A correlated morphological and functional study of the effects of actinomycin D on HeLa cells. I. Effects on the nucleolar and cytoplasmic ribonucleoproteins

The sequence of changes in the ribonucleoproteins (RNP) of HeLa cells caused by low levels (0.01 μg/ml) of actinomycin D was studied. Within a few hours the nucleolini enlarged and began to fuse. They assumed a marginal position in the nucleolus at first producing a mulberry appearance, later fusing to form dark-staining "nucleolar caps". By 16 hr the nucleolar caps either contained no stainable RNA or had disappeared, and the pars amorpha of the nucleolus was diminished in size. After 24 hr of treatment no stainable RNA was present in the nucleoli, although at least some of its protein was still recognizable. Following the nucleolar alterations, two of the species of RNP revealed by the toluidine blue-molybdate methods-cytoplasmic granular RNP and diffuse cytoplasmic RNP, type A-gradually diminished in amount. By 24 hr they had disappeared. Concomitantly there was a 50 per cent fall in the cell content of RNA. Cell division continued, but at a decreasing rate. After 48 hr of treatment cell replication was more severely inhibited. This temporal sequence was reversed on removal of the drug. The pattern of events, taken in conjunction with the parallel biochemical investigation [8], is consistent with a role of the nucleolus as the principal site of synthesis of ribosomal RNA, as has been shown by studies using different methods [29]. This work also provides further support for the identification of the cytoplasmic granular RNP and the diffuse cytoplasmic RNP, type A as ribosomal in nature.