the influence of trp53 in the dose response of radiationinduced ...

Dose-Response: An International Journal Volume 12 | Issue 3

Article 4

9-2014

THE INFLUENCE OF TRP53 IN THE DOSE RESPONSE OF RADIATIONINDUCED APOPTOSIS, DNA REPAIR AND GENOMIC STABILITY IN MURINE HAEMATOPOIETIC CELLS Jennifer A Lemon McMaster University, Hamilton, ON, Canada

Kristina Taylor McMaster University, Hamilton, ON, Canada

Kyle Verdecchia McMaster University, Hamilton, ON, Canada

Nghi Phan McMaster University, Hamilton, ON, Canada

Douglas R Boreham McMaster University, Hamilton, ON, Canada

Follow this and additional works at: http://scholarworks.umass.edu/dose_response Recommended Citation Lemon, Jennifer A; Taylor, Kristina; Verdecchia, Kyle; Phan, Nghi; and Boreham, Douglas R (2014) "THE INFLUENCE OF TRP53 IN THE DOSE RESPONSE OF RADIATIONINDUCED APOPTOSIS, DNA REPAIR AND GENOMIC STABILITY IN MURINE HAEMATOPOIETIC CELLS," Dose-Response: An International Journal: Vol. 12: Iss. 3, Article 4. Available at: http://scholarworks.umass.edu/dose_response/vol12/iss3/4

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Lemon et al.: Trp53 status on induced DNA repair and genomic stability

Dose-Response, 12:365–385, 2014 Formerly Nonlinearity in Biology, Toxicology, and Medicine Copyright © 2014 University of Massachusetts ISSN: 1559-3258 DOI: 10.2203/dose-response.14-008.Lemon

THE INFLUENCE OF TRP53 IN THE DOSE RESPONSE OF RADIATIONINDUCED APOPTOSIS, DNA REPAIR AND GENOMIC STABILITY IN MURINE HAEMATOPOIETIC CELLS

Jennifer A. Lemon, Kristina Taylor, Kyle Verdecchia, Nghi Phan, Douglas R. Boreham 䊐 McMaster University, Department of Medical Physics and Applied Radiation Sciences, Hamilton, ON L8S 4K1 Apoptotic and DNA damage endpoints are frequently used as surrogate markers of cancer risk, and have been well-studied in the Trp53+/- mouse model. We report the effect of differing Trp53 gene status on the dose response of ionizing radiation exposures (0.012 Gy), with the unique perspective of determining if effects of gene status remain at extended time points. Here we report no difference in the dose response for radiationinduced DNA double-strand breaks in bone marrow and genomic instability (MN-RET levels) in peripheral blood, between wild-type (Trp53+/+) and heterozygous (Trp53+/-) mice. The dose response for Trp53+/+ mice showed higher initial levels of radiation-induced lymphocyte apoptosis relative to Trp53+/- between 0 and 1 Gy. Although this trend was observed up to 12 hours post-irradiation, both genotypes ultimately reached the same level of apoptosis at 14 hours, suggesting the importance of late-onset p53-independent apoptotic responses in this mouse model. Expected radiation-induced G1 cell cycle delay was observed in Trp53+/+ but not Trp53+/-. Although p53 has an important role in cancer risk, we have shown its influence on radiation dose response can be temporally variable. This research highlights the importance of caution when using haematopoietic endpoints as surrogates to extrapolate radiation-induced cancer risk estimation.

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Keywords: Trp53, cell cycle arrest, apoptosis, DNA damage, ionizing radiation

INTRODUCTION

Functional p53 is critical for the maintenance of homeostasis and genome integrity of hematopoietic tissue. Following genotoxic damage, p53 protein triggers cell cycle arrest, allowing DNA repair or apoptosis to clear heavily damaged cells (Chaabane et al. 2013, Reinhardt and Schumacher 2012). Cells deficient in p53 protein have abrogated cell cycle arrest and reduced p53-dependent apoptosis (Livingstone et al. 1992, Muller and Vousden 2013). In vivo, reduced levels of functional p53 protein in heterozygous (Trp53+/-) mice results in decreased survival and a shorter tumour latency period in Trp53+/- mice relative to wild-type mice (Jacks et al. 1994, Carlisle et al. 2010). Null mice (Trp53-/-) develop tumours earlier than heterozygotes, with a correspondingly shorter lifespan (Donehower and Lozano 2009). Lymphomas and sarcomas predom-

Address correspondence to Jennifer A. Lemon, Email: [email protected]; Tel: 905525-9140 ext. 27538; Fax: 905-522-5982 365

Published by ScholarWorks@UMass Amherst, 2014

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Dose-Response: An International Journal, Vol. 12 [2014], Iss. 3, Art. 4

J. A. Lemon and others

inate in the tumour spectrum observed in this p53-deficient strain indicating that certain tissues are more predisposed to tumorigenesis caused by the p53 deficiency (Jacks et al. 1994). High dose ionizing radiation exposure (1-4 Gy) has been shown to further reduce the lifespan and tumour latency in null and heterozygous, relative to unirradiated isogenic mice (Kemp 1994). In Trp53+/- mice, the median lifespan of mice with malignant tumours was reduced by 45.4±2.6 days per Gy (Reinhardt and Schumacher 2012). The quantity and activity of p53 in the cell is regulated through post-translational modifications (Fei and El-Deiry 2003), under normal, unstressed conditions, the p53 protein is maintained at a low concentration through a continual degradation process initiated by the ubiquitinylation of p53 by MDM2 (Momand et al. 1992). In response to ionizing radiation, a series of coordinated cellular responses forms what is known as the DNA damage response (DDR) pathway. Within minutes of a DNA double strand break (DSB) created by ionizing radiation exposure, the histone H2A.X is phosphorylated by PIK-kinases (ATM, DNAPK) at the site of nascent DNA DSBs to form γH2A.X foci (Fernandez-Capetillo et al.2004, Stiff 2004, Rothkamm and Horn 2009). This acts as a scaffold for repair proteins to access the DSB. Activated ATM also acts as a transducer of the damage signal and phosphorylates p53 (directly or indirectly) to enable stabilization and accumulation of p53 in the nucleus where it forms a tetrameric complex (Shu et al. 2004). This complex binds to specific downstream gene targets, p53 responsive elements, activating the transcription of proteins that will allow the cell to process the radiation damage via cell cycle arrest, apoptosis, senescence or repair. Cell cycle arrest occurs to enable repair of damage before the cell progresses to division. In mammalian cells, DSBs are repaired using non-homologous end-joining (NHEJ) or homologous recombination (HR). Once the break is repaired, de-phosphorylation of γH2A.X generally occurs (Bonner et al. 2008, Redon et al. 2011). In the event that the damage sustained by the cell is too substantial, the cell undergoes apoptosis. Morphological changes, such as the appearance of phosphatidylserine on the outer membrane of the cell, signals the execution phase of apoptosis (Van Engeland et al. 1998), however the outcome of the cell in response to external signals ultimately depends on the genetic background, microenvironment, tissue type, and the strength/nature of the p53 activating stimuli (Fridman and Lowe 2003). In cells lacking p53 functionality, cells are not able to respond adequately to radiation response signals, enabling the inappropriate survival of damaged cells (Kunugita et al. 2002). As cells continue to cycle, unrepaired or misrepaired DSBs in haematopoietic progenitors (erythroblasts) will lead to micronuclei in newly formed reticulocytes following mitosis (Dertinger et al. 2007). As a biomarker of genetic instability, micronucleated reticulocytes (MN-RETs) are indicative of chromosome damage in bone marrow 366

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Trp53 status on induced DNA repair and genomic stability

progenitor cells, which appears to be an important contributor to tumorigenesis in p53-deficient mice (Symonds et al. 1994, Liu et al. 2004). The aim of this work was to compare the radiation sensitivity of haematopoietic cells in normal Trp53+/+ mice to that of isogenic heterozygous Trp53+/- mice. The spontaneous and radiation-induced responses for various doses and time points post-irradiation were evaluated in order to gain a comprehensive understanding of the role of γH2A.X foci formation, cell cycle delay, apoptosis and micronucleus formation as a surrogate for cancer risk in normal and cancer-prone mice. MATERIALS AND METHODS Mice

Male mice with a single defective copy of Trp53 (B6.129S2Trp53tm1Tyj/1) were bred with wild-type female mice (129X1/SvJ) (Jackson Laboratory, Bar Harbour, Maine). The F1female progeny produced from this cross were genotyped at 4-5 weeks of age and used in experiments at 7-9 weeks of age. All F1 mice were housed five to a cage, in specific pathogen free (SPF) conditions, maintained on a 12 hour light/dark cycle at a room temperature of 24 ± 1°C. Food and water were available ad libitum. Protocols were approved by the Animal Research Ethics Board at McMaster University and carried out in accordance with the Canadian Council on Animal Care. Genotyping

Mice were anaesthetized (2% Isoflurane™ via inhalation) and a tail snip was collected in 95% ethanol to identify mouse genotype as either wild-type (Trp53+/+) or heterozygous (Trp53+/-) using polymerase chain reaction (PCR) as described previously (Jacks et al. 1994). Mice were ear punched at this time for subsequent identification and sorting. Irradiations

All irradiations were performed using a Cs-137 source (662 keV γ-rays) located at McMaster University. For in vivo irradiations, mice were placed in a customized sectioned polycarbonate restraint tube and irradiated at a dose-rate of 0.35 Gy/minute. In vitro samples were irradiated at a dose-rate of 0.19 Gy/minute. Samples were irradiated in ice slurry at 0°C. Reagents

All tissue culture reagents were purchased from Invitrogen Canada (Burlington, ON), unless otherwise stated. All other chemicals were purchased from Sigma-Aldrich (Mississauga, ON) unless otherwise stated. 367


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γH2A.X Assay

Spontaneous and radiation-induced γH2A.X foci were analyzed as mean fluorescence levels measured in lymphocyte-rich bone marrow cultures from Trp53+/+ and Trp53+/- mice using an Epics XL flow cytometer (Beckman Coulter, Miami FL), equipped with a 488 nm argon laser. All samples were maintained at 0°C during preparation unless otherwise specified. The lymphocyte-rich population was identified based on the forward-scatter/side-scatter (FSC/SSC) properties of the sample population. Within this population, only G0/G1 cells were analyzed for mean γH2A.X fluorescence. Cells in G2/M interfere with this assay (MacPhail et al. 2003) and were excluded from analysis based on relative DNA content using propidium iodide (PI). Mice were euthanized via cervical dislocation and a bone marrow sample was harvested by flushing both femurs with cold heparinized RPMI 1640 media (Lonza Inc., Allendale, NJ). Bone marrow suspensions were adjusted 1 x 106 cells/mL with complete media (RPMI 1640 supplemented with 10% Fetal Bovine Serum (FBS), 1% L-Glutamine (2mM), 1% Penicillin (100U/ml)-Streptomycin (100ug/ml)) and irradiated at a range of doses (0, 0.5, 1, 2 or 4 Gy). Following irradiation, samples were incubated at 37°C for the prescribed incubation period. At 0, 15, 30, 60 or 180 minutes post-irradiation, sample aliquots were removed and placed in duplicate flow tubes. These samples were fixed by the addition of cold 70% ethanol (EtOH) and incubated on ice for 1 hour. Samples were then stored at -20°C until analysis. Prior to staining, samples were centrifuged at 200x g and washed once with Tris-buffered saline (TBS). Cell pellets were resuspended in TST (TBS + 4% FBS + 0.1% Triton X-100) and incubated on ice for 10 minutes. Samples were then centrifuged for 7 minutes at 200x g, the supernatant removed and resuspended in TST containing 1:400 dilution of anti-phospho-H2A.X (ser139) antibody (Upstate Cell Signaling, Charlottesville, VA). Following a 2h room temperature (RT) incubation, samples were washed with cold TST, centrifuged and re-suspended in TST containing a 1:500 dilution of AlexaFluor™ 488-conjugated goat anti-rabbit IgG F(ab’)2 antibody (Invitrogen Canada) and incubated for 1 hour. Prior to sample analysis, samples were washed in TBS, centrifuged, and re-suspended in TBS + PI solution. During analysis, cells were gated on the lymphocyte-rich cell population and data from 5x104 cells was acquired. Cell Cycle Analysis

Mice were euthanized via cervical dislocation and a bone marrow sample was harvested by flushing both femurs with cold heparinized RPMI 1640 media (Lonza Inc., Allendale, NJ). Bone marrow suspensions were adjusted 1 x 106 cells/mL with complete media (RPMI 1640 supple368


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mented with 10% Fetal Bovine Serum (FBS), 1% L-Glutamine (2mM), 1% Penicillin (100U/ml)-Streptomycin (100ug/ml), 2.5% 1M HEPES solution) and irradiated with 2 Gy. Following irradiation, samples were incubated at 37°C for the prescribed incubation period. At 0, 2, 4, or 6 hours post-irradiation, sample aliquots were removed and fixed by the addition of cold 70% ethanol (EtOH) and incubated on ice for 1 hour. Samples were then stored at -20°C until analysis. Prior to staining, samples were centrifuged at 200x g and washed once with Tris-buffered saline (TBS). Cell pellets were resuspended in TST (TBS + 4% FBS + 0.1% Triton X-100) and incubated on ice for 10 minutes. Samples were then centrifuged for 7 minutes at 200x g, the supernatant removed and resuspended in TST containing a 1:60 dilution of PI. Samples were incubated for 10 minutes at RT and immediately analyzed by flow cytometry. Data analysis was performed using Multicycle AV software (Phoenix Flow Systems, San Diego CA). Apoptosis Assay

Annexin V and 7-Amino Actinomycin D (Annexin V-FITC/7-AAD kit, Beckman Coulter), in combination with platelet specific antibody CD61 (CD61 PE, Beckman Coulter) and leukocyte specific antibody CD45 (CD45 PE-TR, Caltag Labs), were used to identify apoptotic lymphocyte populations. Events positive for CD61 were eliminated to minimize platelet contamination. Four independent experiments were performed to determine whether differences existed in the spontaneous and radiation-induced responses of peripheral blood lymphocytes from Trp53+/+ and Trp53+/- mice. Mice were anaesthetized, blood was collected by cardiac puncture and diluted with complete medium to a lymphocyte concentration of 1x106 cells/ml. Samples were aliquoted and irradiated on ice in a range of doses (0, 0.25, 0.5, 0.75 or 1 Gy), followed by incubation at 37˚C (5% CO2, 98% humidity) for the requisite time periods (0, 4, 6, 8, 10, 12 or 14 hours). Following incubation, red blood cells were lysed using an ammonium chloride (NH4Cl) solution (0.154M ammonium chloride, 1.5 mM potassium bicarbonate, 0.1mM EDTA), centrifuged and the supernatant removed. Cells were resuspended in an antibody cocktail (CD45 PE-TR, CD61 PE, Annexin V and 7-AAD) in binding buffer. Samples were incubated on ice for 15 minutes, prior to analysis. A total of 2x104 lymphocytes (CD45+/CD61-) were analyzed for each sample. MN-RET Assay

All antibodies, stains and reagents were supplied in Mouse MicroFlowPLUS® (Litron Laboratories, Rochester NY) commercial kit. Newly formed reticulocytes (RETs), are differentiated from mature erythrocytes and platelets in peripheral blood using CD71 and CD61 antibodies. DNA content is then analyzed within the RET population and 369


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MN-RETs are differentially identified through calibration with supplied positive control samples. A total of three independent experiments were performed to assess any effects of genotype on MN-RET formation between Trp53+/+ and Trp53+/- mice. For dose response experiments, mice were irradiated in vivo to absorbed whole body doses of 0, 0.1, 0.25, 0.5, 0.75, 1.0 or 2.0 Gy. Blood was collected 43 hours post-irradiation into heparinized RPMI 1640. For time course experiments, blood was collected using a repeat facial vein bleeding technique at 41, 43, 44, 51, 68 and 137 hours following irradiation with 0 or 1 Gy. Blood samples were fixed within 4 hours of collection as per manufacturer’s instructions. The blood/anticoagulant mix was forcefully added to ultra-cold (-80°C) 100% methanol (MeOH). Fixed blood specimens were stored at -80°C for a minimum of 24 hours prior to preparation and analysis. Fixed blood samples were washed with kit-supplied binding buffer, centrifuged and the supernatant removed. The pellet was resuspended in labelling solution (RNAase, anti-CD-71-FITC and anti-CD61-PE in binding buffer) followed by two incubation periods: 30 minutes at 0°C, followed by 30 minutes at RT. Prior to flow acquisition, cold buffer + PI solution was added to each sample. The flow cytometric sequential gating logic used has been published (Dertinger et al. 1996), for each sample, 2x104 CD71+CD61- events (RETs) were collected and the percentage of MN-RETs was identified within this RET population based on PI content. Statistical Analysis

Statistics were performed using Sigma Plot version 11.0 (Systat Software, Germany). Experimental results represent the mean ± standard error (SEM), with two-sided P values ≤ 0.05 deemed statistically significant. Analysis of Variance (ANOVA) was used to assess the relationship between gene status, dose, time and the resulting biological response for the 3 endpoints. Statistically significant differences were probed using the Bonferroni post-hoc test. Additional t-tests were performed, where appropriate, to determine if significant differences existed between any experimental groups. Dose responses between Trp53+/+ and Trp53+/- mice were compared using multiple linear regression analysis. RESULTS γH2A.X

The spontaneous levels of γH2A.X were measured at 0, 15, 30, 60 and 120 minutes post-irradiation (Figure 1A). There were no significant differences between Trp53+/+ and Trp53+/- mice for spontaneous γH2A.X levels at any time point (P > 0.294). Time, however, was found to affect spontaneous γH2A.X levels (P < 0.006); the 0 minute samples had higher levels (P < 0.01 - 0.05) over all later time points, and were the same for 370


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FIGURE 1. γH2A.X fluorescence in the bone marrow of wild-type Trp53+/+ (closed squares) and heterozygous Trp53+/- (open circles) mice (A) Spontaneous and (B) radiation-induced kinetics were evaluated at 0, 15, 30, 60, 120 minutes post-irradiation. (C) Radiation-induced γH2A.X dose response was evaluated at 30 minutes following 0, 1 or 2 Gy. Data points represent mean ± standard error, n = 3, tested in duplicate.

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both genotypes. Radiation-induced γH2A.X (1 Gy) were calculated by subtracting spontaneous levels from each time point for each genotype to determine radiation-induced values (Figure 1B). Both genotypes showed similar time dependent (P < 0.001) kinetic patterns as fluorescence increased rapidly between 0 and 15 minutes post-irradiation, reached a maximum at 30 minutes post-irradiation of 1.35 ± 0.09 and 1.30 ± 0.07 for Trp53+/+ and Trp53+/- respectively. Levels decreased at a comparatively slower rate between 30 and 120 minutes post-irradiation and p53 status did not appear to influence the kinetics of γH2A.X foci formation or disappearance (P > 0.198). The dose response of Trp53+/+ and Trp53+/bone marrow lymphocytes were investigated at 30 minutes post-irradiation (Figure 1C). Again, spontaneous values were subtracted from each genotype curve. There was a significant dose response for both Trp53+/+ and Trp53+/- (P < 0.001) but genotype did not affect this dose response (P > 0.590). Cell Cycle Analysis

Bone marrow was collected from Trp53+/+ and Trp53+/- mice, aliquoted and irradiated with 0 or 2 Gy γ-radiation and samples were analyzed for cell cycle alterations out to 12 hours post-irradiation. Spontaneous G1 levels were subtracted from the 2Gy values to show only radiation-induced differences in cell cycle and normalize for in vitro culture artifacts. Trp53+/+ mice showed robust G1 cell cycle arrest at 2 or 4h post-irradiation, compared to the diminished response of irradiated Trp53+/- cells (Figure 2). This corresponds to previously published data that indicates transient cell cycle arrest of 1 hour/Gy following low LET exposure (Purrott et al. 1980). Bone marrow from Trp53+/+ mice demon-

FIGURE 2. Radiation-induced G1 cell arrest. Sham-irradiated G1 cells subtracted from 2Gy irradiated G1 cells to derive proportion of radiation-induced G1cells. Filled symbols indicate G1 cell cycle response from Trp53+/+ mice (n=3), empty symbols represent Trp53+/- mice (n=3).

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strated a 10.20 ± 0.41% increase of cells in G1over spontaneous (0 Gy) levels. This was significantly greater than the initial peak value in Trp53+/cells at 1.71 ± 0.31% (P < 0.00008). Interestingly, 8 hours following the initial arrest, there appeared to be a second G1 arrest of lesser magnitude in Trp53+/+ cells (4.93 ± 1.91%; Figure 2). While causation is unclear in the context of this experiment, it may indicate that continued oxidative and/or inflammatory processes from the radiation-insult required additional checkpoint delays to repair any persisting damage. The intermouse variability in cell cycle response in both genotypes was unexpected given the inbred nature of this mouse strain. There were no significant radiation-induced alterations in the G2/M checkpoint in either Trp53+/+ or Trp53+/- cells. Apoptosis

The effect of p53 status on apoptosis has been well characterized; however longer-term kinetics of acute radiation-induced apoptosis has not been characterized in Trp53+/- mice compared to wild-type littermates. Time dependent changes were measured out to 14 hours post-irradiation after 1Gy and sham-irradiated lymphocyte cultures (Figure 3A). Spontaneous levels of apoptosis were found to increase with time (P < 0.001) and were dependent on genotype (P < 0.043). Trp53+/- lymphocytes generally demonstrated higher spontaneous apoptosis levels. Radiation-induced kinetics were determined by subtracting this background level of apoptosis from that induced by a 1 Gy acute radiation exposure for all time points (Figure 3B). Both genotypes displayed similar kinetic patterns throughout the 14 hour incubation period. Radiationinduced apoptosis increased between 4 and 10 hours. Both genotypes decreased at 12 hours and then increased to reach the same level of radiation-induced apoptosis at 14 hours (P > 0.825). The wild-type lymphocytes generally had higher levels of radiation-induced apoptosis at all time points with the exception of 14 hours, although these differences only reached significance at 6 (P < 0.018) and 8 hours (P < 0.001) post-irradiation. The lymphocytes from Trp53+/+ mice were found to be most radiosensitive at 8 hours compared to heterozygous mice. The 8 hour time point was subsequently used in dose response experiments. Spontaneous apoptosis levels (0 Gy) were subtracted from the apoptosis induced by doses of 0, 0.25, 0.5, 0.75 and 1 Gy to assess radiation-induced effects at 8 hours post-irradiation (Figure 3C). Both wild-type and heterozygous lymphocytes showed a dose dependent increase in radiationinduced apoptosis (P < 0.001). The relationship between genotype and radiation-induced apoptosis levels (P < 0.001) was also significant with wild-type lymphocytes displaying consistently higher apoptosis levels for all doses other than 0 Gy (P > 0.624).

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FIGURE 3. Apoptosis in the peripheral blood lymphocytes of wild-type Trp53+/+ (closed squares) and heterozygous Trp53+/- (open circles) mice. (A) Spontaneous and (B) radiation-induced kinetics were evaluated at 0, 4, 6, 8, 10, 12 and 14 hours following 0 or 1 Gy (n=6-16). The radiation-induced values were obtained by subtracting spontaneous apoptosis (0 Gy) from total apoptosis (1 Gy) (C) The apoptotic dose response was evaluated at 8 hours following 0, 0.25, 0.5, 0.75 or 1 Gy (n=10-20). Only radiation-induced apoptosis is displayed; spontaneous apoptosis (0 Gy) has been subtracted from total apoptosis. All data points shown represent mean ± standard error.

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MN-RET

The spontaneous frequency of MN-RET in sham-irradiated mice did not vary with time (P > 0.197) or genotype (P > 0.823) throughout the sampling period (Figure 4A). Radiation-induced kinetics were determined by subtracting this spontaneous MN-RET frequency from that induced by a 1 Gy acute radiation exposure, for all time points (Figure 4B). Radiation-induced MN-RET levels varied with time (P < 0.001). Both genotypes reached a peak level of MN-RETs at 43 hours post-irradiation, subsequently decreasing and returning to spontaneous levels by 137 hours post-irradiation. Genotype appeared to affect the kinetics of MNRET frequency with Trp53+/- mice having higher levels of MN-RET remaining after 44 and 51 hours (Figure 4B). This was particularly evident at 51 hours when the between the different mouse strains reached significance (P < 0.05). Trp53+/+ and Trp53+/- responses however returned to the same level of MN-RET at 68 and 137 hours post-irradiation (P > 0.375). The 43 hour time point was used in subsequent dose response experiments. Trp53+/+ and Trp53+/- mice were irradiated with 0, 0.1, 0.25, 0.75, 1 and 2 Gy in vivo and the spontaneous MN-RET frequency was subtracted to examine radiation-induced effects for each genotype (Figure 4C). There was a dose dependent increase in the frequency of MN-RETs for both genotypes up to 0.75 Gy (P < 0.001) above which dose there was no further increase in micronuclei indicating a saturation level for this endpoint. The lowest dose tested (0.1 Gy) showed a significant increase over spontaneous frequencies (P < 0.012) for both groups of mice. Genotype did not affect the nature of the dose response curve as there was no difference in the frequency of MN-RETs between genotypes at any dose, including 0 Gy (P > 0.520). DISCUSSION

Trp53 acts as a transcriptional activator of genes responsible for maintaining homeostasis and genomic integrity of organisms. Early evidence for the critical role of P53 in tumour growth suppression came from analysis of human colon carcinoma, the majority of which contained mutant Trp53 alleles (Baker et al. 1990). It was later discovered that the Trp53 gene is mutated in over 50% of human cancers (Hollstein et al. 1991). Moreover, individuals with Trp53 germ line mutations are affected by Li-Fraumeni syndrome which is characterized by a high frequency and early onset of tumours, particularly sarcomas (Malkin 2011). A Trp53 gene defect in mice of a C57BL6 x 129SVJ background has been shown to augment cancer risk by increasing the rate of tumorigenesis and consequently decreasing lifespan (Jacks et al. 1994). Moreover, exposure to high doses of radiation (1 - 4 Gy) has been shown to cause a further reduction in the latency period of cancer (Momand et al. 1992, Jacks et al. 375


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FIGURE 4. MN-RET frequency in wild-type Trp53+/+ (closed squares) and heterozygous Trp53+/(open circles) mice. (A) Spontaneous and (B) radiation-induced kinetics were evaluated at 41, 43, 44, 51, 68 and 137 hours post-irradiation (n = 5, analyzed in duplicate)). The radiation-induced values were obtained by subtracting spontaneous MN-RETs from total MN-RETs (C) The dose response of radiation-induced MN-RETS was evaluated at 43 hours following 0, 0.1, 0.25, 0.5, 0.75, 1 or 2 Gy (n = 3-6, analyzed in duplicate)). All data points shown represent mean ±standard error.

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1994), which is associated with an inability to effectively eliminate radiation-induced initiating events via P53 mediated pathways. In contrast, low doses of radiation (0.01-0.1 Gy) have been shown to increase the latency period for lymphoma in Trp53+/- mice (Mitchel et al. 2003). Therefore, we postulated that biological endpoints indicative of DNA damage and repair may be useful surrogates to better understand cancer risk associated with the acute response to low doses of radiation (0.1-2 Gy) were compared between heterozygous Trp53+/- and wild-type Trp53+/+ mice. Cells from the haematopoietic system were evaluated because of their radiosensitivity and because one of the primary cancer types observed in the Trp53+/- mouse model is haematopoietic in origin (lymphoma) (Kemp 1994, Donehower and Lozano 2009). Lymphocytes are often chosen for cytogenetic studies because they are long-lived, radiosensitive, contain DNA and are easy to harvest from circulating blood. While lymphocytes were used to evaluate γH2A.X and apoptosis, erythrocytes were used for cytogenetic analyses. It has been shown that there is a close correlation between the induction of chromosomal aberrations in bone marrow and the production of micronuclei in erythrocytes (Hayashi et al. 1984, Shelby and Witt 1995). Spontaneous Differences between Trp53+/+ and Trp53+/- mice

Despite distinctly different rates of spontaneous tumour formation in Trp53+/- versus Trp53+/+ mice, the spontaneous levels of γH2A.X, apoptosis, MN-RET were not significantly different in the hematopoietic cells of these mice at any of the time points examined. We conclude that any decrease in p53 function associated with Trp53+/- status may not have adversely impacted the cellular processes under normal conditions (sham irradiated) in the tissues and endpoints studied here. Wild-type p53 has a very short half-life (5-20 minutes) and is maintained at very low intracellular levels (Blagosklonny 1997, Giaccia and Kastan 1998). In contrast, upon exposure to ionizing radiation, the half-life of p53, and therefore concentration, increases seven-fold (Giaccia and Kastan 1998). Our findings are congruent with those of Merritt et al. (1994) and Di Masi et al. (2006), who found no difference in spontaneous apoptosis levels in the tissues of mice of differing Trp53 gene status. Radiation exposure was required to potentiate the difference in the apoptotic response of mice with differing Trp53 status. Moreover, Di Masi et al. (2006) studied protein levels in various tissues from Trp53+/+ and Trp53+/- mice (spleen, colon, kidneys, lungs, liver). No significant differences existed in basal levels between the 2 strains but after whole body irradiation (7.5 Gy); the induction of Trp53 was much lower in heterozygous mice (Di Masi et al. 2006). The baseline levels of apoptosis and γH2A.X were found to vary with time whereas the MN-RET values did not. The variation in baseline apoptotic levels could be attributed to ex vivo culture stress. These same conditions 377


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would not be expected to increase the baseline frequency of DNA DSBs (as measured by MN-RET and γH2A.X). This was the case for MN-RET but, unexpectedly, an elevation in γH2A.X foci was seen at 0 hours postirradiation relative to the other time points measured. It is possible this is a technical artifact of the assay (bone marrow collection) which generated γH2A.X foci which disappeared at later time points. It has been shown that not all γH2A.X foci correspond to DSBs (Bonner et al. 2008). Radiation-induced differences in γH2A.X between Trp53+/+ and Trp53+/mice

The initial recognition of DNA damage, as measured by mean γH2A.X fluorescence levels in bone marrow lymphocytes, responded to ionizing radiation in a dose dependent manner. No significant difference was detected between Trp53+/+ and Trp53+/- cells as measured by the kinetic studies of over 120 minutes post-irradiation following 1 Gy. Similarly, there was no significant difference detected between Trp53+/+ and Trp53+/- cells in the magnitude of the radiation response to 1 and 2 Gy at 30 minutes. Consistent with earlier reports, we found that cells exhibited maximum γH2A.X levels at 30 minutes post-irradiation for both strains of mice (Rogakou et al. 1998, MacPhail et al. 2003). The results between 0 and 30 minutes are reflective of damage recognition through foci formation. It has been demonstrated that there is a strong correlation between mean γH2A.X fluorescence detected via flow cytometry and foci formation as detected with microscopy. The fact that no difference was found between Trp53+/+ and Trp53+/- lymphocytes supports the concept that p53 is activated downstream of γH2A.X foci formation in response to ionizing radiation (Fernandez-Capetillo et al. 2004, Stiff 2004). Reduced γH2A.X levels at the 60 and 120 minute time points showed the removal of lesions. Our expectation was that p53-deficient cells would show a slower elimination of γH2A.X foci due to impaired DSB repair. For example, Mirzayans et al. (2006) showed that Li-Fraumeni fibroblast cell lines (heterozygous p53 function) exhibited a reduced rate of DSB joining (comet assay) and removal of γH2A.X foci relative to fibroblast cell lines expressing WT p53 following 8 Gy γ-rays. We found no significant difference in the rate of repair (disappearance of foci) between Trp53+/+ and Trp53+/- bone marrow lymphocytes between 30 and 120 minute following 1 Gy of γ-rays. It is possible that differences in the repair of DSBs would manifest themselves beyond 120 minutes as γH2A.X levels remained significantly elevated beyond background at this time point in both genotypes. Alternatively, it is possible that the magnitude of the stress response induced by 1 Gy was inadequate to demonstrate the difference between the two genotypes.

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Radiation-induced differences in apoptosis between Trp53+/+ and Trp53+/- mice

Apoptosis has been shown to be one of the key mechanisms by which radiation-induced DNA damage is processed and utilizes both p53dependent and independent pathways (Clarke et al. 1993). It is the primary mode of death for peripheral blood lymphocytes. In this work, a dose response was observed for various doses between 0 and 1 Gy at 8 hours post-irradiation in the peripheral blood lymphocytes of Trp53+/+ and Trp53+/- mice. In response to a 1 Gy challenge, wild-type lymphocytes generally experienced higher levels of apoptosis between 0 and 10 hours post-irradiation. These results confirm that peripheral blood lymphocytes undergo p53-mediated apoptosis in a dose dependent manner as p53-deficient cells show a resistance to apoptosis. Similar findings have been demonstrated in the thymocytes, myeloid progenitor cells, splenic cells and intestinal cells (Clarke et al. 1993, Lowe et al. 1993, Merritt et al. 1994, Fujikawa et al. 2000). Moreover, if heterozygous peripheral blood lymphocytes are not undergoing p53-mediated apoptosis in response to ionizing radiation, it is possible that they are evading surveillance. This was shown in the thymocytes of p53-deficient mice which were resistant to apoptosis but maintained higher levels of viability following irradiation relative to wild-type thymocytes (Lowe et al. 1993). In our work, radiationinduced apoptosis in lymphocytes from both genotypes increased to reach the same level at 14 hours despite showing clear differences before that time point. As suggested by Brown and Wouters (1999), examining later time points is crucial in not under- or over-estimating the impact of p53 function. The 14 hour time point suggests that the effect of reduced p53 levels in the lymphocytes becomes less relevant as slower, p53-independent apoptotic mechanisms become more predominant (Lorimore et al. 2013). To our knowledge, there are no reports that investigate the kinetics of radiation-induced apoptosis in the peripheral blood lymphocytes of p53-deficient mice. Radiation-induced differences in cell cycle arrest between Trp53+/+ and Trp53+/- mice

Cellular responses to DNA damage are mediated through highly conserved DNA damage checkpoint mechanisms that are central for tumor suppression by arresting cell cycle progression or inducing cellular senescence and apoptosis (Kastan et al. 1991, Jackson and Bartek 2009). It is well-established that p53 is a key player in the tumor-suppressive DNA damage response (DDR) system, by mediating the fate of damaged cells (Asai et al. 2011, Hanel and Moll 2012). Our data showing dramatically reduced cell cycle delay in the bone marrow of Trp53 +/- mice supports several studies which have shown that Trp53+/- mice have reduced or absent cell cycle arrest (Lee at al. 1994, Attardi and Jacks 1999). Given the 379


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importance of this mechanism as part of the DDR system, it suggests cell cycle delay likely plays a key role in the increased tumorigenesis of this mouse strain. Radiation-induced differences in MN-RET levels between Trp53+/+ and Trp53+/- mice

The frequency of micronuclei in the peripheral blood of mice provides a means by which to evaluate genomic instability in hematopoietic stem cells in vivo. The kinetics of MN-RET formation was evaluated following a 1 Gy acute dose between 41 and 137 hours post-irradiation in the same cohort of mice (n = 5 per genotype) using a serial bleed technique. The radiation-induced MN-RET frequency reached a maximum at 43 hours post-irradiation and declined at 44, 51 and 68 hours, returning to spontaneous levels at 68 and 137 hours. This kinetics pattern is in agreement with previous findings which showed a similar peak in MN-RET frequency at 43 hours post γ-irradiation (Dertinger et al. 2007). Furthermore, no difference was observed in the MN-RET values between Trp53+/+ and Trp53+/- mice at 41 or 43 hours, but Trp53+/- values remained elevated relative to Trp53+/+ values at 44 and 51 hours. This indicates that Trp53 gene status may contribute to the removal of radiation-induced chromosomal aberrations in vivo. Chang et al. (2000) identified a similar trend in mice of differing Trp53 status following exposure to energetic iron particle radiation. Following 1 Gy (1 GeV/amu) irradiation in vivo (or sham irradiation), the frequency (%) of MN-RETs were measured at 3, 9, 38, 56 and 91 days in Trp53+/+, Trp53+/- and Trp53-/mice. At 91 days post-irradiation, MN-RET% in irradiated Trp53-/- mice remained elevated whereas by 9 days, MN-RET% in irradiated Trp53+/+ and Trp53+/- mice had returned to control levels. It was hypothesized that the p53 deficiency altered the ability of DNA damage recognition or repair in the haematopoietic stem cell population which lead to increased MN-RET levels. At 43 hours post-irradiation, both Trp53+/+ and Trp53+/- mice had an identical, dose dependent increase in MN-RET between 0 and 0.75 Gy (Figure 4C) but there was no significant increase in levels of MN-RET at greater than 0.75 Gy. The presence of a saturation point for this assay is consistent with earlier reports (Dertinger et al. 2007, 2009). It has been suggested that a proportion of erythroblasts cannot withstand the damage induced by doses approaching 1 Gy of radiation and therefore undergo apoptosis before dividing and completing the maturation process (Dertinger et al. 2007). Recently, it was shown that various categories of reticulocyte precursors in C57BL6 mice are eliminated in response to 1 Gy by undergoing apoptosis as measured by Annexin V without changes in steady state cell proliferation (Peslak et al. 2011). Based on this hypothesis, we would expect fewer erythroblasts to undergo apoptosis in Trp53+/- mice following radiation exposure to 1 and 2 Gy, 380


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leading to a higher frequency of MN-RETs at 43 hours. However, we found no difference in the MN-RET frequency at 43 hours between Trp53+/+ and Trp53+/- mice at any of the doses tested. This indicates that while apoptosis may be responsible for the removal of precursors, it proceeds by means of a p53-independent pathway, which is consistent with previously reported data which indicates p53-independent cytokine signalling is a critical mediator of radiation-induced apoptosis in bone marrow (Lorimore et al. 2013). It has been shown previously that defective erythroblasts (folate-deficient) also undergo p53-independent apoptosis (Koury et al. 2000) however the relationship between those studies and ours in unclear. Nonetheless, damage to marrow precursor cells and subsequent manifestation of the damage as MN-RETs in circulating blood could be p53-independent. However, it might also be possible that heterozygous mouse cells have sufficient p53 protein to regulate a diminished response similar to wild-type mice. This result could indicate that the signalling mechanisms which trigger changes in MN-RET frequency are very sensitive and may be induced by very low levels of p53 activity. Prior studies have identified that the radiosensitivity of heterozygous cells represents an intermediate response between those of null and wild-type cells, although generally more similar to wild-type (Wang et al. 1996). Linking short term radiation dose responses to increased tumorigenesis in Trp53+/- mice

There is considerable interest in linking short term biological indicators, such as chromosome damage, gene mutation, apoptosis, and DNA damage and repair, to radiation-induced cancer risk in p53-deficient mice. The consensus is that p53 prevents the progression of initiating events (Schmitt et al. 2002, Fei and El-Deiry 2003, Attardi 2005, Kenzelmann Broz and Attardi 2010). These events could be the result of unrepaired DNA damage induced by ionizing radiation, although many other exogenous and endogenous processes are also thought to influence genomic instability in Trp53+/- mice (Attardi and Jacks 1999). The inappropriate recombination of DSBs during V(D) J recombination, for example, is thought to increase the number of potential oncogenic lesions in maturing T-cells in p53-deficient mice which contributes significantly to the development of lymphoma earlier than wild-type mice (Attardi 2005). Many studies have used variations on the p53 knockout model to elucidate the connection between p53 deficiency and the process of carcinogenesis (Kenzelmann Broz and Attardi 2010). Schmitt et al. (2002) showed that apoptosis is selected against during the development of lymphoma in Myc-transgenic Trp53+/- and Trp53+/+ mice. Another study (Lee et al. 1994) used wild-type and transgenic mice expressing p53193Pro or p53Vall35, which are mutant forms of normal p53 protein, to show that irradiated Trp53 transgenic mice and Trp53 null 381


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mice had twice the level of chromosome damage than did irradiated wildtype mice and suggested that this was a means by which tumorigenesis was enhanced. Loss of heterozygosity (LOH) is another important factor governing the development of cancer in Trp53+/- mice. Since many human tumours exhibit loss of heterozygosity, it was assumed that this was a critical step in the cancer phenotype of Trp53+/- mice. The two hit theory proposes that individuals who possess only a single functional copy of a tumour suppressor gene are more likely to progress to carcinogenesis because they already have one inactivated allele (Knudson et al. 1975). This theory assumes that inactivation of both alleles is required for carcinogenesis. Contrary to the two hit theory, Venkatachalam et al. (1998) showed that a reduction in p53 functional gene product was sufficient for the development of tumours in Trp53+/- mice. This conclusion was derived from analysis of tumours in which wild-type characteristics had been maintained by the one allele. Based on our analysis of these common endpoints for cancer risk, the blunted cell cycle arrest in Trp53+/mice appears to be a critical mechanism through which damaged cells perpetuate genomic instability and may be the basis for the increased accumulation of genetic anomalies which increase carcinogenic risk. CONCLUSION

This research offers new insight into the dose response relationship between Trp53 status and radiation-induced γH2A.X foci formation, cell cycle arrest, apoptosis and MN-RET formation in various haematopoietic cell subtypes. The lack of difference in γH2A.X foci formation between mouse strains indicates this process is independent of p53. Trp53+/- mice had lower apoptosis levels per unit dose indicating early apoptosis is p53dependent in the haematopoietic cells, however it also suggests that genomic instability as indicated by persistent γH2A.X foci does not work in concert with inhibited apoptosis to promote carcinogenesis in these mice. Both genotypes of mice had virtually identical responses for MNRET formation, suggesting DNA damage in reticulocyte precursors (erythroblasts in bone marrow) and the subsequent repair or removal of those cells was largely p53-independent, although small non-significant differences in kinetics were observed. The blunted radiation-induced G1cell cycle arrest in Trp53+/- bone marrow provides one of the few clear cut endpoints that further to our understanding of the role of p53 in carcinogenesis. Since the role of p53 in radiation dose responses is highly dependent on cell and tissue types (Komarova et al. 2000, Coates et al. 2003, Kenzelmann Broz and Attardi 2010), the usefulness of using short term biological endpoints, like those used in these experiments, to predict cancer risk may be limited. We have determined that lower levels of radiation-induced apoptosis in lymphocytes and blunted G1 cell cycle arrest presented here, likely elucidate the key mechanism underlying the 382


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higher cancer incidence in Trp53+/- mice, and support previous postulates (Kemp 1994, Mitchel et al. 2003). ACKNOWLEDGEMENTS

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