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PubMed2004

Demethylation of the synuclein gamma gene CpG island in primary gastric cancers and gastric cancer cell lines.

PURPOSE: Whereas synuclein gamma (SNCG) gene expression is usually highly tissue-specific and restricted to the nervous system, SNCG is expressed in advanced-stage breast and ovarian cancers. When overexpressed, SNCG stimulates cancer cell proliferation and metastasis. It is thought that the molecular mechanism of CpG island demethylation may underlie aberrant SNCG expression. To determine whether aberrant SNCG expression and demethylation play a role in gastric carcinogenesis, we examined the expression and methylation status of SNCG in primary gastric cancers, gastric cancer cell lines, and non-neoplastic gastric mucosal tissues. EXPERIMENTAL DESIGN: Ten gastric cancer cell lines, 105 primary gastric cancers, and 10 non-neoplastic gastric mucosal tissues were examined. SNCG expression and methylation status were examined by reverse transcription-PCR and bisulfite-single-strand conformational polymorphism followed by direct sequencing, respectively. The relationship between SNCG methylation status and various clinicopathological factors of the primary gastric cancers was then analyzed. RESULTS: SNCG mRNA expression was observed in 5 of 10 cell lines. Analysis of cell lines positive for SNCG expression revealed that most of the SNCG CpGs were demethylated. SNCG mRNA was not expressed in the 10 non-neoplastic gastric mucosal tissues, although several CpGs were demethylated. Of the 105 primary gastric cancers, 40 (38.1%) showed apparent SNCG demethylation, similar to the result obtained using cell lines. SNCG demethylation was more frequent in primary gastric cancers positive for lymph node metastasis (51%; 26 of 51) than in cancers without lymph node involvement (26%; 14 of 54; P < 0.05), and also more common in stage II-IV (48%; 27 of 56) than in stage I (27%; 13 of 49) cancers (P < 0.05). CONCLUSIONS: Aberrant SNCG gene expression can occur via CpG island demethylation, and tends to occur during the more progressive stages of gastric carcinogenesis.

Adult↗
PubMed2006

Two-color quantitative multiplex methylation-specific PCR.

In recent years, several methylation-specific PCR-based techniques have been developed to identify and characterize hypermethylation of CpG dinucleotides with the primary goal of elucidating a better understanding of the role of DNA methylation in important biological processes, such as chromosome X inactivation and carcinogenesis. The specificity of methylation-specific PCR (MSP) techniques relies on amplifying sodium bisulfite-treated DNA with primers specific to predicted sequences of unmethylated and methylated DNA within the gene of interest. In the past, unmethylated and methylated reactions were singleplex and performed in separate wells. In this paper we report a modification of the real-time quantitative multiplex MSP (QM-MSP) technique of Fackler and colleagues that can be applied to any real-time MSP experiment. Although co-amplification with multiple fluorophores is common in standard reverse transcription PCR (RT-PCR), MSP presents unique challenges both mechanistically and operationally that must be overcome in order to successfully co-amplify two methylation-specific targets. In this two-color modification, unmethylated and methylated primer/probe sets are successfully co-amplified in the same reaction using FAM- and VIC-labeled probes. Our modification decreases the cost and time of each real-time experiment by allowing increased throughput of clinical samples and by doubling either the number of genes or the number of samples that can be analyzed per real-time plate.

Breast Neoplasms↗
PubMed1997

[Molecular analysis of spontaneous and arsenite-induced mutations at the gpt locus in Chinese hamster ovary cells].

In this study, we examined the mutagenicity of sodium arsenite at the xathine-guanine phosphoribosyl transferase locus (gpt) in a pSV2 gpt-transformed CHO cell line AS52. The chemical induced a dose-dependent increase of mutant frequency at the locus. Nested PCR analysis revealed that the majority of arsenite-induced AS52 mutants had totally deleted the gpt gene. For the spontaneous, 50 mumol/L arsenite--and 100 mumol/L arsenite--induced mutants in AS52 cells, the percentages of total deletion of the gpt gene were 36.00%, 54.72% and 66.67% respectively. The PCR products of gpt gene from nine 100 mumo/L sodium bisulfite-induced mutants were analyzed with direct DNA sequence technology. The frameshift mutation was identified in two mutants (2/9). No genomic alteration could be found in the structural gene examined in the other 7 mutants, where their molecular alterations probably located in the promoter region.

Animals↗
PubMed1985

Excision of uracil residues in DNA: mechanism of action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases.

Various octadeoxynucleotides containing uracil at different positions were synthesized and submitted to the action of Escherichia coli and Micrococcus luteus uracil-DNA glycosylases. A uracil residue situated at the 5'-end was excised by the M.luteus enzyme but not by the E.coli one. Uracil residues located at the ultimate and penultimate positions at the 3'-end were not cleaved by either enzymes. At the other central positions, uracil was eliminated with different initial velocities. Single stranded phi X 174 DNA fragments were used to study the influence of the sequence. Cytosine bases were deaminated to give uracil by bisulfite treatment. It was shown that the initial excision velocity of two vicinal uracil residues was decreased. The same observation was made for two uracils separated by one base. A hypothetical scheme is suggested to explain the mechanism of action of uracil-DNA glycosylases.

Base Sequence↗
PubMed2004

A method for cloning and sequencing long palindromic DNA junctions.

DNA sequences containing long adjacent inverted repeats (palindromes) are inherently unstable and are associated with many types of chromosomal rearrangements. The instability associated with palindromic sequences also creates difficulties in their molecular analysis: long palindromes (>250 bp/arm) are highly unstable in Escherichia coli, and cannot be directly PCR amplified or sequenced due to their propensity to form intra-strand hairpins. Here, we show that DNA molecules containing long palindromes (>900 bp/arm) can be transformed and stably maintained in Saccharomyces cerevisiae cells lacking a functional SAE2 gene. Treatment of the palindrome-containing DNA with sodium bisulfite at high temperature results in deamination of cytosine, converting it to uracil and thus reducing the propensity to form intra-strand hairpins. The bisulfite-treated DNA can then be PCR amplified, cloned and sequenced, allowing determination of the nucleotide sequence of the junctions. Our data demonstrates that long palindromes with either no spacer (perfect) or a 2 bp spacer can be stably maintained, recovered and sequenced from sae2Delta yeast cells. Since DNA sequences from mammalian cells can be gap repaired by their co-transformation into yeast cells with an appropriate vector, the methods described in this manuscript should provide some of the necessary tools to isolate and characterize palindromic junctions from mammalian cells.

Base Sequence↗
PubMed1994

Bisulfite induces tandem double CC-->TT mutations in double-stranded DNA. 2. Kinetics of cytosine deamination.

Deamination of cytosine to uracil in double-stranded DNA (ds DNA) by sodium bisulfite has been monitored with a sensitive genetic assay. In this system, reversion of a mutant in the lacZ alpha gene coding sequence of bacteriophage M13mp2 C141 was detected by employing an ung- bacterial strain defective in the enzyme uracil glycosylase. Within the 4-base target, it is possible to measure the rates of induction of C-->T, C-->A, C-->G, and CC-->TT mutations in DNA that has been incubated at physiological temperature and pH and then transfected into ung+ and ung- E. coli cells, respectively, for amplification and detection of the mutation. For concentrations of bisulfite from 1 to 50 mM, the reversion frequency in ung- cells increased linearly with time of incubation. The most interesting features of the bisulfite reaction were as follow: (1) Mutations were reduced 5-fold in ung+ cells, indicating ung is involved in repair of bisulfite-treated transforming DNA. (2) Sequencing of 157 revertants revealed that C-->T and tandem CC-->TT transition mutations comprised 100% of the mutations scored. (3) A unique finding was that, at the highest concentrations and longest incubation times, almost every mutant obtained in ds DNA exposed to bisulfite was found to be a CC-->TT tandem double mutation. (4) The high frequency of tandem double mutants is inconsistent with two random, independent mutational events and, coupled with the observed ung dependence, lends support to the concept of catalytic deamination, wherein bisulfite induces deamination in contiguous cytosines by a concerted mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage M13↗
PubMed1999

Methylation-sensitive, single-strand conformation analysis (MS-SSCA): A rapid method to screen for and analyze methylation.

We have developed methylation-sensitive, single-strand conformation analysis (MS-SSCA) as a method of screening for methylation changes. Bisulfite modification converts cytosines to thymines, but methylated cytosines remain unchanged. This modification creates sequence differences between methylated and unmethylated samples, which can be resolved by SSCA. SSCA is 70-95% efficient at detecting single base changes in a fragment. As bisulfite modification of methylated DNA would typically involve several base changes in a fragment, the efficiency of detecting methylation using MS-SSCA could approach 100%. We applied this method to analyze the BRCA1 promoter CpG island in breast cancer samples. About 20% of sporadic breast cancers are hypermethylated at the BRCA1 promoter CpG island. MS-SSCA rapidly detected those tumors that had previously been shown to be methylated by Southern blotting. The variant bands detected by SSCA were analyzed by sequencing and shown to be methylated. MS-SSCA is a simple method for screening large numbers of samples for methylation and can accelerate genomic sequencing, as all bands can be isolated and sequenced directly.

BRCA1 Protein↗
PubMed2000

Molecular cloning of the gene encoding flavoredoxin, a flavoprotein from Desulfovibrio gigas.

Sulfate-reducing bacteria are rich in unique redox proteins and electron carriers that participate in a variety of essential pathways. Several studies have been carried out to characterize these proteins, but the structure and function of many are poorly understood. Many Desulfovibrio species can grow using hydrogen as the sole energy source, indicating that the oxidation of hydrogen with sulfite as the terminal electron acceptor is an energy-conserving mechanism. Flavoredoxin is an FMN-binding protein isolated from the sulfate-reducing bacteria Desulfovibrio gigas that participates in the reduction of bisulfite from hydrogen. Here we report the cloning and sequencing of the flavoredoxin gene. The derived amino acid sequence exhibits similarity to several flavoproteins which are members of a new family of flavin reductases suggested to bind FMN in a novel mode.

Amino Acid Sequence↗
PubMed2004

Isolation and characterization of site-specific DNA-methyltransferases from Bacillus coagulans K.

Two site-specific DNA methyltransferases, M.BcoKIA and M.BcoKIB, were isolated from the thermophilic strain Bacillus coagulans K. Each of the methylases protects the recognition site 5'-CTCTTC-3'/5'-GAAGAG-3' from cleavage with the cognate restriction endonuclease BcoKI. It is shown that M.BcoKIB is an N6-adenine specific methylase and M.BcoKIA is an N4-cytosine specific methylase. According to bisulfite mapping, M.BcoKIA methylates the first cytosine in the sequence 5'-CTCTTC-3'.

Adenine↗
PubMed1999

DNA methylation at mammalian replication origins.

In Escherichia coli, DNA methylation regulates both origin usage and the time required to reassemble prereplication complexes at replication origins. In mammals, at least three replication origins are associated with a high density cluster of methylated CpG dinucleotides, and others whose methylation status has not yet been characterized have the potential to exhibit a similar DNA methylation pattern. One of these origins is found within the approximately 2-kilobase pair region upstream of the human c-myc gene that contains 86 CpGs. Application of the bisulfite method for detecting 5-methylcytosines at specific DNA sequences revealed that this region was not methylated in either total genomic DNA or newly synthesized DNA. Therefore, DNA methylation is not a universal component of mammalian replication origins. To determine whether or not DNA methylation plays a role in regulating the activity of origins that are methylated, the rate of remethylation and the effect of hypomethylation were determined at origin beta (ori-beta), downstream of the hamster DHFR gene. Remethylation at ori-beta did not begin until approximately 500 base pairs of DNA was synthesized, but it was then completed by the time that 4 kilobase pairs of DNA was synthesized (<3 min after release into S phase). Thus, DNA methylation cannot play a significant role in regulating reassembly of prereplication complexes in mammalian cells, as it does in E. coli. To determine whether or not DNA methylation plays any role in origin activity, hypomethylated hamster cells were examined for ori-beta activity. Cells that were >50% reduced in methylation at ori-beta no longer selectively activated ori-beta. Therefore, at some loci, DNA methylation either directly or indirectly determines where replication begins.

Animals↗
PubMed2002

[The roles of DNA methylation in human neoplasms].

Recent data suggested that cancer appears to be a process that is fuelled both by genetic alterations and by epigenetic mechanisms. Epigenetics refer to the study of changes in gene expression that can be mitotically inherited, but is not associated with the changes in the coding sequence of the affected genes. DNA methylation, the best understood mechanism in epigenetics, is an enzyme-mediated chemical modification that adds methyl (CH3) groups at selected sites on DNA. In humans and most mammals, DNA methylation only affects the cytosine base (C), when it is followed by a guanosine (G). DNA methylation in the promoter regions is a powerful mechanism for the suppression of gene activity. It was thought formerly that two pathways by which tumor suppressor genes became disabled were intragenic mutations and loss of chromosomal material [(loss of heterozygosity (LOH) or homozygous deletion)]. More recent studies have firmly established methylation as one potential hit and represent a third mechanism by which tumor suppressor genes are inactivated, and that, in some cases, methylation is the sole mechanism responsible for the inactivation of a tumor suppressor gene. There is clearly a tumor-specific methylation pattern in many types of tumor. An individual tumor within a single patient may have characteristic set of gene with an increased propensity to become methylated as compared with a tumor of the same type in a different patient. Methylation changes appear to precede apparent malignancy in many cases. Therefore, methylation can distinguish tumor types and subtypes, and should be useful in improving early detection of potentially cancerous cells. Methylation markers may also be used to predict response to chemotherapy or duration of patient survival. The efficacy of hypomethylating agents (e.g. 5-aza-2'-deoxycytidine) as antineoplastic agents has been attributed to two distinct mechanism: cytotoxicity and induction of hypomethylation. Some clinical trials with them have been performed in solid tumors and malignant hematologic disease. In general, the response rates were not satisfactory. A variety of methods are used to evaluate the methylation status of genes. MSP (methylation-specific PCR) is very sensitive and has been widely used. It takes advantage of the DNA sequence difference existing between methylated and unmethylated DNA after bisulfite treatment.

CpG Islands↗
PubMed1999

Extensive intra- and interindividual heterogeneity of p15INK4B methylation in acute myeloid leukemia.

Silencing of the cyclin-dependent kinase inhibitor gene p15INK4B by cytosine methylation of the promoter region has been associated with some types of hematological malignancy. To study in detail the patterns of p15INK4B methylation in patients with acute myeloid leukemia, we adopted a novel approach based on PCR amplification of bisulfite-treated DNA followed by resolution of differentially methylated sequences by denaturing gradient gel electrophoresis. This method visually displays the degree and heterogeneity of DNA methylation and enables the isolation and characterization of distinct clonotypic epigenotypes. A surprisingly high degree of intra- and interindividual heterogeneity of p15INK4B methylation was observed in the 65 acute myeloid leukemia patients examined. Methylation was detected in 46 (71%) of the patients and was observed more frequently in the French-American-British subtypes M1/M2 than in M4/M5 (P < 0.025). Examination of the same panel of samples using a highly sensitive methylation-specific PCR method showed methylated p15INK4B alleles in 61 (94%) of the samples. We present evidence that the higher frequency of p15INK4B methylation determined by methylation-specific PCR may, at least in part, be due to the presence of a small fraction of p15INK4B-methylated lymphocytes in normal blood.

Alleles↗
PubMed2002

Parallel assessment of CpG methylation by two-color hybridization with oligonucleotide arrays.

We have developed a method for the parallel analysis of multiple CpG sites in genomic DNA for their state of methylation. Hypermethylation of CpG islands within the promoters and 5' exons of genes has been found to be a mechanism of transcriptional inactivation associated with a variety of tumors. The method that we developed relies on the differential reactivity of methylated and unmethylated cytosines with sodium bisulfite, which exclusively converts unmethylated cytosines to deoxyuracils. The resulting sequence changes are determined with single-nucleotide resolution by hybridization to an oligonucleotide array. Cohybridization with a reference sample containing a different label provides an internal standard for assessment of methylation state. This method provides advantages in parallelism over existing methods of methylation analysis. We have demonstrated this technique with a region from the promoter of the tumor suppressor gene p16, which is hypermethylated in many cancers.

Base Sequence↗
PubMed1998

Imprinting in Angelman and Prader-Willi syndromes.

Prader-Willi syndrome (PWS) and Angelman syndrome (AS) are caused by deficiencies of gene expression from paternal or maternal chromosome 15q11-q13, respectively. Many advances have occurred during the past year. The gene for necdin was mapped in the PWS candidate region and found to be paternally expressed in mouse and human. The bisulfite method for analysis of methylation was established for genomic sequencing and diagnostics, and the methylation of Snrpn was studied in detail in the mouse. A region near the Snrpn promoter was shown to function as a silencer in Drosophila. Point mutations were found in the gene for E6-AP ubiquitin-protein ligase (UBE3A) identifying it as the AS gene, and tissue-specific imprinting (maternal expression) was shown in the human brain and in hippocampal neurons and Purkinje cells in the mouse.

Angelman Syndrome↗
PubMed2005

Loss of blood group A antigen expression in bladder cancer caused by allelic loss and/or methylation of the ABO gene.

Loss of ABO blood group antigen expression has been reported in transitional cell carcinoma (TCC) of the bladder. Synthesis of the ABO blood group antigen was genetically determined by allelic variants of the ABO gene assigned on 9q34.1. We analyzed loss of heterozygosity (LOH) and promoter hypermethylation of the ABO gene in TCC and compared them with alterations of A antigen expression in TCC, dysplasia and normal urothelium. A total of 81 samples of TCC of the bladder obtained from transurethral resection (TUR) (n=44) and radical cystectomy (n=37) were examined. Expression of the A antigen was evaluated by immunohistochemical staining (IHC) using anti-A antigen monoclonal antibody. LOH of the ABO gene locus was examined by blunt-end single-strand DNA conformational polymorphism (SSCP) analysis using flouresence-based auto sequencer. Promoter hypermethylation of the ABO gene were examined by bisulfite PCR-SSCP (BiPS) analysis and/or methylation-specific PCR (MSP). Loss of A allele and/or hypermethylation were significantly associated with abnormal expression of the A antigen in cases undergoing TUR (P=0.02) and radical cystectomy (P=0.0005). For the analysis of the concomitant dysplasia in 23 cases with TCC of the bladder, the expression of the A antigen was maintained, regardless of the A allelic loss or methylation status in the tumor. In conclusion, A allelic loss and hypermethylation in the promoter region of the ABO gene showed significant correlation with reduction of A antigen expression in TCC, while the expression of the A antigen is maintained in concomitant dysplasia or normal urothelium, suggesting that loss of the ABO gene and/or its promoter hypermethylation is a specific marker for TCC.

ABO Blood-Group System↗
PubMed2002

The human telomerase RNA gene (hTERC) is regulated during carcinogenesis but is not dependent on DNA methylation.

Telomerase, the ribonucleoprotein complex involved in telomere maintenance, is composed of two main components: hTERT and hTERC. hTERT seems to be the rate-limiting factor for telomerase activity, although hTERC expression was also shown to correlate to a certain extent with telomerase reactivation. To determine whether the absence of hTERC expression could be the consequence of DNA methylation, we quantified hTERC RNA in 60 human samples (19 telomerase-negative normal tissues, nine telomerase-positive and 22 telomerase-negative tumor tissues, eight telomerase-positive and two telomerase-negative cell lines) using a quantitative dot blot on RT-PCR products. Most of the normal tissues did not express hTERC whereas, in telomerase-positive cell lines and in telomerase-positive tumor tissues, a strong up-regulation was observed, suggesting that hTERC transcription is up-regulated during tumorigenesis. The two telomerase-negative cell lines did not express hTERC. In a series of 22 telomerase-negative soft tissue sarcomas (STS), half did not express hTERC at all, or only weakly, whereas a wide range of expression was observed in the other half. As methylation might be involved in hTERC silencing, we examined the methylation pattern in all samples by direct sequencing and methylation-specific single stand conformation analysis after bisulfite modification. hTERC methylation was never observed, neither in normal nor in tumor tissues. Furthermore, there was no correlation between hTERC expression and proliferation, telomere length or hTERT expression in telomerase-negative STS. In contrast, three of eight telomerase-positive cell lines and the two telomerase negative cell lines were found to be hypermethylated, suggesting that the methylation observed may occur during cell line establishment. In conclusion, this study shows that hTERC expression is indeed regulated during carcinogenesis, but this regulation is unlikely to depend on hTERC methylation, cell proliferation rate, telomere length or hTERT expression.

Antimetabolites, Antineoplastic↗
PubMed2006

Genetic characterization of fas-associated phosphatase-1 as a putative tumor suppressor gene on chromosome 4q21.3 in hepatocellular carcinoma.

PURPOSE: Allelic loss at chromosome 4q21-23 occurs frequently in human hepatocellular carcinoma, and the putative tumor suppressor gene (TSG) has not yet been identified. We studied the Fas-associated phosphatase-1 (FAP-1) gene as a potential candidate TSG in this region. EXPERIMENTAL DESIGN: The expression level of FAP-1 RNA in hepatocellular carcinomas was evaluated by RNase protection and quantitative PCR. Sodium bisulfite modification and subsequent single-strand conformational polymorphism and sequence analyses were used to assay the methylation of CpGs at FAP-1 promoter. Direct sequencing of the FAP-1 coding region was conducted for detecting the genetic mutations. Two common single nucleotide polymorphisms of FAP-1 were selected for evaluating their association with the hepatocellular carcinoma trait in sporadic and familial hepatocellular carcinomas. Moreover, the functional effect of FAP-1 on cellular proliferation has been evaluated by small interfering RNA approach. RESULTS: Around 50% of hepatocellular carcinomas showed significantly decreased expression of FAP-1 compared with the corresponding nontumorous liver tissues. In most cases, the RNA level was well correlated with the methylation status of promoter CpGs, suggesting that the promoter methylation may contribute to the down-regulation. Several genetic mutations of FAP-1 have been identified in hepatocellular carcinomas. The G/G genotype of FAP-1 cSNP6304 was significantly associated with the increased risk of multiplex familial hepatocellular carcinomas (odds ratio, 2.44; 95% confidence interval, 1.19-5.01). Finally, knockdown expression of FAP-1 was shown to enhance the cellular proliferation in PLC5 cells. CONCLUSIONS: FAP-1 could be inactivated during hepatocarcinogenesis, mainly attributed by allelic loss and promoter methylation. The genetic mutations and polymorphisms may also confront with the higher hepatocellular carcinoma risk. These results first suggested FAP-1 as a putative TSG in hepatocarcinogenesis.

Alleles↗
PubMed2003

Silencing-specific methylation and single nucleotide polymorphism of hMLH1 promoter in gastric carcinomas.

AIM: To investigate CpG methylation and single nucleotide polymorphism (SNP) of a specific promoter region of hMLH1 in primary gastric carcinoma. METHODS: Primary gastric carcinomas (n=80), their corresponding normal mucosal samples, and gastric mucosal biopsies from normal/gastritis control patients (n=54) were used. Hypermethylation at -253 nt and -251 nt in relation with the translational start site and SNP of a silencing specific region (-339 nt-46 nt) in the hMLH1 promoter were analyzed by Bst UI-combined bisulfite assay (COBRA), denaturing high performance liquid chromatogram (DHPLC), and sequencing. RESULTS: (A) The specific methylation at -253 nt and -251 nt was observed in 2 of 60 primary gastric carcinomas, but neither in all of the corresponding mucosa nor in normal/gastritis samples, by Bst UI-COBRA and DHPLC. (B) The hMLH1 promoter was methylated homogeneously in the xenograft of the primary gastric carcinoma with the methylated and unmethylated hMLH1. (C) The pattern of SNP at -93 nt of the hMLH1 promoter in 54 Chinese patients with gastric carcinoma was the same as that in the control patients: 51 % was A/G heteroalleles, 34 % and 15 % were A/A and G/G homoalleles, respectively. CONCLUSION: Biallelic inactivation of hMLH1 by epigenetic silencing existed in human primary gastric carcinoma homogeneously. Hypermethylation of hMLH1 may play a role in the early stage of development of a few gastric carcinomas. The SNP at -93 nt is not related to the susceptibility of gastric carcinomas.

Adaptor Proteins, Signal Transducing↗