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Objective To determine whether pattern of WT1 gene expression is a useful marker for establishing prognosis and tracking disease progression in patients with myelodysplastic syndromes (MDS). Methods RNA were extracted from bone marrow smears of 45 patients with MDS, the WT1 expression were tested by FQ-RT-PCR. Results The degree of WT1 expression was increased during disease progression of MDS. The WT1 expression level was increased more a logarithm grade when a RA patient developed into RAEB, the fluctuation of WT1 expression level was decreased a logarithm grade during RA. Conclusion WT1 gene is a useful marker for assessment in MDS patients. It pointed out may diagnosis RA if the WT1 expression level higher two logarithm grade than the mean of normal control for patients which didn't diagnose by morphology. It may suggest disease progress of MDS if the WT1 expression level increased more a logarithm grade recently. Further confirmation was needed because the case was limited.
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Objective To explore the changes of Par-4 and WT1 genes expression during human leukemic K562 cells apoptosis induced by arsenic trioxide (As2O3). Methods After the K562 cells were treated with arsenic trioxide (2-10 μmol/L) for 24-72 hours, cell survival rate was evaluated by MTT assay and apoptosis was analyzed using flow cytometry. The expressions of mRNA and protein for par-4 and WT1 were tested by Real-time quantitative reverse transcriptase polymerase chain reaction (RQ-RT-PCR) method and Western blotting in K562 cells with various concentrations of arsenic trioxide at different time points. Results After the K562 cells were treated with As2O3 of different concentrations, arsenic trioxide could efficiently inhibit the growth of K562 cells and induce apoptosis with the increase of As2O3 concentration. The same time, the mRNA and protein expressions of Par-4 were up-regulated, while the mRNA and protein expression of WT1 were down-regulated. Conclusion Arsenic trioxide could inhibit the growth of K562 cells and induce apoptosis. Par-4 and WT1 genes could participate in the apoptosis of K562 cells induced by arsenic trioxide.
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Objective To evaluate preliminarily the significance of dynamic detection of Wilms'tumor gene(WT1)expression level on monitoring minimal residual disease(MRD)and predicting clinical relapse in patients of malignancy following allogeneic hematopoietic stem cell transplantation(allo-HSCT).Methods WT1 expression level WaS measured with real.time quantitative reverse transcription polymerase chain reaction(RQ-RT-PCR)method on 102 bone marrow specimens from 31 patients following allo.HSCT.WT1 expression level was determined as the ratio of WT1 mRNA to ABL mRNA times 100%.Resuits The levels of WT1 expression showed significant difference between the relapsed group and the non-relapsed group(P＜0.01),with 0.80%and 0.17%as their median expression level respectively.After dynamic measuring WT1 expression level on patients in the relapsed group.the level was found to increase significantly as compared with those during or before the clinical hematological relapse.both being＞1.0%.The level at the time of relapse Was significantly higher than the latest previous one(P＜0.05).Conclusion Dynamic detection of WT1 expression level with RQ-RT-PCR may be of help in monitoring MRD and warning clinical relapse Oil the patients following allo-HSCT.
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Objective To explore the feasibility of gene therapy targeting on WT1 gene in leukemia.siRNA inhibiting WT1 gene expression was effectively screened out and its affection on proliferation of K562 cell was observed.Methods Three siRNA for WT1 were designed and transfected into HEK293 cells.WT1 mRNA expression was detected by FQ-RT-PCR.WT1 protein expression was detected by Western blotting.The affection of cell proliferation was detected by MTT method. Results The inhibitory effects of siRNA designed different locations were different for wt1 gene.si-wt1-1 gene was the most significant(P＜0.05),but si-wt1-2 and si-wt1-3 had no inhibitory effect on WT1 mRNA expression(P＞0.05).WT1 mRNA expression was reduced to(42.5±1.0)%and(25.3±1.5)%of the controls at 24 h and 48 h transfected with 100 nmol/L si-wt1-1 respectively(P＜0.05),but restored to normal level at 72 h.There was no dose-dependent inhibitory effect for si-wt1-1 by the statistics analysis.The inhibitory effect of si-wt1-1 was obvious and the effect is best at 96h especially(P＜0.05).But si-wt1-2 and si-wt1-3 had no effect on WT1 protein expression by Western blotting analysis(P＞0.05).si-wt1-1 had inhibitory effect for K562 cell proliferation.there was obvious differenee between si-wt1-1 and negative control (P＜0.05).conclusion siRNA can effectively inhibit WT1 gene expression on HEK293 cells and the inhibitory effect on mRNA level is most significant on protein level.si-wt1-1 can effectively inhibit K562 cell proliferation.[Subjeet words]RNA interference;RNA,small interfering;Genes,Wilms tumor
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Here we report two cases of isolated diffuse mesangial sclerosis (IDMS) with early onset end-stage renal failure. These female patients did not show abnormalities of the gonads or external genitalia. Direct sequencing of WT1 PCR products from genomic DNA identified WT1 mutations in exons 8 (366 Arg>His) and 9 (396 Asp>Tyr). These mutations have been reported previously in association with Denys-Drash syndrome (DDS) with early onset renal failure. Therefore we suggest that, at least in part, IDMS is a variant of DDS and that investigations for the WT1 mutations should be performed in IDMS patients. In cases with identified WT1 mutations, the same attention to tumor development should be required as in DDS patients, and karyotyping and serial abdominal ultrasonograms to evaluate the gonads and kidney are warranted.
Subject(s)
Female , Humans , Infant , Infant, Newborn , Base Sequence , DNA/chemistry , DNA Mutational Analysis , Fatal Outcome , Glomerular Mesangium/pathology , Mutation , Nephrosclerosis/genetics , WT1 Proteins/geneticsABSTRACT
T, p.R394W in exon 9.
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AIM: To transfer 4 full-length WT1 isoforms cDNA into the leukemia cell line NB_4 so as to provide a cell model for studying the WT-1 gene function. METHODS: The eukaryotic expression recombinant vectors for WT1 isoforms (pCB6+/WT1) were introduced into the leukemia cell line NB_4 by electroporation. The positive cell clones were screened by G418 culture. The integration of WT1 gene isoforms in NB_4 cells as confirmed by PCR. The mRNA and protein of WT1 were detected by RT-PCR and Western blotting. RESULTS: WT1 gene isoforms were successfully transferred into NB_4 cells. WT1 mRNA and protein expression in the G418-selected cells increased remarkably compared with the control. CONCLUSION: WT1 gene isoforms were effectively transferred into NB_4 cells by electroporation and stably expressed in the transfected cells.
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To elucidate the relationship between WT1 expression and differentiation of leukemia cells and the role of WT1 gene in differentiation of leukemia cells, HL 60 and K562 cell lines were induced for 5 days by all trans retinoic acid(ATRA).Then the degree of differentiation and WT1 expression of cell lines were determined by NBT reduction assay and RT PCR respectively. The resultsshowed that only differentiation of HL 60 cells but no K562 cells could be induced by ATRA. When HL 60 cells were induced to differentiate to granulocytes by ATRA, the expression of WT1 decreased markedly during differentiation. However, WT1 transcripts were not significantly altered in K562 cells in which differentiation wasn't found. It suggested that WT1 gene expression may relate to the differentiation of leukemia cells.