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PubMed2006

Global DNA demethylation in gastrointestinal cancer is age dependent and precedes genomic damage.

We studied the relationships between genetic and epigenetic alterations in gastrointestinal cancer by integrating DNA copy number changes determined by arbitrarily primed PCR (AP-PCR) with DNA methylation variations estimated by methylation-sensitive amplified fragment length polymorphism (MS-AFLP). We analyzed about 100 different chromosomal regions by AP-PCR and over 150 random CpG loci by MS-AFLP in human colon and gastric carcinomas. DNA hypomethylation and hypermethylation alterations distributed gradually and increased with cancer patient age, in contrast with the age-independent genomic alterations. Increased DNA hypomethylation and hypermethylation correlated with increased genomic damage, but only hypomethylation was highly significant in multivariate analyses. We conclude that age-dependent accumulation of DNA demethylation precedes diploidy loss in a significant subset of gastrointestinal cancers.

Adenocarcinoma↗
PubMed2003

Double RNA interference of DNMT3b and DNMT1 enhances DNA demethylation and gene reactivation.

Small interfering RNAs (siRNAs) are newly identified molecules shown to silence genes via targeted mRNA degradation. In this study, we used specific siRNAs as a tool to probe the relationship between two DNA methyltransferase genes, DNMT3b and DNMT1, in the maintenance of DNA methylation patterns in the genome. Levels of DNMT3b or DNMT1 mRNAs and proteins were markedly decreased (up to 80%) on transfecting these siRNAs into the ovarian cancer cell line CP70. The resulting RNA interference showed differential effects on DNA demethylation and gene reactivation in the treated cells. The DNMT1 siRNA treatment led to a partial removal of DNA methylation from three inactive promoter CpG islands, TWIST, RASSF1A, and HIN-1, and restored the expression of these genes. This epigenetic alteration appeared less effective in cells transfected with DNMT3b siRNA. However, the combined treatment of DNMT3b and DNMT1 siRNAs greatly enhanced this demethylation effect, producing 7-15-fold increases in their expression. We also used a microarray approach to examine this RNA interference on 8640 CpG island loci in CP70 cells. The combined siRNA treatment had a greater demethylation effect on 241 methylated loci and selected repetitive sequences than that of the single treatment. Our data thus suggest that whereas DNMT1 plays a key role in methylation maintenance, DNMT3b may act as an accessory to support the function in CP70 cells. This study also shows that siRNA is a powerful tool for interrogating the mechanisms of DNA methylation in normal and pathological genomes.

Cell Division↗
PubMed1994

Macronuclear DNA demethylation is involved in the encystment process of the ciliate Colpoda inflata.

Ciliate encystment is an eukaryotic cell differentiation process which involves a specific gene expression, to form the resting stage. In this study, we investigate, for first time, the DNA methylation pattern changes during encystment in the ciliate Colpoda inflata, and the 5-azacytidine effect on growing cells and encystment. Results indicate that 5-methylcytosine is present in macronuclear DNA of this ciliate and the 5-azacytidine treatment induces encystment in growth conditions. From restriction enzyme digestion and 5-azacytidine experiments, we conclude that a specific DNA demethylation is probably involved in the encystment gene expression of this ciliate.

Animals↗
PubMed2002

Complex relationships between 5-aza-dC induced DNA demethylation and chromosome compaction at mitosis.

A variety of treatments with 5-azadeoxycytidine (5-aza-dC) were applied to cultured human lymphocytes during one to four cell cycles. The effect of 5-aza-dC on DNA methylation was studied by using an antibody against 5-methylcytosine on mitotic chromosomes. 5-Azadeoxycytidine is known to induce strong and permanent demethylation of DNA. Unexpectedly complex relationships were observed between DNA methylation status and chromatid/chromosome compaction. The most dramatic alteration of compaction at mitosis was observed when pre-replicative chromosomes had unifilarly demethylated DNA. The compaction of chromosomes was found to depend only partially on the methylation of their DNA at the time of mitosis. Our results suggest that alteration of DNA methylation prevents the synchronization of chromatin compaction, inducing premature (or delayed) chromosome condensation, and that a crucial step is the DNA methylation status of the pre-replicative chromosome.

Azacitidine↗
PubMed2001

Local DNA demethylation in vertebrates: how could it be performed and targeted?

In vertebrates, cytosine methylation is an epigenetic DNA modification that participates in genome stability and gene repression. Methylation patterns are either maintained throughout cell division, or modified by global or local de novo methylation and demethylation. Site-specific demethylation is a rather elusive process that occurs mainly in parallel to gene activation during development. In light of our studies of the glucocorticoid-dependent DNA demethylation of the tyrosine aminotransferase gene, we discuss the potential biochemical mechanisms allowing DNA demethylation and its targeting to specific sequences by transcription factors as well as possible links to DNA replication and chromatin remodelling.

Animals↗
PubMed2001

5-Aza-2'-deoxycytidine induces histone hyperacetylation of mouse centromeric heterochromatin by a mechanism independent of DNA demethylation.

5-Aza-2'-deoxycytidine (5-azadC) is widely used as a potent inhibitor of DNA methyltransferase. Cells treated with this drug show various phenomena such as the reactivation of repressed genes, change in replication timing, and decondensation of heterochromatin. A number of studies using this drug have been reported so far but it is still controversial whether such changes are due to 5-azadC-induced demethylation itself or the side effects of the drug. Here we report that 5-azadC treatment induces histone hyperacetylation in mouse centromeric heterochromatin which normally contains methylated DNA and hypoacetylated histones. Treatment also affects the intranuclear distribution of histone deacetylase 2 (HDAC2). However, histone hyperacetylation was not observed in DNA methyltransferase 1-deficient cells with a reduced level of genomic DNA methylation. Our results suggest that 5-azadC-induced histone hyperacetylation is independent of DNA demethylation and that DNA methylation is not essential for the maintenance of the histone hypoacetylated state in centromeric heterochromatin.

Acetylation↗
PubMed1987

Sister chromatid exchanges induced by DNA demethylating agents persist through several cell cycles in mammalian cells.

Eukaryotic DNA methylation has been extensively studied in recent years. The ability of many carcinogens to interfere with DNA methylation has not yet been directly related to their tumorigenic activity. Recent data obtained using L-ethionine and 5-azacytidine--both demethylating agents--showed a small but significant increase in the sister chromatid exchange (SCE) rate induced in mammalian cells (human lymphocytes and CHO cells). In this paper we show that the SCE increase induced by both these agents in Chinese hamster ovary (CHO) cells persists for as long as 10 cell cycles. On the other hand mitomycin-C and u.v. light-induced SCEs show a rapid decrease to the control value, as reported for all known SCE inducers. We suggest that DNA demethylation and SCEs are connected through a perturbation of the cell machinery at the level of the replication fork, producing an increase of the error-prone ligation. Since the methylation level is maintained (inherited), the SCE increase produced by these recombinational events will not be corrected through several cell cycles.

Animals↗
PubMed2004

Non-coding RNA directed DNA demethylation of Sphk1 CpG island.

The formation of DNA methylation patterns is one of the epigenetic events that underlie mammalian development. The Sphk1 CpG island is a target for tissue-dependent DNA methylation as well as a template for generating multiple subtypes. The number of mammalian non-coding RNA genes is rapidly expanding. In this study, we found endogenous antisense transcripts, Khps1 subtypes with different sizes (600-20,000nt). A subtype, Khps1a, was a 1290-bp, non-coding, 5'-capped and 3'-polyadenylated RNA that originated from the CpG island and overlapped with a tissue-dependent differentially methylated region (T-DMR) of Sphk1. Intriguingly, overexpression of two fragments of Khps1 caused demethylation of CG sites in the T-DMR. Furthermore, this RNA-directed demethylation was associated with DNA methylation at three CC(A/T)GG sites in the T-DMR. The link between the RNA-directed CG demethylation and non-CG methylation provides a novel mechanism of epigenetic regulation and potential tool for epigenetic manipulation of mammalian cells.

Animals↗
PubMed1997

DNA demethylation and pericentromeric rearrangements of chromosome 1.

Rearrangements in the vicinity of the centromere of chromosome 1 are over-represented in many types of human cancer and are a characteristic feature of a rare genetic disease called ICF (immunodeficiency, centromeric heterochromatin instability, and facial anomalies). Evidence is presented that implicates DNA hypomethylation in the formation of these pericentromeric chromosomal anomalies. The DNA methylation inhibitors 5-azadeoxycytidine and 5-azacytidine, but not other tested genotoxins, induced the preferential formation of pericentromeric rearrangements of chromosome 1 at a very high frequency in a pro-B-cell line (FLEB14) and at a lower frequency in a mature B-cell line (AHH-1). These abnormal chromosomes appear identical to the diagnostic chromosomal aberrations in the ICF syndrome. A major component of the pericentromeric DNA in chromosome 1, satellite 2, was shown to be hypomethylated in an ICF B-cell line, although DNA from this cell line did not display detectable overall hypomethylation. It is hypothesized that demethylation in certain DNA regions, including in pericentromeric satellite DNA, helps lead to pericentromeric chromosomal rearrangements in lymphocytes from ICF patients and in normal lymphoblastoid cells incubated in vitro with DNA demethylating agents.

Adult↗
PubMed1991

5-Azacytidine-induced demethylation of DNA to senescent level does not block proliferation of human fibroblasts.

IMR-90 human diploid fibroblasts (HDF) lose from 30-50% of their genomic 5-methyldeoxycytidine (5mdC) during the cellular aging process. In contrast, immortal SV40-transformed IMR-90 maintain a constant level of 5mdC in culture. Precrisis SV40-transformed HDF (AG3204) represent a stage in between normal cell aging and immortalization because these cells still have a finite proliferative lifespan, but it is longer than that of normal HDF and ends in cell death rather than in G1-arrest. We find that AG3204 cells continue to lose from 12-33% of their 5mdC after a population has become 99% positive for SV40 T-antigen. Both IMR-90 cells and AG3204 cells have similar levels of 5mdC (average of 2.25%) at the end of lifespan. We investigated whether this level of 5mdC is an absolute block to further proliferation by treating IMR-90 and AG3204 cells with 5-azacytidine (5azaC) to reduce their 5mdC levels below the terminal level normally achieved at end of lifespan. We find that both IMR-90 and AG3204 cells undergo extensive proliferation with subterminal levels of 5mdC and that the lifespans of both cell types are shortened by 5azaC treatment. These studies indicate that random genomic DNA demethylation to a specific level of 5mdC is not a direct cause of finite proliferative lifespan. However, the correlation between accelerated DNA demethylation and accelerated aging still suggests that these two phenomena are related. Two ways to explain this relationship are: (1) DNA demethylation during aging is not random, and/or (2) both DNA demethylation and other independent aging processes cooperate to produce finite lifespan. In both cases, accelerated random DNA demethylation could accelerate aging, but not necessarily in direct relationship to the final genomic level of 5mdC achieved during the normal aging process.

Azacitidine↗
PubMed1995

Mechanisms of DNA demethylation in chicken embryos. Purification and properties of a 5-methylcytosine-DNA glycosylase.

We have previously shown that in developing chicken embryos and differentiating mouse myoblasts, the demethylation of 5-metCpGs occurs through the replacement of 5-methylcytosine by cytosine (Jost, J. P. (1993) Proc. Natl. Acad. Sci. U.S.A. 90, 4685-4688; Jost, J. P. & Jost, Y.C. (1994) J. Biol. Chem. 269, 10040-10043). We have now purified over 30,000-fold a 5-methylcytosine-DNA glycosylase from 12-day-old chicken embryos. The enzyme copurifies with a mismatch-specific thymine-DNA glycosylase and an apyrimidic-endonuclease. The reaction product of the highly purified 5-methylcytosine-DNA glycosylase is 5-methylcytosine. The copurified apyrimidic-endonuclease activity cleaves 3' from the apyrimidic sugar. A 52.5-kDa peptide, isolated as a single band from preparative SDS-polyacrylamide gels, has both the 5-methylcytosine-DNA glycosylase and the mismatch-specific thymine-DNA glycosylase activities. 5-Methylcytosine-DNA glycosylase has an apparent pI of 5.5-7.5 and maximal activity between pH 6.5 and 7.5. The Km for hemimethylated oligonucleotide substrate is 8 x 10(-8) M with a Vmax of 4 x 10(-11) mol/h/micrograms proteins. 5-Methylcytosine-DNA glycosylase binds equally well to methylated and non-methylated DNA. The enzyme reacts six times faster with the hemimethylated DNA than with the same bifilarly methylated DNA sequence, and single-stranded methylated DNA is not a substrate. The action of the enzyme is distributive.

Animals↗
PubMed2004

DNA demethylation is necessary for the epigenetic reprogramming of somatic cell nuclei.

Nuclear transplantation experiments in amphibia and mammals have shown that oocyte and egg cytoplasm can extensively reprogram somatic cell nuclei with new patterns of gene expression and new pathways of cell differentiation; however, very little is known about the molecular mechanism of nuclear reprogramming. Here we have used nuclear and DNA transfer from mammalian somatic cells to analyse the mechanism of activation of the stem cell marker gene oct4 by Xenopus oocytes. We find that the removal of nuclear protein accelerates the rate of reprogramming, but even more important is the demethylation of somatic cell DNA. DNA demethylation seems to precede gene reprogramming, and is absolutely necessary for oct4 transcription. Reprogramming by oocytes occurs in the absence of DNA replication and RNA/protein synthesis. It is also selective, operating only on the promoter, but not enhancers, of oct4; both a putative Sp1/Sp3 and a GGGAGGG binding site are required for demethylation and transcription. We conclude that the demethylation of promoter DNA may be a necessary step in the epigenetic reprogramming of somatic cell nuclei.

Animals↗
PubMed2003

DNA demethylating agents and chromatin-remodelling drugs: which, how and why?

DNA hypermethylation at the CpG dinucleotides clustered in "islands" in the promoter regions of genes causes transcriptional repression through the remodelling of chromatin. Aberrant methylation patterns of tumor suppressor genes and their subsequent silencing constitute a common feature of many cancers. Thus, the search for drugs that interfere in methylation-mediated gene repression has become one of the major goals in the design of cancer therapies. The major actors in the mammalian methylation system are DNA-methyltransferases (DNMTs), and methyl-CpG-binding proteins (MBDs), which recognize methylated cytosine and recruit repressor complexes, including histone deacetylases (HDACs). In this context, two major groups of drugs can be distinguished. The first one is constituted by substances that inhibit the action of DNMTs, either competing with cytosine or with S-adenosylmethionine (SAM, AdoMet) or acting over the DNMTs themselves. The second group involves compounds that inhibit subunits of the repressor complexes, such as HDACs. In this manuscript we review these two different groups of drugs, discussing their properties and the side effects that have been described (that occur by interference with other metabolic pathways). We also propose the logical pharmacological extension of these findings to design more specific and effective drugs for the prevention and treatment of human cancer.

Animals↗
PubMed1986

Replacement of 5-methylcytosine by cytosine: a possible mechanism for transient DNA demethylation during differentiation.

In an earlier study it was discovered that when Friend erythroleukemia cells (FELC) were exposed to a variety of chemical agents capable of inducing differentiation, their DNA underwent genome-wide transient demethylation. In an attempt to elucidate the biochemical mechanism responsible for this phenomenon we have induced FELC with 5 mM hexamethylenebisacetamide and labeled the DNA in vivo with a density label, 5-bromodeoxyuridine, and a radioactive label, deoxy[5-3H]cytidine. Newly replicated DNA (heavy-light) was separated from parental DNA (light-light) by isopycnic centrifugation. Incorporation of deoxy[5-3H]cytidine into light-light duplex DNA has been observed only in induced cells concomitantly with the demethylation of the DNA, whereas, in parallel experiments, deoxy[G-3H]adenosine was not incorporated into light-light DNA. It was also found that the labeling of light-light DNA with deoxy[5-3H]cytidine is transient since the 3H label was removed from the DNA during the period of de novo DNA methylation that follows the demethylation. These results, taken together, strongly suggest that the demethylation of the DNA during differentiation is achieved by an enzymatic mechanism whereby 5-methylcytosine is replaced by cytosine.

5-Methylcytosine↗
PubMed2002

Inducible DNA demethylation mediated by the maize Suppressor-mutator transposon-encoded TnpA protein.

Heritable epigenetic inactivation of the maize Suppressor-mutator (Spm) transposon is associated with promoter methylation, and its reversal is mediated by the transposon-encoded TnpA protein. We have developed an assay that permits demethylation of the Spm sequence to be controlled by inducing the expression of TnpA in plant cells. Using this assay, we show that demethylation is a rapid, active process. TnpA is a weak transcriptional activator, and deletions that abolish its transcriptional activity also eliminate its demethylation activity. We show that cell cycle and DNA synthesis inhibitors interfere with TnpA-mediated Spm demethylation. We further show that TnpA has a much lower affinity for fully methylated than for hemimethylated or unmethylated DNA fragments derived from Spm termini. Based on these observations, we suggest that TnpA binds to the postreplicative, hemimethylated Spm sequence and promotes demethylation either by creating an appropriate demethylation substrate or by itself participating in or recruiting a demethylase.

2,4-Dichlorophenoxyacetic Acid↗
PubMed2001

Induction of epithelial differentiation and DNA demethylation in hamster malignant oral keratinocyte by ornithine decarboxylase antizyme.

The hamster ornithine decarboxylase antizyme (ODC-Az) cDNA was transfected into the hamster malignant oral keratinocyte cell line, HCPC-1. Ectopic expression of ODC-Az resulted in the reversion of malignant phenotypes and alteration of DNA methylation status of CCGG sites. The phenotypes examined include ODC enzymatic activity, doubling time, morphological change, anchorage dependent growth, tumorigenicity in nude mice, induction of epithelial differentiation marker protein (involucrin), and change of cell cycle position. Comparison of CCGG DNA methylation status of the ODC-Az and control vector transfectants revealed a significant increase in demethylation of 5-methyl cytosines (m5C) of CCGG sites in the ODC-Az transfectants. Ectopic expression of ODC-Az gene in hamster malignant oral keratinocytes led to reduce ODC activity and the subsequent demethylation of 5-methyl cytosines, presumably via the ODC/ polyamines/ decarboxylated S-adenosylmethionine (dc-AdoMet) pathways. Our data suggest that ODC-Az shared the same pathway of polyamines/ dc-AdoMet/DNA methyltransferase (DNA MTase). We propose that ODC-Az mediates a novel mechanism in tumor suppression by DNA demethylation and presumably re-activation of key cellular genes silenced by DNA hypermethylation during cancer development. Oncogene (2001) 20, 24 - 33.

Agar↗
PubMed1999

Effects of a DNA demethylating agent--5-azacytidine--on testicular morphology during mouse embryo development.

DNA methylation is an epigenetical mechanism that plays crucial roles in cellular differentiation and tissue development in embryogenesis. The aim of the present study was to determine the effects of a demethylating agent, 5-azacytidine, on testicular development during embryonal life in mouse. Ten pregnant mice were administered 5-azacytidine (5-azaC) (i.p. 2 mg/kg of agent dissolved in 0.1 mg/ml PBS) during 8th (Group 1), 11th (Group 2), 14th (Group 3) and 18th (Group 4) days of pregnancy periods and male siblings of these animals were obtained (experimental groups) whereas the control group animals received no treatment and siblings of this group were also obtained. Testicular tissues from all groups were taken 20 days after birth and examined at the light and electron microscopical levels. All pregnancies were terminated in Group 1 animals, therefore no observations could be done in this group. While Group 2 and 3 siblings showed distinctive kongenital abnormalities such as; anancephaly, growth failure, cleft palate, extremity abnormalities, supernumerary ribs and whirled shaped-tails, no such abnormalities were observed in Group 4 when compared to the control group. Microscopical examination of testicular tissues in groups 2 and 3 demonstrated cellular disintegration of spermatocytes in seminiferous tubules. In addition, cytoplasmic vacuoles and thickening of the basement membrane were also evident in both groups 2 and 3. Apoptotic-like cells were seen especially in group 2 and rarely in group 3. There were no structural alterations in group 4 animals, except a decreased number of spermatocytes in seminiferous tubules when compared to the control group, possibly indicating the completion of embryogenesis in this group. In conclusion, it could be suggested that the demethylating agent 5-azacytidine may trigger an unknown gene reactivation during early embryogenesis possibly affecting the cell and tissue differentiation in developing mammalian embryos.

Animals↗