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Promoter Methylation Profiles Between Human Lung 1310 Regular Article Biol. Pharm. Bull. 36(8) 1310–1316 (2013) Vol. 36, No. 8 Promoter Methylation Profiles between Human Lung Adenocarcinoma Multidrug Resistant A549/Cisplatin (A549/DDP) Cells and Its Progenitor A549 Cells Ruiling Guo,*,a Guoming Wu,b,# Haidong Li,a Pin Qian,c Juan Han,d Feng Pan,a Wenbi Li,a Jin Li,b and Fuyun Ji*,b a Department of Respiratory Diseases, 324th Hospital of the People’s Liberation Army; d Department of Emergency Medicine, 324th Hospital of People’s Liberation Army; Chongqing 400020, China: b Institute of Human Respiratory Diseases, Xinqiao Hospital, Third Military Medical University; and c Institute of Field Internal Medicine, Xinqiao Hospital, Third Military Medical University; Chongqing 400037, China. Received February 19, 2013; accepted May 27, 2013 Although aberrant DNA methylation has been implicated in the pathophysiology of lung cancer, the role of methylation in multidrug resistance (MDR) of lung cancer has remained unclear. To investigate whether certain distinct DNA methylation pattern is associated with acquired MDR of lung adenocarcinoma, meth- ylated-DNA immunoprecipitation-chromatin immunoprecipitation (MeDIP-ChIP) was utilised to compare the genome-wide promoter methylation of the human lung adenocarcinoma MDR A549/cisplatin (A549/DDP) cells with its progenitor A549 cells. The comparison identified 3617 genes with differentially methylated promoter, of which 1581 were hypermethylated and 2036 were hypomethylated. Then, bisulphite sequenc- ing polymerase chain reaction (PCR) (BSP) and quantitative reverse transcription (RT)-PCR (Q-PCR) were used to validate the promoter methylation of five candidate genes and to determine whether the expression of genes was associated with the promoter methylation. BSP confirmed that the promoter methylation incidence of the hypermethylated genes, G protein-coupled receptor 56 isoform 3 (GPR56), metallothionein 1G (MT1G), and RAS association domain family gene 1 (RASSF1), was significantly higher in A549/DDP cells compared with A549 cells (p<0.001, p=0.0099, and p=0.0165), whereas no significant difference was found in that of the other two genes, CCNL2 and BAD (p=0.0594 and p=0.5546). Additionally, Q-PCR showed that the mRNA expression of the three hypermethylated genes was significantly lower in A549/DDP cells compared with A549 cells (all p<0.001). In conclusion, this study reported for the first time that a distinct promoter methylation pattern is associated with MDR of lung adenocarcinoma A549/DDP cells and suggested that GPR56, MT1G, and RASSF1 might be the potential methylation markers associated with acquired MDR of lung adenocarcinoma. Key words lung adenocarcinoma; multidrug resistance; DNA methylation; promoter Lung cancer has replaced liver cancer to become the lead- ever, these chemotherapy resistance models are demonstrated ing cause of cancer-related deaths in China, accounting for to be relatively simplistic with the generally poor clinical 22.7% of all cancer deaths.1) The rates of morbidity and mor- outcomes.9,10) Interestingly, the growing evidences of epigen- tality continue to rise rapidly and the lung cancer patients will etic alterations in gene expression have provided an indication reach one million in 2025 if no effective control measures that DNA methylation status of a series of genes changed were taken.2) Currently, lung adenocarcinoma has become the either simultaneously or sequentially might be involved in the major pathologic type of lung cancer, the incidence of which chemotherapy-resistant phenotype of lung adenocarcinoma. accounts for 30 to 40% of all lung cancer cases in China and First, many studies showed that DNA methylation is far more exceeds 50% in many areas of Europe.1–3) Chemotherapy is vulnerable than the DNA sequence to external factors. These still an indispensable key factor in the standardised treatment alterations might be the earliest event in tumourigenesis, of lung adenocarcinoma presently, even though rise of target which could lead to a growth advantage for tumour cells and therapy has been greatly improved the treatment of certain influence the direction of transformation.11) Second, DNA group of lung adenocarcinoma patients such as people with methylation changes can occur rapidly, which results in resis- epidermal growth factor receptor (EGFR) mutation. However, tance arising quickly following chemotherapy treatment.12,13) regretfully, acquired multidrug resistance (MDR) induced by Furthermore, the expression of multiple genes could be simul- chemotherapy is the major barrier to the successful chemo- taneously affected by DNA methylation.14) Therefore, DNA therapeutic treatment.4) Hence, understanding of the potential methylation could be the driving force in acquired MDR,15,16) MDR mechanisms of lung adenocarcinoma is essential to dis- which have been confirmed in resistant tumour cell line of cover novel chemotherapy drugs and improve the efficacy of breast adenocarcinoma,17) murine neuroblastoma cells,18) and chemotherapy treatment. drug-resistant ovarian and colon tumour xenografts.19) These So far, most of MDR studies have been focused on the findings gave us clue that the DNA methylation might also single genes to investigate the MDR mechanism of lung ad- play important roles in the development of the MDR polygenic enocarcinoma and obtained many important results.5–8) How- phenotype of lung adenocarcinoma cells. Until now, except a limited number of genomic regions or 20,21) The authors declare no conflict of interest. genes found to be methylated in MDR of lung cancer, few # Equal contribution with first author. studies have reported a genome-wide promoter methylation * To whom correspondence should be addressed. e-mail: [email protected]; [email protected] © 2013 The Pharmaceutical Society of Japan August 2013 1311 analysis to investigate whether certain distinct promoter meth- whether the surrounding window is enriched for high-intensity ylation pattern is associated with MDR of lung adenocarcino- probes relative to the rest of the array. Each probe has a corre- ma. Thus, in the present study, to investigate the relationship sponding p-value score (−log 10) and a threshold with positive between DNA methylation and MDR of lung adenocarcinoma, signal difference is set to select regions that are enriched (i.e., methylated-DNA immunoprecipitation-chromatin immunopre- methylated) in the test sample. Thus, transfrags or differen- cipitation (MeDIP-ChIP) was utilised to compare the promoter tially methylated regions (DMRs) were generated by interval methylation profiles of the human lung adenocarcinoma MDR analysis with a p-value minimum threshold of 2, maximum A549/DDP cells with its progenitor A549 cells. Then, bisul- spacing between nearby probes within a peak of 500 bp. When phite sequencing polymerase chain reaction (PCR) (BSP) was we get the DMRs data, we map them with genomic transcripts performed to validate the results obtained from MeDIP-ChIP and make comparison analysis between 2 samples using and quantitative reverse transcription (RT)-PCR (Q-PCR) was NimbleGen SignalMap. Annotations of RefSeq were retrieved carried out to investigate whether the expression of the hy- from the NCBI website. The correlation of log 2 MeDIP/Input permethylated/hypomethylated genes was associated with the DNA ratios between replicates were computed using values promoter methylation. The study preliminarily established a from MaxTen calculations as described previously.26) distinct DNA methylation pattern of the lung adenocarcinoma Promoter Methylation Analysis by BSP ​Genomic bisul- MDR cells and revealed several epigenetically inactivated phite sequencing was performed to confirm the sensitivity of genes, which might be the potential candidate genes or meth- the observed DMRs. The genomic DNA was extracted from ylation markers involved in the MDR of lung adenocarcinoma. the A549 and A549/DDP cells using QIAamp DNA Blood Mini Kit according to the manufacturer’s recommendation Materials and MethOds (QIAGEN, Maryland, U.S.A.), respectively, and 400 ng ge- nomic DNA was treated with sodium bisulphite using EZ Cell Lines and Cell Culture ​The MDR cell line A549/ DNA Methylation-Gold Kit (Zymo Research, Orange, CA, DDP was established as described as Guo.22) Briefly, the pro- U.S.A.). Eighty to one hundred nanograms bisulphite-treated genitor A549 cells were first treated with a high-dose shock DNA was used for PCR amplification. The target regions of cisplatin (DDP) (1.0 µg/mL) and then stepwise selected for of the relevant gene promoters and the primers, which were more than 6 months with increasing concentrations of cisplat- designed using the MethPrimer programme (http://www. in at a range of 0.05 to 1.0 µg/mL in RPMI-1640 medium (Hy- urogene.org/methprimer/index1.html), were shown in Table 1. clone, Logan, UT, U.S.A.) with 10% foetal calf serum (Gibco, Then, the BSP products were cloned into a pMD®18-T vector NY, U.S.A.) in a 37°C humidified incubator supplied with 5% according to the manufacturer’s instructions (TaKaRa, Dalian, CO2. Then, the selected cells that demonstrated cross-resis- China). For each cell line and each gene, five positive clones tance to hydroxycamptothecin, vincristine, and 5-fluorouracil were randomly selected for subsequent sequencing. After this, (MDR A549/DDP) and A549 cells were regularly maintained the amplicon sequence data were aligned to the human refer- in RPMI-1640 medium supplemented with 10% foetal calf ence genome, and the extent of methylation (DNA methylation serum in a 37°C humidified incubator supplied with 5% CO2. levels) was determined by comparing the total number of Cs MeDIP-ChIP ​MeDIP was performed as previously (methylated) to Ts (unmethylated) for each CpG site. described.23) Briefly, genomic DNA from A549/DDP cells Gene Expression by Q-PCR ​Total RNA was isolated and A549 cells were extracted using QIAamp DNA Blood using TRIzol according to the manufacturer’s recommenda- Mini Kit according to the manufacturer’s recommendation tion (Invitrogen, Carlsbad, CA, U.S.A.). Two milligrams (QIAGEN, Maryland, U.S.A.), respectively, and fragmented aliquots were reverse transcribed using an AMV First Strand by Bioruptor (Diagenode, Belgium).
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