This thesis explored the association between maternal use of medication during pregnancy and risk of childhood acute lymphocytic leukemia (ALL); specifically, whether use of antibiotics, analgesics, anti-nauseas and/or illicit drugs were associated with an increased risk of ALL in the offspring. All cases of ALL, aged 0--14, diagnosed in Quebec during the period 1994--1997 were identified and matched to population-based controls by age and sex. With an overall response rate of 87%, this resulted in nearly 160 case-control pairs. Information was obtained from parents via telephone interviews, and analyzed using conditional logistic regression. Overall use of medication did not increase risk of childhood ALL (OR = 1.15, 95% CI = 0.66--1.99). Increased risks were observed for illicit drug use in the year prior to birth (OR = 2.44, 95% CI = 0.66--9.00), and for the offspring of women who used pain medication during delivery (OR = 1.88, 95% CI = 1.05--3.31); however, the latter increase was seen for male children only (OR = 3.43, 95% CI = 1.45--8.10).

Intermediate filaments (IFs) are highly regulated and conserved during cell transformation and tumor development. The co-expression of keratin 8, 18 and vimentin has been shown to be related to recurrence and metastasis in both cutaneous and uveal melanomas. This thesis provides the first immunohistochemical evidence that the co-expression of keratin 8, 18 and vimentin is present in one third of conjunctival melanomas. The results show that conjunctival melanomas co-expressing IFs are mixed cell tumors with diffuse growth patterns. It is also shown that melanoma cells co-expressing IFs are mainly located around the peripheral or marginal area of the tumors. In addition, this thesis indicates that the initial tumor thickness significantly increases in conjunctival melanomas co-expressing IFs. Therefore, it is demonstrated that the co-expression of IFs plays an important role in influencing the malignant progression of conjunctival melanomas, and the co-expression of IFs can be used as a prognostic marker for conjunctival melanomas.

Bone marrow toxicity imposes severe limitations on current cancer therapy. To test the feasibility of overcoming chemotherapy-induced hematopoietic suppression, the chemoresistance gene GST was transferred into hematopoietic progenitor cells using the bicistronic retrovirus vectors GIC and GID. As reported herein, GIC transduction of mouse hematopoietic cells resulted in increased in vitro resistance to mechlorethamine (up to 6-fold) and Ara-C (up to 9-fold). Likewise, transduction with GID bicistronic vector conferred increased resistance to mechlorethamine (up to 16-fold) and MTX (up to 12-fold). Furthermore, ex vivo pre-selection of GID-transduced cells was shown to double the fraction of TMTX-resistant progenitor cells. These results show that the retroviral transfer of GIC and GID confers in vitro resistance to nitrogen mustards, cytosine nucleoside analogs and antifolates, and forms an important basis for ongoing in vivo studies to evaluate the therapeutic value of this approach in cancer treatment.

Hepatocellular carcinoma (HCC) is one of the major causes of cancer morbidity and mortality worldwide. However, its incidence is subject to variations: high incidence in sub-Saharan Africa and Asia, intermediate in Mediterranean countries and the Middle East, and low in Western European countries and North America. While different etiological factors have been implicated in the development of HCC, infection with the hepatotropic viruses hepatitis B virus (HBV) and hepatitis C virus (HCV), and exposure to liver carcinogens are the most frequently addressed as directly implicated in HCC development. Epidemiological studies indicate that infection with HBV and exposure to the cyclic mycotoxin Aflatoxin B1 constitute the major risk factors in some regions where the incidence of HCC is elevated. Strikingly, in areas were there is coexistent exposure to both these agents, the incidence of HCC is even higher. In order to elucidate the molecular mechanisms that may lead to a concomitant effect of both HBV and Aflatoxin B1 in HCC development, I hypothesized that HBV may decrease cellular defense mechanisms such as detoxification of active metabolites and/or DNA repair processes increasing the likelihood of mutations. / Detoxification of reactive compounds by phase II enzymes is an important cellular defense process involved in cell susceptibility to carcinogens. I have demonstrated that the activity of human Glutathione Transferase A1 (hGSTAl) enzyme is down-regulated in HBV infected cells. This data correlates with a decrease in protein and mRNA levels. I linked this inhibition---at least partially---to a transcriptional down-regulation of hGSTAl gene expression by the x protein of the HBV (HBx). Strikingly, Oltipraz (4-methyl-5-pyrazinyl-3H-1,2-dithiole-3-thione), which has been found to have cancer chemoprotective properties, overcomes the effect of HBx on the hGSTAl promoter in in-vitro experiments. This result adds new evidence for the importance of the use of Oltipraz as an HCC chemoprotective agent. / I have demonstrated that the HBx is directly involved in the decrease of Nucleotide Excision Repair (NER) activity. Although HBx direct interaction with the tumor suppressor protein p53 was postulated to lead to decreased DNA repair mechanisms, I have shown that this process occurs in both p53-proficient and p53-deficient cells. The results of my work provide evidence that HBx-induced NER inhibition is associated with down-regulation of the expression of the TFIIH factors XPB and XPD. The decreased expression of both genes is regulated by HBx at the transcriptional level. These results were observed in both p53-proficient and p53-deficient cell lines. Interestingly, HBx was found to be capable of transcriptional down-regulation while maintaining its transactivation capacity. In addition, liver tissue from transgenic mice for HBx show decreased levels of XPB and XPD corroborating the results obtained in vitro. / Hence, the HBx protein seems to alter two cellular defense mechanisms that can increase susceptibility to liver carcinogenesis.

Wilms' tumour, a pediatric kidney cancer that affects 1 in 10 000 children, is an excellent paradigm for studying the relationship between cancer and development. The Wilms' tumour suppressor 1 ( WT1) gene was identified through the study of hereditary cases of Wilms' tumour showing cytogenetic deletions at chromosome position 11p13. The WT1 gene encodes a zinc finger transcription factor necessary for the development of the genitourinary system. WT1 functions as an activator or a repressor, interacts with a number of different protein partners and regulates the expression of several genes important for cellular growth and differentiation. WT1 mRNA is present in tissues of mesodermal origin that undergo a mesenchymal to epithelial transition. Expression of WT1 is tightly regulated both temporally and spatially during development of the urogenital system. / We have identified a novel trans-acting factor, named complex D, which shows sequence specific binding to the WT1 promoter. By electrophoretic mobility shift assays (EMSA), we demonstrate that the transcription factor Sp1 binds the WT1 promoter at a site overlapping the complex D binding site. Molecular mass determination experiments and in situ UV crosslinking indicate that complex D is approximately 130 kDa and consists of at least two proteins. Transient transfection assays show that the integrity of the complex D binding site is necessary for maximal activation of a reporter gene, suggesting that complex D may function as an activator. / Similar to WT1, the ETS-domain transcription factor Pea3 is expressed in tissues where mesenchymal-epithelial interactions occur and both gene products are implicated in regulating the expression of genes necessary for the epithelialization of common organs. Transient transfection assays using WT1 promoter-reporter gene constructs identified a Pea3 responsive element in the WT1 promoter. Overexpression of Pea3 transactivates the WT1 promoter and the presence of the intact Pea3 responsive element is necessary for the transactivation. We demonstrate, by EMSA, the sequence specific binding of Pea3 to the responsive element. / WT1 and the paired box domain transcription factor Paired box 2 (Pax2) are expressed at the initial stages of metanephric kidney development and are critical for the initiation of nephrogenesis. We generated WT1/Pax2 compound heterozygous mutant mice to provide an in vivo model for studying the interplay between WT1 and Pax2 during nephrogenesis. WT1+/-/Pax2 1Neu/+ kidneys were 50% smaller that wild type kidneys and displayed a more severe underdevelopment of the medulla, renal calyces and renal pelvis compared to Pax21Neu/+ kidneys. We demonstrate that WT1 and Pax2 proteins physically interact in vitro and in vivo. Our data suggest that WT1 is a modifier of the Pax2 mutant phenotype and that both proteins may be implicated in a common pathway in the transcriptional network governing metanephric development.

Cytogenetic, loss of heterozygosity (LOH) and midrocell-mediated chromosome transfer (MMCT) analyses of epithelial ovarian cancer (EOC) have provided evidence for the role of genes on the short arm of chromosome 3 (3p) in the development of this disease. LOH analysis of ovarian tumors performed in the laboratory of Dr. Tonin identified two minimal regions of deletion (MRD) at 3p25--p26 and 3p24, and a third region extending from 3p14 to the centromere. Based on this study, I hypothesized that there are at least three tumor suppressor genes (TSG)s on the 3p arm that function in suppressing ovarian tumorigenesis. In this thesis, I utilized the novel technology of expression microarrays to identify candidate TSGs based on differential patterns of expression in four well-characterized EOC cell lines compared to a primary culture derived from normal ovarian surface epithelium. MMCT of chromosome 3 into the EOC cell line, OV-90 that harbors complete LOH of one 3p arm resulted in strong suppression of growth. Rapid growth arrest was induced in OV-90 derived hybrids despite the presence of a somatic mutation in TP53 coincident with allele loss at the gene locus. These results suggest that at least one TSG on chromosome 3 is able to function independently of TP53, consistent with the observation made in this thesis that LOH of 3p loci is not significantly associated with somatic mutations in TP53. The acquisition of a growth rate similar to OV-90 in sub-populations of two of the hybrids generated was associated with four regions of deletion on the transferred chromosome 3 at 3p26-pter, 3p14--p21, 3p12--p13, and 3q26--q27, demonstrating the first functional evidence for a TSG important in EOC that maps to each of these regions. Extensive LOH analysis of the 3p25--p26 region of deletion previously identified allowed the definition of a new MRD at 3p26.3 that overlaps the 3p26-pter interval identified in the MMCT study. Expression analysis of genes th

The mechanisms and associated genes implicated in prostate tumorigenesis and cancer progression are still not fully known. Thus, we characterized the expression profile and the role of an attractive candidate, the motility-related protein-1/cluster-of-differentiation antigen 9 (MRP1/CD9) gene (abbreviated as CD9). / We first performed immunohistochemistry studies in a panel of prostate cancer tissues including 107 localized, 60 advanced prostate cancers, 25 bone tissues and 65 lymph nodes with prostate cancer metastasis. CD9 protein, whose expression is inversely correlated with many clinicopathological parameters such as PSA level, differentiation, stage and metastasis status, is inactivated during prostate cancer progression and metastasis. / We then cloned and sequenced CD9 cDNA from prostate cancer cell lines and tissues. This analysis revealed the presence of three major deletions and four commonly occurring point mutations in prostate cancer. All of these genetic aberrations affect the large extracellular and intracellular domains of CD9 protein. We also showed that the overexpression of wild type CD9 protein gives rise to phenotypic changes and loss of replicative potential in an advanced prostate cancer cell line (PC-3). These dying cells demonstrated remarkably enlarged cell surface and prominent cellular protrusions. By carefully defining the time course of these dying cells, we found one third of CD9 overexpressed PC-3 clones died after one and a half months on average after a limited number of cell divisions. In contrast, the other two third of CD9-transfected clones did not die by virtue of loss of exogenous CD9 expression. Furthermore, overexpression of mutated forms of the CD9 protein with one of the major deletions or two of the point mutations did not result in cell mortality and profound morphological changes. / Finally, we compared the RNA expression profile of dying and growing clones using cDNA microarray. Among the differentially expressed genes, the most interesting candidate is MARK1, which is under-expressed by a factor of five in dying clones as compared to growing clones. MAP/microtubule Affinity-Regulating Kinase 1, abbreviated as MARK1, is a member of a family of serine/threonine protein kinases and is able to phosphorylate microtubule-associated proteins (MAPs). The published phenotypic changes reported from overexpression of MARK1 in CHO cells are very similar to what we observed for CD9-overexpressed PC-3 dying cells. Further analysis to confirm the linkage of CD9 protein to cytoskeleton proteins such as MARK1 is compulsory. / These data underline the significance of CD9 expression in prostate cancer progression, uncover major mutations in prostate cancer, and for the first time argue for the connection between CD9 and cytoskeleton-associated proteins. Further studies to confirm the role of CD9 in vivo and to find out the pathway(s) involved in loss of replicative potential caused by CD9 overexpression will definitely improve our understanding of the cell biology of prostate cancer progression.

Objective. To establish a clinically relevant model for the identification of protein serum biomarkers for oral squamous cell carcinoma, and to identify specific candidate proteins. / Methods. Samples of oral cancer and adjacent normal tissue were obtained and were transplanted orthotopically into tongues of immunocompromised mice. When the mice lost 20% of their weight, they were sacrificed by exsanguinations. The serum was analyzed by two separate protocols: DIGE/MALDI and MudPIT/LC/ESI. Preliminary validation was conducted on an established cancer marker. / Results. We identified over one hundred proteins as being differentially expressed between control and cancer-bearing mice (p<0.05); including EGFR, cytokeratin 10, gelsolin, titin, vitronectin, retinoblastoma protein family, bullous pemphigoid antigen, and clusterin. / Conclusion. We report a proteomic approach for the identification of serum biomarkers of oral cancer using an orthotopic mouse model. We identified several proteins that can be exploited as potential markers for diagnosis of oral squamous cell carcinoma.

Non-small cell lung cancer (N-SCLC) is characterized by intrinsic resistance (appearing without drug selection) to chemotherapy treatments. Molecular mechanism(s) of this type of resistance are not fully understood. Using a panel of N-SCLC cell lines, we have demonstrated that upregulation of DNA repair is due, at least in part, to an overexpression of the tyrosine kinase receptor erbB-2. / To explore the mechanism by which erbB-2 affects DNA repair regulation in N-SCLC, I examined the transcriptional regulation of three proteins involved in the early steps of the nucleotide excision repair (NER) pathway, namely XPA, XPB, and XPD, using vectors where a CAT gene was under the control of the promoter for each of the genes. / The second part of my studies was to investigate the effect of potential novel inhibitors of DNA repair synthesis, termed ketonucleosides and keto-C-nucleosides, on cell killing of erbB-2 overexpressing cells compared to cells with low erbB-2. The cytotoxicity and apoptotic induction ability of these drugs was measured in a panel of N-SCLC cell lines. (Abstract shortened by UMI.)

Melanoma-associated antigen (MAA) MART-1/Melan-A has been described in human cutaneous melanoma. The first objective of this study was to investigate the expressivity of MART-1/Melan-A in primary uveal melanomas. 56 formalin-fixed, paraffin-embedded human uveal melanomas were analyzed by immunohistochemistry. MART-1/Melan-A was found to be positive in 73% of cases, with almost 50% of tumours showing a diffuse involvement. The second objective was to investigate the expression of MAA Mart-1/Melan-A and gp100 in primary and metastatic human uveal melanoma lesions developed in albino rabbits. MART-1/Melan-A and gp100 were expressed in all primary lesions, with mainly focal distribution. Conversely, MART-1/Melan-A and gp100 exhibited a homogeneous pattern in the metastatic lesions, which implies upregulation of these antigens during disease progression. It may be postulated that MART-1/Melan-A and gp100 would be effective immunomarkers for immunotherapeutic strategies in human uveal melanoma.