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At least 1,567 records · Page 87Linked to original sources
PubMed2000

The Escherichia coli UVM response is accompanied by an SOS-independent error-prone DNA replication activity demonstrable in vitro.

UVM is an SOS-independent inducible response characterized by elevated mutagenesis at a site-specific 3, N4-ethenocytosine (epsilonC) residue borne on M13 single-stranded DNA transfected into Escherichia coli cells pretreated with DNA-damaging agents. By constructing and using E. coli strain AM124 (polA polB umuDC dinB lexA1[Ind-]), we show here that the UVM response is manifested in cells deficient for SOS induction, as well as for all four of the 'non-replicative' DNA polymerases, namely DNA polymerase I (polA), II (polB), IV (dinB) and V (umuDC). These results confirm that UVM represents a novel, previously unidentified cellular response to DNA-damaging agents. To address the question as to whether the UVM response is accompanied by an error-prone DNA replication activity, we applied a newly developed in vitro replication assay coupled to an in vitro mutation analysis system. In the assay, circular M13 single-stranded DNA bearing a site-specific lesion is converted to circular double-stranded replicative-form DNA in the presence of cell extracts and nucleotide precursors under conditions that closely mimic M13 replication in vivo. The newly synthesized (minus) DNA strand is selectively amplified by ligation-mediated polymerase chain reaction (LM-PCR), followed by a multiplex sequence analysis to determine the frequency and specificity of mutations. Replication of DNA bearing a site-specific epsilonC lesion by cell extracts from uninduced E. coli AM124 cells results in a mutation frequency of about 13%. Mutation frequency is elevated fivefold (to 58%) in cell extracts from UVM-induced AM124 cells, with C --> A mutations predominating over C --> T mutations, a specificity similar to that observed in vivo. These results, together with previously reported data, suggest that the UVM response is mediated through the induction of a transient error-prone DNA replication activity and that a modification of DNA polymerase III or the expression of a previously unidentified DNA polymerase may account for the UVM phenotype.

Bacterial Proteins↗
PubMed2005

STAT3- and DNA methyltransferase 1-mediated epigenetic silencing of SHP-1 tyrosine phosphatase tumor suppressor gene in malignant T lymphocytes.

Expression of SHP-1 phosphatase, a key negative regulator of cell signaling, is lost in T cell lymphomas and other malignancies due to DNA methylation of the SHP-1 promoter by a currently undefined mechanism. We demonstrate that malignant T cells express DNA methyltransferase (DNMT) 1 and that constantly activated signal transducer and activator of transcription (STAT) 3 is capable of binding in vitro to DNA oligonucleotides corresponding to four STAT3 SIE/GAS binding sites identified in the SHP-1 promoter. STAT3, DNMT1, and histone deacetylase 1 form complexes and bind to the SHP-1 promoter in vivo. Treatment with pharmacologic grade DNMT1 anti-sense oligonucleotides and STAT3 small-interfering RNA induces in the malignant T cells DNA demethylation and expression of SHP-1 gene. These data indicate that STAT3 may, in part, transform cells by inducing epigenetic silencing of SHP-1 in cooperation with DNMT1 and, apparently, histone deacetylase 1. Reversal of such gene silencing represents an attractive aim for novel anticancer therapies.

Base Sequence↗
PubMed1996

Increased cytosine DNA-methyltransferase activity is target-cell-specific and an early event in lung cancer.

The association between increased DNA-methyltransferase (DNA-MTase) activity and tumor development suggest a fundamental role for this enzyme in the initiation and progression of cancer. A true functional role for DNA-MTase in the neoplastic process would be further substantiated if the target cells affected by the initiating carcinogen exhibit changes in enzyme activity. This hypothesis was addressed by examining DNA-MTase activity in alveolar type II (target) and Clara (nontarget) cells from A/J and C3H mice that exhibit high and low susceptibility, respectively, for lung tumor formation. Increased DNA-MTase activity was found only in the target alveolar type II cells of the susceptible A/J mouse and caused a marked increase in overall DNA methylation in these cells. Both DNA-MTase and DNA methylation changes were detected 7 days after carcinogen exposure and, thus, were early events in neoplastic evolution. Increased gene expression was also detected by RNA in situ hybridization in hypertrophic alveolar type II cells of carcinogen-treated A/J mice, indicating that elevated levels of expression may be a biomarker for premalignancy. Enzyme activity increased incrementally during lung cancer progression and coincided with increased expression of the DNA-MTase activity are strongly associated with neoplastic development and constitute a key step in carcinogenesis. The detection of premalignant lung disease through increased DNA-MTase expression and the possibility of blocking the deleterious effects of this change with specific inhibitors will offer new intervention strategies for lung cancer.

Animals↗
PubMed1999

Inhibition of DNA methyltransferase stimulates the expression of signal transducer and activator of transcription 1, 2, and 3 genes in colon tumor cells.

Inhibitors of DNA methyltransferase, typified by 5-aza-2'-deoxycytidine (5-Aza-CdR), induce the expression of genes transcriptionally down-regulated by de novo methylation in tumor cells. We utilized gene expression microarrays to examine the effects of 5-Aza-CdR treatment in HT29 colon adenocarcinoma cells. This analysis revealed the induction of a set of genes that implicated IFN signaling in the HT29 cellular response to 5-Aza-CdR. Subsequent investigations revealed that the induction of this gene set correlates with the induction of signal transducer and activator of transcription (STAT) 1, 2, and 3 genes and their activation by endogenous IFN-alpha. These observations implicate the induction of the IFN-response pathway as a major cellular response to 5-Aza-CdR and suggests that the expression of STATs 1, 2, and 3 can be regulated by DNA methylation. Consistent with STAT's limiting cell responsiveness to IFN, we found that 5-Aza-CdR treatment sensitized HT29 cells to growth inhibition by exogenous IFN-alpha2a, indicating that 5-Aza-CdR should be investigated as a potentiator of IFN responsiveness in certain IFN-resistant tumors.

Antimetabolites, Antineoplastic↗
PubMed1995

Specific Alu binding protein from human sperm chromatin prevents DNA methylation.

A protein from human sperm nuclei that specifically binds to Alu DNA repeats has been purified. The specific DNA binding site of this protein within the Alu sequence has been mapped by methylation interference and electrophoretic mobility shift assays. This sperm Alu binding protein selectively protects Alu elements from methylation in vitro and may be responsible for the unmethylated state of Alu sequences in the male germ line resulting in a parent-specific differential inheritance of Alu methylation.

Amino Acid Sequence↗
PubMed2004

Components of the DNA methylation system of chromatin control are RNA-binding proteins.

The view that autosomal gene expression is controlled exclusively by protein trans-acting factors has been challenged recently by the identification of RNA molecules that regulate chromatin. In the majority of cases where RNA molecules are implicated in DNA control, the molecular mechanisms are unknown, in large part because the RNA.protein complexes are uncharacterized. Here, we identify a novel set of RNA-binding proteins that are well known for their function in chromatin regulation. The RNA-interacting proteins are components of the mammalian DNA methylation system. Genomic methylation controls chromatin in the context of transposon silencing, imprinting, and X chromosome dosage compensation. DNA methyltransferases (DNMTs) catalyze methylation of cytosines in CGs. The methyl-CGs are recognized by methyl-DNA-binding domain (MBD) proteins, which recruit histone deacetylases and chromatin remodeling proteins to effect silencing. We show that a subset of the DNMTs and MBD proteins can form RNA.protein complexes. We characterize the MBD protein RNA-binding activity and show that it is distinct from the methyl-CG-binding domain and mediates a high affinity interaction with RNA. The RNA and methyl-CG binding properties of the MBD proteins are mutually exclusive. We speculate that DNMTs and MBD proteins allow RNA molecules to participate in DNA methylation-mediated chromatin control.

Animals↗
PubMed2004

Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase.

DNA methyltransferase Dnmt1 ensures clonal transmission of lineage-specific DNA methylation patterns in a mammalian genome during replication. Dnmt1 is targeted to replication foci, interacts with PCNA, and favors methylating the hemimethylated form of CpG sites. To understand the underlying mechanism of its maintenance function, we purified recombinant forms of full-length Dnmt1, a truncated form of Dnmt1-(291-1620) lacking the binding sites for PCNA and DNA and examined their processivity using a series of long unmethylated and hemimethylated DNA substrates. Direct analysis of methylation patterns using bisulfite-sequencing and hairpin-PCR techniques demonstrated that full-length Dnmt1 methylates hemimethylated DNA with high processivity and a fidelity of over 95%, but unmethylated DNA with much less processivity. The truncated form of Dnmt1 showed identical properties to full-length Dnmt1 indicating that the N-terminal 290-amino acid residue region of Dnmt1 is not required for preferential activity toward hemimethylated sites or for processivity of the enzyme. Remarkably, our analyses also revealed that Dnmt1 methylates hemimethylated CpG sites on one strand of double-stranded DNA during a single processive run. Our findings suggest that these inherent enzymatic properties of Dnmt1 play an essential role in the faithful and efficient maintenance of methylation patterns in the mammalian genome.

Animals↗
PubMed2026

A pragmatic adaptation of the RANO clinical risk score for IDH-Wildtype glioblastoma in the absence of MGMT promoter methylation testing.

BACKGROUND: The novel RANO risk score provides prognostic stratification for patients with IDH-wildtype glioblastoma(GBM) and includes age, Karnofsky Performance Scale(KPS), RANO resection class(RRC), and MGMT promoter methylation(MGMTm). However, MGMTm testing is unavailable in many countries. We aimed to explore the prognostic performance of a RANO-adapted clinical score excluding MGMTm. METHODS: We applied the same scoring system established by original RANO score, excluding MGMTm. Three risk classes were defined as numerical scores derived through tertiles. The primary endpoint was overall survival(OS), and the secondary exploratory endpoint was progression-free survival(PFS). RESULTS: One-hundred twenty patients were included. Three risk classes were identified:low-risk(0-1 points,n:47, 39.2%), intermediate-risk(2-3 points,n:27, 22.5%), and high-risk(&#x2265;4 points,n:46, 38.3%). The median OS was 35.5&#x2009;months(95% CI:21.1-49.9) for the low-risk group, 16&#x2009;months(95% CI:9.9-22.1) for the intermediate-risk group, and 5&#x2009;months(95% CI:3.6-6.3) for the high-risk group(p&#x2009;<&#x2009;0.001). Similarly, the median PFS was 16.3&#x2009;months(95% CI:12.3-20.3) for the low-risk group, 9.9&#x2009;months(95% CI:7.2-12.6) for the intermediate-risk group, and 4.1&#x2009;months(95% CI:3.5-4.7) for the high-risk group(p&#x2009;<&#x2009;0.001). CONCLUSION: This simplified RANO-adapted score demonstrated promising prognostic stratification using basic clinical parameters. As a pragmatic adaptation study, external validation is required before clinical application, particularly in settings where MGMTm testing is unavailable.

Humans↗
PubMed1995

DNA adduct 8-hydroxyl-2'-deoxyguanosine (8-hydroxyguanine) affects function of human DNA methyltransferase.

8-Hydroxyl-2'-deoxyguanosine (also referred to as 8-hydroxyguanine [8-OH-dG] or 7,8-dihydro-8-oxoguanine), a common DNA adduct resulting from injury to DNA via reactive oxygen species, affects the in vitro methylation of nearby cytosine moieties by the human DNA methyltransferase. The exact position of 8-OH-deoxyguanosine relative to a CpG dinucleotide appears important to this effect. Our data indicate that 8-OH-deoxyguanosine diminishes the ability of the methyltransferase to methylate a target cytosine when the 8-OH-deoxyguanosine is one or two nucleotides 3' from the cytosine, on the same strand. On the other hand 8-OH-deoxyguanosine does not diminish the ability of the enzyme to respond to a methyl director (5-methylcytosine) when the 8-OH-deoxyguanosine is on the same strand but one or two nucleotides 3' from the methyl director. Differences in methylation rates as great as 13-fold have been detected using various 8-OH-deoxyguanosine-containing oligonucleotides as substrates in methylation assays. Our findings suggest that oxidative damage of parental strand guanines would permit normal copying of methylation patterns through maintenance methylation, while oxidative damage of guanines in the nascent strand DNA would inhibit such methylation.

Base Sequence↗
PubMed2003

Identification of tumour-specific epigenetic events in medulloblastoma development by hypermethylation profiling.

Medulloblastoma arises in the cerebellum and is the most common malignant brain tumour of childhood, however its molecular basis is not well understood. To assess the role of aberrant epigenetic events in medulloblastoma and identify critical genes in its development, we profiled the promoter methylation status of 11 candidate tumour-suppressor genes (TSGs; p14(ARF), p15(INK4b), p16(INK4a), CASP8, HIC1, EDNRB, TIMP3, TP73, TSLC1, RIZ1 and RASSF1A) in medulloblastoma cell lines, primary tumours and the normal cerebellum. Gene-specific TSG methylation was a significant feature of both medulloblastomas and the cerebellum. Extensive hypermethylation of RASSF1A was detected frequently in medulloblastomas but not in the normal cerebellum (41/44 primary tumours versus 0/5 normal cerebella). In contrast, complete methylation of HIC1 and CASP8 in a subset of primary tumours (17/44 and 14/39) occurred against a consistent background of partial methylation in the normal cerebellum. These data therefore indicate that extensive methylation of RASSF1A, HIC1 and CASP8 are tumour-specific events in medulloblastoma. Moreover, methylation of these genes in medulloblastoma cell lines was associated with their epigenetic transcriptional silencing and methylation-dependent re-expression following treatment with the DNA methyltransferase inhibitor, 5-aza-2'-deoxycytidine. The remaining genes studied showed either low frequency methylation (p14(ARF), p16(INK4a), RIZ1; <7% of cases), no evidence of methylation (p15(INK4b), TIMP3, TP73, TSLC1), or comparable patterns of methylation in the normal cerebellum (EDNRB), suggesting that their hypermethylation does not play a major role in medulloblastoma. Our data demonstrate that tumour-specific hypermethylation affects only a subset of genes, and does not support the existence of a concordant methylation phenotype in this disease. We conclude that epigenetic TSG inactivation is a significant feature of medulloblastoma, and identify RASSF1A, HIC1 and CASP8 as potentially critical genes in its pathogenesis. Furthermore, methylation observed in the normal cerebellum emphasises the requirement for appropriate control tissues when assessing the tumour-specificity of TSG hypermethylation.

Adolescent↗
PubMed1996

Detection of DNA damage after hyperbaric oxygen (HBO) therapy.

Hyperbaric oxygen (HBO) therapy is successfully used for the treatment of a variety of conditions. However, exposure to high concentrations of oxygen is known to induce damage to cells, possibly due to an increased oxygen radical production. As reactive oxygen species also cause DNA damage, we investigated the DNA-damaging effect of HBO with the alkaline version of the single cell gel test (comet assay). Oxidative DNA base modifications were determined by converting oxidized DNA bases to strand breaks using bacterial formamidopyrimidine-DNA glycosylase (FPG), a DNA repair enzyme, which specifically nicks DNA at sites of 8-oxo-guanines and formamidopyrimidines. HBO treatment under therapeutic conditions clearly and reproducibly induced DNA damage in leukocytes of all test subjects investigated. Increased DNA damage was found immediately at the end of the treatment, while 24 h later, no effect was found. Using FPG protein we detected significant oxidative base damage after HBO treatment. DNA damage was detected only after the first treatment and not after further treatments under the same conditions, indicating an increase in antioxidant defences. DNA damage did not occur when the HBO treatment was started with a reduced treatment time which was then increased stepwise.

Adult↗
PubMed1992

Alw26I, Eco31I and Esp3I--type IIs methyltransferases modifying cytosine and adenine in complementary strands of the target DNA.

The specificity of three DNA methyltransferases M.Alw26I, M.Eco31I and M.Esp3I, isolated from Acinetobacter Iwoffi RFL26, Escherichia coli RFL31 and Hafnia alvei RFL3+, respectively, was determined. All the enzymes methylate both strands of asymmetric recognition sites yielding m5C in the top-strand and m6A in the bottom-strand, as below: 5'-GTm5CTC 5'-GGTm5CTC 5'-CGTm5CTC 3'-Cm6AGAG 3'-CCm6AGAG 3'-GCm6AGAG (M.Alw26I) (M.Eco31I) (M.Esp3I) They are the first members of type IIs methyltransferases that modify different types of nucleotides in the recognition sequence.

Acinetobacter↗
PubMed2002

The MT domain of the proto-oncoprotein MLL binds to CpG-containing DNA and discriminates against methylation.

Alterations of the proto-oncogene MLL (mixed lineage leukemia) are characteristic for a high proportion of acute leukemias, especially those occurring in infants. The activation of MLL is achieved either by an internal tandem duplication of 5' MLL exons or by chromosomal translocations that create chimeric proteins with the N-terminus of MLL fused to a variety of different partner proteins. A domain of MLL with significant homology to the eukaryotic DNA methyltransferases (MT domain) has been found to be essential for the transforming potential of the oncogenic MLL derivatives. Here we demonstrate that this domain specifically recognizes DNA with unmethylated CpG sequences. In gel mobility shifts, the presence of CpG was sufficient for binding of recombinant GST-MT protein to DNA. The introduction of 5-methylCpG on one or both DNA strands precluded an efficient interaction. In surface plasmon resonance a KD of approximately 3.3 x 10(-8) M was determined for the GST-MT/DNA complex formation. Site selection experiments and DNase I footprinting confirmed CpG as the target of the MT domain. Finally, this interaction was corroborated in vivo in reporter assays utilizing the DNA-binding properties of the MT domain in a hybrid MT-VP16 transactivator construct.

Amino Acid Sequence↗
PubMed2004

Evolution of dnmt-2 and mbd-2-like genes in the free-living nematodes Pristionchus pacificus, Caenorhabditis elegans and Caenorhabditis briggsae.

Whole genome sequencing of several metazoan model organisms provides a platform for studying genome evolution. How representative are the genomes of these model organisms for their respective phyla? Within nematodes, for example, the free-living soil nematode Caenorhabditis elegans is a highly derived species with unusual genomic characters, such as a reduced Hox cluster (Curr. Biol., 13, 37-40) and the absence of a Hedgehog signaling system. Here, we describe the recent loss of a DNA methyltransferase-2 gene (dnmt-2) in C.elegans. A dnmt-2-like gene is present in the satellite model organism Pristionchus pacificus, another free-living nematode that diverged from C.elegans 200-300 million years ago. In contrast, C.elegans, Caenorhabditis briggsae and P.pacificus all contain an mbd-2-like gene, which encodes another essential component of the methylation system of higher animals and fungi. Cel-mbd-2 is expressed throughout development and RNA interference (RNAi) experiments result in variable phenotypes. In contrast, Cbr-mbd-2 RNAi results in paralyzed larval or adult worms suggesting recent changes of gene function within the genus Caenorhabditis. We speculate that both genes were part of an ancestral DNA methylation system in nematodes and that gene loss and sequence divergence have abolished DNA methylation in C.elegans.

Amino Acid Sequence↗
PubMed2004

Accelerated bisulfite-deamination of cytosine in the genomic sequencing procedure for DNA methylation analysis.

Understanding the biological consequences of DNA methylation is a current focus of intensive studies. A standard method for analyzing the methylation at position 5 of cytosines in genomic DNA involves chemical modification of the DNA with bisulfite, followed by PCR amplification and sequencing. Bisulfite deaminates cytosine, but it deaminates 5-methylcytosine only very slowly, thereby allowing determination of the methylated sites. The determination is usually performed using sodium bisulfite solutions of 3-5 M concentration with an incubation period of 12-16 hr at 50 degrees C. We demonstrate here that this deamination can be speeded up significantly by increasing the bisulfite concentration and the temperature with which the reaction is performed. In an experiment, in which denatured DNA was treated with 9 M bisulfite for 10 min at pH 5.4 and 90 degrees C, deamination of cytosines occurred to an extent of 99.6%, while 5-methylcytosine residues in the DNA were deaminated at less than 10%. Using a plasmid DNA fragment, we observed that the DNA can serve as a template for PCR amplification after the bisulfite treatment. This new procedure is expected to offer an improved genomic sequencing method, leading to the promotion of research on understanding the biological and medical significance of DNA methylation.

Animals↗
PubMed1993

Differential gene expression in fetal mouse germ cells.

We have constructed cDNA libraries representing transcripts from purified populations of germ cells and of somatic cells, isolated from fetal mouse gonads at 12.5 days postcoitum (dpc). We have used these libraries to study differential gene expression in fetal germ cells on the basis of differential representation of specific cDNAs in each library. We show that in addition to expression of housekeeping genes at expected levels, four genes responsible for germ cell-specific phenotypes are expressed at significantly different levels in germ cells and surrounding somatic cells. These include tissue-nonspecific alkaline phosphatase (tnAP), transcription factor Oct-3/4, the c-kit proto-oncogene, and DNA methyltransferase (Mt). The significance of these results is discussed in the context of events contributing to early development of germ cells. It is concluded that fetal germ cells appear to retain a pattern of gene expression resembling that in early pluripotent embryonic cells, and that this may be important for the maintenance of genetic totipotency.

Alkaline Phosphatase↗
PubMed2003

Immortalization of bovine capillary endothelial cells by hTERT alone involves inactivation of endogenous p16INK4A/pRb.

Recent studies show that stable expression of the human telomerase catalytic subunit, hTERT, alone can lead several types of normal human somatic cells to bypass replicative senescence and become immortal. The molecular mechanisms by which telomerase immortalizes cells are not fully understood, although a key function of telomerase is to maintain a critical length of telomeres in order to preserve the stability and integrity of the genome. Here we report that stable transfection of hTERT alone was sufficient to allow bovine capillary endothelial (BCE) cells to bypass senescence and acquire immortality. Surprisingly, telomere lengths in these stable transfectants were progressively shortened during an increasing number of population doublings (PDLs), despite high telomerase activity. The expression of the cyclin-dependent kinase inhibitors (CDKIs) p16INK4A and p21CIP1/WAF1 was concomitantly repressed, and the retinoblastoma protein (pRb) was maintained in a hyperphosphorylated state in the telomerase-expressing cells. Re-expression of p16INK4A in these cells by either treatment with a demethylating agent or by adenovirus-mediated expression reinduced a senescence-like phenotype, suggesting that the inactivation of p16INK4A was due to DNA methylation and was crucial for the immortalization process. In agreement with this finding, the expression levels of the prototypic DNA methyltransferase DNMT1 were elevated in the hTERT-positive cells.

Animals↗