The proposed research will identify modifications to specific CG- rich DNA sequences that occur at the earliest stages of development of human breast cancer. Identification of the scope and scale of these modifications will enable earlier diagnosis and thus increase the efficacy of subsequent treatment. Modifications (specifically changes in methylation) to CpG Islands (CGIs)occurs during the development of a significant subset of breast cancers. These changes affect the regulation of the gene associated with the CGI. Modifications to methylation patterns have also been reported in other CG-rich regions, for example in satellite or repetitive DNA. Disrupted methylation patterns in these sequences may be an early indicator of an aberration in normal cellular functions. Such alterations may either be a primary cause of malignant transformations or occur as a secondary effect. However once established these changes to the expression patterns are faithfully reproduced in progeny cells. A Methyl Binding Domain (MBD) column will isolate all the CG-rich methylated sequences from samples of normal tissue. Subsequent isolation of CG-rich methylated sequences will be from neoplastic samples at an early stage from the same tissue type. In an additional step a process of subtractive hybridisation will isolate those CG-rich sequences, which display divergent methylation patterns between the two types. The sequences will then be examined using a variety of molecular techniques to identify both the numbers of methylation changes during tumour progression and the earliest modification (s).
