Wilms Tumor Protein Research Articles

Cytotoxic CD8 T Cell Immune Responses to Wilms Tumor Protein (WT-1) Characterizes Immunosuppression-Responsive Myelodysplasia (MDS).

Clinical observations and experimental evidence link bone marrow failure in myelodysplastic syndrome (MDS) with a T cell-dominated immunologically-mediated pathophysiology in some patients. Indeed, among 129 patients treated with immunosuppression at NIH, trisomy 8 was associated with hematologic response to immunosuppression. We have demonstrated that trisomy 8 patients have oligoclonal CD8 T cell expansion, as determined by increased proportions of one or more T cell receptor (TCR) V beta subfamilies (Sloand et al. Blood 106:841; 2005) which had cytotoxic activity toward trisomy 8 cell progenitors. Here we examine the response of cytotoxic T cells to cyclin D1 and Wilms tumor protein (WT-1); these candidate autoantigens had earlier been observed to be over-expressed in trisomy 8 CD34 cell in our microarray analysis (Chen et al. Blood 104:4210, 2004 ). Bone marrow mononuclear cells from 19 trisomy 8 patients (defined by cytogenetics and FISH) had significantly increased levels of WT-1 mRNA and protein expression compared to normal CD34 cells by real time PCR and immunoblot, respectively (p<0.001). When patient and control CD8 cells were cultured in the presence of WT-1- and cyclin D1-loaded HLA-A2-restricted antigen-presenting cells (using three different HLA-A0201- restricted epitopes of WT-1 and four different epitopes for CD1), eight patients' CD8 cell tested positive for interferon gamma, indicating a cytotoxic response. Staining with labeled MHC class I A2-restricted tetramers used to measure WT-1-specific cells showed concordant results in all eight patients. Tetramer staining was increased in 12 of 14 trisomy 8 patients examined. Cytotoxic T cells specific for WT-1 were present within the expanded V β subfamilies previously found to suppress trisomy 8 cell proliferation in vitro. CD8 cells from another eight MDS patients without trisomy 8 but who had responded to immunosuppression, sampled prior to treatment, also demonstrated WT1-specific cytotoxic activity, but only two of 18 non-responders showed measurable levels of WT-1-specific cytotoxic T cells (entire treated cohort seen in Fig 1). We infer from these data an autoimmune pathophysiology for trisomy 8 MDS with: 1) over-expression of WT-1 by the aneuploid clone; 2) a specific cytotoxic CD8 T cell immune response to WT-1; 3) subsequent cytotoxic T cell marrow suppression by apoptosis, either by cross reactivity or a bystander effect; and 4) improvement in hematopoiesis after immunosuppression. Other MDS which is responsive to immunosuppression may also be mediated by WT1. [Display omitted]

Clinical and Immunological Activity of WT1 Peptide Vaccination in Patients with Acute Myeloid Leukemia and Myelodysplasia: Results of a Phase II Trial.

The transcription factor Wilms tumor protein (WT) 1 belongs to a new generation of tumor antigens, which are essential for tumor cell proliferation. WT1 is highly expressed in AML and in MDS upon appearance of blasts. A phase II trial of vaccination with the HLA-A2-restricted WT1.126–134 peptide was performed in patients with AML and MDS and overexpression of WT1 to determine immunogenicity and clinical activity. Patients received vaccinations with 0.2 mg WT1.126–134 peptide (day 3), 62.5 mcg dendritic cell-stimulating adjuvant GM-CSF (days 1–4) and 1 mg T helper protein keyhole limpet hemocyanin (day 3). The initial 13 patients were to receive 4 biweekly and subsequent 4-weekly vaccinations, the subsequent 13 patients were continuously vaccinated biweekly. Vaccination was continued in absence of overt disease progression. WT1 levels were assessed by quantitative RT-PCR and WT1-specific T cell responses by tetramer analyses and cytokine flow cytometry. Response assessment following IWG-MDS criteria was used, capturing stable disease and hematologic improvement. A duration of 8 weeks was required for stable disease. Enrolment was completed in June 2006 with 24 patients with AML and 2 with MDS (RAEB). Of the 24 AML patients, 16 had > 5% marrow blasts at study onset (8 without prior chemotherapy, 4 with disease persistence following chemotherapy, 4 with PR), and 8 were in CR at high risk for relapse. A median of 10 (range 4 – 23) vaccinations was administered with 8 patients currently still under treatment. No significant toxicity occurred. To date, 22 patients are evaluable for clinical response. Overall, 8/16 patients with > 5% marrow blasts at study onset displayed clinical efficacy of vaccine treatment (SD or better). One AML patient achieved CR for 12 months after brief initial progression, and 7 patients had disease stabilization (2, 2+, 3, 3, 6, 10+, 14 months). One of these patients with RAEBII had a major response of neutrophils and platelets, and one AML patient had initial progression and subsequent transient complete clearance of peripheral blasts. WT1 transcripts as molecular disease marker decreased at least 3-fold (range 3-fold - >50-fold, median >10-fold) in 12 of 20 currently evaluated patients, including all 8 patients with evidence of clinical efficacy and 4 of 5 AML patients vaccinated in CR. The generation of a WT1-specific T cell response in peripheral blood and bone marrow was detected in 12 of 16 evaluated patients including all 6 of these 16 patients with evidence for clinical efficacy. This study shows that WT1 vaccination has promising antileukemia activity. A multicenter comparative WT1 vaccination study in CR patients at high risk of relapse is currently initiated.

Improved human T-cell responses against synthetic HLA-0201 analog peptides derived from the WT1 oncoprotein

Wilms tumor protein 1 (WT1) is a transcription factor overexpressed in several types of leukemia and solid tumors. For this reason, WT1 is an attractive target for immunotherapy. Four peptide nonamers from WT1 have been identified by others to generate a WT1-specific cytotoxic response in the context of human leukocyte antigen (HLA)-A0201 and A2402. However, as WT1 is a self-antigen, breaking tolerance is a potential obstacle to vaccination. Here, we use a strategy to circumvent tolerance by designing synthetic immunogenic analog peptides that could crossreact to the native peptides (a heteroclitic response). A number of synthetic peptides derived from nonamer sequences of the WT1 protein were designed in which single amino-acid substitutions were introduced at HLA-A0201 major histocompatibility complex (MHC)-binding positions. Several of new peptides could stabilize MHC class I A0201 molecules better than native sequences. Some analogs were also able to elicit WT1-specific T-cell recognition and cytotoxic T-cell lymphocytes more effectively than native sequences. Importantly, T cells stimulated with the new analogs crossreacted with the native WT1 peptide sequence and were able to kill HLA-matched chronic myeloid leukemia cell lines. In conclusion, analog heteroclitic WT1 peptides with increased immunogenicity can be synthesized and are potential cancer vaccine candidates.

WT1 interacts with the splicing protein RBM4 and regulates its ability to modulate alternative splicing in vivo

Wilm's tumor protein 1 (WT1), a protein implicated in various cancers and developmental disorders, consists of two major isoforms: WT1(−KTS), a transcription factor, and WT1(+KTS), a post-transcriptional regulator that binds to RNA and can interact with splicing components. Here we show that WT1 interacts with the novel splicing regulator RBM4. Each protein was found to colocalize in nuclear speckles and to cosediment with supraspliceosomes in glycerol gradients. RBM4 conferred dose-dependent and cell-specific regulation of alternative splicing of pre-mRNAs transcribed from several reporter genes. We found that overexpressed WT1(+KTS) abrogated this effect of RBM4 on splice-site selection, whereas WT1(−KTS) did not. We conclude that the (+KTS) form of WT1 is able to inhibit the effect of RBM4 on alternative splicing.

Clinical and immune responses of WT1-peptide vaccination in patients with acute myeloid leukemia

2511 Background: The transcription factor Wilms tumor protein (WT) 1 belongs to a new generation of tumor antigens, as it is essential for tumor cell proliferation. WT1 is highly expressed both in myeloid leukemias and many carcinomas. This phase 2 proof-of-concept trial was initiated to determine immunogenicity and toxicity of vaccination with a novel HLA-A2-restricted WT1 peptide vaccine. Methods: Sixteen HLA-A2-positive patients with acute myeloid leukemia and one patient with myelodysplasia received 3–18 vaccinations (median 8) of WT1. 126–134 peptide (0.2 mg) together with the T helper protein keyhole limpet hemocyanin (1 mg) and in addition GM-CSF (75 mcg for four days) and. Twelve patients had elevated blast counts at study entry and 5 patients complete remission with high risk for relapse. Results: Six of 12 patients with presence of leukemic blasts had evidence of antileukemic activity. One patient achieved complete remission for 12 months. The patient with myelodysplasia RAEB II had a major response of neutrophils and platelets. Two patients had minor responses with transient clearance of peripheral blasts or improvement of hematopoiesis, and two patients achieved disease stabilization for 3 and 14 months. WT1 transcripts as molecular disease marker decreased in 5 of these 6 patients and also in 4 of 5 high-risk patients. No significant toxicity occurred. The generation of a WT1-specific T cell response in peripheral blood and bone marrow was detected in 9 of 13 patients by tetramer analysis and 8 of 13 patients by intracellular cytokine staining. Conclusion: These results show that WT1 vaccination can induce functional T cell responses associated with antileukemic activity and warrant trials of WT1 vaccination in patients at high risk of relapse and with WT1-expressing carcinomas. No significant financial relationships to disclose.

The serosal mesothelium is a major source of smooth muscle cells of the gut vasculature

Most internal organs are situated in a coelomic cavity and are covered by a mesothelium. During heart development, epicardial cells (a mesothelium) move to and over the heart, undergo epithelial-mesenchymal transition (EMT), and subsequently differentiate into endothelial and vascular smooth muscle cells. This is thought to be a unique process in blood vessel formation. Still, structural and developmental similarities between the heart and gut led us to test the hypothesis that a conserved or related mechanism may regulate blood vessel development to the gut, which, similar to the heart, is housed in a coelomic cavity. By using a combination of molecular genetics, vital dye fate mapping, organ culture and immunohistochemistry, we demonstrate that the serosal mesothelium is the major source of vasculogenic cells in developing mouse gut. Our studies show that the gut is initially devoid of a mesothelium but that serosal mesothelial cells expressing the Wilm's tumor protein (Wt1) move to and over the gut. Subsequently, a subset of these cells undergoes EMT and migrates throughout the gut. Using Wt1-Cre genetic lineage marking of serosal cells and their progeny, we demonstrate that these cells differentiate to smooth muscle of all major blood vessels in the mesenteries and gut. Our data reveal a conserved mechanism in blood vessel formation to coelomic organs, and have major implications for our understanding of vertebrate organogenesis and vascular deficiencies of the gut.

WT1-Peptide Vaccination Shows High Immunogenicity and Clinical Activity in Patients with Acute Myeloid Leukemia.

BACKGROUND: The transcription factor Wilms tumor protein 1 (WT1) holds great promise for immunotherapy of leukemia. WT1 is strongly expressed in the majority of leukemic blasts, is essential for blast proliferation, and is spontaneously immunogenic.METHODS: In the present phase II trial, 12 HLA-A2+ patients with AML without curative treatment option, were vaccinated with WT1.126–134 peptide mixed with adjuvant KLH as T-helper protein and GM-CSF 4 times bi-weekly, then monthly.RESULTS: Patients characteristics, immune responses and clinical outcome are shown in table 1.Patient characteristics, immunologic response, and clinical outcomePatFAB/caryotypeprevious chemotherapydisease status at study onsetno. of vaccinationsclinical outcomeWT1Tetr+ T cells in PB after vaccinationWT1Tetr+ T cells in BM after vaccination1M4yes2.PR15CR 12 months0.49%0.87%2M2 11q23yes1.CR18cCR 30+ months0.43%0.91%3M2noPD4SD 3 months0.42%0.80%4M6yesPD4PDneg.neg.5M2yes1.PR6PD0.37%0.51%6M1yes2. PR9PD0.43%0.40%7M2yes2.PR9PD2.00%1.36%8M7yesPD4PDneg.neg.9M5byes2.CR12cCR 8+ months0.44%0.33%10sAML from MDSnoPD12SD 8 months0.23%0.13%11sAML from MPSnoPD12SD 9+ months0.22%0.53%12M4noPD8SD 3 months1.11%1.35%*PB, peripheral blood; BM, bone marrow; MDS, myelodysplastic syndrome; MPD, myeloproliferative disease.WT1-specific T cells could be detected in 3 patients before vaccination. An induction or enhancement of a T cell response against WT1 was observed in 10 of 12 patients after 2 – 6 vaccinations ranging from 0.22 to 2.00% (median 0.43%) in peripheral blood and from 0.33 to 1.36% (median 0.80%) in bone marrow as analysed by tetramer and cytokine staining. At study onset 6 patients had progressive AML (PD) with 40 – 90% marrow blasts, 4 patients partial remission (PR) following chemotherapy and two patients complete remission (CR) at high risk for relapse. Four of the 6 patients with progressive AML had disease stabilization for 3, 3, 8 and 9 months, which is ongoing in the latter patient. Disease stabilization was accompanied by a decrease/normalization of peripheral blasts in two patients and a >50% decrease in RBC transfusion requirements in a patient with AML evolved from MDS. One patient with PR at study onset had an early relapse and then achieved CR for 12 months (patient 1). Both patients vaccinated in CR are in continuous hematological CR (cCR) for 8+ and 30+ months (patient 2 and 9). The remaining 5 patients had PD after 4 – 9 vaccinations. Bone marrow WT1 RNA levels as molecular disease marker paralleled the clinical course as they decreased 1 – 2 logs in the 3 patients with CR or cCR after 6 vaccinations (Fig. 1A), stabilized or decreased in all 4 patients with SD (Fig. 1B), and increased 1 – 2 logs in 4 of the 5 patients with PD (Fig. 1B). No significant toxic effects were observed.CONCLUSION: WT1 peptide vaccination can efficiently induce a specific immune response and has clinical activity in the absence of significant toxicity. These results warrant further studies of WT1 vaccination in AML patients at high risk for relapse. [Display omitted]

Differential gene regulation of WT1 protein tumor antigen and the components of the antigen processing machinery by interferon-gamma signaling in myeloid leukemia

2587 Background: Expression of high levels of Wilms Tumor Protein-1 (WT1) is associated with poor prognosis in myeloid leukemias and myelodysplastic syndromes.We studied the effects of interferon-γ (Inf-γ) on the myeloid cell line K562 which express the WT1 mRNA and the lymphoid cell line LG2 which does not express the transcripts. The biological effects of Inf-γ are mediated through activating the Janus kinase (JAK)-STAT pathway, which in turn, can activate a large number of genes by binding to the interferon-stimulated response element (ISRE). Methods: The mRNA was detected by reverse transcriptase polymerase chain reaction (RT-PCR) using published primer sequences and quantitated by a real time PCR (RQ-PCR). The WT1 protein expression was detected by western blotting with a commercially available WT1 monoclonal antibody. Intracellular flow cytometry with monoclonal antibodies was used to study the components of the antigen processing machinery (APM) as established before. Results: WT1 is down-regulated in K562 cells by treating K562 cells with Inf-γ. In a timed experiment WT1 mRNA is not seen after 36 hours of treatment both by RT-PCR and RQ-PCR. Western blotting for the protein done at 48 hours confirms the absence of the protein. No apoptosis is seen in these cells as shown by BrDU staining. Intracellular flow cytometry for the APM show- up regulation of the interferon responsive class I heavy chain and Tapasin stabilizing the heavy chain with the beta-2 microglobulin. WT1 Down-stream proteins such as bcl-2 and c-myc are also downregulated. There is no change in the expression of the chaperone proteins such as calreticulin, calnexin and HSP70. However the heat shock proteins HSP90 and HSP27 are downregulated. Similar effects on the WT1 are not seen in the control cell line LG2 or after treatment of the K562 cells with PMA, TNF-alpha or 5-azacytidine. Conclusions: The downregulation of WT1 is Inf-γ specific suggesting involvement of the STAT pathways. WT1 acts a differentiation signal for leukemia cells, which has implications in targeted treatment of leukemia. Preliminary results from patient sample study confirm some of these findings. No significant financial relationships to disclose.

In vitro Stimulation with WT1 Peptide-Loaded Epstein-Barr Virus-Positive B Cells Elicits High Frequencies of WT1 Peptide-Specific T Cells with In vitro and In vivo Tumoricidal Activity

The Wilms tumor protein (WT1) is overexpressed in most acute and chronic leukemias. To develop a practicable, clinically applicable approach for generation of WT1-specific T cells and to comparatively evaluate the immunogenicity of WT1 in normal individuals, we sensitized T cells from 13 HLA-A0201+ and 5 HLA-A2402+ donors with autologous EBV-transformed B cells or cytokine-activated monocytes, loaded with the HLA-A0201-binding WT1 peptides (126-134)RMFPNAPYL or (187-195)SLGEQQYSV or a newly identified HLA-A2402-binding WT1 peptide (301-310)RVPGVAPTL. WT1-specific T cells were regularly generated from each donor. T cells sensitized with peptide-loaded EBV-transformed B cells generated higher numbers of WT1-specific T cells than peptide-loaded cytokine-activated monocytes. Contrary to expectations, the frequencies of WT1 peptide-specific T cells were equivalent to those generated against individual highly immunogenic HLA-A0201-binding EBV peptides. Each of these T-cell lines specifically killed WT1+ leukemias and solid tumors in an HLA-restricted manner but did not lyse autologous or HLA-matched normal CD34+ hematopoietic progenitor cells or reduce their yield of colony-forming unit-granulocyte-macrophage (CFU-GM), burst-forming unit erythroid (BFU-E), or mixed colonies (CFU-mix). Furthermore, WT1 peptide-specific T cells after adoptive transfer into nonobese diabetic-severe combined immunodeficient mice bearing subcutaneous xenografts of WT1+ and WT1- HLA-A0201+ leukemias preferentially accumulated in and induced regressions of WT1+ leukemias that expressed the restricting HLA allele. Such cells are clinically applicable and may prove useful for adoptive cell therapy of WT1+ malignant diseases in humans.

Increased syndecan expression following myocardial infarction indicates a role in cardiac remodeling.

The purpose of this study was to identify essential genes involved in myocardial growth and remodeling following myocardial infarction (MI). Left ventricular noninfarcted tissues from six mice subjected to MI under general anesthesia and from six sham-operated mice were obtained 1 wk after primary surgery and analyzed by means of cDNA filter arrays. Out of a total of 1,176 genes, 641 were consistently expressed, twenty-three were upregulated and thirteen downregulated. Five genes were only expressed following MI. Syndecan-3, a transmembranous heparan sulfate proteoglycan, was found to be upregulated together with a transcriptional activator of syndecans, Wilms tumor protein 1 (WT-1). Northern blotting demonstrated a significant upregulation of syndecan-1, -2, -3, and -4, WT-1, fibronectin, and basic fibroblast growth factor (FGF) receptor 1. Furthermore, Western blot analysis showed statistically significant increases in protein levels for syndecan-3 and -4. In conclusion, we have identified a subset of genes with increased expression in noninfarcted left ventricular tissue following MI, including syndecans 1-4, WT-1, fibronectin, collagen 6A, and FGF receptor 1. Since the syndecans link the cytoskeleton to the extracellular matrix and function as required coreceptors for FGF, we suggest a role for the syndecans in cardiac remodeling following MI.

Novel targeted and immunotherapeutic strategies in chronic myeloid leukemia

The advanced understanding of the molecular biology and immunology of chronic myeloid leukemia (CML) has led to novel therapeutic strategies unique to this disease. CML responds to immune-mediated therapies, including stem cell transplantation, donor lymphocyte infusion (DLI), and interferon alfa. T cells and other immune effectors are implicated in the mechanisms of action of these immune therapies. Recently, clinical observations supported by laboratory data have demonstrated the presence of CML-specific T cells in patients. Several proteins may potentially act as leukemia-specific antigens for major histocompatibility complex (MHC)-restricted cytotoxicity in CML, and active specific therapies (vaccines) are in development. Antigens under investigation include bcr-abl, PR1, Wilms tumor protein (WT1), minor histocompatibility antigens (mH), CML-66, CML-28, and survivin. Other strategies target vascular endothelial growth factor (VEGF) and heat shock protein 90 (Hsp90) inhibitors or make use of CML-derived dendritic cells (DC). Semin Hematol 40:87-96. Copyright 2003, Elsevier Science (USA). All rights reserved.

Novel targeted and immunotherapeutic strategies in chronic myeloid leukemia.

The advanced understanding of the molecular biology and immunology of chronic myeloid leukemia (CML) has led to novel therapeutic strategies unique to this disease. CML responds to immune-mediated therapies, including stem cell transplantation, donor lymphocyte infusion (DLI), and interferon alfa. T cells and other immune effectors are implicated in the mechanisms of action of these immune therapies. Recently, clinical observations supported by laboratory data have demonstrated the presence of CML-specific T cells in patients. Several proteins may potentially act as leukemia-specific antigens for major histocompatibility complex (MHC)-restricted cytotoxicity in CML, and active specific therapies (vaccines) are in development. Antigens under investigation include bcr-abl, PR1, Wilms tumor protein (WT1), minor histocompatibility antigens (mH), CML-66, CML-28, and survivin. Other strategies target vascular endothelial growth factor (VEGF) and heat shock protein 90 (Hsp90) inhibitors or make use of CML-derived dendritic cells (DC).

Calretinin and other mesothelioma markers in synovial sarcoma: analysis of antigenic similarities and differences with malignant mesothelioma.

Synovial sarcoma (SS) is a mesenchymal neoplasm that typically shows epithelial differentiation. SS commonly metastasizes to lung and pleura, and has also been reported as the primary in these locations. The histologic distinction of SS from mesothelioma may be difficult because of the combination of epithelioid and spindle cells, potentially shared locations, and antigenic expression. In this study the authors examined 103 well-documented SSs including 41 biphasic, 44 monophasic, and 18 poorly differentiated SSs in comparison with 23 epithelioid and seven sarcomatous mesotheliomas. Most biphasic SSs (29 of 41, 71%) had fields or foci of calretinin-positive tumor cells. The spindle cell components were more often positive (55%), whereas 14% of tumors had positive epithelial cells. The monophasic and poorly differentiated SSs commonly had foci of calretinin-positive cells (in 52% and 56% of cases respectively). In comparison, all 23 epithelioid mesotheliomas (EM) were extensively calretinin positive and seven sarcomatoid mesotheliomas were variably calretinin positive. HBME-1 positivity was similarly detected in biphasic SS and EM (100% and 87% respectively). Among the other sarcomas, two of 15 malignant peripheral nerve sheath tumors were focally calretinin positive, whereas 16 epithelioid sarcomas, 20 leiomyosarcomas, 20 gastrointestinal stromal tumors, and 20 angiosarcomas were negative. Biphasic SSs differed from mesotheliomas by their more common BerEp4 positivity (90%) whereas EMs showed focal reactivity in 13% cases. Marked CD15 reactivity was rare in both. Wilms tumor protein-1 (WT1) was not detected in SS, but was present in 12 of 17 EMs. CD141 was rare in SS, limited to spindle cell components, whereas EMs typically showed prominent membrane staining in epithelial cells. Simple epithelial keratins were present in all epithelial cells of biphasic SS and mesothelioma (keratin 7[K7], K19), but were only focal in monophasic and poorly differentiated SS. Biphasic SSs were extensively K14 positive (89% of cases), whereas epithelial and sarcomatoid mesotheliomas typically showed only scattered positive cells. The potentially shared calretinin patterns in SS and mesothelioma require the use of other markers. The discriminating features include extensive BerEp4 positivity, rarity of CD141, and lack of WT1 in SS. Global expression of K7 and K19 in mesotheliomas versus focal expression in monophasic and poorly differentiated SSs, and differential patterns of K14 expression may also be helpful.

WT1 staining reliably differentiates desmoplastic small round cell tumor from Ewing sarcoma/primitive neuroectodermal tumor. An immunohistochemical and molecular diagnostic study.

Differentiating desmoplastic small round cell tumor (DSRCT) from another similar small round cell tumor of childhood, the Ewing sarcoma/primitive neuroectodermal tumor (EWS/PNET), can be difficult because morphologic and immunohistochemical features overlap. We studied the predictive value of immunohistochemistry with an antibody to the C-terminal region of the Wilms tumor (WT1) protein for differentiating DSRCT from EWS/PNET in 24 malignant small round cell tumors that had been previously diagnosed as DSRCT or EWS/PNET by standard methods. We performed reverse transcriptase-polymerase chain reaction (RT-PCR) analysis in cases with available tissue as a confirmatory measure: 6 of 13 DSRCTs were informative by RT-PCR, and 6 of 6 showed an EWS-WT1 fusion; all 13 DSRCTs showed strong, definitive nuclear staining with the WT1 antibody. All 11 EWS/PNETs were WT1 antibody negative; 7 of 11 cases classified as EWS/PNET were informative by RT-PCR, and 7 of 7 showed an EWS-FLI-1 fusion. For cases in which the morphologic and immunohistochemical features are consistent with a diagnosis of DSRCT, WT1 antibody staining predicts the EWS-WT1 translocation with high sensitivity and specificity and is, therefore, useful for differentiating DSRCT from EWS/PNET when genetic information is unavailable.

The Wilms tumor protein is persistently associated with the nuclear matrix throughout the cell cycle.

In order to investigate the subnuclear interactions of the WT1 gene product, nuclear fractionation analyses were performed with human osteosarcoma HOS and myelogenous leukemia K562 cells. The WT1 protein was tightly associated with the nucleus and was resistant to high-salt or detergent extraction and DNase I digestion. Both the expression level and stability of WT1 and its resistance to high salt and DNase I treatments remained constant during the cell cycle. In addition, human WT1 ectopically expressed in mouse NIH3T3 cells was also resistant to these treatments. These results suggest that WT1 functions in tight association with the nuclear matrix.

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.