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WT1 Induces Expression of Insulin-Like Growth Factor 2 in Wilms' Tumor Cells'

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WT1 Induces Expression of Insulin-Like Growth Factor 2 in Wilms' Tumor Cells' ICANCER RESEARCH55, 4540-4543. October tS, 19951 Advances in Brief WT1 Induces Expression of Insulin-like Growth Factor 2 in Wilms' Tumor Cells' Kim E. Nichols, Gian G. Re, Yu Xin Yan, A. Julian Garvin, and Daniel A. Haber@ Laboratory of Molecular Genetics, Massachusetts General Hospital Cancer Center and Harvard Medical School, Charlestown, Massachusetts 02129 (K. E. N., Y. X. Y., D. A. HI, and Department of Pathology and Li.iboratory Medicine, Medical University ofSouth Carolina, Qiarleston, South Carolina 1G. G. R., A. J. G.] Abstract by the observation that naturally occurring point mutations arising within the transactivation domain of WT1 convert the encoded protein The Wilma' tumor suppressor gene WTJ encodes a zinc finger tran from a transcriptional repressor to a potent activator of the EGR1 scription factor, whose expression inhibits the growth of the RM1 Wilma' promoter in 3T3 cells (7, 16, 17). The nature of the recipient cells tumor cell line. Transient transfection of WT1 constructs into 3T3 or 293 cells results in transcriptional repression of a number of cotransfected themselves is an important factor in these transient transfection stud promoters containing the early growth response gene 1 consensus se ies. We have demonstrated recently that WT1 and p53 proteins are quence. We now show that WT1 has properties of a transcriptional associated in vivo and that transfection of WT1 into cells with a activator in RM1 cells, an effect that may be associated with the presence deleted p53 gene results in transcriptional activation, an effect that is of a mutated p53 gene In these cells. Stable transfection of wild-type WT1 suppressed by the reintroduction of wild-type p53 (18). Thus, the into RM1 cells results in induction of endogenous insulin-like growth transactivational properties of WT1 appear to be complex, involving factor2 (IGF2)butnotof otherpreviouslypostulatedWT1-targetgenes. specific domains within the protein itself, as well as potential inter The induction of IGF2 is dramatically enhanced by WT1 mutants encod actions with other cellular proteins. ing an altered transactivation domain. We conclude that IGF2 is a poten To define further the transactivational properties of WT1 in an tinily physiological target gene for WT1 and that its induction may appropriate cell type, we analyzed the effect of WT1 expression in the contribute to the growth-stimulating effects of WT1 variants. RM1 Wilms' tumor cell line. This cell line, derived from an anaplastic Introduction Wilms' tumor, has the unique property of indefinite passage in vitro, while retaining the ability to produce tumors with characteristic Wilms' tumor, a pediatric kidney cancer, has been linked to the Wilms' histology following inoculation into nude mice (7, 19). The inactivation of a tumor suppressor gene, WTJ, at the chromosome endogenous WT1 transcript in these cells is expressed at very low I lpl3 locus (reviewed in Ref. 1). WT1 encodes a Cys-His zinc finger levels and is comprised ofthe WT1/del2 variant, an aberrantly spliced protein that is normally expressed in cells of the developing genito transcript that is found in a subset of Wilms' tumors and encodes a urinary system (2—4).Mutations inactivating WT1 have been detected protein with altered transactivational activity (7). We now demon in —10% of sporadic Wilms' tumors (5, 6), and reintroduction of strate that transient transfection of WT1 into RM1 cells, which harbor wild-type WT1 into Wilms' tumor cells expressing an aberrant WT1 a p53 mutation, results in transcriptional activation of a reporter transcript results in growth suppression (7). WT1 has properties of a construct containing the EGR1 consensus site, rather than transcrip transcriptional repressor, based on transient transfection experiments tional repression. Stable transfection of wild-type WT1 in these cells in 3T3 and 293 cells (8). These properties are differentially mediated is associated with an increase in the expression of endogenous IGF2 by alternative splicing variants of wild-type WT1 (9). Whereas the but not of other postulated WT1 target genes with EGR1-containing four zinc finger domains of WT1 bind the 5'-CGCCCCCGC-3' DNA promoters. Transfection of naturally occurring WT1 variants encoding consensus sequence shared by the EGR13 gene product, insertion of an altered transactivation domain leads to a dramatic induction of an alternatively spliced KTS sequence between zinc fingers 3 and 4 endogenous IGF2 mRNA. These results suggest that only a subset of abolishes this DNA-binding activity (10). EGR1-containing, WTI-responsive promoters may be physiologically The EGR1 consensus is found upstream of many transcriptional regulated by WT1. Although we do not detect transcriptional repres start sites, leading to the identification of a number of promoters that sion of endogenous IGF2 by wild-type WT1, the potent induction of bind in vitro-translated WT1 and are repressed in transient trans this growth-inducing gene by naturally occurring WT1 mutants fection assays. WT1 has, therefore, been identified as a potential suggests that these altered proteins may actively contribute to cell transcriptional repressor of genes such as EGR1 (8), IGF2 (1 1), transformation. insulin-like growth factor 1 receptor (12), PDGF-A (13, 14), colony stimulating factor 1 (15), among others. Although transcriptional Materials and Methods suppression of the endogenous genes themselves has not been dem Culture of RM1 Cells. The generation of RM1 cells (also called W4) has onstrated, these experiments have led to the concept that the tumor been described elsewhere (19). RM1 cells were maintained as tumor explants suppressor properties of WT1 might result from its ability to reduce in nude mice by serial s.c. passage. For growth in vitro, tumors were minced expression of growth-inducing gene products. This model is supported and adapted to growth on a collagen matrix and then on standard tissue culture plates and grown in DMEM, 10% FCS, and supplemental glutamine. Sequence Received 7/6/95; accepted 8/30/95. analysis of the WTI transcript has been described previously (7). For p53 The costs of publication of this article were defrayed in part by the payment of page mutational analysis, the p53 transcript was reverse transcribed using random charges. This article must therefore be hereby marked advertisement in accordance with oligonucleotide primers and subjected to PCR amplification using primers 18 U.S.C. Section 1734 solely to indicate this fact. spanning the coding region. The amplified eDNA was analyzed by automated I This grant was supported by NIH Grants CA 37887 (to A. i. G.) and CA 58596 (to D. A. H.). sequencing (ABI), and the mutation observed was confirmed by sequencing 2 To whom requests for reprints should be addressed, at Massachusetts General multiple independent clones. Hospital Cancer Center, CNY 7, Building 149, 13th Street, Charlestown, MA 02129. CAT Assays. To test the transactivational properties of WT1 in RM1 cells, Phone: (617) 726-7805; Fax: (617) 726-5637. cells were transfected by calcium phosphate DNA precipitation with CMV 3 The abbreviations used are: EGRI, early growth response gene 1; IGF2, insulin-like growth factor 2; PDGF, platelet-derived growth factor; CAT, chloramphenicol acetyl driven constructs encoding murine WT1 (with or without the KTS alternative transferase; CMV, cytomegalovirus; RT, reverse transcription. splice), along with the p3X-EGR1-CAT reporter, containing three tandem 4540 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 1995 American Association for Cancer Research. WF1.INDUCED IGF2 IN WILMS' TUMOR CELLS a) EGRI consensus sites upstream of a minimal promoter (10). The total amount (1) of CMV promoter sequence transfected into the cells was equalized with the Ca) 1@ Cd 0 addition of vector DNA, and transfection efficiency was standardized using a cnm@ ci) cotransfected human growth hormone reporter construct (Nichols Institute). @zzz CAT assays were performed by the method of Gorman et a!. (20), and CAT activity was quantitated by scintillation counting of appropriate sections of the TLC plate. Northern Analysis. Total cellular RNA was prepared by extraction with guanidine isocyanate and electrophoresed on a 1% agarose/formaldehyde gel, wT1-j'. followed by transfer to a Genescreen membrane (Amersham). Northern hy bridizations were performed under standard conditions, using eDNA probes for v5rr1, IGF2, EGR1, PDGF-A, Pax 2, and phosphoglutaraldehyde dehydrogen ase (PGAD). Results IGF2 Transcriptional Activation of a WT1-Target Promoter in RM@ Cells. To examine transcriptionalregulationby WT1 in Wilms' tu mor cells, we transfected CMV-driven expression constructs into RM1 cells, along with a reporter containing a WT1 target site. WT1 lacking the KTS alternative splice, WT1(—KTS), has been shown to repress transcription from a promoter containing three tandem EGR1 PGAD — consensus sites (p3X-EGR1-CAT) by 2—3-fold,following transfection into 3T3 cells (10). In contrast, transient transfection of WT1(—KTS) into RM1 cells resulted in 5-fold transcriptional activation of this Fig. 2. Induction ofendogenous IGF2 mRNA in RM1 cells expressing WT1. Northern reporter. Insertion of the KTS splice within the WT1 zinc finger blot of total mRNA from four independent RMI-derived clones, stably transfected with domain, which abrogates binding to the EGR1 consensus, also re antisense WT1, Wi'! lacking the KTS splice (NB! and NB2), and vector alone (Neo), and grown as explants in nude mice. Blots were hybridized with probes for WT1, IGF2, and duced transactivation of the reporter (Fig. 1). PGAD (loading control). The transactivational properties of WT1 appear to be modulated by a number of factors, including promoter structure, cellular context, and the status of p53 (18, 21). To determine the sequence of endog with a role in tumor progression (22, 23).@The absence of wild-type enous p53 in RM1 cells, we amplified the endogenous p53 transcript p53 in RM1 cells may affect the transactivational properties of WT1, by RT-PCR, followed by direct automated sequencing of the ampli as demonstrated for fibroblasts expressing a temperature-sensitive p53 fled product.
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