Hui-Hua Cai, Yue-Ming Sun, Yi Miao, Wen-Tao Gao, Quan Peng, Jie Yao and Han-Lin Zhao

Nanjing, China

Aberrant methylation frequency of TNFRSF10C promoter in pancreatic cancer cell lines

Hui-Hua Cai, Yue-Ming Sun, Yi Miao, Wen-Tao Gao, Quan Peng, Jie Yao and Han-Lin Zhao

Nanjing, China

BACKGROUND:A growing body of evidence suggests that many tumors are initiated by both epigenetic abnormalities and gene mutations, which promote tumor progression. Epigenetic abnormalities include changes in DNA methylation and in the modification of histones. This study aimed to assess the status of methylation in the CpG island (CGI) of the tumor necrosis factor receptor superfamily member 10c (TNFRSF10C) with combined bisulfite restriction analysis (COBRA) and to evaluate its role in the progression of pancreatic cancer (PC).

METHODS:The methylation status of four PC cell lines was assessed using COBRA and/or bisulfite genomic sequencing (BGS). Changes in methylation and TNFRSF10C expression in PC cell lines before and after treatment with 5-aza-2'-deoxycytidine (5-aza-dC) and/or trichostatin A (TSA) were assessed by BGS and real-time RT-PCR. Apoptosis in the four cell lines was tested by flow cytometry (FCM) and TUNEL assay.

RESULTS:The methylation status of the TNFRSF10C promoter was assessed in PC cells (BxPC-3: 68.84±8.71%; CFPAC-1: 0; PANC-1: 96.77±4.57%; SW1990: 54.97±7.33%) with the COBRA assay, which was confirmed by the results of BGS. After treatment with 5-aza-dC and/or TSA, apoptosis was induced in PC cells to different degrees, and the levels of TNFRSF10C transcriptional expression in the PC cell lines (except CFPAC-1) increased markedly after 5-aza-dC treatment.

CONCLUSIONS:A high frequency of CGI methylation in the TNFRSF10C promoter results in inactivation of the gene and enhancement of tumor growth in most PC celllines (except CFPAC-1). Inactivation of TNFRSF10C by CGI hypermethylation can play an important role in PC progression and be potentially useful as a diagnostic marker and a new therapeutic approach for PC.

(Hepatobiliary Pancreat Dis Int 2011; 10: 95-100)

methylation; CpG island; pancreatic cancer; TNFRSF10C

Introduction

Pancreatic cancer (PC) is a malignant tumor associated with a significant degree of morbidity and mortality.[1]It is highly invasive and responds poorly to conventional treatments. Like other tumor types, PC arises from a complex and poorly understood sequence of genetic and epigenetic alterations. Aberrant hypermethylation of CpG islands (CGIs) in gene promoters and histone deacetylation are the main epigenetic changes that lead to gene silencing, but this has not been thoroughly investigated in PC cell lines. Elucidating the mechanisms of these regulatory events is likely to enhance our understanding of this disease and the identification of new treatment targets.

The tumor necrosis factor receptor superfamily member 10c (TNFRSF10C) is one of several TNF-related, apoptosis-inducing ligand (TRAIL)-like decoy receptors. It is a glycosyl phosphatidylinositol-linked membrane molecule that lacks a cytoplasmic region and is located on 8p22-p21.[2]TNFRSF10C is one of the most frequently deleted loci in cancers of the colon,[3]lung,[4]prostate,[5,6]breast[7,8]and bladder.[9]Hypermethylation, specifically of the CGI in the TNFRSF10C promoter, has been reported in neuroblastoma (21%),[10]primary breast cancer (48%), primary lung cancer (37%), malignant mesothelioma (43%),[8]ependymoma (50%), and choroid plexus papilloma (50%).[11]Furthermore, a recent study by Hornstein et al[12]reported a significant decrease in expression of TNFRSF10C in the M0 stage of prostate carcinoma tissue compared to benign prostate tissue.These data suggest that TNFRSF10C is a tumor suppressor gene of prostate carcinoma. However, hypermethylation of the TNFRSF10C promoter in PC has not previously been investigated.

In the present study, we determined the frequency of hypermethylation in the TNFRSF10C promoter in four PC cell lines (BxPC-3, CFPAC-1, PANC-1, and SW1990) by using combined bisulfite restriction analysis (COBRA) and/or bisulfite genomic sequencing (BGS). Changes in methylation status and gene expression of TNFRSF10C before and after treatment with 5-aza-2'-deoxycytidine (5-aza-dC) or trichostatin A (TSA) were also evaluated in these PC cell lines.

Methods

Cell culture and drug treatment

The PC cell lines (BxPC-3, CFPAC-1, PANC-1, and SW1990) were cultured in DMEM (Hyclone, Logan, UT), and all cells were maintained at 37 ℃ and 5% CO2in a humidified incubator. Treatments of cell lines were: 100 nmol/L TSA (Sigma, St. Louis, MO) for 24 hours, 5 μmol/L 5-aza-dC (Sigma, St. Louis, MO) for 72 hours (with fresh drug added every 24 hours), and 5 μmol/L 5-aza-dC for 72 hours followed by 24 hours with 100 nmol/L TSA.[13]The control group was untreated. Apoptosis before and after drug treatment was assayed using flow cytometry (FCM) and TUNEL assay.

Isolation of RNA and genomic DNA

RNA from cells was isolated using Trizol according to the manufacturer's instructions. Recovered RNA was further treated with DNaseiand re-purified using a Micro-to-Midi total RNA purification system (Invitrogen, Carlsbad, CA, USA).

Genomic DNA from cultured cells was isolated from the inter-organic phase of a Trizol-chloroform mixture, then combined with 0.5 ml of back extraction buffer (4 mol/L guanidine thiocyanate, 50 mmol/L sodium citrate, 1 mol/L Tris, pH 8.0). After vigorous mixing by inversion, phase separation was achieved by centrifugation at 12 000×g for 30 minutes. DNA was precipitated from the upper aqueous phase using isopropanol and further purified using Gentra Puregene reagent (Qiagen, Hilden, Germany) with proteinase K treatment.[14]

Bisulfite modification

DNA (4 μg) was denatured with 0.2 mol/L NaOH for 20 minutes at 37 ℃ and treated with sodium bisulfite (Sigma, St Louis, MO) for 16 hours at 50 ℃. Modified DNA was purified using the Wizard DNA Clean-up system (Promega, Madison, WI) and diluted in 50 μl nuclease-free water (NFH2O). Modification was completed by incubation with NaOH (0.3 mol/L final concentration) for 5 minutes at room temperature. DNA was precipitated with ethanol and resuspended in 60 μl NFH2O. DNA from SW1990 cells was treated with CpG methyltransferase (M.SssI) (NEB, Ipswich, MA) according to the manufacturer's protocol and modified by sodium bisulfite for a positive control.

BGS

TNFRSF10C methyl primers were designed using Methyl-Primer-Express software v1.0 (Applied Biosystems, Foster City, CA) to amplify bisulfite-modified DNA. The forward and reverse primer sequences were: 5'-GGA TCC CCA AGA CCC TAA AG-3' and 5'-TGT TGG AAG CGT TGG TGT AA-3', respectively, and a 215-bp product amplified contained 10 CpG dinucleotides. PCR samples contained 36.3 μl NFH2O, 5 μl 10×buffer (Mg2+), 2.5 μl 10 mmol/L dNTP, 2 μl of each primer, 2 μl bisulfite-treated DNA, and 0.2 μl FastTaqTMpolymerase (Qiagen, Hilden, Germany). Amplification was performed with the following cycles: (a) 95 ℃ for 5 minutes; (b) 2 cycles of 95 ℃for 45 seconds, 68 ℃ for 30 seconds, and 72 ℃ for 30 seconds; (c) 3 cycles of 95 ℃ for 45 seconds, 66 ℃ for 30 seconds, and 72 ℃ for 30 seconds; (d) 4 cycles of 95 ℃ for 45 seconds, 64 ℃ for 30 seconds, and 72 ℃for 30 seconds; (e) 5 cycles of 95 ℃ for 45 seconds, 62 ℃ for 30 seconds, and 72 ℃ for 30 seconds; (f) 35 cycles of 95 ℃ for 45 seconds, 60 ℃ for 30 seconds, and 72 ℃ for 30 seconds; with a final extension step for 7 minutes at 72 ℃. Amplified PCR products (5 μl) were separated by electrophoresis in 2% agarose gels and visualized by ethidium bromide staining. Using the Toyobo TA cloning kit (Toyobo, Osaka, Japan), 2 μl of PCR product was subcloned into the pCR4-TOPO vector and transformed into E. coli. Sequencing analysis was performed on 10-20 randomly-selected individual colonies and the data were compared with the UCSC genome reference sequence to assess the methylation status of each CpG site.

COBRA[15]

The restriction enzyme TaqI (NEB, Ipswich, MA) was included in COBRA assay samples as follows: 18 μl NFH2O, 2 μl 10×buffer, 10 μl PCR product, and 2 μl TaqI. Reaction conditions were (a) 65 ℃ for 16 hours and (b) 80 ℃ for 20 minutes. Digested PCR products were separated on an 8% polyacrylamide gel, visualized by ethidium bromide staining, and photographed usinga Bio-Rad gel documentation system. Quantitation was performed using a Molecular Dynamics phosphor imager.

Quantitative RT-PCR

Total RNA was purified from cultured cells before and after treatment with 5-aza-dC or TSA using the RNeasy kit (Qiagen, Hilden, Germany). cDNA was synthesized using SuperScript II reverse transcriptase and Oligo (dT) primers (Invitrogen, Carlsbad, CA) in a 20 μl reaction according to the manufacturer's protocol. For the PCR reactions, Platinum SYBR Green qPCR SuperMix-UDG with ROX (Invitrogen, Carlsbad, CA) was used and reactions were assayed in triplicate using 1 μl of cDNA as a template in a 25 μl reaction. β-actin was used as the reference gene. Primers for TNFRSF10C were: 5'-TAC ACC AAC GCT TCC AAC-3' (forward) and 5'-AAG TGT AGC AGG TGC CCT-3' (reverse). TNFRSF10C PCR was performed with the following cycles: 95 ℃ for 10 minutes, 95 ℃ for 15 seconds, 63 ℃ for 32 seconds, and 72 ℃ for 30 seconds. A total of 40 cycles was performed and each reaction was run in duplicate. Negative controls were included in each experiment. Gene expression was calculated based on the yield of TNFRSF10C relative to β-actin.

Results

Apoptosis before and after drug treatment

The percentage of apoptotic cells after treatment with 5-aza-dC and/or TSA was greater than that of the control in FCM and the TUNEL assay (Fig. 1). Higher levels of apoptosis were measured in CFPAC-1, PANC-1, and SW1990 cells treated with 5-aza-dC than with TSA. In contrast, the cell line BxPC-3 exhibited higher levels of apoptosis after TSA treatment (Fig. 1B).

Methylation frequency of CGI in TNFRSF10C promoter

The methylation frequency of the CGI in the TNFRSF10C promoter was assessed in PC cell lines using the COBRA assay. Methylation was absent in CFPAC-1 cells, 96.77±4.57% in PANC-1 cells, 68.84±8.71% in BxPC-3 cells, and 54.97±7.33% in SW1990 cells (Fig. 2A, B).

At the same time, the DNAs of PANC-1 and CFPAC-1 were subjected to BGS. Ten CpG sites within the CGI of the TNFRSF10C promoter were detected and the percentages of methylation (PANC-1: 98%;CFPAC-1: 0%) were consistent with the results of the COBRA assay (Fig. 2C).

Fig. 1. Evaluation of apoptosis. A: Flow cytometric analysis of apoptosis exhibited by PANC-1 cells after treatment with 5-aza-dC and/or TSA compared to control cells. B: The different apoptosis of PC cell lines treated with the combination of 5-aza-dC and TSA. BxPC-3 was more strongly affected by histone modification than changes in methylation. *, #, △: P<0.05, compared with control group. C-F: Representative images of apoptosis detected by TUNEL assay (original magnification ×400); C: untreated PANC-1 cells; D: negative control; E: PANC-1 cells treated with TSA; F: PANC-1 cells treated with 5-aza-dC. Blue staining indicates the absence of apoptosis and brown staining indicates the presence of apoptosis.

Fig. 2. Methylation frequency of CGI in TNFRSF10C promoter. A: COBRA results from four PC cell lines (M: marker; B: BxPC-3; C: CFPAC-1; P: PANC-1; S: SW1990; SSSI: DNA treated with CpG methyltransferase). B: The methylation rate of TNFRSF10C was calculated from the level of the gray lane (Fig. 2A) according to the following formula: methylation rate (%)=(lane 2+lane 3)/(lane 1+lane 2+lane 3). C: BGS of PANC-1 (98%), CFPAC-1 (0%).

Diversity of expression and changes in methylation before and after drug treatment

Real-time RT-PCR further demonstrated that demethylation was accompanied by a 8.28-fold (5-azadC) and a 5.75-fold (TSA) increase in transcriptional expression of TNFRSF10C in PANC-1, and a 2.86-fold (5-aza-dC) and a 1.70-fold (TSA) increase in SW1990 (Fig. 3A). Unlike the other cell lines, TNFRSF10C was found to be more strongly expressed after TSA treatment (3.64-fold) than 5-aza-dC treatment (2.98-fold) in BxPC-3 cells, which was paralleled by the FCM results (Fig. 1B). These data suggest that silencing of the TNFRSF10C gene is achieved through promoter methylation.

After 5-aza-dC or TSA treatment, BGS was used to re-assess the methylation status of TNFRSF10C in PANC-1 cells. The methylation rate in the untreated group was 98%, in the TSA group 70%, and in the 5-aza-dC group 40% (Fig. 3B).

Fig. 3. Reduced methylation and reactivation of TNFRSF10C expression in PC cell lines after treatment with 5-aza-dC or TSA. A: Quantitative RT-PCR of TNFRSF10C mRNA after treatment of PC cell lines (CFPAC-1 is omitted) with 5-aza-dC or TSA. Untreated cells were used as control in each case. *, #: P<0.05, compared with the control group. B: BGS of the CGI of TNFRSF10C after treatment with 5-aza-dC or TSA in PANC-1 (untreated: 98%; TSA: 70%; 5-aza-dC: 40%).

Discussion

COBRA is a sensitive and quantitative assay to determine DNA methylation levels at specific gene loci in small amounts of genomic DNA. Restriction enzyme digestion is used to reveal methylation-dependent sequence differences in the PCR products of sodium bisulfitetreated DNA, and the results are verified by BGS. We evaluated the aberrant methylation frequency of TNFRSF10C in PC cell lines with the COBRA assay. Using a combined approach of pharmacologic inhibition of epigenetic modifications and gene expression assays, genes subject to epigenetic silencing in PC cell lines were identified. This strategy is also used to identify the targets of epigenetic silencing in other tumor types.[7,16-18]

TNFRSF10C lacks both a cytoplasmic and a transmembrane domain, leaving it unable to participate in apoptotic signaling. Since a direct correlation between TNFRSF10C expression and TRAIL sensitivity has not been demonstrated,[19-21]the role of TNFRSF10C in PC is still unknown.

In our study, hypermethylation of the TNFRSF10C promoter was found in all PC cell lines except CFPAC-1, but the mechanism is unclear. The results demonstrated a high prevalence of TNFRSF10C promoter CGI hyper-methylation in most PC cell lines. And the results of real-time RT-PCR showed low transcription in PC cells because of hypermethylation. After treatment with 5-aza-dC or TSA, we found that the methylation rate of the TNFRSF10C promoter CGI was cut from 98% to 40% (5-aza-dC) and to 70% (TSA) in PANC-1, and the transcriptional expression was enhanced by 8.28-fold (5-aza-dC) and 5.75-fold (TSA). Meanwhile, the apoptotic rate increased markedly. Thus gene silencing of TNFRSF10C is mostly due to aberrant hypermethylation, which plays an important role in the progression of PC. At the same time, 5-aza-dC and TSA have synergistic effects on demethylation to a certain extent, but the mechanism of demethylation by TSA is not clear. In the present study COBRA and BGS showed no methylation of the TNFRSF10C promoter in CFPAC-1 while apoptosis was still induced after 5-aza-dC and/or TSA treatment. The possible reason is that other anti-oncogenes were re-activated because there was no specific demethylation effect of 5-aza-dC. Both apoptosis and transcription of BxPC-3 treated with TSA were greater than that with 5-aza-dC, indicating that histone deacetylation mainly contributes to the progression in BxPC-3. On the contrary, hypermethylation plays a key role in progression in PANC-1 and SW1990. All the above suggest that different mechanisms result in tumorigenesis and progression in different PC cell lines.

With the increasing incidence of morbidity and high mortality, PC responds poorly to conventional treatments. Evaluation of aberrant CGI methylation frequency in the TNFRSF10C promoter may be used as a biomarker of PC. The methylation status of the TNFRSF10C promoter should be investigated in clinical PC specimens before it is used as a new therapeutic approach for PC.

Funding:The study was supported by a grant from the National Natural Science Foundation of China (30471691).

Ethical approval:Not needed.

Contributors:MY proposed the study. CHH and SYM carried out the study, and GWT, PQ and YJ collected and summarized the data. CHH wrote the first draft, which was corrected by MY and ZHL. All authors contributed to the intellectual context and approved the final version. MY is the guarantor.

Competing interest:No benefits in any form have been received or will be received from a commercial party related directly or indirectly to the subject of this article.

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2.科研成果转化过程特征。科研成果转化在不同的阶段表现出不同的性质特点，有些是某一时点上的瞬间转化，有些是一个比较缓慢的过程。不同阶段不同形式的转化，都需要借助一定的条件来完成，研究的任务是找到这些条件，促成转化过程的完成。如表3所示：

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Accepted after revision September 2, 2010

Examine yourself every day. Correct the mistakes, if any, and guard against them, if not.

– (the Song Dynasty) Zhu Xi

May 20, 2010

Author Affiliations: Department of General Surgery, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China (Cai HH, Sun YM, Miao Y, Gao WT, Peng Q, Yao J and Zhao HL); Department of General Surgery, Jiangsu Province Hospital of Traditional Chinese Medicine, Nanjing 210029, China (Cai HH)

Yi Miao, MD, PhD, Department of General Surgery, First Affiliated Hospital, Nanjing Medical University, Nanjing 210029, China (Tel: 86-25-83718836ext6508; Fax: 86-25-83781992; Email: miaoyi@njmu. edu.cn)

© 2011, Hepatobiliary Pancreat Dis Int. All rights reserved.