Mechanisms of DNA damage-Induced Cell Death Radiobiology 2012.
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Transcript of Mechanisms of DNA damage-Induced Cell Death Radiobiology 2012.
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Mechanisms of DNA damage-Induced Cell Death
Radiobiology 2012
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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?
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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)
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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
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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
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Dose and time-dependent death
Shinomiya JCMM 2002
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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
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Apoptotically sensitive Tumor Cells show clonogenic sensitivity
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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
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Bcl2 identified as oncogene because of translocation caused activation
Large survival benefit in hematologic malignancies, less significant in solid tumors
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BH3-only act as trigger for the apoptotic signal
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Mitochondria as Key integrator ofApoptotic signals
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Radiation-Induced Apoptotic Signals
Belka IJROBP (2004)
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Receptor-Mediated Apoptotic Signals
Belka IJROBP (2004)
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Radiation apoptosis is cell type dependent upon either p53 or ceramide
Kolesnick and Fuchs 2002 Science
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Apoptotic Modulators as Radiosensizers
Belka IJROBP (2004)
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In vivo detection of apoptosis
Verheig Can Met Rev 2008
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In vivo detection of apoptosis using 99T Annexin V
Verheig Can Met Rev 2008
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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
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Autophagic machinery
Klionsky Nature 2008
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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
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Doxirubicin Treated Colon Cancer CellsCan be Sorted by Proliferation status
Non-proliferating cells show Senescent morphology
Non-proliferating cells do not Form Colonies
Roninson CR 2003)
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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
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Characteristics of Mitotic Catastrophe
Mitotic catastrophy induced by chk2 inhibitor (debromohymenialdesine).
Castedo Kroemer Onc (2004)
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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
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DSB vs Telomere
Purdy CB 2004
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Defective DSB Repair Causes Cellular &Clinical Radiation Hypersensitivity
From: Hall, “Radiobiology for the Radiologist”From: Hall and Giaccia, “Radiobiology for the Radiologist”
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Radiation-Induced Aberrations
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Radiation-Induced aberrations In metaphase spreads
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FISH to Identify Aberrations
Lymphocytes exposed to 4 Gy Nataragan MR 2003
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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
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Resolution of Dicentric Bridge
Shimizu ECR 2005
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Linear Quadratic relation between Radiation dose and Aberrations
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Cornforth and Bedford RadRes1987
Survival and asymmetric chromosome lesions arelinearly related.
Two linear quadratics make a straight line
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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
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“Bystander effects”
Clonal effects
Mothersill and Seymour 2004
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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