The Role Of Rho GTPases In Abnormal Behaviors Of Leukemic Cells And Aberrant Interaction Of Leukemic Cells With Bone Marrow Microenvironment

Hematopoietic stem cells (HSCs) reside in a complex bone marrow (BM) microenvironment, which is critical for regulating HSCs behavior and fate, including self-renewal, proliferation and differentiation. HSCs homeostasis in each of stem-cell niches is achieved by the delicate balance of cell adhesion and cell motility among distinct location regions and is important for the development of hematopoietic stem cells. Disruption of this homeostasis leads to many detrimental pathological conditions that implicated in HSCs' unbalanced self-renewal and abnormal behaviors.Rho GTPase is a 20-30KD GTP-binding protein that belongs to the Ras superfamily of small G proteins. Rho GTPase family is a key factor that connects extracellular signal and actin cytoskeleton system and regulates cell polarity, cytoskeletal rearrange and actin cytoskeletal dynamic. It has become evident that overexpression and activation of Rho GTPases family are important machanism regulating cell adhesion and migration. To determine the role of abnormal interaction of hematopoitic cells with bone marrow microenvironment in leukemogenesis, we investigated the expression and significance of Rho GTPase family members in leukemic cells. Through detecting the proteins levels of Rac1,RhoA and CDC42 in leukemia patients and determining the role of overexpression and activition of Rac1 in the abnormal behavior of leukemia cells and aberrant interaction of leukemic cells with bone marrow microenvironment, we elucidate that abnormal Rho GTPase associated microenvironment may lead to malignant hematopoiesis.The details are as follows:1. The expression of RhoA. Rac1 and CDC42 proteins in leukemia patients:via Western-blot assay, we detected the relative expression of Rac1 in BMMC samples that were separated from 88 primary acute leukemia patients and 22 normal donors. Meanwhile the relative expressions of RhoA and CDC42 in 54 primary acute leukemia patients were also detected. The results showed that expression levels of RhoA,Rac1 and CDC42 protein in leukemia patients were significantly higher than that of normal samples with P＜0.01.2. The role of high expression of Rac1 in leukemia cells:Through interfering the expression of Rac1 in leukemia cell lines KG1-a and HL-60, we then determined the migration and proliferation ability of leukemic cells. The results showed that siRNA mediated silencing of Rac1 in leukemia cell lines induced inhibition of cell migration, proliferation and colony formation.3. The role of activition of Rac1-GTPase in leukemia cells:Leukemia cells were further treated by Rac GTPase-specific small molecular inhibitor NSC23766. Cell migration and proliferation were then determined. The results showed that blocking Rac1 activity by NSC23766 suppressed cell migration and growth. We further constructed the lentivirus vector carrying cDNA of constitutively active Rac1 or dominant negative Rac1, and infected KG1-a cells. GST-pull down assays confirmed the activity of Rac1-GTPase in infected cells. Then we detected the biological function of infected cells. The results showed that KG1-a cells infected with constitutively active Rac1 lentiviral vector displayed significantly increased cell migration and resistant to VP16. Adhesion of constitutively active Rac1 lentiviral vector infected KG1-a cell with bone marrow matrix cells was significantly descreased. By contrast, the behavior of KG1-a cells that infected by dominant negative Rac1 vector is reverse. Our experiment results indicated that the abnormal activition of Rac1-GTP correlates with accelerated migration,descreased adhesion and resistant to chemotherapeutics VP16 of leukemia cells.The above investigation suggests that overexpression and abnormal activition of Rac1 leads to efficient migration, vigorous proliferation and descreased adhesion. In bone marrow microenvironment, alterations of HSCs' migratory behavior might lead hematopoietic stem cells to escape from the restriction of microenvironment and obtains uncontrolled proliferation ability. Those alterations may contribute to the leukemia development and progression.