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• W2021817265 abstract "Article15 July 1999free access WT1 modulates apoptosis by transcriptionally upregulating the bcl-2 proto-oncogene Marty W. Mayo Marty W. Mayo Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author Cun-Yu Wang Cun-Yu Wang Laboratory of Molecular Signaling and Apoptosis, School of Dentistry, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author S.Scott Drouin S.Scott Drouin Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author Lee V. Madrid Lee V. Madrid Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author Allen F. Marshall Allen F. Marshall Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author John C. Reed John C. Reed the Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Bernard E. Weissman Bernard E. Weissman Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Department of Pathology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author Albert S. Baldwin Albert S. Baldwin Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Department of Biology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author Marty W. Mayo Marty W. Mayo Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author Cun-Yu Wang Cun-Yu Wang Laboratory of Molecular Signaling and Apoptosis, School of Dentistry, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author S.Scott Drouin S.Scott Drouin Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author Lee V. Madrid Lee V. Madrid Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author Allen F. Marshall Allen F. Marshall Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author John C. Reed John C. Reed the Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA Search for more papers by this author Bernard E. Weissman Bernard E. Weissman Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Department of Pathology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author Albert S. Baldwin Albert S. Baldwin Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Department of Biology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA Search for more papers by this author Author Information Marty W. Mayo1, Cun-Yu Wang2, S.Scott Drouin1, Lee V. Madrid1,3, Allen F. Marshall1, John C. Reed4, Bernard E. Weissman1,3,5 and Albert S. Baldwin1,3,6 1Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, NC, 27599-7295 USA 2Laboratory of Molecular Signaling and Apoptosis, School of Dentistry, University of North Carolina, Chapel Hill, NC, 27599-7295 USA 3Curriculum in Genetics and Molecular Biology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA 4the Burnham Institute, 10901 North Torrey Pines Road, La Jolla, CA, 92037 USA 5Department of Pathology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA 6Department of Biology, University of North Carolina, Chapel Hill, NC, 27599-7295 USA The EMBO Journal (1999)18:3990-4003https://doi.org/10.1093/emboj/18.14.3990 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info The Wilms‘ tumor suppressor gene, WT1, encodes a zinc finger transcription factor that has been demonstrated to negatively regulate several growth factor and cognate receptor genes. However, inconsistent with its tumor suppressor function, WT1 has also been demonstrated to be required to inhibit programmed cell death in vitro and in vivo. Moreover, anaplastic Wilms’ tumors, which typically express wild-type WT1, display extreme resistance to chemotherapeutic agents that kill tumor cells through the induction of apoptosis. Although p53 mutations in anaplastic Wilms‘ tumors have been associated with chemoresistance, this event is believed to occur late during tumor progression. Therefore, since dysregulated WT1 expression occurs relatively early in Wilms’ tumors, we hypothesized that WT1 was required to transcriptionally upregulate genes that provide a cell survival advantage to tumor cells. Here we demonstrate that sporadic Wilms' tumors coexpress WT1 and the anti-apoptotic Bcl-2 protein. Using rhabdoid cell lines overexpressing WT1, we show that WT1 activates the endogenous bcl-2 gene through a transcriptional mechanism. Transient transfections and electromobility shift assays demonstrate that WT1 positively stimulates the bcl-2 promoter through a direct interaction. Moreover, WT1 expressing cells displaying upregulated Bcl-2 were found to be resistant to apoptosis induced by staurosporine, vincristine and doxorubicine. These data suggest that in certain cellular contexts, WT1 exhibits oncogenic potential through the transcriptional upregulation of anti-apoptotic genes such as bcl-2. Introduction The Wilms‘ tumor-associated gene WT1 encodes a tumor suppressor gene product that is expressed in the developing kidney and in the adult urogenital system (reviewed in Haber and Housman, 1992; Rauscher, 1993; Reddy and Licht, 1996). Consistent with the role of WT1 as a tumor suppressor, expression of WT1 protein has been shown to suppress cell growth in both Wilms’ tumors and non-Wilms‘ tumors (Haber et al., 1993; Luo et al., 1995; McMaster et al., 1995). Although the WT1 gene is deleted or mutated in nearly all of patients diagnosed with Denys-Drash syndrome (DDS; a syndrome which includes nephropathy, intersex disorders and a predisposition to develop Wilms’ tumors), <10% of all sporadic Wilms‘ tumors contain abnormalities in the WT1 gene (reviewed in Coppes et al., 1993a; Haber and Housman, 1992). Therefore, despite the ability of WT1 to act as a tumor suppressor, 90% of sporadic Wilms’ tumors continue to express this protein either because cells have acquired secondary mutations in other genes which affect WT1 tumor suppressor function, and/or because WT1 provides an important survival function in these tumors. The WT1 protein is a transcription factor which is composed of two functional domains: a proline-glutamine rich domain at the N-terminus, and a zinc finger domain composed of four Cys2-His2 zinc fingers at the C-terminus (Haber and Housman, 1992; Rauscher, 1993; Reddy and Licht, 1996). The WT1 gene yields four alternatively spliced mRNAs: WT1-A, which contains neither alternative splice; WT1-B, which includes an N-terminal splice which codes for an additional 17 amino acids; WT1-C, which includes a C-terminal splice which results in a three amino acid insertion (KTS); and WT1-D, which contains both alternate exons (Haber and Housman, 1992; Rauscher, 1993; Reddy and Licht, 1996). These different isoforms are expressed at a ratio of 1:2.5:3.8:8.3 in the fetal kidney (Haber et al., 1990). WT1-A and WT1-B proteins were originally demonstrated to bind to the same DNA consensus sequence, 5′-GCGGGGGCG-3′, as the structurally related early growth response 1 (Egr-1) protein (Rauscher et al., 1990). However, additional DNA sequences have been identified which display a higher level of affinity for the WT1 protein (Wang et al., 1993b; Hamilton et al., 1995; Nakagama et al., 1995). Because the alternative splice II donor site results in the insertion of three amino acids (KTS) between zinc fingers 3 and 4 of WT1-C and WT1-D proteins, these two isoforms recognize related but distinct DNA sequences (Drummond et al., 1994; Wang et al., 1995). Therefore, not only do differences in DNA-binding sequences suggest that WT1-A and WT1-B may regulate a different subset of genes than KTS-containing WT1 proteins, but WT1 isoforms may also exhibit different biological functions (Englert et al., 1995; Larsson et al., 1995). Consistent with the role of WT1 as a tumor suppressor protein, this transcription factor has been demonstrated in transient co-transfections to repress several cellular promoters, most of which include growth factor and cognate receptor genes (Rauscher, 1993; Reddy and Licht et al., 1996). However, only a few genes, including the epidermal growth factor receptor (EGFR), the insulin-like growth factor receptor (IGFR), and the platelet-derived growth factor (PDGF), have been shown to be repressed endogenously (Gashler et al., 1992; Englert et al., 1995; Werner et al., 1995). Besides being a potent repressor of transcription, WT1 is a transcription factor with strong transactivating potential (Reddy et al., 1995; Wang et al., 1995). Recently, a number of genes have been demonstrated to be positively regulated by WT1-A and WT1-B isoforms, but not by KTS-containing WT1 proteins (Wang et al., 1993a; Nichols et al., 1995; Cook et al., 1996; Kim et al., 1998). The importance of WT1 in normal urogenital development establishes an anti-apoptotic role for WT1. Homozygous deletion of the WT1 gene in the mouse germline results in embryonic lethality at day 11 of gestation due to a failure of kidney and heart development (Kreidberg et al., 1993). In WT1−/− knockout animals, cells of the metanephric blastema, which are required for normal kidney development, fail to proliferate and undergo programmed cell death (Kreidberg et al., 1993). These studies suggest that during kidney development, WT1 either functions to repress pro-apoptotic genes or gene products, or that WT1 acts as potent transcription factor to activate WT1-responsive genes required to overcome programmed cell death. Consistent with this first scenario, WT1 has been reported to modulate programmed cell death by interacting directly with p53 and inhibiting p53-mediated apoptosis (Maheswaran et al., 1995). Since apoptosis is believed to act as a defense against malignant transformation, it is possible that the anti-apoptotic nature of WT1 may potentiate oncogenesis. In support of this idea, WT1 expression has been reported in a number of cancers, including mesotheliomas, erythro- and myeloid-leukemias (Amin et al., 1995; Pritchard-Jones and King-Underwood, 1997). In erythro- and myeloid-leukemia cells, WT1 expression is associated with proliferation and maintenance of an immature cell phenotype (Phelan et al., 1994; Sekiya et al., 1994). Importantly, it has been demonstrated that the loss of WT1 expression sensitizes myeloid cells to undergo programmed cell death, suggesting that WT1 contributes to oncogenesis by inhibiting apoptosis (Algar et al., 1996). In an attempt to identify WT1-regulated genes that provide protection from programmed cell death, we searched for anti-apoptotic genes that were upregulated in response to WT1 expression. Here, we demonstrate that Bcl-2 expression coincides with WT1 protein levels in sporadic Wilms‘ tumors. Additionally, we show that WT1 expression upregulates endogenous bcl-2 mRNA levels by transcriptionally activating the bcl-2 promoter through a high-affinity WT1-binding site. Importantly, we found that cells expressing WT1-B and Bcl-2 are resistant to staurosporine-, vincristine- and doxorubicine-induced apoptosis. Bcl-2 and related proteins have been shown to inhibit apoptosis by regulating the mitochondrial permeability transition, and the subsequent release of apoptosis-inducing factor (AIF) and cytochrome c (reviewed in Reed, 1997; Kroemer, 1997; Green and Reed, 1998; Thornberry and Lazebnik, 1998). This cellular response is then responsible for the activation of caspase-9 and the induction of apoptosis (Green and Reed, 1998; Thornberry and Lazebnik, 1998). Our findings have significant implications not only for understanding Wilms’ tumor progression, but also for normal kidney development, by identifying bcl-2 gene as one of the important proto-oncogenes positively regulated by WT1. Results Primary Wilms' tumors express both WT1 and Bcl-2 More than 90% of all sporadic Wilms‘ tumors express wild-type WT1 (Little et al., 1992; Coppes et al., 1993b), suggesting that WT1 does not function as a tumor suppressor gene product in human nephroblastomas. As late-stage Wilms’ tumors are resistant to apoptotic-inducing chemotherapeutic agents (Beckwith, 1996; Faria et al., 1996), and since cells of the metanephric blastema from the WT1−/− knockout animals are susceptible to programmed cell death (Kreidberg et al., 1993), we were interested in determining whether WT1 expression was associated with the expression of known anti-apoptotic proteins. Although the bcl-2 promoter has been shown to be negatively regulated by the overexpression of WT1 in transient transfection assays (Hewitt et al., 1995; Heckman et al., 1997), we were interested in whether primary sporadic Wilms‘ tumors displayed coordinate expression of WT1 and the anti-apoptotic Bcl-2 protein. Like fetal kidney, Wilms’ tumors typically express all four WT1 isoforms (Haber et al., 1990; see Figure 1A). To address whether Wilms‘ tumors coexpress both WT1 and Bcl-2, total proteins were isolated from nine randomly selected primary sporadic Wilms’ tumors and Western blot analysis was performed. As shown in Figure 1B, eight out of nine Wilms‘ tumors analyzed displayed the full-length WT1 protein (52–54 kDa), while one tumor expressed a smaller (36 kDa) WT1 immunoreactive band. Interestingly, Wilms’ tumors which strongly expressed WT1 immunoreactive bands (52–54 kDa) also displayed high levels of Bcl-2 protein (Figure 1B), while the tumor which displayed the lower WT1 immunoreactive band (36 kDa) failed to express detectable Bcl-2 expression (Figure 1B, lane 3). Only one of the Wilms‘ tumors analyzed, which expressed high levels of WT1 protein (52–54 kDa), failed to express an abundance of Bcl-2 protein (Figure 1B, lane 9). The differences in Bcl-2 protein levels observed in Wilms’ tumor samples were not due to uneven protein loading since re-analysis of the blots demonstrated equal levels of actin protein (Figure 1B). Additionally, Wilms‘ tumor samples, which were known to contain WT1 mutations, failed to display increases in Bcl-2 expression, supporting the idea that WT1 positively regulates Bcl-2 expression (data not shown). These results indicate that many sporadic Wilms’ tumors express both WT1 and Bcl-2 protein and suggest that WT1 does not negatively regulate endogenous bcl-2 expression in nephroblastomas, but rather may positively regulate expression of this gene. Figure 1.Coordinate expression of WT1 and Bcl-2 in sporadic Wilms‘ tumors. (A) A diagrammatic representation of the four alternatively spliced WT1 isoforms. The WT1 transcript contains two alternative splice sites, which results in four distinct transcripts. Alternative splice I, encoded by exon 5, results in a 17 amino acid insertion. Alternative splice II uses an alternative splice donor site located between exons 9 and 10, which results in the insertion of three amino acids [lysine, threonine and serine (KTS)]. Because the KTS insert occurs between zinc fingers 3 and 4, KTS-containing WT1 proteins display altered DNA-binding specificity. (B) Bcl-2 immunoblot analysis was performed using total proteins isolated from nine randomly selected sporadic Wilms’ tumor specimens. Proteins were quantitated using the Bio-Rad assay and 50 μg per lane were resolved on a 10% polyacrylamide gel. Proteins were transferred and immobilized on a nitrocellulose membrane, and protein expression was analyzed using anti-WT1 (C-19), anti-Bcl-2 (100) or anti-Actin (I-19) primary antibodies, all obtained from Santa Cruz Biotech. Horse-radish peroxidase-conjugated anti-mouse, anti-rabbit and anti-goat secondary antibodies (Promega) were detected using ECL (Amersham). Protein molecular weights were determined using pre-stained rainbow markers (Amersham). Download figure Download PowerPoint Cells stably expressing WT1-B display upregulated bcl-2 transcripts and protein To determine more directly whether cells expressing WT1 displayed a coordinate increase in Bcl-2 expression, we analyzed derivatives of the rhabdoid tumor cell line G401 (Weissman et al., 1987) which stably express either the WT1-B or WT1-C isoforms (McMaster et al., 1995). G401 cells fail to express endogenous WT1, and therefore have been used to measure WT1-mediated gene regulation (McMaster et al., 1995; Werner et al., 1995). Since the alternative splice site II inserts or removes the three amino acid sequence (KTS) between zinc fingers III and IV and changes the DNA-binding specificity of WT1 (Drummond et al., 1994; Wang et al., 1995), it was important to compare G401 cells which express either WT1-B, a protein isoform which lacks the KTS, or WT1-C, a protein which contains the KTS amino acid insert (Figure 1A). Total proteins were isolated from Lan-5, a neuroblastoma cell line which expresses high levels of endogenous Bcl-2 protein (Hanada et al., 1993), and from parental G401, G401-Neo control, or from three representative G401 subclones stably expressing either WT1-B or WT1-C proteins. As shown in Figure 2A, G401 clones expressing WT1-B or WT1-C displayed immunoreactive bands (52 and 54 kDa) which were not observed in either the parental or G401-Neo control cells. Interestingly, cells stably expressing the WT1-B protein displayed a higher level of Bcl-2 protein expression than G401-Neo cells (Figure 2A, compare lane 2 with lanes 6–8). Moreover, the two cell lines which displayed the highest levels of WT1-B expression (namely, clones WT1-B.1 and WT1-B.2) also showed the highest levels of Bcl-2 expression (Figure 2A). In contrast, all three cell lines expressing the WT1-C isoform failed to displayed significant differences in Bcl-2 protein expression (Figure 2A, compare lane 2 with lanes 3–5). The increased levels of Bcl-2 protein observed in the WT1-B expressing cells were specific to this proto-oncoprotein, since other anti-apoptotic proteins, namely Bcl-xL, Mcl-1 or A1, failed to show differences in expression (data not shown). Although WT1 has been demonstrated to stabilize p53 and prolong the half-life of this tumor suppressor protein (Maheswaran et al., 1993, 1995), immunoblot analysis failed to display differences in p53 protein levels between G401-Neo and WT1-expressing clones (data not shown). These results indicate that G401 cells expressing WT1-B demonstrate an elevation in Bcl-2 protein expression. In contrast, cells expressing WT1-C, an isoform which contains the alternative splice II (KTS) site and which is known to demonstrate limited DNA-binding activity, failed to positively upregulate endogenous Bcl-2 protein expression (Figure 2A). Figure 2.Rhabdoid cell lines stably expressing WT1-B display elevated endogenous Bcl-2. (A) The Bcl-2 anti-apoptotic protein is upregulated in clones expressing WT1-B, but not in clones expressing the WT1-C isoform. Immunoblots of cellular lysates from parental G401, G401-Neo, and from WT1-B and WT1-C clones were analyzed for the expression of Bcl-2 and WT1 proteins. Total proteins (50 μg per lane) were resolved on a 10% polyacrylamide gel, transferred to a nitrocellulose membrane and analyzed with anti-Bcl-2-, WT1- and actin-specific antibodies (Santa Cruz). (B) Elevated Bcl-2 protein levels, observed in WT1-B expressing cells, is associated with increased bcl-2 transcription. Total RNAs were isolated from Lan-5, G401-Neo control and from WT1-B and WT1-C stable clones, using the TriZol RNA solution (Life Science Inc.). RNAs (10 μg per lane) were resolved on a 0.8% formaldehyde agarose gel, and transferred to a Zeta blot membrane (Bio-Rad, Hercules, CA). Bcl-2 and β actin- specific transcripts were detected by analyzing Northern blots with a 32P-labeled specific random-labeled cDNA probe and blots were subjected to autoradiography. Download figure Download PowerPoint Since our data indicated a differential regulation between the WT1-B and WT1-C isoforms in G401 cells, it was important to determine whether the increased levels of Bcl-2 protein expression (Figure 2A) were due to an elevation in endogenous bcl-2 gene expression. To address this question, total RNAs were isolated from Lan-5, and from G401-Neo cells and G401 cells expressing either WT1-B or WT1-C isoforms. In agreement with our immunoblotting analysis, G401 cells expressing WT1-B displayed an elevation in bcl-2 encoding transcripts, while G401-Neo and clones expressing WT1-C displayed lower levels of bcl-2 transcripts (Figure 2B). Equal amounts of total RNAs were loaded on to the RNA gels, since no significant differences in the levels of transcripts encoding β-actin were detected between the G401-Neo and the WT1-expressing subclones (Figure 2B). Although the WT1-B.3 subclone expressed higher levels of bcl-2 transcripts, for unknown reasons this increase in message did not correlate with Bcl-2 protein expression (Figure 2A and B). Importantly however, these data demonstrate that G401 cells which express WT1-B contain elevated levels of bcl-2 mRNA, while cells expressing WT1-C fail to show significant increases in bcl-2 expression. WT1 positively regulates bcl-2 through a transcriptional mechanism To determine whether the increase in bcl-2 mRNA levels observed in stably expressing WT1-B cells was due to the ability of WT1 to upregulate the bcl-2 promoter region, transient transfection assays were performed. G401 cells expressing either WT1-B (WT1-B.2), WT-1C (WT1-C.1) or vector control (G401-Neo) were transiently co-transfected with a luciferase reporter plasmid containing the bcl-2 promoter (see Figure 4). As shown in Figure 3A, WT1-B.2 cells displayed a significantly higher level of bcl-2 promoter activity than did either G401-Neo or WT1-C.1 cells. To determine whether the increase in bcl-2 promoter activity observed in WT1-B.2 cells was due to WT1-B-mediated transcription, cells were co-transfected with the DDS allele of WT1-B(394 R→W). This mutation disrupts the third zinc finger of WT1 (reviewed in Reddy and Licht, 1996) and creates an isoform which does not bind DNA and actively interferes with WT1 function (Pelletier et al., 1991a). This mutant acts as a dominant-negative inhibitor by heterodimerizing with WT1 and repressing transcriptional activity mediated by wild-type WT1 proteins (Haber et al., 1990; Reddy et al., 1995). As shown in Figure 3A, WT1-B was responsible for elevated bcl-2 promoter activity, since the expression of WT1-B(394 R→W) effectively reduced luciferase activity observed in WT1-B.2 cells. Moreover, expression of the WT1-B(394 R→W) plasmid did not significantly diminish the luciferase activity in either G401-Neo or WT1-C.1 cells, indicating that the dominant-negative protein was not responsible for non-specifically inhibiting basal bcl-2 promoter activity in transfected cells (Figure 3A). The difference in bcl-2 promoter activity observed in WT1-B.1 cells was not due to differences in transfection efficiencies, since similar levels of β-galactosidase expression were observed following transient co-transfection experiments in G401-Neo, WT1-B.2 and WT1-C.1 cells (Figure 3B). Additionally, WT1-B.2 cells transiently expressing the dominant-negative WT1-B(394 R→W) protein also did not show a decrease in β-galactosidase activity, suggesting that the decrease in bcl-2 promoter activity was due to a loss of WT1-B-mediated transcriptional activity and was not due to cytotoxic effects of WT1-B(394 R→W) expression (Figure 3B). These results indicate that WT1-B expression in G401 cells results in increased transcription of the bcl-2 promoter. Figure 3.WT1 positively regulates the bcl-2 regulatory region through a domain located upstream of the P1 promoter. Deletions within the bcl-2 regulatory region were generated and cloned in front of the DNA encoding the luciferase reporter gene (LUC), as described in the Materials and methods. Saos-2 cells were co-transfected with various bcl-2 LUC constructs (2 μg each) along with either the vector control or with an expression vector encoding the WT1-A isoform (1 μg each). Cell extracts were harvested 48 h following transfection and luciferase activity was analyzed. Fold induction was determined by establishing the increase in bcl-2 promoter activity following co-transfection with the WT1-A construct above activity observed following transfection with the empty vector control. Basal promoter expression for each reporter construct was determined by arbitrarily establishing cells co-transfected with bcl-2 XH-LUC and the vector control as 100%. Data presented here represent the average of at least four independent experiments and the standard deviations are shown. Reporter constructs are named based on their restriction fragments. Sites are as indicated:X, XhoI; H, HindIII; S, SmaI; Sa, SacI and St, StuI. Download figure Download PowerPoint Figure 4.WT1 positively regulates bcl-2 through a transcriptional mechanism. (A and B) WT1-B expression is responsible for upregulating the bcl-2 promoter in WT1-B.2 cells. G401-Neo, WT1-B.2 and WT1-C.1 cells were transiently co-transfected with both the bcl-2 XH-LUC reporter and CMV-LacZ (2 μg per 100 mm dish) using lipofectamine reagent (Life Sciences Inc.). In addition, some groups were transfected with plasmids encoding either the dominant-negative mutant WT1-B(394 R→W) or the empty pCMV vector control (1 μg each). Cell extracts were harvested 48 h following the start of transfection. Proteins were quantitated using Bio-Rad reagent and analyzed for both luciferase and β-galactosidase activity, as described in the Material and methods. Data presented in (A) represents the mean ± SD of three independent experiments, while (B) shows data from a single representative experiment. (C) WT1 expression upregulates the bcl-2 promoter in a dose-dependent and cell type-specific manner. CV-1, HeLa and Saos-2 cells were co-transfected with the bcl-2 XH-LUC reporter (2 μg) and either the WT1-A or the empty vector control (pCMV) at varying concentrations (0, 0.05, 0.25, 0.5, 1 and 2 μg). All cell groups were transfected with DNA (4 μg total) and concentrations were normalized using varying amounts of Bluescript SK+/− plasmid (Stratagene). Extracts were harvested 48 h following transfection and analyzed for luciferase activity. Data presented represents the mean ±SD of three independent experiments performed in duplicate. (D) Only the KTS minus WT1 isoforms are capable of positively upregulating the bcl-2 promoter. Saos-2 cells were transfected with the bcl-2 XH-LUC reporter (2 μg) and with plasmids encoding various WT1 isoforms, the mutant WT1-B(394 R→W) or the vector control (1 μg each). Cell extracts were collected 48 h following transfection and luciferase assays were performed. Data represent the average ±SD of three separate experiments. To confirm that expression vectors were encoding the various WT1 transgenes, Western blot analysis was performed on cell extracts. Protein extracts (50 μg/lane), from transiently transfected into Saos-2 cells, were resolved on a 10% polyacrylamide gel, transferred to nitrocellulose and analyzed for WT1 expression using a anti-WT1 antibody (C-19, Santa Cruz). WT1-specific immunoreactive bands, as well as non-specific bands (NS) are indicated with an arrow. Download figure Download PowerPoint Since the bcl-2 promoter has been previously reported to be repressed by WT1 (Hewitt et al., 1995; Heckman et al., 1997), it was important to elucidate whether the ability of WT1 to positively upregulate the bcl-2 promoter region was a cell type-specific phenomenon. Transient transfection experiments were performed in three different cell lines: CV-1, Saos-2 and HeLa. CV-1 is an immortalized green monkey kidney cell line that does not express endogenous WT1. Expression of WT1-A in these cells has been shown to positively upregulate a WT1-responsive reporter in transient transfection assays (Reddy et al., 1995). Saos-2 is an osteosarcoma cell line which fails to express WT1 protein and has been used to demonstrate WT1-mediated transcriptional regulation (Maheswaran et al., 1993, 1995, 1998; Englert et al., 1995). HeLa is a human cervical epithelial line which has been used to characterize the WT1-induced repression of the bcl-2 promoter (Hewitt et al., 1995). As shown in Figure 3C, WT1-A positively upregulated the bcl-2 promoter in a dose-dependent manner in both Saos-2 and CV-1 cells" @default.
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• W2021817265 date "1999-07-15" @default.
• W2021817265 modified "2023-10-18" @default.
• W2021817265 title "WT1 modulates apoptosis by transcriptionally upregulating the bcl-2 proto-oncogene" @default.
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• W2021817265 doi "https://doi.org/10.1093/emboj/18.14.3990" @default.
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