Figure 1
Figure 1. Protein synthesis inhibitor enhanced anthracycline-induced AML cell death, although suppressing death phenotype features in some cells. (A) HL60 cells were treated for 6 hours with DNR (1.6 μmol/L) and CHX (3.6 μmol/L), alone or in combination, and scored for apoptosis (insets; see “Assessment of apoptosis”). (B) NB4 cells were treated for 6 hours with DNR (5 μmol/L) and CHX (3.6 μmol/L), alone or in combination, and scored for apoptosis. (C) Extracts from HL60 cells treated with DNR (8 μmol/L) for 2, 4, or 6 hours in the absence or presence of CHX (3.6 μmol/L, added 1 hour after DNR) were immunoblotted for caspase 3. (D-F) Flow cytometric analysis (representative of 4 separate experiments) of HL60 cells treated for 4 hours with vehicle (D), 8 μmol/L DNR (E) or DNR + 3.6 μmol/L CHX (F). Note that DNR + CHX converted twice as many cells as DNR alone to a compartment with decreased forward (FSC-H) and side (SSC-H) scatter. (G) Scoring of morphologically evident apoptosis in IPC-81 cells treated for 5 hours with DNR (0.1 μmol/L) alone or in combination CHX (3.6 μM). (H) IPC-81 cells were treated for 6 hours with various concentrations of DNR alone or with CHX (3.6 μmol/L) and scored for apoptotic morphology. Note the lack of cell budding, nuclear fragmentation, and chromatin condensation with DNR + CHX and with DNR more than ∼1 μmol/L. The bars show apoptosis in cells incubated for 6 hours with DNR + CHX or CHX alone, washed in excess medium, and incubated without DNR and CHX for another 12 hours before assessment of apoptosis. Note the high proportion of apoptotic cells after washing. (I) As for panel G, except that cells were treated for 3 hours with DNR (0.1 μmol/L) and CHX (3.6 μmol/L), washed and further incubated for 9 hours (as explained in panel H) before scoring of apoptosis. (J) HL60 cells received the protein synthesis inhibitors emetine (10 nmol/L), puromycin (180 nmol/L), rapamycin (10 or 100 nmol/L), or CHX (3.6 μmol/L) at various time points relative to DNR (1.6 μmol/L), and apoptosis scored 4.5 hours after DNR addition. (K) HL60 cells were treated for 6.5 hours with various concentrations of DNR (50-8000 nmol/L) in the absence (○) or presence (●) of CHX (3.6 μmol/L) and scored for apoptosis. (L) The accumulation of apoptotic HL60 cells as a function of time after addition of DNR (8 μmol/L) alone (○), CHX (3.6 μmol/L) alone (□), or a combination of the 2 (●). CHX was added 1 hour after DNR. Apoptosis scores represent the means (± SEM) of 3 to 6 experiments.

Protein synthesis inhibitor enhanced anthracycline-induced AML cell death, although suppressing death phenotype features in some cells. (A) HL60 cells were treated for 6 hours with DNR (1.6 μmol/L) and CHX (3.6 μmol/L), alone or in combination, and scored for apoptosis (insets; see “Assessment of apoptosis”). (B) NB4 cells were treated for 6 hours with DNR (5 μmol/L) and CHX (3.6 μmol/L), alone or in combination, and scored for apoptosis. (C) Extracts from HL60 cells treated with DNR (8 μmol/L) for 2, 4, or 6 hours in the absence or presence of CHX (3.6 μmol/L, added 1 hour after DNR) were immunoblotted for caspase 3. (D-F) Flow cytometric analysis (representative of 4 separate experiments) of HL60 cells treated for 4 hours with vehicle (D), 8 μmol/L DNR (E) or DNR + 3.6 μmol/L CHX (F). Note that DNR + CHX converted twice as many cells as DNR alone to a compartment with decreased forward (FSC-H) and side (SSC-H) scatter. (G) Scoring of morphologically evident apoptosis in IPC-81 cells treated for 5 hours with DNR (0.1 μmol/L) alone or in combination CHX (3.6 μM). (H) IPC-81 cells were treated for 6 hours with various concentrations of DNR alone or with CHX (3.6 μmol/L) and scored for apoptotic morphology. Note the lack of cell budding, nuclear fragmentation, and chromatin condensation with DNR + CHX and with DNR more than ∼1 μmol/L. The bars show apoptosis in cells incubated for 6 hours with DNR + CHX or CHX alone, washed in excess medium, and incubated without DNR and CHX for another 12 hours before assessment of apoptosis. Note the high proportion of apoptotic cells after washing. (I) As for panel G, except that cells were treated for 3 hours with DNR (0.1 μmol/L) and CHX (3.6 μmol/L), washed and further incubated for 9 hours (as explained in panel H) before scoring of apoptosis. (J) HL60 cells received the protein synthesis inhibitors emetine (10 nmol/L), puromycin (180 nmol/L), rapamycin (10 or 100 nmol/L), or CHX (3.6 μmol/L) at various time points relative to DNR (1.6 μmol/L), and apoptosis scored 4.5 hours after DNR addition. (K) HL60 cells were treated for 6.5 hours with various concentrations of DNR (50-8000 nmol/L) in the absence (○) or presence (●) of CHX (3.6 μmol/L) and scored for apoptosis. (L) The accumulation of apoptotic HL60 cells as a function of time after addition of DNR (8 μmol/L) alone (○), CHX (3.6 μmol/L) alone (□), or a combination of the 2 (●). CHX was added 1 hour after DNR. Apoptosis scores represent the means (± SEM) of 3 to 6 experiments.

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