Isolation and characterization of a novel pituitary tumor apoptosis gene.

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.

Loss of expression of the growth inhibitory gene GADD45gamma, in human pituitary adenomas, is associated with CpG island methylation.

Inappropriate expression of cell-cycle regulatory genes and/or their protein products are a frequent finding in pituitary tumours; however, genetic changes associated with or responsible for their dysregulation are in general uncommon. In a search for novel genes, and employing cDNA-representational difference analysis, the gene encoding GADD45gamma was recently isolated and identified as being under-represented in pituitary adenomas. GADD45gamma is a member of a family of genes that are induced by DNA damage and function in the negative regulation of cell growth. In this study, we further confirm this initial report that the majority of pituitary adenomas (22 of 33; 67%) do not express GADD45gamma as determined by RT-PCR analysis. Loss of expression was not associated with either loss of heterozygosity or mutations within the coding region of this gene. In marked contrast, epigenetic change, namely methylation of the GADD45gamma genes CpG island, was a frequent finding (19 of 33 adenoma; 58%) and was significantly associated with tumours in which GADD45gamma transcript was not expressed (18 of 22; 82%; P=0.002). In common with the primary tumours, methylation-associated gene silencing of the GADD45gamma gene was also found in the pituitary tumour cell line AtT20. The treatment of AtT20 cells with the demethylating agent, 5-Aza-2'-deoxycytidine, induced the re-expression of this gene. These findings show that silencing of the GADD45gamma gene in pituitary tumours is primarily associated with methylation of the genes CpG island. Methylation has functional importance since reversal of this epigenetic change in a pituitary-derived cell line is associated with re-expression. Silencing of GADD45gamma, a negative regulator of cell growth, is most likely responsible for conferring a selective growth advantage during tumour evolution and outgrowth.