Radiation targets - cell proliferation, cell death and survival

detailed information about cell proliferation, growth and survival

1. www.radbiol.ucla.edu
Radiation Targets 2:
Cell Proliferation, Cell Death and Survival
Bill McBride
Dept. Radiation Oncology
David Geffen School Medicine
UCLA, Los Angeles, Ca.
[email protected]

2. 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

3. www.radbiol.ucla.edu
Intrinsic Radiosensitivity
The outcome of radiation exposure depends on
•The DNA lesions that are caused and their
persistence
•How cells and tissues ‘sense’danger and
respond by activating cell survival or death
pathways

4. www.radbiol.ucla.edu
FRACTION OF CELLS SURVIVING 2 GY IN VITRO
LYMPHOMA
NEUROBLASTOMA
MYELOMA
SMALL CELL LUNG CANCER
MEDULLOBLASTOMA
BREAST CA
SCC
PANCREATIC CA
COLORECTAL CA
NON-SMALL CELL CA
MELANOMA
OSTEOSARCOMA
GLIOBLASTOMA
HYPERNEPHROMA
0.2 (0.08 -0.37)
0.43 (0.14 -0.75)
0.52 (0.2 -0.86)
Tumor cells vary dramatically in intrinsic radiosensitivity
depending on their tissue of origin. The number of DNA
lesions are the same but the outcome is different.

5. www.radbiol.ucla.edu
20-40GySeminoma, Dysgerminoma, Acute Lymphocytic leukemia,
Wilms’tumor, Neuroblastoma
40-50GyHodgkin's, Lymphosarcoma, Seminoma, Histiocytic cell
sarcoma, Skin ca. (basal and squamous cell)
50-60GySquamous cell ca. (cervix, head and neck), Breast ca., Ovarian
ca.,Medulloblastoma, Retinoblastoma, Ewing's tumor
60-65GyLarynx (<1 cm), breast cancer lumpectomy
70-75GyOral cavity (<2 cm, 2-4 cm), Oro-naso-laryngo-pharyngeal ca.,
Bladder ca., Cervix ca., Uterine ca., Ovarian ca., Lung ca. (<3
cm)
>80GyHead and neck ca. (~4 cm), Breast ca. (~5 cm), Glioblastomas,
Osteogenic sarcomas (bone sarcomas), Melanomas, Soft tissue
sarcomas (~5 cm), Thyroid Ca.
(In Rubin P, et al, eds: Clinical Oncology: A Multidisciplinary Approach,
edition 7, p 72. Saunders, 1993)
Clinically, tumors show the same histological correlation
with respect to sensitivity to RT.

6. www.radbiol.ucla.edu
Not!
Robert Hooke (1635-1703) was the first to use
the term ‘cell’in the 1665 Micrographia
Antony van Leeuwenhoek (1632-1723) -
Made powerful lenses, discovered bacteria
-father of microbiology
Rudolph Virchow (1821-1902) -Recognized
leukemia and mechanism of embolism -
Developed theory that cells come from cells
(“omnis cellula a cellula”)
Walther Flemming (1843-1905) -identified
chromatin and mitosis (Gk, thread)
(“omnis nucleus a nucleo”)
1906Bergonie and Tribandeau. Action des
rayou X sur le testicle Elect. Med.14, 779
-radiosensitivity is related to cell proliferation

7. www.radbiol.ucla.edu
•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 cellsfollowing RT
determines the outcome of cancer treatment.

8. www.radbiol.ucla.edu
(all with distinct, and common, gene patterns)
IR is a pathological signal and can cause senescence
Loss of Proliferative Ability can Occur in
Different Ways
Quiescence Senescence Terminal Death
Differentiation
Property of stem cells
Reversible, physiological
process
Apoptosis and
differentiation is inhibited
High free radical scavenger
levels
Irreversible,
physiological
active process
Cell cycle inhibition is a
secondary effect
Irreversible,
non-physiological
process
Apoptosis
Autophagy
Necrosis

9. www.radbiol.ucla.edu
Stress-induced
(Including radiation)
Proliferation-induced
Cancer-induced
Proliferative
Progenitor
Fibroblast
Post-mitotic
Fibroblast
TGF-b
Radiation-Induced Senescence
p21
Collagen production and fibrosis
Tumor progression
Is particularly relevant to radiation fibrosis, but also
occurs in cells other than fibroblasts.

10. www.radbiol.ucla.edu
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
•Early radiobiologists also discovered that a few cells of specific
types die by interphase death(without dividing)
–This is generally more rapid than mitotic death, occurring 4-
24hrs after irradiation.

11. www.radbiol.ucla.edu
RECURRENCE!
RT
Lethal Sectoring in Mitotic Death
The fear of death is the most unjustified of
all fears, for there's no risk of an accident
for someone who's dead. Albert Einstein

12. www.radbiol.ucla.edu
Control
Cells
Control
-
Nuclei
Stained
Irradiated
Cells
Irradiated
-
Nuclei
Stained
Courtesy:
Randi Syljuasen

13. www.radbiol.ucla.edu
Alternative Deathstyle Mechanisms
Programmed cell death type 1: Apoptosis
Programmed cell death type 2: Autophagy
Pathological Death: Necrosis
•Deathisoftenanactiveprocess:cellsdecidetocommitsuicide
•Deathpathwayspreventcarcinogenesisandmutationsinthemare
associatedwithcancer.Theyprovidepotentialtumor-specific
targetsfortherapeuticintervention.
•Death pathways, and mutations in them, affect intrinsic cellular
radiosensitivity. They provide potential tumor-specific targets for
radiosensitization.

14. www.radbiol.ucla.edu
Alternative Deathstyle Mechanisms
Physiologic Pathologic
Type 1: Apoptosis Type 1: Apoptosis
Type 2: Autophagy Type 2: Autophagy
Type 3: Necrosis
•Type1and2areProgrammed
•Deathislargelyanactiveprocess:cellsdecidetocommitsuicide
•Deathpathwayspreventcarcinogenesisandmutationsin
moleculesinthesepathwaysareassociatedwithcancer.They
providepotentialtumor-specifictargetsfortherapeuticintervention.
•The same death pathways and mutations affect intrinsic cellular
radiosensitivity. They provide potential tumor-specific targets for
radiosensitization.

15. www.radbiol.ucla.edu
Physiologic Programmed Cell Death
Sex differentiation
PCD is involved in:
•Morphogenesis
•Tissue sculpting
•Homeostatic control of
cell numbers
•Preventing
autoimmunity
•PCD is
immunologically
“silent”
“It is a myth to think death is just for
the old. Death is there from the very
beginning”Herman Feifel
Self-reactive
lymphocytes
Irradiation
Fingers
Gut
Tadpole Tails
proliferating cells
This may be why
proliferation often
correlates with
apoptotic index
CELL 88:350, 1997

16. www.radbiol.ucla.edu
Pathologic Programmed Cell Death
•Self sacrifice by infected/damaged cells
•Self sacrifice by immune cells and other normal
cells in the battle zone
•Causes inflammation
–wound healing
–immunity

17. www.radbiol.ucla.edu
The word comes from -from and -falling.
“Like leaves on trees the race of man is found, now green in youth, now withering
on the ground”The Iliad of Homer. Book vi. Line 181
Programmed Cell Death Type I: Apoptosis
Morphology
Apoptosisis a tightly regulated “active”cell
death process that is associated with
Cell and nuclear shrinkage
Nuclear fragmentation with formation of
apoptotic bodies
Blebbing of cell membrane, but no early loss of
membrane integrity
Deletion of single cells in isolation
Lack of an inflammatory response and
phagocytosis by local cells (a silent death!)

18. www.radbiol.ucla.edu
Programmed Cell Death Type I: Apoptosis
Molecular Hallmarks
Histones H2,H3,H4
DNA Spacer Region
(60-100 bp)
Nucleosome DNA Core
(140 bp)
110 A
55 A
Sites of endonuclease cleavage
HISTONE H1
-
+
During apoptosis, endonucleasesare 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.”

19. www.radbiol.ucla.edu
Detection of Apoptosis
-TUNELAssay
•Apoptosiscanbevisualizedintissuesections
usingterminaldeoxynucleotidyltransferase
(TdT)toaddfluorescein-labeled(dUTP)
nucleotidesonto3’-OHendsofDNAthatresult
fromtheactionoftheapoptoticendonuclease
•AnApoptoticIndex(AI)canbederived

20. www.radbiol.ucla.edu
Apoptosis in Gut after IR
Sites of
apoptosis
•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

21. www.radbiol.ucla.edu
Programmed Cell Death Type 2: Autophagy
Morphology
Autophagy
–Atightlyregulatedprocess
–Aresponsetonutrientandgrowthfactor
deprivation,butisalsoseeninphysiologic
processes,egmorphogenesis.
–Organellesandothercellcomponentsare
sequesteredinautophagosomesthatfuse
withlysosomes(self-digestion)
–Increasedendocytosis,vacuolation,
membraneblebbing,nuclearcondensation
–Inessenceitisadefensivereactionthat
eventuallycanleadtocelldeath

22. www.radbiol.ucla.edu
Pathological Cell Death Type 3: Necrosis
Morphology
Necrosisis a rapid non-physiological
process associated with
•Loss of plasma membrane integrity and
deregulated ion homeostasis.
•Swelling and bursting of cells as water enters
•Groups of cells, rather than single cells, are
affected.
•DNA forms a random “smear”on agarose gel.
There is no pattern to its fragmentation.
•Associated with inflammation.

23. www.radbiol.ucla.edu
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

24. www.radbiol.ucla.edu
What Deathstyles are Associated with
Radiation-Induced Death?
Any of them
•Mitotic death after irradiation can be by any molecular
mechanism
•Interphase deathafter 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

25. www.radbiol.ucla.edu
How do cells commit suicide?
•ApoptosisisMediatedbyCaspases-“RoadstoRuin”
•Themorphologicalandbiochemicalhall-marksof
apoptosisaretheresultofcascadicactivationofmembers
ofafamilyofpro-enzymeproteasescalledCaspasesby
–ExtrinsicpathwaythroughTumorNecrosisFactorReceptor
(TNFR)familymembers,whichactivatescaspase8
–Intrinsicpathwaythroughcytochromecleakingfrommitochondria,
whichactivatescaspase9.
•Irrespectiveoftheapoptoticdeathsignal,allcaspases
convergetoactivateaterminalCaspase3-dependent
pathway

26. www.radbiol.ucla.edu
Executioner Caspases
•Executionercaspases 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!
ICAD (inhibitor of caspase activated DNase)
DNA-PK (DNA protein kinase)
PARP (poly-ADP-ribose polymerase)
Caspase 3
Caspase 7Caspase 6
Lamin A Actin
Cell
Shrinkage
iCAD -CAD DNA-PKcs PARP
DNA
Repair
CAD
DNA
Fragmentation

27. www.radbiol.ucla.edu
Sphingomyelin
Ceramide
Members of
TNFR family
with Death
Domains
(TNFR1, Fas,
TRAIL)
p53
ATM
Bax Mitochondria
Cytochrome c
Caspase 9
Apoptosome Complex
Apaf-1
x
Caspase 8
INITIATORS
FADD
EFFECTORS
Caspase 3, 6, 7
TERMINAL PHASE
DNA Damage
JNK
P38 MAPK
Pro-caspase 9
JNK -jun kinase
ATM -mutated in
ataxia
telangiectasia
FADD -Fas
activated death
domain
Apaf -apoptosis
activating factor
Activation of
Pro-caspase 8
Radiation-Induced Apoptosis

28. www.radbiol.ucla.edu
•Thedecisiontocommitapoptosisisdeterminedbyaninternal
“rheostat”withinthecelli.e.cellshaveapro-apoptoticoranti-
apoptoticphenotype
•RadiationincreasestheAI,butdoesnotchangeacellfroman
anti-apoptotictopro-apoptoticphenotype
•Apoptoticcellsreappearbetweenradiationfractions
“There is only one serious philosophical problem. It is suicide. To judge
whether life is, or is not, worth living”Albert Camus

29. www.radbiol.ucla.edu
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

30. www.radbiol.ucla.edu
Sphingomyelin
Ceramide
Members of
TNFR family
With Death
Domains
p53
ATM
Bax
Bcl-2/Bcl-xl
Mitochondria
Cytochrome c
Caspase 9
Apoptosome Complex
Apaf-1
x
Caspase 8
INITIATORS
FADD
EFFECTORS
Caspase 3, 6, 7
TERMINAL PHASE
DNA Damage
Stress
Control Over Radiation-Induced Apoptosis
NF-B
IAPs
IAP -inhibitors of apoptosis
FLIP -FLICE (procaspase
8) inhibitory protein
JNK
P38 MAPK

31. www.radbiol.ucla.edu
Ras
Raf
ERK
P90 RSK
Survival
PI 3-kinase
PDK1
AKT
Bad
TNFR1
NFB
TNFR2
“Survival Pathways”
Bcl-2/Bcl-XL
Growth Factors, Cytokines, Proliferative Signals
Sphingomyelin
Ceramide
Inhibitors of Apoptosis (IAPs)
caspases
Context is everything -
“Location, location, location”
Proliferation
mTOR
Metabolic
Pathway

32. www.radbiol.ucla.edu
Clinical Significance of Cell Death
•Intrinsiccellularradiosensitivityisdeterminedinpartbythebalanceof
thesignalstransducingcelldeathorsurvivalpathways
•ClinicalRTresponseissuperiorintumorswithpathwaysprimedforan
activeformofcelldeath,buttherelationshipbetweenAI(orBAX/Bcl-
2)andlocaltumorcontrolorpatientsurvivalafterRTarecontroversial,
perhapsbecauseexcessivecelldeathoftencorrelateswithhighcell
proliferationorbecausemultiplepathwaystocelldeatharepossible
•ApoptosismayaffecttheclinicalresponseofnormaltissuestoRTe.g.
serouscells-“drymouth”
•Ingeneral,RTincreasestheA.I.onlyincellswithapro-apoptotic
phenotypeandapoptoticcellsreappearbetweenfractionsofRT
•EnhancingPCDinaproportionofcellsdoesnotnecessarilyaffectthe
shapeoftheclonogenicsurvivalcurvesfollowingradiation-this
dependsontheresponseofthesurvivingcells

33. www.radbiol.ucla.edu
•Thepathwaysthatgoverncelldeath/survivalalsogovern
radioresistanceandradiosensitivity!!!!!
•Manipulationofapoptoticpathwaysgenetically,orwith
drugs,canaffectclonogeniccellsurvival
•Survivalpathwaysareappropriatetargetsfortumor
radiosensitization
•EGFR
•Iressa,Tarceva,C225,FarnesylTransferaseInhibitors
•NF-B
•COX-2inhibitors
•Survivalpathwaysformappropriatetargetsfornormal
tissueradioprotection
•Keratinocytegrowthfactor(KGF)inbonemarrow
transplantpatients

34. www.radbiol.ucla.edu
•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.

35. www.radbiol.ucla.edu
Cell Proliferation and Cell Death: Two
Sides of the Same Coin?

36. www.radbiol.ucla.edu
Timeframe of Cellular Life
The Cell Cycle
•Underthemicroscope,Flemmingidentifiedcellsinmitosis(M)andin
interphase-i.e2cellcyclephases
•Howard&Pelc,1951&1953,-beanrootcellsininterphase
incorporate
32
PforDNAsynthesis(Sphase)andthereisatimegap
(G2)beforethebeginningofcelldivision(M)andthereisanothergap
(G1)betweenMandStocompletethecellcycle-i.e.4cellcycle
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 kineticparameters
•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

37. www.radbiol.ucla.edu
Fix and stain
Flash label with
3H-TdR or BdUR for 20 mins
If BdUR
labeled
If 3H-TdR
labeled
Mitotic Index (M.I.)
= lT
M/T
C
Labeling Index (L.I.) = lT
S/T
C
mitosis
*Anti-BdUR
AR film
…
..
…
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…
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…
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..
…
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U.V. microscopy
Mitotic Index Labeling Index
Autoradiography
Where lis a correction factor
for cell division, about 0.69

38. www.radbiol.ucla.edu
Frequency of Labeled Mitosis Technique
(FLM)
•Bycountingthenumberofmitosesthatare
labeledatvarioustimesafter
3
H-thymidine
incorporation,thetimetakenforacellto
traverseaspecificcellcyclephase,andthe
cellcycletime,canbeestimated
•But,itiseasiertouseBUdRandflow
cytometry

39. www.radbiol.ucla.edu
From FLM to FACS
Label cells with
dye and use a
laser to excite it.
Collect output by
photomultiplier
tubes.
E.g. DNA can be
labeled by propidium
iodide (P.I.)
LASER
Cells in fine stream
PM tubes

40. www.radbiol.ucla.edu
Flow Cytometry for DNA Quantity
1. label DNA with propidium iodide
(fluorescent dye)
2. measure light output by flow cytometry
3. analyze DNA histograms
G1
S
G2M
2n
2n + n
4n 4n
2n4n
# cells
degree of fluorescence
G1
S
G2M

41. www.radbiol.ucla.edu
Cell Cycle Kinetic Analysis by Flow Cytometry
G1
G2/M
BrdUrd
green
DNA
red
G1
G2/M
BrdUrd
green
DNA
P.I red
G1
G2/M
BrdUrd
green
DNA
P.I. red
Time
P.I. (DNA -red) combined with Bromodeoxyuridine uptake followed by
staining with fluorescently labeled anti-BrdUrd (green)

42. www.radbiol.ucla.edu
Cell Cycle
G1 phase
variable length
M phase
0.5-1 hr
G2 phase
1-2 hrs
S phase
DNA synthesis
6-8 hrs
Where lis a correction for uneven cell numbers due to mitosis (0.69)
If all cells in a population are dividing
Mitotic Index (M.I.) = lTm / Tc
Labeling Index (L.I.) = lTs /Tc
G0 quiescent

43. www.radbiol.ucla.edu
Cell Cycle Synchronisation
Thebestestimatesofkineticscomefromuseof
cellssynchronizedinaspecificcellcyclephase
•Mitoticcellscanbeshakenofffromsomecelllines-
Mphasecells
•Serumdeprivation-G1phasecells
•HydroxyureasynchronizescellsattheG1/Stransition

44. www.radbiol.ucla.edu
Cell Cycle and Radiosensitivity
S.F.
2016128400
.01
.1
1
Dose (Gy)
LATE S
EARLY S
G1 PHASE
G2/M PHASE
Variationsinsensitivityandin
cellcyclearrestafterirradiation
couldbeimportantinradiation
therapy,becausefractionated
irradiationcan leadto
sensitizationbyreassortment.
Theoxygenenhancementratio(OER)
doesnotvarymuchwiththephaseofthe
cellcycle.
HighLETresponsesarelessaffectedby
cellcyclephasethanlowLETradiation
responses.
G1 S G2 M
Increasing
radioresistance

45. www.radbiol.ucla.edu
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

46. www.radbiol.ucla.edu
•Irradiated (7Gy)
•P.I stain at 9hr
wild-type irradiated
Decrease in S
Increase in G2M
i.e. G1 and G2M arrest
P53 or ATM deficient irradiated
loss of G1/S checkpoint
and only G2M arrest
Cell Cycle Arrest
DNA Damage Dependent Checkpoints

47. www.radbiol.ucla.edu
What Drives Cell Cycle Progression?
GrowthfactorsarerequiredforG0throughG1toS(andcellsurvival)
•ToactivaterestingcellstoenterG1
•ToallowcellstopassthroughG1phase
•TogaincompetencetoprogressintoSphase
Thegrowthfactorsthatarerequiredvarywiththecelltype.Forexample,
forfibroblasts:
•PDGF(plateletderivedGF)activatescells
•EGF(epidermalGF)andinsulinactascompetencefactorstoprogressintoS
phase
•IGF(insulinGF)promotesprogressionintoS
CyclingisgrowthfactorindependentthroughS,G2,M

48. www.radbiol.ucla.edu
Molecular Mechanism of Cell Cycle Progression
Progression through each checkpoint requires:
•Retinoblastoma (Rb) tumor suppressor gene family
• especially G1-S transition
•Regulatory Factors
•Cyclinsthat are synthesized at the appropriate time for each phase and then
degraded to coordinate cell cycle progression. Growth factors induce cyclin
expression in G1.
•Cyclin Dependent Kinases (CDK)are activated by cyclins and
phosphorylate targets required for the next cell cycle phase
•Regulators of CDKs
1.Inhibitory kinases
2.Activated phosphatases
3.Non-kinase inhibitors

49. www.radbiol.ucla.edu
Retinoblastoma Protein pRb
•Cyclin D/cdk4/6 and cyclin E/cdk2 phosphorylate
Rb, which is essential for cell cycle progression into
S
•Phosphorylation of Rb releases E2F, which it
normally is bound to. E2F is a transcription factor
for 20-30 genes that are required for S phase gene
expression.
•pRB mutation often leads to cancer.

50. www.radbiol.ucla.edu
Cyclins
•Have no intrinsic enzymatic activity
•Cyclins A to J have been identified (no I)
•Synthesized and degraded during each cell
cycle phase
•Bind and activate cdks

51. www.radbiol.ucla.edu
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
•E.g. inhibitory and activating phosphates
–Are present throughout cell cycle
–To move cells from G0 to G1 to S
•Cyclin D activates cdks 4/6 and
•Cyclin E activates cdk2
P
P
cdk
Inhibitory phosphate
activating phosphate
Cyclin
kinase
site

52. www.radbiol.ucla.edu
Activating Phosphatases
CDC25 Removes Phosphate from Tyr-15
–CDC25A = cyclin E/CDK2 = G1/S specific
–CDC25B = cyclin A/CDK2 = S-phase exit
–CDC25C = cyclin B/CDK1 = G2/M specific

53. www.radbiol.ucla.edu
Cyclin D
CDK4/6
Cyclin E
CDK2
Responsible for pRb
phosphorylation
Cyclin A
CDK1/2
Cyclin B
CDK1
Cyclin A
CDK1/2
Early -mid G1
Cyclin D
CDK4/6
Responsible for pRb
phosphorylation
cdk1 phosphorylates substrates leads to
•Nuclar envelope breakdown
•Chromosome separation
•Spindle assembly
•Chromosome condensation
Cyclosome (APC)
pRb dephosphorylation
G0 quiescent

54. www.radbiol.ucla.edu
Cyclin Kinase Inhibitors
Phase Complexes Inhibitors
G1 cyclin D-CDK4, 6 p16 (INK 4a),
p19
ARF
(INK 4a)
p15 (INK4b)
G1/S cyclin E-CDK2, 3 p21
CIP1
, p27
KIP1
S cyclin A-CDK2 p21, p57
G2/M cyclin B-CDK1 p21
p53 is a transcription factor for p21, which is why it is
involved in cell cycle arrest after IR
Inhibitors (CKIs) belong to 2 families
•INK4 and KIP/CIP
Generally compete with cyclins for CDKs

55. www.radbiol.ucla.edu
ATM
MRN
complex
53BP1
MDC1
MRN
BRCA1
H2AX
mediators
p53 CHK2
CDC25A phosphorylation
p21
CYCLIN E
CDK2
CYCLIN E
CDK2
P-thr14/tyr15
p21
G1/S Arrest
Sensescence/transient
CDC25A degradation
rapid
slow
transactivation
ATR
53BP1
MDC1
MRN
BRCA1
CHK1
CDC25A phosphorylation
CYCLIN A/E
CDK2
S Phase
Arrest
DSB SSB/Base damage
Replication stress, UV, MMC, hypoxia
Stalled Replication Fork
ATM
ATR
53BP1
MDC1
MRN
BRCA1
CHK2CHK1
CDC25C phosphorylation
and nuclear export
CYCLIN B
CDK!
P-thr14/tyr15
P
G2/M Arrest
DSB Resection
MDM2
sensors
transducers
effectors

56. www.radbiol.ucla.edu
•If p53 or any other molecule governing cell cycle arrest is
mutated, genetic instability results as well as more rapid cell
cycle progression.
•Cyclins, cdks, cdkis and other molecules involved in cell cycle
progression are frequently mutated or have altered expression
in cancer
•e.g. cyclin D amplification and/or p16 deletion or silencing
and/or p53 mutation in Head and Neck Ca
Cell Cycle in Cancer

57. www.radbiol.ucla.edu
Growth Factor/Cytokine
Receptor
Proliferation Cell death
Oncogenes
Ras
Raf
MAPK
PI3K
NF-B
Survival
Signals
Cancer

58. www.radbiol.ucla.edu
DNA damage response
ATM, ATR, MRN
P53, Chk1, Chk2
Initial damage
ROS
Cell cycle arrest Cell death
/survival
DNA repair
JNK
P38 MAPK
NF-kB
Tissue recovery
/lesion formation
Cell death
/survival
Cell proliferation
Cell proliferation
Immediate early
gene response
AU-rich control:
TNF-, IL1b, IL-2, IL-3, GM-
CSF, IL-6, IL-8, IL-12,
IFN/b, VEGF, PDGFB,
NGF, IGFR, DR5, COX-2
Proteasome inhibition
Mitochondrial damage
Activation of EGFR,
TGF-b, etc
Inflammatory
Cytokines and
Growth Factors
P21, Bax, caspase 8,etc.

59. www.radbiol.ucla.edu
Loss of Proliferative Ability can Occur in
Different Ways
Quiescence Senescence Terminal Death
Differentiation
Property of stem cells
Reversible, physiological
process
Apoptosis and
differentiation is inhibited
High free radical scavenger
levels
Irreversible,
physiological
active process
Cell cycle inhibition is a
secondary effect
Irreversible,
non-physiological
process
Apoptosis
Autophagy
Necrosis

60. www.radbiol.ucla.edu
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 = Tpotwhere 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

61. www.radbiol.ucla.edu
Tumor Kinetics
Tc Cell cycle time
G.F. Growth fraction
Tpot Pot. doubling time
Td Actual doubling time
Cell loss factor
Human SCC
36 hrs
0.25
6 days
60 days
0.9
Rate of tumor growth, and the rate of tumor regression, are determined
largely by the cell loss factor!
VARIES GREATLY WITH TUMOR
(36hr x 4)
(1-6/60)

62. www.radbiol.ucla.edu
Tumor Regression
•Therateoftumorgrowthandregressionis
determinedby
•rateofcellloss(
•G.F.
•cellcyclekinetics
•Slowgrowingtumorsmayregressrapidly
•Rapidlygrowingtumorsareexpectedtoregress
andregrowrapidly
•Slowregressionisnotanindicationoftreatment
failure
•TherateoftumorregressionafterTxisnot,in
general,prognostic

63. www.radbiol.ucla.edu
Tumor Regeneration
Rat rhabdomyosarcoma
Hermans and Barendsen, 1969
Tumors can
regenerate at the
same time as
they regress!
Control
Irradiated
Surviving clonogens
measured in vitro
Growth delay
Time
Relative tumor
volume
X-rays

64. www.radbiol.ucla.edu
EVIDENCE FOR ACCELERATED REPOPULATION
IN TUMORS
•Time to tumor recurrence after therapy is shorter
than than would be expected from the original
growth rate
•Split-course radiation therapy often gives poor
results
•Protraction of treatment time often results in poor
results
•Accelerated treatment has been shown to be of
benefit in some circumstances.

65. www.radbiol.ucla.edu
Accelerated Tumor Repopulation
T2 and T3 SCC head and neck(excluding nasopharynx and vocal
cord).TCD
50values are consistent with onset of repopulation at 4
weeksfollowed by accelerated repopulation with a 3-4 day
doubling time, implying a loss in dose of about 0.6 Gy/dy
Withers et al, 1988
T2 T3
local control
no local control

66. www.radbiol.ucla.edu
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.

67. www.radbiol.ucla.edu
Accelerated repopulation in human tumors
provided the rationale for accelerated
fractionation protocols