Effects of actinomycin D analogs on nucleolar phosphoprotein B23 (37,000 daltons/pI 5.1)

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Localization of protein B23 in HeLa cells after treatment with actinomycin D and its analogs was studied using indirect immunofluorescence. Bright nucleolar fluorescence was observed in control HeLa cells. After treatment with actinomycin D (250 ng/ml) for 2 hr, a uniform nucleoplasmic fluorescence was observed. Similar results were obtained with the actinomycin analogs, actinomycin Z5 and actinomycin K2T. Only after a much longer incubation (24 hr) with actinomycin 4-4′-gly was nucleoplasmic fluorescence observed. Actinomycin D, actinomycin Z5, and actinomycin K2T inhibited [ 3H]uridine incorporation into the trichloroacetic acid insoluble fraction of HeLa cells with IC 50 values of 9.5 ± 3.2, 59.1 ± 19.6 and 1423.3 ± 212.2 ng/ml respectively. No inhibition of [ 3H]uridine incorporation was observed using actinomycin 4-4′-gly (2000 ng/ml, 2-hr incubation). The order of potency for the loss of nucleolar fluorescence and the concurrent increase in nucleoplasmic fluorescence was actinomycin D > actinomycin Z5 > actinomycin K2T > actinomycin 4-4′-gly, which correlated with the order of their IC 50 values for inhibition of [ 3H]uridine incorporation. Studies of the effects of actinomycin D and its analogs on RNA synthesis and localization of protein B23 indicated that there is a direct relationship between the B23 “translocation” from nucleolus to nucleoplasm and the inhibition of RNA synthesis. At 45–55% inhibition of RNA synthesis, both nuclear and nucleolar B23 immunofluorescence were observed. At 75–85% inhibition, only a uniform nucleoplasmic fluorescence was observed.