Oncogenic Ral sensitizes immortalized human astrocytes to TRAIL-induced apoptosis via suppression of FLIP_S levels. (A) Normal human astrocytes were serially infected with retroviruses encoding E6, E7, hTERT and either WT RalA, cdc42, E37G, T35S, or Y40C forms of H-Ras12V, after which cells were injected subcutaneously into immunodeficient mice and monitored for growth. The E6/E7/hTERT+cdc42, T35S, and Y40C cells did not form tumors, and as such, the data points for these groups are compressed along the x axis. Error bars indicate standard deviations. (B) Select cells in panel A were exposed to TRAIL (0 to 1,000 ng/ml, 24 h), stained with propidium iodide, and analyzed by flow cytometry for the percentage of cells having a <2N DNA content (apoptotic cells). Error bars indicate standard deviations. (C) Expression of DISC components (FADD, caspase-8, caspase-3, and FLIP_S) was assessed by Western blot analysis from lysates of TRAIL-sensitive G37 and E6/E7/hTERT/Ras cells (lanes 3) or TRAIL resistant E6/E7/hTERT cells and from anti-FADD antibody DISC immunoprecipitates from TRAIL-exposed (800 ng/ml) cells (lanes 4) or TRAIL-exposed cell lysates (lanes 2). Lanes 1, negative control using IgG antibody rather than FADD antibody. (D) TRAIL-resistant/nontransformed (E6/E7/hTERT, C40, and S35) or TRAIL-sensitive/transformed (E6/E7/hTERT +Ras, E6/E7/hTERT/Ral, and G37) cells were lysed, preincubated with vehicle, GDP (negative control to inactivate RalA-GTP), or a nonhydrolyzable form of GTP (GTPγS, positive control to activate RalA-GTP), and incubated with agarose-conjugated RalBP1, a substrate of the activated (GTP bound) form of RalA, or an agarose-conjugated nonspecific substrate (normal rabbit IgG). Substrate-bound (activated) RalA-GTP was then eluted, and levels were assessed by Western blotting (WB) using a RalA antibody and compared to levels of total RalA and alpha tubulin in lysates prior to incubation with RalBP1. , P < 0.05. IP, immunoprecipitate. (E) Western blot analysis of FLIP_S, FLIP_L, and alpha tubulin (loading control) expression in TRAIL-resistant/nontransformed (E6/E7/hTERT, C40, and S35) or TRAIL-sensitive/transformed (E6/E7/hTERT+Ras, E6/E7/hTERT/Ral, and G37) cells. , P < 0.05. (F) TRAIL-sensitive cells were sham infected (CTRL) or stably infected with blank (pFB-Neo), FLIP_S- or FLIP_L-encoding constructs. Cells were then either analyzed for FLIP_S, FLIP_L, and alpha tubulin (loading control) expression by Western blotting (bottom panel), or were exposed to TRAIL (800 ng/ml, 24 h) and analyzed for the percentage of apoptotic cells (top panel). Error bars indicate standard deviations. (G) TRAIL-resistant C40 cells were sham transfected (CTRL) or transiently transfected with either siRNA targeting FLIP_S or FLIP_L, or a nonspecific scrambled siRNA and analyzed for levels of FLIP_S, FLIP_L, or alpha tubulin protein 0 to 72 h after siRNA addition. Alpha tubulin expression shown is derived from FLIP_S siRNA cells and is representative of both cell lines used. (H) Cells created in panel G were exposed to TRAIL (800 ng/ml, 24 h) beginning 48 h after the addition of siRNA and analyzed for the percentage of apoptotic cells. Error bars indicate standard errors of the means.

Ral activation is linked to FLIP_S and the extrinsic cell death pathway via RalBP1. (A) TRAIL-sensitive cells (CTRL) were sham transfected (lipo) or transiently transfected with siRNA targeting RalBP1 or a nonspecific scrambled siRNA and analyzed for levels of RalBP1 and FLIP_S protein 0 to 96 h after siRNA addition. Alpha tubulin expression shown (G37 cell line) is representative of all cell lines. (B) Cells described for panel A (72 h posttransfection) either were incubated with TRAIL (0 or 800 ng/ml, last 24 h of siRNA exposure), stained with propidium iodide, and analyzed by flow cytometry for the percentage of cells having a <2N DNA content (apoptotic cells) (top panel) or were analyzed by Western blotting (WB) for the expression of cdc42, FLIP_S or alpha tubulin or lysed and incubated with agarose-conjugated p21-binding domain of PAK-1 (PAK-1 PBD), a substrate of the activated (GTP-bound) form of cdc42, after which substrate-bound (activated) cdc42-GTP was eluted and levels were assessed by Western blotting using a cdc42 antibody (bottom panel). Error bars indicate standard deviations. IP, immunoprecipitate. (C) TRAIL-resistant cells were retrovirally infected with a blank vector (pcDNA3-neo) or vectors encoding WT RalBP1, Δaa159-214 RalBP1, Δaa65-80 RalBP1, or RALBP1 ΔGAP. Following selection the cells were lysed, preincubated with vehicle, GDP (negative control to inactivate cdc42-GTP), or a nonhydrolyzable form of GTP (GTPγS, positive control to activate cdc42-GTP), and incubated with an agarose-conjugated p21-binding domain of PAK-1 (PAK-1 PBD), a substrate of the activated (GTP bound) form of cdc42, or an agarose-conjugated nonspecific substrate (normal rabbit IgG). Substrate-bound (activated) cdc42 was then eluted, and levels were assessed by Western blotting using a cdc42 antibody and compared to levels of total cdc42 in lysates prior to incubation with PAK-1 PBD (total cdc42 expression shown is for the S35 cells and is representative of all cell lines). , P < 0.05. (D) Cells described for panel C either were analyzed for RalBP1, FLIP_S, or alpha tubulin (loading control) expression by Western blot analysis (bottom panel) or were exposed to TRAIL (800 ng/ml, 24 h) and analyzed for the percentage of apoptotic cells (top panel). Error bars indicate standard errors of the means.

Ral-RalBP1-mediated suppression of cdc42 translationally regulates the extrinsic cell death pathway. (A) Cells were lysed, preincubated with vehicle, GDP (negative control to inactivate cdc42-GTP), or a nonhydrolyzable form of GTP (GTPγS, positive control to activate cdc42-GTP), and incubated with an agarose-conjugated p21-binding domain of PAK-1 (PAK-1 PBD), a substrate of the activated (GTP bound) form of cdc42, or an agarose-conjugated nonspecific substrate (normal rabbit IgG). Substrate-bound (activated) cdc42 was then eluted, and levels were assessed by Western blotting (WB) using a cdc42 antibody and compared to levels of total cdc42 in lysates prior to incubation with PAK-1 PBD. IP, immunoprecipitate. , P < 0.05. (B) TRAIL-sensitive E6/E7/hTERT+Ras, G37, and E6/E7/hTERT/Ral cells (CTRL) were infected with an empty vector or with constructs encoding constitutively active or dominant-negative cdc42 (Q61L and T17N, respectively) and after selection were incubated with rapamycin (0 or 100 nM, 30 min) and analyzed for FLIP_S, FLIP_L, and alpha tubulin expression by Western blotting. (C) Cells described for panel B were analyzed for expression of DISC components as described in the legend for Fig. 1C. (D) Cells described for panel B were analyzed for expression of cdc42 (and alpha tubulin as a loading control) by Western blot analysis (bottom panel) or exposed to TRAIL (800 ng/ml, 24 h) and analyzed for the percentage of apoptotic cells (top panel). Error bars indicate standard deviations. , P < 0.05. (E) E6/E7/hTERT, E6/E7/hTERT + Ral, G37, G37+empty vector, and G37+cdc42Q61L cells were lysed and subjected to sucrose density gradient centrifugation, with subsequent RNA from fractions containing unassembled ribosomal subunits (fractions 1 to 6) or assembled polyribosomes (fractions 7 to 12) spiked with Drosophila RPL3 RNA (to normalize for equal isolation/loading) and analyzed for FLIP_S and RPL3 content by Northern blotting (left panel). The percentage of the FLIP_S signal localized to the polysomal fractions is displayed in the right panel. , P < 0.05. The RPL3 expression shown is for the G37 cells and is representative of all cell lines. Error bars indicate standard deviations. (F) Cells described for panel B were exposed to rapamycin (0 or 100 nM, 30 min) and then TRAIL (800 ng/ml, 24 h) and analyzed for the percentage of apoptotic cells. Error bars indicate standard errors of the means. , P < 0.05.

Ral-RalBP1 suppression of cdc42 translationally regulates the extrinsic cell death pathway via S6K. (A) Western blot analysis of phospho-S6K (activated), phospho-S6, total S6K, and alpha tubulin (loading control) expression in TRAIL-resistant/nontransformed (E6/E7/hTERT, C40, and S35) or TRAIL-sensitive/transformed (E6/E7/hTERT+Ras, E6/E7/hTERT/Ral, and G37) cells. (B) TRAIL-sensitive cells (E6/E7/hTERT+Ras, G37, and E6/E7/hTERT+Ral) that were sham transfected (lipo) or transiently transfected with siRNA targeting RalBP1 or a nonspecific scrambled siRNA (scramble) (72 h), were incubated with rapamycin (0 or 100 nM, last 24 h or siRNA incubation) and analyzed for levels of RalBP1, phospho-S6K (activated), phospho-S6, total S6K, and alpha tubulin (loading control) protein. Alpha tubulin expression shown is for the G37 cells and is representative of all cell lines. (C) E6/E7/hTERT/Ras cells were sham transfected, transiently transfected with either siRNA targeting S6K1 or a nonspecific scrambled siRNA (96 h), or stably transfected with an empty vector control (pRK7-neo or LXSN-neo) or a construct encoding S6K1 or cdc42. Cells were then analyzed by Western blotting for levels of pS6K1, pS6, S6K1, cdc42-GTP, cdc42, FLIP_S, FLIP_L, and alpha tubulin (loading control) (bottom panel), and analyzed for the percentage of apoptotic cells after exposure to rapamycin (0 or 100 nM, 30 min) and TRAIL (800 ng/ml, 24 h) (top panel). Error bars indicate standard deviations. (D) Cells used in panel C were lysed, separated into monosomal and polysomal fractions, and spiked with Drosophila RPL3 RNA, after which RNA was isolated and analyzed for FLIP_S and RPL3 RNA levels by Northern blot analysis (left panel). The percentage of the FLIP_S signal localized to the polysomal fractions is displayed in the right panel. , P < 0.05. The RPL3 expression shown is for the E6/E7/hTERT cells and is representative of all cell lines. Error bars indicate standard errors of the means.

Analysis of the linkage between PTEN, cdc42, phospho-S6K, FLIP_S, and TRAIL sensitivity in primary human glioblastoma multiformes. TRAIL-sensitive xenografted human PTEN WT glioblastoma multiforme cells (lines 6 and 8) were stably infected with an empty vector or with constructs encoding constitutively active or dominant-negative cdc42 (Q61L or T17N, respectively). Cells were then analyzed by Western blotting for levels of cdc42, pS6K, total S6K1, FLIP_S, and alpha tubulin (loading control) (bottom panels) and analyzed for the percentage of apoptotic cells after exposure to rapamycin (0 or 100 nM, 30 min) and TRAIL (800 ng/ml, 24 h) (top panels). Error bars indicate standard errors of the means.