Académique Documents
Professionnel Documents
Culture Documents
Bill McBride
Dept. Radiation Oncology
David Geffen School Medicine
UCLA, Los Angeles, Ca.
wmcbride@mednet.ucla.edu
www.radbiol.ucla.edu
Objectives:
• Know that senescence as well as cell death can lead to loss of
reproductive colongenic cells and affect the outcome of RT
• Be able to distinguish between interphase and mitotic (catastrophic) cell
death following irradiation
• Understand the physiologic, morphologic, and mechanistic differences
between apoptosis, autophagy, and necrosis as deathstyles and how cells
die in response to irradiation
• Understand how survival pathways operate to affect cellular
radiosensitivity and how these can be targeted for radiotherapeutic benefit.
• Know the molecular basis for cell cycle arrest following IR and its
importance in repair and carcinogenesis
• Understand the importance of cell cycle kinetics, cell loss factors in tumor
growth and regression
• Recognize the importance of changes in these parameters during the
course of a fractionated RT regimen
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Intrinsic Radiosensitivity
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FRACTION OF CELLS SURVIVING 2 GY IN VITRO
LYMPHOMA
NEUROBLASTOMA
MYELOMA 0.2 (0.08 - 0.37)
SMALL CELL LUNG CANCER
MEDULLOBLASTOMA
BREAST CA
SCC
PANCREATIC CA 0.43 (0.14 - 0.75)
COLORECTAL CA
NON-SMALL CELL CA
MELANOMA
OSTEOSARCOMA 0.52 (0.2 - 0.86)
GLIOBLASTOMA
HYPERNEPHROMA
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Robert Hooke (1635-1703) was the first to use Not!
the term ‘cell’ in the 1665 Micrographia
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• DSB repair, checkpoint arrest, and cell death are
all part of the DNA damage response to DSBs.
They function synergistically to dictate whether
cells live or die following IR and to prevent
development of chromosome instability.
• The relationship of repair, cell proliferation and cell
death following IR has been the subject of many
studies, primarily because, clinically, loss of
reproductive, clonogenic cells following RT
determines the outcome of cancer treatment.
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Loss of Proliferative Ability can Occur in
Different Ways
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Radiation-Induced Senescence
Is particularly relevant to radiation fibrosis, but also
occurs in cells other than fibroblasts.
TGF-b
Stress-induced
(Including radiation)
Proliferation-induced
Collagen production and fibrosis
Cancer-induced Tumor progression
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Early Observations on Cell Death
after Irradiation
• Radiobiologists like Puck and Marcus (1956) showed that most
reproductive cells die a mitotic death, also known as mitotic
catastrophe, after IR.
– It may take several cell divisions, the number depending on the
radiation dose.
– After 2 Gy, it may average 2-3 cell divisions before death
– This may take several days (as opposed to hours)
– It is due to
• Chromosome loss
• Failure of spindle formation during cytokinesis
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Lethal Sectoring in Mitotic Death
RT
RECURRENCE!
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Courtesy:
Randi Syljuasen
Control
Cells Irradiated
Cells
Control Irradiated
- -
Nuclei Nuclei
Stained Stained
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Alternative Deathstyle Mechanisms
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Alternative Deathstyle Mechanisms
Physiologic Pathologic
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Physiologic Programmed Cell Death
PCD is involved in:
• Morphogenesis Fingers Gut
• Tissue sculpting
• Homeostatic control of
cell numbers
• Preventing
autoimmunity Tadpole Tails Sex differentiation
• PCD is
immunologically
“silent”
This may be why
proliferation often
correlates with
apoptotic index
proliferating cells Self-reactive Irradiation
lymphocytes
“It is a myth to think death is just for
the old. Death is there from the very
beginning” Herman Feifel
CELL 88:350, 1997
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Pathologic Programmed Cell Death
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Programmed Cell Death Type I: Apoptosis
Morphology
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Programmed Cell Death Type I: Apoptosis
Molecular Hallmarks
During apoptosis, endonucleases are induced that cleave
between nucleosomes.
On agarose gel electrophoresis, the DNA separates into
fragments with sizes that are multiples of 180-200 bp. This is
called a “ladder.”
-
Histones H2,H3,H4
DNA Spacer Region Nucleosome DNA Core
(60-100 bp) (140 bp)
55 A
110 A
HISTONE H1
+
Sites of endonuclease cleavage
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Detection of Apoptosis
- TUNEL Assay
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Apoptosis in Gut after IR
• Radiation-induced
apoptosis occurs
in normal tissues
in specific sites
and in cells that
have a pro-
apoptotic
tendency
• In gut this is in the
base of the crypts
Sites of
apoptosis
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Programmed Cell Death Type 2: Autophagy
Morphology
Autophagy
– A tightly regulated process
– A response to nutrient and growth factor
deprivation, but is also seen in physiologic
processes, eg morphogenesis.
– Organelles and other cell components are
sequestered in autophagosomes that fuse
with lysosomes (self-digestion)
– Increased endocytosis, vacuolation,
membrane blebbing, nuclear condensation
– In essence it is a defensive reaction that
eventually can lead to cell death
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Pathological Cell Death Type 3: Necrosis
Morphology
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Triggers for Cell Death
• Type 1 - Apoptosis:
– Extrinsic triggering of “death” receptors (some TNFR family
members)
– Intrinsic DNA damage response pathway
– Alterations in mitochondria membrane permeability
• Type 2 - Autophagy:
• Removal of growth/survival factor signaling. Often called “death by
neglect.” Cells have to receive the appropriate stimuli from their
environment to survive, if not they die often by autophagy. Death is the
default pathway of life! Cells in the wrong microenvironment die of
“homelessness” (anoikis), a form of death by neglect.
• The PI3K/Akt/mTOR pathway is activated by growth factors allowing
increased expression of transporters for glucose, amino acids, etc. Akt
increases glycolysis. mTOR drives protein translation rates.
• Type 3 - Necrosis:
– Extrinsic activation of immune cells leads to release of cytotoxins -
perforins, etc. that cause necrosis
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What Deathstyles are Associated with
Radiation-Induced Death?
Any of them
• Mitotic death after irradiation can be by any molecular
mechanism
• Interphase death after irradiation is by rapid apoptosis
– Prominent in lymphocytes, spermatogonia, oligodendrocytes,
salivary gland
– Occurs in many tumors and tissues, normally in specific sites
• Cells that are most sensitive to radiation considered to
have a pro-apoptotic phenotype
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How do cells commit suicide?
• Apoptosis is Mediated by Caspases - “Roads to Ruin”
• The morphological and biochemical hall-marks of
apoptosis are the result of cascadic activation of members
of a family of pro-enzyme proteases called Caspases by
– Extrinsic pathway through Tumor Necrosis Factor Receptor
(TNFR) family members, which activates caspase 8
– Intrinsic pathway through cytochrome c leaking from mitochondria,
which activates caspase 9.
• Irrespective of the apoptotic death signal, all caspases
converge to activate a terminal Caspase 3-dependent
pathway
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Executioner Caspases
• Executioner caspases cleave >40 substrates (including each other)
leading to the morphological features of apoptosis
• Blocking these caspases does not generally prevent radiation-induced
cell death - by then it is too late!
Caspase 3
Caspase 6 Caspase 7
CAD
Cell DNA DNA
Shrinkage Fragmentation Repair
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• The decision to commit apoptosis is determined by an internal
“rheostat” within the cell i.e. cells have a pro-apoptotic or anti-
apoptotic phenotype
• Radiation increases the AI, but does not change a cell from an
anti-apoptotic to pro-apoptotic phenotype
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Why don’t all cells die by apoptosis
after RTx?
• Mitochondrial Control: Members of the Bcl-2 family (B cell lymphoma
oncogene) localize in the outer membrane of the mitochondria
– Bcl-2 is the prototypical inhibitor of apoptosis
– Bax is from the same family and activates apoptosis
– The balance of pro-apototic (bax) to anti-apoptotic (Bcl-2) factors
control the “leakiness” of the membranes.
• Survival pathways: These affect intrinsic and extrinsic apoptotic and
autophagic pathways and alter the rheostat away from cell death and
towards radioresistancy - acting often through the Bcl-2 family. Major
survival pathways are
– phosphoinositol kinase 3 (PI3K)
– nuclear factor kappa B (NF-B)
• Cancer is associated with mutations in cell death/survival pathways, as
is radioresistance, and these are targets for theraputic intervention
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Control Over Radiation-Induced Apoptosis
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“Survival Pathways”
Growth Factors, Cytokines, Proliferative Signals
PDK1 Proliferation
Metabolic ERK
Pathway
AKT P90 RSK
NFB
Bad
Inhibitors of Apoptosis (IAPs) mTOR
Bcl-2/Bcl-XL
caspases
Context is everything -
“Location, location, location”
Survival
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Clinical Significance of Cell Death
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• The pathways that govern cell death/survival also govern
radioresistance and radiosensitivity!!!!!
• Manipulation of apoptotic pathways genetically, or with
drugs, can affect clonogenic cell survival
• Survival pathways are appropriate targets for tumor
radiosensitization
• EGFR
• Iressa, Tarceva, C225, Farnesyl Transferase Inhibitors
• NF-B
• COX-2 inhibitors
• Survival pathways form appropriate targets for normal
tissue radioprotection
• Keratinocyte growth factor (KGF) in bone marrow
transplant patients
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• Volume 354:567-578 February 9, 2006
• Radiotherapy plus Cetuximab for Squamous-Cell Carcinoma of the Head and Neck
• James A. Bonner, M.D., Paul M. Harari, M.D., Jordi Giralt, M.D., Nozar Azarnia, Ph.D., Dong M.
Shin, M.D., Roger B. Cohen, M.D., Christopher U. Jones, M.D., Ranjan Sur, M.D., Ph.D., David
Raben, M.D., Jacek Jassem, M.D., Ph.D., Roger Ove, M.D., Ph.D., Merrill S. Kies, M.D., Jose
Baselga, M.D., Hagop Youssoufian, M.D., Nadia Amellal, M.D., Eric K. Rowinsky, M.D., and K.
Kian Ang, M.D., Ph.D.
• The median duration of locoregional control was 24.4 months among patients
treated with cetuximab plus radiotherapy and 14.9 months among those given
radiotherapy alone …..
• the median duration of overall survival was 49.0 months among patients treated
with combined therapy and 29.3 months among those treated with radiotherapy
alone …..
• Radiotherapy plus cetuximab significantly prolonged progression-free survival
… With the exception of acneiform rash and infusion reactions, the incidence of
grade 3 or greater toxic effects, including mucositis, did not differ significantly
between the two groups.
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Cell Proliferation and Cell Death: Two
Sides of the Same Coin?
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Timeframe of Cellular Life
The Cell Cycle
• Under the microscope, Flemming identified cells in mitosis (M) and in
interphase - i.e 2 cell cycle phases
• Howard & Pelc, 1951 & 1953, - bean root cells in interphase
incorporate 32P for DNA synthesis (S phase) and there is a time gap
(G2) before the beginning of cell division (M) and there is another gap
(G1) between M and S to complete the cell cycle - i.e. 4 cell cycle
phases
• Taylor et al., 1957 looked at tritiated thymidine uptake (in S) and
measured the time it takes for labeled cells to enter M (= time in G2),
and the other cell cycle kinetic parameters
• More recently, bromodeoxyuridine detected by fluorescent antibody is
used to label cells (in S) and measure cell cycle kinetics by flow
cytometry or U.V. microscopy
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Mitotic Index Labeling Index
If 3H-TdR
If BdUR labeled
labeled AR film
mitosis
*Anti-BdUR
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From FLM to FACS
Label cells with
dye and use a
laser to excite it.
PM tubes Collect output by
photomultiplier
tubes.
E.g. DNA can be
labeled by propidium
iodide (P.I.)
LASER
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Flow Cytometry for DNA Quantity
1. label DNA with propidium iodide
(fluorescent dye)
G2/M
G2/M G2/M
DNA DNA DNA
red P.I. red P.I red
Time
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Cell Cycle
M phase
0.5-1 hr
G2 phase
1-2 hrs
G0 quiescent
S phase
DNA synthesis G1 phase
6-8 hrs variable length
If all cells in a population are dividing
Mitotic Index (M.I.) = lTm / Tc
Labeling Index (L.I.) = lTs /Tc
Where l is a correction for uneven cell numbers due to mitosis (0.69)
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Cell Cycle Synchronisation
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Cell Cycle and Radiosensitivity
1
Variations in sensitivity and in
S.F. cell cycle arrest after irradiation
LATE S
could be important in radiation
.1 therapy, because fractionated
EARLY S irradiation can lead to
G1 PHASE sensitization by reassortment.
G2/M PHASE
.01
0 4 8 12 16 20 The oxygen enhancement ratio (OER)
Dose (Gy) does not vary much with the phase of the
cell cycle.
Increasing
High LET responses are less affected by
radioresistance cell cycle phase than low LET radiation
responses.
G1 S G2 M
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Cell Cycle Arrest
• Cells have “checkpoints” where they “proof-read” DNA for damage
before continuing to cycle. This ensures faithful chromosome replication
and maintains genomic integrity.
• Irradiation causes cells to arrest at these checkpoints
• Cells tend to arrest at
• G1 - especially if they have wt p53. This may lead to apoptosis
• Intra S phase - initiation and elongation stages of DNA
replication are affected by p53 independent mechanisms
• G2 - most cells arrest here - allows chromatid repair prior to
segregation in M
• M phase - block in anaphase until all sister chromatids
have aligned properly on the spindle - Monitors spindle
integrity for cytokinesis
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Cell Cycle Arrest
DNA Damage Dependent Checkpoints
• Irradiated (7Gy)
• P.I stain at 9hr wild-type irradiated
Decrease in S
Increase in G2M
i.e. G1 and G2M arrest
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What Drives Cell Cycle Progression?
Growth factors are required for G0 through G1 to S (and cell survival)
• To activate resting cells to enter G1
• To allow cells to pass through G1 phase
• To gain competence to progress into S phase
The growth factors that are required vary with the cell type. For example,
for fibroblasts:
• PDGF (platelet derived GF) activates cells
• EGF (epidermal GF) and insulin act as competence factors to progress into S
phase
• IGF (insulin GF) promotes progression into S
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Molecular Mechanism of Cell Cycle Progression
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Retinoblastoma Protein pRb
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Cyclins
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Cyclin Dependent Kinases
• Cyclins bind and activate Cdks, which
– Are serine/threonine kinases with multiple
substrates
• e.g. pRb, p53, E2F, etc. that they activate/inactivate
– Have regulatory domains Inhibitory phosphate
• E.g. inhibitory and activating phosphates
– Are present throughout cell cycle P Cyclin
– To move cells from G0 to G1 to S
• Cyclin D activates cdks 4/6 and kinase
• Cyclin E activates cdk2 site P
cdk
activating phosphate
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Activating Phosphatases
CDC25 Removes Phosphate from Tyr-15
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cdk1 phosphorylates substrates leads to
• Nuclar envelope breakdown
• Chromosome separation
• Spindle assembly Cyclin B Cyclosome (APC)
• Chromosome condensation CDK1 pRb dephosphorylation
Cyclin A
CDK1/2
G0 quiescent
Cyclin D
CDK4/6
Cyclin A
CDK1/2
Early - mid G1
Cyclin D
Cyclin E
CDK4/6
CDK2 Responsible for pRb
Responsible for pRb phosphorylation
phosphorylation
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Cyclin Kinase Inhibitors
Inhibitors (CKIs) belong to 2 families
• INK4 and KIP/CIP
Generally compete with cyclins for CDKs
Phase Complexes Inhibitors
G1 cyclin D-CDK4, 6 p16 (INK 4a),
p19ARF (INK 4a)
p15 (INK4b)
G1/S cyclin E-CDK2, 3 p21CIP1, p27KIP1
S cyclin A-CDK2 p21, p57
G2/M cyclin B-CDK1 p21
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DSB SSB/Base damage DSB Resection
Replication stress, UV, MMC, hypoxia
MRN Stalled Replication Fork
complex
ATR
sensors ATM ATM
ATR
H2AX
transactivation
CDC25A degradation
P-thr14/tyr15 P
P-thr14/tyr15
CDK2 CDK!
CDK2
p21 CDK2
CYCLIN E CYCLIN B
CYCLIN A/E
CYCLIN E
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Cancer
Growth Factor/Cytokine
Receptor Survival
Oncogenes Signals
PI3K
Ras NF-B
Raf
MAPK
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Initial damage DNA repair Cell cycle arrest Cell death
/survival
ROS DNA damage response
ATM, ATR, MRN P21, Bax, caspase 8,etc.
P53, Chk1, Chk2
Immediate early
gene response Inflammatory
AU-rich control:
Cytokines and
JNK
P38 MAPK TNF-, IL1b, IL-2, IL-3, GM- Growth Factors
NF-kB CSF, IL-6, IL-8, IL-12,
IFN/b, VEGF, PDGFB,
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Loss of Proliferative Ability can Occur in
Different Ways
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Tissue Kinetics
Kinetics in tumors or normal tissues depend upon
• Cell cycle
• Growth fraction (G.F.)
• G.F. is the proportion of proliferating cells
• G.F. = P / (P + Q) where P = proliferating cells and Q = non-
proliferating cells (quiescent/senescent/differentiated cells)
• Cell loss factor
• Cell Loss Factor is due to death or loss of cells
• If = 0, Td = Tpot where Td is the actual volume doubling time
and Tpot is potential volume doubling time
• = 1 - Tpot / Td
• if G.F. = 1 then Tpot = Tc = lTs / L.I.
• Under steady state conditions, a constant cell number is
maintained by the balance between cell proliferation and cell loss
i.e. = 1.0. In tumors and embryos, < 1.0
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Tumor Kinetics
Human SCC
Rate of tumor growth, and the rate of tumor regression, are determined
largely by the cell loss factor!
VARIES GREATLY WITH TUMOR
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Tumor Regression
• The rate of tumor growth and regression is
determined by
• rate of cell loss (
• G.F.
• cell cycle kinetics
• Slow growing tumors may regress rapidly
• Rapidly growing tumors are expected to regress
and regrow rapidly
• Slow regression is not an indication of treatment
failure
• The rate of tumor regression after Tx is not, in
general, prognostic
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Tumor Regeneration
Relative tumor X-rays
volume
Control
Irradiated Tumors can
Growth delay regenerate at the
same time as
Surviving clonogens they regress!
measured in vitro
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EVIDENCE FOR ACCELERATED REPOPULATION
IN TUMORS
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Accelerated Tumor Repopulation
T2 T3
local control
no local control
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Accelerated Tumor Repopulation
Onset may be about day 21. Repopulation may not be constant and
may increase from 0.6 Gy / day around week 3-4 to even 1.6 – 1.8 Gy /
day around week 6-7 and thereafter.
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Accelerated repopulation in human tumors
provided the rationale for accelerated
fractionation protocols
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