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Introduction: Regulator of chromosome condensation 1 (RCC1) is a critical cell cycle regulator. We firstly identified RCC1 gene hypermethylation in gastric tumor tissues using the differential methylation hybridization (DMH) microarray, but the role of RCC1 in the pathogenesis of gastric carcinoma is largely unknown., Methods: Three gastric cancer cell lines (AGS, MKN45, and TSGH9201) were used to analyze RCC1 gene methylation, mRNA and protein expressions. Furthermore, 85 pairs of matched human gastric carcinoma samples in a tissue microarray were used to analyze RCC1 expression by immunohistochemistry staining., Results: A differential methylation pattern was found in TSGH9201 (100%), MKN45 (87%), and AGS (62%) cell lines at the 9th CpG site of RCC1 exon 1. RCC1 mRNA and protein expressions in AGS cells were significantly higher than in TSGH9201 and MKN45 cell lines (P < 0.05). Tissue array data showed that RCC1 expression was detected in 21% (18/85) of gastric carcinoma tissues and in 80% (76/95) of adjacent non-tumor tissues. The expression of RCC1 in gastric carcinoma tissues was significantly lower than in adjacent non-tumor tissues (P < 0.001). Furthermore, an association between RCC1 expression and clinicopathological features showed that RCC1 expression was closely correlated with tumor differentiation and depth of invasion (P < 0.05)., Conclusions: Our data indicate that RCC1 expression is frequently lost in poorly differentiated gastric cell lines and gastric carcinoma tissues. Loss of RCC1 expression is correlated with tumor differentiation and depth of invasion. These findings suggest that RCC1 may play a tumor suppressor role in gastric carcinoma.

Regarding oral squamous cell carcinoma (OSCC) development, chewing areca is known to be a strong risk factor in many Asian cultures. Therefore, we established an OSCC induced mouse model by 4-nitroquinoline-1-oxide (4-NQO), or arecoline, or both treatments, respectively. These are the main two components of the areca nut that could increase the occurrence of OSCC. We examined the effects with the noncommercial MCGI (mouse CpG islands) microarray for genome-wide screening the DNA methylation aberrant in induced OSCC mice. The microarray results showed 34 hypermethylated genes in 4-NQO plus arecoline induced OSCC mice tongue tissues. The examinations also used methylation-specific polymerase chain reaction (MS-PCR) and bisulfite sequencing to realize the methylation pattern in collected mouse tongue tissues and human OSCC cell lines of different grades, respectively. These results showed that retinoic acid receptor β (RARB) was indicated in hypermethylation at the promoter region and the loss of expression during cancer development. According to the results of real-time PCR, it was shown that de novo DNA methyltransferases were involved in gene epigenetic alternations of OSCC. Collectively, our results showed that RARB hypermethylation was involved in the areca-associated oral carcinogenesis.

Cloned animals usually exhibited many defects in physical characteristics or aberrant epigenetic reprogramming, especially in some important organ development. Osteoponin (OPN) is an extracellular-matrix protein involved in heart and bone development and diseases. In this study, we investigated the correlation between OPN mRNA and its promoter methylation changes by the 5-aza-dc treatment in fibroblast cell and promoter assay. Aberrant methylation of porcine OPN was frequently found in different tissues of somatic nuclear transferred cloning pigs, and bisulfite sequence data suggested that the OPN promoter region -2615 to -2239 nucleotides (nt) may be a crucial regulation DNA element. In pig ear fibroblast cell culture study, the demethylation of OPN promoter was found in dose-dependent response of 5-aza-dc treatment and followed the OPN mRNA reexpression. In cloned pig study, discrepant expression pattern was identified in several cloned pig tissues, especially in brain, heart, and ear. Promoter assay data revealed that four methylated CpG sites presenting in the -2615 to -2239 nt region cause significant downregulation of OPN promoter activity. These data suggested that methylation in the OPN promoter plays a crucial role in the regulation of OPN expression that we found in cloned pigs genome.

In mammals, genome-wide epigenetic reprogramming systems exist in primordial germ cells and zygotes. These reprogramming systems play crucial roles in regulating genome functions during critical stages of embryonic development, and they confer the stability of gene expression during mammalian development. The frequent unexpected loss of progeny from somatic cell nuclear transfer (SCNT) is an ongoing problem. In this study, we used six cloned bovines (named NT-1 to NT-6), which were created by ear fibroblast nuclear transfer and displayed short life spans with multiple organ defects, as an experimental model. We focus here on three imprinted genes (IGF2, H19, and XIST) and four satellite loci (Satellite I, Satellite II, Art2, and VNTR) to investigate their methylation changes. The results revealed that aberrant methylation frequently occurred in the analyzed imprinted genes, but not in the satellite loci, of the cloned bovines. After the bovine fibroblast cells were treated with the 5-aza-2(')-deoxycytidine (5-Aza-dc) demethylation agent, the methylation percentages of the XIST and H19 putative differentially methylated region (DMR) were significantly decreased (XIST, p<0.01; H19, p<0.05) followed by an increase in their mRNA expression levels (p<0.01). Furthermore, we found that five short-lived cloned bovines (NT-1 to NT-5) exhibited more severe aberrant methylation changes in the three imprinted genes examined than the little longer-lived clone (NT-6) compared with wild-type (WT) cows. Our data suggest that the reprogramming of the methylation-controlled regions between the imprinted genes and satellite loci are differences and may be involved with additional mechanisms that need further elucidation.

DNA methylation is a major epigenetic modification in the mammalian genome that regulates crucial aspects of gene function. Mammalian cloning by somatic cell nuclear transfer (SCNT) often results in gestational or neonatal failure with only a small proportion of manipulated embryos producing live births. Many of the embryos that survive to term later succumb to a variety of abnormalities that are likely due to inappropriate epigenetic reprogramming. Aberrant methylation patterns of imprinted genes in cloned cattle and mice have been elucidated, but few reports have analyzed the cloned pig genome. Four surviving cloned sows that were created by ear fibroblast nuclear transfer, each with a different life span and multiple organ defects, such as heart defects and bone growth delay, were used as epigenetic study materials. First, we identified four putative differential methylation regions (DMR) of imprinted genes in the wild-type pig genome, including two maternally imprinted loci (INS and IGF2) and two paternally imprinted loci (H19 and IGF2R). Aberrant DNA methylation, either hypermethylation or hypomethylation, commonly appeared in H19 (45% of imprinted loci hypermethylated vs. 30% hypomethylated), IGF2 (40% vs. 0%), INS (50% vs. 5%), and IGF2R (15% vs. 45%) in multiple tissues from these four cloned sows compared with wild-type pigs. Our data suggest that aberrant epigenetic modifications occur frequently in the genome of cloned swine. Even with successful production of cloned swine that avoid prenatal or postnatal death, the perturbation of methylation in imprinted genes still exists, which may be one of reason for their adult pathologies and short life. Understanding the aberrant pattern of gene imprinting would permit improvements in future cloning techniques.

Angiogenesis is induced in response to ischemia. Thrombospondin-1 (TSP-1) is a potent angiostatic factor. Silencing of TSP-1 expression may contribute to the postischemic angiogenesis. Upregulation of TSP-1, in contrast, may terminate the postischemic angiogenesis. A possible mechanism that silences TSP-1 expression is the DNA methylation of its promoter region. DNA methylation has been reported following cerebral ischemia. The present study aimed to explore whether methylation of the promoter region of TSP-1 regulates its expression after oxygen-glucose deprivation (OGD) in murine cerebral endothelial cells (CECs) in vitro. Sublethal OGD increased the extent of methylation of the promoter region of TSP-1 with a concurrent decrease in TSP-1 mRNA and protein expression in CECs. After reoxygenation, demethylation of the TSP-1 promoter region led to the restoration of TSP-1 mRNA and protein expression. The extent of methylation of the promoter region of TSP-1 was inversely correlated with the extent of TSP-1 gene expression at mRNA and protein levels after OGD. Oxygen-glucose deprivation-induced reduction in the TSP-1 mRNA level was not accompanied by a change in mRNA stability. These findings raise the possibility that OGD downregulation of TSP-1 expression is at least in part due to methylation of its promoter region.

Hypermethylation of multiple CpG islands is a common event in cancer. To assess the prognostic values of this epigenetic alteration, we developed Methylation Target Array (MTA), derived from the concept of tissue microarray, for simultaneous analysis of DNA hypermethylation in hundreds of tissue genomes. In MTA, linker-ligated CpG island fragments were digested with methylation-sensitive endonucleases and amplified with flanking primers. A panel of 468 MTA amplicons, which represented the whole repertoire of methylated CpG islands in 93 breast tumors, 20 normal breast tissues, and 4 breast cancer cell lines, were arrayed on nylon membrane for probe hybridization. Positive hybridization signals detected in tumor amplicons, but not in normal amplicons, were indicative of aberrant hypermethylation in tumor samples. This is attributed to aberrant sites that were protected from methylation-sensitive restriction and were amplified by PCR in tumor samples, while the same sites were restricted and could not be amplified in normal samples. Hypermethylation frequencies of the 10 genes tested in breast tumors and cancer cell lines were 60% for GPC3, 58% for RASSF1A, 32% for 3OST3B, 30% for HOXA5, 28% for uPA, 25% for WT1, 23% for BRCA1, 9% for DAPK1, and 0% for KL. Furthermore, hypermethylation of 5 to 7 loci of these genes was significantly correlated with hormone receptor status, clinical stages, and ages at diagnosis of the patients analyzed. This novel approach thus provides an additional avenue for assessing clinicopathological consequences of DNA hypermethylation in breast cancer.

Differential methylation hybridization (DMH) is a high-throughput microarray technique designed to identify changes in DNA methylation patterns commonly observed in cancer and other disease states. The DMH methodology comprises three fundamental components: the arraying of CpG island clones on glass slides, the preparation of the sample amplicons under investigation, and the hybridization of amplicon targets onto the CpG island microarray. Herein, we outline the DMH protocol and illustrate its utility and the validation approaches used in analyzing the hypermethylation profile of breast tumor and normal samples.

Purpose: The purpose of this study was to profile methylation alterations of CpG islands in ovarian tumors and to identify candidate markers for diagnosis and prognosis of the disease., Experimental Design: A global analysis of DNA methylation using a novel microarray approach called differential methylation hybridization was performed on 19 patients with stage III and IV ovarian carcinomas., Results: Hierarchical clustering identified two groups of patients with distinct methylation profiles. Tumors from group 1 contained high levels of concurrent methylation, whereas group 2 tumors had lower tumor methylation levels. The duration of progression-free survival after chemotherapy was significantly shorter for patients in group 1 compared with group 2 (P < 0.001). Differential methylation in tumors was independently confirmed by methylation-specific PCR., Conclusions: The data suggest that a higher degree of CpG island methylation is associated with early disease recurrence after chemotherapy. The differential methylation hybridization assay also identified a select group of CpG island loci that are potentially useful as epigenetic markers for predicting treatment outcome in ovarian cancer patients.

We present a novel concept by using expressed CpG island sequence tags (ECISTs)for dual analysis of DNA methylation and gene expression in cancer cells. ECISTs are present in the genome and are DNA fragments expected to be located in the promoter and first exon region of genes. Their GC-rich segments can be used for screening hypermethylated CpG sites in cancer cells, and their exon-containing portions can be used for measuring levels of the corresponding transcripts. A total of 1162 loci met the criteria of ECISTs from an initial screening of 7776 CpG island tags. This ECIST panel was used to analyze the breast cancer cell line MDA-MB-231, which was treated with a demethylating agent. Microarray profile analysis identified 30 methylation-silenced genes, the expression of which could be directly reactivated by demethylation. An additional group of 96 up-regulated genes was also identified but appeared to be downstream from this epigenetic cascade. Thus, this study shows that the ECIST microarray can be used to differentiate the primary and secondary causes of demethylation and provides an effective tool to elucidate the mechanisms of aberrant DNA methylation in cancer.

Oligonucleotide microarray-based hybridization is an emerging technology for genome-wide detection of DNA variations. We have extended this principle and developed a novel approach, called methylation-specific oligonucleotide (MSO) microarray, for detecting changes of DNA methylation in cancer. The method uses bisulfite-modified DNA as a template for PCR amplification, resulting in conversion of unmethylated cytosine, but not methylated cytosine, into thymine within CpG islands of interest. The amplified product, therefore, may contain a pool of DNA fragments with altered nucleotide sequences due to differential methylation status. A test sample is hybridized to a set of oligonucleotide (19-23 nucleotides in length) arrays that discriminate methylated and unmethylated cytosine at specific nucleotide positions, and quantitative differences in hybridization are determined by fluorescence analysis. A unique control system is also implemented to test the accuracy and reproducibility of oligonucleotides designed for microarray hybridization. This MSO microarray was applied to map methylated CpG sites within the human estrogen receptor alpha (ERalpha) gene CpG island in breast cancer cell lines, normal fibroblasts, breast tumors, and normal controls. Methylation patterns of the breast cancer cell lines, determined by MSO microarray, were further validated by bisulfite nucleotide sequencing (P <0.001). This proof-of-principle study shows that MSO microarray is a promising technique for mapping methylation changes in multiple CpG island loci and for generating epigenetic profiles in cancer.