Hypermethylation of the p16 Ink4a promoter in B6C3F1 mouse primary lung adenocarcinomas and mouse lung cell lines
Arti C. Patel1,4,
Colleen H. Anna1,
Julie F. Foley2,
Patricia S. Stockton2,
Frederick L. Tyson3,
J.Carl Barrett1 and
Theodora R. Devereux1,5
1 Laboratory of Molecular Carcinogenesis,
2 Laboratory of Experimental Pathology and
3 Division of Extramural Research and Training, Chemical Exposures and Molecular Biology Branch, National Institute of Environmental Health Sciences, PO Box 12233, Mail Drop D4-04, Research Triangle Park, NC 27709, USA and
4 Department of Pathology and Laboratory Medicine, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
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Abstract
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Primary lung tumors from B6C3F1 mice and mouse lung cell lines were examined to investigate the role of transcriptional silencing of the p16 Ink4a tumor suppressor gene by DNA hypermethylation during mouse lung carcinogenesis. Hypermethylation (
50% methylation at two or more of the CpG sites examined) of the p16 Ink4a promoter region was detected in DNA from 12 of 17 (70%) of the B6C3F1 primary mouse lung adenocarcinomas examined, whereas hypermethylation was not detected in normal B6C3F1, C57BL/6 and C3H/He mouse lung tissues. Immunohistochemistry performed on the B6C3F1 lung adenocarcinomas revealed heterogeneous expression of the p16 protein within and among the tumors. Laser capture microdissection was employed to collect cells from immunostained sections of four tumors displaying areas of relatively high and low p16 expression. The methylation status of the microdissected samples was assessed by sodium bisulfite genomic sequencing. The pattern of p16 expression correlated inversely with the DNA methylation pattern at promoter CpG sites in nine of 11 (82%) of the microdissected areas displaying variable p16 expression. To provide further evidence that hypermethylation is involved in the loss of p16 Ink4a gene expression, three mouse lung tumor cell lines (C10, sp6c and CMT64) displaying complete methylation at seven promoter CpG sites and no p16 Ink4a expression were treated with the demethylating agent, 5-aza-2'-deoxycytidine. Re-expression of p16 Ink4a and partial demethylation of the p16 Ink4a promoter were observed in two cell lines (C10 and sp6c) following treatment. These are the first reported studies to provide strong evidence that DNA methylation is a mechanism for p16 inactivation in mouse lung tumors.
Abbreviations: 5-Aza-CdR, 5-aza-2'-deoxycytidine; 6-Aza-C, 6-aza-cytidine; LCM, laser capture microdissection; LOH, loss of heterozygosity; NSCLC, non-small cell lung cancer; TSA, trichostatin A.
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Introduction
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Mouse lung adenocarcinomas share many histological and molecular characteristics with human lung adenocarcinomas (1). Both human adenocarcinomas associated with smoking and mouse lung tumors induced with carcinogens, such as benzo[a]pyrene present in cigarette smoke, possess characteristic codon 12 mutations in the K-ras proto-oncogene (2). Tumors from both species also exhibit loss of heterozygosity (LOH) in homologous regions of the genome that contain putative tumor suppressor genes (1). As a result of these similarities, mouse models for lung carcinogenesis are being used to identify the steps in tumor progression and the relative contributions of genetic, epigenetic and environmental factors in the development of lung cancer (1,35).
Numerous studies report LOH of chromosome 9p in a high frequency of primary non-small cell lung cancers (NSCLCs) (611). The p16Ink4a tumor suppressor, which maps to this region of loss, acts as an inhibitor of CDK4/CDK6 and blocks the G1 to S transition by preventing the phosphorylation of pRb (12). The p16INK4a gene is inactivated in human lung cancer cell lines and primary tumors by homozygous deletion, mutation or hypermethylation (1318). The frequency of homozygous deletions and point mutations in p16INK4a is lower in lung cancers than some other malignancies (19), although analyses of NSCLCs reveal low and often undetectable expression of the p16INK4a gene (19). However, methylation of CpG sites within the p16INK4a promoter region appears to play an important role in transcriptional repression of p16INK4a in NSCLC (17,1921). The role for DNA methylation in lung cancer is supported by studies with human lung cancer cell lines showing that treatment with the demethylating agent 5-aza-2'-deoxycytidine (5-Aza-CdR) restores expression of p16INK4a (17,18).
Evidence from several studies also supports the involvement of p16Ink4a aberrations in mouse lung carcinogenesis. Mouse lung carcinomas show LOH on chromosome 4 in an area that is syntenic to human chromosome 9p21 and corresponds to the location of the mouse tumor suppressor genes p15Ink4b, p16Ink4a and p19Arf (2224). Furthermore, one study reports decreased expression and homozygous codeletion of p15Ink4b, p16Ink4a and p19Arf in some mouse lung tumor cell lines (23). This study also found that several mouse lung tumor cell lines possess the p16Ink4a gene but lack the p16Ink4a transcript (23). Additionally, variable expression of p16Ink4a is found by RTPCR in A/J mouse lung tumors that do not display point mutations in the coding region (25). Immunohistochemistry of A/J mouse lung tumors revealed focal areas that lack p16 staining and RTPCR showed variable expression of p16Ink4a (26). However, homozygous deletions of chromosome 4 and point mutations of p16Ink4a are infrequent (22,23,2527). One study reported that 2/28 (7%) lung tumors from mice treated transplacentally with 3-methylcholanthrene exhibit point mutations in exon 2 of p16Ink4a (27). Based on these observations, it seems likely that hypermethylation of promoter region CpG sites is a mechanism for p16 inactivation in mouse lung tumors. Methylation, like coding region mutations, can lead to the inactivation of tumor suppressor genes, thus providing a selective growth advantage for the affected cells (20).
To gain a better understanding of mouse lung tumorigenesis and the role of DNA methylation, we analyzed primary mouse lung tumors and mouse lung cell lines for the methylation status of the p16Ink4a promoter region. Expression of p16Ink4a by RTPCR and immunohistochemistry was compared with DNA methylation status. Laser capture microdissection (LCM) and bisulfite genomic sequencing were performed to determine the methylation status of the p16Ink4a promoter in tumor areas that express relatively low or high levels of the p16Ink4a transcript. In addition, three mouse lung cell lines (C10, sp6c and CMT64), displaying complete methylation at seven promoter region CpG sites adjacent to the translational start site and lacking p16Ink4a transcript expression, were treated with the demethylating agent 5-Aza-CdR to determine if demethylation would result in re-expression of the p16Ink4a transcript.
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Materials and methods
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Tumor samples
Lung tumors were induced in female B6C3F1 mice by inhalation exposure to 2000 p.p.m. methylene chloride for 6 h/day, 5 days/week. Both chemically induced and spontaneous lung adenocarcinomas were collected over a period of 27 months as described previously (28). At necropsy, sections of the tumors were fixed in 10% neutral buffered formalin and embedded in paraffin. The remaining tumor tissue was frozen and stored at 70°C.
Cell lines
Cell lines, LM1, CMT64, CMT167, CL20I.1, CL20H, sp6c, NNK30pp, sp10 and sp10pp (derived from mouse lung adenocarcinomas), cell line E9 (arising in culture) and cell line C10 (immortalized and non-transformed) were cultured as described previously (23).
DNA isolation
Lung tumors and cells were digested overnight at 37°C in TNE buffer (10 mM Tris, 150 mM NaCl, 2 mM EDTA disodium salt pH 7.5), 1% SDS and 10 µg/ml Pronase. Isolation of DNA was accomplished through successive phenol/chloroform extractions. DNA was ethanol precipitated and resuspended in TE (1 mM Tris, 0.1 mM EDTA pH 7.5).
Loss of heterozygosity (LOH) analysis of primary mouse lung tumors
Microsatellite markers D4Mit77 (Research Genetics, Huntsville, AL) and Mts1 were used to amplify polymorphic regions between strain C57BL/6 and C3H/He mice to identify portions of tumors with LOH at the p16Ink4a locus. The Mts1 forward (5'-GA TTT CTA CGG AAA GCC CTG-3') and reverse (5'-TAT TGT GCA TTT GTG TGT CTG G-3') primers were located 2395 and 2172 bp upstream of the translational start site, respectively. PCR amplification with and without [
-33P]dATP (Amersham, Piscataway, NJ) was performed on 14 methylene-chloride-induced and three spontaneous B6C3F1 lung adenocarcinomas. Controls lacking DNA were included with all amplifications. The PCR products were resolved on either 4% NuSieve® (FMC BioProducts, Rockland, ME):agarose (3:1 ratio) or 6% acrylamide/urea/formamide gels to separate the two alleles. Similar results were obtained in repeat PCR amplifications.
Single-strand conformation polymorphism (SSCP) analysis of primary mouse lung tumors
Mutation analysis of p16Ink4a exons 1 and 2 was performed using the SSCP technique and primers described by Zhuang et al. (29). DNA was amplifed in a nested PCR reaction. Controls lacking DNA were included with all amplifications. The inner PCR product was labeled by incorporation with [
-33P]dATP (Amersham), and two different gel conditions were used to detect mutations. The PCR products were electrophoresed on a 6% non-denaturing polyacrylamide gel containing 10% glycerol at 35 W for 4 h at 4°C and a 0.5x MDE (AT Biochem, Malvern, PA) gel at 3 W for 17 h at room temperature.
Immunohistochemical staining of p16
Protein localization was accomplished using a mouse monoclonal antibody reactive to mouse p16 (Santa Cruz Biotechnology Inc., Santa Cruz, CA) at a 1:40 dilution. Non-immune mouse IgG served as the negative control. Formalin-fixed tissue sections (6 µm) were deparaffinized in xylene and hydrated through a series of graded ethanol and 1x Automation BufferTM (Biomeda, Foster City, CA) washes. The slides were processed on an automated Ventana Medical Systems Nexes Immunohistochemical Automatic Stainer (Ventana Medical Systems Inc., Tucson, AZ) following standard protocol. Slides were visualized using 3,3'-diaminobenzidine as the color-developing reagent. Slides were counterstained with Harris hematoxylin, dehydrated through a graded series of ethanol and xylene washes and cover-slipped using Micromount® (Surgipath, Richmond, IL).
Laser capture microdissection (LCM)
Four tumors (MC152, MC512, MC667 and MC995) displaying heterogeneous expression of p16 were laser capture microdissected using the PixCell IITM LCM System (Arcturus Engineering Inc., Mountain View, CA). Areas exhibiting relatively low and high p16 expression were microdissected by positioning the CapsureTM LCM Transfer Film (Arcturus Engineering Inc.) over the target tissue (30). In general, 500 hits with a laser pulse were used to obtain approximately 1000 cells or 2x106 µm2 of tissue. The cap with the attached transfer film and captured tissue was then fitted onto a 0.5 ml microtube containing 50 µl lysis buffer (0.5% Tween-20, 1 mM EDTA pH 8.0, 50 µM Tris pH 8.5, 0.5 µg/µl Proteinase K). The tube tops were wrapped in paraffin to prevent sample evaporation. The tubes were then inverted, placed in a moist chamber and incubated at 37°C overnight. Following incubation, the samples were incubated for 8 min at 95°C to inactivate the Proteinase K. The DNA was then ethanol precipitated by adding 2 µl glycogen (20 mg/ml; Boehringer Mannheim, Indianapolis, IN), which acts as a carrier, and 1.8 ml of 100% ethanol. The pellet was washed twice with 70% ethanol, dried and resuspended in 10 µl dH2O and stored at 20°C until methylation analysis was performed.
Bisulfite modification of whole primary lung tumor and cell line DNA
Methlyation of the p16Ink4a promoter was evaluated using sodium bisulfite genomic sequencing (31,32). An aliquot of 5 µg of DNA isolated from frozen B6C3F1 lung tumors or mouse lung tumor cell lines was restricted with EcoRI at 37°C overnight in a total volume of 40 and 20 µl, respectively. The digested DNA was denatured at 75°C for 15 min in either 4 (tumor samples) or 2 µl (cell line samples) of 3 M NaOH. For the bisulfite modification, 500 µl of 4.8 M sodium bisulfite and 28 µl of 20 mM hydroquinone were added to the tumor samples, whereas 250 µl of 4.8 M sodium bisulfite and 14 µl of 20 mM hydroquinone were added to the cell line samples. Both sets of samples were overlayed with light mineral oil and incubated at 55°C for 5 h. The modified DNA was purified using Centricon 30 filter units (Millipore Corporation, Bedford, MA) as directed by the manufacturer, and the final volume was adjusted to 100 µl with dH2O. Samples were desulfonated with 4.5 µl of 3 M NaOH and neutralized with 28 µl of 5 M ammonium acetate. Glycogen (1 µg) was added to the DNA isolated from the cell lines, but not the tumor samples. The DNA was ethanol precipitated overnight in 3 vol of 100% ethanol at 20°C. The DNA pellet was washed with 70% ethanol, dried and resuspended in 20 µl dH2O.
Bisulfite modification of laser capture microdissected samples
The LCM samples (10 µl) were restricted with EcoRI at 37°C overnight in a total volume of 20 µl and samples were treated as outlined above for the cell lines, and as described previously (32). Following bisulfite modification, the samples were purified using the Prep-A-Gene DNA Purification System (Bio-Rad, Hercules, CA) as described in the instruction manual. The desulfonation, neutralization and ethanol precipitation steps were carried out as detailed above including glycogen as a carrier. DNA was resuspended in 30 µl TE.
PCR amplification
The bisulfite modified DNA was amplified in nested PCR amplification reactions with modified primers designed using the published sequence for the 5'-untranslated region of p16Ink4a (GenBank accession no. U47018). The modified primers, with the exception of primer RM4 (see sequence below), contained no CpG sites. All cytosines in the primer sequences were replaced by thymines to enable amplification of sodium bisulfite modified DNA. The underlined bases in the primer sequences represent cytosines that are ultimately converted to thymines following bisulfite treatment. The outer reaction was performed using primers FM1 (5'-GTT GTG TAT AGA ATT TTA GTA TTG-3') and RM2 (5'-CCA CCC TAA CCA ATC TAT CTA CAA C-3') located 753 and 5 bp upstream of the translational start site, respectively. The PCR reaction was performed with 1 µl DNA template in a 20 µl reaction subjected to hot start at 85°C and 24 cycles (denaturation at 94°C for 1 min, annealing at 52°C for 35 s, extension at 72°C for 1 min) in a Perkin-Elmer 480 Thermocycler. A 1:10 dilution was made from the outer reaction and 3 µl was used as the template in a 50 µl inner reaction (hot start at 85°C, 33 cycles of: denaturation at 94°C for 1 min, annealing at 56°C for 35 s anneal, 72°C for 1 min and 1 min/cycle extension). Three different primer sets were used for the inner PCR reaction so that CpG sites 1 to 9 and 12 to 20 could be examined for methylation status. The three primer sets included: (i) set 1 (allowed examination of sites 12 to 20): FM3 (5'-TTT TAA TAT TTG GGT GTT GTA TTG-3'), located 506 bp upstream of the translational start site, and RM4 (5'-AC CCA AAC TAC AAA AAA AAT ACA-3'), located 272 bp upstream of the translational start site; (ii) set 2 (allowed examination of sites 8 to 9): 20FM (5'-GGT GTT TAA TTT ATG TTA TAT TTA-3'), located 206 bp upstream of the translational start site, and RM2 (sequence given above); (iii) set 3 (allowed examination of sites 1 to 7): FM6 (5'-TTT TTA GAG GAA GGA AGG AGG GAT TT-3'), located 107 bp upstream of the translational start site, and RM2 (sequence given above). For the paraffin-embedded samples, primer set 2 (20FM and RM2) was used for the outer PCR reaction, and primer set 3 (42FM and 11RM) was used for the inner PCR reaction. DNA from normal lung and no-DNA controls were included in all PCR amplification steps.
Cycle sequencing
PCR products were electrophoresed on a 1.5% low melting point agarose gel and visualized using ethidium bromide. The desired bands were excised and gel purified using Qiagen purification columns (Qiagen Inc., Valencia, CA). The PCR products were sequenced using 33P Thermosequenase Cycle Sequencing KitTM (US Biochemical/Amersham, Cleveland, OH) and primers RM1, RM4 and FM5 (sequence given above) were used as the sequencing primers. Normal lung and no-DNA controls were included in the cycle sequencing reactions. The sequencing reactions were performed in duplicate to verify results.
Southern analysis of cell lines
High molecular weight DNA (10 µg) was either digested with HindIII alone or digested sequentially with both HindIII and HpaII (New England Biolabs). HpaII is a methylation-sensitive restriction enzyme that does not cut if either cytosine in the CCGG sequence is methylated. Digested DNA samples were loaded on 0.7% agarose gels and separated at 45 V for 17 h. The DNA was transferred to GeneScreen Plus® hybridization transfer membranes (NEN Research Products, Boston, MA) and crosslinked. The filters were hybridized overnight at 42°C in 10 ml HybridsolTM (Oncor, Gaithersburg, MD) and probed with a 862 bp gel purified and 32P-labeled PCR product generated with p16Ink4a primers mp16-7F (5'-GTG TAC AGA ATC CTA GCA CTG-3'), located 750 bp upstream of the translational start site, and mp16-16R (5'-GTA CGA CCG AAA GAG TTC G-3'), located 112 bp downstream of the translational start site within exon 1
. This region included three HpaII restriction sites and no HindIII restriction sites. Filters were washed twice in 2x SSC/0.1% SDS at room temperature for 10 min each followed by subsequent washes in 0.1x SSC/0.1% SDS at 50°C. The filters were exposed to BIOMAXTMMR film (Eastman Kodak Company, Rochester, NY) for 3 days at 80°C.
Treatment of cell lines with 5-Aza-CdR
C10 and sp6c cell lines were plated at a density of 5x105 cells and allowed to attach overnight. Cells were then treated with either 0.1 µM 5-Aza-CdR, 1 µM 5-Aza-CdR, 30 nM Trichostatin A (TSA), 0.1 µM 5-Aza-CdR/30 nMT SA or 1 µM 5-Aza-CdR/30 nM TSA for 24 h. Media were changed every 24 h and the cells were harvested on day 3 following treatment. Pellets were frozen and stored at 80°C for subsequent DNA and RNA isolation. The CMT64 cells were cultured in the same manner as described above, except that these cells were treated with TSA following a 24 h treatment with 0.1 µM 5-Aza-CdR and 1 µM 5-Aza-CdR.
RNA isolation and RTPCR of tumors and cell lines
Tumors were homogenized in lysis buffer using a Polytron and cells were lysed using the QIAshredder (Qiagen Inc.). The RNA was isolated from both the tumors and cells using RNeasy spin columns (Qiagen Inc.) according to the manufacturer's instructions. First-strand cDNA was generated from 5 µg total RNA using the SuperScript Kit (Life Technologies). The PCR reaction was performed using promoter- and exon-specific primers for amplification of p15Ink4b, p16Ink4a, p19Arf and ß-actin in separate reactions. Conditions were optimized for individual primer sets to use the lowest number of amplification cycles for detection of the products. The p16Ink4a and p19Arf transcripts were amplified in a Perkin-Elmer 9600 DNA thermal cycler using 27 cycles (denaturation at 94°C for 30 s; annealing at 53°C for 30 s; extension at 72°C for 30 s). The p15Ink4b and ß-actin transcripts were amplified in a Perkin-Elmer 480 DNA thermal cycler for 28 cycles (denaturation at 94°C for 1 min; annealing at 60°C for 30 s; extension at 72°C for 1 min) and 24 cycles (denaturation 94°C for 30 s; annealing for 53°C for 30 s; extension at 72°C for 1 min), respectively. Amplification of p15Ink4b, p19Arf and ß-actin was performed as a comparison for p16Ink4a expression. The following primers were used in the PCR reactions: (i) p16Ink4a: mp16-1F (5'-TCT GGA GCA GCA TGG AGT CC-3') and mp16-2R (5'-TCG CAG TTC GAA TCT GCA CC-3'); (ii) p19Arf: mp19-1F (5'-AGT ACA GCA GCG GGA GCA TG-3') (23) and mp16-2R (same sequence as given above); (iii) p15Ink4b: mp15-1F (5'-TTG TCT CAT GAC GTC ACC AAG-3') and mp15-2R (5'-TG GAT TGG GCG CCT CCC GAA-3'); and (iv) ß-actin: mact-1F (5'-CAC CAC ACC TTC TAC AAT GAG-3') and mact-2R (5'-CAG GAT GGT GTG AGG GAG AG-3'). Products were then electrophoresed together on 8% acrylamide/urea/formamide gels at 40 W. Also, due to a polymorphism in p16Ink4a exon 1
(33) between the C57BL/6 and C3H/He alleles, we were able to examine allele-specific expression by SSCP analysis on a 6% non-denaturing polyacrylamide gel with 10% glycerol at 35 W for 2 h at 4°C. No-RT and no-cDNA controls were included in all amplification reactions. Results were verified in duplicate amplification reactions.
Cell proliferation in cell lines treated with 5-Aza-CdR
Cell lines C10 and sp6c were cultured as described above. Cells were seeded at a density of 2x104 and allowed to attach overnight. Four treatments (five plates per treatment group) were administered for a 24 h period: (i) control, no treatment; (ii) 1 µM 6-aza-cytidine (6-Aza-C); (iii) 0.1 µM 5-Aza-CdR; (iv) 1 µM 5-Aza-CdR. Following the incubation period, the media were removed and the cells were washed gently in PBS. Cells were placed in fresh media and were maintained for an additional 48 h. At this time, the media was removed and the cells were washed gently in PBS. The cells were trypsinized and resuspended in 10 ml media. Four aliquots of cells from each of five plates per treatment were counted using a hemocytometer. The numbers were averaged and the number of cells in each treatment group was recorded as percent of the control. The experiment was repeated with similar results.
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Results
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LOH and mutation analysis in B6C3F1 primary mouse lung tumors
Characterization of LOH and p16Ink4a mutations in the tumor set used in this study was performed prior to beginning an assessment of p16Ink4a methylation status. Five of the methylene-chloride-induced tumors in this study were examined previously for LOH by Hegi et al. (22). The remaining 11 tumors were generated in the same carcinogen bioassay. Previous studies showed that LOH in the chromosome 4 region surrounding the p16Ink4a gene is common in mouse lung tumors (22,24,34,35). Fourteen methylene chloride-induced and three spontaneous B6C3F1 primary mouse lung adenocarcinomas were screened for genetic aberrations in p16Ink4a using LOH and SSCP analyses. LOH of the C3H/He allele was observed in 4/14 (28%) of the chemically induced tumors and 0/3 of the spontaneous tumors examined at two microsatellite markers near and within the p16Ink4a gene (data not shown). SSCP analysis of p16Ink4a exons 1 and 2 revealed no point mutations in any of the 17 B6C3F1 tumors analyzed in our study.
Methylation of the p16Ink4a promoter in primary mouse lung tumors
Because few mutations or homozygous deletions of p16Ink4a were detected in our mouse lung tumors, we decided to investigate by bisulfite genomic sequencing the possible role of p16 inactivation by DNA methylation in mouse lung tumorigenesis. The DNA was modified by treatment with sodium bisulfite, which deaminates unmethylated cytosine residues to uracils that are later replaced by thymines during the PCR amplification process. This method leaves the methylated cytosines intact, allowing site-specific and region-specific methylation to be determined. Direct sequencing of sodium-bisulfite-treated DNA was used to assess the methylation status of CpG sites 1 to 9 (nucleotides 23 to 172 relative to the translational start site, respectively) and 12 to 19 (nucleotides 307 to 433 relative to the translational start site, respectively) in 14 methylene chloride-induced adenocarcinomas, three spontaneous B6C3F1 mouse lung adenocarcinomas, and normal C57BL/6, C3H and B6C3F1 mouse lungs. The methylation status of individual CpG sites in the p16Ink4a promoter region was scored visually according to the following categories: U, unmethylated/undetectable methylation; P, partially methylated, <50% methylation; and M, methylated,
50% methylation (Table I
). All of the tumors possessed at least six partially methylated sites, while 12/17 (70%) of the tumors displayed
50% methylation at two or more CpG sites. The overall pattern of methylation appeared random, and site-specific methylation was not observed. Sites 1 to 7 that are closest to the translational start site were methylated more frequently and to a greater degree than the other CpG sites assessed. One CpG site was partially methylated in 1/5 normal lung tissues examined.
Expression of p16Ink4a by RTPCR and immunohistochemistry
Similar to results obtained by Belinsky et al. (25), analysis of p16Ink4a expression by RTPCR in the 17 B6C3F1 mouse lung tumors and three normal lungs revealed variable expression of the p16Ink4a transcript (data not shown). Due to a polymorphism in exon 1
of p16Ink4a between the C57BL/6 and C3H/He mice, it was possible to assess allele-specific expression by SSCP analysis following RTPCR. Overall differential expression between the C57BL/6 and C3H/He alleles was not observed in these samples (Figure 1
), although one tumor (MC667) exhibited LOH of the C57BL/6 allele. LOH of another region of this tumor sample was not detected in our preliminary study, possibly due to tissue heterogeneity.

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Fig. 1. Expression of C57BL/6 and C3H/He alleles of p16Ink4a in B6C3F1 mouse lung carcinomas by SSCP. Due to a polymorphism in this region between C57BL/6 and C3H/He mice allele-specific expression could be observed. PCR products were resolved on a 0.5x MDE gel (see Materials and methods for details). Lane 1, C57BL/6 normal lung; lane 2, C3H/He normal lung; lanes 3 and 22, B6C3F1 normal lung; lanes 421, B6C3F1 lung adenocarcinomas; lane 23, no cDNA. Lanes 20 and 21 contain cDNA from different parts of the same tumor.
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Immunohistochemical staining of 13 samples, MC478, MC152, MC667, MC995, MC69, MC717, MC698, MC512, MC599, MC242, MC1009, MC1339A and MC1339B for p16 revealed focal areas of relatively high and low expression within each tumor, demonstrating tissue heterogeneity (Figure 2
). Sufficient tumor tissue was not available to perform immunohistochemical analysis on the remaining samples.

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Fig. 2. Immunohistochemical analysis of p16Ink4a expression in B6C3F1 mouse lung adenocarcinomas and subsequent laser capture microdissection of these samples. (A) Tumor sample MC152 incubated with p16Ink4a antibody (100x). Areas of tumor with relatively high and low p16 expression are designated with arrows. (B) Tumor sample MC152 incubated with non-immune mouse IgG (100x). Note the lack of staining in this section. (C) Tumor sample MC995 incubated with p16Ink4a antibody (40x). Note that within the tumor are populations of cells showing high or low expression of the p16 protein (see arrows). (D) Higher magnification of tumor sample MC995 incubated with p16Ink4a antibody (200x). Note that some cells are strongly positive for the p16 protein while others have low levels of the p16 protein (see arrows). (E) Serial section of tumor sample MC995 which corresponds to (C) and shows the areas sampled by LCM and areas of high and low p16 expression (see arrows). (F) Captured cells from LCM procedure representing neoplastic cells with high p16 expression (MC995-2) and low p16 expression (MC995-3) (see arrows).
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Methylation of p16Ink4a in laser capture microdissected samples
In order to demonstrate that the methylation status affected p16 expression, the LCM technique was used to microdissect areas of relatively high versus low p16 staining in tumor samples MC152, MC512, MC667 and MC995 (Figure 2E and F
). Following DNA isolation and sodium bisulfite treatment, methylation was examined at CpG sites 1 to 7. In 9/11 (82%) of the microdissected areas from the four tumors, the pattern of methylation correlated inversely with the pattern of expression (Figure 3
; Table II
). For example, in tumor sample MC152-4 (Figure 3
; Table II
) all seven CpG sites examined in DNA from a region of cells with a low level of p16 expression were completely methylated (no T band in the lane corresponding to the methylated C band), while only one site was partially methylated in DNA from tumor sample MC152-2 in an area expressing a high level of p16 (Figure 3
; Table II
). An exception was tumor MC995 in which one area of high p16 expression exhibited high promoter methylation and another area with low expression displayed low methylation (Table II
). A semi-quantitative analysis of the number of sites displaying
50% methylation (designated by M in Table II
) demonstrates that 31% (11/35) of the sites analyzed are methylated in tumor areas expressing high levels of p16 and 64% (27/42) of the sites analyzed are methylated in tumor areas in expressing low levels of p16 (Table II
).

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Fig. 3. Methylation patterns assessed by bisulfite genomic sequencing at CpG sites 1 to 7 in laser capture microdissected areas from tumor samples MC152 and MC995. The sample numbers correspond to the samples listed in Table II . Note that for all the methylated CpG sites in sample MC152-4 there are no corresponding bands in the T lane, indicating 100% methylation at all CpG sites in this sample.
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Methylation of p16Ink4a in mouse lung cell lines
Cell lines E9, LM1, CMT64, CMT167, C10, CL20I.1, CL20H, sp6c, NNK30pp, sp10 and sp10pp, which were previously shown to possess the p16Ink4a gene but lack p16Ink4a expression by RTPCR (23), were examined for hypermethlyation of the p16Ink4a promoter by both Southern analysis and bisulfite genomic sequencing (Table III
). By Southern analysis either complete or partial methylation was observed in cell lines CMT64, CMT167, E9, C10, sp6c, sp10 and sp10pp at sites 10, 11 and +7 (nucleotides 237, 266 and +916 relative to the translational start site, respectively) (Figure 4
). By sodium bisulfite genomic sequencing, partial methylation was observed in cell lines LM1, CL20I.1, CL20H, sp10 and sp10pp, and complete methylation was observed in cell lines E9, CMT64, CMT167 and sp6c at CpG sites 1 to 7 (Table III
). The bisulfite sequencing method is more sensitive for detecting individual methylated CpG sites. Sites 10 and 11 were not assessed by bisulfite genomic sequencing because primer design in this region was not optimal.

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Fig. 4. Southern blot hybridization of DNA from mouse lung cell lines for methylation of p16Ink4a. Genomic DNA from 11 mouse lung tumor cell lines was restricted with HindIII alone (lane a) or HindIII/HpaII (lane b) to evaluate the methylation status in the promoter region and exon 1 of p16Ink4a. This region contained three HpaII sites. A 7.3 kb product is detected in samples digested with only HindIII. Samples are indicated above each lane. Normal 1, DNA isolated from A/J lung; normal 2, DNA isolated from C57BL/6 lung; normal 3, DNA isolated from BALB/c lung.
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Treatment of mouse lung cell lines with 5-Aza-CdR
To provide further evidence that methylation of promoter region CpG sites affected the expression of the p16Ink4a transcript in mouse lung carcinogenesis, three cell lines (C10, sp6c and CMT64) that displayed complete methylation at sites 1 to 7 by bisulfite genomic sequencing and undetectable p16Ink4a expression prior to treatment were exposed to the demethylating agent, 5-Aza-CdR. The expression of p15Ink4b, p16Ink4a, p19Arf and actin transcripts was assessed by RTPCR (Figure 5
). Re-expression of p16Ink4a was observed in the C10 and sp6c cell lines following treatment with 1 µM 5-Aza-CdR. Evidence from previous studies (36) has shown that treatment with TSA, a histone deacetylase inhibitior, in conjunction with 5-Aza-CdR enhances the expression of some genes. However, treatment with 0.1 µM 5-Aza-CdR or 1 µM TSA, or a combination of the two, did not have an observable effect on p16Ink4a expression in these cells. Expression of p15Ink4b and p19Arf was consistent and unchanged in both cell lines with all treatments (Figure 5
). Bisulfite genomic sequencing of DNA from treated C10 and sp6c cell lines displayed partial methylation in cells treated with 1 µM 5-Aza-CdR in comparison with untreated cells that were methylated completely (data not shown). DNA from all other treatments displayed complete methylation of sites 1 to 7. Interestingly, in the treated and untreated CMT64 cells low levels of p16Ink4a expression were observed but no p15Ink4b and p19Arf transcripts were detected in the untreated CMT64 cells. Enhanced expression of p16 and re-expression of the p15Ink4b and p19Arf transcripts were observed upon treatment with either 0.1 or 1 µM 5-Aza-CdR (data not shown).

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Fig. 5. Expression of p15Ink4b, p16Ink4a, p19Arf and actin by RTPCR in C10 and sp6C mouse lung cell lines following treatment with 5-Aza-CdR. Following reverse transcription of total RNA, cDNA amplification was performed in separate reactions, and then the PCR products were resolved on a 6% acrylamide/urea/formamide gel. The PCR products from the C10 and sp6c cell lines are in lanes 16 and 712, respectively. The specific treatments are noted on the figure. Lanes 3, 6, 9 and 12, re-expression of p16Ink4a following treatment with 5-Aza-CdR; lane 13, normal cDNA from C57BL/6 lung; lanes 1417, sample 3 with PCR products loaded in individual lanes. (**Note that actin and p15Ink4b aliquots were not loaded in lane 8.)
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Effect of 5-Aza-CdR treatment on cell proliferation
Treatment of the C10 and sp6c cells with either 0.1 or 1 µM 5-Aza-CdR decreased cell growth when compared with the untreated cells. In addition, treatment with the 1 µM 6-Aza-C, which is not a demethylating agent like 5-Aza-CdR, did not adversely affect cell growth (Figure 6
). These results are similar to those observed in a study by Otterson et al. (17).

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Fig 6. Cell lines C10 and sp6c were subjected to four treatments: (i) control, no treatment; (ii) 1 µM 6-Aza-C; (iii) 0.1 µM 5-Aza-CdR; (iv) 1 µM 5-Aza-CdR. Cells were plated at a density of 2x104 cells and allowed to attach overnight. The cells were treated for 24 h, washed with PBS, and incubated in fresh media for an additional 48 h. At the end of the incubation period, the cells were trypsinized and counted using a hemocytometer. The cell number was calculated and recorded as percent of the control.
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Discussion
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The detection of methylated CpG sites within the p16Ink4a promoter region in B6C3F1 primary mouse lung tumors and mouse lung cell lines provides the first definitive evidence that DNA hypermethylation and subsequent downregulation of p16Ink4a expression play a role in mouse lung carcinogenesis. The patterns of p16Ink4a expression observed in this study corroborate previous studies with A/J mouse lung tumors, which reported variable expression of both p16Ink4a mRNA and protein (25,26). Studies with mouse lung tumor cell lines (23) and primary A/J mouse lung tumors (25) showed that p16Ink4a may become inactivated by homozygous deletion, although there are some cell lines that lack the p16Ink4a transcript but possess the gene. In general, point mutations in p16Ink4a have not been observed in chemically induced or spontaneous mouse lung tumors (25,26), except in one study that detected a small percentage of point mutations in 3-methylcholanthrene transplacentally induced DBA/2 lung tumors (27). Collectively, these studies provide support for a role for p16 inactivation in mouse lung carcinogenesis, and our study indicates that another mechanism of p16 inactivation in mouse lung tumors is promoter DNA methylation.
Although hypermethylation of CpG islands has been shown to transcriptionally inactivate several tumor suppressor genes, including p16Ink4a, in human cancers (12), the role of DNA methylation in mouse lung carcinogenesis has not been extensively investigated. One study reported no detectable methylation of p16Ink4a in A/J mouse lung tumors (25). However, that study only examined methylation at one ThaI restriction site in the promoter and one HpaII restriction site in exon 1
using Southern analysis and methylation-sensitive restriction enzyme digests (25). Our study used bisulfite genomic sequencing, a more sensitive PCR-based method, to assess the methylation status of multiple CpG sites in the p16Ink4a promoter. In contrast to normal lung tissue, we found at least partial methylation of p16Ink4a in all of the chemically induced and spontaneous mouse lung adenocarcinomas examined.
General patterns of DNA methylation, which include critical site, multiple elements and methylation density models, are compatible with the concept that methylation is a dynamic, stochastic, partial and regional process (20). Unlike some studies that demonstrate abrogation of transcription factor binding sites by critical site methylation (37,38), our study shows that decreased gene expression of p16Ink4a correlates best with a density-dependent methylation model. Evidence from work with p15INK4b in primary acute leukemia demonstrated that the density of methylation at CpG sites strongly correlated with gene silencing (39). In that case, 3040% of the methylation at CpG sites was associated with transcriptional repression (39). Another study using human bladder cancer cell lines provided evidence that density-dependent methylation is one mechanism of p16Ink4a promoter methylation that results in a decrease in gene expression (40). That study (40) and ours found a higher degree of methylation in sites near the 3' end of the promoter. Thus, the pattern of p16Ink4a methylation observed in the mouse lung adenocarcinomas and cell lines in our study is similar to methylation patterns observed for human cell cycle regulatory genes, including p16Ink4a.
Discrepancies between expression and methylation in a few samples can be explained by several scenarios. Tumor areas which possess low or no expression and low methylation may have homozygous deletions of p16Ink4a. Tumor areas that displayed apparent high expression and high methylation of p16Ink4a are more difficult to explain. Levels of the p16 protein increase in response to DNA damage (41), and this may occur early in lung carcinogenesis, possibly as a result of mutations in oncogenes such as K-ras (42). This could help to explain the variable expression of p16Ink4a among tumors and within tumors, especially after chemical treatment. Similar to the regulation of expression of the human telomerase reverse transcriptase (hTERT) gene (43), expression of p16 could be regulated by both methylation-dependent and -independent mechanisms.
It is possible that methylation increases early in neoplastic progression even while cells are subjected to DNA damage and levels of p16 are high (44). Hypermethylation of p16Ink4a has been detected in the preinvasive lesions of individuals with established lung carcinoma (20,44). Thus, increased random CpG methylation of the p16Ink4a promoter that represses gene expression in a progressive (40) manner could be manifested at later stages in tumor development after methylation accumulates and reaches a critical threshold in different parts of the tumor (40). Furthermore, based on the evidence from our study, showing some methylation of the p16Ink4a promoter in all tumors examined, p16Ink4a hypermethylation is likely to precede LOH, which was found in 24% of the tumors in this study and has previously been documented in carcinomas but not adenomas (22). The question of whether methylation exists in mouse lung adenomas remains to be answered since only adenocarcinomas were examined in this study.
The experiments with the 5-Aza-CdR-treated mouse lung cell lines further supports the involvement of p16 in mouse lung carcinogenesis and duplicates observations from studies with human cell lines (17,18). Treatment of the methylated C10 and sp6c cell lines, which lack p16Ink4a expression, with the demethylating agent, 5-Aza-CdR, induced re-expression of the p16Ink4a transcript. Changes in chromatin conformation did not seem to be important, because treatment with the histone deacetylase inhibitor TSA, either alone or in combination with 5-Aza-CdR, had no observable effect on p16Ink4a expression. In previous studies of human cell lines, TSA treatment in the presence of low doses of 5-Aza-CdR, but not high doses, resulted in significant re-expression of MLH1, TIMP3 and p16Ink4a (36). In our study, re-expression of p16Ink4a correlated with a decrease in the methylation status of the p16Ink4a promoter in the sp6c and C10 cell lines treated with 1 µM 5-Aza-CdR, supporting the role for DNA methylation in the inactivation of p16 in mouse lung carcinogenesis.
Interestingly, treatment of the CMT64 cell line, which expressed low levels of p16 and no detectable p19 or p15, with 5-Aza-CdR induced re-expression of p19, a component of the p53 pathway, and p15. This observation provides evidence of a role for p15 and p19 in mouse lung carcinogenesis and is supported by observations that p15Ink4b and p19Arf are frequently inactivated in mouse lung tumor cell lines (23). Together these studies emphasize the importance of not only p16 but also p15 and p19 in mouse lung tumor development.
Overall our study demonstrates an additional similarity between mouse models of lung carcinogenesis and the development of human lung cancer. Hypermethylation of the p16Ink4a promoter at CpG sites has been demonstrated in a variety of primary human tumors, including NSCLC, with the effect of decreased expression of the p16Ink4a transcript (19,21,45,46), thus providing a selective growth advantage for the affected cells (20). Until recently, the paradigm for the inactivation of tumor suppressor genes has involved primarily intragenic mutation and loss of chromosomal material via loss of heterozygosity or homozygous deletion (47). However, the emergence of DNA methylation as a mechanism in carcinogenesis not only adds another pathway that should be considered when attempting to identify mechanisms of gene inactivation (47) in both mouse and human lung carcinogenesis, but also underscores the importance of epigenetic mechanisms in the development of cancer.
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Notes
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5 To whom correspondence should be addressed Email: devereux{at}niehs.nih.gov 
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Acknowledgments
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We thank Catherine M.White for her technical assistance and Drs Roger Wiseman and Robert Sills for their critical review of the manuscript. We also acknowledge Astrid Haugen-Strano for developing the Mts1 primers used for PCR and LOH analyses of the microsatellite sequence in the p16Ink4a promoter.
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Received January 14, 2000;
revised May 18, 2000;
accepted May 24, 2000.