Figure 1.

CPX-351 induces S phase arrest and activation of the ATR/CHK1 pathway. (a) DNA histograms of U937 cells treated for 24 h with diluent or CPX-351 (corresponding to cytarabine at 0.02, 0.04 and 0.08 µM in the fixed combination with doxorubicin) in the presence of the caspase inhibitor Q-VD-OPh²¹ (5 µM) to inhibit apoptosis-associated DNA degradation^19,20. At the completion of the incubation, cells were stained with PI and analyzed by flow microfluorimetry as described in the Methods. (b) Percentage U937 cells in S phase after treatment with the concentrations of CPX-351 shown in panel a as well as additional concentrations. Results are mean ± sd of 3 independent experiments. (c) U937 cells were treated for 4.5 h with diluent (lanes 1–5) or CPX-351 (lanes 6–10) at a concentration equivalent to 2.5 µM cytarabine and 0.5 µM daunorubicin (abbreviated 2.5 µM cytarabine equivalents in subsequent figures) in combination with the indicated concentration of MK-8776 and immunoblotted for the indicated antigens. HSP90β served as a loading control. (d) Dot plots of U937 cells treated for 24 h with diluent or CPX-351 (corresponding to cytarabine at 0.31 µM in the fixed combination with doxorubicin) in the presence of 5 µM Q-VD-OPh and, where indicated, 500 nM MK-8776. At the completion of the incubation, cells were fixed, permeabilized, stained with PI and anti-phospho-Ser¹⁰-Histone H3 (Phospho-H3), and analyzed by flow microfluorimetry. R2 indicates the mitotic population. (e) Percentage U937 cells in M phase after treatment with diluent or CPX-351 along with MK-8776. Results are mean ± sd of 4 independent experiments. *Indicates p < 0.01 compared to diluent or treatment with CPX-351 in the presence of MK-8776.