Wilms Tumor Suppressor Gene Research Articles

WT1 suppresses synthesis of the epidermal growth factor receptor and induces apoptosis.

The Wilms tumor suppressor gene WT1 encodes a developmentally regulated transcription factor that is mutated in a subset of embryonal tumors. To test its functional properties, we developed osteosarcoma cell lines expressing WT1 under an inducible tetracycline-regulated promoter. Induction of WT1 resulted in programmed cell death. This effect, which was differentially mediated by the alternative splicing variants of WT1, was independent of p53. WT1-mediated apoptosis was associated with reduced synthesis of the epidermal growth factor receptor (EGFR), but not of other postulated WT1-target genes, and it was abrogated by constitutive expression of EGFR. WT1 repressed transcription from the EGFR promoter, binding to two TC-rich repeat sequences. In the developing kidney, EGFR expression in renal precursor cells declined with the onset of WT1 expression. Repression of EGFR and induction of apoptosis by mechanism that may contribute to its critical role in normal kidney development and to the immortalization of tumor cells with inactivated WT1 alleles.

G1 phase arrest induced by Wilms tumor protein WT1 is abrogated by cyclin/CDK complexes.

WT1, the Wilms tumor-suppressor gene, maps to the human chromosomal region 11p13 and encodes a transcriptional repressor, WT1, implicated in controlling normal urogenital development. Microinjection of the WT1 cDNA into quiescent cells or cells in early to mid G1 phase blocked serum-induced cell cycle progression into S phase. The activity of WT1 varied significantly depending on the presence or absence of an alternatively spliced region located upstream of the zinc finger domain. The inhibitory activity of WT1 was abrogated by the overexpression of cyclin E/CDK2 as well as cyclin D1/CDK4. Furthermore, both CDK4- and CDK2-associated kinase activities were downregulated in cells overexpressing WT1, whereas the levels of CDK4, CDK2, and cyclin D1 expression were unchanged. These findings suggest that inhibition of the activity of cyclin/CDK complexes may be involved in mediating the WT1-induced cell cycle block.

A rodent model for Wilms tumors: embryonal kidney neoplasms induced by N-nitroso-N'-methylurea.

Embryonal kidney cell tumors develop in rats given the alkylating agent N-nitroso-N'-methylurea as neonates. These tumors resemble the childhood Wilms tumors in their histopathology. Deletions and mutations in the Wilms tumor suppressor gene, WT1, are present in up to 6% of childhood nephroblastomas. To investigate the role of WT1 in rat kidney tumorigenesis, we studied the genetic alterations in WT1 and its target genes. Point mutations were found in WT1 cDNA in 7 of 18 kidney tumors. Mesenchymal tumors contained G-->A transition mutations in codons 128, 364, and 372, typical of the methylating action of N-nitroso-N'-methylurea on DNA. Each of the four nephroblastomas contained the same T-->A mutation at codon 111 of WT1, reflective of transversion mutagenesis by N-nitroso-N'-methylurea in vivo. Like Wilms tumors, mRNA levels of WT1, IGF2, Pax-2, and MK genes were higher than newborn kidney in the majority of the tumors. The histopathology of the rat kidney tumors and the genetic alterations are reminiscent of those observed in Wilms tumors, establishing this as a relevant model system for the human disease.

Autoregulation of the human WT1 gene promoter

The human Wilms tumour suppressor gene, WT1, encodes a zinc-finger protein which can function as a transcriptional activator or suppressor. This study reports the analysis of the human WT1 gene promoter, and demonstrates that high levels of WT1 expression lead to autosuppression of the WT1 promoter. Deletion analyses of the promoter region implicate sequences 5′ and 3′ of the transcriptional start site as being crucial in WT1 autosuppression. Loss or alteration of this function of WT1 may be important in tumourigenesis.

Wilms’ tumor (WT1) gene expression in rat decidual differentiation

Abstract The Wilm's tumor suppressor gene (WT1) encodes a zinc-finger containing transcription factor that is selectively expressed in the developing urogenital tract, where it is thought to play a role in the differentiation of these tissues. We have used immunocytochemistry and in situ hybridization to study WT1 expression in the rat uterus during normal development and pregnancy from 0 to 20 days post coitum (P.c.). WT1 mRNA was abundant in uterine stroma from juvenile rats, but was much less abundant in uterine tissue from sexually mature rats; WT1 expression is not affected by ovariectomy or by treatment with estradiol or estradiol plus progesterone. WT1 gene was highly expressed, however, in the endometrial cells of early pregnancy. On day 6 p.c. WT1 mRNA was detected in anti-mesometrial decidual cells, and WT1 immunoreactivity was concentrated in the nuclei of these cells. All cells of fully-developed deciduoma at 7-8 days p.c. demonstrated WT1 expression. WT1 was not detected in trophoblast/placental tissues but remained abundant in the decidua basalis until parturition. The expression of WT1 was compared with insulin-like growth factor-II (IGF-II) and its receptor in the decidual since it has been shown that IGF-II gene transcription is repressed by WT1 in vitro. However, no spatiotemporal correlation in the expression of these three genes was found in differentiation of the rat decidua. In summary, these data suggest a role for WT1 in decidualization, since its expression is activated during the differentiation of uterine stromal cells into decidual cells.

A second transcriptionally active DNA-binding site for the Wilms tumor gene product, WT1.

The putative Wilms tumor suppressor gene, wt1, encodes a zinc-finger protein that binds to the DNA sequence 5'-GCGGGGGCG-3'. We previously reported that WT1 has separable domains that function either to activate or suppress transcription. We now have identified a second WT1 binding sequence (5'-TCCTCCTCCTCCTCTCC-3') 3' to the transcription initiation site of the platelet-derived growth factor A-chain gene by DNase I footprinting and gel mobility shift assays. WT1 requires both 5' and 3' binding sites for transcriptional suppression; however, WT1 functions as a transcriptional activator when it binds to either the 5' or 3' site alone. This second WT1 binding sequence functions equally well as the previously identified 5'-GCGGGGGCG-3' sequence when analyzed in transient transfection assays. A core DNA sequence recognized by WT1 was defined by using related synthetic oligonucleotides. We also identified sequences similar to the WT1 binding site within the promoter regions of five other growth-related genes and demonstrated that each of these sequences also binds WT1 in gel mobility shift assays. These results thus identify a second WT1 binding site and suggest that additional growth-related genes may be transcriptionally influenced by WT1.

Wilms' tumor (WT1) gene expression in rat decidual differentiation

Abstract The Wilm's tumor suppressor gene (WT1) encodes a zinc-finger containing transcription factor that is selectively expressed in the developing urogenital tract, where it is thought to play a role in the differentiation of these tissues. We have used immunocytochemistry and in situ hybridization to study WT1 expression in the rat uterus during normal development and pregnancy from 0 to 20 days post coitum (p.c.). WT1 mRNA was abundant in uterine stroma from juvenile rats, but was much less abundant in uterine tissue from sexually mature rats; WT1 expression is not affected by ovariectomy or by treatment with estradiol or estradiol plus progesterone. WT1 gene was highly expressed, however, in the endometrial cells of early pregnancy. On day 6 p.c. WT1 mRNA was detected in anti-mesometrial decidual cells, and WT1 immunorcactivity was concentrated in the nuclei of these cells. All cells of fully-developed deciduoma at 7–8 days p.c. demonstrated WT1 expression. WT1 was not detected in trophoblast/placental tissues but remained abundant in the decidua basalis until parturition. The expression of WT1 was compared with insulin-like growth factor-II (IGF-II) and its receptor in the decidual since it has been shown that IGF-II gene transcription is repressed by WT1 in vitro. However, no spatiotemporal correlation in the expression of these three genes was found in differentiation of the rat decidua. In summary, these data suggest a role for WT1 in decidualization, since its expression is activated during the differentiation of uterine stromal cells into decidual cells.

The WT1 Wilms tumor gene product: a developmentally regulated transcription factor in the kidney that functions as a tumor suppressor

Alteration of transcription factor function is becoming a common theme in molecular mechanisms of oncogenesis. A recent example of this trend is the isolation and characterization of the chromosome 11p13 Wilms tumor suppressor gene, WT1. The WT1 protein contains a DNA binding domain consisting of four zinc fingers of the Cys2-His2 class and a proline-glutamine rich region capable of regulating transcription. Deletions of the WT1 gene or point mutations which destroy the DNA binding activity of the protein are associated with the development of the pediatric nephroblastoma Wilms tumor and Denys-Drash syndrome. This article reviews the role of WT1 in normal kidney development processes, the known biochemical functions of the protein and the status of identifying target genes regulated by this potentially oncogenic transcription factor.

Increased expression of the insulin-like growth factor I receptor gene, IGF1R, in Wilms tumor is correlated with modulation of IGF1R promoter activity by the WT1 Wilms tumor gene product.

Wilms tumor is a pediatric neoplasm that arises from the metanephric blastema. The expression of the gene encoding insulin-like growth factor II (IGF-II) is often elevated in these tumors. Since many of the actions of IGF-II are mediated through activation of the IGF-I receptor (IGF-IR), we have measured the levels of IGF-IR mRNA in normal kidney and in Wilms tumor samples using solution hybridization/RNase protection assays. IGF-IR mRNA levels in the tumors were 5.8-fold higher than in adjacent normal kidney tissue. Among the tumors themselves, the levels of IGF-IR mRNA in those containing heterologous stromal elements were 2-fold higher (P < 0.01) than in tumors without these elements. IGF-IR gene (designated IGF1R) expression in the tumors was inversely correlated with the expression of the Wilms tumor suppressor gene WT1, whose inactivation appears to be a key step in the etiology of Wilms tumor. Cotransfection of Chinese hamster ovary cells with rat and human IGF-IR gene promoter constructs driving luciferase reporter genes and with WT1 expression vectors showed that the active WT1 gene product represses IGF-IR promoter activity in a dose-dependent manner. These results suggest that underexpression, deletion, or mutation of WT1 may result in increased expression of the IGF-IR, whose activation by IGF-II may be an important aspect of the biology of Wilms tumor.

Distinct molecular origins for Denys-Drash and Frasier syndromes

The direct involvement of the Wilm's tumor suppressor gene (WT1) in Denys-Drash syndrome through mutations within exons 8 or 9 has recently been established. The absence of such alterations in three patients with Frasier syndrome provides a molecular basis for distinguishing these two syndromes that are associated with streak gonads, pseudohermaphroditism and renal failure.

Homozygous somatic Wt1 point mutations in sporadic unilateral Wilms tumor.

Wilms tumor may be caused by loss of function of genes at different loci. A Wilms tumor suppressor gene, WT1, at chromosome 11 band p13, has recently been cloned and characterized. WT1 has been implicated in the development of Wilms tumor by virtue of mutations in patients with genitourinary anomalies and susceptibility to Wilms tumor. Homozygous intragenic mutations have been reported in Wilms tumors, but usually not in sporadic unilateral Wilms tumors, which constitute the majority of Wilms tumor cases. Using the single-strand conformational polymorphism assay, we have identified three sporadic unilateral Wilms tumors with homozygous point mutations: one with a de novo germ-line nonsense point mutation within WT1 exon 8, and two carrying a somatic mutation within WT1 exon 10. In all three cases loss of the wild-type allele was demonstrated by tumor loss of heterozygosity. This report provides an example of two somatic mutations in the same tumor expected to inactivate WT1 function.

Human platelet-derived growth factor A chain is transcriptionally repressed by the Wilms tumor suppressor WT1.

Wilms tumor, an embryonic kidney malignancy, accounts for approximately 6% of all pediatric neoplasms. A gene implicated in the genesis of this tumor, the Wilms tumor suppressor gene (WT1), encodes a zinc-finger DNA-binding protein (WT1) that functions as a transcriptional repressor. In certain Wilms tumors, the platelet-derived growth factor A chain (PDGF-A) is overexpressed; it has therefore been suggested that it may play an autocrine role in development of these neoplasms. Since the PDGF-A promoter contains putative binding sites for WT1, we explored the role of WT1 in regulating A-chain expression. The major PDGF-A promoter activity was localized in transient transfection assays to a region spanning from -643 to + 8 relative to the transcription start site. WT1 bound to several sites in this region of the promoter, as demonstrated by gel-shift analysis and DNase I footprinting, and functioned as a powerful repressor of PDGF-A transcription in vivo. Maximal repression (> 50-fold) of the PDGF-A promoter was dependent on the presence of multiple WT1 binding sites in transient transfection assays. Our observations suggest a mechanism for normal downregulation of a growth factor gene and of an autocrine growth process of import in kidney development and other biological systems.

Molecular characterization of congenital mesoblastic nephroma and its distinction from Wilms tumor.

Congenital mesoblastic nephroma (CMN) is a rare tumor of the neonatal kidney. It was once thought to be a variant of Wilms tumor that also arises from primitive renal cells. Molecular characteristics of two CMN were studied to clarify their potential relationship to Wilms tumors. Patterns of gene expression were assayed by Northern blot hybridization analysis. Tumors were tested for loss of heterozygosity (LOH) at chromosomes 11p13 and 11p15 using Southern blot analysis. The CMN, like Wilms tumors, demonstrated high-level expression of insulin-like growth factor II. Unlike Wilms tumors, however, the CMN expressed neither the N-myc oncogene nor the putative Wilms tumor suppressor gene, WT1. Using a panel of probes spanning 11p13 and 11p15, no LOH was detected in the CMN, nor was there evidence of deletion or rearrangements of WT1. Although Wilms tumor and CMN both arise from the developing kidney, molecular characterization suggests that different factors are involved in the pathogenesis of these two tumors.

Fenfluramine—induced irreversible pulmonary hypertension

<h3>Background and objectives</h3> The Wilms tumor suppressor gene 1 (<i>WT1</i>) plays an essential role in urogenital and kidney development. Genotype/phenotype correlations of <i>WT1</i> mutations with renal function and proteinuria have been observed in world-wide cohorts with nephrotic syndrome or Wilms tumor (WT). This study analyzed mid-European patients with known constitutional heterozygous mutations in <i>WT1</i>, including patients without proteinuria or WT. <h3>Design, setting, participants &amp; measurements</h3> Retrospective analysis of genotype, phenotype, and treatment of 53 patients with <i>WT1</i> mutation from all pediatric nephrology centers in Germany, Austria, and Switzerland performed from 2010 to 2012. <h3>Results</h3> Median age was 12.4 (interquartile range [IQR], 6–19) years. Forty-four of 53 (83%) patients had an exon mutation (36 missense, eight truncating), and nine of 53 (17%) had an intronic lysine-threonine-serine (KTS) splice site mutation. Fifty of 53 patients (94%) had proteinuria, which occurred at an earlier age in patients with missense mutations (0.6 [IQR, 0.1–1.5] years) than in those with truncating (9.7 [IQR, 5.7–11.9]; <i>P</i>&lt;0.001) and splice site (4.0 [IQR, 2.6–6.6]; <i>P=</i>0.004) mutations. Thirteen of 50 (26%) were treated with steroids and remained irresponsive, while three of five partially responded to cyclosporine A. Seventy-three percent of all patients required RRT, those with missense mutations significantly earlier (at 1.1 [IQR, 0.01–9.3] years) than those with truncating mutations (16.5 [IQR, 16.5–16.8]; <i>P&lt;</i>0.001) and splice site mutations (12.3 [IQR, 7.9–18.2]; <i>P</i>=0.002). Diffuse mesangial sclerosis was restricted to patients with missense mutations, while focal segmental sclerosis occurred in all groups. WT occurred only in patients with exon mutations (<i>n</i>=19). Fifty of 53 (94%) patients were karyotyped: Thirty-one (62%) had XY and 19 (38%) had XX chromosomes, and 96% of male karyotypes had urogenital malformations. <h3>Conclusions</h3> Type and location of <i>WT1</i> mutations have predictive value for the development of proteinuria, renal insufficiency, and WT. XY karyotype was more frequent and associated with urogenital malformations in most cases.