Figure 2. Transient potent rRNA synthesis inhibition with CX-5461 is sufficient to commit ALL cells to cell death despite reactivation of rRNA synthesis.

A. SEM and NALM-6 cells were treated with 250 or 500 nM CX-5461, respectively. An aliquot was harvested after 3 hours, washed twice and cells were suspended in drug free media. Cell-cycle distribution was analyzed after 24 hours by flow cytometry of PI stained cells. Cells show aberrant cell-cycle distribution in drug washout cells compared to DMSO treated control cells. Representative flow cytometry data is shown from one of the three experiments. B. 45S pre-rRNA transcript levels were measured using quantitative PCR and normalized to the expression of GAPDH and Actin. DMSO and CX-5461 washout cells (CX w/o) show no difference in pre-rRNA synthesis at 24 hours. Experiments were repeated three times and data represents mean +/− S.D. C. Schematic of EU labeling of drug washout SEM cells. Newly synthesized EU labeled 45S pre-rRNA transcript levels were measured at 0 and 3 hours after EU removal. D. Cells were treated as in (a) and cell viability was measured using trypan blue staining. Drug washout cells show reduced viability compared to DMSO treated cells. Experiments are repeated three times. Data represents mean +/− S.D. E. SEM and NALM-6 cells were treated as before. NALM-6 cells show an increase in p53 and phospho-p53 levels at 3 hours after CX-5461 treatment. Elevated p53 levels in NALM-6 cells were substantially reduced 24 hours after drug washout.