| BackgroundAcute leukemia (AL) is the most common malignant tumor in children. It can be present in any age of children, but more common in preschool children. The incidence of leukemia was 3/100,000 -4/100,000 per year in children. It is a serious threat to the health of children. The latest two decades, it have been ignificantly improved on the rates of complete remission (CR) and 5-year disease-free survival in children with acute leukemia. If children with acute lymphoblastic leukemia (ALL) were treated with chemotherapy, the CR rate is up to 80% and 5-year survival rate is up to 70% -80%. There is about 20% of patients were treatment failure. The effect of treatment of acute myeloid leukemia (AML) is much worse than ALL. It is less than 60% about the rate of long-term disease-free survival in AML children.Leukemia cell which is resistant to chemotherapeutic drug is one of the main causes for treatment failure. However, It is not very clear of characteristic of leukemia cells about the molecular biolog, and is studying. whether relating to resistant of chemotherapy is In order to further understand the molecular characteristics of leukemia cells, we studied the signaling pathways which are related to leukemia, such as the PI3K/AKT, RAS / RAF / MEK / ERK and JAK / STAT pathway. It has been proved that these pathways promote proliferation, inhibition of apoptosis in leukemia cell and cause leukemia cells resistant to chemotherapy. Some of the molecule drugs targeting Signaling protein are in clinical trials.In recent years, it has developed a new technologies which is use to detect the protein phosphorylated. The new technology is phospho-flow which is detecting protein phosphorylated by flow cytometry. phospho-flow was used to analyze the activation of signaling proteins and the relationship between signal pathways in single leukemia cells. Phospho-flow was applied to risk classification based on the activation of signal pathways in leukemia cell, development of new drugs and monitoring of the efficacy of molecular drug targeted signaling protein. Phospho-flow is potential in clinical applications. we use Phospho-flow to analyze phosphorylation protein of signal pathway in children leukemia cells and study the application in experimental and clinical for leukemia.P-AKT is the activated form of AKT in the PI3K/AKT pathway. P-ERK1 / 2 is the activated form of ERK1 / 2 in the RAS / RAF / MEK / ERK pathway. P-STAT1, P-STAT3, P-STAT5, P-STAT6 is the activated form of STAT1, STAT3, STAT5, STAT6 in the JAK / STAT pathway. A number of studies have found that activation of these pathways were associated with cell survival, apoptosis regulation and related to leukemia cell'drug resistance. Therefore, we use phospho-flow to analyze P-AKT, P-ERK1 / 2 and P-STAT1, P-STAT3, P-STAT5, P-STAT6 in primary children leukemia cells and study the relation between the activation of PI3K/AKT, RAS / RAF / MEK / ERK,JAK / STAT pathway and risk classification ,drug treatment response in children leukemia.Objective1. We aim to establish flow cytometry measurements of phosphorylation protein of leukemia cells in the bone marrow or peripheral blood in our laboratory.2. we use phospho-flow to analyze P-AKT, P-ERK1 / 2 and P-STAT1,P-STAT3, P-STAT5, P-STAT6 in primary children leukemia cells and study the relation between the activation of PI3K/AKT, RAS / RAF / MEK / ERK,JAK / STAT pathway and risk classification ,drug treatment response in children leukemia.Methods1. Flow cytometry analyze the protein phosphorylation of K562 cell line(1) P-AKT, P-ERK1 / 2 positive (positive control): K562 cell line as a positive control cells, K562 cell lines were added U0126 (MEK inhibitor) and LY294002 (PI3K inhibitor) in order to block activation inhibit phosphorylation of the ERK1/2 and AKT ,which is negative control cells.(2) Sample Filtration: Using a final concentration of 4% formaldehyde to fix K562 cell fixed cells,which were incubated at 37℃for 10 minutes. (3) Permeabilize white blood cells:①adding 1mL 90% methanol working solution to permeabilize white blood cells, the k562 cells were incubated in an ice cold for 30min (BD Company under the instructions);②adding 1ml 50% methanol working solution permeabilize white blood cells ,cells were incubated at room temperature for 10min (according to Sue Chow et al (1) Phospho-flow experimental technology).(4) Add antibody: grouped of IGG antibody control tubes and phosphorylation antibodies tubes. IGG control tubes: IGG1-Alexa 488 and IGG1-Alexa 647Phosphorylation antibody tubes: P-ERK1/2- Alexa 488 and P-Akt-Alexa 647 The fluorescent antibody added by 20ul and incubated in the dark at room temperature for 30min, then washed, Finally, We use flow cytometry to measure K562 cells.2. Flow cytometry measure the protein phosphorylated proteinof bone marrow leukemia cells in children with leukemia.(1)Sample fixed and permeabilizedThe bone marrow sample was divided into 2 parts: sample 1 and sample 2.①after extracting mononuclear cells from bone marrow, of leukemia cells fixed: in the use of density gradient centrifugation and mononuclear cells were fixed with 4% formaldehyde, ready to rupture. Rupture of membranes: each tube add 1ml of the rupture of membranes containing 90% methanol working fluid, the cells were incubated in an ice tube 30min.②Sample fixed: fixed white cell and lysis of red cell at the same time: Taking 100ml of bone marrow or blood in the tube and each tube were adding a fixed \ lysis buffer (1×buffer) for 2ml, and then placed at 37°C incubation for 10 minutes (according to reagent instructions BD Company ); Sample permeabilized :Each tube were added 1ml of 90% methanol working solution, and then the cells were incubated in an ice cold for 30min (BD company under the instructions).(2)Staining①Surface staining B-ALL: CD45-Percp/CD10-FITC/CD19-PE/CD34-APC AML: CD45-Percp/CD13-FITC/CD33-PE / CD34-APC T-ALL: CD45-Percp/CD5-FITC/CD7-PE / CD34-APC②Simultaneously label intracellular and surface epitopes IGG control tube: CD45-Percp/IGG1-Alexa 488/IGG1-PE-/IGG1-Alexa 647; Phosphorylated protein tube: CD45Percp/p-ERK1/2-Alexa-488/p-Akt-Alexa 647/CD34-PE or CD45Percp/p-ERK1/2-PE/p-Akt-Alexa 6473. Flow cytometry measure cellsUsing BD FACSCanto to analyze K562 cell line as a positive control and K562 cells were added U0126 and LY294002 were used as negative control for P-AKT, P-ERK1 / 2 .According to the positive and negative K562 control determine P-AKT, P-ERK1 / 2 is positive or negative in children with leukemia. Extracting mononuclear cells were gated cell according to the diagram of FSC / SSC. The tubes Fixing white cell and lysising of red cell were gated the immature leukemia cells accoding to diagram of CD45/SSC. Both sample were combined cell surface CD antigens (exemple: CD10, CD34, CD5, CD7, CD33, etc.) to analyze phosphorylated proteins, such as P-AKT, P-ERK1 / 2.4. Using phospho-flow cytometry to measure P-AKT, P-ERK1 / 2 and P-STAT1, P-STAT3, P-STAT5, P-STAT6The process of phospho-flow cytometry aboute cell treatment is that fixed white cells and lysis the red blood cells at the same time: Taking 100 ml bone marrow or blood for each tube, adding a fixed / lysis buffer (1×buffer) for 2ml, and then incubate at 37°C for 10 minutes. Sample were permeabilized :Each tube were added 1ml of 90% methanol working solution, and then the cells were incubated in an ice cold for 30min .Finally staining antibody for phospho-flow analyze.Results1.Using phosphor-flow cytometry to measure protein phosphorylation in K562 cells:P-AKT, P-ERK is positive expression in K562-positive control; P-AKT, P-ERK1 / 2 is negative expression in the negative control K562 cells which was added with U0126 (MEK inhibitor) and LY294002 (PI3K inhibitor) for treatment.2. The 2 permeabilization approach comparison for K562 cellsThe K562 cells permeabilizing by 90% methanol whose P-AKT and P-ERK1 / 2 positive rates is (52.5±8.7)% and (45.7±10.7)%,respectively; the K562 cells permeabilizing by 50% methanol whose P-AKT and P-ERK1 / 2 positive rates is(36.8±9.3)% and (28.8±12.1)%, respectively;compare the difference of P-AKT and P-ERK1/2 positive rate in the 2 approach,both P <0.05, the difference was statistically significant.3. Compare the rate of protein phosphorylation in sample 1 and sample 2The rates of P-AKT positive of 26 patients were 46.2% and 50.0% in sample 1 and sample 2, respectively (P> 0.05); the difference was no statistically different. The rate of P-ERK1 / 2 positive of 26 patients were 30.8% and 38.5% in sample 1 and sample 2 (P> 0.05), no significant difference between the two methods.4. The expression of phosphorylated proteins in non-M3 AML children:The rate of P-AKT, P-ERK 1/2, P-STAT5, and P-STAT6 was 55.5%, 44.4%, 33.3% and 55.5% respectively. The rate of P-STAT1 and P -STAT3 were 0.0% both. 9 pati- -ents have differences in the expression of phosphorylated proteins.5. The expression of phosphorylated proteins in B-ALL children:The rate of P-AKT, P-ERK1 / 2, P-STAT5 and P-STAT6 were 35.3%, 11.8%, 11.8% and 17.6%. The rate of P-STAT1 and P-STAT3 were both 0.0%; positive rate was 0.0%. 7 of B-ALL children with positive expression, but their protein phosphory- -lation expression were varies. 3 patients have more than 1 protein phosphorylation expression and they express different protein phosphorylation, patient NO.2 was exp- -ression of P-AKT and P-STAT6, patient NO.6 was expression of P-AKT, P-ERK1 / 2, P-STAT5 And P-STAT6; patient NO.7 was expression of P-AKT, P-ERK1 / 2, P-STAT5, P-STAT6. The 3 patients were expression of P-STAT6.6. cases of over one year withdrawal (control group) The expression of P-AKT, P-ERK1 / 2, P-STAT1, P-STAT3, P-STAT5, P-STAT6 in leukemia children who had complete treatment for more then one yearThere is no protein phosphorylation for them.7. Differences between the P-AKT expression positive group and negative group for WBC in the ALL children:WBC were (157.6±131.2)×10~9/mL and (26.6±19.8)×10~9/mL in the P-AKT positive and negative groups, respectively, P <0.05, the difference was statistically significant.8. Difference of expression of protein phosphorylation positive and negative groups in ALL children in early treatment response (D8, D19) :The P-AKT, P-ERK1 / 2, P-STAT1, P-STAT3, P-STAT5, P-STAT6 positive and negative group were no significant differences of sensitivity of prednisone induction therapy in the D8-day and the same to the early responce of induction therapy in D19 . The patients who were expressed two or more protein phosphorylation were correlation with the poor response in the early response of induction therapy, P <0.01.9. Complete remission (CR ) rate of Protein phosphorylation of positive and negative groups in ALL children.10. High risk rate of B-ALL children in P-AKT, P-ERK1 / 2, P-STAT1, P-STAT3, P-STAT5, P-STAT6 positive and negative:High-risk rate were 87.5% and 0% in P-AKT positive and negative children respectively, P <0.05, the difference was statistically significant. High-risk rate between the P-ERK1 / 2, P-STAT1, P-STAT3, P-STAT5, P-STAT6 positive and negative group were no not statistically difference in ALL children.Conclusions1. Experimental program I: The approach which is using 4% formaldehyde to fix phosphorylated proteins and 90% methanol solution to permeabilize cells, can be used to analyze cell lines.2. Experimental Methods II: The approach which is using fix/lysis buffer to fix white cells and lysis red cells the same time and 90% methanol solution to permeabilize cells, can be used to measure the PM or BM for leukemia research.3. Constitutive activation of AKT, ERK1 / 2, STAT5 and STAT6 present in childhood leukemia cells. Our study show that P-AKT, P-ERK1 / 2, STAT5 and STAT6 expression in leukemia cells may be used as molecular markers for leukemia.4. P-AKT may be one of the risk classifications for B-ALL leukemia5. Phosphor-flow analysis of phosphorylated proteins can help the development of molecular drugs and apply to monitoring drug efficacy in clinic trial. It is beneficial for the individual treatment of patients. |
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