Co-activation of ERK, NF-κB, and GADD45β in Response to Ionizing Radiation*

NF-κB has been well documented to play a critical role in signaling cell stress reactions. The extracellular signal-regulated kinase (ERK) regulates cell proliferation and survival. GADD45β is a primary cell cycle element responsive to NF-κB activation in anti-apoptotic responses. The present study provides evidence demonstrating that NK-κB, ERK and GADD45β are co-activated by ionizing radiation (IR) in a pattern of mutually dependence to increase cell survival. Stress conditions generated in human breast cancer MCF-7 cells by the administration of a single exposure of 5 Gy IR resulted in the activation of ERK but not p38 or JNK, along with an enhancement of the NF-κB transactivation and GADD45β expression. Overexpression of dominant negative Erk (DN-Erk) or pre-exposure to ERK inhibitor PD98059 inhibited NF-κB. Transfection of dominant negative mutant IκB that blocks NF-κB nuclear translocation, inhibited ERK activity and GADD45β expression and increased cell radiosensitivity. Interaction of p65 and ERK was visualized in living MCF-7 cells by bimolecular fluorescence complementation analysis. Antisense inhibition of GADD45β strikingly blocked IR-induced NF-κB and ERK but not p38 and JNK. Overall, these results demonstrate a possibility that NF-κB, ERK, and GADD45β are able to coordinate in a loop-like signaling network to defend cells against the cytotoxicity induced by ionizing radiation.

Protein kinase activities play a pivotal role in responses to a variety of stress conditions, e.g. UV light, ionizing radiation (IR), 1 chemotherapy drugs, virus infection, osmotic shock, heat, nitric oxide, lipopolysaccharide, arsenic oxide, and inflammatory cytokines. A predominant feature of molecular responses under such stress conditions is the activation of early responsive transcription factors and signaling elements resulting in a temporary cell cycle arrest and anti-apoptotic responses leading to an increased cell survival (1)(2)(3). Numerous gene products are shown to be inducible in mammalian cells by the stress of IR (4 -6). However, only the key signaling proteins that control cell cycle, DNA repair, and cell growth regulation are believed to be decisive in cell death or survival with the situation of lethal insults (5)(6)(7). Accumulating data have demonstrated that activation of the transcription factor NF-B is critical in defending cells from stressinduced damages and influencing the cell survival rate following different cytotoxic conditions (8 -10).
NF-B, with five Rel family members, i.e. p65/RelA, p50, p52, c-Rel, and Rel B, comprise a stress-sensitive gene regulator. The NF-B heterodimers (mostly p65/p50) are normally sequestered by binding to the inhibitor IB and quickly activated by a stress that causes the phosphorylation and proteolysis of IB (11)(12)(13). Being associated with the regulation of more than 150 effecter genes, NF-B functions in an array of signaling pathways, including cell transformation and antiapoptotic responses (14,15). One of the most characterized features of NF-B activation is to antagonize the signaling network of apoptotic response (16) and to increase cell resistance to radiation and chemotherapy (17). IR-treated HeLa cells demonstrate a transient NF-B activation following IB phosphorylation (18). In breast carcinoma MCF-7 cells (5) and Papillomavirus-transformed human keratinocytes (19), the basal and IR-induced NF-B is paralleled with a reduced cell killing by IR. Interestingly, NF-B is also associated with the mitochondrial stress resulting in the activation of calcineurin and mitochondrial antioxidant enzyme Mn-SOD (20,21). Gene clustering analyses further demonstrate that NF-B is responsible for at least a fraction of genes induced by IR, including Mn-SOD, indicating that the mitochondrial redox imbalance is important for cells to defend IR cytotoxicity (22). Blocking NF-B inhibits the expression of Mn-SOD (22) as well as other IR-induced genes, which enhances IRmediated cell death (19,22). These findings illustrate a possibility that a group of stress-sensitive proteins are required for cell survival. The context of the IR-induced NF-B and other signaling elements needs to be elucidated.
Accumulating results suggest that the group of mitogenactivated protein kinases (MAPKs), i.e. ERK, JNK, p38, functions to signal responses to IR (23)(24)(25) and other stress conditions (26 -28). For instance, ERK is strongly induced by high doses of IR that can activate the membrane-associated tyrosine kinase (25,29). ERK that is originally associated with mitotic response (30,31) has been found to be a key mediator for tyrosine phosphorylation in growth factors and Ras activation (30 -33). Therefore, ERK activation appears to be a necessary event in signaling cell proliferation and survival (26,27,34). In contrast, although JNK, a key member of MAPKs, induces NF-B in anti-apoptotic responses (35), NF-B-targeted genes are shown to inhibit JNK required for initiating tumor necrosis factor-␣-induced apoptosis (8). Recent data further indicate that ERK-mediated anti-apoptotic response is dependent upon its cellular locations and interaction with NF-B⅐IB complexes (36). It remains unclear whether ERK and MAPKs are associated with NF-B-mediated radioprotection.
GADD45␤ (growth arrest and DNA damage-inducible ␤, also named MyD118) is key member of nuclear proteins inducible by DNA-damaging stresses and has been shown to play an active role in cell cycle adjustment that affects cell survival (37). It has been long observed that IR-induced GADD45␤ expression (38) is linked to p53 pathways (39,40). Experiments of antisense transfections find that GADD45␤ interacts with cyclin B1 complex during S and G 2 /M checkpoints following UV irradiation (41). GADD45␤ is also required in T cell receptorinduced responses and ERK, p38, and JNK activation are all substantially suppressed in GADD45␤-deficient CD4(ϩ) T cells (37), suggesting a tight link between GADD45␤ and MAPKs pathways. NF-B is shown to induce Gadd45␤ promoter activity (42) and GADD45␤ induced by NF-B can down-regulate the pro-apoptotic JNK (43,44).
To characterize the signaling network of NF-B activation that appears to play a key role in cell death or survival after high dose IR, we have examined the hypothesis that NF-B, ERK, and GADD45␤ co-operate in radiation response. Inhibition of either factor by transfection of dominant negative mutant or pre-treatment with inhibitor compounds blocks the activity of other two components in a loop-like connection. Because antisense blocking gadd45␤ inhibited NF-B and ERK but not other MAPKs, i.e. p38 and JNK, ERK, and gadd45␤ are likely to be specifically required for signaling NF-B-mediated cell protection. The present study thus reveals a novel connection of NF-B with activation of ERK and GADD45␤ in defending cell survival after IR insults.

EXPERIMENTAL PROCEDURES
Cell Culture and Exposure to Ionizing Radiation-MCF-7 cells were obtained from the American Type Culture Collection (passage number: 126) and maintained in Dulbecco's minimum essential media (Cellgro, Herndon, VA) supplemented with 10% fetal bovine serum (HyClone, Logan, UT), 1% L-glutamine, penicillin (100 units/ml), and streptomycin (100 g/ml) in a humidified incubator (5% CO 2 ) at 37°C. Exponentially growing cells with 60 -80% confluence were exposed to IR at room temperature with a Cs-137 mark I irradiator (dose rate, 436 cGy/min, J. L. Shepherd & Associates). Cells sheltered from IR source were used as the sham-IR control. After IR, cells were cultured at 37°C incubator for further experiments.
Preparation of Nuclear Extracts and Gel Shift Analyses-Subsequent to IR exposure, cells were rinsed with phosphate-buffered saline containing 1 mM EDTA, collected by centrifugation, and resuspended in ice-cold hypotonic lysis buffer supplemented with protease inhibitors (10 mM Hepes, pH 7.9, 0.1 mM EDTA, 0.1 mM EGTA, 10 mM KCl, 0.3% Nonidet P-40, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, and 1 g/ml pepstatin A). Following 15 min of incubation on ice, the cell lysates were vortexed and centrifuged at 15,000 ϫ g for 1 min to obtain the nuclear pellet and the cytosolic protein-containing supernatant. The nuclear pellets were washed using the aforementioned buffer with the exclusion of Nonidet P-40. Then the nuclei were resuspended in the ice-cold solution (20 mM Hepes, pH 7.9, 420 mM NaCl, 1 mM EDTA, 0.1 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 g/ml leupeptin, and 1 g/ml pepstatin A). After 30 min of incubation on ice, the samples were centrifuged at 15,000 ϫ g for 5 min, and the resulting supernatant was saved as nuclear extracts stored in the Ϫ80°C freezer until use.
Gel shift analysis was performed with 3 g of nuclear proteins incubated on ice for 10 min in a total 20 l of DNA binding buffer (10 mM Tris-HCl, pH 7.5, 1 mM MgCl 2 , 0.5 mM EDTA, 0.5 mM dithiothreitol, 5% glycerol, and 50 g/ml polydeoxyinosinnicdeoxycytidylic acid strand DNA) and then incubated for 20 min at room temperature with 5 ϫ 10 4 cpm 32 P-labeled NF-B oligonucleotides probe. The DNA⅐protein complexes were resolved on 10% native polyacrylamide gel electrophoresis and visualized in x-ray film. For supershift assays nuclear extracts were incubated with antibodies p65 (sc-7151) or p50 (sc-7178) at room temperature for 20 min prior to the addition of the radioactive-labeled DNA oligonucleotides.
Immunoblotting Analysis-Cellular extracts were fractionated using a 10% SDS-polyacrylamide gel and transferred by way of a semidry apparatus (Bio-Rad, Hercules, CA) to polyvinylidene difluoride membranes (Bio-Rad). Immunoblot analysis was carried out using antibodies (Santa Cruz Biotechnology, Santa Cruz, CA) and visualized using the horseradish peroxidase-coupled mouse anti-rabbit immunoglobulins followed by the ECL Western blotting detection system (Amersham Biosciences).
Transient Co-transfection of NF-B Reporters with Dominant Negative Mutant IB or Dominant Mutant Erk-Transfection of NF-B luciferase reporter with dominant negative mutant IB-␣ vector (22) or with dominant negative Erk vector (45) were performed as previously detailed (22). Cells in 12-well plates were co-transfected with 0.3 g of NF-B and 0.2 g of ␤-galactosidase reporters for 3 h, and luciferase activity was measured 24 h following exposure to sham or IR. Luciferase activity was measured using an illuminometer (Turner Designs, Sunnyvale, CA). An aliquot of the same cell lysate was used for measurement of ␤-galactosidase activity to normalize luciferase results.
Establishment of Stable Transfectants MCF-7/mIB and MCF-7/DN-Erk-MCF-7 cells were stably transfected with dominant negative mutant mIB or dominant negative Erk (DN-Erk) plasmids, and stable transfectants were obtained using Lipofectamine reagent (Invitrogen) as previously described (22). MCF-7 cells (5 ϫ 10 6 in 100-mm cell culture dishes) were transfected with 15 g of mIB or DN-Erk plasmid, 2 g of G418 marker DNA pcDNA3, and 40 g of Lipofectamine in 6 ml of serum-reduced OPTI-EMEM (Invitrogen). pcDNA3 only was transfected into MCF-7 cells as a vector control. Cells were transfected with transfection solution for 72 h and recovered for 24 h in complete medium, trypsinized, and cultured in the selecting medium for 14 -21 days. The selected mutant and vector control clones were maintained in Dulbecco's modified Eagle's medium, and all transfectants were cultured for at least two passages in G418-free medium before experiments.
Clonogenic Survival Assay-Prior to conducting clonogenic assays, the plating efficiency of the wild type MCF-7, mutant gene transfectants MCF-7/mIB and MCF-7/DN-Erk, and vector control transfectants MCF-7/V were determined. Contingent upon these phycoerythrin findings, the number of cells seeded was calculated to give an identical number of clones for the controls. The number of cells seeded ranged from 1,000 to 200,000 according to the radiation doses 2-12 Gy. 14 -18 days after irradiation, the plates were stained and colonies with more than 50 cells were counted and normalized to the plating efficiency of control and transfected cell lines.
Cell Proliferation Assay-Exponentially growing MCF-7 cells were digested with trypsin, and different cell numbers were plated into multiple well plates for 18 -24 h. Cells were then treated with PD98059 or Gadd45␤ antisense oligonucleotides followed by an exposure to a single dose 5 Gy IR. Cell proliferation was measured by cell numbers calculated with hemocytometers or by MTT (3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide) assay (Promega, Madison, MI).
Protein Kinase Assays-Cell lysates were prepared 24 h after radiation with the lysis buffer provided in the Bio-Rad Cell Lysis Kit by following the user manual. Protein concentration was measured with a DC TM protein assay kit (Bio-Rad Laboratories), and protein concentration was adjusted using the cell lysis buffer. p-JNK, p-p38, and p-ERK1/2 were measured using the Bio-Plex TM phosphoprotein assay (Bio-Rad Laboratories). Briefly, the filter plate was rinsed with 100 l of phosphoprotein wash buffer, followed by adding 50 l of antibodyconjugated beads, 25 l of each cell lysate, and 25 l of phosphoprotein assay buffer. The plate was shaken overnight and incubated with biotinylated anti-phospho-p44/p42 MAPK (ERK1/ERK2; Santa Cruz Biotechnology) antibody, incubated with 50 l of streptavidin-labeled phycoerythrin, and read on the Bio-Plex TM system.
PD98059 and IR Treatment-PD98059 (Sigma) was dissolved in Me 2 SO and MCF-7 cells were incubated with control Me 2 SO or Me 2 SO with a range of concentrations of PD98059 for 2 h. Treatments were terminated by replacing with complete medium without PD98059, and cell pellets were collected 24 h after exposure to a single dose of IR. Proteins were purified from control and PD98059/IR-treated cells, and ERK kinase activity was measured by using the Bio-Plex TM phosphoprotein assay (Bio-Rad) and Western blotting. In a parallel experiment, cells were plated into multiple-well plates, and cell growth was determined after PD98059 and IR treatments.
Antisense Preparation and Treatments-Phosphorothioate-modified antisense oligonucleotides of Gadd45␤ (5Ј-GAAGTTGCGGAAAC-CAACGGT-3Ј) were synthesized with the gene sequence obtained from GenBank TM (AF533019; 1836 -1856). MCF-7 cells grown in a T-75 tissue culture flask were transfected with a specific range of antisense concentrations and Lipofectamine reagent (Invitrogen). Cells were exposed to antisense oligonucleotides for 24 h and then replaced with the complete medium and treated with or without IR. At different time intervals cell pellets were collected for analysis of MAPK activity. In a parallel experiment, cells were plated into multiple well plates, and cell proliferation was determined after antisense and IR treatments.
Imaging of ERK/p65 Interactions in Living Cells-Plasmids with full-length sequence encoding human NF-B p65, ERK1, and ERK2 were fused to N-and C-terminal fragments of enhanced yellow fluorescent protein. The EEK1, ERK2, p65 coding regions were connected by linker sequences as described (46). These expression vectors encoding p65-YC156, ERK1-YN173, and ERK2-YN173 were kind gifts from Dr. C. D. Hu, and co-transfection was performed as previously described (22). Briefly, MCF-7 cells were maintained in complete Dulbecco's modified Eagle's medium with 10% fetal bovine serum (HyClone, Logan, UT), and cells in 24-well plates were co-transfected with the expression vectors indicated in each experiment (a total of 0.4 g of DNA in each well) using Lipofectamine 2000 (Invitrogen). The fluorescence emissions were observed in living cells 14 -16 h after transfection using a Nikon TE300 inverted fluorescence microscope with a cooled chargecoupled device camera.

IR Activates NF-B and ERK but Not p38 or JNK-Accumu-
lating results in literature (47,48) and our previous reports (5,22) have demonstrated that ionizing radiation enhances NF-B DNA binding and transcriptional activities that are associated with reduced cell radiosensitivity. To get insight of the network of NF-B-mediated radioprotection, the activity of a group of MAPKs was measured in irradiated breast carcinoma MCF-7 cells. Agreeing with NF-B activation (Fig. 1A), a single exposure to 5 Gy IR significantly induced the kinase activity of p-ERK (75% increase compared with the sham-IR controls; Fig.  1B). In contrast, no difference was detected in the basal and IR-induced kinase activities of p-p38 and p-JNK. Thus, ERK kinase activity appears to be specifically paralleled with IRinduced NF-B activation.
Overexpression of mIB Blocked IR-induced ERK Activity and GADD45␤ Expression-To test the hypothesis that IRinduced NF-B and ERK activation is mutually dependent, IR-induced ERK activity was measured with the inhibition of NF-B. The dose dependence of NF-B inhibition by overexpression of the dominant negative mutant IB is shown in Fig.  2A. The mutant form of IB␣ that has been shown to inhibit NF-B activity in MCF-7 cells (22) dose-dependently inhibited the basal (MCF-7, Fig. 2A, left panel) and IR-induced (MCF-7 plus 5Gy, Fig. 2A, right panel) NF-B-controlled luciferase activities. IR-mediated NF-B DNA binding activity was assayed by gel shift analysis in stable transfectants (MCF-7/ mIB, with vector control transfectants MCF-7/V as a control). Compared with the wild type MCF-7 and MCF-7/V cells, a striking reduction in IR-induced NF-B DNA binding was induced in MCF-7/mIB transfectants (Fig. 2B). Both NF-B subunits p65 and p50 were found to be present in the IRinduced NF-B⅐DNA binding complexes (Fig. 2C), suggesting that p65 and p50 are the major components of IR-induced NF-B complexes. ERK activity measured by substrate Elk-1 phosphorylation (Fig. 2D) and GADD45␤ measured by Western blot (Fig. 2E) were significantly enhanced by IR in the wild type (MCF-7) and vector control (MCF-7/V) cells. In contrast, ERK activity and GADD45␤ expression were not enhanced by IR in MCF-7 cells overexpressing mIB (MCF-7/mIB).

Overexpression of Dominant Negative ERK (DN-Erk) Blocked IR-induced NF-B Activity and GADD45␤ Expression and Enhanced Cell Radiosensitivity-To determine if IR-induced ERK
is associated with NF-B activation, we analyzed the dosedependence of NF-B inhibition by overexpression of the dominant negative mutant Erk gene that, when overexpressed, inhibits ERK kinase activity (45). IR-induced NF-B luciferase activity was found to be dose-dependently down-regulated by DN-Erk DNA transfection (Fig. 3A). Transfection of 0.3 and 0.6 g of DN-Erk DNA reduced NF-B transcriptional activity to the basal level of wild type MCF-7 cells without IR exposure (Fig. 3A). In addition, DN-Erk stable transfectants (MCF-7/ DN-Erk) demonstrated a predominant reduction in basal and IR-induced NF-B activation (Fig. 3B). Similar to NF-B inhibition, GADD45␤ expression was totally blocked in MCF-7/DN-Erk transfectants (Fig. 3C), suggesting that both NF-B and GADD45␤ can be downstream elements of IR-induced ERK activation. Clonogenic survival of MCF-7/DN-Erk and MCF-7/ mIB transfectants was found to be significantly decreased after the exposure to an array of IR doses (0 -12 Gy; Fig. 3D). Compared to the wild type MCF-7 cells the dose-modifying factors at 10% isosurvival level were 1.43 and 1.39, respectively, for MCF-7/DN-Erk and MCF-7/mIB cells. These results strongly indicate that IR-induced ERK activity is able to induce FIG. 1. NF-B and ERK but not p38 or JNK was activated by IR. A, NF-B activation by 5 Gy IR. MCF-7 cells were co-transfected with NF-B luciferase and ␤-galactosidase reporters for 6 h, and luciferase activity was measured 24 h after exposure to sham-IR (Ϫ) or a single dose of 5 Gy IR (ϩ). Luciferase reporter activity was normalized to ␤-galactosidase (mean Ϯ S.E., n ϭ 3). B, p-ERK but not p-JNK or p-p38 was induced by IR. MCF-7 cells treated with sham-IR (0 Gy) and IR (5 Gy) were collected 24 h after irradiation. p-ERK, p-JNK, and p-p38 antibodies were incubated with 20 g of whole cell lysates, and the immunocomplex was labeled with streptavidin-phycoerythrin and detected by the Bio-Plex (Bio-Rad laboratory) protein array system as described under "Experimental Procedures" (mean Ϯ S.E., n ϭ 3; **, p Ͻ 0.01).
NF-B and GADD45␤ and increase cell clonogenic survival after IR.
PD98059 Inhibited IR-induced ERK and NF-B Activation and Increased Cell Radiosensitivity-PD98059 has been shown to specifically inhibit MAPK activity (49,50) leading to IRinduced apoptosis with a decreased clonogenic survival in MCF-7 cells (51). To investigate if PD98059-mediated radiosensitivity is caused by the inhibition of both NF-B and p-ERK, Fig. 4A shows that IR-induced p-ERK levels (p-MAPK, p44/42) were dose-dependently inhibited by the pre-treatment of cells to PD98059. IR-induced NF-B activity measured by luciferase reporter was inhibited with a similar dose-dependent pattern (Fig. 4B). Radiosensitivity measured by cell proliferation showed that cells pre-exposed to PD98059 increased IRinduced growth inhibition by 24 -40% during the time 48 -72 h after radiation. Without IR, PD98059 did not induce significant growth inhibition (Fig. 4C).
Antisense-blocking Gadd45␤ Inhibited ERK but Not p38 or JNK-GADD45 proteins have been shown to play important roles in maintaining cell genomic stability and regulating the cell cycle (52,53). The GADD45␤-induced anti-apoptotic pathway is believed to occur via NF-B activation (43). To test if GADD45␤ is required for IR induction of ERK and NF-B, p-ERK, p-p38, and p-JNK activities were measured in MCF-7 cells transfected with the antisense of Gadd45␤ (antisense sequences are described under "Experimental Procedures"). Fig. 5A shows that pre-treatment with antisense Gadd45␤ significantly blocked IR-induced ERK activation without affecting the activity of p38 and JNK. These results are consistent with the observation shown in Fig. 1 indicating that ERK is specifically induced in MCF-7 cells by IR. Analyses with Western blotting shown in Fig. 5 (B-D) demonstrated that both basal and IR-induced p-ERK were inhibited by pre-treatment with Gadd45␤ antisense compared to cells without antisense transfection. No detectable difference was induced in p-p38 and p-JNK activities by antisense pre-treatment with or without following IR exposure.
Antisense-blocking GADD45␤ Inhibited IR-induced NF-B Activation Causing Decreased Cell Survival-To determine if GADD45␤ is required for enhancing cell survival after IR insults, the inhibited protein levels of GADD45␤ was verified in irradiated MCF-7 cells transfected with antisense Gadd45␤ (Fig. 6A). Equivalent with GADD45␤ inhibition, IR-induced NF-B luciferase transcription was totally abolished by antisense transfection (Fig. 6B). Without antisense transfection, MCF-7 cells showed ϳ4-fold NF-B luciferase activity induced by IR. Cell growth showed that pre-treatment with Gadd45␤ antisense completely eliminated cell proliferation after IR in contrast to the cell growth treated with 2 Gy IR alone (Fig. 6C). Thus, GADD45␤ expression levels appear to be a sensitive

FIG. 2. Overexpression of mIB inhibited NF-B activity and Elk-1 phosphorylation. A, basal and radiation-induced NF-B activity was dose-dependently inhibited by transfection of dominate negative mutant IB (mIB).
NF-B luciferase reporters were co-transfected with the indicated amounts of mIB plasmids into MCF-7 cells, and luciferase activity was measured 24 h after treatment with or without 5 Gy IR. B, IR-induced NF-B DNA binding activity was inhibited in stable transfectants of mIB. Wild type MFC-7 and stable transfectants of mIB (MCF-7/mIB) or empty vector (MCF-7/V) were treated with or without 5 Gy IR. NF-B DNA binding activity was assayed with DNA retardation gel analysis. MCF-7/Vϩcold oligonucleotide was included as a negative control with the presence of unlabeled NF-B nucleotides. C, NF-B subunits p65 and p50 were detected in the NF-B⅐DNA complexes induced by IR. Nuclear extracts from 5 Gy IR-treated MCF-7 cells were assayed with DNA retardation gel with or without antibody to NF-B subunit p65 and p50 as indicated. NF-B and the antibody super-shifted bands are indicated with arrowheads. D, expression of mIB inhibited IR-induced ERK kinase activity. Wild type MFC-7, empty vector control transfectants (MCF-7/V) and mIB MCF-7 transfectants (MCF-7/mIB) were irradiated with 0 (Ϫ) or 5 Gy (ϩ) IR. Phospho-Elk-1 (sc-355; Santa Cruz Biotechnology) was detected by Western blotting as a downstream ERK kinase substrate. E, expression of mIB inhibited IR-induced GADD45␤. Wild type MFC-7, empty vector control transfectants (MCF-7/V), and mIB MCF-7 transfectants (MCF-7/ mIB) were irradiated with 0 (Ϫ) or 5 Gy (ϩ) IR. GADD45␤ was detected by Western blot (antibody sc-8775; Santa Cruz Biotechnology). factor in mediating IR-induced ERK and NF-B activation and cell survival.
Visualization of Interaction between ERK and NF-B p65bimolecular fluorescence complementation analysis (46) provides the image of protein interactions in living cells. To confirm the direct interactions of NF-B p65 and ERK in MCF-7 cells, expression vectors encoding p65-YC156, Erk1-YN173, and Erk2-YN173 were co-transfected, and protein-protein interaction was analyzed under fluorescence microscopy (46). Fluorescence was observed within 14 -16 h after co-transfection of p65-YC165 with EEK1-YN173 or ERK2-TN173 (Fig. 7,  C and E). In the control experiments, expression of p65-YC156, ERK1-YN173, or ERK2-YN173 alone did not produce significant fluorescence (Fig. 7, A, B, and D). Protein interactions appeared to occur mainly in the nucleus of MCF-7 cells (Fig. 7, C and E), indicating a possibility that NF-B and ERK form complexes to regulate specific genes to increase cell survival. Dynamic analysis of IR-induced protein interactions between NF-B subunits, ERK, and GADD45␤ and nuclear transportation is underway.
IR-Induced G 2 /M Delay Was Reduced by Blocking ERK/NF-B/GADD45␤-We then tested the hypothesis that the increased radiosensitivity, upon inhibiting the ERK/NF-B/ GADD45␤ pathway is caused by alterations in cell cycle adjustment. Cell cycle distribution of wild type MCF-7 cells pre-exposed to the corresponding inhibitors was calculated after exposure to a single dose of 5 Gy (Table I). In contrast to a remarkable G 2 /M delay (3.32-fold) induced in wild type MCF-7 cells by IR, none of MCF-7/DN-Erk transfectants or MCF-7 cells pre-treated with PD98059 and Gadd45␤ antisense showed a significant delay in G 2 /M border. The basal cell accumulation in G 2 /M border without IR insult was slightly increased in cells with the inhibition of the ERK/NF-B/GADD45␤ pathway suggesting an opposite influence of blocking ERK/NF-B/ GADD45␤ pathway versus IR. In addition, compared with the wild type MCF-7 cells, no significant alteration in G 0 /G 1 and S phases was induced by inhibition of ERK/NF-B/GADD45␤. These results strongly imply that a shortage in IR-induced G 2 /M delay causes the radiosensitization induced by inhibition of ERK/NF-B/GADD45␤ pathways. DISCUSSION The present study provides evidence suggesting a novel connection of ERK, NF-B, and GADD45␤ in enhancing cell survival after IR exposure. These factors were found to be coactivated and mutually dependent in radiation responses leading to a protective cell phenotype. Inhibition of ERK by transfection of dominant negative Erk (MCF-7/DN-Erk) or treated with ERK inhibitor PD98059 inhibited NF-B activity and increased cell radiosensitivity. Inhibition of NF-B with transfection of dominant negative IB (MCF-7/mIB) showed a similar pattern of inhibition and radiosensitization. Additionally, antisense-blocking GADD45␤ inhibited NF-B and ERK but not p38 or JNK, causing enhanced IR cytotoxicity. These results illustrate a loop-like connection in IR-induced transcription factors and cell cycle elements required for increasing cell survival.
Identifying the signaling network associated with cell death or survival after IR stress is an essential issue in the research of radioprotection and anticancer radiotherapy. The present study suggests that two key stress-sensitive signaling elements ERK and GADD45␤ are closely related to NF-B activity, forming a loop-like connection to increase cell survival. This is evidenced by the fact that blocking each factor causes the inhibition of the other two and significantly changes cell radiosensitivity. Although the exact mechanisms underlying the formation of the protective network remain to be elucidated, accumulating evidence strongly implicates that a large group of gene regulators and gene products are required for signaling FIG. 4. PD98059 inhibited p-ERK and NF-B activity and increased MCF-7 cell radiosensitivity. A, ERK inhibitor PD98059 dose-dependently reduced IR-induced p-ERK. MCF-7 cells treated with the indicated concentrations of PD98059 for 2 h before exposure to 5 Gy IR and p-ERK activity was determined by Western blotting with antibody of p-ERK (sc-7383; sc-93 for ERK). B, PD98059 dosedependently inhibited NF-B luciferase reporter activity. MCF-7 cells transfected with NF-B luciferase reporters were exposed to the indicated concentrations of PD98059 for 2 h, and luciferase activity was determined 24 h after IR. C, PD98059-increased IR-induced cytotoxicity. MCF-7 cells cultured in 6-well plates were treated with PD98059 by the indicated concentrations of PD98059 for 2 h before 5 Gy IR. Cell proliferation was determined by cell numbers counted 24, 48, and 72 h after radiation (mean Ϯ S.E., n ϭ 5; **, p Ͻ 0.01). cell death or survival (21, 54 -58). The challenge therefore is to identify the specific signaling pattern that plays a decisive role in increasing cell survival. Among all the stress-responsive transcription factors, NF-B has been tightly linked with cell resistance against IR-mediated cytotoxicity and thus have been tested for a possible application in anticancer therapy (22, 58 -60). Our prior data showed that NF-B is causally related to radiosensitivity of human cancer or virus-transformed cells (19,24,56). However, inhibition of NF-B does not always increase cell radiosensitivity. For instance, inhibition of NF-B by overexpressing IB induces little effect in the radiosensitivity of PC3 prostate cancer cells, nor in HD-MyZ Hodgkin's lymphoma cells (61). This apparent paradox strongly implicates that IR-induced signaling elements interact with each other to form a network that provides a by-pass to keep cell survival. The loop-like connection of ERK/NF-B/GADD45␤ found in the present study illustrates a typical interaction of such network.
An important feature in cell response to ionizing radiation is the generation of redox imbalance. Most signaling proteins are known to be sensitive to redox alterations (62,63), and several redox-sensitive transcription factors have been shown to induce the mitochondrial antioxidant enzyme manganese-containing superoxide dismutase (Mn-SOD) that is believed to play a central role in redox regulation (22,54,64). NF-B is actively involved in regulation of Mn-SOD expression (20,65). Notably, both pro-and anti-apoptotic pathways can be activated by NF-B via expression of Mn-SOD (66). We have previously observed that IR-induced mitochondrial Mn-SOD is via NF-B activation and blocking NF-B or Mn-SOD expression down-regulates a group of IR-induced genes and increases cell radiosensitivity (22). Shonai et al. (67) have found that ERKmediated anti-apoptotic signals are activated through inhibition of mitochondrial related caspase-8 activity. Especially, ERK can selectively inhibit IR-induced loss of mitochondrial membrane potential and subsequent cell death. Therefore, IR- Gadd45␤ oligonucleotides for 24 h before IR with 5 Gy. Cell lysate was prepared 24 h after IR, and Western blotting was performed to confirm GADD45␤ inhibition. B, antisense Gadd45␤ inhibited IR-induced NF-B. MCF-7 cells transfected with NF-B luciferase reporters were treated with or without antisense transfection and exposed to IR. Luciferase activity was determined 24 h after IR (**, p Ͻ 0.01, n ϭ 3). C, MCF-7 cells in multiple-well plates were treated with (ϩ) or without (Ϫ) antisense Gadd45␤ oligonucleotides for 24 h followed by 2 Gy IR. Cell proliferation was calculated at different time intervals after IR (data are the combined mean values of three experiments).
induced mitochondrial signaling pathways require ERK activation. The bimolecular fluorescence complementation analysis (46) is applied in the present study to detect the interaction between NF-B subunits and ERK. Interaction of p65 and ERK is detected in living MCF-7 cells after co-transfection (Fig. 7, C  and E). These results and data reported by others (68) indicate but could not prove that activation of ERK/NF-B/GADD45␤ is involved in regulation of Mn-SOD and other specific genes that in turn affect mitochondria-mediated cell death and overall cell survival.
GADD45␤ in the loop of ERK/NF-B/GADD45␤ may provide a specific function for signaling radioprotection. GADD45␤, a key nuclear protein responsive to DNA damage, plays an active role in cell cycle adjustment (37). GADD45 proteins interact with the complex of Cdk1 and CyclinB1, both of which are required for IR-induced cell cycle regulation and radioresistance (5,69). IR strongly induces GADD45 expression with the dependence of p53 activation (39,70,71). IR with a dose as low as 0.5 Gy induces a rapid induction of GADD45␤ (70). NF-B subunit p65 is shown to be sufficient to activate GADD45␤ expression (42). Induction of Gadd45␤ gene expression by p65 is shown to be dependent on three B elements of the Gadd45␤ promoter region (42). Each of these sites is able to bind to NF-B complexes and required for optimal promoter transactivation. Antisense study suggests that all GADD45 proteins are likely to cooperate in the activation of S and G 2 /M checkpoints following exposure to UV irradiation (41). Our present data support that IR-mediated regulation of Gadd45␤ is due to NF-B activation and GADD45␤ further enhances NF-B and ERK activation. A connection of GADD45␤ with activation of ERK and NF-B may be a necessary step to efficiently adjust cell cycle distribution altered by IR stress. Because inhibition of ERK, NF-B, or GADD45␤ significantly reduces G 2 /M delay that is necessary for repairing damaged DNA as to increase cell survival (Table I), the result of forming the loop of ERK/NF-B/GADD45␤ appears to enhance the efficiency of repairing the damaged DNA.
Papa et al. (44) have analyzed the mechanisms by which GADD45␤ inhibits the pathways initiated by activation of JNK. In this case, GADD45␤ is shown to bind to MKK7/ JNKK2, a specific activator of JNK, and block the catalytic activity of MKK7/JNKK2 (44). Interestingly, GADD45␤ is able to inhibit tumor necrosis factor-␣-induced cytotoxicity. Disrupting GADD45␤/MKK7 interaction also blocks GADD45␤ and NF-B and suppresses tumor necrosis factor-␣-induced cytotoxicity. Both results suggest that GADD45␤ interacts with JNK pathways. However, in our present study, a single dose of IR exposure induced little activity of JNK and p38 (Fig. 1) and antisense-blocking GADD45␤ did not induce any changes in p38 and JNK activities (Fig. 5). These findings establish a basis for the hypothesis that the connection of NF-B with GADD45␤ and ERK is a unique network for increasing cell survival under IR stress. JNK and p38 appear not to be directly linked with NF-B-mediated radioresistance.
PD98059 has been shown to inhibit IR-induced ERK (72). Notably, the results reported by Suzuki et al. (72) have demonstrated that IR of a very low dose range, i.e. between 2 and 5 cGy, is able to stimulate the proliferation of normal human diploid cells and tumor cells. Irradiation with less than 1 Gy induces the phosphorylation of ERK, which is decreased if the radiation dose is reduced to 0.5 Gy (72), providing solid evidence that ERK is very sensitive to IR-mediated cytotoxicity. The activated ERK is shown to augment the phosphorylation of Elk-1 protein. As shown in Fig. 2, our present study indicates that Elk-1 phosphorylation is induced by IR and inhibited by overexpression of mIB. The phosphorylation of the ERK is also reduced by pre-treatment with PD98059 (Fig. 4), which inhibits ERK phosphorylation inducible under the stress of IR with 2 cGy or 6 Gy of x-rays (72). In addition, overexpression of ERK in NCI-H1299 human lung carcinoma cells has demonstrated an enhanced proliferation; antisense-blocking ERK abrogates IR-induced protective responses (72). Overall, ERK phosphorylation appears to be an essential step in signaling cell survival under the stress of IR, which obviously requires co-activation of NF-B and GADD45␤.
In summary, the present study demonstrates that two key stress signaling elements, ERK and GADD45␤, are co-activated with NF-B activation in MCF-7 cells exposed to a single dose of ionizing radiation. Blocking NF-B by overexpressing dominant negative mutant IB inhibits ERK activation and decreases cell survival. Inhibition of ERK by overexpression of dominant negative ERK blocks NF-B activation and GADD45␤ expression. Interaction between p65 and ERK proteins was visualized in the nuclei of living MCF-7 cells. Antisense-blocking GADD45␤ inhibits NF-B and ERK but not p38 and JNK. These results suggest that NF-B, ERK, and GADD45␤ are able to coordinate to increase cell survival after the lethal damage induced by ionizing radiation.

TABLE I Cell cycle distribution of MCF-7/DN-Erk and MCF-7/PD98039 cells
The control MCF-7, MCF-7/DN-Erk, and MCF-7 cells treated with PD98039 or antisense GADD45␤ were treated with (ϩ) or without (Ϫ) a single dose of 5-Gy radiation. Cells were collected 48 h after radiation and fixed in 85% ethanol and stained with propidium iodide (PI). PI-stained nuclear DNA content, indicating the distribution of cells in G 0 /G 1 , S, and G 2 , was determined with FACScan plus software (BD Biosciences), and the results of 1 ϫ 10 4 counted cells were analyzed using Multicycle software.