Figure 1.
Everolimus induces a dose dependent cell death in ALL cell lines and patient samples.
(A) Representative dot plots showing the staining with Annexin V and 7AAD on ALL cells treated with vehicle, or 16 µM everolimus for 24 h with the viability indicated on the plots. (B) Dose response curves for ALL cells lines following a 24 h exposure to the indicated concentrations of everolimus. (C) The viability of NALM6 cells treated with everolimus for the indicated time periods. (D) Clonogenic potential of the indicated ALL cell lines following overnight exposure to the indicated concentrations of everolimus. Data has been normalized to control cultures. (E) The effect of a 24 h exposure to the indicated concentrations of everolimus 1 on the viability of patient ALL samples. (F) Western blots showing the phosphorylation of S6RP and 4E-BP1 in NALM6 cells following treatment with indicated concentrations of everolimus over various time intervals.
Figure 2.
Everolimus induces a caspase independent cell death in ALL cells.
(A) NALM6 cells were treated with indicated concentrations of everolimus for the specified time intervals and cell lysates prepared. Western blots for PARP and caspase 3 are shown with b-actin acting as a loading control. (B) NALM6 cells were treated with indicated concentrations of everolimus for 24 h and cell lysates prepared. Western blots for PARP using the C2-10 antibody are shown with b-actin acting as a loading control. (C) NALM6 cells were pretreated with Z-VAD (right panel) or vehicle (left panel) and then exposed to 16 µM everolimus or placebo for 24 hours. Cells were stained for activated caspase 3 and analyzed by flow cytometry and overlay histograms of placebo and everolimus treated cells is shown. (D) The indicated cell lines were treated as in (B) were labeled with Annexin V and 7AAD and analysed by flow cytometry. The percentage of viable cells (dual negative cells) has been plotted. (E) NALM6 cells were treated with placebo or 16 µM everolimus for 6 hours and then stained using TMRM and annexin V. Representative flow plots are shown. The percentage of TMRM positive/annexin V negative and TMRM negative/annexin V positive cells are shown in the upper left and lower right quadrants respectively. (F) Cells treated as in D were stained for cytochrome c. The percentage of cells negative for cytochrome c is shown. (G) NALM6 cells treated as above for the indicated time periods with the specified concentrations of everolimus were assessed by flow cytometry for viability (upper panel), mitochondrial depolarization (middle panel) or cytochrome c release (lower panel) by flow cytometry as described for C, D and E respectively.
Figure 3.
Everolimus-Induced Autophagy is Not Required for Cell Death.
(A–G) NALM6 cells were treated with vehicle or 16 µM everolimus overnight and ultrastructure examined by electron microscopy. Fine arrows indicate autophagic vacuoles containing degrading organelles. Thick arrows indicate stretches of double membrane walling off regions of cytoplasm. Scale bars indicate 100 nm. (H) Cell lysates prepared from NALM6 cells cultured for the indicated time periods with the specified concentration of everolimus were analyzed for LC3-I and LC3-II by Western blotting and sequential blotting for β-actin used as a loading control. (I) The viability of NALM6 cells cultured for 24 h with the indicated concentration of everolimus was assessed with and without the addition of 3MA. The mean ± SD of 3 independent experiments is shown and analyzed using a paired t-test. (J) Analysis of LC3 by Western blotting following a 24 h incubation of NALM6 cells with indicated concentration of everolimus with or without that addition of 3MA. The ratio of LC3-II to LC3-I is indicated below the blot.
Table 1.
Molecular Chaperone and Stress Response Genes Regulated by Everolimus.
Figure 4.
(A) NALM6 cells were treated with 16 µM everolimus overnight and qRT-PCR used to evaluate the expression of the indicated genes. The mean ± SD of the fold change from 3 experiments is shown. (B) NALM6 cells were incubated with the indicated concentrations of everolimus for the specified time periods and cell lysates prepared. Sequential Western blotting was performed on the same membrane to determine the levels of the indicated proteins in each series of panels. Phospho-eIF2α bands were quantitated by densitometry and normalized to β-actin.
Figure 5.
Cell death is dependent on new protein synthesis but not JNK signalling.
(A) NALM6 cells were treated for the indicated time with 16 µM everolimus and cell lysates prepared. Lysates were probed for phosphorylated JNK (p-JNK) and loading assessed using β-actin. (B) NALM6 cells were treated with the indicated concentrations of everolimus for 16 h, with or without a 1 h pre-incubation with the JNK inhibitor SP600125 (5 µM) and viability assessed using annexin V/PI staining. The percentage of viable cells is shown with the bars representing the mean ± SE of 2 experiments. (C) NALM6 or REH cells were incubated with indicated concentrations of cyclohexamide for 1 h prior to the addition of the indicated concentration of everolimus. The mean ± SD of ≥3 independent experiments is shown. *p<0.05 when comparing cells with and without cyclohexamide. (D) NALM6 cells were treated with the indicated concentrations of everolimus for the specified times and cell lysates prepared. The indicated proteins were assessed by Western blotting and where indicated the bands were quantitated by densitometry using β-actin for normalization.