Blocking CD44 And Wnt Signaling Pathway Between The Vicious Cycle Of Inhibition Of CML Progression

Chronic myelocytic leukemia(CML) is a common hematological malignancy.The treatment of CML patients have been significantly improved in these years since the advent of imatinib.But It can not be completely cured because of the leukemia stem cells. Self-renewal.proliferation and differentiation are the main characteristics of stem cells including leukemia stem cells.Its biological characteristics maintenance depends on a number of signaling pathways such as Hedgehog, BMP, Notch and WNT/β-catenin. The role of WNT/β-catenin pathway in normal and cancer stem cells has been well studied. Our study found that,β-catenin was associated with CML significantly. CD44is a cellular adhesion molecule.It is important to the stem cell including leukemia stem cells.Many studies have mentioned the relationship between CD44and CML in recent years.Since CD44and p-catenin both have close relationship with CML, we speculate that CD44and WNT/β-catenin pathways reinforce each other and promote the progress of CML together.Based on this hypothesis, our study focus on CD44monoclonal antibody can block WNT/β-catenin pathway, to prevent the progress of CML.This reserch include two parts.First we want to study the impact on the proliferation of CML cell line K562and intracellular (3-catenin of K562when treated with CD44monoclonal antibody with cell counting, flow cytometry, immunofluorescence. Western Blot, RQ-PCR and other methods.We make the growth curve of K562in different culcture conditions.The results show that, CD44monoclonal antibody can not specifically inhibit the proliferation of K562cell.How the intracellular β-catenin of K562changes when treated with CD44monoclonal antibody are not clear.Maybe the level of β-catenin in K562cells is very low.Second,we studied how the level of CD44changed when activating or blocking the WNT/β-catenin signaling pathway in K562cells with flow cytometry. The data was processed with statistical method,and make correlation analysis.The results show that wether activating or blocking the WNT/β-catenin pathway,therewas no correlation with the level of CD44in K562cells.In summary, CD44monoclonal antibody can not inhibit the proliferation of K562cellsspecifically.Its impact on the WNT/β-catenin pathway inK562cells is unknown.But the WNT/β-catenin pathway had little effect on the expression of CD44in K562cell. So we know that unlikely we first speculated, they do not promote each other. Objective To investigate the effect on the survival.self-renewal, proliferation and totipotency of CD34+CD38-Lin-cell when they were cultured at single cell level without microenvironment in vivo such as cross-talk between cells and different cytokines.etc. Methods Purified UCB CD34+CD38-Lin-cells were separated at single cell level in96-well plates using flow cytometry for four goups:ontrol group [CD34+CD38-Lin-cell+Stem Cell Medium],Shh group [CD34+CD38-Lin-cell+Stem Cell Medium+Shh],BMP-4group [CD34+CD38-Lin-cell+Stem Cell Medium+BMP-4] Jagged-1group [CD34+CD38-Lin-cell+Stem Cell Medium+Jagged-1].Methylcellulose Medium (MethoCult(?) H4434) was using in the Colony-forming experiment which is also in four groups as previously. The number of cells and CFUs each well for the four groups was evaluated at different time points(day1,3,7) with Fluorescence Microscopy counting method. Results Cell division and proliferation can be observed from3day in each group,there was no significant difference between them.But on7day,it changedrCell Viability:Jagged-1>BMP-4> Shh>control, cell number of each well:Jagged-1>BMP-4>control;Colony-forming experiment showed that BFU-E,CFU-G, CFU-M, CFU-GM can be observed in each group,and there was no significant difference between the four groups.But each cell could only form one certain cell colony. Conclusions Microenvironment in vivo can benefit for CD34+CD38-Lin-cell survival,self-renewal and proliferation,but it is not the determining factor,CD34+CD38-Lin-cell can achieve that by itself. However,CD34+CD38-Lin-cell can only maintain cell totipotency in its niche.