Inhibition of nuclear factor-kappaB 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 cytostatic effects on tumor cells, but susceptibility to TNF-alpha varies among different types of cells. TNF-alpha activates a transcription factor, nuclear factor-kappaB (NF-kappaB), which induces a wide variety of genes and causes pleiotrophic responses. In this study, the relationship between susceptibility to TNF-alpha and activation of NF-kappaB was investigated in six human malignant glioma cell lines. Cell proliferation analysis revealed that only one cell line, SK-MG-1, was sensitive to TNF-alpha and that the other five, including U-251MG, were resistant. Electrophoretic mobility-shift assay showed that TNF-alpha strongly activated a subtype of NF-kappaB, the 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-kappaB by TNF-alpha in the resistant cell lines resulted in a significant increase of a reporter gene expression driven by NF-kappaB site, suggesting a possibility that activation of p50-p65 confers resistance to TNF-alpha. To test this hypothesis, we established a stable cell line that expresses an inducible dominant negative NF-kappaB (p65 DN) protein in one of the TNF-alpha-resistant cell lines, U-251MG. In the established clone, induction of p65 DN protein decreased TNF-alpha-dependent increase in the DNA binding of p50-p65 heterodimer and NF-kappaB-dependent reporter gene activity. Although no growth inhibition of this clone was observed by TNF-alpha treatment, induction of p65 DN together with TNF-alpha resulted in a significant decrease in cell number. Cell cycle analysis revealed that this growth inhibition was due to the impairment of cell cycle progression. These results indicate that an active NF-kappaB complex, such as the p50-p65 heterodimer, plays a crucial role in the progression of cell cycle in malignant glioma cells. Refractoriness to TNF-alpha treatment could be prevented by inhibiting NF-kappaB activation.
Otsuka
G
G
Department of Endocrinology and Metabolism, Research Institute of Environmental Medicine, Nagoya University, Japan.
Nagaya
T
T
Saito
K
K
Mizuno
M
M
Yoshida
J
J
Seo
H
H
D002453:Cell Cycle / Q000187:drug effects; D019281:Dimerization; D005910:Glioma / Q000473:pathology*; D006801:Humans; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000502:physiology*; D014407:Tumor Cells, Cultured; D014409:Tumor Necrosis Factor-alpha / Q000494:pharmacology*
D016328:NF-kappa B; D014409:Tumor Necrosis Factor-alpha