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Title: Methylation of the heparan sulphate D-glycosaminyl-3-O-sulphotransferase gene family in a cohort of breast carcinomas
Author: Dick, Craig Peter Charles
Awarding Body: University of Glasgow
Current Institution: University of Glasgow
Date of Award: 2006
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Background. Aberrant methylation of CpG islands is associated with down- regulation or silencing of the associated gene, and it is therefore an alternative mechanism to DNA mutation for silencing tumour suppressor genes in cancer. Methods. Using 'ICEAMP', a technique that utilises a methyl-binding-domain column to isolate methylated DNA, DNA pooled from 36 ductal invasive carcinomas was screened for aberrant CpG island methylation. This screen did not identify any novel CpG island methylation, however, previous screens of pooled DNA from 128 breast carcinomas had identified methylation at the CpG islands of the genes for heparan sulphate 3-0-sulphotransferase 3B (HS3ST3B) and heparan sulphate 3-0-sulphotransferase 1 (HS3ST1). Literature and database interrogation identified four other HS3ST genes, one of which does not have a CpG island (HS3ST5) and another (HS3ST2) that published data indicated was methylated in a high proportion of breast, pancreatic, and colonic cancers. Using MS-PCR a cohort of 80 breast tumours was analysed for methylation at six different CpG islands associated with HS3ST genes, HS3ST3B has two separate CpG islands, one associated with exon 1 (HS3ST3BE1) and one lying 3kb upstream from the promoter (HS3ST3B). Results. HS3ST1 and HS3ST3A showed no CpG island methylation. HS3ST2 was methylated in 45/80 cases, HS3ST4 in 34/80 cases, HS3ST3B in 30/80 cases and HS3ST3BE1 in 15/80 cases. MSPCR sequencing confirmed dense methylation of both HS3ST2 and HS3ST4 CpG islands in 10/10 cases. Using a cell culture system and RTPCR, transcriptional silence of HS3ST2 and HS3ST4 was found to correlate with methylation of the respective gene. Demethylation of the CpG island using 5-aza-2-deoxycytidine lead to reexpression of the associated gene. Statistical analysis showed there were significant correlations between HS3ST2 methylation status and grade (p=0.015), HS3ST3B methylation and lymphovascular invasion (p=0.028), and number of loci methylated and grade (p=0.04). Conclusions. Diverse patterns of promoter methylation involving the HS3ST genes are observed in breast cancer. Some genes in this family are frequently methylated and downregulated while two of the family, HS3ST1 and HS3ST3A, are never methylated. Individual HS3ST enzymes show substrate specificity for oligomeric sequences in heparan sulphate glycosaminoglycan chains, and 3-0-sulphation of heparan sulphate constitutes < 5% of the sulfuryl groups in a heparan sulphate molecule, this means it may be suited to act as a modulator of cell surface biological processes. 3-0-sulphation is essential for antithrombin Ilia functioning, endocytosis of HSV-1, and potentiates bFGF signal transduction. The data presented suggest that 3-0- sulphation of heparan sulphate could play a role in human breast cancer. Further work is required to determine the biological significance of the dissimilarity in methylation of the different family members.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral