FIGURE 3.

Ellipticines rapidly inhibit rDNA transcription in cells, and inhibition mechanism is not linked to known activities of the drugs. A, HeLa, H1299, U2OS, and MCF10A cell lines were incubated for 60 min with various concentrations of 9HE as indicated. The relative level of pre-rRNA was determined by S1 nuclease protection assay. The results were quantified with aid of phosphorimaging and expressed as a percentage of the highest value (set at 100%). The data represent an average from three independent experiments. S.D. is shown. B, U2OS cells were incubated for various times with two concentrations of 9HE as indicated. The relative level of pre-rRNA was determined as in A. C, HTETOP cells were incubated for 48 h with 1 μg/ml (+Tet) or without (no Tet) tetracycline and treated with different 9HE concentrations for 30 min. The relative level of pre-rRNA was determined as in A. D, p53 null H1299 cells were incubated for 2 h with 125 μm caffeine (+caffeine) or without (control) and treated with different 9HE concentrations for 30 min. The relative level of pre-rRNA was determined as in A. E, the level of confluence of growing H1299 (p53^−/−) cells was measured by IncuCyte (Essen) each hour for 43 h in total and expressed as %. Mean values for three independent experiments are represented on the graph, and S.D. are shown. Cells were grown untreated (control) and in the presence of 125 μm caffeine, 50 μm etoposide, 5 μm 9HE and 125 μm caffeine, and 5 μm 9HE combined (9HE + caffeine). Growth media were replaced each 12 h. AU, arbitrary units.