| Popis: |
A new quantitative cytometric technique, termed the ArrayScanTM, is described and used to measure NF-kappaB nuclear translocation induced by interleukin (IL)-1 and tumor necrosis factor-alpha (TNFalpha). The amount of p65 staining is measured in both the nuclei defined by Hoechst 33342 labeling and in the surrounding cytoplasmic area within a preselected number of cells/well in 96-well plates. Using this technique in synchronously activated human chondrocytes or HeLa cells, NF-kappaB was found to move to the nucleus with a half-time of 7-8 min for HeLa and 12-13 min for chondrocytes, a rate in each case about 4-5 min slower than that of Ikappa Balpha degradation. IL-1 receptor antagonist and anti-TypeI IL-1 receptor antiserum on the one hand and anti-TNFalpha and monoclonal anti-TNF receptor 1 antibodies on the other hand could be shown to respectively inhibit IL-1 and TNFalpha stimulation in both cell types. In contrast, a polyclonal anti-TNF receptor 1 antiserum exhibited both a 50% agonism and a 50% antagonism to a TNFalpha stimulation in a dose-dependent fashion, indicating that subtle functional responses to complex agonist and antagonist stimuli could be measured. The effects of different proteasome inhibitors to prevent Ikappa Balpha degradation and subsequent NF-kappaB translocation could also be discriminated; Leu-Leu-Leu aldehyde was only a partial inhibitor with an IC50 of 2 microM, while clastolactacystin beta-lactone was a complete inhibitor with an IC50 of 10 microM. The nonselective kinase inhibitor K252a completely inhibited both IL-1 and TNFalpha stimulation in both cell types with an IC50 of 0.4 microM. This concentration, determined after a 20-min stimulation, was shown to be comparable with that obtained for inhibition of IL-6 production induced by a 100-fold lower IL-1 and TNFalpha concentration measured after 17 h of stimulation. These results suggest that the ArrayScanTM technology provides a rapid, sensitive, quantitative technique for measuring early events in the signal transduction of NF-kappaB. |
