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.

Molecular pathology shows p16 methylation in nonadenomatous pituitaries from patients with Cushing's disease.

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.

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.

Primary colorectal tumors fail to express the proapoptotic mediator PTAG and its reexpression augments drug-induced apoptosis.

Genes implicated in tumor evolution and progression, including those in apoptotic pathways, are associated with methylation-associated gene silencing in different tumor types. By exploiting differential methylation we recently isolated a novel pituitary tumor derived apoptosis gene (PTAG) that augments drug-induced apoptosis. The importance of PTAG was determined in other tumor types, and these studies show that the majority of primary colorectal tumors fail to express the PTAG gene, indicating an important role for PTAG in colorectal tumorigenesis. The effects of expression of PTAG were examined through stable transfection of the colorectal cell lines HCT116 and SW480. Expression of PTAG, per se, had no discernible effects on cell viability or cell kinetics. In contrast to these findings, in cells subject to drug challenges that engaged either a death-receptor mediated or mitochondrial pathway, all of the experiments indicated a role for PTAG in the intrinsic pathway of apoptosis. Loss of PTAG therefore contributes to a blunted apoptotic response and is likely to predispose cells toward malignant transformation and resistance to chemotherapeutic interventions.