Carcinogen exposure differentially modulates RAR-ß promoter hypermethylation, an early and frequent event in mouse lung carcinogenesis

Brian R. Vuillemenot, Leah C. Pulling, William A. Palmisano, Julie A. Hutt and Steven A. Belinsky1

Lung Cancer Program, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108, USA


    Abstract
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The retinoic acid receptor beta (RAR-ß) gene encodes one of the primary receptors for retinoic acid, an important signaling molecule in lung growth, differentiation and carcinogenesis. RAR-ß has been shown to be down-regulated by methylation in human lung cancer. We have used previously lung tumors induced in mice to evaluate the timing and effect of specific carcinogen exposures on targeting genes altered in human lung cancer. These studies were extended to characterize the role of methylation of the RAR-ß gene in murine lung cancers. After treatment with the demethylating agent 5-aza-2'-deoxycytidine (DAC), RAR-ß was re-expressed in silenced cell lines or expressed at a higher rate than without DAC, supporting methylation as the inactivating mechanism. Bisulfite sequencing detected dense methylation in the area of the CpG island that contained the 5' untranslated region and the first translated exon in non-expressing cell lines, compared with minimal and heterogeneous methylation in normal mouse lung. Methylation-specific PCR revealed that this gene is targeted differentially by carcinogen exposures with the detection of methylated alleles in virtually all primary tumors associated with cigarette smoke or 4-methylnitrosamino-1-(3-pyridyl)-butanone (NNK) in contrast to half of tumors induced by methylene chloride or vinyl carbamate. RAR-ß methylation was also detected in 54% of preneoplastic hyperplasias induced by treatment with NNK. Bisulfite sequencing of both premalignant and malignant lesions detected dense methylation in the same area observed in cell lines, substantiating that this gene is functionally inactivated at the earliest histologic stage of adenocarcinoma development. These studies demonstrate that aberrant methylation of RAR-ß is an early and common alteration in murine lung tumors induced by several environmentally relevant exposures.

Abbreviations: DAC, 5-aza-2'-deoxycytidine; ER, estrogen receptor-{alpha}; MSP, methylation-specific PCR; NNK, 4-methylnitrosamino-1-(3-pyridyl)-butanone; RARE, retinoic acid response elements; RAR-ß, retinoic acid receptor-ß


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Epigenetic events, primarily aberrant promoter methylation, are a major mechanism of gene silencing in lung cancer development and progression (1). Genes with diverse functions that include cell cycle control (2), DNA repair (3), carcinogen detoxification (4), transcriptional regulation (5) and cell signaling (6) are all silenced by methylation.

Our recent studies indicate that gene-specific promoter methylation can be differentially modulated by defined carcinogen exposures. For example, the estrogen receptor-{alpha} (ER) gene shows a high incidence of methylation in lung tumors from rodents exposed to plutonium or beryllium, but a low methylation frequency in those induced by NNK [4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone], diesel exhaust or carbon black exposure (7,8). In contrast, p16 methylation was common in rat lung tumors induced by exposure to NNK, diesel exhaust, carbon black or beryllium, but not in tumors from NNK-exposed mice (8,9). Thus, these studies substantiate both exposure and species differences in the targeting of genes for inactivation by methylation.

Studying the effect of specific carcinogens in tobacco with respect to inactivation of critical regulatory pathways could ultimately provide important insight into factors mitigating lung cancer development. There are three retinoic acid receptor family members ({alpha}, ß, {gamma}), all of which mediate retinoid action as heterodimers with retinoid X receptor. The retinoids are viewed as fundamental mediators of cell differentiation and proliferation and represent one class of genes involved in cancer development. Retinoid signaling molecules are involved in lung development, differentiation and carcinogenesis (10). Binding of the retinoid to its receptor results in gene transcription necessary for these processes. RAR-ß exists as multiple isoforms due to the presence of two different promoters, P1 and P2, and alternative splicing (11). The antitumor effects of RAR-ß are mediated, in part, through inhibition of cell proliferation and metastasis (12), as well as induction of apoptosis (13). Inhibition of AP-1 transcription factor activity is one mechanism by which RAR-ß causes these effects (14). RAR-ß expression is also reduced in human and mouse lung tumors. This loss of expression has been associated with methylation of the RAR-ß gene promoter P2 in 41% of non-small cell lung tumors (15). A role for methylation in loss of RAR-ß transcription in murine lung tumors has not been substantiated.

The purpose of the current investigation was to determine the prevalence for RAR-ß inactivation by promoter methylation in murine lung cancer and whether inactivation is influenced by specific carcinogen exposures. We examined RAR-ß methylation in adenocarcinomas induced by exposure to cigarette smoke, the tobacco-specific nitrosamine NNK, vinyl carbamate, a carcinogen within cigarette smoke or methylene chloride. In addition, the timing of RAR-ß methylation in lung carcinogenesis was determined by analysis of precancerous lesions induced by NNK exposure.


    Materials and methods
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Tumor induction
Female A/J mice (6 weeks old) were treated with a single dose (100 mg/kg, i.p.) of NNK and killed at 4-week intervals starting at 14 weeks after treatment until up to 54 weeks (16). Hyperplasias were obtained from lungs of mice killed over the 22–38 week interval, while neoplasms were recovered from lungs collected after 42 weeks or longer. B6C3F1 mice (6–7 weeks old) were exposed to mainstream cigarette smoke for up to 30 months. Two 70 cm2 puffs/min from research cigarettes (type 2R1, Tobacco Health Research Institute, Lexington, KY) were generated by smoking machines (Type 1300, AMESA Electronics, Geneva, Switzerland). Cigarette smoke was diluted with filtered air and delivered to H-2000 whole-body exposure chambers (Hazleton Systems, Aberdeen, MD). The mass concentration of cigarette smoke total particulate material, determined by gravimetric analysis of the filter samples taken during the exposure periods, was 250 mg total particulate matter/m3 (17). Female B6C3F1 mice (8–9 weeks old) were exposed to 2000 p.p.m. methylene chloride delivered to H-2000 whole-body exposure chambers for 6 h/day, 5 days/week, up to 104 weeks (18). C3A mice were treated with a single dose of vinyl carbamate (60 mg/kg, i.p.) and killed ~1 year after treatment (19). All tumors were classified as adenocarcinomas by histological characteristics. A proliferation of type II epithelial cells along intact alveolar septae (16) distinguished hyperplasias induced by NNK treatment.

Cell culture and 5-aza-2'-deoxycytidine treatment
The CL13, CL20, CL25, CL30 and IO33 cell lines were derived from lung tumors induced in A/J mice by treatment with NNK (16); the SPON4 cell line was derived from a spontaneous tumor in the A/J mouse lung. The J303, P212 and P261 cell lines were derived from smoke-induced B6C3F1 lung tumors (unpublished data). CL13 and IO33 cells were grown in RPMI 1640 medium supplemented with 10% fetal bovine serum; CL20, CL25, CL30, J303, P212 and P261 cells were grown in ITRI-1 medium (20). Six cell lines (CL13, CL25, CL30, I033, J303 and SPON4) were treated with 1 µM 5-aza-2'-deoxycytidine (DAC) for 4 days, with media changes and fresh DAC added every 24 h. Cells were then trypsinized and pelleted for RNA isolation.

Nucleic acid isolation
Genomic DNA was extracted from cell pellets, tumors and microdissected hyperplasias by digestion with proteinase K, RNAse A and RNAse T1, followed by phenol–chloroform extraction and ethanol precipitation. DNA from NNK-, methylene chloride-, vinyl carbamate- and approximately half of the cigarette smoke-induced tumors was obtained from frozen tissues. DNA from NNK-induced hyperplasias and additional cigarette smoke-induced tumors was obtained by microdissection. Sequential sections were prepared from tumors and hyperplasias and deparaffinized. A 25-gauge needle attached to a tuberculin syringe was used to remove the lesions under a dissecting microscope. Total cellular RNA was extracted from cell pellets using Tri Reagent (Sigma, St Louis, MO) and treated with DNAse I to destroy any contaminating DNA, followed by phenol extraction and ethanol precipitation.

Reverse transcriptase–PCR
Total cellular RNA (3 µg) was reverse transcribed using a Superscript II kit (Invitrogen, Carlsbad, CA). cDNA was treated with RNAse H to destroy any residual RNA. PCR was performed on 2 µl of cDNA using primers specific for the RAR-ß locus. Primer sequences were as follows: 5'-TTTGACTGTATGGATGTTCTG-3' (forward) and 5'-TACTCTGTGTCTCGATGGATT-3' (reverse). After an initial denaturation at 94°C for 10 min, amplification was carried out for 40 cycles and consisted of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s. This process was followed by a final extension at 72°C for 5 min resulting in a 173 bp product. Primers specific for amplification of the ß-actin gene were used as a template quality control, as described (21). Products were visualized on 3% agarose–TBE gels.

Bisulfite sequencing
Genomic DNA was treated with sodium bisulfite as described (22) to modify unmethylated cytosine residues to uracil, and then to cytosine during subsequent PCR. Modified DNA was amplified using primers specific for the P2 promoter region and the 5' untranslated region extending into exon 3, the first translated exon of the mouse RAR-ß gene. Four tandem sets of primers were used to amplify ~1400 bp that includes 69 CpGs. Primer sequences and PCR conditions are available upon request. The location of each primer set with respect to the transcriptional start site is as follows: Set 1, -544, -235; Set 2, -260, +148; Set 3, +120, +411; Set 4, +362, +786. PCR products were separated on 3% agarose–TBE gels and the band excised. Products were purified using a Gene-clean kit (Bio 101, Carlsbad, CA) and cloned into the pCR II vector using a TA Cloning Kit (Invitrogen). Individual clones were grown, DNA recovered using a Qiaspin kit (Qiagen, Valencia, CA), and then sequenced with an automated sequencer (University of New Mexico Center for Genetics in Medicine, Albuquerque, NM).

Methylation-specific PCR
Bisulfite-modified cell line DNA was subjected to methylation-specific PCR (MSP) to analyze the methylation status of the mouse RAR-ß gene. A nested, two-stage approach was used as described (23) to increase the sensitivity when amplifying products from formalin-fixed tissue where DNA is degraded. A primary set of primers (5'-GGATTAATTATAGGTTTTTAGTTGGTT-3', forward; 5'-ACTTTATTCCAATATCTTTATTACATAC-3', reverse) recognizes a sequence in both methylated and unmethylated alleles of mouse RAR-ß. Two sets of nested primers were used for the secondary PCR. One primer pair recognizes a sequence in which CpG sites are methylated (unmodified by the bisulfite treatment; 5'-TCGTGGTTTTTTTGTGCGGTTC-3', forward; 5'-CAACATACAAAAAAAAAAACTCGCG-3', reverse) and the other recog­nizes a sequence in which CpG sites are unmethylated (modified to UpG by the bisulfite treatment; 5'-TTGTGGATTTTTTTGTGTGGTTTG-3', forward; 5'-CAACATACAAAA AAAAAAACTCACAA-3', reverse). Primers are localized to regions containing frequent CpG dinucleotides in order to distinguish between modified and unmodified DNA. For primary PCR, ~200 ng of DNA was initially denatured at 94°C for 10 min, followed by 40 cycles of amplification consisting of denaturation at 94°C for 30 s, annealing at 60°C for 30 s and extension at 72°C for 30 s. A final extension was performed at 72°C for 5 min to generate a 424 bp product. Primary products were diluted 50-fold and 5 µl used for secondary PCR. Primary template was initially denatured at 94°C for 10 min for the secondary PCR with the methylated primers. Amplification was then carried out for 40 cycles, consisting of denaturation at 94°C for 17 s, annealing at 67°C for 17 s and extension at 72°C for 17 s. This was followed by a final extension at 72°C for 5 min to generate a 158 bp product. PCR with the unmethylated primers used the same conditions, except that the denaturation at 94°C was for 15 s, annealing was at 64°C for 15 s and extension at 72°C was for 15 s. Normal A/J mouse lung DNA was used as a control in all PCRs. Products were visualized on 3% agarose–TBE gels.

Data analysis
The Wilcoxon rank sum test was used for pairwise comparisons of methylation between different environmental exposure groups. All tests were two-sided with significance set at P < 0.05.


    Results
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 References
 
RAR-ß is methylated in cell lines derived from mouse adenocarcinoma induced by exposure to tobacco carcinogens
The RAR-ß transcript was not expressed in two of four cell lines derived from NNK-(CL13, IO33). Expression of this transcript was detected in two cell lines (CL25, CL30) from NNK-derived tumors, one cell line from a smoke tumor (J303), the SPON4 cell line and normal lung (Figure 1A). Treatment with the demethylating agent DAC restored expression of RAR-ß in the cell lines showing no endogenous expression and increased the expression level in the CL25, CL30 and J303 cell lines (see Figure 1A).



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Fig. 1. (A) Re-expression of the RAR-ß gene in lung tumor-derived cell lines following treatment with DAC. Cells were grown for 4 days in either 1 µM DAC (+) or media without DAC (-). Treatment with DAC restored expression of RAR-ß in two lung tumor-derived cell lines (CL13, I033) and increased expression in CL25, CL30 and J303, as shown by the presence of a 171 bp product. There was no substantial change in expression levels within the SPON4 cell line. A/J normal lung (A/J NL) served as a positive control. (B) Methylation-specific PCR analysis of RAR-ß in murine lung tumor-derived cell lines and primary tumors induced by NNK. Tumors and cell lines positive for RAR-ß methylation are depicted by the presence of a 152 bp methylated PCR product. Unmethylated alleles were detected in all tumor samples and some cell lines. Normal A/J mouse lung (NL) served as a negative control for methylation and only unmethylated alleles were present in this sample.

 
Sequencing of DNA modified by sodium bisulfite treatment was performed to determine the precise location and density of CpG methylation in the P2 promoter region, 5' untranslated region and exon 3 of RAR-ß. The CL13 and IO33 cell lines, which re-expressed RAR-ß in the presence of DAC, as well as the CL25 cell line, which showed increased expression following DAC treatment, were chosen for analysis. Sodium bisulfite-modified normal A/J lung DNA was also sequenced. Four sets of nested primers spanning a region of ~1400 bp that contains 69 CpG dinucleotides were sequenced (Figure 2A). The most 5' region of the CpG island, covered by primer Sets 1 and 2, showed sparse methylation in all cell lines analyzed (data not shown). Of 32 CpGs in this region, an average of 3.6 (11%) were methylated within the CL13, CL25 and IO33 cell lines, while no methylation was observed for normal lung. In the CL13 and IO33 cell lines, which did not express the RAR-ß transcript, methylation increased in the 5' untranslated region, whereas the area around the translational start site was almost fully methylated (Figure 2B). The CL25 cell line, in which the RAR-ß transcript increased after DAC treatment, showed less dense methylation than the CL13 and IO33 cell lines, but methylation density also increased in the CpGs surrounding the translational start site. Normal A/J mouse lung showed very sparse methylation. The methylation densities (percentage of methylated CpG sites) for the area sequenced using primer Set 3 were: CL13, 16%; IO33, 14%; CL25, 0%; and A/J normal, 0%. The most densely methylated area, flanking the translational start site of the A2 exon and covered by primer Set 4, showed the following methylation densities: CL13, 99%; IO33, 91%; CL25, 59%; and A/J normal, 14%.



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Fig. 2. Density of methylation within the RAR-ß CpG island in murine lung cancer cell lines and normal mouse lung. The area evaluated for CpG methylation is depicted by the line drawing (A). CpG dinucleotides are indicated by tic marks and the location of a SP-1 binding site and the translational start site are also indicated on the line drawing. The region depicted and amplified included 650 bp starting at the transcriptional start site and extending into exon 3, the first transcribed exon. The two overlapping primer sets used for amplification recognize a bisulfite-modified template but did not discriminate between methylated and unmethylated alleles. Amplified PCR products were cloned and sequenced to determine the methylation status of 37 CpGs within the amplified region of three lung tumor-derived cell lines and normal A/J lung (B). Open and filled circles represent unmethylated or methylated CpGs, respectively. Each row represents one clone.

 
MSP using primers flanking the translational start site verified the results observed with the DAC re-expression experiment. For example, only methylated alleles were detected for the CL13 and IO33 cell lines; however, CL25 contained both methylated and unmethylated alleles (Figure 1B and Table I). Only unmethylated alleles for RAR-ß were detected in normal A/J mouse lung. Overall, nine cell lines derived from tumors induced by NNK or smoke exposure or spontaneously arising were examined by MSP; seven of the nine were methylated (Figure 1B and Table I).


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Table I. Frequency of RAR-ß methylation in murine lung tumor-derived cell lines, hyperplasias and primary tumors

 
Methylation of RAR-ß in primary mouse lung tumors varies with carcinogen exposure
The frequency of RAR-ß methylation was examined by MSP in lung tissue from vehicle-treated A/J (n = 7) and B6C3F1 (n = 6) mice, primary mouse lung tumors induced by exposure to NNK (n = 40), cigarette smoke (n = 21), vinyl carbamate (n = 15) or methylene chloride (n = 18). No methylation was detected in lung tissue from vehicle-treated mice (Table I). The majority of tumors (>=85%) induced by NNK or cigarette smoke showed methylation of the RAR-ß promoter. In contrast, the prevalence for methylation of this gene in vinyl carbamate- and methylene chloride-induced tumors was 60 and 56%, respectively. The methylation prevalence seen in the vinyl carbamate and methylene chloride tumors was significantly less (P < 0.05) when compared with either NNK- or cigarette smoke-induced tumors. Unmethylated RAR-ß alleles were detected in all tumors due to the presence of normal stromal and inflammatory cells.

Sequencing of sodium bisulfite-modified DNA using primer Sets 3 and 4 was performed on tumors induced by exposure to cigarette smoke or NNK to compare the density and location of methylation within the RAR-ß P2 promoter to that seen in cell lines. Three smoke tumors, two that were positive for methylation, and three NNK tumors, all positive for RAR-ß methylation, were sequenced. Similar to the cell lines, the tumors displayed very sparse methylation in the area covered by primer Set 3, with methylation density increasing dramatically around the translational start site (Figure 3A). The average methylation densities for the region covered by primer Set 4 for methylated tumors were NNK, 53% (Figure 3A) and cigarette smoke, 52% (not shown). In contrast, the unmethylated smoke-induced tumor displayed 10% methylation in this region. The fact that these methylation densities are lower than those observed in cell lines, probably stems from the presence of normal stromal and inflammatory cells within the tumor tissue. This assumption is corroborated by the fact that individual clones within the methylated tumors (e.g. NNK tumors 1 or 2) were completely unmethylated (Figure 3A).



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Fig. 3. Density of methylation within the RAR-ß CpG island in murine lung hyperplasias and lung tumors induced by NNK. Conditions are as described in Figure 2. Open and filled circles represent unmethylated or methylated CpGs, respectively. Each row represents one clone.

 
RAR-ß methylation is an early event in NNK-induced mouse lung carcinogenesis
RAR-ß methylation prevalence was assessed in precancerous lesions to determine the timing for inactivation of this gene by methylation. MSP was performed on 13 alveolar hyperplasias induced by NNK. Methylation was seen in 54% of the hyperplasias examined (Table I).

Bisulfite-modified DNA was sequenced from three hyperplasias methylated at the RAR-ß locus. These hyperplasias showed a methylation density around the translational start site of ~77% (Figure 3B). The distribution of methylated CpG dinucleotides within the P2 promoter was similar to that observed for the cell lines and tumors. The increased density for methylation seen within the RAR-ß CpG island in hyperplasias compared with tumors reflects the fact that these lesions were microdissected prior to DNA isolation, thus reducing the contamination by stromal and inflammatory cells.


    Discussion
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 Abstract
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 Materials and methods
 Results
 Discussion
 References
 
This investigation demonstrates that aberrant methylation of the RAR-ß gene, a frequent event in non-small cell lung cancer (15), is also prevalent in murine lung tumors induced by cigarette smoke and several human carcinogens. Methylation and silencing of the RAR-ß gene transcript were associated with dense methylation localized around the translational start site. This gene also appears to be targeted differentially by carcinogen exposures; virtually all tumors associated with cigarette smoke or NNK harbored methylated alleles in contrast to half of the tumors induced by methylene chloride or vinyl carbamate. Finally, this study links methylation of the RAR-ß gene to the earliest histologic stage of adenocarcinoma.

The RAR-ß P2 promoter has a relatively sparse CpG island compared with other genes such as p16INK4a, death associated protein kinase and ER that are silenced through aberrant methylation (7,23,24). There is good conservation with respect to the core RAR-ß P2 promoter and regulatory elements between the human and mouse. Both genes contain four retinoic acid response elements (RAREs) surrounding a TATA box 30 bp upstream of the transcriptional start site (25). The RAREs mediate RAR-ß P2 gene expression in many different cell types (26). In breast cancer cell lines and primary tumors, methylation has been observed in two of three CpG sites within the RAREs and in the 5' untranslated region (27). However, methylation around the RAREs is not obligatory for gene silencing since RAR-ß expression is absent in cell lines or tumors in which methylation was present only in the 5' untranslated region (28). In our study, bisulfite sequencing of a region spanning 1400 bp and including 69 CpGs revealed that only the 13 CpGs most proximal to the translational start site and extending into exon 3, the first transcribed exon, were consistently methylated. The CpGs around the RAREs were not methylated. This region does contain a Sp1 binding site located 134 and 250 bp upstream from the translational start site of the murine and human promoters, respectively, and contains a CpG immediately proximal to the consensus sequence (CGGGGCTGGG). This CpG dinucleotide (CpG #25) was also methylated in the majority of cell lines, tumors and hyperplasias examined by bisulfite sequencing, but has not been examined in human tumors. Sp1 transcription factors contain zinc finger domains and are important components of the transcription machinery (29,30). Thus, methylation around the Sp1 site could profoundly affect RAR-ß transcription due to the recruitment of methyl-CpG binding proteins, cytosine-DNA methyltransferases and deacetylated histones that comprise the transcriptional repression complex associated with aberrant promoter methylation of genes (31).

Previously, we demonstrated that methylation of the ER gene could be modulated differentially depending on the carcinogen exposure. A dramatic difference in prevalence for ER methylation was seen in plutonium- versus NNK-induced tumors (80 versus 17%) (7). In the current study, we observed a significant difference in prevalence of methylation of the RAR-ß gene in cigarette smoke- and NNK-induced tumors versus methylene chloride- and vinyl carbamate-induced tumors. The similar prevalence seen in cigarette smoke and NNK tumors could reflect the fact that NNK is one of the major carcinogens in cigarette smoke. The mechanisms that govern the targeting of genes silenced by promoter methylation remain to be defined precisely, but are probably influenced by effects on DNA chromatin structure that occurs with the formation of DNA adducts. NNK's primary mode of action in carcinogenesis is mediated through the formation of alkylating or pyridyloxobutylating pro-mutagenic DNA adducts (32). The carcinogenicity of vinyl carbamate is also derived from its ability to form pro-mutagenic adducts in DNA, while a DNA adduct has not been characterized for methylene chloride (3336). Thus, differences in rates of adduct formation and/or repair could directly or indirectly influence the propensity to target the RAR-ß gene for silencing by methylation. Irrespective of the precise mechanism underlying this observed difference for RAR-ß methylation, it is clear that inactivation of this pathway is probably a critical component for the development of lung tumors associated with cigarette smoke and NNK in the mouse. Vinyl carbamate- and methylene chloride-induced tumors may arise through pathways that are both dependent and independent of RAR-ß. However, we cannot exclude the possibility that other genes within this signal transduction pathway are altered, thereby alleviating the need to silence the RAR-ß gene. Precedence for gene targeting within a specific pathway is best described for the p16INK4a-retinoblastoma pathway in which mutations in the retinoblastoma gene are common in small cell lung cancer, while p16INK4a methylation is rare; the opposite occurs in non-small cell lung cancer (37,38).

NNK-induced lung cancer in the A/J mouse is characterized by the initial development of focal proliferation of type II cells along the alveolar septae that progress to adenomas and finally to carcinomas. These alveolar hyperplasias exhibit a very high rate of conversion to carcinomas, making them ideal for establishing the timing for genetic and epigenetic changes identified in tumors (16). The timing for inactivation of the RAR-ß gene has not been clearly defined in human lung cancer, although loss of expression was reported in ~30% of bronchial biopsies from former smokers (39). Our current study indicates that inactivation of this gene is common in alveolar hyperplasias. The fact that methylation was observed at a lower frequency (54 versus 90%) in NNK-induced hyperplasias and adenocarcinomas suggests that this gene can be inactivated as both an early and later event in lung cancer development. The similar pattern for methylation seen within the CpG island by bisulfite sequencing between these preneoplastic lesions and tumors supports the fact that this gene can be inactivated at the earliest histologic stage for development of adenocarcinoma. The importance of this gene in tumor development has been supported by intervention studies demonstrating that increased expression of retinoids can reverse neoplastic lesions and prevent second primary tumors in the aerodigestive tract (40).

Impacting the high mortality associated with lung cancer will necessitate early diagnosis and effective preventive intervention in high-risk subjects. The fact that gene silencing through promoter hypermethylation is a common event and occurs early during premalignancy makes it an attractive target for prevention. Previous in vitro studies have demonstrated that treatment with low doses of the demethylating agent DAC combined with a histone deacetylase inhibitor can effectively cause re-expression of genes silenced by aberrant promoter methylation (41). Recently, we extended these findings to an in vivo system in which treatment of mice following exposure to NNK with a low dose of DAC combined with the histone deacetylase inhibitor sodium phenylbutyrate dramatically decreased tumor multiplicity (42). Reversing methylation of the RAR-ß gene in hyperplasias and adenomas within the mouse lung will be a valuable biomarker for establishing the efficacy of this promising preventive approach.


    Notes
 
1 To whom correspondence should be addressed Email: sbelinsk{at}lrri.org Back


    Acknowledgments
 
The authors would like to thank Theodora R.Devereux, MS from the National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina, for providing DNA from the methylene chloride and vinyl carbamate-induced lung tumors. This study was supported by NIEHS ES08801 and P30-ES-012072 in facilities fully accredited by the Association for Assessment and Accreditation of Laboratory Animal Care International.


    References
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received September 5, 2003; revised November 17, 2003; accepted November 19, 2003.