Activated forms of H-RAS and K-RAS differentially regulate membrane association of PI3K, PDK-1, and AKT and the effect of therapeutic kinase inhibitors on cell survival

Expression of H-RAS V12 causes a greater radio-protective/survival effect than K-RAS D13 in HCT116 cells. A, percentage of apoptotic cells after 0, 1, or 4 Gy in parental HCT116 cells (WT), mutant K-RAS-deleted cells (C2), or in H-RAS V12 cells (C10). Cells were serum-starved and 24 h later were mock exposed or irradiated (1 or 4 Gy). After 96 h, cells were harvested, attached to a slide, fixed, and stained with Wright-Giemsa stain and apoptotic cells were counted under microscope as described in Materials and Methods. Columns, % apoptotic cells (n = 4); bars, ±SE. #, P < 0.05 greater than parallel value in parental/WT cells; *, P < 0.05 less than parallel value in parental/WT cells. B, chemical structures of the AKT inhibitor perifosine, the SH series, and ml series of AKT inhibitors and the PDK-1 inhibitor OSU-03012. C, plating efficiency of parental (WT), mutant K-RAS D13-deleted cells (C2), or H-RAS V12 cells (C10) was determined as described in Materials and Methods. For drug treatments, HCT116 cells expressing H-RAS V12 (C10 cells) were serum starved for 24 h before radiation exposure (1 Gy) and treated 30 min before exposure with vehicle, perifosine (PERI; 1, 3, or 10 μmol/L) or SH-5 (1, 3, or 10 μmol/L). Media containing serum was added 24 h after exposure, and 10 to 14 d later colonies were counted as indicated in Materials and Methods. D, numbers of colonies were expressed as a fraction of the respective mock irradiated cells. Columns, means of six separate dishes per experiment from two separate experiments; bars, ±SE. *, P < 0.05 less than corresponding vehicle-treated cell value; **, P < 0.05 less than value treated with 1 μmol/L perifosine. In parallel assays, HCT116 cells expressing H-RAS V12 (C10 cells) were transfected with vector control plasmid or plasmid to express dominant-negative AKT, as described in Materials and Methods. Pools of transfected cells were plated for colony formation assays and irradiated (1 Gy) as described in Materials and Methods. Columns, means of three separate dishes from three separate experiments; bars, ±SE. *, P < 0.05 less than corresponding C10 cell or C10 control plasmid cell value. E, top left, cells were treated with perifosine (PERI, 1 μmol/L; or as indicated) 30 min before irradiation (1 Gy). Cells were isolated 6 h after irradiation and processed to determine P-AKT S473 phosphorylation. Top right, pools of cells transfected with either control vector or vector to express dominant negative AKT were irradiated, isolated 6 h after irradiation, and processed to determine GSK3 (S9/S20) phosphorylation. Bottom left, cells were treated as indicated with SH-5 (1 or 0–10 μmol/L) 30 min before irradiation (1 Gy). Cells were isolated 6 h after irradiation and processed to determine P-AKT S473 phosphorylation. Bottom right, cells were treated with vehicle (VEH), perifosine (PERI, 1–3 μmol/L) or SH-5 (1–3 μmol/L) for 30 min, and 6 and 24 h, at which time all cells were isolated and processed to determine P-AKT S473 phosphorylation (vehicle control was for cells treated with DMSO for 24 h). Representative experiment (n = 3).

The AKT PH domain inhibitors SH-(23-25) and ml-(14-16) reduce the plating efficiency of H-RAS V12 (C10) cells but do not alter cellular radiosensitivity. A, HCT116 cells expressing H-RAS V12 (C10 cells) were serum starved for 24 h before mock radiation exposure and treated 30 min before mock exposure with vehicle, SH-(23-25) (1, 3, or 10 μmol/L) or ml-(14-16) (1, 3, or 10 μmol/L). Media containing serum was added 24 h after exposure, and 10 to 14 d later colonies were counted as indicated in Materials and Methods. Plating efficiency of H-RAS V12 (C10) cells exposed to the various drugs with mock radiation exposure. Columns, means of six separate dishes per experiment from two separate experiments; bars, ±SE. *, P < 0.05 less than corresponding vehicle-treated cell value. B, HCT116 cells expressing H-RAS V12 (C10 cells) were serum starved for 24 h before radiation exposure (1 Gy) and treated 30 min before exposure with vehicle, SH-(23-25) (1, 3, or 10 μmol/L) or ml-(14-16) (1, 3, or 10 μmol/L). Media containing serum was added 24 h after exposure, and 10 to 14 d later colonies were counted as indicated in Materials and Methods. Radiosensitivity of H-RAS V12 (C10) cells exposed to the various drugs with a 1-Gy radiation exposure. Columns, means of six separate dishes per experiment from two separate experiments; bars, ±SE. *, P < 0.05 less than corresponding vehicle-treated cell value.

AKT activation and radiation resistance is dependent on H-RAS V12–dependent membrane localization of PI3K. A, parental (WT), mutant K-RAS D13-deleted (C2), and H-RAS V12 (C10) cells were plated in parallel and serum starved for 24 h. Cells were then either lysed to determine total β-actin expression or lysed with hypotonic buffer prior to preparation of plasma membranes, as described in Materials and Methods. Equal amounts of membrane protein were loaded onto SDS-PAGE and immunoblotting performed against the indicated membrane-associated proteins. Representative experiment (n = 4). B, H-RAS V12 (C10) cells were serum starved for 24 h in the presence or absence of the FTI277 (2 μmol/L) or vehicle (DMSO). Cells were then lysed with hypotonic buffer prior to preparation of membranes, as described above and in Materials and Methods. Equal amounts of membrane protein were loaded onto SDS-PAGE. Representative experiment (n = 3). C, H-RAS V12 (C10) cells were plated in parallel and serum starved for 24 h in the presence or absence of the FTI277 (2 μmol/L). Cells were irradiated (1 Gy) or mock exposed and homogenates taken 0 to 6 h afterwards to determine AKT S473 phosphorylation and total AKT expression, as described in Materials and Methods. Data shown are from 1, 2, 4, and 6 h after irradiation. Representative experiment (n = 3).

Membrane association of PDK-1 is mutated active RAS dependent, whereas membrane association of AKT and PI3K is H-RAS V12 dependent. A, parental (WT), mutant K-RAS D13-deleted (C2), and H-RAS V12 (C10) cells were plated in parallel and serum starved for 24 h. For H-RAS V12 (C10) cells, cells were plated in parallel and serum starved for 24 h in the presence or absence of the FTI277 (2 μmol/L). Cells were then either lysed to determine total β-actin expression or lysed with hypotonic buffer before preparation of plasma membranes, as described in Materials and Methods. Equal amounts of membrane protein were loaded onto SDS-PAGE and immunoblotting performed against the indicated membrane-associated proteins. Representative experiment (n = 4). B, top, H-RAS V12 (C10) cells were plated in parallel and serum starved for 24 h. Cells were treated with either vehicle (DMSO) or OSU-03012 (OSU, 1 μmol/L) 30 min before irradiation (1 Gy). Cells were isolated 6 h after irradiation and subjected to SDS-PAGE and immunoblotting to determine AKT S473 phosphorylation, as described in Materials and Methods. Middle, H-RAS V12 (C10) cells were plated in parallel and serum starved for 24 h. Cells were treated with either vehicle (DMSO) or OSU-03012 (OSU, 0–10 μmol/L) 30 min before mock exposure. Cells were isolated 6 h after mock exposure and subjected to SDS-PAGE and immunoblotting to determine AKT S473 phosphorylation, as described in Materials and Methods. Bottom, H-RAS V12 (C10) cells were plated in parallel and serum starved for 24 h. Cells were treated with either vehicle (DMSO) or PP2 (3 μmol/L) 30 min before irradiation (1 Gy). Cells were isolated 6 h after irradiation and subjected to SDS-PAGE and immunoblotting to determine AKT S473 phosphorylation, as described in Materials and Methods. C, H-RAS V12 (C10) cells were plated in parallel and serum starved for 24 h. Cells were treated with either vehicle (DMSO) or PP2 (3 μmol/L) 30 min before irradiation (1 Gy). Cells were isolated 6 h after irradiation and subjected to SDS-PAGE and immunoblotting to determine PDK-1 Y373/376 phosphorylation, as described in Materials and Methods. D, parental (WT), mutant K-RAS D13-deleted (C2), and H-RAS V12 (C10) cells were plated in parallel and serum starved for 24 h. Cells were isolated and subjected to SDS-PAGE and immunoblotting to determine Total Src family nonreceptor tyrosine kinase protein expression and Total Src family Y416 phosphorylation, as described in Materials and Methods. E, plating efficiency of parental (WT), mutant K-RAS D13-deleted cells (C2), or in H-RAS V12 cells (C10) was determined as described in the Materials and Methods. For drug treatments, HCT116 cells expressing H-RAS V12 (C10 cells) were serum starved for 24 h before radiation exposure (1 Gy) and treated 30 min before exposure with vehicle, OSU-03012 (OSU; 1, 3, or 10 μmol/L) or PP2 (3, 10, or 30 μmol/L). Media containing serum was added 24 h after exposure, and 10 to 14 d later colonies were counted as indicated in Materials and Methods. F, numbers of colonies were expressed as a fraction of the respective mock irradiated cells. Columns, means of six separate dishes per experiment from two separate experiments; bars, ±SE. *, P < 0.05 less than corresponding vehicle-treated cell value. G, plating efficiency of parental (WT), mutant K-RAS D13-deleted cells (C2), and H-RAS V12 cells (C10) was determined as described in Materials and Methods. For drug treatments, parental (WT) HCT116 cells were serum starved for 24 h before radiation exposure (1 Gy) and treated 30 min before exposure with vehicle, OSU-03012 (OSU; 1, 3, or 10 μmol/L) or PP2 (3, 10, or 30 μmol/L). Media containing serum was added 24 h after exposure, and 10 to 14 d later colonies were counted as indicated in Materials and Methods. Columns, means of six separate dishes per experiment from two separate experiments; bars, ±SE. *, P < 0.05 less than corresponding vehicle-treated cell value. H, for drug treatments, parental (WT) HCT116 cells were serum starved for 24 h before radiation exposure (1 Gy) and treated 30 min before exposure with vehicle, OSU-03012 (OSU; 1, 3, or 10 μmol/L). Media containing serum was added 24 h after exposure, and 10 to 14 d later colonies were counted as indicated in Materials and Methods. Columns, means of six separate dishes per experiment from two separate experiments; bars, ±SE. *, P < 0.05 less than corresponding vehicle-treated cell value.

siRNA-mediated inhibition of PDK-1 expression radiosensitizes H-RAS V12 (C10) cells but not parental (WT) HCT116 cells. A, H-RAS V12 (C10) cells were transfected with either a siRNA molecule against PDK-1, a scrambled PDK-1 siRNA, or a sense PDK-1 siRNA (data not shown) as described in Materials and Methods. Twenty-four hours after replating, cells were serum starved for 24 h and then irradiated (1 Gy). Six hours after irradiation, cells were processed for immunoblotting to determine the expression and phosphorylation of various proteins. Representative experiment (n = 3). B, C, D, and E, H-RAS V12 (C10) cells and parental (WT) cells were transfected with either a siRNA molecule against PDK-1 (siPDK-1), a scrambled PDK-1 siRNA (siSCR) as described in Materials and Methods. Forty-eight hours after transfection, cells were replated for colony formation assays and 24 h after plating for these assays, serum starved for 24 h, and then irradiated (1 Gy). Media containing serum was added 24 h after exposure, and 10 to 14 d later colonies were counted as indicated in Materials and Methods. Columns, means of six separate transfection dishes per experiment from two separate experiments; bars, ±SE. *, P < 0.05 less than corresponding siSCR value. B, siPDK-1 did not alter plating efficiency in HCT116 cell lines. C, D, and E, siPDK-1 radiosensitizes H-RAS V12 cells but not parental HCT116 cells.