This study was to determine whether GM-CSF induced WT1 gene expression and to establish an association with markers of proliferation CD71+CD34+ using nPCR and flow cytometry respectively, in samples obtained from 5 ne...This study was to determine whether GM-CSF induced WT1 gene expression and to establish an association with markers of proliferation CD71+CD34+ using nPCR and flow cytometry respectively, in samples obtained from 5 newly diagnosed JMML patients. Overtime (day 0 to day 14) there was an insignificant difference in WT1 gene expression and CD71+CD34+ in JMML samples when compared to peripheral blood of normal volunteers (n = 3). Our study suggests that there is a correlation between WT1 gene expression and cellular proliferation and that GMCSF in vitro does not create a significant difference in JMML samples.展开更多
The overall frequency of WT1 gene alterations in Wilms tumor is still unclear in Taiwan. Here we conducted molecular genetic analysis of the WT1 gene in Taiwan Residents patients with Wilms tumor. Polymerase chain rea...The overall frequency of WT1 gene alterations in Wilms tumor is still unclear in Taiwan. Here we conducted molecular genetic analysis of the WT1 gene in Taiwan Residents patients with Wilms tumor. Polymerase chain reaction and direct sequencing were performed on DNA samples from blood and paraffin-embedded tumor specimens. A constitutional mutation in the WT1 gene was found in one DNA sample from peripheral blood lymphocytes. The remaining DNA samples from peripheral blood lymphocytes and paraffin-embedded tumor specimens were tested negative for both constitutional mutations and somatic mutations. Thus, mutations at other Wilms tumor loci may play an important role in Wilms tumor development.展开更多
The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms＇ tumor 1 suppressor gene （WT1） and CML28 abnormally expressed in malignant diseases of the hematopoietic system and...The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms＇ tumor 1 suppressor gene （WT1） and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the rela- tionship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA （siRNA） against WT1 and CML28 in leukemic cell line K562 to examine the interac- tion between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was de- tected by using RQ-PCR and Western blotting. K562 cells transfected with WTI-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by us- ing immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide im- portant clues for further study on the role of CML28 and WT1 in leukemic cells.展开更多
Background Wilms＇ 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 report...Background Wilms＇ 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. Methods Wilms＇ tumor specimens （blood samples from 70 patients and tumor tissue samples from 52 patients） were used. A long fragment of WTXand 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 W-/-X mutation group and the no mutation group. The relationship between the mutations and clinical phenotype was analyzed. Results W7X 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 WTI mutations were found. No significant differences in clinical parameters were found between patients with and without a W7X mutation. Conclusions WTX 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.展开更多
Aim:The purpose of the present study was to perform a comprehensive analysis of WT1 gene expression in high-risk pediatric acute lymphoblastic leukemia(ALL).Methods:We performed a meta-analysis of WT1 gene expression ...Aim:The purpose of the present study was to perform a comprehensive analysis of WT1 gene expression in high-risk pediatric acute lymphoblastic leukemia(ALL).Methods:We performed a meta-analysis of WT1 gene expression for normal hematopoietic cells vs.primary leukemia cells from 801 pediatric ALL samples deposited in the Oncomine database combined with an in-depth gene expression analysis using our in-house database of gene expression profiles of primary leukemia cells from 1416 pediatric ALL cases.We also examined the expression of WT1 in primary leukemic cells from 299 T-lineage ALL patients in the Oncomine database and 189 T-lineage ALL patients in the archived datasets GSE13159,GSE13351,and GSE13159.Results:Our data provide unprecedented evidence that primary leukemia cells from patients with MLL gene rearrangements(MLL-R)express highest levels of WT1 expression within the high-risk subsets of pediatric B-lineage ALL.Notably,MLL-R^(+)patients exhibited>6-fold higher expression levels of the WT1 gene compared to the other B-lineage ALL subtypes combined(P<0.0001).Our findings in 97 MLL-R^(+)infant B-lineage ALL cases uniquely demonstrated that WT1 is expressed at 1.5-4.2-fold higher levels in MLL-R^(+)infant leukemia cells than in normal hematopoietic cells and revealed that WT1 expression level was substantially higher in steroid-resistant infant leukemia cells when compared to non-leukemic healthy bone marrow cells.Furthermore,our study demonstrates for the first time that the WT1-regulated EWSR1,TP53,U2AF2,and WTAP genes(i.e.,WT1 interactome)were differentially upregulated in MLL-R^(+)leukemia cells illustrating that the MLL-regulatory pathway is aberrantly upregulated in MLL-R^(+)pediatric B-lineage ALL.These novel insights provide a compelling rationale for targeting WT1 in second line treatment of MLL-R^(+)pediatric B-lineage ALL,including MLL-R^(+)infant ALL.Furthermore,our study is the first to demonstrate that leukemia cells from 370 Ph-like patients had significantly higher WT1 expression when compared to normal hematopoietic cells.Finally,our findings demonstrate for the first time that chemotherapy-resistant primarily leukemic cells from relapsed B-lineage ALL patients exhibit higher expression levels of WT1 than primary leukemia cells from newly diagnosed B-lineage ALL patients(P=0.001).Conclusion:Our findings indicate that the WT1 gene product may serve as a target for immunotherapy in high risk/poor prognosis subsets of newly diagnosed as well as relapsed pediatric B-lineage ALL.Our findings also significantly expand the current knowledge of WT1 expression in T-lineage ALL and provide new evidence that WT1 gene and its interactome are expressed in T-lineage ALL cells at significantly higher levels than in normal hematopoietic cells.This previously unknown differential expression profile uniquely indicates that the protein product of WT1 would be an attractive molecular target for treatment of T-lineage ALL as well.展开更多
Objective To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm’ tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells. Methods The H...Objective To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm’ tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells. Methods The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively. Results The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC<sub>50</sub>) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner. Conclusions Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G<sub>0</sub>/G<sub>1</sub> phase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.展开更多
文摘This study was to determine whether GM-CSF induced WT1 gene expression and to establish an association with markers of proliferation CD71+CD34+ using nPCR and flow cytometry respectively, in samples obtained from 5 newly diagnosed JMML patients. Overtime (day 0 to day 14) there was an insignificant difference in WT1 gene expression and CD71+CD34+ in JMML samples when compared to peripheral blood of normal volunteers (n = 3). Our study suggests that there is a correlation between WT1 gene expression and cellular proliferation and that GMCSF in vitro does not create a significant difference in JMML samples.
文摘The overall frequency of WT1 gene alterations in Wilms tumor is still unclear in Taiwan. Here we conducted molecular genetic analysis of the WT1 gene in Taiwan Residents patients with Wilms tumor. Polymerase chain reaction and direct sequencing were performed on DNA samples from blood and paraffin-embedded tumor specimens. A constitutional mutation in the WT1 gene was found in one DNA sample from peripheral blood lymphocytes. The remaining DNA samples from peripheral blood lymphocytes and paraffin-embedded tumor specimens were tested negative for both constitutional mutations and somatic mutations. Thus, mutations at other Wilms tumor loci may play an important role in Wilms tumor development.
文摘The molecular pathogenesis of leukemia is poorly understood. Earlier studies have shown both Wilms＇ tumor 1 suppressor gene （WT1） and CML28 abnormally expressed in malignant diseases of the hematopoietic system and WT1 played an important role in leukemogenesis. However, the rela- tionship between molecular CML28 and WT1 has not been reported. Here we described the use of small interfering RNA （siRNA） against WT1 and CML28 in leukemic cell line K562 to examine the interac- tion between CML28 and WT1. WT1 and CML28 gene expression in transfected K562 cells was de- tected by using RQ-PCR and Western blotting. K562 cells transfected with WTI-siRNA could greatly decrease both mRNA and protein expression levels of WT1 and CML28. In contrast, CML28-siRNA did not exert effect on WT1. Further, subcellular co-localization assay showed that the two proteins could co-localize in the cytoplasm of K562 cells, but WT1/CML28 complexes were not detected by us- ing immunoprecipitation. It was suggested that there exists the relationship between CML28 and WT1. CML28 may be a downstream target molecule of WT1 and regulated by WT1, which will provide im- portant clues for further study on the role of CML28 and WT1 in leukemic cells.
文摘Background Wilms＇ 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. Methods Wilms＇ tumor specimens （blood samples from 70 patients and tumor tissue samples from 52 patients） were used. A long fragment of WTXand 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 W-/-X mutation group and the no mutation group. The relationship between the mutations and clinical phenotype was analyzed. Results W7X 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 WTI mutations were found. No significant differences in clinical parameters were found between patients with and without a W7X mutation. Conclusions WTX 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.
基金This work was supported by departmental funds of the Ares Pharmaceuticals Biotherapy Program.No external funding sources or sponsored research grants were used.
文摘Aim:The purpose of the present study was to perform a comprehensive analysis of WT1 gene expression in high-risk pediatric acute lymphoblastic leukemia(ALL).Methods:We performed a meta-analysis of WT1 gene expression for normal hematopoietic cells vs.primary leukemia cells from 801 pediatric ALL samples deposited in the Oncomine database combined with an in-depth gene expression analysis using our in-house database of gene expression profiles of primary leukemia cells from 1416 pediatric ALL cases.We also examined the expression of WT1 in primary leukemic cells from 299 T-lineage ALL patients in the Oncomine database and 189 T-lineage ALL patients in the archived datasets GSE13159,GSE13351,and GSE13159.Results:Our data provide unprecedented evidence that primary leukemia cells from patients with MLL gene rearrangements(MLL-R)express highest levels of WT1 expression within the high-risk subsets of pediatric B-lineage ALL.Notably,MLL-R^(+)patients exhibited>6-fold higher expression levels of the WT1 gene compared to the other B-lineage ALL subtypes combined(P<0.0001).Our findings in 97 MLL-R^(+)infant B-lineage ALL cases uniquely demonstrated that WT1 is expressed at 1.5-4.2-fold higher levels in MLL-R^(+)infant leukemia cells than in normal hematopoietic cells and revealed that WT1 expression level was substantially higher in steroid-resistant infant leukemia cells when compared to non-leukemic healthy bone marrow cells.Furthermore,our study demonstrates for the first time that the WT1-regulated EWSR1,TP53,U2AF2,and WTAP genes(i.e.,WT1 interactome)were differentially upregulated in MLL-R^(+)leukemia cells illustrating that the MLL-regulatory pathway is aberrantly upregulated in MLL-R^(+)pediatric B-lineage ALL.These novel insights provide a compelling rationale for targeting WT1 in second line treatment of MLL-R^(+)pediatric B-lineage ALL,including MLL-R^(+)infant ALL.Furthermore,our study is the first to demonstrate that leukemia cells from 370 Ph-like patients had significantly higher WT1 expression when compared to normal hematopoietic cells.Finally,our findings demonstrate for the first time that chemotherapy-resistant primarily leukemic cells from relapsed B-lineage ALL patients exhibit higher expression levels of WT1 than primary leukemia cells from newly diagnosed B-lineage ALL patients(P=0.001).Conclusion:Our findings indicate that the WT1 gene product may serve as a target for immunotherapy in high risk/poor prognosis subsets of newly diagnosed as well as relapsed pediatric B-lineage ALL.Our findings also significantly expand the current knowledge of WT1 expression in T-lineage ALL and provide new evidence that WT1 gene and its interactome are expressed in T-lineage ALL cells at significantly higher levels than in normal hematopoietic cells.This previously unknown differential expression profile uniquely indicates that the protein product of WT1 would be an attractive molecular target for treatment of T-lineage ALL as well.
基金Supported by National Natural Science Foundation of China(No.81403223)Zhejiang Provincial Natural Science Foundation of China(No.LQ14H290003)Science and Technology Foundation of Zhejiang Province(No.2015C33173)
文摘Objective To explore the effects of bufalin on inhibiting proliferation, up-regulating methylation of Wilm’ tumor 1 gene (WT1) as well as its possible mechanisms in human erythroid leukemic (HEL) cells. Methods The HEL cells were treated with bufalin at various concentrations to observe cellular morphology, proliferation assay and cell cycle. The mRNA and protein expression levels of WT1 were detected by reverse transcription polymerase chain reaction (RT-PCR), Western blot and immunocytochemistry, DNA methylation of WT1 and protein expression levels of DNA methyltransferase 3a (DNMT3a) and DNMT3b were analyzed by methylation-specific PCR, and Western blot respectively. Results The bufalin was effective to inhibit proliferation of HEL cells in a dose-dependent manner, their suppression rates were from 23.4%±2.1% to 87.2%±5.4% with an half maximal inhibit concentration (IC<sub>50</sub>) of 0.046 μmol/L. Typical apoptosis morphology was observed in bufalin-treated HEL cells. The proliferation index of cell cycle decreased from 76.4%±1.9% to 49.7%±1.3%. The expression levels of WT1 mRNA and its protein reduced gradually with increasing doses of bufalin, meanwhile, the methylation status of WT1 gene changed from unmethylated into partially or totally methylated. While, the expression levels of DNMT3a and DNMT3b protein gradually increased by bufalin treatment in a dose-dependent manner. Conclusions Bufalin can not only significantly inhibit the proliferation of HEL cells and arrest cell cycle at G<sub>0</sub>/G<sub>1</sub> phase, but also induce cellular apoptosis and down-regulate the expression level of WT1. Our results provide the evidence of bufalin for anti-leukemia, its mechanism may involve in increasing WT1 methylation status which is related to the up-regulation of DNMT3a and DNMT3b proteins in erythroid leukemic HEL cells.
