Oncogenic H-Ras Up-Regulates Expression of Ku80 to Protect Cells from ;-Ray Irradiation in NIH3T3 Cells

Research Article

Oncogenic H-Ras Up-regulates Expression of Ku80 to Protect Cells from ;-Ray Irradiation in NIH3T3 Cells

In-Youb Chang,1,3 Cha-Kyung Youn,1,2 Hong-Beum Kim,1,2 Mi-Hwa Kim,1,2 Hyun-Ju Cho,1,2 Young Yoon,1,2 Yun-Sil Lee,4 Myung-Hee Chung,5 and Ho Jin You1,2

1Research Center for Proteineous Materials and Departments of 2Pharmacology and 3Anatomy, School of Medicine, Chosun University, Gwangju, Korea and 4Laboratory of Radiation Effect, Korea Cancer Center Hospital; and 5Department of Pharmacology, School of Medicine, Seoul National University, Seoul, Korea

Abstract The major oncogenic signal from Ras uses the serine- The Ras activation contributes to radioresistance, but the threonine kinase Raf as the effector (13). The activated Ras mechanism is unclear. This article shows that the expression complexes with and promotes Raf phosphorylation. Active Raf is of the dominant-positive H-Ras increased the Ku80 level, the first in a cascade of other kinases, including mitogen- which is one of the key enzymes involved in repairing dsDNA activated protein kinase (MAPK)/extracellular signal-regulated breaks (DSB). After exposing the cells to ionizing radiation kinase (ERK; MEK), which phosphorylates and activates the and analyzing them using an electrophoretic mobility shift MAPK, ERK. Although the Ras/Raf/MEK/ERK cascade plays a assay and pulsed-field gel electrophoresis, it was found that key role in proliferative signaling, there is also evidence activated H-Ras expression in NIH3T3 cells increases the DNA- suggesting that Ras activation can activate MEK kinase (MEKK) and phosphatidylinositol 3-OH kinase (PI3K; ref. 14). MEKK is a binding activity of Ku80 and increases the DSB repair activity. Ku80 small interfering RNA expression was shown to reduce critical component of the stress-associated protein kinase, the oncogenic H-Ras-mediated increase in the DSBs and including the c-Jun NH2-terminal kinase (stress-activated protein suppress the oncogenic H-Ras-mediated resistance of the cells kinase; ref. 15). PI3K activation leads to the production of to ;-ray irradiation, whereas Ku80 overexpression in the phosphorylated phosphatidylinositides with regulatory functions NIH3T3 cells significantly increased the radioresistance. These on the kinases, phosphoinositide-dependent protein kinase and results suggest that the Ku80 expression induced by oncogenic Akt/protein kinase B, which is involved in the survival response H-Ras seems to play an important role in protecting cells (16). Recently, several groups have focused on the downstream against ;-ray irradiation. (Cancer Res 2005; 65(15): 6811-9) mediators for the Ras-induced radioresistance of cancer cells and reported that the PI3K, Raf, and epidermal growth factor receptor (EGFR) signal pathways contribute to the enhanced Introduction radioresistance (17–21). Therefore, there is considerable evidence Radiation therapy is used as a curative treatment for cancer. suggesting a causal relationship between the Ras proteins and However, the radiation responses of tumors differ according to radiation resistance. However, the molecular mechanisms under- histology, doubling time, repair capacity, and other factors (1). lying this effect are unclear. In the present study, we sought to Therefore, understanding the properties of tumor cells that determine the downstream target genes regulated by activated increase or decrease their responsiveness to radiation is the key H-Ras, particularly those that might also be involved in the to improving radiation therapy. Previous studies have shown that radiation resistance, using ponasterone A regulatable oncogenic oncogenic mutations in H-Ras, which frequently occur in many H-Ras-expressing NIH3T3 cells. The results show that the types of cancer, could contribute to an increased radiation survival oncogenic H-Ras-inducible protein Ku80 can function directly rate in transformed cells. It was initially shown that the resistance as a survival effector on the oncogenic H-Ras-expressing cells of NIH3T3 cells to radiation could be enhanced by the expression exposed to g-ray irradiation. of an activated H-Ras gene (2). This was later confirmed independently by several other groups using rodent cells, including Materials and Methods rat embryo fibroblasts and rat rhabdomyosarcomas (3–6), and human tumor cells, including an EJ Ras-transformed bladder Cell culture and DNA constructs. The NIH3T3 cells (American Type carcinoma, DLD-1 colon carcinoma, and HT1080 fibrosarcoma Culture Collection, Manassas, VA) were maintained in EMEM supplemented with 10% fetal bovine serum, 100 units penicillin/mL, and 100 Ag (7, 8). In agreement with such Ras-mediated radioresistance, streptomycin/mL (Invitrogen, Carlsbad, CA). The cells were maintained in several groups have reported that the blocking of Ras activity leads j 5% CO2-95% air at 37 C in a humidified incubator. The constructs of to an increase in radiosensitivity. For example, the expression of an the dominant-positive V12-H-Ras are described elsewhere (22). The antisense vector to Ras as well as the transfection of cells with an murine Ku80 cDNA was amplified by reverse transcription-PCR using the adenoviral vector encoding a single-chain antibody fragment Ku80 oligonucleotide primer (5V-ATGGCGTGGTCGGTAAATAAGGC-3Vand against Ras lead to radiosensitization via the inhibition of Ras 5V-CTATATCATGTCCAGTAAATCA-3V). pIND was supplied by Invitrogen action (9, 10). Similarly, blocking Ras activity using pharmacologic (Carlsbad, CA). inhibitors causes increased radiosensitivity (11, 12). Microarray analysis. The total RNA was isolated using a TriReagent (Sigma-Aldrich, St. Louis, MO) and further purified with RNeasy (Qiagen, Valencia, CA) according to the manufacturer’s instructions. Hybridization was done with the cDNA from the oncogenic H-Ras-expressing cells labeled Requests for reprints: Ho Jin You, Department of Pharmacology, School of with Cy5 and those from the control transfected samples labeled with Cy3. Medicine, Chosun University, 375 Seosuk-dong, Gwangju 501-759, Korea. Phone: 82-62- 230-6337; Fax: 82-62-233-3720; E-mail: [email protected]. Scanning was carried out using a GenePix 4000A scanner (Axon Instru- I2005 American Association for Cancer Research. ments, Inc., Foster City, CA), and image acquisition was done using Axon www.aacrjournals.org 6811 Cancer Res 2005; 65: (15). August 1, 2005

GenePix image software. Analysis of the gene expression data was done Immunofluorescence. The paraformaldehyde-fixed cells were incubated using the GeneSpring software (Silicon Genetics, Inc., Redwood City, CA) with rabbit anti-mouse Ku80 antibodies (Santa Cruz Biotechnology). and PathwayAssist (Ariadne Genomics, Rockville, MD). Staining was visualized by incubation with FITC-conjugated anti-rabbit Statistical analysis of microarray. Intensity-dependent normalization secondary antibodies (Vector Laboratories, Burlingame, CA). The immuno- was also applied, where the ratio was reduced to the residual of the Lowess fluorescence images for the Ku80 proteins were obtained using FV300 laser fit of the intensity versus the ratio curve. Statistical analysis was done using microscopy (Olympus, Japan) at an excitation wavelength appropriate for Student’s t test with a P of 0.05 with the additional criteria of the Ras- FITC (488 nm). expressing cells being either 1.5-fold higher or lower than the control DNA end-binding assay. DNA end-binding assays were carried out transfected cells. The genes that met these variables were classified by a according to a method described elsewhere (25). Briefly, the cell lysates were molecular function using the annotations from Silicon Genetics or Ariadne prepared from f107 cells by lysis in a NP40 lysis buffer [50 mmol/L Tris (pH Genomics. 8.0), 0.5% NP40, 150 mmol/L NaCl, 10 mmol/L EDTA, 0.5 mmol/L PMSF, Northern blotting. The total RNA was prepared using TRIzol (Life 1 Ag/mL leupeptin, 1 Ag/mL pepstatin, 1 Ag/mL aprotinin] on ice for 15 Technologies), separated by electrophoresis, transferred to a nitrocellulose minutes. The NaCl concentration was adjusted to 500 mmol/L by adding filter in 20Â SSC, and then baked at 80jC for 2 hours. The filters were NaCl. The lysates were then precleared by adjusting to 6% polyethylene hybridized using a 32P-labeled mouse Ku80 cDNA probe. After hybridiza- glycol 8000 for 10 minutes on ice and microcentrifugation for 15 minutes tion, the same membrane was reprobed with a 32P-labeled h-actin cDNA at 4jC, yielding extracts of f5 mg/mL. The 56-bp dsDNA (5V-GATCAGTGA probe. Hybridization were carried out in 50% formamide, 10% dextran TGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC-3V) sulfate, 1% SDS, 1 mol/L NaCl at 42jC for 16 hours followed by two was end labeled with T4 polynucleotide kinase in the presence of [g-32P] 10-minute washes at room temperature with 2Â SSC and one 30-minute and then incubated with the complementary oligonucleotide. The radio- wash at 65jCin2Â SSC-1% SDS. labeled fragments were electrophoresed through 5% polyacrylamide gels DNA protein kinase assays. The DNA protein kinase (DNA-PK) ‘‘pull- and subsequently purified. For DNA end binding, the protein extract down’’ assays were done as described previously (23). Briefly, the whole-cell (1-2 Ag) was mixed with 0.175 ng of the radiolabeled probe, 1 Ag of the extract was incubated with pre-swollen dsDNA cellulose (Sigma). The DNA covalently closed circular plasmid DNA, in 1Â binding buffer [20 mmol/L cellulose was washed twice in a buffer and the samples were divided into Tris-HCl (pH 7.5),10 mmol/L EDTA, 10% glycerol], 150 mmol/L NaCl, in a three aliquots. [g-32P]ATP (0.5 AL, 300 Ci/mmol) was added, and the kinase volume of 10 AL at room temperature for 10 minutes. The DNA end-binding assays were conducted in the presence of 4 nmol of the peptide (0.2 mmol/L) reactions were separated on 5% polyacrylamide gels in 1Â TGE [50 mmol/L in a total volume of 20 AL for 10 minutes at 30jC. The reactions were Tris, 380 mmol/L glycine (pH 8.5),10 mmol/L EDTA]. Anti-Ku80 antibodies quenched by adding an equal volume of 30% acetic acid and analyzed by were used for the supershifting experiments. spotting onto phosphocellulose paper, washing, and subjecting them to Small interfering RNAs. The sequences of the 21-nucleotide sense and liquid scintillation counting. The amino acid sequences of the modified p53 antisense RNA with a 2-nucleotide overhang composed of TT(DNA) are as

NH2-terminal substrate (wild-type) and mutant p53 peptides were follows: Ku80-small interfering RNA (siRNA)-1, 5V-ACAAAAUCCAGCCAA- EPPLSQEAFALLKK and EPPLSEQAFALLKK, respectively. All the assays GUUCdTdT-3V for the Ku80 gene (nucleotides 285-305); Ku80-siRNA2, were carried out several times using at least three different extract 5V-ACUGAAGUUUCCAAAGAGGdTdT-3V for the Ku80 gene (nucleotides preparations. 905-925); and LacZ siRNA, 5V-CGUACGCGGAAUACUUCGAdTdT-3V for the Cell survival assays. The exponentially growing cells were irradiated with LacZ gene. These siRNAs were prepared by a transcription-based method g-rays using a g-cell irradiator (Clinac 600C; Varian Medical Systems, Palo using a Silencer siRNA construction kit (Ambion, Austin, TX) according to Alto, CA) at a dose 60 cGy/min. The cells (5,000-15,000 per well) were plated in the manufacturer’s instructions. The cells were transfected with the siRNA six-well Falcon plates and incubated for 2 to 3 weeks. After staining with duplexes using Oligofectamine (Invitrogen). methylene blue, colonies of >50 cells were counted under magnification. The surviving fraction was calculated as follows: number of colonies formed / Results (number of cells plated Â plating efficiency). Each point on the survival curve represents the mean surviving fraction from at least three dishes. Oncogenic H-Ras confers ;-ray resistance to NIH3T3 cells. Pulsed-field gel electrophoresis. Cells were irradiated at a dose of 40 Oncogenic H-Ras has been shown to confer radioresistance to the Gy and then cultured at 37jC. Thirty to 180 minutes later, the cells were majority of cell types examined (2–12). To identify the potential harvested. Samples were resuspended at 107 cells/mL in 1.0% low melting oncogenic H-Ras target genes that might be involved in the onco- point agarose and cast into an agarose plug (80 AL). Clamped homogenous genic H-Ras-mediated increased radioresistance, the effect of electric field (CHEF) gel electrophoresis (CHEF-DRII, Bio-Rad, Hercules, CA) oncogenic H-Ras on the g-ray-induced cytotoxicity was initially was used to separate intact and repaired dsDNA (100 V, pulsed 200-1,800 reevaluated in the NIH3T3 cells. Therefore, the dominant-active seconds for 96 hours). The fraction of DNA migrating from the plug into the V12-H-Ras was subcloned into the vector pIND and formed pIND- lane (% DNA extracted) was measured using a UV transilluminator (312 nmol/L) and image analysis using commercially available software. The Ras to control the generation of oncogenic H-Ras within these fraction of the remaining dsDNA breaks (DSB) was determined as cells. Following the transfection and double selection using G418 the integrated density value of the unrepaired DNA in the lane divided by (selection for pIND-Ras) and zeocin (selection for pVg-retinoid X the total DNA in the lane plus the DNA in the well (24). receptor) for 5 weeks, nine clones were isolated and the oncogenic Western blotting. The cells were washed with PBS and lysed at 0jC for H-Ras expression that could be turned on or off was analyzed 30 minutes in a lysis buffer [20 mmol/L HEPES (pH 7.4), 2 mmol/L EGTA, 50 using ponasterone A. Western blot analysis revealed that treating mmol/L glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mmol/L DTT, the NIH3T3 clone 7 with ponasterone A for 24 hours resulted 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 10 Ag/mL leupeptin, 10 in the efficient induction of oncogenic H-Ras expression in a A g/mL aprotinin, 1 mmol/L Na3VO4, 5 mmol/L NaF]. The protein dose- and time-dependent manner (Fig. 2B; data not shown). To concentration was determined using a Bio-Rad dye-binding microassay examine the effect of the oncogenic H-Ras on the g-ray-induced (Bio-Rad). Twenty micrograms of the protein per lane were electrophoresed g on the 10% SDS-polyacrylamide gels. The proteins were blotted onto the cytotoxicity, the cells were subjected to a range of -ray doses. The Hybond enhanced chemiluminescence (ECL) membranes (Amersham fraction of cells that survived the exposure to g-ray doses ranging Biosciences, Piscataway, NJ) and immunoblotting was done using the from 200 to 800 cGy showed that the NIH3T3 clone 7 cells, which anti-Ku80 and a-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA) and H- generated the oncogenic H-Ras as a result of the ponasterone A Ras antibodies (BD Biosciences, San Diego, CA). The blotted proteins were treatment, were better protected from g-ray irradiation than the detected using an ECL detect system (iNtRON Biotechnology, Seoul, Korea). untreated cells (Fig. 1). In the control experiments, both the empty

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Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Oncogenic H-Ras Up-Regulates Expression of Ku80 vector pIND-transfected NIH3T3 (NIH3T3-P) cells treated with or after treatment with 5 Amol/L ponasterone A for 24 hours (Fig. 2B). without ponasterone A and the NIH3T3 clone 7 cells in the Immunohistochemical analysis showed that Ku80 level was absence of ponasterone A showed a similar low level of protection. significantly higher in the ponasterone A–treated NIH3T3 clone 7 Oncogenic H-Ras mediates an increase in Ku80 expression. cells only (Fig. 2C). This shows that Ku80 is up-regulated via the This study used a DNA chip array to compare the genes expressed oncogenic H-Ras. in the presence or absence of ponasterone A. Two different Expression oncogenic H-Ras enhances dsDNA break repair populations of RNA were isolated, one from the NIH3T3 clone 7 in NIH3T3 cells. The DNA-binding activity of Ku80 was examined cells and the other from similar cells generating the oncogenic to determine the functional significance of the oncogenic H-Ras- H-Ras after being treated with 5 Amol/L ponasterone A for 24 mediated increase in Ku80 expression. Because Ku is known to hours. The cDNA probes, prepared from the total RNA isolated have the major DNA end-binding activity in whole-cell extracts from these cells, were labeled for hybridization with a mouse cDNA (24), the total cell extracts were prepared and examined by an array containing the 7.4K genes (GenePlorer TwinChip Mouse-7.4K, electrophoretic mobility shift assay (EMSA) to determine the Digital Genomics, Seoul, Korea). Among the up-regulated genes dsDNA end-binding activity. As shown in Fig. 3A, the DNA-binding detected, the transcript for Ku80 was found to increase in response activity of Ku80 increased as the ponasterone A concentration was to the oncogenic H-Ras induction. Northern blots were carried out increased. The specificity of the Ku80-dependent DNA end-binding using a probe for Ku80 to confirm that these changes in the activity showed that the supershifted protein-DNA complexes hybridization signal on the DNA chip expression array correspond could be observed after adding the anti-Ku80 antibodies to the to changes in the mRNA abundance. As shown in Fig. 2A, the levels DNA-binding reaction. In contrast to the oncogenic H-Ras- of the Ku80 transcript increased significantly 24 hours after the expressing cells, the DNA binding of Ku80 was unchanged in the 5 Amol/L ponasterone A treatment in the NIH3T3 clone 7 cells. The parental NIH3T3 cells after the ponasterone A treatment. level of Ku80 mRNA in the control cell line, NIH3T3-P, remained This study next used pulsed-field gel electrophoresis to examine unchanged after being treated with 5 Amol/L ponasterone A. the ability of the oncogenic H-Ras-generating cells to repair the Experiments using shorter induction times (2 or 6 hours) also failed DSBs in their DNA following g-ray irradiation at 40 Gy. DSB repair to produce any significant increase in the levels of Ku80 mRNA in the ponasterone A–treated, oncogenic H-Ras-generating NIH3T3 (data not shown). clone 7 cells were significantly enhanced (Fig. 3B). After irradiation, Western blot analysis was done to determine if the increase in the percentage of DSBs remaining decreased rapidly up to 1 hour, the Ku80 mRNA level corresponded to an increase in the Ku80 demonstrating the fast repair in the oncogenic H-Ras-expressing protein level. SDS-PAGE was used to separate the whole-cell cells, and after a 90-minute repair period, these cells showed only extracts of the protein from the untreated cells as well as the 12 F 8% DSBs. In contrast, both NIH3T3-P cells treated with protein from two independent cells generating oncogenic H-Ras ponasterone A and NIH3T3 clone 7 cells in the absence of after being treated with 1 or 5 Amol/L ponasterone A. Western blot ponasterone A revealed similar low levels of DSBs repair. After a 90- analysis with Ku80 antibodies revealed that the level of the Ku80 minute repair period, these cells showed 48 F 7% to 52 F 9% DSBs. protein was higher in the NIH3T3 clone 7 cells in response to Previous work has shown that Ku80 plays an important role in ponasterone A. The largest increase in the Ku80 level was observed activating DNA-PK both in vitro and in vivo (25, 26). Therefore, a DNA-PK pull-down peptide assay was used to examine whether the up-regulation of the Ku80 levels induced by the oncogenic H-Ras affected the DNA-PK activity. To address this issue, the NIH3T3 clone 7 cells were treated with 1 and 5 Amol/L ponasterone A and the DNA-PK activity was subsequently measured. As shown in Fig. 3C, the DNA-PK activity was significantly increased 2.2 F 0.31 and 4.2 F 0.57 times in the extracts from cells generating the oncogenic H-Ras after the 1 and 5 Amol/L ponasterone A treatment, respectively, compared with the extracts from the untreated control cells. Overall, the up-regulation of Ku80 might contribute to the enhancement of the DSB repair induced by oncogenic H-Ras in the NIH3T3 cells. Down-regulation of Ku80 suppresses the oncogenic H-Ras- induced dsDNA break repair. To determine if Ku80 is indeed involved in the oncogenic H-Ras-mediated increase in the DSB repair capacity, siRNAs in the form of two independent, nonoverlapping, 21-bp RNA duplexes, which target Ku80, were used in an attempt to inhibit its expression. The oncogenic H-Ras-expressing NIH3T3 cells and parental NIH3T3 cells were transfected with the mock, control siRNA oligonucleotide, or the Ku80-specific siRNA oligonucleotides. The cells were harvested 48 hours after transfection, and their protein expression levels

Figure 1. Radiation survival in oncogenic H-Ras-expressing NIH3T3 cells. were determined. Western blot analysis revealed that the Ku80- Survival rates of the empty vector NIH3T3-P cells and NIH3T3 clone 7 cells 2 specific siRNA oligonucleotide levels had decreased by >80% in weeks after g-ray irradiation. 5, control NIH3T3-P cells; o, control NIH3T3-P terms of their overall Ku80 protein expression level compared cells treated with 5 Amol/L ponasterone A (Pon A); , untreated NIH3T3 clone 7 cells; ., oncogenic H-Ras-expressing cells treated with 5 Amol/L ponasterone A. with the mock- or control siRNA-transfected cells (Fig. 4A). Points, mean; bars, SD. By 96 hours after transfection, the Ku80 protein levels increased www.aacrjournals.org 6813 Cancer Res 2005; 65: (15). August 1, 2005

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Research back to the levels comparable with the mock- and control siRNA- transfected cells (Fig. 5A). In the control experiments, the NIH3T3 transfected cells (data not shown). This suggests that the Ku80 clone 7 cells in the absence of ponasterone A showed a similar low siRNAs are highly specific and efficient in Ku80 gene silencing in level of protection (Fig. 5B). the oncogenic H-Ras-expressing NIH3T3 clone 7 cells. The DSB To further confirm that Ku80 expression is important for repair capacity after Ku80-siRNA transfection was examined to protection against g-ray irradiation in NIH3T3 cells, the murine determine if Ku80 is involved in the oncogenic H-Ras-mediated Ku80 was subcloned into the vector pcDNA3 to form pcDNA3- increase in the DSB repair. The results showed that the oncogenic Ku80. This construct was transfected into the NIH3T3 cells. Nine H-Ras-expressing cells with reduced Ku80 levels had significantly stably transfected cell lines were established after selection using lower levels of DSB repair activity when compared with the mock- G418 for 5 weeks. Western blot analysis revealed several clones or control siRNA-transfected cells (Fig. 4B), suggesting that Ku80 with a high Ku80 expression level (Fig. 5C). To test the functional expression is important for the oncogenic H-Ras-mediated significance of Ku80 expression, the NIH3T3 cloned cells were increase in the DSB repair activity. irradiated with various g-ray doses and the cellular sensitivity was Ku80 plays an essential role in oncogenic H-Ras-mediated determined by a clonogenic survival assay. The cells transfected cell survival against ;-ray irradiation. To determine if this with Ku80 were more resistant to g-ray irradiation compared with increase in Ku80 expression contributes to the g-radiation the empty vector (pcDNA3)–transfected cells (Fig. 5D), which resistance in the oncogenic H-Ras-transfected cells, the oncogenic suggests that Ku80 expression contributes to the viability of H-Ras-expressing cells and parental NIH3T3 cells were transfected NIH3T3 cells in response to g-ray irradiation. with the mock, control siRNA oligonucleotides, or Ku80-specific siRNA oligonucleotides. Twenty-four hours after transfection, the Discussion cells were irradiated with various g-ray doses, and the cellular In the present study, we showed that Ku80, which is a sensitivity was determined by a clonogenic survival assay. The mammalian DNA repair gene, whose gene product has been oncogenic H-Ras-expressing cells treated with the Ku80 siRNA shown to play an important role in repairing DSBs (27, 28), is the oligonucleotides exhibited hypersensitivity to the lethal effects of ultimate downstream target of oncogenic H-Ras in NIH3T3 cells. g-ray irradiation compared with the mock- or control siRNA- Using pulsed-field gel electrophoresis and cell survival assay, we

Figure 2. Oncogenic H-Ras-induced Ku80 mRNA and protein expression. A, empty vector NIH3T3-P cells and NIH3T3 clone 7 cells were treated with or without 5 Amol/L ponasterone A for 24 hours. Northern blots against the total RNA for the indicated proteins in NIH3T3 and NIH3T3 clone 7 cells grown in the absence or presence of 5 Amol/L ponasterone A. B, NIH3T3-P and NIH3T3 clone 7 cells were treated with or without (indicated concentration) ponasterone A for 24 hours. Western blot experiments were done with the antibodies to Ku80 and Ras and an antibody to a-tubulin as a control for an equal loading. C, NIH3T3-P and NIH3T3 clone 7 cells were treated with or without 5 Amol/L ponasterone A for 24 hours. The cells were then immunostained with the polyclonal antibody for Ku80 and observed by confocal microscopy.

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Figure 3. Oncogenic H-Ras-mediated increase DSB repair. A, DNA end-binding activity of Ku80 in oncogenic H-Ras-expressing NIH3T3 cells as evaluated by EMSA. Lane 1, untreated NIH3T3 clone 7 cells; lane 2, NIH3T3 clone 7 cells treated with 1 Amol/L ponasterone A for 24 hours; lane 3, NIH3T3 clone 7 cells treated with 5 Amol/L ponasterone A for 24 hours; lane 4, NIH3T3 clone 7 cells treated with 5 Amol/L ponasterone A for 24 hours + Ku80 antibody; lane 5, control NIH3T3-P cells; lane 6, NIH3T3-P cells treated with 1 Amol/L ponasterone A for 24 hours; lane 7, NIH3T3-P cells treated with 5 Amol/L ponasterone A for 24 hours; lane 8, NIH3T3-P cells treated with 5 Amol/L ponasterone A for 24 hours + Ku80 antibodies. For the supershifts, the specific antibodies to Ku80 were added to the reaction mixture and incubated for 30 minutes before separating the DNA-protein complexes (lanes 4 and 8). B, DSBs remaining in the empty vector NIH3T3-P cells and NIH3T3 clone 7 cells at the indicated times after a 40 Gy g-ray. 5, control NIH3T3-P cells untreated with 5 Amol/L ponasterone A; o, control NIH3T3-P cells treated with 5 Amol/L ponasterone A; , untreated NIH3T3 clone 7 cells; ., oncogenic H-Ras-expressing cells treated with 5 Amol/L ponasterone A. Points, mean; bars, SD. C, DNA-PK activity in the nuclear extracts from cells grown in the absence (None) or presence (indicated concentration) of ponasterone A. Columns, mean; bars, SD.

have shown that the transient transfection of the activated H-Ras- glycoprotein (p185) with a tyrosine kinase activity homologous to expressing cells with Ku80 siRNA causes the cells to reduce the the EGFR, led to the direct regulation of the DNA repair mechanism, oncogenic H-Ras-mediated increase in the DSB repair activity and and the Ras-coupled pathway is important for modulating the DNA become highly sensitive to g-rays compared with the mock- and repair induced by erbB-2 (31). Using the host cell reactivation of the control siRNA-transfected cells. Subsequent studies revealed that reporter gene expression from the UV-damaged plasmid and the the Ku80 expression in NIH3T3 cells leads to increased radio- unscheduled DNA synthesis following the UV treatment of the cells, resistance. we showed previously that activated H-Ras expression in NIH3T3 Recently, several lines of evidence have suggested that activated cells increased the DNA repair activity (22). Ras may be associated with the regulation of the DNA repair To address the question as to what kind of DNA repair protein activity. For example, transformations by an activated Ras of the might to be involved in the oncogenic H-Ras-mediated increase in human epithelial HBL100 cells resulted in less formation of the DNA repair activity, particularly those that might also be cisplatin-induced interstrand cross-links as well as an increase in involved in the radiation resistance, we analyzed the effect of the DNA repair synthesis (29). Similarly, oncogenic Ras-transfected oncogenic H-Ras on gene expression and have shown that Syrian hamster Osaka-Kanazawa cells exhibited an increase oncogenic H-Ras mediates an increase in levels of Ku80 mRNA resistance to cisplatin as well as a decrease in the intracellular and Ku80 protein, which is a key component of the repair platinum binding to DNA (30). In addition, the expression of the apparatus for DSBs in DNA. The exposure of mammalian cells to erbB-2 proto-oncogene, which encodes a 185-kDa transmembrane ionizing radiation induces lesions in the chromosomal DNA, such www.aacrjournals.org 6815 Cancer Res 2005; 65: (15). August 1, 2005

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Research as strand scissions, single-strand breaks, DSBs, and base cross-links specificity, (b) different expression levels of oncogenic H-Ras, and (32). Among the various forms of DNA damage produced by (c) different status of the v-myc oncogene and p53 anti-oncogene, ionizing radiation, DSBs, if not repaired, seem to be responsible for which may affect the DSB repair activity. Thus, this issue still needs most of the radiation-induced cell death in yeast and mammalian to be studied further to assess the changes of DSB repair with cells, because the DSBs disrupt the integrity of the genome (33, 34). oncogenic Ras in different cells. Homologous recombination and nonhomologous end joining are Deficiencies in the DNA-PKcs activity are responsible for the lack the two principal pathways that mediate the repair of DSBs in of appropriate responses to the DSBs observed in the radiosensitive eukaryotic organisms (35, 36). In yeast, homologous recombination mammalian xrs-6 cell line and severe combined immunodeficient is the major mechanism for DSB repair, whereas in mammalian (scid) mouse strain (23, 48, 49). The mutant phenotypes displayed cells the predominant DSB repair system is the nonhomologous by these cell lines and mice indicate that a loss of the DNA-PKcs end joining pathway. One of the main participants in this pathway catalytic activity results in defective DSB rejoining and an inability is the DNA-PK complex, which is composed of a catalytic subunit, to facilitate V(D)J recombination. One of the phenotypic hallmarks DNA-PKcs, and a regulatory subunit, Ku70 (70 kDa) and Ku80 of scid cells is extreme radiosensitivity, indicating the requirement (86 kDa; refs. 37–39). The Ku proteins were originally identified as for DNA-PK activity in responding appropriately to any genome autoantigens in patients with autoimmune disorders (40) and were damage (50). Similarly, the cells deficient in the Ku80 protein are found to bind tightly to the DNA ends in a manner independent of hypersensitive to ionizing radiation and were deficient in V(D)J the structure of the end. Following the generation of DSBs, the recombination, which is a process that requires the specific Ku70-Ku80 complex binds to the free DNA ends and subsequently formation and rejoining of DSBs (51–53). Moreover, when the recruits and activates the DNA-PKcs at the site of DSBs (41–43). In Chinese hamster ovary cells lacking the functional Ku80 were addition to the regulatory function of the Ku80 protein in DNA-PK, transfected with the human chromosomal fragment coding for Ku80 also has independent DNA repair functions, such as ssDNA- Ku80, V(D)J recombination and radiation sensitivity were restored dependent ATPase activity and the binding and repair of broken to normal levels (54). Therefore, the Ku80 proteins perform single-stranded nicks, gaps in the DNA, and a single-stranded to important role(s) in nonhomologous end joining, which is the double-stranded transition in DNA (44). Using EMSA and pulsed- primary mode of DSB repair in mammalian cells, and control of field gel electrophoresis after exposing the cells to ionizing radioresistance. To determine if the oncogenic H-Ras-mediated radiation, it was found that the activated H-Ras expression in increase in Ku80 expression contributes to radioresistance, the NIH3T3 cells increases the DNA-binding activity of Ku80 and oncogenic H-Ras-expressing cells were transfected with the Ku80- increases the DSB repair activity (Fig. 3). This suggests that Ku80 is specific siRNA, which targets Ku80 and inhibits its expression. It a downstream target protein of the oncogenic H-Ras, which at was found that the transfection of cells with the Ku80-specific higher levels might contribute to g-radiation resistance in siRNA resulted in a decrease in the DSB repair activity following oncogenic H-Ras-transfected cells. However, several studies have g-ray irradiation compared with the mock- and control siRNA- suggested that Ras expression does not affect the DSB repair transfected cells (Fig. 4). In addition, it was also found that the (45–47). This discrepancy with our results and with the findings of Ku80-targeted siRNA oligonucleotides caused the oncogenic previous studies could possibly be because of (a) cell type H-Ras-expressing NIH3T3 cells to be highly sensitive to g-ray

Figure 4. Requirement of Ku80 for the oncogenic H-Ras-mediated increase in the DSBs repair in NIH3T3 cells. A, mock-, control siRNA-, or Ku80 siRNA-transfected cells were incubated with (+) or without (À)5Amol/L ponasterone A. Forty-eight hours after transfection, the total cell extracts were analyzed by Western blotting using the anti-Ku80 and anti-Ras antibodies. For the control experiment to have an equal loading, the membranes were reprobed with the anti-a-tubulin antibody. B, mock-, control siRNA-, or Ku80 siRNA-treated NIH3T3 clone 7 cells were incubated with or without 5 Amol/L ponasterone A (Pon A) for 24 hours. DSBs remaining in NIH3T3 clone 7 cells at the indicated times after irradiation with the 40 Gy g-ray. Columns, mean; bars, SD.

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Figure 5. Ku80 contributes to the oncogenic H-Ras-induced radioresistance. Reduced Ku80 expression results in g-ray irradiation sensitivity in oncogenic H-Ras-expressing NIH3T3 cells. The mock-, control siRNA-, or Ku80 siRNA-transfected cells were incubated with (A) or without (B)5Amol/L ponasterone A for 24 hours. Subsequently, the cells were then treated with the indicated doses of g-ray irradiation, and the cell viability was determined by a clonogenic survival assay. Points, mean; bars, SD. Detection by Western blotting of Ku80 expression in three individual NIH3T3 + Ku80 clones (Ku-clone 1, Ku-clone 3, and Ku-clone 5) and one NIH3T3 + pcDNA3 clone (pcDNA3). C, three Ku80-expressing NIH3T3 clones (Ku-clone 1, Ku-clone 3, and Ku-clone 5) and one control clone (pcDNA3) were irradiated with various g-ray doses, and the cell viability was determined by a clonogenic survival assay (D). Points, mean; bars, SD. irradiation and that Ku80 expression caused the NIH3T3 cells to that EGRF-Ras-PI3K pathway might play an important role in increase the viability following g-ray irradiation (Fig. 5). Therefore, mediating radiation resistance (18, 20). The Ku80 promoter oncogenic H-Ras-mediated up-regulation of Ku80 is involved in contains Sp1-binding sites. Although little is known about what g-radiation resistance when oncogenic H-Ras is expressed. transcription factors actually participate in the Ku80 regulation, a Recent research has suggested a linkage between Ras-mediated prior study suggests that a Sp1 transcription factor contributes to radioresistance and PI3K. For example, expression of active PI3K in the Ku80 expression (55). Sp1 is important for the expression of cells with wild-type Ras results in enhanced radiation resistance, many cellular genes, particularly housekeeping genes. However, Sp1 and this radioresistance could be inhibited by a PI3K inhibitor (17). sites have more recently been found to mediate transcription in In addition, Grana et al. (19) have shown that the activated Ras- response to diverse stimuli, including oncogenes, such as Ras and transformed RIE-1 epithelial cells exhibit resistance to radiation, growth factors and cytokines (56). Regulation of Sp1-dependent and blocking PI3K activity with the inhibitor LY294002 sensitizes transcription may be conveyed by changes in DNA-binding activity, RIE-1 epithelial cells to radiation. Similarly, it has been suggested by association with other transcription factors, by changes in Sp1 www.aacrjournals.org 6817 Cancer Res 2005; 65: (15). August 1, 2005

Downloaded from cancerres.aacrjournals.org on September 25, 2021. © 2005 American Association for Cancer Research. Cancer Research abundance, or in transactivation activity owing to biochemical the action of the Ras and the other members of the Ras pathway modification, such as phosphorylation (56). Overexpression of Ras were strictly regulated during the cell cycle and under different has been shown to mediate Sp1-dependent transcriptional growth conditions (60). However, in a tumor cell, the oncogenic activation, involving PI3K as important intermediary signaling activation of ras is a consequence of point mutations that either molecules (57). PI3K contributes to the phosphorylation of Sp1, impair the GTPase activity or enhance the GTP-binding affinity, and Sp1 is involved in the regulation of gene expression by the resulting in a highly active proliferative signal (61). Furthermore, PI3K/Akt pathway (58, 59). Thus, we speculate that this might ras mutations are found in a variety of human cancers with the occur via phosphorylation of Sp1 leading to its increased binding to highest incidence being observed in f30% of all human tumors and transactivation of the Ku80 promoter. Further experiments are (62). Therefore, the elevated Ku80 levels induced by Ras clearly needed to evaluate the effect of Ras-PI3K pathway on the activation might confer cancer cell resistance to ionizing Ku80 expression in the radioresistance of oncogenic Ras-expressing radiation. These findings, from a pathway involving oncogenic NIH3T3 cells. H-Ras and Ku80, might be relevant to the development of new In summary, the sensitivity of cells to death by ionizing therapeutic approaches, involving the administration of ionizing radiation is a critical determinant of the probability of a cure in radiation together with a Ku80 inhibitor (63, 64). patients receiving radiotherapy for cancer. One factor known to increase the survival of tumor cells after radiation is in the presence of activated oncogenes. Therefore, there is considerable Acknowledgments interest in determining which genes mediate the altered Received 11/12/2004; revised 3/22/2005; accepted 5/22/2005. radioresistance in tumor cells. This study found that Ku80 is Grant support: National Cancer Control R&D Program 2003, Ministry of Health induced by oncogenic H-Ras expression. The results suggest that and Welfare, Republic of Korea, and Nuclear Research and Development Program from Ku80 contributes to the oncogenic H-Ras-mediated increase in the Ministry of Science and Technology of Korea. The costs of publication of this article were defrayed in part by the payment of page the capacity of NIH3T3 cells to repair DSBs and to afford charges. This article must therefore be hereby marked advertisement in accordance protection against ionizing radiation. Under normal conditions, with 18 U.S.C. Section 1734 solely to indicate this fact.

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