Tnf-alpha Induced Apoptosis In B16 Cells, The Nuclear Proteome Research

Apoptosis, also referred to as programmed cell death, is a process in which a cell commits suicide by a mechanism involving de novo gene expression. In the course of the process of apoptosis, protein translocation occurs among different regions of the cell, including plasma membrane, endoplasmic reticulum (ER), mitochondrion and nucleus. Of them, nucleus is the most important effector organelle. Many pro-apoptotic proteins, such as caspases, AIF, and cytochrome c, translocate into nucleus during apoptosis. Investigation of the alterations of proteins in apoptotic nucleus will help to discover mechanisms, pathways and players involved in induction and regulation of apoptosis. In this present study, we will analyze the changes of nuclear protein composition of apoptotic cells, find new apoptosis-related proteins, and investigate their function involved in apoptosis.Tumor necrosis factor a (TNF-a) is mainly produced by activated macrophages during immune and inflammatory responses, it exerts pleiotropic effects on a wide range of cells. In addition to the inflammatory and immunomodulatory activities, TNF-a may trigger apoptosis in many tumor cells in vitro. In this study, TNF-a was used to induce apoptosis of B16 cells. Apoptosis triggered by TNF-a was analyzed by immunocytochemistry, flow cytometry and caspase 3 activity detection. The results indicated that TNF-a could effectively induced apoptosis of B16cells by time-dependent manner. In addition, TNF-a could induce G1 phase arrest of B16 cells. It is possible that induction of Gl phase arrest is one of the mechanisms that TNF-a induces cell apoptosis.In order to find new apoptosis-related proteins, the alterations of nuclear proteins during apoptosis was investigated. The nuclear proteins were extracted from the purified nuclei of TNF-a-induced apoptotic B16 cells, and then analyzed by 2-dimensional electrophoresis. Compared to untreated samples, eleven altered proteins were found in apoptotic nuclei, and identified by MALDI-TOF-MS. Five of them were up-regulated, six of them were down-regulated. Four of the five increased proteins (HSP84, calreticulin, vimentin, GAPDH)may induced apoptosis directly or indirectly, the other one(Plasminogen) has not been shown to be associated with apoptosis; whereas the six identified decreased proteins represent diverse sets ofproteins including regulators of transcription and signal transduction (guanine nucleotide binding protein, laminin receptor 1), proteins participating in mRNA transport (heterochromatin protein 1 alpha, Heterogeneous nuclear ribonucleoprotein A3, heterogeneous nuclear ribonucleoprotein A2/B1), and protein with unknown function(nucleolar protein NO38), they did not exhibit evident relationship with apoptosis. The finding of the altered protein during apoptosis will help to understand the mechanisms involved in apoptosis.Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a classical glycolytic protein and has been used as a"housekeeping" gene in studies of genetic expression and regulation. However, recent advances revealed that GAPDH displays diverse nonglycolytic functions, including apoptosis, tRNA transport, DNA repair, DNA replication and endocytosiso Among those functions, one of the most intriguing is likely to be the induction of apoptosis. In particular, more evidences exhibited that GAPDH is involved in neuronal apoptosis.In the present study, GAPDH was found increased in nuclei during apoptosis induced by TNF-a. This result is consistent with the former findings in studies in neuronal apoptosis. In order to investigate the mechanism of increase of GAPDH in nucleus during TNF-a induced apoptosis, the cytoplasmic and nuclear proteins were separated, respectively, and analyzed by western blot. The results showed that the nuclear GAPDH obviously increased in apoptotic cells compared to control cells, in contrast, the cytoplastic GAPDH displayed decreased. This means that GAPDH translocates to nucleus during apoptosis. In order to further investigate the nuclear translocation of GAPDH during apoptosis, the vector pEGFP-GAPDH, which can express GAPDH with EGFP tag, was constructed, and used to transfect B16 cells. The GAPDH distribution was analyzed by fluorescence microscopy. The results exhibited that GAPDH was mainly localized in cytoplasm in B16 cells. After induction of apoptosis by TNF-a, GAPDH still accumulated in cytoplasm, no obvious nuclear translocation was found. It is possible that only a small fraction of GAPDH tanslocates into nucleus during apoptosis, which is difficult to discern by fluorescence microscopy.In addition, nuclear localized GAPDH-EGFP fusion protein was also constructed. This fusion protein exclusively accumulated in nucleus in B16 cells, but no evidence...