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Aberrant Promoter Hypermethylation of the Death-Associated Protein Kinase Gene Is Early and Frequent in Murine Lung Tumors Induced by Cigarette Smoke and Tobacco Carcinogens

¹ Lung Cancer Program, Lovelace Respiratory Research Institute, Albuquerque, New Mexico, and ² Laboratory of Molecular Toxicology, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina

	ABSTRACT

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Loss of expression of the death-associated protein (DAP)-kinase gene by aberrant promoter methylation may play an important role in cancer development and progression. The purpose of this investigation was to determine the commonality for inactivation of the DAP-kinase gene in adenocarcinomas induced in mice by chronic exposure to mainstream cigarette smoke, the tobacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and vinyl carbamate, and the occupational carcinogen methylene chloride. The timing for inactivation was also determined in alveolar hyperplasias that arise in lung cancer induced in the A/J mouse by NNK. The DAP-kinase gene was not expressed in three of five NNK-induced lung tumor-derived cell lines or in a spontaneously arising lung tumor-derived cell line. Treatment with 5-aza-2'-deoxycytidine restored expression; dense methylation throughout the DAP-kinase CpG island detected by bisulfite sequencing supported methylation as the inactivating event in these cell lines. Methylation-specific PCR detected inactivation of the DAP-kinase gene in 43% of tumors associated with cigarette smoke, a frequency similar to those reported in human non-small cell lung cancer. In addition, DAP-kinase methylation was detected in 52%, 60%, and 50% of tumors associated with NNK, vinyl carbamate, and methylene chloride, respectively. Methylation was observed at similar prevalence in both NNK-induced hyperplasias and adenocarcinomas (46% versus 52%), suggesting that inactivation of this gene is one pathway for tumor development in the mouse lung. Bisulfite sequencing of both premalignant and malignant lesions revealed dense methylation, substantiating that this gene is functionally inactivated at the earliest histological stages of adenocarcinoma development. This study is the first to use a murine model of cigarette smoke-induced lung cancer and demonstrate commonality for inactivation by promoter hypermethylation of a gene implicated in the development of this disease in humans.

	INTRODUCTION

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Loss of expression of death-associated protein (DAP)-kinase is common in human cancers. DAP-kinase is a Ca⁺/calmodulin-regulated serine-threonine kinase involved in IFN--, tumor necrosis factor -, Fas-, transforming growth factor ß-, and ceramide-induced apoptosis (1, 2, 3, 4, 5, 6) . DAP-kinase also is involved in apoptosis triggered by the detachment of cells from the extracellular matrix (7) . Reintroduction of the DAP-kinase gene into highly metastatic mouse lung carcinoma cells delayed tumor growth and strongly reduced their metastatic capacity (8) . DAP-kinase also suppresses c-myc- and E2F-induced oncogenic transformation by activating the p53 apoptotic pathway in a p19^ARF-dependent manner (1) . Thus, loss of this gene at the earliest stages of tumor formation would provide a selective growth advantage, whereas loss at later stages could also facilitate tumor progression and metastasis.

Inactivation of DAP-kinase occurs largely through transcriptional silencing by DNA hypermethylation. Methylation of the DAP-kinase promoter has been observed in bladder, renal, and breast cancer cell lines as well as in head and neck, gastric, and colorectal carcinomas (9, 10, 11, 12, 13) . Additionally, a very high frequency of DAP-kinase promoter methylation has been detected in B-cell malignancies, including 100% of Burkitt’s lymphomas and 84% of B-cell lymphomas (14) . Finally, methylation prevalences ranging from 23% to 44% have been observed in non-small cell lung cancer (NSCLC; Refs. 15, 16, 17 ). The common targeting for inactivation of this gene in many solid tumors reinforces its importance in apoptosis and tumor suppression.

Despite numerous studies demonstrating frequent methylation of DAP-kinase in many human cancers, the commonality for this inactivation in cell lines or tumors from other species has not been examined. Furthermore, the timing of DAP-kinase inactivation by methylation during tumor development has not been established. Our group has used the mouse lung to study mechanisms involved in the development and progression of lung cancer induced by defined exposure to environmental and occupational carcinogens (18 , 19) . The fact that genetic and epigenetic alterations found in human lung adenocarcinomas are also common in murine tumors makes the mouse lung an ideal model for examining pathways involved in lung cancer induced by specific carcinogens (20) . For example, activation of the K-ras oncogene is an early and frequent event in both human lung adenocarcinoma and in spontaneous and chemically induced lung tumors in mice (21, 22, 23, 24, 25) . Furthermore, both human adenocarcinomas associated with smoking and mouse lung tumors induced with the tobacco carcinogen benzo(a)pyrene contain codon 12 G to T mutations in the K-ras oncogene indicative of base mis-pairing associated with the benzo(a)pyrene 7,8-diolexpoxide DNA adduct (21 , 22) . The frequency for methylation of the estrogen receptor gene is also similar in murine and human lung tumors (26) . Finally, the p16^INK4a gene is a common target for inactivation in both human and mouse lung tumors. Interestingly, this gene is silenced primarily by methylation in human tumors, but is both methylated and deleted in murine tumors (27 , 28) .

The purpose of this investigation was to determine whether adenocarcinomas induced in the B6C3F1 mouse by chronic exposure to mainstream cigarette smoke arise in part through inactivation of the DAP-kinase gene. These studies were extended to compare the prevalence for DAP-kinase methylation in tumors induced by the tobacco carcinogens 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and vinyl carbamate and the occupational carcinogen methylene chloride. Finally, the timing for inactivation of this gene was determined in alveolar hyperplasias that arise in our well-characterized model of lung cancer induced in the A/J mouse by NNK.

	MATERIALS AND METHODS

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Tumor Induction.
Female A/J mice (6 weeks of age) were treated with a single dose (100 mg/kg, i.p.) of NNK and sacrificed at 4-week intervals starting at 14 weeks after carcinogen treatment out to 54 weeks (29) . Female 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 (Hazelton Systems, Inc., Aberdeen, MD). The mass concentration of cigarette smoke total particulate material was determined by gravimeteric analysis of the filter samples taken during the exposure periods and determined to be 250 mg total particulate matter/m³ (30) . Female B6C3F1 mice (8–9 weeks of age) were exposed to 2000 ppm methylene chloride delivered to H-2000 whole-body exposure chambers for 6 h/day, 5 days/week, up to 104 weeks (31) . Female C3A mice were treated with a single dose of vinyl carbamate (60 mg/kg, i.p.) and sacrificed approximately 1 year after treatment (32) . All tumors were classified as adenocarcinomas by histological characteristics. Hyperplasias induced by NNK treatment were distinguished by a proliferation of type II epithelial cells along intact alveolar septae (29) .

Cell Culture and 5-Aza-2'-deoxycytidine (DAC) Reexpression Experiments.
The CL13, CL20, CL25, CL30, and IO33 cell lines were developed from A/J mouse lung tumors induced by NNK (33) . Cell lines J303, P212, and P261 were developed from B6C3F1 mouse lung tumors induced by cigarette smoke. The SPON4 cell line was developed from a spontaneous lung tumor in the A/J mouse lung. Cell lines CL13, CL20, and IO33 were grown in RPMI 1640 supplemented with 10% fetal bovine serum; cell lines CL25, CL30, SPON4, J303, P212, and P261 were grown in ITRI-1 medium (33) . Cells were maintained at 37°C in a humid atmosphere containing 5% CO₂. The cell lines were treated with 1 µM DAC for 4 days, with media changes and fresh DAC added every 24 h. After 96 h, cells were trypsinized and pelleted for RNA isolation.

Nucleic Acid Isolation.
Total cellular RNA was extracted from the murine cell lines using TRI reagent (Sigma, St. Louis, MO). Contaminating DNA was removed by digestion with DNase I, followed by phenol extraction and ethanol precipitation. DNA from NNK-induced tumors, methylene chloride-induced tumors, vinyl carbamate-induced tumors, and approximately half of the cigarette smoke-induced tumors was obtained from frozen tumors. DNA from NNK-induced hyperplasias and additional cigarette smoke-induced lung tumors was recovered by microdissection as follows: sequential sections were prepared from tumors and hyperplasias; deparaffinzed; and stained with toluidine blue to facilitate dissection. A 25-gauge needle attached to a tuberculin syringe was used to remove the lesions under a dissecting microscope. Due to the small size of the alveolar hyperplasias, microdissection was used to enrich the samples. Thus, the inclusion of normal-appearing cells was necessary to ensure that enough sample remained to conduct the methylation-specific PCR (MSP) assay after bisulfite modification and column clean-up of the DNA template. Because the goal of the study was to determine whether DAP-kinase methylation was present in these lesions and not to quantitate methylation levels, microdissection was used to enrich the samples. DNA was isolated from microdissected lesions and frozen tumors by overnight digestion with Pronase (1%), followed by standard phenol-chloroform extraction and ethanol precipitation (25) .

Reverse Transcription-PCR.
First-strand cDNA was generated at 42°C from 3 µg of total RNA using the SuperScript II kit (Invitrogen, San Diego, CA). PCR primer sequences were 5'-TCAGGGCGACGAAGCGACA-3' (sense) and 5'-CTGCCTGAACACAG TCATGAT-3' (antisense), and they were designed to cross an exon splice junction. After an initial denaturation at 94°C for 10 min, amplification was carried out for 40 cycles, which consisted of denaturation at 94°C for 30 s, annealing at 64°C for 30 s, and extension at 72°C for 30 s. This was followed by a final extension at 72° for 5 min to generate a 157-bp product. The ß-actin gene was amplified as a control for RNA integrity. Products were visualized on 2% agarose gels.

MSP.
The methylation status of the DAP-kinase promoter in all murine cell lines, hyperplasias, and tumors was determined using a nested, two-stage MSP assay as described previously (34) . The two-stage MSP was used throughout the study because of difficulty in amplifying products from formalin-fixed tissue where DNA is degraded. This enabled results obtained from frozen and fixed tumors to be combined. Stage 1 primers were located at –267 and +93 (+1 was the transcriptional start site) and amplified a region that included a portion of the CpG-rich promoter. Primer sequences were 5'-GATTTTTGGGTTTATATTTTGAGAGG-3' (sense) and 5'-CTCCCRCTCCTCCCACTACCTA-3 prime (antisense; R = G + A). After amplification by stage 1 primers, two different stage 2 PCRs were performed: one amplified unmethylated DNA; and the other amplified methylated DNA. The stage 2 unmethylated and methylated primers were located at –112 and +49 and –108 and +48, respectively. Primer sequences were as follows: stage 2 unmethylated, 5'-TGGGAGGAGTTGTGA GTGT-3' (sense) and 5'-ACAACTATCACTTCATACACC-3' (antisense); and stage 2 methylated, 5'-AGGAGTCGCGAGCGTAGC-3' (sense) and 5'-CAACTATCGCTTCGTACGC-3' (antisense). Normal A/J mouse lung and a murine cell line positive for DAP-kinase methylation (IO33) served as negative and positive controls, respectively. The products were visualized on 2% agarose gels.

Bisulfite Sequencing.
Stage 1 MSP primers were used to amplify the DAP-kinase gene from DNA of A/J mouse normal lung and four cell lines (CL25, CL13, CL30, and IO33). DNA from normal A/J mouse lung, mouse lung hyperplasias derived from exposure to NNK, and mouse lung tumors derived from exposure to NNK and cigarette smoke were amplified using either unmethylated or methylation-specific stage 2 primers. The methylation-specific primers recognize a sequence in which CpGs are methylated; therefore, only methylated alleles are amplified. The 360-bp (stage 1) and 156-bp (stage 2) PCR products were ligated into the PCR II vector using the TA cloning kit (Invitrogen). Four to six clones from each sample were commercially sequenced in both directions [University of New Mexico Center for Genetics in Medicine (Albuquerque, NM) and Sequetech (Mountain View, CA)].

	RESULTS

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The Murine DAP-Kinase CpG Island.
The CpG island within the murine DAP-kinase promoter comprises approximately 84 CpGs over 880 bp that extend through the transcriptional start site (GenBank accession number XM127345). The density of CpGs within this region is similar to that seen in the analogous region in the human DAP-kinase promoter (46 CpGs within 590 bp). In the human promoter, an additional 125 CpGs are located 1000 bp upstream of the initial 590 bp, whereas in the mouse promoter, the density of CpGs is reduced greatly beyond the initial 500 bp. The deduced amino acid sequence of the DAP-kinase gene was 90% homologous to the human sequence present in GenBank (accession number AC007847). Thus, there is good conservation between human and mouse with respect to both amino acid sequence and density of CpGs around the transcriptional start site of the DAP-kinase gene.

DAP-Kinase Expression Correlates with Methylation State in Murine Lung Tumor-Derived Cell Lines.
The DAP-kinase gene was not expressed in three of five NNK-induced mouse lung tumor-derived cell lines (IO33, CL13, and CL30) or in a spontaneously arising mouse lung tumor-derived cell line [SPON4 (Fig. 1) ]. Expression of this gene was detected in the CL25 and CL20 cell lines as well as in lung tissue from the A/J mouse. Treatment with DAC, a demethylating agent that inhibits cytosine DNA-methyltransferases, restored expression of DAP-kinase in all four cell lines showing no endogenous expression and increased the level of expression in the CL20 cell line (Fig. 1) .

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Reexpression of the death-associated protein (DAP)-kinase gene in lung tumor-derived cell lines after treatment with 5-aza-2'-deoxycytidine (DAC). Cells were grown for 4 days in either 1 µM DAC (+) or media without DAC (–). Treatment with DAC restored expression of DAP-kinase in three lung tumor-derived cell lines induced by NNK (IO33, CL13, and CL30) and in one spontaneously arising lung tumor cell line (SPON4) and increased expression of DAP-kinase in CL20, as shown by the presence of a 157-bp product. CL25 showed normal DAP-kinase expression with and without DAC treatment. A/J normal lung (AJ NL) served as a positive control.

View larger version (59K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. Density of methylation within the death-associated protein (DAP)-kinase CpG island in murine lung cancer cell lines and normal mouse lung. Bisulfite-modified DNA was amplified using stage 1 MSP primers specific to the mouse DAP-kinase promoter. The primers used recognized a bisulfite modified template but did not discriminate between methylated and unmethylated alleles. The primers were located at –267 and +93 (+1 was the translational start site). Amplified PCR products were cloned and sequenced to determine the methylation status of the 50 CpGs within the amplified region. Open and filled circles represent unmethylated CpGs and methylated CpGs, respectively. Each row represents one clone.

View larger version (23K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Methylation-specific PCR analysis of death-associated protein (DAP)-kinase in murine lung tumors induced by exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Tumors positive for DAP-kinase methylation (T1, T3, T4, T7, and T10) are indicated by the presence of a 156-bp product. A murine cell line positive for DAP-kinase methylation (IO33) and normal A/J mouse lung (AJ NL) served as a positive control and a negative control, respectively. Unmethylated alleles were detected in all tumor samples and in the mouse normal lung, as depicted by the presence of a 161-bp band. Unmethylated alleles were not detected in IO33.

Finally, to compare methylation densities between premalignant and malignant lesions, bisulfite sequencing was performed on DNA from two alveolar hyperplasias induced by NNK and four adenocarcinomas associated with exposure to either cigarette smoke or NNK. All lesions selected for sequencing were scored as methylated by the MSP assay. DNA was amplified using stage 2 MSP primers that amplified a 156-bp region ranging from –108 to +48 (+1 was the translational start site) and encompassing 16 CpGs. Sequencing of the two hyperplasias revealed dense methylation throughout the region examined, with methylation densities ranging from 63% to 75% (Fig. 4) . Sequencing of tumors associated with exposure to NNK showed densities ranging from 56% to 81%, whereas the density of methylation in cigarette smoke-associated tumors was somewhat lower, at 50–56%. Clones from the same tumor or hyperplasia showed a high degree of homology with respect to methylation density, and although methylation densities varied somewhat between tumors and exposures, similar patterns of methylation were observed in almost all tumors and hyperplasias examined. This pattern consisted of regions of dense CpG methylation, followed by short regions (1–5 CpGs) of little or no methylation. This type of methylation pattern has been seen in other genes, most notably, O⁶-methylguanine-DNA methyltransferase. The methylation pattern in colon and cervical tumor-derived cell lines revealed dense regions of methylation within the O⁶-methylguanine-DNA methyltransferase gene in which nearly every CpG within a small region was methylated, interspersed with regions of very infrequent methylation (35) .

View larger version (44K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Density of methylation within the death-associated protein (DAP)-kinase CpG island in murine lung hyperplasias and lung tumors. Stage 2 methylation-specific PCR primers were used to amplify the DAP-kinase gene in murine hyperplasias derived from exposure to 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) and murine lung tumors derived from exposure to NNK or cigarette smoke. Stage 2 unmethylated primers were used to amplify the normal A/J mouse lung. Primers were located at –112 and +49 (unmethylated) and –108 and +48 (methylated), and +1 was the translational start site. Amplified PCR products were cloned, and four to six individual clones were sequenced to determine the methylation status of the 16 CpGs within the amplified region. CpGs numbered 27 through 42 correspond to the same numbered CpGs depicted in Fig. 2 . Open circles and filled circles represent unmethylated CpGs and methylated CpGs, respectively. Each row represents one clone.

	DISCUSSION

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The prevalence of DAP-kinase methylation was approximately 50% in murine lung tumors associated with cigarette smoke and in two tobacco-related carcinogen exposures, NNK and vinyl carbamate. This prevalence approximates results reported for primary NSCLCs associated with exposure to tobacco (17) . Two of the most likely candidates responsible for the carcinogenicity of cigarette smoke are the polyaromatic hydrocarbons and the tobacco-specific nitrosamines, such as NNK (36 , 37) . The primary mode of action of NNK in carcinogenesis is mediated through the formation of alkylating or pyridyloxobutylating promutagenic DNA adducts (38) . Vinyl carbamate, a potent carcinogen in the mouse lung, is an intermediate formed by the bioactivation of the tobacco carcinogen ethyl carbamate (38 , 39) . The carcinogenicity of vinyl carbamate is also derived from its ability to form promutagenic adducts in DNA (18 , 40) . In addition to the tobacco-related compounds, the frequency of DAP-kinase methylation was also high in tumors induced by exposure to the occupational carcinogen methylene chloride. Although methylene chloride is associated with the development of lung tumors in mice, the mechanism by which it induces these tumors is uncertain. Some studies suggest it is a genotoxic carcinogen causing sister chromatid exchange or loss of heterozygosity (41) , whereas others fail to demonstrate direct interactions with DNA, and no specific DNA adducts have been identified (42 , 43) . Thus, carcinogens inducing lung cancer through distinct effects on DNA show a similar propensity to arise in part through inactivation of the DAP-kinase gene. Currently, the mechanisms by which carcinogen exposure leads to aberrant gene-specific promoter hypermethylation are unknown. The presence of DNA adducts has been shown to block transcription factor binding (44) . The disruption of replication timing that would follow such inhibition has been proposed as a mechanism by which normally unmethylated regions of DNA become aberrantly methylated (45) .

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 malignant tumors (46) . The timing for inactivation of the DAP-kinase gene has not been clearly defined in human lung cancer. Previously, we reported a low frequency of DAP-kinase methylation in nonmalignant bronchial epithelial cells from the airways of smokers (47) . Our current study indicates that inactivation of this gene is common in alveolar hyperplasias. The fact that methylation was observed at a similar frequency (46% versus 52%) in NNK-induced hyperplasias and adenocarcinomas suggests that one pathway for tumor progression in the mouse lung involves the clonal selection of cells with inactivation of the DAP-kinase gene. The similar pattern for methylation seen within the promoter region by bisulfite sequencing between these preneoplastic lesions and tumors supports the argument that inactivation of this gene is occurring at the earliest histological stage for development of adenocarcinoma. The importance of this gene in tumor development has been supported by its association with reduced survival in persons diagnosed with stage I NSCLC (17) . Furthermore, because DAP-kinase is an integral component of several apoptotic pathways, its silencing should facilitate expansion of premalignant clones of cells.

Lung cancer is currently the leading cause of cancer death in both men and women in the United States with a 2-year survival of 12% for persons with unresectable disease (48) . A recent comparison of four chemotherapy regimens considered the "best approach" revealed no difference in survival, leading the investigators to conclude that chemotherapy for NSCLC has reached a therapeutic plateau (49) . The development of effective preventive interventions could prove invaluable for reducing the high mortality associated with this disease. One promising molecular strategy for the treatment of lung cancer involves the reactivation of genes that have been silenced by aberrant methylation. Previous in vitro studies have demonstrated that treatment with low doses of the demethylating agent DAC in combination with a histone deacetylase inhibitor can effectively cause reexpression of genes silenced by aberrant promoter methylation (50) . Recently, we extended these findings to an in vivo system in which treatment of mice after exposure to NNK with a low dose of DAC in combination with the histone deacetylase inhibitor sodium phenylbutyrate dramatically decreased tumor multiplicity.³ Reversing methylation of the DAP-kinase gene in hyperplasias and adenomas within the mouse lung will be a valuable biomarker for establishing the efficacy of this promising preventive approach.

	FOOTNOTES

 
Grant support: Supported by National Institute of Environmental Health Sciences Grants ES08801 and P30-ES-012072 in facilities fully accredited by the Association for the Assessment and Accreditation of Laboratory Animal Care International.

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.

Requests for reprints: Steven A. Belinsky, Lovelace Respiratory Research Institute, 2425 Ridgecrest Drive SE, Albuquerque, NM 87108. Phone: (505) 348-9465; Fax: (505) 348-4990; E-mail: sbelinsk{at}LRRI.org

³ S. A. Belinsky, unpublished observations.

Received 7/15/03. Revised 2/10/04. Accepted 2/13/04.

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