Altered subcellular distribution of nucleolar protein fibrillarin by actinomycin D in HEp-2 cells.

AIM To study the effects of actinomycin D on subcellular distribution of nucleolar protein fibrillarin in HEp-2 (human esophageal epithelial type 2) cells, and molecular mechanisms for maintenance of fibrillarin in nucleolus. METHODS Indirect immunofluorescence assay was employed to investigate subcellular distribution of nucleolar protein fibrillarin and immunoblotting analysis was used to detect the total cellular amount of fibrillarin. RESULTS Control cells with no drug treatment showed bright clumpy nucleolar staining, which indicated that fibrillarin decorated the nucleolus only. Treatment with actinomycin D caused dislocation of fibrillarin from nucleoli to nucleoplasm with numerous stained small nucleoplasmic entities. Immunoblotting showed that neither total cellular amount of fibrillarin nor the integrity of fibrillarin was changed upon the treatment. The dislocation of fibrillarin in cells treated at a lower concentration (0.05 mg/L) of actinomycin D, was totally reversible after removal of the drug from the medium. However, this reversion was not observed at a high drug concentration (1 mg/L). CONCLUSION Actinomycin D induced dislocation of fibrillarin from nucleoli to nucleoplasm in HEp-2 cells. The retention of fibrillarin within the nucleolus was related to active RNA synthesis.