Transcriptional factors NF-chi B and AP-1/c-fos in cell response on nocodazole treatment

Nuclear concentration of NF-kappaB p65 and p50 subunits and of AP-1/c-fos w as studied using immunofluorescence and Western blot assays in cultured CHO -K1 and HeLa control proliferating, serum starved and nocodazole treated ce lls. In addition, NF-kappaB and AP-1 activity was investigated in T-cell hy bridoma 2B4 cells using gel-retardation assay. In all the cells studied, at concentrations of 1 mcg/ml or 5 mcg/ml nocodazole induced rapid depolymeri zation of microtubules during 10-20 min. It appeared in Western blot assay that nocodazole treatment of cells always decreased the amount of proteolit ic degradation products of p65, p50 and, especially, c-fos. Amount of p65 a nd p50 determined in western blots of nuclear extracts was not significally changed after nocodazole treatment except elimination of p50 in nocodazole -treated serum-starved CHO-K1, and transient lack of p65 in 1 mug/ml nocoda zole-treated CHO-K1 cells. Immunofluorescence assay demonstrates that nocodazole treated CHO-K1 and He La cells concentration of c-fos in nuclei increased dramatically compared t o control cells. Intensity of anti-NF-kappaB-p50 staining in proliferating CHO-K1 and HeLa cells increased moderately after 60 min or even 30 min of n ocodazole action or did not change, whereas anti-p65 staining intensity tra nsiently decreased and then restored, or stayed almost constant. In serum-s tarved CHO-K1 cells nocodazole caused elimination of residual NF-kappaB fro m nuclei (inactivation of Nf-kappaB?), Gel-shift assay revealed that nocodazole treatment did not affect NF-kappaB activity in T-hybridoma nuclear extracts, but dramaticaly increased activi ty of AP-1/c-fos. Thus, activation of AP-1/c-fos is universal and strong response of cells on nocodazole treatment, while activation of NF-kappaB is not universal and i s not strong. AP-1 activation can be used in further investigation of micro tubule participation in signal transduction. Aberration of genome expressio n under nocodazole action must be taken into account in experiments with th is drug or another anti-microtubule drugs.