Multiple studies, initially employing candidate gene approaches have described inappropriate methylation of genes responsible for cell cycle arrest and more recently those involved in apoptotic pathways. Indeed, early studies showed that the p16 (CDKN2A) gene is silenced in up to 70% of sporadic pituitary tumours and enforced expression leads to cell cycle arrest.
More recent investigations have taken a whole genome approach where differences in methylation in tumours versus normal tissue were exploited to identify novel targets. Using one of these approaches we identified a gene of previous unknown function that was silenced in the majority of sporadic pituitary tumours and one of the mechanisms associated with silencing was methylation of its associated CpG island. Although enforced expression of this gene in pituitary cells did no lead to cell cycle arrest we found that it sensitised cells to the apoptotic effects of bromocriptine challenge. On this basis we assigned the gene and its protein product the acronym PTAG, for pituitary tumour apoptosis gene.
Still more recently, and the focus of our current research we have employed siRNA technology to knock-down the gene transcripts of proteins responsible for maintaining methylation patterns in cells through successive cell divisions. Since these techniques require actively proliferating cells we used the murine pituitary cell line AtT20 as a model system. These experiments provided convincing data that we were able to reverse methylation and hence silencing. Whole genome microarray analysis of cells in which we had inhibited methylation revealed multiple re-expressed genes compared to cells challenged with a scrambled control. In addition our studies showed that many of these genes, prior to siRNA challenge were silenced through methylation mediated mechanisms.
For several of the genes identified in the model system we extended our investigation to pituitary tumours of human origin. We found that this model, across a species boundary, was able to identify aberrant novel genes that were methylated in tumours of human origin. As proof of principle, and for one ofthe identified genes silenced through methylation in human tumours we performed function analysis. These studies showed that enforced re-expression was responsible for inhibition of proliferation in these cells. Identification and functional analysis of novel genes on a genome wide scale is an important step in our understanding of the aberrations responsible for the initiation and progression of this tumour type. Importantly, since these genes are not deleted or mutated, it will be possible to reverse the methylation through genetic and or pharmacological interventions thus offering exciting new avenues for medical management and intervention.