Bisulfite-modified DNA Research Articles

Hypermethylated 14-3-3-σ and ESR1 gene promoters in serum as candidate biomarkers for the diagnosis and treatment efficacy of breast cancer metastasis

BackgroundNumerous hypermethylated genes have been reported in breast cancer, and the silencing of these genes plays an important role in carcinogenesis, tumor progression and diagnosis. These hypermethylated promoters are very rarely found in normal breast. It has been suggested that aberrant hypermethylation may be useful as a biomarker, with implications for breast cancer etiology, diagnosis, and management. The relationship between primary neoplasm and metastasis remains largely unknown. There has been no comprehensive comparative study on the clinical usefulness of tumor-associated methylated DNA biomarkers in primary breast carcinoma and metastatic breast carcinoma. The objective of the present study was to investigate the association between clinical extension of breast cancer and methylation status of Estrogen Receptor1 (ESR1) and Stratifin (14-3-3-σ) gene promoters in disease-free and metastatic breast cancer patients.MethodsWe studied two cohorts of patients: 77 patients treated for breast cancer with no signs of disease, and 34 patients with metastatic breast cancer. DNA was obtained from serum samples, and promoter methylation status was determined by using DNA bisulfite modification and quantitative methylation-specific PCR.ResultsSerum levels of methylated gene promoter 14-3-3-σ significantly differed between Control and Metastatic Breast Cancer groups (P < 0.001), and between Disease-Free and Metastatic Breast Cancer groups (P < 0.001). The ratio of the 14-3-3-σ level before the first chemotherapy cycle to the level just before administration of the second chemotherapy cycle was defined as the Biomarker Response Ratio [BRR]. We calculated BRR values for the "continuous decline" and "rise-and-fall" groups. Subsequent ROC analysis showed a sensitivity of 75% (95% CI: 47.6 - 86.7) and a specificity of 66.7% (95% CI: 41.0 - 86.7) to discriminate between the groups for a cut-off level of BRR = 2.39. The area under the ROC curve (Z = 0.804 ± 0.074) indicates that this test is a good approach to post-treatment prognosis.ConclusionsThe relationship of 14-3-3-σ with breast cancer metastasis and progression found in this study suggests a possible application of 14-3-3-σ as a biomarker to screen for metastasis and to follow up patients treated for metastatic breast cancer, monitoring their disease status and treatment response.

Abstract 847: LINE-1 hypomethylation is a marker of poor prognosis in stage IA non-small cell lung cancer

Abstract Introduction: Non-small cell lung cancer (NSCLC) has a relatively poor prognosis and is a leading cause of cancer death worldwide. A substantial proportion of NSCLC patients suffer a recurrence following curative tumor resection, even when they have early stage disease. Molecular markers that are able to predict patient prognosis after surgery are therefore of clinical relevance, especially for early stage NSCLC. Global hypomethylation and the hypermethylation of gene promoter regions are common events in tumor DNA and are possible markers to predict clinical course of NSCLC patients. The aim of this study was to evaluate the prognostic significance of both global hypomethylation and gene promoter hypermethylation in DNA from NSCLC. Methods: Genomic DNA was obtained from tumor tissue of 379 NSCLC patients who underwent surgery. Methylation levels were measured by real-time PCR following bisulfite modification of DNA and were correlated with clinicopathological parameters and patient prognosis. Methylation of long interspersed nuclear element 1 (LINE-1) was used as a surrogate marker for global methylation. Hypermethylation of the APC, CDH13 and RASSF1 promoter regions was also evaluated. Results: Tumor tissue showed significantly higher CDH13 and RASSF1 methylation levels compared to normal lung tissue, but lower LINE-1 methylation levels. APC, RASSF1 and LINE-1 methylation levels were significant prognostic factors in univariate analysis of an initial cohort of 234 cases. APC and LINE-1 methylation remained significant prognostic factors in multivariate analysis that included age, gender, smoking history, histological type and pathological stage. LINE-1 methylation showed marginally significant prognostic value in stage IA and IB disease. To examine whether LINE-1 methylation was an independent prognostic factor, the study cohort was expanded to 364 cases. In sub-group analysis of stage IA cases with expanded cohort, survival was significantly worse for patients with LINE-1 hypomethylation. Multivariate analysis also revealed that LINE-1 methylation had significant prognostic value for stage IA NSCLC patients. Sub-group analysis failed to show prognostic value for LINE-1 methylation in all other NSCLC disease stages. Conclusion: LINE-1 hypomethylation was an independent marker of poor prognosis in stage IA NSCLC. Validation of this finding in additional tumor cohorts could have clinical relevance for the management of early stage NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 847.

Abstract 156: Implications of LINE1 methylation for bladder cancer risk in women

Abstract Bladder cancer is the ninth most incident form of cancer in the United States and is an exposure-related disease which contributes significantly to morbidity and health care costs. Of concern is the fact that incidence rates have been stable in men in the last twenty years but have been increasing in women at a rate of 0.2% a year. Although tobacco use and occupational exposures have been linked to bladder cancer, much of the etiology of this disease remains unexplained. Epigenetic alterations including changes to cellular DNA methylation levels contribute to carcinogenesis and may serve as powerful biomarkers of this disease. This investigation sought to determine whether hypomethylation at the long interspersed nuclear elements (LINE1), reflective of the level of global DNA methylation, in peripheral blood-derived DNA is associated with increased risk of bladder cancer. LINE1 methylation was measured from blood-derived DNA obtained from participants of a population-based incident case control study of bladder cancer in New Hampshire. Cases included white, 25-74 year olds, with a histopathologically confirmed diagnosis of bladder cancer while controls were selected from records of the Department of Transportation and the Medicare Program. Bisulfite-modified DNA was pyrosequenced to determine LINE1 methylation status; a total of 285 cases and 465 controls were evaluated for methylation and for a number of different risk factors for bladder cancer including arsenic. Being in the lowest LINE1 methylation decile was associated with a 1.8-fold increased risk of bladder cancer (95% CI, 1.12-2.90) in models controlling for gender, age and smoking, with the association being stronger in women than in men (ORs = 2.48, 95% CI 1.19-5.17 in women and 1.47, 95% CI 0.79-2.74 in men). Amongst non-invasive cancers, being in the lowest LINE1 methylation decile conferred an almost two fold risk of bladder cancer (OR 1.94, 95% CI, 1.17-3.22) while the risk was non-significant amongst invasive cancers after adjusting for gender, age and smoking. Amongst controls, women were more likely to have lower LINE1 methylation than men (p-value 0.04), and levels of arsenic in the 90th percentile were associated with reduced LINE1 methylation (p-value 0.04). This investigation has identified a novel epigenetic marker of bladder cancer which has the potential for utility as a screening strategy for this disease, and which may aid in providing a more comprehensive understanding of the etiology of bladder cancer. In addition, this study shows an increased risk of LINE1 hypomethylation with high levels of arsenic, raising important questions about the nature of the causal pathway for alterations in DNA repeat methylation to contribute to cancer risk. These results also suggest that women demonstrate lower levels of LINE1 methylation, which may also aid in explaining the disparate incidence of this disease in men and women. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 156.

Abstract 144: A microarray-based study of DNA methylation changes in prostate cancer

Abstract Background: Prostate cancer is the most common cancer in men in the United States. Prior work, based on analyses of selected genes, has shown that epigenetic changes are important in disease progression and pathogenesis. We hypothesized that a global comparative analysis of DNA methylation changes in neoplastic prostate cells would provide an opportunity to discover novel genes involved in prostate carcinogenesis. We designed such a study using DNA from paraffin embedded normal and cancer specimens and from commonly used prostate cancer cell lines. Methods: DNA from LNCaP, VCaP, DU145, DUPRO, PC3, LAPC4, and 22RV cells, paraffin-embedded clinical samples including 78 cancer samples and 3 normal prostate samples from radical prostatectomies, positive control in vitro methylated DNA, and negative control DKO cells, in which the DNMT1 and DNMT3b enzymes were genetically disrupted, were modified with sodium bisulfite. Along with these samples, non bisulfite-modified genomic DNA, which served as an additional negative control, was also hybridized to Illumina Golden Gate BeadArrays after labeling and amplification, which allowed for the assessment of 1505 CpG sites in 807 genes. A Beta methylation value from the BeadStudio Software is assigned to each CpG site. Probes with Beta&amp;gt;0.25 were considered methylated (Beta&amp;gt;0.5 represented a strong methylation level). In order to enrich for cancer-specific methylation, we excluded probes with methylation in normal prostate samples, genomic DNA samples, or DKO negative control samples. Probes unmethylated in the positive control in vitro methylated DNA samples were also excluded. Results: 101 genes were methylated in at least one prostate cancer cell line and clinical sample and in the positive control samples but not in the negative control samples or normal prostate samples. Pathways whose members were silenced by DNA methylation included: growth factor signaling, cell-cell adhesion, differentiation, and apoptosis. The methylation status of single genes or combinations of 2 or more methylated genes resulted in 78%-90% sensitivity and 100% specificity for prostate cancer tissues versus normal tissues. Conclusions: Our results demonstrate that high density 96-well BeadArrays are an efficient and cost-effective method to ascertain the DNA methylation status of many genes simultaneously in cell lines and in paraffin-embedded clinical samples. Of equal importance, our study demonstrates the importance of including positive and negative controls both for DNA methylation and for bisulfite conversion in such studies to more specifically identify cancer-specific DNA methylation changes that will most likely result in cancer- specific gene silencing. Our results have implications not only for improving prostate cancer detection but also for understanding the genes, whose silencing by DNA methylation, may be critical for prostate cancer development and progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 144.

Long Interspersed Nuclear Element 1 Hypomethylation Is a Marker of Poor Prognosis in Stage IA Non–Small Cell Lung Cancer

Global hypomethylation and the hypermethylation of gene promoter regions are common events in tumor DNA. The aim of this study was to evaluate the prognostic significance of both global hypomethylation and gene promoter hypermethylation in DNA from non-small cell lung cancer (NSCLC). Genomic DNA was obtained from the tumor tissue of 379 NSCLC patients who underwent surgery. Methylation levels were measured by real-time PCR following bisulfite modification of DNA and were correlated with clinicopathologic parameters and patient prognosis. Methylation of long interspersed nuclear element 1 (LINE-1) was used as a surrogate marker for global methylation. Hypermethylation of the APC, CDH13, and RASSF1 promoter regions was also evaluated. Tumor tissue showed significantly higher CDH13 and RASSF1 methylation levels compared with normal lung tissue, but lower LINE-1 methylation levels. APC, RASSF1, and LINE-1 methylation levels were significant prognostic factors in univariate analysis of an initial cohort of 234 cases. APC and LINE-1 methylation remained significant prognostic factors in multivariate analysis that included age, gender, smoking history, histologic type, and pathologic stage. LINE-1 methylation showed marginally significant prognostic value in stage IA and IB disease. Expansion of the study cohort to 364 cases revealed that LINE-1 methylation had significant prognostic value for stage IA NSCLC patients in multivariate analysis. LINE-1 hypomethylation was an independent marker of poor prognosis in stage IA NSCLC. Validation of this finding in additional tumor cohorts could have clinical relevance for the management of early-stage NSCLC.

Implications of LINE1 Methylation for Bladder Cancer Risk in Women

Epigenetic alterations including changes to cellular DNA methylation levels contribute to carcinogenesis and may serve as powerful biomarkers of the disease. This investigation sought to determine whether hypomethylation at the long interspersed nuclear elements (LINE1), reflective of the level of global DNA methylation, in peripheral blood-derived DNA is associated with increased risk of bladder cancer. LINE1 methylation was measured from blood-derived DNA obtained from participants of a population-based incident case-control study of bladder cancer in New Hampshire. Bisulfite-modified DNA was pyrosequenced to determine LINE1 methylation status; a total of 285 cases and 465 controls were evaluated for methylation. Being in the lowest LINE1 methylation decile was associated with a 1.8-fold increased risk of bladder cancer [95% confidence interval (95% CI), 1.12-2.90] in models controlling for gender, age, and smoking, and the association was stronger in women than in men (odds ratio, 2.48; 95% CI, 1.19-5.17 in women; and odds ratio, 1.47; 95% CI, 0.79-2.74 in men). Among controls, women were more likely to have lower LINE1 methylation than men (P = 0.04), and levels of arsenic in the 90th percentile were associated with reduced LINE1 methylation (P = 0.04). LINE1 hypomethylation may be an important biomarker of bladder cancer risk, especially among women.

DISAPPEARANCE OF MALE MITOCHONDRIAL DNA AFTER THE FOUR-CELL STAGE IN SPOROPHYTES OF THE ISOGAMOUS BROWN ALGA SCYTOSIPHON LOMENTARIA (SCYTOSIPHONACEAE, PHAEOPHYCEAE)1

Mitochondrial DNA (mtDNA) of the isogamous brown alga Scytosiphon lomentaria (Lyngb.) Link is inherited maternally. We used molecular biological and morphological analyses to investigate the fate of male mitochondria. Ultrastructural observations showed that the number of 25 mitochondria in a zygote coincided with the number of mitochondria derived from male and female gametes. This number remained almost constant during the first cell division. Strain-specific PCR in single germlings suggested that mtDNA derived from the female gamete remained in the germling during development, while the male mtDNA gradually and selectively disappeared after the four-cell stage. One week after fertilization, male mtDNA had disappeared in sporophytic cells. Using bisulfite DNA modification and methylation mapping assays, we found that the degree of methylation on three analyzed sites of mtDNA was not different between male and female gametes, suggesting that maternal inheritance of mtDNA is not defined by its methylation. This study indicates that the mechanism of selective elimination of male mtDNA is present in each cell of a four-celled sporophyte and that it does not depend on different degrees of DNA methylation between male and female mtDNA.

Methylation Induced Gene Silencing of HtrA3 in Smoking-Related Lung Cancer

Some 85% of lung cancers are smoking related. Here, we investigate the role of serine protease HtrA3 in smoking-related lung cancer. We assess HtrA3 methylation and its corresponding expression in the human bronchial cell line BEAS-2B following cigarette smoke carcinogen treatment, in lung cancer cell lines and in primary lung tumors from light, moderate, and heavy smokers. We also show the effects of HtrA3 downregulation on MTT reduction and clonogenic survival with etoposide and cisplatin treatment and the corresponding effects of HtrA3 re-expression during treatment. We show for the first time that HtrA3 expression is reduced or completely lost in over 50% of lung cancer cell lines and primary lung tumors from heavy smokers. Treatment of HtrA3-deficient cell lines with 5-aza-2'-deoxycytidine resulted in a dose-dependent increase in HtrA3 transcription. Further, sequence analysis of bisulfite-modified DNA from lung cancer cell lines and from primary lung tumors showed an increased frequency of methylation within the first exon of HtrA3 with a corresponding loss of HtrA3 expression, particularly in tumors from smokers. In BEAS-2B, treatment with the cigarette smoke carcinogen 4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone resulted in HtrA3 downregulation with a corresponding increase in methylation. Additional studies indicate resistance to etoposide and cisplatin cytotoxicity as a functional consequence of HtrA3 loss. Finally, immunohistochemical analysis of primary lung tumors revealed a strong correlation between low HtrA3 expression and heavy smoking history. Collectively, these results suggest that cigarette smoke-induced methylation of HtrA3 could contribute to the etiology of chemoresistant disease in smoking-related lung cancer.

DNA Methylation in Patients with Colorectal Cancer - Correlation with Some Clinical and Morphological Features and with Local Tumour Invasion

To study quantitatively the promoter methylation of hMLH1, p16INK, TIMP3 and TPEF genes in patients with colorectal cancer and synchronous polyps, and correlate it with some clinicomorphological features. DNA was extracted from all studied tumours and the corresponding normal mucosa. Microsatellite instability was analysed using two mononucleotide (BAT 25 and BAT 26) and three dinucleotide markers (D2S123, D5S356, D17S250) and automated DNA sequencing. Quantitative analysis of methylation was performed using DNA bisulfite modification, PCR with biotinylated primers, visualisation by 2% agarose gel electrophoresis and pyrosequencing. High methylation levels of hMLH1 and p16INK were found in elderly patients (mean age 73.8 +/- 9.5 years and 65.7 +/- 16.6 years, p < 0.03, t-test). Proximal tumours were more often associated with microsatellite instability (p < 0.05, Fisher's test) and higher level of methylation of hMLH1, p16INK and TIMP3 (p < 0.02, Kruskal-Wallis test), while tumours with poor differentiation tended to have higher methylation of the p16INK gene (p < 0.02, Kruskal-Wallis test). Local tumour invasion was correlated with the level of methylation of hMLH1, TIMP3 and the CpG island methylator phenotype (CIMP) status. Tumours with liver metastases showed a lower level of TIMP3 methylation than tumours with no systemic invasion (p < 0.05, Kruskal-Wallis test). We found concordance of methylation in 56% of the cases with colonic cancer and synchronous adenomas; the remaining 44% were discordant. Tumours with microsatellite instability, high level methylation and CIMP have distinctive clinical and morphological features. The level of hMLH1 and TIMP3 methylation and CIMP status can be correlated with the local tumour invasion. Different mechanisms, even for one and the same patient, can be responsible for the development of more than one third of the synchronous polyps and carcinomas.

Stool Methylation-Specific Polymerase Chain Reaction Assay for the Detection of Colorectal Neoplasia in Korean Patients

To investigate the feasibility of detecting hypermethylation in stool samples as a noninvasive screening tool for colorectal cancer and precancerous lesions, we evaluated the hypermethylation of three genes in the stool samples of patients with colorectal cancer, patients with colorectal adenomas, and healthy control subjects. Stool samples were obtained from 37 endoscopically diagnosed healthy controls, 52 patients with adenomas, and 60 patients with colorectal cancer. The methylation status of the methylguanine DNA methyltransferase, human Mut L homolog-1, and vimentin promoter in bisulfite-modified DNA was investigated in a blinded manner by methylation-specific polymerase chain reaction with primer pairs designed to amplify specifically the methylated or unmethylated alleles. The methylated methylguanine DNA methyltransferase, human Mut L homolog-1, and vimentin were detected in 51.7%, 30.0%, and 38.3% of colorectal cancer, and in 36.5%, 11.%, and 15.4% of colorectal adenomas, respectively. The sensitivities of the combined study, using three markers for the detection of colorectal cancer and colorectal adenomas, were 75.0% and 59.6%. The specificity was 86.5%. Our results have demonstrated that the promoter hypermethylation for methylguanine DNA methyltransferase, human Mut L homolog-1, and vimentin in stool samples is a feasible epigenetic marker that is a sensitive, specific, and noninvasive alternative for colorectal cancer screening. This method of screening for colorectal cancer may be useful for patients that decline screening because of fear or inconvenience.

Genome-wide screen of promoter methylation identifies novel markers in melanoma

DNA methylation is an important component of epigenetic modifications, which influences the transcriptional machinery aberrant in many human diseases. In this study we present the first genome-wide integrative analysis of promoter methylation and gene expression for the identification of methylation markers in melanoma. Genome-wide promoter methylation and gene expression of eight early-passage human melanoma cell strains were compared with newborn and adult melanocytes. We used linear mixed effect models (LME) in combination with a series of filters based on the localization of promoter methylation relative to the transcription start site, overall promoter CpG content, and differential gene expression to discover DNA methylation markers. This approach identified 76 markers, of which 68 were hyper- and eight hypomethylated (LME, P < 0.05). Promoter methylation and differential gene expression of five markers (COL1A2, NPM2, HSPB6, DDIT4L, MT1G) were validated by sequencing of bisulfite-modified DNA and real-time reverse transcriptase PCR, respectively. Importantly, the incidence of promoter methylation of the validated markers increased moderately in early and significantly in advanced-stage melanomas, using early-passage cell strains and snap-frozen tissues (n = 18 and n = 24, respectively) compared with normal melanocytes and nevi (n = 11 and n = 9, respectively). Our approach allows robust identification of methylation markers that can be applied to other studies involving genome-wide promoter methylation. In conclusion, this study represents the first unbiased systematic effort to determine methylation markers in melanoma and revealed several novel genes regulated by promoter methylation that were not described in cancer cells before.

Discovery of the methylation of the metastasis suppressor gene KiSS-1 in bladder cancer

5023 Background: In an attempt to uncover the mechanisms by which the metastasis suppressor gene, KiSS1, is lost in bladder progression, we tested the hypothesis of epigenetic silencing. An enriched 5’-CpG islands was identified around the transcription start site of KiSS1, supporting its susceptibility to be epigenetically modified by hypermethylation. To the best of our knowledge, KiSS1 had not been reported to be epigenetically altered in bladder cancer or any other tumor type. In this report, the impact and clinical relevance of KiSS1 methylation along bladder cancer progression was evaluated using in vitro strategies as well as on tissue specimens. Methods: The methylation status of KiSS1 was analyzed by two polymerase chain reaction (PCR) analysis strategies of bisulfite-modified genomic DNA. First, by genomic sequencing of both strands of KiSS1 promoter and second, by methylated specific PCR (MS-PCR). The epigenetic silencing of KiSS1 by hypermethylation was tested in bladder cancer cells (n = 12) before and after azacytidine treatment. The methylation status of KiSS1 promoter was then evaluated by MS-PCR in a large series of 524 bladder tumors. KiSS1 transcript expression were analyzed by RTPCR on bladder tumors (n = 177) for which KiSS1 methylation was assessed by MS-PCR. Results: KiSS1 hypermethylation was frequent in the bladder cancer cells analyzed and associated with low gene expression by RT-PCR. KiSS-1 expression was increased in vitro by the demethylating agent azacytidine in methylated cells. KiSS1 was found to be frequently methylated in a large series of 524 bladder tumors (68.1%). KiSS1 methylation was significantly associated with tumor stage (p &lt; 0.001) and tumor grade (p &lt; 0.001). Interestingly, a significant association was found between transcript levels by quantitative RT-PCR in bladder tumors and increasing tumor stage (p &lt; 0.001). Moreover, the presence of KiSS1 methylation was associated with low transcript expression (p &lt; 0.001). Conclusions: KiSS1 was identified to be epigenetically modified in bladder cancer. The association of KiSS1 methylation with cancer progression suggests the utility of incorporating KiSS1 methylation assessment in the clinical management of patients affected by uroepithelial neoplasias. No significant financial relationships to disclose.

DNA methylation biomarkers of prostate cancer: Confirmation of candidates and evidence urine is the most sensitive body fluid for non‐invasive detection

A prostate cancer (PCa) biomarker with improved specificity relative to PSA is a public health priority. Hypermethylated DNA can be detected in body fluids from PCa patients and may be a useful biomarker, although clinical performance varies between studies. We investigated the performance of candidate PCa DNA methylation biomarkers identified through a genome-wide search. Real-time PCR was used to measure four DNA methylation biomarkers: GSTP1 and three previously unreported candidates associated with the genes RASSF2, HIST1H4K, and TFAP2E in sodium bisulfite-modified DNA. Matched plasma and urine collected prospectively from 142 patients referred for prostate biopsy and 50 young asymptomatic males were analyzed. Analysis of all biomarkers in urine DNA significantly discriminated PCa from biopsy negative patients. The biomarkers discriminated PCa from biopsy negative patients with AUCs ranging from 0.64 for HIST1H4K (95% CI 0.55-0.72, P < 0.00001) to 0.69 for GSTP1 (95% CI 0.60-0.77, P < 0.00001). All biomarkers showed minimal correlation with PSA. Multivariate analysis did not yield a panel that significantly improved performance over that of single biomarkers. All biomarkers showed greater sensitivity for PCa in urine than in plasma DNA. Analysis of the biomarkers in urine DNA significantly discriminated PCa from biopsy negative patients. The biomarkers provided information independent of PSA and may warrant inclusion in nomograms for predicting prostate biopsy outcome. The biomarkers' PCa sensitivity was greater for urine than plasma DNA. The biomarker performances in urine DNA should next be validated in formal training and test studies.

MS-qFRET: A quantum dot-based method for analysis of DNA methylation

DNA methylation contributes to carcinogenesis by silencing key tumor suppressor genes. Here we report an ultrasensitive and reliable nanotechnology assay, MS-qFRET, for detection and quantification of DNA methylation. Bisulfite-modified DNA is subjected to PCR amplification with primers that would differentiate between methylated and unmethylated DNA. Quantum dots are then used to capture PCR amplicons and determine the methylation status via fluorescence resonance energy transfer (FRET). Key features of MS-qFRET include its low intrinsic background noise, high resolution, and high sensitivity. This approach detects as little as 15 pg of methylated DNA in the presence of a 10,000-fold excess of unmethylated alleles, enables reduced use of PCR (as low as eight cycles), and allows for multiplexed analyses. The high sensitivity of MS-qFRET enables one-step detection of methylation at PYCARD, CDKN2B, and CDKN2A genes in patient sputum samples that contain low concentrations of methylated DNA, which normally would require a nested PCR approach. The direct application of MS-qFRET on clinical samples offers great promise for its translational use in early cancer diagnosis, prognostic assessment of tumor behavior, as well as monitoring response to therapeutic agents.

CPG HYPERMETHYLATION OF COL1A2 CONTRIBUTES TO PROLIFERATION AND MIGRATION ACTIVITY OF BLADDER CANCER

INTRODUCTION AND OBJECTIVE: Gene silencing due to aberrant DNA methylation plays an important role in carcinogenesis. For genome-wide screening for methylated genes in bladder cancer (BC), we performed cDNA microarray analysis of the BC cell line (BOY) treated with demethylating agent (5-Aza-dC). Collagen type 1 alpha 2 (COL1A2) gene was identified as the most up-regulated one of the 30,144 genes screened. We hypothesize that inactivation of the COL1A2 gene through CpG methylation contributes to proliferation and migration activity of human BC by changing extra-cellular matrix (ECM). METHODS: To test this hypothesis, we extracted DNA and RNA from 67 BC samples and 10 normal bladder epitheliums (NBEs) from cystectomy and transurethral resection. The CpG hypermethylation of COL1A2 promoter was analyzed by quantitative methylation-specific PCR (QMSP) and confirmed by bisulfite-modified DNA sequencing. Real-time RT-PCR was carried out to evaluate COL1A2 mRNA expression. We established a stable COL1A2 transfectant for gain of function studies, for which cellular proliferation and migration were examined by MTT assay and wound healing assay, respectively. The protein secretion of type 1 collagen in culture medium was detected by immunoblot. RESULTS: Bisulfite DNA sequencing demonstrated that the promoter hypermethylation of COL1A2 was a frequent event in clinical BCs. The methylation index was significantly higher in the 67 BCs than in the10 NBEs (42.3 ± 28.9 vs. 0.07 ± 0.01, p = 0.0011). Conversely, COL1A2 mRNA expression was significantly lower in the BCs than in the NBEs (15.8 ± 3.8 vs. 32.0 ± 22.0, p = 0.0052). In vitro, after 5-aza-dC treatment, the COL1A2 mRNA expression was markedly increased in BOY. Our cell proliferation assays consistently demonstrated a significant growth inhibition in the COL1A2 transfectant compared with control and wild-type BOY cells (on day 5; 0.791 ± 0.020, 1.056 ± 0.034, and 1.107 ± 0.027, respectively, p < 0.0001). Wound healing assays also showed a significant wound healing inhibition in the COL1A2 transfectant compared to the counterparts (% of wound closure; 75.5 ± 0.2, 94.3 ± 2.9, and 96.9 ± 3.1, respectively, p = 0.0016). Immunoblot demonstrated that type 1 collagen protein markedly increased in culture medium of the COL1A2 transfectant. CONCLUSIONS: Our data suggest that inactivation of the COL1A2 gene through CpG methylation may change an ECM binding on epithelial collagen and contributes to proliferation and migration activity of human bladder cancer.

Large-Scale Profiling of Archival Lymph Nodes Reveals Pervasive Remodeling of the Follicular Lymphoma Methylome

Emerging technologies allow broad profiling of the cancer genome for differential DNA methylation relative to benign cells. Herein, bisulfite-modified DNA from lymph nodes with either reactive hyperplasia or follicular lymphoma (FL) were analyzed using a commercial C/UpG genotyping assay. Two hundred fifty-nine differentially methylated targets (DMT) distributed among 183 unique genes were identified in FL. Comparison of matched formalin-fixed, paraffin-embedded and frozen surgical pathology replicates showed the complete preservation of the cancer methylome among differently archived tissue specimens. Analysis of the DMT profile is consistent with a pervasive epigenomic remodeling process in FL that affects predominantly nonlymphoid genes.

BTG3 tumor suppressor gene promoter demethylation, histone modification and cell cycle arrest by genistein in renal cancer

BTG3/ANA/APRO4 has been reported to be a tumor suppressor gene in some malignancies. It constitutes important negative regulatory mechanism for Src-mediated signaling, a negative regulator of the cell cycle and inhibits transcription factor E2F1. We report that BTG3 is downregulated in renal cancer and that the mechanism of inactivation is through promoter hypermethylation. Quantitative real-time polymerase chain reaction (PCR) showed that BTG3 was downregulated in cancer tissues and cells. Genistein and 5-aza-2'-deoxycytidine (5Aza-C) induced BTG3 messenger RNA (mRNA) expression in A498, ACHN and HEK-293 renal cell carcinoma (RCC) cell lines. Bisulfite-modified PCR and DNA sequencing results showed complete methylation of BTG3 promoter in tumor samples and cancer cell lines. Genistein and 5Aza-C treatment significantly decreased promoter methylation, reactivating BTG3 expression. Chromatin immunoprecipitation assay revealed that genistein and 5Aza-C increased levels of acetylated histones 3, 4, 2H3K4, 3H3K4 and RNA polymerase II at the BTG3 promoter indicative of active histone modifications. Enzymatic assays showed genistein and 5Aza-C decreased DNA Methyltransferase, methyl-CpG-binding domain 2 activity and increased HAT activity. Cell cycle and 3-(4,5-dimethylthiazole-2-yl)-2,5-biphenyl tetrazolium bromide cell proliferation assays showed that genistein has antiproliferative effect on cancer cell growth through induction of cell cycle arrest. This is the first report to show that BTG3 is epigenetically silenced in RCC and can be reactivated by genistein-induced promoter demethylation and active histone modification. Genistein had similar effects to that of 5Aza-C, which is a potent demethylating agent with high toxicity and instability. Genistein being a natural, non-toxic, dietary isoflavone is effective in retarding the growth of RCC cells, making it a promising candidate for epigenetic therapy in renal carcinoma.

Fas Expression in Metastatic Osteosarcoma Cells Is Not Regulated by CpG Island Methylation

Fas expression in osteosarcoma (OS) cells is inversely correlated with the metastatic potential of OS to the lung. The purpose of this study was to determine whether loss of Fas expression in metastatic OS cells is secondary to DNA methylation of CpG islands in the Fas gene. SAOS-2 cells have high levels of Fas expression and do not form lung metastases when injected intravenously, whereas LM7 cells have low levels of Fas expression and do produce lung metastases. Using the endonucleases HpaII and MspI and a polymerase chain reaction-based methylation assay, we found that all four CpG sites in the CCGG sequence in the Fas promoter region were unmethylated in both SAOS-2 and LM7 cells. We performed detailed analysis of the 28 and 46 CpG sites in the Fas promoter and first intron region, respectively, using bisulfite-modified genomic DNA sequencing. More than 99.8% of the examined CpG sites were unmethylated and there was no difference of CpG methylation in SAOS-2 and LM7 cells as well as LM7 metastatic lung tumor tissue samples. Treatment of LM7 cells and another OS cell line, DLM8 with low levels of Fas expression, with demethylation agent, 5-azadeoxycitidine (AzadC), did not change the Fas expression and did not increase sensitivity of AzadC-treated cells to Fas ligand (FasL) treatment. In conclusion, our data indicate that decreased Fas expression in OS cells is not secondary to DNA methylation of CpG islands in the Fas gene and that Fas expression cannot be increased by using demethylation agents.

Methylation-sensitive high-resolution melting

The base composition of PCR products derived from sodium bisulfite-modified templates is methylation dependent. Hence, methylated and unmethylated, PCR products show different melting profiles when subjected to thermal denaturation. The methylation-sensitive high-resolution melting (MS-HRM) protocol is based on the comparison of the melting profiles of PCR products from unknown samples with profiles specific for PCR products derived from methylated and unmethylated control DNAs. The protocol consists of PCR amplification of bisulfite-modified DNA with primers designed to proportionally amplify both methylated and unmethylated templates and subsequent high-resolution melting analysis of the PCR product. The MS-HRM protocol allows in-tube determination of the methylation status of the locus of interest following sodium bisulfite modification of template DNA in less than 3 h. Here, we provide a protocol for MS-HRM, which enables highly sensitive, labor- and cost-efficient single-locus methylation studies on the basis of DNA high-resolution melting technology.

The Research of Phenylhexyl Isothiocyanate's Influence on p16 Gene Methylation of Leukaemia Cell

Objective Aberrant DNA methylation of CpG site is among the earliest and most frequent alterations in cancer. The aim of this study was to study whether phenylhexyl isothiocyanate can reduce the methylation level of P16 gene. This study used a microarray-based method for quantificationally detecting changes of P16 gene methylation in leukemia patient and U266 cell line. And to simply discuss the effect of phenylhexyl isothiocyanate on tumor methylation.Methods This method used bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. One set of oligonucleotide probes were designed to fabricate a DNA microarray to detect the methylation changes of P16 gene CpG islands. Each set contained a pair of methylated and unmethylated oligonucleotides for interrogating 3 CpG sites in close proximity. By TA cloning, PCR, sequencing, positive and negative DNA targets were required. Next drawing a standard curve by fluorescence intensity. Leukemia samples DNA were abstracted and bisulfite-modified. Sample DNA targets were required by PCR amplification and were hybridized with the microarry. Finally the microarry was scanned with ScanArray Lite microarray analysis systems.Results The linear relationship (R2=0.9660) was established and it could be used to eliminate background noise. The methylation level of U266 is hypermethylation, after cultured with PHI, the level reduce. Eleven patients have P16 gene hypermethylation in thrity. After culture with PHI, seven patients showed level reduce, one patient showed raise, three patients showed remaining.Conclusion PHI can reduce the methylation level of P16 gene in U266 cell line, and also can reduce the methylation level of P16 gene of leukemia patient samples in vitro culture.