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.

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Purpose: The majority of cases of Cushing's disease are due to the presence of a corticotroph microadenoma. Less frequently no adenoma is found and histology shows either corticotroph hyperplasia, or apparently normal pituitary. In this study we have used molecular pathology to determine whether the tissue labeled histologically as "normal" is indeed abnormal.Experimental Design: Tissue from 31 corticotroph adenomas and 16 nonadenomatous pituitaries were subject to methylation-sensitive PCR to determine the methylation status of the p16 gene CpG island. The proportion of methylated versus unmethylated CpG island was determined using combined bisulphite restriction analysis. Methylation status was correlated with immunohistochemical detection of p16.Results: Seventeen of 31 adenomas (54.8%), 4 of 6 cases of corticotroph hyperplasia, and 7 of 10 apparently normal pituitaries showed p16 methylation. Ten of 14 (71%; P ‫؍‬ 0.01) adenomas and 2 of 3 cases of corticotroph hyperplasia, which were methylated, failed to express p16 protein. However, only 2 of 7 apparently normal pituitaries that were methylated failed to express p16 protein. Quantitative analysis of methylation using combined bisulphite restriction analysis showed only unmethylated CpG islands in postmortem normal pituitaries; however, in adenomas 80 -90% of the cells within a specimen were methylated. The reverse was true for corticotroph hyperplasia and apparently normal pituitaries where only 10 -20% of the cells were methylated. Thus, the decreased proportion of cells that were methylated, particularly in those cases of apparently normal pituitary, is the most likely explanation for the lack of association between this change and loss of cognate protein in these cases.Conclusions: To our knowledge this is the first report that describes an intrinsic molecular change, namely methylation of the p16 gene CpG island, common to all three histological patterns associated with Cushing's disease. Thus, the use of molecular pathology reveals abnormalities undetected by routine pathological investigation. In cases of "apparently" normal pituitaries it is not possible to determine whether the change is associated with adenoma cells "scattered" throughout the gland, albeit few in number, or with the ancestor-clonal origin of these tumor cells.