Fig. 2. Characterization of fadraciclib-induced CLL cell death.

A Fadraciclib induced loss of mitochondrial membrane potential and apoptosis in the CLL cells. A representative flow cytometry result of CLL cells incubated with DMSO control or fadraciclib at 0.8 and 1.6 μM for 24 h. The cells were stained with annexin V-Cy5/DiOC6(3) and analyzed by flow cytometry. Cells with intact mitochondrial membrane potential were stained positive for DiOC6(3), and apoptotic cells were stained positive for annexin V. The percentages of live cells (annexin-–negative/DiOC6 + ) were shown in the upper left quadrant. B Induction of cell death in CLL cells after 6 h (●) and 24 h (■) exposure to increasing concentrations of fadraciclib. Cell viability was measured by annexin V/PI double staining and quantitated by flow cytometry analysis. Data represent mean ± SE of 5 samples. C Comparison of fadraciclib toxicity to CLL cells and to normal B and T cells from healthy donors. Cell survival (mean ± SE) was compared after 24 h incubation with increasing concentrations of fadraciclib in 5 CLL cell samples (○) and normal B cells (■), T cells (▲), and other cells (neither B nor T, ●) from 3 healthy donors. D Comparison of cell death induced by fadraciclib in CLL cells from patients with different prognostic characteristics and treatment histories. Cell death induced by 1.5 μM fadraciclib after 24 h incubation was compared in CLL cells from patients with favorable or poor prognostic factors. The number of samples in each group is shown below the columns. None of the comparisons were significantly different (p > 0.05). E Time course of induction of cell death by the CDK inhibitors and dactinomycin. CLL cells were incubated with approximately 2 × IC₅₀ concentrations of each compound for a duration of 22 h, and viability was measured by annexin V/PI double staining every 2 h. Data represent mean viability ± SE in 4 CLL samples. F Dose-response curves of the CDK inhibitors and dactinomycin. CLL cells were incubated with various concentrations of each compound for 24 h, and viability was measured by annexin V/PI. Data represent the mean viability ± SE of 5 to 10 samples). G Correlation analysis of the IC₅₀ values of the CDK inhibitors to induce apoptosis in the CLL cells and their IC₅₀ for each CDK. A 2-tailed Spearman analysis showed that the IC₅₀ values against CLL cells correlated significantly to their IC₅₀ values against CDK9, but not to that of the other CDKs.