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Cell Growth & Differentiation Vol. 13, 297-305, July 2002
© 2002 American Association for Cancer Research

The Wilms’ Tumor Suppressor Wt1 Is Associated with the Differentiation of Retinoblastoma Cells1

Nicole Wagner2, Kay-Dietrich Wagner2, Gunnar Schley, Sarah E. Coupland, Heinrich Heimann, Rosemarie Grantyn and Holger Scholz3

Johannes-Müller-Institut für Physiologie, Humboldt-Universität, Charité, 10117 Berlin [N. W., K. D. W., G. S., R. G., H. S.]; Medizinische Klinik I, Humboldt-Universität, Charité, 10117 Berlin [N. W.]; and Institut für Pathologie [S. E. C.] and Klinik für Ophthalmologie [H. H.], Universitätsklinikum Benjamin Franklin, Freie Universität Berlin, 12200 Berlin, Germany


    Abstract
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
We have demonstrated recently that Wilms’ tumor suppressor 1 (Wt1),in addition to its role in genitourinary formation,is required for the differentiation of ganglion cells in the developing retina. Here we provide further evidence that Wt1 is associated with neuronal differentiation. Thus, the retinoblastoma-derived human cell line, Y-79, contained robust amounts of Wt1 mRNA and protein. Wt1 expression was down-regulated upon laminin-induced differentiation of Y-79 into neuron-like cells. Inhibition of Wt1 with antisense oligonucleotides dramatically reduced the capacity of undifferentiated Y-79 cells to undergo neuronal differentiation, whereas sense and missense oligonucleotides had no effect. Wt1 immunoreactivity was also detected in solid retinoblastomas, in which it resided mainly in areas with moderate proliferative activity. These findings suggest a role for Wt1 in the differentiation of retinoblastoma cells. Furthermore, Wt1 expression in retinoblastoma may reflect the potential of these tumors to initiate the early steps of neuronal differentiation.


    Introduction
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Retinoblastoma is the most common form of pediatric ocular malignancy affecting ~1 in 20,000 live births (1 , 2) . The tumors can occur sporadically, but they are more prevalent in children with a family history of retinoblastoma. Retinoblastomas are considered to develop from the pluripotent retinal progenitor cells, which, instead of differentiating into their neuronal and glial progenies, acquire a malignant phenotype (3 , 4) . Histological examination revealed that the tumor cells attempt to differentiate into photoreceptors and Müller cells or may even become apoptotic (5, 6, 7) . It is unknown whether this phenomenon is accounted for by a subpopulation of cells that retained their responsiveness to environmental signals or whether a limited number of tumor cells exist that can reactivate, although incompletely, their endogenous differentiation program. Statistical analysis of sporadic versus hereditary cases of retinoblastoma led Knudson to propose his "two-hit hypothesis," according to which both copies of the retinoblastoma susceptibility gene (RB1) must be inactivated for this tumor to arise (8) .

We have shown recently that another tumor suppressor gene, Wt1,4 is required for normal development of the retina (9) . Wt1 belongs to the early growth response (egr) family of zinc finger proteins (10, 11, 12) and is mutationally inactivated in a subset of nephroblastoma (Wilms’ tumor; Refs. 13 , 14 ). In addition to its inhibitory effect on tumor growth, Wt1 is required for normal embryonic development. Thus, mouse embryos with targeted disruption of the Wt1 gene exhibit a lack of formation of the kidneys, gonads, spleen, and adrenal glands in addition to defects of mesothelial structures (15, 16, 17) . Furthermore, our recent findings indicate that Wt1-/- embryos have markedly thinner retinas than their wild-type counterparts with apoptotic loss of a large fraction of retinal ganglion cell precursors (9) . These observations demonstrated for the first time that Wt1, besides its predicted role in mesenchyme to epithelial conversion (15 , 17) , can also act as an important regulator for the development of certain neurons.

In this study, we examined whether the capacity of embryonic retinal cells to express Wt1 is maintained in their malignantly transformed counterparts, the retinoblastoma cells. If so, we investigated whether Wt1 expression in retinoblastoma cells would be related to their commitment to differentiate into neuron-like cells. As an in vitro model we used Y-79 cells, a tumor line derived from human retinoblastoma (18 , 19) , which has been found previously to undergo neuronal differentiation in response to treatment with laminin (20, 21, 22) . By demonstrating that Wt1 is indeed associated with the neuronal conversion of Y-79 cells, our findings further support a relationship between Wt1 and neuron differentiation.


    Results
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Wt1 mRNA and Protein Is Expressed in Human Retinoblastoma.
To explore whether Wt1 expression in the normally developing retina (9) is recapitulated in pediatric retinal tumors, Wt1 mRNA and protein was analyzed with the use of RT-PCR and immunohistochemistry in tissue specimens obtained from six patients with moderate to poorly differentiated retinoblastoma. The tumors were classified according to Nork et al. (23) based on the presence of cells with high nuclear:cytoplasmic ratios and the occurrence of pseudorosettes and Homer-Wright rosettes. As shown in Fig. 1ACitation , Wt1 mRNA was detected by RT-PCR in all tumor specimens analyzed. Wt1 expression in retinal tumors was confirmed at the protein level. A representative Wt1 immunohistochemistry of retinoblastoma is depicted in Fig. 1, B–ECitation . Wt1 immunoreactivity (purple staining), which was detected with an alkaline phosphatase technique, predominated in the more differentiated parts of the tumors as indicated by the presence of Flexner-Wintersteiner rosettes (Fig. 1D)Citation . Remarkably, Wt1 was not restricted to the nuclei but was also evident in the cytoplasm of the tumor cells (Fig. 1D)Citation . Double-immunolabeling of Wt1 and Ki-67 (brown color) revealed that Wt1 was expressed mainly in those parts of the tumors that exhibited decreased proliferative activity (Fig. 1E)Citation . No staining was seen when the tissue sections were incubated with normal serum instead of primary antibody (Fig. 1F)Citation . For validation of our immunohistochemistry technique, we performed Wt1 labeling of tissue sections from human kidney in the same set of experiments. Consistent with the results by others (24 , 25) , Wt1 immunoreactivity was confined to the nuclei of the glomerular podocytes but was not detected in the tubules (Fig. 1G)Citation .



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Fig. 1. Expression of Wt1 mRNA and protein in human retinoblastoma. Tissue samples were obtained from six patients diagnosed with moderate to poorly differentiated retinoblastoma (RB1 to RB6) according to the classification of Nork et al. (23) . RT-PCR was performed with 1 µg of total RNA each. The number of PCR cycles was adjusted to 28 to be within the exponential phase of the semiquantitative PCR. Note that Wt1 transcripts were detected in all tumor samples examined (A). B–E, representative Wt1 immunohistochemistry of human retinoblastoma. Wt1 protein was detected using a second antibody conjugated with alkaline phosphatase as a substrate (purple staining). Nuclei were counterstained with hematoxylin (blue staining). The low power magnification (B) shows normal (right) and neoplastic retina (left). High power magnifications (C and D) revealed Wt1-positive cells in the more differentiated parts of retinoblastomas, especially in areas with Flexner-Wintersteiner rosettes (D). Note also that Wt1 immunoreactivity is restricted to tumor regions with low proliferative activity, as demonstrated by double-labeling with the proliferation marker Ki-67 (E), which is visualized by a brown color reagent. Wt1-positive cells (purple) were detected in regions with low Ki-67 immunoreactivity. For negative controls, the primary antibody was replaced by normal serum (F). The immunohistochemistry technique was validated by demonstration of nuclear Wt1 staining of glomerular podocytes in tissue sections from human kidney (G).

 
Wt1 mRNA and Protein Is Down-Regulated upon Induced Differentiation of Retinoblastoma-derived Y-79 Cells.
Next, we established a tissue culture model to address the potential role of Wt1 in neuronal differentiation at the cellular level. We and others (20, 21, 22) have reported previously that retinoblastoma-derived Y-79 cells can be grown under conditions that promote their transition into neuron-like cells. Indeed, immunoblotting with a polyclonal anti-Wt1 antibody revealed that Y-79 cells grown as suspension cultures contained high amounts of Wt1 protein. As shown in Fig. 2ACitation , Wt1 protein levels were higher in retinoblastoma-derived Y-79 cells than in the human embryonic kidney (HEK) 293 cell line and in rat kidney at postnatal day 3 (P3). Comparable amounts of Wt1 protein were found in Y-79 and K562 leukemia cells (Fig. 2A)Citation . Only very low levels of Wt1 mRNA were detected by RT-PCR in Weri-Rb-1 cells, another retinoblastoma line.5 We therefore decided to use Y-79 instead of Weri-Rb-1 cells for the experiments. We next examined whether Wt1 expression would change upon differentiation of Y-79 cells into neuron-like cells. For this purpose, Y-79 cells were induced to acquire a neuronal phenotype by the addition of soluble laminin to the cultures as described below (see "Materials and Methods"). Undifferentiated Y-79 cells and cultures enriched in differentiated cells were compared for their levels of Wt1 mRNA with the use of a semiquantitative RT-PCR. As shown in Fig. 2BCitation , Wt1 mRNA was dramatically down-regulated in cultures containing >60% neuron-like cells. The decrease of Wt1 transcripts in differentiated Y-79 cells was specific as ß-actin, and AP-2{alpha} mRNA levels were not significantly changed upon laminin-induced differentiation (Fig. 2C)Citation . Notably, soluble laminin had no effect on Wt1 mRNA in undifferentiated Y-79 cells that were grown as suspension cultures (data not shown).



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Fig. 2. A, Wt1 protein in different cell lines and in kidneys from 3-day-old rats (P3). Twenty µg of protein from total cell lysates were loaded per lane and separated on a 10% SDS-PAGE. Immunoblotting was performed with polyclonal rabbit anti-Wt1 antibody. The membrane was reprobed with a polyclonal anti-ß-actin antibody to detect differences in protein loading. Note the high Wt1 protein content (Mr {approx}55,000 band) in retinoblastoma-derived Y-79 cells as compared with HEK293 cells and 3-day-old rat kidney. Wt1 protein levels were similar in Y-79 and in K562 leukemia cells. B, analysis of mRNA levels in undifferentiated Y-79 cells and in cultures containing >60% neuron-like cells. Wt1 transcripts were dramatically down-regulated in differentiated versus undifferentiated cells as demonstrated by semiquantitative RT-PCR (B). In contrast, mRNA levels of ß-actin and the AP-2{alpha} transcription factor were similar in undifferentiated and neuron-like Y-79 cells (C). undiff., undifferentiated; diff., differentiated.

 
Western blot analysis was performed to explore whether down-regulation of Wt1 mRNA in differentiated cells was accompanied by a decrease in Wt1 protein. It is evident that Wt1 protein levels were markedly reduced in cultures enriched in neuronal cells as compared with undifferentiated Y-79 cells (Fig. 3A)Citation . We used immunostaining to distinguish whether down-regulation of Wt1 protein was restricted to a subclass of Y-79 cells or whether it occurred in all (undifferentiated and neuron-like) retinoblastoma cells. As shown in Fig. 3, B and CCitation , incubation with a polyclonal anti-Wt1 antibody clearly stained the round-shaped undifferentiated Y-79 cells, whereas the process-bearing, neuronal cells remained unlabeled. No cells were stained when normal rabbit serum instead of specific primary antibody was used (Fig. 3, D and E)Citation .



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Fig. 3. Analysis of Wt1 protein in Y-79 cells. Twenty µg of protein in total lysates obtained from undifferentiated Y-79 cells and from cultures containing ~60% neuron-like cells were separated on a 10% SDS-PAGE and analyzed by immunoblotting with a polyclonal anti-Wt1 antibody (A). Down-regulation of Wt1 protein in differentiated Y-79 cells was confirmed by immunocytochemistry. A representative example of Wt1 immunostaining is shown in C. Note the strong labeling of the round-shaped undifferentiated Y-79 cells, whereas the neuron-like cell (arrowhead) does not stain with anti-Wt1 antibody (C). B shows a phase contrast view of C to make the process-bearing (Wt1-negative) cell more visible (arrow). No specific staining was obtained when the primary anti-Wt1 antibody was replaced by normal rabbit serum (phase contrast view in D and E).

 
Wt1 Levels Are Not Closely Linked to the Proliferation Rates of Y-79 Cells.
Which process is targeted by Wt1 in Y-79 cells? The large amounts of Wt1 protein could reflect the high proliferation rates of undifferentiated Y-79 cells, or they could be a prerequisite for entering a differentiation pathway. To distinguish between these possibilities, cell proliferation rates were adjusted to different levels by incubating the cultures for 3 days in the presence of variable (0–15%) FCS concentrations. Thereafter, equal aliquots of the cells were counted in a Neubauer chamber to estimate cell proliferation. The cell counts correlated very closely with the concentration of FCS in the culture medium (Fig. 4A)Citation . However, stepwise serum depletion down to 0% FCS did not significantly change Wt1 transcript levels (Fig. 4B)Citation , suggesting that Wt1 expression and proliferation rates were not intimately linked. Instead, it becomes more likely that down-regulation of Wt1 mRNA and protein was associated with neuronal differentiation of the Y-79 cells.



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Fig. 4. Effect of variable concentrations of FCS in the culture medium on cell numbers (A) and on Wt1 and ß-actin transcript levels (B). The Y-79 cells were suspended in 24-well plates at a density of 104 cells/ml and cultured for 3 days in RPMI 1640 containing the indicated concentration of FCS. Wt1 and ß-actin mRNA levels were measured by a semiquantitative RT-PCR. Note that Wt1 transcripts, unlike the cell proliferation rates reflected by the cell counts, did not change in proportion to the FCS concentration in the tissue culture medium. Bars, SD.

 
Antisense Inhibition of Wt1 Prevents the Laminin-induced Differentiation of Y-79 Cells.
To answer the question of whether Wt1 expression is indeed required for the neuronal differentiation of Y-79 cells, de novo synthesis of Wt1 was gradually inhibited by transfection with variable amounts (0.04–1.3 µM) of specific fluorescein-conjugated phosphorothioate antisense oligonucleotides. Transfection efficiencies ranged from 30 to 50%, as estimated by fluorescence microscopy (data not shown). The transfected cells were grown for 3 days under conditions that favored neuronal differentiation. Western blot analysis was performed with immunoprecipitates of Y-79 cell lysates and showed that treatment with specific antisense oligonucleotides indeed reduced the synthesis of Wt1 protein (Fig. 5A)Citation . The fraction of differentiated cells increased in a graded manner from 0.5 to >8% with decreasing concentrations (from 1.3 to 0.04 µM) of the Wt1 antisense oligonucleotides (Fig. 5, C and D)Citation . Notably, all cells on the plate were included into the cell counts, no matter whether they had taken up the fluorescein-tagged oligonucleotides. Because the transfection efficiencies ranged between 30 and 50% as estimated by fluorescence microscopy (data not shown), one may therefore even underestimate the potency of Wt1 antisense treatment to suppress the neuronal differentiation of Y-79 cells. In contrast, Wt1 sense and missense oligonucleotides, even at the highest concentrations (1.3 µM), did not significantly change the percentage of neuronal Y-79 cells (Fig. 5, C and D)Citation .



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Fig. 5. Antisense inhibition of Wt1 gradually reduces the conversion of Y-79 into neuron-like cells. Transfections were performed with the indicated concentrations (between 0.04 and 1.3 µM) of either missense or specific Wt1 sense and antisense oligonucleotides (39) , yielding transfection efficiencies between 30 and 50%. The transfected cells were grown for 3 days under conditions that permitted neuronal differentiation. Treatment with Wt1 antisense oligonucleotides (1.3 µM) significantly reduced de novo synthesis of Wt1, as demonstrated by immunoprecipitation with a polyclonal rabbit anti-Wt1 antibody (A). B and C show representative phase contrast views of Y-79 cells treated either with Wt1 antisense (B) or sense (C) oligonucleotides. The transfected undifferentiated and neuron-like Y-79 cells were counted under the microscope (10 optical fields, x400). The effect of variable concentrations of oligonucleotides on the fraction of neuron-like Y-79 cells is indicated in (D). Values are means of five experiments performed as duplicates; bars, SE. ***, statistical significance (P < 0.01) between cells transfected with sense and antisense oligonucleotides.

 

    Discussion
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
In this study, we show for the first time that the Wilms’ tumor suppressor Wt1 is expressed in retinoblastoma tissue and cultured retinoblastoma cells. Retinoblastoma is a pediatric retinal tumor, which is thought to arise when the pluripotent progenitor cells in the developing retina fail to differentiate along the neuronal and glial lineage. Retinoblastomas have occasionally been reported to undergo spontaneous regression (26, 27, 28) , thus maintaining the hope that malignant transformation of the tumor cells can be reversed, at least in part, by activation of differentiation pathways. Several findings of this study suggest that Wt1 may have a role in the differentiation of retinoblastoma cells. Wt1 immunoreactivity was detected mainly in the better-differentiated areas of the tumors that exhibited low proliferative activity. Furthermore, the fraction of cultured Y-79 retinoblastoma cells that were capable of neuronal differentiation correlated inversely with the concentration of specific antisense oligonucleotides used to inhibit Wt1 expression. Cytoplasmic Wt1 immunolabeling of retinoblastoma cells may be unexpected at first sight, because Wt1 has originally been identified as a nuclear transcription factor. However, cytoplasmic Wt1 has also been observed recently in a significant fraction of mesotheliomas and in the majority of pulmonary adenocarcinomas (29) . The molecular mechanisms that direct Wt1 to the nucleus and/or cytoplasm are still unclear. Remarkably, phosphorylation of Wt1 resulted in cytoplasmic retention of the protein and greatly reduced its DNA binding affinity (30) , suggesting that the subcellular distribution of Wt1 depends on posttranscriptional modification of the protein.

Detection of Wt1 immunoreactivity in the tumor rosettes is seemingly in conflict with down-regulation of Wt1 in neuron-like Y-79 cells. In this regard, it must be considered that the Wt1-negative Y-79 cells were terminally differentiated postmitotic cells, whereas the tumor rosettes in retinoblastoma did not reach the same level of neuronal differentiation. We therefore assume that expression of Wt1 in retinoblastoma may permit the tumor cells to undergo the initial steps of differentiation. However, expression of Wt1 neither in solid retinoblastoma nor in cultured Y-79 cells was sufficient for the tumor cells to acquire a fully differentiated state. Thus, the characteristic phenotypic changes of Y-79 cells were seen only after addition of soluble laminin-1 to the cultures. Laminin treatment did not significantly change Wt1 mRNA levels in undifferentiated Y-79 cells, indicating that its effect was secondary to neuronal differentiation rather than resulting from a direct inhibitory action on Wt1 expression. Laminin-1 has been characterized previously as a differentiating agent acting both on normal retinal precursors (31) and their malignant counterparts, the retinoblastoma cells (20 , 21) . The requirement for Wt1 in laminin-induced neuronal differentiation points to the possibility that Wt1 sensitizes Y-79 cells to laminin-1 and/or other extracellular matrix components. A number of novel target genes for Wt1 including integrin {alpha}8 have been identified recently in mesenchymal fibroblasts (32) . In the developing chick retina, expression of the integrin subunit {alpha}6 was enhanced by insulin-like growth factor I, which stimulated the conversion of retinal precursors into ganglion cells (33) . It will be of interest to explore whether up-regulation of integrin {alpha}8 and/or other members of the integrin family of laminin receptors by Wt1 activates a neuronal differentiation pathway in retinoblastoma cells.

Significant amounts of Wt1 mRNA and protein have been detected previously in several tumor lines whose normally developing counterparts expressed Wt1 only at a low level. For instance, Wt1 transcripts dramatically decreased when hematopoietic tumor cells were induced to mature into their megakaryocytic and granulocytic progenies (34 , 35) . Our recent findings indicate that down-regulation of Wt1 is not restricted to the differentiation of transformed tumor cells but is recapitulated during normal embryonic development (9) . Thus, Wt1 mRNA was abundantly expressed in the majority of retinal neuroblasts during the early embryonic stages. Later during development, Wt1 expression became restricted to the presumptive retinal ganglion cell layer and was completely absent from adult retinas (9) . Along with our present findings, these data favor the view that Wt1 is not required to maintain retinal cells in a differentiated state but may be important for the initiation of a neuronal differentiation program instead. In support of the latter possibility, Wt1 has been shown recently to mediate the transcriptional response of PC12 pheochromocytoma cells to stimulation with nerve growth factor (36) .

In summary, together with our previous observations (9) , these findings indicate that Wt1, in addition to its critical role in mesenchyme to epithelial conversion, is also involved in the differentiation of certain neurons. Moreover, our data raise the possibility that the molecular signaling pathways for the maturation of the pluripotent retinal progenitors are preserved, at least in part, in retinoblastoma cells.


    Materials and Methods
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 
Cell Cultures.
All cell lines used in this study were obtained from American Type Culture Collection. Retinoblastoma-derived Y-79 cells (ATCC no. HTB-18) were grown in suspension at 37°C in a humidified 95% air, 5% CO2 atmosphere in RPMI 1640 (Life Technologies, Inc., Eggenstein, Germany) supplemented with 10% FCS (Biochrom KG, Berlin, Germany), 2 mM L-glutamine (Life Technologies, Inc.), 100 units/ml penicillin (Life Technologies, Inc.), and 100 µg/ml streptomycin (Life Technologies, Inc.). The K562 and HEK293 cell lines were maintained in DMEM (Life Technologies, Inc.) containing 10% FCS and antibiotics. The medium was routinely renewed three times/week. Monolayer cultures were split at a 1:10 ratio before reaching confluence.

Differentiation and Isolation of Neuron-like Y-79 Cells.
Differentiation of Y-79 cells was obtained according to a protocol described previously (22) but without using the tyrosine kinase inhibitor K252a. Briefly, attachment cultures were prepared by seeding the cells at an initial density of 3 x 104 cells/cm2 on the bottom of tissue culture flasks coated with 0.1% poly-D-lysine (Sigma, Deisenhofen, Germany). Within 3 days after addition of 20 µg/ml laminin-1 (Becton Dickinson, Heidelberg, Germany) to the culture medium, ~12% of the cells displayed structural signs of differentiation, i.e., neurite-like processes that exceeded the soma diameter by a factor of 2. These morphological criteria for neuron differentiation were confirmed by immunostaining. Thus, in contrast to round-shaped undifferentiated Y-79 cells, the process-bearing cells were immunonegative for GFAP, while staining of the neuronal marker neurofilament (NF) 68 was maintained (Fig. 6Citation ; Ref. 22 ). Because neuron-like cells exhibited enhanced adhesiveness to the plastic dishes, they could be isolated by removing the nonadherent undifferentiated Y-79 cells during three washes with PBS. Microscopic inspection (x200) revealed that ~60% of the remaining cells displayed a neuron-like morphology.



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Fig. 6. Immunocytochemistry of Y-79 cells grown either in the absence (A) or presence (B) of soluble laminin-1 in the culture medium. The cells were seeded at an initial density of 3 x 104 cells/cm2 on poly-D-lysine-coated tissue culture flasks. After treatment with soluble laminin-1 (20 µg/ml) for 3 days, double immunofluorescence labeling of GFAP and NF 68 was performed using the Cy3 and FITC reagents for visualization, respectively. Undifferentiated Y-79 cells, which express both marker proteins, exhibit a yellow color because of the overlap of the green (NF 68) and red (GFAP) fluorescence signal. Note the intensive green color of the neuron-like cell in B, reflecting loss of GFAP immunoreactivity.

 
Cell Counts and Statistics.
Cells in unfixed monolayer cultures were counted under phase-contrast illumination (x200). Each data point represents the average from 15 optical fields (0.418 mm2) in two different dishes and at least three different experiments. Results are shown as mean ± SD.

RT-PCR.
Total RNA was extracted from Y-79 cells with the Trizol reagent (Life Technologies, Inc.). The RNA pellet was dissolved in diethyl pyrocarbonate-treated H2O at a concentration of 1 µg/µl. First-strand cDNA synthesis was performed with 3 µg of total RNA using oligo(dT) primers and superscript II reverse transcriptase (Life Technologies, Inc.). One-tenth of the reaction product was taken for PCR amplification. Semiquantitative PCR was performed according to Di Fulvio et al. (37) . In a first series of experiments, the optimal number of PCR cycles was determined for each primer set so that the reactions were carried out during the course of exponential amplification. The Wt1 and AP-2{alpha} primers were used at final concentrations of 200 2nM. The primer concentration for amplification of ß-actin transcripts, which served as an internal control, was 20 nM. Under these conditions, the efficiency of the RT-PCR reaction for each gene did not plateau, and the numbers of cycles used in these experiments were kept to 28. The PCR reactions were carried out in a thermal cycler (Biometra, UNO-II, Göttingen, Germany) according to the following routine protocol: DNA denaturation at 94°C, primer annealing at 58°C, extension of double-stranded DNA at 72°C, and each step lasting 45 s. The following primers were used for PCR amplification: human Wt1, 5'-AAGCTGTCCCACTTACAGATG-3' (forward primer), 5'-CAGCTGGAGTTTGGTCATGTT-3' (reverse primer); human ß-actin, 5'-TTCTACAATGAGCTGCGTGTG-3' (forward primer), 5'-CGTCACACTTCATGATGGAGT-3' (reverse primer); and human AP-2{alpha}, 5'-CGTGGTGAACCCCAACGAAGT-3' (forward primer), 5'-GAAGTGGGTCAAGCAG-CTCTG-3' (reverse primer).

Transfection of Y-79 Cells with Oligonucleotides.
Accepted procedures for the use of antisense oligonucleotides were applied (38) . The Y-79 cells were seeded on 0.1% poly-D-lysine-coated 24-well tissue culture plates at a density of 3 x 104 cells/cm2 for transfection with Wt1 sense, antisense, and missense oligonucleotides at concentrations between 0.04 and 1.3 µM. Fluorescein-conjugated phosphorothioate oligonucleotides (Metabion, Martinsried, Germany) were transfected in serum-free medium using a 1:1 liposome formulation of the cationic lipid DMRIE and cholesterol (DMRIE-C reagent) according to the manufacturer’s instructions (Life Technologies, Inc.). Transfection efficiencies ranged from 30 to 50%, as determined by inspection under a fluorescence microscope (Axiovert 100; Zeiss, Göttingen, Germany). On the day after the transfection, the medium was replaced, and the cells were grown for 3 days in RPMI 1640 (Life Technologies, Inc.) supplemented with 10% FCS and 20 µg/ml laminin (Becton and Dickinson, Heidelberg, Germany) to permit conversion into neuron-like cells. The fraction of differentiated cells was determined by counting at least 10 optical fields (x400) in two different dishes and five separate experiments. The following oligonucleotides spanning the region from -3 bp to +12 bp of the putative Wt1 translation initiation codon were used (39) : 5'-CAAATGGGCTCCGAC-3' (sense), 5'-GTCGGAGCCCATTTG-3' (antisense). The sequence of the missense oligonucleotide was 5'-CCGTTGTAGGCCAGT-3'.

SDS-PAGE and Immunoprecipitation.
Undifferentiated Y-79 suspension cultures and cultures enriched (>60%) in neuron-like Y-79 cells were obtained as described above and washed twice in PBS. Total cell lysates were prepared by heating the cells to 95°C for 3 min in TBS/1% SDS buffer. Twenty µg of protein were loaded per lane and separated on a denaturing 10% SDS-PAGE. Protein transfer onto polyvinylidene difluoride membranes (Amersham Pharmacia Biotech, Freiburg, Germany) was performed with a semidry blotting apparatus (Bio-Rad). A polyclonal antibody raised in rabbit against the NH2 terminus of Wt1 protein (WT 180; Santa Cruz Biotechnology, Heidelberg, Germany) was used at a 1:1.000 dilution, followed by incubation with goat antirabbit secondary antibody (1:5.000) and detection by an enhanced chemiluminescence system (ECL; Amersham). The membranes were reprobed with a goat anti-ß-actin polyclonal antibody (C-11; Santa Cruz Biotechnology) to detect differences in protein loading.

After transfection with Wt1 sense, antisense, and missense oligonucleotides, the Y-79 cells were grown for 3 days under conditions that permitted neuronal differentiation (see above). After 72 h, the cells were harvested, collected by brief centrifugation (500 x g at room temperature), and washed twice with L-methionine-free RPMI 1640 to deplete intracellular methionine stores. Pulse-labeling of the Y-79 cells (2 x 106 cells/ml) was done by a 60-min incubation at 37°C in L-methionine-free culture medium supplemented with 0.2 µCi/ml of [35S]methionine (specific activity, >1000 Ci/mmol; ICN). After removal of the labeling medium, the cells were washed in ice-cold PBS and lysed in a buffer containing 150 mM NaCl, 100 mM Tris (pH 7.5), 1 mM EDTA, 1 mM EGTA, 0.2 mM sodium vanadate, 0.2 mM phenylmethylsulfonyl fluoride, and 0.5% NP40. The lysates were precleared by incubation for 30 min at 4°C with 1.0 µg of normal rabbit IgG and 20 µl of protein A-Sepharose (Pharmacia Biotech, Freiburg, Germany). After pelleting the lysates by centrifugation at 1500 rpm (4°C), the supernatants were incubated for 1 h at 4°C with 1 µg of a polyclonal rabbit anti-Wt1 antibody (WT 180; Santa Cruz Biotechnology). Twenty µl of protein A-Sepharose conjugate were added to the reaction, and incubation was continued overnight at 4°C. The immunoprecipitates were collected and washed four times in lysis buffer. After resuspension in electrophoresis buffer, the samples were boiled and analyzed by autoradiography after separation on a 10% SDS-PAGE.

Immunohistochemistry and Tissue Samples.
For Wt1 immunostaining, monolayer cultures were fixed with a mixture of 1.5% paraformaldehyde and 1% glutaraldehyde in PBS. The cells were permeabilized with 0.1% Triton X-100 in TBS, and endogenous peroxidase activities were blocked in a solution of 3% H2O2 in methanol (1:4) for 5 min, washed, and incubated with the primary antibody for 16 h at 4°C. A rabbit polyclonal anti-Wt1 antibody (C-19; Santa Cruz Biotechnology) in TBS containing 5% normal goat serum was applied at the dilution 1:150, followed by incubation with a biotinylated secondary antibody (goat antirabbit, 1:150 in TBS + 1% BSA; Vector Laboratories, Inc.) and the streptavidin-peroxidase complex (Sigma). The reaction product was visualized with diaminobenzidine and hydrogen peroxide (Sigma). For GFAP/NF68 double-staining, monolayer cultures were fixed with 3% paraformaldehyde (Sigma). The cells were preincubated for 1 h in PBS containing 0.1% Triton X-100, 10% normal goat serum, and then incubated with anti-GFAP mouse IgG1 monoclonal antibody (Sigma; clone G-A-5, 1:400 dilution in PBS) for 48 h at 4°C. This step was followed by incubation with a biotinylated secondary antibody (goat antimouse, 1:100 in PBS containing 1% BSA; Sigma) and application of streptavidin-Cy3. After another preincubation with 10% normal goat serum, the cells were treated for 16 h at 4°C with an anti-NF 68 mouse IgG1 monoclonal antibody (Sigma; clone NR4, 1:400 dilution in PBS). A FITC-conjugated secondary antibody (goat antimouse, 1:25 in PBS; Dianova, Hamburg, Germany) was used for visualization of the reaction products. Cy3 (red color) and FITC (green fluorescence) double-labeling produced a yellow staining of cells that coexpressed NF-68 and GFAP proteins (Fig. 6)Citation . No specific fluorescence signal could be detected when normal mouse serum instead of primary antibodies was used (not shown).

Five eyes were enucleated from patients diagnosed with moderate to poorly differentiated retinoblastoma according to the classification of Nork et al. (23) . Tissues were fixed in 4% buffered formalin and embedded in paraffin. Immunohistology was performed with polyclonal anti-Wt1 antibody that is reactive in paraffin sections as described above (C-19, 1:50 dilution in TBS; Santa Cruz Biotechnology). An antigen retrieval method using a pressure cooker was performed before immunohistochemical staining (40) . The alkaline phosphatase anti-alkaline phosphatase method was used to demonstrate the binding of the primary antibodies (41) . Nuclei were counterstained with hematoxylin. Wt1 immunostaining of tissue samples from human kidney was performed accordingly. Paraffin sections of some tumors were also double-stained for the proliferation marker Ki-67 using goat polyclonal antibody (C-20, 1:150 dilution in TBS; Santa Cruz Biotechnology). The Ki-67 protein was detected by an avidin-biotin peroxidase technique using diaminobenzidine/hydrogen peroxide as substrate. The Wt1 antigen was revealed by the alkaline phosphatase anti-alkaline phosphatase procedure with the use of naphthol AS-MX plus Fast Red TR (Sigma-Aldrich) as substrates. The primary antibody was replaced by normal serum in the negative controls.


    Acknowledgments
 
The expert technical assistance of I. Grätsch and A. Richter is gratefully acknowledged. We thank Dr. M. Gessler for the gift of human Wt1 cDNA.


    Footnotes
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 This study was supported by Grants Scho 634/3-1 and GRK 238 from the Deutsche Forschungsgemeinschaft. Back

2 These authors contributed equally to this work. Back

3 To whom requests for reprints should be addressed, at Johannes-Müller-Institut für Physiologie, Humboldt-Universität, Charité, Tucholskystrasse 2, 10117 Berlin, Germany. Phone: 49-30-450-528213; Fax: 49-30-450-528972; E-mail: holger.scholz{at}charite.de Back

4 The abbreviations used are: Wt1, Wilms’ tumor suppressor 1; RT-PCR, reverse transcription-PCR; HEK, human embryonic kidney; GFAP, glial fibrillary acidic protein. Back

5 N. Wagner and K.-D. Wagner, unpublished observations. Back

Received for publication 11/30/01. Revision received 3/29/02. Accepted for publication 5/27/02.


    References
 TOP
 Abstract
 Introduction
 Results
 Discussion
 Materials and Methods
 References
 

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