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A hallmark of cancer is aberrant DNA methylation, consisting of global hypomethylation and regional hypermethylation of tumor suppressor genes. DNA methyltransferase inhibitors have been recognized as promising candidate anticancer drugs. Drug development has focused on DNA methylation inhibitors with the goal of activating tumor suppressor genes silenced by DNA methylation. 5-azacytidine (5-AC; Vidaza), a global DNA methyltransferase inhibitor, was Food and Drug Administration approved to treat myelodysplastic syndromes and is clinically tested for solid tumors. In this paper, it was demonstrated that 5′-aza-2′-deoxycytidine (5-azaCdR) activated both silenced tumor suppressor genes and pro-metastatic genes by demethylation, raising the concern that it would promote metastasis. 5-AzaCdR treatment increased the invasiveness of non-invasive breast cancer cell lines MCF-7 cells and ZR-75-1 and dramatically induced pro-metastatic genes; Urokinase plasminogen activator (uPA), matrix metalloproteinase 2 (MMP2), metastasis-associated gene (H-MTS1; S100A4) and C-X-C chemokine receptor 4 (CXCR4). The hypothesis that the blocking of cellular transformation activity of DNA methyltransferase inhibitor could be separated from the pro-metastatic activity was tested using short interfering RNA (siRNA) targeted to the different DNA methyltransferase (DNMT) genes. Although depletion of DNMT1 had the strongest effect on colony growth suppression in cellular transformation assays, it did not result in demethylation and activation of uPA, S100A4, MMP2 and CXCR4 in MCF-7 cells. Depletion of DNMT1 did not induce cellular invasion in MCF-7 and ZR-75-1 non-invasive breast cancer cell lines. These data have implications on the design of new DNA methyltransferase inhibitor and on the proper utilization of current inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: DNA methylation is a covalent modification of DNA that plays an important role in setting gene expression programs during development. Recent evidence suggests that changes in DNA methylation patterns are involved in human disease through altering normal gene expression programming. In contrast to genetic changes aberrant DNA methylation patterns are potentially reversible raising the hope for DNA methylation based therapeutics. It was previously believed that the only relevant DNA methylation reaction in mature cells is DNA methyltransferase (DNMT), which accurately copies the DNA methylation pattern during cell division. The major effort in the field has therefore focused on developing DNMT inhibitors for cancer a disease of mitotic cells. However, recent evidence suggests that the DNA methylation state in both mitotic and postmitotic cells is a balance of DNA methylating and demethylating activities. This expands the scope of DNMT inhibitors to postmitotic tissues such as the brain. Since the identity of the DNA demethylating activity is still a mystery, the development of DNA demethylation inhibitors has been lagging. This review will discuss DNA methylation and demethylation machineries, and their therapeutic potentials as targets for small molecule inhibitors. [ABSTRACT FROM AUTHOR]

Abstract: Several lines of evidence point to the early origin of adult onset disease. A key question is: what are the mechanisms that mediate the effects of the early environment on our health? Another important question is: what is the impact of the environment during adulthood and how reversible are the effects of early life later in life? The genome is programmed by the epigenome, which is comprised of chromatin, a covalent modification of DNA by methylation and noncoding RNAs. The epigenome is sculpted during gestation, resulting in the diversity of gene expression programs in the distinct cell types of the organism. Recent data suggest that epigenetic programming of gene expression profiles is sensitive to the early-life environment and that both the chemical and social environment early in life could affect the manner by which the genome is programmed by the epigenome. We propose that epigenetic alterations early in life can have a life-long lasting impact on gene expression and thus on the phenotype, including susceptibility to disease. We will discuss data from animal models as well as recent data from human studies supporting the hypothesis that early life social-adversity leaves its marks on our epigenome and affects stress responsivity, health, and mental health later in life. [Copyright &y& Elsevier]

The article details approaches used for the isolation, the identification of a RNA binding protein and the validation of its role. It mentions the role of DNA methyltransferase 1 (DNMT1) in the maintenance of DNA methylation patterns during cell division. It cites the use of RNA affinity capture with the 3'-UTR of DNMT1 mRNA and matrix-assisted laser desorption-time of flight tandem mass spectrometry (MALDI-TOF-MS-MS) analysis in isolating and identifying AUF 1 (AU-rich element ARE:poly-(U)-binding/degradation factor) as the binding protein.

Suggests that DNA methyltransferase 1 (DNMT1) is a multifunctional protein that has regulatory activities in addition to DNA methylation activity. Explanation on the reversibility of the DNA methylation pattern; Functional consequences of the coordinate regulation of DNMT1 and DNA replication; Therapeutic implications for the design of DNMT1 inhibitors for anti-cancer therapy.

Studies the feedback regulation of DNA methyltransferase gene expression by a methylation-sensitive DNA element. Induction of hypomethylation challenge by a pharmacological inhibitor of DNA methyltransferase; Role of DNA methylation in the regulation of genome functions.

Tests the hypothesis that cytosine DNA methyltransferase (DNA MeTase) is a candidate target for anticancer therapy. Inhibition of DNA MeTase mRNA, DNA MeTAse activity, and tumorigenesis ex vivo by antisense oligodeoxynucleotide to the translation initiation region of the murine DNA MeTase.

DNA methylation is a major determinant of epigenetic inheritance. DNA methyltransferase 1 (DNMT1) is the enzyme responsible for the maintenance of DNA methylation patterns during cell division, and deregulated expression of DNMT1 leads to cellular transformation. We show herein that AU-rich element/poly(U)-binding/degradation factor 1 (AUF1)/heterogenous nuclear ribonucleoprotein D interacts with an AU-rich conserved element in the 3′ untranslated region of the DNMT1 mRNA and targets it for destabilization by the exosome. AUF1 protein levels are regulated by the cell cycle by the proteasome, resulting in cell cycle-specific destabilization of DNMT1 mRNA. AUF1 knock down leads to increased DNMT1 expression and modifications of cell cycle kinetics, increased DNA methyltransferase activity, and genome hypermethylation. Concurrent AUF1 and DNMT1 knock down abolishes this effect, suggesting that the effects of AUF1 knock down on the cell cycle are mediated at least in part by DNMT1. In this study, we demonstrate a link between AUF1, the RNA degradation machinery, and maintenance of the epigenetic integrity of the cell. [ABSTRACT FROM AUTHOR]

DNA methyltransferase 1 (DNMT1) is an important component of the epigenetic machinery and is responsible for copying DNA methylation patterns during cell division. Coordination of DNA methylation and DNA replication is critical for maintaining epigenetic programming. Knockdown of DNMT1 leads to inhibition of DNA replication, but the mechanism has been unclear. Here we show that depletion of DNMT1 with either antisense or small interfering RNA (siRNA) specific to DNMT1 activates a cascade of genotoxic stress checkpoint proteins, resulting in phosphorylation of checkpoint kinases 1 and 2 (Chk1 and -2), γ H2AX focus formation, and cell division control protein 25a (CDC25a) degradation, in an ataxia telangiectasia mutated-Rad3-related (ATR)-dependent manner. siRNA knockdown of ATR blocks the response to DNMT1 depletion; DNA synthesis continues in the absence of DNMT1, resulting in global hypomethylation. Similarly, the response to DNMT1 knockdown is significantly attenuated in human mutant ATR fibroblast cells from a Seckel syndrome patient. This response is sensitive to DNMT1 depletion, independent of the catalytic domain of DNMT1, as indicated by abolition of the response with ectopic expression of either DNMT1 or DNMT1 with the catalytic domain deleted. There is no response to short-term treatment with 5-aza-deoxycytidine (5-aza-CdR), which causes demethylation by trapping DNMT1 in 5-aza-CdR-containing DNA but does not cause disappearance of DNMT1 from the nucleus. Our data are consistent with the hypothesis that removal of DNMT1 from replication forks is the trigger for this response. [ABSTRACT FROM AUTHOR]

Abstract: Recent observations suggest that DNA methylation plays an important role in memory and long-term potentiation (LTP) in the hippocampus and is involved in programming the offspring epigenome in response to maternal care. Global DNA methylation is believed to be stable postnatally and to be similar across tissues in the adult mammal. It has also been a long held belief that DNA methyltransferases (DNMTs) play a very limited role in postmitotic tissues. Recent data suggests a more dynamic role for DNA methylation in the brain postnatally, therefore we examined the global state of methylation and the expression of the known DNMTs in the different regions of the hippocampus. We observed strikingly different levels of global methylation in the adult rat dentate gyrus (DG) and CA1 region in comparison with the CA2 and CA3 regions. mRNA levels of DNA methyltransferases exhibited similar regional specificity and were correlated with global DNA methylation levels. These regional differences in global methylation and expression of the DNA methylation machinery in the adult brain are consistent with the emerging hypothesis that DNA methylation may play a dynamic physiological role in the adult brain. [Copyright &y& Elsevier]

DNA methyltransferase 1 (DNMT1) catalyzes the postreplication methylation of DNA and is responsible for maintaining the DNA methylation pattern during cell division. A long list of data supports a role for DNMT1 in cellular transformation and inhibitors of DNMT1 were shown to have antitumorigenic effects. It was long believed that DNMT1 promoted tumorigenesis by maintaining the hypermethylated and silenced state of tumor suppressor genes. We have previously shown that DNMT1 knock down by either antisense oligonucleotides directed at DNMT1 or expressed antisense activates a number of genes involved in stress response and cell cycle arrest by a DNA methylation-independent mechanism. In this report we demonstrate that antisense knock down of DNMT1 in human lung carcinoma A549 and embryonal kidney HEK293 cells induces gene expression by a mechanism that does not involve either of the known epigenomic mechanisms, DNA methylation, histone acetylation, or histone methylation. The mechanism of activation of the cell cycle inhibitor p21 and apoptosis inducer BIK by DNMT1 inhibition is independent of the mechanism of activation of the same genes by histone deacetylase inhibition. We determine whether DNMT1 knock down activates one of the nodal transcription activation pathways in the cell and demonstrate that DNMT1 activates Sp1 response elements. This activation of Sp1 response does not involve an increase in either Sp1 or Sp3 protein levels in the cell or the occupancy of the Sp1 elements with these proteins. The methylation-independent regulation of Sp1 elements by DNMT1 unravels a novel function for DNMT1 in gene regulation. DNA methylation was believed to be a mechanism for suppression of CG-rich Sp1-bearing promoters. Our data suggest a fundamentally different and surprising role for DNMT1 regulation of CG-rich genes by a mechanism independent of DNA methylation and histone acetylation. The implications of our data on the biological roles of DNMT1 and the therapeutic potential of DNMT1 inhibitors as anticancer agents are discussed. [ABSTRACT FROM AUTHOR]

Evaluates the hypothesis that vertebrate cells possess mechanisms that protect them from epigenomic stress similar to DNA damage checkpoints. Effect of DNA methyltransferase knockdown by an oligonucleotide antisense on the intra-S-phase arrest of DNA replication; Implication on the arrest of cells at different positions throughout the S-phase of the cell cycle; Proposition on intra-S-phase arrest of DNA replication.