Participation of an abnormality in the transforming growth factor–β signaling pathway in resistance of malignant glioma cells to growth inhibition induced by that factor

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Object

Malignant glioma cells secrete and activate transforming growth factor–β (TGFβ) and are resistant to growth inhibition by that factor. Nevertheless, the mechanism underlying this effect remains poorly understood. In this study, the mechanism of the resistance to growth inhibition induced by TGFβ was investigated.

Methods

The authors examined the expression of downstream components of the TGFβ receptor, including Smad2, Smad3, Smad4, and Smad7, and the effect of TGFβ1 treatment on the phosphorylation of Smad2 and the nuclear translocation of Smad2 and Smad3 by using 10 glioma cell lines and the A549 cell line, which is sensitive to TGFβ-mediated growth inhibition. The expression of two transcriptional corepressor proteins, SnoN and Ski, and the effect of TGFβ1 treatment on the expression of the SnoN protein and the cell cycle regulators p21, p15, cyclin-dependent kinase–4 (CDK4), and cyclin D1 were also examined.

Expression of the Smad2 and Smad3 proteins was lower in the glioma cell lines than in the A549 cell line and in normal astrocytes. In particular, Smad3 expression was low or very low in nine of the 10 malignant glioma cell lines. Expression of Smad4 was low in four glioma cell lines, and expression of the Smad7 protein was similar when compared with protein expression in the A549 cell line and in normal astrocytes. The levels of Smad2 phosphorylation after TGFβ1 treatment were lower in glioma cell lines than in the A549 cell line, except for one glioma cell line. Seven of the 10 glioma cell lines exhibited lower levels of nuclear translocation of Smad2 and Smad3, and two cell lines that expressed very low levels of Smad3 protein showed no nuclear translocation. All glioma cell lines expressed the SnoN protein and its expression was unaltered by treatment with TGFβ1. Three glioma cell lines expressed high levels of the Ski protein. The expression of the p21cip1, p15INK4B, CDK4, and cyclin D1 proteins was not altered by TGFβ1 treatment, except in one cell line that displayed a slight increase in p21 protein. Overall, the expression of the Smad2 and Smad3 proteins was low in the glioma cell lines, the phosphorylation and nuclear translocation of Smad2 and Smad3 were impaired, and the TGFβ receptor signal did not affect the expression of the SnoN, p21, p15, cyclin D1, and CDK4 proteins.

Conclusions

These results suggest that the ability to resist TGFβ-mediated growth inhibition in malignant glioma cells is due to abnormalities in the TGFβ signaling pathway.

Abbreviations used in this paper:CDK = cyclin-dependent kinase; GBM = glioblastoma multiforme; GFAP = glial fibrillary acidic protein; PCNA = proliferating cell nuclear antigen; TGFβ = transforming growth factor–β.

Article Information

Address reprint requests to: Hirofumi Naganuma, M.D., Ph.D., Department of Neurosurgery, Faculty of Medicine, University of Yamanashi, 1110 Shimokato, Chuo City, Yamanashi 409-3898, Japan. e-mail: nagahiro@yamanashi.ac.jp.

© AANS, except where prohibited by US copyright law.

Headings

Figures

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    A schematic drawing summarizing the TGFβ signaling pathway. P = phosphorylation.

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    Line graphs showing the effect of TGFβ1 on the growth of the Mv1Lu and A549 cell lines, which are sensitive to TGFβ-induced growth inhibition. Transforming growth factor–β1 (5 ng/ml) was placed in a 96-well plate in triplicate and serial twofold dilutions were made in the plate. The cell suspension (2000 cells) was added to the 96-well plates and cultured for 72 hours. The values of optical density correlate to the number of cells in each well. The values are expressed as means ± standard deviations.

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    Bar graph demonstrating the effect of TGFβ1 on the growth of 10 malignant glioma cell lines and the A549 cell line. The glioma cell lines (2000 cells/well) were cultured in 96-well plates with or without serially diluted TGFβ1. The values are expressed as means ± standard deviations.

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    Results of a Western blot analysis of Smad2, Smad3, Smad4, and Smad7 proteins in 10 malignant glioma cell lines. The A549 cell line, which is sensitive to TGFβ, and normal human astrocytes were used as controls. Forty micrograms of protein was loaded onto each lane; the molecular mass (kD) is indicated. A: A positive control for Smad4 using Jurkat cell lysate. B: A positive control for Smad7 using HepG2 cell lysate. NHA = normal human astrocyte.

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    Results of a Western blot analysis of the cellular location of Smad7 protein. Each cell lysate was fractionated into cytoplasmic (Cyto) and nuclear (Nuc) fractions. Forty micrograms of protein was loaded onto each lane. The efficacy of the fractionation was examined using antiactin and anti–heat shock protein 70 (HSP70) antibodies as internal controls.

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    Results of a Western blot analysis of the effect of TGFβ1 on the phosphorylation of Smad2 and on the translocation of Smad2 and Smad3 to the nucleus. Cells from the A549 line, normal astro-cytes, and 10 malignant glioma cell lines were incubated with (+) or without (−) TGFβ1 (5 ng/ml) for 30 minutes and examined for induction of phosphorylated Smad2 (P-Smad2) by Western blotting. Tumor cells were also incubated with or without TGFβ1 (5 ng/ml) for 1 hour, and the cytoplasmic and nuclear fractions were examined for Smad2 and Smad3. Forty micrograms of cytoplasmic proteins, or 10 or 20 μg of nuclear proteins were loaded onto each lane. For the A549 cell line, 20 μg of cytoplasmic protein and 10 μg of nuclear protein were loaded. The protein contents of actin and PCNA were examined as internal controls for cytoplasmic and nuclear fractions, respectively. fr. = fraction.

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    Results of a Western blot analysis of the transcriptional corepressor proteins SnoN and Ski in 10 malignant glioma cell lines, the A549 cell line, and normal astrocytes. Hela cell lysate was used as a positive control for SnoN and Ski. Forty micrograms of protein was loaded onto each lane.

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    Results of a Western blot analysis of the effect of TGFβ1 on the expression of SnoN protein. A: Normal astrocyte A549 cells were treated with (+) or without (−) TGFβ1 (5 ng/ml) for 0.5 or 12 hours and whole-cell lysates were examined for SnoN. B: Ten types of malignant glioma A549 cells were treated with (+) or without (−) TGFβ1 (5 ng/ml) for 60 minutes and the nuclear fractions were examined for SnoN. The protein content of PCNA was examined as an internal control for the nuclear fraction.

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    Results of a Western blot analysis of the effect of TGFβ1 on the expression of p21, cyclin D1, and CDK proteins. Glioma cells and A549 cells were incubated with (+) or without (−) TGFβ1 (5 ng/ml) for 12 hours. Forty micrograms of protein was loaded onto each lane.

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    Results of a Western blot analysis of the effect of TGFβ1 on the expression of p15 protein. Glioma cells (five lines), A549 cells, and normal astrocytes were incubated with (+) or without (−) TGFβ1 (5 ng/ml) for 12 hours. The TM2, YMG3, and YMG4 glioma cell lines expressed p15 protein, and the T98G and U251 glioma cell lines showed no signs of p15 expression. Forty micrograms of protein was loaded onto each lane.

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