To determine mechanisms for pituitary neoplasia we used methylation sensitive arbitrarily primed-PCR (MsAP-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 bisulphite 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 qRT-PCR of individual adenoma irrespective of subtype showed the majority (30 of 38; 79%) failed to express this gene relative to normal pituitary. Sodium bisulphite sequencing of individual adenomas showed that 6 of 30 (20%) that failed to express PTAG were methylated, however, genetic change as determined by LOH and sequence analysis was not apparent in the remaining tumors that failed to express this gene. In those cases where this genes CpG island 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 µM) 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 co-incubation experiments with the general caspase inhibitor z-VAD-fmk. In addition, in time course experiments, direct measurement of active caspases by FLICA labeling, showed an augmented increase (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.