Characterization of RNA aptamer binding by the Wilms' tumor suppressor protein WT1

The interaction of the zinc finger protein WT1 with RNA aptamers has been investigated using a quantitative binding assay, and the results have been compared to those from a previous study of the DNA binding properties of this protein. A recombinant peptide containing the four zinc fingers of WT1 (WT1-ZFP) binds to representatives of three specific families of RNA aptamers with apparent dissociation constants ranging from 13.8 +/- 1.1 to 87.4 +/- 10.4 nM, somewhat higher than the dissociation constant of 4.12 +/- 0.4 nM for binding to DNA. An isoform that contains an insertion of three amino acids between the third and fourth zinc fingers (WT1[+KTS]-ZFP) also binds to these RNAs with slightly reduced affinity (the apparent dissociation constants ranging from 22.8 to 69.8 nM) but does not bind to DNA. The equilibrium binding of WT1-ZFP to the highest-affinity RNA molecule was compared to the equilibrium binding to a consensus DNA molecule as a function of temperature, pH, monovalent salt concentration, and divalent salt concentration. The interaction of WT1-ZFP with both nucleic acids is an entropy-driven process. Binding of WT1-ZFP to RNA has a pH optimum that is narrower than that observed for binding to DNA. Binding of WT1-ZFP to DNA is optimal at 5 mM MgCl(2), while the highest affinity for RNA was observed in the absence of MgCl(2). Binding of WT1 to both nucleic acid ligands is sensitive to increasing monovalent salt concentration, with a greater effect observed for DNA than for RNA. Point mutations in the zinc fingers associated with Denys-Drash syndrome have dramatically different effects on the interaction of WT1-ZFP with DNA, but a consistent and modest effect on the interaction with RNA. The role of RNA sequence and secondary structure in the binding of WT1-ZFP was probed by site-directed mutagenesis. Results indicate that a hairpin loop is a critical structural feature required for protein binding, and that some consensus nucleotides can be substituted provided proper base pairing of the stem of the hairpin loop is maintained.     

Cancer immunotherapy targeting Wilms' tumor gene WT1 product

The Wilms' tumor gene WT1 is expressed at high levels not only in acute myelocytic and lymphocytic leukemia and in chronic myelocytic leukemia but also in various types of solid tumors including lung cancers. To determine whether the WT1 protein can serve as a target Ag for tumor-specific immunity, three 9-mer WT1 peptides (Db126, Db221, and Db235), which contain H-2Db-binding anchor motifs and have a comparatively higher binding affinity for H-2Db molecules, were tested in mice (C57BL/6, H-2Db) for in vivo induction of CTLs directed against these WT1 peptides. Only one peptide, Db126, with the highest binding affinity for H-2Db molecules induced vigorous CTL responses. The CTLs specifically lysed not only Db126-pulsed target cells dependently upon Db126 concentrations but also WT1-expressing tumor cells in an H-2Db-restricted manner. The sensitizing activity to the Db126-specific CTLs was recovered from the cell extract of WT1-expressing tumor cells targeted by the CTLs in the same retention time as that needed for the synthetic Db126 peptide in RP-HPLC, indicating that the Db126-specific CTLs recognize the Db126 peptide to kill WT1-expressing target cells. Furthermore, mice immunized with the Db126 peptide rejected challenges by WT1-expressing tumor cells and survived for a long time with no signs of autoaggression by the CTLs. Thus, the WT1 protein was identified as a novel tumor Ag. Immunotherapy targeting the WT1 protein should find clinical application for various types of human cancers.     

Nephropathy and defective spermatogenesis in mice transgenic for a single isoform of the Wilms' tumour suppressor protein, WT1-KTS, together with one disrupted Wt1 allele

The Wilms' tumour suppressor protein, WT1, is a zinc finger protein essential for the development of several organs, including the kidney and gonads. In each of these tissues WT1 is required at multiple stages of development and its persistent expression in podocytes and Sertoli cells suggests WT1 may also have a role in the maintenance of kidney and testis function throughout adult life. Naturally occurring isoforms of WT1 are generated by alternative mRNA splicing. An altered ratio of the splice isoforms WT1-KTS and WT1 + KTS appears to be sufficient to account for the developmental abnormalities (pseudohermaphroditism and nephropathy) characteristic of Frasier syndrome. We show that mice with a transgene encoding WT1-KTS do not differ from their wild-type littermates unless they are also heterozygous for a null mutation at the endogenous Wt1 locus. Animals with both genetic modifications develop proteinuria, together with multiple glomerular cysts, and male infertility. These pathologic changes may be explained as a consequence of altering the WT1 isoform ratio in tissues that express WT1 during adulthood. The results suggest WT1 misexpression could contribute to human glomerulocystic kidney disease.     

Physical and functional interaction between the HCMV IE2 protein and the Wilms' tumor suppressor WT1

Human cytomegalovirus (HCMV) is a major renal pathogen in congenitally infected infants and renal allograft recipients. It has been shown that human kidney cells of glomerular, tubular, and vascular origin were all infected by HCMV in vitro. It has previously been demonstrated that the IE2 protein of HCMV directly associates with the zinc finger domain of Egr-1. The zinc finger region of WT1 is a sequence-specific DNA-binding domain which also recognizes the consensus DNA binding site (5'-CGCCCCCGC-3') of Egr-1, thus suggesting a possible interaction between WT1 and IE2. Here we demonstrate that HCMV IE2 binds to the C-terminal region of WT1 containing zinc finger domain in vivo as well as in vitro and that WT1 can inhibit IE2-driven transactivation of the responsive promoter. Our results suggest that WT1 may be able to regulate the functional activity of HCMV IE2. Furthermore, these data may provide new insights into the possible involvement of HCMV in WT1-related pathogeneses.     

WT1 (Wilms' tumor 1) peptide immunotherapy for renal cell carcinoma

Tumor-specific immunotherapy with a Wilms' tumor 1 (WT1) peptide has been on clinical trial for leukemia, myelodysplastic syndrome, breast and lung cancers and is producing promising results. In this study, we treated three patients with renal cell carcinoma with an anchor modified, HLA-A*2402 binding WT1 peptide which was emulsified in Freund's incomplete adjuvant. In two patients tumor growth was suppressed and clinical response was evaluated as stable disease by the RECIST criteria after 3 months of weekly immunizations. Notably, development of new metastases has stopped in these patients for a prolonged period. No deleterious side effects were observed. Peptide-specific T cells were expanded in PBMCs of the patients and a substantial fraction of them bore the surface phenotype consistent with a CD8+ cytotoxic effector population. Although established tumors did not regress further, considering the component of the vaccine, i.e. peptide alone, the stabilization effect suggested the potential of WT1 peptide to develop into a more effective vaccine. To our knowledge, this is the first report of WT1 immunotherapy for renal cell carcinoma. Hopefully, the results will stimulate more extensive clinical studies.     

Regulation of ornithine decarboxylase gene expression by the Wilms' tumor suppressor WT1.

The importance of ornithine decarboxylase (ODC) to cell proliferation is underscored by the complex array of cell-specific mechanisms invoked to regulate its synthesis and activity. Misregulation of ODC has severe negative consequences on normal cell function, including the acquisition of tumorigenic growth properties by cells overexpressing ODC. We hypothesize that ODC gene expression is a candidate target for the anti-proliferative function of certain tumor suppressors. Here we show that the Wilms' tumor suppressor WT1 binds to multiple sites within the human ODC promoter, as determined by DNase I protection and methylation interference assays. The expression of WT1 in transfected HCT 116, NIH/3T3 and HepG2 cells represses activity of the ODC promoter controlling expression of a luciferase reporter gene. In contrast WT1 expression enhances ODC promoter activity in SV40-transfected HepG2 cells. Both the extent of modulation of ODC gene expression and the mediating WT1 binding elements are cell specific. Constructs expressing WT1 deletion mutants implicate two regions required for repressor function, as well as an intrinsic activation domain. Understanding the regulation of ODC gene expression by WT1 may provide valuable insights into the roles of both WT1 and ODC in development and tumorigenesis.     

Localization of the Antennapedia protein in Drosophila embryos and imaginal discs

[This corrects the article on p. 3327 in vol. 5.].     

Localization of the Antennapedia protein in Drosophila embryos and imaginal discs

Antibodies have been raised against a fusion protein containing the 3' region of the coding sequence of the Antennapedia (Antp) gene fused to β-galactosidase. The distribution of the protein on whole mount embryos and imaginal discs of third instar larvae was examined by immunofluorescence. In young embryos, expression of the Antp protein was limited to the thoracic segments in the epidermis, whereas it was found in all neuromeres of head, thorax and abdomen. At the end of embryogenesis, the Antp protein mainly accumulated in the ventral nervous system in certain parts of the thoracic neuromeres, from posterior T1 to anterior T3, with a gap in posterior T2. Comparison of Antp protein distribution in nervous systems from wild-type and Df P9 embryos, lacking the genes of the Bithorax-complex (BX-C), revealed a pattern of expression which indicated that the BX-C represses Antp in the posterior segments with the exception of the last abdominal neuromeres (A8-9) which are regulated independently. The protein pattern in nervous systems from Sex combs reduced(Scr^xF9) mutant embryos was indistinguishable from that found in wild-type embryos; thus, neurogenic expression of Antp in T1 and the more anterior segments does not appear to be under the control of Scr⁺. All imaginal discs derived from the three thoracic segments express Antp protein. The distribution was distinct in each disc; strongest expression was observed in the proximal parts of the discs. In the leg discs the protein distribution seemed to be compartmentally restricted, whereas in the wing disc this was not the case. Antp protein was not detected in the eye-antennal disc. In embryos, as well as in imaginal discs, the protein is localized in the nucleus.     

The Wilms' tumor gene WT1 is a common marker of progenitor cells in fetal liver

It is well known that the Wilms' tumor gene WT1 plays an important role in cell proliferation and differentiation, and in organ development. In this study, to examine the role of the WT1 gene in lineage determination, fetal liver cells from LacZ-transgenic mice, in which WT1 expression was marked by the expression of the LacZ gene driven by WT1 promoter, were FACS-sorted according to LacZ expression of high (LacZ(++)) or undetectable (LacZ(-)) levels, which paralleled endogenous WT1 expression levels. LacZ(++) fetal liver cells were enriched by hepatocyte and endothelial progenitor cells. These results indicated that WT1 expression is a common marker of both hepatocyte and endothelial progenitors. These results also implied a role of the WT1 gene in lineage determination.     

Significant reduction of WT1 gene expression,possibly due to epigenetic alteration in Wilms' tumor

WT1 at 11p13 is a tumor suppressor gene, an aberration of which causes Wilms' tumor (WT). Since WT1 expression is reduced in a certain proportion of WTs and its mutation is found only in 10-20% of WTs, we examined WT1 gene silencing due to epigenetic alteration in a total of 22 WTs. WT1 expression was significantly reduced in half of WTs without any mutation in the WT1 gene itself, suggesting that the reduction of expression was possibly epigenetic. We found promoter hypermethylation in one WT with loss of heterozygosity (LOH) and showed that promoter methylation reduced reporter gene activity by a reporter assay. These data suggested that methylation was an epigenetic mechanism leading to WT1 silencing and that the expression-reduced allele by hypermethylation combined with LOH was consistent with the revised two-hit model. In addition, as the beta-catenin mutation is frequently associated with the WT1 mutation, the association of WT1 silencing with the beta-catenin mutation was also investigated. beta-catenin mutated in only one WT without WT1 silencing, suggesting that the beta-catenin mutation was not associated with the reduction of WT1 expression.     

Netrin-1 inhibits sprouting angiogenesis in developing avian embryos

Netrin-1 is a bifunctional axonal guidance cue, capable of attracting or repelling developing axons via activation of receptors of the deleted in colorectal cancer (DCC) and uncoordinated 5 (UNC5) families, respectively. In addition to its role in axon guidance, Netrin-1 has been implicated in angiogenesis, where it may also act as a bifunctional cue. Attractive effects of Netrin-1 on endothelial cells appear to be mediated by an as yet unknown receptor, while repulsion of developing blood vessels in mouse embryos is mediated by the UNC5B receptor. To explore evolutionary conservation of vascular UNC5B expression and function, we have cloned the chick unc5b homologue. Chick and quail embryos showed unc5b expression in arterial EC and sprouting angiogenic capillaries. To test if Netrin-1 displayed pro- or anti-angiogenic activities in the avian embryo, we grafted cell lines expressing recombinant chick or human Netrin-1 at different stages of development. Netrin-1 expressing cells inhibited angiogenic sprouting of unc5b expressing blood vessels, but had no pro-angiogenic activity at any stage of development examined. Netrin-1 also had no effect on the recruitment of circulating endothelial precursor cells. Taken together, these data indicate that vascular unc5b expression and function is conserved between chick and mice.     

c-Ha-ras-1 alleles in bladder cancer,Wilms' tumour and malignant melanoma

Summary Polymorphism of the human c-Ha-ras-1 gene has been analysed in DNA from 168 individuals using the enzymes MspI and HpaII. In all, 35 bladder cancer patients, 28 melanoma patients, 22 Wilms' tumour patients, 24 first-degree relatives of Wilms' tumour or melanoma patients and 59 unaffected controls were studied. A total of 13 different fragment sizes was detected, 4 common and 9 unusual. Of the latter, 4 were observed only in cancer patients or their first-degree relatives. The frequency of unusual alleles was significantly greater in bladder cancer patients and in the combined tumour group than in controls, thus providing support for the association of unique Ha-ras alleles and cancer. Some unaffected relatives of patients carried unusual alleles, and thus there is no absolute relationship between Ha-ras genotype and disease.