Cell Death Douglas Brash HRT 213A 52988 douglas'brashyale'edu

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Cell DeathDouglas BrashHRT
213A5-2988douglas.brash_at_yale.edu
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3 Modes of cell death
Necrosis Apoptosis mitotic
catastrophe Autophagy
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Necrosis
groups of cells die cell swells
passive inflammatory response
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Apoptosis
individual cells die cell shrinks
active process energy specific genes
required no inflammation
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Autophagy
cell self-digests large double-membrane
vacuoles nucleus cytoskeleton remain intact
until late
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Necrosis



Knee replacement / Warfarin
Silicone implant
Burn
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Sequence of Events in Necrosis
Membrane becomes permeable Ca enters or
is released from ER stores Cell swells due to
osmosis Ca activates calpain, ruptures
lysosomes Lysosomal enzymes are released and
activated by Ca eg cathepsin DNA is degraded
to nucleotides (random smear on gel)
Inflammatory response
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Sequence of Events in Necrosis
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Necrosis - g
Parent cell was g-irradiated in G1 and it divided
43 hrs after irradiation. Daughter cell is shown
(arrow). It changes from a normal-looking flat
cell (at 10 min) to a cell in which the membrane
ruptures (20 min) and the cell "explodes" (30
min).
UCSF Rad Oncol
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Apoptosis
TUNEL staining for apoptosis in chick limb bud
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Apoptosis of the polliwog tail
www/offwell.info www.calarts.edu
Day 121
Day 131
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Apoptotic keratinocytes after UV
H E
TUNEL
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Apoptosis DNA Ladders
Camptothecin Jurkat cells
Hypoxia rat cerebellar cells
g-ray (3, 6 Gy) mammary cells
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Apoptosis prevents mutations
p53 codon 270
p53 codon 275
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8
6
4
2
0
C3H/HeJ
C3H/gld apoptosis- defective
C3H/HeJ
C3H/gld apoptosis- defective
Hill et al, Science 285 898, 1999
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Apoptosis prevents developmental defects
Brash, Nat Med.2525, 1996
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Sequence of Events in Apoptosis
Cell rounds up, communication with neighbors
stops Cell membrane forms blebs
Phosphatidylserine flips over (inner membrane ?
outer) Proteins crosslinked (transglutaminase,
stabilizes blebs) b-tubulin, actin
polymerize Cell shrinks Chromatin
condenses ('pyknotic nuclei') DNA cleaved
stepwise (DNA ladder) 50,000 - 300,000 bp then
185 bp (internucleosomal cuts) Macrophages
recognize altered carbohydrates via lectin
receptors phagocytize apoptotic bodies No
inflammatory response Series of genes are
involved, including p53
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Apoptosis
stops moving
0 hr cell g-irradiated in late S/G2 6.5
hr divides 14.50 hr daughter normal 14.83
hr (8.3 hr after division) rounds up and
blebs 15.17 hr dies 29.00 hr membrane
ruptures
UCSF Rad Oncol
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Different cell types are wired differentlythymoc
ytes are g-sensitive ( are p53-dependent)
100
WT
p53-/-
80
Survival ()
60
40
20
0
g 0
g 5
g 10
g 20
UVB 0
1200
3000
600
5000
?????Gy)
UV (J/m2)
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Different cell types are wired differentlykerati
nocytes are g-resistant ( g is p53-independent)
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WT
p53-/-
10
Apoptosis
0
5
20
Gy
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Necrosis vs. Apoptosis
www.celldeath.de/encyclo/aporev/aporev.htm
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Mitotic Catastrophe
Occurs during mitosis, 1-3 cell divisions
after g-irradiation (necrosis and apoptosis
can occur at interphase) Giant, multinucleate
cells with uncondensed chromatin DNA damage
response ? centrosome amplification ?
abnormal chromosome segregation Lethal
chromosome aberrations (rings dicentrics)
Dominates in apoptosis-defective cells, eg tumor
cell lines Often followed by apoptosis. "Is
not a mode of cell death but a means to cell
death." Lehnert It's unclear to me what
happens in tumors in a patient.
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Mitotic Cell Death
Aberrant mitosis in a L5178 cell after 4 Gy
12 hr after irradiation blocked in G2 20
hr attempts division 22 hr divides and
fuses 45 minutes later 24 hr apoptosis starts
UCSF Rad Oncol
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Clonogenic Cell Death in C. elegans
g-irradiate larvae, esp. during mitosis ? cells
die 3 cell generations later, during
organogenesis organ abnormalities chromosome
aberrations / micronuclei unknown note
holocentric chromosomes (spindle binds entire
xsome) not apoptosis no pyknosis caspase-i
ndependent active process requires DNA damage
response ? RAS pathway Ca role unknown
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The Two Apoptosis Pathways
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Apoptosis Pathways
Extrinsic
Intrinsic
g-ray, UV
(DSB)
DFF43 (Ca-activated DNAse)
R.W. Johnstone et al. Cell 108, 153, 2002
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Death receptor pathway
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Death ligands and receptors
FASL
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DNA is a target for cell death after
g-irradiation
Nucleus is more sensitive than cytoplasm to g
microbeam Cells are killed by tritiated
thymidine incorporated into DNA Halogenated
thymidine analogs in DNA radiosensitize but
substituted deoxyuridines don't
Radiosensitivity correlates with interphase
chromosome volume with number of chromosomes
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Apoptosis Pathways
Intrinsic
Extrinsic
g-ray, UV
(DSB)
DFF43 (Ca-activated DNAse)
apoptosome
R.W. Johnstone et al. Cell 108, 153, 2002
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The apoptosome
www.sghms.ac.uk
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Inhibiting apoptosis IAP proteins
Extrinsic
Intrinsic
g-ray, UV
(DSB)
DFF43 (Ca-activated DNAse)
R.W. Johnstone et al. Cell 108, 153, 2002
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Inhibiting apoptosis survival factors
Extrinsic
Intrinsic
g-ray, UV
(DSB)
NFk-B
DFF43 (Ca-activated DNAse)
NFk-B
R.W. Johnstone et al. Cell 108, 153, 2002
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Radiation can also activate apoptosis without
ligand or DNA damage
Extrinsic
Intrinsic
g-ray, UV
SH
phosphatase
(DSB)
NFk-B
ceramide
DFF43 (Ca-activated DNAse)
NFk-B
R.W. Johnstone et al. Cell 108, 153, 2002
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Receptor kinases
ligand
domain that binds phosphotyrosine
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Kinases Phosphatases
kinase
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Caspase-independent apoptosis
Extrinsic
Intrinsic
g -ray, UV
(DSB)
NFk-B
DFF43 (Ca-activated DNAse)
AIF endo G lysozome, proteasome, ER
NFk-B
R.W. Johnstone et al. Cell 108, 153, 2002
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Caspase-independent cell death
Nuclear fragmentation without chromatin
condensation or cell shrinkage. Caspase-indepen
dent nucleases AIF (non-DFF45) endo
G Other proteases ER calpain (like
necrosis) autophagy inhibited by
DNA-PK, survival pathways (Akt,
mTOR)
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Defects in apoptosis and autophagy underlie
tumorigenesis and drug resistance
Inactivating apoptosis genes, or
overexpressing survival genes, allow tumors
to escape apoptosis that kills abnormal cells.
apoptosis Fas p53, ARF, ATM
survival NFk-B, Bcl-2, IAP's, PTEN, Ras, Akt
autophagy beclin 1 These mutations also
make tumor cells more resistant to
chemotherapy and in some cases radiation therapy.
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Senescence
Giant cells that metabolize but do not
proliferate Induced by telomere shortening
P21 overexpression g-radiation
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Senescent cells(stained for b-galactosidase)
www.molecular-cancer.com
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Senescence
Giant cells that metabolize but do not
proliferate Induced by telomere shortening,
P21 overexpression, and g-radiation
Derails tumor formation from within the cell
itself (good) Interferes with chemotherapy
(bad) Senescent cells stimulate proliferation
of adjacent premalignant and malignant cells
(bad)
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T Mak Nat Revs Ca
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Reading
Lehnert 199, Chapter 12, Chapter 14.1 Hall
Giaccia 35-41, 45, 303-305 Perez 48-49,
97-99 If you're curious www.celldeath.de/encycl
o/aporev/aporev.htm Okada H, Mak TW. Pathways of
apoptotic and non-apoptotic death in tumour
cells. Nat Rev Cancer. 2004,
4592-603. Johnstone RW, Ruefli AA, Lowe SW.
Apoptosis a link between cancer genetics and
chemotherapy. Cell. 2002 , 108153-64. Löffler
H, Lukas J, Bartek J, Krämer A. Structure meets
function--centrosomes, genome
maintenance and the DNA damage response. Exp Cell
Res. 3122633- 40, 2006. Mitotic cell
death Giaccia AJ, Kastan MB. The complexity of
p53 modulation emerging patterns from
divergent signals. Genes Dev. 1998,
122973-83. Brown JM, Wouters BG. Apoptosis,
p53, and tumor cell sensitivity to anticancer
agents. Cancer Res. 1999, 591391-9 (Stanford
radiation therapy dept)