Effect Of Inhibition Of Ubiquitin-proteasome Pathway On Human Leukemic Cells And Normal Bone Marrow & Study Of Its Molecular Mechanism

The ubiquitin-proteasome (UP) pathway is the principal mechanism for the degradation of short-lived proteins in eukaryotic cells. It is involved in a broad range of cellular processes, such as DNA repair, cell cycle progression, transcriptional regulation, antigen presentation and control of gene expression. Recently, the UP pathway has also been found to have an important role in apoptosis, but the exact role of the UP pathway in regulating this process is far from clear. Specific proteasome inhibitors have been shown to in7duce apoptosis in many kinds of human malignant cells through inhibiting of the UP pathway, which provides clues for human antineoplastic therapy. On the other hand, the effect of inhibition of the UP pathway on different cells may vary from each other. Though many studies used proteasome inhibitor to demonstrate the induction of apoptosis, others found that these agents inhibited programmed cell death. Objective: 1, Examine the different effects of proteasome inhibitor Z-TL-CHO on human leukemic cells and normal bone marrow, study the possible mechanism of the ~different sensitivity through the observation of the expression of Bcl-.2 during apoptosis. 2, Using the apoptosis model established by the inhibition of the UP pathway in leukemia cell line and the technology of proteomics to study the changes in protein profile that define the execution phase of the apoptotic response. Method: MTT assay or trypanblue staining was used to evaluate the cytotoxic effects of Z-TL-CHO. Immunomagnetic cell separation and colony formation were performed to investigate the effect of Z-TL-CHO on the proliferation and differentiation of normal hematopoietic progenitor cells. The cell cycle was analyzed by P1 staining in flow cytometry assay. Morphology study was performed through electron or optical microscope. DNA ladder was used to prove apoptosis. The effect of Z-TL-CHO on the expression of Bcl-2 and Caspase-3 druing apoptosis was evaluated by Western blotting. A highly sensitive colorimetric assay was employed to detect the activation of caspase-3. Two- dimensional polyacrylamide gel electrophoresis (2-D PAGE) coupled with analysis of gel image were employed to decide differently expressed proteins during apoptosis, which were waiting for further identification by mass spectrometry. Result: â‘ The proteasome inhibitor Z-TL-CHO could induce many human leukemic cell lines to undergo apoptosis through a dose-dependent manner. Apoptosis was associated with a gradual cleavage of Bcl-2 into a shortened 22kDa fragment as well as the activation of Caspase-3. By comparison, the leukemic cell line M-07e and KG-i a, which both express Bcl-2 at a relatively high level, had different susceptibility to undergo apoptosis, which possibly due to their different levels of activation of Caspase-3 precursor, as well as their different degree of Bcl-2 cleavage after treated by Z-TL-CI-IO. â‘¡Z-TL- CHO had little cytotoxic effect on normal BM MNCs. The cytotoxic effects of Z-TL-CHO were altered in leukemia patients?BM MNCs. Western blotting showed that the expression of Bcl-2 in those primary leukemic cells of high sensitivity was relatively high and ?cleavage of 26kDa Bcl-...