We have shown previously that the transduction of a number of human tumor cell lines with an adenovirus (AV1Y28) expressing a single-chain antibody fragment (scFv) directed against Ras proteins results in radiosensitization. Because Ras is involved in the regulation of a number of transcription factors, we have determined the effects of this adenovirus on the activation of nuclear factor-κB (NF-κB), a radiation-responsive transcription factor associated with cell survival. In U251 human glioma cells, radiation-induced NF-κB was significantly attenuated by prior transduction of the anti-Ras scFv adenovirus. This effect appeared to involve an inhibition of IκB kinase activity and IκBα phosphorylation. Inhibitors to the Ras effectors mitogen-activated protein kinase kinase, phosphatidylinositol 3-kinase, and p38, however, did not reduce radiation-induced NF-κB. Whereas AV1Y28 inhibited NF-κB activation by hydrogen peroxide and ferricyanide, it had no effect of tumor necrosis factor-α-induced NF-κB activation. These results are consistent with a novel Ras-dependent, oxidant-specific signaling pathway mediating the activation of NF-κB. In additional cell lines radiosensitized by AV1Y28, radiation-induced NF-κB activation was also inhibited by the anti-Ras scFv, whereas in cell lines not radiosensitized, radiation did not activate NF-κB. This correlation suggested that AV1Y28-mediated radiosensitization involved the inhibition of radiation-induced NF-κB activation. However, inhibition of NF-κB activation via the expression of a dominant-negative form of IκBα in U251 cells had no effect on radiation-induced cell killing and did not influence AV1Y28-mediated radiosensitization. Therefore, whereas AV1Y28 inhibits radiation-induced NF-κB activation, this process does not appear to play a direct role in its radiosensitizing actions.

Ras proteins comprise a relative early component in a number of signaling pathways involved in the regulation of such fundamental cellular processes as proliferation and differentiation (1). In addition, these small GTP-binding proteins have been implicated as determinants of cellular sensitivity to IR.2 It was initially shown that the resistance of NIH 3T3 cells to radiation could be enhanced by the expression of a mutant H-ras gene (2). Subsequent studies demonstrated that the mutation or overexpression of ras genes increased the radioresistance of both tumorigenic and nontumorigenic cell lines (3, 4). Pirollo et al.(5) showed that transfection of cells expressing an activated H-ras with an antisense oligonucleotide directed against this ras gene resulted in an enhanced level of radiation-induced cell death. More recently, Bernhard et al.(6) reported that the genetic inactivation of oncogenic N- or K-ras in human tumor cells leads to increased radiosensitivity. Thus, there is considerable evidence suggesting a causal relationship between Ras proteins and radiation resistance.

However, the signaling pathways through which Ras influences cellular radioresponse have not been defined. Bernhard et al.(7, 8) used FTIs and geranylgeranyltransferase inhibitors to prevent Ras association with the cell membrane and found an increase in radiation-induced cell killing. Although effective in disrupting Ras function, these pharmacological agents inhibit protein prenylation in general and, consequently, affect the activity of a number of additional prenylated proteins including Rho and Rac (9). Moreover, these agents are selective for a given species of Ras proteins: FTIs for H-Ras and geranylgeranyltransferase inhibitors for K-Ras. Yet, data are available indicating that each of the Ras species (H-, K-, and N-) can influence radiosensitivity (2, 6). Given their lack of specificity for Ras and yet selectivity for specific Ras species, use of these compounds in defining the process involved in Ras-mediated radiosensitization could lead to ambiguous results.

In search of a less restrictive (i.e., with respect to ras mutational status) and yet specific strategy for compromising Ras activity, we have investigated the application of an adenovirus expressing a gene coding for a single-chain antibody fragment (scFv) directed against Ras proteins (10). The scFv expressed by this adenovirus was derived from the neutralizing monoclonal antibody Y13–259 and recognizes all four of the mammalian Ras proteins (H, N, K-4A, and K-4B; Ref. 11). The plasmid-mediated expression of Y13–259-scFv in mammalian cells and Xenopus oocytes inhibited Ras-induced transactivation of a reporter gene as well as Ras-mediated DNA synthesis (11, 12). As a means of delivering the anti-Ras scFv to tumor cells, Cochet et al.(13) inserted the gene for Y13–259-scFv into an adenoviral vector downstream from a CMV promoter. The intratumoral injection of this adenovirus into human colon carcinoma xenografts generated in nude mice significantly inhibited tumor growth. These results suggested that the adenovirus-mediated expression of Y13–259-scFv is an effective means of compromising Ras function. We have shown recently that this anti-Ras adenovirus (AV1Y28) enhances radiation-induced cell killing in four human tumor cell lines (10), which is consistent with a role for Ras in regulating cellular radioresponse. This tumor cell radiosensitization occurred independently of ras gene mutational status and did not involve an increase in radiation-induced apoptosis or a redistribution of cells into a more radiosensitive phase of the cell cycle (10).

Because AV1Y28 targets each of the Ras p21 species and yet is specific for Ras, it seems appropriate for fundamental investigations into the mechanisms through which Ras influences radiosensitivity. Ras activity is mediated via interactions with a number of critical molecules including guanine nucleotide exchange factors, guanine nucleotide activator proteins, and a variety of potential downstream effector proteins, which currently number 18 known proteins (14). However, the ultimate function of Ras is to transduce environmental signals to the nucleus, resulting in modifications of gene expression and, consequently, cell behavior. A critical step in this process is the activation of specific transcription factors. Whereas Ras has been shown to regulate a number of transcription factors, one of particular interest is NF-κB (15), which has been implicated as a determinant of radiosensitivity (16, 17). Therefore, as an initial investigation into the mechanism responsible for AV1Y28-induced radiosensitization, we have determined the effects of this adenovirus on radiation-induced NF-κB activation in a number of human cell lines. Data presented here indicate that AV1Y28 inhibits the radiation-mediated activation of NF-κB in those cell lines in which this adenovirus enhances radiosensitivity. This inhibition is specific for radiation and other sources of oxidative stress in that AV1Y28 has no effect on NF-κB activation by TNF-α. However, whereas AV1Y28-mediated inhibition of radiation-induced NF-κB correlated with AV1Y28-mediated radiosensitization, transfection with a dominant-negative IκBα did not result in enhanced radiation-induced cell killing.

Antibodies and Reagents.

Pan-Ras (Ab-2) antibody was obtained from Oncogene Research (Cambridge, MA); c-Myc (9E10) antibody, which recognizes Myc-tagged proteins, and a Raf-RBD assay kit were obtained from Upstate Biotechnology (Lake Placid, NY). The Raf antibody was obtained from BD Transduction Laboratories/PharMingen (San Diego, CA). The antibody for phospho-IκBα (ser32) was obtained from Cell Signaling Technology (Beverly, MA). Antibodies to RalGDS, IκBα, IKKα, NF-κB p50, and NF-κB p65 and the IκBα-GST fusion protein were acquired from Santa Cruz Biotechnology (Santa Cruz, CA). Human TNF-α, wortmannin, ferricyanide [K3Fe(CN)6], DMSO, and H2O2 were purchased from Sigma Chemical Co. (St. Louis, MO). Kinase inhibitors PD98059 (MEK) and SB203580 (p38) were obtained from Biomol (Plymouth Meeting, PA).

Cell Lines and Culture Conditions.

Human cell lines used were those described previously (10) including U251 glioblastoma, SW620 and HT29 colon carcinoma and C-29A normal fibroblasts. In addition, the SF539 glioblastoma and BXPC-3 pancreatic carcinoma cell lines (American Type Culture Collection) were used in this study. Each was grown in MEM or RPMI 1640, respectively, containing gentamicin (10 μg/ml), sodium pyruvate (1 mm), and 10% fetal bovine serum.

Recombinant Adenovirus and Infection Conditions.

The AV1Y28 and control adenoviral vectors used were those described previously (13). Briefly, AV1Y28 is an E1A-deleted, replication-defective recombinant adenovirus containing the anti-Ras Y13–259-scFv expression construct driven by a CMV promoter. As a control virus, an adenovirus of a similar construction was used, except that the expression cassette contained the luciferase gene (Ad-Luc). The adenovirus-mediated gene transfer efficiency for each cell type was determined by using β-galactosidase staining after exposure to an adenovirus expressing the LacZ gene (Ad5CMV-LacZ). Viral titers to achieve >80% transfection efficiency were 103 vp/cell for U251 cells, 3 × 103 vp/cell for SW620, HT29, and C-29A cells, and 104 vp/cell for the SF539 and BXPC-3 cell lines. In vitro infections were performed 24 h after plating. Monolayer cultures were washed in PBS and incubated with purified virus in 1 ml of growth medium without serum for 1 h at 37°C with brief agitation every 15 min. After 1 h, fresh growth medium with 10% fetal bovine serum was added.

Radiation.

Cultured monolayer cells were irradiated using a 137Cs source at a dose rate of 3.7 Gy/min (United States Nuclear, Burbank, CA).

Ras Activity.

GTP-bound Ras (active) proteins were identified according to a pull-down assay as described (18) using a commercially available kit (Upstate Biotechnology, Lake Placid, NY). Briefly, the peptide fragment corresponding to the RBD of c-Raf-1, attached to agarose beads, was incubated with cell lysates overnight at 4°C. The beads were then isolated, washed, and subjected to SDS-PAGE. Proteins were transferred to nitrocellulose membranes and subjected to immunoblot with the pan-Ras antibody.

Cell Fractionation.

Cellular fractionation was performed as described with several modifications (19). Briefly, cell cultures were washed twice with PBS, scraped into 10 ml of PBS, and pelleted. The supernatant was aspirated, and the cell pellet was resuspended in three volumes of hypotonic lysis buffer [10 mm Tris (pH 7.4), 0.26 m sucrose, 100 mm NaCl, 1 mm DTT, 1 mm PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 250 mg/ml benzamide]. The suspension was incubated on ice for 10 min, subjected to three rounds of freezing/thawing, and vortexed to insure complete cell breakage. Samples were spun at 100,000 × g for 60 min at 4°C. The supernatant (S100) was collected as the soluble fraction, treated with 1% NP40, and stored at −80°C. The pellet (P100) was resuspended in hypotonic lysis buffer with 1% NP40, incubated on ice for 30 min, and vortexed for 30 s at 10-min intervals. The insoluble fraction was then stored at −80°C. Protein concentrations were determined using the Bio-Rad DC protein assay kit.

Immunoblot Analysis.

Cell cultures (2 × 106) were washed twice with PBS, scraped into 10 ml of PBS, and pelleted by centrifugation at 500 × g. The supernatant was aspirated, and the cell pellet was resuspended in three volumes of cell lysis buffer [20 mm HEPES (pH 7.9), 400 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% NP40, 1 mm DTT, 1 mm PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, 250 mg/ml benzamide, 50 mm NaF, and 1 mm NaO3V4]. The resulting suspension was incubated on ice for 30 min and vortexed for 30 s at 10-min intervals. Samples were spun at 16,000 × g for 10 min at 4°C and stored at −80°C. Protein concentrations were determined using the Bio-Rad DC protein assay kit (Hercules, CA). Cell lysates combined with 2× SDS-PAGE loading buffer were applied to a 5% stacking, 10% SDS-polyacrylamide gel and resolved at a constant current of 20 mA/gel for 1 h. After electrophoresis, the gel was electroblotted onto a Hybond-ECL nitrocellulose membrane (Amersham/Pharmacia, Uppsala, Sweden). The nonspecific sites on the membrane were blocked at room temperature for 2 h with either 5% nonfat milk (unphosphorylated proteins) in Tris-buffered saline supplemented with 0.1% Tween 20 (TBS-T) or 5% BSA (phosphorylated proteins) in TBS-T. Membranes were then probed with the appropriate primary antibody at 1 μg/ml in blocking solution overnight at 4°C, washed three times in TBS-T, and incubated with the appropriate secondary antibody at a 1:2000 dilution in blocking solution for 1 h at room temperature. Membranes were then washed three times in TBS-T and incubated with ECL+ Western blotting detection reagents (Amersham/Pharmacia) to detect the secondary antibody. Visualization and quantification were performed using the Storm 860 chemifluorescence scanner (Molecular Dynamics, Sunnyvale, CA).

Preparation of Nuclear Extracts.

Cells (2 × 106) plated in 10-cm dishes were washed twice with ice-cold PBS, scraped into 10 ml of PBS, and pelleted by centrifugation at 500 × g for 8 min. The supernatant was aspirated, and the pellet was resuspended in 200 μl of ice-cold lysis buffer [50 mm Tris (pH 7.6), 50 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1% NP40, 1 mm DTT, 1 mm PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 250 mg/ml benzamide]. After incubation on ice for 10 min, samples were vortexed for 10 s and centrifuged for 5 min at 16,000 × g at 4°C. The supernatant was discarded, and the nuclear pellet was resuspended in 25 μl of ice-cold extraction buffer [20 mm HEPES (pH 7.9), 400 mm NaCl, 1 mm EDTA, 1 mm EGTA, 1 mm DTT, 1 mm PMSF, 1 mg/ml aprotinin, 1 mg/ml leupeptin, and 250 mg/ml benzamide]. The pellet was incubated on ice for 30 min with vigorous vortexing every 10 min, followed by centrifugation at 16,000 × g for 10 min at 4°C. The supernatant (nuclear extract) was collected and frozen at −80°C. Protein concentrations were determined using a Bio-Rad DC protein assay kit.

EMSA.

EMSA was performed using the gel shift assay system from Promega Corp. (Madison, WI) as described in the protocol provided by the manufacturer but with some modifications. Briefly, an oligonucleotide containing the NF-κB consensus sequence (5′-GT TGA GGG GAC TTT CCC AGG C-3′) was end-labeled with T4 polynucleotide kinase and [γ-32P]ATP. Nuclear extracts were incubated with or without unlabeled oligo competitor for 15 min at room temperature in the presence of 30 mm HEPES, 1.5 mm EDTA, 5 mm DTT, 7.5% glycerol, and 20 ng/ml poly(deoxyinosinic-deoxycytidylic acid) in a total volume of 20 μl. The labeled probe (1 μl) was then added to the samples and incubated at 37°C for 15 min. The reaction was stopped with 2 μl of gel loading dye, and samples were resolved on a 7% native polyacrylamide gel in a Tris-boric acid-EDTA buffer at 175 V for 2 h. The gel was then dried and evaluated using a Storm 860 scanner (Molecular Dynamics, Sunnyvale, CA).

Immunoprecipitation.

Equal protein concentrations (400 μg) from each sample were aliquoted to a total volume of 200 μl in lysis buffer. Samples were precleared with 1 μl of an antibody of the same species as the IP antibody and 20 μl of Sepharose agarose beads (Amersham/Pharmacia). The mixture was lightly agitated at 4°C for 3 h. Samples were spun at 16,000 × g for 5 min. The supernatant was transferred to a new Eppendorf tube, and the IP antibody was added (0.5 μg). The mixture was lightly agitated overnight at 4°C; 20 μl of agarose beads were added, and the samples were mixed for 3 h. Samples were then spun at 16,000 × g for 5 min, and the supernatant was removed. The pellet was washed three times in a wash buffer [400 mm NaCl, 50 mm Tris (pH 7.6), and 0.1% NP40] and once in PBS, each time centrifuging the sample and discarding the supernatant. After adding 20 μl of 2× SDS-PAGE gel loading buffer, samples were boiled for 5 min at 100°C and subjected to SDS-PAGE and immunoblotting.

IκB Kinase Assay.

IKKα was immunoprecipitated from whole cell lysates as described (20) with the following modifications. Washed immunocomplexes were combined with 20 μl of kinase buffer [25 mm Tris-HCl (pH 7.5), 5 mm β-glycerophosphate, 2 mm DTT, 0.1 mm Na3VO4, and 10 mm MgCl2], 1 mm cold ATP, 1 μg IκBα-GST fusion protein (substrate protein), and 10 μCi [γ-32P]ATP. Samples were then incubated for 20 min at 37°C, and the reaction was stopped by adding 5 μl of 2× SDS buffer. Samples were boiled at 100°C for 5 min, spun down at 10,000 × g for 5 min at 4°C, and run out on 10% SDS-PAGE gel. The gel was then dried, exposed to a phosphorimaging screen overnight, and scanned on a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

Stable Transfection of U251 Glioma Cells with IκBα-Mut Vector.

U251 glioma cells (1 × 106) were transfected with 10 μg of pLXSN-IκBα-Mut (provided by Dr. P. Chiao, M. D. Anderson Cancer Center, Houston, TX), an expression vector containing IκBα cDNA with serine to alanine mutations at S32 and S36 and the neomycin resistance gene (21), using the standard calcium phosphate technique according to the manufacturer’s instructions (Promega). Twenty-four h later, cells were washed once in PBS and placed in neomycin selection medium containing 800 μg/ml G418. After 10 days, individual neomycin-resistant colonies were isolated by trypsinization and established as subcultures.

Clonogenic Survival Assay.

Cell cultures were trypsinized to generate a single cell suspension, and specified numbers were seeded into 60-mm tissue culture dishes. Dishes were then returned to the incubator for 4–6 h after which, in the radiation experiments, they were removed and exposed to specified doses of γ irradiation using a 137Cs source (3.7 Gy/min). At this time after plating, cells have attached to the dishes but have not yet divided. After irradiation, dishes were returned to the incubator for 10–14 days (depending on cell line) before staining with crystal violet. Colonies that contain >50 cells were then counted. Radiation survival curves were constructed after normalization for the cytotoxicity induced by virus alone. Data from three to four independent experiments were used to generate the survival curves.

We had shown previously that AV1Y28 enhanced radiation-induced cell killing in a number of human tumor cell lines but not in a normal human fibroblast cell line (10). This study has been extended to include the SF539 glioma and BXPC-3 pancreatic carcinoma cell lines. Radiation clonogenic survival curves with and without AV1Y28 were generated for each cell line (Ref. 10; data not shown for the SF539 and BXPC-3 cell lines); the surviving fraction after 2 Gy (SF2) for each of the cell lines with and without AV1Y28 pretreatment in shown in Table 1. Clonogenic survival analysis indicated that AV1Y28 enhanced the radiosensitivity of the U251 and SF539 glioblastoma cell lines and the HT29 and SW620 colon carcinoma cell lines; in contrast, this adenovirus had no effect on the radiosensitivity of C-29A normal human fibroblasts or the pancreatic carcinoma cell line BXPC-3.

These data illustrate that the AV1Y28-mediated radiosensitization occurs independently of ras gene mutational status. Although mutant Ras proteins are constitutively active, the activity of Ras proteins in the wild-type ras cells used in this study remained to be determined. To evaluate the level of GTP-bound (active) Ras protein, a pull-down assay was performed using the c-Raf-1 RBD, followed by immunoblotting with a pan-Ras antibody. As shown in Fig. 1, each of the cell lines with mutant ras contained activated Ras proteins, as expected. However, in addition, SF539 and HT29 cells, which contain wild-type ras, also contained a significant level of activated Ras. Interestingly, activated Ras proteins were found in each of the cell lines radiosensitized by AV1Y28. In contrast, cell lines not sensitized by AV1Y28 (BXPC-3 and C-29A normal fibroblasts) contained very low or nondetectable levels of activated Ras. Thus, whereas independent of ras mutational status (Table 1), AV1Y28-mediated radiosensitization appears to require the presence of active (GTP-bound) Ras protein. AV1Y28 and/or irradiation with 10 Gy had no effect on Ras activity in any of the cell lines evaluated (data not shown).

Initial studies of the mechanisms of AV1Y28-mediated radiosensitization focused on U251 human glioblastoma cells (N-ras mutant). To verify that the anti-Ras scFv expressed by AV1Y28 interacts with Ras proteins in these cells, a combined immunoprecipitation and immunoblot protocol was followed that took advantage of the c-Myc tag included on the scFv expressed by AV1Y28 (13). U251 whole cell lysates were immunoprecipitated with a pan-Ras antibody (Ab-2), which recognizes a different epitope than the peptide sequence targeted by the scFv, and immunoblotted using an antibody to c-Myc (Fig. 2,A). Immunoblotting detected expression of the c-Myc tag only in the AV1Y28-treated sample (Lane 4), confirming that the adenovirus expresses the scFv. When cell lysates were first subjected to immunoprecipitation with the pan-Ras antibody, the c-Myc tag was detected only in the adenovirus-transduced cells (Lane 2), demonstrating that the scFv interacts with Ras proteins. Because the functional form of Ras requires membrane association, U251 lysates were separated into soluble and insoluble fractions, and the same IP and IB protocol was applied. As shown in Fig. 2 B, the c-Myc tag was detected in the soluble and insoluble fractions of the AV1Y28-treated cells (Lanes 6 and 8), indicating that scFv was located in both cellular compartments. However, the Ras and scFv interaction was found only in the insoluble fraction (Lane 2), consistent with the location of functional Ras proteins.

Whereas a number of transcription factors are influenced by Ras (22, 23), the one that has been most closely associated with radioresponse is NF-κB (16, 17). Therefore, to pursue the mechanisms mediating the radiosensitization induced by the anti-Ras scFv expressed by the AV1Y28 adenovirus, we determined the effects of this adenovirus on radiation-induced NF-κB activity in the U251 human glioma cell line. Exposure of U251 cells to AV1Y28 has been shown to result in an increase in radiation-induced clonogenic cell death (Ref. 10; Table 1). EMSA analyses showed that radiation activated NF-κB DNA binding in U251 cells in a time-dependent manner, reaching a maximum at 2 h and returning to control levels by 8 h (Fig. 3,A). Supershift analysis using p50 and p65 antibodies demonstrated that radiation-induced NF-κB consisted of a mixture of p50/p50 homodimer and p50/p65 heterodimer complexes (data not shown). In U251 cells transduced with AV1Y28, however, the activation of NF-κB DNA binding after irradiation was significantly reduced (Fig. 3,B, Lanes 9–12). In contrast, transduction of U251 cells with an adenovirus expressing the luciferase gene in place of the anti-Ras scFv expression cassette had no effect on radiation-induced activation of NF-κB (Fig. 3 B, Lanes 3–6).

NF-κB can be activated by a variety of stimuli; the most frequently investigated stimulus is the cytokine TNF-α. To determine whether AV1Y28 also inhibits NF-κB activation induced by this cytokine, U251 cells were treated with TNF-α with and without prior exposure to AV1Y28. As shown in Fig. 4,A, AV1Y28 has no effect on TNF-α-induced NF-κB-DNA binding. The effects of AV1Y28 on NF-κB activation by H2O2, a cell-permeable oxidizing agent (24), and ferricyanide, a non-cell-permeable molecule that generates intracellular ROS through an interaction occurring at the cell membrane (25), were also investigated in U251 cells. As shown in Fig. 4 B, these free radical-generating compounds induced NF-κB activity, and in each case AV1Y28 significantly attenuated the activation. These data suggest that inhibition of NF-κB activation by AV1Y28 was selective for those treatments that generate ROS.

To determine whether the more frequently investigated Ras effectors were involved in radiation-induced NF-κB activation, U251 cells were subjected to a 1-h pretreatment with the inhibitors of PI3K (wortmannin), MEK (PD98059), and p38 (SB203580). The 1-h exposure to each inhibitor was found sufficient to inhibit their respective kinase activity (data not shown). As shown in Fig. 5, these inhibitors had no effect on radiation-induced activation of NF-κB. These results indicate that the activation of NF-κB by radiation does not require the PI3K, MEK, or p38 signaling pathways.

One of the critical steps in initiating NF-κB activation is the phosphorylation of IκBα on serines 32 and 36 (26). To determine whether AV1Y28 influences this event, U251 cells were transduced with the adenovirus, irradiated, and analyzed for the presence of phosphorylated IκBα. IB analysis of whole cell lysates indicated the presence of a slower migrating species of IκBα 2 h after exposure to radiation (Fig. 6,A, Lane 3). In contrast, cells transduced with AV1Y28 did not exhibit the slower migrating species of IκBα (Fig. 6,A, Lane 6). TNF-α treatment served as a positive control and resulted in the strong induction of a slower migrating form of IκBα, indicating the presence of a phosphorylated species (Fig. 6,A, Lane 7). These data suggest that IR induces the phosphorylation of IκBα, albeit to a lesser extent than TNF-α. As an additional approach for evaluating IκBα phosphorylation, cell lysates were initially immunoprecipitated for IκBα, and the resulting immunocomplexes were separated by SDS-PAGE and subjected to IB analyses with an antibody that specifically recognizes the serine 32 phosphorylated form of IκBα. As shown in Fig. 6,B, irradiation induced the phosphorylation of IκBα (Lane 2), although at a level considerably lower than that induced by TNF-α (Lane 5). Treatment with AV1Y28, however, prevented the phosphorylation of IκBα by radiation (Lane 4). In contrast, AV1Y28 did not affect serine phosphorylation induced by TNF-α (Fig. 6 C). These data suggest that AV1Y28 suppresses the activation of radiation-induced NF-κB through the inhibition of IκBα serine phosphorylation.

Previously, Li et al.(27) demonstrated that the IKKs directly phosphorylate IκBα. In addition, IR has been shown to increase IKK activity in mammalian cells (20). To determine whether the AV1Y28-mediated inhibition of radiation-induced NF-κB involved an effect on IKK, U251 whole cell lysates were immunoprecipitated with an antibody to IKKα, and kinase activity was evaluated. Constitutive IKK activity was readily apparent in U251 cells with a slight but reproducible increase induced by IR (Fig. 7). However, exposure to AV1Y28 significantly reduced the constitutive as well as IR-induced IKK activity (Fig. 7). These results suggest that Ras plays a role in regulating IKK activity.

As an initial evaluation of the relationship between AV1Y28-mediated suppression of radiation-induced NF-κB activation and radiosensitization, NF-κB activity was analyzed in cell lines that were or were not sensitized by the adenovirus (Table 1). According to EMSA analyses, the sensitized cell lines SF539, HT29, and SW620 exhibited a radiation-induced, time-dependent increase in NF-κB DNA binding (Fig. 8,A, Lanes 3–6). In each cell line, as for U251, the radiation-induced activation of NF-κB was significantly suppressed by pretreatment with AV1Y28 (Fig. 8,A, Lanes 9–12). In contrast, the C-29A fibroblast cell line, which was not radiosensitized by AV1Y28, failed to activate NF-κB after doses up to 10 Gy by 2 h after irradiation (Fig. 8,B) or at times out to 8 h after 10 Gy (data not shown). Treatment of C-29A cells, with TNF-α, however, did result in NF-κB DNA binding. In the nonradiosensitized tumor cell line BXPC-3, there was a high level of constitutive NF-κB p50/p50 homodimer DNA binding (Fig. 8 C). Although IR induced a slight increase in NF-κB p50/p50 homodimer binding that was reduced by AV1Y28, the NF-κB p50/p65 heterodimer complex was not induced in the BXPC-3 cell line. These data are indicative of a correlation between radiosensitization induced by AV1Y28 and its ability to inhibit radiation-induced NF-κB.

To specifically determine the role of NF-κB in AV1Y28-mediated radiosensitization, a dominant-negative IκBα construct (IκBα-Mut) was transfected into U251 cells. Subsequently, 15 neomycin-resistant clones were selected, grown out as individual subclones, and analyzed for IκBα-Mut expression by IB. The two highest expressing clones (clones 4 and 6) and, as a control, the two lowest expressing clones (clones 10 and 15) were selected for additional studies. Expression of IκBα-Mut in the selected clones is shown in Fig. 9,A. IκBα-Mut is present as a truncated isoform of the full-length IκBα protein and runs at a lower molecular weight on SDS-PAGE gels.3 To determine whether expression of the IκBα-Mut inhibits radiation-induced NF-κB DNA binding, EMSA analysis was performed on lysates collected from irradiated cultures. As shown in Fig. 9,B, irradiation resulted in NF-κB DNA binding in the parental cells (Lane 4) and in the low IκBα-Mut-expressing clones (Lanes 10 and 12). In contrast, in clones expressing high levels of IκBα-Mut, IR did not significantly induce NF-κB DNA binding (Lanes 6 and 8). Consistent with the function of IκBα as an inhibitor of NF-κB, constitutive levels of NF-κB binding were also slightly reduced in the high IκBα-Mut-expressing clones as compared with the parental cell line (Lanes 5 and 3, respectively). The ability of NF-κB activation to influence radiosensitivity was then determined by clonogenic survival analysis. As shown in Fig. 10,A, the high expressing IκBα-Mut clones (clones 4 and 6) had a similar radioresponse as the low expressing clones (clones 10 and 15) and as the parental cell line. Thus, at least in the U251 cell line, the inhibition of radiation-induced NF-κB was not sufficient to modify cellular radiosensitivity. To determine whether the inhibition of NF-κB was necessary for AV1Y28-mediated radiosensitization, clonogenic cell survival analysis after 2 Gy was performed on IκBα-Mut-expressing cells transduced with AV1Y28 or a control vector (Ad-Luc). As shown in Fig. 10 B, treatment of AV1Y28 sensitized the high and low expressing IκBα-Mut clones to a comparable level as the parental cell line. These data indicate that NF-κB activation is also not necessary for the radiosensitizing actions of AV1Y28.

We had shown previously that the anti-Ras scFv delivered by AV1Y28 enhances the radiosensitivity of a number of tumor cell lines, consistent with a role for Ras in regulating radiosensitivity (2, 4). However, as shown in Fig. 1, the radiosensitization induced by AV1Y28, whereas independent of ras gene mutational status, does require the presence of Ras proteins in the active (GTP-bound) form. The presence of active Ras proteins in tumor cells with wild-type ras is consistent with previous studies (28, 29). In contrast to the H-ras mutation required for radiosensitization by FTIs (7), the requirement for activated Ras proteins suggests that AV1Y28 will have radiosensitizing capabilities against a broader range of tumors. Furthermore, the correlation between Ras activation and AV1Y28 radiosensitization reinforces the previous survival data from normal human fibroblasts, suggesting that this adenovirus (and the Ras pathway affected) will have at least some degree of specificity for tumor over normal cells.

The goal of these studies was to use the anti-Ras scFv expressed by the AV1Y28 to investigate the relationship between Ras, NF-κB, and radiosensitization. The data presented here show clearly that AV1Y28 inhibits radiation-induced activation of NF-κB in a number of tumor cell lines, indicating that Ras proteins are involved in this process. Although Ras has been implicated previously in regulating NF-κB activity under other treatment conditions (22, 30, 31), the results described here suggest that the Ras pathway mediating this radiation effect is unique. That is, as shown in Fig. 5, the most frequently studied Ras effectors MAPK, PI3K, and p38 are not involved in radiation-induced NF-κB activity. Furthermore, on the basis of IP/IB studies, AV1Y28 has no effect on the constitutive interaction between Ras and Raf or RalGDS in U251 cells (data not shown). Thus, it appears that an alternative Ras-mediated pathway is involved in the activation of NF-κB by radiation.

Whereas AV1Y28 clearly abrogated IKK activity and NF-κB activation after irradiation, it had no effect on the activation of this transcription factor in response to TNF-α. The inability of AV1Y28 to affect TNF-α-induced activation is in agreement with the absence of Ras proteins in the well-defined TNF-α-mediated pathway of NF-κB activation (32). However, Pahan et al.(23) reported recently that the transient expression of a dominant-negative form of Ras (Δp21ras), which antagonizes RasGEF function (33), inhibits NF-κB activation by TNF-α in rat astrocytes. The disparity between these results and those presented here is unclear but may be accounted for by the use of normal versus tumor cells (34) or may reflect different modes of action for the dominant-negative construct and AV1Y28. Although AV1Y28 did not affect NF-κB DNA binding by TNF-α, it clearly inhibited the activation induced by H2O2 and ferricyanide. Combined with its effects on radiation-induced activation of NF-κB, these results suggest that the inhibitory action of AV1Y28 is specific for a pathway mediated by ROS. On the basis of different time courses of activation, it was previously proposed that the oxidant- and cytokine-induced NF-κB activation proceeded along different pathways (35). In addition, a number of studies have implicated ROS as regulators of Ras activity (36, 37). Thus, it appears that a significant difference between the oxidant- and cytokine-mediated pathways is the participation of Ras proteins.

A critical step in the activation of NF-κB is the phosphorylation of its inhibitor IκBα (38). Whereas distinct initial signaling pathways may regulate NF-κB activation by particular stimuli, most appear to converge at the level of IKK as the immediate regulator of NF-κB. Arsura et al. found increased NF-κB activity in Ras-transformed rat liver epithelial cells as a result of increased phosphorylation of IκBα. Furthermore, transfection of these cells with a dominant-negative IKK inhibited NF-κB activation suggesting Ras-induced activation of NF-κB is mediated by the IKK complex (39). In addition, platelet-derived growth factor stimulation of normal human fibroblasts and rat fibroblast-like synoviocytes resulted in the activation of a Ras→PI3K→Akt→IKK→NF-κB signaling cascade (31). As shown in Fig. 7, IR results in a slight increase in IKK activity, which is consistent with the small level of IκBα phosphorylation induced by radiation (Fig. 6, A and B). AV1Y28 pretreatment prevented this increase and reduced constitutive IKK activity, suggesting that some aspect of Ras function is involved in the regulation of IKK activity. Because inhibition of PI3K, MEK, and p38 failed to inhibit IR-induced NF-κB, the specific Ras effector associated with the IKK complex and thus mediates radiation-induced NF-κB activation remains to be identified.

NF-κB activation after other types of insults has been associated with decreased apoptosis and enhanced cell survival (40, 41). Furthermore, inhibition of NF-κB activation has been suggested as a means of increasing tumor cell sensitivity to standard cytotoxic agents (42, 43, 44). However, with respect to radiation, the effects of inhibiting NF-κB activation have been contradictory. Wang et al.(16) reported that the stable expression of a dominant-negative IκBα, which results in an inhibition of NF-κB activation, enhanced apoptotic killing by radiation in a human fibrosarcoma cell line. In addition, Yamagishi et al.(17) showed that the overexpression of IκBα, which also inhibited NF-κB activation, enhanced the radiosensitivity of two glioblastoma cell lines as determined by clonogenic survival, although apoptosis levels were not reported. In contrast to these results, Pajonk et al.(45) reported that treatment of a prostate carcinoma and a Hodgkin’s lymphoma cell line with an adenovirus expressing a gene coding for a dominant-negative form of IκBα, although cytotoxic alone, had no effect on radiosensitivity. The data presented here clearly indicate a correlation between the ability of AV1Y28 to induce radiosensitization and to inhibit radiation-induced NF-κB. However, an alternative strategy to attenuate NF-κB activation after irradiation in U251 cells, (i.e., stable expression a dominant-negative mutant of IκBα) failed to affect the level of radiation-induced cell killing. In addition, AV1Y28 retained its ability to radiosensitize cells expressing the dominant-negative IκBα. These data indicate that, at least in U251 cells, that the inhibition of NF-κB activation is not necessary or sufficient for AV1Y28-induced radiosensitization. Given that NF-κB is typically associated with an apoptotic response, the lack of a role for its inhibition is consistent with our previous observations regarding the lack of a change in apoptosis frequency in irradiated cells treated with AV1Y28 (10).

However, because there is a correlation between AV1Y28-mediated radiosensitization and its ability to inhibit NF-κB activation after irradiation, it would appear that there might be some aspect of NF-κB signaling or metabolism involved in the AV1Y28-mediated effect on radioresponse. Along these lines, IKKα has recently been found to have activities in addition to phosphorylating IκBα (46, 47). Given the significant effect of AV1Y28 on constitutive IKK activity and the apparent lack of involvement in NF-κB in U251 radiosensitivity, it may be that IKKα independent of NF-κB plays a role in regulating radioresponse. Clearly, this possibility will require additional studies.

Fig. 1.

Ras activation in cell lines sensitized and nonsensitized by AV1Y28. Cell lysates were subjected to a pull-down assay using the Raf RBD peptide fragment and immunoblotted with the pan-Ras antibody. (+), U251 whole cell lysate subjected to IB only to serve as a positive control.

Fig. 1.

Ras activation in cell lines sensitized and nonsensitized by AV1Y28. Cell lysates were subjected to a pull-down assay using the Raf RBD peptide fragment and immunoblotted with the pan-Ras antibody. (+), U251 whole cell lysate subjected to IB only to serve as a positive control.

Close modal
Fig. 2.

Interaction between the anti-Ras scFv and Ras proteins in U251 cells. Cell cultures were transduced with 103 vp/cell of AV1Y28 and 48 h later collected for IP and/or IB analysis. IP was performed using an anti-Ras antibody (Ab-2), followed by IB using an antibody (9E10) to the c-Myc tag of the anti-Ras scFv. A, whole cell lysates; B, U251 lysates were separated into soluble (S) and insoluble (I) fractions. Arrows, cellular c-Myc protein and the scFv c-Myc tag. For IP lanes, IgH and IgL represent the heavy and light immunoglobulin chains, respectively.

Fig. 2.

Interaction between the anti-Ras scFv and Ras proteins in U251 cells. Cell cultures were transduced with 103 vp/cell of AV1Y28 and 48 h later collected for IP and/or IB analysis. IP was performed using an anti-Ras antibody (Ab-2), followed by IB using an antibody (9E10) to the c-Myc tag of the anti-Ras scFv. A, whole cell lysates; B, U251 lysates were separated into soluble (S) and insoluble (I) fractions. Arrows, cellular c-Myc protein and the scFv c-Myc tag. For IP lanes, IgH and IgL represent the heavy and light immunoglobulin chains, respectively.

Close modal
Fig. 3.

Effect of AV1Y28 on radiation-induced NF-κB DNA binding in U251 cells. A, cells were irradiated (10 Gy) and collected at the indicated time points for evaluation by EMSA. B, 48 h after being transduced with 103 vp/cell of an adenovirus expressing either the anti-Ras scFv (AV1Y28) or the luciferase gene (Ad-Luc), cells were irradiated (10 Gy) and collected at the indicated time points for evaluation by EMSA. ∗, lanes containing a 100-fold excess of cold NF-κB oligonucleotide competitor. C, unirradiated control sample.

Fig. 3.

Effect of AV1Y28 on radiation-induced NF-κB DNA binding in U251 cells. A, cells were irradiated (10 Gy) and collected at the indicated time points for evaluation by EMSA. B, 48 h after being transduced with 103 vp/cell of an adenovirus expressing either the anti-Ras scFv (AV1Y28) or the luciferase gene (Ad-Luc), cells were irradiated (10 Gy) and collected at the indicated time points for evaluation by EMSA. ∗, lanes containing a 100-fold excess of cold NF-κB oligonucleotide competitor. C, unirradiated control sample.

Close modal
Fig. 4.

Effect AV1Y28 on NF-κB DNA binding activity induced by H2O2, ferricyanide, or TNF-α. Forty-eight h after being transduced with 103 vp/cell of AV1Y28 (Y28) or mock-transduced (control), U251 cells were exposed to TNF-α (10 ng/ml, 30 min); A) or H2O2 (100 μm, 2 h) or ferricyanide (10 nm, 0.5 h); B) before collection for EMSA analysis. C, unirradiated control sample.

Fig. 4.

Effect AV1Y28 on NF-κB DNA binding activity induced by H2O2, ferricyanide, or TNF-α. Forty-eight h after being transduced with 103 vp/cell of AV1Y28 (Y28) or mock-transduced (control), U251 cells were exposed to TNF-α (10 ng/ml, 30 min); A) or H2O2 (100 μm, 2 h) or ferricyanide (10 nm, 0.5 h); B) before collection for EMSA analysis. C, unirradiated control sample.

Close modal
Fig. 5.

Inhibition of PI3K, MEK, and p38 and the radiation-induced activation of NF-κB in U251 cells. Cultures were treated with the PI3K inhibitor wortmannin (100 nm, 1 h), the MEK inhibitor PD98059 (50 μm, 1 h), or the p38 inhibitor SB203580 (10 μm, 1 h), followed by irradiation (10 Gy) and 2 h later collected for evaluation by EMSA. DMSO treatment alone was used as the vehicle control.

Fig. 5.

Inhibition of PI3K, MEK, and p38 and the radiation-induced activation of NF-κB in U251 cells. Cultures were treated with the PI3K inhibitor wortmannin (100 nm, 1 h), the MEK inhibitor PD98059 (50 μm, 1 h), or the p38 inhibitor SB203580 (10 μm, 1 h), followed by irradiation (10 Gy) and 2 h later collected for evaluation by EMSA. DMSO treatment alone was used as the vehicle control.

Close modal
Fig. 6.

Effect of AV1Y28 on radiation or TNF-α induced serine phosphorylation of IκBα in U251 cells. A, 48 h after being transduced with 103 vp/cell of AV1Y28 or mock transduced (control), cells were irradiated (10 Gy) and collected 1 and 2 h later or treated with TNF-α (10 ng/ml) and collected 5 and 30 min later for IB analysis using an antibody to IκBα. C, unirradiated sample. B, following the same treatment protocols (10 Gy, 2 h; 10 ng/ml TNF-α, 5 min), cell lysates were subjected to IP with the IκBα antibody and IB with a phosphospecific (serine 32) IκBα antibody (1:200 dilution). C, cells were exposed to AV1Y28 or mock transduced and 48 h later treated with TNF-α (10 ng/ml, 5 min). Lysates then were subjected to the IP/IB procedure as in B. To insure band specificity in B and C, Ab designates lanes in which analysis was performed in the absence of cell lysates containing only the appropriate antibodies.

Fig. 6.

Effect of AV1Y28 on radiation or TNF-α induced serine phosphorylation of IκBα in U251 cells. A, 48 h after being transduced with 103 vp/cell of AV1Y28 or mock transduced (control), cells were irradiated (10 Gy) and collected 1 and 2 h later or treated with TNF-α (10 ng/ml) and collected 5 and 30 min later for IB analysis using an antibody to IκBα. C, unirradiated sample. B, following the same treatment protocols (10 Gy, 2 h; 10 ng/ml TNF-α, 5 min), cell lysates were subjected to IP with the IκBα antibody and IB with a phosphospecific (serine 32) IκBα antibody (1:200 dilution). C, cells were exposed to AV1Y28 or mock transduced and 48 h later treated with TNF-α (10 ng/ml, 5 min). Lysates then were subjected to the IP/IB procedure as in B. To insure band specificity in B and C, Ab designates lanes in which analysis was performed in the absence of cell lysates containing only the appropriate antibodies.

Close modal
Fig. 7.

Inhibition of IKK Activity by AV1Y28. Forty-eight h after being transduced with 103 vp/cell of AV1Y28 or mock transduced (control), U251 cells were exposed to 20 Gy and collected at 1 h (A). Lysates were initially subjected to immunoprecipitation with an anti-IKKα antibody and combined with [γ-32P]ATP and IκBα-GST substrate protein. Anti-IKKα antibody conjugated to agarose beads reacted with [γ-32P]ATP and IκBα-GST substrate protein in the absence of cell lysates was used as the control. B, quantification of IKK activity in A using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Fig. 7.

Inhibition of IKK Activity by AV1Y28. Forty-eight h after being transduced with 103 vp/cell of AV1Y28 or mock transduced (control), U251 cells were exposed to 20 Gy and collected at 1 h (A). Lysates were initially subjected to immunoprecipitation with an anti-IKKα antibody and combined with [γ-32P]ATP and IκBα-GST substrate protein. Anti-IKKα antibody conjugated to agarose beads reacted with [γ-32P]ATP and IκBα-GST substrate protein in the absence of cell lysates was used as the control. B, quantification of IKK activity in A using ImageQuant software (Molecular Dynamics, Sunnyvale, CA).

Close modal
Fig. 8.

Effect of AV1Y28 on radiation-induced NF-κB DNA binding in radiosensitized and nonsensitized cell lines. A, cell lines were exposed to a viral titer sufficient to transduce at least 80% of cells. Forty-eight h after being transduced with AV1Y28 or Ad-Luc, cells were irradiated (10 Gy), and lysates were collected at the indicated time points for evaluation by EMSA. B, C-29A fibroblasts were exposed to increasing doses of radiation and collected 2 h later for evaluation by EMSA. In addition, cells were treated with TNF-α (10 ng/ml, 30 min). C, BXPC-3 cells were exposed to AV1Y28 or Ad-Luc, 48 h later irradiated (10 Gy), and lysates were collected at the specified times for evaluation by EMSA. ∗, lanes containing a 100-fold excess of cold NF-κB oligonucleotide competitor. C, unirradiated control sample.

Fig. 8.

Effect of AV1Y28 on radiation-induced NF-κB DNA binding in radiosensitized and nonsensitized cell lines. A, cell lines were exposed to a viral titer sufficient to transduce at least 80% of cells. Forty-eight h after being transduced with AV1Y28 or Ad-Luc, cells were irradiated (10 Gy), and lysates were collected at the indicated time points for evaluation by EMSA. B, C-29A fibroblasts were exposed to increasing doses of radiation and collected 2 h later for evaluation by EMSA. In addition, cells were treated with TNF-α (10 ng/ml, 30 min). C, BXPC-3 cells were exposed to AV1Y28 or Ad-Luc, 48 h later irradiated (10 Gy), and lysates were collected at the specified times for evaluation by EMSA. ∗, lanes containing a 100-fold excess of cold NF-κB oligonucleotide competitor. C, unirradiated control sample.

Close modal
Fig. 9.

IκBα-Mut Expression. U251 cells (1 × 106) were transfected with a plasmid expressing a dominant-negative IκBα and neomycin resistance gene. Cells were grown in the presence of G418 (800 μg/ml) for 10 days. Resistant clones were isolated and analyzed for expression of IκBα-Mut protein. A, immunoblot of selected clones expressing high (Lanes 2 and 3) and low (Lanes 4 and 5) levels of IκBα-Mut. Actin was used as a loading control. B, high and low expressing IκBα-Mut clones were irradiated (10 Gy), collected after 2 h, and analyzed by EMSA. Samples containing a 100-fold excess of cold NF-κB oligonucleotide (Cold Oligo) competitor were run as an indicator of specificity (Lanes 1 and 2).

Fig. 9.

IκBα-Mut Expression. U251 cells (1 × 106) were transfected with a plasmid expressing a dominant-negative IκBα and neomycin resistance gene. Cells were grown in the presence of G418 (800 μg/ml) for 10 days. Resistant clones were isolated and analyzed for expression of IκBα-Mut protein. A, immunoblot of selected clones expressing high (Lanes 2 and 3) and low (Lanes 4 and 5) levels of IκBα-Mut. Actin was used as a loading control. B, high and low expressing IκBα-Mut clones were irradiated (10 Gy), collected after 2 h, and analyzed by EMSA. Samples containing a 100-fold excess of cold NF-κB oligonucleotide (Cold Oligo) competitor were run as an indicator of specificity (Lanes 1 and 2).

Close modal
Fig. 10.

Radioresponse of U251 IκBα-Mut clones. Parental and designated IκBα-Mut clones were trypsinized, plated at specified cell numbers, and irradiated at selected doses 6 h later. Colony-forming efficiency was determined 10 days later. A, clonogenic survival analysis of high (clones 4 and 6) and low (clones 10 and 15) expressing IκBα-Mut clones and the parental line. Values represent the mean of two independent experiments. B, parental and IκBα-Mut-expressing clones were transduced with AV1Y28 or Ad-Luc (103 vp/cell), and clonogenic survival after 2 Gy was determined as in A. Values represent the means for three independent experiments; bars, SE. ∗, the surviving fraction at 2 Gy for AV1Y28-treated cells is significantly lower than that for the Ad-Luc-treated cells (Student’s t test; P < 0.05).

Fig. 10.

Radioresponse of U251 IκBα-Mut clones. Parental and designated IκBα-Mut clones were trypsinized, plated at specified cell numbers, and irradiated at selected doses 6 h later. Colony-forming efficiency was determined 10 days later. A, clonogenic survival analysis of high (clones 4 and 6) and low (clones 10 and 15) expressing IκBα-Mut clones and the parental line. Values represent the mean of two independent experiments. B, parental and IκBα-Mut-expressing clones were transduced with AV1Y28 or Ad-Luc (103 vp/cell), and clonogenic survival after 2 Gy was determined as in A. Values represent the means for three independent experiments; bars, SE. ∗, the surviving fraction at 2 Gy for AV1Y28-treated cells is significantly lower than that for the Ad-Luc-treated cells (Student’s t test; P < 0.05).

Close modal

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

2

The abbreviations used are: IR, ionizing radiation; FTI, farnesyltransferase inhibitor; CMV, cytomegalovirus; NF-κB, nuclear factor-κB; TNF, tumor necrosis factor; MEK, mitogen-activated protein kinase kinase; vp, viral particle(s); RBD, Ras binding domain; PMSF, phenylmethylsulfonyl fluoride; EMSA, electrophoretic mobility shift assay; IP, immunoprecipitation; IB, immunoblot; GST, glutathione S-transferase; ROS, reactive oxygen species; PI3K, phosphatidylinositol 3-kinase; IKK, IκB kinase.

3

P. Chiao, personal communication.

Table 1

AV1Y28-mediated radiosensitization (SF2)a

Cell linesSensitized SF2Cell linesNonsensitized SF2
Ad-LucY28Ad-LucY28
U251b 0.69 0.40 C-29A 0.18 0.20 
SF539 0.82 0.53 BXPC-3 0.57 0.61 
SW620b 0.64 0.35    
HT29 0.73 0.50    
Cell linesSensitized SF2Cell linesNonsensitized SF2
Ad-LucY28Ad-LucY28
U251b 0.69 0.40 C-29A 0.18 0.20 
SF539 0.82 0.53 BXPC-3 0.57 0.61 
SW620b 0.64 0.35    
HT29 0.73 0.50    
a

Cultures were either transduced with AV1Y28 (Y28) or a control virus (Ad-Luc). The next day, cultures were irradiated, trypsinized, and plated at specified cell numbers. Colony-forming efficiency was determined 10–14 days later, and survival curves were constructed after normalizing the data to the cytotoxicity induced by viral vectors. SF2 represents the surviving fraction at 2 Gy. Data for the U251, SW629, HT29, and C-29A cell lines was reported previously (10).

b

Cell lines containing mutant ras genes (U251, N-ras; SW620, K-ras).

1
Vojtek A. B., Der C. J. Increasing complexity of the Ras signaling pathway.
J. Biol. Chem.
,
273
:
19925
-19928,  
1998
.
2
Sklar M. D. The ras oncogenes increase the intrinsic resistance of NIH 3T3 cells to ionizing radiation.
Science (Wash. DC)
,
239
:
645
-647,  
1988
.
3
Ling C. C., Endlich B. Radioresistance induced by oncogenic transformation.
Radiat. Res.
,
120
:
267
-279,  
1989
.
4
Samid D., Miller A. C., Rimoldi D., Gafner J., Clark E. P. Increased radiation resistance in transformed and nontransformed cells with elevated ras proto-oncogene expression.
Radiat. Res.
,
126
:
244
-250,  
1991
.
5
Pirollo K. F., Hao Z., Rait A., Ho C. W., Chang E. H. Evidence supporting a signal transduction pathway leading to the radiation-resistant phenotype in human tumor cells.
Biochem. Biophys. Res. Commun.
,
230
:
196
-201,  
1997
.
6
Bernhard E. J., Stanbridge E. J., Gupta S., Gupta A. K., Soto D., Bakanauskas V. J., Cerniglia G. J., Muschel R. J., McKenna W. G. Direct evidence for the contribution of activated N-ras and K-ras oncogenes to increased intrinsic radiation resistance in human tumor cell lines.
Cancer Res.
,
60
:
6597
-6600,  
2000
.
7
Bernhard E. J., Kao G., Cox A. D., Sebti S. M., Hamilton A. D., Muschel R. J., McKenna W. G. The farnesyltransferase inhibitor FTI-277 radiosensitizes H-ras- transformed rat embryo fibroblasts.
Cancer Res.
,
56
:
1727
-1730,  
1996
.
8
Bernhard E. J., McKenna W. G., Hamilton A. D., Sebti S. M., Qian Y., Wu J. M., Muschel R. J. Inhibiting Ras prenylation increases the radiosensitivity of human tumor cell lines with activating mutations of ras oncogenes.
Cancer Res.
,
58
:
1754
-1761,  
1998
.
9
Cox A. D., Der C. J. Farnesyltransferase inhibitors and cancer treatment: targeting simply Ras?.
Biochim. Biophys. Acta
,
1333
:
F51
-F71,  
1997
.
10
Russell J. S., Lang F. F., Huet T., Janicot M., Chada S., Wilson D. R., Tofilon P. J. Radiosensitization of human tumor cell lines induced by the adenovirus-mediated expression of an anti-Ras single-chain antibody fragment.
Cancer Res.
,
59
:
5239
-5244,  
1999
.
11
Werge T. M., Baldari C. T., Telford J. L. Intracellular single chain Fv antibody inhibits Ras activity in T-cell antigen receptor stimulated Jurkat cells.
FEBS Lett.
,
351
:
393
-396,  
1994
.
12
Biocca S., Pierandrei-Amaldi P., Cattaneo A. Intracellular expression of anti-p21ras single chain Fv fragments inhibits meiotic maturation of Xenopus oocytes.
Biochem. Biophys. Res. Commun.
,
197
:
422
-427,  
1993
.
13
Cochet O., Kenigsberg M., Delumeau I., Virone-Oddos A., Multon M. C., Fridman W. H., Schweighoffer F., Teillaud J. L., Tocque B. Intracellular expression of an antibody fragment-neutralizing p21 ras promotes tumor regression.
Cancer Res.
,
58
:
1170
-1176,  
1998
.
14
McFall A., Ulku A., Lambert Q. T., Kusa A., Rogers-Graham K., Der C. J. Oncogenic Ras blocks anoikis by activation of a novel effector pathway independent of phosphatidylinositol 3-kinase.
Mol. Cell. Biol.
,
21
:
5488
-5499,  
2001
.
15
Finco T. S., Westwick J. K., Norris J. L., Beg A. A., Der C. J., Baldwin A. S., Jr. Oncogenic Ha-Ras-induced signaling activates NF-κB transcriptional activity, which is required for cellular transformation.
J. Biol. Chem.
,
272
:
24113
-24116,  
1997
.
16
Wang C. Y., Mayo M. W., Baldwin A. S., Jr. TNF- and cancer therapy-induced apoptosis: potentiation by inhibition of NF-κB.
Science (Wash. DC)
,
274
:
784
-787,  
1996
.
17
Yamagishi N., Miyakoshi J., Takebe H. Enhanced radiosensitivity by inhibition of nuclear factor κB activation in human malignant glioma cells.
Int. J. Radiat. Biol.
,
72
:
157
-162,  
1997
.
18
de Rooij J., Bos J. L. Minimal Ras-binding domain of Raf1 can be used as an activation-specific probe for Ras.
Oncogene
,
14
:
623
-625,  
1997
.
19
Bergo M. O., Leung G. K., Ambroziak P., Otto J. C., Casey P. J., Young S. G. Targeted inactivation of the isoprenylcysteine carboxyl methyltransferase gene causes mislocalization of K-Ras in mammalian cells.
J. Biol. Chem.
,
275
:
17605
-17610,  
2000
.
20
Li N., Karin M. Ionizing radiation and short wavelength UV activate NF-κB through two distinct mechanisms.
Proc. Natl. Acad. Sci. USA
,
95
:
13012
-13017,  
1998
.
21
Feig B. W., Lu X., Hunt K. K., Shan Q., Yu D., Pollock R., Chiao P. Inhibition of the transcription factor nuclear factor-κB by adenoviral-mediated expression of IκBαM results in tumor cell death.
Surgery
,
126
:
399
-405,  
1999
.
22
Janssen-Heininger Y. M., Macara I., Mossman B. T. Cooperativity between oxidants and tumor necrosis factor in the activation of nuclear factor (NF)-κB: requirement of Ras/mitogen-activated protein kinases in the activation of NF-κB by oxidants.
Am. J. Respir. Cell Mol. Biol.
,
20
:
942
-952,  
1999
.
23
Pahan K., Liu X., McKinney M. J., Wood C., Sheikh F. G., Raymond J. R. Expression of a dominant-negative mutant of p21(ras) inhibits induction of nitric oxide synthase and activation of nuclear factor-κB in primary astrocytes.
J. Neurochem.
,
74
:
2288
-2295,  
2000
.
24
Quesada A. R., Byrnes R. W., Krezoski S. O., Petering D. H. Direct reaction of H2O2 with sulfhydryl groups in HL-60 cells: zinc-metallothionein and other sites.
Arch. Biochem. Biophys.
,
334
:
241
-250,  
1996
.
25
Kaul N., Choi J., Forman H. J. Transmembrane redox signaling activates NF-κB in macrophages.
Free Radical Biol. Med.
,
24
:
202
-207,  
1998
.
26
Brown K., Gerstberger S., Carlson L., Franzoso G., Siebenlist U. Control of IκBα proteolysis by site-specific, signal-induced phosphorylation.
Science (Wash. DC)
,
267
:
1485
-1488,  
1995
.
27
Li J., Peet G. W., Pullen S. S., Schembri-King J., Warren T. C., Marcu K. B., Kehry M. R., Barton R., Jakes S. Recombinant IκB kinases α and β are direct kinases of IκBα.
J. Biol. Chem.
,
273
:
30736
-30741,  
1998
.
28
Guha A., Feldkamp M. M., Lau N., Boss G., Pawson A. Proliferation of human malignant astrocytomas is dependent on Ras activation.
Oncogene
,
15
:
2755
-2765,  
1997
.
29
Feldkamp M. M., Lau N., Rak J., Kerbel R. S., Guha A. Normoxic and hypoxic regulation of vascular endothelial growth factor (VEGF) by astrocytoma cells is mediated by Ras.
Int. J. Cancer
,
81
:
118
-124,  
1999
.
30
Koong A. C., Chen E. Y., Mivechi N. F., Denko N. C., Stambrook P., Giaccia A. J. Hypoxic activation of nuclear factor-κB is mediated by a Ras and Raf signaling pathway and does not involve MAP kinase (ERK1 or ERK2).
Cancer Res.
,
54
:
5273
-5279,  
1994
.
31
Romashkova J. A., Makarov S. S. NF-κB is a target of AKT in anti-apoptotic PDGF signalling.
Nature (Lond.)
,
401
:
86
-90,  
1999
.
32
Fischer C., Page S., Weber M., Eisele T., Neumeier D., Brand K. Differential effects of lipopolysaccharide and tumor necrosis factor on monocytic IκB kinase signalsome activation and IκB proteolysis.
J. Biol. Chem.
,
274
:
24625
-24632,  
1999
.
33
Shields J. M., Pruitt K., McFall A., Shaub A., Der C. J. Understanding Ras. “it ain’t over ’til it’s over.”.
Trends Cell Biol.
,
10
:
147
-154,  
2000
.
34
Serrano M., Lin A. W., McCurrach M. E., Beach D., Lowe S. W. Oncogenic ras provokes premature cell senescence associated with accumulation of p53 and p16INK4a.
Cell
,
88
:
593
-602,  
1997
.
35
Anderson M. T., Staal F. J., Gitler C., Herzenberg L. A., Herzenberg L. A. Separation of oxidant-initiated and redox-regulated steps in the NF-κB signal transduction pathway.
Proc. Natl. Acad. Sci. USA
,
91
:
11527
-11531,  
1994
.
36
Deora A. A., Win T., Vanhaesebroeck B., Lander H. M. A redox-triggered ras-effector interaction. Recruitment of phosphatidylinositol 3′-kinase to Ras by redox stress.
J. Biol. Chem.
,
273
:
29923
-29928,  
1998
.
37
Irani K., Goldschmidt-Clermont P. J. Ras, superoxide and signal transduction.
Biochem. Pharmacol.
,
55
:
1339
-1346,  
1998
.
38
Karin M., Ben Neriah Y. Phosphorylation meets ubiquitination: the control of NF-κB activity.
Annu. Rev. Immunol.
,
18
:
621
-663,  
2000
.
39
Arsura M., Mercurio F., Oliver A. L., Thorgeirsson S. S., Sonenshein G. E. Role of the IκB kinase complex in oncogenic Ras- and Raf-mediated transformation of rat liver epithelial cells.
Mol. Cell. Biol.
,
20
:
5381
-5391,  
2000
.
40
Brach M. A., Hass R., Sherman M. L., Gunji H., Weichselbaum R., Kufe D. Ionizing radiation induces expression and binding activity of the nuclear factor κB.
J. Clin. Investig.
,
88
:
691
-695,  
1991
.
41
Mohan N., Meltz M. L. Induction of nuclear factor κB after low-dose ionizing radiation involves a reactive oxygen intermediate signaling pathway.
Radiat. Res.
,
140
:
97
-104,  
1994
.
42
Bertrand F., Atfi A., Cadoret A., L’Allemain G., Robin H., Lascols O., Capeau J., Cherqui G. A role for nuclear factor κB in the antiapoptotic function of insulin.
J. Biol. Chem.
,
273
:
2931
-2938,  
1998
.
43
Heck S., Lezoualc’h F., Engert S., Behl C. Insulin-like growth factor-1-mediated neuroprotection against oxidative stress is associated with activation of nuclear factor κB.
J. Biol. Chem.
,
274
:
9828
-9835,  
1999
.
44
Mattson M. P., Culmsee C., Yu Z., Camandola S. Roles of nuclear factor κB in neuronal survival and plasticity.
J. Neurochem.
,
74
:
443
-456,  
2000
.
45
Pajonk F., Pajonk K., McBride W. H. Inhibition of NF-κB, clonogenicity, and radiosensitivity of human cancer cells.
J. Natl. Cancer Inst.
,
91
:
1956
-1960,  
1999
.
46
Hu Y., Baud V., Oga T., Kim K. I., Yoshida K., Karin M. IKKα controls formation of the epidermis independently of NF-κB.
Nature (Lond.)
,
410
:
710
-714,  
2001
.
47
Lamberti C., Lin K. M., Yamamoto Y., Verma U., Verma I. M., Byers S., Gaynor R. B. Regulation of β-catenin function by the IκB kinases.
J. Biol. Chem.
,
276
:
42276
-42286,  
2001
.