WT1 Promoter Research Articles

Rreb1 is a key transcription factor in Sertoli cell maturation and function and spermatogenesis in mouse.

Spermatogenesis is a developmental process driven by interactions between germ cells and Sertoli cells. This process depends on appropriate gene expression, which might be regulated by transcription factors. This study focused on Rreb1, a zinc finger transcription factor, and explored its function and molecular mechanisms in spermatogenesis in a mouse model. Our results showed that RREB1 was predominantly expressed in the Sertoli cells of the testis. The decreased expression of RREB1 following injection of siRNA caused impaired Sertoli cell development, which was characterized using a defective blood-testis barrier structure and decreased expression of Sertoli cell functional maturity markers; its essential trigger might be SMAD3 destabilization. The decreased expression of RREB1 in mature Sertoli cells influenced the cell structure and function, which resulted in abnormal spermatogenesis, manifested as oligoasthenoteratozoospermia, and we believe RREB1 plays this role by regulating the transcription of Fshr and Wt1. RREB1 has been reported to activate Fshr transcription, and we demonstrated that the knockdown of Rreb1 caused a reduction in follicle-stimulating hormone receptor (FSHR) in the testis, which could be the cause of the increased sperm malformation. Furthermore, we confirmed that RREB1 directly activates Wt1 promoter activity, and RREB1 downregulation induced the decreased expression of Wt1 and its downstream polarity-associated genes Par6b and E-cadherin, which caused increased germ-cell death and reduced sperm number and motility. In conclusion, RREB1 is a key transcription factor essential for Sertoli cell development and function and is required for normal spermatogenesis.

Deletion of an intronic HIF-2α binding site suppresses hypoxia-induced WT1 expression

Hypoxia-inducible factors (HIFs) play a key role in the adaptation to low oxygen by interacting with hypoxia response elements (HREs) in the genome. Cellular levels of the HIF-2α transcription factor subunit influence the histopathology and clinical outcome of neuroblastoma, a malignant childhood tumor of the sympathetic ganglia. Expression of the Wilms tumor gene, WT1, marks a group of high-risk neuroblastoma. Here, we identify WT1 as a downstream target of HIF-2α in Kelly neuroblastoma cells. In chromatin immunoprecipitation assays, HIF-2α bound to a HRE in intron 3 of the WT1 gene, but not to another predicted HIF binding site (HBS) in the first intron. The identified element conferred oxygen sensitivity to otherwise hypoxia-resistant WT1 and SV40 promoter constructs. Deletion of the HBS in the intronic HRE by genome editing abolished WT1 expression in hypoxic neuroblastoma cells. Physical interaction between the HRE and the WT1 promoter in normoxic and hypoxic Kelly cells was shown by chromosome conformation capture assays. These findings demonstrate that binding of HIF-2α to an oxygen-sensitive enhancer in intron 3 stimulates transcription of the WT1 gene in neuroblastoma cells by hypoxia-independent chromatin looping. This novel regulatory mechanism may have implications for the biology and prognosis of neuroblastoma.

Co-activation of WT1 and AP-1 proteins on WT1 gene promoter to induce WT1 gene expression in K562 cells

Earlier studies have revealed one function of the inhibitory mechanism of curcumin. Activating PKCα induces WT1 gene expression via signalling through downstream JNK and c-JUN. In the present study, the effect of c-JUN/AP-1 binding and transcriptional regulation of the WT1 gene promoter was investigated in K562 leukaemic cells. The non-cytotoxic dose (IC20 values) of curcumin (WT1 and AP-1 inhibitors) was employed to examine its effect on WT1 gene-mediated WT1 and AP-1 protein expression. Non-cytotoxic doses of both tanshinone IIA (AP-1 DNA-binding inhibitor) and SP600125 (JNK inhibitor) were used to test the role of c-JUN/AP-1 in WT1 gene expression. Curcumin, tanshinone IIA, and SP600125 inhibited WT1 protein expression in a dose-dependent manner (5–15 μM) at 24 h as shown by immunoblotting. A ChIP assay showed that curcumin and tanshinone IIA inhibited AP-1 and WT1 binding to the proximal WT1 promoter (−301 bp), and a luciferase reporter assay showed that the WT1 luciferase gene reporter activity was decreased after curcumin, tanshinone IIA, and SP600126 treatments. Furthermore, depletion of c-JUN abrogated WT1 gene expression. In summary, AP-1 contributes to the WT1 autoregulation of WT1 gene expression in leukaemic K562 cells.

Genomic and epigenetic signatures associated with survival rate in oral squamous cell carcinoma patients.

Purpose: Although oral squamous cell carcinoma (OSCC) presents great mortality and morbidity worldwide, the mechanisms behind its clinical behavior remain unclear. Biomarkers are needed to forecast patients' survival and, among those patients undergoing curative therapy, which are more likely to develop tumor recurrence/metastasis. Demonstrating clinical relevance of these biomarkers could be crucial both for surveillance and in helping to establish adjuvant therapy strategies. We aimed to identify genomic and epigenetic biomarkers of OSCC prognosis as well as to explore a noninvasive strategy to perform its detection.Methods: OSCC tumor and non-tumor tissue samples and cells scrapped from the tumor surface were genomic and epigenetically evaluated by Methylation-Specific Multiplex Ligation-dependent Probe Amplification technique.Results: Copy number alterations in ATM, CASR, TP73, CADM1, RARB, CDH13, PAX5, RB1 genes and GATA5, PAX6, CADM1 and CHFR promoter methylation were shown to be associated with worse OSCC patients' survival. Copy number alterations in BRCA1, CDKN2A, CHFR, GATA5, PYCARD, STK11, TP53, VHL genes and GATA5, CADM1, KLLN, MSH6, PAX5, WT1 promoter methylation were shown to be associated with development of metastasis/relapses during or after OSCC patients' treatment. We also found a good agreement in the status of CDKN2A promoter methylation evaluated noninvasively or in the tumor tissue.Conclusions: Genomic and epigenetic signatures were validated in a larger and geographically separate cohort, from TCGA database, which reinforce their clinical applicability. Noninvasive methodologies for detection of these signatures require further studies before translation in to clinical practice.

The stress-response molecule NR4A1 resists ROS-induced pancreatic β-cells apoptosis via WT1

Pancreatic β-cells often face endoplasmic reticulum stress and/or ROS-associated oxidative stress under adverse conditions. Our previous work has verified that NR4A1 protects pancreatic β-cells from ER-stress induced apoptosis. However, It remains unknown whether NR4A1 is able to protect pancreatic β-cells against ROS-associated oxidative stress. In the present study, our data showed that NR4A1 protein expression rapidly increased in MIN6 cells upon H2O2 treatment, and overexpression of NR4A1 in MIN6 cells conferred resistance to cell apoptosis induced by H2O2. These results were further substantiated in isolated islets from mice infected with an adenovirus overexpressing NR4A1. 8-hydroxy-2′-deoxyguanosine (8-OHdG) was used as a biomarker for oxidative stress or a marker for ROS damage. We found that the 8-OHdG level in the islets from NR4A1 knockout mice fed with high-fat diet was much higher than that in the islets from parental control mice; and higher apoptotic rate was observed in the islets from NR4A1 KO mice compared to control mice. Further investigation of underlying mechanisms of NR4A1’s protective effects showed that NR4A1 overexpression in MIN6 cells reduced Caspase 3 activation caused by H2O2, and increased expression of WT1 and SOD1. There is a putative NR4A1 binding site (−1118bp to −1111bp) in WT1 promoter; our data demonstrated that NR4A1 protein physically associates with the WT1 promoter, and enhanced WT1 promoter transactivation and knockdown of WT1 in MIN6 cells induced apoptosis. These findings suggest that NR4A1 protects pancreatic β-cells against H2O2 mediated apoptosis by up-regulating WT1 expression.

Downregulation of the WT1 gene expression via TMPyP4 stabilization of promoter G-quadruplexes in leukemia cells.

The WT1 gene is an important oncogene, and its overexpression is considered as an effective target for anticancer therapy. Regulation of its gene transcription is one way for WT1-targeting drug design. Recently, in silico analysis of some oncogene promoters like WT1 showed some guanine-rich regions with the ability to form G-quadruplex structures. Ligands like 5,10,15,20-tetra(N-methyl-4-pyridyl)-porphine (TMPyP4) have predominant effect on G-quadruplex stabilization. The aim of this study was to understand the effect of TMPyP4 on WT1 gene transcription via stabilization of promoter G-quadruplexes. We examined the formation of new G-quadruplex motifs in WT1 promoter in the presence of TMPyP4. In order to understand the nature of its interaction with WT1 promoter quadruplexes, differential pulse voltammetry (DPV), circular dichroism (CD), polyacrylamide gel electrophoresis, electrophoretic mobility shift assay (EMSA), polymerase chain reaction (PCR) stop assays, and quantitative RT-PCR were performed. According to the results, the WT1 promoter can form stable intramolecular parallel G-quadruplexes. In addition, after 48 and 96h of incubation, 100μM TMPyP4 reduced the WT1 transcription to 9 and 0.4%, respectively, compare to control. We report that ligand-mediated stabilization of G-quadruplexes within the WT1 promoter can silence WT1 expression. This study might offer the basis for the reasonable design and improvement of new porphyrin derivatives as effective anti-leukemia agents for cancer therapy.

The Role of WT1 in Embryonic Development and Normal Organ Homeostasis.

The Wilms' tumor suppressor gene 1 (Wt1) is critically involved in a number of developmental processes in vertebrates, including cell differentiation, control of the epithelial/mesenchymal phenotype, proliferation, and apoptosis. Wt1 proteins act as transcriptional and post-transcriptional regulators, in mRNA splicing and in protein-protein interactions. Furthermore, Wt1 is involved in adult tissue homeostasis, kidney function, and cancer. For these reasons, Wt1 function has been extensively studied in a number of animal models to establish its spatiotemporal expression pattern and the developmental fate of the cells expressing this gene. In this chapter, we review the developmental anatomy of Wt1, collecting information about its dynamic expression in mesothelium, kidney, gonads, cardiovascular system, spleen, nervous system, lung, and liver. We also describe the adult expression of Wt1 in kidney podocytes, gonads, mesothelia, visceral adipose tissue, and a small fraction of bone marrow cells. We have reviewed the available animal models for Wt1-expressing cell lineage analysis, including direct Wt1 expression reporters and systems for permanent Wt1 lineage tracing, based on constitutive or inducible Cre recombinase expression under control of a Wt1 promoter. Finally we provide a number of laboratory protocols to be used with these animal models in order to assess reporter expression.

WT1 Protects Leukemic Cells from Cytotoxic Replicative Stress

BACKGROUND:The transcription factor WT1 is a “pan-leukemic marker”. WT1 is overexpressed in a significant number of AML, CML, ALL, and MDS cells. Moreover, the persistence of WT1 expression after chemotherapy indicates residual leukemic stem cells. Hydroxyurea can decrease the white blood cell count in leukemic patients. Hydroxyurea inhibits ribonucleotide reductase and consequently depletes the cellular dNTP pool. This process induces replicative stress and activates the intra-S phase checkpoint. The prolonged arrest of DNA replication leads to double strand DNA breaks and apoptosis. The aim of our study was to investigate whether WT1 has an impact on the survival of AML and CML cells treated with hydroxyurea. Furthermore, we analyzed whether replicative stress affects WT1 expression. We also addressed whether clinically relevant drugs modulating WT1 influence apoptosis, autophagy, and DNA damage signaling in leukemic cells.METHODS:We used human AML and CML cells (MV4-11, RS4-11, NB4, HEL, K562) and primary AML cells with various genetic lesions (FLT3-ITD, MLL-AF4, PML-RARα, JAK2V617F, BCR-ABL; p53 mutant or null) or a complex karyotype. Cells were exposed to 0,5 mM hydroxyurea and analyzed by FACS, Western Blot and mRNA expression analyses. RNAi with morpholinos targeting the WT1 mRNA as well as the tyrosine kinase inhibitor imatinib (0,1-1 µM) and epigenetic modifiers of the histone deacetylase inhibitor family (HDACi: 1,5-5 µM entinostat and 10-100 nM panobinostat) were used to decrease WT1 levels.RESULTS:1.The stability of WT1 is causally linked to the survival of leukemic cells undergoing replicative stress. Hydroxyurea-treated leukemic cells retaining WT1 (K562 and RS4-11 cells) accumulate in S phase and survive. However, cells that lose WT1 (NB4, MV4-11, HEL, and primary AML cells) succumb to apoptosis (57-70% apoptosis after 24 h; p<0,001).2.The specific elimination of WT1 in K562 cells with morpholino antisense nucleotides (10 nM) significantly sensitizes the cells to hydroxyurea-induced apoptosis (26-30% increased apoptosis rates after 24 h; p<0,005).3.The hydroxyurea-induced loss of WT1 in sensitive cells is caspase-dependent but does not require the p53 tumor suppressor. The pan-caspase inhibitor ZVAD-FMK (20-50 µM) and the ectopic overexpression of mitochondrial anti-apoptotic proteins (BCL-XL/BCL2) prevents the hydroxyurea-induced depletion of WT1 and apoptosis in NB4 cells (from 62-69% to 9-14%; p<0,001).4.Moreover, our data reveal that hydroxyurea promotes the occurrence of previously unrecognized WT1 cleavage products. These are different from the known HTRA2-induced WT1 fragments, as they contain the WT1 zinc fingers required for DNA binding. These novel cleavage products also contain the domain for transcriptional activation, but lack the domain for transcriptional repression. We demonstrate that caspases cleave the full-length WT1 protein into such fragments, which accumulate in the nucleus of hydroxyurea-treated NB4 cells.5.Resistance of BCR-ABL-positive K562 CML cells to pro-apoptotic effects of hydroxyurea can be overcome with the tyrosine kinase inhibitor imatinib. This drug inhibits the WT1 promoter and sensitizes the cells to hydroxyurea (22-48% increased apoptosis rate after 24 h; p<0,005).6.The HDACi entinostat and panobinostat suppress the WT1 gene and accelerate the proteasomal degradation of WT1 by an increased expression of the E2 ubiquitin conjugase UBCH8. The depletion of WT1 by HDACi renders K562 cells sensitive towards hydroxyurea-induced killing and causes a synergistic activation of cell death (apoptosis increased by 43-55% after 24 h; p<0,001).7.In K562 cells hydroxyurea/entinostat combinations lead to a caspase-dependent cleavage of the DNA repair enzyme PARP-1 and to the accumulation of double-strand DNA breaks.8.We further show that entinostat significantly shifts the balance from a hydroxyurea-induced autophagy program to apoptosis (from 60% to 16% autophagy; p<0,001) in K562 cells. We reveal that entinostat depletes proteins critical for autophagy and autophagosome-lysosome fusion (Beclin-1 and HDAC6) in these cells.CONCLUSIONS:Our data illustrate that WT1 is a novel biological target of hydroxyurea, independent of a specific genetic lesion and irrespective of a functional p53 pathway. The inhibition of WT1 might be therapeutically exploitable for targeting approaches against leukemic cells. DisclosuresNo relevant conflicts of interest to declare.

Methylation-specific multiplex ligation-dependent probe amplification identifies promoter methylation events associated with survival in glioblastoma

DNA methylation plays an important role in cancer biology and methylation events are important prognostic and predictive markers in many tumor types. We have used methylation-specific multiplex ligation-dependent probe amplification to survey the methylation status of MGMT and 25 tumor suppressor genes in 73 glioblastoma cases. The data obtained was correlated with overall survival and response to treatment. The study revealed that methylation of promoter regions in TP73 (seven patients), THBS1 (eight patients) and PYCARD (nine patients) was associated with improved outcome, whereas GATA5 (21 patients) and WT1 (24 patients) promoter methylation were associated with poor outcome. In patients treated with temozolomide and radiation MGMT and PYCARD promoter methylation events remained associated with improved survival whereas GATA5 was associated with a poor outcome. The identification of GATA5 promoter methylation in glioblastoma has not previously been reported. Furthermore, a cumulative methylation score separated patients into survival groups better than any single methylation event. In conclusion, we have identified specific methylation events associated with patient outcome and treatment response in glioblastoma, and these may be of functional and predictive/prognostic significance. This study therefore provides novel candidates and approaches for future prospective validation.

RNAa-mediated overexpression of WT1 induces apoptosis in HepG2 cells

AimRecent studies have reported that double-stranded RNA (dsRNA) can activate gene expression by targeting promoter sequence in a process termed RNA activation. The present study was conducted to evaluate the potential of WT1 induction by small activating RNA targeting the WT1 promoter (dsWT1) in the treatment of hepatocellular carcinoma.MethodsThe human hepatocellular carcinoma cell line HepG2 was transfected with dsRNA by liposomes. The expression of mRNA and protein in cells were investigated using real-time reverse real-time quantitative PCR and Western blot, respectively. Cell viability and clonogenicity were determined by MTT assay and clonogenicity assay, respectively. Cell apoptosis was evaluated by flow-cytometric analysis.ResultsExpressions of WT1 mRNA and protein in dsWT1 treated HepG2 cells were significantly elevated. Inhibition of cell viability by dsWT1 was dose-dependent and time-dependent. Reduction of the number and size of colonies formed were found in dsWT1 treated cells. dsWT1 induced significant apoptosis in HepG2 cells. The decreased anti-apoptotic protein Bcl-2 and elevated pro-apoptotic protein Bak expression were detected in dsWT1 treated cells. The level of pro-caspase-3 remarkably decreased and cleaved caspase-3 and PARP fragment were also detected in dsWT1 treated cells.ConclusionThese data show that RNAa-mediated overexpression of WT1 may have therapeutic potential in the treatment of hepatocellular carcinoma.

Reactivation of Silenced WT1 Transgene by Hypomethylating Agents - Implications forin vitroModeling of Chemoimmunotherapy

BackgroundA cell line with transfected Wilms' tumor protein 1 (WT1) is has been used for the preclinical evaluation of novel treatment strategies of WT1 immunotherapy for leukemia due to the lack of appropriate murine leukemia cell line with endogenous WT1. However, silencing of the transgene occurs. Regarding the effects of hypomethylating agents (HMAs) on reactivation of silenced genes, HMAs are considered to be immune enhancers.MethodsWe treated murine WT1- transfected C1498 (mWT1-C1498) with increasing doses of decitabine (DAC) and azacitidine (AZA) to analyze their effects on transgene reactivation.ResultsDAC and AZA decreased the number of viable cells in a dose- or time-dependent manner. Quantification of WT1 mRNA level was analyzed by real-time polymerase chain reaction after mWT1-C1498 treated with increasing dose of HMA. DAC treatment for 48 h induced 1.4-, 14.6-, and 15.5-fold increment of WT1 mRNA level, compared to untreated sample, at 0.1, 1, and 10µM, respectively. Further increment of WT1 expression in the presence of 1 and 10µM DAC was evident at 72 h. AZA treatment also induced up-regulation of mRNA, but not to the same degree as with DAC treatment. The correlation between the incremental increases in WT1 mRNA by DAC was confirmed by Western blot and concomitant down-regulation of WT1 promoter methylation was revealed.ConclusionThe in vitro data show that HMA can induce reactivation of WT1 transgene and that DAC is more effective, at least in mWT1-C1498 cells, which suggests that the combination of DAC and mWT1-C1498 can be used for the development of the experimental model of HMA-combined WT1 immunotherapy targeting leukemia.

Tumor Suppressor Menin Represses Paired Box Gene 2 Expression via Wilms Tumor Suppressor Protein-Polycomb Group Complex

Tumor suppressor menin, the product of the MEN1 gene, plays a key role in controlling histone 3 lysine 4 trimethylation (H3K4me3) and gene transcription, which can regulate proliferation, apoptosis, and differentiation. However, little is known as to whether menin controls gene expression and cell proliferation and survival via regulating Polycomb group (PcG) protein complex/H3K27me3. Here we show that menin specifically represses transcription factor Paired box gene 2 (Pax2) through PcG-mediated H3K27me3 and Wilms tumor suppressor protein (WT1), a zinc finger domain-containing DNA-binding protein. Menin does not directly bind to the Pax2 locus, instead, it up-regulates WT1 expression. WT1 recruits PcG complex to the Pax2 promoter and represses expression of Pax2 through PcG-dependent H3K27me3. Moreover, WT1 also interacts with DNA methyltransferase 1 (DNMT1), and recruits DNMT1 to the Pax2 promoter, resulting in hypermethylation of CpG in the Pax2 promoter. Together, these studies have uncovered a novel epigenetic mechanism whereby menin regulates H3K27me3 and promoter DNA methylation via WT1 and suggest that WT1 protein plays an important, yet previously unappreciated role in regulating the function of the menin/PcG axis, H3K27 methylation, and DNA methylation, resulting in repression of gene transcription.

TGF-beta1 reduces Wilms' tumor suppressor gene expression in podocytes

Wilms' tumor suppressor gene (WT1) is essential for normal podocyte function, and transforming growth factor (TGF)-beta contributes to focal segmental glomerulosclerosis (FSGS). We aimed to address whether TGF-beta affects WT1 expression in podocytes. A human podocyte cell line treated with TGF-beta1 and kidneys in Alb/TGF-beta1-transgenic mice were analyzed for WT1 expression. In cultured podocytes, TGF-beta1 reduced WT1 protein expression determined by western blotting beginning at 8 h and decreased WT1 messenger RNA (mRNA) expression measured by quantitative reverse transcription-polymerase chain reaction beginning at 3 h. Knockdown of Smad4 by small hairpin (sh) RNA partially rescued the TGF-beta1-induced reduction of both WT1 protein and mRNA expressions in the cultured podocytes. TGF-beta1 did not alter luciferase activity of the reporter construct for a human WT1 promoter but reduced that for a human WT1 5' enhancer construct, suggesting that TGF-beta1 may regulate WT1 expression by altering the 5' enhancer activity. In the transgenic mice, WT1 protein expression in podocytes was decreased at 1 and 3 weeks of age, while glomeruloclerosis developed after 3 weeks. TGF-beta1 reduces WT1 expression in cultured human podocytes and podocytes in mice before overt glomerulosclerosis begins. The effects are at least partially Smad4 dependent. Our findings identify a novel pathway linking TGF-beta1 to podocyte injury and FSGS. The WT1 reduction may be a useful marker for early podocyte injury.

A dual epigenomic approach for the search of obesity biomarkers: DNA methylation in relation to diet‐induced weight loss

Epigenetics could help to explain individual differences in weight loss after an energy-restriction intervention. Here, we identify novel potential epigenetic biomarkers of weight loss, comparing DNA methylation patterns of high and low responders to a hypocaloric diet. Twenty-five overweight or obese men participated in an 8-wk caloric restriction intervention. DNA was isolated from peripheral blood mononuclear cells and treated with bisulfite. The basal and endpoint epigenetic differences between high and low responders were analyzed by methylation microarray, which was also useful in comparing epigenetic changes due to the nutrition intervention. Subsequently, MALDI-TOF mass spectrometry was used to validate several relevant CpGs and the surrounding regions. DNA methylation levels in several CpGs located in the ATP10A and CD44 genes showed statistical baseline differences depending on the weight-loss outcome. At the treatment endpoint, DNA methylation levels of several CpGs on the WT1 promoter were statistically more methylated in the high than in the low responders. Finally, different CpG sites from WT1 and ATP10A were significantly modified as a result of the intervention. In summary, hypocaloric-diet-induced weight loss in humans could alter DNA methylation status of specific genes. Moreover, baseline DNA methylation patterns may be used as epigenetic markers that could help to predict weight loss.

PPARβ activation inhibits melanoma cell proliferation involving repression of the Wilms’ tumour suppressor WT1

Peroxisome proliferator-activated receptors (PPARs) are ligand-activated transcription factors that strongly influence molecular signalling in normal and cancer cells. Although increasing evidence suggests a role of PPARs in skin carcinogenesis, only expression of PPARγ has been investigated in human melanoma tissues. Activation of PPARα has been shown to inhibit the metastatic potential, whereas stimulation of PPARγ decreased melanoma cell proliferation. We show here that the third member of the PPAR family, PPARβ/δ is expressed in human melanoma samples. Specific pharmacological activation of PPARβ using GW0742 or GW501516 in low concentrations inhibits proliferation of human and murine melanoma cells. Inhibition of proliferation is accompanied by decreased expression of the Wilms’ tumour suppressor 1 (WT1), which is implicated in melanoma proliferation. We demonstrate that PPARβ directly represses WT1 as (1) PPARβ activation represses WT1 promoter activity; (2) in chromatin immunoprecipitation and electrophoretic mobility shift assays, we identified a binding element for PPARβ in the WT1 promoter; (3) deletion of this binding element abolishes repression by PPARβ and (4) the WT1 downstream molecules nestin and zyxin are down-regulated upon PPARβ activation. Our findings elucidate a novel mechanism of signalling by ligands of PPARβ, which leads to suppression of melanoma cell growth through direct repression of WT1.

Bortezomib in a myelodysplastic cell line: A gene expression study

18018 Background: NF-kB has been reported to be constitutively activated in P39 cell line (a myelomonocytic cell line) and in MDS cases with higher risk of transformation. Another gene whose expression levels have been reported to play a relevant prognostic role in MDS is WT1. Two NF-kB binding sites within the WT1 promoter have been reported; thus, we decided to assess if a compound able to inactivate NF-kB would be also able to down-regulate the WT1 expression. Finally, we performed microarray and real-time quantitative PCR assays for understanding the molecular pathways more significantly affected by this treatment. Methods: For gene expression studies, samples were hybridized on Whole Human Genome Microarray (Agilent). The genes identified as de-regulated by bortezomib were then analyzed for network and gene ontology by Ingenuity Pathway Analysis software. Results have been confirmed by using the TaqMan Low Density Array Human Apoptosis Panel. Results: Bortezomib inactivated NF-kB, and exerted an anti-proliferative and pro-apoptotic effect by blocking cell cycle in the G2 phase. It increased the release of reactive oxygen species and down-regulated the WT1 expression. In the untreated P39, 84 of the 93 genes involved in the apoptosis pathway and represented in the Taqman Low-Density Arrays were expressed. After 12h-treatment, bortezomib up-regulated DIABLO and NFKBIB, (a NF-kB inhibitor), and down-regulated NF-kB1, NF-kB2, and BIRC1, an anti- apoptotic gene. Moreover, microarray assays showed that the PPAR, P53, IL6, IL2, hypoxia, Huntington’s disease, TLR and cell cycle were the pathways more significantly modified. Among down-regulated genes, JUN, HSP70 and HSP90 would be relevant. Indeed, high levels of HSPs have been reported to negatively condition the overall survival of patients with high-risk MDS. Other genes down-regulated were CREBBP, PMAIP1, SPP1, and some adhesion molecules, such as ICAM1. Even reduction of integrins could be a relevant effect exerted by the proteasome inhibitor, because higher serum levels of ICAM1 have been reported to be associated with high-risk MDS and to negatively condition the survival. Conclusions: These results suggest the possible use of proteasome inhibitors in treatment of high-risk MDS. In vivo trials will be useful to confirm this hypothesis. No significant financial relationships to disclose.

The transcriptional activity of WT1 gene promoter and enhancer in cell lines with diverse tissue origin

In this study, tissue-specific transcriptional activity of WT1 promoter and enhancer was investigated in a hope to design gene therapy strategy based on the transcriptional regulatory elements of WT1 gene. WT1 promoter and/or enhancer were ligated into pEGFP-1 to construct recombinant vectors with EGFP gene as a reporter and analyzed using Flow cytometry. The results showed that pEWP containingWT1 promoter induced the highest EGFP expression in ECV304 at 16.54 +/- 2.45 times, mildly higher in MCF-7 and SHG44 at 9.46 +/- 1.10 and 7.29 +/- 0.73 times, and in K562 cell line at 2.93 +/- 0.27 times, compared to that of pEGFP-1. However, pEWPA, with WT1 enhancer inserted at Afl II site, increased basal transcription levels of the WT1 promoter in HT-29, SHI-1 and K562 cells by 4.81, 3.06 and 1.01-fold, respectively. pEWPD inserted at NotI and pEWPE inserted at BamI had no ability to increase the transcriptional activity of WT1 promoter. Moreover, reporter gene silenced was observed in transfected host cells by flow cytometryt and real-time PCR. These results suggested that transcriptional activities of WT1 promoter in the recombinant vector seemed not correlated to the constitutional expression level of endogenous WT1 gene. The WT1 enhancer could promote the transcriptional activities of WT1 promoter in some of the cell lines regardless of the hematopoietic tissue origin. The inserted site of enhancer in vector influenced the transcriptional activity of promoter and the extent of reporter gene silencing exerted the influence on the analysis of transcriptional activity.

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.

The zinc finger domain of Wilms' tumor 1 suppressor gene (WT1) behaves as a dominant negative, leading to abrogation of WT1 oncogenic potential in breast cancer cells

IntroductionThere is growing evidence that the Wilms' tumor 1 suppressor gene (WT1) behaves as an oncogene in some forms of breast cancer. Previous studies have demonstrated that the N-terminal domain of WT1 can act as a dominant negative through self-association. In the studies presented here we have explored the potential for the zinc finger domain (ZF) of WT1 to also have dominant-negative effects, and thus further our understanding of this protein.MethodsUsing full-length and ZF-only forms of WT1 we assessed their effect on the WT1 and c-myc promoter using luciferase and chromatin immunoprecipitation assays. The gene expression levels were determined by quantitative real-time RT-PCR, northern blot and western blot. We also assessed the effect of the ZF-only form on the growth of breast cancer cell lines in culture.ResultsTransfection with WT1–ZF plasmids resulted in a stronger inhibition of WT1 promoter than full-length WT1 in breast cancer cells. The WT1–ZF form lacking the lysine–threonine–serine (KTS) insert (ZF - KTS) can bind to the majority of WT1 consensus sites throughout the WT1 promoter region, while the ZF containing the insert (ZF + KTS) form only binds to sites in the proximal promoter. The abundances of endogenous WT1 mRNA and protein were markedly decreased following the stable expression of ZF - KTS in breast cancer cells. The expressions of WT1 target genes, including c-myc, Bcl-2, amphiregulin and TERT, were similarly suppressed by ZF - KTS. Moreover, WT1–ZF - KTS abrogated the transcriptional activation of c-myc mediated by all four predominant isoforms of WT1 (including or lacking alternatively spliced exons 5 and 9). Finally, WT1–ZF - KTS inhibited colony formation and cell division, but induced apoptosis in MCF-7 cells.ConclusionOur observations strongly argue that the WT1–ZF plasmid behaves as a dominant-negative regulator of the endogenous WT1 in breast cancer cells. The inhibition on proliferation of breast cancer cells by WT1–ZF - KTS provides a potential candidate of gene therapy for breast cancer.

The WT1 Wilms' tumor suppressor gene is a downstream target for insulin‐like growth factor‐I (IGF‐I) action in PC12 cells

The biological actions of the insulin-like growth factors, IGF-I and IGF-II, are mediated by the ligand-induced activation of the IGF-I receptor (IGF-IR), a transmembrane heterotetramer linked to the ras-raf-mitogen-activated protein kinase (MAPK) and phosphatidyl inositol 3 kinase (PI3K)-protein kinase B (PKB)/Akt signal transduction cascades. The Wilms' tumor suppressor gene (wt1) encodes a zinc finger transcription factor, WT1, which has been implicated in various cellular processes including proliferation, differentiation and apoptosis. In the present study we demonstrated that IGF-I modulates the WT1 gene expression in neurally derived PC12 cells in a dose- and time-dependent manner. This effect was mediated through both the MAPK and PI3-kinase signaling pathways, as shown by the ability of the specific inhibitors UO126 and LY294002 to abrogate IGF-I action. Moreover, using RT-PCR and transient transfection assays, we demonstrated that the IGF-I effect was associated with corresponding changes in WT1 mRNA levels and WT1 promoter activity. In addition, the results of the present study revealed that high WT1 levels were associated with the induction of apoptosis, whereas low WT1 levels were correlated with the inhibition of apoptosis, as demonstrated by poly ADP ribose polymerase (PARP) cleavage, Bax expression, Annexin V-FITC staining, and by the use of antisense oligonucleotides against WT1. In summary, our results show that the wt1 gene is a novel target for IGF-I action in neurally derived cells.