Humans ; Kidney Diseases ; genetics ; Mutation ; WT1 Proteins ; genetics

Humans ; Infant ; Intracellular Signaling Peptides and Proteins ; genetics ; Membrane Proteins ; genetics ; Mutation ; Nephrotic Syndrome ; etiology ; genetics ; WT1 Proteins ; genetics

Objective: To monitor the WT1 mRNA level and its dynamic changes in patients with myelodysplastic syndromes (MDS) after hypomethylating agents (HMA) , as well as to assess the significance of WT1 mRNA levels and its dynamic changes in evaluating the efficacy of HMA and distinguishing the disease status of heterogeneous patients with stable disease (SD) . Methods: Bone marrow or peripheral blood samples of 56 patients with MDS who underwent hypomethylating agents (≥4 cycles) from November 2009 to March 2018 were tested by real-time quantitative polymerase chain reaction (PCR) to detect the expression of WT1 mRNA, and to observe the correlation between the dynamic changes of WT1 mRNA expression and clinical efficacy and prognosis of patients. Results: WT1 mRNA expression levels of MDS patients decreased significantly after 3 cycles of hypomethylating agent treatment. Besides, the WT1 mRNA expression levels of patients increased significantly after diseases progression. According to the dynamic changes of WT1 mRNA expression levels during SD, 45 cases could be further divided into increased group and non-increased group. In those SD patients with increased WT1 mRNA expression level, the ratio of suffering disease progression or transformation to AML was 95.65% (22/23) , whereas the ratio turned to be 9.09% (2/22) for the non-increased group (χ(2)=33.852, P<0.001) . Compared with those SD patients reporting no increase in WT1 mRNA expression level, the overall survival[17 (95%CI 11-23) months vs not reached, P<0.001] and progression-free survival [13 (95%CI 8-18) months vs not reached, P<0.001] of those SD patients reporting increase in WT1 mRNA expression level were significantly shorter. Conclusion: WT1 mRNA expression level is a useful indicator to assess the efficacy of hypomethylating agents in MDS patients. Especially in patients with SD, detection of the changes in WT1 mRNA expression level is able to predict disease progression and help to make clinical decision.
Bone Marrow ; Humans ; Myelodysplastic Syndromes/genetics* ; Prognosis ; RNA, Messenger ; WT1 Proteins/genetics*

In order to investigate the feasibility of using EGFP as a reporter gene in WT1 transcriptional regulation study. WT1 promoter and enhancer were ligated into the vector pEGFP-1 by recombinant DNA technique and confirmed by restriction enzymes digestion. The resultant constructs were transfected into K562 cell line by DMRIE-C reagent and the function of these WT1 gene elements was detected by using a fluorescent microscope after transfection for 48 hours. The results indicated that the recombinant vectors, pEWP containing WT1 promoter, and pEWPE, pEWPA and pEWPD harboring both WT1 enhancer and promoter, had been successfully constructed. Fluorescence was observed in K562 cells transfected by pEWP, pEWPE, pEWPA and pEWPD, while no fluorescence could be detected in cells transfected by pEGFP-1. It is concluded that EGFP gene as a reporter gene can be applied to the WT1 transcriptional regulation study, which provides the basis for gene therapy.
Enhancer Elements, Genetic ; genetics ; Genes, Reporter ; genetics ; Green Fluorescent Proteins ; genetics ; Humans ; K562 Cells ; Transfection ; WT1 Proteins ; genetics

OBJECTIVETo study the change of WT1 gene expression during human leukemic K562 cell differentiation and apoptosis induced by bufalin.

METHODSCell viability was determined by trypan blue exclusion, cell differentiation and apoptosis by nitro blue tetrazolium (NBT) reduction test, morphology and flow cytometry, expression of WT1 protein by Western blot analysis, and expression of WT1 mRNA by reverse transcriptase-polymerase chain reaction (RT-PCR).

RESULTS(1) The cell proliferation was inhibited by bufalin and the IC(50) at 24, 48, 72 h were 0.026, 0.032 and 0.006 micro mol/L, respectively. (2) Bufalin induced K562 cell differentiation towards macrophage/monocyte within concentration from 0.01 to 0.05 micro mol/L and apoptosis at higher than 0.026 micro mol/L. (3) The expression of WT1 protein and mRNA were downregulated by bufalin in the initial stage of differentiation and apoptosis induced by bufalin.

CONCLUSIONK562 cell differentiation and apoptosis induced by bufalin might relate to the downregulation of WT1 expression.

Apoptosis ; Cell Differentiation ; Cell Proliferation ; Down-Regulation ; Humans ; WT1 Proteins ; genetics

The aim of this study was to investigate the expression of wt1 gene and the changes of gene expression in minimal residual disease (MRD) models (K562, HL-60 cell lines) and acute leukemia (AL) patients through inhibiting the expression of wt1 gene by antisense oligonucleotides (ASO). The bone marrow (BM) of 56 AL patients with complete remission (CR) was collected, then the BM samples with positive expression of wt1 gene were screened by RT-PCR. The cells of MRD model and screened wt1 gene positive samples were cultured and treated by ASO, then the changes of wt1 gene expression were detected. The results indicated that the sensitivity of wt1 gene was 10(-3)-10(-4), and the positive rate of BM wt1 gene expression in 56 AL patients with CR was 16%. After BM of 9 AL CR patients with MRD and MRD model (K562, HL-60 cells) expressing wt1 gene were treated by ASO, it was found that the wt1 expression in ASO group was blocked, while wt1 gene could be still detected in both sense oligonucleotides (SO) and control groups. It is concluded that ASO can obstruct the expression of wt1 gene on the residual leukemia cells in vitro.
Gene Expression ; HL-60 Cells ; Humans ; K562 Cells ; Neoplasm, Residual ; genetics ; Oligonucleotides, Antisense ; genetics ; WT1 Proteins ; genetics

OBJECTIVETo study the DNA methylation status of WT1 gene promoter region in hematologic malignancy cell lines and its correlation with WT1 gene expression.

METHODS1. RT-PCR and methylation-specific PCR were performed for detecting WT1 gene expression and DNA methylation status in its promoter region in 8226, HL-60, Jurkat, K562, KG-1, NB4, Raji, SHI-1, U266 and U937 cell lines. 2. Treatment of U937 cells with 5-aza-CdR, a demethylation inducing agent and the changes in WT1 gene expression level and its promoter region methylation status were determined.

RESULTS1. HL-60, K562, KG-1, NB4 and SHI-1 cells showed higher levels while 8226, Jurkat, Raji, U266 and U937 cells showed extremely low levels of WT1 expression. DNA hypermethylation in WT1 gene promoter region was identified in 8226, Jurkat, Raji, U266 and U937 cells. 2. The WT1 gene expression in U937 was enhanced after treatment with 5-aza-CdR accompanied with the decrease of methylated and the increase of unmethylated levels in its promoter region.

CONCLUSIONModulation of the DNA methylation status in WT1 promoter region is one of the epigenetic mechanisms for regulating its expression.

Cell Line, Tumor ; DNA Methylation ; Gene Expression Regulation, Neoplastic ; Hematologic Neoplasms ; genetics ; Humans ; Promoter Regions, Genetic ; genetics ; WT1 Proteins ; genetics

This study was purposed to construct a fusion DNA vaccine containing WT1 multi-epitope and stimulating epitope of mycobacterium tuberculosis heat shock protein 70 and to detect its expression and immunogenicity. On the basis of published data, a multi-epitope gene (Multi-WT1) containing three HLA *0201-restricted CTL epitopes: one HLA*2402-restricted CTL epitope, two Th epitopes and one universal Th Pan-DR epitope (PADRE) was constructed. DNA-coding sequence was modified by Computer-Aided Design (CAD) to optimize proteasome-mediated epitope processing through the introduction of different amino acid spacer sequences. The synthetic nucleotide sequence was then inserted into an eukaryotic vector to construct the plasmid pcDNA3.1-WT1.For enhancing CTL activity, HSP70 fragment including stimulatory domain P407-426 was amplified by PCR from mycobacterial HSP70 gene and cloned into pcDNA3.1(+). Then Multi-WT1 was fused to the N-terminal of pcDNA3.1-mHSP70(407-426) to make the multi-epitope fusion gene vaccine pcDNA3.1-WT1-mHSP70(407-426). HEK-293T cells were transfected with this vaccine and the expressed product was identified by RT-PCR. Enzyme-linked immunospot assay (ELISPOT) was used to evaluate the immunological responses elicited by vaccine. The results showed that the most of WT1 epitopes could be correctly cleaved which was confirmed by software Net Chop 3.1 and PAPROCIanalysis. RT-PCR showed correct expression of target gene in HEK293T cells and ELISPOT showed specific T-cell responses. It is concluded that the eukaryotic expression vector PcDNA3.1-WT1-mHSP70(407-426) fusion gene has been successfully constructed and the immunity response is also elicited, which is a good candidate for further research of DNA vaccine.
Bacterial Proteins ; genetics ; immunology ; Epitopes ; genetics ; immunology ; Genetic Vectors ; HSP70 Heat-Shock Proteins ; genetics ; immunology ; Humans ; Immunodominant Epitopes ; Vaccines, DNA ; genetics ; immunology ; WT1 Proteins ; genetics ; immunology

Child ; Child, Preschool ; Female ; Humans ; Infant ; Intracellular Signaling Peptides and Proteins ; genetics ; Male ; Membrane Proteins ; genetics ; Nephrotic Syndrome ; ethnology ; genetics ; WT1 Proteins ; genetics

BACKGROUNDWilms' tumor (nephroblastoma) is the most common pediatric kidney cancer. Only one Wilms' tumor gene is known, WT1 at 11p13, which is mutated in 5% - 10% of Wilms' tumors. Recently, mutations were reported in WTX at Xq11.1 in Wilms' tumors. This study investigated the mutation proportion, type, and distribution in WTX and WT1 in children with Wilms' tumor. The role of WTX/WT1 in the development of Wilms' tumor, and the relationship between clinical phenotype and genotype, were also studied.

METHODSWilms' tumor specimens (blood samples from 70 patients and tumor tissue samples from 52 patients) were used. A long fragment of WTX and 10 exons and intron sequences of WT1 were amplified by polymerase chain reaction (PCR) from extracted genomic DNA and sequenced. A chi-square test compared the difference between the WTX mutation group and the no mutation group. The relationship between the mutations and clinical phenotype was analyzed.

RESULTSWTX mutations were found in 5/52 tumor tissues and in 2/70 peripheral blood samples (five cases in total, all point mutations). Two patients had a WTX mutation in both samples. WT1 mutations were found in 2/52 tumor tissues and in 4/70 peripheral blood samples (five cases in total, all point mutations). One patient had a WT1 mutation in both samples. Ten cases had WTX or WT1 mutation (19.2% of Wilms' tumors). No overlapping WTX and WT1 mutations were found. No significant differences in clinical parameters were found between patients with and without a WTX mutation.

CONCLUSIONSWTX mutations occur early in Wilms' tumor development, but at a low proportion. There was no evidence that WTX is the main cause of Wilms' tumor. Clinical parameters of patients with WTX mutations are not related to the mutation, indicating a limited impact of WTX on tumor progression. WTX and WT1 mutations occur independently, suggesting a relationship between their gene products.

Adaptor Proteins, Signal Transducing ; genetics ; Child, Preschool ; Female ; Humans ; Male ; Mutation ; Tumor Suppressor Proteins ; genetics ; WT1 Proteins ; genetics ; Wilms Tumor ; genetics