Growth Inhibition Of Acute Leukemia Cells By WT1 Antisense Oligonucleotides

WTl is located chromosome 11 p13,which originally isolated from Wilms' tumor cells by a cloning technique. WTl gene spans about 50kb. WTl mRNA encodes transcription regulatory proteins, which have molecular masses of 52 -54Kda. WT1 proteins have two functions of repressing and activating transcription factors. In recent years, researchers founded that WTl gene is overex-pressed in various types of leukemia. The levels of WTl gene expression inversely correlated with the prognosis and correlated with the relapse of acute leukemia. The potential function of WTl gene in the genesis and development of leukemias remains controversial.Antisense oligonucleotides technique is a kind of gene therapy methodology that uses selective nucleotide sequences against the target gene to treat some diseases.The object of this experiment is to inhibit the expression of the WTl protein by antisense oligonucleotides, to observe the changes of morphology and proliferation behavior of the acute leukemia cells, to research the role of the WTl in the genesis and development of leukemias and discuss the application of antisense oligonucleotides on antisense therapy.Materials1. K562 cell line and acute leukemia cells2. WTl antisense and sense oligonucleotides3. Reagents for Western BlottingMethods1. Examine the expression of the WT1 protein in leukemia cells1. 1 Purification and cultivate of leukemia cellsHeparinized bone marrow cells were diluted by three or four times, put on Ficoll - Isopaque solution and centrifuged. The leukemia cells were recovered from the interface, washed twice, cultured in RPMI 1640 medium containing 10% fetal bovine serum ( FBS) 100IU/ml penicillin and 100IU/ml streptomycin. Microscopic examination showed the purity of the leukemia cells to be more than 90%.1. 2 Preparation of protein sampleThe cells in the logarithmic growth phase were lysed with RIPA buffer on ice and centrifuged. A protein extract was obtained.1. 3 Examine the expression of the WT1 protein by Western blot analysis2. The growth inhibition of leukemia cells by WT1 antisense oligonucleoti-des.The cells, which expressed WT1 protein, were cultured in 24 - well plate. Three group were established; (1) AS group; (2) SE group ; (3) blank control group. The cells were treated with the antisense oligonucleotides , sense oligo-nucleotides and RPMI 1640 medium, respectively, the terminal concentrations of oligomers were 200ug/ml. The reagents were added with 100ug/ml per 24 hours until 96 hours.2. 1 Assays for the changes of morphologyDyed the cells and observed the morphology changes under the microscope.2.2 Examine the expression of the WT1 protein in the cells treated with antisense oligonucleotides by Western blot analysisResults1. WT1 protein expression was determined in acute leukemia patients by Western blot analysis. 6 patients were found to have positive results among 9 pa-tients, and in 8 AML patients, 6 cases got positive results, in one ALL patient, no case got positive result.2. The growth inhibition of leukemia cells by WTl antisense oligonucleotides.2. 1 Observed the karyopyknosis or karyorrhexis of the cells in AS group. The quantity of the cells did not increase. The cells in both SE and blank control group did not change.2. 2 WTl protein expression was detected by Western blot analysis. The WTl antisense oligonucleotides produced a significant decrease in the WTl protein levels. In contrast, the sense oligonucleotides did not affect the WTl protein levels.ConclusionWTl is a kind of tumor gene in leukemia. The WTl antisense oligonucleotides produce a significant decrese in the WTl protein levels. WTl antisense oligonucleotides can inhibit the proliferation of the leukemia cells and promote the death of the cells. WTl antisense oligonucleotides may become an effective reagent for antisense therapy and a method for depletion of bone marrow.