Downregulation Of WT1 Research Articles

Pure curcumin decreases the expression of WT1 by upregulation of miR-15a and miR-16-1 in leukemic cells

BackgroundPure curcumin has been reported to down-regulate the expression of WT1 in leukemic cells. However, the molecular mechanism underlying the down-regulation of WT1 by curcumin is not completely delineated. The purpose of this present study is to identify a new miRNA-mediated mechanism which plays an important role in the anti-proliferation effects of curcumin in leukemic cells.MethodsK562 and HL-60 cells were treated with different concentrations of curcumin for 24 and 48 hours, the level of miR-15a/16-1 and WT1 were detected by qRT-PCR and Western blotting. WT1 expression and cell proliferation were detected by Western blotting and CCK-8, after curcumin treated-K562 and HL-60 cells were transfected with anti-miR-15a/16-1 oligonucleotides.ResultsWe found that pure curcumin upregulated the expression of miR-15a/16-1 and downregulated the expression of WT1 in leukemic cells and primary acute myeloid leukemia (AML) cells. Overexpression of miR-15a/16-1 deduced the protein level of WT1 in leukemic cells, but downregulation of WT1 by siRNA-WT1 could not increase the expression of miR-15a/16-1 in leukemic cells. These results reveal that curcumin induced-upregulation of miR-15a/16-1 is an early event upstream to downregulation of WT1. Furthermore, anti-miR-15a/16-1 oligonucleotides (AMO) partly reversed the downregulation of WT1 induced by pure curcumin in leukemic cells and AMO promoted the growth of curcumin treated-K562 and HL-60 cells.ConclusionThus, these data suggest for the first time that pure curcumin downregulated the expression of WT1 partly by upregulating the expression of miR-15a/16-1 in leukemic cells. miR-15a/16-1 mediated WT1 downregulation plays an important role in the anti-proliferation effect of curcumin in leukemic cells.

Abstract 4015: Flavopiridol-induced upregulation of HtrA1 is associated with suppression of its negative transcriptional regulator WT-1 and with enhanced chemosensitivity

Abstract We previously identified serine protease HtrA1 as a down-regulated gene in epithelial ovarian cancer (EOC) that modulates cisplatin-induced cytotoxicity. However, the mechanism by which HtrA1 sensitizes cells to cisplatin-induced cytotoxicity remained unknown. We have shown previously that the pan-cyclin dependent kinase inhibitor flavopiridol (FP) sensitizes cells to cisplatin-induced cytotoxicity; we hypothesized that flavopiridol might therefore modulate the expression of pro-apoptotic proteins. Microarray data indicated that while flavopiridol treatment alone or in combination with cisplatin up-regulated HtrA1 expression by 2-3 fold (providing a means to upregulate HtrA1 expression), treatment with cisplatin alone had no effect. Interestingly, in searching for underlying mechanisms, we noted that WT-1 transcription factor binding elements that is present in HtrA1 promoter is down-regulated by flavopiridol in our microarray studies. Immunoblot analysis revealed that WT-1 is highly expressed in HtrA1-deficient OV167, OV202, PE04 and Ovcar3 cells but not in HtrA1-expressing SKOV3, TOV21G and PE01 cells, suggesting WT-1 negatively regulates HtrA1 expression in EOC. Immunoblot analysis showed FP treatment resulted in down-regulation of WT-1 with a corresponding increase in HtrA1 protein levels in OV202 cells. Additionally, stable down-regulation of WT-1 by short hairpin RNA (shRNA) in OV202 cells increased HtrA1 expression. Conversely, enhanced expression of full length WT-1 isoform in SKOV3 cells downregulated HtrA1 expression. Reporter assay showed that FP treatment upregulated HtrA1 promoter activity in OV202 cells. Reporter assay and chromatin immunoprecipitation experiments showed that WT-1 is specifically recruited to the HtrA1 promoter. Moreover, downregulation of WT-1 expression in OV202 cells increased cisplatin-mediated cytotoxicity as determined by both 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide and clonogenic assays – an effect reversed by enhanced expression of HtrA1. Collectively, our studies demonstrate a linkage between the effects of flavopiridol on HtrA1 and its regulatory transcription factor WT-1 that appears in part to explain why flavopiridol has chemosensitizing properties Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4015. doi:10.1158/1538-7445.AM2011-4015

Effect of WT1 gene silencing on the tumorigenicity of human glioblastoma multiforme cells

Wilms tumor 1 (WT1) is overexpressed in many human cancers, including glioblastoma multiforme (GBM). In another study, the authors showed that transient WT1 silencing increases the radiosensitivity of glioma cells. Studies of nonglioma cell lines have demonstrated that WT1 promotes cell proliferation and survival; however, this ability has not been rigorously analyzed in human GBM. The authors tested the efficacy of 2 sequences of short hairpin RNA (shRNA) directed against WT1 in U251MG human GBM cells and found that 1 sequence was capable of stably silencing WT1 expression. They then evaluated the effect of WT1 silencing on cellular proliferation, invasion, and in vivo tumor formation. Stable WT1-shRNA expression significantly decreased the proliferation of U251MG cells in vitro as demonstrated by both an adenosine 5'-triphosphate-based viability assay and tritiated thymidine uptake. Furthermore, stable WT1 silencing caused significantly slower growth after the subcutaneous inoculation of tumor cells in the flanks of athymic nude mice and was associated with an increased latency period. Data in this study provide proof of the principle that downregulation of WT1 causes decreased tumorigenicity of a GBM cell line in vitro and in vivo and suggest that WT1 is a promising target for novel molecular GBM therapies, perhaps in combination with standard treatment modalities.

Wilms' Tumor 1 Suppressor Gene Mediates Antiestrogen Resistance via Down-Regulation of Estrogen Receptor-α Expression in Breast Cancer Cells

The antiestrogen tamoxifen has been used in the treatment of hormone-responsive breast cancer for over a decade. The loss of estrogen receptor (ER) expression is the most common mechanism for de novo antiestrogen resistance. Wilms' tumor 1 suppressor gene (WT1) is a clinically useful marker that is associated with poor prognosis in breast cancer patients; its high level expression is frequently observed in cases of breast cancer that are estrogen and progesterone receptor negative. The lack of expression of these receptors is characteristic of tumor cells that are not responsive to hormonal manipulation. To determine whether there is a linkage between WT1 expression and antiestrogen resistance in breast cancer cells, we studied the effect of WT1 on tamoxifen responsiveness in ERalpha-positive MCF-7 cells. We found that overexpression of WT1 in MCF-7 markedly abrogated tamoxifen-induced cell apoptosis and 17beta-estradiol (E(2))-mediated cell proliferation. The expressions of ERalpha and its downstream target genes were significantly repressed following overexpression of WT1, whereas the down-regulation of WT1 by WT1 shRNA could enhance ERalpha expression and the sensitivity to tamoxifen treatment in ERalpha-negative MDA468 and HCC1954 cells that express high levels of WT1. Furthermore, we have confirmed that the WT1 protein can bind to endogenous WT1 consensus sites in the proximal promoter of ERalpha and thus inhibit the transcriptional activity of the ERalpha promoter in a WT1 site sequence-specific manner. Our study clearly implicates WT1 as a mediator of antiestrogen resistance in breast cancer through down-regulation of ERalpha expression and supports the development of WT1 inhibitors as a potential means of restoring antiestrogen responsiveness in breast cancer therapy.

Interferons Downregulate the Expression of WT1 in Myeloid Leukemia through the JAK/STAT Pathway and This Potentiates Chemotherapy Induced Apoptosis.

Introduction: Expression of the WT1 gene is tightly regulated during normal hematopoietic development, and dysregulation of WT1 expression can lead to leukemia. However, relatively little is known about how the WT1 gene is regulated. Whereas WT1 overexpression blocks myeloid progenitor differentiation, the interferons (IFN, both type I and type II) have been shown to promote this differentiation, which underlies their clinical use in the myeloproliferative disorders. We hypothesized that the effect of IFN on myelopoeisis was via regulation of WT1 expression.Materials and Methods: We examined the effect of interferon-(both α and γ) treatment on WT1 mRNA expression on the CML cell line K562 and the AML cell line KG1. We studied the gene expression by RT-PCR and the protein expression by western blotting and flow cytometry by methods previously described. WT1 gene was knocked down using shRNA transfected by standard lipofectamine method. Cell viability was measured by annexin V staining by FACS.Results: Interferon (α and γ) treatment resulted in downregulation of WT1 mRNA expression indicating a common IFN-induced mechanism(s). Importantly, downregulation of WT1 was also associated with downregulation of downstream WT1 targets, such as the bcl-2 and c-myc. Blocking STAT1 phosphorylation by epigallocatechin (EGCG) completely abrogated this effect of IFNg on WT1 protein expression. These findings also suggested that this regulation was primarily at the transcription level, which lead us to initially examine the WT1 promoter. Analysis of the promoter region revealed no canonical STAT1 binding sites; however IFN-induced STAT1 signaling transcriptionally regulates the expression of the transcription factor IRF-1. We first analyzed the WT1 promoter (Transcription Element Search Software, www.cbil.upenn.edu/tess) for possible IRF-1 binding sites and identified a putative site at -222 to - 240 of transcription start site. The actual interaction was confirmed by Electromobility gel shift assay (EMSA) using oligonucleotides derived from the putative sequence. Treatment with IFNg substantially increased the amount of shifted probe, consistent with increased oligonucleotide binding by IRF-1 protein. IFNg treatment alone had little effect on cell viability. However, treatment with IFNg or INFa resulted in significant sensitization of both K562 and KG1 to chemotherapy and significantly increases apoptosis in both cell lines. Since it is possible that IFN-mediated sensitization is due to one of the myriad of interferon responses we specifically down regulated WT1 expression by shRNA in K 562 cells. The chosen shRNA clone alone induced some apoptosis but the combination of WT1 silencing plus etoposide markedly increased cell death.Conclusions: In summary our data strongly support the hypothesis that downregulation of WT1 expression impairs the survival of leukemic blasts, and sensitizes these cells to chemotherapy-induced apoptosis. The data also demonstrate that WT1 expression can be modulated by clinically available agents (INFa), and point to potentially new strategies to target AML. Ongoing studies in primary AML cells will not only confirm the findings above but also lay the foundation for therapeutic targeting of a key protein necessary for leukemia cell survival in the face of cytotoxic therapy.

Down-regulation of WT1/+17AA gene expression using RNAi and modulating leukemia cell chemotherapy resistance

We have shown that inhibition of WT1/+17AA protein expression following transfection with a vector-based small interfering RNA expression construct in K562 cell lines, leads to a decrease in MDR1 and P-glycoprotein levels, accumulation of Rh123, and enhancement of the doxorubicin cytotoxicity. Our findings suggest that WT1/+17AA exerts its oncogenic function by modulating multidrug resistance in leukemia cells.

Inhibition of WT1 and bcl2 in leukemic cell lines by small interfering RNA (siRNA)

16504 The expression of Wilm‘s tumor gene-1 (wt1) and bcl-2 is considered to have a proliferating and survival supporting effect in leukemia blast cells. The downregulation of wt1 by means of antisense-oligonucleotides and ribozymes revealed an inhibition of cell proliferation and induction of cell death. Here we describe the effect of siRNA against wt1 and bcl-2 in leukemic cell lines. RT-PCR and western blot analyses were performed to examine wt1 and bcl-2 gene expression in transfected leukemia cell lines. Apoptosis was detected with FACS analysis. K562 and HL-60 cell lines transfected with wt1 siRNA showed decreasing levels of wt1 mRNA and protein expression after 24 and 48 hours. The cell proliferation was reduced between 45% and 76% 48 hours after transfection, and apoptosis increased from 6.6 % in control cells to 12.2 % 24 hours after transfection in transfected cells. 48 hours after transfection the amount of apoptotic cells increased up to 45 % in transfected cells. Bcl-2 siRNA only induced apoptosis in about 15% of the cells. The combination of wt1 and bcl-2 siRNA had no additive effect on the induction of apoptosis. The expression of wt1 seems to be more important for cell survival than expression of the anti-apoptotic gene bcl-2. We therefore consider siRNA targeting human wt1 as possible tool against leukemic cells overexpressing wt1. No significant financial relationships to disclose.

WT1 and DAX-1 inhibit aromatase P450 expression in human endometrial and endometriotic stromal cells.

The orphan nuclear receptor steroidogenic factor-1 (SF-1) induces the expression of Müllerian inhibiting substance (MIS) and many steroidogenic genes, including aromatase P450 (P450arom). Dosage-sensitive sex reversal adrenal hypoplasia congenita critical region on the X chromosome gene 1 (DAX-1) inhibits SF-1-mediated induction of MIS and other steroidogenic genes, whereas Wilms' tumor suppressor gene (WT1) augments SF-1-mediated MIS expression. The effects of WT1 on steroidogenesis or P450arom expression have not been explored to date. In human endometriotic stromal cells, extremely high levels of P450arom mRNA and enzyme activity are present. Prostaglandin E(2) stimulates cAMP formation, SF-1 binding activity, P450arom mRNA levels, and estrogen synthesis in endometriotic stromal cells. Stromal cells of eutopic endometrium from disease-free women, on other hand, do not contain readily detectable levels of P450arom mRNA. Thus, we evaluated herein the possible roles of WT1 and DAX-1 in cAMP/SF-1-mediated regulation of P450arom expression in endometriotic and endometrial stromal cells. We also determined the cellular distribution and levels of these transcription factors in pathological endometriotic vs. normal eutopic endometrial tissues by immunohistochemistry to understand their in vivo roles. In vitro transcriptional regulation studies showed that both WT1 and DAX-1 inhibited cAMP and/or SF-1-induced P450arom promoter activity in a dose-dependent fashion in cultured human endometriotic and endometrial stromal cells. Site-directed disruption of the SF-1 binding site (-136/-124 bp) in the P450arom promoter abolished basal or cAMP/SF-1-induced promoter activity in the presence or absence of WT1 or DAX-1. Immunohistochemistry and H-scoring showed that DAX-1 was ubiquitously present in epithelial and stromal cells of both tissues. WT1, on the other hand, was preferentially expressed in stromal (vs. epithelial) cells. Moreover, WT1 levels in endometriotic stromal cells are significantly down-regulated compared with normal endometrial stromal cells. In summary, WT1 or DAX-1 inhibits cAMP-SF-1 pathway-dependent P450arom expression in cultured human endometriotic and endometrial stromal cells. In vivo down-regulation of WT1 in endometriotic stromal cells (vs. normal endometrial stromal cells) may in part be responsible for aberrantly increased P450arom expression and estrogen formation in this pathological tissue.

Ribozyme-mediated cleavage of wt1 transcripts suppresses growth of leukemia cells.

The Wilms' tumor gene product (WT1) was identified as a tumor suppressor in pediatric kidney tumors. Conversely, acute leukemias express WT1 at a high frequency, and leukemias with high levels of WT1 expressed by leukemic blast cells have a significantly worse prognosis, suggesting an oncogenic function of WT1 in leukemic cells. To address this issue, we developed five hammerhead ribozymes (RZ1-RZ5) designed to cleave various wt1-mRNA GUC-recognition sites and thus suppress wt1 expression. Using in vitro transcribed ribozymes and truncated wt1 target RNAs as substrates, we performed in vitro cleavage assays. The sequence of two ribozymes was then cloned into the pCDNA3 expression vector containing a self-processing ribozyme cassette. Downregulation of wt1 due to ribozyme expression was analyzed in the human 293 embryonic kidney and the K562 chronic myeloid leukemia cell line by Western blotting and RT-PCR. Growth of stable transfected K562 cells was determined by proliferation analysis and 3H-thymidine incorporation. In vitro, the anti-wt1 ribozymes were able to recognize and cleave the target RNA in a highly sequence-specific and time-dependent manner. The ribozymes showed different catalytic activity. Coexpression of wt1 and the self-processing ribozymes pRZ3 and pRZ5, respectively, resulted in a significantly downregulated WT1 protein level when transiently transfected in 293 cells. Furthermore, stable transfection of pRZ3 and pRZ5 resulted in considerably reduced expression of endogenous wt1 in K562 cells, correlating with the inhibition of cell proliferation and the induction of cell death. Our data suggest that anti-wt1 ribozymes are a potent inhibitor of wt1 expression with possible implications for the inhibition of cell proliferation in leukemic cells.

Downregulation of Wilms’ tumor gene (WT1) is not a prerequisite for erythroid or megakaryocytic differentiation of the leukemic cell line K562

The Wilms’ tumor gene (WT1) encodes a transcription factor of the zinc finger type. A high expression of WT1 has been detected in a range of acute leukemias, and WT1 is downregulated during induced differentiation of some leukemic cell lines. Overexpression of WT1 in some myeloid cell lines confers resistance to differentiation induction. These observations suggest that a high WT1 expression in hematopoietic cells is incompatible with differentiation. In this study, each of the four different isoforms of WT1 was constitutively overexpressed in the leukemic cell line K562. K562 cells express endogenous WT1, which is downregulated as a response to induced differentiation along the erythroid and megakaryocytic pathways. We now demonstrate that a forced exogenous expression of the four different isoforms of WT1 in K562 does not affect the differentiation response, as judged by accumulation of hemoglobin in response to hemin or the expression of megakaryocytic cell surface markers in response to 12-O-tetradecanoylphorbol-13-acetate (TPA). We conclude that downregulation of WT1 during induced differentiation of K562 cells is not a prerequisite for erythroid or megakaryocytic differentiation of these cells.

Wilms Tumor Gene Expression in Acute Myeloid Leukemias

The Wilm's tumor gene product (WT-1) is suggested to act as a tumor suppressor in childhood malignancies of the kidney and as a transcription factor with regulating activity on a number of growth and differentiation factors. Wt-1 has been shown to be expressed in blast cells of the vast majority of patients with acute myeloid and lymphoblastic leukemias (AL) by a number of workers.High levels of wt-1 mRNA expression in blast cells of newly diagnosed AML patients predict worse prognosis when compared to patients with no or low wt-1 mRNA expression. Patients achieving complete responses after chemotherapy usually lose detectable signals of wt-1. In relapse of the disease reoccurrence of wt-1 mRNA can be determined in almost all patients with initially detectable wt-1 mRNA. Using sensitive techniques such as reverse transcription poly-merase chain reaction (RT-PCR) relapses are preceded by wt-1 expression in some cases. Although a subpopulation of normal hematopoietic precursor cells has also been shown to express message for wt-1, detectable levels of wt-1 during follow-ups in AML patients have been shown to be useful as a marker for residual blast populations or even to predict relapse of AML.Whether the high level of wt-1 expression is a non-specific phenomenon resulting from malignant transformation or whether it has an impact on the pathophysiology of AML or the uncontrolled growth of AML blasts is still controversial. However, there are indicators for an involvement of wt-1 in malignant events of AML blasts such as the downregulation of wt-1 in chemically induced differentiation of AML blast cell lines or the interactions of wt-1 with the protooncogene bcl-2 and the tumor suppressor gene p53. In conclusion, its possible relevance as an AML marker and its role in pathophysiological mechanisms in AML will still have to be defined in the future.