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DNA methylation studies in cattle. J Appl Genet 2021; 62:121-136. [PMID: 33400132 DOI: 10.1007/s13353-020-00604-1] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2020] [Revised: 10/23/2020] [Accepted: 12/22/2020] [Indexed: 12/12/2022]
Abstract
Investigation of the role of epigenetics in cattle breeding is gaining importance. DNA methylation represents an epigenetic modification which is essential for genomic stability and maintenance of development. Recently, DNA methylation research in cattle has intensified. The studies focus on the definition of methylomes in various organs and tissues in relation to the expression of genes underlying economically important traits, and explore methylome changes under developmental, environmental, disease, and diet influences. The investigations further characterize the methylation patterns of gametes in connection with their quality, and study methylome alterations in the developing naturally or assisted produced zygotes, embryos, and fetuses, considering their viability. A wide array of technologies developed for accurate and precise analysis of DNA methylation patterns is employed for both single-gene and genome-wide studies. Overall, the research is directed towards the identification of single methylation markers or their combinations which may be useful in the selection and breeding of animals to ensure cattle improvement.
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Ma WR, Xu P, Liu ZJ, Zhou J, Gu LK, Zhang J, Deng DJ. Impact of GFRA1 gene reactivation by DNA demethylation on prognosis of patients with metastatic colon cancer. World J Gastroenterol 2020; 26:184-198. [PMID: 31988584 PMCID: PMC6962434 DOI: 10.3748/wjg.v26.i2.184] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 10/08/2019] [Revised: 12/14/2019] [Accepted: 12/23/2019] [Indexed: 02/06/2023] Open
Abstract
BACKGROUND The expression of the membrane receptor protein GFRA1 is frequently upregulated in many cancers, which can promote cancer development by activating the classic RET-RAS-ERK and RET-RAS-PI3K-AKT pathways. Several therapeutic anti-GFRA1 antibody-drug conjugates are under development. Demethylation (or hypomethylation) of GFRA1 CpG islands (dmGFRA1) is associated with increased gene expression and metastasis risk of gastric cancer. However, it is unknown whether dmGFRA1 affects the metastasis of other cancers, including colon cancer (CC).
AIM To study whether dmGFRA1 is a driver for CC metastasis and GFRA1 is a potential therapeutic target.
METHODS CC and paired surgical margin tissue samples from 144 inpatients and normal colon mucosal biopsies from 21 noncancer patients were included in this study. The methylation status of GFRA1 islands was determined by MethyLight and denaturing high-performance liquid chromatography and bisulfite-sequencing. Kaplan-Meier analysis was used to explore the effect of dmGFRA1 on the survival of CC patients. Impacts of GFRA1 on CC cell proliferation and migration were evaluated by a battery of biological assays in vitro and in vivo. The phosphorylation of AKT and ERK proteins was examined by Western blot analysis.
RESULTS The proportion of dmGFRA1 in CC, surgical margin, and normal colon tissues by MethyLight was 68.4%, 73.4%, and 35.9% (median; nonparametric test, P = 0.001 and < 0.001), respectively. Using the median value of dmGFRA1 peak area proportion as the cutoff, the proportion of dmGFRA1-high samples was much higher in poorly differentiated CC samples than in moderately or well-differentiated samples (92.3%% vs 55.8%, Chi-square test, P = 0.002) and significantly higher in CC samples with distant metastasis than in samples without (77.8% vs 46.0%, P = 0.021). The overall survival of patients with dmGFRA1-low CC was significantly longer than that of patients with dmGFRA1-high CC (adjusted hazard ratio = 0.49, 95% confidence interval: 0.24-0.98), especially for 89 CC patients with metastatic CC (adjusted hazard ratio = 0.41, 95% confidence interval: 0.18-0.91). These data were confirmed by the mining results from TCGA datasets. Furthermore, GFRA1 overexpression significantly promoted the proliferation/invasion of RKO and HCT116 cells and the growth of RKO cells in nude mice but did not affect their migration. GFRA1 overexpression markedly increased the phosphorylation levels of AKT and ERK proteins, two key molecules in two classic GFRA1 downstream pathways.
CONCLUSION GFRA1 expression is frequently reactivated by DNA demethylation in CC tissues and is significantly associated with a poor prognosis in patients with CC, especially those with metastatic CC. GFRA1 can promote the proliferation/growth of CC cells, probably by the activation of AKT and ERK pathways. GFRA1 might be a therapeutic target for CC patients, especially those with metastatic potential.
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Affiliation(s)
- Wan-Ru Ma
- Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Beijing 100143, China
| | - Peng Xu
- Shihezi University School of Medicine, Shihezi 832000, Xinjiang Uygur Autonomous Region, China
- Morphological Center of Basic Medical School of Xinjiang Medical University, Urumqi 830011, Xinjiang Uygur Autonomous Region, China
| | - Zhao-Jun Liu
- Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Beijing 100143, China
| | - Jing Zhou
- Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Beijing 100143, China
| | - Lian-Kun Gu
- Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Beijing 100143, China
| | - Jun Zhang
- Shihezi University School of Medicine, Shihezi 832000, Xinjiang Uygur Autonomous Region, China
| | - Da-Jun Deng
- Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Beijing 100143, China
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Li P, Gan Y, Qin S, Han X, Cui C, Liu Z, Zhou J, Gu L, Lu ZM, Zhang B, Deng D. DNA hydroxymethylation increases the susceptibility of reactivation of methylated P16 alleles in cancer cells. Epigenetics 2019; 15:618-631. [PMID: 31790633 DOI: 10.1080/15592294.2019.1700004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/16/2022] Open
Abstract
It is well established that 5-methylcytosine (5mC) in genomic DNA of mammalian cells can be oxidized into 5-hydroxymethylcytosine (5hmC) and other derivates by DNA dioxygenase TETs. While conversion of 5mC to 5hmC plays an important role in active DNA demethylation through further oxidation steps, a certain proportion of 5hmCs remain in the genome. Although 5hmCs contribute to the flexibility of chromatin and protect bivalent promoters from hypermethylation, the direct effect of 5hmCs on gene transcription is unknown. In this present study, we have engineered a zinc-finger protein-based P16-specific DNA dioxygenase (P16-TET) to induce P16 hydroxymethylation and demethylation in cancer cells. Our results demonstrate, for the first time, that although the hydroxymethylated P16 alleles retain transcriptionally inactive, hydroxymethylation could increase the susceptibility of reactivation of methylated P16 alleles.
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Affiliation(s)
- Paiyun Li
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute , Beijing, China
| | - Ying Gan
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute , Beijing, China
| | - Sisi Qin
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute , Beijing, China
| | - Xiao Han
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute , Beijing, China
| | - Chenghua Cui
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute , Beijing, China
| | - Zhaojun Liu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute , Beijing, China
| | - Jing Zhou
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute , Beijing, China
| | - Liankun Gu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute , Beijing, China
| | - Zhe-Ming Lu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute , Beijing, China
| | - Baozhen Zhang
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute , Beijing, China
| | - Dajun Deng
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute , Beijing, China
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Liu Z, Lin H, Gan Y, Cui C, Zhang B, Gu L, Zhou J, Zhu G, Deng D. P16 Methylation Leads to Paclitaxel Resistance of Advanced Non-Small Cell Lung Cancer. J Cancer 2019; 10:1726-1733. [PMID: 31205528 PMCID: PMC6547999 DOI: 10.7150/jca.26482] [Citation(s) in RCA: 18] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/04/2018] [Accepted: 01/12/2019] [Indexed: 01/07/2023] Open
Abstract
Paclitaxel-based chemotherapy is widely used as the first-line treatment for non-small cell lung cancer (NSCLC). However, only 20%-40% of patients have shown sensitivity to paclitaxel. This study aimed to investigate whether P16 methylation could be used to predict paclitaxel chemosensitivity of NSCLC. Advanced NSCLC (N=45) were obtained from patients who were enrolled in a phase-III randomized paclitaxel-based clinical trial. Genomic DNA samples were extracted from the biopsies prior to chemotherapy. P16 methylation was detected using MethyLight. The association between P16 methylation and the sensitivity of paclitaxel in cell lines was determined by in vitro assay using a P16-specific DNA demethylase (P16-TET) and methyltransferase (P16-Dnmt). The total response rate of the low-dose paclitaxel-based chemo-radiotherapy was significantly lower in P16 methylation-positive NSCLCs than that in the P16 methylation-negative NSCLCs (2/15 vs. 16/30: adjusted OR=0.085; 95%CI, 0.012-0.579). Results revealed that P16 demethylation significantly decreased paclitaxel resistance of lung cancer H1299 cells (IC50 values decreased from 2.15 to 1.13 µg/ml, P<0.001). In contrast, P16-specific methylation by P16-Dnmt significantly increased paclitaxel resistance of lung cancer HCC827 cells and gastric cancer BGC823 cells (IC50 values increased from 18.2 to 24.0 ng/ml and 0.18 to 0.81 µg/ml, respectively; P=0.049 and <0.001, respectively). The present results suggest that P16 methylation may lead to paclitaxel resistance and be a predictor of paclitaxel chemosensitivity of NSCLC.
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Affiliation(s)
- Zhaojun Liu
- Key Laboratory of Carcinogenesis and Translational Research, Division of Etiology
| | - Hongmei Lin
- Key Laboratory of Carcinogenesis and Translational Research, Department of Radiation Oncology, Peking University Cancer Hospital and Institute, Beijing, 100142
- National Clinical Research Center for Respiratory Diseases, Department of Radiation Oncology, China-Japan Friendship Hospital, Beijing, 100029, China
| | - Ying Gan
- Key Laboratory of Carcinogenesis and Translational Research, Division of Etiology
| | - Chenghua Cui
- Key Laboratory of Carcinogenesis and Translational Research, Division of Etiology
| | - Baozhen Zhang
- Key Laboratory of Carcinogenesis and Translational Research, Division of Etiology
| | - Liankun Gu
- Key Laboratory of Carcinogenesis and Translational Research, Division of Etiology
| | - Jing Zhou
- Key Laboratory of Carcinogenesis and Translational Research, Division of Etiology
| | - Guangying Zhu
- Key Laboratory of Carcinogenesis and Translational Research, Department of Radiation Oncology, Peking University Cancer Hospital and Institute, Beijing, 100142
- National Clinical Research Center for Respiratory Diseases, Department of Radiation Oncology, China-Japan Friendship Hospital, Beijing, 100029, China
| | - Dajun Deng
- Key Laboratory of Carcinogenesis and Translational Research, Division of Etiology
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A sensitive fluorometric DNA nanobiosensor based on a new fluorophore for tumor suppressor gene detection. Talanta 2018; 190:140-146. [DOI: 10.1016/j.talanta.2018.07.042] [Citation(s) in RCA: 11] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/05/2018] [Revised: 07/11/2018] [Accepted: 07/12/2018] [Indexed: 12/13/2022]
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Liu H, Liu Z, Liu XW, Xu S, Wang L, Liu Y, Zhou J, Gu L, Gao Y, Liu XY, Shi H, Sun Z, Deng D. A similar effect of P16 hydroxymethylation and true-methylation on the prediction of malignant transformation of oral epithelial dysplasia: observation from a prospective study. BMC Cancer 2018; 18:918. [PMID: 30249192 PMCID: PMC6154879 DOI: 10.1186/s12885-018-4787-6] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/12/2018] [Accepted: 08/31/2018] [Indexed: 01/18/2023] Open
Abstract
Background Total P16 methylation (P16M), including P16 hydroxymethylation (P16H) and true-P16M, correlates with malignant transformation of oral epithelial dysplasia (OED). Both true-P16M and P16H are early events in carcinogenesis. The aim of this study is to prospectively determine if discrimination of true-P16M from P16H is necessary for prediction of cancer development from OEDs. Methods Patients (n = 265) with mild or moderate OED were recruited into the double blind two-center cohort. Total-P16M and P16H were analyzed using the 115-bp MethyLight, TET-assisted bisulfite (TAB) methylation-specific PCR (MSP), and TAB-sequencing. Total-P16M-positive and P16H-negative samples were defined as true-P16M-positive. Progression of OEDs was monitored for a minimum 24 months follow-up period. Results P16H was detected in 23 of 73 (31.5%) total-P16M-positive OEDs. Follow-up information was obtained from 247 patients with an ultimate compliance rate of 93.2%. OED-derived squamous cell carcinomas were observed in 13.0% (32/247) patients during follow-up (median, 41.0 months). The cancer progression rate for total-P16M-positive patients was significantly increased when compared to total-P16M-negative patients [23.3% vs 8.6%; adjusted odds ratio = 2.67 (95% CI: 1.19–5.99)]. However, the cancer progression rates were similar between P16H- and true-P16M-positive OEDs [26.1% (6/23) vs 22.0% (11/50); odds ratio = 0.80 (95% CI: 0.22–2.92)]. The cancer-free survival was also similar for these patients. Conclusion P16H and true-P16M are similar biomarkers for determining malignant potential of OEDs. Discrimination of P16H from true-P16M, at least in OED, may be not necessary in clinical applications. Trial registration This study is registered prospectively in the U.S. National Institutes of Health Clinical Trials Protocol Registration System (trial number NCT02967120, available at https://ClinicalTrials.gov/ct2/show/NCT02967120). Electronic supplementary material The online version of this article (10.1186/s12885-018-4787-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Hongwei Liu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Peking University School of Stomatology, Beijing, 100081, China
| | - Zhaojun Liu
- Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Aetiology, Peking University Cancer Hospital and Institute, Beijing, 100142, China
| | - Xue-Wei Liu
- Capital Medical University School of Stomatology, Beijing, 100050, China
| | - Si Xu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Peking University School of Stomatology, Beijing, 100081, China
| | - Lei Wang
- Capital Medical University School of Stomatology, Beijing, 100050, China
| | - Yang Liu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Peking University School of Stomatology, Beijing, 100081, China
| | - Jing Zhou
- Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Aetiology, Peking University Cancer Hospital and Institute, Beijing, 100142, China
| | - Liankun Gu
- Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Aetiology, Peking University Cancer Hospital and Institute, Beijing, 100142, China
| | - Yan Gao
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Peking University School of Stomatology, Beijing, 100081, China
| | - Xiao-Yong Liu
- Capital Medical University School of Stomatology, Beijing, 100050, China
| | - Huidong Shi
- Georgia Cancer Center, Medical College of Georgia, Augusta University, Augusta, GA, 30912, USA
| | - Zheng Sun
- Capital Medical University School of Stomatology, Beijing, 100050, China.
| | - Dajun Deng
- Key Laboratory of Carcinogenesis and Translational Research (MOE/Beijing), Division of Aetiology, Peking University Cancer Hospital and Institute, Beijing, 100142, China.
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Zhang B, Zhou J, Liu Z, Gu L, Ji J, Kim WH, Deng D. Clinical and biological significance of a - 73A > C variation in the CDH1 promoter of patients with sporadic gastric carcinoma. Gastric Cancer 2018; 21:606-616. [PMID: 29168119 DOI: 10.1007/s10120-017-0778-6] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 08/22/2017] [Accepted: 11/12/2017] [Indexed: 02/07/2023]
Abstract
BACKGROUND CDH1 germline mutations lead to hereditary diffuse gastric carcinomas. However, it is unclear whether genetic variations in the CDH1 promoter affect the progression of sporadic gastric carcinomas (SGCs). METHODS SGC patients in two independent cohorts with follow-up data were enrolled. The CDH1 genotypes, including the - 73A > C polymorphism (rs28372783), were determined by PCR sequencing. The CDH1 promoter activity was determined using reporter assays. SNAIL bound to CDH1 alleles was determined by chromatin immunoprecipitation primer extension PCR. CDH1 DNA methylation was determined by bisulfite-based PCR analyses. RESULTS Kaplan-Meier analyses showed that the overall survival (OS) of the - 73C/C patients was significantly longer than that of the - 73A/C or - 73A/A patients in a Chinese cohort [n = 526; hazard ratio 0.68 (95% CI 0.47-1.00)], which was validated in an independent Korea cohort [n = 215; hazard ratio 0.49 (95% CI 0.26-0.94)]. Moreover, the transcription activity of the - 73C alleles was significantly higher than that of the - 73A alleles in vitro and in vivo. The ratio of SNAIL recruited to the promoter regions of the - 73C and - 73A alleles was 1:10, indicating a strong influence of this polymorphism on the recruitment of SNAIL to the flanking E-box. The prevalence of DNA methylation of the CpG island and shore within the promoter of the - 73C allele was much less than that of the - 73A allele in both gastric tissues and cancer cell lines. CONCLUSION The - 73A > C variation may lead to differences in the overall survival of SGC patients and allele-specific repressions of CDH1.
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Affiliation(s)
- Baozhen Zhang
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu #52, Haidian District, Beijing, 100142, China.
| | - Jing Zhou
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu #52, Haidian District, Beijing, 100142, China
| | - Zhaojun Liu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu #52, Haidian District, Beijing, 100142, China
| | - Liankun Gu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu #52, Haidian District, Beijing, 100142, China
| | - Jiafu Ji
- Department of Surgery, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu #52, Haidian District, Beijing, 100142, China
| | - Woo Ho Kim
- Department of Pathology, Seoul National University College of Medicine, Jongnogu, Seoul, Korea
| | - Dajun Deng
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu #52, Haidian District, Beijing, 100142, China.
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Yotani T, Yamada Y, Arai E, Tian Y, Gotoh M, Komiyama M, Fujimoto H, Sakamoto M, Kanai Y. Novel method for DNA methylation analysis using high-performance liquid chromatography and its clinical application. Cancer Sci 2018; 109:1690-1700. [PMID: 29520901 PMCID: PMC5980336 DOI: 10.1111/cas.13566] [Citation(s) in RCA: 17] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/08/2017] [Revised: 02/17/2018] [Accepted: 02/25/2018] [Indexed: 12/21/2022] Open
Abstract
The aim of this study was to develop a new methodology that is suitable for DNA methylation diagnostics and to demonstrate its clinical applicability. We developed a new anion-exchange column for high-performance liquid chromatography (HPLC) with electrostatic and hydrophobic properties. Both cytosine and thymine, corresponding to methylated and unmethylated cytosine after bisulfite modification, respectively, are captured by electrostatic interaction and then discriminated from each other by their hydrophobic interactions. The DNA methylation levels of synthetic DNA were quantified accurately and reproducibly within 10 minutes without time-consuming pretreatment of PCR products, and the measured values were unaffected by the distribution of methylated CpG within the synthetic DNA fragments. When the DNA methylation status of the FAM150A gene, a marker of the CpG island methylator phenotype specific to clear cell renal cell carcinoma (ccRCC), was examined in 98 patients with ccRCC, bulk specimens of tumorous tissue including cancer cells showing DNA methylation of the FAM150A gene were easily identifiable by simply viewing the differentiated chromatograms, even when the cancer cell content was low. Sixteen ccRCC showing DNA methylation more frequently exhibited clinicopathological parameters reflecting tumor aggressiveness (ie, a larger diameter, higher histological grade, vascular involvement, renal vein tumor thrombi, infiltrating growth, tumor necrosis, renal pelvis invasion and higher pathological TNM stage), and had significantly lower recurrence-free and overall survival rates. These data indicate that HPLC analysis using this newly developed anion-exchange column could be a powerful tool for DNA methylation diagnostics, including prognostication of patients with cancers, in a clinical setting.
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Affiliation(s)
- Takuya Yotani
- Department of PathologyKeio University School of MedicineTokyoJapan
- Tsukuba Research InstituteResearch and Development DivisionSekisui Medical Co., Ltd.RyugasakiJapan
- Division of Molecular PathologyNational Cancer Center Research InstituteTokyoJapan
| | - Yuriko Yamada
- Tsukuba Research InstituteResearch and Development DivisionSekisui Medical Co., Ltd.RyugasakiJapan
- Division of Molecular PathologyNational Cancer Center Research InstituteTokyoJapan
| | - Eri Arai
- Department of PathologyKeio University School of MedicineTokyoJapan
- Division of Molecular PathologyNational Cancer Center Research InstituteTokyoJapan
| | - Ying Tian
- Department of PathologyKeio University School of MedicineTokyoJapan
| | - Masahiro Gotoh
- Division of Molecular PathologyNational Cancer Center Research InstituteTokyoJapan
| | | | | | - Michiie Sakamoto
- Department of PathologyKeio University School of MedicineTokyoJapan
| | - Yae Kanai
- Department of PathologyKeio University School of MedicineTokyoJapan
- Division of Molecular PathologyNational Cancer Center Research InstituteTokyoJapan
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Gan Y, Ma W, Wang X, Qiao J, Zhang B, Cui C, Liu Z, Deng D. Coordinated transcription of ANRIL and P16 genes is silenced by P16 DNA methylation. Chin J Cancer Res 2018; 30:93-103. [PMID: 29545723 DOI: 10.21147/j.issn.1000-9604.2018.01.10] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/25/2022] Open
Abstract
Objective To investigate the relationship between the transcription of ANRIL, P15, P14 and P16 at the same locus and the regulation mechanism of ANRIL. Methods Publicly available database of Cancer Cell Line Encyclopedia (CCLE) was used in bioinformatic analyses. Methylation of CpG islands was detected by denaturing high performance liquid chromatography (DHPLC). Gene transcript levels were determined using quantitative real-time polymerase chain reaction (qRT-PCR) assays. An engineered P16-specific transcription factor and DNA methyltransferase were used to induce P16-specific DNA demethylation and methylation. Results The expression level of ANRIL was positively and significantly correlated with that of P16 but not with that of P15 in the CCLE database. This was confirmed in human cell lines and patient colon tissue samples. In addition, ANRIL was significantly upregulated in colon cancer tissues. Transcription of ANRIL and P16 was observed only in cell lines in which the P16 alleles were unmethylated and not in cell lines with fully methylated P16 alleles. Notably, P16-specific methylation significantly decreased transcription of P16 and ANRIL in BGC823 and GES1 cells. In contrast, P16-specific demethylation re-activated transcription of ANRIL and P16 in H1299 cells (P<0.001). Alteration ofANRIL expression was not induced by P16 expression changes. Conclusions ANRIL and P16 are coordinately transcribed in human cells and regulated by the methylation status of the P16 CpG islands around the transcription start site.
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Affiliation(s)
- Ying Gan
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China
| | - Wanru Ma
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China
| | - Xiuhong Wang
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China
| | - Juanli Qiao
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China
| | - Baozhen Zhang
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China
| | - Chenghua Cui
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China
| | - Zhaojun Liu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China
| | - Dajun Deng
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital & Institute, Beijing 100142, China
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Liu Z, Zhou J, Gu L, Deng D. Significant impact of amount of PCR input templates on various PCR-based DNA methylation analysis and countermeasure. Oncotarget 2016; 7:56447-56455. [PMID: 27487147 PMCID: PMC5302926 DOI: 10.18632/oncotarget.10906] [Citation(s) in RCA: 8] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/06/2016] [Accepted: 07/20/2016] [Indexed: 12/21/2022] Open
Abstract
Methylation changes of CpG islands can be determined using PCR-based assays. However, the exact impact of the amount of input templates (TAIT) on DNA methylation analysis has not been previously recognized. Using COL2A1 gene as an input reference, TAIT difference between human tissues with methylation-positive and -negative detection was calculated for two representative genes GFRA1 and P16. Results revealed that TAIT in GFRA1 methylation-positive frozen samples (n = 332) was significantly higher than the methylation-negative ones (n = 44) (P < 0.001). Similar difference was found in P16 methylation analysis. The TAIT-related effect was also observed in methylation-specific PCR (MSP) and denatured high performance liquid chromatography (DHPLC) analysis. Further study showed that the minimum TAIT for a successful MethyLight PCR reaction should be ≥ 9.4 ng (CtCOL2A1 ≤ 29.3), when the cutoff value of the methylated-GFRA1 proportion for methylation-positive detection was set at 1.6%. After TAIT of the methylation non-informative frozen samples (n = 94; CtCOL2A1 > 29.3) was increased above the minimum TAIT, the methylation-positive rate increased from 72.3% to 95.7% for GFRA1 and 26.6% to 54.3% for P16, respectively (Ps < 0.001). Similar results were observed in the FFPE samples. In conclusion, TAIT critically affects results of various PCR-based DNA methylation analyses. Characterization of the minimum TAIT for target CpG islands is essential to avoid false-negative results.
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Affiliation(s)
- Zhaojun Liu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Haidian District, Beijing, 100142, China
| | - Jing Zhou
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Haidian District, Beijing, 100142, China
| | - Liankun Gu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Haidian District, Beijing, 100142, China
| | - Dajun Deng
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Division of Etiology, Peking University Cancer Hospital and Institute, Haidian District, Beijing, 100142, China
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Cui C, Gan Y, Gu L, Wilson J, Liu Z, Zhang B, Deng D. P16-specific DNA methylation by engineered zinc finger methyltransferase inactivates gene transcription and promotes cancer metastasis. Genome Biol 2015; 16:252. [PMID: 26592237 PMCID: PMC4656189 DOI: 10.1186/s13059-015-0819-6] [Citation(s) in RCA: 54] [Impact Index Per Article: 5.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2015] [Accepted: 10/30/2015] [Indexed: 12/14/2022] Open
Abstract
Background P16 DNA methylation is well known to be the most frequent event in cancer development. It has been reported that genetic inactivation of P16 drives cancer growth and metastasis, however, whether P16 DNA methylation is truly a driver in cancer metastasis remains unknown. Results A P16-specific DNA methyltransferase (P16-dnmt) expression vector is designed using a P16 promoter-specific engineered zinc finger protein fused with the catalytic domain of dnmt3a. P16-dnmt transfection significantly decreases P16 promoter activity, induces complete methylation of P16 CpG islands, and inactivates P16 transcription in the HEK293T cell line. The P16-Dnmt coding fragment is integrated into an expression controllable vector and used to induce P16-specific DNA methylation in GES-1 and BGC823 cell lines. Transwell assays show enhanced migration and invasion of these cancer cells following P16-specific DNA methylation. Such effects are not observed in the P16 mutant A549 cell line. These results are confirmed using an experimental mouse pneumonic metastasis model. Moreover, enforced overexpression of P16 in these cells reverses the migration phenotype. Increased levels of RB phosphorylation and NFκB subunit P65 expression are also seen following P16-specific methylation and might further contribute to cancer metastasis. Conclusion P16 methylation could directly inactivate gene transcription and drive cancer metastasis. Electronic supplementary material The online version of this article (doi:10.1186/s13059-015-0819-6) contains supplementary material, which is available to authorized users.
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Affiliation(s)
- Chenghua Cui
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Aetiology, Peking University Cancer Hospital & Institute, Beijing, 100142, China. .,Department of Pathology, Institute of Hematology & Hospital of Blood Diseases, Chinese Academy of Medical Sciences, Tianjin, 300020, China.
| | - Ying Gan
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Aetiology, Peking University Cancer Hospital & Institute, Beijing, 100142, China.
| | - Liankun Gu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Aetiology, Peking University Cancer Hospital & Institute, Beijing, 100142, China.
| | - James Wilson
- GRU Cancer Center, Georgia Regents University, Augusta, GA30912, USA.
| | - Zhaojun Liu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Aetiology, Peking University Cancer Hospital & Institute, Beijing, 100142, China.
| | - Baozhen Zhang
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Aetiology, Peking University Cancer Hospital & Institute, Beijing, 100142, China.
| | - Dajun Deng
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education/Beijing), Department of Aetiology, Peking University Cancer Hospital & Institute, Beijing, 100142, China.
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12
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Sun R, Li J, Wang B, Guo Y, Ma L, Quan X, Chu Z, Li T. Liver kinase B1 promoter CpG island methylation is related to lung cancer and smoking. Int J Clin Exp Med 2015; 8:14070-14074. [PMID: 26550370 PMCID: PMC4613055] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/15/2015] [Accepted: 05/20/2015] [Indexed: 06/05/2023]
Abstract
The aim of this study was to explore the association of CpG islands methylation of liver kinase B1 (LKB1) with primary lung cancer and smoking, providing a theoretical basis for the demethylating drug to treat lung cancer by detecting the LKB1 promoter CpG methylation. mRNA expression of LKB1 were detected by in situ hybridization and methylation status on Hap II locus of the promoter of LKB1 was analyzed by methylation-specific polymerase chain reaction (PCR). 7 of 80 LKB1 positive cases had methylation on CpG islands while 18 of 44 LKB1 negative cases had methylation on CpG islands. The difference was significant between CpG island methylation and LKB1 expression. 8 of 54 cases of early and middle lung cancer were detected LKB1 promoter CpG island methylation while 30 controls were not detected, the difference was significant. 5 of 64 more-than-5-year cases had methylation on CpG islands while 20 of 60 less-than-5-year cases had methylation. The difference was significant between of 5-year survival and CpG island methylation of LKB1. 22 of 74 smoking cases of lung cancer had methylation on CpG islands of LKB1 while only 3 of 50 non-smoking cases had methylation. The difference of smoking and CpG island methylation of LKB1 was significant. LKB1 promoter CpG islands aberrant methylation is closely associated with the occurrence, development and prognosis of lung cancer, especially with smoking history including clinical early diagnosis and prognosis. CpG islands methylation in the promoter of LKB1 is likely important one of the mechanism of smoking-associated lung cancer.
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Affiliation(s)
- Rongju Sun
- Department of Emergency, General Hospital of PLABeijing 100853, China
- Department of Emergency, Affiliated Hainan Hospital, General Hospital of PLASanya 572000, China
| | - Jie Li
- Department of Thoracic Surgery, General Hospital of PLABeijing 100853, China
| | - Bo Wang
- Department of Thoracic Surgery, General Hospital of PLABeijing 100853, China
| | - Yingfei Guo
- Department of Emergency or Respiration, The First Affiliated Hospital of General Hospital of PLABeijing 100048, China
| | - Lingyun Ma
- Department of Emergency or Respiration, The First Affiliated Hospital of General Hospital of PLABeijing 100048, China
| | - Xiaojiao Quan
- Department of Emergency, Affiliated Hainan Hospital, General Hospital of PLASanya 572000, China
| | - Zhixiang Chu
- Department of Emergency, Affiliated Hainan Hospital, General Hospital of PLASanya 572000, China
| | - Tanshi Li
- Department of Emergency, General Hospital of PLABeijing 100853, China
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13
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Cui C, Lu Z, Yang L, Gao Y, Liu W, Gu L, Yang C, Wilson J, Zhang Z, Xing B, Deng D, Sun ZS. Genome-wide identification of differential methylation between primary and recurrent hepatocellular carcinomas. Mol Carcinog 2015; 55:1163-74. [PMID: 26138747 DOI: 10.1002/mc.22359] [Citation(s) in RCA: 14] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/16/2015] [Revised: 05/19/2015] [Accepted: 06/15/2015] [Indexed: 01/27/2023]
Affiliation(s)
- Chenghua Cui
- Division of Cancer Etiology; Key Laboratory of Carcinogenesis and Translational Research Ministry of Education; Peking University Cancer Hospital Institute; Beijing China
| | - Zheming Lu
- Division of Cancer Etiology; Key Laboratory of Carcinogenesis and Translational Research Ministry of Education; Peking University Cancer Hospital Institute; Beijing China
| | - Liu Yang
- Beijing Institutes of Life Science; Chinese Academy of Sciences; Beijing China
| | - Yanhong Gao
- Division of Cancer Etiology; Key Laboratory of Carcinogenesis and Translational Research Ministry of Education; Peking University Cancer Hospital Institute; Beijing China
| | - Wei Liu
- Department of Surgery; Peking University Cancer Hospital and Institute; Beijing China
| | - Liankun Gu
- Division of Cancer Etiology; Key Laboratory of Carcinogenesis and Translational Research Ministry of Education; Peking University Cancer Hospital Institute; Beijing China
| | - Chen Yang
- Division of Cancer Etiology; Key Laboratory of Carcinogenesis and Translational Research Ministry of Education; Peking University Cancer Hospital Institute; Beijing China
| | - James Wilson
- GRU Cancer Center; Georgia Regents University; Augusta Georgia
| | - Zhiqian Zhang
- GRU Cancer Center; Georgia Regents University; Augusta Georgia
| | - Baocai Xing
- Department of Surgery; Peking University Cancer Hospital and Institute; Beijing China
| | - Dajun Deng
- Division of Cancer Etiology; Key Laboratory of Carcinogenesis and Translational Research Ministry of Education; Peking University Cancer Hospital Institute; Beijing China
| | - Zhong Sheng Sun
- Beijing Institutes of Life Science; Chinese Academy of Sciences; Beijing China
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14
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Detection of DNA methylation by hyperbranched rolling circle amplification and DNA microarray. CHINESE CHEM LETT 2014. [DOI: 10.1016/j.cclet.2014.09.010] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/22/2022]
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Liu Z, Zhang J, Gao Y, Pei L, Zhou J, Gu L, Zhang L, Zhu B, Hattori N, Ji J, Yuasa Y, Kim W, Ushijima T, Shi H, Deng D. Large-scale characterization of DNA methylation changes in human gastric carcinomas with and without metastasis. Clin Cancer Res 2014; 20:4598-612. [PMID: 25009298 PMCID: PMC4309661 DOI: 10.1158/1078-0432.ccr-13-3380] [Citation(s) in RCA: 72] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/20/2022]
Abstract
PURPOSE Metastasis is the leading cause of death for gastric carcinoma. An epigenetic biomarker panel for predicting gastric carcinoma metastasis could have significant clinical impact on the care of patients with gastric carcinoma. The main purpose of this study is to characterize the methylation differences between gastric carcinomas with and without metastasis. EXPERIMENTAL DESIGN Genome-wide DNA methylation profiles between 4 metastatic and 4 nonmetastatic gastric carcinomas and their surgical margins (SM) were analyzed using methylated-CpG island amplification with microarray. The methylation states of 73 candidate genes were further analyzed in patients with gastric carcinoma in a discovery cohort (n=108) using denatured high performance liquid chromatography, bisulfite-sequencing, and MethyLight. The predictive values of potential metastasis-methylation biomarkers were validated in cohorts of patients with gastric carcinoma in China (n=330), Japan (n=129), and Korea (n=153). RESULTS The gastric carcinoma genome showed significantly higher proportions of hypomethylation in the promoter and exon-1 regions, as well as increased hypermethylation of intragenic fragments when compared with SMs. Significant differential methylation was validated in the CpG islands of 15 genes (P<0.05) and confirmed using bisulfite sequencing. These genes included BMP3, BNIP3, CDKN2A, ECEL1, ELK1, GFRA1, HOXD10, KCNH1, PSMD10, PTPRT, SIGIRR, SRF, TBX5, TFPI2, and ZNF382. Methylation changes of GFRA1, SRF, and ZNF382 resulted in up- or downregulation of their transcription. Most importantly, the prevalence of GFRA1, SRF, and ZNF382 methylation alterations was consistently and coordinately associated with gastric carcinoma metastasis and the patients' overall survival throughout discovery and validation cohorts in China, Japan, and Korea. CONCLUSION Methylation changes of GFRA1, SRF, and ZNF382 may be a potential biomarker set for prediction of gastric carcinoma metastasis.
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Affiliation(s)
- Zhaojun Liu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu, Beijing, China
| | - Jun Zhang
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu, Beijing, China. Shihezi University School of Medicine, Shihezi, China
| | - Yanhong Gao
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu, Beijing, China
| | - Lirong Pei
- GRU Cancer Center, Georgia Regents University, Augusta, Georgia
| | - Jing Zhou
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu, Beijing, China
| | - Liankun Gu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu, Beijing, China
| | - Lianhai Zhang
- Department of Surgery, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu, Beijing, China
| | - Budong Zhu
- Department of Oncology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu, Beijing, China
| | - Naoko Hattori
- Division of Epigenetics, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan
| | - Jiafu Ji
- Department of Surgery, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu, Beijing, China
| | - Yasuhito Yuasa
- Department of Molecular Oncology, Tokyo Medical and Dental University, Bunkyo-ku, Tokyo, Japan
| | - Wooho Kim
- Department of Pathology, Seoul National University College of Medicine, Jongno-gu, Seoul, Korea
| | - Toshikazu Ushijima
- Division of Epigenetics, National Cancer Center Research Institute, Chuo-ku, Tokyo, Japan
| | - Huidong Shi
- GRU Cancer Center, Georgia Regents University, Augusta, Georgia.
| | - Dajun Deng
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu, Beijing, China.
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16
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Yang C, Gu L, Deng D. Distinct susceptibility of induction of methylation of p16ink4a and p19arf CpG islands by X-radiation and chemical carcinogen in mice. MUTATION RESEARCH-GENETIC TOXICOLOGY AND ENVIRONMENTAL MUTAGENESIS 2014; 768:42-50. [DOI: 10.1016/j.mrgentox.2014.04.012] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Received: 03/04/2014] [Revised: 04/17/2014] [Accepted: 04/19/2014] [Indexed: 11/26/2022]
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Qin S, Li Q, Zhou J, Liu ZJ, Su N, Wilson J, Lu ZM, Deng D. Homeostatic maintenance of allele-specific p16 methylation in cancer cells accompanied by dynamic focal methylation and hydroxymethylation. PLoS One 2014; 9:e97785. [PMID: 24828678 PMCID: PMC4020935 DOI: 10.1371/journal.pone.0097785] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2014] [Accepted: 04/22/2014] [Indexed: 12/16/2022] Open
Abstract
AIM p16 Methylation frequently occurs in carcinogenesis. While it has been hypothesized that the p16 methylation states are dynamically maintained in cancer cells, direct evidence supporting this hypothesis has not been available until now. METHODS A fusion cell model was established which reprogrammed the native DNA methylation pattern of the cells. The methylation status of the p16 alleles was then repeatedly quantitatively analyzed in the fusion monoclonal, parental cancer cell lines (p16-completely methylated-AGS and unmethylated-MGC803), and HCT116 non-fusion cell using DHPLC and bisulfite sequencing. Histone methylation was analyzed using chromatin immuno-precipitation (ChIP)-PCR. P16 expression status was determined using immuno-staining and RT-PCR. RESULTS The methylation status for the majority of the p16 alleles was stably maintained in the fusion monoclonal cells after up to 60 passages. Most importantly, focal de novo methylation, demethylation, and hydroxymethylation were consistently observed within about 27% of the p16 alleles in the fusion monoclones, but not the homozygously methylated or unmethylated parental cells. Furthermore, subclones of the monoclones consistently maintained the same p16 methylation pattern. A similar phenomenon was also observed using the p16 hemi-methylated HCT116 non-fusion cancer cell line. Interestingly, transcription was not observed in p16 alleles that were hydroxymethylated with an antisense-strand-specific pattern. Also, the levels of H3K9 and H3K4 trimethylation in the fusion cells were found to be slightly lower than the parental AGS and MGC803 cells, respectively. CONCLUSION The present study provides the first direct evidence confirming that the methylation states of p16 CpG islands is not only homeostatically maintained, but also accompanied by a dynamic process of transient focal methylation, demethylation, and hydroxymethylation in cancer cells.
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Affiliation(s)
- Sisi Qin
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Cancer Etiology, Peking University Cancer Hospital & Institute, Beijing, China
| | - Qiang Li
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Cancer Etiology, Peking University Cancer Hospital & Institute, Beijing, China
| | - Jing Zhou
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Cancer Etiology, Peking University Cancer Hospital & Institute, Beijing, China
| | - Zhao-jun Liu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Cancer Etiology, Peking University Cancer Hospital & Institute, Beijing, China
| | - Na Su
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Cancer Etiology, Peking University Cancer Hospital & Institute, Beijing, China
| | - James Wilson
- GRU Cancer Center, Georgia Regents University, Augusta, Georgia, United States of America
| | - Zhe-ming Lu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Cancer Etiology, Peking University Cancer Hospital & Institute, Beijing, China
- * E-mail: (ZML); (DD)
| | - Dajun Deng
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Division of Cancer Etiology, Peking University Cancer Hospital & Institute, Beijing, China
- * E-mail: (ZML); (DD)
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18
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Hegde M, Ferber M, Mao R, Samowitz W, Ganguly A. ACMG technical standards and guidelines for genetic testing for inherited colorectal cancer (Lynch syndrome, familial adenomatous polyposis, and MYH-associated polyposis). Genet Med 2013; 16:101-16. [PMID: 24310308 DOI: 10.1038/gim.2013.166] [Citation(s) in RCA: 110] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/17/2013] [Accepted: 09/17/2013] [Indexed: 12/28/2022] Open
Abstract
Lynch syndrome, familial adenomatous polyposis, and Mut Y homolog (MYH)-associated polyposis are three major known types of inherited colorectal cancer, which accounts for up to 5% of all colon cancer cases. Lynch syndrome is most frequently caused by mutations in the mismatch repair genes MLH1, MSH2, MSH6, and PMS2 and is inherited in an autosomal dominant manner. Familial adenomatous polyposis is manifested as colonic polyposis caused by mutations in the APC gene and is also inherited in an autosomal dominant manner. Finally, MYH-associated polyposis is caused by mutations in the MUTYH gene and is inherited in an autosomal recessive manner but may or may not be associated with polyps. There are variants of both familial adenomatous polyposis (Gardner syndrome--with extracolonic features--and Turcot syndrome, which features medulloblastoma) and Lynch syndrome (Muir-Torre syndrome features sebaceous skin carcinomas, and Turcot syndrome features glioblastomas). Although a clinical diagnosis of familial adenomatous polyposis can be made using colonoscopy, genetic testing is needed to inform at-risk relatives. Because of the overlapping phenotypes between attenuated familial adenomatous polyposis, MYH-associated polyposis, and Lynch syndrome, genetic testing is needed to distinguish among these conditions. This distinction is important, especially for women with Lynch syndrome, who are at increased risk for gynecological cancers. Clinical testing for these genes has progressed rapidly in the past few years with advances in technologies and the lower cost of reagents, especially for sequencing. To assist clinical laboratories in developing and validating testing for this group of inherited colorectal cancers, the American College of Medical Genetics and Genomics has developed the following technical standards and guidelines. An algorithm for testing is also proposed.
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Affiliation(s)
- Madhuri Hegde
- Department of Human Genetics, Emory University School of Medicine, Atlanta, Georgia, USA
| | | | - Rong Mao
- Mayo Clinic, Salt Lake City, Utah, USA
| | | | - Arupa Ganguly
- University of Pennsylvania, Philadelphia, Pennsylvania, USA
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Deng D, Lu Z. Differentiation and adaptation epigenetic networks: Translational research in gastric carcinogenesis. CHINESE SCIENCE BULLETIN-CHINESE 2012. [DOI: 10.1007/s11434-012-5578-0] [Citation(s) in RCA: 10] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023]
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Zhang H, Shan L, Wang X, Ma Q, Fang J. A novel bisulfite-microfluidic temperature gradient capillary electrophoresis platform for highly sensitive detection of gene promoter methylation. Biosens Bioelectron 2012; 42:503-11. [PMID: 23246658 DOI: 10.1016/j.bios.2012.10.013] [Citation(s) in RCA: 15] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/20/2012] [Revised: 10/02/2012] [Accepted: 10/03/2012] [Indexed: 01/30/2023]
Abstract
The hypermethylated tumor suppressor gene promoters are widely recognized as promising markers for cancer screening and ideal targets for cancer therapy, however, a major obstacle in their clinical study is highly sensitive screening. To address this limitation, we developed a novel bisulfite-microfluidic temperature gradient capillary electrophoresis (bisulfite-μTGCE) platform for gene methylation analysis by combining bisulfite treatment and slantwise radiative heating system-based μTGCE. Bisulfite-treated genomic DNA (gDNA) was amplified with universal primers for both methylated and unmethylated sequences, and introduced into glass microfluidic chip to perform electrophorectic separation under a continuous temperature gradient based on the formation of heteroduplexes. Eight CDKN2A promoter model fragments with different number and location of methylation sites were prepared and successfully analyzed according to their electrophoretic peak patterns, with high stability, picoliter-scale sample consumption, and significantly increased detection speed. The bisulfite-μTGCE could detect methylated gDNA with a detection limit of 7.5pg, and could distinguish as low as 0.1% methylation level in CDKN2A in an unmethylated background. Detection of seven colorectal cancer (CRC) cell lines with known and unknown methylation statuses of CDKN2A promoter and 20 tumor tissues derived from CRC patients demonstrated the capability of detecting hypermethylation in real-world samples. The wider adaptation of this platform was further supported by the detection of the CDKN2A and MLH1 promoters' methylation statuses in combination. This highly sensitive, fast, and low-consumption platform for methylation detection shows great potential for future clinical applications.
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Affiliation(s)
- Huidan Zhang
- Department of Cell Biology, Key Laboratory of Cell Biology, Ministry of Public Health, and Key Laboratory of Medical Cell Biology, Ministry of Education, China Medical University, 92 Beier Road, Heping District, Shenyang, Liaoning 110001, PR China
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Hutzler M, Geiger E, Jacob F. Use of PCR-DHPLC (Polymerase Chain Reaction-Denaturing High Performance Liquid Chromatography) for the Rapid Differentiation of IndustrialSaccharomyces pastorianusandSaccharomyces cerevisiaeStrains. JOURNAL OF THE INSTITUTE OF BREWING 2012. [DOI: 10.1002/j.2050-0416.2010.tb00798.x] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 11/06/2022]
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Du Y, Liu Z, Gu L, Zhou J, Zhu BD, Ji J, Deng D. Characterization of human gastric carcinoma-related methylation of 9 miR CpG islands and repression of their expressions in vitro and in vivo. BMC Cancer 2012; 12:249. [PMID: 22703336 PMCID: PMC3517451 DOI: 10.1186/1471-2407-12-249] [Citation(s) in RCA: 26] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2012] [Accepted: 05/04/2012] [Indexed: 12/14/2022] Open
Abstract
Background Many miR genes are located within or around CpG islands. It is unclear whether methylation of these CpG islands represses miR transcription regularly. The aims of this study are to characterize gastric carcinoma (GC)-related methylation of miR CpG islands and its relationship with miRNA expression. Methods Methylation status of 9 representative miR CpG islands in a panel of cell lines and human gastric samples (including 13 normal biopsies, 38 gastritis biopsies, 112 pairs of GCs and their surgical margin samples) was analyzed by bisulfite-DHPLC and sequencing. Mature miRNA levels were determined with quantitative RT-PCR. Relationships between miR methylation, transcription, GC development, and clinicopathological characteristics were statistically analyzed. Results Methylation frequency of 5 miR CpG islands (miR-9-1, miR-9-3, miR-137, miR-34b, and miR-210) gradually increased while the proportion of methylated miR-200b gradually decreased during gastric carcinogenesis (Ps < 0.01). More miR-9-1 methylation was detected in 62%-64% of the GC samples and 4% of the normal or gastritis samples (18/28 versus 2/48; Odds ratio, 41.4; P < 0.01). miR-210 methylation showed high correlation with H. pylori infection. miR-375, miR-203, and miR-193b methylation might be host adaptation to the development of GCs. Methylation of these miR CpG islands was consistently shown to significantly decrease the corresponding miRNA levels presented in human cell lines. The inverse relationship was also observed for miR-9-1, miR-9-3, miR-137, and miR-200b in gastric samples. Among 112 GC patients, miR-9-1 methylation was an independent favourable predictor of overall survival of GC patients in both univariate and multivariate analysis (P < 0.02). Conclusions In conclusion, alteration of methylation status of 6 of 9 tested miR CpG islands was characterized in gastric carcinogenesis. miR-210 methylation correlated with H. pylori infection. miR-9-1 methylation may be a GC-specific event. Methylation of miR CpG islands may significantly down-regulate their transcription regularly.
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Affiliation(s)
- Yantao Du
- Key Laboratory of Carcinogenesis and Translational Research, Ministry of Education, Division of Cancer Etiology, Peking University Cancer Hospital and Institute, Fu-Cheng-Lu, No,52 Haidian District, Beijing 100142, China.
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Mikeska T, Candiloro ILM, Dobrovic A. The implications of heterogeneous DNA methylation for the accurate quantification of methylation. Epigenomics 2012; 2:561-73. [PMID: 22121974 DOI: 10.2217/epi.10.32] [Citation(s) in RCA: 114] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/11/2022] Open
Abstract
DNA methylation based biomarkers have considerable potential for molecular diagnostics, both as tumor specific biomarkers for the early detection or post-therapeutic monitoring of cancer as well as prognostic and predictive biomarkers for therapeutic stratification. Particularly in the former, the accurate estimation of DNA methylation is of compelling importance. However, quantification of DNA methylation has many traps for the unwary, especially when heterogeneous methylation comprising multiple alleles with varied DNA methylation patterns (epialleles) is present. The frequent occurrence of heterogeneous methylation as distinct from a simple mixture of fully methylated and unmethylated alleles is generally not taken into account when DNA methylation is considered as a cancer biomarker. When heterogeneous DNA methylation is present, the proportion of methylated molecules is difficult to quantify without a method that allows the measurement of individual epialleles. In this article, we critically assess the methodologies frequently used to investigate DNA methylation, with an emphasis on the detection and measurement of heterogeneous DNA methylation. The adoption of digital approaches will enable the effective use of heterogeneous DNA methylation as a cancer biomarker.
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Affiliation(s)
- Thomas Mikeska
- Molecular Pathology Research & Development Laboratory, Department of Pathology, Peter MacCallum Cancer Centre, Locked Bag 1, A'Beckett Street, Melbourne, Victoria 8006, Australia.
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Rodríguez JL, Pascual J, Viejo M, Valledor L, Meijón M, Hasbún R, Yrei NY, Santamaría ME, Pérez M, Fernández Fraga M, Berdasco M, Rodríguez Fernández R, Cañal MJ. Basic procedures for epigenetic analysis in plant cell and tissue culture. Methods Mol Biol 2012; 877:325-341. [PMID: 22610639 DOI: 10.1007/978-1-61779-818-4_25] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/01/2023]
Abstract
In vitro culture is one of the most studied techniques, and it is used to study many developmental processes, especially in forestry species, because of growth timing and easy manipulation. Epigenetics has been shown as an important influence on many research analyses such as cancer in mammals and developmental processes in plants such as flowering, but regarding in vitro culture, techniques to study DNA methylation or chromatin modifications were mainly limited to identify somaclonal variation of the micropropagated material. Because in vitro culture is not only a way to generate plant material but also a bunch of differentially induced developmental processes, an approach of techniques and some research carried out to study the different changes regarding DNA methylation and chromatin and translational modifications that take place during these processes is reviewed.
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Affiliation(s)
- José L Rodríguez
- Área de Fisiología Vegetal, Departamento BOS, Facultad de Biología, Universidad de Oviedo, Oviedo, Spain.
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25
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Sun LL, Cao DY, Yang JX, Li H, Zhou XR, Song ZQ, Cheng XM, Chen J, Shen K. Population-based case–control study on DAPK1, RAR-β2 and MGMT methylation in liquid-based cytology. Arch Gynecol Obstet 2011; 285:1433-9. [DOI: 10.1007/s00404-011-2149-6] [Citation(s) in RCA: 13] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/24/2011] [Accepted: 11/11/2011] [Indexed: 11/25/2022]
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Ming S, Gao J, Sun T. [The relationship between the TSLC1 silencing and DNA methylation in human lung cancer cells]. ZHONGGUO FEI AI ZA ZHI = CHINESE JOURNAL OF LUNG CANCER 2010; 13:464-9. [PMID: 20677643 PMCID: PMC6000693 DOI: 10.3779/j.issn.1009-3419.2010.05.16] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.1] [Reference Citation Analysis] [Abstract] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Indexed: 11/19/2022]
Abstract
背景与目的 TSLC1在多种肿瘤中表达下调或失活，其表达下调与DNA高甲基化有很大关系。本研究旨在探索TSLC1在肺癌细胞中的表达缺失与其启动子区DNA甲基化的关系。 方法 采用RT-PCR和Real-time PCR方法检测TSLC1在正常肺组织和3种肺癌细胞系（A549、NCI-H446和Calu-3）中的表达谱；运用亚硫酸氢盐修饰后测序（bisulfte sequencing）方法检测上述正常肺组织和肺癌细胞中TSLC1启动子区的甲基化状态；应用甲基化转移酶抑制剂5-氮-2-脱氧胞苷（5-Aza-dC）处理上述细胞后，采用Real-time PCR方法检测处理前后TSLC1的表达变化。 结果 TSLC1在正常肺组织和A549细胞中表达，其启动子区DNA未发生甲基化；而在NCI-H446和Calu-3细胞中表达缺失，其启动子区DNA发生高甲基化，并且5-Aza-dC处理NCI-H446和Calu-3细胞后可促进TSLC1的表达。 结论 TSLC1在肺癌细胞中的表达缺失是由其启动子区的DNA高甲基化引起。
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Affiliation(s)
- Shuhong Ming
- Department of Respiratory Medicine, Beijing Hospital Ministry of Health, Beijing 100730, China.
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Wen XZ, Akiyama Y, Pan KF, Liu ZJ, Lu ZM, Zhou J, Gu LK, Dong CX, Zhu BD, Ji JF, You WC, Deng DJ. Methylation of GATA-4 and GATA-5 and development of sporadic gastric carcinomas. World J Gastroenterol 2010; 16:1201-8. [PMID: 20222162 PMCID: PMC2839171 DOI: 10.3748/wjg.v16.i10.1201] [Citation(s) in RCA: 43] [Impact Index Per Article: 2.9] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To understand the implication of GATA-4 and GATA-5 methylation in gastric carcinogenesis.
METHODS: Methylation status of GATA-4 and GATA-5 CpG islands in human gastric mucosa samples, including normal gastric biopsies from 45 outpatients, gastric dysplasia [low-grade gastric intraepithelial neoplasia (GIN), n = 30; indefinite, n = 77], and 80 paired sporadic gastric carcinomas (SGC) as well as the adjacent non-neoplastic gastric tissues was analyzed by methylation specific polymerase chain reaction (MSP) and confirmed by denatured high performance liquid chromatography (DHPLC). Immunohistochemical staining was used to detect protein expression. The correlation between GATA-4 and GATA-5 methylation and clinicopathological characteristics of patients including Helicobacter pylori (H. pylori) infection was analyzed.
RESULTS: GATA-4 and GATA-5 methylation was frequently observed in SGCs (53.8% and 61.3%, respectively) and their corresponding normal tissues (41.3% and 46.3%) by MSP. The result of MSP was consistent with that of DHPLC. Loss of both GATA-4 and GATA-5 proteins was associated with their methylation in SGCs (P = 0.01). Moreover, a high frequency of GATA-4 and GATA-5 methylation was found in both gastric low-grade GIN (57.1% and 69.0%) and indefinite for dysplasia (42.9% and 46.7%), respectively. However, GATA-4 and GATA-5 methylation was detected only in 4/32 (12.5%) and 3/39 (7.7%) of normal gastric biopsies. GATA-4 methylation in both normal gastric mucosa and low-grade GIN was also significantly associated with H. pylori infection (P = 0.023 and 0.027, two-sides).
CONCLUSION: Epigenetic inactivation of GATA-4 (and GATA-5) by methylation of CpG islands is an early frequent event during gastric carcinogenesis and is significantly correlated with H. pylori infection.
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Xiang S, Liu Z, Zhang B, Zhou J, Zhu BD, Ji J, Deng D. Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas. BMC Cancer 2010; 10:44. [PMID: 20163738 PMCID: PMC2834620 DOI: 10.1186/1471-2407-10-44] [Citation(s) in RCA: 26] [Impact Index Per Article: 1.7] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/28/2009] [Accepted: 02/17/2010] [Indexed: 11/10/2022] Open
Abstract
BACKGROUND Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation. METHODS Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0. RESULTS From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A approximately Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%). CONCLUSIONS Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively.
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Affiliation(s)
- Shengyan Xiang
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Beijing Cancer Hospital and Institute, Peking University School of Oncology, Fu-Cheng-Lu, No.52, Haidian District, Beijing, 100142, China
| | - Zhaojun Liu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Beijing Cancer Hospital and Institute, Peking University School of Oncology, Fu-Cheng-Lu, No.52, Haidian District, Beijing, 100142, China
| | - Baozhen Zhang
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Beijing Cancer Hospital and Institute, Peking University School of Oncology, Fu-Cheng-Lu, No.52, Haidian District, Beijing, 100142, China
| | - Jing Zhou
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Beijing Cancer Hospital and Institute, Peking University School of Oncology, Fu-Cheng-Lu, No.52, Haidian District, Beijing, 100142, China
| | - Bu-Dong Zhu
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Beijing Cancer Hospital and Institute, Peking University School of Oncology, Fu-Cheng-Lu, No.52, Haidian District, Beijing, 100142, China
| | - Jiafu Ji
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Beijing Cancer Hospital and Institute, Peking University School of Oncology, Fu-Cheng-Lu, No.52, Haidian District, Beijing, 100142, China
| | - Dajun Deng
- Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education), Beijing Cancer Hospital and Institute, Peking University School of Oncology, Fu-Cheng-Lu, No.52, Haidian District, Beijing, 100142, China
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Mei M, Deng D, Liu TH, Sang XT, Lu X, Xiang HD, Zhou J, Wu H, Yang Y, Chen J, Lu CM, Chen YJ. Clinical implications of microsatellite instability and MLH1 gene inactivation in sporadic insulinomas. J Clin Endocrinol Metab 2009; 94:3448-57. [PMID: 19567531 DOI: 10.1210/jc.2009-0173] [Citation(s) in RCA: 41] [Impact Index Per Article: 2.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/07/2023]
Abstract
CONTEXT The molecular pathogenesis of sporadic insulinomas is unknown. There is a lack of biomarker to distinguish benign and malignant form of insulinoma. OBJECTIVE Our objective was to confirm the occurrence of microsatellite instability (MSI) in insulinomas, to identify alterations of mismatch repair (MMR) genes in the tumors, and to evaluate the possibility to distinguish benign and malignant insulinoma or to predict the clinical outcome of patients with these alterations. DESIGN AND PATIENTS We detected MSI and inactivation of MLH1 gene in 55 sporadic insulinomas by PCR, immunohistochemical staining, allelic typing, analysis of promoter methylation, and exon mutations. Their correlations with clinicopathological characteristics were analyzed with univariate and multivariate statistic analysis. RESULTS A high rate of MSI (MSI-H) was found in 33% of sporadic insulinomas. Reduced expression of mutL homolog 1 (MLH1) protein was observed in 36% of insulinomas and correlated with MSI-H (P = 0.008). Promoter methylation and loss of heterozygosity of MLH1 gene was found in 31 and 49% of insulinomas, respectively. Reduced expression of MLH1 and MSI-H were significantly associated with both tumor malignancy (P = 0.033 and P = 4.8 x 10(-6), respectively) and incurable disease (P = 0.006 and P = 0.001, respectively). CONCLUSION High frequency of MSI occurred in sporadic insulinomas. The silencing of MLH1 gene may partially contribute to the MSI-H in the tumors. Assessing MSI-H and expressions of MLH1 could be used to distinguish benign and malignant insulinomas and to predict the outcome of patients.
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Affiliation(s)
- Mei Mei
- Department of Gastroenterology, Peking Union Medical College Hospital, Peking Union Medical College, Chinese Academy of Medical Sciences, Beijing 100730, China
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Gao J, Chen T, Liu J, Liu W, Hu G, Guo X, Yin B, Gong Y, Zhao J, Qiang B, Yuan J, Peng X. Loss of NECL1, a novel tumor suppressor, can be restored in glioma by HDAC inhibitor-Trichostatin A through Sp1 binding site. Glia 2009; 57:989-99. [PMID: 19062177 DOI: 10.1002/glia.20823] [Citation(s) in RCA: 29] [Impact Index Per Article: 1.8] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/08/2023]
Abstract
Nectin-like molecule 1 (NECL1)/CADM3/IGSF4B/TSLL1/SynCAM3 is a neural tissue-specific immunoglobulin-like cell-cell adhesion molecule downregulated at the mRNA level in 12 human glioma cell lines. Here we found that the expression of NECL1 was lost in six glioma cell lines and 15 primary glioma tissues at both RNA and protein levels. Re-expression of NECL1 into glioma cell line U251 would repress cell proliferation in vitro by inducing cell cycle arrest. And also NECL1 could decrease the growth rate of tumors in nude mice in vivo. To further investigate the mechanism why NECL1 was silenced in glioma, the basic promoter region located at -271 to +81 in NECL1 genomic sequence was determined. DNA bisulfite sequencing was performed to study the methylation status of CpG islands in NECL1 promoter; however, no hypermethylated CpG site was found. Additionally, the activity of histone deacetylase (HDACs) in glioma was higher than that in normal brain tissues, and the expression of NECL1 in glioma cell lines could be reactivated by HDACs inhibitor-Trichostatin A (TSA). So the loss of NECL1 in glioma was at least partly caused by histone deacetylation. Luciferase reporter assays, chromatin immunoprecipitation and co-immunoprecipitation (co-IP) assays indicated that Sp1 played an important role in this process by binding to either HDAC1 in untreated glioma cells or p300/CBP in TSA treated cells. Our finding suggests that NECL1 may act as a tumor suppressor in glioma and loss of it in glioma may be caused by histone deacetylation.
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Affiliation(s)
- Jing Gao
- National Laboratory of Medical Molecular Biology, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences and Peking Union Medical College, National Human Genome Center, Beijing 100005, China
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31
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Sepulveda AR, Jones D, Ogino S, Samowitz W, Gulley ML, Edwards R, Levenson V, Pratt VM, Yang B, Nafa K, Yan L, Vitazka P. CpG methylation analysis--current status of clinical assays and potential applications in molecular diagnostics: a report of the Association for Molecular Pathology. J Mol Diagn 2009; 11:266-78. [PMID: 19541921 DOI: 10.2353/jmoldx.2009.080125] [Citation(s) in RCA: 40] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/27/2023] Open
Abstract
Methylation of CpG islands in gene promoter regions is a major molecular mechanism of gene silencing and underlies both cancer development and progression. In molecular oncology, testing for the CpG methylation of tissue DNA has emerged as a clinically useful tool for tumor detection, outcome prediction, and treatment selection, as well as for assessing the efficacy of treatment with the use of demethylating agents and monitoring for tumor recurrence. In addition, because CpG methylation occurs early in pre-neoplastic tissues, methylation tests may be useful as markers of cancer risk in patients with either infectious or inflammatory conditions. The Methylation Working Group of the Clinical Practice Committee of the Association of Molecular Pathology has reviewed the current state of clinical testing in this area. We report here our summary of both the advantages and disadvantages of various methods, as well as the needs for standardization and reporting. We then conclude by summarizing the most promising areas for future clinical testing in cancer molecular diagnostics.
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Affiliation(s)
- Antonia R Sepulveda
- Methylation Working Group of the Association for Molecular Pathology Clinical Practice Committee, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania, USA
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32
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Huang KH, Huang SF, Chen IH, Liao CT, Wang HM, Hsieh LL. Methylation of RASSF1A, RASSF2A, and HIN-1 Is Associated with Poor Outcome after Radiotherapy, but not Surgery, in Oral Squamous Cell Carcinoma. Clin Cancer Res 2009; 15:4174-80. [DOI: 10.1158/1078-0432.ccr-08-2929] [Citation(s) in RCA: 57] [Impact Index Per Article: 3.6] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
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Hung CC, Lin SY, Lin SP, Niu DM, Lee NC, Cheng WF, Chen CP, Lin WL, Lee CN, Su YN. Identification of CpG methylation of the SNRPN gene by methylation-specific multiplex PCR. Electrophoresis 2009; 30:410-6. [PMID: 19137525 DOI: 10.1002/elps.200800225] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022]
Abstract
In this article, we show that methylation-specific multiplex PCR (MS-multiplex PCR) is a sensitive and specific single assay for detecting CpG methylation status as well as copy number aberrations. We used MS-multiplex PCR to simultaneously amplify three sequences: the 3' ends of the SNRPN gene (for unmethylated sequences), the KRITI gene (as internal control), and the promoter of the SNRPN gene containing CpG islands (for methylated sequences) after digestion with a methylation-sensitive restriction enzyme (HhaI). We established this duplex assay for the analysis of 38 individuals with Prader-Willi syndrome, 2 individuals with Angelman syndrome, and 28 unaffected individuals. By comparing the copy number of the three regions, the methylation status and the copy number changes can be easily distinguished by MS-multiplex PCR without the need of bisulfite treatment of the DNA. The data showed that MS-multiplex PCR allows for the estimation of the methylation level by comparing the copy number aberrations of unknown samples to the standards with a known methylated status. The in-house-designed MS-multiplex PCR protocol is a relatively simple, cost-effective, and highly reproducible approach as a significant strategy in clinical applications for epigenetics in a routine laboratory.
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Affiliation(s)
- Chia-Cheng Hung
- Institute of Biomedical Engineering, National Taiwan University, Taipei, Taiwan
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Chou LS, Lyon E, Mao R. Molecular diagnosis utility of multiplex ligation-dependent probe amplification. ACTA ACUST UNITED AC 2008; 2:373-85. [DOI: 10.1517/17530059.2.4.373] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/14/2022]
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35
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Hattermann K, Mehdorn HM, Mentlein R, Schultka S, Held-Feindt J. A methylation-specific and SYBR-green-based quantitative polymerase chain reaction technique for O6-methylguanine DNA methyltransferase promoter methylation analysis. Anal Biochem 2008; 377:62-71. [PMID: 18384736 DOI: 10.1016/j.ab.2008.03.014] [Citation(s) in RCA: 47] [Impact Index Per Article: 2.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2007] [Revised: 03/05/2008] [Accepted: 03/07/2008] [Indexed: 01/28/2023]
Abstract
The O(6)-methylguanine DNA methyltransferase (MGMT) gene encodes a DNA repair enzyme whose activity is a major mechanism of resistance to alkylating drugs in glioblastoma treatment. Hypermethylation of the MGMT promoter is associated with chemosensitivity because it reduces MGMT activity. Here we present a method combining methylation-specific and SYBR-green-based quantitative PCR (MSQP) for MGMT promoter methylation analysis. This highly specific, sensitive, and reproducible method allows the quantification of fully methylated and fully unmethylated MGMT DNA species in terms of percentage. Values are related to standard curves, corrected for DNA input by an internal standard, and calculated in relation to methylated and unmethylated control DNAs as a percentage share. Finally, values are defined relative to the sum of fully methylated and unmethylated MGMT DNA sample amount to obtain percentage of methylated reference and percentage of unmethylated reference results. We have used this technique to investigate MGMT promoter methylation in relation to MGMT mRNA expression in nine tumor cell lines and 15 primary glioblastoma patients. Presented data confirm that this assay is suitable for detection of low amounts of methylated and unmethylated MGMT promoter DNA. Carefully validated quantitative MSQP assays will be useful in both research and clinical molecular diagnosis.
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Affiliation(s)
- Kirsten Hattermann
- Department of Anatomy, University of Kiel, Olshausenstr. 40, 24098 Kiel, Germany
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36
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Dikow N, Nygren AO, Schouten JP, Hartmann C, Krämer N, Janssen B, Zschocke J. Quantification of the methylation status of the PWS/AS imprinted region: Comparison of two approaches based on bisulfite sequencing and methylation-sensitive MLPA. Mol Cell Probes 2007; 21:208-15. [PMID: 17303379 DOI: 10.1016/j.mcp.2006.12.002] [Citation(s) in RCA: 37] [Impact Index Per Article: 2.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/03/2006] [Revised: 11/23/2006] [Accepted: 12/21/2006] [Indexed: 12/13/2022]
Abstract
Standard methods used for genomic methylation analysis allow the detection of complete absence of either methylated or non-methylated alleles but are usually unable to detect changes in the proportion of methylated and unmethylated alleles. We compare two methods for quantitative methylation analysis, using the chromosome 15q11-q13 imprinted region as model. Absence of the non-methylated paternal allele in this region leads to Prader-Willi syndrome (PWS) whilst absence of the methylated maternal allele results in Angelman syndrome (AS). A proportion of AS is caused by mosaic imprinting defects which may be missed with standard methods and require quantitative analysis for their detection. Sequence-based quantitative methylation analysis (SeQMA) involves quantitative comparison of peaks generated through sequencing reactions after bisulfite treatment. It is simple, cost-effective and can be easily established for a large number of genes. However, our results support previous suggestions that methods based on bisulfite treatment may be problematic for exact quantification of methylation status. Methylation-specific multiplex ligation-dependent probe amplification (MS-MLPA) avoids bisulfite treatment. It detects changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in one simple reaction. Once established in a laboratory setting, the method is more accurate, reliable and less time consuming.
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Affiliation(s)
- Nicola Dikow
- Institute of Human Genetics, University of Heidelberg, Im Neuenheimer Feld 366, D-69120 Heidelberg, Amsterdam, The Netherlands
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37
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Edelman EJ, Maksimova Y, Duru F, Altay C, Gallagher PG. A complex splicing defect associated with homozygous ankyrin-deficient hereditary spherocytosis. Blood 2007; 109:5491-3. [PMID: 17327413 PMCID: PMC1890827 DOI: 10.1182/blood-2006-09-046573] [Citation(s) in RCA: 9] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/02/2023] Open
Abstract
Defects in erythrocyte ankyrin are the most common cause of typical, dominant hereditary spherocytosis (HS). Detection of ankyrin gene mutations has been complicated by allelic heterogeneity, large gene size, frequent de novo mutations, and associated mRNA instability. Using denaturing high-performance liquid chromatography (DHPLC)-based mutation detection, a mutation in the splice acceptor of exon 17 was discovered in a Turkish family. Reticulocyte RNA and functional minigene splicing assays in heterologous cells revealed that this mutation was associated with a complex pattern of aberrant splicing, suggesting that removal of intron 16 is important for ordered ankyrin mRNA splicing. As predicted by clinical, laboratory, and biochemical studies, the parents were heterozygous and the proband was homozygous for this mutation. These data indicate that DHPLC offers a highly sensitive, economic, and rapid method for mutation detection and, unlike previously suggested, homozygosity for a mutation associated with dominant ankyrin-linked HS may be compatible with life.
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Affiliation(s)
- E Jennifer Edelman
- Department of Pediatrics, Yale University School of Medicine, New Haven, CT 06520-8064, USA
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38
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Deligezer U, Esin Akisik E, Dalay N. A novel application of melting curves: utility of peak area calculation for relative methylation quantification. ACTA ACUST UNITED AC 2007; 45:867-73. [PMID: 17617029 DOI: 10.1515/cclm.2007.146] [Citation(s) in RCA: 6] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/15/2022]
Abstract
AbstractClin Chem Lab Med 2007;45:867–73.
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Affiliation(s)
- Ugur Deligezer
- Department of Basic Oncology, Oncology Institute, Istanbul University, Istanbul, Turkey
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Judson H, Stewart A, Leslie A, Pratt NR, Baty DU, Steele RJC, Carey FA. Relationship between point gene mutation, chromosomal abnormality, and tumour suppressor gene methylation status in colorectal adenomas. J Pathol 2006; 210:344-50. [PMID: 16902913 DOI: 10.1002/path.2044] [Citation(s) in RCA: 18] [Impact Index Per Article: 0.9] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/19/2023]
Abstract
Epigenetic mechanisms in carcinogenesis may have a significant role in the development of colorectal cancer. To investigate this phenomenon in early-stage disease, promoter methylation status in the tumour suppressor genes APC, MGMT, hMLH1, P14/P14ARF, and CDKN2A/P16 was investigated in 78 colorectal adenomas. These had previously been characterized for mutations of APC, KRAS, and TP53 genes and for chromosomal abnormality by comparative genomic hybridization (CGH). APC hypermethylation was seen in 52 tumours (66.7%). APC showed either methylation or mutation in 66 lesions (84.6%), but these events were not statistically associated. MGMT methylation was detected in 39 cases (50%). Adenomas with this abnormality showed a significantly lower number of chromosomal changes by CGH (p < 0.02), confirming that DNA repair defect of this type is associated with a lower level of chromosomal instability. An hMLH1 methylation defect was seen in only one adenoma (1.3%), from a patient who had a synchronous cancer showing the same defect. Methylation of P14 (P14ARF) was seen in 31 adenomas (39.7%) and CDKN2A (P16) abnormality in 25 (32.1%). DNA methylation at two or more loci was seen in 46 tumours (59%), while 11 lesions (14.1%) showed no evidence of hypermethylation at any of the loci studied. Methylation at any or all of MGMT, P14 or P16 was significantly associated with APC methylation (p = 0.01). Those neoplasms with more than two methylated genes showed significantly fewer chromosomal abnormalities than adenomas with one or no methylated loci (p < 0.001). There was no association between specific individual chromosomal abnormalities, APC, KRAS or TP53 mutations and any pattern of methylation abnormality. We conclude that methylation abnormality is very common in pre-invasive colorectal neoplasia, and that high level methylation is associated with low level chromosomal instability.
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Affiliation(s)
- H Judson
- Department of Human Genetics, Ninewells Hospital and University of Dundee, Dundee, UK
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40
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Ogino S, Kawasaki T, Brahmandam M, Cantor M, Kirkner GJ, Spiegelman D, Makrigiorgos GM, Weisenberger DJ, Laird PW, Loda M, Fuchs CS. Precision and performance characteristics of bisulfite conversion and real-time PCR (MethyLight) for quantitative DNA methylation analysis. J Mol Diagn 2006; 8:209-17. [PMID: 16645207 PMCID: PMC1867588 DOI: 10.2353/jmoldx.2006.050135] [Citation(s) in RCA: 358] [Impact Index Per Article: 18.8] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/30/2022] Open
Abstract
Assays to measure DNA methylation, which are important in epigenetic research and clinical diagnostics, typically rely on conversion of unmethylated cytosine to uracil by sodium bisulfite. However, no study has comprehensively evaluated the precision and performance characteristics of sodium bisulfite conversion and subsequent quantitative methylation assay. We developed quantitative real-time polymerase chain reaction (MethyLight) to measure percentage of methylated reference (PMR, ie, degree of methylation) for the MGMT, MLH1, and CDKN2A (p16) promoters. To measure the precision of bisulfite conversion, we bisulfite-treated seven different aliquots of DNA from each of four paraffin-embedded colon cancer samples. To assess run-to-run variation, we repeated MethyLight five times. Bisulfite-to-bisulfite coefficient of variation (CV) of PMR ranged from 0.10 to 0.38 (mean, 0.21), and run-to-run CV of PMR ranged from 0.046 to 0.60 (mean, 0.31). Interclass correlation coefficients were 0.74 to 0.84 for the three loci, indicating good reproducibility. DNA mixing study with methylated and unmethylated DNA showed good linearity of the assay. Of 272 colorectal cancers evaluated, most showed PMR either <1 or >10, and promoter methylation (PMR >4) was tightly associated with loss of respective protein expression (P < 10(-16)). In conclusion, sodium bisulfite conversion and quantitative MethyLight assays have good precision and linearity and can be effectively used for high-throughput DNA methylation analysis on paraffin-embedded tissue.
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Affiliation(s)
- Shuji Ogino
- Department of Pathology, Brigham and Women's Hospital, Harvard Medical School, 75 Francis St., Boston, MA 02115, USA.
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41
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Luo D, Zhang B, Lv L, Xiang S, Liu Y, Ji J, Deng D. Methylation of CpG islands of p16 associated with progression of primary gastric carcinomas. J Transl Med 2006; 86:591-8. [PMID: 16534497 DOI: 10.1038/labinvest.3700415] [Citation(s) in RCA: 48] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/31/2022] Open
Abstract
Inactivation of p16 by methylation of CpG islands is a frequent early event in gastric carcinogenesis. The positive relationship between p16 methylation and the clinical characteristics of gastric carcinomas (GC) has not been reported to date. In the present study, a DHPLC assay to quantify p16 methylation was established (detection limit by fluorescence detector: 1:255 (Methlyated vs Unmethylated)). The proportion of methylated p16 in the representative samples was confirmed and standardized by clone sequencing. Then, the DHPLC and two regular methylation-specific PCR (MSP) assays were used to detect p16 methylation in 82 paired, resected GCs and their adjacent normal tissues. Results showed that the average proportion of methylated p16 in GCs was significantly higher than that in their adjacent samples (12.90 vs 0.63%; t-test P=0.005). A much higher proportion of methylated p16 was detected in GCs with metastases (local or distant) than without metastases (14.76 vs 2.61%; t-test P=0.014). A proportional relationship was observed between clinical stages and positive rates of p16 methylation in GCs and/or adjacent tissues: 27.3, 37.5, and 58.8% (by DHPLC) for stage-I, -II, -III-IV of GCs, respectively (two-sided Fisher's exact test P=0.016). To confirm the data obtained by DHPLC, two MSP primer sets (p16-M and p16-M2) were also used to analyze p16 methylation in the same set of samples simultaneously. Data of MSP assay using the primer set p16-M2, but not p16-M, correlated with that of DHPLC. These results imply that the primer set p16-M2 might be more suitable than p16-M to detect p16 methylation in gastric tissues. In conclusion, the present data indicates that p16 methylation correlates with progression of GCs significantly.
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Affiliation(s)
- Daya Luo
- Peking University School of Oncology, Beijing Cancer Hospital and Beijing Institute for Cancer Research, Beijing, China
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42
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Nygren AOH, Ameziane N, Duarte HMB, Vijzelaar RNCP, Waisfisz Q, Hess CJ, Schouten JP, Errami A. Methylation-specific MLPA (MS-MLPA): simultaneous detection of CpG methylation and copy number changes of up to 40 sequences. Nucleic Acids Res 2005; 33:e128. [PMID: 16106041 PMCID: PMC1187824 DOI: 10.1093/nar/gni127] [Citation(s) in RCA: 280] [Impact Index Per Article: 14.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
Copy number changes and CpG methylation of various genes are hallmarks of tumor development but are not yet widely used in diagnostic settings. The recently developed multiplex ligation-dependent probe amplification (MLPA) method has increased the possibilities for multiplex detection of gene copy number aberrations in a routine laboratory. Here we describe a novel robust method: the methylation-specific MLPA (MS-MLPA) that can detect changes in both CpG methylation as well as copy number of up to 40 chromosomal sequences in a simple reaction. In MS-MLPA, the ligation of MLPA probe oligonucleotides is combined with digestion of the genomic DNA–probe hybrid complexes with methylation-sensitive endonucleases. Digestion of the genomic DNA–probe complex, rather than double-stranded genomic DNA, allowed the use of DNA derived from the formalin treated paraffin-embedded tissue samples, enabling retrospective studies. To validate this novel method, we used MS-MLPA to detect aberrant methylation in DNA samples of patients with Prader–Willy syndrome, Angelman syndrome or acute myeloid leukemia.
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Affiliation(s)
- Anders O. H. Nygren
- MRC-Holland bvAmsterdam, The Netherlands
- Department of Clinical and Human Genetics, VU University Medical CenterAmsterdam, The Netherlands
| | - Najim Ameziane
- Department of Clinical and Human Genetics, VU University Medical CenterAmsterdam, The Netherlands
| | | | | | - Quinten Waisfisz
- Department of Hematology, VU University Medical CenterAmsterdam, The Netherlands
| | - Corine J. Hess
- Department of Hematology, VU University Medical CenterAmsterdam, The Netherlands
| | | | - Abdellatif Errami
- MRC-Holland bvAmsterdam, The Netherlands
- To whom correspondence should be addressed. Tel: +31 20 4891248; Fax: +31 20 6891149;
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43
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Shen J, Zhang JZ, Ke Y, Deng D. Formation of A2143G mutation of 23S rRNA in progression of clarithromycin resistance in Helicobacter pylori 26695. Microb Drug Resist 2005; 11:100-6. [PMID: 15910222 DOI: 10.1089/mdr.2005.11.100] [Citation(s) in RCA: 5] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
The aim of this work was to investigate the link between 23S rRNA mutation and clarithromycin (CLR)-resistant Helicobacter pylori (H. pylori). CLR-resistant (CLRr) H. pylori strains were selected from the parent strain, H. pylori 26695, using medium containing CLR. Point mutations of 23S rRNA were analyzed by denaturing high-performance liquid chromatography and sequencing and restriction fragment length polymorphism (RFLP). Protein profiles of these strains were obtained by surface-enhanced laser/desorption ionization time-of-flight mass spectrometry technology. Two phenotype-stable L-CLRr resistant strains (MIC 8, 10 microg/ml) and two subsequent CLRr strains (MIC 32 microg/ml) were obtained. An A2143G mutation of 23S rRNA was detectable in two CLRr strains, but in neither the CLRs parent strain nor the L-CLRr strains. Four clinical CLRr H. pylori strains were obtained from 41 Chinese H. pylori isolates. The A2143G mutation was observed in all four CLRr isolates, but not in the six analyzed CLRs ones. Protein profiling showed that the pattern of protein expression was changed from the CLRs parent strain to the L-CLRr strains and then to the CLRr strains progressively. The formation of A2143G mutations of 23S rRNA might be a late event in the development of CLR-resistance of H. pylori 26695. Early events related to alteration of the pattern of protein expression might be involved in the development of CLR-resistance too.
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Affiliation(s)
- Jing Shen
- Peking University School of Oncology and Beijing Institute for Cancer Research, Beijing, China
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Sun Y, Deng D, You WC, Bai H, Zhang L, Zhou J, Shen L, Ma JL, Xie YQ, Li JY. Methylation of p16 CpG islands associated with malignant transformation of gastric dysplasia in a population-based study. Clin Cancer Res 2005; 10:5087-93. [PMID: 15297411 DOI: 10.1158/1078-0432.ccr-03-0622] [Citation(s) in RCA: 62] [Impact Index Per Article: 3.1] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/16/2022]
Abstract
PURPOSE Inactivation of p16 by aberrant methylation of CpG islands is a frequent event in carcinomas and precancerous lesions of various organs, including the stomach. The aim of this study is to investigate the relationship between p16 methylation and malignant transformation of human gastric dysplasia (DYS) based on follow-up endoscopic screening in a high-risk population. EXPERIMENTAL DESIGN Genomic DNA samples were extracted from paraffin blocks of gastric mucosal biopsies that were histopathologically diagnosed as low-grade DYS from patients who developed gastric carcinomas [GCs (n = 21)] and those that did not do so (n = 21) during 5 years of follow-up. The methylation status of p16 CpG islands of each sample was detected by methylation-specific PCR, denatured high-performance liquid chromatography, and sequencing. RESULTS Aberrant p16 methylation was observed in 5 of 21 samples of DYS that progressed to GC but in 0 of 21 samples that did not progress to GC (P = 0.048, two-sided). Sequencing results confirmed that all CpG sites were methylated in the analyzed sequence from these five p16-methylated cases. Furthermore, p16 methylation was also observed in the five subsequent GCs. Unmethylated p16 CpG islands were detected in all of the samples without p16 methylation. CONCLUSIONS Our findings suggest p16 methylation is correlated with the malignant transformation of gastric DYS, and p16 methylation might be a useful biomarker for prediction of malignant potential of gastric DYS.
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Affiliation(s)
- Yu Sun
- Peking University School of Oncology and Beijing Institute for Cancer Research, Beijing, People's Republic of China
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45
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Gao L, Cheng L, Zhou JN, Zhu BL, Lu ZH. DNA microarray: a high throughput approach for methylation detection. Colloids Surf B Biointerfaces 2005; 40:127-31. [PMID: 15708500 DOI: 10.1016/j.colsurfb.2004.10.024] [Citation(s) in RCA: 11] [Impact Index Per Article: 0.6] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 10/26/2022]
Abstract
We described a DNA microarray-based method combined with bisulphite treatment of DNA and regular PCR to examine hyper-methylation in promoter 1A of APC gene. A set of oligonucleotide probes were designed and immobilized on the aldehyde-coated glass slides for detecting the methylation pattern of 15 selected CpG sites in the region. The methylation status of 30 colorectal tumor samples have been examined by both of methylation-specific PCR (MS-PCR) and the present microarray method. The methylation pattern of the 15 CpG sites for the samples have been obtained with the microarray. A total of 19 samples out of 30 were methylated by microarray, in which five samples cannot be detected by MS-PCR due to the methylated CpG patterns not accordant to the MS-PCR primers. The detecting ratio for methylation of APC gene of colorectal tumor samples increased from 46.7% with MS-PCR to 63.3% with the microarray, which successfully demonstrated that DNA microarray-based method not only can obtained the methylation patterns for the related genes, but also decrease the false-negative results of methylation status by the conventional MS-PCR for the investigated genes.
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Affiliation(s)
- L Gao
- Key Laboratory for Molecular and Biomolecular Electronics of Ministry of Education, Southeast University, Nanjing 210096, China
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46
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Simmons DL, Botting RM, Hla T. Cyclooxygenase isozymes: the biology of prostaglandin synthesis and inhibition. Pharmacol Rev 2004; 56:387-437. [PMID: 15317910 DOI: 10.1124/pr.56.3.3] [Citation(s) in RCA: 1205] [Impact Index Per Article: 57.4] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
Nonsteroidal anti-inflammatory drugs (NSAIDs) represent one of the most highly utilized classes of pharmaceutical agents in medicine. All NSAIDs act through inhibiting prostaglandin synthesis, a catalytic activity possessed by two distinct cyclooxygenase (COX) isozymes encoded by separate genes. The discovery of COX-2 launched a new era in NSAID pharmacology, resulting in the synthesis, marketing, and widespread use of COX-2 selective drugs. These pharmaceutical agents have quickly become established as important therapeutic medications with potentially fewer side effects than traditional NSAIDs. Additionally, characterization of the two COX isozymes is allowing the discrimination of the roles each play in physiological processes such as homeostatic maintenance of the gastrointestinal tract, renal function, blood clotting, embryonic implantation, parturition, pain, and fever. Of particular importance has been the investigation of COX-1 and -2 isozymic functions in cancer, dysregulation of inflammation, and Alzheimer's disease. More recently, additional heterogeneity in COX-related proteins has been described, with the finding of variants of COX-1 and COX-2 enzymes. These variants may function in tissue-specific physiological and pathophysiological processes and may represent important new targets for drug therapy.
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Affiliation(s)
- Daniel L Simmons
- Department of Chemistry and Biochemistry, E280 BNSN, Brigham Young University, Provo, UT 84604, USA.
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47
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Abstract
DNA methylation is the main epigenetic modification in humans, and changes in methylation patterns play an important role in tumorigenesis. Hypermethylation of normally unmethylated CpG islands in the promoter regions often occurs in important tumor suppressor genes, DNA repair genes, and metastasis inhibitor genes. The changes of methylation status of various gene promoters seem to be a common feature of malignant cells and these changes can occur early in the progression process. Therefore detection of aberrant promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or detection of cancer recurrence. The purpose of this review is to provide a summary of the most commonly used techniques for the study of DNA methylation. Current scientific literature involving methylation detection methods was reviewed with an emphasis on polymerase chain reaction (PCR)-based detection methods. The current methodologies may be broadly classed into PCR-based methylation assays and non-PCR-based methylation assays. The problems and advantages of the different methods for detecting aberrant methylation are discussed. As the number of genes known to be hypermethylated in cancer is growing, the detection of aberrant promoter region methylation will be a promising approach for using DNA-based markers for the early detection of human cancers. Many techniques, especially PCR-based methylation assay techniques, make it practical to use these new methylation biomarkers in early cancer diagnosis.
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Affiliation(s)
- Ze-Jun Liu
- Department of Laboratory Medicine, Southwest Hospital, Third Military Medical University, Chongqing 400038, People's Republic of China.
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Yue CM, Deng DJ, Bi MX, Guo LP, Lu SH. Expression of ECRG4, a novel esophageal cancer-related gene, downregulated by CpG island hypermethylation in human esophageal squamous cell carcinoma. World J Gastroenterol 2003; 9:1174-8. [PMID: 12800218 PMCID: PMC4611778 DOI: 10.3748/wjg.v9.i6.1174] [Citation(s) in RCA: 66] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Indexed: 02/06/2023] Open
Abstract
AIM: To study the mechanisms responsible for inactivation of a novel esophageal cancer related gene 4 (ECRG4) in esophageal squamous cell carcinoma (ESCC).
METHODS: A pair of primers was designed to amplify a 220 bp fragment, which contains 16 CpG sites in the core promoter region of the ECRG 4 gene. PCR products of bisulfite-modified CpG islands were analyzed by denaturing high-performance liquid chromatography (DHPLC), which were confirmed by DNA sequencing. The methylation status of ECRG 4 promoter in 20 cases of esophageal cancer and the adjacent normal tissues, 5 human tumor cell lines (esophageal cancer cell line-NEC, EC109, EC9706; gastric cancer cell line- GLC; human embryo kidney cell line-Hek293) and 2 normal esophagus tissues were detected. The expression level of the ECRG 4 gene in these samples was examined by RT-PCR.
RESULTS: The expression level of ECRG 4 gene was varied. Of 20 esophageal cancer tissues, nine were unexpressed, six were lowly expressed and five were highly expressed compared with the adjacent tissues and the 2 normal esophageal epithelia. In addition, 4 out of the 5 human cell lines were also unexpressed. A high frequency of methylation was revealed in 12 (8 unexpressed and 4 lowly expressed) of the 15 (80%) downregulated cancer tissues and 3 of the 4 unexpressed cell lines. No methylation peak was observed in the two highly expressed normal esophageal epithelia and the methylation frequency was low (3/20) among the 20 cases in the highly expressed adjacent tissues. The methylation status of the samples was consistent with the result of DNA sequencing.
CONCLUSION: These results indicate that the inactivation of ECRG 4 gene by hypermethylation is a frequent molecular event in ESCC and may be involved in the carcinogenesis of this cancer.
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Affiliation(s)
- Chun-Mei Yue
- Department of Etiology and Carcinogenesis, Cancer Institute, Chinese Academy of Medical Sciences, Beijing 100021, China
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Frueh FW, Noyer-Weidner M. The use of denaturing high-performance liquid chromatography (DHPLC) for the analysis of genetic variations: impact for diagnostics and pharmacogenetics. Clin Chem Lab Med 2003; 41:452-61. [PMID: 12747586 DOI: 10.1515/cclm.2003.068] [Citation(s) in RCA: 56] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/13/2022]
Abstract
Over the past five years, denaturing high-performance liquid chromatography (DHPLC) has emerged as one of the most versatile technologies for the analysis of genetic variations. With the benefit of novel polymer chemistries used for separation, the accuracy, sensitivity, and the throughput of DHPLC for DNA and RNA analysis have greatly improved. DHPLC has been adopted in many laboratories for the screening of mutations and single-nucleotide polymorphisms (SNPs). The ability of DHPLC to detect known and unknown mutations simultaneously has put this technology at the forefront of genetic analysis for a wide variety of diseases. In addition, the high sensitivity of DHPLC combined with the accuracy of the heteroduplex analysis has allowed the development of applications beyond the scope of traditional sequencing or genotyping, e.g., the early detection of cancer. This article reviews the methods, which made DHPLC a widely used tool for diagnosis in molecular genetics and pharmacogenetics. The article provides an overview of current applications in these fields and points to novel applications in areas like epigenetics and the analysis of heteroplasmic mitochondrial DNA, in which DHPLC is becoming the leading technology.
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50
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Bai H, Gu L, Zhou J, Deng D. p16 hypermethylation during gastric carcinogenesis of Wistar rats by N-methyl-N'-nitro-N-nitrosoguanidine. Mutat Res 2003; 535:73-8. [PMID: 12547284 DOI: 10.1016/s1383-5718(02)00288-7] [Citation(s) in RCA: 22] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/04/2023]
Abstract
Inactivation of the tumor suppressor gene, p16 by CpG hypermethylation is a common event in various tumors including gastric carcinoma. The aim of this study is to investigate if p16 hypermethylation is an early and frequent event in gastric carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). The frequency and timing of p16 hypermethylation during the multistep gastric carcinogenesis in Wistar rats were analyzed in various microdissected gastric lesions. The p16 methylation status and the presence of p16 protein were analyzed by methylation-specific PCR and immunohistochemistry, respectively. Results showed that p16 methylation frequency was correlated with the severity of gastric pathologic lesions, positively. For instance, p16 methylation was found in 2.7% of normal gastric epithelium (n = 36), 16.7% of chronic atrophy gastritis (n = 24), 37.5% of dysplasia (n = 24), 67.4% of gastric adenoma (n = 43), and 85.2% of gastric carcinoma (n = 27). The p16 methylation in the distal stomach epithelium was higher than that in the proximal stomach. p16 protein was expressed in all of 15 p16 unmethylated gastric epithelial samples, but not expressed in all of 12 p16 methylated samples. These results suggest that CpG island hypermethylation may account for the silencing of p16 in rat stomach and is an early event whose accumulation will finally lead to gastric carcinogenesis.
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Affiliation(s)
- Hua Bai
- Department of Cancer Etiology, Peking University Health Science Center and Beijing Institute for Cancer Research, Da-Hong-Luo-Chang Street, Western District, Beijing 100034, China
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