Abstract #405: Caspase-8 and -9 interdependency in glucocorticoid-induced apoptosis

Glucocorticoids, like prednisolone (PRED) and dexamethasone, have been used extensively in the treatment of childhood acute lymphoblastic leukemia (ALL). Despite that the mechanism by which glucocorticoids induce apoptosis in leukaemic cells remains largely elusive. The key to maximize the potential of these drugs is to elucidate and understand the processes that are responsible for treatment failure in childhood ALL. We examined the mechanism of PRED induced apoptosis using 4 clinically important pre-B ALL cell lines: 697, Rs4:11, SUPB-15 and REH. After treatment with PRED (10.0µg/mL) for 24 hours, the 4 cell lines can be divided into PRED sensitive (697, Rs4:11, SUPB-15) as well as PRED resistant (REH) cell lines using apoptosis assay (Cell Death Detection ELISA) and MTS assay (Promega). Apoptosis is mediated by caspases that cleave at aspartate residues of their substrates and are responsible for inactivation of important cellular proteins that lead to biochemical and morphological changes. Using western blots and caspase activity luciferase assay kit (Promega), we examined 3 members of the caspase family: caspase-3, caspase-8 and caspase-9. Western blot analyses show that PRED induces the cleavage of all three caspase members in the 3 sensitive cell lines. On the other hand, no caspase cleavage was observed in REH resistant cell line. Upon co-treatment with RU486, a glucocorticoid receptor antagonist, PRED induced apoptosis was repressed together with failure of cleavage of the 3 caspases using the caspase activity assay kit and western blot. Using specific caspase inhibitors, we explored the role of each of these caspases in apoptosis induced by PRED. Apoptosis is not prevented by treatment with a specific caspase-3 inhibitor in sensitive cell lines. In contrast, the addition of either specific caspase-8 or caspase-9 inhibitors was able to individually inhibit PRED induced apoptosis. Interestingly, we observed that the inhibition of caspase-8 activity was followed by a concurrent suppression in caspase-9 activity and vice versa, which indicates that PRED induced apoptosis may involve caspase-8 and -9 interdependency. The knowledge of the pivotal caspases mediating PRED induced apoptosis provides the foundation for further studies with the objective of resolving the complexity of glucocorticoid sensitivity and resistance and maximizing the therapeutic efficacy of glucocorticoid in ALL.

Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 405.