pervanadate and acetylleucyl-leucyl-norleucinal

To assess the role of nuclear factor kappaB (NFKB) in cellular radiosensitivity, three different IkappaB-alpha (also known as NFKBIA) expression plasmids, i.e., S-IkappaB (mutations at (32, 36)Ser), Y-IkappaB (a mutation at (42)Tyr), and SY-IkappaB, were constructed and introduced into human brain tumor M054 cells. The clones were named as M054-S8, M054-Y2 and M054-SY4, respectively. Compared to the parental cell line, M054-S8 and M054-Y2 cells were more sensitive to X rays while M054-SY4 cells exhibited the greatest sensitivity. After treatment with N-acetyl-Leu-Leu-norleucinal, a proteasome inhibitor, the X-ray sensitivity of M054-S8 and M054-SY4 cells did not change, while that of M054-Y2 cells and the parental cells was enhanced. An increase in X-ray sensitivity accompanied by a decrease in translocation of NFKB to the nucleus in parental cells was observed after treatment with pervanadate, an inhibitor of tyrosine phosphatase, as well as in M054-S8 and M054-SY4 cells. Repair of potentially lethal damage (PLD) was observed in the parental cells but not in the clones. Four hours after irradiation (8 Gy), the expression of TP53 and phospho-p53 ((15)Ser) was induced in the parental cells but not in M054-S8, M054-Y2 or M054-SY4 cells. Our data suggest that inhibition of IkappaB-alpha phosphorylation at serine or tyrosine acts independently in sensitizing cells to X rays. NFKB may play a role in determining radiosensitivity and PLD repair in malignant glioma cells; TP53 may also be involved.

Topics: Cell Survival; Cloning, Molecular; DNA Repair; Gene Expression Regulation, Neoplastic; Glioma; Humans; Leupeptins; Mutagenesis, Site-Directed; NF-kappa B; Phosphorylation; Radiation Tolerance; Transfection; Tumor Cells, Cultured; Vanadates

Tumor necrosis factor activates nuclear transcription factor kappaB (NF-kappaB) by inducing serine phosphorylation of the inhibitory subunit of NF-kappaB (IkappaBalpha), which leads to its ubiquitination and degradation. In contrast, pervanadate (PV) activates NF-kappaB and induces tyrosine phosphorylation of IkappaBalpha (Singh, S., Darney, B. G., and Aggarwal, B. B. (1996) J. Biol. Chem. 271, 31049-31054; Imbert, V., Rupec, R. A., Antonia, L., Pahl, H. L., Traenckner, E. B.-M., Mueller-Dieckmann, C., Farahifar, D., Rossi, B., Auderger, P., Baeuerle, P. A., and Peyron, J.-F. (1996) Cell 86, 787-798). Whether PV also induces IkappaBalpha degradation and whether degradation is required for NF-kappaB activation are not understood. We investigated the effect of PV-induced tyrosine phosphorylation on IkappaBalpha degradation and NF-kappaB activation. PV activated NF-kappaB, as determined by DNA binding, NF-kappaB-dependent reporter gene expression, and phosphorylation and degradation of IkappaBalpha. Maximum degradation of IkappaBalpha occurred at 180 min, followed by NF-kappaB-dependent IkappaBalpha resynthesis. N-Acetylleucylleucylnorlucinal, a proteasome inhibitor, blocked both IkappaBalpha degradation and NF-kappaB activation, suggesting that the IkappaBalpha degradation is required for NF-kappaB activation. PV did not induce serine phosphorylation of IkappaBalpha but induced phosphorylation at tyrosine residue 42. Unlike tumor necrosis factor (TNF), PV did not induce ubiquitination of IkappaBalpha. Like TNF, however, PV induced phosphorylation and degradation of IkappaBalpha, and subsequent NF-kappaB activation, which could be blocked by N-tosyl-L-phenylalanine chloromethyl ketone, calpeptin, and pyrrolidine dithiocarbomate, suggesting a close link between PV-induced NF-kappaB activation and IkappaBalpha degradation. Overall, our studies demonstrate that PV activates NF-kappaB, which, unlike TNF, requires tyrosine phosphorylation of IkappaBalpha and its degradation.

Topics: Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; HeLa Cells; Humans; I-kappa B Proteins; Leupeptins; Multienzyme Complexes; NF-kappa B; NF-KappaB Inhibitor alpha; Phosphorylation; Proteasome Endopeptidase Complex; Protein Tyrosine Phosphatases; Transcription, Genetic; Tumor Necrosis Factor-alpha; Tyrosine; U937 Cells; Vanadates