MEK-1 inhibition sensitizes AML blasts to ATO-induced apoptosis. (A) Primary AML blasts were seeded at 2.5 × 105 in the presence of DMSO (vehicle) or PD184352 (1 μM) for 3 hours and then were incubated for 18 hours with the indicated concentration of ATO. Endogenous TA-p73α, TA-p73β, and ΔN-p73 proteins were revealed by immunoblotting analysis using a mouse monoclonal anti-p73 (clone 1288) or a mouse monoclonal anti-ΔNp73 (clone 38C674) antibody. Endogenous p53 was revealed by immunoblotting using a mouse monoclonal anti-p53 (DO-1) antibody. Anti–actin immunoblotting was performed as loading control. (B) TA-p73α, TA-p73β, ΔN-p73, and β-actin bands were subjected to densitometric scanning using the TINA 2 software, and the TA-(p73α+p73β)/ΔN-p73 ratio was calculated. (C) Expression of PARP cleavage, Bax, PUMA, and p53AIP1 was revealed after 48 hours of treatment. Cell lysates were analyzed by immunoblotting analysis using mouse monoclonal anti-PARP (F2), rabbit polyclonal anti-Bax, rabbit polyclonal anti-PUMA, rabbit polyclonal anti-p53AIP1 (CT), and goat polyclonal anti–human actin. Antiactin immunoblotting was performed as loading control. (D) Primary AML blasts were cultured as described. After 48 hours of treatment, the cells were harvested for ΔΨm detection by flow cytometry. Values are expressed as percentage of cells with low ΔΨm. (inset) CI plot for the combination of escalating doses of PD184352 and ATO at a 1:1 ratio, obtained as described in the Table 1 footnote. The dashed line indicates a CI value of 1. CI, 1. (E) Primary AML blasts were cultured as described and were stained for annexin V binding. CTR indicates control; PD, PD184352 (1 μM).
