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 ofApoptotic 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 CellsCan 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

Cornforth and Bedford RadRes1987

Survival and asymmetric chromosome lesions arelinearly related.

Two linear quadratics make a straight line

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

“Bystander effects”

Clonal 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