Biochemical and Biophysical Research Communications 387 (2009) 132–138 Contents lists available at ScienceDirect Biochemical and Biophysical Research Communications journal homepage: www.elsevier.com/locate/ybbrc Frequent promoter hypermethylation and transcriptional downregulation of BTG4 gene in gastric cancer Wenjie Dong a, Shuiping Tu b, Jing Xie c, Pinghu Sun a, Yunlin Wu a, Lifu Wang a,* a Department of Gastroenterology, Rui-jin Hospital, Shanghai Jiao Tong University, Shanghai, China b Department of Medicine, College of Physicians and Surgeons, Columbia University, NY, USA c Department of Pathology, Rui-jin Hospital, Shanghai Jiao Tong University, Shanghai, China article info abstract Article history: The BTG4 gene belongs to the BTG family of genes endowed with antiproliferative properties. In this Received 26 June 2009 study, we have found that BTG4 undergoes promoter CpG island hypermethylation-associated inactiva- Available online 1 July 2009 tion in gastric cancer and 50-aza-20-deoxycytidine (DAC) treatment restores BTG4 expression. We also found BTG4 levels were significantly reduced in primary gastric cancer but not in normal gastric tissues. BTG4 reexpression in gastric cancer causes growth inhibition of colony assays and nude mice. Taken Keywords: together, our data support BTG4 as a candidate tumor suppressor gene that is epigenetically silenced BTG4 in the majority of gastric cancers. Gastric cancer Ó 2009 Elsevier Inc. All rights reserved. Hypermethylation The BTG4 gene maps to 11q23 and encodes a 26 kDa protein of Materials and methods the BTG family of growth inhibitor genes. BTG2 (PC3) was the found- ing member of the BTG family of genes endowed with antiprolifera- Cell lines and tissue specimens. The SV40-transformed immortal tive properties, namely, BTG1, ANA/BTG3, PC3B, TOB, and TOB2 [1]. human gastric mucosal epithelial cell line (GES-1) and five gastric PC3 was originally isolated as a gene induced by nerve growth factor cancer cell lines (AGS, SGC-7901, MKN-28, MKN-45, BCG-823) during neuronal differentiation of rat PC12 cells, or by TPA in NIH3T3 were all preserved in our laboratory and maintained in RPMI cells (named TIS21), and is a marker for neuronal birth in vivo. BTG2, 1640 with 10% FBS. BCG-823 is an adherent, poorly differentiated, the human homolog of PC3, regulated by p53, was likely relevant to human gastric cancer cell line with mutant p53, and MKN-45 cell cell cycle control and cellular response to genotoxic damage [2]. line expresses wild-type p53 [6]. Cells were treated with 5 lM/L BTG2 was also identified as an important downstream effector of 50-aza-20-deoxycytidine (DAC; Sigma) for 48 h. The Adriamycin p53-dependent arrest of proliferation in mouse and human fibro- (ADR) was added at a concentration of 0.5 lg/ml for 24 h. 38 gas- blasts transduced by oncogenic Ras [3]. Similarly, BTG3, another tar- tric cancer samples were obtained from the Department of Pathol- get of p53, was reported to negatively regulate cell proliferation by ogy, Rui-jin Hospital. Samples of adjacent normal gastric mucosa binding to and inhibiting E2F1 [4]. from 15 patients, at least 2 cm distant from the tumor, were also Although much remains unknown about the function of BTG4, collected. The adjacent non-tumor area subsequently was verified the BTG4 gene has been found to be completely absent or barely by histology to be free of tumor infiltration. The clinicopathological detectable in colorectal cancer cell lines, and BTG4 overexpression characteristics were analyzed according to tumor size, histological suppresses colony formation in colorectal cancer cells [5]. To char- grading and presence of nodal metastasis. acterize the role of BTG4 in gastric cancer, we examined a series of DNA methylation analysis of the BTG4 gene. Genomic DNA (2 lg) primary gastric cancers and control tissues for BTG4 expression was modified with sodium bisulfite using EpiTect Bisulfite kit (Qia- and promoter methylation. Our results confirm frequent downreg- gen). Methylation status was analyzed by bisulfite genomic ulation of BTG4 expression in primary gastric cancers and identify sequencing of the CpG islands. The fragment covering 15 CpG sites hypermethylation of the BTG4 gene as a common epigenetic aber- from BTG4 promoter region was amplified from bisulfite-modified ration in these tumors. DNA. The primers used were 50-TTTTCGAGGGGTATAAGGAGAGTTT ATTTT-30 (sense) and 50-CCAAAACTCTAACTCCTAAATAAACA-30 (antisense). Amplified products were cloned into pMD18-T simple * Corresponding author. Address: Rui-jin Hospital Affiliated to Shanghai Jiao Tong vector (Takara), 6 independent clones were sequenced. University School of Medicine, 197 Rui-jin Road, Shanghai 200025, China. Fax: +86 21 64150773. Semiquantitative polymerase chain reaction. Total RNA was E-mail address: [email protected] (L. Wang). extracted from gastric cancer or normal tissues using TRIZOL 0006-291X/$ - see front matter Ó 2009 Elsevier Inc. All rights reserved. doi:10.1016/j.bbrc.2009.06.140 W. Dong et al. / Biochemical and Biophysical Research Communications 387 (2009) 132–138 133 (Invitrogen). Reverse transcription was carried out using Moloney (128 bp), the sense primer was 50-ATTCGTTTCGTTTCGCGTTCGTT Murine Leukemia Virus Reverse Transcriptase (M-MLV RT, Prome- TC-30 and the antisense primer was 50-TTTTTATTTGTTTTGTTTTG ga). The polymerase chain reaction was performed using SYBR TGTTTGTTTTG-30. For the unmethylated allele (134 bp), the sense Green kit following the manufacture’s protocol (Takara). Primers primer was 50-CCTAAAACTAACTCTCTC AACCCCA-30 and the anti- used in RT-PCR: BTG4-F: 50-GTTTCTCTTTCTGATCTAGCAGGA-30; sense primer was 50-AATACTAAAATACAACATCAC CTCCA-30. The BTG4-R: 50-TCAGAGTGCCAGTGA CTTCTGTA-30. Actin-F: 50-AGATGT annealing temperatures for the methylated and unmethylated GGATCAGCAAGCGGAGT-30; Actin-R: 50-GCAATCAAAGTCCTCGG DNA were 67 °C and 61 °C, respectively, for 40 s each. Hot-start CCACATT-30. A 7900HT Fast Real-time PCR System (Applied Biosys- PCR with a total cycle number of 30 was used in all MSP DNA tems) was used for testing. Following the protocol of the manufac- amplifications. Denaturation and extension cycles were main- turer, the amount of BTG4 expression, normalized to a human actin tained for 30 and 45 s, respectively. The methylation status of the endogenous reference is given by: 2ÀDDCt Real-time RT-PCR was p16 promoter was determined by MSP as described previously repeated at least three times for each specimen, and mean was [8]. For the methylated allele, the sense primer was 50-TTATTA- obtained. GAGGGTGGGGCGGATCGC-30 and the antisense primer was 50- DNA extraction from paraffin block. The DNA extraction was per- GACCCCGAACCGCGACCGTAA-30. For the unmethylated allele, the formed as previously described [7]. Briefly, formalin-fixed, paraffin sense primer was 50-TTATTAGAGGGTGGGGTGGATTGT-30 and the wax-embedded tissues were cut into 10 mm thick sections. Before antisense primer was 50-CCACCTAAATCAACCTCCAACCA-30. DNA extraction, the sections were placed on slides and stained Construction of plasmids and stable cell line generation. For con- with hematoxylin–eosin to evaluate the admixture of non-tumor- struction of pCMV4-flag-BTG4, the BTG4 cDNA was generated by ous tissues. Areas corresponding to tumor or surrounding normal reverse transcription PCR using BTG4 forward primer (50-AAAG gastric tissue were microdissected separately. Microdissected tis- CCTTATGAGAGATGAAATTGCAACAA-30) and reverse primer (50-GG sues were collected in 15 ml centrifuge tubes, and deparaffinized ATCCCTACAGTTTTGTTCTTTCCA-30). The sequence was confirmed overnight at 63 °C in xylene. After centrifugation at full speed for by DNA sequencing, and ligated into the HindIII and BamHI sites 5 min, the supernatant was removed. Ethanol was added to the of pCMV-flag vector (Invitrogen). For transfection experiments, pellet to remove residual xylene, and then removed by centrifuga- BCG-823 and MKN45 cells were plated into 6-well plates 24 h be- tion. The genomic DNA was isolated using DNeasy Tissue Kit fore transfection. The cells were transfected with 5 lg/well of according to the manufacturer’s instruction. empty pCMV-flag or pCMV4-flag-BTG4 using Superfect (Qiagen, Methylation-specific PCR (MSP). To study BTG4 promoter hyper- Germany) according to manufacturer’s instructions. For 48 h after methylation, we also performed MSP as described previously. For transfection, the cells were passaged at 1:5 and cultured in med- the BTG4 promoter methylation study, we used two sets of primers ium supplemented with G418 at 500 lg/ml for 4 weeks. One that could amplify the modified DNA of either the methylated or BCG-823 clone and one MKN45 clone reexpressing BTG4 (BCG- unmethylated alleles separately. For the methylated allele 823/BTG4, MKN45/BTG4) were selected for further study, with Fig. 1. Demonstration of BTG4 promoter methylation by sequencing of sodium bisulfite–modified DNA from GES-1 and the indicated gastric cancer cell lines. Methylated and unmethylated CpG dinucleotides are shown by closed and open circles, respectively. Each line of circles represents analysis of a single cloned allele. An illustrative fragment of the sequencing electropherogram is shown for GES-1 and MKN-28, the CpG sites are underlined. 134 W. Dong et al. / Biochemical and Biophysical Research Communications 387 (2009) 132–138 BTG4 expression verified by western blot. As a control group, cells immediately analyzed by flow-cytometry. The data are presented stably transfected with an empty vector pCMV-flag were also gen- as bi-parametric dot plots showing Annexin V–FITC green fluores- erated (BCG-823/vector, MKN45/vector). cence versus PI red fluorescence. Western blotting. Cells were harvested and samples (20 lg) of Anchorage-independent growth. For anchorage-independent the cell lysate were subjected to 10% SDS–PAGE gel electrophore- growth assay, 2 Â 104 cells were plated in 0.3% low melting point sis, after which the resolved proteins were transferred to nitrocel- agar/growth medium onto six-well plates with a 0.6% agar under- lulose membranes (Amersham Biosciences). The membranes were lay. After 3 weeks, colonies that were >1 mm in diameter were then blocked with 5% non-fat milk and 0.1% Tween 20 in Tris-buf- counted.