A hallmark of DNA methylation is the fact that 60 to 80% of CpG dinucleotide sequences in the vertebrate genome are methylated at the 5th-position of cytosine while the remaining unmethylated sequences are nonrandomly distributed throughout the genome generating a pattern of methylation that is both tissue and gene specific. Several lines of evidence suggest that methylation patterns correlate with the expression level of eukaryotic genes and that DNA methylation plays an important role in regulating the state of differentiation of mammalian cells. The methylation of DNA is catalyzed by the DNA methyltransferase (DNA MeTase) enzyme, which transfers a methyl group from S-adenosylmethionine to DNA. Cells that exhibit different DNA methylation patterns express similar mRNA levels and DNA MeTase activities. It has therefore been suggested that patterns of methylations are the result of an interplay between the level of the nonspecific DNA MeTase enzyme and other site- or tissue-specific nuclear factors; changing either one of these parameters will result in a change in DNA methylation patterns. In accordance with this hypothesis it has been proposed that regulation of DNA methyltransferase gene expression plays a role in the maintenance and generation of DNA methylation patterns, as is the case in prokaryotic systems. Bestor et al., cloned the cDNA encoding the mouse DNA MeTase gene but nothing was known about the elements regulating its expression. To further understand regulation of the mouse DNA MeTase gene expression I cloned and sequenced the 5$ sp prime$-upstream region of the gene and demonstrated that: (A) The mouse DNA MeTase promoter is a unique housekeeping promoter lacking the classical binding sites for known transcription factors. (B) The mouse DNA MeTase is induced by the Ras-AP-1 pathway. (C) Induction of the mouse DNA MeTase by the Ras-AP-1 leads to profound changes in cell morphology, methylation patterns and tumorigenicity all of which can be inhib

Since the beginning of this work in 1998, it is estimated that 1780 men died of prostate cancer in Quebec. Molecular analysis of prostate cancer will eventually lead to the discovery of key genes involved in its onset and progression. The present project was to compare gene expression profiles in human non-tumorigenic versus tumorigenic prostate cell lines generated in our laboratory. A putative tumor suppressor gene present on 12q13 would be responsible for the non-tumorigenic phenotype of one cell line as discovered earlier by our team. / In order to compare gene expression patterns, expression arrays from Clontech, bearing 588 genes known to be involved in human cancers, were hybridized with cDNA derived from two related cell lines available in our laboratory. This one experiment provided interesting hints on differentially expressed genes that could be involved in human prostate cancer. Interesting clones were confirmed by Northern blots. When commercial antibody was available, analysis was extended at the protein level. A combination of these analyses revealed no striking difference in the level of expression for the genes previously identified by the arrays hybridization. / Simultaneously, differential display PCR techniques, allowing the discovery of unknown differentially expressed molecules and thus complementing the previous approach, were applied to compare related cell lines and unique hybrids. Cloning and sequencing of differential fragments brought us to what could be a new cDNA expressed in many human cell lines. / Prostate cancer is not well characterized enough to allow accurate diagnosis or appropriate therapy strategies. Differentially expressed molecules analyzed in this project as well as the putative new cDNA might fulfil part of this lack in the understanding of this disease.

Activin, a member of the TGFbeta superfamily, regulates a variety of biological effects including induction of terminal differentiation and growth arrest. Malignancy is characterized by loss of the differentiated phenotype and uncontrolled cell growth, therefore investigating the activin signalling pathways and the mechanisms by which it regulates its biological functions have important implications for tumourigenesis. / In my project, I investigated the role of activin on cell differentiation and growth inhibition in different tumoural cell lines. Our results indicate that activin is a potent inducer of cellular differentiation in transformed cell lines of erythroid origin. Furthermore, our studies also indicate that activin is an inhibitor of cell growth in both the erythroleukemia K562 and hepatoma HepG2 cell lines. Proliferation studies indicate that activin mediates its anti-proliferative effects by upregulating the cyclin-dependent kinase inhibitors (CDIs), p15Ink4 and p21cip1/Waf1, which in turn, induce cell cycle arrest. The activin-mediated transcriptional induction of p15Ink4b gene promoter requires the two transcription factors, Sp1or Fast-1, and is mediated through Smad2, 3 and 4. TGFbeta, however, only requires the presence of Sp1 to induce the p15Ink4b gene promoter, highlighting for the first time a difference in the signalling pathways of the two related growth factors, activin and TGFbeta. / In addition to Sp1 and Fast-l, the involvement of the co-activator MSG-1 in the activin signalling pathway was also studied and preliminary results indicate that MSG-1 can increase activin's induction of target genes, suggesting that MSG-1 may have a potentiating effect in the regulation of activin's biological effects.

<p> Although breast cancer is typically considered a disease that affects women at an older age, younger women are also impacted by breast cancer. Additionally, current literature suggests that women who are diagnosed at a younger age face greater challenges adjusting to breast cancer survivorship. Young survivors are often in the midst of starting their career, beginning a family, and planning for their future. Thus, a cancer diagnosis drastically interrupts these plans. This study examined the relationship between different variables that are commonly studied in young breast cancer survivorship. Women were eligible if they were diagnosed between the ages of 19-45 years, were post-treatment at the time of the study, and first-time survivors of breast cancer. Participants were recruited from social media websites, online support and advocacy group, and cancer centers across the United States. Participants completed an online survey. This study examines the structure of the hypothesized latent variables included in a proposed structural model of psychosocial adjustment to breast cancer survivorship. Two of the four hypothesized latent factors were supported by the data, and a revised structural model of psychosocial adjustment to young breast cancer survivorship is provided. Follow-up research should examine the structural model and determine the most important factors in predicting young breast cancer survivors' quality of life and life satisfaction. </p>

This dissertation explores the question “how does a person who is a cancer patient finds their way to being a dying person?” Through the lens of modernism (Giddens), discourse analysis (Foucault), and philosophical hermeneutics (Gadamer) the author examines how the institution of cancer control is constituted, and how the cancer patient is co-constructed by this system and people entering into it as people needing cancer treatment. Language is explored to uncover meanings and discourses which help shape this experience and self-narrative of the cancer patients who face leaving the cancer control system and do or do not find their way to palliative care systems. From this perspective the more solitary and less shaped experience of ‘unbecoming a cancer patient’ is explored for those cancer patients whose treatment has failed. The liminal space between the expert systems of cancer control and palliative care is what is revealed and problematized. What is explored is what this liminal space between these two systems is, and how people who find or lose themselves in this space at this time might be met, without succumbing to the modernist temptation to create yet another expert system to manage what is explored. What is at stake for people at this time is their own self-narrative going on, and it was found for some people in a liminal space this self-narrative faltered. It is revealed that nurses are best positioned epistemologically to support people at this time, and the question of where this support ought to happen is explored in terms of the ideological fit within current health system alignments. This work adds an important theoretical rendering of the term liminality and has important implications for person centred nursing care and health system redesign. / Graduate

Cis-Pt(NH$ sb3) sb2$Cl$ sb2$ is now well established as an antitumor drug. There are still difficulties in its widespread administration, however, because of its numerous side effects and toxicity. Replacement of the NH$ sb3$ groups by amines more compatible to the human system might possibly be a way of surmounting these problems. One such amine is 1-adamantanamine which has been demonstrated to exibit both antiviral and anticancer activity. / New platinum(II) complexes of the type (Pt(A$ sp prime$)Cl$ sb3 rbrack sp-$, Pt(A)(A$ sp prime$)Cl$ sb2$ and Pt(A)(A$ sp prime$)I$ sb2$, where A = methylamine, ethylamine, cyclobutylamine and cyclopentylamine and A$ sp prime$ = 1-adamantanamine, 2-adamantanamine and 1-methyladamantanamine have been synthesized. The structure of (2-adamH) (Pt(EtNH$ sb2$)Cl$ sb3$) complex has been determined by X-ray diffraction. / The synthesis of platinum(II) complexes containing the dimethylformamide ligand (DMF) was undertaken to study the reactivity of DMF with chloro-bridged dimers and trimers. When platinum complexes of the type (Pt(A)Cl$ sb3 rbrack sp-$ were stirred in perchloric acid, oligomeric species were formed which can be cleaved by O-donor ligands such as DMF, water or acetone. A series of complexes of the type (Pt(A)Cl$ sb2 rbrack sb{ rm n}$ and (Pt(A)(DMF)Cl$ sb2$), where A = methylamine, ethylamine, cyclobutylamine, cyclopentylamine, dimethylamine and DMF has been synthesized. The crystal structure of a cyclic trimer, cyclo-tri-$ mu$-chloro-tri- (chloro(dimethylamine)platinum(II)) was determined. All the compounds have been characterized by infrared and Raman spectroscopy, by $ sp{195}$Pt- and $ sp1$H-NMR spectroscopy (when soluble) and occasionally by X-ray diffraction when suitable crystals could be obtained.

Tumors develop and progress as a result of alterations in oncogene and tumor suppressor genes. Knudson's two hit model predicts that cancer arises when both alleles of a tumor suppressor gene are inactivated. The insulin-like growth factor 2 receptor gene (IGF2R) is known to be a tumor suppressor for breast cancer, as mutations inactivating both alleles have been found in these tumors, consistent with its known functions in degrading insulin-like growth factor 2 (IGF2) and activating transforming growth factor beta1 (TGF-beta1), a growth inhibitor. Tumor suppressors can be inactivated by several means, such as genomic imprinting (a preferential silencing of a particular parental chromosome), large deletions resulting in loss of heterozygosity (LOH), and point mutations. In both humans and mice, IGF2R has gamete-of-origin dependent methylation which results in exclusive maternal expression in mice, while in humans imprinted expression is seen only in rare individuals and could be a predisposing factor for cancer. Since it is known that LOH plays a role in the genesis of breast cancer, we hypothesized that in some cases the event inactivating the other gene copy is parental imprinting. Our hypothesis predicted that this LOH is preferentially maternal and, therefore, the remaining allele is unmethylated. We tested this prediction in 20 breast cancer samples, thirteen of which (65%) were heterozygous and 9/13 (69%) had LOH. Two of 9 tumors (~22%) showed complete lack of methylation, consistent with LOH of the maternal allele. However, surprisingly and potentially significantly, normal tissue from the breast cancer patients was completely methylated in 4 samples and completely unmethylated in 2. This suggests that IGF2R methylation disruption may be a pre-existing, cancer-predisposing molecular lesion in the breast of these patients. More work is needed to exclude technical artifacts in reaching this conclusion.

The regulation of the genome by the epigenetic modifications of DNA methylation and histone modification is increasingly recognized as a vital factor in the development, physiology and pathology of vertebrates. There is mounting evidence suggesting that both aberrant DNA methylation and histone modifications are common events in cancer. This has lead to the establishment of both DNMTs and HDACs as important targets in cancer therapy. There are currently several clinical trials that are testing inhibitors of both DNMTs and HDACs as anticancer agents. This thesis attempts to understand the roles that DNMT1 and HDAC1 play in the regulation of gene expression and epigenomic inheritance. In the first chapter, we examined the effects of DNMT1 inhibitors on gene expression and found that DNMT1 can regulate gene expression independent of its DNA methyltransferase activity. This novel role of DNMT1 has challenged a widely accepted theory that the mechanism of DNMT1 inhibitors involves inhibition of the catalytic activity of DNMT1, thus leading to demethylation and reexpression of tumor suppressors previously silenced by methylation. In chapter 2, we further examined different roles of DNMT1. We showed that different DNMT1 inhibitors inhibit different DNMT1 functions and produce different effects on gene expression. Our data suggests that inhibition of DNMT1 enzymatic activity can produce serious long-term effects as a result of massive non-selective demethylation of the genome. In contrast, reduction of DNMT1 levels was shown to result in a rapid arrest of cell growth, limited demethylation and induction of stress-response genes. We hypothesize that these effects are a result of the activation of an epigenetic check point that has evolved to protect the cell from undergoing replication in the absence of DNMT1. In chapter 3, we further explore the roles of DNMT1 in methylation independent regulation of gene expression, which has been suggested to in

Mitochondrial background. Recent studies on cancer have detected many mutations and much variability in the mitochondrial genome, particularly in the non-coding region (D-loop). The present study set out to sequence and examine the hypervariable region I (HVR-I) of the D-loop and transfer RNA Leucine (tRNA Leu) for mutations in breast cancer patients. Methods. Tumor and normal tissues from 17 patients that carry mutations in either BRCA1 (n = 11), BRCA2 (n = 3) or are non-carriers of mutations in either gene (n = 3) were examined by direct genomic sequencing of PCR products. Results. We found 44 variants in the HVR-I of 16 patients, twenty-six were polymorphisms, four somatic variants, and fourteen variations were undetermined because corresponding (unaffected) normal tissue was not available. One BRCA1 mutated tumor had four somatic tumor variants (1/14). All other BRCA1/BRCA2 mutated tumors had no somatic variants. Nine out of 14 (64%) of these patients had a total 22 germline variants. One out of three (33%) non-carriers had four germ-line variants. No variants were found in tRNA Leu. A five kilobase deletion was also found as a germ-line variant in two of seven (29%) patients. There were no obvious differences in the frequency of homoplasmic variants in the mitochondrial genome between the BRCA1/BRCA2 mutation carriers and non-carriers. Conclusion. Direct genomic sequencing of PCR products showed that there were no striking differences in homoplasmic variants between tumor and normal tissue, thus homoplasmic variants in mtDNA did not have a role in tumorigenesis in our samples. We speculate that the marked differences in mutation frequencies observed amongst various studies could be the result of differences in the techniques used to generate and analyze the data. / EGFR background. Recent studies have identified mutations in the tyrosine kinase (TK) domain of the epidermal growth factor receptor (EGFR) in lung carcinomas. These mutations make the tumors sensitive to a molecular targeted drug called getifinib. Methods. We also sequenced the TK domain of EGFR in 16 breast cancer patients (BRCA1 = 9, BRCA2 = 4, non-carriers = 3) for mutations. Results. We did not find mutations reported previously in lung carcinomas but we identified other variants, in exons 18, 20 and 21 of the mutation carvers. We found one out of thirteen (eight percent) of the BRCA1/BRCA2 mutation carriers had somatic tumor variants, three out of thirteen (23%) patients had somatic variants found only in the adjacent normal tissue, and four out of thirteen (31%) patients had germ-line variants. The non-carriers did not have any variants. The variants found in the exons were two missense variants in exon 18 of two patients, three 'silent' substitutions in exon 20 of three patients, and two patients had exon 21 variants; a missense variant and a 'silent' substitution. Intronic variants were also found in three patients. Patient 5420 harbored more than one variant in the tumor tissue and patient 5483 harbored more than one somatic variant in the adjacent normal tissue. Although the sample size is small, these preliminary results seem to show a difference in EGFR variants between BRCA1/BRCA2 mutation carriers and non-carriers. Conclusion . EGFR variants found in this study were not the same ones found in lung cancer, but other variants could be significant in breast cancer progression and could possibly represent drug targets for future therapy.

Invasion and metastasis largely determine the clinical course of cancer. A better understanding of the mechanisms required for metastases formation in secondary sites will lead to the development of new and more successful treatment strategies. The present work describes our results with a murine Lewis lung carcinoma model which consists of two cell lines, H-59 and M-27, with different patterns of metastasis in vivo. Using this model, a positive correlation was found between the expression of insulin-like growth factor I receptor (IGF-IR) and the ability of the tumor cells to metastasize to the liver. The highly invasive, highly metastatic, liver colonizing cell line H-59 expressed significantly higher levels (5-fold) of IGF-IR than the poorly invasive M-27 cells which metastasize to the lung only. When expression of IGF-IR in H-59 cells was suppressed by transfection with a plasmid vector expressing IGF-IR cDNA in an antisense orientation, the cells lost their proliferative response to IGF-I in vitro, their ability to migrate in response to IGF-I and their metastatic potential. To further study the role of IGF-IR in the process of liver metastasis, IGF-IR was overexpressed in the lung metastasizing M-27 cells by transfection with a plasmid vector expressing full length human IGF-IR cDNA. The stable transfectants had an enhanced proliferative response to IGF-I and hepatocyte conditioned medium (HCM) and acquired an invasive potential as demonstrated in the Matrigel invasion assay. When inoculated via the splenic/portal route in vivo, these cells but not the wild-type or mock-transfected cells gave rise to multiple liver nodules. To further investigate the link between IGF-IR and invasion, metalloproteinase 2 (MMP-2) expression in the tumor cells was investigated. It was found that the antisense transfected H-59 cells expressed significantly lower levels of MMP-2 as assessed by RT-PCR, Western blot analysis and gelatin zymography. M-27 cells overexpressing IGF-IR