Can we predict radiation carcinogenesis from first principles?

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.

7. 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)

8. CHO cells 0.12 Gy h-1 0.5 Gy h-1 45 Gy h-1 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).

9. 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.

10. How are DSBs rejoined? • Homologous recombination (HR) • Gene conversion • Single-strand annealing • Non-homologous end joining (NHEJ)

11. Homologous DNA • Sister chromatid • Homologous chromosome • Repetitive DNA sequences

12. 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)

13. 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 Signaling14, 969-975 (2002)

14. 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.

15. Local damage complexity DNA organized into a chromatin fiber x Simple double strand break Track x Complex double strand break Track

16. 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.

17. Pairwise damage interaction

18. Proximity effects • 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. Regional Multiply Damaged Sites x x x Track

19. 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.8:S77-S95 (1985).

20. 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).

21. CHO cells 0.12 Gy h-1 0.5 Gy h-1 45 Gy h-1 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

22. Dose rate (Gy h-1) 4 hour Time (h) S = 0.12 Split-dose Experiment RMR parameters for CHO cells

23. External beam radiation therapy S (SF2)30 = 7.27 × 10-3 S = 5.4×10-3 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

24. Brachytherapy S = 3.9×10-4 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. Sebrt × Sbrachy= 4.8×10-5 Scombined = 7.4×10-6 Sbrachy(100 Gy) = 8.9×10-3 Sebrt(60 Gy) = 5.4×10-3 Combined radiation treatments Hypothetical combined external beam and brachytherapy radiation treatment (160 Gy total delivered dose). RMR parameters for CHO cells

26. 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, 3197–3209 (2002) and RK Sachs, P. Hahnfeld, DJ Brenner. Int. J. Radiat. Biol.72(4), 351-74 (1997).

27. 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 implies • A small fraction of the initial damage is unrepairable (< 2%) • complex DSBs? • Rapid DSB rejoining • G → 0 (large ) • (/) << 1 • Rapid DSB fixation competes with 1st and 2nd order repair. • Other possibilities 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.

31. Tumor growth kinetics • Exponential cell kinetics are sometimes observed Cell death rate (h-1) Cell birth rate (h-1) Doubling time

32. 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).

33. 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.

35. 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 < ~ 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.

38. 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. I’ve caught a virus and must leave you. Maybe I’ve got the virus and just don’t know it yet Direct cell-to-cell communication through gap junctions Autocrine and paracrine signaling through excreted messengers

39. 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.

40. Radiation killing 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

41. Microevolution of a tumor • Normal and transformed cells vie for resources Normal cells Tumor cells Crowding effects: Over-expression of growth factor receptors

42. 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.

43. Mechanisms and parameters Can we simulate cancer from first principles? • Yes! • No! • Maybe… Computational issues What’s a first principle?

44. http://healthsciences.purdue.edu/vc/ 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.