Effects of actinomycin D analogs on nucleolar phosphoprotein B23 (37,000 daltons/pI 5.1).
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 IC50 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 greater than actinomycin Z5 greater than actinomycin K2T greater than actinomycin 4-4'-gly, which correlated with the order of their IC50 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.
B Y
BY
Yung
R K
RK
Busch
H
H
Busch
A B
AB
Mauger
P K
PK
Chan
D002466:Cell Nucleolus / Q000032:analysis*; D003609:Dactinomycin / Q000031:analogs & derivatives* / Q000494:pharmacology; D006367:HeLa Cells; D006801:Humans; D009687:Nuclear Proteins; D000090243:Nucleophosmin; D009698:Nucleoproteins / Q000032:analysis*; D010750:Phosphoproteins / Q000032:analysis*; D012313:RNA / Q000096:biosynthesis; D012261:Ribonucleoproteins / Q000032:analysis*; D013997:Time Factors
C000716967:NPM1 protein, human; D009687:Nuclear Proteins; D009698:Nucleoproteins; D010750:Phosphoproteins; D012261:Ribonucleoproteins; D000090243:Nucleophosmin; D003609:Dactinomycin; D012313:RNA