Rob Stewart, Ph.D.

1
Can we predict radiation carcinogenesis from
first principles?

• Rob Stewart, Ph.D.
• School of Health Sciences
• Purdue University
• 550 Stadium Mall Drive
• West Lafayette, IN 47907-2051
• April 11, 2003

2
Nature of The Beast
In many situations, cells and tissues are exposed
to temporally and spatially complex radiation
fields
3
Radiation field depends on particle energy
5,000 mm
25,000 mm
1 MeV e- in water
5 MeV e- in water
4
the type of radiation
500 mm
500 mm
250 keV e in water
250 keV e- in water
5
and the initial direction(s) of flight
500,000 mm (50 cm)
500,000 mm (50 cm)
1 MeV photons in water
10 MeV photons in water
6
Dose is only the first step
Cancer develops through physical, chemical,
biochemical and microevolutionary processes that
happen over hours, days, months and even years.
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The Infamous Double Strand Break (DSB)

• Pose a major threat to integrity of the genome
• Created by
• Certain chemotherapeutic drugs (e.g., bleomycine)
• Spontaneously as a by-productive of cellular
  processes
• Oxidative metabolism
• Replication fork encounters a single-strand break
• Ionizing radiation (including of course cosmic
  rays, dental x-rays, radon, 40K, etc.)

Cytosine (C)
Guanine (G)
Adenine (A)
Thymine (T)
Complementary base pairs encode genetic
information and provide opportunities for
error-free repair.
Double strand break (DSB)
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Are all DSBs lethal?
DSBs are distributed among identically irradiated
cells according to a Poisson distribution
Some cells survive because they do not sustain
critical DNA damage (i.e., a DSB).
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Most DSBs are rejoined and non-lethal

• Radiation creates 25-40 DSB Gy-1 cell-1.
• Less than 4 of the initial DSBs are lethal.

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How are DSBs rejoined?

• Homologous recombination (HR)
• Gene conversion
• Single-strand annealing
• Non-homologous end joining (NHEJ)

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Homologous DNA

• Sister chromatid
• Homologous chromosome
• Repetitive DNA sequences

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Homologous Recombination (HR)

• Requires extensive regions of homology
• Allelic recombination
• Sister chromatid
• Homologous chromosome
• Ectopic recombination
• Other regions of genome with sequence homology
• Holiday junction resolution is not random
• Gene conversion without cross over more frequent
  than gene conversion with cross over.

HR has the potential to rejoin DSBs with no loss
in genetic information (error-free repair)
Adapted from M. van den Bosch, P.H.M Lohman, and
A. Pastink, DNA Double-Strand Break Repair by
Homologous Recombination, Biol. Chem. 383,
873-892 (2002)
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Non-Homologous End Joining (NHEJ)

• DSB is recognized by DNA protein kinase (DNA-PK)
• KU80/KU70 heterodimer
• Catalytic sub-unit DNA-PKcs
• Ligase IV and XRCC4 co-factor promote ligation of
  the DNA break ends

NHEJ is an error prone DSB restitution pathway
Adapted from F. Daboussi, A. Dumay, F. Delacote,
and B.S. Lopez, DNA double-strand break repair
signaling The case of RAD51 post-translational
regulation, Cellular Signaling 14, 969-975 (2002)
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DSB repair in mammalian cells

• HR is potentially error-free. But inappropriate
  HR can lead to large DNA rearrangements
  (chromosome aberrations).
• Impaired or increased HR has been associated with
  a predisposition towards cancer
• NHEJ is highly mutagenic but consequences are
  usually less severe.
• NHEJ predominates in G0 (quiescent cells) and in
  G1/early S phase cells. HR is important in late
  S/G2 phase.
• DSB repair is not the same in quiescent and
  actively dividing cells.
• DSB repair is a function of cell cycle phase.
• HR and NHEJ are regulated through a complex set
  of signaling pathways.
• Overall rate and fidelity of DSB repair can be
  disrupted in many different ways.

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Local damage complexity
DNA organized into a chromatin fiber
x
Simple double strand break
Track
x
Track
Complex double strand break
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DSB repair may be affected by damage complexity

• Not difficult to imagine that collateral damage
  near the site of two opposing strand breaks could
  impair
• Resection of damaged break ends
• DNA synthesis
• Branch migration and Holiday junction formation
• Disruptions in HR would likely depend on the
  spatial configuration and types of nearby damage
  sites.

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Pairwise damage interaction
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Proximity effects
Regional Multiply Damaged Sites

• One radiation track can create multiple DSB.
• Some DSBs may be in close spatial proximity.
• Break ends in close temporal and spatial
  proximity are more likely to interact than ones
  separated in time or space.
• Frequency of pairwise damage interaction
  increases with increasing particle LET.

x
x
x
Track
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Repair-misrepair (RMR) model
DSBs are created and rejoined
Gy h-1 at time t
pairwise damage interaction
Repair processes convert fraction (1-a) of the
initial DSBs to lethal or non-lethal mutations
Non-lethal
Lethal
C.A. Tobias, The repair-misrepair model in
radiobiology comparison to other models. Radiat.
Res. Suppl. 8S77-S95 (1985).
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Surviving fraction
S(t) is the fraction of cells free of lethal
damage at time t. Lethal damage created during a
short time interval dt, whose average is dF/dt,
are randomly distributed among cells without
regard for which cells already have lethal damage.
Surviving fraction at time t
For a review, see R.K. Sachs, P. Hahnfeld, and
D.J. Brenner, Review The link between low-LET
dose-response relations and the underlying
kinetics of damage production/repair/misrepair.
Int. J. Radiat. Biol. 72(4) 351-374 (1997).
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Virtual Cell (VC) Software

• Simulates the repair and misrepair of DNA damage
• LPL model (Curtis 1986)
• RMR model (Tobias (1985)
• TLK model (Stewart 2001)
• Predicts endpoints such as
• Expected number of DSB as a function of time
• Fraction of cells that survive irradiation
• Fraction of cells that acquire genetic
  instability and become unstable (transformed)
• Tumor control probability after radiation therapy
• Expected time of tumor reoccurrence after
  radiation therapy

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Split-dose Experiment
RMR parameters for CHO cells
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External beam radiation therapy
S ? (SF2)30 7.27 10-3
S 5.410-3
SF2 0.849
A 60 Gy radiation treatment (2 Gy 30) delivered
over 6 weeks (M-F skipping weekends). The 2 Gy
daily doses are delivered at 6 Gy h-1 ( 2 Gy/20
minutes).
RMR parameters for CHO cells
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Brachytherapy
S 3.910-4
A 125I seed that delivers 150 Gy in 1.1 years.
Dose rate decreases exponentially with a
half-life of 1,443 h (peak dose rate 72.4 mGy
h-1).
RMR parameters for CHO cells
25
Combined radiation treatments
Hypothetical combined external beam and
brachytherapy radiation treatment (160 Gy total
delivered dose).
RMR parameters for CHO cells
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RMR and LQ survival models are related

• The widely used linear-quadratic (LQ) survival
  model may be written as

Equating S(D) and S(?) gives
See M. Guerrero, R.D. Stewart, J. Wang, and X.A.
Li. Phys. Med. Biol. 47, 31973209 (2002) and RK
Sachs, P. Hahnfeld, DJ Brenner. Int. J. Radiat.
Biol. 72(4), 351-74 (1997).
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A mechanistic interpretation of the LQ
Accuracy of repair process
Rate of DSB rejoining

Pairwise damage interaction process
always creates a mutation (chromosome
aberration). But not all of them are lethal.
Expect a/b ratio to increase as rate of DSB
rejoining (l) increases.
28
Prediction of LQ parameters from first
principles a tantalizing possibility

• Small black filled symbols generated using Monte
  Carlo sampling methods
• Large red symbols parameter values obtained from
  the direct analysis of measured survival data

29
Lack of a dose rate effect is insufficient
evidence to infer no repair
dose rate effects
30
Damage formation and repair is still only the
beginning
Cancer develops through physical, chemical,
biochemical and microevolutionary processes that
happen over hours, days, months and even years.
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Tumor growth kinetics

• Exponential cell kinetics are sometimes observed

Cell birth rate (h-1)
Cell death rate (h-1)
Doubling time
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Radiation therapy for the treatment of prostate
cancer

• Prostate tumor composed of 107 tumor cells.
• Wang et al. (2003) radiosensitivity parameters

JZ Wang, M. Guerrero, XA Li. How low is the
alpha/beta ratio for prostate cancer? Int. J.
Radiat. Oncol. Biol. Phys. 55(1), 194-203 (2003).
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Tumor control probability (TCP)
Dose in parentheses is the treatment dose that
gives a TCP of 90

• Prostate tumor composed of 107 tumor cells.
• Wang et al. (2003) radiosensitivity parameters

JZ Wang, M. Guerrero, XA Li. How low is the
alpha/beta ratio for prostate cancer? Int. J.
Radiat. Oncol. Biol. Phys. 55(1), 194-203 (2003).
34
Multi-stage cancer model(s)

• Through a series of mutational events, stem cells
  acquire minor and enhanced genetic instability
  and other traits.
• Tumor forms through the clonal expansion of the
  unstable cell population.
• Cell birth/death processes may change as cells
  progress towards malignancy.

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Incidence of lung cancer

• At background radiation levels (75 to 225 mGy),
  endogenous processes may account for 70 to 90 of
  lung cancers.
• At 1 Gy, endogenous processes may account for as
  much as 30 of lung cancers.

Estimated lung cancer incidence with and without
DNA damage caused by endogenous processes.
H. Schöllnberger, R.D. Stewart, R.E.J. Mitchel,
and W. Hofmann, An examination of radiation
hormesis mechanisms using a multi-stage
carcinogenesis model. In progress. Abstract
submitted to ICRR 2003 Brisbane, Australia (2003).
36
Induction of cellular defense mechanisms

• A 3-fold low dose (rate) enhancement in DNA
  repair and radical scavenging would provide
  support for an effective threshold.

Estimated lung cancer incidence with and without
low dose (rate) adaptations in radical scavenging
and DNA repair.
H. Schöllnberger, R.D. Stewart, R.E.J. Mitchel,
and W. Hofmann, An examination of radiation
hormesis mechanisms using a multi-stage
carcinogenesis model. In progress. Abstract
submitted to ICRR 2003 Brisbane, Australia (2003).
37
Comments

• Multi-stage models that use exponential cell
  growth kinetics are extremely sensitive to the
  selection of a net cell birth rate (a-b) and
  have conceptual difficulties
• For lung cancer, (a-b) 0.012 0.001.
• Cell density lt 108 to 109 cells cm-3.
• Tumor size has a finite upper bound 1014 or
  1015 cells.
• Over extended periods of time (months or years),
  age, health status, etc., etc., will impact on
  cancer development
• Cell birth/death parameters will change over time
  and most likely has a stochastic (chaotic)
  element.
• Wounds or disease may temporarily alter the
  tissue microenvironment and accelerate the clonal
  expansion of aberrant cells.
• Normal cells affect behavior of transformed cells
  and vice versa
• Cell signaling (bystander) effects.

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Cell signaling

• Cells in higher animals coordinate cellular
  activities using hundreds of different kinds of
  signaling molecules
• Proteins, small peptides, amino acids,
  nucleotides, steroids, retinoids, fatty acid
  derivatives, and gases such as nitric oxide (NO)
  and carbon monoxide
• Signaling can be long range (synaptic and
  endocrine signaling) or short range (autocrine
  and paracrine signaling)

Goodbye friends. Ive caught a virus and must
leave you.
Maybe Ive got the virus and just dont know it
yet
Direct cell-to-cell communication through gap
junctions
Autocrine and paracrine signaling through
excreted messengers
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Why do we care?

• Medium transfer and single-cell irradiator
  experiments demonstrate that radiation-damaged
  cells emit signals that cause radiation-like
  changes in nearby undamaged cells
• changes in gene expression, mutations, increases
  in sister chromatid exchanges, induction of
  chromosomal instability, and cell transformation

• Cell birth, differentiation and death processes
  are highly regulated through multiple signaling
  networks.

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Growth Inhibition

• Cell growth in vivo is limited (at a minimum) by
  the availability of space, nutrients, and growth
  factors
• Cells compete with each other for resources

Life support capacity
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Microevolution of a tumor

• Normal and transformed cells vie for resources

Normal cells
Tumor cells
Crowding effects
Over-expression of growth factor receptors
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Virtual Tissue Model (VTM)

• System of 6 or 7 differential equations describe
  the in vivo cell system (i.e., the Virtual
  Tissue).
• Normal cells and transformed cells vie for
  resources.
• Tissue microenvironment is re-shaped as the
  relative number of normal and transformed cells
  changes.

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Can we simulate cancer from first principles?

• Yes!
• No!
• Maybe

Computational issues
Whats a first principle?
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A lot more can be done
Acknowledgement The Virtual Cell software
development effort is supported in part by the
U.S. Department of Energy's Low Dose Radiation
Research Program through the Office of Science
(BER), Grant Number DE-FG02-03ER63541.