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1.  Expression of undifferentiated embryonic cell transcription factor-1 (UTF1) in breast cancers and their matched normal tissues 
Cancer Cell International  2014;14(1):116.
Objectives
Undifferentiated embryonic cell transcription factor-1 (UTF1) plays a critical role in the developmental timing during embryonic development. However, there is little paper dealing with UTF1 expressed in adult tissues. In the present study, we evaluate the expression of UTF1 in breast cancer and its correlation with clinicopathological parameters.
Methods
Real-time polymerase chain reaction (real-time PCR) was applied to detect the expression of UTF1 mRNA in the 55 pairs of samples of breast cancer tissues and match normal tissues. △△CT method was used to evaluate the relative quantity of target mRNA expression.
Results
Among the 55 pairs of samples of breast cancer tissues and match normal tissues adjacent to the tumor, the UTF1 mRNA levels in normal tissues were significantly higher than those observed in breast cancer tissues (p < 0.001). UTF1 mRNA levels expression correlated with lymph node metastasis (p = 0.002) and tumor size (p < 0.001).
Conclusions
Expression of UTF1 in breast cancer tissues were confirmed in this study. Decreased expression of UTF1 mRNA in breast cancer tissues was maybe one of the factors impact on tumorigenes in breast cancer patients.
doi:10.1186/s12935-014-0116-6
PMCID: PMC4247222  PMID: 25435811
Breast cancer; UTF1; Polymerase chain reaction (PCR); Metastasis; Lymph node
2.  A Genome-Wide Screen for Promoter Methylation in Lung Cancer Identifies Novel Methylation Markers for Multiple Malignancies  
PLoS Medicine  2006;3(12):e486.
Background
Promoter hypermethylation coupled with loss of heterozygosity at the same locus results in loss of gene function in many tumor cells. The “rules” governing which genes are methylated during the pathogenesis of individual cancers, how specific methylation profiles are initially established, or what determines tumor type-specific methylation are unknown. However, DNA methylation markers that are highly specific and sensitive for common tumors would be useful for the early detection of cancer, and those required for the malignant phenotype would identify pathways important as therapeutic targets.
Methods and Findings
In an effort to identify new cancer-specific methylation markers, we employed a high-throughput global expression profiling approach in lung cancer cells. We identified 132 genes that have 5′ CpG islands, are induced from undetectable levels by 5-aza-2′-deoxycytidine in multiple non-small cell lung cancer cell lines, and are expressed in immortalized human bronchial epithelial cells. As expected, these genes were also expressed in normal lung, but often not in companion primary lung cancers. Methylation analysis of a subset (45/132) of these promoter regions in primary lung cancer (n = 20) and adjacent nonmalignant tissue (n = 20) showed that 31 genes had acquired methylation in the tumors, but did not show methylation in normal lung or peripheral blood cells. We studied the eight most frequently and specifically methylated genes from our lung cancer dataset in breast cancer (n = 37), colon cancer (n = 24), and prostate cancer (n = 24) along with counterpart nonmalignant tissues. We found that seven loci were frequently methylated in both breast and lung cancers, with four showing extensive methylation in all four epithelial tumors.
Conclusions
By using a systematic biological screen we identified multiple genes that are methylated with high penetrance in primary lung, breast, colon, and prostate cancers. The cross-tumor methylation pattern we observed for these novel markers suggests that we have identified a partial promoter hypermethylation signature for these common malignancies. These data suggest that while tumors in different tissues vary substantially with respect to gene expression, there may be commonalities in their promoter methylation profiles that represent targets for early detection screening or therapeutic intervention.
John Minna and colleagues report that a group of genes are commonly methylated in primary lung, breast, colon, and prostate cancer.
Editors' Summary
Background.
Tumors or cancers contain cells that have lost many of the control mechanisms that normally regulate their behavior. Unlike normal cells, which only divide to repair damaged tissues, cancer cells divide uncontrollably. They also gain the ability to move round the body and start metastases in secondary locations. These changes in behavior result from alterations in their genetic material. For example, mutations (permanent changes in the sequence of nucleotides in the cell's DNA) in genes known as oncogenes stimulate cells to divide constantly. Mutations in another group of genes—tumor suppressor genes—disable their ability to restrain cell growth. Key tumor suppressor genes are often completely lost in cancer cells. But not all the genetic changes in cancer cells are mutations. Some are “epigenetic” changes—chemical modifications of genes that affect the amount of protein made from them. In cancer cells, methyl groups are often added to CG-rich regions—this is called hypermethylation. These “CpG islands” lie near gene promoters—sequences that control the transcription of DNA into RNA, the template for protein production—and their methylation switches off the promoter. Methylation of the promoter of one copy of a tumor suppressor gene, which often coincides with the loss of the other copy of the gene, is thought to be involved in cancer development.
Why Was This Study Done?
The rules that govern which genes are hypermethylated during the development of different cancer types are not known, but it would be useful to identify any DNA methylation events that occur regularly in common cancers for two reasons. First, specific DNA methylation markers might be useful for the early detection of cancer. Second, identifying these epigenetic changes might reveal cellular pathways that are changed during cancer development and so identify new therapeutic targets. In this study, the researchers have used a systematic biological screen to identify genes that are methylated in many lung, breast, colon, and prostate cancers—all cancers that form in “epithelial” tissues.
What Did the Researchers Do and Find?
The researchers used microarray expression profiling to examine gene expression patterns in several lung cancer and normal lung cell lines. In this technique, labeled RNA molecules isolated from cells are applied to a “chip” carrying an array of gene fragments. Here, they stick to the fragment that represents the gene from which they were made, which allows the genes that the cells express to be catalogued. By comparing the expression profiles of lung cancer cells and normal lung cells before and after treatment with a chemical that inhibits DNA methylation, the researchers identified genes that were methylated in the cancer cells—that is, genes that were expressed in normal cells but not in cancer cells unless methylation was inhibited. 132 of these genes contained CpG islands. The researchers examined the promoters of 45 of these genes in lung cancer cells taken straight from patients and found that 31 of the promoters were methylated in tumor tissues but not in adjacent normal tissues. Finally, the researchers looked at promoter methylation of the eight genes most frequently and specifically methylated in the lung cancer samples in breast, colon, and prostate cancers. Seven of the genes were frequently methylated in both lung and breast cancers; four were extensively methylated in all the tumor types.
What Do These Findings Mean?
These results identify several new genes that are often methylated in four types of epithelial tumor. The observation that these genes are methylated in multiple independent tumors strongly suggests, but does not prove, that loss of expression of the proteins that they encode helps to convert normal cells into cancer cells. The frequency and diverse patterning of promoter methylation in different tumor types also indicates that methylation is not a random event, although what controls the patterns of methylation is not yet known. The identification of these genes is a step toward building a promoter hypermethylation profile for the early detection of human cancer. Furthermore, although tumors in different tissues vary greatly with respect to gene expression patterns, the similarities seen in this study in promoter methylation profiles might help to identify new therapeutic targets common to several cancer types.
Additional Information.
Please access these Web sites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.0030486.
US National Cancer Institute, information for patients on understanding cancer
CancerQuest, information provided by Emory University about how cancer develops
Cancer Research UK, information for patients on cancer biology
Wikipedia pages on epigenetics (note that Wikipedia is a free online encyclopedia that anyone can edit)
The Epigenome Network of Excellence, background information and latest news about epigenetics
doi:10.1371/journal.pmed.0030486
PMCID: PMC1716188  PMID: 17194187
3.  Promoter Hypermethylation of KLF4 Inactivates Its Tumor Suppressor Function in Cervical Carcinogenesis 
PLoS ONE  2014;9(2):e88827.
Objective
The KLF4 gene has been shown to be inactivated in cervical carcinogenesis as a tumor suppressor. However, the mechanism of KLF4 silencing in cervical carcinomas has not yet been identified. DNA methylation plays a key role in stable suppression of gene expression.
Methods
The methylation status of the KLF4 promoter CpG islands was analyzed by bisulfite sequencing (BSQ) in tissues of normal cervix and cervical cancer. KLF4 gene expression was detected by RT-PCR, immunohistochemistry and western blot. KLF4 promoter methylation in cervical cancer cell line was determined by BSQ and methylation-specific polymerase chain reaction (MS-PCR). Cell proliferation ability was detected by cell growth curve and MTT assay.
Results
The methylated allele was found in 41.90% of 24 cervical cancer tissues but only in 11.11% of 11 normal cervix tissues (P<0.005). KLF4 mRNA levels were significantly reduced in cervical cancer tissues compared with normal cervix tissues (P<0.01) and KLF4 mRNA expression showed a significant negative correlation with the promoter hypermethylation (r = −0.486, P = 0.003). Cervical cancer cell lines also showed a significant negative correlation between KLF4 expression and hypermethylation. After treatment with the demethylating agent 5-Azacytidine (5-Aza), the expression of KLF4 in the cervical cancer cell lines at both mRNA and protein levels was drastically increased, the cell proliferation ability was inhibited and the chemosensitivity for cisplatin was significantly increased.
Conclusion
KLF4 gene is inactivated by methylation-induced silencing mechanisms in a large subset of cervical carcinomas and KLF4 promoter hypermethylation inactivates the gene’s function as a tumor suppressor in cervical carcinogenesis.
doi:10.1371/journal.pone.0088827
PMCID: PMC3925171  PMID: 24551169
4.  Epigenetic Silencing of Somatostatin in Gastric Cancer 
Digestive diseases and sciences  2010;56(1):125-130.
Background
Somatostatin (SST), a primary inhibitor of gastrin-stimulated gastric acid secretion, has potent antitumor and anti-secretory activities in several human cancers.
Aims
This study was performed to investigate the SST gene expression levels and possible epigenetic mechanisms that regulate its expression in gastric adenocarcinomas.
Methods
Quantitative real time-RT PCR and quantitative bisulfite pyrosequencing technologies were applied to study primary gastric cancer tissue samples and cell lines.
Results
Quantitative real-time RT-PCR analysis demonstrated down-regulation of SST transcript in 93% of gastric carcinoma samples (30/32), as compared to 21 normal samples (P<0.001). Because of the presence of a large CpG island in the SST promoter, we next examined its promoter DNA methylation levels using quantitative bisulfite pyrosequencing technology. The results demonstrated a significant increase in SST promoter DNA methylation levels in tumor samples as compared to normal samples (P<0.05). Promoter DNA hypermethylation and silencing of SST was also detected in seven gastric cancer cell lines that we tested. To confirm the role of promoter DNA methylation as an epigenetic mechanism regulating SST expression, AGS gastric cancer cells were treated with 5-Aza-deoxycytidine. This treatment led to reduction in the promoter DNA methylation levels of SST accompanied by restoration of its mRNA expression.
Conclusions
Our results indicate that promoter DNA methylation levels play a critical role in regulating SST expression in gastric cancer. This finding provides a foundation for further studies on the role of SST in gastric carcinogenesis and its potential as a biomarker for gastric cancers.
doi:10.1007/s10620-010-1422-z
PMCID: PMC3082506  PMID: 20927589
methylation; expression; somatostatin; gastric cancer
5.  In Vivo and In Vitro Dynamics of Undifferentiated Embryonic Cell Transcription Factor 1 
Stem Cell Reports  2014;2(3):245-252.
Summary
Pluripotent stem cells retain the ability to differentiate into the three germ layers and germline. As a result, there is a major interest in characterizing regulators that establish and maintain pluripotency. The network of transcription factors continues to expand in complexity, and one factor, undifferentiated embryonic cell transcription factor 1 (UTF1), has recently moved more into the limelight. To facilitate the study of UTF1, we report the generation and characterization of two reporter lines that enable efficient tracking, mapping, and purification of endogenous UTF1. In particular, we include a built-in biotinylation system in our targeted locus that allows efficient and reliable pulldown. We also use this reporter to show the dynamic regulation of Utf1 in distinct stem cell conditions and demonstrate its utility for reprogramming studies. The multipurpose design of the reporter lines enables many directions of future study and should lead to a better understanding of UTF1’s diverse roles.
Highlights
•Generation and characterization of two multipurpose knockin Utf1 reporter lines•Real-time imaging of UTF1 levels in vitro and in vivo•Highly efficient genome-wide mapping of UTF1 using built-in biotinylation•Highly dynamic UTF1 levels in 2i versus serum/LIF conditions
The network of transcription factors that are involved in the regulation of pluripotency continues to expand in complexity, and one factor, undifferentiated embryonic cell transcription factor 1 (UTF1), has recently moved more into the limelight. To facilitate the study of UTF1, Meissner and colleagues report the generation and characterization of two reporter lines that enable efficient tracking, mapping, and purification of endogenous UTF1.
doi:10.1016/j.stemcr.2014.01.007
PMCID: PMC3964277  PMID: 24672748
6.  hTERT promoter activity and CpG methylation in HPV-induced carcinogenesis 
BMC Cancer  2010;10:271.
Background
Activation of telomerase resulting from deregulated hTERT expression is a key event during high-risk human papillomavirus (hrHPV)-induced cervical carcinogenesis. In the present study we examined hTERT promoter activity and its relation to DNA methylation as one of the potential mechanisms underlying deregulated hTERT transcription in hrHPV-transformed cells.
Methods
Using luciferase reporter assays we analyzed hTERT promoter activity in primary keratinocytes, HPV16- and HPV18-immortalized keratinocyte cell lines and cervical cancer cell lines. In the same cells as well as cervical specimens we determined hTERT methylation by bisulfite sequencing analysis of the region spanning -442 to +566 (relative to the ATG) and quantitative methylation specific PCR (qMSP) analysis of two regions flanking the hTERT core promoter.
Results
We found that in most telomerase positive cells increased hTERT core promoter activity coincided with increased hTERT mRNA expression. On the other hand basal hTERT promoter activity was also detected in telomerase negative cells with no or strongly reduced hTERT mRNA expression levels. In both telomerase positive and negative cells regulatory sequences flanking both ends of the core promoter markedly repressed exogenous promoter activity.
By extensive bisulfite sequencing a strong increase in CpG methylation was detected in hTERT positive cells compared to cells with no or strongly reduced hTERT expression. Subsequent qMSP analysis of a larger set of cervical tissue specimens revealed methylation of both regions analyzed in 100% of cervical carcinomas and 38% of the high-grade precursor lesions, compared to 9% of low grade precursor lesions and 5% of normal controls.
Conclusions
Methylation of transcriptionally repressive sequences in the hTERT promoter and proximal exonic sequences is correlated to deregulated hTERT transcription in HPV-immortalized cells and cervical cancer cells. The detection of DNA methylation at these repressive regions may provide an attractive biomarker for early detection of cervical cancer.
doi:10.1186/1471-2407-10-271
PMCID: PMC2904279  PMID: 20534141
7.  DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines 
Molecular Cancer  2008;7:15.
Background
DNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of methylation-sensitive genes were evaluated among breast cancer cell lines.
Results
The relationship between gene expression (assessed by RT-PCR and quantitative real-time PCR), promoter methylation (assessed by methylation-specific PCR, bisulfite sequencing, and 5-aza-2'deoxycytidine treatment), and the DNA methyltransferase machinery (total DNMT activity and expression of DNMT1, DNMT3a, and DNMT3b proteins) were examined in 12 breast cancer cell lines. Unsupervised cluster analysis of the expression of 64 methylation-sensitive genes revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes (CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A), whereas the low-frequency methylator cell lines do not methylate these genes. Hypermethylator cell lines coordinately overexpress total DNMT activity and DNMT3b protein levels compared to normal breast epithelial cells. In contrast, most low-frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from normal. Microarray data mining identified a strong cluster of primary breast tumors that express the hypermethylation signature defined by CDH1, CEACAM6, CST6, ESR1, LCN2, and SCNN1A. This subset of breast cancers represents 18/88 (20%) tumors in the dataset analyzed, and 100% of these tumors were classified as basal-like, suggesting that the hypermethylator defect cosegregates with poor prognosis breast cancers.
Conclusion
These observations combine to strongly suggest that: (a) a subset of breast cancer cell lines express a hypermethylator phenotype, (b) the hypermethylation defect in these breast cancer cell lines is related to aberrant overexpression of DNMT activity, (c) overexpression of DNMT3b protein significantly contributes to the elevated DNMT activity observed in tumor cells expressing this phenotype, and (d) the six-gene hypermethylator signature characterized in breast cancer cell lines defines a distinct cluster of primary basal-like breast cancers.
doi:10.1186/1476-4598-7-15
PMCID: PMC2246151  PMID: 18221536
8.  Expression Pattern of Stemness-Related Genes in Human Endometrial and Endometriotic Tissues 
Molecular Medicine  2009;15(11-12):392-401.
Endometriosis is a chronic disease characterized by the presence of ectopic endometrial tissue outside of the uterus with mixed traits of benign and malignant pathology. In this study we analyzed in endometrial and endometriotic tissues the differential expression of a panel of genes that are involved in preservation of stemness status and consequently considered as markers of stem cell presence. The expression profiles of a panel of 13 genes (SOX2, SOX15, ERAS, SALL4, OCT4, NANOG, UTF1, DPPA2, BMI1, GDF3, ZFP42, KLF4, TCL1) were analyzed by reverse transcription–polymerase chain reaction in human endometriotic (n = 12) and endometrial samples (n = 14). The expression of SALL4 and OCT4 was further analyzed by immunohistochemical methods. Genes UTF1, TCL1, and ZFP42 showed a trend for higher frequency of expression in endometriosis than in endometrium (P < 0.05 for UTF1), whereas GDF3 showed a higher frequency of expression in endometrial samples. Immunohistochemical analysis revealed that SALL4 was expressed in endometriotic samples but not in endometrium samples, despite the expression of the corresponding mRNA in both the sample groups. This study highlights a differential expression of stemness-related genes in ectopic and eutopic endometrium and suggests a possible role of SALL4-positive cells in the pathogenesis of endometriosis.
doi:10.2119/molmed.2009.00068
PMCID: PMC2727462  PMID: 19690622
9.  Regulation of pluripotency and self-renewal of ES cells through epigenetic-threshold modulation and mRNA pruning 
Cell  2012;151(3):576-589.
SUMMARY
ES cell pluripotency requires bivalent epigenetic modifications of key developmental genes regulated by various transcription factors and chromatin modifying enzymes. How these factors coordinate with one another to maintain the bivalent chromatin state so that ES cells can undergo rapid self-renewal while retaining pluripotency is poorly understood. We report that Utf1, a target of Oct4 and Sox2, is a bivalent chromatin component that buffers poised states of bivalent genes. By limiting PRC2 loading and Histone 3 lysine-27 trimethylation, Utf1 sets proper activation thresholds for bivalent genes. It also promotes nuclear tagging of mRNAs transcribed from insufficiently silenced bivalent genes for cytoplasmic degradation through mRNA de-capping. These opposing functions of Utf1 promote coordinated differentiation. The mRNA degradation function also ensures rapid cell proliferation by blocking the Myc-Arf feedback control. Thus, Utf1 couples the core pluripotency factors with Myc and PRC2 networks to promote the pluripotency and proliferation of ESCs.
doi:10.1016/j.cell.2012.09.023
PMCID: PMC3575637  PMID: 23101626
Utf1; PRC2; Myc; bivalency; pluripotency; self-renewal; ES cells; epigenetics; mRNA degradation; differentiation
10.  Epigenetic inactivation of secreted frizzled-related protein 2 in esophageal squamous cell carcinoma 
AIM: To investigate the expression and methylation status of the secreted frizzled-related protein 2 (SFRP2) in esophageal squamous cell carcinoma (ESCC) and explore its role in ESCC carcinogenesis.
METHODS: Seven ESCC cell lines (KYSE 30, KYSE150, KYSE410, KYSE510, EC109, EC9706 and TE-1) and one immortalized human esophageal epithelial cell line (Het-1A), 20 ESCC tissue samples and 20 paired adjacent non-tumor esophageal epithelial tissues were analyzed in this study. Reverse-transcription polymerase chain reaction (RT-PCR) was employed to investigate the expression of SFRP2 in cell lines, primary ESCC tumor tissue, and paired adjacent normal tissue. Methylation status was evaluated by methylation-specific PCR and bisulfite sequencing. The correlation between expression and promoter methylation of the SFRP2 gene was confirmed with treatment of 5-aza-2’-deoxycytidine. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2-transfected cells and examined them with regard to cell proliferation, colony formation, apoptosis and cell cycle in vivo and in vitro.
RESULTS: SFRP2 mRNA was expressed in the immortalized normal esophageal epithelial cell line but not in seven ESCC cell lines. By methylation-specific PCR, complete methylation was detected in three cell lines with silenced SFRP2 expression, and extensive methylation was observed in the other four ESCC cell lines. 5-aza-2’-deoxycytidine could restore the expression of SFRP2 mRNA in the three ESCC cell lines lacking SFRP2 expression. SFRP2 mRNA expression was obviously lower in primary ESCC tissue than in adjacent normal tissue (0.939 ± 0.398 vs 1.51 ± 0.399, P < 0.01). SFRP2 methylation was higher in tumor tissue than in paired normal tissue (95% vs 65%, P < 0.05). The DNA methylation status of the SFRP2 correlated inversely with the SFRP2 expression. To assess the potential role of SFRP2 in ESCC, we established stable SFRP2 transfectants and control counterparts by introducing pcDNA3.1/v5 hisA -SFRP2 or pcDNA3.1/v5 hisA -empty vector into KYSE30 cells lacking SFRP2 expression. After transfection, the forced-expression of SFRP2 was confirmed by the RT-PCR. In comparison with the control groups, stably-expressed SFRP2 in KYSE 30 cells significantly reduced colony formation in vitro (47.17% ± 15.61% vs 17% ± 3.6%, P = 0.031) and tumor growth in nude mice (917.86 ± 249.35 mm3 vs 337.23 ± 124.43 mm3, P < 0.05). Using flow cytometry analysis, we found a significantly higher number of early apoptotic cells in SFRP2-transfected cells than in the control cells (P = 0.025). The mean cell number in the S and G2-M phases of the cell cycle was also significantly lower in SFRP2-transfected KYSE30 cells compared with mock transfected counterparts.
CONCLUSION: Silencing of SFRP2 expression through promoter hypermethylation may be a factor in ESCC carcinogenesis through loss of its tumor-suppressive activity.
doi:10.3748/wjg.v18.i6.532
PMCID: PMC3280398  PMID: 22363119
Esophageal squamous cell carcinoma; Secreted frizzled-related protein 2; Methylation; Tumor suppressor gene; Wnt signaling pathway
11.  Identification of guanine nucleotide-binding protein γ-7 as an epigenetically silenced gene in head and neck cancer by gene expression profiling 
International Journal of Oncology  2013;42(4):1427-1436.
Silencing of tumor suppressor genes plays a vital role in head and neck carcinogenesis. Aberrant hypermethylation in the promoter region of some known or putative tumor suppressor genes occurs frequently during the development of various types of cancer including head and neck squamous cell carcinoma (HNSCC). In this study we used an expanded mRNA expression profiling approach followed by microarray expression analysis to identify epigenetically inactivated genes in HNSCC. Two HNSCC cell lines were treated with 5-aza-2′-deoxycytidine followed by microarray analysis to identify epigenetically silenced genes in HNSCC. We found 1,960, 614 and 427 genes were upregulated in the HNSCC cell lines JHU-012, JHU-011 and the combination of both cell lines, respectively. HNSCC tumor and normal mucosal samples were used for gene profiling by a 47K mRNA gene expression array and we found 7,140 genes were downregulated in HNSCC tumors compared to normal mucosa, as determined by microarray analysis, and were integrated with cell line data. Integrative analysis defined 126 candidate genes, of which only seven genes showed differential methylation in tumors and no methylation in normal mucosa after bisulfite sequencing. Following validation by QMSP, one gene, guanine nucleotide-binding protein γ-7 (GNG7), was confirmed to be highly methylated in tumors and unmethylated in normal mucosal and salivary rinse samples demonstrating cancer-specific methylation in HNSCC tissues. TXNIP and TUSC2 were partially methylated in tumors and normal salivary rinses but unmethylated in normal mucosa. We concluded that GNG7 is a highly specific promoter methylated gene associated with HNSCC. In addition, TXNIP and TUSC2 are also potential biomarkers for HNSCC.
doi:10.3892/ijo.2013.1808
PMCID: PMC3981008  PMID: 23403885
guanine nucleotide-binding protein γ-7; gene expression; silencing; head and neck squamous cell carcinoma; epigenetics
12.  Profiling epigenetic inactivation of tumor suppressor genes in tumors and plasma from cutaneous melanoma patients 
Oncogene  2004;23(22):4014-4022.
Aberrant methylation of CpG islands in promoter regions of tumor suppressor genes (TSG) has been demonstrated in epithelial origin tumors. However, the methylation profiling of tumor-related gene promoter regions in cutaneous melanoma tumors has not been reported. Seven known or candidate TSGs that are frequently hyper-methylated in carcinomas were assessed by methylation-specific polymerase chain reaction (MSP) in 15 melanoma cell lines and 130 cutaneous melanoma tumors. Four TSGs were frequently hypermethylated in 86 metastatic tumor specimens: retinoic acid receptor-β2 (RAR-β2) (70%), RAS association domain family protein 1A (RASSF1A) (57%), and O6-methylguanine DNA methy-latransferase (MGMT) (34%), and death-associated protein kinase (DAPK) (19%). Hypermethylation of MGMT, RASSF1A, and DAPK was significantly lower in primary melanomas (n = 20) compared to metastatic melanomas. However, hypermethylation of RAR-β2 was 70% in both primary and metastatic melanomas. Cell lines had hypermethylation profiles similar to those of metastatic melanomas. The analysis of these four markers of metastatic tumors demonstrated that 97% had ≥1 gene(s) and 59% had ≥2 genes hypermethylated. The methylation of genes was verified by bisulfite sequencing. The mRNA transcripts could be re-expressed in melanoma cell lines having hypermethylated genes following treatment with 5′-aza 2′-deoxycytidine (5Aza-dC). Analysis of melanoma patients’ plasma (preoperative blood; n = 31) demonstrated circulating hypermethylated MGMT, RAR-β2, and RASSF1A DNA for at least one of the markers in 29% of the patients. Our findings indicate that the incidence of TSG hypermethylation increases during tumor progression. Methylation of TSG may play a significant role in cutaneous melanoma progression.
doi:10.1038/sj.onc.1207505
PMCID: PMC2856469  PMID: 15064737
MGMT; RAR-β2; RASSF1A; methylation; melanoma
13.  Progression to metastatic stage in a cellular model of prostate cancer is associated with methylation of the androgen receptor gene and transcriptional suppression of the insulin-like growth factor-I receptor gene 
Experimental cell research  2010;316(9):1479-1488.
The progression of prostate cancer from an organ-confined, androgen-sensitive disease to a metastatic one is associated with dysregulation of androgen receptor (AR)-regulated target genes and with a decrease in insulin-like growth factor-I receptor (IGF1R) expression. DNA methylation of CpG islands is an epigenetic mechanism associated with gene silencing. Recent studies have demonstrated that methylation occurs early in prostate carcinogenesis and, furthermore, may contribute to androgen independence. The methylation status of the AR and IGF1R genes was evaluated in a series of prostate cancer cell lines corresponding to early (benign) and advanced (metastatic) stages of the disease. Results of 5-Aza-2'-deoxycytidine (5-Aza) experiments, methylation specific PCR, and sodium bisulfite-direct DNA sequencing revealed that the AR promoter is hypermethylated in metastatic M12, but not in benign P69, cells. On the other hand, no methylation was seen in the IGF1R promoter at any stage of the disease. We show, however, that 5-Aza treatment, which caused demethylation of the AR promoter, led to a significant increase in IGF1R mRNA levels, whereas addition of the AR inhibitor flutamide decreased the IGF1R mRNA levels to basal values measured prior to the 5-Aza treatment. Given that the IGF1R gene has been identified as a downstream target for AR action, our data is consistent with a model in which the AR gene undergoes methylation during progression of the disease, leading to dysregulation of AR targets, including the IGF1R gene, at advanced metastatic stages.
doi:10.1016/j.yexcr.2010.03.007
PMCID: PMC2873092  PMID: 20338164
insulin-like growth factor-I receptor (IGF1R); androgen receptor; DNA methylation; prostate cancer; epigenetic regulation
14.  Genome-wide methylation profiling identifies hypermethylated biomarkers in high-grade cervical intraepithelial neoplasia 
Epigenetics  2012;7(11):1268-1278.
Epigenetic modifications, such as aberrant DNA promoter methylation, are frequently observed in cervical cancer. Identification of hypermethylated regions allowing discrimination between normal cervical epithelium and high-grade cervical intraepithelial neoplasia (CIN2/3), or worse, may improve current cervical cancer population-based screening programs. In this study, the DNA methylome of high-grade CIN lesions was studied using genome-wide DNA methylation screening to identify potential biomarkers for early diagnosis of cervical neoplasia. Methylated DNA Immunoprecipitation (MeDIP) combined with DNA microarray was used to compare DNA methylation profiles of epithelial cells derived from high-grade CIN lesions with normal cervical epithelium. Hypermethylated differentially methylated regions (DMRs) were identified. Validation of nine selected DMRs using BSP and MSP in cervical tissue revealed methylation in 63.2–94.7% high-grade CIN and in 59.3–100% cervical carcinomas. QMSP for the two most significant high-grade CIN-specific methylation markers was conducted exploring test performance in a large series of cervical scrapings. Frequency and relative level of methylation were significantly different between normal and cancer samples. Clinical validation of both markers in cervical scrapings from patients with an abnormal cervical smear confirmed that frequency and relative level of methylation were related with increasing severity of the underlying CIN lesion and that ROC analysis was discriminative. These markers represent the COL25A1 and KATNAL2 and their observed increased methylation upon progression could intimate the regulatory role in carcinogenesis. In conclusion, our newly identified hypermethylated DMRs represent specific DNA methylation patterns in high-grade CIN lesions and are candidate biomarkers for early detection.
doi:10.4161/epi.22301
PMCID: PMC3499328  PMID: 23018867
cervical precancerous lesion; DNA methylation; MeDIP-chip; cervical scraping
15.  Alcohol dehydrogenase, iron containing, 1 promoter hypermethylation associated with colorectal cancer differentiation 
BMC Cancer  2013;13:142.
Background
The aberrant methylation of CpG islands in the promoter is associated with colorectal cancer (CRC) carcinogenesis. In our previous study, the promoter of alcohol dehydrogenase, iron containing, 1 (ADHFE1) was most highly methylated in CRC compared to normal colorectal mucosa. In this study, we examined the expression and function of the ADHFE1 in CRC.
Methods
We examined the promoter methylation and mRNA expression of ADHFE1 with 5-aza-2′-deoxycytidine (5-Aza-2-dC) in 12 CRC cell lines, 124 paired CRC and adjacent normal mucosa, and 59 advanced adenomas. To confirm methylation of ADHFE1, we performed bisulfite genomic sequencing in 3 CRC cell lines, 6 paired CRC and adjacent normal mucosa. ADHFE1 protein expression was studied using western blot and immunohistochemistry, respectively in the 36 and 243 paired CRC and adjacent normal tissue. We transfected the DLD-1 with pcDNA3.1 vector containing ADHFE1 and examined the expression of differentiation marker, such as ALP, CEA and Cdx2. We examined the ADHFE1 expression at distinct developmental stages in mouse embryos.
Results
The ADHFE1 promoter was hypermethylated in all CRC cell lines, 81.8% in CRCs, and 84.7% in advanced adenomas, with reciprocal change by 5-Aza-2-dC. The expression of ADHFE1 mRNA was down-regulated in all CRC cell lines and 96.3% in CRC tissues. The expression of ADHFE1 protein was down-regulated in 91.7% of CRC tissues. In the immunohistochemistry, normal epithelial cells at the crypt top showed very strong ADHFE1 expression, whereas they were much weaker at the crypt base. In CRC, the good differentiation was significantly associated with high ADHFE1 expression. The activity of differentiation marker, such as ALP and CEA, was higher in pcDNA3.1-ADHFE1 transfected CRC cells with consistent correlation with ADHFE1 protein than control. In mouse embryos, ADHFE1 in the large intestine was the first detected at E15.5. At E18.5, ADHFE1 was predominantly expressed in the top of the mature crypt epithelium.
Conclusions
It showed that the hypermethylation of ADHFE1 promoter in CRC is concordance with down-regulation of ADHFE1 mRNA and ADHFE1 protein. ADHFE1 has an important role of differentiation in CRC, as well as normal colorectal mucosa and embryonic developmental processes.
doi:10.1186/1471-2407-13-142
PMCID: PMC3618294  PMID: 23517143
ADHFE1; Promoter methylation; Colorectal cancer; Differentiation
16.  Epigenetic Silencing of Maspin Expression Occurs Early in the Conversion of Keratocytes to Fibroblasts 
Experimental eye research  2008;86(4):586-600.
Maspin, a 42 kDa non-classical serpin (serine protease inhibitor) that controls cell migration and invasion, is mainly expressed by epithelial-derived cells but is also expressed in corneal stromal keratocytes. Upon culture of stromal keratocytes in the presence of FBS, maspin is down regulated to nearly undetectable levels by passage two. DNA methylation is one of several processes that controls gene expression during cell differentiation, development, genetic imprinting, and carcinogenesis but has not been studied in corneal stromal cells. The purpose of this study was to determine whether DNA methylation of the maspin promoter and histone H3 dimethylation are involved in the mechanism of down regulation of maspin synthesis in human corneal stromal fibroblasts and myofibroblasts. Human donor corneal stroma cells were immediately placed into serum-free defined medium or cultured in the presence of FBS and passed into serum-free medium or medium containing FBS or FGF-2 to induce the fibroblast phenotype or TGF-β1 for the myofibroblast phenotype. These cell types are found in wounded corneas. The cells were used to prepare RNA for semi-quantitative or quantitative RT-PCR or to extract protein for western analysis. In addition, P4 FBS cultured fibroblasts were treated with the DNA demethylating agent, 5-aza-2′-deoxycytidine (5-Aza-dC), and the histone deacetylase inhibitor, trichostatin A (TSA). Cells with and without treatment were harvested and assayed for DNA methylation using sodium bisulfite sequencing. The methylation state of histone H3 associated with the maspin gene in the P4 fibroblast cells was determined using a ChIP assay. Freshly harvested corneal stromal cells expressed maspin but upon phenotypic differentiation, maspin mRNA and protein were dramatically down-regulated. Sodium bisulfite sequencing revealed that the maspin promoter in the freshly isolated stromal keratocytes was hypomethylated while both the P0 stromal cells and the P1 cells cultured in the presence of serum free defined medium, FGF-2 and TGF-β1 were hypermethylated. Down regulation of maspin synthesis was also associated with histone H3 dimethylation at Lysine 9. Both maspin mRNA and protein were reexpressed at low levels with 5-Aza-dC but not TSA treatment. Addition of TSA to 5-Aza-dC treated cells did not increase maspin expression. Treatment with 5-Aza-dC did not significantly alter demethylation of the maspin promoter but did demethylate histone H3. These results show maspin promoter hypermethylation and histone methylation occur with down regulation of maspin synthesis in corneal stromal cells and suggest regulation of genes upon conversion of keratocytes to wound healing fibroblasts can involve promoter and histone methylation.
doi:10.1016/j.exer.2008.01.003
PMCID: PMC2374753  PMID: 18291368
Cornea; Stromal Cells; Maspin; DNA Methylation; Stromal Fibroblasts; Stromal Myofibroblasts; Keratocytes
17.  Epigenetic inactivation of the MIR129-2 in hematological malignancies 
Background
MIR129-2 has been shown to be a tumor suppressor microRNA hypermethylated in epithelial cancers.
Patients and methods
Epigenetic inactivation of MIR129-2 was studied by methylation-specific PCR (MSP) in 13 cell lines (eight myeloma and five lymphoma), 15 normal controls and 344 primary samples including acute myeloid leukemia (AML), acute lymphoblastic leukemia (ALL), chronic myeloid leukemia (CML), chronic lymphocytic leukemia (CLL), non-Hodgkin’s lymphoma (NHL), multiple myeloma (MM) at diagnosis, MM at relapse/progression, and monoclonal gammopathy of undetermined significance (MGUS). Expression of MIR129 and its target, SOX4, in cell lines was measured before and after hypomethylating treatment and MIR129 overexpression. MIR129 expression was correlated with MIR129-2 methylation status in primary lymphoma samples. Tumor suppressor function of MIR129 was demonstrated by MTT and trypan blue exclusion assay after MIR129 overexpression.
Results
The sensitivity of the methylated-MSP was one in 103. Different MSP statuses, including complete methylation, partial methylation, and complete unmethylation, were verified by quantitative bisulfite pyrosequencing. All five lymphoma and seven of eight myeloma cell lines showed complete and partial MIR129-2 methylation. In primary samples, MIR129-2 methylation was absent in AML and CML, but detected in 5% ALL, 45.9% CLL, 49.5% MM at diagnosis, and 59.1% NHL. In CLL, MIR129-2 methylation adversely impacted on survival (p=0.004). In MM, MIR129-2 methylation increased from 27.5% MGUS to 49.5% MM at diagnosis and 41.5% at relapse/progression (p=0.023). In NHL, MIR129-2 methylation was associated with MIR124-1 and MIR203 methylation (p<0.001), and lower MIR129 expression (p=0.009). Hypomethylation treatment of JEKO-1, homozygously methylated for MIR129-2, led to MIR129-2 demethylation and MIR129 re-expression, with downregulation of SOX4 mRNA. Moreover, MIR129 overexpression in both mantle cell lines, JEKO-1 and GRANTA-519, inhibited cellular proliferation and enhanced cell death, with concomitant SOX4 mRNA downregulation.
Conclusions
MIR129-2 is a tumor suppressive microRNA frequently methylated in lymphoid but not myeloid malignancies, leading to reversible MIR129-2 silencing. In CLL, MIR129-2 methylation was associated with an inferior survival. In MM, MIR129-2 methylation might be acquired during progression from MGUS to symptomatic MM. In NHL, MIR129-2 methylation might collaborate with MIR124-1 and MIR203 methylation in lymphomagenesis.
doi:10.1186/1756-8722-6-16
PMCID: PMC3576298  PMID: 23406679
microRNA; Tumor suppressor; Hypermethylation; MIR129; Hematological cancers
18.  Hypermethylation of genomic 3.3-kb repeats is frequent event in HPV-positive cervical cancer 
BMC Medical Genomics  2009;2:30.
Background
Large-scale screening methods are widely used to reveal cancer-specific DNA methylation markers. We previously identified non-satellite 3.3-kb repeats associated with facioscapulohumeral muscular dystrophy (FSHD) as hypermethylated in cervical cancer in genome-wide screening. To determine whether hypermethylation of 3.3-kb repeats is a tumor-specific event and to evaluate frequency of this event in tumors, we investigated the 3.3-kb repeat methylation status in human papilloma virus (HPV)-positive cervical tumors, cancer cell lines, and normal cervical tissues. Open reading frames encoding DUX family proteins are contained within some 3.3-kb repeat units. The DUX mRNA expression profile was also studied in these tissues.
Methods
The methylation status of 3.3-kb repeats was evaluated by Southern blot hybridization and bisulfite genomic sequencing. The expression of DUX mRNA was analyzed by RT-PCR and specificity of PCR products was confirmed by sequencing analysis.
Results
Hypermethylation of 3.3-kb repeats relative to normal tissues was revealed for the first time in more than 50% (18/34) of cervical tumors and in 4 HPV-positive cervical cancer cell lines. Hypermethylation of 3.3-kb repeats was observed in tumors concurrently with or independently of hypomethylation of classical satellite 2 sequences (Sat2) that were hypomethylated in 75% (15/20) of cervical tumors. We have revealed the presence of transcripts highly homologous to DUX4 and DUX10 genes in normal tissues and down-regulation of transcripts in 68% of tumors with and without 3.3-kb repeats hypermethylation.
Conclusion
Our results demonstrate that hypermethylation rather than hypomethylation of 3.3-kb repeats is the predominant event in HPV-associated cervical cancer and provide new insight into the epigenetic changes of repetitive DNA elements in carcinogenesis.
doi:10.1186/1755-8794-2-30
PMCID: PMC2695481  PMID: 19473516
19.  XAF1 is frequently methylated in human esophageal cancer 
AIM: To explore epigenetic changes in the gene encoding X chromosome-linked inhibitor of apoptosis-associated factor 1 (XAF1) during esophageal carcinogenesis.
METHODS: Methylation status of XAF1 was detected by methylation-specific polymerase chain reaction (MSP) in four esophageal cancer cell lines (KYSE30, KYSE70, BIC1 and partially methylated in TE3 cell lines), nine cases of normal mucosa, 72 cases of primary esophageal cancer and matched adjacent tissue. XAF1 expression was examined by semi-quantitative reverse transcriptional polymerase chain reaction and Western blotting before and after treatment with 5-aza-deoxycytidine (5-aza-dc), a demethylating agent. To investigate the correlation of XAF1 expression and methylation status in primary esophageal cancer, immunohistochemistry for XAF1 expression was performed in 32 cases of esophageal cancer and matched adjacent tissue. The association of methylation status and clinicopathological data was analyzed by logistic regression.
RESULTS: MSP results were as follows: loss of XAF1 expression was found in three of four esophageal cell lines with promoter region hypermethylation (completely methylated in KYSE30, KYSE70 and BIC1 cell lines and partially in TE3 cells); all nine cases of normal esophageal mucosa were unmethylated; and 54/72 (75.00%) samples from patients with esophageal cancer were methylated, and 25/72 (34.70%) matched adjacent tissues were methylated (75.00% vs 34.70%, χ2 = 23.5840, P = 0.000). mRNA level of XAF1 measured with semi-quantitative reverse transcription polymerase chain reaction was detectable only in TE3 cells, and no expression was detected in KYSE30, KYSE70 or BIC1 cells. Protein expression was not observed in KYSE30 cells by Western blotting before treatment with 5-aza-dc. After treatment, mRNA level of XAF1 was detectable in KYSE30, KYSE70 and BIC1 cells. Protein expression was detected in KYSE30 after treatment with 5-aza-dc. Immunohistochemistry was performed on 32 cases of esophageal cancer and adjacent tissue, and demonstrated XAF1 in the nucleus and cytoplasm. XAF1 staining was found in 20/32 samples of adjacent normal tissue but was present in only 8/32 samples of esophageal cancer tissue (χ2= 9.143, P = 0.002). XAF1 expression was decreased in cancer samples compared with adjacent tissues. In 32 cases of esophageal cancer, 24/32 samples were methylated, and 8/32 esophageal cancer tissues were unmethylated. XAF1 staining was found in 6/8 samples of unmethylated esophageal cancer and 2/24 samples of methylated esophageal cancer tissue. XAF1 staining was inversely correlated with XAF1 promoter region methylation (Fisher’s exact test, P = 0.004). Regarding methylation status and clinicopathological data, no significant differences were found in sex, age, tumor size, tumor stage, or metastasis with respect to methylation of XAF1 for the 72 tissue samples from patients with esophageal cancer.
CONCLUSION: XAF1 is frequently methylated in esophageal cancer, and XAF1 expression is regulated by promoter region hypermethylation.
doi:10.3748/wjg.v18.i22.2844
PMCID: PMC3374990  PMID: 22719195
X chromosome-linked inhibitor of apoptosis-associated factor 1; Esophageal cancer; Methylation; Methylation-specific polymerase chain reaction; Semi-quantitative reverse transcriptional polymerase chain reaction
20.  Epigenetic silencing of glutaminase 2 in human liver and colon cancers 
BMC Cancer  2013;13:601.
Background
Glutaminase 2 (Gls2) is a p53 target gene and is known to play an important role in energy metabolism. Gls2 has been reported to be downregulated in human hepatocellular carcinomas (HCC). However, the underlying mechanism responsible for its downregulation is still unclear. Here, we investigated Gls2 expression and its promoter methylation status in human liver and colon cancers.
Methods
mRNA expression of Gls2 was determined in human liver and colon cancer cell lines and HCC tissues by real-time PCR and promoter methylation was analyzed by methylation-specific PCR (MSP) and validated by bisulfite genome sequencing (BGS). Cell growth was determined by colony formation assay and MTS assay. Statistical analysis was performed by Wilcoxon matched-pairs test or non-parametric t test.
Results
First, we observed reduced Gls2 mRNA level in a selected group of liver and colon cancer cell lines and in the cancerous tissues from 20 HCC and 5 human colon cancer patients in comparison to their non-cancerous counter parts. Importantly, the lower level of Gls2 in cancer cells was closely correlated to its promoter hypermethylation; and chemical demethylation treatment with 5-aza-2′-deoxycytidine (Aza) increased Gls2 mRNA level in both liver and colon cancer cells, indicating that direct epigenetic silencing suppressed Gls2 expression by methylation. Next, we further examined this correlation in human HCC tissues, and 60% of primary liver tumor tissues had higher DNA methylation levels when compared with adjacent non-tumor tissues. Detailed methylation analysis of 23 CpG sites at a 300-bp promoter region by bisulfite genomic sequencing confirmed its methylation. Finally, we examined the biological function of Gls2 and found that restoring Gls2 expression in cancer cells significantly inhibited cancer cell growth and colony formation ability through induction of cell cycle arrest.
Conclusions
We provide evidence showing that epigenetic silencing of Gls2 via promoter hypermethylation is common in human liver and colon cancers and Gls2 appears to be a functional tumor suppressor involved in the liver and colon tumorigenesis.
doi:10.1186/1471-2407-13-601
PMCID: PMC3878668  PMID: 24330717
Gls2; Methylation; Hepatocellular carcinoma; Colon cancer
21.  In Vivo Function and Evolution of the Eutherian-Specific Pluripotency Marker UTF1 
PLoS ONE  2013;8(7):e68119.
Embryogenesis in placental mammals is sustained by exquisite interplay between the embryo proper and placenta. UTF1 is a developmentally regulated gene expressed in both cell lineages. Here, we analyzed the consequence of loss of the UTF1 gene during mouse development. We found that homozygous UTF1 mutant newborn mice were significantly smaller than wild-type or heterozygous mutant mice, suggesting that placental insufficiency caused by the loss of UTF1 expression in extra-embryonic ectodermal cells at least in part contributed to this phenotype. We also found that the effects of loss of UTF1 expression in embryonic stem cells on their pluripotency were very subtle. Genome structure and sequence comparisons revealed that the UTF1 gene exists only in placental mammals. Our analyses of a family of genes with homology to UTF1 revealed a possible mechanism by which placental mammals have evolved the UTF1 genes.
doi:10.1371/journal.pone.0068119
PMCID: PMC3706607  PMID: 23874519
22.  Expression of GBGT1 is epigenetically regulated by DNA methylation in ovarian cancer cells 
BMC Molecular Biology  2014;15:24.
Background
The GBGT1 gene encodes the globoside alpha-1,3-N-acetylgalactosaminyltransferase 1. This enzyme catalyzes the last step in the multi-step biosynthesis of the Forssman (Fs) antigen, a pentaglycosyl ceramide of the globo series glycosphingolipids. While differential GBGT1 mRNA expression has been observed in a variety of human tissues being highest in placenta and ovary, the expression of GBGT1 and the genes encoding the glycosyltransferases and glycosidases involved in the biosynthesis of Fs as well as the possible involvement of DNA methylation in transcriptional regulation of GBGT1 expression have not yet been investigated.
Results
RT-qPCR profiling showed high GBGT1 expression in normal ovary surface epithelial (HOSE) cell lines and low GBGT1 expression in all (e.g. A2780, SKOV3) except one (OVCAR3) investigated ovarian cancer cell lines, a finding that was confirmed by Western blot analysis. Hierarchical cluster analysis showed that GBGT1 was even the most variably expressed gene of Fs biosynthesis-relevant glycogenes and among the investigated cell lines, whereas NAGA which encodes the alpha-N-acetylgalactosaminidase hydrolyzing Fs was not differentially expressed. Bisulfite- and COBRA-analysis of the CpG island methylation status in the GBGT1 promoter region demonstrated high or intermediate levels of GBGT1 DNA methylation in all ovarian cancer cell lines (except for OVCAR3) but marginal levels of DNA methylation in the two HOSE cell lines. The extent of DNA methylation inversely correlated with GBGT1 mRNA and protein expression. Bioinformatic analysis of GBGT1 in The Cancer Genome Atlas ovarian cancer dataset demonstrated that this inverse correlation was also found in primary ovarian cancer tissue samples confirming our cell line-based findings. Restoration of GBGT1 mRNA and protein expression in low GBGT1-expressing A2780 cells was achieved by 5-aza-2’-deoxycytidine treatment and these treated cells exhibited increased helix pomatia agglutinin-staining, reflecting the elevated presence of Fs disaccharide on these cells.
Conclusions
GBGT1 expression is epigenetically silenced through promoter hypermethylation in ovarian cancer. Our findings not only suggest an involvement of DNA methylation in the synthesis of Fs antigen but may also explain earlier studies showing differential GBGT1 expression in various human tissue samples and disease stages.
doi:10.1186/1471-2199-15-24
PMCID: PMC4193910  PMID: 25294702
Globo series; Glycosphingolipids; Forssman antigen; Epigenetics; DNA methylation; Transcriptional regulation
23.  Detection and analysis of the methylation status of PTCH1 gene involved in the hedgehog signaling pathway in a human gastric cancer cell line 
The aim of this study was to investigate the correlation between patched 1 (PTCH1) expression and its methylation in a human gastric cancer cell line, in order to provide new information regarding carcinogenesis and the development of gastric cancer. Quantitative reverse transcription polymerase chain reaction (qPCR) and the immunocytochemical S-P method were used to identify the changes in PTCH1 mRNA and protein expression prior to and following the treatment of the AGS human gastric cancer cell line with 5-aza-2′-deoxycytidine (5-Aza-dc), a methylation inhibitor. The methylation status of the promoter region of the PTCH1 gene in the AGS gastric cancer cell line was examined using methylation-specific PCR (MSP), while CpG island methylation in the PTCH1 gene 5′ regulatory sequence was analyzed using DNA methylation analysis software. The expression of PTCH1 mRNA and protein was absent in the AGS gastric cancer cell line prior to 5-Aza-dc treatment. However, the expression of PTCH1 mRNA and protein appeared in the AGS cells treated with 5-Aza-dc. CpG island hypermethylation of the PTCH1 gene was observed in the AGS gastric cancer cell line using MSP combined with DNA sequencing. PTCH1 expression was negatively correlated with the level of promoter methylation in the AGS cells. In conclusion, the high level of methylation in the PTCH1 gene promoter region may be involved in carcinogenesis and the development of gastric cancer, and may provide a new biomarker for gastric cancer.
doi:10.3892/etm.2013.1334
PMCID: PMC3829737  PMID: 24255663
gastric neoplasms; tumor cell line; PTCH1 gene; DNA methylation; 5-aza-2′-deoxycytidine
24.  Clinical significance of promoter region hypermethylation of microRNA-148a in gastrointestinal cancers 
OncoTargets and therapy  2014;7:853-863.
Background
MicroRNAs are associated with tumor genesis and progression in various carcinomas. MicroRNA-148a (miR-148a) was reported to have low expression in gastrointestinal cancers, and might be regulated by promoter region DNA methylation.
Methods
Bisulfite-modified sequencing was used to determine the promoter region DNA methylation status of human gastrointestinal cancer cell lines. Expression levels of miR-148a in cell lines treated with 5-aza-2′-deoxycytidine were determined by quantitative real-time polymerase chain reaction. Total DNA was extracted from the tissues of 64 patients with gastric cancer and 51 patients with colorectal cancer. Methylation status was determined by methylation-specific polymerase chain reaction. All statistical analyses were performed with SPSS 17.0 software.
Results
The promoter regions of genes in human gastrointestinal cancer cell lines were all hypermethylated, except for HT-29, and the expression of miR-148a tended to be higher than in controls after treatment with 5-aza-2′-deoxycytidine. The methylation-specific polymerase chain reaction results showed that 56.25% of gastric cancer tissues and 19.61% of colorectal cancer tissues were hypermethylated. A strong correlation was found between the expression of miR-148a and the methylation status of promoter regions (P<0.001, chi-square test and Pearson’s correlation). Furthermore, promoter region CpG site hypermethylation of miR-148a was correlated with increased tumor size (P=0.01) in gastric cancer after analyzing the correlation between methylation status and clinicopathologic characteristics.
Conclusion
The promoter region CpG sites were hypermethylated in gastrointestinal cancers. Promoter region hypermethylation status was associated with the expression of miR-148a and tumor invasiveness in gastric cancer, and may prove to be a new biomarker and method for treating gastric cancer.
doi:10.2147/OTT.S60888
PMCID: PMC4045258  PMID: 24920927
microRNAs; methylation; CpG site; clinicopathologic characteristics
25.  Epigenetic inactivation of mir-34b/c in addition to mir-34a and DAPK1 in chronic lymphocytic leukemia 
Background
TP53 mutation/deletion is uncommon in chronic lymphocytic leukemia (CLL). We postulated that components of TP53-centered tumor suppressor network, miR-34b/c, in addition to DAPK1 and miR-34a might be inactivated by DNA hypermethylation. Moreover, we tested if miR-34b/c methylation might correlate with miR-203 or miR-124-1 methylation in CLL.
Methods
miR-34b/c, miR-34a and DAPK1 methylation was studied in 11 normal controls, 7 CLL cell lines, and 78 diagnostic CLL samples by methylation-specific polymerase chain reaction. MEC-1 cells were treated with 5-Aza-2’-deoxycytidine for reversal of methylation-associated miRNA silencing. Tumor suppressor properties of miR-34b were demonstrated by over-expression of precursor miR-34b in MEC-1 cells.
Results
miR-34b/c promoter was unmethylated in normal controls, but completely methylated in 4 CLL cell lines. miR-34b/c expression was inversely correlated with miR-34b/c methylation. Different MSP statuses of miR-34b/c, including complete methylation and complete unmethylation, were verified by quantitative bisulfite pyrosequencing. 5-Aza-2’-deoxycytidine treatment resulted in promoter demethylation and miR-34b re-expression in MEC1 cells. Moreover, over-expression of miR-34b resulted in inhibition of cellular proliferation and increased cell death. In primary CLL samples, miR-34a, miR-34b/c and DAPK1 methylation was detected in 2.6%, 17.9% and 34.6% of patients at diagnosis respectively. Furthermore, 39.7%, 3.8% and 2.6% patients had methylation of one, two or all three genes respectively. Overall, 46.2% patients had methylation of at least one of these three genes. Besides, miR-34b/c methylation was associated with methylation of miR-34a (P = 0.03) and miR-203 (P = 0.012) in CLL.
Conclusions
Taken together, miR-34b/c is a tumor suppressor miRNA frequently methylated, and hence silenced in CLL. Together with DAPK1 methylation, miR-34b/c methylation is implicated in the disruption of the TP53-centered tumor suppressor network. Moreover, the association of miRNA methylation warrants further study.
doi:10.1186/1479-5876-12-52
PMCID: PMC3941938  PMID: 24559316
MicroRNA; TP53 network; Tumor suppressor; DNA methylation; Chronic lymphocytic leukemia

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