Article Figures & Data
Figures
- Fig. 1.
Status of the Akt pathway and effect of PI3K inhibition in breast cancer cells. A, cells were plated at 5 × 105 per well and exposed to HS (10% FBS) or LS (0.1% FBS) overnight or LS + IGF-I (50 ng/ml) for 15 min. Immunoblotting was performed as described. The upper panel shows levels of S473 phosphorylation, middle panel levels of T308 phosphorylation, and lower panel levels of total Akt. B, levels of PTEN protein under normal growth conditions (HS). *, cell lines with mutant PTEN. α-Tubulin levels are shown to indicate comparable loading. C, cells were plated as above and exposed to LS or LS + LY294002 (25 μm) for 2 h. Active Akt was immunoprecipitated with anti-phospho S473 antibodies, and in vitro kinase assays were performed as described using GSK3α/β as a substrate. Levels of phosphorylated GSK3α/β are shown. D, cells were plated at 1.25 × 105 per well and exposed to LS (▪) or LS +25 μm LY294002 (□) for 48 h. Cells were harvested, and apoptosis was quantified via flow cytometry as described. Columns are the means from four independently performed experiments; bars, SE. *, two-tailed Ps comparing LY294002 treatment to control were <0.05. E, cells were plated at 3 × 105 per well in triplicate, and parallel samples were transiently cotransfected with plasmids encoding GFP and pSG5 (▪) or GFP and Akt-CAAX ([cjs2108]) as described. Subsequently, cells were exposed to LS for 48 h. Whole cell lysates from ungated samples were harvested for immunoblotting to assess HA levels (inset). Samples were also fixed, stained with propidium iodide, and prepared for flow cytometry. Apoptosis was quantified by gating on GFP-positive cells and performing cell cycle analysis. Columns are the means from three independently performed experiments; bars, SE. *, two-tailed P comparing apoptosis with CAAX to vector alone was <0.05.
- Fig. 2.
LY294002 potentiates chemotherapy-induced apoptosis and correlates with early induction of Akt activity by chemotherapeutic agents. A, cells were plated at 1.25 × 105/well and placed in 0.1% FBS. LY294002 and chemotherapeutic agents were added simultaneously, and cells were incubated for 48 h. Apoptosis was measured using flow cytometry as described. Lanes C, control; Lanes P, paclitaxel (5 μm), Lanes E, etoposide (100 μm for all but MB468, 1 μm for MB468 cells); Lanes T, trastuzumab (10.5 ng/ml); Lanes D, doxorubicin (5 μm). [cjs2108], control; □, LY294002 (25 μm). Columns are the means from four independently performed experiments for each cell line; bars, SE. *, two-tailed Ps comparing LY294002 + chemotherapy to chemotherapy alone were <0.05. B, left panels, time-dependent induction of Akt phosphorylation. Cells were plated at 5 × 105 per well and were treated with doxorubicin (5 μm) in LS for 0, 1, 6, or 24 h. Samples were harvested for immunoblotting as described. Representative experiments showing levels of phosphorylated S473 and total Akt are shown. Where levels of total Akt differed significantly, regions of fast green-stained membranes that encompassed Mr 60,000 (molecular weight of Akt) are shown to demonstrate comparable loading. Middle panels, quantification of band intensity at the time point of maximal induction of Akt phosphorylation with 5 μm doxorubicin for each cell line was performed using NIH Image software. Columns are the means from three independently performed experiments; bars, SE. *, P < 0.05. Right panels, dose-dependent induction of Akt phosphorylation. Cells were incubated in LS with the doses of doxorubicin shown for the period of time that yielded maximal induction of Akt phosphorylation for each cell line (24 h for MB468 and MB231 cells; 6 h for ZR75-1). Representative immunoblots showing levels of phospho-S473 and total Akt are shown. C, cells were treated as in B but with trastuzumab (10.5 ng/ml) for the time course (left panels). Dose responses were assessed at 1 h for all cell lines (right panels).
- Fig. 3.
LY294002 potentiates tamoxifen-induced apoptosis in ER+ cells and correlates with early induction of Akt activity by tamoxifen. Cells (A, ZR75-1; B, MCF7; C, MB231) were plated at 1 × 104/well and exposed to LS alone (Control) or 40 or 80 nm tamoxifen with (□) or without ([cjs2108]) LY294002 (25 μm). Cell Death ELISAs were performed at 48 h as described. Ratios of apoptosis were established using untreated cell extracts as having a value of 1. Columns are the means from three experiments; bars, SE. Representative immunoblots from three experiments for phospho-S473 and total Akt in cells treated with 80 nm tamoxifen in LS for 0, 1, 6, or 24 h (or ZR75-1 cells treated with various doses of tamoxifen for 6 h (A, right panels, inset) are shown in left insets. Middle insets, quantification of band intensity at the time point of maximal induction of Akt phosphorylation with 80 nm tamoxifen for each cell line was performed using NIH Image software. Bars are the means from three independently performed experiments; bars, SE. *, P < 0.05.
- Fig. 4.
Modulating Akt activity with dominant-negative or constitutively active Akt mutants alters the sensitivity of breast cancer cells to chemotherapy. A, cells were plated at 3 × 105 per well, and parallel samples in triplicate were transiently cotransfected with plasmids encoding GFP and pSG5 (▪) or GFP and Akt-CAAX ([cjs2108]) or GFP alone (not shown) as described. Cells were allowed to recover from transfection for 24 h and then exposed to LS ± doxorubicin (5 μm) for 48 h. Whole cell lysates from ungated samples were harvested for immunoblotting to assess HA levels (top insets) and cleavage of PARP (lower insets). Samples were also fixed, stained with propidium iodide, and prepared for flow cytometry. Apoptosis was quantified by gating on GFP-positive cells and performing cell cycle analysis. Levels of apoptosis were equivalent in cells transfected with GFP alone or with GFP and pSG5 (data not shown). Columns are the means from triplicate samples; bars, SD. A representative experiment from three independent experiments is shown for each cell line. B, cells were stably transfected, and individual clones were expanded under antibiotic selection as described. Kinase assays were performed and are shown in top panels. Immunoblotting was performed to assess HA expression (middle panels). To measure apoptosis, clones were plated in triplicate at 1.25 × 105/well, exposed to 0.1% FBS, and treated with or without doxorubicin (500 nm) for 48 h, and CellDeath ELISAs were performed as described. Increased viability in the Myr Akt clones is expressed in relation to the vectors clones, where viability after doxorubicin treatment was set to 1. Columns are means from triplicate samples; bars, SD. A representative experiment from three independent experiments is shown for each cell line. *, P < 0.05.
Volume 1, Issue 9
