Akt suppresses apoptosis by stimulating the transactivation potential of the RelA/p65 subunit of NF-kappaB

Abstract

It is well established that cell survival signals stimulated by growth factors, cytokines, and oncoproteins are initiated by phosphoinositide 3-kinase (PI3K)- and Akt-dependent signal transduction pathways. Oncogenic Ras, an upstream activator of Akt, requires NF-kappaB to initiate transformation, at least partially through the ability of NF-kappaB to suppress transformation-associated apoptosis. In this study, we show that oncogenic H-Ras requires PI3K and Akt to stimulate the transcriptional activity of NF-kappaB. Activated forms of H-Ras and MEKK stimulate signals that result in nuclear translocation and DNA binding of NF-kappaB as well as stimulation of the NF-kappaB transactivation potential. In contrast, activated PI3K or Akt stimulates NF-kappaB-dependent transcription by stimulating transactivation domain 1 of the p65 subunit rather than inducing NF-kappaB nuclear translocation via IkappaB degradation. Inhibition of IkappaB kinase (IKK), using an IKKbeta dominant negative protein, demonstrated that activated Akt requires IKK to efficiently stimulate the transactivation domain of the p65 subunit of NF-kappaB. Inhibition of endogenous Akt activity sensitized cells to H-Ras(V12)-induced apoptosis, which was associated with a loss of NF-kappaB transcriptional activity. Finally, Akt-transformed cells were shown to require NF-kappaB to suppress the ability of etoposide to induce apoptosis. Our work demonstrates that, unlike activated Ras, which can stimulate parallel pathways to activate both DNA binding and the transcriptional activity of NF-kappaB, Akt stimulates NF-kappaB predominantly by upregulating of the transactivation potential of p65.

FIG. 1

Stimulation of NF-κB-dependent transcription by the H-Ras(V12, C40) effector mutant. NIH 3T3 cells were transiently cotransfected with an NF-κB-responsive reporter (3x-κB-Luc, 0.5 μg) and with expression plasmids bearing the gene encoding H-Ras(V12), H-Ras effector mutants, or an empty vector control (VC) (1 μg each). Cell lysates were harvested 24 h posttransfection, and luciferase activity was assayed as described in Materials and Methods. Data are presented as multiples of the level of activation obtained for the vector control group, which was normalized to 1. Results are the means ± standard deviations of results of three independent experiments. (Gel) Total protein was isolated from a representative transfection experiment, and immunoblot analysis was performed for transgene expression. Protein samples (50 μg per lane) were resolved on a 10% polyacrylamide gel, transferred to nitrocellulose, and probed for hemagglutinin-tagged p21^Ras proteins with a hemagglutinin-specific antibody (BABCO, Berkeley, Calif.). Immunoblot assays for actin expression confirmed that relatively equal amounts of proteins were loaded in each lane.

FIG. 2

H-Ras(V12) stimulates NF-κB-dependent transcription through PI3K- and Akt-dependent pathways. (A) Activated forms of PI3K (PI3K*) and Akt stimulate the transcriptional activity of NF-κB as effectively as H-Ras(V12). NIH 3T3 cells were cotransfected with the 3x-κB-Luc reporter (0.5 μg), an internal control plasmid reporter (pCMV-LacZ, 1 μg), and various expression constructs (1 μg each). Luciferase and β-Gal activities were assayed 24 h posttransfection, and fold luciferase activity was determined by normalizing values to total protein levels and to β-Gal enzyme levels. Results are expressed as multiples of the level of activation obtained with the mutant 3x-κB-Luc reporter, which contains mutated cis elements that are no longer capable of binding NF-κB. Data are representative of results of at least three independent experiments, and the means ± standard deviations are shown. (Gel) Immunoblot analysis demonstrates that transfected cells effectively express the various transgenes. (B) H-Ras(V12)-induced NF-κB transcriptional activity requires PI3K- and Akt-dependent signaling pathways. NIH 3T3 cells were transiently transfected with the 3x-κB-Luc reporter (0.5 μg), in either the absence (−) or presence (+) of H-Ras(V12) (1 μg). Additionally, cells were transfected with the empty vector control (VC) or with expression constructs bearing genes encoding dominant negative PI3K (ΔPI3K) or Akt (Akt T308A and Akt K179A, 1 μg each). Luciferase levels were measured 24 h posttransfection in order to avoid potential pitfalls associated with cell death. Data are multiples of the level of activation obtained with the mutant 3x-κB-Luc control, as described above. Results are representative of at least three independent experiments and were normalized to an internal β-Gal-expressing plasmid. (Gel) Immunoblot analysis shows that transfected NIH 3T3 cells express the dominant negative P13K (ΔPI3K) and Akt (DN Akt) constructs. (C) PI3K requires Akt to stimulate NF-κB-dependent transcription. NIH 3T3 cells were transfected with the 3x-κB-Luc reporter (0.5 μg), a vector control (VC), activated PI3K (PI3K*), or dominant negative Akt (Akt K179A) (1 μg each). Forty-eight h posttransfection whole-cell extracts were harvested and assayed for luciferase activity. Results are plotted as multiples of the level of activation of the vector control and are representative of three independent experiments. The means ± standard deviations are shown.

FIG. 2

H-Ras(V12) stimulates NF-κB-dependent transcription through PI3K- and Akt-dependent pathways. (A) Activated forms of PI3K (PI3K*) and Akt stimulate the transcriptional activity of NF-κB as effectively as H-Ras(V12). NIH 3T3 cells were cotransfected with the 3x-κB-Luc reporter (0.5 μg), an internal control plasmid reporter (pCMV-LacZ, 1 μg), and various expression constructs (1 μg each). Luciferase and β-Gal activities were assayed 24 h posttransfection, and fold luciferase activity was determined by normalizing values to total protein levels and to β-Gal enzyme levels. Results are expressed as multiples of the level of activation obtained with the mutant 3x-κB-Luc reporter, which contains mutated cis elements that are no longer capable of binding NF-κB. Data are representative of results of at least three independent experiments, and the means ± standard deviations are shown. (Gel) Immunoblot analysis demonstrates that transfected cells effectively express the various transgenes. (B) H-Ras(V12)-induced NF-κB transcriptional activity requires PI3K- and Akt-dependent signaling pathways. NIH 3T3 cells were transiently transfected with the 3x-κB-Luc reporter (0.5 μg), in either the absence (−) or presence (+) of H-Ras(V12) (1 μg). Additionally, cells were transfected with the empty vector control (VC) or with expression constructs bearing genes encoding dominant negative PI3K (ΔPI3K) or Akt (Akt T308A and Akt K179A, 1 μg each). Luciferase levels were measured 24 h posttransfection in order to avoid potential pitfalls associated with cell death. Data are multiples of the level of activation obtained with the mutant 3x-κB-Luc control, as described above. Results are representative of at least three independent experiments and were normalized to an internal β-Gal-expressing plasmid. (Gel) Immunoblot analysis shows that transfected NIH 3T3 cells express the dominant negative P13K (ΔPI3K) and Akt (DN Akt) constructs. (C) PI3K requires Akt to stimulate NF-κB-dependent transcription. NIH 3T3 cells were transfected with the 3x-κB-Luc reporter (0.5 μg), a vector control (VC), activated PI3K (PI3K*), or dominant negative Akt (Akt K179A) (1 μg each). Forty-eight h posttransfection whole-cell extracts were harvested and assayed for luciferase activity. Results are plotted as multiples of the level of activation of the vector control and are representative of three independent experiments. The means ± standard deviations are shown.

FIG. 3

PI3K and Akt activate NF-κB by upregulating the transactivation potential of the p65 subunit. (A) Activated H-Ras(V12) and MEKK stimulate cellular pathways which result in nuclear translocation and increased DNA binding of NF-κB. Human 293T cells were transiently transfected with the vector control (VC) or activated forms of MEKK, H-Ras, Akt, or PI3K (PI3K*) (3 μg each). Nuclear extracts were prepared and EMSAs were performed to assess the presence of NF-κB DNA binding activity. Nuclear extracts isolated from TNF-stimulated 293T cells (15 ng/ml for 15 min) served as a positive control for NF-κB DNA binding activity. (Bottom) Nuclear extracts analyzed for NF-κB DNA binding activity were reanalyzed using a ³²P-labeled Oct-1-specific double-stranded probe to confirm the quality of the nuclear proteins. Note that the Oct-1-specific DNA-protein complex but not the free probe is shown. No Add, no addition. (B) IKKβ or H-Ras(V12) expression stimulates IκBα degradation. NIH 3T3 cells were transfected with plasmids bearing the genes encoding the Flag-tagged IκBα and His-tagged LacZ (2 μg each). Additionally, cells were transfected with the vector control (VC) or with plasmids bearing genes encoding wild-type IKKβ or activated forms of H-Ras, MEKK, PI3K, or Akt (1 μg each). Forty-eight hours posttransfection, cells were lysed and proteins (80 μg per lane) were resolved by PAGE. Transfected IκBα protein was detected using a Flag-specific antibody (M2; Sigma). Protein loading and transfection efficiencies were controlled by performing immunoblot analysis for His-tagged LacZ expression within each experimental group (data not shown). (C) Activated PI3K and Akt stimulate the transactivation domain of NF-κB. NIH 3T3 cells were transiently transfected with the Gal4-luciferase reporter (100 ng) and with plasmids bearing the genes encoding either Gal4-p65 or Gal4–Elk-1 (100 ng each). Cells were also transfected with the empty vector control (VC) or activated H-Ras(V12), PI3K, or Akt. Luciferase levels were measured and expressed as multiples of the level of activation of the empty vector control. Data presented are the means ± standard deviations of results of three independent experiments.

FIG. 3

PI3K and Akt activate NF-κB by upregulating the transactivation potential of the p65 subunit. (A) Activated H-Ras(V12) and MEKK stimulate cellular pathways which result in nuclear translocation and increased DNA binding of NF-κB. Human 293T cells were transiently transfected with the vector control (VC) or activated forms of MEKK, H-Ras, Akt, or PI3K (PI3K*) (3 μg each). Nuclear extracts were prepared and EMSAs were performed to assess the presence of NF-κB DNA binding activity. Nuclear extracts isolated from TNF-stimulated 293T cells (15 ng/ml for 15 min) served as a positive control for NF-κB DNA binding activity. (Bottom) Nuclear extracts analyzed for NF-κB DNA binding activity were reanalyzed using a ³²P-labeled Oct-1-specific double-stranded probe to confirm the quality of the nuclear proteins. Note that the Oct-1-specific DNA-protein complex but not the free probe is shown. No Add, no addition. (B) IKKβ or H-Ras(V12) expression stimulates IκBα degradation. NIH 3T3 cells were transfected with plasmids bearing the genes encoding the Flag-tagged IκBα and His-tagged LacZ (2 μg each). Additionally, cells were transfected with the vector control (VC) or with plasmids bearing genes encoding wild-type IKKβ or activated forms of H-Ras, MEKK, PI3K, or Akt (1 μg each). Forty-eight hours posttransfection, cells were lysed and proteins (80 μg per lane) were resolved by PAGE. Transfected IκBα protein was detected using a Flag-specific antibody (M2; Sigma). Protein loading and transfection efficiencies were controlled by performing immunoblot analysis for His-tagged LacZ expression within each experimental group (data not shown). (C) Activated PI3K and Akt stimulate the transactivation domain of NF-κB. NIH 3T3 cells were transiently transfected with the Gal4-luciferase reporter (100 ng) and with plasmids bearing the genes encoding either Gal4-p65 or Gal4–Elk-1 (100 ng each). Cells were also transfected with the empty vector control (VC) or activated H-Ras(V12), PI3K, or Akt. Luciferase levels were measured and expressed as multiples of the level of activation of the empty vector control. Data presented are the means ± standard deviations of results of three independent experiments.

FIG. 4

Activated PI3K and Akt stimulate the p65 transactivation domain of NF-κB in a manner dependent on IκB kinase. (A) Akt requires IKKβ to activate NF-κB-dependent transcription. NIH 3T3 cells were transfected with the 3x-κB-Luc reporter (0.5 μg) and with the empty vector control (VC), M-Akt, or dominant negative IKKβ (DN IKKβ) alone or with M-Akt plus DN IKKβ (1 μg each). Forty-eight hours posttransfection, whole-cell extracts were harvested and assayed for luciferase activity. Results are plotted as multiples of the level of activation obtained with the vector control and are averages ± standard deviations of results of three independent experiments. (B) Akt requires IKK to stimulate TAD 1 of the p65 subunit of NF-κB. NIH 3T3 cells were transfected with the Gal4-luciferase reporter, Gal4-p65 (100 ng each), and the indicated constructs described above (1 μg each). Results are expressed as multiples of the level of activation obtained with the vector control. The data are the means ± standard deviations of results of three independent experiments. (Gel) Western blot analysis of transfected Gal4-p65. Whole-cell extracts of the transfections described above (25 μg of protein each) were separated by SDS–10% PAGE, transferred to nitrocellulose, and assayed with an antibody specific for the Gal4 DNA binding domain (sc-510; Santa Cruz Biotech, Santa Cruz, Calif.). Primary antibodies were detected using an HRP-labeled secondary antibody and by performing ECL.

FIG. 5

Characterization of Rat-1:iRas cells expressing a dominant negative Akt protein. (A) Characterization of the Rat-1:iRas–dominant negative Akt cells. Rat-1:iRas cells etopically expressing a plasmid bearing the gene encoding the dominant negative Akt(K179A) protein (DN Akt) or the vector control were generated, as described in the Materials and Methods. Total protein (50 μg) was isolated from Rat-1:iRasV cells (vector control cells), three Rat-1:iRas–dominant negative Akt clones (.5, .7, and .15), and Rat-1:iRas–dominant negative Akt-P cells (Pool) in the absence or presence of IPTG (5 mM). Protein samples were resolved on an SDS–10% polyacrylamide gel, transferred to membrane, and analyzed for the presence of Akt, Ras, and α-tubulin. Akt(K179A) protein was detected using a hemagglutinin-specific antibody (BABCO). IPTG-induced p21^Ras expression was detected using a pan-Ras monoclonal antibody (Calbiochem, San Diego, Calif.). To ensure equal levels of protein loading, blots were reanalyzed with an α-tubulin-specific antibody (Sigma). Primary antibodies were detected using an HRP-labeled secondary antibody and by performing ECL. (B) Expression of the dominant negative Akt protein blocks H-Ras(V12)-induced endogenous Akt activity. Subconfluent Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells were grown overnight in medium containing 2% FBS. Eighteen hours later cells were washed and cultured for 4 h without serum and with or without IPTG (5 mM). Some groups received LY 294002 (10 μM) 3 h after serum deprivation. Immunocomplex kinase assays for Akt were performed as described in Materials and Methods. The fold Akt activity was determined by obtaining values for Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells (grown in the absence of IPTG) and normalizing these numbers to 1. Data presented are representative of results of at least three different assays, which generated similar results. (Gel) Immunoblot analysis demonstrating that relatively equal amounts of total Akt protein were immunoprecipitated during the course of the experiment. Both endogenous Akt and hemagglutinin-tagged dominant negative Akt(K179A) proteins are shown.

FIG. 6

The inhibition of endogenous Akt activity sensitizes cells to H-Ras(V12)-induced apoptosis. (A) Expression of H-Ras(V12) is associated with an increase in apoptosis in cells expressing the dominant negative Akt protein (DN Akt). Rat-1:iRasV, Rat-1:iRas–dominant negative Akt clones (.5, .7, and .15), and Rat-1:iRas–dominant negative Akt-P cells (pooled clone) were grown overnight in medium containing a reduced concentration of serum (2% FBS). Eighteen hours later, cells were treated in either the absence or presence of IPTG (5 mM). Apoptotic cells were harvested from the supernatants 48 h after IPTG addition, fixed in paraformaldehyde, and stained with Hoechst dye, and cells displaying nuclear fragmentation and condensation were counted, as described in Materials and Methods. Results are expressed as numbers of apoptotic cells (means ± standard deviations). Assays were repeated at least three independent times. (B) Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells were cultured and stimulated with IPTG as described above. Cell morphologies were analyzed 48 h after IPTG addition. Paraformaldehyde-fixed cells were analyzed for apoptosis by performing TUNEL analysis (Boehringer Mannheim). The upper four images show phase-contrast microscopy (magnification, ×20). The bottom four images show fluorescence microscopy of TUNEL-positive cells (magnification, ×40). (C) H-Ras(V12) no longer stimulates the p65 transactivation domain in cells stably expressing the dominant negative Akt protein. Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells (pooled clone) were transfected with the Gal4-luciferase reporter (100 ng) and with constructs bearing the gene encoding the Gal4-p65 fusion protein (100 ng). Eighteen hours following transfections, cells were stimulated with IPTG (5 mM). Twenty-four hours following IPTG addition, cell extracts were isolated and luciferase activities were determined. Results are the averages ± standard deviations from three independent experiments performed in triplicate. (D) H-Ras(V12) stimulates Gal4–Elk-1 in the vector control and cells expressing dominant negative Akt [Akt(K179A)]. Cells were transfected with Gal4-luciferase and Gal4–Elk-1 (100 ng each). Twenty-four hours posttransfection, cells were incubated in either DMEM containing 10% serum and IPTG (5 mM) or DMEM plus 10% serum alone for an additional 24 h. Whole-cell extracts were isolated and assayed for luciferase levels. Results are expressed as multiples of the level of activation obtained with the vector control or dominant negative Akt-P without IPTG incubation and are the averages ± standard deviations of results of three independent experiments performed in triplicate.

FIG. 6

The inhibition of endogenous Akt activity sensitizes cells to H-Ras(V12)-induced apoptosis. (A) Expression of H-Ras(V12) is associated with an increase in apoptosis in cells expressing the dominant negative Akt protein (DN Akt). Rat-1:iRasV, Rat-1:iRas–dominant negative Akt clones (.5, .7, and .15), and Rat-1:iRas–dominant negative Akt-P cells (pooled clone) were grown overnight in medium containing a reduced concentration of serum (2% FBS). Eighteen hours later, cells were treated in either the absence or presence of IPTG (5 mM). Apoptotic cells were harvested from the supernatants 48 h after IPTG addition, fixed in paraformaldehyde, and stained with Hoechst dye, and cells displaying nuclear fragmentation and condensation were counted, as described in Materials and Methods. Results are expressed as numbers of apoptotic cells (means ± standard deviations). Assays were repeated at least three independent times. (B) Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells were cultured and stimulated with IPTG as described above. Cell morphologies were analyzed 48 h after IPTG addition. Paraformaldehyde-fixed cells were analyzed for apoptosis by performing TUNEL analysis (Boehringer Mannheim). The upper four images show phase-contrast microscopy (magnification, ×20). The bottom four images show fluorescence microscopy of TUNEL-positive cells (magnification, ×40). (C) H-Ras(V12) no longer stimulates the p65 transactivation domain in cells stably expressing the dominant negative Akt protein. Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells (pooled clone) were transfected with the Gal4-luciferase reporter (100 ng) and with constructs bearing the gene encoding the Gal4-p65 fusion protein (100 ng). Eighteen hours following transfections, cells were stimulated with IPTG (5 mM). Twenty-four hours following IPTG addition, cell extracts were isolated and luciferase activities were determined. Results are the averages ± standard deviations from three independent experiments performed in triplicate. (D) H-Ras(V12) stimulates Gal4–Elk-1 in the vector control and cells expressing dominant negative Akt [Akt(K179A)]. Cells were transfected with Gal4-luciferase and Gal4–Elk-1 (100 ng each). Twenty-four hours posttransfection, cells were incubated in either DMEM containing 10% serum and IPTG (5 mM) or DMEM plus 10% serum alone for an additional 24 h. Whole-cell extracts were isolated and assayed for luciferase levels. Results are expressed as multiples of the level of activation obtained with the vector control or dominant negative Akt-P without IPTG incubation and are the averages ± standard deviations of results of three independent experiments performed in triplicate.

FIG. 6

The inhibition of endogenous Akt activity sensitizes cells to H-Ras(V12)-induced apoptosis. (A) Expression of H-Ras(V12) is associated with an increase in apoptosis in cells expressing the dominant negative Akt protein (DN Akt). Rat-1:iRasV, Rat-1:iRas–dominant negative Akt clones (.5, .7, and .15), and Rat-1:iRas–dominant negative Akt-P cells (pooled clone) were grown overnight in medium containing a reduced concentration of serum (2% FBS). Eighteen hours later, cells were treated in either the absence or presence of IPTG (5 mM). Apoptotic cells were harvested from the supernatants 48 h after IPTG addition, fixed in paraformaldehyde, and stained with Hoechst dye, and cells displaying nuclear fragmentation and condensation were counted, as described in Materials and Methods. Results are expressed as numbers of apoptotic cells (means ± standard deviations). Assays were repeated at least three independent times. (B) Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells were cultured and stimulated with IPTG as described above. Cell morphologies were analyzed 48 h after IPTG addition. Paraformaldehyde-fixed cells were analyzed for apoptosis by performing TUNEL analysis (Boehringer Mannheim). The upper four images show phase-contrast microscopy (magnification, ×20). The bottom four images show fluorescence microscopy of TUNEL-positive cells (magnification, ×40). (C) H-Ras(V12) no longer stimulates the p65 transactivation domain in cells stably expressing the dominant negative Akt protein. Rat-1:iRasV and Rat-1:iRas–dominant negative Akt-P cells (pooled clone) were transfected with the Gal4-luciferase reporter (100 ng) and with constructs bearing the gene encoding the Gal4-p65 fusion protein (100 ng). Eighteen hours following transfections, cells were stimulated with IPTG (5 mM). Twenty-four hours following IPTG addition, cell extracts were isolated and luciferase activities were determined. Results are the averages ± standard deviations from three independent experiments performed in triplicate. (D) H-Ras(V12) stimulates Gal4–Elk-1 in the vector control and cells expressing dominant negative Akt [Akt(K179A)]. Cells were transfected with Gal4-luciferase and Gal4–Elk-1 (100 ng each). Twenty-four hours posttransfection, cells were incubated in either DMEM containing 10% serum and IPTG (5 mM) or DMEM plus 10% serum alone for an additional 24 h. Whole-cell extracts were isolated and assayed for luciferase levels. Results are expressed as multiples of the level of activation obtained with the vector control or dominant negative Akt-P without IPTG incubation and are the averages ± standard deviations of results of three independent experiments performed in triplicate.

FIG. 7

M-Akt-transformed cells require NF-κB to block etoposide-induced apoptosis. (A) Generation of H-Ras(V12)- and M-Akt-transformed Rat-1 cells. Rat-1 cells stably expressing activated H-Ras(V12) or M-Akt were generated as described in Materials and Methods. Total protein (50 μg) was isolated from Rat-1:Hygro, Rat-1:H-Ras(V12), and Rat-1:M-Akt cells, resolved by performing PAGE, and transferred to nitrocellulose membrane. Immunoblot analysis was performed to detect transgenic expression of p21^ras and M-Akt using the pan-Ras antibody or the hemagglutinin antibody, respectively. (B) The p65 transactivation domain is activated in Rat-1 cells transformed with either H-Ras(V12) or M-Akt. Rat-1:Hygro, Rat-1:H-Ras(V12), and Rat-1:M-Akt cells were transiently transfected with a Gal4-luciferase reporter (100 ng) and with constructs bearing the genes encoding Gal4-p65 (100 ng). Forty-eight hours following the start of transfection, cell extracts were harvested and luciferase activities were determined. Data are the averages ± standard deviations of results of three experiments performed in triplicate. (C) Rat-1:M-Akt cells are resistant to apoptotic induction agents. Rat-1:Hygro and Rat-1:M-Akt cells were either left untreated (No Add) or given etoposide (15 μM) or staurosporine (50 μM). Apoptotic cell numbers were determined 18 h following the addition of either etoposide or staurosporine (Stauro). Results presented here are the means ± standard deviations of results of two independent experiments performed in duplicate. (D) M-Akt requires NF-κB to overcome etoposide-induced apoptosis. Rat1:M-Akt cells were cultured overnight in medium containing 2% FBS, after which cells were infected with either Ad-CMV or Ad-SRIκBα (50 PFU/cell). Six hours following adenovirus-mediated gene delivery, cells were either left untreated (No Add) or treated with either etoposide (5 μM) or staurosporine (25 μM). Apoptotic cell numbers were analyzed in etoposide-treated Rat-1:M-Akt cells over the time course indicated, while the apoptotic cell numbers detected for staurosporine were analyzed 60 h after the drug addition. Data presented are the averages ± standard deviations of results of two different experiments where the numbers of apoptotic cells were counted in triplicate. (Gel) Immunoblot analysis demonstrating that Ad-SRIκBα is effectively expressed in the Rat-1 cell lines. IκBα proteins were detected using a rabbit polyclonal antibody (C-21; Santa Cruz Biotech). Protein samples analyzed in lanes 1, 3, 5, and 7 are from Rat-1:M-Akt cells infected with Ad-CMV, while those in lanes 2, 4, 6, and 8 are from cells infected with Ad-SRIκBα.

FIG. 7

M-Akt-transformed cells require NF-κB to block etoposide-induced apoptosis. (A) Generation of H-Ras(V12)- and M-Akt-transformed Rat-1 cells. Rat-1 cells stably expressing activated H-Ras(V12) or M-Akt were generated as described in Materials and Methods. Total protein (50 μg) was isolated from Rat-1:Hygro, Rat-1:H-Ras(V12), and Rat-1:M-Akt cells, resolved by performing PAGE, and transferred to nitrocellulose membrane. Immunoblot analysis was performed to detect transgenic expression of p21^ras and M-Akt using the pan-Ras antibody or the hemagglutinin antibody, respectively. (B) The p65 transactivation domain is activated in Rat-1 cells transformed with either H-Ras(V12) or M-Akt. Rat-1:Hygro, Rat-1:H-Ras(V12), and Rat-1:M-Akt cells were transiently transfected with a Gal4-luciferase reporter (100 ng) and with constructs bearing the genes encoding Gal4-p65 (100 ng). Forty-eight hours following the start of transfection, cell extracts were harvested and luciferase activities were determined. Data are the averages ± standard deviations of results of three experiments performed in triplicate. (C) Rat-1:M-Akt cells are resistant to apoptotic induction agents. Rat-1:Hygro and Rat-1:M-Akt cells were either left untreated (No Add) or given etoposide (15 μM) or staurosporine (50 μM). Apoptotic cell numbers were determined 18 h following the addition of either etoposide or staurosporine (Stauro). Results presented here are the means ± standard deviations of results of two independent experiments performed in duplicate. (D) M-Akt requires NF-κB to overcome etoposide-induced apoptosis. Rat1:M-Akt cells were cultured overnight in medium containing 2% FBS, after which cells were infected with either Ad-CMV or Ad-SRIκBα (50 PFU/cell). Six hours following adenovirus-mediated gene delivery, cells were either left untreated (No Add) or treated with either etoposide (5 μM) or staurosporine (25 μM). Apoptotic cell numbers were analyzed in etoposide-treated Rat-1:M-Akt cells over the time course indicated, while the apoptotic cell numbers detected for staurosporine were analyzed 60 h after the drug addition. Data presented are the averages ± standard deviations of results of two different experiments where the numbers of apoptotic cells were counted in triplicate. (Gel) Immunoblot analysis demonstrating that Ad-SRIκBα is effectively expressed in the Rat-1 cell lines. IκBα proteins were detected using a rabbit polyclonal antibody (C-21; Santa Cruz Biotech). Protein samples analyzed in lanes 1, 3, 5, and 7 are from Rat-1:M-Akt cells infected with Ad-CMV, while those in lanes 2, 4, 6, and 8 are from cells infected with Ad-SRIκBα.