The family of Crk adaptor proteins is integral to the molecular signaling of cellular migration. Only limited research has demonstrated a role in human carcinogenesis. Through the analysis of human breast cancers with immunohistochemistry, there is a near significant association implicating Crk proteins over-expression and a worsened overall survival. There was no statistical difference in a relation to nodal status. The RNA interference of Crk in human cancer cell lines caused a significant decrease in cell migration. This in vitro suppression of a malignant characteristic could provide a new avenue towards the molecular targeting of breast cancer that may eventually benefit patient outcomes.

The hormonal form of vitamin D3, 1,25-dihydroxyvitamin D 3 (1,25(OH)2D3) is well known for its role in regulating calcium homeostasis. However, many studies have shown 1,25(OH) 2D3 and its analogue EB1089 have broad potential as anticancer agents due to their antiproliferative and pro-differentiating activities. Here, we examined the effects of 1,25(OH)2D3 and EB1089 on proliferation and target gene regulation of human head and neck squamous cell carcinoma (SCC) lines SCC4, SCC9, SCC15 and SCC25 and mouse AT84 SCC cells. A range of sensitivities to 1,25(OH)2D3 and EB1089 was observed, from complete G0/G1 arrest of SCC25 cells to only 50% inhibition of SCC9 cell growth. We have analyzed the molecular mechanisms underlying the antiproliferative effects of EB1089 on SCC25 cells by screening over 10,000 genes on cDNA and oligonucleotide arrays. These studies have identified ~200 novel target genes of 1,25(OH)2D3/EB1089, many of which are essential for normal DNA repair, are markers of cellular differentiation, or are key cell cycle regulators. One of these is the growth arrest and DNA damage (GADD45alpha) gene. Induction of the GADD45alpha gene and its encoded protein in EB1089-treated cells was confirmed by northern and western blotting, respectively. Moreover, while expression of proliferating cell nuclear antigen (PCNA) was reduced in EB1089-treated cells, coimmunoprecipitation studies revealed increased association between GADD45alpha and PCNA in treated cells, consistent with the capacity of GADD45alpha to stimulate DNA repair. Our data also show that amphiregulin, a member of the epidermal growth factor family, is a 1,25(OH)2D3 target gene, and suggest that its induction may contribute to the growth inhibitory effects of 1,25(OH) 2D3. In addition, we show that 1,25(OH)2D 3 analogues induce expression of the cyclin-dependent kinase inhibitor p27KIP1 in different cell types by inhibiting expression of subunits of the SCFSKP2 ubiquitin liga / These studies (1) suggest that differences in action of the EB1089 and 1,25(OH)2D3 arise more from their relative sensitivities to metabolism and than from differing effects on VDR function, (2) substantially expand the number of known D3 target genes in cancer cells (3) enhance our understanding of the molecular mechanism controlling the antiproliferative effects of 1,25(OH)2D3 analogs in cancer (4) provide a number of marker genes that will be useful in assessing the sensitivity of tumor samples to the antiproliferative effects of 1,25(OH)2D 3.

The Wilms' tumor suppressor gene, WT1, encodes a zinc finger protein thought to be involved in transcriptional regulation. It has been shown in a yeast two-hybrid system that wt1 can form homodimers. In this study, random mutations that abrogate this interaction in yeast were generated and characterized as a preliminary means of defining the amino acid residues required for wt1 homodimerization. Five types of mutations were identified: mutations that alter the 5$ prime$ Untranslated Region (UTR), and silent, frameshift, nonsense and missense mutations. Further examination of the position and effect of the frameshift, nonsense and missense mutations suggested the amino terminus, specifically exons 1 and 2, as the region of the protein required for homodimerization. It is hoped that a better understanding of the wt1-wt1 interaction may shed light on the role of this protein in normal kidney development and tumorigenesis.

The regional lymph nodes are frequently the first site to be invaded by disseminating cancer cells and the extent of lymph node involvement is an important determinant of prognosis and treatment for the most common carcinomas. To study the possible role of the lymph node stroma in regulating the growth of breast cancer cells, rat peripheral lymph node stromal cells (STC) were isolated and their interaction with rat and human breast carcinoma cells was investigated. Initially we found that media conditioned by STC was mitogenic for human and rat breast cancer cells and this effect could be enhanced in the presence of extracellular matrix proteins such as fibronectin and type IV collagen, or through stromal-tumor cell contact. Several morphologically distinct clonal sublines of STC were then obtained for further analysis. Using a combination of RT-PCR, Northern blot analysis, immunochemistry and functional assays, two functional subpopulations were subsequently identified; one producing predominantly hepatocyte growth factor (HGF) and the other insulin-like growth factor-I (IGF-I). Monoclonal antibodies generated to these cells indicated that these subpopulations were also antigenically distinct. The results suggest that carcinoma cell growth in regional nodes may be regulated through the cooperative activities of distinct STC populations.

Mammalian Cut proteins appear to function as transcriptional repressors that play a role in determining cell type specificity. Recent evidence suggests that cut genes are tumour suppressor genes. This hypothesis was further examined by using functional assays to evaluate the abilities of human Cut to: (a) suppress oncogene-mediated cell transformation, and (b) interact with the SV40 Large T (LT) antigen. The results indicate that: (a) there was a decrease in the number of transformed colonies and foci when rat embryo fibroblasts (REFs) were transfected with ras/c-myc or ras/E1A and cut; (b) the homeodomain region of Cut interacted with SV40 LT in vitro; and (c) Cut protein levels were elevated and Cut DNA binding was increased in cells transfected or transformed with SV40LT. These observations suggest that Cut proteins may regulate cell proliferation and that the Cut homeodomain may mediate protein interactions.

Wilms' tumor, a childhood malignancy of the kidney, is one of the most common pediatric tumors. The disease occurs in both sporadic and hereditary forms and is associated with a number of congenital disorders. Wilms' tumor appears to be genetically heterogeneous although only a single Wilms' tumor suppressor gene, designated WT1, has been isolated to date. WT1 encodes a zinc finger protein and is mutated in a subset of Wilms' tumors and in patients with Denys-Drash syndrome (DDS), an association of Wilms' tumor and severe genitourinary defects. This thesis reports a mutational analysis of WT1, detailing the spectrum and frequency of mutations in sporadic Wilms' tumors. Most WT1 mutations were homozygous and were predicted to cause premature termination of translation, suggesting that tumorigenesis associated with WT1 involves a two-hit mechanism. The mutational status of WT1 was determined in patients with variable expressivity of the DDS phenotype in order to assess potential genotype/phenotype correlations. Patients with less severe developmental anomalies had germline mutations predicted to result in truncated WT1 proteins, while those with full manifestation of DDS were characterized by missense mutations in the zinc finger region. This demonstrates that different mutations in WT1 are associated with specific effects on genitourinary development. / We investigated the biochemical properties of WT1 and assessed the effects of some naturally occurring mutations. A number of studies have provided evidence that WT1 may be involved in RNA metabolism. We used an iterative selection method to identify potential RNA ligands to WT1. Specific, high affinity ligands were isolated. Mutational analysis elucidated structural features necessary for the RNA-protein interaction. These ligands may reflect RNA sequences targeted by WT1 in vivo. / Since our studies demonstrated that WT1 mutations are restricted to a minority of Wilms' tumors, the understanding of mechanisms underlying Wilms' tumorigenesis required the identification of other genes which contribute to this malignancy. We studied the involvement of the p53 gene tumor suppressor, a critical regulator of abnormal cellular growth and genetic stability. P53 mutations were restricted to the rare anaplastic subtype of Wilms' tumor, a genetically unstable histological variant associated with poor prognosis and resistance to therapy. P53 mutations occurred as late events, associated with malignant progression. The association of p53 mutations with anaplasia allows rationalization of the clinicopathological features of this histological variant, indicating that p53 may regulate genome integrity and chemosensitivity of human cancer cells in vivo.

The malignant phenotype, by definition, is composed of two facets, consisting of the tumor's ability to proliferate as well as its ability to invade the surrounding normal tissue. We propose that in malignant gliomas the protein kinase C (PKC) family of enzymes, which has been shown to be overexpressed in these tumours, plays a pivotal role in both their proliferative and invasive capacity. In the first genes of experiments where the expression of the alpha isoform of PKC was significantly decreased following antisense oligonucleotide treatment, we were able to report the novel finding that this inhibition not only inhibits glioma proliferation in a dose-dependent manner but that this inhibition of growth was the consequence of programmed cell death (1). In the second focus of this thesis, we addressed the postulate that the high PKC activity present in glioblastoma cells may also contribute to the invasive nature of this tumor by modulating the expression of proteolytic enzymes, the matrix metalloproteinases, (MMPs). In contrast to previous studies implicating MMP-9, our data demonstrated that MMP-2 was the metalloproteinase most closely associated with glioma invasion. Furthermore, we were also able to report that the regulation of MMP-2, and thus glioma invasion, could be modulated by PKC (2). In summary, this work presents evidence to support a role for PKC in both the proliferative and invasive phenotypes which characterize malignant gliomas. The translational nature of this research is readily illustrated by the emergence of clinical trials in which PKC and MMPs constitute the principal chemotherapeutic targets.

Met is a transmembrane receptor tyrosine kinase whose ligand is hepatocyte growth factor/scatter factor (HGF/SF). HGF/SF stimulates epithelial cell mitogenesis, motility and branching tubulogenesis. Met and HGF/SF are essential for embryonic development, and are implicated in oncogenesis. In this thesis, the possible role of Met in the development of human breast cancer is examined. The MET gene is located at chromosome 7q31, a region frequently deleted in breast cancer. In this thesis, I demonstrate that MET is included in the smallest common region of deletion of chromosome 7q31 in breast cancer, and is therefore a candidate for a breast cancer tumor suppressor gene. To investigate this further, I elucidated the intron-exon structure of the MET gene. The MET gene is composed of 21 exons, spanning approximately 130 kilobases(kb) of chromosomal DNA. Interestingly, the first coding exon of MET, exon 2, is unusually large, at 1214 nucleotides (nt). Exon 2 is subject to alternative splicing in all epithelial cells studied, and is skipped to produce a 7kb mRNA species which does not encode a protein product. Furthermore, in seven of 13 human breast cancer cell lines studied, exon-skipping of MET exon 2 occurs more frequently when compared to two immortalized breast epithelial cell lines. These seven breast cancer cell lines have an altered ratio of Met mRNA isoforms, with increased levels of the non-protein-encoding 7kb exon 2-skipped mRNA, and a corresponding decrease in the expression of Met receptor in these cells. Significantly, in all other carcinoma types studied to date, the Met receptor is expressed at high levels, as in normal epithelial cells, indicating that loss of Met protein is specific to breast carcinoma cells. The alteration in the normal splicing pattern of MET is correlated with alterations in the normal pattern of expression of SR splicing proteins, which are responsible for regulating alternative splicing. I propose that loss of Met is an event s

Cell transformation and malignancy may modulate some regulatory parameters of DNA replication, resulting in altered features of initiation of DNA replication in these cells. The objectives of this research were to examine the possibility for tumor-specific DNA replication origins and activation of replication origins in human transformed cells. / Conventional PCR was used to detect chromosomal activities of several known and putative replication origins in four human cell lines (HeLa, NSF, WI38 and SK-MG-1). Quantitative comparison of origin activities demonstrates that origins associated with c-myc and NOA3 were approximately twice as active in HeLa cells as in NSF cells. / To test whether the differential origin activities at certain loci in HeLa and NSF cells were due to shifts in origin usage among multiple initiation sites, DNA replication initiation sites and initiation frequencies over 12.5 kb of the human c-myc locus, including 4.6 kb of new 5 ' sequence, were determined by competitive PCR. In this study, one predominant site was located at ∼0.5 kb upstream of the exon 1, and a second new major site was identified in exon 2. The relative usage of origins over the same region of the c-myc locus in the two types of cells was similar, but activities of all origins in HeLa cells were approximately twice those in NSF cells. / In order to better determine if cell type (Hela vs. NSF) or transformation were most likely responsible for altered origin activities, we studied initiation of replication in an isogenic cell pair, WI38 and SV40-transformed WI38 (WI38 VA13 2RA). We found, once again, that the activities of all origins at the c-myc locus were approximately twice as high in immortalized and transformed WI38(SV40) cells as in WI38 cells. This also suggests that the increased activities of origins at the c-myc locus in HeLa cells are caused by the processes leading to malignancy. Although there was significantly increased activity of promoter P2 (7.5--8.0-fold), it had no preferential influence on origin activities at the major sites. Finally, the potential associations between the DNA replication origin activity and transcription activity through higher order regulation by nuclear structure have been discussed.

Glycophosphatidyl-inositol (GPI)-linked carcinoembryonic antigen (CEA) family members, including CEA itself, are absent in rodents and seem to have evolved recently. We have shown previously that CEA-CEA binding required for intercellular adhesion involves a double reciprocal interaction between the N-terminal (N domain) and internal A3B3 domains of anti-parallel molecules on apposed cell surfaces. To answer the question of whether this binding mechanism also evolved with CEA, the adhesive binding mechanism for transmembrane biliary glycoprotein (BGP), presumably more primordial, was investigated. Since CEA and BGP can be expressed on the same cells, and can have opposite effects on the cell phenotype, heterotypic interactions between them were also of interest. A construct with a deletion (17 amino acids) in the N domain of BGP was created which is unable to mediate intercellular adhesion. This $ Delta$NBGP construct, along with BGP splice variant containing only the N domain and various chimeric CEA/NCAM (neural cell adhesion molecule) constructs, was used to establish the fact that both BGP-BGP and BGP-CEA binding are obligate N-N domain. Thus fundamentally different molecular adhesion mechanisms exist in the CEA family.