| Description |
The transcriptional regulation of the cyclooxygenase-two gene is the focus of this dissertation. An important feature of the carcinogenic process of the colon involves the constitutive activation of the cyclooxgenase-2 (COX-2) gene instead of its typical mode of induction by growth factors, tumor promoters, and inflammatory cytokines. Here we test the role that a basal regulatory protein Pontin52 plays in this process along with another protein, LEF-1, which is important in Wnt signaling. We find that overexpression of LEF-1 cDNA is sufficient to increase cyclooxygenase-2 promoter activity 1.8-fold by a reporter assay. Similarly, overexpression of Pontin52 increases COX-2 promoter activity 3- to 5-fold; a strong synergistic response is observed with the overexpression of LEF-1 and Pontin52 together. Deregulation of Wnt pathway proteins, LEF-1 and Pontin52, therefore, contributes to the up-regulation of COX-2 transcriptionally. Increased mRNA levels for COX-2 and Pontin52 are observed for cancer tissues as compared to normal colonic mucosa by RT-PCR and in situ hybridization. COX-2 is a downstream target of Wnt and TGF-beta; pathways. We find that the COX-2 promoter is activated synergistically by the overexpression of LEF-1/beta-catenin, and a constitutively active TGF-beta; type I receptor. Mutational analysis of putative Smad and LEF-1 binding sites suggests that the effect mediated on the COX-2 promoter is through basal transcriptional machinery proteins. Thus, a downstream consequence of dysregulation is the overexpression of COX-2 which then contributes to the carcinogenic potential of the cell. Additionally, we test the role of a variant allele of the COX-2 promoter in transcriptional regulation. Although a significantly higher frequency of homozygosity for the variant allele is found in colon cancer cell lines as compared to a collection of random parent DNAs in the Utah and CEPH family sets, no increased frequency of homozygosity is observed in a tissue analysis of normal mucosa, polyp, and colon cancer DNA. The presence of this variant allele, therefore, plays no role in conferring an increased susceptibility in developing colon carcinoma and fails to increase promoter activity. Finally, models are proposed to address the mechanisms by which these regulatory proteins act to effect the COX-2 promoter. |
