis can lead to
auto-immune diseases
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Links between apoptosis and cancer

• Some cancer-causing viruses use tricks to prevent
  the programmed death of the cells they have
  transformed into malignant or immortalized cells
• Some cancer cells produce high levels of
  anti-apoptosis proteins (e.g. IAP or Bcl family
  proteins)
• Certain agents used in cancer chemotherapy
  achieve their effects by inducing apoptosis in
  cancer cells

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Apoptosis
Necrosis
Tightly regulated and controlled
Not regulated or controlled
Active participation of cellular components
Passive process
Follows a specific ordered pattern of events
Cell swells and disintegrates in a disordered
manner
No leakage of cellular contents
Rupture of cell membrane results in the leakage
of cellular contents into extracellular space
No inflammation
Associated with inflammation
Induced by cell signaling or slight damage to the
cell
Induced by massive cellular injury
Kerr, Wyllie, and Currie, Brit. J. Cancer 26239,
1972
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Cellular changes associated with
apoptosis Overview
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Cellular changes associated with apoptosis Early
events
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Cellular changes associated with apoptosis DNA
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Cellular changes associated with apoptosis DNA
Nucleus becomes convoluted and buds into
several fragments
Apoptotic cell
Normal cell
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Cellular changes associated with apoptosis
Terminal events
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The intracellular machinery responsible for
apoptosis is conserved in animal cells

• Key players Caspase family of proteases
• Cysteine at active site
• Cleave target proteins at specific ASpartic acid
  residues
• --gt CASpases

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The caspase cascade of apoptosis
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Induction of apoptosis by either extracellular or
intracellular stimuli
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Regulators of procaspases and caspases Two major
families

• Bcl-2 family
• Members inhibit or promote caspase activation
• Block or stimulate cytochrome c release from
  mitochondria (directly or indirectly)
• Examples
• -Bcl-2 blocks cytochrome c release
• -Bad binds inactivates death-inhibiting
  members of Bcl-2 family

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Regulators of procaspases and caspases Two major
families

• IAP family (Inhibitor of APoptosis)
• Inhibit apoptosis by preventing procaspase
  activation and/or directly binding/inhibiting
  caspases
• Originally discovered as proteins produced by
  insect viruses-prevent infected cell from
  killing itself before virus has time to
  replicate

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Press Release The 2002 Nobel Prize in Physiology
or Medicine 7 October 2002 The Nobel Assembly at
Karolinska Institute has today decided to award
The Nobel Prize in Physiology or Medicine for
2002 jointly to Sydney Brenner, H. Robert
Horvitz, and John E. Sulston for their
discoveries concerning "genetic regulation of
organ development and programmed cell death"
1942
1927
1947
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Caenorhabditis elegans Why study worms?

• Reproduce very rapidly (3 week life span)
• Easy to induce mutations with ethyl
  methylsulfonate (EMS)
• Capable of reproducing as hermaphrodites
• Simple organism with only 1090 somatic cells
• Development is invariant and has been mapped
  such that the fates of all cells are known

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Caenorhabditis elegans Apoptosis

• 131 of 1090 somatic cells normally undergo PCD
  (105 of the 131 PCDs are in the nervous
  system)-cell fate
• Death of these cells is not required for
  viability
• Special optics can be used to observe PCD in
  live worms

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Premise PCD is an active biological process that
requires the function of specific genes
Goal To define how PCD occurs by using genetics
EMS treat worms
Examine for excess live cells
Characterize mutant gene
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Genetic analysis of PCD in C. elegans
Cell death (ced) defects ced-3 ced-4
ced-9 Egg-laying (egl) defects egl-1
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Great system for studying PCD 131 PCD events
during somatic development
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First PCD mutants identified Defects in
engulfment of dying cells
Phenotype of ced-1 or ced-2 mutants Accumulation
of apoptotic corpses
Wanted these genes
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Problem difficult to find PCD mutants that lack
dying cells because few corpses at any moment in
time
Solution Perform screen for PCD mutants in
ced-1 mutant background
ced-1 mutant, normal PCD corpses
accumulate ced-1 mutant, PCD mutant lack of
corpses
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Screen for PCD mutants in ced-1 background
Identified the killer gene ced-3

• In ced-3 mutants, 131 cells that normally undergo
  apoptosis instead survive
• Is ced-3 required in cells that die or elsewhere?
• Genetic mosaic analysis performed
• ced-3 acts within dying cells themselves
• Confirmed that PCD is a process of cellular
  suicide

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What is the function of CED-3 protein?

• Insight came 2 years after cloning
• Similar in sequence to the protease
  interleukin-1-beta converting enzyme
  (ICE)-purified biochemically by drug companies
  working on inflammatory disease
• Founding members of a family of cysteine
  proteases now known as caspases
• Beginning of understanding of mechanism of
  process of PCD involves proteolysis by caspases

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Conservation of function

• Expression of CED-3 in mammalian cells induces
  them to undergo PCD
• Strongly suggested that mammalian cells also
  contain similar machinery (CED-3/ICE-like
  cysteine proteases) for cellular suicide

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Similar screen for PCD mutants
Identified the anti-killer gene ced-9

• Gain-of-function (dominant) ced-9 allele
  identified in screen no PCD of cells that
  should die
• Identified loss-of-function ced-9 alleles later
  cells that should live undergo PCD

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What is the function of CED-9 protein?

• Encodes protein similar to product of human
  proto- oncogene Bcl-2 (B cell lymphoma)
• Bcl-2 was already of great interest in the field
  of oncology misexpression causes follicular
  lymphoma
• Prior studies of Bcl-2 indicated that it could
  protect cells in the immune system from
  undergoing apoptosis
• Later family of CED-9/Bcl-2-like proteins
  identified that regulate apoptosis in mammalian
  cells

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Conservation of function
Human Bcl-2 rescues C. elegans ced-9
mutants Suggested that a conserved function of
CED-9/Bcl-2 is to antagonize the death process
this pathway was no longer simply an abstract
formalism derived from complicated genetic
studies of a microscopic soil-dwelling roundworm,
but rather a framework for a process fundamental
to human biology and human disease. Bob Horvitz,
Nobel lecture
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Ordering genes in the PCD pathway

• Observations
• 1. ced-9 is required to prevent cell death
• 2. ced-3 is required for cell death to occur

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Ordering genes in the PCD pathway
Genotype PCD Wild-type Normal ced-3
(lf) Reduced ced-9 (lf) Excessive ced-3
(lf) ced-9 (lf) Reduced
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Identification of a PCD gene from a screen for
egg-laying (egl) defective mutants
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egl-1 (gf)
Rare dominant gain-of-function mutation in
egl-1 causes an egg-laying defect because HSN
vulva neurons die inappropriately

• Suggested that activation of egl-1 promotes cell
  death
• Generated loss-of-function allele of egl-1
• --gt No cell death (like ced-3 and ced-4)
• egl-1 encodes homolog of pro-apoptotic regulator
  Bad

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Mutation of ced-3 suppresses the effects of an
egl-1 (gain-of-function) mutation
Genotype HSN? Egg-laying defects? Wild-type
Yes No egl-1 (gf) No Yes egl-1 (gf)
ced-3 (lf) Yes No
Used as basis for new screen for PCD genes
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New screen for PCD genes suppressors of
egg-laying defect of egl-1 (gf)

• Identified ced-4
• Human counterpart identified 5 yrs later
• -Apaf-1 (Apoptotic protease activating factor)
• -Identified in biochemical screen for cell
  death regulators (X. Wangs lab)
• -Pro-apoptotic function-aggregates and
  activates procaspases

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Without the insights that came from detailed
analysis of the development of the transparent,
genetically tractable nematode worm, it would
have been very much harder to discover these
genes and understand the cell-death process in
vertebrates. Molecular Biology of the Cell
(Alberts) textbook
Worms Vertebrates EGL-1 Bad CED-9 Bcl-2 CED
-3 Caspase homolog CED-4 Apaf-1
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Core molecular genetic pathway for PCD in C.
elegans
ced-9
ced-3
PCD
ced-4
egl-1
Bcl-2
Caspase
Apaf-1
Bad
(Genetic arrows/bars)
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A model for the activation of programmed cell
death in C. elegans
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Apoptotic pathway
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The next wave of screens for regulators of
apoptosis in C. elegans
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Identifying negative regulators of growth in
Drosophila
Iswar Hariharans lab UC Berkeley
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Screen logic compare the relative proliferation
of wild-type and mutant cells
Mutant tissue underrepresented
Mutant tissue overrepresented
neutral
common
rare
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Screening for negative regulators of cell growth
and proliferation
Mutant
Control
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Newly hatched larva
eye imaginal disc, 20 cells
10 rounds of cell division
Adult eye (20,000 cells)
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Newly hatched larva 20 cells give rise to eye
disc
Third instar larva
Adult eye
Pupal lattice
early larva
proliferation and differentiation
proliferation
apoptosis
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Clonal analysis using the Flp/FRT system
Flp Flp recombinase FRT Flp recombinase target

• Flp mediates site-specific recombination between
  FRT sites (in yeast)
• System works efficiently when engineered into
  Drosophila

Transgenic flies with FRT sites at identical
positions on homologous chromosomes (one
mutagenized chromosome)
Site-specific recombination
Can make a mutagenized chromosome arm homozygous
in clones of cells
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Advantages of making mitotic clones using Flp/FRT
system

• Can screen F1 progeny for recessive
  loss-of-function phenotypes (no need to go to
  F3 as with traditional screens)
• Only cells of interest are homozygous for
  mutagenized chromosomes-rest of organism is
  heterozygous (gets around problem of only being
  able to analyze the earliest phenotype of a
  mutation)

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The Screen
mutagenize
V
X
males
/ females
mutant/
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Mutations in the Drosophila Tsc1 homolog cause
increased organ and cell size
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Overexpression of Tsc1 and Tsc2 reduces organ size
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Tuberous Sclerosis Complex (TSC)

• TSC is a human disease characterized by tumorous
  growths in a variety of tissues, mental
  retardation, epilepsy
• One-third of cases are familial (autosomal
  dominant) and two-thirds of cases are sporadic
  mutations
• Mutations in either of two genes result in
  disease Tsc1 and Tsc2
• TSC1 and TSC2 are large novel proteins that form
  a complex that restricts cellular growth

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Increased tissue in hippo eye clones
Wild-type Control
hippo (hpo)
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Increased tissue in hippo eye clones
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• During development, to control/restrict growth
• cells stop dividing
• (b) cells die (apoptosis)
• When a mutation results in extra cells, it can be
  because
• cells keep dividing and/or
• (b) cells dont die

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Sav-Hpo-Wts form a complex
Auto-inhibitory domain
Kinase
Hippo (kinase)
DxxD
Dimerization domain Salvador binding domain
CC
W W
Salvador (scaffold)
Hpo binding domain
wts binding domain
Hpo binding domain
Kinase
Warts (kinase)
Bryant et al.(1993) Xu et al. (1995) Justice et
al. (1995)
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Sav-Hpo-Wts complex linked to human
cancer Human sav (hWW45) is mutated in cancer
cell lines N. Tapon, K.F. Harvey, D.W. Bell,
D.C. Wahrer, T.A. Schiripo, D.A. Haber, I.K.
Hariharan. salvador Promotes both cell cycle
exit and apoptosis in Drosophila and is mutated
in human cancer cell lines. Cell (2002)
110467-478. Lats1 (wts) KO mice develop soft
tissue sarcomas M.A. St John, W. Tao, X. Fei, R.
Fukumoto, M.L. Carcangiu, D.G. Brownstein, A.F.
Parlow, J. McGrath, T. Xu.Mice deficient of Lats1
develop soft-tissue sarcomas, ovarian tumours
and pituitary dysfunction. Nat Genet (1999)
21182-186.
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Sav-Hpo-Wts complex promotes apoptosis by
promoting degradation of DIAP1
Ubiquitin Ligase
26S proteasome

• IAP family (Inhibitor of APoptosis)
• Inhibit apoptosis by preventing procaspase
  activation and/or directly binding/inhibiting
  caspases
• Originally discovered as proteins produced by
  insect viruses-prevent infected cell from
  killing itself before virus has time to
  replicate

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Cell Proliferation Survival in Development
C. elegans Screen for regulators of apoptosis
D. melanogaster Screen for negative regulators of
cell proliferation
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WednesdaySandy Zinkel Cell proliferation
survival in cancer
Identification of important cancer-related
regulatory pathways through cloning genes at the
breakpoint of chromosomal translocations in human
leukemia and lymphoma Focus on BCL-2 -Identificati
on of BCL-2 at the breakpoint of t(1418) in
follicular lymphoma -Characterization of
BH3-domain (death domain) -Strategies for
targeted therapy using peptide BH3-mimetics