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Expression of Dominant-negative Form of Ets-1 Suppresses Fibronectin-stimulated Cell Adhesion and Migration Through Down-Regulation of Integrin 5 Expression in U251 Glioma Cell Line¹

Department of Molecular Virology and Oncology [D. K., Ta. T., M. N., To. T., H. S.], Center for the Development of Molecular Target Drugs, Cancer Research Institute [H.S.], Department of Neurosurgery, School of Medicine [D. K., M. N., To. T., J. Y.] Kanazawa University, Kanazawa, Ishikawa 920-0934, Japan

Ets transcription factors are associated with tumor malignancy. We reported previously that the stable transfection of the dominant-negative form of Ets-1 (Ets-DN) in the glioma cell line U251 induced down-regulation of urokinase-type plasminogen activator mRNA expression and invasiveness (M. Nakada et al., J. Neuropathol. Exp. Neurol., 58: 329–334, 1999). Here we analyzed effects of Ets-DN expression on cell adhesion, migration, and phosphorylation of focal adhesion kinase. U251 cells expressing Ets-DN (U251-DN) showed reduced cell adhesion, spreading, and extension of actin stress fibers on dishes coated with fibronectin but not on dishes coated with collagen. Migration of U251-DN cells was found to be significantly inhibited compared with that of parental cells when examined by wound-induced migration assay on fibronectin-coated dishes. Phosphorylation levels of focal adhesion kinase in U251-DN cells were also attenuated on dishes coated with fibronectin. Reduced expression level of integrin 5 subunit in U251-DN cells was demonstrated by semiquantitative reverse transcription-PCR analysis. Semiquantitative reverse transcription-PCR of surgical samples of brain tumors revealed that the expression level of Ets-1 mRNA correlated with that of integrin 5 mRNA in glioma. The experimental metastatic ability of U251-DN cells examined in chick embryo was considerably lower than that of parental cells. These results suggest that Ets-1 contributes to glioma malignancy by up- regulating expression of the integrin 5 subunit, which composes integrin 5ß1 and mediates intracellular signaling and the subsequent acceleration of the invasive process, including cell adhesion and migration.

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