Inhibition of nuclear factor-kappa B activation confers sensitivity to tumor necrosis factor-alpha by impairment of cell cycle progression in human glioma cells

Tumor necrosis factor (TNF)-alpha has been shown to exert cytotoxic or cyto static effects on tumor cells, but susceptibility to TNF-alpha varies among different types of cells. TNF-alpha activates a transcription factor, nucl ear factor-kappa B (NF-kappa B), which induces a wide variety of genes and causes pleiotrophic responses. In this study, the relationship between susc eptibility to TNF-alpha and activation of NF-kappa B was investigated in si x human malignant glioma cell lines. Cell proliferation analysis revealed t hat only one cell line, SK-MG-1, was sensitive to TNF-alpha and that the ot her five, including U-251MG, were resistant. Electrophoretic mobility-shift assay showed that TNF-alpha strongly activated a subtype of NF-kappa B, th e p50-p65 heterodimer, in all of the resistant cell lines tested. However, this activation was weak in the sensitive cell line, SK-MG-1. Activation of NF-kappa B by TNF-alpha in the resistant cell lines resulted in a signific ant increase of a reporter gene expression driven by NF-kappa B site, sugge sting a possibility that activation of p50-p65 confers resistance to TNF-al pha. To test this hypothesis, we established a stable cell line that expres ses an inducible dominant negative NF-kappa B (p65 DN) protein in one of th e TNF-alpha-resistant cell lines, U-251MG. In the established clone, induct ion of p65 DN protein decreased TNF-alpha-dependent increase in the DNA bin ding of p50-p65 heterodimer and NF-kappa B-dependent reporter gene activity . Although no growth inhibition of this clone was observed by TNF-alpha tre atment, induction of p65 DN together with TNF-alpha resulted in a significa nt decrease in cell number. Cell cycle analysis revealed that this growth i nhibition was due to the impairment of cell cycle progression. These result s indicate that an active NF-kappa B complex, such as the p50-p65 heterodim er, plays a crucial role in the progression of cell cycle in malignant glio ma cells. Refractoriness to TNF-alpha treatment could be prevented by inhib iting NF-kappa B activation.