Biomedical subjects

Moshe Szyf

Publications and source records attributed to Moshe Szyf.

At least 37 records · Page 2Linked to original sources

PubMed2004

Early dysregulation of hippocampal proteins in transgenic rats with Alzheimer's disease-linked mutations in amyloid precursor protein and presenilin 1.

The response of the hippocampal proteome to expression of mutant proteins present in familial forms of Alzheimer's disease (AD) was studied using transgenic rats. These animals carry both the amyloid precursor protein Swedish and 717 mutation (APP(SW+717)) as well as the presenilin 1 Finnish mutation (PS1(FINN)). This transgenic rat model displays intracellular amyloid beta (Abeta) in neurons of the neocortex and the hippocampus (CA2 and CA3). The hippocampus was selected as it is one of the first brain regions affected in AD and is involved in the processing of short-term memory and spatial memory. Applying a proteomic approach, we demonstrate that the expression of APP(SW+717) and PS1(FINN) transgenes causes changes in expression of hippocampal proteins, some of which have been previously linked to learning and memory formation. The protein alterations documented here occur in the absence of plaque formation and prior to the onset of cognitive deficits later observed in these transgenic rats. This indicates that molecular changes take place in the hippocampal neurons in response to expression of mutant proteins APP(SW+717) and PS1(FINN), which precede the occurrence of overt extracellular accumulation of extracellular amyloid. The implications of these findings on our understanding of the early stages of AD are discussed.

Alzheimer Disease↗

PubMed2004

DNA methylation and breast cancer.

DNA methylation and chromatin structure patterns are tightly linked components of the epigenome, which regulate gene expression programming. Two contradictory changes in DNA methylation patterns are observed in breast cancer; regional hypermethylation of specific genes and global hypomethylation. It is proposed here that independent mechanisms are responsible for these alterations in DNA methylation patterns and that these alterations deregulate two different processes in breast cancer. Regional hypermethylation is brought about by specific regional changes in chromatin structure, whereas global demethylation is caused by a general increase in demethylation activity. Hypermethylation silences growth regulatory genes resulting in uncontrolled growth whereas hypomethylation leads to activation of genes required for metastasis. DNA methylation inhibitors activate silenced tumor suppressor genes resulting in arrest of tumor growth and are now being tested as candidate anticancer drugs. Demethylation inhibitors are proposed here to be potential novel candidate antimetastatic agents, which would bring about methylation and silencing of metastatic genes. Future therapeutic application of either methylation or demethylation inhibitors in cancer therapy would require understanding of the relative role of these processes in the evolution of cancer.

Animals↗

PubMed2004

DNA demethylation and cancer: therapeutic implications.

The epigenome, which is comprised of chromatin and its associated proteins and the patterns of covalent modification of DNA by methylation, sets up and maintains gene expression programs. A hallmark of cancer is a paradoxical aberration of DNA methylation patterns, a global loss of DNA methylation, that coexists with regional hypermethylation of certain genes. The hypermethylation of tumor-suppressor genes has attracted significant attention recently and DNA methylation inhibitors are being tested as potential anticancer agents. However, emerging data suggests that hypomethylation plays a role in activating genes required for metastasis and invasion. It is proposed here that hypermethylation and hypomethylation in cancer are independent processes, which target different programs at different stages in tumorigenesis. Understanding the relative roles of hypomethylation and hypermethylation in cancer has clear implications on the therapeutic use of agents targeting the DNA methylation machinery, which are discussed in this review.

Breast Neoplasms↗

PubMed2004

Epigenetic programming by maternal behavior.

Here we report that increased pup licking and grooming (LG) and arched-back nursing (ABN) by rat mothers altered the offspring epigenome at a glucocorticoid receptor (GR) gene promoter in the hippocampus. Offspring of mothers that showed high levels of LG and ABN were found to have differences in DNA methylation, as compared to offspring of 'low-LG-ABN' mothers. These differences emerged over the first week of life, were reversed with cross-fostering, persisted into adulthood and were associated with altered histone acetylation and transcription factor (NGFI-A) binding to the GR promoter. Central infusion of a histone deacetylase inhibitor removed the group differences in histone acetylation, DNA methylation, NGFI-A binding, GR expression and hypothalamic-pituitary-adrenal (HPA) responses to stress, suggesting a causal relation among epigenomic state, GR expression and the maternal effect on stress responses in the offspring. Thus we show that an epigenomic state of a gene can be established through behavioral programming, and it is potentially reversible.

Acetylation↗

PubMed2004

Reversal of the hypomethylation status of urokinase (uPA) promoter blocks breast cancer growth and metastasis.

Metastasis is a leading cause of mortality and morbidity in cancer. Urokinase (uPA), only expressed by the highly invasive cancer cells, has been implicated in invasion, metastases, and angiogenesis of several malignancies including breast cancer. Because uPA expression is strongly correlated with its hypomethylated state, we utilized the uPA gene in the highly invasive MDA-231 human breast cancer cells as a model system to test the hypothesis that pharmacological reversal of the uPA promoter hypomethylation would result in its silencing and inhibition of metastasis. S-Adenosyl-l-methionine (AdoMet) has previously been shown to cause hypermethylation and inhibit demethylation. Treatment of MDA-231 cells with AdoMet, but not its unmethylated analogue S-adenosylhomocysteine, significantly inhibits uPA expression and tumor cell invasion in vitro and tumor growth and metastasis in vivo. The effects of AdoMet on uPA expression were reversed by the demethylating agent 5'-azacytidine, supporting the conclusion that AdoMet effects are caused by hypermethylation. Knockdown of the methyl-binding protein 2 also causes a significant inhibition of uPA expression in vitro and tumor growth and metastasis in vivo. These treatments did not have any effects on estrogen receptor expression, suggesting that inhibition of hypomethylation will not affect genes already silenced by hypermethylation. These data are consistent with the hypothesis that hypomethylation of critical genes like uPA plays a causal role in metastasis. Inhibition of hypomethylation can thus be used as a novel therapeutic approach to silence the pro-metastatic gene uPA and block breast cancer progression into the aggressive and metastatic stages of the disease.

Animals↗

PubMed2004

DNA methyltransferase 1 knock down induces gene expression by a mechanism independent of DNA methylation and histone deacetylation.

DNA methyltransferase 1 (DNMT1) catalyzes the post-replication 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.

Acetylation↗

PubMed2004

Early environmental regulation of hippocampal glucocorticoid receptor gene expression: characterization of intracellular mediators and potential genomic target sites.

Environmental conditions in early life permanently alter the development of glucocorticoid receptor gene expression in the hippocampus and hypothalamic-pituitary-adrenal responses to acute or chronic stress. In part, these effects can involve an activation of ascending serotonergic pathways and subsequent changes in the expression of transcription factors that might drive glucocorticoid receptor expression in the hippocampus. This paper summarizes the evidence in favor of these pathways as well as recent studies describing regulatory targets within the chromatin structure of the promoter region of the rat hippocampal glucocorticoid receptor gene.

Animals↗

PubMed2004

Epigenetic programming of stress responses through variations in maternal care.

Early life experiences shape an individual's physical and mental health across the lifespan. Not surprisingly, an upbringing that is associated with adversity can produce detrimental effects on health. A central theme that arises from studies in human and nonhuman species is that the effects of adversity are mediated by the interactions between a mother and her young. In this review we describe some of the long-term effects of maternal care on the offspring and we focus on the impact of naturally occurring variations in the behavior of female rats. Of particular interest are mothers that engage in high or low amounts of licking/grooming (LG) and arched-back nursing (ABN) of their pups, but do so within the normal range for this species. Such variations in LG-ABN can alter the function of the hypothalamic-pituitary-adrenal (HPA) axis, and cognitive and emotional development by directly affecting the underlying neural mechanisms. At the heart of these mechanisms is gene expression. By studying the hippocampal glucocorticoid receptor gene, we have identified that maternal care regulates its expression by changing two processes: the acetylation of histones H3-K9, and the methylation of the NGFI-A consensus sequence on the exon 1(7) promoter. Sustained "maternal effects" appear elsewhere in biology, including plants, insects, and lizards, and may have evolved to program advantages in the environments that the offspring will likely face as adults. Given the importance of early life and parent-child interactions to later behavior, prevention and intervention programs should target this critical phase of development.

Affect↗

PubMed2004

Rat transgenic models with a phenotype of intracellular Abeta accumulation in hippocampus and cortex.

In this communication we report the characterization of several transgenic rat lines expressing human AbetaPP carrying the Swedish and Indiana mutations (coded UKUR28), the human presenilin 1 transgene with the 'Finn' mutation (coded UKUR19) and double transgenic rats expressing both transgenes (coded UKUR25). In these Tg rats, the AbetaPP and PS1 transgene expression was largely restricted to the hippocampus and neocortex. The PS1 transgenic rats did not produce visible changes in Abeta immunoreactivity. The AbetaPP transgenic rats (both the single Tg UKUR28, and double Tg UKUR25) generated a phenotype of intra-neuronalbeta accumulation without plaque formation and with no increased immunoreactivity for AbetaPP amino and carboxyl-terminal epitopes. This phenotype was apparent as early as 6 months of age in the transgenic rat lines carrying the human AbetaPP transgene. No senile plaques of aggregated Abeta were observed in any of the transgenic lines generated, up to 24 months of age. The hAbetaPP single homozygous Tg line (UKUR28) showed an increase in ERK2, without changes in glycogen synthase kinase 3 (GSK3) activity. A preliminary protein analysis of the hippocampus of the double transgenic rat (UKUR25) by mass spectrometry showed differences in the protein profile between this transgenic line and controls.

Alzheimer Disease↗

PubMed2003

Methylated DNA-binding protein 2 antisense inhibitors suppress tumourigenesis of human cancer cell lines in vitro and in vivo.

Methylated DNA-binding protein 2 (MBD2) has been proposed to function both as a silencer of methylated genes and as a DNA demethylase. Our previous data indicated that knockdown of MBD2 inhibited tumourigenesis of human cancer lines and MBD2-deficient mice were recently shown to be resistant to intestinal tumourigenesis. MBD2 is an attractive anticancer target since MBD2-deficient mice were previously shown to be viable and fertile and knockdown of MBD2 was reported to have no effect on cellular growth parameters of non-transformed cells. In this paper we test the hypothesis that pharmacological inhibition of MBD2 inhibits cancer growth in vivo using human tumour lines implanted in mice as a model. We develop sequence-specific antisense inhibitors of MBD2 and we show that these agents inhibit anchorage-independent growth of human lung (A549) and colorectal (HCT116) cancer cell lines in vitro and tumourigenic growth of human cancer cell xenografts in vivo. MBD2 antisense oligonucleotide does not inhibit the growth of normal and transformed cell lines and does not alter cell cycle parameters in vitro and does not exhibit overt toxicity in vivo in comparison with a scrambled control oligonucleotide, as determined by measuring body mass, blood cell parameters and liver and kidney enzymes. Our data provide a proof of principle that MBD2 is a new anticancer target and that pharmacological inhibition of MBD2 by agents such as the antisense inhibitors described in this paper is a potential new anticancer therapy, which in contrast to the vast majority of current approaches does not target normal progression of the cell cycle.

Animals↗

PubMed2003

Valproate induces replication-independent active DNA demethylation.

In this report, we demonstrate that valproic acid (VPA), a drug that has been used for decades in the treatment of epilepsy and as a mood stabilizer, triggers replication-independent active demethylation of DNA. Thus, this drug can potentially reverse DNA methylation patterns and erase stable methylation imprints on DNA in non-dividing cells. Recent discoveries support a role for VPA in the regulation of methylated genes; however, the mechanism has been unclear because it is difficult to dissociate active demethylation from the absence of DNA methylation during DNA synthesis. We therefore took advantage of an assay that measures active DNA demethylation independently from other DNA methylation and DNA replication activities in human embryonal kidney 293 cells. We show that VPA induces histone acetylation, DNA demethylation, and expression of an ectopically methylated CMV-GFP plasmid in a dose-dependent manner. In contrast, valpromide, an analogue of VPA that does not induce histone acetylation, does not induce demethylation or expression of CMV-GFP. Furthermore, we illustrate that methylated DNA-binding protein 2/DNA demethylase (MBD2/dMTase) participates in this reaction since antisense knockdown of MBD2/dMTase attenuates VPA-induced demethylation. Taken together, our data support a new mechanism of action for VPA as enhancing intracellular demethylase activity through its effects on histone acetylation and raises the possibility that DNA methylation is reversible independent of DNA replication by commonly prescribed drugs.

Blotting, Southern↗

PubMed2003

The methyl donor S-Adenosylmethionine inhibits active demethylation of DNA: a candidate novel mechanism for the pharmacological effects of S-Adenosylmethionine.

S-Adenosylmethionine (AdoMet) is the methyl donor of numerous methylation reactions. The current model is that an increased concentration of AdoMet stimulates DNA methyltransferase reactions, triggering hypermethylation and protecting the genome against global hypomethylation, a hallmark of cancer. Using an assay of active demethylation in HEK 293 cells, we show that AdoMet inhibits active demethylation and expression of an ectopically methylated CMV-GFP (green fluorescent protein) plasmid in a dose-dependent manner. The inhibition of GFP expression is specific to methylated GFP; AdoMet does not inhibit an identical but unmethylated CMV-GFP plasmid. S-Adenosylhomocysteine (AdoHcy), the product of methyltransferase reactions utilizing AdoMet does not inhibit demethylation or expression of CMV-GFP. In vitro, AdoMet but not AdoHcy inhibits methylated DNA-binding protein 2/DNA demethylase as well as endogenous demethylase activity extracted from HEK 293, suggesting that AdoMet directly inhibits demethylase activity, and that the methyl residue on AdoMet is required for its interaction with demethylase. Taken together, our data support an alternative mechanism of action for AdoMet as an inhibitor of intracellular demethylase activity, which results in hypermethylation of DNA.

Cells, Cultured↗

PubMed2003

Epigenomic stress response. Knockdown of DNA methyltransferase 1 triggers an intra-S-phase arrest of DNA replication and induction of stress response genes.

The DNA methylation pattern is an important component of the epigenome that regulates and maintains gene expression programs. In this paper, we test the hypothesis that vertebrate cells possess mechanisms protecting them from epigenomic stress similar to DNA damage checkpoints. We show that knockdown of DNMT1 (DNA methyltransferase 1) by an antisense oligonucleotide triggers an intra-S-phase arrest of DNA replication that is not observed with control oligonucleotide. The cells are arrested at different positions throughout the S-phase of the cell cycle, suggesting that this response is not specific to distinct classes of origins of replication. The intra-S-phase arrest of DNA replication is proposed to protect the genome from extensive DNA demethylation that could come about by replication in the absence of DNMT1. This protective mechanism is not induced by 5-aza-2'-deoxycytidine, a nucleoside analog that inhibits DNA methylation by trapping DNMT1 in the progressing replication fork, but does not reduce de novo synthesis of DNMT1. Our data therefore suggest that the intra-S-phase arrest is triggered by a reduction in DNMT1 and not by demethylation of DNA. DNMT1 knockdown also leads to an induction of a set of genes that are implicated in genotoxic stress response such as NF-kappaB, JunB, ATF-3, and GADD45beta (growth arrest DNA damage 45beta gene). Based on these data, we suggest that this stress response mechanism evolved to guard against buildup of DNA methylation errors and to coordinate inheritance of genomic and epigenomic information.

Cell Division↗

PubMed2003

Enhanced antitumor activity of a combination of MBD2-antisense electrotransfer gene therapy and bleomycin electrochemotherapy.

BACKGROUND: MBD2 is a methylated DNA-binding protein that has been previously suggested to have transcriptional silencing as well as DNA demethylase activities. We have previously shown that electrotransfer of an MBD2-antisense encoding plasmid inhibits tumor growth in vivo. In this study we tested whether a combination of MBD2-antisense gene therapy and bleomycin chemotherapy has an augmented antitumor effect in comparison with either monotherapy. METHODS: Mice bearing human non-small-cell lung carcinoma line H1299 xenoplants were treated with electrotransfer of either bleomycin or MBD2-antisense expression plasmid or a combination of both therapies and tumor growth following treatment was monitored. RESULTS: A combination of electrotransfer of MBD2-antisense and bleomycin electrochemotherapy has an additive inhibitory effect on the rate of tumor growth and a synergistic effect on the number of tumor-free animals when compared with either monotherapy. CONCLUSIONS: Our results suggest that a combination of MBD2-antisense electrotransfer gene therapy and chemotherapy with bleomycin is a candidate new approach to anticancer therapy.

Animals↗

PubMed2003

DNA methylation and cancer therapy.

Vertebrate DNA is modified by methyl moieties at the 5'-position of cytosine rings residing in the di-nucleotide sequence CpG. Approximately 80% of CpG dinucleotide sequences are methylated. The pattern of distribution of methylated CGs is cell-type specific and correlates with gene expression programming and chromatin structure. Three kinds of seemingly contradictory aberrations in DNA methylation are observed in cancer, global hypomethylation, and regional hypermethylation and deregulated level of expression of DNA methyltransferases. It was previously proposed that the DNA methylation machinery is a candidate target for anticancer therapy. Inhibition of hypermethylation was the first therapeutic target. However, recent data suggests that inhibition of DNA methylation might have untoward effects such as induction of genes involved in metastasis. This review discusses the relative role of the three levels of alteration in the DNA methylation in cancer, proposes a unified hypothesis on the relative roles of increased DNA methyltransferase as well as the coexistence of hypo -and hyper- methylation in cancer and its possible implications on anticancer therapy.

Animals↗

PubMed2003

Targeting DNA methylation in cancer.

There is overwhelming evidence that DNA methylation patterns are altered in cancer. Methylation of CG-rich islands in regulatory regions of genes marks them for transcriptional silencing. Multiple genes, which confer selective advantage upon cancer cells such as tumor suppressors, adhesion molecules, inhibitors of angiogenesis and repair enzymes are silenced. In parallel, tumor cell genomes are globally less methylated than their normal counterparts. In contrast to regional hypermethylation, this loss of methylation in cancer cells occurs in sparsely distributed CG sequences. We now understand that DNA methylation machineries might include a number of DNA methyltransferases, proteins that direct DNA methyltransferases to specific promoters, chromatin modifying enzymes as well as demethylases. There is also data to suggest that pharmacological down regulation of some members of the DNA methylation machinery could inhibit cancer in vitro, in vivo and in clinical trials. Understanding which functions of DNA methylation machinery are critical for cancer is essential for the design of inhibitors of the DNA methylation machinery as anticancer agents. This review discusses the possible role of DNA methyltranferases and demethylases in tumorigenesis and the possible pharmacological and therapeutic implications of the DNA methylation machinery.

Animals↗

PubMed2003

Human DNA methyltransferase gene DNMT1 is regulated by the APC pathway.

Epigenomic changes in DNA methylation patterns are evident in a variety of cancers, including colorectal cancer (CRC). In addition, a large proportion of CRC tumors and cell lines harbor genetic mutations in the APC/beta-catenin/TCF transcription activation pathway. While several target genes have been proposed, a causal downstream agent between APC mutation and cancer has not been fully established. Because previous work implicates DNA methyltransferase (DMNT1) as a critical point in tumorigenesis and recent studies suggest that familial CRC also exhibits epigenetic alterations, we sought to investigate whether this gene might be regulated by APC in CRC. Reconstitution of wild type APC in HT-29 CRC cell lines reduced the expression of both a reporter gene driven by the minimal DNMT1 promoter and DNMT1 mRNA that is independent of cell growth stasis. We also provide evidence for a causal role of DNMT1 in CRC by demonstrating that antisense-driven reduction of DNMT1 mRNA inhibits anchorage-independent growth, an indicator of tumorigenesis, of CRC cells. These data support future consideration of DNMT1 as a target in the treatment of CRC.

Adenomatous Polyposis Coli Protein↗

PubMed2002

Regulation of DNA methylation in human breast cancer. Effect on the urokinase-type plasminogen activator gene production and tumor invasion.

Urokinase-type plasminogen activator (uPA) is a member of the serine protease family and can break down various components of the extracellular matrix to promote growth, invasion, and metastasis of several malignancies including breast cancer. In the current study we examined the role that the DNA methylation machinery might be playing in regulating differential uPA gene expression in breast cancer cell lines. uPA mRNA is expressed in the highly invasive, hormone-insensitive human breast cancer cell line MDA-MB-231 but not in hormone-responsive cell line MCF-7. Using methylation-sensitive PCR, we show that 90% of CpG dinucleotides in the uPA promoter are methylated in MCF-7 cells, whereas fully demethylated CpGs were detected in MDA-MB-231 cells. uPA promoter activity, which is directly regulated by the Ets-1 transcription factor, is inhibited by methylation as determined by uPA promoter-luciferase reporter assays. We then tested whether the state of expression and methylation of the uPA promoter correlates with the global level of DNA methyltransferase and demethylase activities in these cell lines. We show that maintenance DNA methyltransferase activity is significantly higher in MCF-7 cells than in MDA-MB-231 cells, whereas demethylase activity is higher in MDA-MB-231 cells. We suggest that the combination of increased DNA methyltransferase activity with reduced demethylase activity contributes to the methylation and silencing of uPA expression in MCF-7 cells. The converse is true in MDA-MB-231 cells, which represents a late stage highly invasive breast cancer. The histone deacetylase inhibitor, Trichostatin A, induces the expression of the uPA gene in MDA-MB-231 cells but not in MCF-7 cells. This supports the hypothesis that DNA methylation is the dominant mechanism involved in the silencing of uPA gene expression. Taken together, these results provide insight into the mechanism regulating the transcription of the uPA gene in the complex multistep process of breast cancer progression.

Base Sequence↗