Mechanisms of
DNA damage-Induced Cell Death
Radiobiology 2012
Radiation-Induced
Cellular Damage
• Direct DNA damage – easy to recognize that
one target must be repaired
• Membrane damage – Signaling through
ceramide/Growth Factor receptors?
• Postmitotic (secondary) DNA damage - due to
loss or alteration in genetic material
• Bystander Damage – through soluble factors?
Characterization of Cellular death
(in vitro)
• Biochemical death – trypan blue positive
– Cells unable to exclude dye (loss of membrane
potential or membrane)
• Reproductive death – colony formation
– cells unable to form a colony (may sit very happily
on the dish)
Classification of death processes
• Programmed cell death (classical apoptosis and
autophagic)
– Requires energy, activation of caspases (proteolysis)
and nucleases (chromatin fragment)
• Necrotic death
– Loss of energy leads to ion imbalance across plama
membrane (Na/K ATPase)
• Mitotic death
– Acute mitotic catastrophe, or loss of essential genetic
material
Tumor cell reproductive death
(Clonogenic death)
Fraction of Cells
Unable to Grow
Fraction that Apoptose
+
Fraction that undergo autophagy
=
+
Fraction that undergo mitotic catastrophy
+
Fraction that enter permanent senescence
+
Fraction that suffer late mitotic death
+
Fraction that get lethal bystander damage
Dose and time-dependent death
Shinomiya JCMM 2002
Radiation-Induced Apoptosis
• 1992 Lowe showed oncogenically transformed
mouse cells apoptosed in response to DNA
damage in a p53-dependent manner
• Apoptotic p53 effector genes identified: bax,
PUMA, noxa, perp (and others)
• However, p53 sensitizes to hypoxia without
transactivation
Apoptotically sensitive
Tumor Cells show
clonogenic sensitivity
Apoptotic Cascades
• Receptor Mediated
– Fas/TNFR family, FADD, Caspase 8, effector
caspases
• Mitochondrially Mediated
– Cytochrome C, APAF-1, Caspase 9, effector
caspases
• Crosstalk in pathways at C8-Bid acitvation
Bcl2 identified as oncogene because of
translocation caused activation
Large survival benefit in hematologic malignancies, less significant in solid tumors
BH3-only act as
trigger for the
apoptotic signal
Mitochondria as
Key integrator of
Apoptotic signals
Radiation-Induced Apoptotic Signals
Belka IJROBP (2004)
Receptor-Mediated Apoptotic Signals
Belka IJROBP (2004)
Radiation apoptosis is cell type dependent
upon either p53 or ceramide
Kolesnick and Fuchs 2002 Science
Apoptotic Modulators as
Radiosensizers
Belka IJROBP (2004)
In vivo detection of apoptosis
Verheig Can Met Rev 2008
In vivo detection of apoptosis
using 99T Annexin V
Verheig Can Met Rev 2008
Autophagic Death
• Autohagy is active “self eating”
• Process for degradation of cellular constituents
that do not fit in the proteasome (Aggregates or
organelles)
• Tagging of proteins through ATG5-ATG12
system
• Beclin-1 is a haploinsufficient tumor suppressor
gene
Autophagic machinery
Klionsky Nature 2008
Radiation Senescence
• First described in normal cells
• Later determined to occur in tumor cells
• Senescence-associated B-galactosidase as a
marker
• p53 and p21 as major contributors
Doxirubicin Treated
Colon Cancer Cells
Can be Sorted by
Proliferation status
Non-proliferating cells show
Senescent morphology
Non-proliferating cells do not
Form Colonies
Roninson CR 2003)
Mitotic catastrophe
• Caused by a combination of deficient cell cycle
checkpoint and cellular damage at mitosis
• Has characteristics of apoptosis (capase 2
activation)
• Thought to prevent aneuploidy, so loss could
contribute to tumor progression
Characteristics of Mitotic Catastrophe
Mitotic catastrophy induced by chk2 inhibitor (debromohymenialdesine).
Castedo Kroemer Onc (2004)
Radiation-Induced
Chromosome Aberrations
• Exact mechanisms not established, but
involves DNA damage and Misrepair
• ATM fragile chromosomes and radiosensitivity
• Multiple lesions needed for complex
aberrations – the D2 component to survival
curves
• Linear relationship between “lethal” lesions and
survival
DSB vs Telomere
Purdy CB 2004
Defective DSB Repair Causes Cellular &
Clinical Radiation Hypersensitivity
From: Hall, “Radiobiology for the Radiologist”
From: Hall and Giaccia, “Radiobiology
for the Radiologist”
Radiation-Induced Aberrations
Radiation-Induced aberrations
In metaphase spreads
FISH to Identify Aberrations
Lymphocytes exposed to 4 Gy
Nataragan MR 2003
Asymmetric chromosome aberrations
from DSBs
• Occur primarily in unreplicated cells (G1)
• Can result in direct loss of genetic material
– Acentric fragment, internal deletion
• Can lead to future generation events
– Dicentric bridge, breakage, fusion, bridge
• G2 breaks lead to chromatid events, and often
one daughter cell has complete DNA content
Resolution of Dicentric Bridge
Shimizu ECR 2005
Linear Quadratic relation between
Radiation dose and Aberrations
Two linear quadratics make a straight line
Survival and asymmetric
chromosome lesions are
linearly related.
Cornforth and Bedford
RadRes1987
Bystander Role in Radiation Induced
Cellular Death
• Photon does not pass through the cell, but
there is still a biologic effect
• Due to secreted substance (paracrine), or one
passed through gap junctions
• Can lead to late effect mutation or even cell
death
• Not really dose-dependent
Clonal effects
“Bystander effects”
Mothersill and Seymour 2004
Summary
• Radiation can induce “death” through a number of
established pathways
– Most significant fraction is due aberrations in solid
tumors
• Oncogenic transformation can alter inherent
sensitivity to PCD (more myc, less bcl2)
• Radiation sensitizers very difficult to achieve
because they have to be specific for tumor cells