Purpose: Death-associated protein kinase (DAPK) is a pro-apoptotic serine/threonine kinase involved in apoptosis. Aberrant methylation of DAPK was reported in lung cancers by methylation-specific PCR. However, we were unable to relate methylation with gene silencing with the same methodology. Our goals were to develop a methodology that related methylation with gene silencing and use it to study the state of the gene in lung cancers.

Experimental Design and Results: Using a semiquantitative real-time reverse transcription-PCR, DAPK expression was lower in lung cancers than in corresponding nonmalignant bronchial epithelial cells in five of six primary short-term cultures. In continuous cell lines, mRNA expression was down-regulated, as well as compared with nonmalignant bronchial epithelial cells, and its protein was not detected by Western blotting in 17 of 23 (74%) cell lines. We investigated methylation status of 5′ flanking region of DAPK by combined bisulfite restriction analysis and bisulfited DNA sequencing. Aberrant methylation was detected in 21 of 48 (44%) cell lines, 2 of 6 primary cultured tumors, and 14 of 38 (37%) primary lung cancers, although varying degrees of methylation were noticed. Furthermore, bisufite sequence data suggested that aberrant methylation might occur selectively at some CpG dinucleotides in cell lines which had absent expression. Treatment with 5-aza-2′-deoxycytidine restored DAPK expression in heavily methylated cell lines tested, and histone deacetylase inhibitor trichostatin A alone restored DAPK expression in some methylated cell lines as well.

Conclusions: Our major findings are: (a) DAPK expression is frequently down-regulated in lung cancers; (b) aberrant methylation of DAPK is frequent in lung cancers, although considerable heterogeneity of methylation is present, and some specific CpG dinucleotides are often methylated in expression negative lung cancers; and (c) besides methylation and histone deacetylation, there may be other mechanisms for down-regulation of DAPK expression.

The DAPK3 family is a novel subfamily of pro-apoptotic serine/threonine kinases, consisting of at least five family members which are ubiquitously expressed in various tissues and capable of inducing apoptosis (1, 2). The sequence homology of the five kinases is largely restricted to the NH2-terminal kinase domain, whereas the adjacent COOH-terminal regions are very diverse and link individual family members to specific signal transduction pathways. The DAPK family members are involved in both extrinsic and intrinsic pathways of apoptosis. In addition, inactivation of DAPK decreases the induction of p19ARF/p53, resulting in inactivation of the p53-dependent apoptotic pathway (3). The original and best studied family member is DAPK, which contains a death domain at its COOH-terminal region and was initially isolated as a positive mediator of apoptosis induced by IFN-γ (4, 5). It plays a role in tumor pathogenesis and metastasis (5).

Loss of DAPK expression has been documented in many cancer types, including lymphomas, nasopharyngeal carcinomas, pituitary adenomas, and cancers of the gastrointestinal tract and cervix (6, 7, 8, 9). Functional loss of tumor suppressor genes may occur via point mutations, allelic deletions, homozygous deletions, or by aberrant methylation of the promoter region. Loss of DAPK kinase usually occurs by aberrant methylation, although other mechanisms have been described. In lung cancer, methylation of DAPK has been reported at frequencies ranging from 19 to 44% (10, 11, 12, 13). Methylation of NSCLC has been reported to be associated with poor prognosis (12), advanced pathologic stage increased tumor size, and lymph node involvement (11), but not with tobacco or asbestos exposure, or with k-ras or p53 mutations (11). Methylation may occasionally be detected in the bronchial epithelium of smokers (14). DAPK is located on chromosome 9q34.1 (15), a region of frequent allelic loss in both NSCLC and SCLCs (50–64%; Ref. 16).

For methylation to be biologically important, it should be related to gene silencing (17). However, the precise relationship between methylation and gene expression in lung cancers and their cell lines is not documented. All of the above cited references to DAPK methylation and lung cancer used the methodology and primers designed by Herman et al.(6, 10). These primers target the noncoding region of exon 1 instead of the usually used promoter region because the sequence of the promoter region was not available at that time.4 Our goals were to develop the methodology which related methylation of the promoter region with gene silencing and to use it to study the methylation status of the gene in lung cancer cell lines and tumors.

Lung Cancer Cell Lines.

Forty-eight lung cancer cell lines (23 NSCLCs and 25 SCLCs) established by us (18) were used for this study. Cell cultures were grown in RPMI 1640 (Life Technologies, Inc., Rockville, MD), supplemented with 5% fetal bovine serum and incubated in 5% CO2 at 37°C.

Primary Culture of Resected Lung Cancer and NBEC.

Six NSCLC primary cells (two cases each of adenocarcinoma, squamous cell carcinoma, and large cell carcinoma) and their corresponding NBECs were cultured as described previously (19). In brief, tissues were diced and placed in trypsin at 4°C for 22–24 h. The next day, the specimens were cultured in MCDB153++ medium, consisting of MCDB 153 basal medium (Sigma, St. Louis, MO), supplemented with growth factors in a collagen-coated dish. The epithelial nature of the cultured cells was confirmed by immunostaining. Early passages were preserved in liquid nitrogen until tested.

Clinical Samples.

Surgically resected specimens from 38 primary lung tumors and 15 corresponding nonmalignant lung tissues were obtained for methylation studies. Nonmalignant control samples included bronchial brushes from smoker subjects, buccal swabs, and peripheral blood lymphocytes from healthy never-smoker volunteers (Table 3). Appropriate Institutional Review Board permission was obtained from both participating centers, and written informed consent was obtained from all subjects.

mRNA Expression of DAPK by Semiquantitative Real-time RT-PCR.

Expression of DAPK was analyzed by semiquantitative real-time RT-PCR. Total RNA was extracted from the cell lines with TRI Reagent (Molecular Research Center, Inc., Cincinnati, OH) following the manufacturer’s instructions. Reverse transcription reaction was performed on 2 μg of total RNA with the SuperScript II First-Strand Synthesis using oligo (dT) primer (Life Technologies, Inc.). cDNAs were semiquantified by fluorescence-based real-time RT-PCR by using TaqMan technology (Perkin-Elmer Corp., Foster City, CA), with the Gene Amp 5700 Sequence Detection System (Perkin-Elmer Corp.). As an internal reference gene, TBP(20) was used to normalize the expression of DAPK. The expression ratio was defined as the ratio of the fluorescence emission intensity values for the DAPK PCR products compared with those of the TBP, multiplied by 100. This ratio was used as a measure for the relative level of DAPK expression in the particular sample. The sequences of the primers and probe used to quantify DAPK were as follows: 5′-TTCAGGCAGGAAAACGTGGAT-3′ (Forward primer), 6FAM-5′-ACACCGGCGAGGAACTTGGCAGT-3′-TAMRA (probe), and 5′-TTTTCTCACGGCATTTCTTCACA-3′ (reverse primer) and TBP were 5′-TGCTGCGGTAATCATGAGGAT-3′ (forward primer), 6FAM-5′-AGAGAGCCACGAACCACGGCACTG-3′-TAMRA (probe), and 5′-TGGAAAACCCAACTTCTGTACAAC-3′ (reverse primer). Semiquantitative real-time RT-PCR was performed in a reaction volume of 50 μl. The final reaction mixtures contained the forward and reverse primers at 300 nm each; the probe at 100 nm; 200 μm each of dATP, dCTP, and dGTP; 400 μm deoxyuridinetriphosphate, 5 mm MgCl2, 1 × PCR buffer, 1 unit of HotStarTaq DNA polymerase (Qiagen, Inc., Valencia, CA), and 2 μl of cDNA. PCR was performed under the following conditions: 95°C for 12 min, followed by 50 cycles of 95°C for 15 s and 60°C for 1 min. Semiquantitative real-time RT-PCR for both DAPK and TBP were performed in duplicate. We used serial dilutions of the positive control cDNA to create a standard curve.

Protein Expression of DAPK by Western Blotting.

Proteins (30 μg of total) were separated by SDS-PAGE in 5% polyacrylamide gels and transferred to nitrocellulose membranes. The membranes were incubated with monoclonal antibody to DAPK (BD Transduction, Laboratories, Lexington NY) and then with secondary antibody coupled with horseradish peroxidase, according to the manufacturer’s instructions. The membrane was developed by SuperSignal West Pico Chemiluminescent Substrate (Pierce, Rockford, IL).

DNA Extraction and COBRA.

Genomic DNA was obtained from cell lines, primary tumors, and nonmalignant cells by digestion with proteinase K (Life Technologies, Inc.) followed by two extractions with phenol/chloroform (1:1; Ref. 21) and treated with sodium bisulfite. Briefly, 1 μg of genomic DNA was denatured by NaOH and modified by sodium bisulfite (Sigma). The modified DNA was purified using Wizard DNA purification kit (Promega, Madison, WI) and treated with NaOH to desulfonate, precipitated with ethanol and resuspended in water. COBRA was performed as described previously (22). Sodium bisulfite-treated DNA was amplified by PCR using the following primers: (a) DAPK forward 5′-AGGGTAGTTTAGTAATGTGTTATAGG-3′ and (b) DAPK reverse 5′-CCTTAACC-TTCCCAATTACTC-3′. These primers were designed to exclude binding to any CpG dinucleotide to ensure amplification of both methylated and unmethylated forms. The resultant 659-bp amplicon of the DAPK was termed RCOBRA (Fig. 1). PCR products were digested with BstUI (CGCG) and TaqI (TCGA) separately. There are nine sites for BstUI and five sites for TaqI in RCOBRA (Fig. 1). Digested PCR products were visualized on 2% agarose gels stained with ethidium bromide.

5-Aza-CdR and TSA Treatment.

Lung cancer cell lines were incubated in culture medium with 5-Aza-CdR or TSA (23). Drug treatment was accomplished by adding reagents to the culture medium to final concentrations as follows: (a) 5-Aza-CdR alone for 6 days, 4 μm, and (b) TSA alone for 24 h, 300 nm. Media were changed every 48 h for 5-Aza-CdR and 24 h for TSA.

Map of 5′ Flanking Region of DAPK and Sodium Bisulfite-treated DNA Sequencing.

The RCOBRA was sequenced to determine its methylation status. Nine cell lines and two pairs of primary tumor cells and corresponding NBEC culture were cloned (seven cell lines were methylated, and two were unmethylated by COBRA). The locations of CpG sites in the RCOBRA are shown in Fig. 1 A. PCR products for RCOBRA were cloned into plasmid vectors using the Topo TA cloning kit (Invitrogen), following the manufacturer’s instructions. Plasmid DNAs were purified using the Wizard Plus miniprep kit (Promega) and then sequenced by Applied Biosystems PRISM dye terminator cycle sequencing method (Perkin-Elmer Corp.). This region encompassed 74 CpG dinucleotides, which included exon 1 (24).

Statistical Analysis.

The quantitative ratios of different groups were compared using the Mann-Whitney U nonparametric test. Probability values of P < 0.05 were regarded as statistically significant. All statistical tests were two sided.

DAPK mRNA and Protein Expression.

We performed semiquantitative real-time RT-PCR to examine DAPK mRNA in six pairs of short-term cultures of NSCLC tumors and corresponding NBECs, lung cancer cell lines, and two cases of freshly obtained bronchial mucosa (Table 1 and Fig. 2). The mean DAPK expression ratio in fresh bronchial mucosa specimens (mean = 901) and cultures (mean = 922) and mean ratio for all bronchial specimens was 917. We also compared mRNA expression ratio and protein expression in 23 lung cancer cell lines (Figs. 2 and 3 and Table 2). The mean ratios of the protein-negative and -positive groups were 17 and 645, and protein was not detected by Western blotting if the expression ratio was <100. On the basis of these results, we considered a ratio < 100 as down-regulation of DAPK expression. Sixteen of 25 (64%) lung cancer cell lines and three of six (50%) primary NSCLC cultures were down-regulated (Tables 1 and 2). Furthermore, DAPK expression of five of the six paired samples was lower in tumors than in corresponding NBECs (Table 2). The expression ratios of two bronchial brush samples were 850 and 951, suggesting that DAPK expression in short-term culture samples was not affected by culture condition, and short-term culture samples (mean = 922) were good models for DAPK expression study (Fig. 2).

5-Aza-CdR and TSA Treatment.

We classified 25 cell lines into three patterns based on DAPK expression and methylation status: (a) cell lines with pattern A consisted of 10 (40%) cell lines with aberrant methylation and down-regulated expression status; (b) pattern B consisted of six (24%) cell lines which were methylation negative and down-regulated for expression; and (c) pattern C consisted of nine (36%) cell lines that were methylation negative and had normal expression. Eight cell lines of pattern A, three of pattern B, and four of pattern C were treated with 5-Aza-CdR or TSA. DAPK expression of pattern A cell lines was up-regulated >10-fold by 5-Aza-CdR in seven of eight cell lines, although the degree of up-regulation varied considerably (Table 1). In three cell lines with pattern A (NCI-H1963, NCI-H2195, and HCC15), DAPK expression was up-regulated >10-fold by TSA alone. By contrast, DAPK expression was not up-regulated dramatically (<2-fold) in patterns B and C by treatment with either drug. Expression levels of some cell lines decreased compared with baseline (no treatment) level. A cytotoxic effect from drug exposure was observed in some of these cell lines, which may be the cause for decreased expression.

Aberrant Promoter Methylation of DAPK.

We performed COBRA for DAPK in lung cancers and nonmalignant samples. (Table 3 and Fig. 4). There were no samples which were not amplified by PCR, indicating no examples of homozygous deletion in the examined region. Because the degree of digestion by restriction enzymes was very variable, considerable heterogeneity of methylation may exist, although the possibility of incomplete digestion by restriction enzymes was not excluded. Although some digested bands showed a weak intensity by agarose gel analysis, we considered samples as being methylation-positive alleles if the PCR products were digested by either BstUI or TaqI. By these criteria, aberrant methylation was present in 21 of 48 (44%) cell lines. Twenty samples were digested by both BstUI and TaqI, and one cell line was digested by BstUI but not TaqI. Two of 6 primary cell-cultured lung tumors were digested by BstUI and TaqI, indicating positive methylation. In primary lung cancers, 14 of 38 (37%) cases showed the presence of aberrant methylation. Eleven samples were digested by both BstUI and TaqI, 2 were digested only by BstUI, and 1 was digested only by TaqI. There were no significant differences in frequencies of aberrant methylation among the histological types of primary NSCLC. Aberrant methylation was present in 1 of 12 peripheral blood lymphocytes and none of the buccal swabs from healthy volunteers. Aberrant methylation was present in 1 of 15 corresponding nonmalignant lung tissues and none of 5 bronchial-blushing samples (Table 3).

Sodium-bisulfited Genomic DNA Sequencing.

Randomly selected DAPK alleles in samples were examined for the methylation status of 74 CpG dinucleotides within RCOBRA (Fig. 1,B). PCR amplicons were cloned to sequence the RCOBRA region that included the MSP primer attachment sites reported previously (Fig. 1). The data confirmed the existence of methylation in cell lines, which revealed aberrant methylation by COBRA; however, there was considerable heterogeneity of methylation, and methylation hot spots were identified, e.g., cell line NCI-H60, which was regarded as methylation positive by COBRA, although bisulfite sequence data showed the degree of methylation was relatively low. Of interest, the MSP primer sites were not frequently methylated in the samples. Two unmethylated cell lines by COBRA showing high (NCI-H1994) or low (HCC366) expression levels were hardly methylated in 10 cloned alleles, confirming the results of COBRA. Although there was considerable heterogeneity, alleles from COBRA-positive primary cancer cell cultures were frequently methylated compared with corresponding NBECs. Furthermore, the frequently methylated sites in primary tumor cultures were similar to those methylated in established cancer cell lines. Regarding the fidelity of bisulfite treatment, the complete conversion of all cytosines not consisting of CpG sites to uracils in all sequenced clones indicates the high fidelity of bisulfite treatment. This result strongly suggests the reason for heterogenesity is not incomplete bisulfite modification but represents the presence of variously methylated alleles in samples.

Apoptosis is one of the hallmarks of cancer (25), and lung cancers develop resistance to apoptosis at multiple levels (26). DAPK is an important pro-apoptotic gene inactivated in many tumor types. Although several studies of DAPK inactivation in lung cancers have been published (10, 11, 12, 13), for reasons discussed previously, the true status of the gene in lung cancer was unknown. To study the DAPK gene in lung cancers, we developed or used the following: (a) real-time RT-PCR assay for RNA expression; (b) a Western blot method for protein expression; (c) determined the methylation status of promoter region and exon 1; and (d) a COBRA method for the methylation status that correlated with gene expression.

The reported frequencies of DAPK methylation in NSCLC are highly variable (19–44%; Refs. 10, 11, 12, 13). These reports used the standard MSP method for DAPK methylation as originally reported by Esteller et al.(10). The standard method examines the methylation status of part of the noncoding first exon of the gene, because the sequence of the promoter region was not known at that time.5 For these reasons, we precisely investigated the methylation status of 5′ flanking region in lung cancer samples, using COBRA, which allowed us to investigate the methylation status of a wide region of DAPK gene, including a region reported previously for MSP. Because considerable heterogeneity of methylation was suggested by our COBRA results, we performed sequencing of bisulfite-treated DNA and confirmed the heterogeneity and identified specific methylated sites which were related to gene silencing. Furthermore, we noted that CpG dinucleotides included in the MSP primer sites reported previously were not frequently methylated in lung cancers with absent expression. As we and others have indicated previously, bisulfite sequencing is essential for the design of MSP primers for methylation results to reflect gene silencing (23, 27).

We demonstrate that the expression of DAPK is variable but frequently low in both NSCLC and SCLC cell lines and primary NSCLC cultures compared with NBECs, indicating that DAPK is down-regulated in many lung cancers. To clarify the mechanism for down-regulation of DAPK expression, cell lines were classified into three patterns (patterns A, B, and C) based on expression and methylation status, and we treated samples of each three groups with 5-Aza-CdR or TSA. In cell lines of group A (defined as down-regulated expression with aberrant methylation), the expression was up-regulated by 5-Aza-CdR in seven of eight cell lines or TSA in three of eight cell lines, suggesting that aberrant methylation is the major cause for down-regulation of DAPK expression in this category and that histone deacetylation contributes to DAPK down-regulation as well. In group B cell lines (defined as down-regulated expression without methylation), the expression was not altered by drug treatment with either 5-Aza-CdR or TSA, suggesting the presence of another, as yet unidentified mechanism for down-regulation of gene expression. Group C cell lines (defined as normal expression without methylation) had little or no effect on expression by drug treatment. Of interest, NCI-H60 was not heavily methylated, and the expression was not up-regulated by 5-Aza-CdR. This result suggests that partial methylation was unlikely to be a major cause for gene down-regulation and that other mechanisms may be involved in this cell line. Our data indicate that aberrant methylation and histone deacetylation of the 5′ flanking region are correlated with DAPK silencing in lung cancers. Furthermore, our results indicate that DAPK down-regulation may be dependent on the extent and, possibly, location of methylation. Thus, expression of some cell lines could be restored only by TSA if the promoter was not completely methylated. Recently, Suzuki et al.(28) analyzed the expression of >10,000 genes by microarray analysis and found that 74 genes were up-regulated in expression after treatment with 5-Aza-CdR or TSA. Their conclusion, which is consistent with our findings, is that transcriptional silencing is mediated by both methylation and histone deacetylation, with methylation being dominant.

Previous work has indicated that, in epithelial cells, CpG island methylation increases with age (29, 30), and it is possible that CpG dinucleotide methylation was present in some nonmalignant respiratory epithelial cells. To confirm the tumor specificity of DAPK methylation, we sequenced tumor cells and corresponding NBECs of primary cultured cells (consisting of pure malignant and nonmalignant epithelial cells, respectively). Only occasional methylation was present in NBECs, indicating the aberrant methylation of DAPK was a tumor-dependent phenomenon.

Expression was low or absent in many lung cancer cell lines and primary cultures compared with bronchial epithelium. Sequencing of promoter region indicated that methylation of the first exon was only partially related to gene expression. Methylation of the promoter region correlated more accurately with gene expression. An appropriate MSP assay could not be designed because the relevant methylated region was very limited; thus, a COBRA was used, and all methylated lines, as determined by sequencing, were COBRA positive. Methylated lines reactivated by 5-Aza-CdR or TSA, confirming that methylation, in conjunction with histone deacetylation, was the mechanism in these lines. All expression-positive cell lines show no methylation. However, several unmethylated lines did not express DAPK, indicating that other as yet unknown mechanisms of inactivation exist. Limited data from primary tumor and bronchial mucosa samples confirmed these observations and indicate that the cell line data applied to tumors.

We extended our study to SCLC tumors and cell lines for which little or no data exist. Previously, we have reported differences of methylation status between SCLC and NSCLC for several genes, including P16, RASSF1A, APC, and caspase 8(31, 32). However, there were no significant differences between SCLC and NSCLC tumors and cell lines in the methylation status of DAPK.

Our results demonstrate that DAPK is frequently down-regulated in lung cancers by a combination of methylation of specific CpG dinucleotides in DAPK 5′ flanking region and histone deacetylation; although as yet unidentified mechanism(s) may play a role. Thus, our study confirms that the crucial pro-apoptotic molecule DAPK is inactivated in an important subset of SCLC and NSCLC tumors and cell lines, and we have developed and validated an improved method to document methylation. Our findings are of biological and possibly clinical relevance.

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.

1

Supported by Grant P50CA70907 from the University of Texas Specialized Program of Research Excellence in Lung Cancer and Grant 5U01CA8497102 from the Early Detection Research Network, NCI (Bethesda, MD).

3

The abbreviations used are: DAPK, death-associated protein kinase; MSP, methylation-specific PCR; 5-Aza-CdR, 5-aza-2′-deoxycytidine; TSA, trichostatin A; NSCLC, non-small cell lung cancer; SCLC, small cell lung cancer; TBP, TATA box-binding protein; NBEC, nonmalignant bronchial epithelial cell culture; RT-PCR, reverse transcription-PCR; COBRA, combined bisulfite restriction analysis; RCOBRA, region for COBRA; NCI, National Cancer Institute.

4

J. Herman, personal communication.

5

J. Herman, personal communication.

Fig. 1.

Positions of CpG dinucleotides in the 659-bp-long region of DAPK gene used for COBRA and bisulfite sequencing. The region (named RCOBRA) includes the 5′ flanking region, the untranslated exon 1, and part of intron 1. A, the positions of CpG dinucleotides in the genomic sequence are indicated by thin vertical lines. Bent arrow, transcription start site (TSS; +1). Two horizontal arrows, the locations of the MSP primers reported previously (within exon 1). B, the sites for BstUI and T indicate the sites for TaqI restriction enzymes (also see Fig. 1B). B, map of methylated CpG dinucleotides in individual cloned DNA fragments of lung cancer samples [nine lung cancer cell lines and two pairs of short-term cultures of NSCLC tumors (4T and 6T) and corresponding NBECs (4N and 6N)]. Each row, one sequenced allele. Each circle, a CpG dinucleotide, with • indicating methylation and ○ lack of methylation. Numbers on top, the CpG dinucleotides in the amplicon (5′ to 3′). The positions of CpG dinucleotides for MSP primers reported previously are indicated by horizontal arrows.

Fig. 1.

Positions of CpG dinucleotides in the 659-bp-long region of DAPK gene used for COBRA and bisulfite sequencing. The region (named RCOBRA) includes the 5′ flanking region, the untranslated exon 1, and part of intron 1. A, the positions of CpG dinucleotides in the genomic sequence are indicated by thin vertical lines. Bent arrow, transcription start site (TSS; +1). Two horizontal arrows, the locations of the MSP primers reported previously (within exon 1). B, the sites for BstUI and T indicate the sites for TaqI restriction enzymes (also see Fig. 1B). B, map of methylated CpG dinucleotides in individual cloned DNA fragments of lung cancer samples [nine lung cancer cell lines and two pairs of short-term cultures of NSCLC tumors (4T and 6T) and corresponding NBECs (4N and 6N)]. Each row, one sequenced allele. Each circle, a CpG dinucleotide, with • indicating methylation and ○ lack of methylation. Numbers on top, the CpG dinucleotides in the amplicon (5′ to 3′). The positions of CpG dinucleotides for MSP primers reported previously are indicated by horizontal arrows.

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Fig. 2.

Relationship between DAPK mRNA expression levels, protein expression, and methylation status in lung cancer cell lines. Quantitative ratios were obtained by semiquantitative real-time RT-PCR. Expression of DAPK protein was determined by Western blotting. ○, samples without methylation; •, methylation-positive samples. Thick lines, the mean of each group. n = sample number. Because values are expressed on a log scale, completely negative values are expressed as values of 0.01.

Fig. 2.

Relationship between DAPK mRNA expression levels, protein expression, and methylation status in lung cancer cell lines. Quantitative ratios were obtained by semiquantitative real-time RT-PCR. Expression of DAPK protein was determined by Western blotting. ○, samples without methylation; •, methylation-positive samples. Thick lines, the mean of each group. n = sample number. Because values are expressed on a log scale, completely negative values are expressed as values of 0.01.

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Fig. 3.

Representative examples of Western blot analysis for DAPK in lung cancer cell lines. Expression of the housekeeping gene actin was used as a control for protein loading. NS, non-small cell lung cancer; SC, small cell lung cancer.

Fig. 3.

Representative examples of Western blot analysis for DAPK in lung cancer cell lines. Expression of the housekeeping gene actin was used as a control for protein loading. NS, non-small cell lung cancer; SC, small cell lung cancer.

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Fig. 4.

Representative examples of COBRA of DAPK in lung cancers. PCR products for the RCOBRA illustrated in Fig. 1 A were digested by BstUI or TaqI. a, cell line samples; NS, non-small cell lung cancer; SC, small cell lung cancer. b, primary lung cancer samples (T) and NBEC. M, DNA size marker.

Fig. 4.

Representative examples of COBRA of DAPK in lung cancers. PCR products for the RCOBRA illustrated in Fig. 1 A were digested by BstUI or TaqI. a, cell line samples; NS, non-small cell lung cancer; SC, small cell lung cancer. b, primary lung cancer samples (T) and NBEC. M, DNA size marker.

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Table 1

DAPK mRNA expression and methylation status in primary NSCLCs and corresponding NBECsa

Pair no.SampleHistologybMethylationmRNA quantitative ratioT/N ratio
Tumor LC − 364 0.1 
 NBEC   2992  
Tumor LC − 409 0.6 
 NBEC   695  
Tumor SQ 82 0.3 
 NBEC   244  
Tumor AD 11 0.03 
 NBEC   331  
Tumor SQ − 78 0.5 
 NBEC   173  
Tumor AD − 1710 1.6 
 NBEC   1096  
Pair no.SampleHistologybMethylationmRNA quantitative ratioT/N ratio
Tumor LC − 364 0.1 
 NBEC   2992  
Tumor LC − 409 0.6 
 NBEC   695  
Tumor SQ 82 0.3 
 NBEC   244  
Tumor AD 11 0.03 
 NBEC   331  
Tumor SQ − 78 0.5 
 NBEC   173  
Tumor AD − 1710 1.6 
 NBEC   1096  
a

Primary cultures of resected lung cancer and corresponding NBECs were examined expression and methylation status. Quantitative ratio was obtained by semiquantitative real-time RT-PCR. Methylation status was examined by COBRA.

b

AD, adenocarcinoma; SQ, squamous cell carcinoma; LC, large cell carcinoma.

Table 2

DAPK expression and methylation status in lung cancer cell linesa

Cell lineTypeMethylation statusGroupmRNA expression ratioRatio (treated/base)Protein
BaseAzaTSAAza/baseTSA/baseBase
H1963 SC 231 50 ∞ ∞ − 
H1299 NS 120 ∞ − − 
H1870 SC 82 − − 
H1514 SC 48 2.5 ∞ 25 − 
HCC15 NS 0.1 38 29 376 293 − 
H157 NS 3.0 86 10 29 − 
H2195 SC 99 12 − 
H60 SC 33 45 28 − 
H2141 SC ND ND ND ND − 
H2170 NS 0.1 ND ND ND ND − 
H2107 SC − 0.1 0.1 0.1 − 
H524 SC − 83 33 15 0.4 0.2 − 
HCC366 NS − 21 27 19 1.3 0.9 − 
H1395 NS − 28 ND ND ND ND − 
H2009 NS − 32 ND ND ND ND − 
H82 SC − 64 ND ND ND ND − 
H592 SC − 165 229 105 0.6 
H1450 SC − 604 1180 692 
H1819 NS − 1083 1704 392 1.6 0.4 ND 
H1770 NS − 3264 3335 2852 0.9 ND 
H740 SC − 214 ND ND ND ND 
H1703 NS − 287 ND ND ND ND 
H211 SC − 461 ND ND ND ND 
H1994 SC − 1100 ND ND ND ND 
H2171 SC − 1681 ND ND ND ND 
Cell lineTypeMethylation statusGroupmRNA expression ratioRatio (treated/base)Protein
BaseAzaTSAAza/baseTSA/baseBase
H1963 SC 231 50 ∞ ∞ − 
H1299 NS 120 ∞ − − 
H1870 SC 82 − − 
H1514 SC 48 2.5 ∞ 25 − 
HCC15 NS 0.1 38 29 376 293 − 
H157 NS 3.0 86 10 29 − 
H2195 SC 99 12 − 
H60 SC 33 45 28 − 
H2141 SC ND ND ND ND − 
H2170 NS 0.1 ND ND ND ND − 
H2107 SC − 0.1 0.1 0.1 − 
H524 SC − 83 33 15 0.4 0.2 − 
HCC366 NS − 21 27 19 1.3 0.9 − 
H1395 NS − 28 ND ND ND ND − 
H2009 NS − 32 ND ND ND ND − 
H82 SC − 64 ND ND ND ND − 
H592 SC − 165 229 105 0.6 
H1450 SC − 604 1180 692 
H1819 NS − 1083 1704 392 1.6 0.4 ND 
H1770 NS − 3264 3335 2852 0.9 ND 
H740 SC − 214 ND ND ND ND 
H1703 NS − 287 ND ND ND ND 
H211 SC − 461 ND ND ND ND 
H1994 SC − 1100 ND ND ND ND 
H2171 SC − 1681 ND ND ND ND 
a

Lung cancer cell lines of which quantitative ratio is available are listed. Quantitative ratio was obtained by semiquantitative real-time RT-PCR. Methylation status was examined by COBRA. NS, non-small cell lung cancer; SC, small cell lung cancer; ND, not done; base, baseline value (no drug treatment); Aza, 5-aza-2′-deoxycytidine; TSA, trichostatin A. For cell lines with the prefix H, the full prefix is NCI-H.

Table 3

Aberrant methylation of DAPK in samples by combined bisulfite restriction analysisa

SamplesNo. methylated (%)
Lung cancers  
 Cell lines (n = 48) 21 (44%) 
  NSCLC (n = 23) 10 (43%) 
  SCLC (n = 25) 11 (44%) 
 Tumors (n = 38) 14 (37%) 
  AD (n = 20) 8 (40%) 
  SQ (n = 12) 4 (33%) 
  SCLC (n = 6) 2 (33%) 
Nonmalignant specimens (n = 46) 2 (4%) 
 Primary NBEC cultures (n = 6) 
 Bronchial brushes (n = 5) 
 Peripheral lung tissues (n = 15) 1 (7%) 
 Peripheral lymphocytes (n = 12) 1 (8%) 
 Buccal brushes (n = 8) 
SamplesNo. methylated (%)
Lung cancers  
 Cell lines (n = 48) 21 (44%) 
  NSCLC (n = 23) 10 (43%) 
  SCLC (n = 25) 11 (44%) 
 Tumors (n = 38) 14 (37%) 
  AD (n = 20) 8 (40%) 
  SQ (n = 12) 4 (33%) 
  SCLC (n = 6) 2 (33%) 
Nonmalignant specimens (n = 46) 2 (4%) 
 Primary NBEC cultures (n = 6) 
 Bronchial brushes (n = 5) 
 Peripheral lung tissues (n = 15) 1 (7%) 
 Peripheral lymphocytes (n = 12) 1 (8%) 
 Buccal brushes (n = 8) 
a

AD, adenocarcinoma; SQ, squamous cell carcinoma.

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