The Degradation Of BCR/ABL By Chimeric Ubiquitin Ligase And Its Growth-inhibitory Effect On Chronic Myelogenous Leukaemia Cells

Chronic myelogenous leukemia (CML) is a malignant tumor derived from hematopoietic stem cell, which accounting for20%of the adult leukemia. The annual incidence of CML is1.6/100000. There are three phases:chronic phase, accelerated phase and snap phase. The median survival duration of the untreated patients for three stages is4～5years,6～9months and3～6months, respectively. Thus, CML is a kind of malignant blood system disease that seriously affects human health. Ph chromosome t (9,22)(q34. qll) exist in more than90%of malignant cells in CML patients. Because of such a chromosome translocation, different fusion genes involving BCR and ABL are formed, among them p210BCR-ABL is the most common form. The fusion protein deletes N-terminal Cap domain of c-ABL kinase and thus loss the inhibitory effect on its own; meanwhile, N-terminal coiled-coil domain of BCR mediates homologous dimerization or tetramerization of BCR-ABL, leading to the constitutive phosphorylation of BCR-ABL and the excessive activation of its protein tyrosine kinase activity. Activated BCR-ABL, through recruiting the downstream adaptors, activates multiple signaling pathways and networks incide cells, leading to excessive cellular proliferation and apoptosis resistance. Many clinical and experimental studies demonstrated that excessive activation of BCR-ABL fusion gene is not only a characteristic of CML, but also a very important reason for the occurrence, maintainance and the development of CML. Thus, BCR-ABL is a very good therapeutic target in CMLUbiquitin-proteasome pathway is found to be a highly efficient pathway for degradation of intracellular proteins. This pathway, depending on ATP and ubiquitin, degradates the cytoplasm-and nucleo-protein with highly selectivity and is involved in almost all aspects of cellular processes. Ubiquitination is carried out by three enzymies as following:in the presence of ATP, ubiquitin binds to El and is activated; then ubiquitin is transferred from El-ubiquitin complexe to E2; due to the catalysis of E3, ubiquitin is attached to the target substrate by a isopeptide bond. Upon tagged by ubiquitin, the target protein will be degradated in proteosomes. The E3is the most important enzyme that determines the substrate specificity. RING-finger E3s is named as such due to the catalytic RING domain and is the biggest family.U-box E3s contains U-box domain in structure. These two E3families, which do not forme covalent bonds with ubiquitin, directly or indirectly bind with the substrates and catalyze transfer of ubiquitin from E2-Ub to the substrate, resulting their ubiquitination and degradation. Our group attempt to design and generate the chimeric ubiquitin ligases, so as to selectively recognize and degrade the oncogenic Bcr-Abl in CML.At the first part of the experiment in this study, we constructed the eukaryotic expressing vector of human Bcr-Abl and Bcr-AblT315I by fragmental PCR and tested their expression in HEK293T cells. This laid the foundation for the following study. At the second part of the experiment, we examined the effect of chimeric ubiquitin ligase on target protein in K562and HEK-293T. At the third part, we determined role of the chimeric ubiquitin ligase in proliferation-inhibition and apoptosis-induction in K562cells.Part1:Two fragments of Bcr-Abl (Bcr-a, Abl-c) were amplified from pGD210by PCR. These two fragments were digested using double restriction enzymes NheⅠ/XhoⅠ and KpnⅠ/HindⅢ respectively, and then inserted into NheⅠ/XhoⅠ and KpnⅠ/HindⅢ sites of the eukaryotic expression vector pcDNA3.1(-). The internal fragment between Bcr-a and Abl-c was digested with XhoⅠ/KpnⅠ from pGD210and then subcloned into XhoⅠ/KpnⅠ site of pcDNA3.1(-) that already contains Bcr-a and Abl-c. After confirmation by enzyme digestion and sequencing, pcDNA3.1(-)-Bcr-Abl was transfected into293T cells and the expression of Bcr-Abl was determined by Western Blot. The mutational part was amplified with designed site-mutagenesis primers by PCR from pcDNA3.1(-)-Bcr-Abl, then replaced the original part in pcDNA3.1(-)-Bcr-Abl.Part2:The chimeric ubiquitin ligase pFLAG-CMV-SH2-U or the control pFLAG-CMV-SH2was electronically transfected into K562cells, or transfected into HEK-293T cells together with the eukaryotic expression vector pcDNA3.1(-)-Bcr-Abl or pcDNA3.1(-)-Bcr-Abl T315I, to determine the chimeric ubiquitin ligase-mediated Bcr-Abl degradation We found that either in K562or HEK-293T cells, the chimeric ubiquitin ligase caused downregulation of its target Bcr-Abl protein levels. Real-time PCR further confirmed that the target protein levels was decreased in post-translation level.Part3:The chimeric ubiquitin ligase pFLAG-CMV-SH2-U or the control pFLAG-CMV-SH2was electronically transfected into K562cells, then we determined the cell proliferation by MTT assay and cell apoptosis by flowcytometry. The results implie that the chimeric ubiquitin ligase is able to inhibit proliferation and promote apoptosis of K562cells.To sum up, CML cells have overactivated BCR-ABL fusion protein. With the well-designed ubiquitin ligase that is able to degrade BCR-ABL and BCR-ABLT315I, we will selectively attenuate or eliminate the oncoproteins. This may provide a novel therapeutic strategy for CML treatment.