Tumor Gene WT1 Research Articles

WT1 as a novel target antigen for cancer immunotherapy.

Wild-type Wilms' tumor gene WT1 is expressed at high levels not only in most of acute myelocytic, acute lymphocytic, and chronic myelocytic leukemia, but also in various types of solid tumors including lung cancer. We tested the ability of the gene product (WT1) to serve as a target antigen for tumor specific immunotherapy both in human in vitro system and mouse in vivo system. In the latter, we can evaluate the efficacy and the side effects of WT1 vaccination in vivo. In the human in vitro system, two WT1 peptides that contain HLA-A2.1 binding anchor motifs were determined to bind to HLA-A2.1 molecules. Peripheral blood mononuclear cells (PBMC) from an HLA-A2.1-psitive donor were repeatedly stimulated in vitro with TAP-deficient T2 cells pulsed with each of these two peptides, and CD8-positive cytotoxic T lymphocytes (CTLs) that specifically lyse WT1-expressing, HLA-A2.1-positive tumor cells were induced. Other groups also have succeeded in generating CTLs which specifically lyse WT1-expressing leukemia cells, and which do not inhibit colony-formation of normal hematopoietic cells that express WT1 at physiological levels. In the mouse in vivo system, immunization of C57BL/6 mice with one WT1 peptide with relatively high binding affinity for H-2D(b) molecules, which contain H-2D(b) binding anchor motifs, induced CTLs, which specifically lysed WT1-expressing tumor cells in an H-2D(b)-restricted manner. Furthermore, mice immunized with the WT1 peptide (peptide vaccination) or WT1 cDNA (DNA vaccination) rejected challenges by WT1-expressing tumor cells and survived with no signs of auto-aggression to WT1-expressing normal organs by the induced CTLs. The WT1 protein has been identified as a novel tumor antigen and recent investigations provide a rationale for developing WT1-based adoptive T cell therapy and vaccination against various kinds of malignant neoplasms.

A Functional Interaction with CBP Contributes to Transcriptional Activation by the Wilms Tumor Suppressor WT1

The Wilms tumor gene WT1 encodes a zinc finger transcription factor that is required for normal kidney development. WT1 was identified as a transcriptional repressor, based on its suppression of promoter reporters, but analysis of native transcripts using high density microarrays has uncovered transcriptional activation, rather than repression, of potential target genes. We report here that WT1 binds to the transcriptional coactivator CBP, leading to synergistic activation of a physiologically relevant promoter. The physical interaction between WT1 and CBP is evident in vitro and in vivo, and the two proteins are co-immunoprecipitated from embryonic rat kidney cells. The WT1-CBP association requires the first two zinc fingers of WT1 and the adenovirus 5 E1A-binding domain of CBP. Overexpression of this domain of CBP is sufficient to inhibit WT1-mediated transcriptional activation of a promoter reporter, as is co-transfection of E1A. Retrovirally driven expression of either the CBP fragment or of E1A in human hematopoietic cells suppresses the induction by WT1 of its endogenous target gene, p21(Cip1). These observations support a model of WT1 as a transcriptional activator of genes required for cellular differentiation.

Wilms' tumor gene WT1: its oncogenic function and clinical application.

The Wilms' tumor gene WT1 is a gene responsible for the childhood renal tumor. Wilms' tumor, and is defined as a tumor suppressor gene. However, the wild-type WT1 gene is highly expressed in leukemic blast cells of myeloid and lymphoid origin, and thus, WT1 messenger RNA provides a novel tumor marker for detection of minimal residual disease of leukemias and for monitoring disease progression of myelodysplastic syndromes. The WT1 gene exerts an oncogenic function rather than a tumor-suppressor gene function in solid tumors as well as leukemias, and the WT1 gene product is an attractive tumor antigen capable of eliciting cytotoxic T lymphocytes against WT1-expressing tumors.

The Wilms' Tumor Gene Product (WT1) Modulates the Response to 1,25-Dihydroxyvitamin D3 by Induction of the Vitamin D Receptor

The Wilms' tumor gene (wt1) encodes a transcription factor involved in urogenital development, in particular in renal differentiation, and in hematopoietic differentiation. Differentiation of a number of solid tumor and leukemic cells lines can be mediated by 1,25-dihydroxyvitamin D(3). This is predominantly mediated by the nuclear receptor for 1,25-dihydroxyvitamin D(3), the vitamin D receptor (VDR). In initial experiments addressing a possible link between WT1 and VDR, we observed a correlated expression of WT1 and VDR mRNA in samples from renal tissues. HT29 colon carcinoma cells, stably transfected to express WT1, exhibited elevated endogenous VDR levels compared with control cells transfected with a control construct. Elevated VDR expression was found in wt1-transfected human embryonic kidney 293 cells, as well. In transient cotransfection experiments, we observed an activation of a vdr promoter reporter by WT1 through a WT1 recognition element, indicating transcriptional regulation of the vdr gene expression by WT1. The responsive sequence element was specifically bound by wild-type, but not by mutated WT1, in electrophoretic mobility shift assays. HT29 colon carcinoma cells, which respond to 1,25-dihydroxyvitamin D(3) with slow induction of growth arrest, were investigated for the influence of WT1 on 1,25-dihydroxyvitamin D(3)-mediated growth suppression. Although HT29 cells transfected with a control construct responded moderately to 1,25-dihydroxyvitamin D(3), the response of HT29 cells expressing WT1 was strikingly enhanced. Stimulation with dihydroxyvitamin D(3) caused an up to 3-fold reduction in the growth rate of different HT29 clones expressing WT1 as compared with control cells lacking WT1 expression. Thus, induction of VDR by WT1 leads to an enhanced response to 1,25-dihydroxyvitamin D(3). We conclude that the vitamin D receptor gene is a target for transcriptional activation by WT1, suggesting a possible physiological role of this regulatory pathway.

Hox11 is required to maintain normal Wt1 mRNA levels in the developing spleen.

Mice deficient in Hox11 are asplenic. As Hoxll can function as a transcription factor, we examined the spatial and temporal mRNA expression patterns of Hox11 and a candidate target gene, the Wilm's tumor gene Wt1, in the developing spleen. Hox11 mRNA first appears at approximately dE10.5 in the dorsal mesogastrium while Wt1 mRNA is expressed from dE11.5, approximately 24 hours after Hox11 mRNA first appears. Wt1 mRNA was significantly reduced in the spleen anlage of Hox11-null mice suggesting that Wt1 acts downstream of Hox11 in a transcriptional cascade. Additionally, Hox11 protein is able to transactivate the WT1 promoter in a Hox11-null fibroblast cell line. As Wt1-null embryos have recently been reported to be asplenic, these findings suggest that Wt1 and Hox11 may be components common to a genetic hierarchy that is required for spleen development.

Immunohistochemical detection of the Wilms' tumor gene (WT1) in epithelial ovarian tumors.

The Wilms' tumor gene WT1 plays multiple roles in the development of the genitourinary organs and Wilms' tumors. The aims of this study were to immunohistochemically evaluate WT1 expression in normal female genital tissues and in epithelial ovarian tumors and to look for correlations between WT1 expression and histologic subtypes and cell proliferation in epithelial ovarian tumors. In normal female genital organs, WT1 expression was recognized in ovarian surface epithelium, the lining of inclusion cysts, and tubal epithelium, but not in the cervical or endometrial epithelium. In epithelial ovarian tumors, serous tumors generally revealed a high WT1 expression. Among adenocarcinomas, serous carcinoma revealed a significantly higher WT1 expression than the other histologic subtypes. There were no significant correlations between the WT1 labeling index and the Ki-67 labeling index, and no significant difference in survival between those showing high and low WT1 expression among the malignant cases. These results suggest that WT1 expression may be related to cell differentiation, and that the histologic subtypes of epithelial ovarian carcinomas may differ considerably in their biological characteristics.

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.

Human cytotoxic T-lymphocyte responses specific for peptides of the wild-type Wilms' tumor gene (WT1 ) product.

The product of the Wilms' tumor gene WT1 is a transcription factor overexpressed not only in leukemic blast cells of almost all patients with acute myeloid leukemia, acute lymphoid leukemia, and chronic myeloid leukemia, but also in various types of solid tumor cells. Thus, it is suggested that the WT1 gene plays an important role in both leukemogenesis and tumorigenesis. Here we tested the potential of WT1 to serve as a target for immunotherapy against leukemia and solid tumors. Four 9-mer WT1 peptides that contain HLA-A2.1-binding anchor motifs were synthesized. Two of them, Db126 and WH187, were determined to bind to HLA-A2.1 molecules in a binding assay using transporter associated with antigen processing-deficient T2 cells. Peripheral blood mononuclear cells from an HLA-A2.1-positive healthy donor were repeatedly sensitized in vitro with T2 cells pulsed with each of these two WT1 peptides, and CD8(+) cytotoxic T lymphocytes (CTLs) that specifically lyse WT1 peptide-pulsed T2 cells in an HLA-A2.1-restricted fashion were induced. The CTLs also exerted specific lysis against WT1-expressing, HLA-A2.1-positive leukemia cells, but not against WT1-expressing, HLA-A2.1-negative leukemia cells, or WT1-nonexpressing, HLA-A2. 1-positive B-lymphoblastoid cells. These data provide the first evidence of human CTL responses specific for the WT1 peptides, and provide a rationale for developing WT1 peptide-based adoptive T-cell therapy and vaccination against leukemia and solid tumors.

Cytotoxic T-lymphocyte responses elicited to Wilms' tumor gene WT1 product by DNA vaccination.

We recently have reported that Wilms' tumor gene WT1 is highly expressed not only in leukemias but also in various types of solid tumors and that WT1 protein is a novel tumor antigen against which cytotoxic T lymphocytes (CTLs) can be elicited by immunization with 9-mer WT1 peptides capable of binding to major histocompatibility complex (MHC) class I molecules. In the present study, plasmid DNA encoding murine full-length WT1 protein was injected intramuscularly into C57BL/6 mice. The mice vaccinated with the WT1 plasmid DNA elicited CTLs against the WT1 protein, and the CTLs specifically killed WT1-expressing tumor cells in a MHC class I-restricted manner. Furthermore, the vaccinated mice rejected the challenges of WT1-expressing tumor cells and survived with no signs of autoimmunity caused by the CTLs. These results demonstrated that vaccination with the WT1 plasmid DNA can elicit CTL responses specific for the WT1 protein, resulting in the acquisition of rejection activity against challenges of WT1-expressing tumor cells. This WT1 DNA vaccination may find clinical application for various types of solid tumors as well as leukemias.

Wt1 Gene Expression in Childhood Leukemias

The expression of the Wilms’ tumor gene (wt1) was detected in various tissues during embryonic development. Mutations in the wt1 gene probably play an important role in certain tumors, e.g. the Wilms’ tumor. Furthermore the expression of wt1 gene was found in some human leukemias. In the present study we investigated the expression of wt1 gene in several types of childhood leukemia by reverse transcriptase-polymerase chain reaction. Bone marrow or peripheral blood of 61 pediatric patients (48 at initial diagnosis, 13 at first or second relapse) were analyzed. wt1 gene expression was detected in 35/48 patients (73%) with newly diagnosed leukemias and in 12/13 cases (92%) who had suffered from relapse. The expression levels were higher for AML than for ALL. The frequency of wt1 expression in different subtypes of acute leukemia was compared with results found in adult patients. Our results show that the frequency of wt1 gene expression in acute childhood leukemias is similar to previous data reported for adults.

Wilms' tumour gene (wt1) expression at diagnosis has no prognostic relevance in childhood acute lymphoblastic leukaemia treated by an intensive chemotherapy protocol.

Expression of the Wilms' tumour gene (wt1) has been demonstrated in a large proportion of human acute leukaemias and is thought to play a role in leukaemogenesis. Recent observations in adult patients with acute leukaemia suggest that wt1 gene expression is a poor prognostic factor. In childhood acute leukaemia, the clinical role of wt1 gene expression has not been established. We have therefore investigated bone marrow samples from 50 children with acute lymphocytic leukaemia at the time of diagnosis for the presence of wt1 transcripts to determine whether wt1 gene expression is associated with specific characteristics of leukaemic cells and whether it is predictive of response to treatment. All patients were treated according to the ALL-BFM 90 protocol. The median observation time was 30 months. Wt1 transcripts were detected by RT-PCR in 60% of the diagnostic samples. Wt1 PCR positive patients showed a higher median leukocyte and peripheral blast cell count than wt1 negative patients. High and intermediate risk patients more frequently displayed wt1 transcripts than low risk patients. No correlation between wtl gene expression and FAB type, immunophenotype, co-expression of myeloid antigens or karyotype has been observed. Furthermore, there was no correlation between wt1 gene expression at diagnosis and achievement of complete remission (CR) and no difference in disease-free survival (DFS) or overall survival (OS) between wt1 positive and negative patients (p > 0.1). These data indicate that (1) wt1 gene expression at diagnosis is detected more frequently in patients with high leukocyte and peripheral blast cell counts, but is not associated with specific characteristics of leukaemic cells, (2) wt1 gene expression is not an independent prognostic factor for CR, DFS or OS in childhood ALL treated by an intensive therapy protocol.

Mutations of theWT1 gene in childhood nonlymphoid hematological malignancies

The Wilms' tumor gene WT1, whose loss of function accounts for the genesis of about 10% of Wilms' tumors, is expressed in hematopoietic stem cells and leukemia. By analogy with the relationship between the kidney stem cell and Wilms' tumor, it is probable that WT1 is mutated in leukemia. WT1 mutations have been found in only eight cases of primary leukemia, mainly in acute myeloid leukemia (AML) and rarely in acute lymphoblastic leukemia. However, two other studies have demonstrated the absence of WT1 mutations in leukemia. To determine if WT1 mutations are associated with leukemias, we screened childhood nonlymphoid malignancies for WT1 mutation. WT1 mutations were found in 6 of 46 (13%) AMLs, but not in other nonlymphoid hematological malignancies. In addition, the presence of WT1 mutations in AML caused by chromosomal translocations suggests that mutations of WT1 may lead to the progression of leukemia.

YAC complementation shows a requirement for Wt1 in the development of epicardium, adrenal gland and throughout nephrogenesis.

The Wilms' Tumour gene WT1 has important functions during development. Knock-out mice were shown to have defects in the urogenital system and to die at embryonic day E13.5, probably due to heart failure. Using a lacZ reporter gene inserted into a YAC construct, we demonstrate that WT1 is expressed in the early proepicardium, the epicardium and the subepicardial mesenchymal cells (SEMC). Lack of WT1 leads to severe defects in the epicardial layer and a concomitant absence of SEMCs, which explains the pericardial bleeding and subsequent embryonic death observed in Wt1 null embryos. We further show that a human-derived WT1 YAC construct is able to completely rescue heart defects, but only partially rescues defects in the urogenital system. Analysis of the observed hypoplastic kidneys demonstrate a continuous requirement for WT1 during nephrogenesis, in particular, in the formation of mature glomeruli. Finally, we show that the development of adrenal glands is also severely affected in partially rescued embryos. These data demonstrate a variety of new functions for WT1 and suggest a general requirement for this protein in the formation of organs derived from the intermediate mesoderm.

Constitutive expression of the Wilms’ tumor gene WT1 inhibits the differentiation of myeloid progenitor cells but promotes their proliferation in response to granulocyte-colony stimulating factor (G-CSF)

Bone marrow (BM) cells that were concentrated for hematopoietic progenitor cells by in vivo treatment with 5-FU were infected with a recombinant retrovirus containing a human full-sized, non-spliced type WT1 (Wilms’ tumor gene 1) cDNA and then colony-assayed in the presence of granulocyte-colony stimulating factor (G-CSF). Significantly more colony-forming units granulocyte-monocyte (CFU-GM), colony-forming units granulocyte (CFU-G), and colony-forming units monocyte (CFU-M) colonies were formed in response to G-CSF from the BM cells infected with the WT1-containing retrovirus than from the control BM cells infected with an empty vector. Furthermore, FACS analysis of cell surface differentiation markers showed the inhibition of differentiation by constitutive WT1 expression resulting from the infection with the WT1-containing retrovirus. These results thus showed that the constitutive WT1 expression promoted the proliferation of myeloid progenitor cells but inhibited their differentiation in response to G-CSF, suggesting the alteration of G-CSF signaling pathway. The results also supported our hypothesis that the WT1 gene performs an oncogenic rather than a tumor suppressor gene function in hematopoietic progenitor cells, although the WT1 gene potentially performs both functions. This finding implies an important role of the WT1 gene in leukemogenesis.

The Wilms' tumor gene WT1 is a good marker for diagnosis of disease progression of myelodysplastic syndromes.

The Wilms' tumor gene, WT1, is a tumor marker for leukemic blast cells. The WT1 expression levels were examined for 57 patients with myelodysplastic syndromes (MDS) (refractory anemia (RA), 35; RA with excess of blasts (RAEB) 14; RAEB in transformation (RAEB-t), six; and MDS with fibrosis, two) and 12 patients with acute myeloid leukemia (AML) evolved from MDS. These levels significantly increased in proportion to the disease progression of MDS from RA to overt AML via RAEB and RAEB-t in both bone marrow (BM) and peripheral blood (PB). WT1 expression levels in PB significantly correlated with the evolution of RAEB or RAEB-t to overt AML within 6 months. Therefore, WT1 expression levels in PB were superior to those in BM for early prediction of the evolution to AML by means of quantitation of the WT1 expression levels. Furthermore, WT1 expression in PB of patients with overt AML evolved from MDS was significantly decreased by effective chemotherapy or allogeneic stem cell transplantation and became undetectable in long-term survivors. These results clearly showed that WT1 expression levels are a tumor marker for preleukemic or leukemic blast cells of MDS and thus reflect the disease progression of MDS. Therefore, monitoring of WT1 expression levels has made continuous assessment of the disease progression of MDS possible, as well as the prediction of the evolution of RAEB or RAEB-t to overt AML within 6 months. The results also showed that quantitation of WT1 expression levels is useful for diagnosis of minimal residual disease of MDS with high sensitivity, thus making it possible to evaluate the efficacy of treatment for MDS.

Expression of the Wilms' Tumor Gene WT1 in Solid Tumors and Its Involvement in Tumor Cell Growth

To determine the role of the Wilms' tumor gene WT1 in tumorigenesis of solid tumors, expression of the WT1 gene was examined in 34 solid tumor cell lines (four gastric cancer cell lines, five colon cancer cell lines, 15 lung cancer cell lines, four breast cancer cell lines, one germ cell tumor cell line, two ovarian cancer cell lines, one uterine cancer cell line, one thyroid cancer cell line, and one hepatocellular carcinoma cell line) by means of quantitative reverse transcriptase‐polymerase chain reaction. WT1 gene expression was detected in three of the four gastric cancer cell lines, all of the five colon cancer cell lines, 12 of the 15 lung cancer cell lines, two of the four breast cancer cell lines, the germ cell tumor cell line, the two ovarian cancer cell lines, the uterine cancer cell line, the thyroid cancer cell line, and the hepatocellular carcinoma cell line. Therefore, of the 34 solid tumor cell lines examined, 28 (82%) expressed WT1. Three cell lines expressing WT1 (gastric cancer cell line AZ‐521, lung cancer cell line OS3, and ovarian cancer cell line TYK‐nu) were further analyzed for mutations and/or deletions in the WT1 gene by means of single‐strand conformation polymorphism analysis. However, no mutations or deletions were detected in the region of the WT1 gene ranging from the 3/end of exon 1 to exon 10 (the WT1 gene consists of 10 exons) in these three cell lines. Furthermore, when AZ‐521, OS3, and TYK‐nu cells were treated with WT1 antisense oligomers, the growth of these cells was significantly inhibited in association with a reduction in WT1 protein levels. Furthermore, constitutive expression of the transfected WT1 gene in cancer cells inhibited the antisense effect of WT1 antisense oligomer on cell growth. These results indicated that the WT1 gene plays an essential role in the growth of solid tumors and performs an oncogenic rather than a tumor‐suppressor gene function.

Frasier syndrome is caused by defective alternative splicing of WT1 leading to an altered ratio of WT1 +/-KTS splice isoforms.

The Wilms' tumor gene WT1 plays a key role in genitourinary development and subsequent normal function. Homozygous mutations of WT1 can be found in approximately 15% of Wilms' tumors. Furthermore, somatic heterozygous loss of WT1 is known to lead to cryptorchidism and hypospadias in males. A much more severe phenotype is seen in patients with Denys-Drash syndrome which results from heterozygous dominant-negative mutations of the gene. Characteristic features are mesangial sclerosis with early kidney failure, varying degrees of gonadal dysgenesis and high risk of Wilms' tumors. Here we show that a related disease, Frasier syndrome, characterized by focal glomerular sclerosis, delayed kidney failure and complete gonadal dysgenesis, is probably caused by specific intronic point mutations of WT1 that preferentially affect a CpG dinucleotide. Disruption of alternative splicing at the exon 9 splice donor site prevents synthesis of the usually more abundant WT1 +KTS isoform from the mutant allele. In contrast to Denys-Drash syndrome, no mutant protein is produced. The splice mutation leads to an imbalance of WT1 isoforms in vivo , as detected by RT-PCR on streak gonadal tissue. Thus, WT1 isoforms must have quite different functions, and the pathology of Frasier syndrome suggests that especially gonadal development may be particularly sensitive to imbalance or relative underrepresentation of the WT1 +KTS isoform.

Desmoplastic Small Round Cell Tumor: RT-PCR Analysis and Immunohistochemical Detection of the Wilm's Tumor Gene WT1

Desmoplastic small round cell tumor is an aggressive neoplasm first described in 1991. Recently, a reciprocal translocation t(11;22)(p13;q12) has been characterized by conventional cytogenetic studies and molecular analysis. This translocation involves the Ewing's sarcoma gene on chromosome 22 and the Wilms' tumor gene WT1 on chromosome 11. The chimeric transcript corresponding to the fusion gene could be detected by the reverse transcriptase-polymerase chain reaction (RT-PCR). Using an anti-WT1 antibody, the WT1 part of the putative chimeric protein could be recognized by immunohistochemistry. We describe two well-characterized cases of intraabdominal desmoplastic small round cell tumor in two male patients aged 14 and 28 with both RT-PCR analysis and immunostaining for WT1. In this report, we insist on the necessity to increase the RT-PCR analysis in DSRCT in order to obtain a precise differential diagnosis. In addition, WT1 immunostaining may serve as a useful diagnostic marker for DSRCT.

Differential Effects of Wilms Tumor WT1 Splice Variants on the Insulin Receptor Promoter

The Wilms tumor gene WT1 has been implicated in the early development of the kidney. Mutations in WT1 are found in a small fraction of Wilms tumor, a pediatric nephroblastoma, and Denys–Drash syndrome, characterized by genitourinary abnormalities. The WT1 gene product functions as a transcriptional repressor of growth factor-related genes. The kidney is one of the major sites of insulin actionin vivoand expresses high levels of insulin receptors (IR). IR expression has been detected during early embryogenesis, suggesting that it may play a role in development. We investigated whether two WT1 splice variants lacking or including a three-amino-acid (KTS) insertion between the third and fourth zinc finger in the DNA-binding domain could repress the IR promoterin vitro.We show that the +KTS variant effectively represses promoter activity under all conditions tested but the −KTS variant was only able to repress in the presence of cotransfected C/EBPβ or a dominant-negative p53 mutation. Deletional mapping indicated that distinct regions of the IR promoter mediated the effects of the two isoforms and DNaseI footprint analysis identified potential WT1 binding sites within these regions.

WT1 contributes to leukemogenesis: expression patterns in 7,12-dimethylbenz[a]anthracene (DMBA)-induced leukemia.

The Wilms'-tumor gene WT1 may have a different function from a tumor-suppressor gene in some leukemias. Using the 7,12-dimethylbenz[a]anthracene (DMBA)-induced rat leukemia system, we examined whether WT1 expression was involved during leukemogenesis, since this model enabled us to analyze cells altered by DMBA at various stages of leukemogenesis. By the semi-quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) method, WT1 expression was detected in 15 (71%) of 21 DMBA-induced erythroblastic leukemias. Among 15 WT1-expressing leukemias, GATA-1, which is an erythroid-specific transcription factor and might regulate WT1 expression, was also expressed in 13 cases (p < 0.05). On the other hand, WT1 expression was not detected in any normal or early pre-leukemic rats and was detected in 1 of 8 rats in late pre-leukemic stages. These results showed that cells with a high expression level of WT1 tended to develop into leukemia and that WT1 contributed to leukemogenesis in the late stage, suggesting that the expression of WT1 plays an important role in cell proliferation and in maintaining the viability of some leukemia cells.