Figure 1. Constitutive Akt Activation Promotes Myeloid Maturation and Apoptosis of Leukemic Cells.

(A) Lineage^low, Sca-1⁻, cKit^high, CD34⁺ cells purified from healthy and leukemic mice were stimulated with or without mSCF and then subjected to flow cytometry with phospho-Akt^Ser473 (CD34⁺ myeloid progenitors [MP] versus CD34⁺ leukemic progenitors [LP], p = 0.0478). Right panel is a histogram from a single experiment with the left panel representing mean ± standard error of the mean (SEM) from three experiments.

(B) Mononuclear bone marrow cells (MNBCs) recovered from MLL-AF9 leukemic mice were infected with MSCV-IRES-GFP control (Ctrl) or myr-Akt-expressing retroviruses. Cells from each condition were then treated with vehicle or 10 nM or 100 nM rapamycin and evaluated for numbers of GFP⁺ cells every 2 days using flow cytometry (day 6 *Ctrl versus myr-Akt, vehicle, p = 0.0005; **Ctrl versus myr-Akt, 10 nM rapamycin, p = 0.0008; ***Ctrl versus myr-Akt, 100 nM rapamycin, p = 0.0046; n = 3). Data are represented as the mean ± standard deviation (SD).

(C) GFP⁺ cells treated as described above were analyzed for CD11b expression (*Ctrl versus myr-Akt, vehicle, p < 0.0001; **Ctrl versus myr-Akt, 10 nM rapamycin, p < 0.0001; *** Ctrl versus myr-Akt, 100 nM rapamycin, p < 0.0001; n = 3) or (D) stained with May-Grünwald Giemsa. Data are represented as the mean ± SD.

(E and F) Flow cytometric analysis of control and myr-Akt-infected cells incubated with pHrodo fluorescent-labeled E. coli particles. (E) Flow cytometric histogram plot of pHrodo-stained Ctrl versus myr-Akt GFP⁺ cells and (F) graphical representation of three replicates (*Ctrl versus myr-Akt, vehicle, p < 0.0001; **Ctrl versus myr-Akt, 10 nM rapamycin, p < 0.0001; ***Ctrl versus myr-Akt, 100 nM rapamycin, p = 0.0009; n = 3). Data are represented as the mean ± SD.

(G) Flow cytometric analysis of Annexin V expression on Ctrl and myr-Akt-expressing cells (*Ctrl versus myr-Akt, p = 0.0006; n = 3). Data are represented as the mean ± SD.

See also Figure S1.