The role of Wilms' tumor genes Masao Hirose Division of Transfusion Medicine, The University of Tokushima School of Medicine, Tokushima, Japan Abstract:The constitutional chromosomal deletion within the short arm of one copy of chromosome 11, at band p13, which often correlated with WAGR syndrome consisting of Wilms' tumor with aniridia, genitourinary malformation, and mental retardation, provided the first clue to the genetic events in the development of Wilms' tumor. WT1 gene is encoded by 10 exons, resulting in messenger RNA subject to a complex pattern of alternative splicing. WT1 gene encodes a zinc finger transcription factor, which binds to GC-rich sequences and functions as a transcriptional activator or repressor for many growth factor genes. WT1protein is mainly expressed in developing kidney, testis, and ovary, indicating that it is involved in the differentiation of genitourinary tissues, all thought to be the sites of origin of Wilms' tumor. The point mutation of WT1 results in Denys-Drash syndrome. The other Wilms' tumor gene, WT2 at 11p15.5, is linked to Beckwith-Wiedemann syndrome. The possibility that WT1 is involved in the etiology of rhabdoid tumor of the kidney was discussed. WT1 is expressed in immortalized hematologic cells such as EBV-LCL and hematologic malignancies, but not in PBL or IL-2L. High level WT1 expression in leukemia cells and a poor prognosis are linked in patients with leukemia, making the gene a novel marker for leukemia cells. A correlated expression between WT1 and mdr-1 in vincristine resistant cells indicates a close relation with multi-drug resistance and is a promising diagnositic marker for chemoresistance in hematologic malignancies. J. Med. Invest. 46:130-140, 1999 Keywords:WT, tumor suppressor gene, Wilms' tumor, rhabdoid tumor, leukemia, chemoresistance Etiology of WAGR syndrome In1964, Miller et al. reported an association of Wilms' tumor with aniridia, hemihypertrophy and other congenital malformations (1). A new disease entity, WAGR syndrome consisting of Wilms' tumor with aniridia, genitourinary malformations, and mental retardation was subsequently proposed (2). Wilms' tumor is the most common childhood intraabdominal solid tumor of the kidney (3). Thereafter, Riccardi et al. found a correlation between WAGR syndrome and karyotypic abnormality within the short arm of one copy of chromosome11, at band p13 (4). This constitutional chromosomal deletion provided the first clue to the genetic events in the development of Wilms' tumor. In1971, Knudson compared cases showing the earlier age of onset and the bilateral presentation of retinoblastomas (RB) in children with a family history of this disease with those showing the unilateral tumors and proposed a model to explain the etiology of this disease, the two-event hypothesis (5). This model predicts that tumor formation depends on two-rate limiting genetic events. Tumorigenesis in children who have a constitutional lesion, either inherited from a parent or resulting from a spontaneous mutation, needed only one new genetic event. Contrary to this, sporadic cases required two independent somatic mutations. A similar model for Wilms' tumor was proposed later (6). Subsequent genetic studies of a number of tumors confirmed that the two postulated genetic events are caused by the inactivation of both alleles of a tumor suppressor gene (7). Now, that Wilms' tumor is caused by the Wilms' tumor suppressor gene WT1 is demonstrated. The Wilms' tumor gene related diseases are summarized in Table1. Structure and regulatory mechanisms of WT1 The WT1 gene that is mapped to chromosome locus 11p13 is encoded by 10 exons, resulting in messenger RNA subject to a complex pattern of alternative splicing (8, 9). The WT1 promoter has three transcription initiation sites and the gene is a member of the GC-rich, TATA-less and CCAAT-less class of RNA polymerase II genes (10, 11). The WT1 transcript is - 3.5 kb long and encodes a zinc-finger protein, WT1, with a predicted molecular weight Mr of 47kd to 49kd, depending on the presence or absence of two alternatively spliced exons. The first alternative splicing introduces exon 5, encoding 17 amino acids (+17aa), just proximal to the first of four zinc fingers. The second results in an insertion of three amino acids (+KTS) between the third and fourth zinc fingers. The most prevalent WT1 mRNA variant has both of the insertions present (+KTS and +17aa), and the least common form is missing both insertions (-KTS and -17aa) (9). The insertion of three amino acids (+KTS) disrupts the distance between zinc fingers 3 and 4 and thus alters its DNA-binding specificity (10). The four patterns of alternative splicing are shown in Figure1. The elements responsible for regulating the tissue specific expression of WT1 are not known, although Sp1 and GATA-1 have been shown to positively modulate WT1 expression (11-13). In addition, a cell type-specific enhancer has been identified within the 3'end of the human WT1 gene (14). Recently, Pax 2 and Pax 8 were shown to contribute to the regulation of WT1. When Pax 2 and Pax 8 expression becomes maximal, WT1 levels begin increasing (15). Recently, it was reported that the ectopic expression of p50 and p65 subunits of NF-κB stimulated the murine WT1 promoter activity by 10-30-fold in a transient transfection assay (16). Subsequently, the regulators or signal cascades that could modulate the function of WT1 were studied. Evidence was obtained that WT1 protein expressed exogenously in fibroblasts was phosphorylated in vivo and that treatment with forskolin, which activates the cAMP-dependent protein kinase (PKA) in vivo, induced phosphorylation of additional sites at Ser-365 and Ser-393 in WT1, resulting in abolishment of the DNA binding capacity of WT1 in vitro (17). In addition, Maheswaran and coworkers presented evidence that p53 physically interacts with WT1 in transfected cells and that WT1 transcriptional activity might be modulated by p53 (18). Whereas in NIH-3T3 cells wild type WT1 repressed expression, its transfection into Saos-2 cells, which lack endogenous p53, resulted in increased transcription from a promoter into which epidermal growth factor receptor 1 (EGR-1) consensus binding sites had been added (18). However, the WT1 transcriptional activity was independent of p53 genotype in the induction of P3 promoter activity of mouse insulin-like growth factor II gene (IGF-2) in primary cultures derived from p53 wild type (p53+/+) and knock-out (p53+/-) mouse embryo (19). There is a report that upregulation of WT1 expression is associated with an expression marker for the differentiation into monocytes/macrophages of c-fms and Mac-1 in murine myeloblastic cell line, M1, cells cultured in leukemia inhibitory factor (LIF), resulting in apoptotic cell death (20). Functions of WT1 The WT1 gene encodes a zinc finger transcription factor, which binds to GC-rich sequences (5'-GCGGGGGCG-3') and functions as a transcriptional activator or repressor (21). It represses transcription of growth factor [platelet derived growth factor-A (PDGF-A) chain, colony stimulating factor-1 (CSF-1), and IGF-2] and growth factor receptor (IGF-1R) genes, and the other genes [retinoic acid receptor-alpha (RAR-α), c-myc, and bcl-2](22-27). In addition, it's been known that WT1 mediates the expression of genes such as EGR-1, EGF-R, inhibin-α, Pax2, transforming growth factor-beta (TGF-β) and WT1 itself (28-33). The transient transfection of WT1 constructs into NIH-3T3 or 293 cells results in the transcriptional repression of a number of co-transfected promoters containing the EGR-1 consensus sequence (34). The EGR-1 consensus is found upstream of many transcriptional start sites, leading to the identification of a number of promoters that bind in vitro-translated WT1 and are repressed in transient transfection assay. Haber and coworkers transfected the four wild type WT1 isoforms into RM1 human anaplasia of the kidney cell line, a variant form of Wilms'tumor, inoculated into nude mice and observed that each isoform independently suppressed the emergence of colony formation (35). Subsequently, they found that stable transfection of WT1 into RM1 cells results in induction of endogenous IGF-2 but not of other previously postulated WT1 target genes (34). WT1 is expressed only in specific types of cells, which is a major difference from RB or p53 (36). The pattern of normal WT1 expression has provided important clues to the function of WT1 during differentiation. The WT1 protein is mainly expressed in developing kidney, testis, and ovary, indicating that it is involved in the differentiation of genitourinary tissues. Other organs expressing the protein are spleen and the methothelial cells of heart, lung and abdomen (36-38). In the kidney, WT1 is expressed only in condensing blastemic cells, renal vesicles, and glomerular epithelium, all thought to be sites of origin of Wilms'tumor. Renal WT1 expression peaks around the time of birth and then rapidly declines as the organ matures (37, 38). In contrast to its transient expression in the developing kidney, WT1 is expressed continuously in methothelial cells, sertoli cells of testis, and granulosa cells of the ovary (39). The critical developmental role of WT1 is evident in the severe genitourinary and methothelial abnormalities of mice whose WT1 alleles have been deleted. WT1 knock-out mice die before birth with failure of kidney and gonadal development in addition to hypoplasia of the heart and lungs (40). The WT1 protein is present in normal breast tissue and appears to be developmentally regulated in that a high percentage of breast tumor cells express little or no WT1 protein. It is known that breast tumor growth is controlled by the genes encoding components of the IGF and TGF-β signaling system and, recently, altered expression of WT1 in breast cancer was also reported (41). WT2 at 11p15 The second Wilms'tumor gene was identified at chromosome locus 11p15 and designated WT2 (42, 43). This locus is linked to the Beckwith-Wiedemann syndrome (BWS), showing manifestations such as visceromegary, macroglossia, or hemihypertrophy in some patients (43, 44). p57 was recently identified as a cyclin-dependent kinase (CDK) inhibitor, which is found to have the strongest tumor suppressor activity among the genes of the p21 family (45, 46). As shown in Figure 2, like related family members such as p21 and p27, p57 binds to several G1-cyclin/CDK complexes and arrests the cell cycle at the G1 phase (47). Human p57 is found at chromosome locus 11p15.5, a region implicated in the etiology of embryonal tumors including Wilms'tumor and BWS. Frequent loss of 11p15.5 in Wilms' tumor indicates that WT2 is located in this region (45, 48). Role in rhabdoid tumor of the kidney The author established two cell lines of rhabdoid tumor of the kidney (RTK), SWT-1 and SWT-2, as shown in Figure 3 (49). RTK was once proposed to be a variant of Wilms'tumor and subsequently segregated from an unfavorable type of sarcomatous renal tumor that was grouped with clear cell sarcoma (CCSK) and anaplasia of the kidney (50-53). It is well known that RTK has a much poorer prognosis than Wilms'tumor (50, 52, 53). In an attempt to identify the etiology of RTK, the mRNA expression of genes including WT1, p57, IGF-2, p53, N-myc, and c-myc was tested using reverse transcriptase-polymerase chain reaction (RT-PCR) in the two cell lines. The expressions were compared with those in a biopsied kidney tissue and a normal mesangeal cell line, MCP-3. Coinciding with the karyotype of SWT-2 cell lines, del 11p13 as shown in Figure 4, no mRNA expression of WT1 was found. Interestingly, SWT-1, which showed a normal karyotype at least on the G-banding analysis, also lacked the WT1 expression as shown in Figure 5. In addition, SWT-2 lacked the expression of two genes, p57 and IGF-2, while SWT-1 expressed them. Both N-myc and c-myc expression was detected in the two cell lines (data not shown). These results strongly suggest that WT1 is involved in the etiology of RTK. However, transient transfection of WT1 did not reduce colony formation in the other RTK cell line, SM2 (35). The etiology of RTK may need other genetic abnormalities in addition to WT1. The presence of p57 mRNA in SWT-1 implies that WT2 is not involved in the etiology of this disease. Other functions of WT1 cDNA constructs encoding the four human WT1 splice variants caused programmed cell death, which was associated with a reduced synthesis of EGF-R in human osteosarcoma cell lines, U2OS and Saos-2 (54). The effect of this assay was independent of the presence of the p53 gene. However, the nature of the recipient cells themselves is an important factor in these transient transfection studies. WT1 and p53 proteins are associated in vivo and transfection of WT1 into cells deleted of the p53 gene results in transcriptional activation, an effect that is suppressed by the reinduction of wild-type p53 (18).WT1 was transiently transfected into the p53-negative Hep3B and the p53-positive Hep2G hepatoma cell lines, resulting in the induction of apoptosis in both cell lines by the wild type splice variant, whereas the WT1 (+KTS) isoform did not induce apoptosis (55). This result is independent of the effect of p53, which seems to contradict a previous publication in which the expression of the WT1 (-/-) isoform was reported to suppress p53-induced apoptosis in both U2OS and E1A-transformed baby rat kidney (18). Thus, the observation that the effect of the WT1 isoforms is cell type dependent may explain these discrepancies. Subsequently, it was demonstrated that inducible expression of WT1 in osteosarcoma cells triggers programmed cell death through an effect that is associated with transcriptional repression of endogenous EGFR. This WT1 mediated apoptosis was preceded by induction of the CDK inhibitor p21, associated with G1 phase arrest(56). 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, from which inhibition of the activity of cyclin/CDK complexes may be involved in mediating the WT1-induced cell cycle block (57). Mutation of WT1 Mutations in the WT1 gene underlie5 % to 10 %of cases of sporadic Wilms'tumor (58). Athough the majority follow Knudson's two-hit hypothesis for tumor suppressor genes, it is now clear that a substantial minority ( - 30 %) of Wilms'tumors retain one normal WT1 allele, suggesting that in some cases, heterozygous mutation is sufficient for tumorigenesis. Five different types of mutations are commonly found in Wilms'tumors:large deletions of part of the gene, nonsense or frameshift mutations affecting amino acids in the zinc fingers critical for DNA binding, missense mutations affecting the putative activation or repression domains, and mutations preventing correct soliciting. Approximately 75 % of the WT1 mutations found in sporadic Wilms'tumor produce a truncated protein, whereas missense mutations in the zinc finger region predominate in Denys-Drash syndrome (DDS)(58).Wilms'tumor patients have an increased frequency of leukemias as second primary tumors, some of which may be due to WT1 mutation (59). The biological significance of DNA binding and transcriptional regulation by WT1 is underscored by the finding of small deletions and point mutations in the WT1 zinc-fingers that abolish DNA binding in a number of Wilms'tumors, especially in tumors associated with the DDS (60). Consequently, the ability of the mutant WT1 allele, containing an in-frame deletion within the DNA-binding domain, to transform baby rat kidney was tested. The mutant WT1 gene was found to cooperate with the adenoviral E1A gene in transforming baby rat kidney cells, whereas the wild-type WT1 gene in all of its alternatively spliced forms neither suppressed E1A-induced focus formation in soft agar nor cooperated with E1A (61). Expression in hemopoetic cells The WT1 gene is strongly over-expressed in leukemic blasts compared with immature hematopoetic progenitors with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, making it a novel marker for leukemia cells. The poor prognosis and the higher level of WT1 gene expression are linked in patients with leukemia. Thus, it was supposed that the aberrant over-expression of WT1 contributed to the pathogenesis of AML (62, 63). In addition, WT1 was shown to be useful in the monitoring of minimal residual disease in patients with hematologic malignancy. However, a consequential result that normal CD34 positive stem cells as well as acute myeloid leukemia cells equally express WT1 mRNA was obtained (64). It was reported that K562 cells down regulate WT1 mRNA during induced erythroid and megakaryocytic differentiation (65). The same phenomenon was observed in HL60 cells, suggesting that sustained high levels of WT1 are incompatible with differentiation (66). Likewise, the expression of WT1 inversely correlated with the differentiation level of acute leukemias (67). WT1 antisense oligomers inhibit the cell growth of both leukemic cell lines and fresh leukemic blasts from patients with acute leukemia or chronic myeloid leukemia, indicating that the WT1 protein may be important for the sustained proliferation of leukemia cells (68). Contrary to these findings, the monoblastic cell line, U937, constitutively expressing either of the isoforms, (-KTS) or (+KTS), did not respond to differentiation induction by retinoic acid or vitamin D3, whereas the cell line unable to express this gene responded to these substances (69). A recent result indicated that WT1 expression competes with the differentiation-inducing signal mediated by G-CSF receptor and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation (70). WT1 mutation is associated with a failure to achieve complete remission and a lower survival rate in AML, confirming that WT1 mutations underlie a similar proportion of cases of AML to that seen in Wilms'tumors and its normal role in hemopoesis is at a very early progenitor stage (71). Relation between WT1 and multidrug resistance (MDR) The drug resistance that is a major obstacle to cancer treatment develops at the initial stage of the therapy or during chemotherapy after relapse (72). p170-kd permeability-related glycoprotein (P-GP) encoded by the mdr-1 gene has ATPase activity which reduces the intracellular drug accumulation by increasing the efflux of anti-cancer drugs from cells (73, 74). p190-kd multidrug resistance-associated protein (MRP) encoded by the mrp gene is a member of the ATP-binding cassette super family of membrane transporter proteins that displays minor homology to P-GP(75). It is known that both P-GP and MRP are expressed in normal peripheral blood lymphocytes (PBL), monocytes, and granulocytes as well as cells of hematologic disease. However, the spectrum of cells expressing P-GP is narrow as compared with those expressing MRP, which exists in a wider spectrum of cells or tissues than P-GP (76-79). In recent years, a 110-kd lung resistance-related protein (LRP) has been found(80), which is reported to be expressed in monocytes among the bone marrow cells (81). In an attempt to understand the cause of the poor prognosis of leukemia patients whose leukemia cells expressed a high level of WT1, the author investigated the relation between WT1 and multidrug resistance (MDR). The expression level of the WT1 gene in human hemopoetic cells such as peripheral blood lymphocytes (PBL), interleukin-2 activated lymphocytes (IL-2L), the lymphoblastoid cell lines immortalized by EBV (EBV-LCL), and the cultured cell lines derived from human hematologic malignancies was compared with that of mdr-1, mrp, and lrp in Table 2. Positive expressions of MDR-related genes as above were detected in all PBL, IL-2L, and EBV-LCL, while the WT-1 expression was found in immortalized cell lines such as EBV-LCL and hematologic malignancies. 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Received for publication May 25, 1999;accepted June 24, 1999. Address correspondence and reprint requests to Masao Hirose, M.D., Division of Transfusion Medicine, The University of Tokushima School of Medicine, Kuramoto-cho, Tokushima770-8503, Japan and Fax:+88-633-7211.