Isolation and Characterization of a Novel Pituitary Tumor Apoptosis Gene

Abstract

To determine mechanisms for pituitary neoplasia we used methylation-sensitive arbitrarily primed-PCR to isolate novel genes that are differentially methylated relative to normal pituitary. We report the isolation of a novel differentially methylated chromosome 22 CpG island-associated gene (C22orf3). Sodium bisulfite sequencing of pooled tumor cohorts, used in the isolation of this gene, showed that only a proportion of the adenomas within the pools were methylated; however, expression analysis by quantitative RT-PCR of individual adenoma irrespective of subtype showed the majority (30 of 38; 79%) failed to express this gene relative to normal pituitary. Sodium bisulfite sequencing of individual adenomas showed that 6 of 30 (20%) that failed to express pituitary tumor apoptosis gene (PTAG) were methylated; however, genetic change as determined by loss of heterozygosity and sequence analysis was not apparent in the remaining tumors that failed to express this gene. In those cases where the CpG island of these genes was methylated it was invariably associated with loss of transcript expression. Enforced expression of C22orf3 in AtT20 cells had no measurable effects on cell proliferation or viability; however, in response to bromocriptine challenge (10-40 microm) cells expressing this gene showed a significantly augmented apoptotic response as determined by both acridine orange staining and TUNEL labeling. The apoptotic response to bromocriptine challenge was inhibited in coincubation experiments with the general caspase inhibitor z-VAD-fmk. In addition, in time course experiments, direct measurement of active caspases by fluorochrome-labeled inhibition of caspases, showed an augmented increase (approximately 2.4 fold) in active caspases in response to bromocriptine challenge in cells expressing C22orf3 relative to those harboring an empty vector control. The pituitary tumor derivation and its role in apoptosis of this gene led us to assign the acronym PTAG to this gene and its protein product. The ability of cells, showing reduced expression of PTAG, to evade or show a blunted apoptotic response may underlie oncogenic transformation in both the pituitary and other tumor types.

Pituitary tumours: findings from whole genome analyses

“…The growth suppressive properties of this gene, in a pituitary context, were investigated in vitro; transfection of GADD45g significantly reduced tumour cell line proliferation as determined by colony-forming efficiency (CFE) assays. Similar conclusions, with respect to reduced expression of GADD45g, in a significant proportion of pituitary tumours were reached in an independent study (Bahar et al 2004b). This study also investigated the mechanisms responsible for or associated with loss of transcript expression and showed that loss of GADD45g expression was significantly associated with methylation of this gene CpG island.…”

“…Interestingly, the murine homologue of this gene is not expressed in the mouse pituitary cell line AtT20 and the CpG island associated with murine GADD45g is also heavily methylated. Treatment of these cells with the demethylating agent 5-Aza 2 0 -deoxycytodine resulted in robust re-expression of this gene (Bahar et al 2004b). It is important to note that these findings do not show that methylation per se is responsible for gene silencing; however, they do show this change is causal in maintaining the silent epigenetic state.…”

Epidrug mediated re-expression of miRNA targeting the HMGA transcripts in pituitary cells

“…cDNA was synthesized using 200U of Maloney mouse leukemia virus reverse transcriptase (Promega, Southampton, UK) according to the manufacturer's instructions and as previously described. [26] Quantitative RT-PCR:…”

Make way for the ‘next generation’: application and prospects for genome-wide, epigenome-specific technologies in endocrine research

“…These techniques would permit, for the first time, the whole genome analysis for identification of novel methylated genes (reviewed in Esteller (2007)). Indeed, our own studies exploiting methylationsensitive digestion and subsequent PCR were successful at identifying novel and inappropriately methylated genes in pituitary adenomas (Bahar et al 2004). However, a concern with these types of studies is that the majority of techniques rely on methylation-sensitive restriction digest, where incomplete digestion can confound the interpretation of derived data.…”