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PubMed2004

Coordinate downregulation of a novel imprinted transcript ITUP1 with PEG3 in glioma cell lines.

The human paternally expressed gene 3 (PEG3) on chromosome 19q13.4 is one of the candidate tumor suppressor genes for glioma. We have previously reported that the epigenetic silencing of PEG3 expression in glioma cell lines is dependent on aberrant DNA methylation of an exonic CpG island. Here, we have identified three expressed sequence tags (ESTs), H80201, H78825 and AW197312, that exhibit paternal allele-specific expression, using human monochromosomal hybrids containing the paternal or maternal origin of PEG3 locus. The EST H80201 was shown to be expressed only from the paternal allele in normal human lymphoblasts by utilizing a single nucleotide polymorphism (SNP). Monoallelic expression of EST H80201 was also detected in non-tumor adult human brain tissues of gliomas. These ESTs were located directly adjacent to PEG3 in a head-to-head orientation. We have named this new transcript, imprinted transcript 1, which is located upstream but oppositely oriented to PEG3 (ITUP1). The ITUP1 showed a similar expression profile with PEG3 in glioma cell lines. Bisulfite genomic sequencing and reverse transcription (RT)-PCR analysis indicated that hypermethylation of the promoter region correlated with the absence of these transcripts. This suggests that ITUP1 and PEG3 are coordinately regulated, and that downregulation of the both genes may be important in the development of glioma.

Alleles↗

PubMed2005

Identification of molecular markers for metastasis-related genes in primary breast cancer cells.

Comparing differential gene expression profiles established by cDNA microarray between normal cells (N), primary carcinoma cells (T), and metastatic carcinoma cells (M) may determine those critical genes directly associated with progression and metastasis of breast cancer. Total RNA was extracted by laser microdissection (LMD) from 20 slices of T, N and M from 6 cases. After amplification by a T7-based system, differentially expressed genes between T, N and M were identified by cDNA microarray. In addition, to clarify the mechanism for altered gene expression, we determined the methylation status by sequencing after bisulfite treatment for intriguing genes. As a result, the expression of motility related protein-1 (MRP-1/CD9), peripheral myelin protein-22 (PMP-22), and caspase 3 (CASP-3) were down-regulated in M compared to T. We focused especially on MRP-1 and found that the expression status of MRP-1 was significantly inversely associated with stage of disease in 56 cases of breast cancer (P<0.05), and the relapse free survival in 5 years was significantly higher in MRP-1 positive cases than those negative cases (P<0.05). Conversely, overexpression, by 11-fold, of signal transduction and translation factors were observed in T compared to N. The cancer specific methylation was observed only in CASP-3 in a case. In conclusion, the establishment of the present assay allows us to detect genes directly associated with each cell population within tumor tissue and gives us clues to identify metastasis-related genes comprehensively in clinical breast cancer cases.

Antigens, CD↗

PubMed2001

Silencing of HTR1B and reduced expression of EDN1 in human lung cancers, revealed by methylation-sensitive representational difference analysis.

Aberrantly hypermethylated genes in human lung cancers were searched for by a genome scanning technique, methylation-sensitive-representational difference analysis (MS-RDA). A total of 59 DNA fragments were isolated as those methylated more heavily in either/both of two lung squamous cell carcinoma cell lines, EBC-1 and LK-2, than in a primary culture of normal human bronchial epithelium, NHBE. Thirty-four DNA fragments, whose hypermethylation was confirmed in primary squamous cell carcinomas, were sequenced. By database searches, 17 of them were shown to be located within 2 kb of putative CpG islands, and five of the 17 DNA fragments had transcribed regions of known genes in their vicinities. By RT-PCR of the five genes in the carcinoma cell lines and NHBE, decreased expression of HTR1B (5-hydroxytryptamine receptor 1B) and EDN1 (endothelin-1) was observed. Sequencing after bisulfite modification showed that the CpG island in the promoter region of HTR1B was hypermethylated, while that of EDN1 was not. Demethylation and re-expression of HTR1B were observed after treatment of LK-2 cells with 5-aza-2'-deoxycytidine. In primary lung cancers, decreased mRNA expression of HTR1B was observed in 11 of 20 cases, and that of EDN1 was in 16 of 20 cases. Immunohistochemical analysis of endothelin-1 confirmed that its immunoreactivity was reduced in squamous cell carcinoma cells compared with that in normal bronchial epithelial cells. Considering that endothelin-1 induces apoptosis in melanoma cells and that silencing of endothelin receptor B is observed in prostate cancers, its reduced expression was speculated to confer a growth advantage to lung cancer cells. MS-RDA was shown to isolate DNA fragments that are hypermethylated and silenced, such as HTR1B, and those whose expressions are altered and the methylation statuses outside the promoter region are altered, such as EDN1.

Aged↗

PubMed2026

Nonadditive gene expression and reduced homoeolog expression bias in an intraspecific hexaploid wheat hybrid.

BACKGROUND: Intraspecific hybridization in allopolyploid plants can generate additive and nonadditive changes in gene expression through interactions between divergent parental genomes. However, how it simultaneously affects gene expression and the relative expression of homoeologs in higher-order polyploids is less well understood. To study this, we sequenced seedling leaf transcriptomes and profiled gene body methylation in two hexaploid wheat (Triticum aestivum L.) cultivars and their F₁ hybrids. RESULTS: Although only 4.3% of genes differed in expression between the parents, 22.3% deviated from mid-parent expression in the hybrids, with many showing transgressive expression. 32.1% of triads contained at least one homoeolog that deviated from mid-parent expression, and all three homoeologs deviated in 11% of triads, substantially more than expected by chance. Triads in which all three homoeologs were overexpressed also showed reduced differences in expression among homoeologs. Greater parental divergence in relative homoeolog expression was associated with nonadditive expression. Genes lacking gene body methylation were also more likely to show dominant or transgressive expression, whereas gene body methylation was associated with more balanced homoeolog expression and additive or conserved expression. CONCLUSIONS: Intraspecific hybridization in hexaploid wheat, even without a change in ploidy, was associated with widespread nonadditive gene expression and altered relative homoeolog expression within triads. These responses were associated with parental differences in homoeolog expression and the absence of gene body methylation. Although our findings are limited to seedling leaves from a single intraspecific cross, they provide a basis for testing the generality of these patterns across tissues, developmental stages, and genetic backgrounds.

Triticum↗

PubMed2005

Promoter hyper-methylation of calcium binding proteins S100A6 and S100A2 in human prostate cancer.

BACKGROUND: S100A6 and S100A2 are members of the S100 family of calcium binding proteins, which are down regulated in prostate cancer, however the molecular mechanism(s) underlying their loss of expression is unknown. METHODS: The promoter and exon 1 region of the S100A6 and S100A2 genes was sequenced in bisulfite modified DNA from non-malignant, benign prostatic hyperplasia (BPH), malignant and metastatic prostate tissues and in cell lines. Immunohistochemistry was performed to correlate S100A2 expression with methylation status. RESULTS: S100A6 methylation was absent or occurred at isolated sites in 14/14 cases of non-malignant epithelium and 5/5 cases of BPH tissues, whereas methylation was seen in 14/27 (52%) cases of prostatic cancer (P<0.0001), 2/2 cases of metastatic cancer and in the CWR22 prostatic cancer xenograft. Critical CpG sites within the S100A2 promoter were methylated in LNCaP, LNCaP-LN3, and CWR22 cells but not in Du145, PC3 or BPH45 cells. In tissues, S100A2 methylation was seen in 32/34 (94%) cases of adenocarcinoma and 5/5 cases of metastatic cancer. However, S100A2 methylation was also seen in 9/12 (75%) cases of non-malignant tissues and in 5/5 cases of BPH. Immunostaining, showed absent S100A2 expression all 41 cases of prostatic cancer, whereas staining was seen in the basal cells of non-malignant epithelium. CONCLUSIONS: Loss of S100A6 and S100A2 proteins is frequent in human prostatic cancer. A major mechanism underlying the loss of S100A6 expression appears to involve promoter hyper-methylation. However, mechanisms other than methylation of the known promoter are involved in silencing S100A2 in the prostate.

Adenocarcinoma↗

PubMed2000

Transcriptional inactivation of the tissue inhibitor of metalloproteinase-3 gene by dna hypermethylation of the 5'-CpG island in human gastric cancer cell lines.

The tissue inhibitor of metalloproteinase-3 (TIMP-3), a recently cloned member of TIMP gene family, has been implicated in the negative regulation of tumor cell invasion and tumor growth. Down-regulation of this gene has been shown to occur in a mouse carcinogenesis model, suggesting that it might play a role in the tumor progression of some cancers. In this study, we used human gastric cancer cell lines to investigate whether TIMP-3 gene expression is suppressed in human gastric cancer. We examined whether aberrant DNA methylation of the 5'-CpG island of the TIMP-3 gene is involved in this cancer. Nine of 10 human gastric cancer cell lines completely lost TIMP-3 gene expression compared with normal samples. Southern blot analysis and bisulfite genomic sequencing revealed aberrant hypermethylation near the transcription-start site of the TIMP-3 gene in all cell lines lacking TIMP-3 expression. Treatment of these cell lines with the demethylating agent 5-aza-2'-deoxycytidine restored TIMP-3 gene expression. Our results suggest that the TIMP-3 gene is another early target of tumor-associated aberrant DNA methylation in human gastric carcinogenesis. Consequently, genetic silencing of TIMP-3 may lead to a more malignant and invasive phenotype in these cancer cells.

Animals↗

PubMed2001

Hypermethylation of the caveolin-1 gene promoter in prostate cancer.

BACKGROUND: Hypermethylation of CpG islands in the promoter regions of tumor suppressor genes is one mechanism of tumorigenesis. Caveolin-1 (Cav-1), a gene coding for the structural component of cellular caveolae, is involved in cell signaling and has been proposed to be a tumor suppressor gene in several malignancies. This gene maps to 7q31.1, a site known to be deleted in some prostate tumors. We chose to examine the methylation status of the promoter region of Cav-1 to determine whether this gene could function as a tumor suppressor in prostate cancer METHODS: Genomic DNA from both tumor and normal prostate epithelial cells was obtained from paraffin-embedded prostate sections by laser capture microdissection (LCM). The methylation status of 24 CpG sites at the 5' promoter region of Cav-1 was analyzed by bisulfite-direct-sequencing after amplification by PCR using primers specific for bisulfate modified DNA. Immunohistochemistry staining with a cav-1-specific antibody was also performed to evaluate the expression of the gene RESULTS: Twenty of the 22 (90.9%) informative cases showed promoter hypermethylation in the tumor cell population when compared with adjacent normal prostate cells with an average Methylation Index (potential frequency of total possible methylated Cs) from tumor cells equal to 0.426 vs. 0.186 for normal cells (P = 0.001). While no association with Gleason grade was found, overall increased methylation correlated with PSA failure (P = 0.016), suggestive of clinical recurrence. Elevated immunoreactivity with a Cav-1 antibody was observed in tumor cells from 7 of 26 prostate samples tested; this was associated with a Gleason score but not correlated with PSA failure or Methylation Index CONCLUSIONS: CpG sites at the 5' promoter of Cav-1 are more methylated in tumor than in adjacent normal prostate cells. Hypermethylation of the Cav-1 promoter supports the notion that Cav-1 may function as a tumor suppressor gene in prostate cancer and evidence is presented suggesting that methylation status of this gene is not only a marker for cancer but also may be predictive of outcome.

Aged↗

PubMed2001

CpG hypermethylation of the promoter region inactivates the estrogen receptor-beta gene in patients with prostate carcinoma.

BACKGROUND: The down-regulation of the estrogen receptor-beta (ERbeta) gene is associated with several malignancies, including prostate carcinoma. The purpose of the current study was to investigate the mechanisms of ERbeta inactivation through the analysis of CpG methylation of the promoter region of ERbeta gene. METHODS: ERbeta protein expression was examined by immunohistochemistry in 23 cases of human prostate carcinoma and 40 cases of benign prostatic hyperplasia (BPH). DNA was extracted from these tissues and processed for sodium bisulfite genomic sequencing. The percentage of methylation of CpG sites in the promoter region of ERbeta (-376 to -117), which contains 19 CpG sites, was determined from genomic sequencing data. The prostate carcinoma cell lines DU145 and ND1 were treated with the demethylating agent 5-AZAC and ERbeta mRNA expression was analyzed by reverse transcriptase-polymerase chain reaction. RESULTS: In BPH tissues, ERbeta protein expression was found mainly in epithelial cells. ERbeta protein expression was lacking in 83% of prostate carcinoma samples (19 of 23 samples) whereas all cases of BPH (40 of 40) demonstrated expression of ERbeta protein. The mechanism of inactivation of the ERbeta gene in prostate carcinoma was CpG methylation because the degree of methylation at all CpG sites within the promoter region between -376 and -117 was higher in prostate carcinoma samples compared with BPH tissues. Nine of 19 CpG sites within the promoter region of ERbeta displayed significant differences in methylation between prostate carcinoma and BPH samples. The prostate carcinoma cell lines appeared to lack ERbeta expression. However, 5-AZAC treatment restored ERbeta expression in those cell lines, suggesting that methylation inactivates the ERbeta gene in prostate carcinoma. CONCLUSIONS: The results of the current study demonstrate, for what we believe to be the first time, that the inactivation of the ERbeta gene in prostate carcinoma occurs through CpG methylation of the promoter region of this gene.

Azacitidine↗

PubMed2006

Hypermethylated promoter region of DR3, the death receptor 3 gene, in rheumatoid arthritis synovial cells.

OBJECTIVE: To examine the promoter activity and protein expression of the death receptor 3 gene DR3, a member of the apoptosis-inducing Fas gene family, with particular reference to the methylation status of its promoter region in rheumatoid arthritis (RA). METHODS: Genomic DNA was prepared from peripheral blood mononuclear cells obtained from healthy individuals and from patients with RA and synovial cells obtained from patients with RA and osteoarthritis. The methylation status of the DR3 promoter was analyzed by bisulfite genomic sequencing and methylation-specific polymerase chain reaction techniques. Gene promoter activity and protein expression were examined using the luciferase reporter and Western blotting techniques. RESULTS: The promoter region of the DR3 gene contained many CpG motifs, including one CpG island that was specifically hypermethylated in synovial cells from patients with RA. Promoter assays showed that the promoter CpG island was essential for the transactivation of the DR3 gene and that forced hypermethylation of the CpG island with the bacterial methylase Sss I in vitro resulted in inhibition of the DR3 gene expression. Furthermore, the expression of DR-3 protein was down-modulated in association with methylation of the promoter CpG island in RA synovial cells. CONCLUSION: The CpG island in the DR3 gene promoter was specifically methylated to down-modulate the expression of DR-3 protein in rheumatoid synovial cells, which may provide resistance to apoptosis in RA synovial cells.

Adult↗

PubMed2005

CpG methylation at promoter site -140 inactivates TGFbeta2 receptor gene in prostate cancer.

BACKGROUND: The action of transforming growth factor beta (TGF-beta) is mediated through type 1 (TbetaRI) and type 2 (TbetaRII) receptors. Prostate cancer cells are often resistant to TGF-beta signaling due to loss of TbetaRII expression. The authors of the current study hypothesized that CpG methylation of the TbetaRII promoter at the Sp1 binding site -140 mediates this loss of TbetaRII expression in prostate cancer. METHODS: Sixty-seven prostate cancer (PC) samples, 8 benign prostatic hyperplasia (BPH) samples, and 4 prostate cancer cell lines (DUPro, LNCaP, ND-1 and PC-3) were analyzed for 1) TbetaRII mRNA expression by semiquantitative RT-PCR, 2) TbetaRII protein expression by immunohistochemistry, and 3) TGFbetaRII promoter methylation at CpG site -140 by methylation specific PCR and bisulfite DNA sequencing. Prostate cancer cell lines were treated with the demethylating agent 5aza2'deoxycytidine to determine if TbetaRII gene expression could be increased by blocking promoter methylation. RESULTS: mRNA and protein expression of TbetaRII was lower in the PC samples than in the BPH samples. CpG methylation at site -140 was higher in PC than in BPH (P < 0.01). Promoter methylation was inversely correlated with TbetaRII mRNA expression in the PC and BPH samples (P < 0.0001). PC3, ND1, and DUPro TbetaRII mRNA expression increased following treatment of cells with 5-aza-2'-deoxycytidine. CONCLUSION: CpG methylation of the TbetaRII promoter at CPG site -140 leads to functional loss of the TbetaRII gene in prostate cancer. Treatment with 5-aza-2' deoxycytidine can restore gene expression. The current study results report the first association between prostate cancer and loss of the TGF- beta signaling pathway by TbetaRII DNA promoter methylation.

Azacitidine↗

PubMed2006

Smoking influences aberrant CpG hypermethylation of multiple genes in human prostate carcinoma.

BACKGROUND: Aberrant CpG methylation profiles of gene promoters and their correlation with advanced pathologic features have been well investigated in prostate carcinoma (PC). Several case-control and prospective studies have revealed a positive association between current smoking and PC. The authors hypothesized that smoking influences both progression and prognosis of PC through CpG hypermethylation of related genes. METHODS: A total of 164 PC patients (52 current, 30 former, and 82 never smokers) and 69 benign prostatic hyperplasia (BPH) patients were examined by methylation-specific PCR (MSP) for 3 genes: adenomatous polyposis coli (APC), glutathione S-transferase pi (GSTP1), and multidrug resistance one (MDR1). The methylation status of representative samples was confirmed by bisulfite DNA sequencing analysis. The newly defined methylation score (M-score) of each sample is the sum of the corresponding log hazard ratio (HR) coefficients derived from multivariate logistic regression analysis for pathology (BPH vs. PC), and was related to clinical and pathologic outcome including smoking status. RESULTS: The M-score was significantly higher in the current smokers than in never smokers (P = 0.008). Spearman rank correlation test demonstrated a significant correlation between pack-years smoked and M-score in PCs (P = 0.039). Significant correlation of the M-score methylation was observed with high pT category (P < 0.001), high Gleason sum (P < 0.001), high preoperative prostate-specific antigen (PSA) (P = 0.041), and advanced pathologic features. In addition, Gleason sum was significantly associated with PSA failure-free probability as a poor outcome (P = 0.020). CONCLUSIONS: This is the first study to demonstrate significant correlation of the methylation status of multigenes with smoking status in PC. Smoking status may influence both progression and prognosis of PC through CpG hypermethylation of related genes.

ATP Binding Cassette Transporter, Subfamily B, Mem↗

PubMed2006

Caspase-8 levels correlate with the expression of signal transducer and activator of transcription 1 in high-grade but not lower grade neuroblastoma.

BACKGROUND: Previous studies in advanced-stage neuroblastoma (NB) have shown a link between the silencing of caspase-8 and methylation of a regulatory region at the boundary between caspase-8 exon 3 and intron 3. However, a number of recent studies from NB cell lines have shown that the transcriptional regulation of caspase-8 may reside with interferon gamma-sensitive promoters through the action of transcription factors, such as signal transducer and activator of transcription 1 (STAT-1). In this study, the authors tested the hypothesis that there is a correlation between caspase-8 and STAT-1 protein expression levels that may be linked to methylation of the regulatory elements of either of these genes. METHODS: Thirty clinical tumor samples of all stages, including 13 samples from patients with Stage 4 disease, were analyzed by quantitative immunoblotting for caspase-8 and STAT-1. The DNA methylation status of putative caspase-8 and STAT-1 regulatory elements were determined by bisulfite-modified sequencing analysis. RESULTS: A significant correlation was observed between caspase-8 and STAT-1 protein levels in Stage 4 NB samples but not in lower stage NB samples. Caspase-8 and STAT-1 protein levels varied widely across all stages of NB and did not correlate with methylation of these genes. CONCLUSIONS: A strong correlation was observed between STAT-1 levels and caspase-8 levels in clinical Stage 4 NB. This suggests that STAT-1 or similar transcription factors, and not methylation, may play a role in controlling caspase-8 levels in this illness. No evidence of such a correlation between caspase-8 and STAT-1 levels was observed in lower clinical stages, suggesting that mechanisms controlling caspase-8 expression in NB vary with clinical stage.

Caspase 8↗

PubMed2002

Densely methylated MLH1 promoter correlates with decreased mRNA expression in sporadic colorectal cancers.

It has been reported that MLH1 is silenced by promoter methylation, and that this phenomenon is associated with microsatellite instability (MSI) in sporadic colorectal cancer (CRC). To clarify the significance of MLH1 promoter methylation in sporadic CRC, we examined the correlation between methylation status over the entire promoter region and mRNA expression in cases showing high-frequency MSI (MSI-H). MLH1 promoter methylation was analyzed using the bisulfite modification sequencing in 48 MSI-H cases. We also screened for somatic mutation, loss of heterozygosity, and immunohistochemical staining of MLH1. The results showed that methylation patterns could be subdivided into three types: methylation of more than 80% of the CpG sites analyzed (type 1 methylation), methylation of less than 20% (type 2 methylation), and methylation mainly in the region 500 to 921 bases upstream from the translation start site (type 3 methylation). Of the three types, only type 1 methylation correlated with decreased mRNA expression. The frequency of type 1 methylation was significantly higher in cases involving the proximal colon (66.7%, 18/27) compared to that of the distal colon and rectum (23.8%, 5/21, P = 0.004). Immunohistochemical staining of MSI-H cases showed that decreased MLH1 was found in 77.1% (37/48). Of the cases with decreased MLH1, type 1 methylation was present in 59.5% (22/37). Overall, our data suggested that the type 1 methylation pattern may affect MLH1 mRNA expression, such that the majority of MSI-H cases in sporadic CRC, especially proximal colon cancer, exhibited type 1 methylation.

Adaptor Proteins, Signal Transducing↗

PubMed2004

Mapping of a candidate colorectal cancer tumor-suppressor gene to a 900-kilobase region on the short arm of chromosome 8.

Loss of heterozygosity (LOH) on 8p occurs at high frequencies in many tumor types, including colorectal carcinoma (CRC). We previously used microcell-mediated chromosome transfer (MMCT) into the CRC cell line SW620 to map a approximately 7.7-Mb colorectal cancer-suppressor region (CRCSR) at 8p22-23.1. In the current study, we transferred small fragments of this CRCSR into SW620 to refine the region further. Two microcell hybrids containing a 321- to 898-kb region around the D8S552 marker at 8p23.1 showed suppression of soft agar clonicity and tumorigenicity in athymic mice when compared to control cell lines. These data suggest that the putative colorectal tumor-suppressor gene is within this small region. We analyzed two candidate genes within this region: FLJ23749 and KIAA1456. Expression of both genes was detected in normal colonic crypt cells and in mucosa. Quantitative reverse transcriptase polymerase chain reaction showed downregulation of KIAA1456 in 9 of 12 primary colorectal tumors compared to matching normal mucosa, but normal or increased expression of FLJ23749. FLJ23749 was expressed in all CRC cell lines tested; however, KIAA1456 was downregulated in three cell lines, including SW620, and was restored in the suppressed microcell hybrids. 5'aza-2'Deoxycytidine treatment of the downregulated cell lines restored expression of KIAA1456, but bisulfite genomic sequencing did not show a correlation between promoter methylation and expression. Forty percent of the primary tumors showed LOH at this CRCSR locus, and mutation analysis revealed somatic mutations in 1 of 88 primary colorectal tumors for both KIAA1456 and FLJ23749. Despite the rarity of somatic mutations, the expression data suggest that KIAA1456 is still a candidate for the putative 8p colorectal cancer tumor-suppressor gene.

Animals↗

PubMed2004

General implications for CpG hot spot mutations: methylation patterns of the human iduronate-2-sulfatase gene locus.

The methylation pattern at CpG sites of a housekeeping gene correlates with the likelihood of mutation. Mucopolysaccharidosis (MPS) type II, an X-linked disorder, results from the deficiency of iduronate-2-sulfatase (IDS). In these patients, over 35% of independent point mutations at the IDS gene locus were found at CpG sites as transitional events. To gain insight into the relationship between methylation status and CpG hot spot mutations, we investigated patterns of cytosine methylation in the entire IDS gene, except for introns 4-8. Bisulfite genomic sequencing was performed on the normal leukocyte DNA. Our data show that: 1) cytosine methylation at the CpG sites was extensive, except for those present from the promoter region to a portion of intron 3; 2) a sharp boundary of methylated-nonmethylated regions was observed at the 5'-flanking region, whereas a gradual change in methylation was observed in the 2.0-kb segment in the 3'-flanking region; 3) the boundary of the 5'-flanking region contained multiple Sp1 sites and the TATA box; 4) the CpG sites in exons 1 and 2 were hypomethylated and were associated only with rare transitional mutations, while the CpG sites in exon 3 were also hypomethylated, yet were associated with a high rate of transitional mutations; 5) there was no striking sex difference in the methylation patterns in active alleles; and, 6) the methylation in both strands was symmetrical, except at the boundary of methylated-unmethylated regions.

Amino Acid Sequence↗

PubMed2002

Analysis of mutations and identification of several polymorphisms in the putative promoter region of the P34CDC2-related CDC2L1 gene located at 1P36 in melanoma cell lines and melanoma families.

Chromosome 1 abnormalities are the most commonly detected aberrations in many cancers including malignant melanoma. Partial deletions and an allelic loss of the chromosome 1p36 region observed in melanoma indicate the presence of putative tumor suppressor gene(s) in this region. A candidate gene, CDC2L1, which encodes PITSLRE proteins related to p34(cdc2), is mapped to 1p36. To determine whether CDC2L1 mutation is involved in melanoma development, we examined 20 melanoma cell lines and 11 members of melanoma-prone families linked to chromosome 1p36. Mutation analysis throughout the entire coding region of the CDC2L1 gene revealed only 1 mutation (C-->T at nucleotide location 97 of exon 7, Ser-->Leu) in the melanoma cell line UACC 903 out of 20 melanoma cell lines and 6 melanoma cases. However, 4 polymorphic nucleotide changes, C-48T, G-53C, T-103C and T-210C, in the putative promoter region of CDC2L1 were identified. The 4 variants were located within or beside the conserved binding sites of transcription factors TCF11, MZF1 and TAAC box, indicating their potential effects on the regulation of CDC2L1 expression. No aberrant methylation of the CDC2L1 CpG island in the promoter region was observed by sodium bisulfite genomic sequencing. These results indicate that mutations are rare in the CDC2L1 gene in these melanoma cell lines and melanoma families and that the aberrant cytosine methylation of the CDC2L1 CpG island is not the mechanism of CDC2L1 repression in melanoma. The contribution of 4 promoter polymorphisms to the transcriptional regulation of the gene and its association with melanoma warrants further investigation.

CDC2 Protein Kinase↗

PubMed2003

ASC/TMS1, a caspase-1 activating adaptor, is downregulated by aberrant methylation in human melanoma.

ASC/TMS1 is an adaptor protein activating caspase-1 that stimulates processing of proIL-1beta and proIL-18. ASC was reported to be aberrantly methylated and silenced in human breast cancers. In our present study, ASC expression was examined in 12 melanoma cell lines by Western blot analysis and in 18 benign melanocytic nevi and 32 melanoma tissues by immunohistochemical staining. ASC expression was absent or reduced in 7 of 12 (58.3%) cell lines and in 20 of 32 (62.5%) melanoma tissues, whereas all 18 benign melanocytic nevi showed intensive ASC expression. To investigate whether ASC silencing in melanoma is involved in aberrant methylation, methylation specific PCR was carried out. Five of ten (50%) melanoma tissues exhibited methylation in CpG island of ASC companied with reduced ASC expression. Six of twelve (50%) melanoma cell lines showed aberrant methylation in the ASC gene, and 4 of the 6 (66.7%) methylation positive cell lines exhibited reduced ASC expression. We characterized methylation patterns in melanoma cell lines by using bisulfite genomic sequencing, and found that the degree of aberrant methylation correlated with the level of reductive ASC expression. Treatment with demethylating agent 5-aza-2'-deoxycytidine resulted in both demethylation of the ASC gene and the upregulation of ASC expression in the methylation positive melanoma cell lines. Our study shows that ASC is downregulated in melanoma, and that its suppression is partially mediated by aberrant methylation.

Apoptosis↗

PubMed2006

Analysis of genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine in gastric cancer cell lines.

In gastric cancer, increasing numbers of genes have been reported to be silenced by aberrant methylation. However, global analysis of epigenetic inactivation in cancer cells has rarely been performed. For screening the genes upregulated by the demethylating agent 5-aza-2'-deoxycytidine (DAC), cDNA microarray analysis (AceGene(R), containing 30,000 genes) was performed using gastric cancer cell lines (AGS, MKN74, MKN1, MKN45 and Kato3) treated with DAC. The candidate upregulated genes were confirmed by real-time PCR, and the methylation status of 5'CpG islands was determined by bisulfite DNA sequencing or methylation-specific PCR. Among the upregulated genes considered to have CpG island in their promoter regions, we selected 5 genes (BCL2L10, DKK1, DNAJD1, GAGED2 and NMU) that exhibited a greater than 3-fold increase in at least 2 cell lines. Of these, we could determine the methylation status of 5'CpG islands of BCL2L10, DKK1 and DNAJD1. 5'CpG of BCL2L10 and DNAJD1 was hypermethylated in 4 of 5 gastric cancer cell lines, whereas 5'CpG of DKK1 was hypermethylated in only 1 cell line. MSP analysis for BCL2L10 revealed that the CpG island was demethylated after DAC treatment. In addition, we observed that overexpression of BCL2L10 could promote apoptosis and growth-inhibitory effect in gastric cancer cell lines. In conclusion, some of the genes upregulated by DAC treatment may be transcriptionally repressed by promoter hypermethylation. These genes might be related to gastric carcinogenesis. In particular, the suppression of BCL2L10, which could induce apoptosis and inhibit proliferation of cancer cells, might be one of the underlying mechanisms for gastric carcinogenesis.

Azacitidine↗