©1996 by The American Society for Biochemistry and Molecular Biology, Inc.
LLC-PK Cell Growth Is Repressed by WT1 Inhibition of G-protein Protooncogene Transcription (*)

(Received for publication, October 17, 1995)

T. Bernard Kinane Jonathan D. Finder Akira Kawashima Dennis Brown Mauro Abbate William J. Fredericks (1) Vikas P. Sukhatme (2) Frank J. Rauscher III (1) Louis Ercolani (§)

From the  (1)Wistar Institute of Anatomy and Biology, Philadelphia, Pennsylvania 19104, (2)Renal and the Pediatric Pulmonary Units, Departments of Medicine and Pediatrics, Massachusetts General Hospital, Renal Unit, Beth Israel Hospital, Boston, Massachusetts 02114, and Harvard Medical School, Boston, Massachusetts 02129

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS AND DISCUSSION
FOOTNOTES
REFERENCES

ABSTRACT

The temporal expression of the early growth response gene (EGR-1) is one molecular mechanism for both maximal activation of the Galpha gene and accelerated growth in mitotically active predifferentiated LLC-PK(1) renal cells. These events are dependent on an enhancer area in the 5`-flanking region of the Galpha gene that contains an EGR-1 motif (5`-CGCCCCCGC-3`). However, acquisition of the polarized phenotype in LLC-PK(1) cells is accompanied by loss of EGR-1 expression and occupancy of the EGR-1 site by nuclear binding proteins other than EGR-1. We now demonstrate that one of these binding proteins is the Wilms' tumor suppressor (WT1). Furthermore, the temporal expression of WT1 in LLC-PK(1) cells acquiring the polarized phenotype represses both Galpha gene activation and growth in these cells. These findings suggest the existence of differentiation-induced pathways in LLC-PK(1) cells that alternatively abrogrates EGR-1 and promotes WT1 gene expression, thereby modulating a target protooncogene Galpha that is participatory for growth and differentiation in renal cells. These studies emphasize the usefulness of the LLC-PK(1) renal cell as a model to elucidate normal programs of genetic differentiation in which WT1 participates.


INTRODUCTION

Wilms' tumor (WT) (^1)is a pediatric nephric neoplasm arising from the continued proliferation of embryonic blastemal cells that fail to differentiate(1) . WT occurs in both sporadic and hereditary forms and as part of the WAGR syndrome (Wilms' tumor, aniridia, genitourinary abnormalities, and mental retardation)(2) . Chromosome 11p13 contains a region encoding the tumor suppressor gene WT1, which is commonly deleted in heritable WT(3, 4) . Internal deletions and mutations of the WT1 gene are found in some WTs (reviewed in (5) ). Although WT1 is expressed in uterus, spinal cord, spleen, abdominal wall musculature, and the mesothelial lining of organs within the thoracic cavity, its highest expression is in the developing urogenital system(6) . The WT1 gene encodes four splice variants of a transcription factor containing a glutamine/proline-rich N terminus and four carboxyl Cys(2)His(2) type zinc fingers, which recognize 5`-GCGGGGGCGGTG-3` and redundant 5`-TCC-3` cis motifs (7, 8, 9) . Alternative splice I allows the variable insertion of exon 5 between the transactivating and DNA binding domains of WT1. Alternative splice II allows the variable insertion of nine nucleotides encoding lysine, threonine, and serine (+KTS or -KTS) between the third and fourth zinc fingers(7) . WT1 cis motifs are also recognized by another Cys(2)His(2) type zinc finger transcription factor EGR-1 also known as NGF1-A, Krox 24, TIS-8, and Zif 268, which is induced after mitogenic and differentiation cues in several cell types (reviewed in (10) ). In contrast to EGR-1, WT1 appears to predominantly function as a transcriptional repressor of several growth-related genes including EGR-1, insulin-like growth factor II, platelet-derived growth factor alpha chain, insulin-like growth factor I receptor, colony-stimulating factor 1, transforming growth factor beta1, and Pax2 (reviewed in (11) ). WT1 mutations prevent DNA binding or transcriptional repressive functions that are postulated to allow the constitutive expression of growth factors leading to renal neoplasia. Consistent with this possibility, suppression of colony formation occurs in WT cells transfected with wild type WT1 isoforms(12) .

Further insights into molecular cascades linking WT1 to target genes in renal cells would clearly be advantageous. WTs occur with greater frequency in pediatric kidneys containing persistent renal stem cells, a condition designated nephroblastomatosis or nephrogenic rests. WT1 mutations occur in these rests, suggesting they may represent a transitional cell preceding malignancy(13) . Embryonal kidney cell tumors reminiscent of WT can be induced in rats given the alkylating agent N-nitroso-N`-methyl urea(14) . However, cultured cells have yet to be developed from these sources. We have previously identified genes participatory for epithelial cell growth and differentiation events in LLC-PK(1) cells, an extensively characterized cultured epithelial cell line derived from juvenile male pig kidney(15) . These studies indicated that as in lower eukaryotic organisms such as Dictyostelium(16, 17, 18) and Drosophila(19) , heterotrimeric guanine nucleotide binding (G) proteins are involved in signal transduction pathways required for both growth and cellular differentiation programs in renal cells.

G proteins are composed of individual alpha, beta, and subunits that are encoded by gene superfamilies that have been conserved by eukaryotes throughout evolution (reviewed in (20) ). Most of the transducing activities of G proteins in mammalian cells are associated with the state of activation of the alpha subunit, which is involved in GDP/GTP exchange and GTP hydrolysis (reviewed in (21) and (22) ). G proteins can alter cell growth or differentiation by participation in growth factor receptor signaling pathways that converge in the nucleus to alter gene expression. Mutations in Galpha comparable with Ha-ras GP21, which decrease GTPase activity, are found in tumors of the adrenal cortex and ovary(23) . Such mutations, which convert the Galpha gene into the oncogene gip2 induce increased growth and oncogenic transformation in Rat-1a cells(24) . Increased growth may be a consequence of persistent activation of pathways coupled to mitogen-activated protein kinase (25) . The Galpha subunit interacts with pathways required for differentiation of F9 teratocarcinoma cells(26) . Even modest repression of Galpha expression is associated with renal developmental and morphologic abnormalities in transgenic mice, underscoring its important role in renal differentiation events(27) . Polarized LLC-PK(1) cells contain two Galpha(i) isoforms, Galpha and Galpha, which are involved, respectively, in the regulation of hormone-stimulated adenylyl cyclase and constitutive proteoglycan secretion through the Golgi complex(28, 29) . The genes encoding both Galpha(i) subunits are transcriptionally activated in these cells in a coordinated manner during growth and differentiation but differ in response to glucocorticoids and cAMP(30, 31, 32) . We recently determined in mitotically active predifferentiated LLC-PK(1) cells that the temporal expression of EGR-1 is one molecular mechanism for both maximal activation of the Galpha gene and accelerated growth in these cells. These events were dependent on an enhancer area in the 5`-flanking region of the Galpha gene that contains an EGR-1 motif (5`-CGCCCCCGC-3`)(33) . Notably, acquisition of the polarized phenotype in LLC-PK(1) cells was accompanied by loss of EGR-1 expression and occupancy of the EGR-1 site by nuclear binding proteins other than EGR-1. In the present study we determine whether one of these binding proteins is WT1.


EXPERIMENTAL PROCEDURES

Cell Culture Cells

Wild type LLC-PK(1) cells are a polarized epithelial cell line derived from pig kidney. Cells were grown as confluent monolayers and maintained in Dulbecco's modified Eagle's medium containing 10 or 0.1% fetal calf serum in a 5% CO(2) atmosphere as described previously(15) . Cells were plated at a density of 1 times 10^6/10 cm^2, achieving confluence at approximately culture day 7.

Cellular Transfections

Plasmids

pRSV (WT1) and pRSV (Galpha) are plasmids containing, respectively, the entire coding sequence of human WT1 and the entire coding sequence of rodent G-protein alpha subunit driven by a Rous sarcoma virus promoter. Two plasmids m-14 LUC or m-4 LUC containing or lacking a putative EGR-1 binding sequence (5`-CGCCCCCGC-3`) were generated from nested deletions of plasmid 10-4 LUC (XmaIII), which contains an alpha 5`-flanking sequence fused to a firefly luciferase reporter gene as described previously(30) . A third plasmid-mutated m-14 LUC that replaced the nucleotides 1-5 of the EGR-1 site with adenosines was generated by polymerase chain reaction mutagenesis as described previously (33) utilizing the mutagenizing primer, 5`-CGGGCTACGAGATCCGCCAAAAACCGCCGTCGGGCAGCGGAG-3`. Plasmids containing this DNA segment were confirmed by dideoxynucleic acid sequencing.

Transient Transfections

Plasmids were transfected in equimolar amounts into LLC-PK(1) cells by calcium phosphate precipitation as described previously(30) . Optimum transfection efficiency was obtained by the addition of 20 µg of total plasmid DNA/55-cm^2 p10 plate (Falcon) followed by incubation for 20 h without glycerol shock. When required, this amount of DNA was achieved by the addition of ``carrier plasmid'' Bluescript II KS+. Transfection efficiency was normalized by co-transfection with 2.5 µg of pSV2Apap, a plasmid carrying a human placental alkaline phosphatase reporter gene driven by a Rous sarcoma virus promoter (generously provided by T. Kadesch, University of Pennsylvania).

Transfection Assays

Forty-eight to 98 h after transfection, LLC-PK(1) cells were washed twice in phosphate-buffered saline (without calcium or magnesium) and then lysed by addition of 1.0 ml of lysis buffer A (1% Triton, 25 mM glycylglycine, pH 7.8, 15 mM MgSO(4), 4 mM EGTA, and 1 mM fresh dithiothreitol). Scraped lysates were transferred to Eppendorf microfuge tubes and centrifuged at 10,000 times g for 5 min at 4 °C. The supernatants were transferred to fresh Eppendorf tubes and briefly vortexed prior to each assay. In some experiments the cell number of each plate was determined by direct cell count of trypsinized cells utilizing inverted phase microscopy.

Firefly Luciferase and Human Placental Alkaline Phosphatase Assays

These were performed as described previously(30) . Results are expressed as percent increase ± S.E. in luciferase activity normalized for heat-insensitive alkaline phosphatase activity. Data were analyzed by the paired Student's t test.

Protein Assay

This was performed by the dye binding assay of Bradford as described by the manufacturer (Bio-Rad).

Mobility Shift Assays

Nuclear Extract Preparation

Nuclear proteins were extracted from LLC-PK(1) cells as described previously (33) .

Binding Assays

6 µg of nuclear extract was preincubated for 30 min in the presence of P end-labeled double-stranded DNA, 4-6 µg of poly(dI-dC), 140 mM KCl, 9% glycerol, 18 mM Tris, pH 7.3, and 1 mM EDTA at 4 °C. Complexes were separated on 5-6% polyacrylamide gel with 89 mM Tris borate, 89 mM boric acid, 2 mM EDTA buffer (TBE) at 0.2 times concentration. The electrophoresis was carried out at 10 V/cm for 3-5 h in 0.2 times TBE buffer at 4 °C.

RNA Gel Blots

RNA from LLC-PK(1) cells was separated by electrophoresis in 1.0% formaldehyde/agarose gels and then transferred to GeneScreen Plus membranes (DuPont NEN). Membranes were prehybridized for 2 h at 42 °C in the presence of 1% SDS, 1 M NaCl, 10% dextran sulfate, and 50% deionized formamide. Hybridization was performed under similar conditions for 24 h in the presence of a human WT1 cDNA labeled with [alpha-P]ATP by priming with random hexamers followed by extension of these primers with the Klenow fragment of DNA polymerase(30) . After hybridization, membranes were washed twice for 30 min each in 0.3 M NaCl, 0.03 M sodium citrate at 23 °C, then in the same buffer with 1% SDS at 65 °C, followed by 15 mM NaCl, 1.5 mM sodium citrate at 65 °C. The membranes were dried and autoradiographed with Kodak XAR film at -80 °C for 6-96 h with or without Cronex Lightning Plus intensifying screens. Quantification of hybridization signals was performed by densitometry of the autoradiograms with an LKB ultroscan XL enhanced laser densitometer.

Immunoblotting and Immunofluorescence of EGR-1 and WT1

LLC-PK(1) cells were washed twice in phosphate-buffered saline (without calcium or magnesium) and then lysed by addition of 1.0 ml of lysis buffer A. Scraped lysates were solubilized by boiling in sample buffer (1% SDS, 30 mM Tris, pH 6.8, 12% glycerol) and loaded onto a 10% acrylamide gel with 150 µg of protein loaded per lane. Following SDS-polyacrylamide gel electrophoresis, proteins were transferred onto Immobilon membrane (Millipore), and the membrane was then stained with Coomassie Blue to ensure that all lanes contained equivalent amounts of transferred protein. The destained membrane was then blocked in blotting buffer (5% nonfat dry milk in 20 mM Tris, pH 7.4, with 0.15 M NaCl and 1% Triton X-100), incubated with either preimmune or immune rabbit IgG anti-EGR-1 Wi 21 (alpha 1) (which detects the non-zinc finger region of the EGR-1 protein corresponding to residues 29-117) or rabbit polyclonal alpha6F or murine monoclonal H7 antibody (which detects the 173-residue amino-terminal non-zinc finger region of the WT1 protein) diluted 1/1000 in blotting buffer and washed. In other experiments cell lysates were initially reacted with these antisera followed by precipitation with protein A prior to electrophoresis as described previously(8) . EGR-1 and WT1-bound proteins were reacted with an enhanced chemiluminescent detection system as described by the manufacturer (Amersham Corp.) followed by autoradiography.

LLC-PK(1) cells plated on glass coverslips were fixed for immunofluorescent staining on Days 1-7. Cells were fixed in 4% paraformaldehyde for 1 h, permeabilized in Triton X-100 for 4 min, and then incubated in PBS containing 0.1% bovine serum albumin for 5 min to reduce nonspecific background staining. The cells were incubated for 2 h in anti-EGR-1 Wi 21 (alpha 1), anti-WT1 alpha6F or H7, preimmune rabbit IgG, or non-immune murine ascites at 1:50 or 1:100 dilutions, washed three times in 0.1% bovine serum albumin in PBS, and then incubated for 1 h with goat anti-rabbit or anti-murine IgG conjugated to fluorescein isothiocyanate (Kirkegaard and Perry). Cells were washed three times in PBS, then mounted in 100 mM Tris-HCl:glycerol, 50:50, 2% n-propyl gallate, pH 8, and viewed on an Olympus BHS photomicroscope equipped for epifluorescence.

Autoradiography

For mobility shift and immunoblotting studies the dried gels or membranes were autoradiographed with Kodak XAR film at -80 °C for 0.5-96 h with Cronex Lightning Plus intensifying screens (DuPont). Quantification of signals was performed by densitometry of the autoradiograms with an LKB Ultroscan XL enhanced laser densitometer (Pharmacia Biotech Inc.).


RESULTS AND DISCUSSION

During culture, LLC-PK(1) cells differentiate from a rounded cell type to a fully polarized epithelium. Prior to their polarization and tight junction formation, these cells undergo several rounds of cell division coincident with the maximal EGR-1 expression and activation of the Galpha gene(31) . To determine whether the WT1 gene also participates in these events a full-length human WT1 cDNA and a polyclonal (alpha6F) or monoclonal (H7) antibody to the 173-residue amino-terminal non-zinc finger region of the WT1 protein were used to detect its expression in LLC-PK(1) cells. As seen in Fig. 1C, immunofluorescence of dividing non-confluent non-polarized cells with this antibody revealed a bright nuclear staining pattern in virtually every cell for EGR-1 whereas WT1 was not detected. By contrast, in fully polarized confluent cells this staining pattern was reversed. Quantification of EGR-1 and WT1 proteins in cells at Days 1-7 after plating by immunoblotting revealed a reciprocal pattern of expression for each protein in these cells. EGR-1 was barely detectable immediately after plating whereas WT1 was well expressed. However, by 36-48 h, when the cells were actively dividing, EGR-1 protein was maximally expressed whereas WT1 was not detectable. On later culture days 5-7, following the full polarization of these cells, EGR-1 expression fell whereas WT1 protein and mRNA expression increased (Fig. 1, A and B). In day 7 cultures there was sporadic lifting of some quiescent cell monolayers (data not shown). Ingrowth of cells occurred in the margins of denuded areas of the culture plates with a corresponding re-expression of EGR-1 protein. The pattern of maximal expression of the EGR-1 protein coincides with the temporal maximal activation of the Galpha gene during growth, whereas its repression coincided with the maximal expression of the WT1 protein during cell quiescence(31) .


Figure 1: Detection of WT1 gene expression in cultured LLC-PK(1) cells. A, Northern blot analysis of a tRNA(-), Wilms' tumor (+), and LLC-PK(1) RNA from culture days 0 (day of trypsinization) through day 7 with a full-length human WT1 cDNA detected a predominant 3-kilobase transcript. B, quiescent LLC-PK(1) cells from day 10 of culture were trypsinized (day 0) and cultured for successive days. Immunoblotting of total cell extract from each of these cultures was performed to compare the relative amounts of EGR-1 or WT1 protein present. Each lane represents 150 µg of protein quantitated. Autoradiograms of these proteins from the same acrylamide gel show in the upper panel EGR-1 induction (80 kDa) on culture day 1. The lower panel II shows maximal induction of WT1 protein (50 kDa) on culture days 5-7. Immunoprecipitation of WT1 was concurrently performed in these cultures, which also showed maximal induction of WT1 protein on culture days 5-7 seen in panel I. C, cell monolayers at days 1-10 were fixed, permeabilized, and stained by immunofluorescence with immune rabbit IgG anti-EGR-1 Wi 21 (alpha 1) (which detects the non-zinc finger region of the EGR-1 protein corresponding to residues 29-117) or alpha6F and H7 antibodies (which detect the non-zinc finger amino-terminal region of the WT1 protein corresponding to residues 1-173). Representative images of cell cultures at days 2 (left panel) and 7 (right panel) are shown. No staining was observed with preimmune rabbit IgG or non-immune mouse ascites (data not shown).



In other cell types the overexpression of the Galpha subunit can activate signaling pathways that contribute to accelerated cell growth. Likewise overexpression of Galpha or EGR-1 also accelerates LLC-PK(1) cell growth. As WT1 expression corresponded to culture times when LLC-PK(1) cells were both differentiated and growth arrested, we questioned whether expression of WT1 in predifferentiated LLC-PK(1) cells would antagonize the effects of EGR-1 on cell growth during this period. LLC-PK(1) cells were transiently transfected with Bluescript or plasmids encoding cDNAs for Galpha (pRSV Galpha) or WT1 (pRSV WT1), driven by a viral Rous sarcoma promoter-enhancer to overexpress each protein. As seen in Fig. 2, by day 3 of culture predifferentiated cells transfected with plasmids encoding Galpha had growth rates that were 1.8 times faster than cells transfected with Bluescript, whereas LLC-PK(1) cells transfected with WT1 had growth rates that were 35% lower than Bluescript-transfected cells. Transfection efficiency in all these experiments was comparable at approximately 10-20% efficiency. Despite the initial altered growth rates of Galpha and WT1-transfected cells, they still developed a normal polarized phenotype upon achieving confluence. These data suggested that both Galpha and WT1 participate in signaling pathways that normally contribute to renal cell growth.


Figure 2: Growth curves of LLC-PK(1) cells in 10% fetal calf serum. Squares depict cells transfected with plasmids encoding Galpha, triangles depict cells transfected with plasmids encoding WT1, and circles depict cells transfected with Bluescript II KS. Results are expressed as the mean cell number ± S.E. of 12 independent observations.



We previously documented in rapidly dividing non-polarized cells a 135-bp enhancer area in the -200 to -335 region of the Galpha gene. This region contains a binding site (5`-CGCCCCCGC-3`) for the EGR-1 transcription factor that provides a genomic signaling pathway for mitogenesis(33) . To assess whether WT1 was repressing cell growth by repressing maximal transcriptional activation of the Galpha gene, cells were co-transfected with the pRSV WT1 plasmid, and plasmids encoding firefly luciferase reporter genes fused to 5`-flanking areas of the Galpha gene with (M14) or without the EGR-1 site (mutated M14 and M4). As seen in Fig. 3, a 60% repression of Galpha transcription was only found in renal cells following their transfection with the M14 plasmid that contained both an intact EGR-1 binding site and also overexpressed a functional WT1 protein. These data suggested that the WT1 protein was contributing to the temporal transcriptional repression of the Galpha gene in LLC-PK(1) cells during culture.


Figure 3: Transcription of Galpha genes in polarized LLC-PK(1) cells. Upper panel, plasmids M14 (black bars), mutated M14 (gray bars), and M4 (striped bars), which represent serial deletions of 10-4 LUC-(XmaIII) fused to a firefly luciferase cDNA, were co-transfected with Bluescript II KS or a plasmid encoding WT1, pRSV WT1. These cells were then examined for luciferase activity. Luciferase activity results are expressed as percent of control in light units ± S.E. of constructs transfected with WT1 above those transfected with Bluescript II KS, normalized for human placental alkaline phosphatase activity in each of six individual deletion experiments (100% indicates unity or no change from control). Lower panel, sequence of the Galpha gene indicating the respective start sites for deletions of M14 and M4 plasmids. The EGR-1 consensus sequence ``CGCCCCCCGC'' is underlined. Sites of the created mutation in M14 are shown above.



To determine whether the WT1 protein was directly contributing to the activation of the Galpha gene, a double-stranded 23-bp DNA segment derived from the 5`-flanking sequence of the gene, which also contained the EGR-1 consensus sequence (5`-ATCCGCC CGCCCCCGCCGTCGGG-3`), was synthesized and P end-labeled for direct binding studies in mobility shift assays. Nuclear extracts from LLC-PK(1) cells that were either actively dividing and non-polarized (culture day 1) or LLC-PK(1) cells that were relatively quiescent and fully polarized (culture day 7) were examined. As seen in Fig. 4, the binding patterns of nuclear extracts from culture day 1 were different from those on culture day 7. Nuclear extracts from day 7 cells consistently demonstrated an additional faster mobility complex. We previously identified the EGR-1 protein as a predominant component of nuclear binding proteins in day 1 dividing cells but not day 7 quiescent cells (33) . Based on our immunocytochemical studies it would be anticipated that the WT1 protein should be present in nuclear extracts of quiescent fully polarized LLC-PK(1) cells on culture day 7. To determine whether WT1 was one of the proteins interacting with the 23-bp probe, nuclear extracts from culture days 1 and 7 were preincubated with H7 antibody. Following electrophoresis, retarded mobility of only the additional complex in nuclear extracts from culture day 7 was found. The specificity of this interaction was demonstrated by competition with the 28-kDa protein expressing the amino-terminal 173 residues of the WT1 protein. These data demonstrate that the WT1 protein was one component of these nuclear complexes. Detectability of the WT1 protein was consistent with its pattern of maximal expression in quiescent polarized LLC-PK(1) cells that also have a corresponding repression of the Galpha gene.


Figure 4: Mobility shift assays with the 23-bp DNA segment of the Galpha gene containing the EGR-1 binding site identify WT1 as a component of an induced nuclear complex in quiescent polarized LLC-PK(1) cells. Nuclear extracts were prepared from LLC-PK(1) cells 24 or 144 h after trysinization and culture. A 23-bp DNA segment containing the EGR-1 consensus sequence (5`-ATCCGCCCGCCCCCGCCGTCGGG-3`) was used as a probe in mobility shift assays. Lane 1, probe alone; lane 2, probe with 24-h nuclear extracts; lane 3, probe with 24-h nuclear extracts preincubated with non-immune ascites; lane 4, probe with 24-h nuclear extracts preincubated with immune ascites alpha 6F; lane 5, probe with 144-h nuclear extracts; lane 6, probe with 144-h nuclear extracts preincubated with non-immune ascites; lane 7, probe with 144-h nuclear extracts preincubated with immune ascites H7; lane 8, probe with 144-h nuclear extracts preincubated with immune ascites H7 and albumin; lane 9, probe with 144-h nuclear extracts preincubated with immune ascites H7 and competed with recombinant WT1 protein to which H7 was generated. Note similar data were obtained with the alpha6F antibody. The arrow indicates the position of the faster mobility complex detected in nuclear extracts 144 h after trypsinization and culture.



These findings suggest the existence of differentiation-induced pathways in LLC-PK(1) cells that alternatively abrogrates EGR-1 and promotes WT1 gene expression, thereby modulating a target protooncogene Galpha that is participatory for growth and differentiation in these renal cells. These studies emphasize the usefulness of the LLC-PK(1) renal cell as a model to elucidate normal programs of genetic differentiation in which WT1 participates. Further examination these pathways may provide significant insights into the molecular events involved in renal hypertrophy, nephrogenesis, and oncogenesis.


FOOTNOTES

*
This work was supported by National Institutes of Health Grant DK-42543, American Heart Association Established Investigatorship Award 94003090 (to L. E.), National Institutes of Health Grant F32 DK-08838 (to J. D. F.), National Institutes of Health Grant T32 DK-07540 (to T. B. K.), National Institutes of Health Grants CA52009, CA47983, and CA10815 and Pew Scholar (to F. J. R.), National Institutes of Health Grant CA09171 (to W. J. F.), and National Institutes of Health Grant DK-38452 (to L. E. and D. B.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Renal Unit, 8th Floor, Massachusetts General Hospital, East, 149 13th St., Charlestown, MA 02129. Tel.: 617-726-5666; Fax: 617-726-5669.

(^1)
The abbreviations used are: WT, Wilms' tumor; PBS, phosphate-buffered saline; bp, base pair(s).


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