Effect of WT1 gene silencing on the tumorigenicity of human glioblastoma multiforme cells

Laboratory investigation

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Object

Wilms tumor 1 (WT1) is overexpressed in many human cancers, including glioblastoma multiforme (GBM). In another study, the authors showed that transient WT1 silencing increases the radiosensitivity of glioma cells. Studies of nonglioma cell lines have demonstrated that WT1 promotes cell proliferation and survival; however, this ability has not been rigorously analyzed in human GBM.

Methods

The authors tested the efficacy of 2 sequences of short hairpin RNA (shRNA) directed against WT1 in U251MG human GBM cells and found that 1 sequence was capable of stably silencing WT1 expression. They then evaluated the effect of WT1 silencing on cellular proliferation, invasion, and in vivo tumor formation.

Results

Stable WT1-shRNA expression significantly decreased the proliferation of U251MG cells in vitro as demonstrated by both an adenosine 5′-triphosphate–based viability assay and tritiated thymidine uptake. Furthermore, stable WT1 silencing caused significantly slower growth after the subcutaneous inoculation of tumor cells in the flanks of athymic nude mice and was associated with an increased latency period.

Conclusions

Data in this study provide proof of the principle that downregulation of WT1 causes decreased tumorigenicity of a GBM cell line in vitro and in vivo and suggest that WT1 is a promising target for novel molecular GBM therapies, perhaps in combination with standard treatment modalities.

Abbreviations used in this paper: ANOVA = analysis of variance; ATP = adenosine 5′-triphosphate; GBM = glioblastoma multiforme; GFP = green fluorescent protein; PBS = phosphate-buffered saline; SDS = sodium dodecyl sulfate; shRNA = short hairpin RNA; WT1 = Wilms tumor 1; [3H]Td = tritiated thymidine.

Article Information

Address correspondence to: William C. Broaddus, M.D., Ph.D., Department of Neurosurgery, Virginia Commonwealth University, P.O. Box 980631, Richmond, Virginia 23298-0631. email: wcbroadd@vcu.edu.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    Gel blots demonstrating LN-18 and U251MG GBM cell expression of WT1 protein. Forty micrograms of protein extracted from the U251MG and PC3 cells was separated using SDS–polyacrylamide gel electrophoresis and examined for WT1 expression by Western blot. The PC3 prostate carcinoma cell extract was used as a positive control for WT1 expression. The U251MG cells expressed high levels of WT1 protein. The anti–cyclophilin A (CypA) antibody demonstrated equal protein loading in all lanes.

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    Silencing of WT1 decreased in vitro cell proliferation. A: Gel blots showing U251MG and LN-18 cells virally transduced with expression vectors containing GFP and 1 of 2 sequences (2N and 4N) of WT1-shRNA. After several weeks of selection pressure, protein was extracted from the pooled population and analyzed for WT1 silencing by Western blot. B: Phase-contrast microscopy images demonstrating the structure of parental and virally transduced cells. Original magnification × 10. C: Fluorescence microscopy images (upper) revealing that the majority of U251MG cells transduced with the expression vector stably expressed GFP. Light microscopy images (lower) for comparison. Original magnification × 40. D: Graph of the ATP-based cell viability assay performed on U251MG cells on Days 0, 1, 3, 5, and 7 after plating showing the decrease in proliferation of the U251.Sh cells relative to cells transduced with empty vector (U251.VC). *p < 0.0003. E: Bar graph of results of the [3H]TdR uptake assay performed on Day 5 after plating confirming that the U251.Sh cells proliferated more slowly. *p = 0.0003. F: Graph of an ATP-based cell viability assay demonstrating the proliferation of LN18.Sh cells on Days 0, 1, 3, and 5 after plating relative to the LN18.VC cells. *p < 0.05.

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    Bar graph indicating that the silencing of WT1 decreased in vitro invasion. The U251 parental, U251.VC, and U251.Sh cells were plated in the top chamber of a modified chemotaxis manifold on a Matrigel-coated porous membrane. Two days after plating, the number of cells that had invaded the membrane was quantified by an ATP viability assay and demonstrated that U251.Sh cells exhibited lower invasion through the membrane. RLU = relative luciferase units. *p = 0.01.

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    Silencing of WT1 decreased in vivo growth rate. A: Photographs of representative animals from each group on Day 16 of postinoculation demonstrating the difference in tumor size of the U251.Sh tumors relative to the U251 parental (U251.UT) and U251.VC tumors. The scalpel handle is shown for scale. B: Bar graph of growth curve demonstrating the slower growth rate of U251.Sh tumors (15 mice, left). The in vivo growth experiment was repeated with similar results (15 mice, right). Error bars represent the standard error. *p < 0.0001, relative to U251 parental tumor growth. C: Photomicrographs demonstrating densely cellular tumors. Arrows indicate mitotic bodies. H & E. D: Representative Western blots of excised tumor protein lysates demonstrating that WT1 was expressed by all tumors. Expression of WT1 in protein extracts from cells growing in culture is shown for comparison.

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