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.
Subject(s)
Humans , Apoptosis , Blotting, Western , Caspase 3 , Caspase 8 , Cell Death , Cell Line , Cell Survival , Chromatin , Cytochromes c , Cytosol , Electrophoresis , Flow Cytometry , Gels , Guanosine , Guanosine Triphosphate , IMP Dehydrogenase , Inosine Monophosphate , Membrane Potential, Mitochondrial , Membrane Potentials , Mitochondria , Oxidoreductases , Peptide Hydrolases , Phosphorylation , T-LymphocytesABSTRACT
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.
Subject(s)
Humans , Apoptosis , Blotting, Western , Caspase 3 , Caspase 8 , Cell Death , Cell Line , Cell Survival , Chromatin , Cytochromes c , Cytosol , Electrophoresis , Flow Cytometry , Gels , Guanosine , Guanosine Triphosphate , IMP Dehydrogenase , Inosine Monophosphate , Membrane Potential, Mitochondrial , Membrane Potentials , Mitochondria , Oxidoreductases , Peptide Hydrolases , Phosphorylation , T-LymphocytesABSTRACT
PURPOSE: To determine accurately the extent of the disease and risk of recurrence is important in enhancing the therapeutic success rate of breast cancer. Primary tumor state and axillary node invasion were some well known factors to predict the prognosis of breast cancer. However, some patients with early stage cancer developed systemic metastasis later despite of little possibility of recurrence based on some previously establised prognostic system. These results demand another approach to predict systemic metastases in patient without gross evidence of further recurrence. Micrometastases is a promising key to explain the recurrence in these patients and micrometastases in bone marrow could raised the ongoing recurrence in skeletal system which is the most frequent metastatic site in breast cancer. Therefore we tricd to determine the rate of micrometastasis in surgically resectable Korean breast cancer patients and the relationship with clinicopathological characteristics of the cancer. MATERIALS AND METHODS: We studied bone marrow aspirate specimens from 38 patients with breast cancer who underwent curative resection at Chonnam University Hospital from January 1996 to February 1997. And reverse transcription polymerase chain reaction (RT-PCR) to detect messenger RNA for cytokeratin 19 was performed. RESULTS: Metastases in bone marrow were detected in 8/38 patients (21.1%). No sta- tistically significant relationship existed between bone marrow metastasis and clinicopa- thological parameters for predicting prognosis that consisted of tumor state, lymph node invasion, histologic grade, steroid receptor, and c-erbB2 overexpression. In particular, bone marrow metastasis developed even from ductal carcinoma in situ. CONCLUSION: Bone marrow metastasis may be developed from the extremely early stage of breast cancer and we can not make the corelationship between the bone marrow metastasis and establised some prognostic factors. Based on these results, we recommand the evaluation of bone marrow metastasis in all breast cancer patients and require the close follow-up to allow more sensitive prediction of ongoing recurrence and higher curability.