Sodium Bisulfite Modification Research Articles

High-Resolution Melting (HRM) analysis of DNA methylation using semiconductor chip-based digital PCR.

Digital PCR (dPCR) technology allows absolute quantification and detection of disease-associated rare variants, and thus the use of dPCR technology has been increasing in clinical research and diagnostics. The high-resolution melting curve analysis (HRM) of qPCR is widely used to distinguish true positives from false positives and detect rare variants. In particular, qPCR-HRM is commonly used for methylation assessment in research and diagnostics due to its simplicity and high reproducibility. Most dPCR instruments have limited fluorescence channels available and separate heating and imaging systems. Therefore, it is difficult to perform HRM analysis using dPCR instruments. A new digital real-time PCR instrument (LOAA) has been recently developed to integrate partitioning, thermocycling, and imaging in a single dPCR instrument. In addition, a new technique to perform HRM analysis is utilized in LOAA. The aim of the present study is to evaluate the efficiency and accuracy of LOAA dPCR on HRM analysis for the detection of methylation. In this study, comprehensive comparison with Bio-Rad qRT-PCR and droplet-based dPCR equipment was performed to verify the HRM analysis-based methylation detection efficiency of the LOAA digital PCR equipment. Here, sodium bisulfite modification method was applied to detect methylated DNA sequences by each PCR method. Melting curve analysis detected four different Tm values using LOAA and qPCR, and found that LOAA, unlike qPCR, successfully distinguished between different Tm values when the Tm values were very similar. In addition, melting temperatures increased by each methylation were about 0.5℃ for qPCR and about 0.2 ~ 0.6℃ for LOAA. The melting temperature analyses of methylated and unmethylated DNA samples were conducted using LOAA dPCR with TaqMan probes and EvaGreen, and the result found that Tm values of methylated DNA samples are higher than those of unmethylated DNA samples. The present study shows that LOAA dPCR could detect different melting temperatures according to methylation status of target sequences, indicating that LOAA dPCR would be useful for diagnostic applications that require the accurate quantification and assessment of DNA methylation.

Delineating the mechanism of fragility at BCL6 breakpoint region associated with translocations in diffuse large B cell lymphoma.

BCL6 translocation is one of the most common chromosomal translocations in cancer and results in its enhanced expression in germinal center B cells. It involves the fusion of BCL6 with any of its twenty-six Ig and non-Ig translocation partners associated with diffuse large B cell lymphoma (DLBCL). Despite being discovered long back, the mechanism of BCL6 fragility is largely unknown. Analysis of the translocation breakpoints in 5' UTR of BCL6 reveals the clustering of most of the breakpoints around a region termed Cluster II. In silico analysis of the breakpoint cluster sequence identified sequence motifs that could potentially fold into non-B DNA. Results revealed that the Cluster II sequence folded into overlapping hairpin structures and identified sequences that undergo base pairing at the stem region. Further, the formation of cruciform DNA blocked DNA replication. The sodium bisulfite modification assay revealed the single-strandedness of the region corresponding to hairpin DNA in both strands of the genome. Further, we report the formation of intramolecular parallel G4 and triplex DNA, at Cluster II. Taken together, our studies reveal that multiple non-canonical DNA structures exist at the BCL6 cluster II breakpoint region and contribute to the fragility leading to BCL6 translocation in DLBCL patients.

Assessment of Aberrant Promoter Hypermethylation of RASSF1a and P16 in Endometrial Carcinoma

Endometrial carcinoma is one of the most common gynecological malignancies known to affect around 142000 women worldwide. Aberrant promoter hypermethylation of tumor suppressor genes is a common epigenetic alteration reported in cancers. In the present study we report the aberrant promoter hypermethylation of RASSF1a and p16 in 78 endometrial cancer patients. Methods: Endometrial carcinoma samples were collected after pathological examination and DNA was extracted. The DNA was subjected to sodium bisulfite modification and then used to perform methylation specific PCR. The PCR was performed using a two step procedure of nested and methylation specific PCR. The PCR product was visualized using agarose gel electrophoresis. The results obtained were correlated with various clinicopathological parameters. Chi square test was used for statistical analysis and a p-value of <0.05 was considered statistically significant. Result: 60 of the 78 (76.92%) samples showed methylation for RASSF1a gene. 28 of the 78 (35.8%) samples showed methylation for p16 gene. A higher methylation frequency was observed for RASSF1a in the endometrioid and poorly differentiated histological subtypes and stage I and II of the disease. Conclusion: Aberrant promoter hypermethylation of RASSF1a and p16 is known to play an important role in endometrial cancer initiation and progression. The methylation patterns can be used as an important molecular marker for early diagnosis and prognosis of endometrial cancer. Keywords: Epigenetic alterations, DNA Hypermethylation, Endometrial cancer, RASSF1a, p16, Methylation-specific PCR.

Early detection of the major male cancer types in blood-based liquid biopsies using a DNA methylation panel

BackgroundLung (LC), prostate (PCa) and colorectal (CRC) cancers are the most incident in males worldwide. Despite recent advances, optimal population-based cancer screening methods remain an unmet need. Due to its early onset, cancer specificity and accessibility in body fluids, aberrant DNA promoter methylation might be a valuable minimally invasive tool for early cancer detection. Herein, we aimed to develop a minimally invasive methylation-based test for simultaneous early detection of LC, PCa and CRC in males, using liquid biopsies.ResultsCirculating cell-free DNA was extracted from 102 LC, 121 PCa and 100 CRC patients and 136 asymptomatic donors’ plasma samples. Sodium-bisulfite modification and whole-genome amplification was performed. Promoter methylation levels of APCme, FOXA1me, GSTP1me, HOXD3me, RARβ2me, RASSF1Ame, SEPT9me and SOX17me were assessed by multiplex quantitative methylation-specific PCR.SEPT9me and SOX17me were the only biomarkers shared by all three cancer types, although they detected CRC with limited sensitivity. A “PanCancer” panel (FOXA1me, RARβ2me and RASSF1Ame) detected LC and PCa with 64% sensitivity and 70% specificity, complemented with “CancerType” panel (GSTP1me and SOX17me) which discriminated between LC and PCa with 93% specificity, but with modest sensitivity. Moreover, a HOXD3me and RASSF1Ame panel discriminated small cell lung carcinoma from non-small cell lung carcinoma with 75% sensitivity, 88% specificity, 6.5 LR+ and 0.28 LR–. An APCme and RASSF1Ame panel independently predicted disease-specific mortality in LC patients.ConclusionsWe concluded that a DNA methylation-based test in liquid biopsies might enable minimally invasive screening of LC and PCa, improving patient compliance and reducing healthcare costs. Moreover, it might assist in LC subtyping and prognostication.

The von Hippel Lindau tumour suppressor gene is a novel target of E2F4-mediated transcriptional repression in preeclampsia

The von Hippel Lindau tumour suppressor (VHL) protein is essential for proper placental development and is downregulated in preeclampsia (PE), a devastating disorder of pregnancy typified by chronic hypoxia. To date, knowledge on VHL genetic and epigenetic regulation is restricted to inactivating mutations and loss-of-heterozygosity in renal cell carcinomas. Herein, we sought to examine whether VHL DNA is subject to differential methylation, and if so, whether it is altered in early-onset PE (E-PE). Sodium bisulfite modification and methylation-specific PCR analysis revealed that VHL is subject to extensive methylation in a CpG-rich region within its promoter in the human placenta. Notably, we detected significant differences in methylation in E-PE placentae relative to normotensive age-matched controls at key transcription factor binding sites, including that of the transcriptional repressor E2F4. Treatment of JEG3 cells with 5-Aza-2′-deoxycytidine, revealed that loss of DNA methylation promoted VHL transcription by attenuating VHL association with E2F4. RNAi knockdown of E2F4 in vitro confirmed its function on VHL repression. Exposure of JEG3 cells to transforming growth factor beta (TGFβ) downregulated VHL mRNA. In line with elevated levels of TGFβ3 in E-PE, chromatin immunoprecipitation assays revealed that E2F4-VHL association was enhanced upon TGFβ3 treatment, indicative of VHL transcriptional inhibition. In line with decreased VHL expression in E-PE, augmented E2F4-VHL association was also observed in E-PE placental tissue relative to controls. In conclusion, we demonstrate for the first time that hypomethylation of VHL DNA at a key transcription factor binding site has significant consequences for its transcriptional repression in early-onset preeclampsia.

Molecular combing reveals complex 4q35 rearrangements in Facioscapulohumeral dystrophy.

Facioscapulohumeral dystrophy (FSHD), one of the most common hereditary neuromuscular disorders, is associated with a complex combination of genetic variations at the subtelomeric 4q35 locus. As molecular diagnosis relying on Southern blot (SB) might be challenging in some cases, molecular combing (MC) was recently developed as an additional technique for FSHD diagnosis and exploration of the genomic organization of the 4q35 and 10q26 regions. In complement to the usual SB, we applied MC in a large cohort of 586 individuals with clinical FSHD. In 332 subjects, the two 4q alleles were normal in size, allowing exclusion of FSHD1 while we confirmed FSHD1 in 230 patients. In 14 patients from 10 families, we identified a recurrent complex heterozygous rearrangement at 4q35 consisting of a duplication of the D4Z4 array and a 4qA haplotype, irresolvable by the SB technique. In five families, we further identified variations in the SMCHD1 gene. Impact of the different mutations was tested using a minigene assay and we analyzed DNA methylation after sodium bisulfite modification and NGS sequencing. We discuss the involvement of this rearrangement in FSHD since all mutations in SMCHD1 are not associated with D4Z4 hypomethylation and do not always segregate with the disease.

Alteration in Methylation Pattern of Retinoblastoma 1 Gene Promotor Region in Intestinal Metaplasia with or without Helicobacter pylori and Gastric Cancer Patients.

Helicobacter pylori, intestinal metaplasia (IM), and gene methylation play important roles in gastric carcinogenesis. However, the association among H. pylori infection, IM, gastric cancer (GC), and gene methylation is not fully understood. Cell cycle control involving retinoblastoma 1 (RB1) gene is one of the main regulatory pathways reported to be altered in gastric carcinogenesis. The purpose of this research is to assess the methylation status of RB1 gene in GC and IM with or without H. pylori infection, and to discuss the possible role of H. pylori-induced RB1 gene methylation in the mechanism of gastric carcinogenesis. The methylation profile of RB1 gene was analyzed by sodium bisulfite modification and methylation-specific PCR in GC (n = 24), IM patients with H. pylori positive (n = 20) and negative (n = 20), and control subjects (n = 20). According to methylation levels in RB1 gene; the high correlation values were detected between H. pylori positive-IM group and GC group, and between H. pylori positive-IM and H. pylori negative-IM groups (p < 0.05). No correlations between H. pylori negative-IM and GC groups and between GC and control groups were detected in methylation status of RB1 gene. High methylation levels in RB1 gene in H. pylori positive individuals may suggest an elevated risk of gastric cancer occurrence.

Methylation ofDAPKandTHBS1genes in esophageal gastric-type columnar metaplasia

To explore methylation of DAPK, THBS1, CDH-1, and p14 genes, and Helicobacter pylori (H. pylori) status in individuals harboring esophageal columnar metaplasia. Distal esophageal mucosal samples obtained by endoscopy and histologically diagnosed as gastric-type (non-specialized) columnar metaplasia, were studied thoroughly. DNA was extracted from paraffin blocks, and methylation status of death-associated protein kinase (DAPK), thrombospondin-1 (THBS1), cadherin-1 (CDH1), and p14 genes, was examined using a methyl-sensitive polymerase chain reaction (MS-PCR) and sodium bisulfite modification protocol. H. pylori cagA status was determined by PCR. In total, 68 subjects (33 females and 35 males), with a mean age of 52 years, were included. H. pylori cagA positive was present in the esophageal gastric-type metaplastic mucosa of 18 individuals. DAPK, THSB1, CDH1, and p14 gene promoters were methylated by MS-PCR in 40 (58.8%), 33 (48.5%), 46 (67.6%), and 23 (33.8%) cases of the 68 esophageal samples. H. pylori status was associated with methylation of DAPK (P = 0.003) and THBS1 (P = 0.019). DNA methylation occurs in cases of gastric-type (non-specialized) columnar metaplasia of the esophagus, and this modification is associated with H. pylori cagA positive infection.

In Situ Sodium Bisulfite Modification of Genomic DNA from Microdissected Melanoma Paraffin-Embedded Archival Tissues.

DNA methylation analysis of paraffin-embedded archival tumor tissues (PEAT) is important in clinical and translational research studies. Efficient identification and isolation of homogeneous cell population, optimal DNA extraction, and sodium bisulfite modification (SBM) are essential, particularly in small tumor lesions. Laser capture microdissection (LCM) coupled with an in situ SBM improves the specificity, through histopathology accuracy, and the amount of genomic DNA modified for downstream methylation assays.

Viability of Lymph Node Samples Obtained by Echobronchoscopy in the Study of Epigenetic Alterations in Patients With Lung Cancer

IntroductionThe diagnosis of microscopic lymph node metastasis in lung cancer is challenging despite the constant advances in tumor staging. The analysis of the methylation status of certain genes in lymph node samples could improve the diagnostic capability of conventional cyto-histological methods. The aim of this study was to demonstrate the feasibility of methylation studies using cytological lymph node samples. MethodsA prospective study including 88 patients with a diagnosis or strong suspicion of non-small cell lung cancer, in which an echobronchoscopy was performed on mediastinal or hilar lymph nodes for diagnosis and/or staging purposes. DNA was extracted from cytological lymph node samples and sodium bisulfite modification was performed. Methylation studies for p16/INK4a and SHOX2 were accomplished by MS-qPCR and pyrosequencing. ResultsThe methodology used in our study yielded optimal/good DNA quality in 90% of the cases. No differences in DNA concentration were observed with respect to the lymph node biopsied and final diagnosis. Methylation analyses using MS-qPCR and pyrosequencing were not possible in a small number of samples mainly due to low DNA concentration, inadequate purity, fragmentation and/or degradation as a consequence of bisulfite conversion. ConclusionMethylation quantification using MS-qPCR and pyrosequencing of cytological lymph node samples obtained using echobronchoscopy is feasible if an appropriate DNA concentration is obtained, notably contributing to the identification of epigenetic biomarkers capable of improving decision making for the benefit of potentially curable lung cancer patients.

Viabilidad de las muestras ganglionares obtenidas por ecobroncoscopia para el estudio de alteraciones epigenéticas en pacientes con cáncer de pulmón

IntroductionThe diagnosis of microscopic lymph node metastasis in lung cancer is challenging despite the constant advances in tumor staging. The analysis of the methylation status of certain genes in lymph node samples could improve the diagnostic capability of conventional cyto-histological methods. The aim of this study was to demonstrate the feasibility of methylation studies using cytological lymph node samples. MethodsProspective study including 88 patients with a diagnosis or strong suspicion of non-small cell lung cancer, in which an echobronchoscopy was performed on mediastinal or hilar lymph nodes for diagnostic and/or staging. DNA was extracted from cytological lymph node samples and sodium bisulfite modification was performed. Methylation studies for p16/INK4a and SHOX2 were accomplished by MS-qPCR and pyrosequencing. ResultsThe methodology used in our study yielded optimal/good DNA quality in 90% of the cases. No differences in DNA concentration were observed with respect to the lymph node biopsied and final diagnosis. Methylation analyses using MS-qPCR and pyrosequencing were not possible in a small number of samples mainly due to low DNA concentration, inadequate purity, fragmentation and/or degradation as a consequence of bisulfite conversion. ConclusionMethylation quantification using MS-qPCR and pyrosequencing of cytological lymph node samples obtained using echobronchoscopy is feasible if an appropriate DNA concentration is obtained, notably contributing to the identification of epigenetic biomarkers capable of improving decision-making for the benefit of potentially curable lung cancer patients.

G-Quadruplex Structures Formed at the HOX11 Breakpoint Region Contribute to Its Fragility during t(10;14) Translocation in T-Cell Leukemia

The t(10;14) translocation involving the HOX11 gene is found in several T-cell leukemia patients. Previous efforts to determine the causes of HOX11 fragility were not successful. The role of non-B DNA structures is increasingly becoming an important cause of genomic instability. In the present study, bioinformatics analysis revealed two G-quadruplex-forming motifs at the HOX11 breakpoint cluster. Gel shift assays showed formation of both intra- and intermolecular G-quadruplexes, the latter being more predominant. The structure formation was dependent on four stretches of guanines, as revealed by mutagenesis. Circular dichroism analysis identified parallel conformations for both quadruplexes. The non-B DNA structure could block polymerization during replication on a plasmid, resulting in consistent K(+)-dependent pause sites, which were abolished upon mutation of G-motifs, thereby demonstrating the role of the stretches of guanines even on double-stranded DNA. Extrachromosomal assays showed that the G-quadruplex motifs could block transcription, leading to reduced expression of green fluorescent protein (GFP) within cells. More importantly, sodium bisulfite modification assay showed the single-stranded character at regions I and II of HOX11 in the genome. Thus, our findings suggest the occurrence of G-quadruplex structures at the HOX11 breakpoint region, which could explain its fragility during the t(10;14) translocation.

OP196: HPV-16 and CDKN2A hypermethylation in oral squamous cell carcinoma

Purpose Human papillomaviruses have been implicated in the pathogenesis of a subset of squamous cell carcinoma of the head and neck. Some high risk HPV types, especially HPV 16 and 18, and epigenetic silencing of cancer-related genes have been correlated with the genesis of oral squamous cell carcinomas (OSCC). The aim of this work was to investigate the incidence of HPV infection and its correlation with methylation status of CDKN2A in OSCC. Material and methods The samples consisted of 45 paraffin-embedded or fresh specimens of OSCC. Demographic and risk factors exposure information were collected during the patient interview and clinical information was obtained through medical chart reviews. HPV DNA was detected by nested PCR (external primers MY09/MY11 and internal primers GP5/GP6). All reactions included HPV positive and negative controls, as well as a no-template and beta-globin controls. HPV genotype was determined by direct sequencing of PCR fragments and GeneBank BLAST alignment. For methylation assessment, sodium bisulfite modification of DNA (270 ng) was performed using the methylSEQrTM Bisulfite Conversion Kit (Applied Biosystems, Foster City, CA, USA). The modified DNA was used as template for methylation-specific polymerase chain reaction (MSP) using primers specific to methylated and un-methylated sequences. Statistical analyses of associations between variables were performed by the Fisher’s exact test ( p Results HPV (subtype 16) was detected in three out of 45 patients (6%). Promoter hypermethylation of the CDKN2A gene was observed in 24.4% of the cases. Statistical analysis showed an association trend between CDKN2A methylation and nodal involvement ( p = 0.079). Conclusions This study suggesting that methylation of CDKN2A in OSCCs is a potential predictive marker of malignant transformation, independent of HPV infection. Financial Support: CAPES, CNPq and FAPES.

Association of BRCA1 promoter methylation with rs11655505 (c.2265C&gt;T) variants and decreased gene expression in sporadic breast cancer

Breast cancer is the most common cancer and the main cause of cancer morbidity for women worldwide and is manifestation of abnormal genetic as well as epigenetic changes. Therefore, our aim was to study the association of BRCA1 promoter methylation with rs11655505 (c.-2265C/T) variants and gene expression in sporadic breast cancer. Twenty-nine sporadic breast cancer tissues and 26 normal biopsies were used for this study. Genomic DNA and total RNA were extracted from paraffin-embedded tissue and SNP analysis performed. Methylation status of the BRCA1 promoter region was determined by methylation-specific PCR after sodium bisulfite modification of DNA. Among all clinical-pathological parameters only estrogen receptor -ve and +ve samples were significantly different for methylation status (P = 0.04). The genotypic (CC, CT and TT), allelic frequencies and methylation status had not been found to be significantly different from that of healthy controls (P = 0.67, 0.71 and 0.17, respectively). Similarly, methylated BRCA1 promoter was not found to be significantly different in different genotypes from unmethylated promoters between patients and controls. Interestingly, only heterozygous (CT) genotypes with low and normal expression of BRCA1 were significantly different for the differential expression of BRCA1 compared to controls (P = 0.004). However, in tumor samples decreased expression of gene is associated with methylated state of BRCA1 promoter [OR (95 % CI) = 25.09 (2.17-29.75); P = 0.01]. Our data suggest that both single nucleotide variations rs11655505 (c.-2265C/T) and the methylation status of BRCA1 are not associated significantly with the occurrence of sporadic breast cancer in studied population. However, decreased expression of gene is associated with the CT genotypes and the disease. But, in case of tumor samples, an association of methylation of the promoter to the decreased expression of BRCA1 gene suggests the possible role of methylation in gene silencing.

A systematic evaluation of whole genome amplification of bisulfite-modified DNA

BackgroundStudying DNA methylation profiles in detail should be the first step in epigenetic research. Although sodium bisulfite modification of genomic DNA is the gold standard method for DNA methylation analysis, this method results in the loss of the majority of the DNA material. Whole genome amplification (WGA) of bisulfite-modified DNA is expected to provide a rich source of materials, but its validity has not been thoroughly evaluated. In this study, we evaluated the extent of biased amplification in the WGA of bisulfite-modified DNA and the reproducibility of independent WGA reactions. We performed the multiple displacement amplification-based WGA separately three times. Each experiment included two reactions using 10 or 50 ng of bisulfite-modified DNA as template. DNA methylation levels were compared between WGA products and original bisulfite-modified DNA at about 450,000 CpG sites.ResultsUsing a sufficient amount of bisulfite-modified DNA for WGA was critical for downstream application. The considerable deviations from original bisulfite-modified DNA were found in the middle range of DNA methylation levels. Distribution of hyper- and hypomethylation were equal, which suggested that the deviation at each CpG site occurred randomly. Averaging the data from independently amplified WGA products dramatically improved the overall quality.ConclusionsWGA of bisulfite-modified DNA could be a valuable tool for epigenetic research, but careful experimental design and data interpretation are required.

Methylation of the APAF-1 and DAPK-1 promoter region correlates with progression of renal cell carcinoma in North Indian population

Aberrant promoter hypermethylation of cancer associated genes occur frequently during carcinogenesis and may serve as a cancer biomarker. The aim of this study was to investigate the occurrence and relevance of promoter methylation of the tumor suppressor DAPK-1, APAF-1 () and SPARC in relation to different pathological stages and histological grades of tumor progression that might act as possible independent prognostic factor in the susceptibility towards renal cell carcinoma (RCC) in North Indian population. Three tumor suppressor gene promoters namely APAF-1, DAPK-1 and SPARC were assessed by methylation-specific PCR (MS-PCR) in 196 primarily resected renal cell tumors paired with the corresponding normal tissue samples. After genomic DNA isolation and sodium bisulfite modification, methylation levels were determined and correlated with standard clinicopathological parameters, pathological stage and Fuhrman nuclear grade of RCC. Significant differences in methylation frequency among the four subtypes of renal tumors were found for APAF-1 (p < 0.001), DAPK-1 (p < 0.001) and SPARC (p = 0.182), when compared with the corresponding normal tissue. Male subjects showed stronger association of methylation frequency of all the three genes with RCC than the female subjects. Additionally, higher frequency of APAF-1, DAPK-1 and SPARC promoter methylation were directly correlated with higher tumor stage (p (trend) < 0.001). Higher frequency of promoter methylation of APAF-1 and SPARC were also associated with higher nuclear grade (p < 0.001 and p = 0.036, respectively). This gene panel might contribute to a more optimal diagnostic coverage and information, improving preoperative assessment and therapeutic decision-making in patients harboring suspicious renal masses.

Physicochemical Properties of Soy Protein Adhesives Obtained by In Situ Sodium Bisulfite Modification During Acid Precipitation

AbstractSuccessful industrial applications of soy protein adhesives require high adhesion strength and low viscosity at high solid protein concentration. This study examined the effects of β‐conglycinin (7S) and glycinin (11S) ratios on the physicochemical properties of soy protein adhesives. Soy protein adhesives with various 7S/11S ratios were extracted from soy flour slurry modified with sodium bisulfite using the acid precipitation method, which is based on the different solubilities of 7S and 11S globulins. Seven glycinin‐rich soy protein fractions and six β‐conglycinin‐rich soy protein fractions were obtained. The external morphology of the samples changed from the viscous cohesive phase to the clay‐like phase without cohesiveness. The viscous cohesive samples had good flowability and good water resistance with a wet adhesion strength of 2.0–2.8 MPa. They were stable for up to several months without phase separation at room temperature. Based on the results, we suggest that proper protein–protein interaction, hydration capacity (glycinin‐rich soy protein fractions), and certain ratios of 7S and 11S (β‐conglycinin rich soy protein fractions) in the soy protein sample are crucial to continuous protein phase formation. Hydrogen bonding, electrostatic forces, and hydrophobic interactions are involved in maintaining the protein viscous cohesive network, whereas disulfide bonds do not exert significant effects. This study describes a new way to investigate viscous cohesive soy protein systems with high solid protein content, thus alleviating the disadvantages of traditional methods for studying the adhesive properties of soy protein isolates, which tend to have poor water resistance, low solid contents, and short storage life.

LINE-1 Hypomethylation During Primary Colon Cancer Progression

BackgroundMethylation levels of genomic repeats such as long interspersed nucleotide elements (LINE-1) are representative of global methylation status and play an important role in maintenance of genomic stability. The objective of the study was to assess LINE-1 methylation status in colorectal cancer (CRC) in relation to adenomatous and malignant progression, tissue heterogeneity, and TNM-stage.Methodology/Principal FindingsDNA was collected by laser-capture microdissection (LCM) from normal, adenoma, and cancer tissue from 25 patients with TisN0M0 and from 92 primary CRC patients of various TNM-stages. The paraffin-embedded tissue sections were treated by in-situ DNA sodium bisulfite modification (SBM). LINE-1 hypomethylation index (LHI) was measured by absolute quantitative analysis of methylated alleles (AQAMA) realtime PCR; a greater index indicated enhanced hypomethylation. LHI in normal, cancer mesenchymal, adenoma, and CRC tissue was 0.38 (SD 0.07), 0.37 (SD 0.09), 0.49 (SD 0.10) and 0.53 (SD 0.08), respectively. LHI was significantly greater in adenoma tissue compared to its contiguous normal epithelium (P = 0.0003) and cancer mesenchymal tissue (P<0.0001). LHI did not differ significantly between adenoma and early cancer tissue of Tis stage (P = 0.20). LHI elevated with higher T-stage (P<0.04), was significantly greater in node-positive than node-negative CRC patients (P = 0.03), and was significantly greater in stage IV than all other disease stages (P<0.05).Conclusion/SignificanceBy using in-situ SBM and LCM cell selection we demonstrated early onset of LINE-1 demethylation during adenomatous change of colorectal epithelial cells and demonstrated that LINE-1 demethylation progression is linear in relation to TNM-stage progression.

Epigenetic Regulation of MicroRNA Expression In Hodgkin Lymphoma

AbstractAbstract 3885 Background:In recent years, microRNAs (miRNAs) have emerged as key regulators of carcinogenesis. miRNA expression is deregulated in multiple hematological malignancies. One of the mechanisms that can affect miRNA expression is methylation in the promoter regions of miRNA genes. The main objective of the present study was to identify tumor suppressor miRNAs that are silenced by alterations in gene methylation in Hodgkin Lymphoma (HL). In addition, since demethylating agents could affect miRNA expression, we evaluated the in vitro effectiveness of 5-Aza-2-deoxycytidine (AZA), a DNA methyltransferase inhibitor, in HL cell lines. Methods:To detect miRNAs regulated by methylation, we analyzed the expression of 670 mature miRNAs in two HL cell lines, L-428 and L-1236, before and after AZA treatment, by TaqMan Human MicroRNA Arrays V2.0 (Applied Biosystems) in an ABI 7900 HT sequence detection system. miRNA expression data was analyzed by the 2-ΔΔCt method using RNU48 as endogenous control. To validate the methylation status, genomic DNA samples from four HL cell lines (L-428, L-1236, HDMYZ and L-540) and peripheral B-cells from healthy donors were modified by sodium bisulfite using the EZ DNA Methylation kit (Zymo Research). The DNA methylation status of these samples was analyzed by methylation specific PCR (MSP) after sodium bisulfite modification of DNA. To evaluate AZA in vitro effectiveness, we treated the four HL cell lines daily with 20 nM, 250 nM, 1 μM and 5 μM of AZA or DMSO (vehicle control), and we assayed proliferation with CellTiter 96 AQueous One Solution Cell Proliferation Assay (MTS) after 72 hours of treatment. Results:After AZA treatment in the L-428 and L-1236 HL cell lines, 32 miRNAs (of 670 analyzed) were expressed de novo, 15 of which were present in both cell lines. miR-34a, miR-203, miR-342, miR-105, miR-490, miR-375 and the miR-500 family had well-defined CpG islands in their promoter regions. The MSP analysis showed that miR-34a, miR-203, miR-490 and miR-375 were methylated in L-428 and L-1236 HL cell lines, but not in the normal B-cells. miR-342 was methylated in L-1236 HL cell line, but not in L-428 nor normal B-cells. To further validate this we analyzed the methylation pattern in HDMYZ and L-540 HL cell lines and found that only miR-203, miR-375 and miR-490 were methylated in all 4 HL cell lines analyzed. We observed an AZA dose-dependent reduction in proliferation in all four HL cell lines. After 72 hours of treatment with AZA at 5μM, proliferation significantly decreased by 29% in L-428, 36% in L-1236, 28% in HDMYZ and 22% in L-540. Moreover, the analysis of miRNA levels during the treatment showed a re-expression of methylated miRNAs after 48 hours. The methylation analysis of these miRNAs in laser captured microdisected HL cells from patients and functional analysis are ongoing and complete data will be reported. Conclusions:In summary, HL exhibits a characteristic epigenetic pattern which includes the methylation of key miRNAs during the carcinogenesis process, such as miR-34a and miR-203, which have previously been shown to act as tumor suppressors in lung, colorectal and other cancers. Moreover, we present evidence that demethylating agents allow the re-expression of these tumor suppressor miRNAs, suggesting that they could be a promising novel treatment for HL patients.Supported by FIS grant (PS09/00547). Disclosures:No relevant conflicts of interest to declare.

Cancer/Testis Antigen CAGE Exerts Negative Regulation on p53 Expression through HDAC2 and Confers Resistance to Anti-cancer Drugs

The role of the cancer/testis antigen CAGE in drug resistance was investigated. The drug-resistant human melanoma Malme3M (Malme3M(R)) and the human hepatic cancer cell line SNU387 (SNU387(R)) showed in vivo drug resistance and CAGE induction. Induction of CAGE resulted from decreased expression and thereby displacement of DNA methyltransferase 1(DNMT1) from CAGE promoter sequences. Various drugs induce expression of CAGE by decreasing expression of DNMT1, and hypomethylation of CAGE was correlated with the increased expression of CAGE. Down-regulation of CAGE in these cell lines decreased invasion and enhanced drug sensitivity resulting from increased apoptosis. Down-regulation of CAGE also led to decreased anchorage-independent growth. Down-regulation of CAGE led to increased expression of p53, suggesting that CAGE may act as a negative regulator of p53. Down-regulation of p53 enhanced resistance to drugs and prevented drugs from exerting apoptotic effects. In SNU387(R) cells, CAGE induced the interaction between histone deacetylase 2 (HDAC2) and Snail, which exerted a negative effect on p53 expression. Chromatin immunoprecipitation assay showed that CAGE, through interaction with HDAC2, exerted a negative effect on p53 expression in Malme3M(R) cells. These results suggest that CAGE confers drug resistance by regulating expression of p53 through HDAC2. Taken together, these results show the potential value of CAGE as a target for the development of cancer therapeutics.