Mutant K-rasV12 increases COX-2, peroxides and DNA damage in lung cells
Anna Maciag1,3,
Gunamani Sithanandam2 and
Lucy M. Anderson1
1 Laboratory of Comparative Carcinogenesis, National Cancer Institute at Frederick and 2 Basic Research Program SAIC-Frederick, Inc., Frederick, MD 21702, USA
3 To whom correspondence should be addressed Email: amaciag{at}mail.ncifcrf.gov
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Abstract
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K-ras is frequently mutated in lung adenocarcinomas. Recent discovery that wild-type K-ras is tumor suppressive in the lung raises a question: how is mutant K-ras aggressively oncogenic? We hypothesized that mutant K-ras might lead to generation of reactive oxygen species (ROS) and DNA damage, contributing to malignant transformation. We stably transfected human mutant K-rasV12 into non-transformed peripheral mouse lung epithelial cells (E10 line). Constitutively active mutant K-rasV12 in E10 cells led to a highly significant (P < 0.001) increased level of peroxides, and a corresponding increase in the amount of DNA strand-break damage, compared with the parental line E10 and the vector control. Levels of superoxide were not increased, suggesting a direct source of peroxides, such as cyclooxygenase-2 (COX-2). COX-2 protein and activity measured as prostaglandin E2 level were up-regulated in cells expressing mutant K-rasV12; COX-2 activity correlated with K-ras activity (K-ras p21-GTP). Both peroxide generation and DNA single strand breaks were significantly reduced by pre-treatment with COX-2-specific inhibitor SC 58125, confirming COX-2 as the source of the ROS. COX-2 has been repeatedly implicated in lung cancer, and is known to be regulated by ras and to release ROS. Our data suggest that up-regulation of COX-2, with a consequent increase in peroxides and DNA damage, contributes to the dominant oncogenicity of mutant K-ras.
Abbreviations: CM-H2DCF-DA, 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate; COX, cyclooxygenase; DCF, 2',7'-dichlorofluorescein; HBSS, Hanks' balanced salt solution; NBT, nitroblue tetrazolium; PGE2, prostaglandin E2; ROS, reactive oxygen species
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Introduction
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The K-ras gene is frequently mutated in lung adenocarcinoma as well as in cancers of the pancreas, colon and other tissues. Transgenic expression of mutant K-ras in the mouse lung led to rapid development of multiple tumors (14). However, wild-type K-ras is a tumor suppressor in mouse lung (5). How then does mutant K-ras act as a dominant oncoprotein? Several studies demonstrated that expression of a constitutively active mutant of H-ras led to an increase in reactive oxygen species (ROS) generation in fibroblasts (611), keratinocytes (12) and kidney epithelial cells (13,14). V-Ki-ras also caused a marked increase in ROS in fibroblasts (9). Malignant transformation of kidney cells by v-H-ras was suppressed by over-expression of anti-oxidant enzymes superoxide dismutase or glutathione peroxidase (14). ROS are known to react with various intracellular targets, including lipids, proteins and DNA. ROS may contribute to the transformation by causing DNA damage. These observations led us to the hypothesis that mutant K-ras might cause an increase in ROS and DNA damage in lung epithelial cells, and in this way lead to transformation.
Preliminary experiments supported this hypothesis: ROS as peroxides were significantly increased in peripheral lung epithelial cells expressing mutant K-rasV12, compared with controls. However, superoxides were not increased, and an inhibitor of flavoproteins had no effect, eliminating several usual sources of ROS, including the mitochondrial electron transport system and NAD(P)H oxidase. An attractive remaining candidate was cyclooxygenase-2 (COX-2), expected to be a direct producer of peroxides. Several studies have reported the induction of COX-2 in association with activated H-ras (1518). A relationship between COX-2 expression and K-ras mutation has been found in colorectal adenomas (19). We therefore hypothesized that the observed increase in ROS in K-rasV12 transfected lung cells was due to the up-regulation of COX-2.
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Materials and methods
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Cell lines and stable transfection with human K-rasV12 and human WT-K-ras
The E10 cell line was originally derived from explants of BALB/c female mouse lung (20,21) and was obtained from Dr Randall Ruch, Medical College of Ohio. The cells and the clones derived from E10 transfected with human K-rasV12 or wild-type K-ras in pCMV-Tag1 Vector (Stratagene, La Jolla, CA) were maintained at 37°C and 5% CO2 in Connaught Medical Research Laboratories (CMRL) 1066 medium (BioFluids, Rockville, MD) supplemented with 4 mM glutamine, penicillin (100 U/ml), streptomycin (100 µg/ml) and 10% fetal bovine serum (Gemini Bio-Products, Woodland, CA). For transfection, the E10 cells were seeded in 10 cm Petri dishes and allowed to grow for 24 h to
60% confluence. The cells were transfected with 3 µg human K-rasV12 or WT K-ras construct in 6 µl FuGENE 6 Transfection Reagent (Roche Diagnostics, Indianapolis, IN). Individual neomycin-resistant colonies were isolated by ring cloning. Clones with high expression of the hK-rasV12 construct were identified by RTPCR with human K-ras specific primers (upper primer: 5' GAC TGA ATA TAA ACT TGT GGT A 3', lower primer: 5' CTA TTG TTG GAT CAT ATT GC 3') with 40 cycles utilized for semi-quantitative assessment.
Determination of peroxides generation
The intracellular level of peroxides was quantified by the oxidation of the peroxide-sensitive fluorophore 5,6-chloromethyl-2',7'-dichlorodihydrofluorescein diacetate (CM-H2DCF-DA) (Molecular Probes, Eugene, OR). Cells were loaded with 5 µM CM-H2DCF-DA in Hanks' balanced salt solution (HBSS) at 37°C and 5% CO2. After 20 min of incubation the cells were harvested with trypsinEDTA and 2',7'-dichlorofluorescein (DCF) fluorescence was analyzed immediately by flow cytometry with the excitation source at 488 nm and emission at 530 nm. All experiments were performed three times, each time in triplicate.
Determination of superoxide production
A modified version of a previously described assay for the intracellular conversion of nitroblue tetrazolium (NBT) to formazan by superoxide anion was used to measure the level of superoxide (22). Briefly, the cells were incubated with 0.5 mg/ml NBT (Roche) in HBSS for 30 min, rinsed three times with methanol to remove unreduced NBT, fixed in absolute ethanol, and allowed to air dry. The formazan content of the cells was then solubilized with 480 µl 2 M KOH and 560 µl dimethyl sulfoxide (DMSO) and the absorbance at 630 nm was measured spectrophotometrically. A standard curve was prepared by adding KOH and DMSO to known amounts of NBT.
The NBT assay results were confirmed by dihydroethidium (DHE, Molecular Probes) staining. DHE is oxidized by superoxide to yield fluorescent ethidium (excitation 475 nm/emission 610 nm). The cells were incubated with 5 µM DHE for 30 min, then the dye containing HBSS was removed, cells rinsed and fresh medium was added. The ethidium fluorescence was observed under an inverted fluorescent microscope Zeiss Axiovert 200 M (Carl Zeiss, Thornwood, NY).
Prostaglandin E2 assay
The cells were seeded into 6-well culture dishes and allowed to grow for 24 h to 70% confluence. The culture medium was replaced with fresh medium (3 ml/well) and after desired time intervals (indicated in the figure legends) an aliquot of culture medium was removed from each well and any particulate was removed by centrifugation. Prostaglandin E2 (PGE2) level in the culture medium was measured using a PGE2 competitive enzyme immunoassay (Cayman Chemicals, Ann Arbor, MI) according to the manufacturer's protocol. Each experiment was performed in triplicate and the mean PGE2 values are shown as the final results.
COX-2 protein immunoblotting
For protein immunoblotting the cells were harvested in lysis buffer [25 mM HEPES buffer containing 150 mM NaCl, 10 mM MgCl2, 1% Nonidet P-40, 0.25% sodium deoxycholate, 10% glycerol, 2.5 mM EDTA, 0.28 TIU/ml aprotinin, 10 mM NaF, 2 mM phenylmethylsulfonyl fluoride (PMSF) and 2 mM Naorthovanadate]. Cell lysates (5 µg) were resolved by SDSpolyacrylamide gel electrophoresis (12% Trisglycine gel, Invitrogen Life Technologies, Carlsbad, CA) and immunoblotted with polyclonal antibody to COX-2 (1:2000) (Santa Cruz Biotechnology, Santa Cruz, CA).
Assay for active K-ras p21
K-ras activity assay was performed as described previously (23) with modification. Total cell lysate (500 µg of protein) was incubated with 30 µg of Raf1-RBD (Upstate Biotechnology, Lake Placid, NY) for 1 h at 4°C. The beads were pelleted by centrifugation and washed three times with lysis buffer. The bound proteins were eluted by boiling in Laemmli's buffer containing 50 mM dithiothreitol and subjected to SDSPAGE followed by immunoblotting with monoclonal antibody to K-ras (1:2000) (Oncogene Science, Uniondale, NY).
Comet assay
The alkaline comet assay was performed using a modification of the method of Singh (24). The cells suspended in HBSS at 2 x 105/ml were mixed 1:10 with 0.8% low melting point agarose and layered onto comet slides (Trevigen, Gaithersburg, MD). The slides were placed into a chilled lysing solution (2.5 M NaCl, 100 mM Na2EDTA, 10 mM Tris-base, 1% sarcosyl, 10% DMSO and 1% Triton X-100, pH 10) and lysed overnight at +4°C in the dark. After 15 min of unwinding in alkaline solution (300 mM NaOH, 1 mM Na2EDTA, pH 12.1) the slides were subjected to electrophoresis at 0.8 V/cm for 20 min. The slides were neutralized with 0.4 M TrisHCl, pH 7.4, dehydrated with ethanol and allowed to air-dry, then stained with SYBR Gold (1:10 000, Molecular Probes, Eugene, OR). Each experiment was performed in triplicate. Typically 100 cells were analyzed per sample. Comets were observed under fluorescent microscope Zeiss Axiovert 200 M and images were captured with AxioCam MRm digital camera. The comet tail intensity was quantified using Labworks 4.0 software (UVP, Upland, CA).
Treatment with chemicals
The cells were seeded into 6-well culture dishes and allowed to grow to 7080% confluence. For the experiment with the COX-2-specific inhibitor SC58125 (25) (Calbiochem Biosciences, La Jolla, CA) the cells were treated with 10 nM, 100 nM, 1 µM or 10 µM SC58125 in complete medium for time periods indicated in the figure legends, then subjected to assay for PGE2, peroxides or Comet assay. For the experiments with diphenylene iodonium (DPI) (Sigma Chemical, St Louis, MO), cells were treated with 20 µM DPI for 20 min, followed by DCF assay in the presence of DPI.
Statistical analyses
Data in all experiments are represented as mean ± SE. Statistical comparisons were carried out using ANOVA with Bonferroni correction using GraphPad InStat 3.0 (GraphPad Software, San Diego, CA). P values <0.05 were considered to be statistically significant. Regression analysis was performed using the Pearson test.
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Results
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We stably transfected mouse lung E10 epithelial cells with human mutant K-rasV12. Individual derived clones carrying the mutant K-ras gene (designated 61-28, 61-29 and 73-13) were compared with a clone carrying empty vector only (60-1) and the parental E10 line. Expression of constitutively active human mutant K-rasV12 in these cells resulted in a significant, 2-fold increase in intracellular level of peroxides, as indicated by fluorescence of DCF, an oxidized derivative of H2DCF-DA (Figure 1). To check whether wild-type K-ras over-expression can also induce production of intracellular ROS, we used the 8-10 cell line, which is a stable transfectant of E10 cells with human WT K-ras. We did not observe any increase in DCF fluorescence in 8-10 cells (Figure 1). This strongly indicates that induction of peroxides generation in these lung cells is a specific effect of mutant K-ras.

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Fig. 1. Effect of K-rasV12 expression on the level of intracellular peroxides. The clonal cell lines indicated were loaded with 5 µM CM-H2DCF-DA for 20 min, then immediately assessed by flow cytometry with the excitation source at 488 nm and emission at 530 nm.
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To determine if the activated K-rasV12 induced DNA damage in our lung epithelial cells we employed the comet assay to measure DNA strand breaks. K-rasV12 transfected cells showed a significant increase in DNA single strand break level in comparison with the parental E10 line and to the vector control (Figure 2).

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Fig. 2. Comet assay. The level of single strand breaks was significantly higher in K-rasV12 transfected cells in comparison with parental line (E10) and vector control (60-1).
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The next question was the source of the peroxides. H2O2 can be generated by spontaneous or superoxide dismutase-catalyzed dismutation of superoxide. We used two different methods to determine if superoxide was involved: the nitroblue tetrazolium reduction method and dihydroethidium staining. We did not observe any increased NBT reduction (Figure 3A), or any increase in the level of ethidium fluorescence (Figure 3B) in the cells expressing K-rasV12. This indicates that superoxide was not the source of H2O2 giving DCF signal. We also found no evidence that a flavoprotein is involved in ROS production in K-rasV12 transfectants: treatment of the cells with DPI, a flavoprotein inhibitor, did not affect peroxides' level (Figure 3C).

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Fig. 3. Superoxide level remained the same in K-rasV12 transfectants and controls, as determined by NBT reduction method (A) and dihydroethidium staining (B). (C) The cells were pretreated with 20 µM DPI for 20 min then subjected to the assay for peroxides by flow cytometry. DPI treatment did not affect intracellular level of peroxides.
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Peroxides may also be directly generated by COX-2 (26), an enzyme that has frequently been observed as induced by ras expression (1518). We therefore attempted to determine whether COX-2 could be a source of peroxides in the K-rasV12 transfectants. Expression of COX-2 protein was elevated in the cells transfected with human K-rasV12 compared with parental line and vector controls, as demonstrated by protein immunoblotting (Figure 4A). COX-2 activity measured as prostaglandin E2 level was significantly higher in K-rasV12 transfectants than in controls (Figure 4B). COX-2 activity in these cells correlated significantly with K-ras activity (Figure 4C and D).

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Fig. 4. Effect of mutant K-rasV12 expression on COX-2 protein level and activity. (A) Five micrograms of total cell lysate protein was immunoblotted with anti-COX-2 antibody, then re-probed for ß-actin (42 kDa) as a loading control (representative blot). (B) The amount of prostaglandin E2 secreted into the culture media within 30 min was significantly higher in K-rasV12 transfectants than in controls. (C) K-ras activity (K-ras p21GTP complex) in E10 cells, vector control and K-rasV12 transfected (representative blot). (D) Correlation between K-ras p21 activity and COX-2 protein expression in K-rasV12 transfectants and controls. Filled circle, K-rasV12 transfectants; clear square, vector control; clear circle, parental line E10.
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From these results, we concluded that transfection of constitutively active K-rasV12 mutant into E10 cells led to up-regulation of COX-2 and postulated that the increased level of peroxides was a by-product of COX enzymatic activity.
To confirm that COX-2 was the source of peroxides in the K-rasV12 transfected cells, we treated the cells with COX-2-specific inhibitor, SC58125. We used four different concentrations of SC58125, within the range of between 10 nM and 10 µM. The cells were counted after treatment with inhibitor to confirm that SC58125 did not cause any cell loss due to its toxicity. The cell count was unaltered with all used concentrations and after 30 min of treatment (data not shown).
Treatment with SC58125 resulted in a significant and concentration-dependent reduction of DCF fluorescence in all cell lines (Figure 5B). Even the lowest dose of SC58125, 10 nM, reduced PGE2 production to control levels (Figure 5A) and significantly reduced DCF fluorescence (Figure 5B). This strongly confirms that COX-2 was responsible for the increased production of peroxides resulting from mutant K-rasV12 expression. The decrease in DCF fluorescence strongly correlated with the decrease in COX-2 activity measured as PGE2 level (r = 0.90 at P < 0.0001) (Figure 5C). The residual DCF fluorescence is probably due to the fact that CM-H2DCF-DA and SC58125 had to be added simultaneously to the cells; thus some oxidation of H2DCF-DA occurred before the full inhibitory effect of SC58125 was achieved.

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Fig. 5. Effect of COX-2 specific inhibitor, SC58125, on COX-2 activity and intracellular level of peroxides. The cells were seeded onto 6-well culture dishes and allowed to grow to 70% confluence. The cells were treated with 10 nM, 100 nM, 1 µM and 10 µM SC58125 for 30 min. (A) Treatment with SC58125 inhibited prostaglandin E2 production (measured as PGE2 secreted into the culture medium within 30 min). (B) DCF staining for intracellular peroxides. Cells were loaded with 5 µM H2DCF-DA for 30 min in the presence of between 10 nM and 10 µM SC58125. Peroxide signal in K-rasV12 transfectants decreased after 30 min treatment with inhibitor in a concentration-dependent manner. (C) Correlation between decrease ( ) in COX-2 activity/PGE2 level and decrease in peroxides generation measured as DCF fluorescence. Values are relative to cells not treated with SC58125. Filled circle, K-rasV12 transfectants; clear square, vector control; clear circle, parental line E10.
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Furthermore, the increase in DNA strand break damage was significantly reduced by treatment with the COX-2-specific inhibitor SC58125 (Figure 6), confirming COX-2 involvement in the mutant K-ras-dependent DNA damage. Within 30 min after addition of 10 µM SC58125, most of the increase in DNA damage related to K-ras mutant has been corrected; after 30 min of treatment the level of DNA strand breaks in mutant K-ras carrying line 73-13 did not differ significantly from that observed for vector control (Figure 6). About 50% of the repair occurred between 5 and 15 min after addition of SC58125. Lower concentrations of SC58125 were less effective in reducing DNA damage detected by Comet assay (not shown).

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Fig. 6. Treatment with COX-2-specific inhibitor SC58125 significantly reduced the level of DNA single strand breaks. The cells (at 70% confluence, growing on 6-well culture dishes) were treated with 10 µM SC58125. At intervals of 5, 15 and 30 min after addition of the inhibitor the cells were collected by scraping into ice-cold HBSS and immediately processed for the Comet assay. (a) P < 0.001 versus 60-1 (vector control); (b) P < 0.001 versus 73-13 at time point 0. The level of DNA strand break damage in the line 73-13 after 30 min with inhibitor was not significantly different from that observed for 60-1 (vector control).
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In conclusion, the results suggest that ROS generation, through COX-2 up-regulation, may contribute to the active oncogenicity of mutant K-ras in the lung as a result of DNA damage.
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Discussion
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In this study we show that expression of human mutant K-rasV12 in mouse lung epithelial E10 cells led to generation of peroxides. Several previous studies reported generation of ROS as an effect of activated ras signaling. Fibroblasts or keratinocytes stably transfected with a constitutively active H-ras produced large amounts of superoxide (68) and NADPH oxidase was a possible source of ROS. We also show that mutant K-ras-induced ROS generation leads to DNA single strand break damage. This may cause mutations and possibly other genetic changes resulting in cell transformation.
COX-2 appeared to be the source of the increased level of peroxides in the K-rasV12 transfected cells. COX-2 has frequently been observed as induced by ras expression, including activated H-ras and K-ras transfected into fibroblasts or rat intestinal epithelial cells (1518). Morphological transformation was associated with rapid induction of COX-2 in fibroblasts and intestinal epithelial cells transformed by mutated H-ras, and in the latter cells the specific COX-2 inhibitor SC58125 reduced tumor growth (15). However, there has been no report showing an increase in ROS generation in H-ras or K-ras transfectants with elevated COX-2 expression. COX-2 is an intracellular enzyme that directly generates peroxides, as was evidently occurring in our cells. Furthermore, COX-2 has been implicated in lung cancer, and lung cancer clinical trials of COX-2 inhibitors are in progress (2734).
Although a relationship between the over-expression of COX-2 and carcinogenesis has been suggested by several studies, the precise mechanism for this process is the subject of ongoing investigation. Most work has focused on the signaling effects of the eicosanoid products of COX-2 activity, related to cell division, apoptosis, invasion, etc. (3234). However, it has also been shown that DNA and/or nucleosides can be oxidized when incubated in vitro with COX-2 and arachidonic acid (26). Inhibitor studies implicated COX-2 in DNA damage in human lung adenocarcinoma CL-3 cells (35). In smokers, the oxidative activity of COX could also lead to bioactivation of smoke carcinogens such as polycyclic aromatic hydrocarbons and aromatic amines (32). This mechanism of carcinogen activation is particularly significant in cells/tissues with low cytochrome P450 activity, such as lung. A role for COX-2 in the initiation stages of lung cancer development and in association with K-ras, via oxidative DNA damage and/or carcinogen activation, is supported by its up-regulation in atypical alveolar epithelium (27), in pre-neoplastic lesions (27,29), and in early stage lung cancers and surrounding tissue, especially adenocarcinomas (31,33,36) where K-ras is frequently mutated. Similar observations were made in lung tumor-prone A/J mice (37). Three human lung cancer cell lines with K-ras mutation expressed COX-2 highly and their growth was reduced by the COX-2 inhibitor sulindac sulfide (38). Reduced incidence of lung carcinoma has been reported in cases of prolonged use of aspirin, a non-selective COX inhibitor (39), and both the non-specific COX inhibitor acetylsalicylic acid and the COX-2-specific inhibitor NS-398 inhibited lung tumor causation in mice by the tobacco carcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (40). On the other hand, celecoxib, a COX-2 inhibitor, did not suppress development of mouse lung tumors caused by urethane or 3-methylcholantrene plus butylated hydroxytoluene (41). The nature and incidence of K-ras mutations were not reported for the latter study.
In conclusion, our results associate, for the first time, ROS release by COX-2 with DNA damage in an important cancer target cell. Induction of COX-2 by mutant K-ras, with resultant increase in ROS and DNA damage, may be part of its mechanism of active oncogenicity in the lung. These results support COX-2 as an attractive target for chemoprevention and intervention strategies.
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Acknowledgments
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We thank Louise R.Finch and Refika B.Turnier for flow cytometric analysis and Dr Alan O.Perantoni for critical comments on the manuscript. This publication has been funded in part with Federal funds from the NIC, NIH, under contract no. NO1-CO-12400.
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References
|
---|
- Johnson,L., Mercer,K., Greenbaum,D., Bronson,R.T., Crowley,D., Tuveson,D.A. and Jacks,T. (2001) Somatic activation of the K-ras oncogene causes early onset lung cancer in mice. Nature, 410, 11111116.[CrossRef][ISI][Medline]
- Jackson,E.L., Willis,N., Mercer,K., Bronson,R.T., Crowley,D., Montoya,R., Jacks,T. and Tuveson,D.A. (2001) Analysis of lung tumor initiation and progression using conditional expression of oncogenic K-ras. Genes Dev., 15, 32433248.[Abstract/Free Full Text]
- Fisher,G.H., Wellen,S.L., Klimstra,D., Lenczowski,J.M., Tichelaar,J.W., Lizak,M.J., Whitsett,J.A., Koretsky,A. and Varmus,H.E. (2001) Induction and apoptotic regression of lung adenocarcinomas by regulation of a K-Ras transgene in the presence and absence of tumor suppressor genes. Genes Dev., 15, 32493262.[Abstract/Free Full Text]
- Meuwissen,R., Linn,S.C., van der Valk,M., Mooi,W.J. and Berns,A. (2001) Mouse model for lung tumorigenesis through Cre/lox controlled sporadic activation of the K-Ras oncogene. Oncogene, 20, 65516558.[CrossRef][ISI][Medline]
- Zhang,Z., Wang,Y., Vikis,H.G. et al. (2001) Wildtype Kras2 can inhibit lung carcinogenesis in mice. Nature Genet., 29, 2533.[CrossRef][ISI][Medline]
- Sundaresan,M., Yu,Z.X., Ferrans,V.J., Sulciner,D.J., Gutkind,J.S., Irani,K., Goldschmidt-Clermont,P.J. and Finkel,T. (1996) Regulation of reactive-oxygen-species generation in fibroblasts by Rac1. Biochem. J., 318 (Pt 2), 379382.[ISI][Medline]
- Irani,K., Xia,Y., Zweier,J.L., Sollott,S.J., Der,C.J., Fearon,E.R., Sundaresan,M., Finkel,T. and Goldschmidt-Clermont,P.J. (1997) Mitogenic signaling mediated by oxidants in Ras-transformed fibroblasts. Science, 275, 16491652.[Abstract/Free Full Text]
- Lee,A.C., Fenster,B.E., Ito,H., Takeda,K., Bae,N.S., Hirai,T., Yu,Z.X., Ferrans,V.J., Howard,B.H. and Finkel,T. (1999) Ras proteins induce senescence by altering the intracellular levels of reactive oxygen species. J. Biol. Chem., 274, 79367940.[Abstract/Free Full Text]
- Liou,J.S., Chen,C.Y., Chen,J.S. and Faller,D.V. (2000) Oncogenic ras mediates apoptosis in response to protein kinase C inhibition through the generation of reactive oxygen species. J. Biol. Chem., 275, 3900139011.[Abstract/Free Full Text]
- Liu,R., Li,B. and Qiu,M. (2001) Elevated superoxide production by active H-ras enhances human lung WI-38VA-13 cell proliferation, migration and resistance to TNF-
. Oncogene, 20, 14861496.[CrossRef][ISI][Medline]
- Chuang,J.I., Chang,T.Y. and Liu,H.S. (2003) Glutathione depletion-induced apoptosis of Ha-ras-transformed NIH3T3 cells can be prevented by melatonin. Oncogene, 22, 13491357.[CrossRef][ISI][Medline]
- Yang,J.-Q., Li,S., Domann,F.E., Buettner,G.R. and Oberley,L.W. (1999) Superoxide generation in v-Ha-ras-transduced human keratinocyte HaCaT cells. Mol. Carcinogen., 26, 180188.[CrossRef][ISI][Medline]
- Yang,J.Q., Li,S., Huang,Y., Zhang,H.J., Domann,F.E., Buettner,G.R. and Oberley,L.W. (2001) V-Ha-Ras overexpression induces superoxide production and alters levels of primary antioxidant enzymes. Antioxid. Redox Signal., 3, 697709.[CrossRef][ISI][Medline]
- Yang,J.Q., Buettner,G.R., Domann,F.E., Li,Q., Engelhardt,J.F., Weydert,C.D. and Oberley,L.W. (2002) v-Ha-ras mitogenic signalling through superoxide and derived reactive oxygen species. Mol. Carcinogen., 33, 206218.[CrossRef][ISI][Medline]
- Sheng,G.G., Shao,J., Sheng,H., Hooton,E.B., Isakson,P.C., Morrow,J.D., Coffey,R.J.Jr, DuBois,R.N. and Beauchamp,R.D. (1997) A selective cyclooxygenase 2 inhibitor suppresses the growth of H-ras-transformed rat intestinal epithelial cells. Gastroenterology, 113, 18831891.[ISI][Medline]
- Sheng,H., Williams,C.S., Shao,J., Liang,P., DuBois,R.N. and Beauchamp,R.D. (1998) Induction of cyclooxygenase-2 by activated Ha-ras oncogene in Rat-1 fibroblasts and the role of mitogen-activated protein kinase pathway. J. Biol. Chem., 273, 2212022127.[Abstract/Free Full Text]
- Sheng,H., Shao,J., Dixon,D.A., Williams,C.S., Prescott,S.M., DuBois,R.N. and Beauchamp,R.D. (2000) Transforming growth factor-beta1 enhances Ha-ras-induced expression of cyclooxygenase-2 in intestinal epithelial cells via stabilization of mRNA. J. Biol. Chem., 275, 66286635.[Abstract/Free Full Text]
- Sheng,H., Shao,J. and Dubois,R.N. (2001) K-Ras-mediated increase in cyclooxygenase 2 mRNA stability involves activation of the protein kinase B1. Cancer Res., 61, 26702675.[Abstract/Free Full Text]
- Fujita,M., Fukui,H., Kusaka,T., Morita,K., Fujii,S., Ueda,Y., Chiba,T., Sakamoto,C., Kawamata,H. and Fujimori,T. (2000) Relationship between cyclooxygenase-2 expression and K-ras gene mutation in colorectal cancer. J. Gastroenterol. Hepatol., 11, 12771281.[CrossRef]
- Smith,G.J., Le Mesurier,S.M., de Montfort,M.L. and Lykke,A.W. (1984) Development and characterization of type 2 pneumocyte-related cell lines from normal adult mouse lung. Pathology, 16, 401405.[ISI][Medline]
- Bentel,J.M., Lykke,A.W. and Smith,G.J. (1989) Cloned murine non-malignant, spontaneously transformed and chemical tumour-derived cell lines related to the type 2 pneumocyte. Cell Biol. Int. Rep., 13, 729738.[ISI][Medline]
- Rook,G.A., Steele,J., Umar,S. and Dockrell,H.M. (1985) A simple method for the solubilisation of reduced NBT, and its use as a colorimetric assay for activation of human macrophages by gamma-interferon. J. Immunol. Methods, 82, 161167.[CrossRef][ISI][Medline]
- Kammouni,W., Ramakrishna,G., Sithanandam,G., Smith,G.T., Fornwald,L.W., Masuda,A., Takahashi,T. and Anderson,L.M. (2002) Increased K-ras protein and activity in mouse and human lung epithelial cells at confluence. Cell Growth Differ., 13, 441448.[Abstract/Free Full Text]
- Singh,N.P., McCoy,M.T., Tice,R.R. and Schneider,E.L. (1988) A simple technique for quantitation of low levels of DNA damage in individual cells. Exp. Cell Res., 175, 184191.[ISI][Medline]
- Reitz,D.B., Li,J.J., Norton,M.B. et al. (1994) Selective cyclooxygenase inhibitors: novel 1,2-diarylcyclopentenes are potent and orally active COX-2 inhibitors. J. Med. Chem., 37, 38783881.[ISI][Medline]
- Nikolic,D. and van Breemen,R.B. (2001) DNA oxidation induced by cyclooxygenase-2. Chem. Res. Toxicol., 14, 351354.[CrossRef][ISI][Medline]
- Hida,T., Yatabe,Y., Achiwa,H., Muramatsu,H., Kozaki,K., Nakamura,S., Ogawa,M., Mitsudomi,T., Sugiura,T. and Takahashi,T. (1998) Increased expression of cyclooxygenase 2 occurs frequently in human lung cancers, specifically in adenocarcinomas. Cancer Res., 58, 37613764.[Abstract]
- Wolff,H., Saukkonen,K., Anttila,S., Karjalainen,A., Vainio,H. and Ristimaki,A. (1998) Expression of cyclooxygenase-2 in human lung carcinoma. Cancer Res., 58, 49975001.[Abstract]
- Hosomi,Y., Yokose,T., Hirose,Y., Nakajima,R., Nagai,K., Nishiwaki,Y. and Ochiai,A. (2000) Increased cyclooxygenase 2 (COX-2) expression occurs frequently in precursor lesions of human adenocarcinoma of the lung. Lung Cancer, 30, 7381.[CrossRef][ISI][Medline]
- Xu,X.-C. (2002) COX-2 inhibitors in cancer treatment and prevention, a recent development. Anti-Cancer Drugs, 13, 127137.[CrossRef][ISI][Medline]
- Sweeney,C.J., Marshall,M.S., Barnard,D.S., Heilman,D.K., Billings,S.D., Cheng,L., Marshall,S.J. and Yip-Schneider,M.T. (2002) Cyclo-oxygenase-2 expression in primary cancers of the lung and bladder compared to normal adjacent tissue. Cancer Detect. Prev., 26, 238244.[CrossRef][ISI][Medline]
- Gridelli,C., Maione,P., Airoma,G. and Rossi,A. (2002) Selective cyclooxygenase-2 inhibitors and non-small cell lung cancer. Curr. Med. Chem., 9, 18511858.[ISI][Medline]
- Ermert,L., Dierkes,C. and Ermert,M. (2003) Immunohistochemical expression of cyclooxygenase isoenzymes and downstream enzymes in human lung tumors. Clin. Cancer Res., 9, 16041610.[Abstract/Free Full Text]
- Richardson,C.M., Sharma,R.A., Cox,G. and O'Byrne,K.J. (2003) Epidermal growth factor receptors and cyclooxygenase-2 in the pathogenesis of non-small cell lung cancer: potential targets for chemoprevention and systemic therapy. Lung Cancer, 39, 113.[CrossRef][ISI][Medline]
- Lin,S.-Y., Tsai,S.-J., Wang,L.-H., Wu,M.-F. and Lee,H. (2002) Protection by quercetin against cooking oil fumes-induced DNA damage in human lung adenocarcinoma CL-3 cells: role of COX-2. Nutr. Cancer, 44, 95101.[CrossRef][ISI][Medline]
- Niki,T., Kohno,T., Iba,S. et al. (2002) Frequent co-localization of Cox-2 and laminin-5 gamma2 chain at the invasive front of early-stage lung adenocarcinomas. Am. J. Pathol., 160, 11291141.[Abstract/Free Full Text]
- Wardlaw,S.A., March,T.H. and Belinsky,S.A. (2000) Cyclooxygenase-2 expression is abundant in alveolar type II cells in lung cancer-sensitive mouse strains and in premalignant lesions. Carcinogenesis, 21, 13711377.[Abstract/Free Full Text]
- Heasley,L.E., Thaler,S., Nicks,M., Price,B., Skorecki,K. and Nemenoff,R.A. (1997) Induction of cytosolic phospholipase A2 by oncogenic Ras in human non-small cell lung cancer. J. Biol. Chem., 272, 1450114504.[Abstract/Free Full Text]
- Harris,R.E., Beebe-Donk,J. and Schuller,H.M. (2002) Chemoprevention of lung cancer by non-steroidal anti-inflammatory drugs among cigarette smokers. Oncol. Rep., 9, 693695.[ISI][Medline]
- Rioux,N. and Castonguay,A. (1998) Prevention of NNK-induced lung tumorigenesis in A/J mice by acetylsalicylic acid and NS-398. Cancer Res., 58, 53545360.[Abstract]
- Kisley,L.R., Barrett,B.S., Dwyer-Nield,L.D., Bauer,A.K., Thompson,D.C. and Malkinson,A.M. (2002) Celecoxib reduces pulmonary inflammation but not lung tumorigenesis in mice. Carcinogenesis, 23, 16531660.[Abstract/Free Full Text]
Received October 31, 2003;
revised June 24, 2004;
accepted July 21, 2004.