The Role Of Anaphase-Promoting-Complex Cdh1 On Biological Behaviour Research In Blast Crisis Of Chronic Myelogenous Leukemia

BACKGROUD AND OBJECTIVEExosome is a single complex of multiple 3'→5' exonucleases, which is involved in the processing and degradation of several RNA species. CML28 is identical to hRpr46p, a constituent of the human exosome which is a novel 28 kDa TAA identified by serological analysis of cDNA expression libraries (SEREX) screening of a chronic myeloid leukemia (CML) cDNA expression library. As such, CML28 locates in 19q13 which is 28kD, it is overexpressed in tumor cells, such as leukemia, lung cancer, melanoma, and prostate cancer, but not in normal hematopoietic or other tissues, with the exception of testis. Our previously research result has described that CML28 is expressed at high levels in CML-BP primary cells, its expression maybe related with the proliferation regulation of CML cells. Bioinformatics result showed that there maybe an interaction between CML28 and APCcdhl, as a result, we built the relationship between CML28 and APCcdhl in the role of cell proliferation and metabolism.Being one of the important paths for selective protein degradation in organisms, Ubiquitin-proteasome Path-way (UP Path-way) regulates almost all the vital activates in animal and plant bodies, including not only proliferation, differentiation, apoptosis of cells, but also replication, repair, transcription of DNA, as well as control of protein quality. It is also concerned with the courses of pathogens intrusion and immune response to pathogenic organism and human organism. It controls various vital processes from procreation, development, growth to ageing. As a major regulator and terminator of protein function, ubiquitin-mediated protein degradation path-way consists of two processes, i.e. substrate protein ubiquitination and ubiquitin-proteasome dependented protein degradation. Ubiquitin is a highly conservative polypeptide chain composed of 76 amino acids, which is so named because of its wide existence in various kinds of cells. The process of covalent union between ubiquitin and Lys residue of its substrate protein is so-called as ubiquitination, which is a multienzyme cascade reaction mainly mediated by ubiquitin kinase E1, ubiquitin-carrier protein E2 and ubiquitin ligase E3. Ubiquitin can directly transfer from E2 to particular target protein to form ubiquitin-protein complex; however, in most cases, specific binding of target protein and E3 happens at first for E3 can make E2 and target protein get closer to each other, and then target protein combines with the ubiquitin linked to E2. In such a way ubiquitination of target protein is completed. E3 plays an extremely important role in this process. It is the determinative factor for the timeliness and specificity of substrate ubiquitination. In human genome, there is only one kind of E1-coded gene, less than 60 kinds of E2-coded gene and more than 400 kinds of E3-coded gene. E3 makes ubiquitin select substrate protein in specific ways. In most ubiquitination process, selection of substrate protein and connection of ubiquitin are realized depending on specific E3, so E3 is regarded as the most important enzyme in ubiquitination process and it is reputed as the 'brain' of the ubiquitination system. Many E3 existing in complex form have been found gradually, mainly including two categories, i.e. APC/C and SCF complex, which play an important role in regulating genetic transcription, cell cycle and signal transduction, etc.APC/C polyubiquitin ligases E3 have two substrate-linked proteins, APCcdc20 and APCcdh1, both of which are important in cell cycle regulation. Staying in active state from mitosis anaphase to G1 phase, APCcdh1 takes part in regulating cellular mitosis plays a key role especially in the phase of Gl/S transition, whereas APCcdc20 plays an important role from the metaphase to the anaphase of mitosis. Accurate DNA replication and chromatid segregation are key factors for cell division and maintenance of genetic stability. APCcdhl, the anaphase-promoting complex in mitosis, participates in regulation of accurate DNA repair from Gl phase to S phase, acting as cell-cycle regulatory protein.Expression of APCcdh1 exists in the cells of solid tumors such as non-small cell lung cancer, gastrointestinal stromal tumor, breast cancer and colorectal tumor; moreover, there is high expression of APCcdh1 in hematological tumors, especially in chronic myeloid leukemia (CML), which indicates the important regulatory effect of APCcdhl on the come-out and growth of solid tumors and hematological tumors. In recent years, APCcdh1 protein has becoming a focus of tumor study. As the abovementioned, many researches have been made on its effect on solid tumors, but there is no solid evidence about the effect of APCcdh1 on maintenance of genetic stability in CML-blast crisis (BC), and there is no related research about how UP Path-way is influenced by traditional drugs against chronic myelogenous leukemia.Skp2 is one of the subunits of SCF ubiquitin ligase complex, having up-regulated expression in various kinds of human tumors, so it is considered as a potential oncoprotein. Its over-expression is correlated with the development of many tumors, including breast cancer, gastric cancer and lung cancer, etc. SCF/Skp2 participates in regulation of cell cycle through inducing the degradation of CDK inhibitor p27 and p21. However, different E3 may have interaction relationship with each other. Many researches show that, APCcdh1 can influence the development of many tumors via regulating Skp2-p27 pathway, such as gastrointestinal stromal tumor, breast cancer and malignant tumor of colon.According to our findings of previous research, CML28/hRrp46p participates in cell proliferation regulation of K562.1. We plan to use more bioinformatics methods to search downstream, which interact with CML28, to obtain some instructive clues for the further study about the action mechanism and related signaling pathway in blast crisis of chronic myelogenous leukemia.2. According to the result of bioinformatics, we will study the expression and the cellular location of anaphase-promoting complex substrate-linked protein APCcdh1 in K562 and CML-BC primary cells.3. Treated K562 and K562-IM cells with Tyrosine kinase inhibitor (TKI) and protease inhibitor bortezomib, then study the change of the APCcdh1-Skp2-p27 cascade after treated with these drugs, as well as the change of the localization of APCcdh1 protein in CML-BC cells and variation of cell cycle and apoptosis. Discuss the mechanism of imatinib and bortezomib on influencing the biological functions of CML-BC cells.4. Detected the expression of silencing APCcdh1 in human CML-BC cell line K562. Discuss the influence of APCcdh1 expression on biological functions in K562, and also discuss APCcdh1-skp2-p27 cascade for APCcdh1 effect in K562 cells as well as the regulatory effect on APCcdc20.METHOD1. Using bioinformatics methods, we made prediction studies and analysis on the characteristics, functions and domain of CML28 with the help of on-line bioinformatics software such as UCSC human genome database, InterProScan, ProtoNet, Motifscan and ELM, etc. We forecasted the protein APCcdhl which may have interactions with CML28, and further verified the interactions existing between APCcdhl and CML28 with methods of CO-IP and immunofluorescence.2. Western-blot method was used to detect the expression of APCcdhl in CML-BC cells accompanied with additional chromosome karyotype or drug-resistance to imatinib. Immunofluorescence was applied to detecting localization of APCcdhl and Skp2 in CML-BC on the level of protein localization in cells, and moreover, the relation between APCcdhl and pathogenesis of CML-BC was discussed.3. K562 and K562-IM cells were treated with imatinib, nilotinib and bortezomib respectively. Western-blot method was used to detect the variation of protein expression level in APCcdhl-Skp2-p27 pathway and FACS method was used to examine the influence of imatinib and bortezomib on apoptosis and on cell cycle. How tyrosine kinase inhibitor (TKI) and protease inhibitor accelerate cell apoptosis and induce arrest of cell cycle was also discussed.4. Based on full-length cDNA of APCcdhl and Skp2, targeting was designed and prepared for siRNA sequence of APCcdhl and Skp2 respectively. Sequences are shown as follows:APCcdhl siRNA:5'-UGAGAAGUCUCCCAGUCAG-3'Skp2 siRNA:5'-GCAUGUACAGGUGGCUGUU-3'Western-blot and immunofluorescence methods were used to detect the silencing efficiency of APCcdhl and Skp2 interference fragments on the APCcdhl and Skp2 in K562 cells. FACS was adopted to detect the cell-cycle variation of K562 cells after silencing expression of APCcdhl. And more, Western-blot method was employed to detect the regulatory effect of APCcdhl interference on APCcdhl-Skp2-p27 pathway and APCcdc20 expression in K562 cells. RESULT1. Using bioinformatics methods, we made prediction studies and analysis on the characteristics, functions and domain of CML28 with the help of on-line bioinformatics software such as UCSC human genome database, InterProScan, ProtoNet, Motifscan, ELM and PDB etc. The analysis results showed that CML28 protein has a number of (Motif:126-131/210-215/222-227/257-262) highly-conservative and interactive domains D-box, which could be identified by APCcdhl/APCdcc20, activate factors of APC/C, and perhaps there exist interactions and regulatory effect between CML28 and APCcdhl/APCcdc20. With methods of CO-IP and immunofluorescence, we preliminarily proved that certain interactions may exist between APCcdhl and CML28.2. APCcdhl is differently expressed in K562 and CML-BC primary cells, APCcdhl was highly expressed in K562 cells. Lower expression of APCcdhl is detected in CML-BC primary cells accompanied with additional chromosome abnormalities or imatinib-resistance, but relatively higher in such cells without additional chromosome abnormalities or imatinib-sensitive.3. Imatinib or bortezomib can accelerate the apoptosis of K562 cells, and make the cells stay in quiescence in G0/G1 or arrest at G2/M respectively. Both of them can increase the expression of APCcdhl, and lead to degradation of Skp2 and recruitment of p27 at meanwhile, we get the same result when treated the K562/IM cells with nilotinib or bortezomib. We also found that after treated with imatinib and bortezomib, subcellular relocalization of APCcdhl in K562 cells changes from mainly distributing in cytoplasm to nucleus.4. After temporarily down-regulating expression of APCcdhl with a siRNA, the results of Western-blot and immunofluorescence showed that the expression of APCcdhl is significantly down-regulated. The results of FACS analysis indicated that interfering APCcdhl can accelerate the progress of Gl-S. Comparing with the negative control group, cells in G0/G1 was obviously decreased. The results of Western-blot showed that, down-regulating APCcdh1 can obviously recruit Skp2 and APCcdc20, further decrease the expression of p27, and then proceed to participate in regulation of cell cycle.CONCLUSION1. Using bioinformatics methods, we made prediction studies and analysis on the characteristics, functions and domain of CML28 with the help of on-line bioinformatics software. The analysis results showed that there maybe interactions between CML28 and APCcdh1. With methods of CO-IP and immunofluorescence, we preliminarily proved that certain interactions may exist between APCcdhl and CML28.2. Lower expression of APCcdhl is detected in CML-BC primary cells accompanied with additional chromosome abnormalities or drug-resistance to imatinib, but relatively higher in such cells without additional chromosome abnormalities or drug-sensitive to imatinib.3. Our study demonstrates both TKIs and proteasome exert a BCR-ABL-independent effect on control cell cycle progression and apoptosis, apart from that, expression and dynamic distribution of Cdhl, induced by imatinib and bortezomib, provide a novel mechanism to inhibit BCR-ABL downstream cascade via BCR-ABL independent pathway.4. Furthermore, we demonstrated bortezomib still exert the regulation effect on expression and relocation of APCcdh1 even in IM-resistant CML-BC cells.5,Our study demonstrated APCcdh1 silencing leads to promotion of G1-S transition resulting from dysregulation of APCcdhl-Skp2-p27 pathway and regulates the cellular proliferation dynamics.