Regulation Of Proliferation And Imatinib-resistance Of Chronic Myeloid Leukemia By Notch Signaling Pathway

For the majority of patients with chronic myelogenous leukemia(CML),the ABL1 kinase tyrosine kinase inhibitor(TKI) therapy has successfully changed the current treatment situation of CML, and it has the milestone significance within the treatment history. Epidemiological studies show that the widespread use of imatinib therapy in CML has the same age as the healthy crowd control about nearly 90% in the relative survival rates of patients with chronic myelogenous leukemia since 2001 [1].However, patients with CML for two years imatinib treatment showed that the complete cytogenetic ease(CCyR) rates of patients has no differences with other treatments [2], at the same time,from the European clinical trials had confirmed that withdrawal of the chronic myelogenous leukemia drug most leaded to relapse at the molecular level, and long-term use of tyrosine kinase inhibitors was no cure for most of the patients with CML. These resultes made the present study focused on eliminating residual lesions the malignant Ph +chronic myelogenous leukemia stem cells, therefore the realization of this results would lead to a permanent cure and long-term disease-free survival. Previous studies focused on the elimination of chronic myelogenous leukemia stem cell research, and the mainlyapproach was through the use of pharmacology and immunological methods to achieve this goal. A growing body of research points out that combined with other important signaling pathways drugs at the same time, those might be prompted to eradication of chronic myelogenous leukemia stem cells.Ph + leukemia stem cells express the same cell-surface markers of HSC, but they are located in the presences of different activated signaling pathways.BCR-ABL fusion gene can activate the myeloid cells signaling pathways. However, not all the pathways help for leukemogenesis. It is more likely that only some critical signaling pathways are the most important key factors of maintaining the CML stem cell. What’re the functions of those pathways in leukemia stem cells?Which signaling pathways could be therapeutic targets?These were the researchers must focus on the core of the problems.The Notch signaling pathway is a highly conservative signaling pathway, and plays a prominent role in cell growth, proliferation, differentiation and apoptosis.Among a wide variety of organisms and the different types of tissue differentiation, Notch signaling pathway plays a significant regulatory role, and it is considered as oncogenesis, including the process of the leukemia occurrence and development. A large number of studies had shown that functions of Notch signaling in normal hematopoietic stem cells HSC were to preserve and promote the self-renewal of HSC, and through the effective activation of Notch signaling could contribute to the expansion of HSC. Furthermore, Notch signaling pathway is a part of the most important signaling pathways, which regulates stem cell proliferation and differentiation. In recent years, there were many researches about the relations with Notch signaling and myeloid leukemia/leukemia stem cell. Some experiments proved that Notch signaling in myeloid leukemia played the role of oncogene,and the others reckoned as the role of tumor suppressor genes.Objective:To reveal the Notch signaling in CML development and molecular mechanism of TKI resistance,and provide a theoretical basis for targeting therapy of Notch signaling pathway,we explore the functions of Notch signaling in chronic myelogenous leukemia and leukemia stem cells,,Methods:1.Comparative and analysis of Notch1 and HES1 expression in gene expression profile chips for Notch signaling related genes in the CML cell line K562, distinct types of leukemia patients with different stages of CML patient’s expression,Imatinib treatment of CML cell lines K562, and imatinib-resistant K562 cell lines, we validated the changes of expression in CML patients and K562 cells by Real time PCR;2.To observe the function of Notch signaling in CML cell line and primitive LSC CML cell biology, We overexpressed Notch ligand DLL1 to activation of Notch signaling in co-cultured cells by using OP9 cells as support cell lines, and applied Notch signaling activator m D1 R to activate Notch signaling in BA+(BCR/ABL) mice for discovering the function of mD1 R in BA + mouse disease development process;3.To reveal the mechanisms of Notch signaling in CML, we used the gene expression profile chip by bioinformatics method to analyze the regulation relationship between Notch signaling pathway and important genes of CML.Finally we established the important molecular interactions network, using Realtime PCR and Western blot to validate the results.Results:1. Compared with other leukemia cell lines, the Notch signaling expression levels of K562 cells were in the middle level; The Expressions of Notch1 in CML patients were higher than other styles of leukemia, and the expressions of HES1 were no obviously different; Notch1 receptor’ expressions in AP of CML were higher than other periods of CML, while HES1’ expressions in patients with relapse and blast crisis increased significantly; Imatinib could obviously decrease the mRNA level of Notch and HES1 in CML cells;the expressions of Notch1 and Hes1 in CML drug-resistant cell lines also increased significantly,and we verified the results by the methods of Realtime PCR.2. The activation of Notch signaling had a certain role in promoting survival rate and the cell cycle in K562; In the co-culture system of Notch signaling activation was beneficial to K562 cloning formation and reducing apoptosis; Activatation of Notch signaling was adverse to against to maintain self-renewal of leukemia stem cells.3. K562 cells co-culturing with OP9 cells experiments with or without Imatinib were demonstrated that activation of the NOTCH promoted K562 cell growth and proliferation.In adding GSIs,K562 cells were lesser than control,and G0 cells increased. After adding Imatinib, the activation of the NOTCH signaling could protect K562 from apoptosis, to avoid killing effect;4.Using gene chip technology and bioinformatics methods, we analyzed the Imatinib-K562 and CML mouse chip data with Genetic variations, GO analysis, and Pathway analysis, for establishing the molecular network diagram.Then,we used qPCR and Western blot methods to validate the expression levels of gene chip data.Finally,we predicted the functions of Notch signaling pathway in proliferation of K562 cells and resistance mechanisms of LSC.Conclusion1.The Notch signaling may participate in the carcinogenesis and progression of CML;2.The Notch signaling may play a different role in different periods of CML cells,activation of the Notch signaling on CML cells to promote growth, on the other hand, play an opposite role in leukemic stem cells. This leads activation of the Notch signaling in CML may complicate.