Mizoribine-mediated Apoptosis Signaling in Jurkat T Cells

ABSTRACT

PURPOSE: Mizoribine (MZR), an inhibitor of Inosine monophosphate (IMP) dehydrogenase which depletes cellular GTP, is clinically used as an immunosuppressive drug. This study was designed to evaluate the mechanism by which MZR exerts the cytotoxic effect on Jurkat T cells. METHODS: Jurkat T cell is a human T lymphocytic cell line. It was obtained from the Korean Type Culture Collection. Cell viability was measured by the MTT assay and flow cytometry. Caspase activity assay, Western blotting, 2-D PAGE, and mitochondrial membrane potential were detected using biochemical analysis. Morphologic finding was observed by Hoechst staining. RESULTS: The data demonstrated that the treatment of MZR decreased cell viability in a dose- and time-dependent manner. MZR-induced cell death was confirmed as apoptosis, which was characterized by chromatin condensation and H2AX phosphorylation. MZR increased the catalytic activity of caspase-3 protease, -8 protease and -9 proteases. The activation of caspase-3 protease was further confirmed by the degradation of polymerase (PARP), a substrate of caspase-3 protease by MZR in Jurkat T cells. Furthermore, MZR induced the changes of the mitochondrial transmembrane potential (MTP) and the cytosolic release of cytochrome c from the mitochondria. In addition, MZR induced the decrease of Bcl-X(L) expression whereas the increase of Bcl-X(S), Bak and Bim expression. Guanosine markedly inhibited cell viability and apoptosis through consistent suppression of the activity of caspase-8 protease, an upstream caspase among the caspase family, H2AX phosphorylation and PARP cleavage in MZR-treated cells. Also, I have screened the expression profile of proteins in the Jurkat T cells by using two-dimensional (2-D) gel electrophoresis. Among 300 spots resolved in the 2-D gels, the comparison of the control versus apoptotic cells revealed that the signal intensity of 10 spots was decreased and 5 spots was increased. CONCLUSION: The results suggest that MZR functions as an inhibitor of IMP dehydrogenase in apoptosis of Jurkat T cells via activation of intrinsic caspase cascades as well as mitochondrial dysfunction.