Number Of Methylated CpG Sites Research Articles

Epigenetic Regulation of Phenotypic Sexual Plasticity Inducing Skewed Sex Ratio in Zebrafish.

The plasticity of sexual phenotype in response to environmental conditions results in biased sex ratios, and their variation has an effect on population dynamics. Epigenetic modifications can modulate sex ratio variation in species, where sex is determined by genetic and environmental factors. However, the role of epigenetic mechanisms underlying skewed sex ratios is far from being clear and is still an object of debate in evolutionary developmental biology. In this study, we used zebrafish as a model animal to investigate the effect of DNA methylation on sex ratio variation in sex-biased families in response to environmental temperature. Two sex-biased families with a significant difference in sex ratio were selected for genome-wide DNA methylation analysis using reduced representation bisulfite sequencing (RRBS). The results showed significant genome-wide methylation differences between male-biased and female-biased families, with a greater number of methylated CpG sites in testes than ovaries. Likewise, pronounced differences between testes and ovaries were identified within both families, where the male-biased family exhibited a higher number of methylated sites than the female-biased family. The effect of temperature showed more methylated positions in the high incubation temperature than the control temperature. We found differential methylation of many reproduction-related genes (e.g., sox9a, nr5a2, lhx8a, gata4) and genes involved in epigenetic mechanisms (e.g., dnmt3bb.1, dimt1l, hdac11, h1m) in both families. We conclude that epigenetic modifications can influence the sex ratio variation in zebrafish families and may generate skewed sex ratios, which could have a negative consequence for population fitness in species with genotype-environment interaction sex-determining system under rapid environmental changes.

Extent, pattern, and prognostic value of MGMT promotor methylation: does it differ between glioblastoma and IDH-wildtype/TERT-mutated astrocytoma?

IntroductionThe cIMPACT-NOW update 6 first introduced glioblastoma diagnosis based on the combination of IDH-wildtype (IDHwt) status and TERT promotor mutation (pTERTmut). In glioblastoma as defined by histopathology according to the WHO 2016 classification, MGMT promotor status is associated with outcome. Whether this is also true in glioblastoma defined by molecular markers is yet unclear.MethodsWe searched the institutional database for patients with: (1) glioblastoma defined by histopathology; and (2) IDHwt astrocytoma with pTERTmut. MGMT promotor methylation was analysed using methylation-specific PCR and Sanger sequencing of CpG sites within the MGMT promotor region.ResultsWe identified 224 patients with glioblastoma diagnosed based on histopathology, and 54 patients with IDHwt astrocytoma with pTERTmut (19 astrocytomas WHO grade II and 38 astrocytomas WHO grade III). There was no difference in the number of MGMT methylated tumors between the two cohorts as determined per PCR, and also neither the number nor the pattern of methylated CpG sites differed as determined per Sanger sequencing. Progression-free (PFS) and overall survival (OS) was similar between the two cohorts when treated with radio- or chemotherapy. In both cohorts, higher numbers of methylated CpG sites were associated with favourable outcome.ConclusionsExtent and pattern of methylated CpG sites are similar in glioblastoma and IDHwt astrocytoma with pTERTmut. In both tumor entities, higher numbers of methylated CpG sites appear associated with more favourable outcome. Evaluation in larger prospective cohorts is warranted.

PATH-32. EXTENT, PATTERN, AND PROGNOSTIC VALUE OF MGMT PROMOTOR METHYLATION: DOES IT DIFFER BETWEEN GLIOBLASTOMA AND IDH-WILDTYPE/TERT-MUTATED ASTROCYTOMA?

Abstract INTRODUCTION The cIMPACT-NOW update 6 introduced glioblastoma diagnosis based on the combination of IDH-wildtype (IDHwt) status and TERT promotor mutation (pTERTmut). In glioblastoma as defined by histopathology according to the WHO 2016 classification, MGMT promotor status is associated with outcome. Whether this is also true in glioblastoma defined by molecular markers is yet unclear. METHODS We searched the institutional database for patients with: 1.) glioblastoma defined by histopathology; and 2.) IDHwt astrocytoma with pTERTmut. MGMT promotor methylation was analysed using methylation-specific PCR and Sanger sequencing of CpG sites within the MGMT promotor region. RESULTS We identified 224 patients with glioblastoma diagnosed based on histopathology, and 71 patients with IDHwt astrocytoma with pTERTmut (32 astrocytomas WHO grade II and 39 astrocytomas WHO grade III). There was no difference in the number of MGMT methylated tumors between the two groups as determined per PCR, and also neither the number nor the pattern of methylated CpG sites differed as determined per Sanger sequencing. Progression-free (PFS) and overall survival (OS) was similar between the two groups. Surgery was associated with improved overall survival in IDHwt astrocytoma with pTERTmut. In patients treated with radiochemotherapy or radiotherapy, higher numbers of methylated CpG sites were associated with favourable outcome in both groups. CONCLUSION Extent and pattern of methylated CpG sites are similar in glioblastoma and IDHwt astrocytoma with pTERTmut. In both groups, higher numbers of methylated CpG sites are associated with favourable outcome when radio/chemotherapy is administered. Surgery may form the basis for favourable outcome.

BIOM-07. QUANTITATIVE MGMT PROMOTER METHYLATION INDEX INDICATES A NON-LINEAR PROGNOSTIC EFFECT IN GLIOBLASTOMA, SUGGESTING THAT USE OF OPTIMAL CUTOFF POINTS MAY BE CLINICALLY DISADVANTAGEOUS

Abstract BACKGROUND Epigenetic inhibition of the O6-methylguanine-DNA-methyltransferase (MGMT) gene has emerged as a clinically relevant prognostic marker in glioblastoma (GBM). Methylation of the MGMT promoter has been shown to increase chemotherapy efficacy. While traditionally reported as a binary marker, recent methodological advancements have led to quantitative approaches that measure methylation, providing clearer insights into methylation’s functional relationship with survival. METHODS A CLIA assay and bisulfite sequencing was utilized to develop a quantitative, 17-point MGMT promoter methylation index derived from the number of methylated CpG sites. Retrospective review of 240 newly diagnosed GBM patients was performed in order to discern how risk for mortality transforms as promoter methylation increases. Non-linearities were captured by fitting splines to Cox proportional hazard models, plotting smoothed residuals, and creating survival plots. Covariates included age, KPS, IDH1 mutation, and extent of resection. RESULTS Median follow-up time and progression free survival were 16 and 9 months, respectively. 176 subjects experienced death. A one-unit increase in CpG methylation on a scale of 1-17 resulted in a 4% reduction in hazard (95% CI 0.93–0.99, P< 0.005). Moreover, GBM patients with low-levels of methylation (1-6 CpG sites) fared markedly worse (HR=1.62, 95% CI 1.03-2.54, P< 0.036) than individuals who were unmethylated (reference group). Subjects with medium-levels of methylation (7-12 CpG sites) had the greatest reduction in hazard (HR=0.48, 95% CI 0.29-0.80, P< 0.004), followed by individuals in the highest methylation tertile (HR=0.62, 95% CI 0.40-0.97, P< 0.035). CONCLUSION This novel approach offers greater bisulfite conversion efficiency when compared to alternative methods, reducing the likelihood of false positives. Analysis of the resulting methylation index scores demonstrates a non-linear relationship between MGMT methylation and survival, suggesting conformation of the marker’s protective effect. These findings challenge the current understanding of MGMT’s functional form and underline why implementing an “optimal cutoff point” may be disadvantageous.

NGMA-6. Quantitative MGMT Promoter Methylation Index Indicates Non-Linear, Prognostic Effect in Glioblastoma

BackgroundEpigenetic inhibition of the O6-methylguanine-DNA-methyltransferase (MGMT) gene has emerged as a clinically relevant prognostic marker in glioblastoma (GBM). Methylation of the MGMT promoter has been shown to increase chemotherapy efficacy. While traditionally reported as a binary marker, recent methodological advancements have led to quantitative methods of measuring methylation, allowing for clearer insights into methylation’s functional relationship with survival.MethodsA CLIA assay and bisulfite sequencing was utilized to develop a quantitative, 17-point MGMT promoter methylation index derived from the number of methylated CpG sites. Retrospective review of 242 newly diagnosed GBM patients was performed in order to discern how risk for mortality transforms as promoter methylation increases. Non-linearities were captured by fitting splines to Cox proportional hazard models, plotting smoothed residuals, and creating survival plots. Covariates included age, KPS, IDH1 mutation, and extent of resection.ResultsMedian follow-up time and progression free survival were 15.9 and 9 months, respectively. 176 subjects experienced death. A one-unit increase in CpG methylation resulted in a 4% reduction in hazard (95% CI 0.93–0.99, P<0.005). Moreover, GBM patients with low levels of methylation (1–6 CpG islands) fared markedly worse (HR=1.62, 95% CI 1.03–2.54, P<0.036) than individuals who were unmethylated (reference group). Subjects with medium levels of methylation (7–12 sites) had the greatest reduction in hazard (HR=0.48, 95% CI 0.29–0.80, P<0.004), followed by individuals in the highest methylation tertile (HR=0.62, 95% CI 0.40–0.97, P<0.035).ConclusionThe extent of MGMT methylation shares a non-linear relationship with survival, suggesting conformation of the marker’s protective effect. This finding challenges the current understanding of MGMT and underlines the clinical importance of determining its prognostic utility. Potential limitations include censoring, sample size, and extraneous mutations. Future research is warranted to examine whether the location of CpG site methylation contributes to a reduction in mortality hazard.

The number of methylated CpG sites within the MGMT promoter region linearly correlates with outcome in glioblastoma receiving alkylating agents

MGMT-promoter methylation is associated with favorable outcome in glioblastoma. The aim of this study was to determine whether the absolute number of methylated Cytosine-Guanine-dinucleotide-(CpG-)sites within the DMR-2 island of the MGMT-promoter may correlate with outcome in a qualitative or quantitative fashion. In a cohort of newly diagnosed glioblastoma patients treated with stereotactic biopsy or open tumor resection plus concomitant chemoradiotherapy, we assessed MGMT-promoter methylation by methylation-specific polymerase-chain-reaction (MSP). Methylation of the CpG-sites 74–98 within the MGMT-promoter region was additionally analysed by Sanger sequencing, and the total number of methylated CpG-sites was correlated with outcome using proportional hazards models. 215 patients with glioblastoma were identified and stratified per MSP (positive: 53%, negative: 47%). Among MSP-positive tumors, hierarchical clustering identified three subgroups with different methylation rates (median: 80% vs. 52% vs. 47%), indicating a site-dependent methylation propagation. The methylation status of a given CpG-site indicated a neighborhood-dependent methylation propagation. Survival was linearly associated with the cumulative number of methylated CpG-sites. This was particularly true in patients who received at least one adjuvant cycle of temozolomide. Notably, all CpG-sites analyzed contributed similarly to effect size; this enabled a further predictive substratification of MSP-positive tumors with median OS ranging from as low as 17.1 months (< 18 methylated CpG-sites) to as high as 26.2 months (≥ 18 methylated CpG-sites) in the overall cohort. All in all, total number of methylated CpG-sites may correlate with outcome in a linear fashion. Such analysis may therefore add further predictive value to conventional methods of determining the MGMT-promoter status.

Extent and prognostic value of MGMT promotor methylation in glioma WHO grade II

MGMT promotor methylation is associated with favourable outcome in high-grade glioma. In glioma WHO grade II, it is unclear whether the extent of MGMT promotor methylation and its prognostic role is independent from other molecular markers. We performed a retrospective analysis of 155 patients with glioma WHO grade II. First, all 155 patients were assigned to three molecular groups according to the 2016 WHO classification system: (1) oligodendroglioma, IDH-mutant and 1p19q co-deleted (n = 81); (2) astrocytoma, IDH-mutant and 1p19q non-codeleted (n = 54); (3) astrocytoma, IDH-wildtype (n = 20). MGMT promotor methylation was quantified using Sanger sequencing of the CpG sites 74–98 within the MGMT promotor region. Highest numbers of methylated CpG sites were found for oligodendroglioma, IDH-mutant and 1p19q co-deleted. When 1p19q co-deletion was absent, numbers of methylated CpG sites were higher in the presence of IDH-mutation. Accordingly, lowest numbers were seen in the IDH-wildtype subpopulation. In the entire cohort, larger numbers of methylated CpG sites were associated with favourable outcome. When analysed separately for the three WHO subgroups, a similar association was only retained in astrocytoma, IDH-wildtype. Collectively, extent of MGMT promotor methylation was strongly associated with other molecular markers and added prognostic information in astrocytoma, IDH-wildtype. Evaluation in prospective cohorts is warranted.

PATH-28. EXTENT AND PROGNOSTIC VALUE OF MGMT PROMOTOR METHYLATION DEPEND ON IDH MUTATION AND 1p19q CO-DELETION IN GLIOMA WHO GRADE II

Abstract INTRODUCTION Methylation of the promotor region of the O6-methylguanin-DNA-methyltransferase (MGMT) gene is associated with increased survival in low- and high-grade glioma. It is unknown whether this association also applies to the 2016 WHO categories of glioma WHO grade II. MATERIAL AND METHODS We retrospectively searched the institutional database of the Center for Neuro-Oncology for patients with glioma WHO grade II. Patients were assigned to one of three groups according to the 2016 WHO classification system: 1. 1p19q co-deleted oligodendroglioma, IDH mutant; 2. 1p19q non-codeleted astrocytoma, IDH mutant; 3. 1p19q non-codeleted astrocytoma, IDH wild-type. MGMT methylation status was analysed using Sanger sequencing of the CpG sites 74-98 within the MGMT promotor region. The total number of methylated CpG sites was calculated for each patient. RESULTS 155 patients with glioma WHO grade II were encountered, including 81 1p19q co-deleted, IDH mutant oligodendrogliomas; 54 IDH mutant astrocytomas; and 20 IDH wild-type astrocytomas. The mean number of methylated CpG sites among oligodendrogliomas was significantly higher when compared to IDH mutant astrocytomas (18.9 ± 0.4 vs. 16.3 ± 0.6; p = 0.001). In turn, the number of methylated CpG sites among IDH mutant astrocytomas was higher when compared to IDH wild-type astrocytomas (16.3 ± 0.6 vs. 12.3 ± 1.9; p = 0.007). Median follow-up was estimated at 35 months. Median time to malignant progression was 87 months for all patients, and median overall survival was not reached. In the entire cohort, a larger number of methylated CpG sites was prognostic of overall survival and time to malignant progression. When analysed separately for the three WHO subgroups, a similar association was only retained in IDH wild-type astrocytoma. CONCLUSION In our series of WHO II gliomas, MGMT promotor methylation appeared strongly associated with 1p19q codeletion and IDH mutations. MGMT promotor methylation was only prognostic in IDH wild-type astrocytoma.

Alleviation of Symptoms and Improvement of Endometrial Receptivity Following Laparoscopic Adenomyoma Excision and Secondary Therapy with the Levonorgestrel-releasing Intrauterine System.

To assess the treatment efficacy of adenomyoma and improvement in implantation receptivity associated with the levonorgestrel-releasing intrauterine system (LNG-IUS) combined with adenomyoma resection. Fifty subjects (control group) underwent laparoscopic adenomyoma excision and received gonadotropin-releasing hormone (GnRH) agonists, and 201 subjects (experimental group) underwent laparoscopic adenomyoma resection and received GnRH agonists combined with the LNG-IUS. Pipelle endometrial biopsies were obtained in the mid-luteal phase, before the operation, and 19months after the operation. Menstrual blood loss was measured using a pictorial blood loss assessment chart. Pain intensity during menstruation was evaluated on a 10-point visual analog scale (VAS). The volume of uterus was measured through ultrasound. Quantification of HOXA10 promoter methylation was performed through bisulfite sequencing polymerase chain reaction (BSP). Real-time polymerase chain reaction analyzed the expression levels of endometrial HOXA10-mRNA and leukemia inhibitory factor (LIF)-mRNA. After surgery, the scores for dysmenorrhea and menorrhagia were significantly improved, and the volume of the uterus was obviously reduced (all p < 0.01). The mean number of methylated CpG sites, level of endometrial HOXA10-mRNA, and level of endometrial LIF-mRNA were 5.6 ± 1.7 versus 3.9 ± 1.3, 0.8 ± 0.2 versus 0.9 ± 0.3, and 0.8 ± 0.2 versus 1.0 ± 0.2, respectively, in the control group versus the experimental group at 19months after surgery, and significant improvements were observed in the experimental group (p < 0.001, p = 0.034, p < 0.001). Laparoscopic adenomyoma excision and GnRH agonists can alleviate the symptoms, reduce the number of methylated CpG sites in HOXA10, and improve endometrial HOXA10-mRNA and endometrial LIF-mRNA levels. When combined with subsequent use of the LNG-IUS, better efficacy can be achieved.

P14.02 Influence of individual CpG methylation status on outcome in adult patients with glioblastoma multiforme receiving alkylating agent treatment

Abstract Background: Methylation of the O-6-methylguanine-DNA methyltransferase (MGMT) promotor causes gene silencing and has been associated with a favourable prognosis in patients with glioblastoma multiforme (GBM) receiving alkylating chemotherapy. However, analysis of MGMT promotor methylation is usually reported as a cut-off depending on the results of the correspondent CpG site testing. This approach disregards a possible heterogeneity concerning the methylation status within the individual CpG sites and its possible association with prognosis in GBM patients. The current study aimed at elucidating the association between methylation of CpG sites 74–98 within the MGMT promotor region and outcome in GBM patients receiving alkylating agents. Material and Methods: Individual methylation status of 230 patients with histologically proven GBM following concomitant radio-chemotherapy with TMZ after stereotactic biopsy or open tumor resection (OTR) was assessed by the Sanger sequencing (Sseq) approach. Methylation of CpG sites 74–98 within the MGMT promotor region was defined according to a ratio of cytosine /thymine peak &gt;50%. The total number of methylated CpG sites as well as clinical factors such as age, Karnofsky Performance Score (KPS) and mode of surgical procedure were correlated with outcome using proportional hazards models. In a subset of 34 patients, a correlation between individual CpG methylation and MGMT mRNA expression was performed. Results: Median progression-free (PFS) and overall survival (OS) were 7.8 and 14.6 months, respectively. Alongside younger age, KPS&gt; 80 and OTR, the cumulative total number of methylated loci within the CpG sites 74–98 was strongly associated with both PFS and OS and retained its prognostic influence on outcome in multivariate models (p &lt;0.001). Furthermore, a linear coherence between the total number of methylated CpG sites 74–98 and survival parameters could be observed. Moreover, low number of methylated CpG sites was observed in tumor specimen with a high mRNA expression and vice versa (Spearman correlation coefficient: -0.62). Conclusion: In contrast to the concept of dichotomizing the MGMT promotor status into ‘methylated’ and ‘non-methylated’, our approach shows a clear heterogeneity within the methylation status of the CpG sites 74–98 within the GBM tumor specimens. Our data suggest a strong correlation between outcome and the total number of methylated CpG sites, thus an up-front analysis of the individual GpC site methylation status prior to initiation of alkylating chemotherapy might help to improve treatment response in GBM patients.

Abstract 116: Epigenetic Regulation of Human Angiotensinogen (hAGT) Gene by SNPs.

Hypertension is a complex disease caused by a combination of genetic and environmental factors. Renin angiotensin system (RAS) dysfunction leads to essential hypertension. Polymorphisms in the AGT gene increase RAS activity and cause blood pressure elevation. We and others have shown that SNPs in the promoter of hAGT gene can be arranged mainly in two haplotypes. Variants -1670A, -1562C, and -1561T always occur with -6A and form the haplotype-l (Hap-l), while variants -1670G, -1562G, and -1561G always occur with -6G and constitute haplotype-ll (Hap-ll). We have made transgenic animals containing these two haplotype blocks and shown that Haplotype-I is the risk haplotype and increases hAGT expression in TG animals. We hypothesized that these SNPs, when present together as Hap-I or Hap-II in transgenic mice (TG) may lead to haplotype dependent DNA methylation of CpG sites in the promoter of the hAGT gene. Methylation patterns may modulate gene transcription at an epigenetic level and this, in turn, could be dependent on individual polymorphisms. Hypo-methylation is associated with higher gene expression and hyper methylation is associated with lower gene expression. We have found that the number of methylated CpG sites in the hAGT promoter is decreased after high salt (HS) treatment in the liver of Hap-I TG animals as compared to the liver of Hap-I animals. In the liver of Hap-I animals, the hAGT promoter CpG sites are methylated at -460,-434,-386,-261,+42, whereas after high salt treatment, the promoter DNA methylation is observed at -460,-386,+65. On the other hand methylation of hAGT promoter CpG sites in the liver of Hap-II are increased as compared to the liver of Hap-II after high salt treatment. In the liver of Hap-II animals, the CpG sites are methylated at -386,-346,+65, whereas after high salt treatment, methylate sites are observed at -386,-261. Results of this experiment show that methylation of CpG sites is reduced in the DNA obtained from high salt treated liver of both haplotypes. However, DNA from high salt treated liver of Hap-II animals had least number of methylated CpG sites and therefore open chromatin that leads to higher expression of the hAGT gene in this haplotype.

Effect of O6-methylguanine-DNA methyltransferase methylation in medulloblastoma.

Medulloblastoma is a highly malignant brain tumor that predominately affects children and requires multimodal treatment, including chemotherapy with alkylating agents. O6-methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that plays an important role in tumor resistance to alkylating agents. Recent studies demonstrated that MGMT promoter methylation suppresses the expression of MGMT and is associated with favorable outcomes of malignant glioma patients. However, the MGMT methylation status and its prognostic impact on medulloblastoma have not been fully elucidated to date. The objective of the present study was to investigate the association between MGMT status and clinical outcomes of pediatric medulloblastoma patients. The records of 15 patients with medulloblastoma treated at our institution were reviewed, and the methylation status of 18 CpG sites in the MGMT promoter region was determined using bisulfite sequencing analysis. A larger number of methylated CpG sites was identified in 9 patients with complete remission (median, 5 sites; range, 2-9 sites) compared with that in 6 patients with relapse (median, 2 sites, range, 1-4 sites; P=0.041). These results suggest that a higher number of methylated CpG sites in the MGMT promoter region are associated with a favorable outcome of medulloblastoma.

Estrogen-mediated downregulation of AIRE influences sexual dimorphism in autoimmune diseases.

Autoimmune diseases affect 5% to 8% of the population, and females are more susceptible to these diseases than males. Here, we analyzed human thymic transcriptome and revealed sex-associated differences in the expression of tissue-specific antigens that are controlled by the autoimmune regulator (AIRE), a key factor in central tolerance. We hypothesized that the level of AIRE is linked to sexual dimorphism susceptibility to autoimmune diseases. In human and mouse thymus, females expressed less AIRE (mRNA and protein) than males after puberty. These results were confirmed in purified murine thymic epithelial cells (TECs). We also demonstrated that AIRE expression is related to sexual hormones, as male castration decreased AIRE thymic expression and estrogen receptor α-deficient mice did not show a sex disparity for AIRE expression. Moreover, estrogen treatment resulted in downregulation of AIRE expression in cultured human TECs, human thymic tissue grafted to immunodeficient mice, and murine fetal thymus organ cultures. AIRE levels in human thymus grafted in immunodeficient mice depended upon the sex of the recipient. Estrogen also upregulated the number of methylated CpG sites in the AIRE promoter. Together, our results indicate that in females, estrogen induces epigenetic changes in the AIRE gene, leading to reduced AIRE expression under a threshold that increases female susceptibility to autoimmune diseases.

Epigenetic mechanisms of peptidergic regulation of gene expression during aging of human cells.

Expression levels of genes encoding specific transcription factors and other functionally important proteins vary upon aging of pancreatic and bronchial epithelium cell cultures. The peptides KEDW and AEDL tissue-specifically affect gene expression in pancreatic and bronchial cell cultures, respectively. It is established in this work that the DNA methylation patterns of the PDX1, PAX6, NGN3, NKX2-1, and SCGB1A1 gene promoter regions change upon aging in pancreatic and bronchial cell cultures in correlation with variations in their expression levels. Thus, stable changes in gene expression upon aging of cell cultures could be caused by changes in their promoter methylation patterns. The methylation patterns of the PAX4 gene in pancreatic cells as well as those of the FOXA1, SCGB3A2, and SFTPA1 genes in bronchial cells do not change upon aging and are unaffected by peptides, whereas their expression levels change in both cases. The promoter region of the FOXA2 gene in pancreatic cells contains a small number of methylated CpG sites, their methylation levels being affected by cell culture aging and KEDW, though without any correlation with gene expression levels. The promoter region of the FOXA2 gene is completely unmethylated in bronchial cells irrespective of cell culture age and AEDL action. Changes in promoter methylation might be the cause of age- and peptide-induced variations in expression levels of the PDX1, PAX6, and NGN3 genes in pancreatic cells and NKX2-1 and SCGB1A1 genes in bronchial cells. Expression levels of the PAX4 and FOXA2 genes in pancreatic cells and FOXA1, FOXA2, SCGB3A2, and SFTPA1 genes in bronchial cells seem to be controlled by some other mechanisms.

Abstract 991: Integrated analysis of gene expression, DNA copy number, and CpG island methylation of 5-fluorouracil-resistant human gastric cancer cell lines.

Abstract Background: A major problem in chemotherapy is treatment failure due to anticancer drug resistance. A better understanding of mechanisms underlying acquired resistance may facilitate the development of an optimal second-line therapy for recurrent cancer. This study was designed to elucidate mechanisms underlying acquisition of 5-fluoroucacil (5-FU) resistance by human gastric cancer cells. Method: 5-FU resistant cell lines were established by continuously exposing parent cell lines (MKN45, MKN-74, KATO-III, and NCI-N87) to escalating concentrations (1-5 μM) of 5-FU over 1 year. To elucidate mechanisms of resistance, gene expression, DNA copy number, and DNA methylation were measured by Whole Human Genome 44K oligo DNA microarray, 244K CGH microarray, and 244K CpG Island Microarray (Agilent Technologies, Santa Clara, CA), respectively. Data analysis was done with GeneSpring GX™ software, version 11.0 (Agilent Technologies) and Partek genomic suite software, version 6.5 (Partek Inc., St. Louis, MO). Results: Each of the cell lines acquired high resistance to 5-FU, although the degree of resistance varied. IC50 of the 5-FU-resistant cell lines was 3.8- to 11.6-fold higher than that of the parent cell lines. On gene expression analysis, 3.0%, 2.3%, 1.4%, and 1.4% of 41,093 probes were differentially expressed with more than 5-fold changes in MKN45/FU, KATOIII/FU, NCI-N87/FU, and MKN74/FU as compared with their parental cell lines, respectively. Changes occurring in at least 3 cell lines were expressed by only 0.2% of probes. Copy number analysis revealed that DNA copy gains occurred at 16.6%, 6.7%, 5.1%, and 5.6% of all genomic segments elucidated in MKN45/FU, KATOIII/FU, NCI-N87/FU and MKN74/FU, respectively. The frequencies of DNA copy losses were 35.6%, 14.0%, 3.2%, and 8.9%, respectively. Methylation analysis revealed that the number of methylated CpG sites in NCI-N87/FU and MKN74/FU increased by 1.5 times as compared with the parental cell lines. In contrast, the numbers of methylated CpG sites in MKN45/FU and KATOIII/FU rarely changed (around 40% each). Conclusions: Mechanisms leading to acquired resistance against 5-FU differed among the 4 gastric cancer cell lines. Changes in copy number most frequently occurred in MKN45/FU cells. In NCI-N87/FU and MKN74/FU, the numbers of methylated CpG sites increased. Such genome-wide integrated analysis will facilitate an understanding of mechanisms for 5-FU acquired resistance and help to identify therapeutic target genes. Citation Format: Takashi Kobunai, Ayako Nakamura, Mamoru Nukatsuka, Kazuhiko Hayashi, Teiji Takechi. Integrated analysis of gene expression, DNA copy number, and CpG island methylation of 5-fluorouracil-resistant human gastric cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 991. doi:10.1158/1538-7445.AM2013-991

Molecular mechanism of an individual with weaken B phenotype in ABO blood group

To elucidate the serological characteristics and molecular mechanism of a blood donor with weaken B antigen. The ABO blood group antigens on red blood cells were identified by monoclonal antibodies, the ABO antibodies in serum were detected by standard A, B, O cells and the activity of the B glycosyltransferase was analyzed. The full-length sequence and 5'-untranslated region (5'-UTR) sequence of ABO gene were amplified by polymerase chain reaction (PCR) respectively and direct sequencing. The alternative splicing isoforms of ABO cDNA were obtained by reverse transcription-PCR(RT-PCR) and analyzed with cloning and sequencing techniques. The level of methylation of the CpG island in ABO gene promoter was analyzed by bisulfite sequencing method. The serological characteristic of the donor showed that the B antigen was decreased obviously without anti-B antibodies in serum and the B glycosyltransferase activity was decreased as well. The genotype of the donor was B101/O01 without any other mutations in the full-length coding sequences and splice receptor sites. The nucleotide characteristics of the 5'-UTR was consistent with B101/O01 and no any abnormity was identified in the promoter, enhancer and the negative regulatory sequence regions. The integrative cDNA transcript of ABO gene was obtained and no new splicing isoform was found. Compared with the normal B phenotype, a number of methylated CpG sites were found near the promoter of ABO gene in this sample. The methylation in the CpG island of ABO gene promoter region may cause weak expression of the B antigen.

An epigenetic mechanism for capecitabine resistance in mesothelioma

Mesothelioma is an uncommon malignancy whose global incidence continues to rise. The therapeutic standard for advanced disease is intravenous pemetrexed and cisplatin. The anti-folate capecitabine is significantly less effective than pemetrexed. The balance between thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD), and thymidine phosphorylase (TP) is critical to the efficacy of capecitabine. DNA from mesothelioma cell lines was bisulfite treated and examined by MS-PCR, RNA was obtained for real-time PCR analysis, and protein lysates were obtained for Western immunoblot analysis. Cytotoxicity was assessed by MTT assay, comparing 5-aza-CdR pretreated or untreated cells with 5′-deoxy-5-fluorouridine (DFUR), 5-FU, and pemetrexed. Finally bisulfite sequencing of the extracellular growth factor-1 ( ECGF-1) gene was performed on 4 mesothelioma samples and pericardial tissue. One of the four cell lines tested (H290) was methylated for ECGF-1. This corresponded to a lack of TP expression by real-time PCR and Western immunoblot. Treatment with 1 μM 5-aza-CdR increased TP mRNA and protein expression in H290. DFUR, the substrate for TP, showed increased cytotoxicity when delivered after 5-aza-CdR exposure in the methylated cell line. There was no difference in any of the unmethylated cell lines when cells were exposed to 5-FU or pemetrexed with or without 5-aza-CdR. Patient tumor samples revealed an increased number of methylated CpG sites in ECGF-1 compared to normal pericardium. Methylation of ECGF-1, leads to transcriptional silencing of TP and may explain the lack of any effect of capecitabine, especially when compared to pemetrexed.

Increased number of methylated CpG islands correlates with Helicobacter pylori infection, histological and serological severity of chronic gastritis

Promoter hypermethylation of tumor suppressor genes is one of the major events in gastric carcinogenesis. Promoter hypermethylation is also present in non-neoplastic gastric epithelium as age-related phenomenon and some reports suggest the potential association between promoter hypermethylation and Helicobacter pylori infection. Here, we examined whether methylation of multiple promoter CpG islands would occur by H. pylori infection and correlate with histological or serological severity of chronic gastritis. One hundred and ninety-one gastric mucosa samples were obtained by endoscopy. The promoter methylation status of the p14, p16, DAP-kinase and CDH1 genes were determined by methylation-specific-polymerase chain reaction. The degree of gastritis in the antrum was assessed according to the updated Sydney system in 150 participants. The pepsinogen (PG) I/II ratio was calculated based on the data of serum PG I and PG II levels measured by radioimmunoassay in 54 selected cases. CpG island methylation was found in 32.5% for p14, 35.1% for p16, 43.5% for DAP-kinase and 36.1% for CDH1, whereas non, 1, 2, 3, and all methylation of four promoter CpG sites were present in 46 (24.1%), 59 (30.9%), 46 (24.1%), 30 (15.7%), and 10 (5.2%) participants, respectively. A strong association between the increased number of methylated CpG islands and H. pylori infection was observed (P<0.0001). An increased number of methylated CpG islands was also associated with severity of neutrophil infiltration (P<0.0001), mononuclear cell infiltration (P<0.0001) and atrophy (P=0.0021) in all, and severity of neutrophil infiltration (P=0.0177) and mononuclear cell infiltration (P=0.0004) in H. pylori-positive participants. An increased number of methylated CpG islands correlated with lower PG I/II ratio in all (P=0.0105) and H. pylori-infected participants (P=0.074). Multiple promoter CpG islands would be methylated by H. pylori infection, and an increased number of methylated CpG sites correlate with histological and serological severity of chronic gastritis.

Evaluation of MGMT promoter methylation status and correlation with temozolomide response in orthotopic glioblastoma xenograft model

CpG methylation within the O6-methylguanine-DNA-methyltransferase (MGMT) promoter is associated with enhanced survival of glioblastoma multiforme (GBM) patients treated with temozolomide (TMZ). Although MGMT promoter is methylated in approximately 50% of GBM, several studies have reported a lack of correlation between MGMT methylation and protein expression levels and consequently inaccurate discrimination of TMZ sensitive and resistant patients. To understand the limitations of currently used assays, TMZ responsiveness of 13 GBM xenograft lines was correlated with MGMT protein expression and MGMT promoter methylation determined by (1) standard methylation-specific polymerase chain reaction (MS-PCR), (2) quantitative MS-PCR (qMS-PCR), and (3) bisulfite sequencing. For each xenograft line, mice with established intracranial xenografts were treated with vehicle control or TMZ (66 mg/kgx5 days), and TMZ response was defined as relative prolongation in median survival for TMZ-treated versus control-treated mice. The relative survival benefit with TMZ was inversely related to MGMT protein expression (r=-0.75; P=0.003) and directly correlated with qMS-PCR (r=0.72; P=0.006). There was a direct correlation between MGMT methylation signal by qMS-PCR and the number of methylated CpG sites within the region amplified by MS-PCR (r=0.78, P=0.002). However, bisulfite sequencing revealed heterogeneity in the extent of CpG methylation in those tumors with a robust qMS-PCR signal. Three of the 4 GBM lines with a qMS-PCR signal greater than 10% had at least 1 unmethylated CpG site, while only one line was fully methylated at all 12 CpG sites. These data highlight one potential limitation of the evaluation of MGMT methylation by MS-PCR assay and suggest that more detailed evaluation of methylation at individual CpG sites relative to TMZ response may be worth pursuing.

Modulation by celecoxib and difluoromethylornithine of the methylation of DNA and the estrogen receptor-alpha gene in rat colon tumors.

The ability of celecoxib and alpha-difluoromethylornithine (DFMO) to modulate the DNA hypomethylation and the hypermethylation of the estrogen receptor (ER)-alpha gene in colon tumors was evaluated as potential biomarkers for chemoprevention. Colon tumors were induced in rats by azoxymethane. Celecoxib (500 mg/kg), DFMO (100, 1000 and 3000 mg/kg) or celecoxib + 1000 mg/kg DFMO were administered in the diet for 7 or 28 days prior to death at week 37. Relative to the normal colon mucosa, colon tumors contained global hypomethylated DNA but simultaneous hypermethylation of the promoter plus exon-1 region of the ER-alpha gene. Limited treatment with celecoxib (500 p.p.m. in diet) or DFMO (1000 or 3000 p.p.m. in diet) reversed the DNA hypomethylation. Administering 1000 and 3000 p.p.m. DFMO for 7-days decreased the number of methylated CpG sites in the ER-alpha gene from 5.00 +/- 0.95 to 3.83 +/- 0.75 and 1.75 +/- 0.49 these levels were further reduced to 0.50 +/- 0.26 following administration of 1000 mg/kg for 28 days. Celecoxib administered for 7 and 28 days reduced the number of methylated sites to 4.25 +/- 0.48 and 1.5 +/- 0.50. The combination containing celecoxib and DFMO reduced the number of methylated sites to 0.20 +/- 0.20 at both 7 and 28 days. In parallel with the hypermethylation of the ER-alpha gene, the mRNA expression of the gene was decreased in colon tumors and was increased by celecoxib, DFMO or the combination. Celecoxib and DFMO reversed DNA hypomethylation and the hypermethylation of the ER-alpha gene in colon tumors supporting the hypothesis that modulation of methylation is a biomarker of chemoprevention.