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.