Positive energy imbalance occurs when energy intake exceeds energy expenditure. In modern society, increased consumption of caloric-dense food and reduced energy expenditure due to low physical activity and sedentary lifestyle drive positive energy imbalance. Manifested as weight gain, positive energy imbalance is implicated in the development and progression of several cancers. This dissertation focuses on reduced energy expenditure, sedentary behavior and weight gain in relation to cancer risk. In chapter 1, we prospectively evaluated the relationship between physical activity and incident cancers of the digestive system encompassing the digestive tract (mouth, throat, esophagus, stomach, small intestine, and colorectum) and digestive accessory organs (pancreas, gallbladder, and liver) among men in the Health Professionals Follow-Up Study. In men, a higher level of physical activity, regardless of its intensity, was associated with a decreased risk of digestive system cancers, particularly digestive tract cancers. The association was independent of obesity and diabetes. In chapter 2, we examined whether sedentary lifestyle, as indicated by time spent sitting watching TV, has an effect on colorectal cancer risk independent of physical activity. The relationship was prospectively investigated among women in the Nurses’ Health Study and men in the Health Professionals Follow-Up Study. In the combined population of women and men, prolonged sitting watching TV was an independent risk factor for colorectal cancer. The elevated risk associated with prolonged sitting was not completely offset by participation in physical activity. In chapter 3, we assessed the strength and shape of the dose-response relationships between adult weight gain and risk of adiposity-related cancers (e.g., cancers of the breast, endometrium, ovary, prostate, colon, pancreas) using dose-response meta-analysis. Adult weight gain was linearly associated with risk of breast, endometrial, and ovarian cancers among postmenopausal women not using hormone therapy; with risk of colon cancer, particularly among men. Results of this dissertation suggest that increasing the overall amount of physical activity, reducing sedentary time, particularly sitting watching TV, and avoiding adult weight gain should be widely encouraged for the prevention of several major cancers. / Nutrition

Although testicular cancer is a relatively rare cancer in Canada, accounting for only 1.1% of all malignant neoplasms in males, it is the most common cancer among men 20 to 45. Understanding of the causes of testicular cancer risk in general, and the association with diet in particular, remains limited. Data from the National Enhanced Cancer Surveillance System were used to explore the relationship of diet and testicular cancer risk. There were 601 cases of testicular cancer and 744 controls available for study. We systematically examined 17 food groups, 15 nutrients, and 7 individual foods based on data collected through a 69-item food-frequency questionnaire. Our results suggest that higher dairy product intake, specifically cheese, is associated with a higher risk of testicular cancer in Canadian males. Risk differences were observed between histological subtypes of testicular cancer. This thesis provides a basis for future studies designed to address testicular cancer etiology.

Background. Standard traditional parameters relied on for estimating the risk of disease recurrence after curative radiotherapy in prostate cancer are stage, Gleason score and PSA. Objectives. To elucidate the prognostic role of biomarkers: P-53, MIB-1, MVD, Bax and BCL2 in prostate cancer. Method. Cox proportional hazard model was used to estimate the risk of disease progression associated with these biomarkers and develop models based on traditional parameters only or incorporating biomarkers. Models were compared for their predictive potential using Akaike information criteria and concordance index. Results. Statistically significant associations were found between all biomarkers and risk of progression. MVD, Bax and Bax/BCL2 were independent predictors of outcome. Models incorporating biomarkers were superior to the traditional one in identifying patients at risk of local progression. Conclusions. Biomarkers may be useful in forecasting the risk of local recurrence following radiotherapy in prostate cancer patients. The results require validation in a separate cohort.

Bladder cancer is the second most frequently diagnosed neoplasm of the urinary tract. Effects of physical activity and body fat mass on risk of bladder cancer are not well studied. Physical activity and/or body weight may exert their effects on bladder cancer risk through hormonal, immune, or yet other unknown causal pathways. This thesis, using a case-control study design, investigated the association between recreational physical activity, relative body weight (BMI), and risk of bladder cancer in men and women separately. The analysis was based on a portion of the National Enhanced Cancer Surveillance System dataset. The self-reported data had been collected from bladder cancer cases and population controls during 1994--1997 in seven Canadian Provinces. The analyzed samples were 2,312 males (670 cases and 1,642 controls) and 1,824 women (359 cases and 1,465 controls). In men and women, the adjusted ORs (highest vs. lowest quartile) for physical activity were 1.24 (95% CI: 0.93, 1.66) and 0.76 (95% CI: 0.52, 1.21), respectively. The corresponding ORs for BMI were 1.29 (95% CI: 0.96, 1.72) and 1.07 (95% CI: 0.74, 1.55), respectively. This study found that parous women were at reduced risk of bladder cancer compared to nulliparous women (OR = 0.56. 95% confidence interval: 0.39, 0.80). In agreement with other studies, certain occupations and cigarette smoking were associated with increased risk of bladder cancer. In men, but not in women, coffee consumption was associated with a slightly increased risk of bladder cancer (≥1 cup per day vs. never: OR = 1.30, 95% CI: 1.00, 1.68). There was not enough evidence in this study to conclude that recreational physical activity and BMI were related to risk of bladder cancer.

The presence of allergy may reduce cancer risk. Literature searches identified 142 epidemiological studies on this association. Data from the American Cancer Society Cancer Prevention Study-II cohort were used to explore the relationship between self-reported asthma and/or hay fever and cancer mortality in 508 318 men and 483 079 women who were cancer-free at baseline. During 18 years of follow-up from 1982--2000, there were 44 524 cancer deaths in men and 36 567 in women. Cox proportional hazards models were used to obtain adjusted relative risks for overall cancer mortality and for cancer mortality at 12 sites. There was approximately a 10% reduction in overall cancer mortality among people with asthma and hay fever. Asthma and/or hay fever were also associated with a reduced risk of cancer at a number of other sites, and some of the associations were modified by gender and smoking status.

Ovulation is a putative risk factor for ovarian cancer, and may be attributed to proliferating ovarian surface epithelial (OSE) cells interacting with the extracellular matrix (ECM) during ovulatory wound repair. We investigated the effects of ECM and cellular density on OSE cell morphology, proliferation, and expression of c-Kit, a proto-oncogene expressed in 70% of ovarian tumours. Fibrillar collagen I caused significant changes in morphology and proliferation but monomeric ECM had no effect. Normal human ovaries co-expressed KIT, collagen I and fibronectin in 75% of abnormal OSE structures. At increased cellular densities, rat OSE cells expressed increased levels of Kit. Retrovirus-mediated expression of c-Kit in OSE caused a significant increase in proliferation. Additionally, sequence analysis of c-KIT in 21 ovarian tumours revealed no mutations. These results suggest a relationship between KIT and collagen I in OSE and that cellular density in the OSE regulates KIT, which increases cell proliferation and may contribute to transformation.

Most human tumors exhibit accumulation of mutations throughout their progression. In addition, many human tumors are infiltrated with inflammatory cells, such as neutrophils and macrophages. These cells can generate potentially mutagenic species, including nitric oxide-derived species. We hypothesize that these species contribute to the accumulation of mutations in tumors. Our lab previously developed the 'Mutatect' mouse tumor model to study this hypothesis. Mutatect fibrosarcoma cells can form solid tumors in C57BL/6 mice when injected subcutaneously. These tumors are variably infiltrated with neutrophils. The neutrophils contain inducible nitric oxide synthase, and tumor cells contain nitrotyrosine, an indicator of nitric oxide-derived damage. Neutrophil number correlates with mutation arising in vivo in Mutatect cells at the hypoxanthine phosphoribosyltransferase (hprt) locus, a marker of genetic damage. Dietary vitamin E significantly lowers the hprt mutations. I genetically engineered Mutatect cells to produce interleukin-8, a neutrophil attractant. Cells expressing high levels of interleukin-8 formed small tumors, whereas cells expressing lower levels produced large tumors. A biochemical assay was developed to quantify neutrophil content in tumors. Interleukin-8-expressing tumors had a significantly higher neutrophil content and hprt mutations than non-expressing tumors. Interleukin-8 gene instability was observed in high interleukin-8-expressing tumors. Dietary vitamin E dramatically inhibited both hprt mutations and interleukin-8 instability. It also affected neutrophil distribution in tumors. High level of protein nitrotyrosine was seen in tumors with high neutrophil content (and mutations). Using proteomics tools, a significant amount of nitrotyrosine could be identified in histones, the major nuclear proteins associated with DNA. Mechanism of nitric oxide-derived mutagenicity was also examined. Previous studies implicate the role of intracellular S-nitrosoglutathione, which was recently found to be detoxified by formaldehyde dehydrogenase. I developed several cell-lines with down-regulated formaldehyde dehydrogenase levels using RNA interference and antisense techniques. A metabolic cycle, involving formaldehyde dehydrogenase, capable of regulating protein S-nitrosation was identified. This thesis has contributed significantly towards understanding the role of inflammatory cells and the nitric oxide-derived factors in solid tumors. It has also allowed us to understand the mechanism(s) of S-nitrosation and tyrosine nitration in proteins, both of which may have physiological and pathological relevance.

X&barbelow;IAP a&barbelow;ssociated f&barbelow;actor 1&barbelow; (XAF1) was initially isolated as novel 34 kDa protein which bound XIAP in a two-hybrid screening. The XAF1A protein consists of 301 a.a. and contains seven potential zinc finger domains. Two alternatively splice variants of XAF1 were later isolated. One isoform (XAF1B) was formed by the removal of a 57 bp exon, which leads to an in-frame deletion of the third zinc finger and the creation of a shorter 32.5 kDa protein. The other splice variant (XAF1C) contains a 154 bp exon insertion, which truncates the sixth and seventh zinc fingers to produce an 18.7 kDa protein. XAF1A and XAF1B, but not XAF1C, bound XIAP in in vitro pull down assays. Northern blot analysis showed at least four distinct sizes of xaf1 mRNA ranging between 3.9 and 7.0 kb, which may indicate other XAF1 isoforms yet to be discovered. Though the possible role of these zinc fingers on the XAF1/XIAP interaction has yet to be determined, recent experiments indicate that XAF1A can block the ability of XIAP to inhibit caspase-3 in vitro. Furthermore, overexpression of XAF1A in HEL299 cells triggered a G1 cell cycle arrest. This G1 arrest coincides with an increase in p21, but not p53. The ability of XAF1 to block XIAP function and induce cell cycle arrest suggests a role for XAF1 in the control of both apoptosis and cell growth. The coding regions of XAF1A, B and C are encoded on a total of 9 exons within a span of 20 kb. The single copy xaf1 gene has been mapped, using FISH analysis, distal to the TP53 gene on 17p13.2. Southern blot analysis of YACS within this region further localizes the xaf1 gene on YAC 746 C 10, which contains the markers D17S1831, D17S796, and D17S1881. These markers are located approximately 3 cM telomeric to the TP53 gene. Since the xaf1 gene is located in a region commonly deleted in numerous types of cancers, this may suggest a tumour suppressor role for XAF1 in cancer. To test this theory, a 60 cell line panel from the NCl was analyzed for xaf1 RNA expression by Taqman and heterozygosity status of markers proximal to xaf1. Taqman analysis indicated that the majority of cell lines expressed little or no xaf1 RNA while xiap levels were relatively high. A PCR study of markers near xaf1 showed significant loss of heterozygosity (LOH) in this region. The loss of xaf1 expression and significant LOH near the xaf1 gene indicate that the down-regulation of XAF1 may be important in the development of the transformed phenotype.

Thymidylate Synthase (TS) catalyzes the final step in the synthesis of thymidylate, a nucleotide required for DNA synthesis. This enzyme is the target enzyme for several important anti-cancer drugs including 5-fluorouracil (5-FU) and 5-fluorodeoxyuridine (5-FUdR). 5-FU and 5-FUdR are metabolized to FdUMP which, in the presence of a folate cofactor, inhibits TS by forming a tight covalent "ternary complex". TS is usually considered to be a cytoplasmic enzyme, but some reports suggest it may also be present in the nucleus. One recent report has indicated that high levels of nuclear TS were associated with poorer clinical response to 5-FU. Using a combination of immunocytochemistry, confocal microscopy, immunohistochemistry, western blotting and cell fractionation techniques, evidence is presented that a fraction of cellular TS is indeed located in the nucleus of some colorectal cancer cell lines and tissues. The specificity for TS of the polyclonal antibody used was first confirmed. The effect of TS overexpression on cellular localization was studied in HeLa-55 cells, a 5-FUdR-resistant line containing an amplified TS gene. Nuclear TS was clearly evident in HeLa-55 but not in parental HeLa cells. Fractionation of HeLa-55 growing in 5-FUdR showed that both the ternary complex and free TS were present in the cytoplasm, while only free TS was present in the nucleus. From a panel of 6 colorectal cancer lines and normal fibroblasts, RKO and HCT-116 clearly showed nuclear TS. Strong evidence of nuclear TS in some colorectal cancer tissues is also presented. A putative leucine-rich nuclear export signal sequence (NES) was identified in TS protein. TS constructs lacking this sequence accumulated more nuclear TS, but the sequence did not function as a nuclear export signal when fused to GFP. A recent report suggests that p53 status can affect the sensitivity of HCT-116 to 5-FUdR. I examined HCT-116 p53 wt and HCT-116 p53 null cells and observed that p53 null cells, in contrast to p53 wt cells, failed to form a ternary complex when exposed to 5-FUdR. p53 wt cells were also more sensitive to killing by 5-FUdR. However, the differences observed were in fact independent of p53 status. 5-FUdR-sensitive cell lines became resistant after several passages in culture. Current evidence suggests that the basis of resistance is related to either drug metabolism or uptake. This thesis has contributed towards a better understanding of TS, a cellular target for an important class of anti-cancer drugs.

Failure of radiotherapy for treatment of neoplastic disease can be due to selection of radioresistant S-phase cells within tumours. However, S-phase cells can be targeted with a type-1 DNA topoisomerase inhibitor, camptothecin (CPT) or one of its analogs. The first objective of this study was to understand the mechanism of cellular death of human tumour and normal cells following treatment to CPT and single dose (nonfractionated) X-radiation. We found that necrotic cell death was more important than apoptotic cell death during concurrent CPT and radiation treatment in melanoma (SkMel-3) cells, but not in normal fibroblast (AG1522) cells. Further experiments involving fractionated X-radiation pre-treatment gave rise to the presence of radioresistant cells in the human tumour colorectal cell line (HCT116). Therefore, the second objective of this study was to assess whether the radioresistant cells occurred by selection or adaptation to the X-radiation treatment. We discovered that the resistant subpopulation of cells surviving a previous fractionated irradiation exposure was likely due to the selection of a radioresistant subpopulation, whereas the radiationsensitive clone manifested a potential inducible radiation response. The third objective was to produce a model system for distinguishing the genetic factors that may be involved in the radiosensitive or radioresistant phenotype. To this end, a set of untreated genetically-related human cell clones of varying radioresponses, with non-overlapping drug sensitivities, were generated from the HCT116 cell line, suggesting the presence of clonal heterogeneity and providing an excellent model. Lastly, using the clones derived from objective 3, the fourth objective was to exploit this model system and determine the genetic basis of radiosensitivity. To do this, we used human cDNA microarrays containing 19,200 ESTs and verified these gene expression changes with Q-PCR. Of a number of genes identified by the array, only spermidine/spermine N1-acetyltransferase (SSAT), an enzyme that catabolizes radioprotective polyamines had a known potential link to radiosensitivity. Our findings establish the potential importance of intrinsic gene expression in radioresponsiveness, and the identification of SSAT as a possible modulator of radiosensitivity, providing a potential tool for understanding and predicting X-radiation response.