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.

The cytotoxic effects of doxorubicin (DOX) and daunorubicin (DAU) on HeLa cells cultured under different serum conditions were analyzed by the "nucleophosmin translocation" assay using immunofluorescence. Bright nucleolar fluorescence was observed in untreated cells. A shift from nucleolar to nuclear fluorescence was observed with increasing doses of DOX or DAU, with longer incubation times. A lesser degree of nucleophosmin translocation from nucleoli to nucleoplasm was observed in serum-deprived cells under the same DOX or DAU treatment. These results correlated well with those of cell-growth-reversibility and colony-formation studies, showing decreased inhibitory effects of growth on cells cultured in medium without serum. Furthermore, cells cultured in medium supplemented with the lipoprotein-free serum responded to DOX in a similar way to cells cultured without serum. High-density lipoprotein (HDL) and low-density lipoprotein (LDL) were then added to the lipoprotein-free serum. Cells cultured in medium with the HDL-supplemented, serum showed increased sensitivity to DOX. Inhibition of cell growth and colony formation was observed in such HDL-supplemented cells upon DOX treatment (30 min). LDL, on the other hand, did not show an increase in the anti-cancer response. These results suggested that the variation in response of cells to DOX anti-cancer treatment under different growth conditions may be due to their varied concentrations of HDL. "Nucleophosmin translocation", which is useful for monitoring and ensuring the efficacy of the drug during anti-cancer treatment, provides an improved potential for successful chemotherapy.

No abstract

During continuous exposure, cells were more responsive to doxorubicin (DOX) in the presence of dipyridamole (DPM). Translocation of nucleolar phosphoprotein B23 and inhibition of cell growth occurred with a lower dose of DOX and in a shorter incubation time in the presence of DPM. DPM did not change translocation induced by actinomycin D (Act-D). Short exposure of HeLa cells to Act-D induced "reversible" translocation of protein B23 as well as "reversible" inhibition of cell growth. DPM included in the cell culture after removal of Act-D inhibited the recovery of cell growth as well as the corresponding relocalization of protein B23 from the nucleoplasm to nucleoli. DPM administered in the fresh medium after 30 min exposure to DOX had little effect on the potentiation of the induced translocation of protein B23 and inhibition of cell growth. Our results indicated that "B23 translocation" is closely associated with states of cell growth. The potentiation of the inhibition of cell growth by DPM is associated with the extent of enhanced protein B23 translocation. "B23 translocation" may therefore be a simple and rapid method for assessing the inhibition of cell growth and for determining the efficacy of combination cancer chemotherapy.