Radiation Targets 2:

Cell Proliferation, Cell Death and Survival

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

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

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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

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.
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20-40Gy Seminoma, Dysgerminoma, Acute Lymphocytic leukemia,
Wilms’ tumor, Neuroblastoma
40-50Gy Hodgkin's, Lymphosarcoma, Seminoma, Histiocytic cell
sarcoma, Skin ca. (basal and squamous cell)
50-60Gy Squamous cell ca. (cervix, head and neck), Breast ca., Ovarian
ca.,Medulloblastoma, Retinoblastoma, Ewing's tumor
60-65Gy Larynx (<1 cm), breast cancer lumpectomy
70-75Gy Oral cavity (<2 cm, 2-4 cm), Oro-naso-laryngo-pharyngeal ca.,
Bladder ca., Cervix ca., Uterine ca., Ovarian ca., Lung ca. (<3
cm)
>80Gy Head 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.

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 Robert Hooke (1635-1703) was the first to use Not!
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”)
1906 Bergonie and Tribandeau. Action des
rayou X sur le testicle Elect. Med.14, 779
- radiosensitivity is related to cell proliferation

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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

Quiescence Senescence Terminal Death

Differentiation
Property of stem cells Irreversible, Irreversible, Apoptosis
Reversible, physiological non-physiological physiological Autophagy
process process active process Necrosis
Apoptosis and Cell cycle inhibition is a
differentiation is inhibited secondary effect
High free radical scavenger
levels

(all with distinct, and common, gene patterns)

IR is a pathological signal and can cause senescence

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 Radiation-Induced Senescence
Is particularly relevant to radiation fibrosis, but also
occurs in cells other than fibroblasts.

TGF-b

Proliferative p21 Post-mitotic

Progenitor Fibroblast
Fibroblast

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

• 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.

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 Lethal Sectoring in Mitotic Death
RT

RECURRENCE!

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

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Courtesy:
Randi Syljuasen

Control
Cells Irradiated
Cells

Control Irradiated
- -
Nuclei Nuclei
Stained Stained

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 Alternative Deathstyle Mechanisms

Programmed cell death type 1: Apoptosis

Programmed cell death type 2: Autophagy
Pathological Death: Necrosis
• Death is often an active process: cells decide to commit suicide
• Death pathways prevent carcinogenesis and mutations in them are
associated with cancer. They provide potential tumor-specific
targets for therapeutic intervention.
• Death pathways, and mutations in them, affect intrinsic cellular
radiosensitivity. They provide potential tumor-specific targets for
radiosensitization.

www.radbiol.ucla.edu
 Alternative Deathstyle Mechanisms

Physiologic Pathologic

Type 1: Apoptosis Type 1: Apoptosis

Type 2: Autophagy Type 2: Autophagy
Type 3: Necrosis
• Type 1 and 2 are Programmed
• Death is largely an active process: cells decide to commit suicide
• Death pathways prevent carcinogenesis and mutations in
molecules in these pathways are associated with cancer. They
provide potential tumor-specific targets for therapeutic intervention.
• The same death pathways and mutations affect intrinsic cellular
radiosensitivity. They provide potential tumor-specific targets for
radiosensitization.

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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

• Self sacrifice by infected/damaged cells

• Self sacrifice by immune cells and other normal
cells in the battle zone
• Causes inflammation
– wound healing
– immunity

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 Programmed Cell Death Type I: Apoptosis
Morphology

Apoptosis is 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!)

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

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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

• Apoptosis can be visualized in tissue sections

using terminal deoxynucleotidyl transferase
(TdT) to add fluorescein-labeled (dUTP)
nucleotides onto 3’-OH ends of DNA that result
from the action of the apoptotic endonuclease
• An Apoptotic Index (AI) can be derived

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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

Necrosis is 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.

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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

Lamin A Actin iCAD - CAD DNA-PKcs PARP

CAD
Cell DNA DNA
Shrinkage Fragmentation Repair

ICAD (inhibitor of caspase activated DNase)

DNA-PK (DNA protein kinase)
PARP (poly-ADP-ribose polymerase)
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 Radiation-Induced Apoptosis

INITIATORS DNA Damage Members of

Sphingomyelin TNFR family
with Death
FADD Domains
JNK ATM Ceramide
(TNFR1, Fas,
P38 MAPK x TRAIL)
Activation of
Pro-caspase 8
EFFECTORS
p53 Bax Mitochondria
JNK - jun kinase

Cytochrome c ATM - mutated in

ataxia
Pro-caspase 9 Caspase 9 Caspase 8 telangiectasia
Apaf-1
FADD - Fas
activated death
Apoptosome Complex domain
TERMINAL PHASE Apaf - apoptosis
Caspase 3, 6, 7 activating factor

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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

• Apoptotic cells reappear between radiation fractions

“There is only one serious philosophical problem. It is suicide. To judge

whether life is, or is not, worth living” Albert Camus

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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

DNA Damage Members of

INITIATORS
Stress TNFR family
Sphingomyelin
With Death
Ceramide Domains
JNK ATM
FADD
P38 MAPK x
NF-B
EFFECTORS
p53 Bax Mitochondria IAPs
Bcl-2/Bcl-xl
Cytochrome c
Caspase 9 Caspase 8
Apaf-1
Apoptosome Complex
IAP - inhibitors of apoptosis
TERMINAL PHASE
FLIP - FLICE (procaspase
Caspase 3, 6, 7 8) inhibitory protein

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 “Survival Pathways”
Growth Factors, Cytokines, Proliferative Signals

TNFR2 TNFR1 Sphingomyelin

Ceramide PI 3-kinase Ras

Raf

PDK1 Proliferation
Metabolic ERK
Pathway
AKT P90 RSK
NFB
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

• Intrinsic cellular radiosensitivity is determined in part by the balance of

the signals transducing cell death or survival pathways
• Clinical RT response is superior in tumors with pathways primed for an
active form of cell death, but the relationship between AI (or BAX/Bcl-
2) and local tumor control or patient survival after RT are controversial,
perhaps because excessive cell death often correlates with high cell
proliferation or because multiple pathways to cell death are possible
• Apoptosis may affect the clinical response of normal tissues to RT e.g.
serous cells - “dry mouth”
• In general, RT increases the A.I. only in cells with a pro-apoptotic
phenotype and apoptotic cells reappear between fractions of RT
• Enhancing PCD in a proportion of cells does not necessarily affect the
shape of the clonogenic survival curves following radiation - this
depends on the response of the surviving cells

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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

Flash label with

Fix and stain 3H-TdR or BdUR for 20 mins

If 3H-TdR
If BdUR labeled
labeled AR film
mitosis
*Anti-BdUR

Mitotic Index (M.I.)

= l TM/TC
U.V. microscopy Autoradiography
…
.... …
… .. .... …
…
… …
.. ..
…
…
.... …
…
……
.. … …
..…
………
.. .. …
..
.. .. ........
…
..

Where l is a correction factor

Labeling Index (L.I.) = l TS/TC for cell division, about 0.69
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Frequency of Labeled Mitosis Technique
(FLM)

• By counting the number of mitoses that are

labeled at various times after 3H-thymidine
incorporation, the time taken for a cell to
traverse a specific cell cycle phase, and the
cell cycle time, can be estimated
• But, it is easier to use BUdR and flow
cytometry

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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

Cells in fine stream

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 Flow Cytometry for DNA Quantity
1. label DNA with propidium iodide
(fluorescent dye)

2. measure light output by flow cytometry

3. analyze DNA histograms

4n 4n
G1
G2 M
# cells G2M
S
G1
S 2n
2n 4n
2n +  n
degree of fluorescence
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 Cell Cycle Kinetic Analysis by Flow Cytometry
P.I. (DNA - red) combined with Bromodeoxyuridine uptake followed by
staining with fluorescently labeled anti-BrdUrd (green)

BrdUrd BrdUrd BrdUrd

green green green
G1
G1 G1

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

The best estimates of kinetics come from use of

cells synchronized in a specific cell cycle phase
• Mitotic cells can be shaken off from some cell lines -
M phase cells
• Serum deprivation - G1 phase cells
• Hydroxyurea synchronizes cells at the G1/S transition

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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

P53 or ATM deficient irradiated

loss of G1/S checkpoint
and only 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

Cycling is growth factor independent through S, G2, M

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 Molecular Mechanism of Cell Cycle Progression

Progression through each checkpoint requires:

• Retinoblastoma (Rb) tumor suppressor gene family
• especially G1-S transition
• Regulatory Factors
• Cyclins that 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

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 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.

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 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

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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

– CDC25A = cyclin E/CDK2 = G1/S specific

– CDC25B = cyclin A/CDK2 = S-phase exit
– CDC25C = cyclin B/CDK1 = G2/M specific

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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

p53 is a transcription factor for p21, which is why it is

involved in cell cycle arrest after IR

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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

53BP1 53BP1 53BP1

MDC1 MDC1 MDC1
mediators
MRN MRN MRN
BRCA1 BRCA1 BRCA1
rapid
slow
MDM2
transducers p53 CHK2 CHK1 CHK2 CHK1

transactivation

effectors CDC25A phosphorylation CDC25A phosphorylation CDC25C phosphorylation

p21 and nuclear export

CDC25A degradation
P-thr14/tyr15 P
P-thr14/tyr15
CDK2 CDK!
CDK2
p21 CDK2
CYCLIN E CYCLIN B
CYCLIN A/E
CYCLIN E

S Phase G2/M Arrest

G1/S Arrest
Sensescence/transient Arrest
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 Cell Cycle in Cancer

• 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

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 Cancer

Growth Factor/Cytokine
Receptor Survival

Oncogenes Signals
PI3K
Ras NF-B
Raf
MAPK

Proliferation Cell death

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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,

NGF, IGFR, DR5, COX-2 Cell proliferation

Proteasome inhibition
Mitochondrial damage
Activation of EGFR,
TGF-b, etc

Tissue recovery Cell death

/lesion formation Cell proliferation /survival

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 Loss of Proliferative Ability can Occur in
Different Ways

Quiescence Senescence Terminal Death

Differentiation
Property of stem cells Irreversible, Irreversible, Apoptosis
Reversible, physiological non-physiological physiological Autophagy
process process active process Necrosis
Apoptosis and Cell cycle inhibition is a
differentiation is inhibited secondary effect
High free radical scavenger
levels

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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

Tc Cell cycle time 36 hrs

G.F. Growth fraction 0.25

Tpot Pot. doubling time 6 days (36hr x 4)

Td Actual doubling time 60 days

Cell loss factor 0.9 (1-6/60)

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

Time Rat rhabdomyosarcoma

Hermans and Barendsen, 1969

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 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.

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 Accelerated Tumor Repopulation
T2 T3
local control

no local control

T2 and T3 SCC head and neck (excluding nasopharynx and vocal

cord). TCD50 values are consistent with onset of repopulation at 4
weeks followed 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

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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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