Investigation of chromosomal abnormalities during colorectal tumour development by somatic cell hybrid and fluorescence in-situ hybridisation analysis

Familial adenomatous polyposis (FAP) is a dominantly inherited disorder where affected individuals usually develop hundreds of polyps in the colon. Cancer inevitably follows as a result of an accumulation of mutations within the adenoma, (the adenoma to carcinoma sequence.) The events leading to colorectal cancer in individuals with FAP, and individuals with 'sporadic' colorectal polyps or cancers were investigated for this thesis. Two approaches were undertaken: Somatic cell hybrid studies in individuals with FAP, and Fluorescence In Situ Hybridisation (FISH), with alpha-satellite probes. Somatic cell hybrids were generated from 6 separate fusions of human tissue from four different FAP patients with the Chinese hamster cell line A23. The human tissues used were one secondary cancer of the liver, two adenomas, two lymphocyte preparations, and one fibroblast cell line. The lymphocyte preparations and the fibroblast cell line were from two FAP patients one known to carry a constitutional abnormality and the other with a somatic clonal abnormality involving chromosome 5. Somatic cell analysis was carried out by PCR using human specific primers, and by FISH painting experiments. The aim was to try to rescue possible chromosomes of interest from the fresh human tissue samples, since the setting up of primary cultures proved unsuccessful. However somatic cell hybrids made from fresh human tissue resulted in a high degree of fragmentation of human chromosomes and therefore these hybrids did not appear to be of any use, at least in the isolation of chromosome 5 marker chromosomes derived from the tumour tissue. For the lymphocyte preparations and for the fibroblast cell line fusions, the aim was to try to separate the normal chromosome 5 from the rearranged one. From these fusions only one somatic cell hybrid clone, P2c, was generated with possible future use in mapping experiments in the region of the gene causing FAP (the APC gene). A second approach was to use FISH with alpha-satellite centromeric probes in order to determine the number of chromosomes 7, 17, and 18 within colorectal polyp and cancer tissue samples from FAP and non FAP individuals. These chromosomes have been implicated in the development of colorectal cancer. Normal mucosa was used as a control to determine the significance of the results. Three colon adenoma cell lines, LIM1215, HCA7, and JW2 were also investigated and compared with their karyotypes. These results were also compared to DNA analysis that had been carried out on the same samples. The results show that although loss of heterozygosity can be detected by DNA probes on these chromosomes, this is not due to whole chromosome loss in many cases, since the centromeric probes did not pick up this loss. In some cases however, chromosome loss was detected by FISH in a significant percentage of cells, but not by DNA analysis, indicating that more than one clone of cells was present in the tissue. The presence of different clones of cells within a single tumour may be important in the development of the tumour as a whole. The FISH data seemed to reflect the basic principle that tumours develop independently even when they are derived from a single individual. The results did not show any strong relationship between patient age, sex, tumour type, size, or site and the presence of cells with numerical chromosomal abnormalities. The main numerical chromosomal change detected by FISH in this study were an increase in chromosome 7 and a loss of chromosome 17 in colorectal tumours from sporadic and FAP patients. Normal mucosa from FAP patients also showed numerical chromosomal changes compared to mucosa from sporadic colorectal patients, indicating that FAP patients may suffer an inherent chromosome instability due to germline mutations in the APC gene. Numerical changes in chromosome 18 were rarely detected but, monosomy of this chromosome appeared to be associated with feeder independence in cell line JW2