Thesis (M. Sc.)--Carleton University, 2002. / Includes bibliographical references (p. 69-74). Also available in electronic format on the Internet.

BACKGROUND: Radiation exposure to the abdominal region causes intestinal toxicity and is also capable of inducing colorectal cancers (CRC). Genotype-phenotype studies provide some evidence explaining the variation in familial adenomatous polyposis (FAP) patients caused by modifiers of adenomatous polyposis coli (APC). This study aims to extend our understanding of irradiation-induced modifiers of ApcMin/+ mice and CRC. METHODS: By using a pre-existing backcross between recombinant inbred line of ApcMin/+ mice to the irradiation sensitive inbred BALB/c mouse, we obtained panels of 2Gy-irradiated and sham-irradiated N2 ApcMin/+ mice for genotyping with a genome-wide panel of microsatellites markers. Using the number of adenomas in different intestinal segments to represent polyp multiplicity, we carried out a genome wide quantitative trait loci (QTL) scan followed by statistical epistasis modelling and bioinformatics analysis. RESULTS: We identified five significant QTLs responsible for radiation induced tumour multiplicity in the upper small intestine defined as Mom (Modifier of Min) radiation-induced polyposis (Mrip1-5) on chromosome 2 (LOD 2.8, p = 0.0003), two regions within chromosome 5 (LOD 5.2, p=0.00001, 6.2, p=0.00001) and two regions within chromosome 16 (LOD 4.1, p=4x10-5 and 4.8, p=0.00001). Suggestive QTLs were found for sham-irradiated mice on chromosomes 3, 6 and 13 (LOD 1.7, 1.5 and 2.0 respectively; p,0.005). Two significant QTLs were detected in the 2large intestine on chromosome 2 and 7 (LOD 2.7, p=1.2x10-3 and 2.2, p=1.2x10-3, 12 respectively). Using statistical epistasis modelling and logical selection of target genes though in silico sequence based on BALB/c specific non-synonymous polymorphisms which are predicted deleterious we selected target genes and further eliminated genes by sequencing and mRNA expression. CONCLUSIONS: Our study locates the QTL regions responsible for increased radiation-induced intestinal tumorigenesis in ApcMin/+ mice and identifies candidate genes with predicted functional polymorphisms that are involved in spindle checkpoint and chromosomal stability (Bub1b, Bub1r, and Casc5), Wnt pathway (Tiam1, Rac1), DNA repair (Recc1 and Prkdc) and inflammation (Duox2, Itgb2l and Cxcl5).

Parthenolide (PN), a major active component of the traditional herbal medicine feverfew, has been shown to have anti-inflammatory and anti-tumor properties. More remarkably, the cytotoxicity of PN seems selective to tumor cells but not their normal cell counterparts. In the present study, we investigate whether and how PN selectively enhances tumor sensitivity to radiation therapy by using prostate cancer cells LNCaP, DU145 and PC3, as well as normal prostate epithelial cells PrEC. Our study demonstrates that inhibition of NF-κB pathway and suppression of its downstream target MnSOD are common mechanisms for the radiosensitization effect of PN in prostate cancer cells. The differential susceptibility to PN in two radioresistant cancer cells, DU145 and PC3, is due, in part, to the fact that in addition to NF-κB inhibition, PN activates the PI3K/Akt pro-survival pathway in both cell lines. The presence of PTEN in DU145 cells enhances the radiosensitization effect of PN by suppression of the steady state level of activated p-Akt. We also demonstrate that PN selectively exhibits a radiosensitization effect on prostate cancer PC3 cells but not on normal prostate epithelial PrEC cells. PN causes oxidative stress in PC3 cells but not in PrEC cells, as determined by the oxidation of the ROS-sensitive probe H2DCFDA and intracellular reduced thiol and disulfide levels. In PC3 but not PrEC cells, PN activates NADPH oxidase leading to a decrease in the level of reduced thioredoxin, activation of PI3K/Akt and consequent FOXO3a phosphorylation, which results in the downregulation of FOXO3a targets, antioxidant enzyme MnSOD and catalase. Importantly, when combined with radiation, PN further increases ROS levels in PC3 cells, while it decreases radiation-induced oxidative stress in PrEC cells, possibly by increasing GSH level. Overall, our data support the concept that increasing oxidative stress in cancer cells, which are already under high constitutive oxidative stress, will lead to cell death, while the same stress may allow normal cells to maintain redox homeostasis through adaptive response. Thus, modulating cell redox status may be a novel approach to efficiently and selectively kill cancer cells.

Thesis (Ph.D.) - University of St Andrews, January 2008.

The BEIR V preferred relative risk models and standard life-table techniques are used to project lifetime fatal cancer risk factors for average members of the Canadian population. Uncertainties associated with projections are evaluated for: (1) sampling variation (statistical error), (2) extrapolation of risks to low doses and low dose rates, (3) projection of excess lifetime cancer risks beyond the current periods of human observation in epidemiological studies, (4) the transfer of site-specific excess risk coefficients between populations with differing baseline cancer rates, and (5) the effect of differences in the age and sex distributions among occupations in the Canadian "radiation" workforce. Results are used to assess the applicability of the fatal cancer risk estimates recommended in ICRP publication 60 to the Canadian population. It was found that sampling variation, extrapolating to low doses and dose rates, projecting excess risks beyond current periods of observation, and the uncertainty in how to transfer site-specific excess risks between populations all cause substantial variations in lifetime cancer risk projections. Site-specific cancer risk projections may be expected to vary by factors of 2 to 5, depending on the source of uncertainty. Site-specific differences were found in the fatal cancer risk factors projected for "average" male and female workers among different occupations in the Canadian workforce. Site-specific worker averages differed by as much as a factor 3. Female average risk factors for digestive cancers were substantially higher than male workers, while male average risk factors tended to be higher for leukemia and respiratory cancer. Overall however, the majority of worker risk factors were within 2.5% of the site-specific projections for the workforce as a whole. The ICRP-60 nominal fatal cancer risk estimates, tissue weighting factors, and lifetime risk projections for prolonged radiation exposure were all in good agreement with equivalent values derived in this report for the Canadian population. In view of the uncertainties, the results suggest the ICRP estimated cancer risks are as good as any presently available and supports the use of the ICRP recommended values for the planning and regulation of radiation protection in Canada. / Thesis / Master of Science (MS)

It is increasingly recognised that individual risk of cancer may be related to genetically determined differences in the ability of cells to identify and repair DNA damage. Cell cycle based assays of chromosomal radiosensitivity provide the greatest power for discriminating differences in response to DNA damage and it has been suggested that individuals who are genetically susceptible to cancer show increased chromosomal radiosensitivity. The relationship between chromosomal radiosensitivity and early onset cancer was investigated in a population of Danish survivors of childhood and adolescent cancer and a control group comprising of their partners using the G₂ assay of chromosomal radiosensitivity. Heritability was also examined in the offspring. No significant differences in radiosensitivity profiles were found between partner controls and either the cancer survivors or offspring. However, when compared to the Westlakes Research Institute control population, significant differences were observed with the cancer survivors (P = 0.002) and offspring (P < 0.001), supporting an association of chromosomal radiosensitivity with cancer predisposition. Heritability studies suggested the majority of phenotypic variance of chromosomal radiosensitivity was attributable to a putative major gene locus with dominant effect. Since G2 chromosomal radiosensitivity indirectly measures the ability of cells to repair DNA damage induced by ionising radiation exposure, variants in DNA repair genes may explain inter-individual variation observed. Sixteen polymorphisms in nine genes from four DNA repair pathways were investigated. Genotype frequencies at the Asp148Glu polymorphism were associated with childhood cancer in survivors. Analysis of variance and FBAT analysis suggested significant associations at both the Thr241Met and Ser326Cys polymorphism sites with G₂ radiosensitivity, but neither remained significant after multiple-test adjustment. This study invites further exploration of the predictive capacity of G₂ chromosomal radiosensitivity in cancer predisposition. Clearly, further work is needed to correlate radiosensitivity with genetic polymorphisms, which may underlie cancer susceptibility and variation in radiosensitivity.

Introduction: Some past breast cancer radiotherapy regimens led to an increased risk of death from heart disease. Although heart dose from breast cancer radiotherapy has generally reduced over the past few decades, there may still be some cardiac risk. Estimation of future risk for women irradiated today requires both measurement of their cardiac dose and dose-response relationships, which depend on cardiac dosimetry of past regimens, in conjunction with long-term follow-up data. Methods: Virtual simulation and computed tomography 3-dimensional treatment planning on a representative patient were used to estimate mean heart and coronary artery doses for women irradiated since 1950 in 71 randomised trials in the Early Breast Cancer Trialists’ Collaborative Group (EBCTCG) overview. Patient-to-patient variability in cardiac dose was assessed. Heart and coronary artery doses were also calculated for breast cancer radiotherapy regimens used since the 1950s in Sweden. Cardiac doses from contemporary (year 2006) radiotherapy were assessed for 55 patients who received tangential breast cancer irradiation at a large UK radiotherapy centre. The maximum heart distance (i.e. the maximum distance between the anterior cardiac contour and the posterior tangential field edges) was measured for the left-sided patients, and its value as a predictor of cardiac doses assessed. Results: Mean heart dose for women irradiated in the EBCTCG trials varied from <1 to 18 Gray, and mean coronary artery dose from <1 to 57 Gray. Patient-to-patient variability was moderate. Mean heart dose for women irradiated in Sweden since the 1950s varied from <1 to 24 Gray, and mean coronary artery dose from <1 to 46 Gray. Heart dose from tangential irradiation has reduced over the past four decades. However, mean heart dose for left-sided patients irradiated in 2006 was 2 Gray and around half of them still received >20 Gray to parts of the heart and left anterior descending coronary artery. For these patients, maximum heart distance was a reliable predictor of cardiac doses. For the other patients, mean heart dose varied little and was usually less than 2 Gray. Conclusions: Cardiac doses from breast cancer radiotherapy can be estimated reliably and are now available for use in deriving dose-response relationships in the EBCTCG data and in a Scandinavian case-control study. Cardiac dose has reduced over the past four decades. Therefore the cardiac risk is also likely to have reduced. Nevertheless, for some patients, parts of the heart still receive >20 Gray in the year 2006.

Medical radiation is estimated to contribute to over 200,000 deaths annually. Recent increases in the use of radiation-producing medical imaging examinations have led to increasing cumulative radiation dose to the general public. Multiple measures have been taken to address this alarming trend, including physician education, technologist education on dose reduction, and equipment-facilitated dose reduction techniques. Shield use can reduce the primary beam by up to 95%. Medical imaging technologists are the primary individuals responsible for applying shielding during an examination. Currently, literature shows that technologists are not shielding individuals as often as they should. After pilot testing, medical imaging technologists were recruited via email to participate in a national cross-sectional survey in September 2017. The survey contained items related to technologists’ demographics, shielding behaviors, and attitudes and beliefs measured at four social-ecological levels – intrapersonal, interpersonal, organizational, and community. The American Registry of Radiologic Technologists (ARRT) provided a list of technologists’ email addresses from their directory. One thousand six-hundred and sixty-one email notifications were sent out in the summer of 2017. Of those, 218 technologists (13%) completed the survey. Among technologists who considered their primary modality to be computed tomography (CT), organizational level factors were a positive significant predictor of shielding behavior. None of the four levels were significant in predicting shielding behavior among diagnostic radiological technologists (x-ray). Individual factors were significantly correlated to shielding behavior among radiologic technologists in the intrapersonal, organizational, and community levels. Study results indicated that interventions implemented at the organizational level may be most effective in increasing shield use among CT technologists. Additional research is needed to better understand factors affecting shield use among medical imaging technologists.

Radiation-induced damage to the genomic DNA of cells may lead to errors in transcription and replication and, if not repaired correctly, these may result in mutations, genomic instability and cell death. Laser microbeams have generally been used by many research groups to investigate the real-time dynamics of protein recruitment in response to DNA insults in mammalian cells; however, such irradiations induce a plethora of DNA damage (including UV base damage, base damage, SSBs and DSBs and complex damage). A novel experimental setup has been designed capable of following the dynamics of protein recruitment in response to DNA insults in mammalian cells shortly following submicrosecond- pulsed electron irradiation of living mammalian cells, not possible using conventional irradiation techniques. This arrangement was developed based on a 6 MeV electron pulse linear accelerator, to deliver sparsely ionising radiation, coupled to an automated, time-lapse inverted epifluorescence microscope imaging system. An integrated robotic system contained within a physiological environment of 37°C and 5&percnt; CO₂ was used to transfer remotely and repetitively custom-designed cell dishes containing the mammalian cells between irradiation and imaging locations. Following the development of the linear accelerator and associated imaging devices, preliminary ‘proof-of-principle’ investigations were carried out using living HT1080 mammalian cells containing YFP-tagged 53BP1, an established biomarker of DSB, to follow the recruitment and loss of 53BP1 to sites of radiation-induced DNA damage in real-time. This novel experimental setup has allowed for the first time observations of the appearance and disappearance of radiation-induced foci in the same cell population at very early times. These single-foci studies have provided evidence for the formation of not only promptly formed DSBs but also late appearing DNA damage signalled by 53BP1. These data highlight different classes of DSBs formed in response radiation damage. Additionally, the role of cell cycle on the repair kinetics was undertaken using HT1080- 53BP1-YFP cells which also express Geminin-mCherry under appropriate selection. Geminin is increasingly expressed from early S-phase onwards, but is degraded following mitosis. Geminin-associated fluorescence can be used as a marker of progression through the cell cycle. 53BP1 repair kinetics were characterised in response to radiation damage in combination with ATM and PARP inhibitors. These studies provided supporting evidence for the existence of different classes of DSBs, potentially assigned to radiation-induced replication breaks and DSBs formed by enzymatic conversion of clustered damage. These preliminary ‘proof-of-principle’ findings using DNA damage repair as an example, emphasize the use of this novel technology to explore the dynamics of numerous other biochemical processes in living cells in real-time with the knowledge of being able to quantify the range of damage induced by IR coupled with accurate dosimetry. The knowledge obtained may be used to identify potential biological targets coupled with drug discovery for translation into adjuncts for radiotherapy.

DNA is wrapped around proteins called histones, whose modification regulates numerous cellular processes. Therefore it is not surprising that mutations in the genes that modify the histones are frequently associated with human cancer. For example, mutations in SETD2, encoding the sole enzyme that catalyses histone H3 lysine 36 trimethylation (H3K36me3), occur frequently in multiple cancer types. This identifies H3K36me3 loss as an important event in cancer development, and also as a potential therapeutic target. This thesis investigates the following questions: (1) how does the loss of H3K36me3 contribute to cancer development; and (2) what therapy can be used to kill cancers that have already lost H3K36me3. To answer the first question, this thesis shows that H3K36me3 facilitates the accurate repair of DNA double-stranded breaks (DSBs) by homologous recombination (HR). H3K36me3 promotes HR by recruiting CtIP to the site of DSBs to carry out resection, allowing the binding of HR proteins (such as RPA and RAD51) to the damage sites. Thus it is proposed that error-free HR repair within H3K36me3-decorated transcriptionally active genomic regions suppresses genetic mutations which could promote tumourigenesis. To answer the second question, this thesis reveals a clinically relevant synthetic lethal interaction between H3K36me3 loss and WEE1 inhibition. WEE1 inhibition selectively kills H3K36me3-deficient cells by inhibiting DNA replication, and subsequent fork stalling results in MUS81 endonuclease-dependent DNA damage and cell death. The mechanism is found to be synergistic depletion of RRM2 (ribonucleotide reductase small subunit), the enzyme that generates deoxyribonucleotides (dNTPs). This work reveals two pathways that regulate RRM2: one involves transcriptional activation of RRM2 by H3K36me3, and the other involves RRM2 degradation regulated by Cyclin-Dependent Kinase, CDK1 (which is controlled by WEE1, CHK1 and ATR). Based on this mechanism, the synthetic lethal interaction is expanded, from between two genes, to between two pathways. Supported by in vivo experiments, the study suggests that patients with cancers that have lost H3K36me3 could benefit from treatment with the inhibitors of WEE1, CHK1 or ATR.