MIZORIBINE-MEDIATED APOPTOTIC SIGNALING PATHWAY IN HUMAN T CELL LINE

Abstract

P778 Propose: Mizoribine (MZR), an inhibitor of IMP dehydrogenase which depletes cellular GTP, is used clinically as an immunosuppressive drug. This study was designed to evaluate the mechanism by which MZR exerts the cytotoxic effect on Jurkat T cells. Methods: The data demonstrated that treatment of MZR decreased cell viability in a dose- and time-dependent manner. MZR-induced cell death was confirmed as apoptosis characterized by chromatin condensation and H2AX phosphorylation. Results: MZR increased the catalytic activity of caspase-3 protease -8 protease and -9 proteases. Activation of caspase-3 protease was further confirmed by degradation of polymerase (PARP), a substrate of caspase-3 protease by MZR in Jurkat cells. Furthermore, MZR induced the changes of mitochondrial transmembrane potential (MTP) and the cytosolic release of cytochrome c from mitochondria. In addition, MZR induced the decrease of Bcl-XL expression whereas increase of Bcl-XS, Bak and Bim expression. Guanosine markedly inhibited cell viability and apoptosis with consistent suppression of activity of caspase-8 protease, upstream caspase among caspase family, H2AX phosphorylaion and PARP cleavage in MZR-treated cells. Also, we have screened the expression profile of proteins in Jurkat cells by using two-dimensional (2-D) gel electrophoresis. Among 300 spots resolved in 2-D gels, the comparison of control versus apoptotic cells revealed that signal intensity of 10 spots was decreased and 5 spots was increased. Conclusions: The results suggest that MZR functions as an inhibitor of IMP dehydrogenase in apoptosis of Jurkat cells via activation of intrinsic caspase cascades as well as mitochondrial dysfunction.