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Title: Characterisation of a novel pituitary tumour apoptosis gene identified by methylation sensitive screening
Author: Bahar, Adil.
Awarding Body: University of Keele
Current Institution: Keele University
Date of Award: 2005
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Epigenetic changes associated with gene silencing are a frequent finding in multiple tumour types including those of pituitary origin. Several techniques have been described that exploit differential methylation in tumour versus normal tissue to identify novel differentially methylated sequences. One of these techniques, methylation-sensitive arbitrarily primed PeR (MsAP-PCR), was applied to pooled pituitary adenoma and post-mortem normal pituitary DNA, to isolate and characterise novel differentially methylated sequences. A novel chromosome 22 CpG island-associated gene (C220rf3), was identified as differentially methylated, determined by sodium bisulphite sequencing. Although the analysis of pooled tumour DNA showed that only a small proportion of tumours harboured this epigenetic modification, expression analysis, employing quantitative RTPCR (qRT -PCR), showed that the majority of pituitary tumours (-80%) failed to express this gene relative to normal pituitary. Northern blotting confirmed the expression status of this gene as determined by qRT-PCR. Sodium bisulphite sequencing of individual tumours showed that 6 of 30 (20%) that failed to express this gene were methylated; however genetic changes as determined by loss of heterozygosity (LOH) and sequence analysis, was not apparent in the remaining tumours that failed to express this gene. In tumours that were methylated this change was invariably associated with loss of transcript expression. Enforced expression of C220rf3 in AtTt20 cells had no measurable effects on cell proliferation or viability, however in response to bromocriptine challenge (lO-40IlM), cells expressing this gene showed a significantly augmented apoptotic response as determined by both acridine orange staining and TUNEL labelling. The apoptotic response to bromocriptine challenge was inhibited in co-incubation experiments with the general caspase inhibitor z-VAD-fmk. Furthermore, measurement of active caspases by flourochrome labelling, showed an augmented caspase activity (-2.4 fold) in response to bromocriptine challenge in cells expressing C220rf3 relative to those harbouring an empty vector control. The .e.ituitaryJumour derivation and a role in apoptosis of this gene led us to assign the acronym PTAG to this novel gene and its protein product. To further define the role of PTAG in apoptotic pathways, AtT20 cells were challenged with drugs that activate either the intrinsic (etoposide) apoptotic pathway or that are known to induce apoptosis through a receptor-mediated pathway (the PPARy receptor ligand rosiglitazone). An augmented apoptotic response was only apparent in PTAG expressing cells to challenge with etoposide, and apoptosis was effectively inhibited in co-incubation experiments with a caspase-9 specific inhibitor. Conversely, apoptosis induced in rosiglitazone challenged cells was inhibited with a caspase-8 specific inhibitor whereas the caspase-9 inhibitor was without effect. These findings suggest that these two agents (etoposide and rosiglitazone) activate an intrinsic and receptor-mediated pathway respectively. Collectively, these finding place PTAG in an intrinsic apoptotic pathway in these pituitary tumour derived cells. The ability of cells, showing reduced expression of PT AG, to evade or exhibit a blunted apoptotic response may underlie oncogenic transformation or perhaps a subsequent drug resistant phenotype in both the pituitary and perhaps other tumour types. It will be important to define mechanisms responsible for its loss since re-expression may act to "chemo-sensitize" cells and thereby lead to more efficacious chemotherapeutic interventions.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available