Expression and structural analysis of glucocorticoid receptor isoform gamma in human leukaemia cells using an isoform-specific real-time polymerase chain reaction approach
التفاصيل البيبلوغرافية
العنوان:
Expression and structural analysis of glucocorticoid receptor isoform gamma in human leukaemia cells using an isoform-specific real-time polymerase chain reaction approach
Glucocorticoids are broadly used for chemotherapy in childhood acute lymphoblastic leukaemia (ALL). The intracellular effects of glucocorticoids are mediated through the glucocorticoid receptor. The human glucocorticoid receptor gamma isoform (hGR-gamma) differs from the main isoform (hGR-alpha) by an additional amino acid within the DNA binding domain of the receptor protein. This may decrease hGR-alpha-mediated transcriptional activation. The importance of hGR-gamma expression in childhood ALL is unknown. To evaluate hGR-gamma mRNA expression levels, a real-time polymerase chain reaction (PCR)-based approach, allowing the selective amplification of hGR-gamma, was developed and optimized. We were able to demonstrate target selectivity of hGR-gamma amplification using sequence-specific primers. Studying the structure of the 3' end of hGR-gamma, a combination of this isoform with other hGR isoforms could be demonstrated. Using analysis of hGR-gamma-specific amplification in comparison with the expression of hGR-total (all isoforms) in leukaemic blasts from patients with either a good response to prednisone (PGR) or poor-prednisone response (PPR) in vivo, relative hGR-gamma expression was observed to be lower in cells from patients with PGR compared with PPR, in particular after 10 h of dexamethasone stimulation. These data were correlated with cell survival, demonstrating a more pronounced induction of apoptosis in cells from patients with PGR as compared with PPR.