Schedule-dependent effects of two consecutive, divided, low doses of actinomycin D on translocation of protein B23, inhibition of cell growth and RNA synthesis in HeLa cells.

Abstract

The effects of 2 consecutive, divided, low doses of actinomycin-D (Act-D) on cellular localization of protein B23, inhibition of cell growth, RNA synthesis and colony formation were studied in HeLa cells. The second dose of Act-D was administered at various times after removal of the first dose. One short exposure of HeLa cells to Act-D had previously been shown to induce "reversible" translocation of protein B23, inhibition of cell growth, and RNA synthesis. Relocalization of protein B23 from the nucleoplasm to nucleoli as well as "reversible" inhibition of cell growth and RNA synthesis were still observed in cells that had been treated with a second dose of Act-D administered as early as 0-2 hr or as late as 30 hr after removal of the first dose of Act-D. In contrast, no relocalization of protein B23 from the nucleoplasm to nucleoli was observed in cells that had been treated with a second dose of Act-D administered 9 hr after removal of the first dose. A second exposure to Act-D, administered 9 hr after removal of the first dose, caused irreversible inhibition of cell growth and RNA synthesis; a significant inhibitory effect on colony formation was also observed. RNA synthesis in HeLa cells after 2 sequential exposures to Act-D was further analyzed by 1% agarose gel electrophoresis. There were higher-molecular-weight bands above 28S RNA, which may be the 45S and 32S RNA, observed in the controls and in the cells that had been exposed to Act-D treatment once or in the cells that underwent Act-D exposure twice, in which the second dose was administered as early as 0-2 hr or as late as 30 hr after removal of the first dose. These high-molecular-weight bands were not observed in the cells that underwent Act-D exposure twice, in which the second dose was administered 9 hr after removal of the first. These results indicated that cells at different stages of inhibition or that have recovered from the first exposure to Act-D respond differently to the second short Act-D exposure.