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Cancer Research 67, 9248, October 1, 2007. doi: 10.1158/0008-5472.CAN-07-1764
© 2007 American Association for Cancer Research
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Cell, Tumor, and Stem Cell Biology

Glutathione Transferase {pi} Plays a Critical Role in the Development of Lung Carcinogenesis following Exposure to Tobacco-Related Carcinogens and Urethane

Kenneth J. Ritchie1, Colin J. Henderson1, Xiu Jun Wang1, Olga Vassieva1, Dianne Carrie1, Peter B. Farmer2, Margaret Gaskell2, Kevin Park3 and C. Roland Wolf1

1 Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Ninewells Hospital and Medical School, Dundee, United Kingdom; 2 Cancer Biomarkers and Prevention Group, Biocentre, University of Leicester, Leicester, United Kingdom; and 3 Department of Pharmacology and Therapeutics, University of Liverpool, Liverpool, United Kingdom

Requests for reprints: C. Roland Wolf, Cancer Research UK Molecular Pharmacology Unit, Biomedical Research Centre, Level 5, Ninewells Hospital and Medical School, Dundee DD1 9SY, United Kingdom. E-mail: Roland.Wolf{at}cancer.org.uk.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Human cancer is controlled by a complex interaction between genetic and environmental factors. Such environmental factors are well defined for smoking-induced lung cancer; however, the roles of specific genes have still to be elucidated. Glutathione transferase {pi} (GSTP) catalyzes the detoxification of electrophilic diol epoxides produced by the metabolism of polycyclic aromatic hydrocarbons such as benzo[a]pyrene (BaP), a common constituent of tobacco smoke. Activity-altering polymorphisms in Gstp have therefore been speculated to be potential risk modifiers in lung cancer development. To clearly establish a role for GSTP in lung tumorigenesis, we investigated whether deletion of the murine Gstp genes (Gstp1 and Gstp2) alters susceptibility to chemically induced lung tumors following exposure to BaP, 3-methylcholanthrene (3-MC), and urethane. Gstp-null mice were found to have substantially increased numbers of adenomas relative to wild-type mice following exposure to all three compounds (8.3-, 4.3-, and 8.7-fold increase for BaP, 3-MC, and urethane, respectively). In Gstp-null mice, the capacity of pulmonary cytosol to catalyze conjugation of the BaP diol epoxide was significantly reduced. Concomitant with this, a significant increase in the level of BaP DNA adducts was measured in the lungs of null animals; however, no increase in DNA adducts was measured in the case of 3-MC exposure, suggesting that an alternative protective pathway exists. Indeed, significant differences in pulmonary gene expression profiles were also noted between wild-type and null mice. This is the first report to establish a clear correlation between Gstp status and lung cancer in vivo. [Cancer Res 2007;67(19):9248–57]


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Smoking is the leading cause of lung cancer–related death in the world. Each cigarette contains a complex mixture of polycyclic aromatic hydrocarbons (PAH) along with other lung carcinogens, tumor promoters, and cocarcinogens (1). PAHs are activated by the mammalian cytochrome P450 system and epoxide hydrolase to electrophilic reactive metabolites, which function as carcinogenic initiators (2, 3). Glutathione S-transferases (GST) are a multigene family of dimeric enzymes (EC 2.5.1.18; GST {alpha}, µ, {pi}, {theta}, {sigma}, {zeta}, {kappa}, and {omega}) identified on the basis of their amino acid sequence and substrate specificity (4). GSTs are regarded as being important detoxification enzymes due to their capacity to catalyze the addition of reduced glutathione (GSH) to reactive electrophiles produced by cytochrome P450 metabolism. As a consequence, there has been a significant interest in elucidating the relationship between GSTP function and resistance to cancer chemotherapeutic agents and the development of cancer (5–7). In a genetic approach to study GST functions, we have generated mice nulled at the Gstp gene locus (8). These mice develop normally, are fertile, and show no obvious abnormalities. Topical application of the skin tumor–inducing PAH 7,12-dimethylbenz(a)anthracene followed by the tumor-promoting agent 12-O-tetradecanoylphorbol-13-acetate resulted in a significant increase in the number of papillomas in null animals, showing that GSTP is an important determinant in PAH-induced skin cancer susceptibility (8).

There are many reports speculating on a relationship between GSTP and susceptibility to lung cancer in humans (for review, see ref. 9). These studies are mostly epidemiologic in nature and, for this reason, are complex and the results obtained are equivocal. We have therefore used the Gstp-null mouse model to study the roles of GSTP in lung tumorigenesis under controlled conditions that are not affected by the variables that confound epidemiologic studies. Primary lung tumors in mice, whether chemically or genetically induced, share key biochemical, morphologic, and histologic features with the human disease (10, 11). Consequently, to investigate the role of GSTP in lung cancer, we exposed Gstp-null mice to two PAHs and the carbamate urethane. The absence of GSTP resulted in significantly increased rates of lung adenoma formation for all compounds tested. However, it seemed that the change in lung cancer susceptibility could not be simply rationalized by the capacity of GSTP to reduce carcinogen DNA adduct levels.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents. 7R,8S,9S-Trihydroxy-10S-(N2-deoxyguanosyl-3'-phosphate)-7,8,9,10-tetrahydrobenzo[a]pyrene (BPDE adduct standard) was purchased from National Cancer Institute (NCI) Chemical Carcinogen Repository, Midwest Research Institute. Genomic DNA extraction kits were obtained from Qiagen Ltd. Calf spleen phosphodiesterase was obtained from Merck Biosciences Ltd. and T4 polynucleotide kinase (3'-phosphatase-free) was purchased from Roche. [{gamma}-32P]ATP (>185 TBq/mmol, >5,000 Ci/mmol) was purchased from Amersham International. TLC plates were obtained from VWR and Camlab. (+/–)-Anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE) was purchased from the NCI Chemical Repository. [3H]GSH (specific activity, 52 µCi/nmol) was supplied by New England Nuclear. Complete protease inhibitor tablets were purchased from Roche. All other chemicals were of the highest grade available and were purchased from Sigma or Fisher Scientific Ltd.

Animals. All experiments were undertaken in accordance with the criteria outlined in a license granted under the Animals (Scientific Procedures) Act 1986 and approved by the Animal Ethics Committees of the University of Dundee and Cancer Research UK. Gstp1/p2-null and wild-type mouse lines, on a 129 x MF1 background, were generated and maintained by random intercrossing to sustain a heterogeneous mixed genetic background as previously reported (8).

Administration of PAHs. Eight-week-old, male and female wild-type and Gstp-null mice were given a single i.p. dose of benzo[a]pyrene (BaP), 3-methylcholanthrene (3-MC), or urethane at 200 mg/kg, 100 mg/kg, and 10 mg/mouse, respectively. Control mice received equivalent doses of the tricaprylin vehicle only. At time of death or 5 months later, mice were sacrificed and lungs harvested directly into Tellyesniczky's fluid (for 24 h at 4°C to affect fixation and to reveal lung surface adenomas). To allow histologic assessment of lung tumors, lung tissue was then processed for H&E stains using standard procedures.

Isolation and culture of mouse lung fibroblasts. Lungs from adult male Gstp-null and wild-type mice were harvested under sterile conditions, homogenized, and digested with trypsin (0.25%) at 37°C for 30 min with stirring. The resultant single-cell suspension was then cultured in a 95% air/5% CO2 37°C atmosphere with a 50:50 DMEM/Ham's F-12 medium containing 10% FCS. Confluent monolayers were then passaged and maintained in the above medium using standard techniques.

Cell lysis. For harvest, the medium was aspirated and the cells were washed with 5 mL of ice-cold PBS and then lysed in 50 mmol/L Tris-HCl (pH 7.5), 1 mmol/L EDTA, 1 mmol/L EGTA, 1 mmol/L sodium orthovanadate, 10 mmol/L sodium ß-glycerophosphate, 50 mmol/L sodium fluoride, 5 mmol/L sodium pyrophosphate, 0.27 mol/L sucrose, 1% (v/v) Triton X-100, 0.1% (v/v) 2-mercaptoethanol, and protease inhibitor cocktail (1 tablet/50 mL). The lysates were then centrifuged at 16,000 x g for 10 min at 4°C and the supernatants stored at –20°C.

Immunoblotting. Samples were denatured in SDS before being run on polyacrylamide gels, after which they were transferred onto nitrocellulose membranes. The membranes were then blocked for 1 h at room temperature in 50 mmol/L Tris-HCl (pH 7.5), 150 mmol/L NaCl, 0.2% Tween (TBST), and 5% (w/v) bovine serum albumin (BSA), for Biosource antibodies, or 5% skimmed milk powder, for Cell Signaling antibodies. Membranes were then incubated overnight at 4°C with gentle rocking with either a phosphospecific antibody, which recognized the activated forms of c-jun NH2-terminal kinase (JNK)-1 and JNK2 (Biosource), in TBST containing 3% BSA or with an antibody, which recognized the inactive JNK isoforms (Cell Signaling Technology), in TBST containing 5% BSA. The following day, membranes were washed thrice with TBST (5 min per wash) and then exposed to secondary antibody in TBST containing 5% (w/v) skimmed milk powder. Membranes were then washed four times with TBST (5 min per wash) and immunoreactive proteins visualized with enhanced chemiluminescence plus (Amersham) reagent according to the manufacturer's instructions. Stripping of membranes, if required, was carried out using Re-Blot Plus mild antibody stripping solution (Chemicon International).

DNA adducts. For investigation of DNA adduct formation, 8-week-old male and female wild-type and Gstp-null mice were given a single i.p. dose of either BaP or 3-MC at 200 and 100 mg/kg, respectively. Five days later, lungs were harvested and DNA was extracted using anion-exchange resin columns according to the manufacturer's instructions (Qiagen). Samples were analyzed by 32P postlabeling in a minimum of three independent experiments. DNA samples (10 µg) were enzymatically digested to deoxyribonucleoside 3'-monophosphates using micrococcal nuclease (350 mU) and calf spleen phosphodiesterase (20 mU), at 37 °C for 15 h. DNA samples from 3-MC–treated animals underwent adduct enrichment based on the nuclease P1 digestion method originally described by Reddy and Randerath (12). In brief, a proportion of the DNA was retained for 32P postlabeling (using 20 µCi of [{gamma}-32P]ATP and 5 units of T4 polynucleotide kinase) of "total" nucleotides and the remainder was further digested with nuclease P1 (18 µg; 2 µg/µL in 0.28 mol/L sodium acetate, 0.5 mmol/L zinc chloride, pH 5.0) at 37°C for 1 h. DNA samples from BaP-treated animals underwent adduct enhancement by butanol extraction using the modified version as described by Phillips and Castegnaro (13). Adducted nucleotides including the BPDE adduct standard were radiolabeled by 5'-phosphorylation using 12.5 µCi of [{gamma}-32P]ATP and 1.25 units of T4 polynucleotide kinase per microgram of DNA. Incubation was carried out for 1 h at 37°C. 32P postlabeled adducts were subjected to separation by either TLC (3-MC–treated samples) or high-pressure liquid chromatography (BaP-treated DNA samples) as described below.

High-pressure liquid chromatography of adducts. High-pressure liquid chromatography was carried out as previously published (14). For quantification of adducts, known amounts of [{gamma}-32P]ATP were injected onto the high-pressure liquid chromatography. Eluate corresponding to the ATP peak was collected and radioactivity was determined by scintillation counting [measured in disintegrations per minute (dpm)] and compared with theoretical dpm values. A calibration line based on peak areas from high-pressure liquid chromatography and corresponding values from scintillation counting (or theoretical values) was plotted. Dpm values for the actual samples were read from the calibration line and relative adduct labeling was calculated based on the method previously described (12).

TLC of adducts. 32P postlabeled adduct samples were applied to 10 x 15-cm PEI-cellulose TLC plates. TLC plates were initially developed in D1 (2.3 mol/L sodium phosphate, pH 5.8) onto filter paper wicks. Adducts were then separated by two-dimensional chromatography using solvent systems D2 (8.5 mol/L urea, 4.0 mol/L lithium formate, pH 3.5) and D3 (8.5 mol/L urea, 0.8 mol/L lithium chloride, 0.5 mol/L Tris-HCl, pH 8.5). Finally, plates were developed in D4 (1.7 mol/L sodium phosphate, pH 6.0) onto a filter paper wick. Radiolabeled total nucleotides were applied to TLC plates and developed in one direction using 0.12 mol/L sodium phosphate, pH 6.8. Visualization of TLC plates was carried out using phosphorimager cassettes, which were scanned using a phosphorimager, and analysis of the TLC plates was carried out using ImageQuant software (Molecular Dynamics). Adduct quantitation was carried out using the method of Gupta et al. (15).

Determination of GST-mediated GSH conjugation of BPDE. Incubations contained 1 mg/mL of lung cytosolic protein and 2 mmol/L GSH in 50 mmol/L Tris-HCl buffer (pH 7.5) to give a final volume of 200 µL. Spontaneous conjugation was determined in the absence of cytosol. BPDE or GSH was omitted from control incubations. Samples were placed in a 37°C water bath and reactions initiated by the addition of BPDE (final concentration, 1–50 µmol/L). After 1 min, the reaction was terminated by the addition of ice-cold acetonitrile (600 µL). Following incubation on ice for 10 min and centrifugation at 18,000 x g (5 min), the combined supernatants of two extractions of the protein pellet were evaporated to dryness under a stream of nitrogen gas. The residue was reconstituted in 50 mmol/L Tris-HCl (pH 7.5; 100 µL) for immediate analysis by high-pressure liquid chromatography.

Aliquots (20 µL) were eluted from a HyPurity 5-µm C-18 column (150 x 4.6 mm; ThermoHypersil) with acetonitrile in trifluoroacetic acid (0.1%) at 1 mL/min. Peak area measurements of absorbance (247 nm) were used to quantify the BPDE-GSH conjugates formed and were compared with authentic radiolabeled standards [prepared from a 2-h incubation containing BPDE (125 µmol/L), 5 mg/mL wild-type hepatic cytosol, and [3H]GSH (20 µCi)]. The identification of the BPDE-GSH conjugates was confirmed by liquid chromatography-mass spectrometry.

Microarray analysis. Mice were sacrificed by an increasing concentration of CO2 and lungs were immediately removed for preparation of RNA. Total RNA was isolated using a phenol-guanidine isothiocyanate reagent, TRIzol (Invitrogen Ltd.), and further purified with an RNeasy Mini Kit (Qiagen) in accordance with the manufacturer's instructions. RNA was pooled from three animals of each genotype, and subsequent hybridizations were carried out in duplicate. The A260/280 ratio of total RNA used was typically ≥1.9. The quality of RNA was assessed by using an Agilent 2100 Bioanalyzer (Agilent Technologies).

The probe labeling and hybridization procedures were conducted by following the Affymetrix Technical Manual (Affymetrix). cDNA was synthesized from the total RNA by using Superscript ds-cDNA synthesis kit (Invitrogen) with a T7-(dT)24 primer incorporating a T7 RNA polymerase promoter. The cRNA was prepared and biotin labeled by in vitro transcription by using BioArray High Yield RNA Transcript Labeling Kit (ENZO Biochemical). Labeled cRNA was fragmented by incubation at 94°C for 35 min in the presence of 40 mmol/L Tris-acetate (pH 8.1), 100 mmol/L potassium acetate, and 30 mmol/L magnesium acetate. The samples were tested with hybridization to GeneChip Test3 arrays and analyzed by Agilent Bioanalyzer. Fragmented cRNA (15 µg) was hybridized for 16 h at 45°C to an MG_U74Av2 array (Affymetrix). After hybridization, the gene chips were washed and stained with streptavidin-phycoerythrin by using a fluidics station (Affymetrix). The arrays were scanned in an Agilent G2500A scanner. Affymetrix oligonucleotide microarrays use multiple perfect-match and mismatch oligonucleotides to determine expression levels, and Affymetrix GCOS software was used to scan, determine the presence and the average difference value, and assess signal intensity of each probe set. Chip fluorescence was normalized by scaling total chip fluorescence intensity to a common value of 100 before comparison, and a normalization value was set at 1. Data and statistical analyses were done with Genespring v6.1 (Silicon Genetics) bioinformatics algorithms.

Statistical analysis. We carried out statistical analyses using StatView 4.5 for Macintosh. Significant differences when comparing two groups were determined by unpaired t test. P < 0.05 was considered to be significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Gstp-null mice have increased incidence and multiplicity of lung adenomas. Following i.p. administration of the carcinogens to be tested, murine lung adenomas were readily identified by the naked eye as yellowish-white, discrete nodules located in the lung periphery, just below the visceral pleura (Fig. 1A ). Histologic examination of such tissue revealed mainly papillary and mixed solid/papillary lesions that did not differ in type between Gstp-null and wild-type mice following treatment with 3-MC, BaP, and urethane (Fig. 1B and C). Gstp-null mice exposed to BaP and urethane exhibited a marked increase in lung tumor incidence (the number of mice with at least a single adenoma) relative to wild-type mice (71% versus 29% and 92% versus 29%, respectively; Table 1 ). This, however, was not the case following 3-MC exposure as both Gstp-null and wild-type mice exhibited approximately equivalent incidences of at least a single tumor (75% versus 80%; Table 1). In addition to the increased incidence, an 8-fold increase in tumor multiplicity (average number of tumors per mouse) was also observed in Gstp-null mice treated with BaP, consistent with the role of Gstp in the conjugation of the highly carcinogenic metabolite of BaP, (+)-anti-benzo[a]pyrene-7,8-diol-9,10-epoxide (BPDE; Table 1; ref. 16). A statistically significant increase in tumor multiplicity relative to wild-type mice also occurred in null mice following exposure to both 3-MC (4-fold) and urethane (9-fold; Table 1), indicating that GSTP also plays an important role in the tumorigenesis induced by these compounds in vivo. Further analysis indicated that there was a sex difference in the effects observed in BaP-treated Gstp nulls in that 100% of females developed at least a single adenoma compared with only 43% of males (Table 2 ). Tumor multiplicity was also significantly increased in both BaP- and 3-MC–treated Gstp-null females relative to males by ~5-fold (Table 2). Additionally, in the case of 3-MC, there was a profound increase in lethality, with only 44% of Gstp-null mice surviving by week 22 (Fig. 1D). This effect was not observed in the case of the other two carcinogens and may be related to the larger number of adenomas formed. Significantly, apart from the noted pulmonary adenomas, no other tumors were observed in the liver, kidney, spleen, bladder, or gastrointestinal tract of either Gstp-null or wild-type mice following dosing with 3-MC, BaP, or urethane.


Figure 1
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  		Figure 1. Increased adenoma number and decreased survival in Gstp1/p2-null mice following carcinogen exposure. A, adenomas (arrowheads) are visible on the surface of the lungs of Gstp1/p2+/+ (left) and Gstp1/p2–/– (right) mice following exposure to 3-MC. B, gross histology of lung tumor found in Gstp1/p2+/+ (left) and Gstp1/p2–/– (right) mice following 3-MC exposure. Magnification, x4. C, detailed histology of lung tumor found in Gstp1/p2+/+ (left) and Gstp1/p2–/– (right) mice following 3-MC exposure. Magnification, x20. D, survival curve of Gstp1/p2+/+ and Gstp1/p2–/– mice following a single i.p. dosing with 3-MC (100 mg/kg; n = 7–16 per group).

 

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  		Table 1. Incidence and multiplicity of lung tumorigenesis in Gstp1/p2+/+ and Gstp1/p2–/– mice treated with BaP, 3-MC, and urethane

 

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  		Table 2. Sex differences in incidence and multiplicity of lung tumorigenesis in Gstp1/p2+/+ and Gstp1/p2–/– mice treated with BaP, 3-MC, and urethane

 
Increased DNA adducts in the lungs of Gstp-null mice. To investigate the mechanism of the increased tumor incidence of lung adenomas, we examined the levels of PAH DNA adducts. Following BaP administration, a 4-fold increase in DNA adducts was observed in Gstp nulls relative to controls (Fig. 2A ). This difference was more marked in male (2-fold) than in female Gstp-null animals (Fig. 2B). In the case of 3-MC, however, no differences in adduct levels were observed (Fig. 2C). The higher BPDE adduct levels in Gstp-null animals is consistent with a role for GSTP in the detoxification of electrophilic species produced by metabolic activation. However, the larger increase in DNA adducts observed in null males is not consistent with the higher level of tumorigenesis in females.


Figure 2
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  		Figure 2. DNA adduct levels in mice treated with PAH carcinogens for 5 d. A, representative chromatograms of BPDE DNA adducts in Gstp1/p2+/+ and Gstp1/p2–/– male mice lungs following a single 200 mg/kg i.p. dose of BaP. Peak 1 represents the major BPDE adduct (top), untreated Gstp1/p2–/– mice (top middle), BaP-treated Gstp1/p2–/– mice (bottom middle), and BaP-treated Gstp1/p2+/+ mice (bottom); note difference in scale in bottom graph versus other graphs. Quantification of DNA adduct levels in lungs of male and female Gstp1/p2+/+ and Gstp1/p2–/– mice. B, BPDE-DNA adducts following a single 200 mg/kg i.p. dose of BaP (n = 8). C, 3-MC DNA adducts following a single 100 mg/kg i.p. dose of 3-MC (n = 4). Columns, mean; bars, SE. *, P < 0.05; **, P < 0.01, Gstp1/p2+/+ versus Gstp1/p2–/– (unpaired Student's t test). D, BPDE-GSH conjugate formation in lung cytosolic fractions from Gstp1/p2+/+ and Gstp1/p2–/– mice. Values, mean following subtraction of the spontaneous rate; bars, SE. **, P < 0.01; *, P < 0.05, for Gstp1/p2+/+ versus Gstp1/p2–/– (unpaired Student's t test; n = 4).

 
Gstp-null animals have reduced capacity to conjugate BPDE. To further investigate the change in detoxification capacity of wild-type and Gstp-null animals, we examined the production of BPDE-GSH conjugates in mouse lung cytosol fractions in vitro. The addition of cytosolic fractions to the incubations significantly increased the rate of BPDE-GSH conjugate formation over the spontaneous rate, indicating an enzymatic reaction. The conjugation of BPDE in pulmonary cytosol from Gstp-null mice was, however, markedly reduced relative to wild-type controls for both diol epoxide enantiomers (Fig. 2D). At 25 µmol/L (+)-anti-BPDE, the rate was 3-fold lower in Gstp-null samples (0.25 ± 0.02 pmol/min) compared with wild-type animals (0.74 ± 0.07 pmol/min).

For (–)-anti-BPDE, a similar reduction in detoxification capacity was observed. At 25 µmol/L, there was a 2.2-fold decrease in the rate of (–)-anti-BPDE-GSH formed in null samples relative to wild-type samples (0.26 ± 0.01 versus 0.57 ± 0.06 pmol/min; Fig. 2D). The above data show that GSTP plays a major role in BPDE conjugation and detoxification in the murine lung.

Increased JNK activity in lung cells from Gstp-null mice. Half of all human lung adenocarcinomas are found to contain activating mutations of the K-ras oncogene. In the mouse, K-ras mutations are found in more than 90% of spontaneous and chemically induced lung tumors (11). Further, studies suggest that c-jun NH2-terminal kinase (JNK) is important in ras-mediated cellular transformation (17, 18). Previously, it has been reported that Gstp-null mice show increased activity of JNK in both liver and lung (19). In view of this, and the suggested role of JNK activation in carcinogenesis (for review, see ref. 20), we investigated JNK activity in isolated lung fibroblasts from Gstp-null and wild-type animals. Intriguingly, significantly increased phospho-JNK levels were observed in Gstp-null samples (Fig. 3 ). The increase in JNK activity in cells derived from the lungs of Gstp1/p2-null mice is consistent with the role of GSTP acting to inhibit JNK activity (21). This raises the possibility that GSTP may affect lung tumorigenesis by affecting downstream signaling pathways rather than affecting the levels of DNA carcinogen adducts.


Figure 3
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  		Figure 3. Deficiency of GSTP results in increased JNK phosphorylation levels in mouse lung fibroblasts. A, Western blot analysis of phosphorylated JNK levels in lung fibroblast cells from Gstp1/p2+/+ and Gstp1/p2–/– mice. Blots were then stripped and probed for total JNK levels (B). The same results were obtained in several independent experiments. Samples were run on 10% PAGE gels using 20 µg of total cellular protein per track. Western blots were carried out with anti–phospho-JNK antibody (1:1,000) and JNK antibody (1:1,000) as described in Materials and Methods.

 
Changes in gene expression in Gstp-null mouse lungs. To further characterize how pulmonary physiologic gene expression may be altered in Gstp-null mice, we carried out mRNA expression profiling by microarray analysis. In Gstp-null lungs, the expression of 61 genes was increased, and that of 14 genes was decreased, by a factor of 2-fold or greater relative to wild-type lung (Table 3 ). Gstp2 mRNA was found to be absent from Gstp-null lung tissue, consistent with deletion of the gene.


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  		Table 3. Genes differentially expressed in Gstp-null mouse lungs relative to wild-type controls

 
Interestingly, genes involved in mucin production (Gob-5 and GOB-4) and inflammation (Reg3g, Saa3, Hspa1a, and Hspa1b) featured prominently in the 61 genes found to be up-regulated, as did genes involved in sterol metabolism (cytochrome P450) and general metabolism (Ucp1 and Etfdh). Differential expression relative to wild-type mice was also noted for several genes that show an association with cancer (pip5k2c, Psca, and Oaz1). Among those genes whose expression was down-regulated, cell motility and cytoskeletal genes (Dynlt1 and Myl2) and genes involved in ion transport (Kcjn15) and signal transduction (Zfp68) were found. Clearly, deletion of the murine Gstp1/p2 genes has a fundamental effect on the pulmonary environment, which may facilitate the development of cancer following administration of PAHs and urethane.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In common with other organs that encounter environmental pollutants (liver, intestinal tract, and the skin), the pulmonary system has evolved a number of defenses to detoxify and remove such chemicals. Here, we have investigated the consequence of the removal of one these defense mechanisms, GSTP, on the development of lung cancer as induced by exposure to three chemical compounds. The results reported herein unequivocally show that GSTP has a key role in determining susceptibility to lung cancer following exposure to carcinogens.

The pulmonary system is composed of a heterogeneous cellular population, reflecting the requirement for many specialized tasks within the organ as a whole. Interestingly, alveolar type II cells (ATII cells), which produce surfactant and serve as progenitors for alveolar type I cell (ATI cells), and Clara cells, which make up 70% of the bronchiolar epithelium, are found to contain both cytochrome P450s (and hence the potential for metabolic activation of carcinogens) and GSTP (22–24). Indeed, alveolar type II cells and Clara cells are widely thought to be the originating cells of lung cancer (25). Gstp-null mice thus provided an excellent model with which to study lung tumor development.

Murine lung cancer progresses through a multistage developmental lineage from hyperplasia to adenoma and then on to carcinoma (26). In this study, we used adenoma number as an index of tumor susceptibility, based on a mouse lung adenoma bioassay (27). The study involved two PAHs, benzo[a]pyrene and 3-MC, representing the class of chemicals found in tobacco smoke and conclusively established as the source of DNA adducts responsible for coding mutations within critical regions of oncogenes such as RAS and MYC and in tumor suppressor genes such as TP53 and CDKN2A, the loss of normal cellular growth control mechanisms, and the development of cancer (28). The study also included the carbamate urethane, which has been extensively used to produce pulmonary adenomas in mice. Each compound tested increased tumor burden in both genotypes relative to control animals receiving vehicle only. Consistent with a protective role for GSTP, Gstp-null mice were found to have increased numbers of adenomas relative to wild-type mice. This study, however, also reveals that 3-MC induced significantly more adenomas per null mouse compared with either BaP or urethane, and may reflect a difference in the importance of GSTP for the detoxification of different carcinogens. This would seem to be substantiated by the increased lethality observed in Gstp-null mice following 3-MC exposure (Fig. 1D). We also show that there is a significant sex difference in Gstp-null mice in response to BaP; whether this relates directly to the Gstp gene requires further investigation. Interestingly, increased lung carcinogenesis in female CD-1 mice following BaP exposure has previously been reported as has lower expression of GSTP in female mouse liver (29, 30).

Consistent with the evidence that GSTs may be involved in the detoxification of urethane via the metabolism of its carcinogenic derivative vinyl carbamate epoxide, the results reported herein are also the first to implicate GSTP as an important determinant of tumor development following exposure to urethane, a carcinogen commonly encountered via the consumption of fermented foods and alcohol (31, 32).

The use of Gstp-null mice in this study has allowed the investigation of the consequences of GSTP1 deletion in a stable genetic background under controlled conditions of carcinogenic exposure. Consistent with the observation of increased adenoma numbers in Gstp-null mice, Gstp1 gene deletion is found to significantly increase the formation of pulmonary DNA adducts in vivo following exposure to BaP. Furthermore, analysis of the generation of the BPDE-GSH conjugate shows that the loss of Gstp1 and Gstp2 in null mice leads to a significant decrease in levels of this conjugate relative to wild-type mice. Importantly, however, GSTP only accounts for 17% of the total GST activity toward the model substrate 1-chloro-2,4-dinitrobenzene in mouse tissue (33). In contrast, GSTP is responsible for more than 90% of the GST activity within the adult human lung epithelial cell population (34), suggesting that the role of GSTP in BPDE detoxification in human lung is of great importance. Interestingly, in the liver, the rate of BPDE-GSH formation in Gstp1/p2-null mice is equivalent to the spontaneous rate, indicating that GSTP is the sole determinant of hepatic BPDE-GSH formation (data not shown). In this regard, it is feasible that the difference in pulmonary DNA adduct levels, and therefore lung tumors, observed between Gstp-null and wild-type mice in the case of BaP may be due to an increased circulating level of the diol epoxide metabolite in the null. In the case of 3-MC exposure, the lack of increased DNA adduct levels in the lungs of Gstp-nulls relative to wild-type mice is, however, intriguing and suggests that Gstp may influence carcinogenesis, particularly in the case of 3-MC, by an alternative mechanism. This is also substantiated by the finding that in the case of BaP, the effects of Gstp deletion, in terms of adduct formation, were much greater in males, yet tumor incidence was higher in females treated with this compound.

In murine skin tumorigenesis studies, 3-MC has been found to cause a distinct mutation pattern (codons 13 and 61) in the Ha-ras gene (35, 36). In primary lung tumors in mice, Ki-ras has been found to be mutated, specifically in codon 12 or 13, following exposure to 3-MC (37), and polymorphism in the Ki-ras gene has been shown to be predictive of lung tumor susceptibility in mice (10). Importantly, it is worthy to note that we have also reported in this study that lung fibroblasts derived from Gstp-null mice show increased phosphorylation of JNK relative to wild-type mice, consistent with previously published data (19). Significantly, studies suggest that JNK activity is required for efficient tumor transformation and tumorigenesis by Ras (17, 38). The possibility thus exists that tumors with the same tissue origin may undergo tumorigenesis, independent of DNA adduct status but dependent on the carcinogen used and the level of JNK activity as controlled by GSTP expression.

To further investigate the basal pulmonary environment that exists within the Gstp-null mouse lung, we carried out microarray analysis on lung tissue derived from both Gstp-null and wild-type tissues. Interestingly, we report that the deletion of Gstp results in the creation of an inflamed, protumorigenic environment within the lung. Several of the most up-regulated genes were found to be associated with asthma. Indeed, the most up-regulated gene, Gob-5 (24-fold up-regulated), has been identified as a key molecule in the induction of murine asthma and has been strongly associated with mucin gene regulation and goblet cell hyperplasia (39, 40). Further evidence of inflammation is indicated by the 7.7-fold up-regulation of Trefoil factor 2, which is strongly induced by allergens and interleukins, and serum amyloid A3 (up-regulated 5.9-fold), which is thought to have a role in local responses to injury and inflammation (41). Interestingly, several P450s (Cyp3A11, Cyp3A16, and Cyp4A14) were also found to be up-regulated (2.3-, 2.9-, and 3.2-fold, respectively) in the lungs of the null mouse. Evidence of a protumorigenic environment was seen in the 2-fold up-regulation of phosphatidylinositol-4-phosphate 5-kinase, type II-{gamma}, which has been correlated with malignancies (42). We also noted that there is a 2.9-fold repression of ornithine decarboxylase (ODC) antizyme, a polyamine-induced cellular protein that binds to ODC and targets it for rapid ubiquitin-independent degradation by the 26S proteasome (43). Interestingly, increased activity of ornithine decarboxylase is widely believed to be a contributing factor in tumorigenesis (44).

Analysis of the microarray data presented here clearly reveals an elevated level of basal inflammation in the lungs of Gstp-null animals and an environment that is enabled for cancer development. Importantly, however, the lack of tumor incidence observed in control (untreated) animals indicates that this environment is not in itself sufficient for cancer development and further highlights the importance of GSTP in prevention of lung cancer following exposure to PAHs.

Epidemiologic evidence has hypothesized that GSTP is an important factor in individual susceptibility to smoking-induced lung cancer. Consequently, it has been of key importance to verify this in vivo. By using the Gstp-null mouse model, we have significantly substantiated this hypothesis and underlined the fundamental importance of GSTP in the detoxification of BaP, a major constituent of tobacco smoke, in vivo. Further, the microarray data suggest that Gstp1/p2 plays an important role in lung inflammatory responses in a manner that may influence the protumorigenic microenvironment. There are now several reports that indicate novel functions for GSTP that are not related to its catalytic activities; the surprising findings that Gstp-null mice are less susceptible to paracetamol toxicity and less likely to develop colon cancer are of particular interest (45, 46). Further, we have found that Gstp-null mice have increased tumor multiplicity and decreased life expectancy following 3-MC exposure relative to wild-type mice in the absence of elevated DNA adduct levels. Additionally, we also report that GSTP confers resistance to tumorigenesis induced by the non-PAH urethane. Alternative mechanisms for the effects of GSTP on tumorigenesis induced by PAHs therefore seem to exist. Indeed, the noncatalytic modulation of the JNK cell signaling cascade by GSTP and the recent report suggesting that GSTP can suppress tumor necrosis factor-{alpha}– and TRAF2-ASK1–triggered cell death may give some clues about alternative functions (21, 47). On the basis of the current work, it is predicted that variability in GSTP expression or function will influence lung tumorigenesis. Despite uncertainties about the mechanism of these effects, the results reported here indicate that Gstp plays a key role in vivo in determining susceptibility to lung cancer following exposure to chemical carcinogens of the type commonly found in tobacco smoke.


   Acknowledgments
 
Grant support: Cancer Research UK.

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.

We thank Mark Chamberlain for help with microarray analysis and Prof. Stewart Fleming for advice on mouse lung pathology.


   Footnotes
 
Note: K.J. Ritchie and C.J. Henderson contributed equally to this work.

Received 5/15/07. Revised 7/ 3/07. Accepted 7/31/07.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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