Expression and mutation analysis of the Wilms' tumor 1 gene in human neural tumors.

The Wilms' tumor 1 gene, WT1, encodes a zinc-finger protein that is implicated in the development of Wilms' tumor. Mutant or aberrantly expressed WT1 isoforms have also been described in desmoplastic small round cell tumor, acute leukemias, mesothelioma, breast tumors and melanoma. During early development, WT1 is expressed in the brain and spinal cord, however its role in the malignancies that affect these tissues has not been previously investigated. In our study we have examined neural tumors including brain tumors and neuroblastomas for WT1 expression and for mutations affecting the zinc-fingers. Although WT1 expression was detected in gliomas, medulloblastomas and neuroblastomas, neither zinc-finger region mutations nor splicing anomalies affecting the KTS site were detected. We therefore conclude that WT1 does not play a significant role in the etiology of human neural tumors.     

Wilms' tumor gene WT1 17AA(-)/KTS(-) isoform induces morphological changes and promotes cell migration and invasion in vitro

The wild-type Wilms' tumor gene WT1 is overexpressed in human primary leukemia and in a wide variety of solid cancers. All of the four WT1 isoforms are expressed in primary cancers and each is considered to have a different function. However, the functions of each of the WT1 isoforms in cancer cells remain unclear. The present study demonstrated that constitutive expression of the WT1 17AA(-)/KTS(-) isoform induces morphological changes characterized by a small-sized cell shape in TYK-nu.CP-r (TYK) ovarian cancer cells. In the WT1 17AA(-)/KTS(-) isoform-transduced TYK cells, cell-substratum adhesion was suppressed, and cell migration and in vitro invasion were enhanced compared to that in mock vector-transduced TYK cells. Constitutive expression of the WT1 17AA(-)/KTS(-) isoform also induced morphological changes in five (one gastric, one esophageal, two breast and one fibrosarcoma) of eight cancer cell lines examined. No WT1 isoforms other than the WT1 17AA(-)/KTS(-) isoform induced the phenotypic changes. A decrease in alpha-actinin 1 and cofilin expression and an increase in gelsolin expression were observed in WT1 17AA(-)/KTS(-) isoform-transduced TYK cells. In contrast, co-expression of alpha-actinin 1 and cofilin or knockdown of gelsolin expression by small interfering RNA restored WT1 17AA(-)/KTS(-) isoform-transduced TYK cells to a phenotype that was comparable to that of the parent TYK cells. These results indicated that the WT1 17AA(-)/KTS(-) isoform exerted its oncogenic functions through modulation of cytoskeletal dynamics. The present results may provide a novel insight into the signaling pathway of the WT1 gene for its oncogenic functions.     

Wilms' tumor gene WT1-shRNA as a potent apoptosis-inducing agent for solid tumors

Wilms' tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an important role in leukemogenesis and tumorigenesis. We tested apoptosis-inducing ability of short hairpin RNAs targeting exon 5 (shWTE5), exon10 (shWTE10) and 3'UTR (shWT3U) of the WT1 gene. Among the three WT1-shRNAs, since shWTE5 most effectively induced apoptosis, its ability as an apoptosis-inducing agent was intensively examined. shWTE5 induced mitochondrial damage and resultant apoptosis in five WT1-expressing solid cancer cells originated from gastric (AZ-521), lung (LU99B), ovarian (TYKnuCPr) cancers, fibrosarcoma (HT-1080) and glioblastoma (A172). Moreover, shWTE5 significantly enhanced apoptosis induced by chemotherapeutic agents, doxorubicin (DOX) and etoposide (ETP), or by death ligand TRAIL in all of the four solid tumor cells examined (HT-1080, LU99B, TYK and A172). Transduction of one each of WT1 isoforms with exon 5 [17AA(+)KTS(+) and 17AA(+)KTS(-)] prevented mitochondrial damage induced by ETP or TRAIL and inhibited apoptosis. These results showed that shWTE5 induced apoptosis through the suppression of the WT1 isoform with exon 5. Furthermore, shWTE5 increased expression of proapoptotic Bak and Bax proteins and decreased antiapoptotic Bcl-xL and Bcl-2 proteins in WT1-expressing HT-1080 cells, indicating that WT1 isoforms with exon 5 might play an antiapoptotic role through regulation of Bcl-2 family genes in solid tumor cells. The results presented here demonstrated that WT1-shRNA targeting exon 5 should serve as a potent anti-cancer agent for various types of solid tumors.     

WT1 is a tumor-associated antigen in colon cancer that can be recognized by in vitro stimulated cytotoxic T cells

The Wilms' tumor suppressor gene (WT1) has been shown to be overexpressed in acute and chronic leukemias and in a variety of solid human malignancies, including cancers of the breast and lung. In our present study, we investigated the potential role of WT1 gene in human colon cancer. WT1 mRNA and protein expression was analyzed in a panel of human colon cancer cell lines and primary colon carcinomas by RT-PCR and Western blot analysis, respectively. A mutational screen of WT1' zinc-finger region was carried out by sequence analysis. Finally, using peptide-stimulated cytotoxic T cells it was investigated whether WT1-expressing colon tumor cells are a potential target for antigen-specific immunotherapy. Medium to high abundant levels of WT1 mRNA were detected by RT-PCR in 10 of 12 (83%) colon cell lines and by quantitative, real-time RT-PCR in 13 of 15 (87%) primary tumors, whereas only very low levels of expression were found in 2 primary tumors. Interestingly, however, low levels of WT1 mRNA were also detected in all samples derived from normal colon mucosa. When RT-PCR products were examined by sequence analysis, both +KTS and -KTS splice isoforms but no zinc-finger mutations were found, suggesting that the wild-type form of the WT1 gene is expressed. To determine whether the WT1 protein can serve as a target antigen for immunotherapy, 2 HLA-A2.1-restricted WT1 peptides (Db126 and WH187) were used for the in vitro induction of WT1-specific cytotoxic T lymphocytes (CTLs). The WH187-specific CTLs not only lysed target cells pulsed exogenously with cognate peptide but also WT1-expressing colon tumor cells in a HLA-restricted manner. These findings identify the WT1 protein as an attractive target for the development of antigen-specific immunotherapy in human colon cancer.     

Exogenous expression of WT1 gene influences U937 cell biological behaviors and activates MAPK and JAK-STAT signaling pathways

Wilms’ tumor 1 (WT1) gene plays important roles in leukemogenesis. To further explore its underlying mechanisms, we transfected two WT1 isoforms, WT1(+17AA/−KTS) and WT1(+17AA/+KTS) into U937, a WT1-null monoblastic cell line, studied their effects on migration, colony formation, apoptosis, gene expression and pertinent signaling pathways of U937 cells. The results showed that WT1(+17AA/−KTS), but not WT1(+17AA/+KTS), enhanced migration and colony forming abilities of U937 cells, and suppressed etoposide-induced U937 cell apoptosis. Transfection of WT1 isoforms activated gene expressions of chemokine, and induced up-regulation of signaling molecules involved in JAK-STAT and MAPK signaling pathways. This study showed that exogenous expression of WT1 gene remarkably affected biological behaviors of U937 cells, and these effects are possibly mediated by up-regulation of genes related to chemokine, JAK-STAT and MAPK signaling pathways.     

WT1基因对肾胚瘤细胞系G401细胞肿瘤逆转的研究

目的 肾胚瘤是一种较常见的儿科实体肿瘤，该肿瘤的发生与抑癌基因WT1密切相关。本研究的目的是利用脂质体介导转染法将WT1cDNA导入G401细胞，研究WT1基因对G401细胞生长凋亡及致瘤性的作用。方法 通过Western blot检测WT1基因在肾胚瘤中的表达、采用碘化丙锭(PI)染色法检测G401细胞的凋亡情况、并且对G401的致瘤性进行分析，探讨WT1基因在肾胚瘤发生及治疗中的作用。结果 WT1 DNA可诱导G401细胞凋亡，并且使其致瘤性有所降低。结论 该结果为确定WT1在肾胚瘤病因方面的作用、为肾胚瘤的基因治疗提供重要的依据。     

Expression of the WT1 gene -KTS domain isoforms suppresses the invasive ability of human lung squamous cell carcinoma cells

Although the WT1 gene was originally isolated as a tumor suppressor gene from Wilms' tumor, oncogenic roles for WT1 have been reported in several tumors. Here, we present new findings of high levels of WT1 expression associated with the suppression of lymph node metastasis in patients with human lung squamous cell carcinoma (SCC). We investigated the effect of down-regulated WT1 gene expression on the invasive phenotype of the SCC cell line RERF-LC-AI. Invasive ability was enhanced in WT1-specific siRNA-transfected cells, and a WT1 target gene p21(Waf1/Cip1) was isolated by comprehensive gene expression analysis. As several isoforms are produced from the WT1 gene, we isolated eight major WT1 isoforms from a cDNA library and cloned each variant into an expression vector. Luciferase reporter assays revealed that p21(Waf1/Cip1) expression was enhanced only by the WT1 cDNA variants that included a three-amino acid deletion (-KTS). Our results suggested that the -KTS-containing variants of WT1 are directly involved in the regulation of p21(Waf1/Cip1) expression and the subsequent suppression of lymph node metastasis in human lung squamous cell carcinoma.     

Antiapoptotic function of 17AA(+)WT1 (Wilms' tumor gene) isoforms on the intrinsic apoptosis pathway

The WT1 gene is overexpressed in human primary leukemia and a wide variety of solid cancers. The WT1 gene is alternatively spliced at two sites, yielding four isoforms: 17AA(+)KTS(+), 17AA(+)KTS(-), 17AA(-)KTS(+), and 17AA(-)KTS(-). Here, we showed that 17AA(+)WT1-specific siRNA induced apoptosis in three WT1-expressing leukemia cell lines (K562, HL-60, and Kasumi-1), but not in WT1-non-expressing lymphoma cell line (Daudi). 17AA(+)WT1-specific siRNA activated caspase-3 and -9 in the intrinsic apoptosis pathway but not caspase-8 in the extrinsic one. On the other hand, 17AA(-)WT1-specific siRNA did not induce apoptosis in the three WT1-expressing cell lines. The apoptosis was associated with activation of proapoptotic Bax, which was activated upstream of the mitochondria. Constitutive expression of 17AA(+)WT1 isoforms inhibited apoptosis of K562 leukemia cells induced by apoptosis-inducing agents, etoposide and doxorubicin, through the protection of mitochondrial membrane damages, and DNA-binding zinc-finger region of 17AA(+)WT1 isoform was essential for the antiapoptotic functions. We further studied the gene(s) whose expression was altered by the expression of 17AA(+)WT1 isoforms and showed that the expression of proapoptotic Bak was decreased by the expression of 17AA(+)KTS(-)WT1 isoform. Taken together, these results indicated that 17AA(+)WT1 isoforms played antiapoptotic roles at some points upstream of the mitochondria in the intrinsic apoptosis pathway.     

WT1 in acute leukemia, chronic myelogenous leukemia and myelodysplastic syndrome: therapeutic potential of WT1 targeted therapies.

Among clinicians, initial awareness of the Wilms' tumor gene was limited mostly to pediatric oncologists. Almost a decade ago, overexpression of Wilms' tumor 1 (WT1) was observed in adult acute leukemia. Subsequent studies indicated that WT1 overexpression occurs in most cases of acute myelogenous leukemia, acute lymphoblastic leukemia, chronic myelogenous leukemia (CML), and myelodysplastic syndrome (MDS). Limited tissue expression of WT1 in adults suggests that WT1 can be a target for leukemia/MDS therapy. WT1 expression in stem/progenitor cells remains unsettled. However, lack of progenitor cell suppression by WT1 antisense or WT1-specific cytotoxic T cells provide some assurance that WT1 expression in progenitor cells is minimal or absent. Immunotherapy-based WT1 approaches are furthest along in preclinical development. WT1-specific cytotoxic lymphocytes can be generated from normals and leukemic patients. In mice, WT1 vaccines elicit specific immune responses without evidence of tissue damage. In this paper, we review studies validating the immunogenicity of WT1 and propose that leukemia and MDS may be a good clinical model to test the efficacy of a WT1 vaccine.     

Wilms’ tumor gene WT1 promotes homologous recombination‐mediated DNA damage repair

The Wilms’ tumor gene WT1 is overexpressed in leukemia and various types of solid tumors and plays an oncogenic role in these malignancies. Alternative splicing at two sites yields four major isoforms, 17AA(+)KTS(+), 17AA(+)KTS(?), 17AA(?)KTS(+), and 17AA(?)KTS(?), and all the isoforms are expressed in the malignancies. However, among the four isoforms, function of WT1[17AA(?)KTS(+)] isoform still remains undetermined. In the present study, we showed that forced expression of WT1[17AA(?)KTS(+)] isoform significantly inhibited apoptosis by DNA‐damaging agents such as Doxorubicin, Mitomycin, Camptothesisn, and Bleomycin in immortalized fibroblast MRC5SV and cervical cancer HeLa cells. Knockdown of Rad51, an essential factor for homologous recombination (HR)‐mediated DNA repair canceled the resistance to Doxorubicin induced by WT1[17AA(?)KTS(+)] isoform. GFP recombination assay showed that WT1[17AA(?)KTS(+)] isoform alone promoted HR, but that three other WT1 isoforms did not. WT1[17AA(?)KTS(+)] isoform significantly upregulated the expression of HR genes, XRCC2, Rad51D, and Rad54. Knockdown of XRCC2, Rad51D, and Rad54 inhibited the HR activity and canceled resistance to Doxorubicin in MRC5SV cells with forced expression of WT1[17AA(?)KTS(+)] isoform. Furthermore, chromatin immunoprecipitation (ChIP) assay showed the binding of WT1[17AA(?)KTS(+)] isoform protein to promoters of XRCC2 and Rad51D. Immunohistochemical study showed that Rad54 and XRCC2 proteins were highly expressed in the majority of non‐small‐cell lung cancer (NSCLC) and gastric cancer, and that expression of these two proteins was significantly correlated with that of WT1 protein in NSCLCs. Our results presented here showed that WT1[17AA(?)KTS(+)] isoform had a function to promote HR‐mediated DNA repair. © 2014 The Authors. Molecular Carcinogenesis published by Wiley Periodicals, Inc.

     

RNAi silencing of the WT1 gene inhibits cell proliferation and induces apoptosis in the B16F10 murine melanoma cell line

The Wilms' tumor protein 1 (WT1) is essential for tumor cell proliferation and is highly expressed in various hematological and solid malignancies including human malignant melanoma. We investigated whether WT1 expression is essential for growth in the B16F10 murine melanoma cell line. Toward this end, we examined WT1 protein expression and WT1 isoforms (17AA+/17AA-, KTS+/KTS-) in this cell line. WT1 was silenced by two RNA interference constructs, designated WT1-1 and WT1-2. RNA interference-mediated reduction of WT1 protein expression significantly inhibited B16F10 cell viability. Loss of WT1 also increased caspase-3 and poly-ADP-ribose polymerase activation, as well as apoptotic body formation, chromatin condensation, and DNA fragmentation. Together, these findings implicate decreased WT1 protein levels in the induction of apoptosis. These results imply that WT1 plays a distinct role in B16F10 melanoma growth.     

RNA expression of the WT1 gene in Wilms' tumors in relation to histology.

BACKGROUND: On the basis of accumulating data, the recently isolated WT1 gene is a Wilms' tumor gene and a putative tumor suppressor gene. These findings include expression in developing fetal kidney, intragenic deletions in tumors, and germline mutations in predisposed individuals. Wilms' tumors, which exhibit a broad range of differentiation, are composed of three cell types: blastema, epithelium, and stroma. PURPOSE: The purpose of this study was to investigate the relationship between WT1 gene expression and histologic composition in Wilms' tumors in an effort to elucidate how the WT1 gene functions in proliferation of these histologic components. METHODS: We used Northern blot hybridization to study WT1 gene expression by messenger RNA (mRNA) accumulation in 20 tumors of varying histology and in adjacent uninvolved kidney tissue. In two patients, tumors were also compared before and after therapy. RESULTS: Tumors that were predominantly blastemal expressed high amounts of WT1 mRNA, whereas predominantly stromal tumors expressed either low or undetectable amounts. Blastemal tumors that were predominantly poorly differentiated expressed WT1 mRNA at higher levels than those that were more well differentiated. Although we expected that a putative tumor suppressor gene like WT1 would generally be expressed at lower levels in tumor than in normal kidney, this was true only in predominantly stromal cells. One of the two patients studied before and after therapy had a dramatic response to therapy accompanied by a decline in WT1 gene expression and disappearance of blastemal and epithelial elements. CONCLUSIONS: A correlation was observed between WT1 gene expression and histology of the tumors. Level of expression was inversely related to the degree of differentiation in blastemal tumors and in the patient with a dramatic response to therapy. These results, in conjunction with the observation that WT1 mRNA is abundant in normal fetal kidney, suggest that WT1 gene expression is related to kidney development, especially in differentiation of blastemal components. IMPLICATIONS: Further studies to search for alterations of the WT1 gene in tumors and to identify regulatory factors in gene expression will increase understanding of the role of this gene in normal development and tumorigenesis.     

Suppression of growth and dissemination in human pre-B leukemia cells by tumor necrosis factor-alpha in scid mice

Tumor necrosis factor (TNF) has been shown to inhibit the growth of ALL cells. Since the systemic administration of TNF for malignancy results in poor response and severe toxicity, future efforts should concentrate on local treatment. Here we examined the suppressive effect of TNF alpha on leukemic cells engrafted in scid mice. NALM6 cells derived from pre-B ALL were injected in scid mice subcutaneously with or without Matrigel. In mice with Matrigel, subcutaneous tumors rapidly increased with time, whereas none of the mice without Matrigel showed any obvious signs of disease or apparent tumors. High levels of leukemic infiltration were observed in peripheral organs in mice with Matrigel by flow cytometry and PCR for human beta-actin mRNA expression, while mice without Matrigel showed low or undetectable infiltration in these organs. Human TNF alpha was also coinjected subcutaneously with NALM6 cells and Matrigel into scid mice. Mice with 10 ng of TNF alpha showed small subcutaneous tumors at 8 weeks, which slowly increased. They were found to have a small number of leukemic cells in peripheral organs by flow cytometry. By PCR, all organs with the exception of lung and brain showed low or undetectable expression of beta-actin. However, a large dose of TNF alpha (100 ng) had no suppressive effect on tumor growth and leukemic infiltration in mouse organs. Similar results were obtained in colony formation of leukemic cells in vitro. To examine the mechanism of the suppressive effect of TNF alpha, the expression of TNF receptors in tumor cells was analyzed by flow cytometry. Parental NALM6 expressed both TNF alpha receptors I (TNFR60) and II (TNFR80), but these expressions were suppressed in tumor cells from mice with Matrigel. Only TNFR80 expression was induced in tumor cells of mice with 10 ng of TNF alpha. The induction of Fas expression was also detected, whereas neither DNA fragmentation nor apoptotic change in histology was observed in tumor cells of mice with TNF alpha. These results suggest that the suppressive effect of TNF alpha on the growth of leukemic cells in scid mice is mediated through the activation of TNFR80 without apoptotic signal.