Endocrine Abstracts (2002) 3 P119

Identification of novel epigenetically silenced sequences in pituitary tumours

DJ Simpson, SJ Cutty, RN Clayton & WE Farrell

Postgraduate Medicine, University of Keele, Stoke on Trent, Staffordshire, UK

In numerous tumour types, including those of pituitary origin, gene silencing is frequently mediated through inappropriate methylation of CpG islands. In studies designed to identify novel methylated sequences we used a technique termed arbitrarily primed methylation sensitive PCR (MsAP-PCR) to identify DNA in its hypermethylated forms. The technique relies on cutting the DNA, with methylation sensitive restriction enzymes, prior to PCR amplification. In a similar way in which differential display identifies sequence-specific expression differences, this technique employs MsAP-PCR to generate differential fragments that reflect their methylation status.

We have incorporated several modifications (primarily, subtraction techniques) that have eliminated the majority of false positive results. Our investigations, of pooled tumour DNA identified a total of 14 differentially methylated amplicons. Amplicons were resolved, eluted, subcloned and sequenced. Differential methylation was initially confirmed by methylation sensitive sequencing of the pooled tumour DNA (used to isolate the fragment) in comparison to normal pituitary. In some cases [6] these were common to each tumour subtype and for others [8] subtype specific. Analysis across genome databases shows that we have isolated sequences associated with p53 regulated apoptosis, zinc finger binding proteins, glioma TSG region protein 1 and other sequences of unknown function. Preliminary data on two of these amplicons, in non-functioning tumours and somatotrophinoma subtypes, revealed methylation frequencies of 20 and 50% respectively and this was associated with gene silencing. We are currently exploring the function of these frequently methylated sequences in cell line model systems. The information gained will provide targets for anti-tumour therapy.

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