Ding, Shi-fa; (1994) Study of tumour suppressor genes in malignant tumours of liver, pancreas and biliary system. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

The importance of tumour suppressor gene inactivation by gene deletion and/or mutation in tumour development has been established. Loss of heterozygosity (LOH), or allele loss, in tumour DNA is thought to represent the loss of such genes. Detection of LOH in particular tumours has been widely used for searching for the loci of tumour suppressor genes in the tumours. Few studies on tumour suppressor genes in tumours of liver, pancreas and biliary system have been performed. The work in this thesis was undertaken to test the hypothesis that loss of tumour suppressor genes by LOH occurred in these tumours. Eighty-two such tumours (liver tumours: 53; carcinoma of the pancreas: 15 and cholangiocarcinoma: 14) were investigated to search for the consistent LOH. Paired normal and tumour DNA were analyzed by Southern hybridisation using a panel of restriction fragment length polymorphism (RFLP) DNA probes, previously assigned to different chromosomal regions. In informative patients, band losses in tumour DNA compared with bands of normal DNA on autoradiographs were scored as LOH or allele loss. In hepatocellular carcinoma (HCC) without liver cirrhosis, LOH in the chromosome region 5q35-qter was found in 6 out of 6 informative patients detected by the probe ?MS8, and the loss was distinct from the adenomatous polyposis coli (APC) locus in the region of 5q21-22. The results strongly suggested that a putative tumour suppressor gene for HCC without liver cirrhosis was located in the region 5q35-qter. A high frequency of loss was also found in 17p13, the chromosome region encompassing the p53 tumour suppressor gene. HCC with liver cirrhosis had no loss in the region of 5q35-qter, but in 1q42-43, 5p and 17p13. LOH occurred at a low frequency in fibrolamellar carcinomas and in none of the hepatocellular adenomas studied. In contrast, multiple allelic losses were found in two sarcomatoid liver carcinomas. Cholangiocarcinoma had a similar LOH pattern to that in HCC without liver cirrhosis, ie, losses in 5q35-qter and 17p13. Liver metastases from colorectal cancers showed a high incidence of allele loss on chromosomes 5q21-22, 17p13 and 18q. In carcinoma of the pancreas, LOH was detected in 1p33-35, 6q27 and 11q13. An extensive pilot study was performed to isolate the putative tumour suppressor gene in chromosomal region 5q35-qter for HCC without liver cirrhosis and cholangiocarcinoma. Effort was made to isolate DNA sequences from chromosome 5 that were expressed in normal human liver tissue, on the assumption that tumour suppressor genes are transcribed in normal tissues. This was performed by cross-screening a human chromosome 5 genomic DNA library in phage Charon 21A with a normal human liver cDNA library. Twenty-one positive clones in phages were obtained after the third round of screening. These clones were recloned to plasmid Bluescript KS+ for further use since clones would be easier to use in plasmid vectors than in phages. A proposal to identify the putative tumour suppressor gene from these clones was made.

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