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[Cancer Research 65, 6811-6819, August 1, 2005]
© 2005 American Association for Cancer Research

Cell and Tumor Biology

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

In-Youb Chang1,3, Cha-Kyung Youn1,2, Hong-Beum Kim1,2, Mi-Hwa Kim1,2, Hyun-Ju Cho1,2, Young Yoon1,2, Yun-Sil Lee4, Myung-Hee Chung5 and Ho Jin You1,2

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

Requests for reprints: Ho Jin You, Department of Pharmacology, School of Medicine, Chosun University, 375 Seosuk-dong, Gwangju 501-759, Korea. Phone: 82-62-230-6337; Fax: 82-62-233-3720; E-mail: hjyou{at}.chosun.ac.kr.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
The Ras activation contributes to radioresistance, but the mechanism is unclear. This article shows that the expression of the dominant-positive H-Ras increased the Ku80 level, which is one of the key enzymes involved in repairing dsDNA breaks (DSB). After exposing the cells to ionizing radiation and analyzing them using an electrophoretic mobility shift assay and pulsed-field gel electrophoresis, it was found that activated H-Ras expression in NIH3T3 cells increases the DNA-binding activity of Ku80 and increases the DSB repair activity. Ku80 small interfering RNA expression was shown to reduce the oncogenic H-Ras-mediated increase in the DSBs and suppress the oncogenic H-Ras-mediated resistance of the cells to {gamma}-ray irradiation, whereas Ku80 overexpression in the NIH3T3 cells significantly increased the radioresistance. These results suggest that the Ku80 expression induced by oncogenic H-Ras seems to play an important role in protecting cells against {gamma}-ray irradiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Radiation therapy is used as a curative treatment for cancer. However, the radiation responses of tumors differ according to histology, doubling time, repair capacity, and other factors (1). Therefore, understanding the properties of tumor cells that increase or decrease their responsiveness to radiation is the key to improving radiation therapy. Previous studies have shown that oncogenic mutations in H-Ras, which frequently occur in many types of cancer, could contribute to an increased radiation survival rate in transformed cells. It was initially shown that the resistance of NIH3T3 cells to radiation could be enhanced by the expression of an activated H-Ras gene (2). This was later confirmed independently by several other groups using rodent cells, including rat embryo fibroblasts and rat rhabdomyosarcomas (3–6), and human tumor cells, including an EJ Ras-transformed bladder carcinoma, DLD-1 colon carcinoma, and HT1080 fibrosarcoma (7, 8). In agreement with such Ras-mediated radioresistance, several groups have reported that the blocking of Ras activity leads to an increase in radiosensitivity. For example, the expression of an antisense vector to Ras as well as the transfection of cells with an adenoviral vector encoding a single-chain antibody fragment against Ras lead to radiosensitization via the inhibition of Ras action (9, 10). Similarly, blocking Ras activity using pharmacologic inhibitors causes increased radiosensitivity (11, 12).

The major oncogenic signal from Ras uses the serine-threonine kinase Raf as the effector (13). The activated Ras complexes with and promotes Raf phosphorylation. Active Raf is the first in a cascade of other kinases, including mitogen-activated protein kinase (MAPK)/extracellular signal-regulated kinase (ERK; MEK), which phosphorylates and activates the MAPK, ERK. Although the Ras/Raf/MEK/ERK cascade plays a key role in proliferative signaling, there is also evidence suggesting that Ras activation can activate MEK kinase (MEKK) and phosphatidylinositol 3-OH kinase (PI3K; ref. 14). MEKK is a critical component of the stress-associated protein kinase, including the c-Jun NH2-terminal kinase (stress-activated protein kinase; ref. 15). PI3K activation leads to the production of phosphorylated phosphatidylinositides with regulatory functions on the kinases, phosphoinositide-dependent protein kinase and Akt/protein kinase B, which is involved in the survival response (16). Recently, several groups have focused on the downstream 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 radioresistance (17–21). Therefore, there is considerable evidence suggesting a causal relationship between the Ras proteins and radiation resistance. However, the molecular mechanisms underlying this effect are unclear. In the present study, we sought to determine the downstream target genes regulated by activated H-Ras, particularly those that might also be involved in the radiation resistance, using ponasterone A regulatable oncogenic H-Ras-expressing NIH3T3 cells. The results show that the oncogenic H-Ras-inducible protein Ku80 can function directly as a survival effector on the oncogenic H-Ras-expressing cells exposed to {gamma}-ray irradiation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell culture and DNA constructs. The NIH3T3 cells (American Type Culture Collection, Manassas, VA) were maintained in EMEM supplemented with 10% fetal bovine serum, 100 units penicillin/mL, and 100 µg streptomycin/mL (Invitrogen, Carlsbad, CA). The cells were maintained in 5% CO2-95% air at 37°C in a humidified incubator. The constructs of the dominant-positive V12-H-Ras are described elsewhere (22). The murine Ku80 cDNA was amplified by reverse transcription-PCR using the Ku80 oligonucleotide primer (5'-ATGGCGTGGTCGGTAAATAAGGC-3' and 5'-CTATATCATGTCCAGTAAATCA-3'). pIND was supplied by Invitrogen (Carlsbad, CA).

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 with Cy5 and those from the control transfected samples labeled with Cy3. Scanning was carried out using a GenePix 4000A scanner (Axon Instruments, Inc., Foster City, CA), and image acquisition was done using Axon GenePix image software. Analysis of the gene expression data was done using the GeneSpring software (Silicon Genetics, Inc., Redwood City, CA) and PathwayAssist (Ariadne Genomics, Rockville, MD).

Statistical analysis of microarray. Intensity-dependent normalization was also applied, where the ratio was reduced to the residual of the Lowess fit of the intensity versus the ratio curve. Statistical analysis was done using Student's t test with a P of 0.05 with the additional criteria of the Ras-expressing cells being either 1.5-fold higher or lower than the control transfected cells. The genes that met these variables were classified by a molecular function using the annotations from Silicon Genetics or Ariadne Genomics.

Northern blotting. The total RNA was prepared using TRIzol (Life Technologies), separated by electrophoresis, transferred to a nitrocellulose filter in 20x SSC, and then baked at 80°C for 2 hours. The filters were hybridized using a 32P-labeled mouse Ku80 cDNA probe. After hybridization, the same membrane was reprobed with a 32P-labeled ß-actin cDNA probe. Hybridization were carried out in 50% formamide, 10% dextran sulfate, 1% SDS, 1 mol/L NaCl at 42°C for 16 hours followed by two 10-minute washes at room temperature with 2x SSC and one 30-minute wash at 65°C in 2x SSC-1% SDS.

DNA protein kinase assays. The DNA protein kinase (DNA-PK) "pull-down" assays were done as described previously (23). Briefly, the whole-cell extract was incubated with pre-swollen dsDNA cellulose (Sigma). The DNA cellulose was washed twice in a buffer and the samples were divided into three aliquots. [{gamma}-32P]ATP (0.5 µL, 300 Ci/mmol) was added, and the kinase assays were conducted in the presence of 4 nmol of the peptide (0.2 mmol/L) in a total volume of 20 µL for 10 minutes at 30°C. The reactions were quenched by adding an equal volume of 30% acetic acid and analyzed by spotting onto phosphocellulose paper, washing, and subjecting them to liquid scintillation counting. The amino acid sequences of the modified p53 NH2-terminal substrate (wild-type) and mutant p53 peptides were EPPLSQEAFALLKK and EPPLSEQAFALLKK, respectively. All the assays were carried out several times using at least three different extract preparations.

Cell survival assays. The exponentially growing cells were irradiated with {gamma}-rays using a {gamma}-cell irradiator (Clinac 600C; Varian Medical Systems, Palo Alto, CA) at a dose 60 cGy/min. The cells (5,000-15,000 per well) were plated in six-well Falcon plates and incubated for 2 to 3 weeks. After staining with methylene blue, colonies of >50 cells were counted under magnification. The surviving fraction was calculated as follows: number of colonies formed / (number of cells plated x plating efficiency). Each point on the survival curve represents the mean surviving fraction from at least three dishes.

Pulsed-field gel electrophoresis. Cells were irradiated at a dose of 40 Gy and then cultured at 37°C. Thirty to 180 minutes later, the cells were harvested. Samples were resuspended at 107 cells/mL in 1.0% low melting point agarose and cast into an agarose plug (80 µL). Clamped homogenous electric field (CHEF) gel electrophoresis (CHEF-DRII, Bio-Rad, Hercules, CA) was used to separate intact and repaired dsDNA (100 V, pulsed 200-1,800 seconds for 96 hours). The fraction of DNA migrating from the plug into the lane (% DNA extracted) was measured using a UV transilluminator (312 nmol/L) and image analysis using commercially available software. The fraction of the remaining dsDNA breaks (DSB) was determined as the integrated density value of the unrepaired DNA in the lane divided by the total DNA in the lane plus the DNA in the well (24).

Western blotting. The cells were washed with PBS and lysed at 0°C for 30 minutes in a lysis buffer [20 mmol/L HEPES (pH 7.4), 2 mmol/L EGTA, 50 mmol/L glycerol phosphate, 1% Triton X-100, 10% glycerol, 1 mmol/L DTT, 1 mmol/L phenylmethylsulfonyl fluoride (PMSF), 10 µg/mL leupeptin, 10 µg/mL aprotinin, 1 mmol/L Na3VO4, 5 mmol/L NaF]. The protein concentration was determined using a Bio-Rad dye-binding microassay (Bio-Rad). Twenty micrograms of the protein per lane were electrophoresed on the 10% SDS-polyacrylamide gels. The proteins were blotted onto the Hybond enhanced chemiluminescence (ECL) membranes (Amersham Biosciences, Piscataway, NJ) and immunoblotting was done using the anti-Ku80 and {alpha}-tubulin (Santa Cruz Biotechnology, Santa Cruz, CA) and H-Ras antibodies (BD Biosciences, San Diego, CA). The blotted proteins were detected using an ECL detect system (iNtRON Biotechnology, Seoul, Korea).

Immunofluorescence. The paraformaldehyde-fixed cells were incubated with rabbit anti-mouse Ku80 antibodies (Santa Cruz Biotechnology). Staining was visualized by incubation with FITC-conjugated anti-rabbit secondary antibodies (Vector Laboratories, Burlingame, CA). The immunofluorescence images for the Ku80 proteins were obtained using FV300 laser microscopy (Olympus, Japan) at an excitation wavelength appropriate for FITC (488 nm).

DNA end-binding assay. DNA end-binding assays were carried out according to a method described elsewhere (25). Briefly, the cell lysates were prepared from ~107 cells by lysis in a NP40 lysis buffer [50 mmol/L Tris (pH 8.0), 0.5% NP40, 150 mmol/L NaCl, 10 mmol/L EDTA, 0.5 mmol/L PMSF, 1 µg/mL leupeptin, 1 µg/mL pepstatin, 1 µg/mL aprotinin] on ice for 15 minutes. The NaCl concentration was adjusted to 500 mmol/L by adding NaCl. The lysates were then precleared by adjusting to 6% polyethylene glycol 8000 for 10 minutes on ice and microcentrifugation for 15 minutes at 4°C, yielding extracts of ~5 mg/mL. The 56-bp dsDNA (5'-GATCAGTGATGGAGTTGGCCACTCCCTCTCTGCGCGCTCGCTCGCTCACTGAGGCC-3') was end labeled with T4 polynucleotide kinase in the presence of [{gamma}-32P] and then incubated with the complementary oligonucleotide. The radiolabeled fragments were electrophoresed through 5% polyacrylamide gels and subsequently purified. For DNA end binding, the protein extract (1-2 µg) was mixed with 0.175 ng of the radiolabeled probe, 1 µg of the covalently closed circular plasmid DNA, in 1x binding buffer [20 mmol/L Tris-HCl (pH 7.5),10 mmol/L EDTA, 10% glycerol], 150 mmol/L NaCl, in a volume of 10 µL at room temperature for 10 minutes. The DNA end-binding reactions were separated on 5% polyacrylamide gels in 1x TGE [50 mmol/L Tris, 380 mmol/L glycine (pH 8.5),10 mmol/L EDTA]. Anti-Ku80 antibodies were used for the supershifting experiments.

Small interfering RNAs. The sequences of the 21-nucleotide sense and antisense RNA with a 2-nucleotide overhang composed of TT(DNA) are as follows: Ku80-small interfering RNA (siRNA)-1, 5'-ACAAAAUCCAGCCAAGUUCdTdT-3' for the Ku80 gene (nucleotides 285-305); Ku80-siRNA2, 5'-ACUGAAGUUUCCAAAGAGGdTdT-3' for the Ku80 gene (nucleotides 905-925); and LacZ siRNA, 5'-CGUACGCGGAAUACUUCGAdTdT-3' for the LacZ gene. These siRNAs were prepared by a transcription-based method using a Silencer siRNA construction kit (Ambion, Austin, TX) according to the manufacturer's instructions. The cells were transfected with the siRNA duplexes using Oligofectamine (Invitrogen).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Oncogenic H-Ras confers {gamma}-ray resistance to NIH3T3 cells. Oncogenic H-Ras has been shown to confer radioresistance to the majority of cell types examined (2–12). To identify the potential oncogenic H-Ras target genes that might be involved in the oncogenic H-Ras-mediated increased radioresistance, the effect of oncogenic H-Ras on the {gamma}-ray-induced cytotoxicity was initially reevaluated in the NIH3T3 cells. Therefore, the dominant-active V12-H-Ras was subcloned into the vector pIND and formed pIND-Ras to control the generation of oncogenic H-Ras within these cells. Following the transfection and double selection using G418 (selection for pIND-Ras) and zeocin (selection for pVg-retinoid X receptor) for 5 weeks, nine clones were isolated and the oncogenic H-Ras expression that could be turned on or off was analyzed using ponasterone A. Western blot analysis revealed that treating the NIH3T3 clone 7 with ponasterone A for 24 hours resulted in the efficient induction of oncogenic H-Ras expression in a dose- and time-dependent manner (Fig. 2B; data not shown). To examine the effect of the oncogenic H-Ras on the {gamma}-ray-induced cytotoxicity, the cells were subjected to a range of {gamma}-ray doses. The fraction of cells that survived the exposure to {gamma}-ray doses ranging from 200 to 800 cGy showed that the NIH3T3 clone 7 cells, which generated the oncogenic H-Ras as a result of the ponasterone A treatment, were better protected from {gamma}-ray irradiation than the untreated cells (Fig. 1). In the control experiments, both the empty vector pIND-transfected NIH3T3 (NIH3T3-P) cells treated with or without ponasterone A and the NIH3T3 clone 7 cells in the absence of ponasterone A showed a similar low level of protection.



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  		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 µmol/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 µmol/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 {alpha}-tubulin as a control for an equal loading. C, NIH3T3-P and NIH3T3 clone 7 cells were treated with or without 5 µmol/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 1. Radiation survival in oncogenic H-Ras-expressing NIH3T3 cells. Survival rates of the empty vector NIH3T3-P cells and NIH3T3 clone 7 cells 2 weeks after {gamma}-ray irradiation. {square}, control NIH3T3-P cells; {circ}, control NIH3T3-P cells treated with 5 µmol/L ponasterone A (Pon A); {04-4065fx01g}, untreated NIH3T3 clone 7 cells; {bullet}, oncogenic H-Ras-expressing cells treated with 5 µmol/L ponasterone A. Points, mean; bars, SD.

 
Oncogenic H-Ras mediates an increase in Ku80 expression. This study used a DNA chip array to compare the genes expressed in the presence or absence of ponasterone A. Two different populations of RNA were isolated, one from the NIH3T3 clone 7 cells and the other from similar cells generating the oncogenic H-Ras after being treated with 5 µmol/L ponasterone A for 24 hours. The cDNA probes, prepared from the total RNA isolated from these cells, were labeled for hybridization with a mouse cDNA array containing the 7.4K genes (GenePlorer TwinChip Mouse-7.4K, Digital Genomics, Seoul, Korea). Among the up-regulated genes detected, the transcript for Ku80 was found to increase in response to the oncogenic H-Ras induction. Northern blots were carried out using a probe for Ku80 to confirm that these changes in the hybridization signal on the DNA chip expression array correspond to changes in the mRNA abundance. As shown in Fig. 2A, the levels of the Ku80 transcript increased significantly 24 hours after the 5 µmol/L ponasterone A treatment in the NIH3T3 clone 7 cells. The level of Ku80 mRNA in the control cell line, NIH3T3-P, remained unchanged after being treated with 5 µmol/L ponasterone A. Experiments using shorter induction times (2 or 6 hours) also failed to produce any significant increase in the levels of Ku80 mRNA (data not shown).

Western blot analysis was done to determine if the increase in the Ku80 mRNA level corresponded to an increase in the Ku80 protein level. SDS-PAGE was used to separate the whole-cell extracts of the protein from the untreated cells as well as the protein from two independent cells generating oncogenic H-Ras after being treated with 1 or 5 µmol/L ponasterone A. Western blot analysis with Ku80 antibodies revealed that the level of the Ku80 protein was higher in the NIH3T3 clone 7 cells in response to ponasterone A. The largest increase in the Ku80 level was observed after treatment with 5 µmol/L ponasterone A for 24 hours (Fig. 2B). Immunohistochemical analysis showed that Ku80 level was significantly higher in the ponasterone A–treated NIH3T3 clone 7 cells only (Fig. 2C). This shows that Ku80 is up-regulated via the oncogenic H-Ras.

Expression oncogenic H-Ras enhances dsDNA break repair in NIH3T3 cells. The DNA-binding activity of Ku80 was examined to determine the functional significance of the oncogenic H-Ras-mediated increase in Ku80 expression. Because Ku is known to have the major DNA end-binding activity in whole-cell extracts (24), the total cell extracts were prepared and examined by an electrophoretic mobility shift assay (EMSA) to determine the dsDNA end-binding activity. As shown in Fig. 3A, the DNA-binding activity of Ku80 increased as the ponasterone A concentration was increased. The specificity of the Ku80-dependent DNA end-binding activity showed that the supershifted protein-DNA complexes could be observed after adding the anti-Ku80 antibodies to the DNA-binding reaction. In contrast to the oncogenic H-Ras-expressing cells, the DNA binding of Ku80 was unchanged in the parental NIH3T3 cells after the ponasterone A treatment.



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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 µmol/L ponasterone A for 24 hours; lane 3, NIH3T3 clone 7 cells treated with 5 µmol/L ponasterone A for 24 hours; lane 4, NIH3T3 clone 7 cells treated with 5 µmol/L ponasterone A for 24 hours + Ku80 antibody; lane 5, control NIH3T3-P cells; lane 6, NIH3T3-P cells treated with 1 µmol/L ponasterone A for 24 hours; lane 7, NIH3T3-P cells treated with 5 µmol/L ponasterone A for 24 hours; lane 8, NIH3T3-P cells treated with 5 µmol/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 {gamma}-ray. {square}, control NIH3T3-P cells untreated with 5 µmol/L ponasterone A; {circ}, control NIH3T3-P cells treated with 5 µmol/L ponasterone A; {04-4065fx01g}, untreated NIH3T3 clone 7 cells; {bullet}, oncogenic H-Ras-expressing cells treated with 5 µmol/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.

 
This study next used pulsed-field gel electrophoresis to examine the ability of the oncogenic H-Ras-generating cells to repair the DSBs in their DNA following {gamma}-ray irradiation at 40 Gy. DSB repair in the ponasterone A–treated, oncogenic H-Ras-generating NIH3T3 clone 7 cells were significantly enhanced (Fig. 3B). After irradiation, the percentage of DSBs remaining decreased rapidly up to 1 hour, demonstrating the fast repair in the oncogenic H-Ras-expressing cells, and after a 90-minute repair period, these cells showed only 12 ± 8% DSBs. In contrast, both NIH3T3-P cells treated with ponasterone A and NIH3T3 clone 7 cells in the absence of ponasterone A revealed similar low levels of DSBs repair. After a 90-minute repair period, these cells showed 48 ± 7% to 52 ± 9% DSBs. Previous work has shown that Ku80 plays an important role in 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 µmol/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 ± 0.31 and 4.2 ± 0.57 times in the extracts from cells generating the oncogenic H-Ras after the 1 and 5 µmol/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 were determined. Western blot analysis revealed that the Ku80-specific siRNA oligonucleotide levels had decreased by >80% in terms of their overall Ku80 protein expression level compared with the mock- or control siRNA-transfected cells (Fig. 4A). By 96 hours after transfection, the Ku80 protein levels increased back to the levels comparable with the mock- and control siRNA-transfected cells (data not shown). This suggests that the Ku80 siRNAs are highly specific and efficient in Ku80 gene silencing in the oncogenic H-Ras-expressing NIH3T3 clone 7 cells. The DSB repair capacity after Ku80-siRNA transfection was examined to determine if Ku80 is involved in the oncogenic H-Ras-mediated increase in the DSB repair. The results showed that the oncogenic H-Ras-expressing cells with reduced Ku80 levels had significantly lower levels of DSB repair activity when compared with the mock- or control siRNA-transfected cells (Fig. 4B), suggesting that Ku80 expression is important for the oncogenic H-Ras-mediated increase in the DSB repair activity.



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  		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 (–) 5 µmol/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-{alpha}-tubulin antibody. B, mock-, control siRNA-, or Ku80 siRNA-treated NIH3T3 clone 7 cells were incubated with or without 5 µmol/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 {gamma}-ray. Columns, mean; bars, SD.

 
Ku80 plays an essential role in oncogenic H-Ras-mediated cell survival against {gamma}-ray irradiation. To determine if this increase in Ku80 expression contributes to the {gamma}-radiation resistance in the oncogenic H-Ras-transfected cells, the oncogenic H-Ras-expressing cells and parental NIH3T3 cells were transfected with the mock, control siRNA oligonucleotides, or Ku80-specific siRNA oligonucleotides. Twenty-four hours after transfection, the cells were irradiated with various {gamma}-ray doses, and the cellular sensitivity was determined by a clonogenic survival assay. The oncogenic H-Ras-expressing cells treated with the Ku80 siRNA oligonucleotides exhibited hypersensitivity to the lethal effects of {gamma}-ray irradiation compared with the mock- or control siRNA-transfected cells (Fig. 5A). In the control experiments, the NIH3T3 clone 7 cells in the absence of ponasterone A showed a similar low level of protection (Fig. 5B).



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  		Figure 5. Ku80 contributes to the oncogenic H-Ras-induced radioresistance. Reduced Ku80 expression results in {gamma}-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) 5 µmol/L ponasterone A for 24 hours. Subsequently, the cells were then treated with the indicated doses of {gamma}-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 {gamma}-ray doses, and the cell viability was determined by a clonogenic survival assay (D). Points, mean; bars, SD.

 
To further confirm that Ku80 expression is important for protection against {gamma}-ray irradiation in NIH3T3 cells, the murine Ku80 was subcloned into the vector pcDNA3 to form pcDNA3-Ku80. This construct was transfected into the NIH3T3 cells. Nine stably transfected cell lines were established after selection using G418 for 5 weeks. Western blot analysis revealed several clones with a high Ku80 expression level (Fig. 5C). To test the functional significance of Ku80 expression, the NIH3T3 cloned cells were irradiated with various {gamma}-ray doses and the cellular sensitivity was determined by a clonogenic survival assay. The cells transfected with Ku80 were more resistant to {gamma}-ray irradiation compared with the empty vector (pcDNA3)–transfected cells (Fig. 5D), which suggests that Ku80 expression contributes to the viability of NIH3T3 cells in response to {gamma}-ray irradiation.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we showed that Ku80, which is a mammalian DNA repair gene, whose gene product has been shown to play an important role in repairing DSBs (27, 28), is the ultimate downstream target of oncogenic H-Ras in NIH3T3 cells. Using pulsed-field gel electrophoresis and cell survival assay, we have shown that the transient transfection of the activated H-Ras-expressing cells with Ku80 siRNA causes the cells to reduce the oncogenic H-Ras-mediated increase in the DSB repair activity and become highly sensitive to {gamma}-rays compared with the mock- and control siRNA-transfected cells. Subsequent studies revealed that the Ku80 expression in NIH3T3 cells leads to increased radioresistance.

Recently, several lines of evidence have suggested that activated Ras may be associated with the regulation of the DNA repair activity. For example, transformations by an activated Ras of the human epithelial HBL100 cells resulted in less formation of cisplatin-induced interstrand cross-links as well as an increase in the DNA repair synthesis (29). Similarly, oncogenic Ras-transfected Syrian hamster Osaka-Kanazawa cells exhibited an increase resistance to cisplatin as well as a decrease in the intracellular platinum binding to DNA (30). In addition, the expression of the erbB-2 proto-oncogene, which encodes a 185-kDa transmembrane glycoprotein (p185) with a tyrosine kinase activity homologous to the EGFR, led to the direct regulation of the DNA repair mechanism, and the Ras-coupled pathway is important for modulating the DNA repair induced by erbB-2 (31). Using the host cell reactivation of the reporter gene expression from the UV-damaged plasmid and the unscheduled DNA synthesis following the UV treatment of the cells, we showed previously that activated H-Ras expression in NIH3T3 cells increased the DNA repair activity (22).

To address the question as to what kind of DNA repair protein might to be involved in the oncogenic H-Ras-mediated increase in the DNA repair activity, particularly those that might also be involved in the radiation resistance, we analyzed the effect of oncogenic H-Ras on gene expression and have shown that oncogenic H-Ras mediates an increase in levels of Ku80 mRNA and Ku80 protein, which is a key component of the repair apparatus for DSBs in DNA. The exposure of mammalian cells to ionizing radiation induces lesions in the chromosomal DNA, such as strand scissions, single-strand breaks, DSBs, and base cross-links (32). Among the various forms of DNA damage produced by ionizing radiation, DSBs, if not repaired, seem to be responsible for most of the radiation-induced cell death in yeast and mammalian cells, because the DSBs disrupt the integrity of the genome (33, 34). Homologous recombination and nonhomologous end joining are the two principal pathways that mediate the repair of DSBs in eukaryotic organisms (35, 36). In yeast, homologous recombination is the major mechanism for DSB repair, whereas in mammalian cells the predominant DSB repair system is the nonhomologous end joining pathway. One of the main participants in this pathway is the DNA-PK complex, which is composed of a catalytic subunit, DNA-PKcs, and a regulatory subunit, Ku70 (70 kDa) and Ku80 (86 kDa; refs. 37–39). The Ku proteins were originally identified as autoantigens in patients with autoimmune disorders (40) and were found to bind tightly to the DNA ends in a manner independent of the structure of the end. Following the generation of DSBs, the Ku70-Ku80 complex binds to the free DNA ends and subsequently recruits and activates the DNA-PKcs at the site of DSBs (41–43). In addition to the regulatory function of the Ku80 protein in DNA-PK, Ku80 also has independent DNA repair functions, such as ssDNA-dependent ATPase activity and the binding and repair of broken single-stranded nicks, gaps in the DNA, and a single-stranded to double-stranded transition in DNA (44). Using EMSA and pulsed-field gel electrophoresis after exposing the cells to ionizing radiation, it was found that the activated H-Ras expression in NIH3T3 cells increases the DNA-binding activity of Ku80 and increases the DSB repair activity (Fig. 3). This suggests that Ku80 is a downstream target protein of the oncogenic H-Ras, which at higher levels might contribute to {gamma}-radiation resistance in oncogenic H-Ras-transfected cells. However, several studies have suggested that Ras expression does not affect the DSB repair (45–47). This discrepancy with our results and with the findings of previous studies could possibly be because of (a) cell type specificity, (b) different expression levels of oncogenic H-Ras, and (c) different status of the v-myc oncogene and p53 anti-oncogene, which may affect the DSB repair activity. Thus, this issue still needs to be studied further to assess the changes of DSB repair with oncogenic Ras in different cells.

Deficiencies in the DNA-PKcs activity are responsible for the lack of appropriate responses to the DSBs observed in the radiosensitive mammalian xrs-6 cell line and severe combined immunodeficient (scid) mouse strain (23, 48, 49). The mutant phenotypes displayed by these cell lines and mice indicate that a loss of the DNA-PKcs catalytic activity results in defective DSB rejoining and an inability to facilitate V(D)J recombination. One of the phenotypic hallmarks of scid cells is extreme radiosensitivity, indicating the requirement for DNA-PK activity in responding appropriately to any genome damage (50). Similarly, the cells deficient in the Ku80 protein are hypersensitive to ionizing radiation and were deficient in V(D)J recombination, which is a process that requires the specific formation and rejoining of DSBs (51–53). Moreover, when the Chinese hamster ovary cells lacking the functional Ku80 were transfected with the human chromosomal fragment coding for Ku80, V(D)J recombination and radiation sensitivity were restored to normal levels (54). Therefore, the Ku80 proteins perform important role(s) in nonhomologous end joining, which is the primary mode of DSB repair in mammalian cells, and control of radioresistance. To determine if the oncogenic H-Ras-mediated increase in Ku80 expression contributes to radioresistance, the oncogenic H-Ras-expressing cells were transfected with the Ku80-specific siRNA, which targets Ku80 and inhibits its expression. It was found that the transfection of cells with the Ku80-specific siRNA resulted in a decrease in the DSB repair activity following {gamma}-ray irradiation compared with the mock- and control siRNA-transfected cells (Fig. 4). In addition, it was also found that the Ku80-targeted siRNA oligonucleotides caused the oncogenic H-Ras-expressing NIH3T3 cells to be highly sensitive to {gamma}-ray irradiation and that Ku80 expression caused the NIH3T3 cells to increase the viability following {gamma}-ray irradiation (Fig. 5). Therefore, oncogenic H-Ras-mediated up-regulation of Ku80 is involved in {gamma}-radiation resistance when oncogenic H-Ras is expressed.

Recent research has suggested a linkage between Ras-mediated radioresistance and PI3K. For example, expression of active PI3K in cells with wild-type Ras results in enhanced radiation resistance, and this radioresistance could be inhibited by a PI3K inhibitor (17). In addition, Grana et al. (19) have shown that the activated Ras-transformed RIE-1 epithelial cells exhibit resistance to radiation, and blocking PI3K activity with the inhibitor LY294002 sensitizes RIE-1 epithelial cells to radiation. Similarly, it has been suggested that EGRF-Ras-PI3K pathway might play an important role in mediating radiation resistance (18, 20). The Ku80 promoter contains Sp1-binding sites. Although little is known about what transcription factors actually participate in the Ku80 regulation, a prior study suggests that a Sp1 transcription factor contributes to the Ku80 expression (55). Sp1 is important for the expression of many cellular genes, particularly housekeeping genes. However, Sp1 sites have more recently been found to mediate transcription in response to diverse stimuli, including oncogenes, such as Ras and growth factors and cytokines (56). Regulation of Sp1-dependent transcription may be conveyed by changes in DNA-binding activity, by association with other transcription factors, by changes in Sp1 abundance, or in transactivation activity owing to biochemical modification, such as phosphorylation (56). Overexpression of Ras has been shown to mediate Sp1-dependent transcriptional activation, involving PI3K as important intermediary signaling molecules (57). PI3K contributes to the phosphorylation of Sp1, and Sp1 is involved in the regulation of gene expression by the PI3K/Akt pathway (58, 59). Thus, we speculate that this might occur via phosphorylation of Sp1 leading to its increased binding to and transactivation of the Ku80 promoter. Further experiments are clearly needed to evaluate the effect of Ras-PI3K pathway on the Ku80 expression in the radioresistance of oncogenic Ras-expressing NIH3T3 cells.

In summary, the sensitivity of cells to death by ionizing radiation is a critical determinant of the probability of a cure in 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 interest in determining which genes mediate the altered radioresistance in tumor cells. This study found that Ku80 is induced by oncogenic H-Ras expression. The results suggest that Ku80 contributes to the oncogenic H-Ras-mediated increase in the capacity of NIH3T3 cells to repair DSBs and to afford protection against ionizing radiation. Under normal conditions, the action of the Ras and the other members of the Ras pathway were strictly regulated during the cell cycle and under different growth conditions (60). However, in a tumor cell, the oncogenic activation of ras is a consequence of point mutations that either impair the GTPase activity or enhance the GTP-binding affinity, resulting in a highly active proliferative signal (61). Furthermore, ras mutations are found in a variety of human cancers with the highest incidence being observed in ~30% of all human tumors (62). Therefore, the elevated Ku80 levels induced by Ras activation might confer cancer cell resistance to ionizing radiation. These findings, from a pathway involving oncogenic H-Ras and Ku80, might be relevant to the development of new therapeutic approaches, involving the administration of ionizing radiation together with a Ku80 inhibitor (63, 64).


   Acknowledgments
 
Grant support: National Cancer Control R&D Program 2003, Ministry of Health and Welfare, Republic of Korea, and Nuclear Research and Development Program from the Ministry of Science and Technology of Korea.

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.

Received 11/12/04. Revised 3/22/05. Accepted 5/22/05.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

  1. Peters LJ, Withers HR, Thames HD Jr, Fletcher GH. Tumor radioresistance in clinical radiotherapy. Int J Radiat Oncol Biol Phys 1982;8:101–8.[Medline]
  2. Sklar MD. The ras oncogenes increase the intrinsic resistance of NIH 3T3 cells to ionizing radiation. Science 1988;239:645–7.[Abstract/Free Full Text]
  3. Ling CC, Endlich B. Radioresistance induced by oncogenic transformation. Radiat Res 1989;120:267–79.[Medline]
  4. McKenna WG, Weiss MC, Endlich B, et al. Synergistic effect of the v-myc oncogene with H-ras on radioresistance. Cancer Res 1990;50:97–102.[Abstract/Free Full Text]
  5. Hermens AF, Bentvelzen PA. Influence of the H-ras oncogene on radiation responses of a rat rhabdomyosarcoma cell line. Cancer Res 1992;52:3073–82.[Abstract/Free Full Text]
  6. Pirollo KF, Tong YA, Villegas Z, Chen Y, Chang EH. Oncogene-transformed NIH 3T3 cells display radiation resistance levels indicative of a signal transduction pathway leading to the radiation-resistant phenotype. Radiat Res 1993;135:234–43.[Medline]
  7. Mille AC, Kariko K, Myers CE, Clark EP, Samid D. Increased radioresistance of EJ Ras-transformed human osteosarcoma cells and its modulation by lovastatin, an inhibitor of p21ras isoprenylation. Int J Cancer 1993;53:302–7.[Medline]
  8. Bernhard EJ, Stanbridge EJ, Gupta S, et al. 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 2000;60:6597–600.[Abstract/Free Full Text]
  9. Russell JS, Lang FF, Huet T, et al. Radiosensitization of human tumor cell lines induced by the adenovirus-mediated expression of an anti-Ras single-chain antibody fragment. Cancer Res 1999;59:5239–44.[Abstract/Free Full Text]
  10. Rait A, Pirollo K, Will DW, et al. 3'-End conjugates of minimally phosphorothioate-protected oligonucleotides with 1-O-hexadecylglycerol: synthesis and anti-ras activity in radiation-resistant cells. Bioconjug Chem 2000;11:153–60.[CrossRef][Medline]
  11. Bernhard EJ, Kao G, Cox AD, et al. The farnesyltransferase inhibitor FTI-277 radiosensitizes H-ras-transformed rat embryo fibroblasts. Cancer Res 1996;56:1727–30.[Abstract/Free Full Text]
  12. Bernhard EJ, McKenna WG, Hamilton AD, et al. Inhibiting Ras prenylation increases the radiosensitivity of human tumor cell lines with activating mutations of ras oncogenes. Cancer Res 1998;58:1754–61.[Abstract/Free Full Text]
  13. Campbell SL, Khosravi-Far R, Rossman KL, Clark GJ, Der CJ. Increasing complexity of Ras signaling. Oncogene 1998;17:1395–413.[CrossRef][Medline]
  14. Downward J. Ras signalling and apoptosis. Curr Opin Genet Dev 1998;8:49–54.[CrossRef][Medline]
  15. Hagemann C, Blank JL. The ups and downs of MEK kinase interactions. Cell Signal 2001;13:863–75.[CrossRef][Medline]
  16. Cantley LC. The phosphoinositide 3-kinase pathway. Science 2002;296:1655–7.[Abstract/Free Full Text]
  17. Gupta AK, Bakanauskas VJ, Cerniglia GJ, et al. The Ras radiation resistance pathway. Cancer Res 2001;61:4278–82.[Abstract/Free Full Text]
  18. Gupta AK, McKenna WG, Weber CN, et al. Local recurrence in head and neck cancer: relationship to radiation resistance and signal transduction. Clin Cancer Res 2002;8:885–92.[Abstract/Free Full Text]
  19. Grana TM, Rusyn EV, Zhou H, Sartor CI, Cox AD. Ras mediates radioresistance through both phosphatidylinositol 3-kinase-dependent and Raf-dependent but mitogen-activated protein kinase/extracellular signal-regulated kinase kinase-independent signaling pathways. Cancer Res 2002;62:4142–50.[Abstract/Free Full Text]
  20. McKenna WG, Muschel RJ, Gupta AK, Hahn SM, Bernhard EJ. The RAS signal transduction pathway and its role in radiation sensitivity. Oncogene 2003;22:5866–75.[CrossRef][Medline]
  21. Grana TM, Sartor CI, Cox AD. Epidermal growth factor receptor autocrine signaling in RIE-1 cells transformed by the Ras oncogene enhances radiation resistance. Cancer Res 2003;63:7807–14.[Abstract/Free Full Text]
  22. Cho HJ, Jeong HG, Lee JS, et al. Oncogenic H-Ras enhances DNA repair through the Ras/phosphatidylinositol 3-kinase/Rac1 pathway in NIH3T3 cells. Evidence for association with reactive oxygen species. J Biol Chem 2002;277:19358–66.[Abstract/Free Full Text]
  23. Finnie NJ, Gottlieb TM, Blunt T, Jeggo PA, Jackson SP. DNA-dependent protein kinase activity is absent in xrs-6 cells: implications for site-specific recombination and DNA double-strand break repair. Proc Natl Acad Sci U S A 1995;92:320–4.[Abstract/Free Full Text]
  24. Lees-Miller SP, Godbout R, Chan DW, et al. Absence of p350 subunit of DNA-activated protein kinase from a radiosensitive human cell line. Science 1995;267:1183–5.[Abstract/Free Full Text]
  25. Gottlieb TM, Jackson SP. The DNA-dependent protein kinase: requirement for DNA ends and association with Ku antigen. Cell 1993;72:131–42.[CrossRef][Medline]
  26. Dvir A, Peterson SR, Knuth MW, Lu H, Dynan WS. Ku autoantigen is the regulatory component of a template-associated protein kinase that phosphorylates RNA polymerase II. Proc Natl Acad Sci U S A 1992;89:11920–4.[Abstract/Free Full Text]
  27. Taccioli GE, Gottlieb TM, Blunt T, et al. Ku80: product of the XRCC5 gene and its role in DNA repair and V(D)J recombination. Science 1994;265:1442–5.[Abstract/Free Full Text]
  28. Tuteja R, Tuteja N. Ku autoantigen: a multifunctional DNA-binding protein. Crit Rev Biochem Mol Biol 2000;35:1–33.[CrossRef][Medline]
  29. Levy E, Baroche C, Barret JM, et al. Activated ras oncogene and specifically acquired resistance to cisplatin in human mammary epithelial cells: induction of DNA cross-links and their repair. Carcinogenesis 1994;15:845–50.[Abstract/Free Full Text]
  30. Kinashi Y, Akaboshi M, Masunaga S, Ono K, Watanabe M. Resistance to 195mPt-radiolabeled cis-diaminedichloroplatinum (II) of SHOK cells transfected with various oncogenes. Radiat Med 1998;16:233–7.[Medline]
  31. Yen L, Nie ZR, You XL, Richard S, Langton-Webster BC, Alaoui-Jamali MA. Regulation of cellular response to cisplatin-induced DNA damage and DNA repair in cells overexpressing p185(erbB-2) is dependent on the ras signaling pathway. Oncogene 1997;14:1827–35.[CrossRef][Medline]
  32. Ward JF. DNA damage produced by ionizing radiation in mammalian cells: identities, mechanisms of formation, and reparability. Prog Nucleic Acid Res Mol Biol 1988;35:95–125.[Medline]
  33. Olive PL. The role of DNA single- and double-strand breaks in cell killing by ionizing radiation. Radiat Res 1988;150:42–51.
  34. Iliakis G. The role of DNA double strand breaks in ionizing radiation-induced killing of eukaryotic cells. Bioessays 1991;13:641–8.[CrossRef][Medline]
  35. Jeggo PA. DNA breakage and repair. Adv Genet 1998;38:185–218.[Medline]
  36. Chu G. Double strand break repair. J Biol Chem 1997;272:24097–100.[Free Full Text]
  37. Smith GC, Jackson SP. The DNA-dependent protein kinase. Genes Dev 1999;13:916–34.[Free Full Text]
  38. Meek K, Gupta S, Ramsden DA, Lees-Miller SP. The DNA-dependent protein kinase: the director at the end. Immunol Rev 2004;200:132–4.[CrossRef][Medline]
  39. Thacker J, Zdzienicka MZ. The XRCC genes: expanding roles in DNA double-strand break repair. DNA Repair (Amst) 2004;3:1081–90.
  40. Mimori T, Hardin JA, Steitz JA. Characterization of the DNA-binding protein antigen Ku recognized by autoantibodies from patients with rheumatic disorders. J Biol Chem 1986;261:2274–8.[Abstract/Free Full Text]
  41. Jeggo PA, Taccioli GE, Jackson SP. Menage a trois: double strand break repair, V(D)J recombination and DNA-PK. Bioessays 1995;17:949–57.[CrossRef][Medline]
  42. Jackson SP, Jeggo PA. DNA double-strand break repair and V(D)J recombination: involvement of DNA-PK. Trends Biochem Sci 1995;20:412–5.[CrossRef][Medline]
  43. Bliss TM, Lane DP. Ku selectively transfers between DNA molecules with homologous ends. J Biol Chem 1997;272:5765–73.[Abstract/Free Full Text]
  44. Featherstone C, Jackson SP. Ku, a DNA repair protein with multiple cellular functions. Mutat Res 1999;434:3–15.[Medline]
  45. Iliakis G, Metzger L, Muschel RJ, McKenna WG. Induction and repair of DNA double strand breaks in radiation-resistant cells obtained by transformation of primary rat embryo cells with the oncogenes H-ras and v-myc. Cancer Res 1990;50:6575–9.[Abstract/Free Full Text]
  46. Diem C, Runger TM. Processing of three different types of DNA damage in cell lines of a cutaneous squamous cell carcinoma progression model. Carcinogenesis 1997;18:657–62.[Abstract/Free Full Text]
  47. Bristow RG, Hu Q, Jang A, et al. Radioresistant MTp53-expressing rat embryo cell transformants exhibit increased DNA-dsb rejoining during exposure to ionizing radiation. Oncogene 1998;16:1789–802.[CrossRef][Medline]
  48. Blunt T, Finnie NJ, Taccioli GE, et al. Defective DNA-dependent protein kinase activity is linked to V(D)J recombination and DNA repair defects associated with the murine scid mutation. Cell 1995;80:813–23.[CrossRef][Medline]
  49. Kirchgessner CU, Patil CK, Evans JW, et al. DNA-dependent kinase (p350) as a candidate gene for the murine SCID defect. Science 1995;267:1178–83.[Abstract/Free Full Text]
  50. Biedermann KA, Sun JR, Giaccia AJ, Tosto LM, Brown JM. scid mutation in mice confers hypersensitivity to ionizing radiation and a deficiency in DNA double-strand break repair. Proc Natl Acad Sci U S A 1991;88:1394–7.[Abstract/Free Full Text]
  51. Nussenzweig A, Chen C, da Costa Soares V, et al. Requirement for Ku80 in growth and immunoglobulin V(D)J recombination. Nature 1996;382:551–5.[CrossRef][Medline]
  52. Nussenzweig A, Sokol K, Burgman P, Li L, Li GC. Hypersensitivity of Ku80-deficient cell lines and mice to DNA damage: the effects of ionizing radiation on growth, survival, and development. Proc Natl Acad Sci U S A 1997;94:13588–93.[Abstract/Free Full Text]
  53. Wachsberger PR, Li WH, Guo M, et al. Rejoining of DNA double-strand breaks in Ku80-deficient mouse fibroblasts. Radiat Res 1999;151:398–407.[Medline]
  54. Ross GM, Eady JJ, Mithal NP, et al. DNA strand break rejoining defect in xrs-6 is complemented by transfection with the human Ku80 gene. Cancer Res 1995;55:1235–8.[Abstract/Free Full Text]
  55. Ludwig DL, Chen F, Peterson SR, Nussenzweig A, Li GC, Chen DJ. Ku80 gene expression is Sp1-dependent and sensitive to CpG methylation within a novel cis element. Gene 1997;199:181–94.[CrossRef][Medline]
  56. Black AR, Black JD, Azizkhan-Clifford J. Sp1 and kruppel-like factor family of transcription factors in cell growth regulation and cancer. J Cell Physiol 2001;188:143–60.[CrossRef][Medline]
  57. Pal S, Datta K, Khosravi-Far R, Mukhopadhyay D. Role of protein kinase C{zeta} in Ras-mediated transcriptional activation of vascular permeability factor/vascular endothelial growth factor expression. J Biol Chem 2001;276:2395–403.[Abstract/Free Full Text]
  58. Reisinger K, Kaufmann R, Gille J. Increased Sp1 phosphorylation as a mechanism of hepatocyte growth factor (HGF/SF)-induced vascular endothelial growth factor (VEGF/VPF) transcription. J Cell Sci 2003;116:225–38.[Abstract/Free Full Text]
  59. Pore N, Liu S, Shu HK, et al. Sp1 is involved in Akt-mediated induction of VEGF expression through an HIF-1-independent mechanism. Mol Biol Cell 2004;15:4841–53.[Abstract/Free Full Text]
  60. Macaluso M, Russo G, Cinti C, Bazan V, Gebbia N, Russo A. Ras family genes: an interesting link between cell cycle and cancer. J Cell Physiol 2002;192:125–30.[CrossRef][Medline]
  61. White MA, Nicolette C, Minden A, et al. Multiple Ras functions can contribute to mammalian cell transformation. Cell 1995;80:533–41.[CrossRef][Medline]
  62. Bos JL. Ras oncogenes in human cancer: a review. Cancer Res 1989;49:4682–9.[Abstract/Free Full Text]
  63. Ismail IH, Martensson S, Moshinsky D, et al. SU11752 inhibits the DNA-dependent protein kinase and DNA double-strand break repair resulting in ionizing radiation sensitization. Oncogene 2004;23:873–82.[CrossRef][Medline]
  64. Leahy JJ, Golding BT, Griffin RJ, et al. Identification of a highly potent and selective DNA-dependent protein kinase (DNA-PK) inhibitor (NU7441) by screening of chromenone libraries. Bioorg Med Chem Lett 2004;14:6083–7.[CrossRef][Medline]



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