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Carcinogenesis Advance Access originally published online on January 19, 2008
Carcinogenesis 2008 29(6):1267-1275; doi:10.1093/carcin/bgn012
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© The Author 2008. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Epigenetic silencing of O6-methylguanine DNA methyltransferase gene in NiS-transformed cells

Weidong Ji1,2,3, Linqing Yang2, Lei Yu2, Jianhui Yuan2, Dalin Hu1, Wenjuan Zhang1, Jianping Yang1, Yaqin Pang1, Wenxue Li1, Jiachun Lu3, Juan Fu3, Jiakun Chen3, Zhongning Lin1, Wen Chen1 and Zhixiong Zhuang1,2,*

1 Faculty of Preventive Medicine, School of Public Health, Sun Yat-Sen University, 74 Zhongshan Road 2, Guangzhou 510080, People's Republic of China
2 Department of toxicology Shenzhen Center for Disease Control and Prevention, 21 Tianbei Road 1, Shenzhen 518020, People's Republic of China
3 Institute for Chemical Carcinogenesis, Guangzhou Medical College, 195 Dongfeng Xi Road, Guangzhou 510182, People's Republic of China

* To whom correspondence should be addressed. Tel/Fax: +86 755 25639066; Email: zxzhuang2007{at}126.com

Correspondence may also be addressed to Wen Chen. Tel: +86 20 87330599; Fax: +86 20 87330446; Email: wenchen1107{at}163.com


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussions
 Supplementary material
 Funding
 References
 
Nickel (Ni) compounds are potent carcinogens and can induce malignant transformation of rodent and human cells. To uncover the molecular mechanisms of nickel sulfide (NiS)-induced cell transformation, we investigated epigenetic alterations in a set of DNA repair genes. The silencing of the O6-methylguanine DNA methyltransferase (MGMT) gene locus and upregulation of DNA methyltransferase 1 (DNMT1) expression was specifically detected in NiS-transformed human bronchial epithelial (16HBE) cells. In addition, we noted epigenetic alterations including DNA hypermethylation, reduced histone H4 acetylation and a decrease in the ratio of Lys-9 acetylated/methylated histone H3 at the MGMT CpG island in NiS-transformed 16HBE cells. Meanwhile, we identified concurrent binding of methyl-CpG-binding protein 2, methylated DNA-binding domain protein 2 and DNMT1 to the CpG island of the MGMT promoter, demonstrating that these components collaborate to maintain MGMT methylation in NiS-transformed cells. Moreover, depletion of DNMT1 by introduction of a small hairpin RNA construct into NiS-transformed cells resulted in a 30% inhibition of cell proliferation and led to increased MGMT gene expression by reversion of the epigenetic modifications at the MGMT promoter region. MGMT suppression and hypermethylation at the CpG island of the MGMT promoter occurred 6 days after NiS treatment, indicating that epigenetic modifications of MGMT might be an early event in tumorigenesis. Taken together, these observations demonstrate that epigenetic silencing of MGMT is associated with DNA hypermethylation, histone modifications and DNMT1 upregulation, which contribute to NiS-induced malignant transformation.

Abbreviations: ACTB, β-actin; ChIP, chromatin immunoprecipitation; DAC, 5-aza-2-deoxycytidine; DNMT1, DNA methyltransferase 1; H3K9ac, histone H3 Lys-9 acetylation; H3K9me2, histone H3 Lys-9 methylation; H4ac, histone H4 acetylation; MBD, methylated DNA-binding domain protein; MeCP2, methyl-CpG-binding protein 2; MGMT, O6-methylguanine DNA methyltransferase; mRNA, messenger RNA; Ni, nickel; NiS, nickel sulfide; NSTC, nickel sulfide-transformed cell; PCR, polymerase chain reaction; Q-PCR, quantitative polymerase chain reaction; TSA, trichostatin A; 16HBE, human bronchial epithelial


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussions
 Supplementary material
 Funding
 References
 
Nickel (Ni) is a widely used metal in manufacturing. Epidemiological studies from occupationally exposed populations and experimental animals implicate exposure to both water-insoluble and water-soluble Ni with an increased incidence of lung and nasal cancers (1–3). However, the molecular mechanisms of tumorigenesis are still not well understood. Previous studies have demonstrated that epigenetic alterations, oxidative stress, DNA damage, defects in DNA repair and the activation of certain transcription factors might be associated with Ni compound-induced carcinogenicity (4). Recently, several lines of evidence revealed that DNA methylation and histone modifications are involved in Ni compound-induced gene modification at specific loci (5–9), such as p16, serpin, transgenic gpt and others, supporting the notion that epigenetic changes contribute to Ni compound-induced cellular toxicity and tumorigenicity.

Gene expression programs governing tumorigenesis involve multiple epigenetic changes including DNA methylation, histone modification and non-coding RNA (10,11) Epigenetic regulation of gene expression is controlled by the posttranslational modification of histones and DNA methylation, resulting in the alteration of chromatin structure and function at gene loci (11,12). To date, methylation of CpG dinucleotides is known to be mediated by at least three DNA methyltransferases, including DNA methyltransferase 1 (DNMT1), DNMT3a, and DNMT3b (13,14). Constitutive expression of exogenous DNMT1 is sufficient to transform NIH3T3 cells (15), while depletion of DNMT1 has also been shown to reactivate methylation-silenced tumor suppressor genes in human cancer cells (16). DNMT1 is also overexpressed in many tumor types (17,18). It is clear that DNMT1 functions to maintain CpG methylation and aberrant gene silencing in human cancer cells (19).

Histone modifications also play critical roles in cancer development (12). DNA methylation changes are linked with the presence of an aberrant pattern of histone modification (20). In addition, it has been proposed that methyl-CpG-binding proteins [methylated DNA-binding domain proteins (MBDs)] serve as a bridge between histone-modifying enzymes and hypermethylated DNA associated with gene inactivation. In addition, MBDs can interact directly with DNMT1 and are associated with the repression of tumor suppressor genes in cell models (21). These observations make it clear that significant cross talk exists between different epigenetic pathways. Identification of the components of these functioning molecular pathways and elucidation of the relationship between DNMTs, histone modifications, MBDs and aberrant methylation patterns will eventually reveal how the epigenetic network is regulated.

Previously, we established a model of human bronchial epithelial (16HBE) cell transformation induced by nickel sulfide (NiS) (22). Treatment of immortal 16HBE cells with NiS six times (every 20 days) resulted in anchorage-independent cell growth and the formation of tumors in immunodeficient mice 100 days after first treatment. Transcriptional suppression of a human tumor suppressor, fragile histidine triad gene and genomic instability were also observed specifically in NiS-transformed cells (22,23). Given the result that NiS treatment caused genomic instability, we hypothesized that the inactivation of DNA repair genes resulting from abnormal epigenetic modifications might play an important role in NiS-induced transformation.

To test this hypothesis, we first examined the messenger RNA (mRNA) levels of a set of DNA repair genes including O6-methylguanine DNA methyltransferase (MGMT), hMLH1, hMSH6 and BRCA1 in NiS-transformed 16HBE cells and untreated cells. Interestingly, despite robust expression in normal cells, we found that MGMT expression was undetectable at both mRNA and protein levels in NiS-transformed cells. In order to determine what lead to the silencing of MGMT, we examined DNA methylation status, the dynamics of histone acetylation and methylation. Here, we show that suppression of MGMT in NiS-transformed cells is mediated by aberrant epigenetic regulation.


   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussions
 Supplementary material
 Funding
 References
 
Chemicals
Crystalline NiS was purchased from Johnson Matthey Catalog (Royston, Hertfordshire, UK). Trichostatin A (TSA) was obtained from BioVision (Mountain View, CA). The 5-aza-deoxycytidine (DAC) was purchased from Sigma Chemical Company (St Louis, MO).

Cell culture and treatment
The human bronchial epithelial cell line (16HBE) was a gift from Dr D.C.Gruenert (University of California, San Francisco, CA). 16HBE cells were maintained in minimum essential medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine. Cells were treated with crystalline NiS at 0.25, 0.5, 1.0 or 2.0 µg/cm2 for 24 h. The cultures were split 1:4 and subjected to another round of treatment. The cells were treated with NiS for one, two or three times. DAC treatment was given at 3 µM for 72 h and TSA treatment was given at 100 ng/ml for 24 h.

Plasmids and retroviral infections
The small hairpin RNA-expressing vectors, pLKO.1-small hairpin green fluorescent protein (control vector) and pLKO.1-shDNMT1, were generously provided by Dr W.C.Hahn (Dana Farber Cancer Institute, Boston, MA) from the RNAi Consortium at the Broad Institute. pLKO.1-small hairpin green fluorescent protein and pLKO.1-shDNMT1 were introduced into transformed nickel sulfide-transformed cell (NSTC) 2 using lentiviral infection followed by selection with puromycin (1.0 µg/ml) to generate stable shDNMT1-expressing cell lines, NSTC2shGFP and NSTC2shDNMT1 as described previously (24).

Cell proliferation
Cells (1 x 104) were plated in triplicate and harvested at the indicated time points. The number of viable cells was determined using a Z2 Particle Count and Size Analyzer (Beckman Coulter, Miami, FL). Triplicate plates were counted for each cell line.

Quantitation of gene expression by reverse transcription–quantitative polymerase chain reaction
Total RNA was reverse transcribed and quantitative polymerase chain reaction (Q-PCR) was performed using Brilliant SYBR® Green QPCR kit (Stratagene, La Jolla, CA) at the following conditions: 95°C for 15 min, followed by 40 cycles of 94°C for 30 s and 60°C for 1 min. Samples were analyzed in triplicate. The primers used are shown in supplementary Table 1 (available at Carcinogenesis Online). Gene expression values were calculated based on the comparative quantitative method (the {Delta}{Delta}CT method) and normalized to values obtained from the amplification of β-actin (ACTB).

Immunoblotting
Cells were extracted with lysis buffer containing 1% NP-40, 1% sodium dodecyl sulfate, 0.5% sodium deoxycholate, 10 µg/ml phenylmethyl sulfonylfluoride, 0.2 mM ethylenediaminetetraacetic acid and 0.5 mM DL-Dithiothreitol. Cell lysates were centrifuged at 13 000g for 15 min at 4°C and insoluble debris was discarded. Soluble proteins (20 µg) were subjected to 10% sodium dodecyl sulfate–polyacrylamide gel electrophoresis prior to immunoblotting. Antibodies used include MGMT, MBD1 and glyceraldehyde-3-phosphate dehydrogenase (Santa Cruz Biotechnology, Santa Cruz, CA), DNMT1 and methyl-CpG-binding protein 2 (MeCP2) (Sigma–Aldrich Company, St Louis, MO) and MBD2 (Upstate Biotechnology, Lake Placid, NY).

Sodium bisulfite conversion
Genomic DNA was prepared from the cultured cells using DNAzol (Invitrogen, Hongkong, China), according to the manufacturer's instructions. Genomic DNA (1 µg) was denatured by adding freshly prepared 3 M NaOH and incubated at 55°C for 20 min. For complete denaturation, the samples were incubated at 95°C for 5 min and immediately cooled on ice. Bisulfite solution was prepared by dissolving 5.4 g of sodium bisulfite in 10 ml water, adding 666 µl of a 40 mM hydroquinone solution and adjusting the pH to 5.0 with 10 M NaOH. The bisulfite solution was added to the denatured DNA, mixed and incubated at 55°C for 16 h in the dark. The DNA was recovered by using the Wizard DNA Clean-Up System (Promega, Madison, WI) followed by elution in 40 µl of water. Subsequently, 4 µl of 3 M NaOH was added, and the samples were incubated for 15 min at 37°C. The solution was then neutralized by adding 6 M NH4OAc (pH 7.0). The DNA was ethanol precipitated, washed in 70% ethanol, dried and resuspended in 20 µl of water.

MethyLight analysis
After sodium bisulfite conversion, methylation analysis was performed by a fluorescence-based, real-time polymerase chain reaction (PCR) assay (MethyLight) as described previously (25). Two sets of primers and probes, designed specifically for bisulfite-converted DNA, were used: a methylated set for the gene of interest and a reference set for ACTB to normalize for input DNA. The reference primers and the probe were designed in a region of the ACTB gene that lacks any CpG dinucleotides to allow for equal amplification, regardless of methylation levels. Parallel TaqMan PCR reactions were performed with primers specific for the bisulfite-converted methylated sequence for a particular locus and with the ACTB reference primers. Specificity of the reactions for methylated DNA was confirmed separately using human sperm DNA (with very low levels of CpG island methylation) and M.SssI-treated sperm DNA (heavily methylated) as described previously (25). The percentage of fully methylated molecules at a specific locus was calculated by dividing the specific GENE/ACTB ratio of a sample by the GENE/ACTB ratio of M.SssI-treated sperm DNA and multiplying by 100. We used the abbreviation ‘percentage of methylated reference’ to indicate this measurement. Each measurement was performed independently three times. The primer and probe sequences used are listed in supplementary Table 1 (available at Carcinogenesis Online). Since methylation of MGMT gene is concentrated in two hot spots which has been proved to be significantly involved in MGMT gene silencing (26), a 122 bp fragment (+74 to +196 relative to transcription start site), located in methylated hotspot region 2# of MGMT gene (Figure 1), was chosen to amplify by MethyLight assay.


Figure 1
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  		Fig. 1. Schematic diagram of the CpG island of MGMT promoter. (GenBank coordinates: 2498780-2499620. GenBank coordinates refer to locus NT_008818 [GenBank] ). The distribution of CpGs crosses over the promoter and the first exon of MGMT locus. Each vertical bar represents the presence of a CpG dinucleotide. The CpGs in the two hot spot regions as indicated are usually methylated in MGMT-silenced cells. F1, F2, F3 and F4 represent regions selected for ChIP analysis, while M1 and M2 represent regions analyzed by MethyLight assay and bisulfite sequencing.

 
Bisulfite sequencing
Genomic DNA was treated with bisulfite as described above. A 161 bp fragment of the MGMT promoter representing nucleotides –292 to –131 relative to the transcription start site (Figure 1), located in methylated hotspot region 1# (26), was amplified using primers which can anneal independent of methylation state and recognize bisulfite-modified DNA. The PCR primers used are shown in supplementary Table 1 (available at Carcinogenesis Online). The PCR fragment was cloned into pMD20-T vector (TaKaRa, Biotechnology Co., Dalian, China) and transfected into Escherichia coli JM109; 10 clones from each cell line were selected followed by sequencing with M13 primers. The number of methylated CpGs at a specific site was divided by the number of clones analyzed (n = 10 in all cases) to yield a percentage of methylation at each site. Average percent methylation across all CpG sites was calculated by the number of methylated CpGs divided by the total number of CpGs (n = 160 in all cases).

Chromatin immunoprecipitation assay
Chromatin immunoprecipitation (ChIP) assay was performed using EZ ChIP Assay Kit (Upstate Biotechnology) with minor modifications. Briefly, protein extract from 1 x 106 cells was cross-linked to DNA by addition of formaldehyde directly to the culture medium to a final concentration of 1% for 10 min at room temperature. The cross-linking reaction was quenched by adding glycine solution to a final concentration of 0.125 M for 5 min at room temperature. The medium was then removed and cells were collected and suspended in sodium dodecyl sulfate lysis buffer containing 1x protease inhibitor cocktail (Upstate Biotechnology). Cells were sonicated to yield fragments of 500 bp average size. The sonicated samples were precleared with 60 µl of salmon sperm DNA/protein G agarose beads for 1 h at 4°C with agitation. The soluble chromatin fraction was collected, and 1% of the supernatant was applied for input normalization. Five microliters of either anti-acetyl-histone H3 (Lys 9) (no.07-352), anti-dimethyl-histone H3 (Lys 9) (no.07-212), anti-acetyl-histone H4 (no.06-866), anti-MBD2 (no.07-198), normal mouse IgG (no.12-371B) (the negative control), anti-RNA polymerase II (no.05-623B) (the positive control) (Upstate Biotechnology), anti-MBD1 (no.ab3753), anti-MeCP2 (no.ab3752) (Abcam, Cambridge, MA) or anti-DNMT1 (no.IMG-261A) (Imgenex, San Diego, CA) was added and incubated overnight with rotation. Immune complexes were collected with 60 µl of salmon sperm DNA/protein G agarose beads. After washing and elution, the cross-links were reversed and the samples were digested with proteinase K for 2 h at 45°C. DNA was recovered and purified using spin columns (Mo Bio Laboratories, Carlsbad, CA).

Q-PCR analysis of immunoprecipitated DNA
To allow accurate measurement of the amount of DNA precipitated, Q-PCR was performed using a SYBR® Green QPCR kit (Stratagene). The PCR conditions were 95°C for 15 min, then 40 cycles of 94°C for 30 s, 56°C for 1 min and 72°C for 30 s. Four fragments of MGMT were amplified by F1, F2, F3 and F4 primer pairs (supplementary Table 1, available at Carcinogenesis Online), of which F1, F2 and F3 are located within promoter and first exon CpG islands and F4 within a downstream promoter. The relative differences among cells were determined using the {Delta}{Delta}CT method. A {Delta}CT value was calculated for each sample using the CT values for the input DNA samples to normalize the ChIP assay results. A {Delta}{Delta}CT value was then calculated by subtracting the {Delta}CT for the control cells from each treatment {Delta}CT within an experiment. The {Delta}{Delta}CT values were converted to fold differences compared with the control by raising 2 to the {Delta}{Delta}CT power.


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussions
 Supplementary material
 Funding
 References
 
Downregulation of MGMT expression in NiS-treated or NiS-transformed human cells
Previously, we obtained a transformed 16HBE cell line by treatment of immortal 16HBE cells with NiS (22). To examine the epigenetic alterations of DNA repair genes in these transformed cells, we first compared the mRNA levels from a set of genes including MGMT, hMLH1, hMSH6 and BRCA1 between NiS-transformed 16HBE cells and control cells treated with vehicle alone using real-time PCR. While there were no detectable changes in the mRNA levels of hMLH1, hMSH6 and BRCA1, we found that MGMT expression was reduced to undetectable levels by both mRNA and protein expression in the NiS-transformed cell lines NSTC1 and NSTC2 (Figure 2A and B). In order to define whether the suppression of MGMT expression occurred at an early stage of tumorigenesis, we examined MGMT expression in cells treated with NiS at doses of 0.25, 0.5, 1.0 or 2.0 µg/cm2 for 24 h administered one, two or three times every 2 days. No significant changes were observed in cells treated with NiS one or two times. However, significant dose-dependent downregulation of MGMT expression was found in cells treated with NiS three times (Figure 2A and B), suggesting that downregulation of MGMT may be an early event involved in NiS-induced cell transformation. These results are consistent with clinical findings showing that ~20% of human tumor cells are completely deficient in MGMT function (26).


Figure 2
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  		Fig. 2. MGMT expression in NiS-treated or NiS-transformed human 16HBE cells. mRNA level was measured by real-time PCR with input normalization by ACTB mRNA level. The results are expressed as percentage of the controls (mean ± SEM) from three independent experiments. (A) mRNA levels of MGMT in NiS-treated cells or two NiS-transformed cell lines (NSTC1 and NSTC2). Bars labeled as NiS0.025, NiS0.5, NiS1 or NiS2 represent the dose of NiS treatment (µg/cm2). (B) The protein levels of MGMT in NiS-treated cells and NiS-transformed cells as indicated were detected by immunoblotting. Dose-dependent downregulation of MGMT protein expression was found in cells treated with NiS three times. MGMT expression in transformed cells (NSTC2) treated with DAC or TSA, or after shDNMT1-induced silencing detected by Q-PCR (C) and immunoblotting (D).

 
In order to determine whether the suppression of MGMT was reversible, we treated NSTC2 cells with a DNA methyltransferase inhibitor, DAC or histone deacetylase inhibitor, TSA. We found that two chemicals significantly induced the expression of MGMT determined by Q-PCR (Figure 2C) and immunoblotting (Figure 2D), implying that the suppression of MGMT was due to aberrant epigenetic regulation.

DNA hypermethylation at CpGs of MGMT promoter
To assess whether CpGs hypermethylation was involved in transcriptional silencing of MGMT in NiS-transformed cells, we performed a MethyLight assay and bisulfite sequencing for quantitative analysis of DNA methylation. First, we examined the specificity of the MethyLight assay by PCR using forward and reverse primers and a fluorogenic probe to distinguish between fully methylated and fully unmethylated molecules of bisulfite-converted DNA as described in Materials and Methods. As shown in supplementary Table 2 (available at Carcinogenesis Online), the sperm DNA yielded a positive MethyLight product with the unmethylated primers and probe, whereas there were no amplifications detected in a set of negative controls. In contrast, M.SssI-treated sperm DNA and universal Methylated DNA with predominantly methylated MGMT alleles gave a positive amplification in the methylated reaction, but not in the unmethylated controls. A bisulfite-treated DNA sample gave no detectable signal with oligonucleotides designed to recognize a non-bisulfite-converted ACTB control sequence (supplementary Table 2, available at Carcinogenesis Online). These results demonstrate that the MethyLight assays are specific to bisulfite-converted DNA, reducing the possibility of false-positive results caused by incomplete bisulfite conversion, which can be obtained when using bisulfite sequencing.

Next, we detected DNA hypermethylation at the CpGs of MGMT in NiS-treated or NiS-transformed human 16HBE cells. The percentage of methylated reference, representing the percentage of fully methylated molecules at a specific locus, was 74.6 and 53.8% in two NiS-transformed cell lines, NSTC1 and NSTC2, respectively (Figure 3A). Methylation at the MGMT promoter was decreased after DAC or TSA treatment, which is consistent to the previous finding that DAC or TSA treatment led to recovery of MGMT expression. Treatment of NiS resulted in increase of percentage of methylated reference in a dose-dependent manner (Figure 3B). Similarly, DAC or TSA treatment led to decrease of MGMT methylation.


Figure 3
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  		Fig. 3. Methylation analysis on CpG island of MGMT gene. Quantitative methylation analysis of MGMT CpG island in NiS-transformed cells (A) or NiS-treated cells (B) with or without DAC or TSA treatment was assessed by MethyLight assay. The results were expressed as the percentage of methylated reference (PMR, mean ± SEM) from three independent experiments. (C) Bisulfite sequencing on CpG island (–292 to –131) of the MGMT gene. For each cell line, 10 clones were selected for sequencing. Methylated CpG site and unmethylated CpG site were marked as solid circle and open circle, respectively. The CpG position relative to transcription start site was shown at top of each CpG site. (D) At each CpG site, the area filled with black represents the average percentage of methylation across all CpG sites tested in cells indicated. The value of average percentage was shown at the end of each row.

 
To confirm the CpGs methylation status of the MGMT promoter, bisulfite sequencing was used to determine the percentage of methylation at 16 CpG sites within the MGMT promoter and first exon area. The average rate of methylation was 25.6% in NiS-treated cells and 68.9% in NiS-transformed human 16HBE cells, whereas it was only 7.5% in control cells. In addition, treatment with DAC or shDNMT1 suppression led to decreased cytosine methylation by 77.4 or 70.4%, respectively, in NiS-transformed cells (Figure 3C and D). Taken together, these observations indicate that the hypermethylation at the promoter region of MGMT gene plays a causal role in the downregulation of MGMT expression in NiS-transformed human 16HBE cells.

Downregulation of MGMT expression is associated with increased DNMT1 expression
To further define the regulatory complex that maintains hypermethylation of MGMT, we measured the expression of DNMT1, DNMT3a, DNMT3b, MeCP2 and MBD2 in NiS-treated and NiS-transformed cells using both reverse transcription–Q-PCR and immunoblotting. While no significant changes in DNMT3a, DNMT3b, MeCP2 and MBD2 expression were detected in NiS-treated or NiS-transformed cells at both mRNA and protein levels, 1.6~2.0- or 2.6~3.4-fold upregulation of DNMT1 expression was observed in NiS-treated or NiS-transformed cells (Figure 4A and B).


Figure 4
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  		Fig. 4. DNMT1 expression in NiS-treated or NiS-transformed human 16HBE cells. (A) mRNA levels of DNMT1 in NiS-treated cells and NiS-transformed cells as indicated. (B) Protein levels of the cells indicated were detected by immunoblotting with specific antibodies against DNMT1, MBD2 and MeCP2. Expression of glyceraldehyde-3-phosphate dehydrogenase was served as an internal control. (C) Protein levels of DNMT1 in NCTC2 cells treated with DAC or TSA or after shDNMT1-induced silencing were detected by immunoblotting. (D) mRNA levels of DNMT1 in transformed cells (NSTC2) treated with DAC or TSA or after shDNMT1-induced silencing.

 
To determine whether upregulation of DNMT1 was directly associated with MGMT silencing, we introduced a DNMT1-specific small hairpin RNA or a control small hairpin RNA targeting green fluorescent protein (small hairpin green fluorescent protein) into NiS-transformed 16HBE cells, generating cell line NSTC2shDNMT1 and NSTC2shGFP. As shown in Figure 4C, DNMT1 expression was suppressed by 82% in shDNMT1-expressing cells (NSTC2shDNMT1) compared with control cells (NSTC2shGFP). The suppression of DNMT1 induced a 30% decrease in cell proliferation (data not shown). In addition, we detected MGMT reexpression in NSTC2shDNMT1 cells, demonstrating that DNMT1 downregulation was sufficient to reverse MGMT silencing (Figure 2C and D). Moreover, treatment of DAC or TSA resulted in suppression of DNMT1 mRNA and protein levels, confirming that DNA methylation and histone acetylation both contribute to the control of transcriptional activity.

Histone modifications in MGMT CpG island promoter
To investigate whether histone modifications at the MGMT promoter area are associated with MGMT expression in NiS-treated and NiS-transformed human cells, we performed a quantitative-ChIP assay to analyze histone modifications.

Previous studies have shown that histone H4 acetylation (H4ac) and histone H3 Lys-9 acetylation (H3K9ac) are associated with open chromatin and active transcription, whereas histone H3 Lys-9 methylation (H3K9me2) serves as a marker of condensed and inactive chromatin (27). Here, we examined H4ac, H3K9ac and H3K9me2 in four different regions within the MGMT gene. Three out of four regions, defined as F1, F2, F3, are located within CpG island promoter, whereas region F4 is located downstream of CpG island. As shown in Figure 5A, the F3 region of MGMT CpG island promoter in NiS-treated cell (NiS2) and NiS-transformed cells (NSTC1 and NSTC2) showed a significantly higher level of H3K9me2, but lower levels of H4ac and H3K9ac compared with untreated control cells. In addition, the ratio of Lys-9 acetylated/methylated H3 (H3K9ac/H3K9me2) was markedly decreased. The fold differences compared with the control were shown in Figure 5A. NSTC2 or NiS2 cells treated with DAC or TSA led to reversion of the states of all histone modifications examined (Figure 5B and C). Similar patterns of histone modifications were found at region F1 and F2 (supplementary Figure 1, available at Carcinogenesis Online). In contrast, all cells exhibited low levels of H4ac, H3K9ac and H3K9me2 at region F4 compared with those at region F3 (Figure 5D). Although amount of H3K9ac and H3K9me2 were significantly affected by DAC or TSA treatment at region F3, no obvious changes were found at region F4 in NSTC2 cells. It appears that region F4 does not involve in epigenetic regulation of MGMT. Taken together, these results indicate that high levels of H3K9me2 and low levels of H4ac and H3K9ac at CpG island could be the epigenetic features that lead to the suppression of MGMT expression.


Figure 5
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  		Fig. 5. Histone modifications at the promoter region of MGMT gene. The quantitative-ChIP assay was performed to analyze histone modifications including H3K9ac, H3K9me2 and H4ac at the promoter of MGMT gene. The data were expressed as mean ± SEM, representing the relative levels of specific histone modification state at region F3 of MGMT after chromatin immune precipitation with specific antibodies against H3K9ac, H3K9me2 or H4ac with normalization by total input DNA. The fold differences compared with the control were calculated and shown in the figures. The states of different histone modifications in NiS-treated cells (NiS2) and NiS-transformed cells (NSTC2) (A) or NSTC2 cells treated with DAC or TSA (B) or NiS2 cells treated with DAC or TSA (C) were depicted. (D) Representative images resulted from Q-PCR assay at region F3, F4 of MGMT locus after ChIP using specific antibodies indicated in NSTC2 cells with or without treatment with DAC or TSA. IgG was served as a negative control.

 
Identification of binding proteins in MGMT CpG island promoter
MBDs are interpreters of the DNA methylation signal and function as transcriptional repressors through interactions with Histone deacetylase complexes and DNMTs (28). To detect which proteins were involved in MGMT regulation, we performed the quantitative-ChIP assay to identify the binding of MBDs or DNMT1 to the CpGs of MGMT gene using specific antibodies against MeCP2, MBD2, MBD1 or DNMT1. The amount of MGMT CpGs binding in the MeCP2, MBD2 and DNMT1 immune complexes at region F3 was 3.3-, 4.6- and 2.4-fold greater in NiS-treated cells and 4.7-, 3.3- and 4.3-fold greater in NiS-transformed cells than those in control cells (Figure 6A). In contrast, in MBD1 immune complexes, we detected weak binding to the CpGs of MGMT in NiS-transformed cells with no difference compared with that in control cells (Figure 6A and C). Inhibition of DNMT by DAC or shDNMT1 suppression not only reduced the binding of DNMT1 to the CpGs of MGMT promoter but also released MeCP2 and MBD2 from the CpGs of MGMT in NSTC2 cells (Figure 6B and C). The same pattern of protein binding was also observed at region F1, F2, but not region F4 (Figure 6C, supplemental Figure 2, available at Carcinogenesis Online). Taken together, these findings demonstrated that DNMT1, together with MeCP2 and MBD2, is recruited to the CpGs region of MGMT promoter. Moreover, it appears that DNMT1 is required for forming stable complexes, which are responsible for maintaining MGMT hypermethylation state.


Figure 6
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  		Fig. 6. The binding of MBDs and DNMT1 to the promoter region of MGMT gene. quantitative-ChIP assay was performed to detect the binding of MBDs or DNMT1 to the CpGs of MGMT gene using specific antibodies against MeCP2, MBD2, MBD1 or DNMT1. The data were expressed as mean ± SEM, representing the relative levels of MGMT amplification at region F3 after chromatin immune precipitation with specific antibodies against MeCP2, MBD2, MBD1 or DNMT1 with normalization by total input DNA. The fold differences compared with the control were calculated and shown in the figures. The amount of MGMT CpGs binding in the MeCP2, MBD2, MBD1 and DNMT1 immune complexes at region 3 in NiS-treated cells (NiS2) and NiS-transformed cells (NSCT2) (A) or in NSCT2 cells treated with DAC or silenced by shDNMT1 (B). (C) Representative images resulted from Q-PCR assay at region F3, F4 of MGMT locus after ChIP assay using specific antibodies indicated in NSTC2 cells treated with DAC or silenced by shDNMT1 introduction.

 

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Several lines of evidence support the hypothesis that carcinogen-induced epigenetic changes play key roles during carcinogenesis (11,29,30). Using Methylight, ChIP assays and RNA interference, we found that the transcriptional suppression of MGMT expression in NiS-transformed cells was mediated by specific combinations of epigenetic modifications, revealing an epigenetic pattern governing the malignant phenotype of human cells.

In this study, the suppression of MGMT expression was specifically found in NiS-transformed 16HBE cells, consistent with prior findings that Ni (II) inhibits the repair of O6-methylguanine in mammalian cells via suppression of MGMT activity (27). In primary human cancers, prior work has demonstrated that inactivation of MGMT is a common event, preventing both MGMT expression and repair of alkylation damage (31). Moreover, ~20% of human tumor cells appear to be completely deficient in MGMT and these cells are hypersensitive to the carcinogen-induced O6-guanine adduct formation (32). Taken together, although only weakly mutagenic (9,33), Ni compounds distinctly interfere with the repair of DNA base modifications, which might be involved in Ni compound-induced tumorigenicity.

Tumor cells have long been known to have high DNA methyltransferase levels (34,35). Increased DNA methyltransferase expression was demonstrated to be an essential molecular step in c-fos-mediated transformation in vitro (36), suggesting that abnormalities in DNMT1 expression probably contribute to the development of human cancer cells (35). Our findings that upregulation of DNMT1 expression associates with MGMT hypermethylation and lack of DNMT1 leading to inhibition of cell proliferation and renewal of MGMT expression supports the general set of mechanistic hypotheses that emphasize a malfunctioning of a maintenance DNA methyltransferase in the acquisition of abnormal CpG dinucleotide patterns characteristic of neoplastic transformation (35).

It has been reported that Ni compounds cause chromatin condensation and that dense heterochromatic regions of the genome are a preferential site for Ni action (37). In this study, we provide evidence that a condensed and inactive state of chromatin, characterized by a high level of histone methylation and a low level of histone acetylation exists at the MGMT promoter region in NiS-transformed human cells. These observations are consistent with previous findings that gpt transgenic silencing induced by Ni compounds is accompanied by DNA methylation and histone modifications (5). Collectively, these findings indicate that the epigenetic suppression of the specific genes mediated by DNA methylation and chromatin remodeling is crucial to Ni compound-associated tumorigenicity.

Methylated CpG sites are recognized by a family of protein factors containing a methyl-CpG-binding domain (MBD), which serves as a bridge between histone-modifying enzymes and hypermethylated DNA associated with gene inactivation (38). To date, five family members have been identified in mammals: MeCP2, MBD1, MBD2, MBD3 and MBD4 (39). Each of these proteins, with the exception of MBD3, is capable of binding specifically to methylated DNA. MeCP2, MBD1 and MBD2 can repress transcription from methylated gene promoters (39). As determined by quantitative-ChIP assay, we demonstrated that MeCP2 and MBD2 were associated with CpGs of the MGMT gene locus in NiS-transformed cells, indicating that these proteins are recruited into a functional complex responsible for MGMT silencing. Similar results were found in the MGMT-negative cancer cell lines, LU65 and MDA-MB-231, which show that MeCP2 or MBD2 are bound preferentially to the MGMT CpG island (40,41). Moreover, in the Ni-induced gpt transgene silenced cells, MeCP2 binds specifically to the promoter region of the gpt transgene (7). In addition to MBD2 and MeCP2, we also found that DNMT1 was physically and functionally associated with the CpGs of MGMT. Inhibition of DNMT or lack of DNMT1 expression lead to reduced binding of MeCP2 and MBD2 to the CpGs, confirming that DNMT1 interacts with MeCP2 directly and that this interaction is essential for the maintenance of DNA methylation (21). These results demonstrate that DNMT1 is targeted to specific regions via a DNMT1–MeCP2 complex, leading to a stable long-term hypermethylation of MGMT in NiS-transformed cells.

In this study, we demonstrated that treatment of DAC, a potent DNMTs inhibitor, leads to epigenetic alterations similar to what we observed by shDNMT1-induced gene suppression, indicating that the action of DAC was, at least in part, mediated by inhibition of DNMT1 activity. Several lines of evidence reveals that DAC irreversibly binds to DNMTs (42,43), leading to reductions in global 5-methylcytosine levels (42) and methylation of CpG islands of epigenetically silenced tumor suppressor genes (44). Our findings provide compelling evidence that DAC restored expression of MGMT through inhibition of DNMT1 and histone modification. TSA is a histone deacetylase inhibitor that cause hyperacetylation on histone (45). Low concentration of TSA and its structural analog, suberoyl anilide hydroxamic acid, can induce cell differentiation and inhibit growth in tumors, with little effects on normal cells (46). In our study, treatment with TSA restored expression of MGMT suggest that TSA functioned on epigenetic reactivation of silenced MGMT gene through cross talk between DNA methylation and histone modification. Taken together, our observations reinforce the notion that multiple layers of epigenetic modification interplay and control the states of transcription activity of a set of specific genes.

It has been suggested that DNA methylation changes occur at the early stages of cancer development (47). Analysis of transgenic adenocarcinoma mouse prostate lesions revealed greatly elevated DNMT1 mRNA and protein levels beginning in prostatic intraepithelial neoplasia and continuing through advanced prostate cancer and metastasis (18). Hypermethylation of p16 and MGMT in patients with squamous cell lung carcinoma was detected in sputum samples 3 years prior to disease diagnosis (48). Methylation of MGMT can also be used for discriminating between squamous cell carcinoma, adenocarcinoma and normal tissue (49). In our study, 6 days after NiS treatment, we could detect MGMT suppression. The MGMT methylation together with upregulation of DNMT1 existed throughout the entire process of NiS-induced cell transformation (data not shown). These observations confirm that NiS-associated epigenetic alterations occur at an early stage of malignant transformation. MGMT methylation may be a valuable biomarker for Ni compound exposure and early detection of lung cancer.

Indeed, the epigenetic process is highly complex and multifaceted. It is important to determine the temporal relationship of these events, DNA methylation, histone modification and gene silencing in cancer cells. In one of proposed models, DNA methylation precedes histone modifications. Histone deacetylation is triggered by seeds of DNA methylation, followed by the spread of DNA hypermethylation across the island, histone methylation and extensive DNA methylation of the CpG island (50). Our results, at least in part, support this model. In present study, we found that the average rate of methylation was increased in NiS-treated cells. Interestingly, some CpG sites (–186, –195, –228 relative to transcription start site) displayed preferential hypermethylation in NiS-treated cells similar to the transformed cells NSTC2, suggesting that methylation of these sites might be an important precursor to further dense methylation of MGMT CpG island. In addition, we found that the histone H3K9 were gradually deacetylated and methylated from the NiS-treated cells to the transformed cells. Thus, we speculated that overexpression of DNMT1 induced by Ni2+ resulted in hypermethylation of critial CpG sites in MGMT CpG island promoter in NiS-treated cells. Methylation of these CpG sites (seeds of DNA methylation) might trigger the binding of MBDs, which in turn promotes recruitment of histone deacetylases, histone methytransferase and DNA methytransferases, leading to inactivative chromatin and spread of DNA methylation. This process may be gradual, with the density of DNA methylation increasing by a self-reinforcing system, to achieve a stable long-term silencing state.

In summary, we extend the proposition that histone and DNA modifications represent important characteristics of cell malignant transformation and further our understanding of the mechanisms by which DNA methylation is targeted to specific regions of the genome and interpreted by specific methyl-CpG-binding proteins. The findings of these epigenetic features in transformed cells may underlie the molecular mechanisms that govern multistage tumorigenesis.


   Supplementary material
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 Supplementary material
Funding
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Supplementary Figures 1 and 2 and Tables 1 and 2 can be found at http://carcin.oxfordjournals.org/


   Funding
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussions
 Supplementary material
 Funding
References
 
Key Program of Natural Science Foundation of China (30630055); NSFC (30571588, 30571592, 30671746); New Century Excellent Talents (05-0722); National Key Basic Research and Development Program (2002CB512903); Natural Science Foundation of Guangdong (5001763, 04002730, 07003055); Doctoral Program of Higher Education of China (20060558024); Key Program of High Technology Research and Development Program of Shenzhen (JH200505300503A).


   Acknowledgments
 
We thank Richard Possemato and Dr William C.Hahn for critical reading of the manuscript.

Conflict of Interest Statement: None declared.


   References
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 References
 

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Received September 20, 2007; revised December 4, 2007; accepted December 27, 2007.


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