Figure 2
Figure 2. MEK and Akt inactivation plays a key role in perifosine/PD-mediated lethality, and this treatment induces apoptosis in and diminishes the colony-forming ability of primary AML cells. (A) Two clones (Akt-CA4 and Akt-CA7) of U937 cells expressing constitutively active Akt (myristoylated Akt) and the empty-vector pUSEamp were left untreated or treated with perifosine and PD184352 for 16 hours, after which protein lysates were prepared and subjected to Western blot analysis to monitor the levels of total Akt and the degree of its phosphorylation. Endo-Akt indicates endogenous Akt. Alternatively, extent of apoptosis was determined using annexin V analysis (B). Values represent the means for 3 separate experiments ± SD. *Significantly lower than values obtained for empty-vector pUSEamp cells (P < .01). (C) U937 cells inducibly expressing dominant-negative Akt were left untreated or treated for 48 hours with 2 μg/mL doxycycline, after which the cells were either lysed and Western blot performed (C inset) or exposed to PD184352 (10 or 15μM) for an additional 24 hours and then subjected to annexin V/PI analysis. Values represent the means for 3 separate experiments ± SD. *Significantly higher than values obtained in the absence of doxycycline (P < .02). (D) U937 cells were transiently transfected with siRNA against MEK1 (siMEK1) or negative control siRNA (siNC) for 48 hours and then treated with 3μM perifosine or 10μM PD184352. The extent of apoptosis was monitored at 48 hours after treatment using annexin V staining assay. Values represent the means for 3 separate experiments ± SD. *Significantly higher than values obtained for siNC-transfected cells (P < .05). (E) Leukemic blasts were isolated, as described in “Isolation of patient-derived leukemic blasts,” from the bone marrow of 4 patients with AML (FAB classification M2), exposed to perifosine (7.5μM) and PD (10μM) for 24 hours, after which the extent of apoptosis was determined using annexin V analysis. (F) Alternatively, protein lysates were prepared and subjected to Western blot analysis. (G) Normal CD34+ cells were isolated, as described in “Isolation of CD34+ cells,” from the bone marrow of normal subjects (nonleukemic) and exposed to perifosine (7.5μM) and PD184352 (10μM) alone or in combination for 24 hours, after which cell viability was determined by flow cytometry using the annexin V staining assay. (H) Primary AML (patient nos. 2 and 4) and normal CD34+ cells (Lonza) were plated in methylcellulose in the presence of perifosine (7.5μM) and PD184352 (10μM) alone or in combination for 10 to 14 days, after which the CFUs were enumerated and expressed as a percentage of untreated cells, as described in “Methylcellulose colony formation assays.” (E-G) Each sample was analyzed in triplicate; values represent the means ± SD.

MEK and Akt inactivation plays a key role in perifosine/PD-mediated lethality, and this treatment induces apoptosis in and diminishes the colony-forming ability of primary AML cells. (A) Two clones (Akt-CA4 and Akt-CA7) of U937 cells expressing constitutively active Akt (myristoylated Akt) and the empty-vector pUSEamp were left untreated or treated with perifosine and PD184352 for 16 hours, after which protein lysates were prepared and subjected to Western blot analysis to monitor the levels of total Akt and the degree of its phosphorylation. Endo-Akt indicates endogenous Akt. Alternatively, extent of apoptosis was determined using annexin V analysis (B). Values represent the means for 3 separate experiments ± SD. *Significantly lower than values obtained for empty-vector pUSEamp cells (P < .01). (C) U937 cells inducibly expressing dominant-negative Akt were left untreated or treated for 48 hours with 2 μg/mL doxycycline, after which the cells were either lysed and Western blot performed (C inset) or exposed to PD184352 (10 or 15μM) for an additional 24 hours and then subjected to annexin V/PI analysis. Values represent the means for 3 separate experiments ± SD. *Significantly higher than values obtained in the absence of doxycycline (P < .02). (D) U937 cells were transiently transfected with siRNA against MEK1 (siMEK1) or negative control siRNA (siNC) for 48 hours and then treated with 3μM perifosine or 10μM PD184352. The extent of apoptosis was monitored at 48 hours after treatment using annexin V staining assay. Values represent the means for 3 separate experiments ± SD. *Significantly higher than values obtained for siNC-transfected cells (P < .05). (E) Leukemic blasts were isolated, as described in “Isolation of patient-derived leukemic blasts,” from the bone marrow of 4 patients with AML (FAB classification M2), exposed to perifosine (7.5μM) and PD (10μM) for 24 hours, after which the extent of apoptosis was determined using annexin V analysis. (F) Alternatively, protein lysates were prepared and subjected to Western blot analysis. (G) Normal CD34+ cells were isolated, as described in “Isolation of CD34+ cells,” from the bone marrow of normal subjects (nonleukemic) and exposed to perifosine (7.5μM) and PD184352 (10μM) alone or in combination for 24 hours, after which cell viability was determined by flow cytometry using the annexin V staining assay. (H) Primary AML (patient nos. 2 and 4) and normal CD34+ cells (Lonza) were plated in methylcellulose in the presence of perifosine (7.5μM) and PD184352 (10μM) alone or in combination for 10 to 14 days, after which the CFUs were enumerated and expressed as a percentage of untreated cells, as described in “Methylcellulose colony formation assays.” (E-G) Each sample was analyzed in triplicate; values represent the means ± SD.

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