Methylation-sensitive Representational Difference Analysis Research Articles

CpG methylated ribosomal RNA genes in relation to wing polymorphism in the rice pest Sogatella furcifera

Sogatella furcifera is a major rice pest that exhibits wing dimorphism. The methylation-sensitive representational difference analysis (MS-RDA) is widely used in plants and some animals to identify methylation differences in genomic DNA regions. However the method has been applied to insects very less. The objective of the current research was to monitor differential cytosine methylations at CCGG sequences in macropterous and brachypterous adults of female S. furcifera to determine the association between gene methylation and wing phenotypes using MS-RDA. After the second subtractive hybridization, two differentially methylated DNA fragments were obtained. These DNA fragments were then cloned into the pGEM-T vector and sequenced. One fragment from the positive hybridization was 464 bp, and highly similar to the 18S ribosomal RNA (rRNA) genes from some animals. Another fragment from the reverse hybridization was 453 bp, and homologous to the 28S rRNA gene of S. furcifera with a similarity rate as high as 99%. These results provide potential evidence that DNA methylation of rRNA genes may be related to wing phenotype variations in S. furcifera, and will facilitate future studies on the epigenetic mechanisms of insect wing polymorphism.

Differentially methylated genomic fragments related with sexual dimorphism of rice pests, Sogatella furcifera.

Sogatella furcifera (Hovarth) is a major rice pest with sexual dimorphism. The objective of the current research was to monitor differentially cytosine methylation at CCGG sequences in male and female adults of S. furcifera to determine the association between gene methylation and sexual phenotypes using methylation-sensitive representational difference analysis. After the second subtractive hybridization, four differentially methylated DNA bands were obtained and sequenced. Ten different fragments were found. One fragment from the positive hybridization was 120 bp, and highly similar to the tramtrack genes from Nasonia vitripennis. Another fragment from the reverse hybridization was 414 bp, and homologous to the 28S rRNA gene of S. furcifera with a similarity rate as high as 99%. We also discussed how DNA methylation of tramtrack and 28S rRNA genes produced effects on sexual differentiation and development. These results provide potential evidence that DNA methylation of some genes may be related to sexual phenotype variations in S. furcifera and will facilitate future studies on the epigenetic mechanisms of insect sexual dimorphism.

Paired Box Gene 5 Is a Novel Tumor Suppressor in Hepatocellular Carcinoma Through Interaction With p53 Signaling Pathway §Δ

The paired box 5 (PAX5) is a member of PAX transcription factors family involved in the regulation of embryonic development. However, the role of PAX5 in carcinogenesis is largely unclear. We identified that PAX5 is involved in human cancer by methylation-sensitive representational difference analysis. We examined the biological functions and related molecular mechanisms of PAX5 in hepatocellular carcinoma (HCC). Promoter methylation of PAX5 was evaluated by methylation-specific polymerase chain reaction (PCR) and bisulfite genomic sequencing (BGS). The functions of ectopic PAX5 expression were determined by viability assay, colony formation, and cell cycle analyses, along with in vivo tumorigenicity assays. The PAX5 target signal pathway was identified by promoter luciferase assay, chromosome immunoprecipitation (ChIP), and pathway PCR array. PAX5 is expressed in normal human liver tissue, but silenced or down-regulated in 83% (10/12) of HCC cell lines. The mean expression level of PAX5 was significantly lower in primary HCCs as compared to their adjacent normal tissues (P < 0.0001). The promoter methylation contributes to the inactivation of PAX5. Restoring PAX5 expression in silenced HCC cell lines suppressed cell proliferation, induced apoptosis in vitro, and inhibited tumor growth in nude mice (P < 0.0001). The pathway luciferase reporter assay indicated that PAX5 activated p53 and p21 signaling. ChIP analysis demonstrated that PAX5 directly bound to the p53 promoter. The antitumorigenic function of PAX5 was at least up-regulated by p53 and its downstream targets including tumor necrosis factor, Fas ligand, leucine-rich repeats, and death domain-containing, poly(rC) binding protein 4, p21, and growth arrest and DNA-damage-inducible alpha. PAX5 is frequently inactivated by promoter methylation in HCC. PAX5 appears to be a functional tumor suppressor involved in liver carcinogenesis through direct regulation of the p53 signaling pathway.

MicroRNAs and epigenetics in human breast cancer.

Abstract Abstract #4051 MicroRNAs (miRNAs), which regulate gene expression at the post-transcriptional level, are suggested to play important roles in carcinogenesis. To clarify the miRNAs dysregulation and the relation to epigenetic mechanisms in human breast cancer, we analyzed a breast cancer cell line, MDA-MB-468 that has previously been used for the genome-wide epigenetic analysis: methylation-sensitive-representational difference analysis (MS-RDA). Normal human mammary epithelial cells (HMEC) were compared with MDA-MB-468. The global expression profile (471 miRNAs) of these samples was examined using 3D-Gene Human miRNA oligo chip (Toray, Tokyo, Japan). We identified 4 miRNAs (0.8%), hsa-miR-200a, hsa-miR-429, hsa-miR-652 and hsa-miR-200b, which were expressed at ten-times higher levels in cancer cells and, conversely, 12 miRNAs (2.5%), hsa-miR-137, hsa-miR-138, hsa-miR-34b, hsa-miR-34c, hsa-miR-374, hsa-miR-376a, hsa-miR-422b, hsa-miR-452, hsa-miR-520e, hsa-miR-455, hsa-miR-554 and hsa-miR-574 that were silenced in the cancer cells. Expression analyses using other breast cancer cells (MCF-7, YMB-1-E, T-47D, MDA-MB-231) revealed a significant overexpression of 4 miRNAs, hsa-miR-200a, hsa-miR-429, hsa-miR-652 and hsa-miR-200b and frequent silencing of 5 miRNAs, hsa-miR-138, hsa-miR-34c, hsa-miR-376a, hsa-miR-452, and hsa-miR-455 in breast cancer cells. As for epigenetic mechanisms, overexpressed miRNAs were found to target silenced genes in the MDA-MB-468 cell line, and treatment of MDA-MB-468 with a demethylating agent, 5-aza-2'-deoxycytidine, restored the expression of 5 miRNAs, hsa-miR-137, hsa-miR-374, hsa-miR-520e, hsa-miR-554 and hsa-miR-574. Our results show the existence of a novel miRNA signature in breast cancer, suggesting that epigenetic mechanisms may be involved in the regulation of miRNA expression. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 4051.

Methylation of Protocadherin 10, a Novel Tumor Suppressor, Is Associated With Poor Prognosis in Patients With Gastric Cancer

By using methylation-sensitive representational difference analysis, we identified protocadherin 10 (PCDH10), a gene that encodes a protocadherin and is silenced in a tumor-specific manner. We analyzed its epigenetic inactivation, biological effects, and prognostic significance in gastric cancer. Methylation status was evaluated by combined bisulfite restriction analysis and bisulfite sequencing. The effects of PCDH10 re-expression were determined in growth, apoptosis, proliferation, and invasion assays. PCDH10 target genes were identified by complementary DNA microarray analysis. PCDH10 was silenced or down-regulated in 94% (16 of 17) of gastric cancer cell lines; expression levels were restored by exposure to demethylating agents. Re-expression of PCDH10 in MKN45 gastric cancer cells reduced colony formation in vitro and tumor growth in mice; it also inhibited cell proliferation (P < .01), induced cell apoptosis (P < .001), and repressed cell invasion (P < .05), up-regulating the pro-apoptosis genes Fas, Caspase 8, Jun, and CDKN1A; the antiproliferation gene FGFR; and the anti-invasion gene HTATIP2. PCDH10 methylation was detected in 82% (85 of 104) of gastric tumors compared with 37% (38 of 104) of paired nontumor tissues (P < .0001). In the latter, PCDH10 methylation was higher in precancerous lesions (27 of 45; 60%) than in chronic gastritis samples (11 of 59; 19%) (P < .0001). After a median follow-up period of 16.8 months, multivariate analysis revealed that patients with PCDH10 methylation in adjacent nontumor areas had a significant decrease in overall survival. Kaplan-Meier survival curves showed that PCDH10 methylation was associated significantly with shortened survival in stage I-III gastric cancer patients. PCDH10 is a gastric tumor suppressor; its methylation at early stages of gastric carcinogenesis is an independent prognostic factor.

Identification of Genes Targeted by CpG Island Methylator Phenotype in Neuroblastomas, and Their Possible Integrative Involvement in Poor Prognosis

Background/Aims: CpG island (CGI) methylator phenotype (CIMP) is strongly associated with poor prognosis in neuroblastomas (NBLs; hazard ratios 7–22). Methylation of nonpromoter CGIs is useful to detect the presence of the CIMP, while the poor prognosis is considered to be caused by gene silencing due to promoter methylation. Here, promoter CGIs targeted by the CIMP were searched for. Methods: A genome-wide screening was performed by methylation-sensitive representational difference analysis of CIMP(+) and CIMP(–) NBLs. Results: Promoter CGIs of 9 genes were methylated in CIMP(+) NBL cell lines and caused silencing of their downstream genes. On analysis of 90 clinical specimens, CYP26C1,FERD3L (N-TWIST), CRYBA2 and PCDHGC4 were methylated at significantly higher incidences in CIMP(+) NBLs than CIMP(–) NBLs, while the difference was unclear for NPY, SPAG6, DDIT4L, CHR3SYT and C6Orf141. Methylation of CYP26C1 and FERD3L was significantly associated with poor prognosis, but weaker than the presence of the CIMP. Treatment of an NBL cell line with a demethylating agent caused demethylation of multiple promoter CGIs, and enhanced 13-cis-retinoic acid-induced neuronal differentiation. Conclusion: Our results indicate that the CIMP causes poor prognosis of NBLs by inducing methylation of multiple promoter CGIs with various incidences.

Methylation perturbations in retroelements within the genome of a Mus interspecific hybrid correlate with double minute chromosome formation

A reduction in the DNA modification of cytosine methylation has been linked directly to chromosome rearrangements concomitant with retroelement amplification in several marsupial hybrid genomes. While phenotypes observed for interspecific eutherian hybrids are suggestive of methylation perturbations and retroelement instability, no link between retroelements, DNA methylation, and chromosome instability has yet been identified. Previous studies in eutherian hybrids, however, have been limited to a gross examination of methylation using methylation-sensitive restriction enzyme analysis or focused on single-copy genes and/or have avoided examination of repetitive DNA. Methylation changes and retroelements are proposed as mechanisms for double minute chromosome formation and oncogene amplification, both present in the genome of a Mus hybrid model, thus making it an ideal system to evaluate methylation status more closely. We have used the PCR-based methodologies methylation-sensitive amplicon subtraction (MS-AS) and methylation-sensitive representational difference analysis (MS-RDA) to detect differentially methylated sequences between three complex genomes and to isolate methylation perturbations in a Mus musculus × Mus caroli hybrid. This novel application of MS-AS and MS-RDA resulted in the isolation of differentially methylated retroelements surrounding the locus on Chromosome 10 responsible for double minute chromosome formation within this interspecific eutherian hybrid.

Isolation and bioinformatics analysis of differentially methylated genomic fragments in human gastric cancer

To isolate and analyze the DNA sequences which are methylated differentially between gastric cancer and normal gastric mucosa. The differentially methylated DNA sequences between gastric cancer and normal gastric mucosa were isolated by methylation-sensitive representational difference analysis (MS-RDA). Similarities between the separated fragments and the human genomic DNA were analyzed with Basic Local Alignment Search Tool (BLAST). Three differentially methylated DNA sequences were obtained, two of which have been accepted by GenBank. The accession numbers are AY887106 and AY887107. AY887107 was highly similar to the 11th exon of LOC440683 (98%), 3' end of LOC440887 (99%), and promoter and exon regions of DRD5 (94%). AY887106 was consistent (98%) with a CpG island in ribosomal RNA isolated from colorectal cancer by Minoru Toyota in 1999. The methylation degree is different between gastric cancer and normal gastric mucosa. The differentially methylated DNA sequences can be isolated effectively by MS-RDA.

Aberrant DNA methylation associated with bipolar disorder identified from discordant monozygotic twins

To search DNA methylation difference between monozygotic twins discordant for bipolar disorder, we applied a comprehensive genome scan method, methylation-sensitive representational difference analysis (MS-RDA) to lymphoblastoid cells derived from the twins. MS-RDA isolated 10 DNA fragments derived from 5' region of known genes/ESTs. Among these 10 regions, four regions showed DNA methylation differences between bipolar twin and control co-twin confirmed by bisulfite sequencing. We performed a case-control study of DNA methylation status of these four regions by pyrosequencing. Two regions, upstream regions of spermine synthase (SMS) and peptidylprolyl isomerase E-like (PPIEL) (CN265253), showed aberrant DNA methylation status in bipolar disorder. SMS, a gene on X chromosome, showed significantly higher DNA methylation level in female patients with bipolar disorder compared with control females. However, there was no difference of mRNA expression. In PPIEL, DNA methylation level was significantly lower in patients with bipolar II disorder than in controls. The expression level of PPIEL was significantly higher in bipolar II disorder than in controls. We found strong inverse correlation between gene expression and DNA methylation levels of PPIEL. These results suggest that altered DNA methylation statuses of PPIEL might have some significance in pathophysiology of bipolar disorder..

Identification of PRTFDC1 silencing and aberrant promoter methylation of GPR150, ITGA8 and HOXD11 in ovarian cancers

Methylated promoter CpG islands (CGIs) can be used to find novel tumor-suppressor genes and disease markers. In this study, to identify promoter CGIs aberrantly methylated in human ovarian cancers, we performed a genome-wide screening for differentially methylated DNA fragments using methylation-sensitive-representational difference analysis (MS-RDA). MS-RDA isolated 185 DNA fragments specifically methylated in an ovarian cancer cell line (ES-2), compared with a normal human ovarian surface epithelial cell line (HOSE6-3), and 33 of them were derived from putative promoter CGIs. Ten ovarian cancer cell lines were analyzed by methylation-specific PCR, and seven ( GPR150, LOC222171, PRTFDC1, LOC339210, ITGA8, C9orf64 and HOXD11) of the 33 CGIs were methylated in one or more of the cell lines. Their downstream genes were barely expressed in cell lines without unmethylated DNA molecules by quantitative reverse-transcription-PCR. Demethylation of methylated cell lines with 5-aza-2′-deoxycytidine restored expression of two genes ( PRTFDC1 and C9orf64). In primary ovarian cancers, CGIs of GPR150 (in 4 of 15 cancers), ITGA8 (2/15), PRTFDC1 (1/15) , and HOXD11 (1/15) were methylated. Silencing of PRTFDC1 was revealed here for the first time, and aberrant methylation of GPR150, ITGA8 and HOXD11 could be candidate tumor markers.

Frequent reduced expression of alpha-1B-adrenergic receptor caused by aberrant promoter methylation in gastric cancers

Recent studies have suggested that epigenetic inactivation of tumour-related genes by promoter methylation participates in the development of gastric cancer. We newly identified the frequently aberrant promoter methylation of alpha-1B-adrenergic receptor (ADRA1B) in colorectal cancer by methylation-sensitive representational difference analysis (MS-RDA) and examined the methylation status of the ADRA1B promoter in 34 paired samples of colorectal cancer and surrounding epithelial tissue, and 34 paired samples of gastric cancer and surrounding epithelial tissue. In colorectal cancers, only four of 34 (11.8%) tumours showed ADRA1B promoter methylation. In contrast, ADRA1B promoter methylation was detected in 24 of 34 (70.6%) gastric cancers and in 14 of 34 (41.2%) surrounding epithelial tissues. The frequency of ADRA1B promoter methylation was higher in gastric epithelial tissues with intestinal metaplasia (41.6%) than in those without intestinal metaplasia (25.0%). Reverse transcription–PCR detected reduced ADRA1B expression in 12 of 18 (66.7%) gastric cancers, and its promoter methylation was detected in 11 of these 12 (91.7%) gastric cancers with reduced ADRA1B expression. Thus, ADRA1B promoter is frequently methylated in gastric cancer. Our results suggest that the ADRA1B gene is an important tumour-related gene frequently involved in the development and progression of gastric cancer.

Silencing of Peroxiredoxin 2 and Aberrant Methylation of 33 CpG Islands in Putative Promoter Regions in Human Malignant Melanomas

Aberrant methylation of promoter CpG islands (CGI) is involved in silencing of tumor suppressor genes and is also a potential cancer biomarker. Here, to identify CGIs aberrantly methylated in human melanomas, we did a genome-wide search using methylation-sensitive representational difference analysis. CGIs in putative promoter regions of 34 genes (ABHD9, BARHL1, CLIC5, CNNM1, COL2A1, CPT1C, DDIT4L, DERL3, DHRS3, DPYS, EFEMP2, FAM62C, FAM78A, FLJ33790, GBX2, GPR10, GPRASP1, HOXA9, HOXD11, HOXD12, HOXD13, p14ARF, PAX6, PRDX2, PTPRG, RASD1, RAX, REC8L1, SLC27A3, TGFB2, TLX2, TMEM22, TMEM30B, and UNC5C) were found to be methylated in at least 1 of 13 melanoma cell lines but not in two cultured normal melanocytes. Among these genes, Peroxiredoxin 2 (PRDX2) was expressed in normal melanocytes, and its expression was lost in melanomas with methylation. The loss of expression was restored by treatment of melanomas with a demethylating agent 5-aza-2'-deoxycytidine. In surgical melanoma specimens, methylation of PRDX2 was detected in 3 of 36 (8%). Furthermore, immunohistochemical analysis of PRDX2 showed that disappearance of immunoreactivity tends to associate with its methylation. PRDX2 was recently reported to be a negative regulator of platelet-derived growth factor signaling, and its silencing was suggested to be involved in melanomas. On the other hand, 12 CGIs were methylated in >or=9 of the 13 melanoma cell lines and are considered as candidate melanoma biomarkers.

The Stress-Responsive Gene GADD45G Is a Functional Tumor Suppressor, with Its Response to Environmental Stresses Frequently Disrupted Epigenetically in Multiple Tumors

The CpG island of GADD45G was identified as a target sequence during the identification of hypermethylated genes using methylation-sensitive representational difference analysis combined with 5-aza-2'-deoxycytidine demethylation. Located at the commonly deleted region 9q22, GADD45G is a member of the DNA damage-inducible gene family. In response to stress shock, GADD45G inhibits cell growth and induces apoptosis. Same as other GADD45 members, GADD45G is ubiquitously expressed in all normal adult and fetal tissues. However, its transcriptional silencing or down-regulation and promoter hypermethylation were frequently detected in tumor cell lines, including 11 of 13 (85%) non-Hodgkin's lymphoma, 3 of 6 (50%) Hodgkin's lymphoma, 8 of 11 (73%) nasopharyngeal carcinoma, 2 of 4 (50%) cervical carcinoma, 5 of 17 (29%) esophageal carcinoma, and 2 of 5 (40%) lung carcinoma and other cell lines but not in any immortalized normal epithelial cell line, normal tissue, or peripheral blood mononuclear cells. The silencing of GADD45G could be reversed by 5-aza-2'-deoxycytidine or genetic double knockout of DNMT1 and DNMT3B, indicating a direct epigenetic mechanism. Aberrant methylation was further frequently detected in primary lymphomas although less frequently in primary carcinomas. Only one single sequence change in the coding region was detected in 1 of 25 cell lines examined, indicating that genetic inactivation of GADD45G is very rare. GADD45G could be induced by heat shock or UV irradiation in unmethylated cell lines; however, this stress response was abolished when its promoter becomes hypermethylated. Ectopic expression of GADD45G strongly suppressed tumor cell growth and colony formation in silenced cell lines. These results show that GADD45G can act as a functional new-age tumor suppressor but being frequently inactivated epigenetically in multiple tumors.

Identification of 27 5' CpG islands aberrantly methylated and 13 genes silenced in human pancreatic cancers.

Aberrantly methylated DNA fragments were searched for in human pancreatic cancers, using the genome scanning technique: methylation-sensitive-representational difference analysis (MS-RDA). MS-RDA isolated 111 DNA fragments derived from CpG islands (CGIs), and 35 of them were from CGIs in the 5' regions of known genes. Methylation-specific PCR (MSP) of the CGIs in seven pancreatic cancer cell lines and two pancreatic ductal epithelial cell lines showed that 27 CGIs in the 5' regions were aberrantly methylated in at least one of the cancer cell lines. Quantitative reverse-transcription-PCR analysis showed that downstream genes of all the CGIs were either not expressed or only very weakly expressed in cancer cell lines with the aberrant methylation. In the pancreatic ductal epithelial cell lines, 18 genes were expressed at various levels, and nine genes were not expressed at all. Treatment of a cancer cell line with a demethylating agent, 5-aza-2'-deoxycytidine, restored the expression of 13 genes, RASGRF2, ADAM23, NEF3, NKX2-8, HAND1, EGR4, PRG2, FBN2, CDH2, TLL1, NPTX1, NTSR1 and THBD, showing their silencing by methylation of their 5' CGIs. MSP of 24 primary pancreatic cancers showed that all these genes, except for THBD, were methylated in at least one cancer. Some of those were suggested to be potentially involved in pancreatic cancer development and progression.

Reduced Expression of GNA11 and Silencing of MCT1 in Human Breast Cancers

Alteration in the methylation status of a gene is often associated with its altered expression. Based on a genome scanning technique for differences in CpG methylations, methylation-sensitive representational difference analysis, DNA fragments hypermethylated in a human breast cancer were isolated. A DNA fragment was isolated from intron 1 of guanine-nucleotide-binding protein α-11 (GNA11). mRNA expression of GNA11 was shown to be decreased in 10 of 16 breast cancers by RT-PCR analysis, and the immunoreactivity of the GNA11 product, Gα11 subunit of heterotrimeric G-protein, was observed to be reduced in 14 of the 16 cancers by immunohistochemistry. Methylation of a CpG island (CGI) in the 5′ region of GNA11 or that of intron 1 did not show a clear correlation with its decreased expression. Another DNA fragment was isolated from a CGI in the 5′ upstream region of monocarboxylate transporter 1 (MCT1), and was methylated in 4 of 20 breast cancers. The CGI was also methylated in a human breast cancer cell line, MDA-MB-231, and quantitative RT-PCR showed that its expression was almost lost in the cell line. By treatment of the cells with a demethylating agent, 5-aza-2′-deoxycytidine, the methylation was removed and the expression was restored. GNA11 is involved in signalling of gonadotropin-releasing hormone receptor, which negatively regulates cell growth. MCT1 is involved in cellular transportation of butyrate, which induces cellular differentiation. Downregulation of these two genes was suggested to be involved in human breast cancers.

Identification of Novel Imprinted Genes in a Genome-Wide Screen for Maternal Methylation

A characteristic of imprinted genes is that the maternal and paternal alleles show differences in methylation. To perform a genome-wide screen for novel imprinted loci, we applied methylation-sensitive representational difference analysis (Me-RDA) to parthenogenetic mouse embryos, to identify differentially methylated regions (DMRs) methylated specifically on the maternal allele. We isolated a total of 26 distinct clones from known and novel DMRs and identified three novel imprinted genes. Nap1l5 is located on proximal chromosome 6 and encodes a protein with homology with nucleosome assembly proteins (NAPs); it has tissue-specific imprinting with expression from the paternal allele. We identified two DMRs on chromosome 15, a chromosome that was not thought to contain imprinted loci, and demonstrated that each is associated with a paternally expressed transcript. Peg13 gives rise to a noncoding RNA that is highly expressed in the brain and imprinted in all tissues examined. A DMR was also identified at the chromosome 15 Slc38a4 gene, which encodes a system A amino acid transporter; we show that Slc38a4 is imprinted in a tissue-specific manner. Interestingly, two of the three novel genes identified in this screen are located within the introns of other genes; their identification indicates that such "microimprinted" domains may be more common than previously thought.

Methylation-sensitive representational difference analysis and its application to cancer research.

Methylation-sensitive representational difference analysis (MS-RDA) was previously established to detect differences in the methylation status of two genomes. This method uses the digestion of genomic DNA with a methylation-sensitive restriction enzyme, HpaII, and PCR to prepare "HpaII-amplicons," followed by RDA. An HpaII-amplicon prepared using betaine and reverse electrophoresis was enriched 3.6-fold (compared with the HpaII-amplicon prepared by the original method) with DNA fragments originating from CpG islands (CGIs). As for the specificity of MS-RDA, it was shown that DNA fragments that are unmethylated in the tester and almost completely methylated in the driver are efficiently isolated. This indicated that genes that are in biallelic methylation or in monoallelic methylation with loss of the other allele are efficiently isolated. Further, by use of two additional methylation-sensitive six-base recognition restriction enzymes, SacII and NarI, more DNA fragments were isolated from CGIs in the 5' regions of genes. After analysis of human lung, gastric, and breast cancers, 12 genes were seen to be silenced and additional genes seen to show decreased expression in association with methylation of genomic regions outside CGIs in the 5' regions of genes. MS-RDA is effective in identifying silenced genes in various cancers.

Methylation-associated silencing of heparan sulfate D-glucosaminyl 3-O-sulfotransferase-2 (3-OST-2) in human breast, colon, lung and pancreatic cancers.

Aberrant CpG methylations play important roles in cancer development and progression. In this study, aberrant methylations in human breast cancer were searched for using methylation-sensitive representational difference analysis (MS-RDA). A CpG island (CGI) in the 5' region of the heparan sulfate D-glucosaminyl 3-O-sulfotransferase-2 (3-OST-2) gene was found to be hypermethylated, while its exon 2 was hypomethylated. In seven breast cancer cell lines, hypermethylation of the 5' region and loss of 3-OST-2 expression were observed. Treatment with a demethylating agent, 5-aza-2'-deoxycytidine, removed the methylation of the CGI in the 5' region and restored its expression, demonstrating silencing of the 3-OST-2 gene. Methylation-specific PCR (MSP) analysis in 85 primary breast cancers showed that the hypermethylation of the CGI in the 5' region was present in 75 (88%) of them. Quantitative reverse transcriptase-PCR (RT-PCR) analysis in 37 primary breast cancers showed that the average expression level was decreased in them. Further, MSP analysis in primary colon, lung and pancreatic cancers showed that hypermethylation of the CGI in the 5' region was present in the colon (8/10, 80%), lung (7/10, 70%) and pancreatic (10/10, 100%) cancers. These results showed that silencing of 3-OST-2 was present in a wide range of human cancers. The 3-OST-2 gene encodes an enzyme involved in the final modification step of heparan sulfate proteoglycans (HSPGs), and its silencing is expected to result in abnormal modification of HSPGs and abnormal signal transduction. From the high incidence, silencing of the 3-OST-2 gene is expected to have high diagnostic, and potentially therapeutic, values.

Reduced expression of the insulin‐induced protein 1 and p41 Arp2/3 complex genes in human gastric cancers

Aberrantly methylated DNA fragments in a human gastric cancer were searched for by a genome-scanning method, methylation-sensitive-representational difference analysis (MS-RDA). Six DNA fragments flanked by CpG islands (CGIs) and hypermethylated in the cancer were isolated. Four of the 6 fragments possessed genes in their vicinities. Quantitative RT-PCR analysis of the 4 genes showed reduced expression of 2 genes in cancers: Insulin-induced protein 1 (INSIG1/CL-6) and p41 Arp2/3 complex (p41-Arc). As for INSIG1, a DNA fragment was derived from the edge of a CGI in the promoter region. The edge was methylated in 11 of 22 primary gastric cancers, whereas the center was not methylated in any cancer. INSIG1 expression was markedly reduced in 19 cancers, including the 11 cancers with the methylation. By 5-aza-2'-deoxycytidine treatment of 5 cell lines with the methylation of the edge, partial restoration of INSIG1 expression was detected only in 2 of them. These data indicated that, although the reduced INSIG1 expression in cancers was associated with the methylation at the edge of the CGI in the promoter region, the methylation was likely to be a secondary change. As for p41-Arc, a DNA fragment was derived from a CGI overlapping exon 8, and its methylation did not correlate with its expression. However, methylation of a CGI in the promoter region with a marked reduction of its expression was observed in 1 of the 22 primary cancers. INSIG1 and p41-Arc are known to be involved in cellular differentiation and morphology, respectively, and it was suggested that their reduced expressions might be involved in gastric cancer development or progression.

WTH3, a new member of the Rab6 gene family, and multidrug resistance

The WTH3 gene was obtained by a DNA fragment isolated by the methylation-sensitive representational difference analysis technique due to its hypermethylation in the human multidrug resistant (MDR) breast cancer cell line MCF7/AdrR. The WTH3 gene product is 89% and 91% identical to the human Rab6 and Rab6c proteins, but possesses an elongated C-terminal region which contains 46 extra amino acids. Nevertheless, we consider the WTH3 gene a new member of the Rab6 gene family. Semi-quantitative reverse transcriptase–polymerase chain reaction results showed that WTH3 was 15 and 4 times downregulated in MCF7/AdrR and MES-SA/Dx5, a human MDR uterine sarcoma cell line, as compared to their non-MDR parental cell lines. Permanent expression of the WTH3 transgene in MDR cell lines increased to varying degrees their sensitivity to several anticancer drugs, which included doxorubicin, taxol, vinblastine, vincristine, and etoposide, as compared to the control sublines transfected with the empty vector. Flow cytometry and fluorescence microscope experiments suggest that the WTH3 transgene stimulated the host’s uptake and retention of DOX. Our results imply that the WTH3 gene plays a role(s) in MDR phenotype development in vitro.