Gene expression in carcinogenesis

Sammanfattning: It is important to understand the processes of carcinogenesis and to find out which genes are involved . One way to study the process on a molecular basis is to identify differences in gene expression. The method mRNA differential display was used to compare normal tissues to different cancer tissues and cell lines in three different experiments to study migration, colon cancer and breast cancer. The first experiment was designed to isolate genes involved in migration of cancer cells by studying glioma cells migrating on different matrices in vitro. In the internal experiment a novel gene, C7, was upregulated in cells migrating on laminin and was isolated and cloned. C7 is the homologue of Drosophila L82, a late puff gene and human OXR1, a gene, which protects cells against oxidation. Additional members of this novel gene family are found in a number of eukaryotic species. In the adult, the C7 gene is most highly expressed in brain and testis. It is upregulated in a cancer cell line, SW480, and a prostate cancer cell line. C7 has at least 4 transcripts expressed in the mouse embryo and was localized to the nucleoli of the cell, by immunohistochemistry, suggesting that it may be involved in the formation or function of this important organelle. In the next experiment, colorectal cancer tissue was compared with normal colon mucosa. The alpha- chain of type 11 collagen (COL11A1) was found to be upregulated and the water channel protein aquaporin 8 (A QP8) was down regulated in colorectal cancer. COL11A is normally not expressed in colon but was here shown to be expressed in 79% of the colorectal carcinomas. Analyses of other collagens showed that COL5A2 was not expressed in normal colon but was co-expressed with COL11A1 in the tumors, suggesting that stromal expression of COL11A1 and COL5A2 is associated with malignancy in colorectal cancer. Patients with germline mutations in the APC gene show, besides colonic polyposis, symptoms of stromal fibroblast involvement, which could be related to COL11A1 expression. To find out if the COL11A was an early event in the carcinogenesis, 14 polyps from one FAP patient were studied. A weak, though statistically significant upregulation in COL11A1 expression was found in adenomas from one FAP patient. It was suggested that expression of COL11A1 in colorectal tumors is associated with the APC pathway in FAP and perhaps also in sporadic colorectal cancer. In situ hybridization demonstrated that AQP8 was expressed in the cells facing the lumen in the normal colonic epithelium. These cells are the mature cells of the migrating epithelial cells of the colon crypt. The result suggests that the expression of AQP8 is a marker of normal proliferating colonic epithelial cells and that tumors arise in cells not expressing this protein. In the third experiment, differential display was used to compare breast cancer tissue, breast cancer cell lines and two normal breast cell lines. The cyclin D2 gene was downregulated in the tumor tissues and in the cancer cell lines. Significantly low cyclin D2 expression was seen in 10/39 (25%) sporadic breast cancers, and in 38/70 (54%) of familial breast cancers. In total, low expression was seen in 48/109 (44%) tumors. In order to find the explanation for the down-regulation of cyclin D2 we screened for inactivating mutations in tumors with low expression and studied methylation of the promoter region in breast cancer cell lines. Growth factors were used in an attempt to stimulate cell lines to express cyclin D2, and we transfected the cell lines with a vector expressing cyclin D2. The results suggest that methylation could be associated with the low expression of cyclin D2 in breast cancer.