Purging Of Leukemia Minimal Residual Disease With WT1 Antisense Oligonucleotides In Vitro

PrefaceAlong with the application of the new chemotherapy and the development of haemopoietic stem cell transplantation, the complete remission rate of acute leukemia has been greatly enhanced. The longevity time of leukemia patients has obviously been elongated, but the minimal residual disease of complete remission in patients is still the main cause of leukemia relapse.Proliferation of hematopoietic cells is regulated by various factors. Development of leukemia is associated with abnormal expression of genes. The gene of Wilms' tumor located at chromosome 11p13, is regulatory factor. It possesses double functions of inhibition and activation of transcription. It possibly conducts as a regulatory factor to control proliferative and/or differential gene of hematopoietic cell. WT1 play a certain role in genesis, development and prognosis of leukemia.Antisense oligonucleotides are nucleotide sequences complementing with mRNAs. They have effect on genetic transcription, mRNA shearing and mRNA translation, etc to educe specifically blocking target gene expression.The objectives of this study are to inhibit the expression of WT1 gene by antisense oligonucleotides,to observe the changes of the gene expression in minimal residual disease model and patients, to investigate the role of the WT1 in the genesis and development of leukemia and to study the application of anti-sense oligonucleotides in the treatment of leukemia minimal residual disease.Materials1. Reagents: Ficoll lymphocyte separative solution, Trizol, chloroform,avantin, dNTP, RNA enzyme inhibitor, reverse transcriptase, Taq DNA poly-merase, RPMI1640, are provided from Infectious Disease Experiment Lab. Primers and oligonucleotides are purchased from Takara company.2. Equipment;C02 gas incubator, high speed refrigerated centrifuge, elec-trophoresis apparatus, PCR equipment, -152^0 medical freezer, -30T! medical freezer.Methods1. The establishment and cultivation of leukemia minimal residual disease model with WT1 gene ( + )The sensitivity of WT1 gene are detected by RT - PCR on K562 and HL60 cell line, using the mononuclear cells of normal person's marrow with K562N HL60 cells pro rata to prepare leukemic minimal residual disease model according to the lowest sensitivity.2. The screening of the bone marrow of complete remission patient with WT1 gene ( + ) acute leukemia56 complete remission patients with acute leukemia followed up to by doctors. The expression conditions of WT1 gene were examined with RT - PCR to screen out WT1 gene ( + ) bone marrow.3. WT1( +) cell cultureThe bone marrow of complete remission patients with acute leukemia and leukemia minimal residual disease model, expressing the WT1 gene, were cultured in RPMI1640 which contain 10% fetal bovine serum, 50|xg/ml streptomycin and lOOIU/ml penicillin, and incubated at 31°C in 5% CO2 atmosphere.4. Cell treatment with WT1 antisense oligonucelotidesCell suspension of the bone marrow of complete remission patients with a-cute leukemia and leukemia minimal residual disease model, expressing the WT1 gene, were cultured to 24 wells of cultural discus. The concentration is 2 x lOVml. The total volume of cell suspension for every cultural discus is lml, which is divided into three groups and separately cultured with antisense oligonucleotides, sense oligonucleotides and RPMI1640. Final concentration of oligonucleotides is 120|xg/ml. We added 60|xg/ml of each reagent to the cultures, per 24 hours until 72 hours.5. Assays for WTl gene expressionCollecting cells of each group after 72 hours and determining the expression of WTl gene by RT - PCR.Results1. The sensitivity of WTl is 10 "3 - 10 "4 by RT - PCR.2. The establishment of leukemia minimal residual disease model with WTl gene ( + ) : K562, HL60 cells were mixed with mononuclear cells of normal persons marrow to prepare leukemia minimal residual disease model according to the proportion of 1;103.3. The screening result of the bone marrow of acute leukemia complete remission patients with WTl gene ( + ) : 9 patients expressed WTl gene positive out of 56 complete remission patients with acute leukemia, hinting the existence of minimal residual disease. The positive rate of WTl gene is 16%.4. The effect of antisense oligonucleotids on WTl gene;Collecting each group cells after treatment with WTl antisense oligonucleotides for 72 hours, we detected the WTl gene by RT - PCR. The result is that WTl gene in ASO group had disappeared, while in both SO and control groups, WTl gene could still be detected. We can conclude that antisense oligonucleotides can inhibit the expression of WTl gene effectively.ConclusionWTl antisense oligonucleotides can inhibit the proliferation of the residual leukemia cells of leukemia minimal residual disease and promote the death of the cells. This antisense oligonucleotide may become an effective agent for antisense therapy and a method to purging of autologous bone marrow transplantation.