Silencing Of Multiple Genes Research Articles

Enhancement of efficiency of chitosan-based complexes for gene transfection with poly(γ-glutamic acid) by augmenting their cellular uptake and intracellular unpackage.

As a cationic polysaccharide, chitosan (CS) has been identified for its potential use as a non-viral vector for exogenous gene transfection. However, owing to their electrostatic interactions, CS complexes may cause difficulties in gene release upon their arrival at the site of action, thus limiting their transfection efficiency. In this work, an attempt is made to facilitate the release of a gene by incorporating a negatively-charged poly(γ-glutamic acid) (γPGA) into CS complexes in order to diminish their attractive interactions. The mechanisms of exploiting γPGA to enhance the transfection efficiency of CS complexes are elucidated. The feasibility of using this CS/γPGA-based system for DNA or siRNA transfer is explored as well. Additionally, potential of the CS/γPGA formulation to deliver disulfide bond-conjugated dual PEGylated siRNAs for multiple gene silencing is also examined. Moreover, the genetic use of pKillerRed-mem, delivered using complexes of CS and γPGA, to express a membrane-targeted KillerRed as an intrinsically generated photosensitizer for photodynamic therapy is described.

SOCS1 hypermethylation mediated by DNMT1 is associated with lipopolysaccharide-induced inflammatory cytokines in macrophages

Macrophages activation which releases the pro-inflammatory cytokines is an essential event in the process of inflammation. SOCS1 has been shown to act as a negative regulator of cytokine signals and plays a key role in the suppression of tissue injury and inflammatory diseases. DNA methylation mediated by specific DNA methyltransferases1 (DNMT1) which contributes to the epigenetic silencing of multiple genes. SOCS1 promoter hypermethylation is by far the best categorized epigenetic change in tumors. Our study with a view to investigate whether the loss of SOCS1 due to SOCS1 promoter methylation was involved in the course of inflammatory cytokines released from lipopolysaccharide (LPS)-stimulated macrophages. Here, we found that treatment of LPS-induced RAW264.7 macrophage cells with the DNA methylation inhibitor 5-aza-2′-deoxycytidine (5-azadC) reduced aberrant promoter hypermethylation of SOCS1 and prevented the loss of the expression of SOCS1 in macrophages which secret inflammatory cytokines. Knockdown of DNMT1 gene not only attenuated the SOCS1 gene promoter methylation but also up-regulated the expression of SOCS1 in activated RAW264.7 cells. Furthermore, silencing of DNMT1 prevented the activation of JAK2/STAT3 pathway in LPS-induced RAW264.7 cells. These studies demonstrated that DNMT1-mediated SOCS1 hypermethylation caused the loss of SOCS1 expression results in negative regulation of activation of the JAK2/STAT3 pathway, and enhanced the release of LPS-induced pro-inflammatory cytokines such as TNF-α and IL-6 in macrophages.

A Vector Library for Silencing Central Carbon Metabolism Genes with Antisense RNAs in Escherichia coli

We describe here the construction of a series of 71 vectors to silence central carbon metabolism genes in Escherichia coli. The vectors inducibly express antisense RNAs called paired-terminus antisense RNAs, which have a higher silencing efficacy than ordinary antisense RNAs. By measuring mRNA amounts, measuring activities of target proteins, or observing specific phenotypes, it was confirmed that all the vectors were able to silence the expression of target genes efficiently. Using this vector set, each of the central carbon metabolism genes was silenced individually, and the accumulation of metabolites was investigated. We were able to obtain accurate information on ways to increase the production of pyruvate, an industrially valuable compound, from the silencing results. Furthermore, the experimental results of pyruvate accumulation were compared to in silico predictions, and both sets of results were consistent. Compared to the gene disruption approach, the silencing approach has an advantage in that any E. coli strain can be used and multiple gene silencing is easily possible in any combination.

Disulfide bond-conjugated dual PEGylated siRNAs for prolonged multiple gene silencing

Many human diseases carry at least two independent gene mutations, further exacerbating clinical disorders. In this work, disulfide bond-conjugated dual PEGylated siRNAs were synthesized, capable of specifically targeting and silencing two genes simultaneously. To achieve efficient delivery, the conjugated siRNAs were formulated with the cationic chitosan together with an anionic polymer, poly(γ-glutamic acid) (γPGA), to form a ternary complex. Experimental results indicate that the incorporated γPGA could significantly enhance their intracellular delivery efficiency, allowing for reduction of the disulfide bond-conjugated PEGylated siRNAs delivered to the PEGylated siRNAs in the reductive cytoplasmic environment. The PEGylated siRNAs could more significantly increase their enzymatic tolerability, effectively silence multiple genes, and prolong the duration of their gene silencing capability than the unmodified siRNAs could. Silencing of different genes simultaneously significantly contributes to the efforts to treat multiple gene disorders, and prolonged duration of gene silencing can reduce the need for frequent administrations.

Single or Multiple Gene Silencing Directed by U6 Promoter-Based shRNA Vectors Facilitates Efficient Functional Genome Analysis in Medicago truncatula

Short hairpin RNA (shRNA) expression vectors are an effective means to deliver double-stranded RNA that mediates sequence-specific cleavage of target transcripts in RNA interference (RNAi). Previously, we constructed shRNA expression vectors based on U6 small nuclear RNA (snRNA) gene promoters of Medicago truncatula. To facilitate screening of shRNA candidates, a protoplast-based dual luciferase assay system was developed, which consists of a shRNA-expressing effector plasmid and a target gene-carrying reporter plasmid. RNAi efficiency of a given shRNA could be measured by comparing two luciferase activities derived from transcripts with an intact or interrupted 3′ untranslated region as a result of RNAi. Using a multiple U6-shRNA cassette plasmid that enables targeting of multiple genes simultaneously, the expression of two Δ1-pyrroline-5-carboxylate synthetase genes (MtP5CS1 and MtP5CS2) was silenced with a U6 plasmid carrying two 27-nucleotide-stem shRNA cassettes targeting each gene. The decreased transcript levels were accompanied by decreased protein levels and decreased free proline contents. To examine possible U6-shRNA-mediated RNAi in nodules, two remorin-encoding MtREM genes were targeted by single and double U6-shRNA plasmids. Silencing of MtREM2.2, an essential nodulation gene, MtREM1.1, an uncharacterized gene, or both inhibited nodule formation in transgenic roots. Moreover, M. truncatula roots transformed with a suppressor-overexpresser construct wherein MtREM2.2-targeting U6-shRNA and GmREM2.3-overexpressing cassettes were placed tandemly showed decreased MtREM2.2 transcripts yet survived nitrogen-free medium, indicating complementation of MtREM2.2 with the overexpressed soybean gene. These results demonstrate that U6 promoter-based shRNA-expressing plasmids are valuable for multiple gene functional analyses in protoplasts and nodules of M. truncatula.

Expression of DNA Methyltransferases in Breast Cancer Patients and to Analyze the Effect of Natural Compounds on DNA Methyltransferases and Associated Proteins

PurposeThe DNA methylation mediated by specific DNA methyltransferases (DNMTs), results in the epigenetic silencing of multiple genes which are implicated in human breast cancer. We hypothesized that the natural compounds modulate the expression of DNMTs and their associated proteins in the breast cancer cell lines and affect the methylation mediated gene silencing.MethodsThe DNMTs transcript expression was analyzed by reverse transcription-polymerase chain reaction (RT-PCR) in the tumors and the adjacent normal breast tissues of the patients with invasive ductal breast carcinoma. We tested the hypothesis that the natural compounds, viz., epigallocatechin gallate (EGCG), genistein, withaferin A, curcumin, resveratrol, and guggulsterone, have demethylation potential. To investigate this hypothesis, we analyzed the DNMTs expression at the transcript levels, followed by the analysis of DNMT1 and its associated proteins (HDAC1, MeCP2, and MBD2).ResultsThe increased DNMTs transcripts expression, viz., DNMT1, DNMT3a, and DNMT3b, in the breast cancer tissues suggest involvement of the DNMTs in the breast carcinogenesis. Quantitative RT-PCR analysis revealed that the treatment with natural compounds, viz., EGCG, genistein, withaferin A, curcumin, resveratrol, and guggulsterone, resulted in a significant decrease in the transcript levels of all the DNMTs investigated. Importantly, these natural compounds decreased the protein levels of DNMT1, HDAC1, and MeCP2.ConclusionOur results demonstrate that the natural compounds, EGCG, genistein, withaferin A, curcumin, resveratrol, and guggulsterone, have the potential to reverse the epigenetic changes. Moreover, their lack of toxicity makes these natural compounds promising candidates for the chemoprevention of the breast cancer. In-depth future mechanistic studies aimed to elucidate how these compounds affect the gene transcription are warranted.

Concerted suppression of all starch branching enzyme genes in barley produces amylose-only starch granules.

BackgroundStarch is stored in higher plants as granules composed of semi-crystalline amylopectin and amorphous amylose. Starch granules provide energy for the plant during dark periods and for germination of seeds and tubers. Dietary starch is also a highly glycemic carbohydrate being degraded to glucose and rapidly absorbed in the small intestine. But a portion of dietary starch, termed “resistant starch” (RS) escapes digestion and reaches the large intestine, where it is fermented by colonic bacteria producing short chain fatty acids (SCFA) which are linked to several health benefits. The RS is preferentially derived from amylose, which can be increased by suppressing amylopectin synthesis by silencing of starch branching enzymes (SBEs). However all the previous works attempting the production of high RS crops resulted in only partly increased amylose-content and/or significant yield loss.ResultsIn this study we invented a new method for silencing of multiple genes. Using a chimeric RNAi hairpin we simultaneously suppressed all genes coding for starch branching enzymes (SBE I, SBE IIa, SBE IIb) in barley (Hordeum vulgare L.), resulting in production of amylose-only starch granules in the endosperm. This trait was segregating 3:1. Amylose-only starch granules were irregularly shaped and showed peculiar thermal properties and crystallinity. Transgenic lines retained high-yield possibly due to a pleiotropic upregualtion of other starch biosynthetic genes compensating the SBEs loss. For gelatinized starch, a very high content of RS (65 %) was observed, which is 2.2-fold higher than control (29%). The amylose-only grains germinated with same frequency as control grains. However, initial growth was delayed in young plants.ConclusionsThis is the first time that pure amylose has been generated with high yield in a living organism. This was achieved by a new method of simultaneous suppression of the entire complement of genes encoding starch branching enzymes. We demonstrate that amylopectin is not essential for starch granule crystallinity and integrity. However the slower initial growth of shoots from amylose-only grains may be due to an important physiological role played by amylopectin ordered crystallinity for rapid starch remobilization explaining the broad conservation in the plant kingdom of the amylopectin structure.

Virus‐induced multiple gene silencing to study redundant metabolic pathways in plants: Silencing the starch degradation pathway in Nicotiana benthamiana

Virus-induced gene silencing (VIGS) is a rapid technique that allows for specific and reproducible post-transcriptional degradation of targeted mRNA. The method has been proven efficient for suppression of expression of many single enzymes. The metabolic networks of plants, however, often contain isoenzymes and gene families that are able to compensate for a mutation and mask the development of a silencing phenotype. Here, we show the application of multiple gene VIGS repression for the study of these redundant biological pathways. Several genes in the starch degradation pathway [disproportionating enzyme 1; (DPE1), disproportionating enzyme 2 (DPE2), and GWD] were silenced. The functionally distinct DPE enzymes are present in alternate routes for sugar export to the cytoplasm and result in an increase in starch production when silenced individually. Simultaneous silencing of DPE1 and DPE2 in Nicotiana benthamiana resulted in a near complete suppression in starch and accumulation of malto-oligosaccharides.

Simultaneous gene transduction and silencing using stimuli-responsive viral/nonviral chimeric nanoparticles

Despite viral vectors’ predominant use in clinical trials, due to higher gene delivery efficiency than nonviral counterparts, intrinsic immunogenicity and limited tunability for multi-modal effects are major concerns for their usage in gene therapy. An adeno-associated viral (AAV) particle was shielded with acid-degradable, siRNA-encapsulating polyketal (PK) shell, resulting in core–shell viral/nonviral chimeric nanoparticles (ChNPs). The AAV core of a ChNP is protected from immune responses by the PK shell which also facilitates the intracellular trafficking of the AAV core and efficiently releases the encapsulated siRNA into the cytoplasm. ChNPs led to significantly enhanced gene transduction, compared to unmodified free AAVs, and simultaneous silencing of a target gene, while avoiding inactivation by recognition from the immune system. Furthermore, conjugation of sialic acid (SA) on the surface of ChNPs enabled receptor-mediated targeted gene delivery to CD22-expressing cells. The ChNPs developed in this study combine the advantages of both viral and nonviral vectors and are a promising platform for targeted co-delivery of DNA and siRNA in inducing synergistic therapeutic effects by simultaneous expression and silencing of multiple genes.

Novel animal models for studying complex brain disorders: BAC-driven miRNA-mediated in vivo silencing of gene expression

In schizophrenia, glutamic acid decarboxylase 1 (GAD1) disturbances are robust, consistently observed, cell-type specific and represent a core feature of the disease. In addition, neuropeptide Y (NPY), which is a phenotypic marker of a sub-population of GAD1-containing interneurons, has shown reduced expression in the prefrontal cortex in subjects with schizophrenia, suggesting that dysfunction of the NPY+ cortical interneuronal sub-population might be a core feature of this devastating disorder. However, modeling gene expression disturbances in schizophrenia in a cell type-specific manner has been extremely challenging. To more closely mimic these molecular and cellular human post-mortem findings, we generated a transgenic mouse in which we downregulated GAD1 mRNA expression specifically in NPY+ neurons. This novel, cell type-specific in vivo system for reducing gene expression uses a bacterial artificial chromosome (BAC) containing the NPY promoter-enhancer elements, the reporter molecule (eGFP) and a modified intron containing a synthetic microRNA (miRNA) targeted to GAD1. The animals of isogenic strains are generated rapidly, providing a new tool for better understanding the molecular disturbances in the GABAergic system observed in complex neuropsychiatric disorders such as schizophrenia. In the future, because of the small size of the silencing miRNAs combined with our BAC strategy, this method may be modified to allow generation of mice with simultaneous silencing of multiple genes in the same cells with a single construct, and production of splice-variant-specific knockdown animals.

Emerging similarities in epigenetic gene silencing by long noncoding RNAs

Long noncoding RNAs (lncRNAs) such as Xist, Air, and Kcnq1ot1 are required for epigenetic silencing of multiple genes in cis within large chromosomal domains, including distant genes located hundreds of kilobase pairs away. Recent evidence suggests that all three of these lncRNAs are functional and that they silence gene expression, in part, through an intimate interaction with chromatin. Here we provide an overview of lncRNA-dependent gene silencing, focusing on recent findings for the Air and Kcnq1ot1 lncRNAs. We review molecular evidence indicating that these lncRNAs interact with chromatin and correlate their presence with specific histone modifications associated with gene silencing. A general model for a lncRNA-dependent gene-silencing mechanism is presented based on the apparent ability of lncRNAs to recruit histone-modifying activities to chromatin. However, alternate mechanisms may be required to explain the silencing of some lncRNA-dependent genes. Finally, we discuss unanswered questions and future perspectives associated with these enigmatic lncRNA molecules.

Conditional gene silencing of multiple genes with antisense RNAs and generation of a mutator strain of Escherichia coli

In this study, we describe a method of simultaneous conditional gene silencing of up to four genes in Escherichia coli by using antisense RNAs. We used antisense RNAs with paired termini, which carried flanking inverted repeats to create paired double-stranded RNA termini; these RNAs have been proven to have high silencing efficacy. To express antisense RNAs, we constructed four IPTG-inducible vectors carrying different but compatible replication origins. When the lacZ antisense RNA was expressed using these vectors, lacZ expression was successfully silenced by all the vectors, but the expression level of the antisense RNA and silencing efficacy differed depending on the used vectors. All the vectors were co-transformable; the antisense RNAs against lacZ, ackA, pta and pepN were co-expressed, and silencing of all the target genes was confirmed. Furthermore, when antisense RNAs were targeted to the mutator genes mutS, mutD (dnaQ) and ndk, which are involved in DNA replication or DNA mismatch repair, spontaneous mutation frequencies increased over 2000-fold. The resulting mutator strain is useful for random mutagenesis of plasmids. The method provides a robust tool for investigating functional relationships between multiple genes or altering cell phenotypes for biotechnological and industrial applications.

Dysregulation of MicroRNAs that Regulate DNMT3b Expression in Primary Breast Cancers

A subset of breast cancer cell lines express a hypermethylation defect related to DNMT3b overexpression resulting from loss of expression of regulatory microRNAs (miRs). Most primary breast cancers overexpress DNMT3b compared to normal breast epithelia, and certain subsets of breast cancer (basal‐type) display gene expression signatures that reflect methylation‐dependent silencing of multiple genes, consistent with the expression of a hypermethylation defect in vivo. Several miRs have been implicated in the regulation of DNMT3b. We examined the expression of miRs‐29a, 29b, 29c, 148a, and 148b, in a group of 15 primary breast cancers (n=6 luminal A, n=2 luminal B, n=2 Her2+, n=5 basal) and two normal breast tissues. miR expression levels <50% of normal were found in 9/15 (60%) tumors for 29a/29c, 10/15 (67%) tumors for 29b, 12/15 (80%) tumors for 148a, and 8/15 (53%) for 148b. Further, 12/15 (80%) tumors lost expression =2 miRs, 9/15 (60%) lost =3 miRs, and 8/15 (53%) lost =4 miRs. The aggregate miR expression score did not differ for luminal A versus basal breast cancers, suggesting that loss of additional factors may govern the hypermethylation defect. These results suggest that loss of expression of miRs‐29a, 29b, 29c, 148a, and 148b occurs commonly among primary breast cancers, but may contribute to the expression of a hypermethylation defect in a subset of these neoplasms.Support: NIH CA 78343

Prognostic Relevance of Promoter Hypermethylation of Multiple Genes in Breast Cancer Patients

Background: Methylation-mediated suppression of detoxification, DNA repair and tumor suppressor genes has been implicated in cancer development. This study was designed to investigate the impact of concurrent methylation of multiple genes in breast tumors on disease prognosis.Methods: Methylation specific PCR was carried out to analyze the methylation status of seven genes in archived breast tissues and determine the effect of aberrant methylation of multiple genes on disease prognosis and patients’ survival.Results: Promoter hypermethylation was observed in PRB 67%, ERα 64%, RASSF1A 63%, p16INK4A 51%, PRBβ2 22%, GSTP1 25% and BRCA1 27% of the breast cancers, respectively. Concurrent methylation of BRCA1, ERα, GSTP1 and RARβ2, was observed in a large proportion of breast cancers analyzed, suggesting that these genes do not appear to be methylated alone. Patients with high methylation indices had poor prognosis (p < 0.001, Hazards ratio = 14.58). Cox regression analysis showed RARβ2 promoter methylation to be an independent important determinant of breast cancer prognosis.Conclusions: Our results suggest that methylation of multiple genes plays an important role in prognosis of breast cancer. Our study not only describes the association of methylation mediated silencing of multiple genes with the severity of disease, but also drives to speculate the molecular crosstalk between genes or genetic pathways regulated by them individually.

Prognostic relevance of promoter hypermethylation of multiple genes in breast cancer patients.

Background: Methylation-mediated suppression of detoxification, DNA repair and tumor suppressor genes has been implicated in cancer development. This study was designed to investigate the impact of concurrent methylation of multiple genes in breast tumors on disease prognosis. Methods: Methylation specific PCR was carried out to analyze the methylation status of seven genes in archived breast tissues and determine the effect of aberrant methylation of multiple genes on disease prognosis and patients’ survival. Results: Promoter hypermethylation was observed in PRB 67%, ERα 64%, RASSF1A 63%, p16INK4A 51%, PRBβ2 22%, GSTP1 25% and BRCA1 27% of the breast cancers, respectively. Concurrent methylation of BRCA1, ERα, GSTP1 and RARβ2, was observed in a large proportion of breast cancers analyzed, suggesting that these genes do not appear to be methylated alone. Patients with high methylation indices had poor prognosis (p < 0.001, Hazards ratio = 14.58). Cox regression analysis showed RARβ2 promoter methylation to be an independent important determinant of breast cancer prognosis. Conclusions: Our results suggest that methylation of multiple genes plays an important role in prognosis of breast cancer. Our study not only describes the association of methylation mediated silencing of multiple genes with the severity of disease, but also drives to speculate the molecular crosstalk between genes or genetic pathways regulated by them individually.

A tightly regulated Pol III promoter for synthesis of miRNA genes in tandem

A compelling tool for functional genetics is to silence the expression of multiple related genes concomitantly and reversibly. Such a tool will accelerate the understanding on gene interaction in signaling pathway and the development of comprehensive animal models for human diseases. Multiple gene silencing may be achieved by concurrent expression of multiple miRNA from a Pol II promoter. By comparison, Pol III promoters possess greater capacity to synthesize RNA of high yield and are consisted of compact elements and simple terminators to be convenient for handling. The miRNA-induced gene silencing is a dose-dependent event, and thus, Pol III promoter as a miRNA driver increases the chance to induce phenotypes subsequent to the gene silencing. As a Pol III promoter, endogenous U6 promoter synthesizes small nuclear RNA of high yield and is commonly adapted for miRNA synthesis. Whether U6 promoter is effective to synthesize multiple miRNA in tandem remains to be determined. This study exploited a possibility to express multiple miRNA genes from U6 promoter and also tested the inducibility of varying types of Tet-regulatable U6 promoters. With miR-30a backbone, two miRNA genes were functionally and efficiently expressed from a U6 promoter. The transcriptional activity of Tet-regulatable U6 promoter was tightly regulated by Tetracycline system after sufficient repeats of Tetracycline Operator sequence were introduced within the promoter regions and also between U6 promoter and miRNA gene. This newly developed U6 miRNA system would make multi-gene silencing efficient and reversible.

DNMT3b overexpression contributes to a hypermethylator phenotype in human breast cancer cell lines

BackgroundDNA hypermethylation events and other epimutations occur in many neoplasms, producing gene expression changes that contribute to neoplastic transformation, tumorigenesis, and tumor behavior. Some human cancers exhibit a hypermethylator phenotype, characterized by concurrent DNA methylation-dependent silencing of multiple genes. To determine if a hypermethylation defect occurs in breast cancer, the expression profile and promoter methylation status of methylation-sensitive genes were evaluated among breast cancer cell lines.ResultsThe relationship between gene expression (assessed by RT-PCR and quantitative real-time PCR), promoter methylation (assessed by methylation-specific PCR, bisulfite sequencing, and 5-aza-2'deoxycytidine treatment), and the DNA methyltransferase machinery (total DNMT activity and expression of DNMT1, DNMT3a, and DNMT3b proteins) were examined in 12 breast cancer cell lines. Unsupervised cluster analysis of the expression of 64 methylation-sensitive genes revealed two groups of cell lines that possess distinct methylation signatures: (i) hypermethylator cell lines, and (ii) low-frequency methylator cell lines. The hypermethylator cell lines are characterized by high rates of concurrent methylation of six genes (CDH1, CEACAM6, CST6, ESR1, LCN2, SCNN1A), whereas the low-frequency methylator cell lines do not methylate these genes. Hypermethylator cell lines coordinately overexpress total DNMT activity and DNMT3b protein levels compared to normal breast epithelial cells. In contrast, most low-frequency methylator cell lines possess DNMT activity and protein levels that are indistinguishable from normal. Microarray data mining identified a strong cluster of primary breast tumors that express the hypermethylation signature defined by CDH1, CEACAM6, CST6, ESR1, LCN2, and SCNN1A. This subset of breast cancers represents 18/88 (20%) tumors in the dataset analyzed, and 100% of these tumors were classified as basal-like, suggesting that the hypermethylator defect cosegregates with poor prognosis breast cancers.ConclusionThese observations combine to strongly suggest that: (a) a subset of breast cancer cell lines express a hypermethylator phenotype, (b) the hypermethylation defect in these breast cancer cell lines is related to aberrant overexpression of DNMT activity, (c) overexpression of DNMT3b protein significantly contributes to the elevated DNMT activity observed in tumor cells expressing this phenotype, and (d) the six-gene hypermethylator signature characterized in breast cancer cell lines defines a distinct cluster of primary basal-like breast cancers.

Action at a distance: epigenetic silencing of large chromosomal regions in carcinogenesis

Despite the completion of the Human Genome Project, we are still far from understanding the molecular events underlying epigenetic change in cancer. Cancer is a disease of the DNA with both genetic and epigenetic changes contributing to changes in gene expression. Epigenetics involves the interplay between DNA methylation, histone modifications and expression of non-coding RNAs in the regulation of gene transcription. We now know that tumour suppressor genes, with CpG island-associated promoters, are commonly hypermethylated and silenced in cancer, but we do not understood what triggers this process or when it occurs during carcinogenesis. Epigenetic gene silencing has always been envisaged as a local event silencing discrete genes, but recent data now indicates that large regions of chromosomes can be co-coordinately suppressed; a process termed long range epigenetic silencing (LRES). LRES can span megabases of DNA and involves broad heterochromatin formation accompanied by hypermethylation of clusters of contiguous CpG islands within the region. It is not clear if LRES is initiated by one critical gene target that spreads and conscripts innocent bystanders, analogous to large genetic deletions or if coordinate silencing of multiple genes is important in carcinogenesis? Over the next decade with the exciting new genomic approaches to epigenome analysis and the initiation of a Human Epigenome Project, we will understand more about the interplay between DNA methylation and chromatin modifications and the expression of non-coding RNAs, promising a new range of molecular diagnostic cancer markers and molecular targets for cancer epigenetic therapy.

Epigenetic silencing of multiple genes in primary CNS lymphoma

Epigenetic silencing of functionally important genes is important in the development of malignancies and is a source of potential markers for molecular detection. Primary central nervous system lymphoma (PCNSL) is an increasingly common tumor that has not been extensively examined for changes in promoter region methylation. We examined 14 tumor suppressor genes in 25 cases of PCNSL using methylation-specific PCR. Methylation was observed in DAPK (84%), TSP1 (68%), CRBP1 (67%), p16(INK) (4a) (64%), p14(ARF) (59%), MGMT (52%), RARbeta2 (50%), TIMP3 (44%), TIMP2 (42%), p15(INK) (4b) (40%), p73 (28%), hMLH1 (12%), RB1 (8%) and GSTP1 (8%). Promoter methylation of p14(ARF), p16(INK) (4a) and MGMT was correlated with loss of expression by immunohistochemical staining. The methylation of many of these genes in PCNSL is similar to that reported in other high-grade B-cell lymphomas. All 25 cases of PCNSL had methylation of at least 2 genes. Methylation of DAPK, p16(INK) (4a) or MGMT was found in 96% of the tumors, suggesting simple marker strategies to detect circulating methylated DNA in serum that might facilitate early tumor detection. Our study provides insight into the epigenetic alterations in PCNSL and provides potential biomarkers of disease.

DNA hypermethylation of TIMP3 gene in invasive breast ductal carcinoma

Background. - Neoplastic cells often display aberrant methylation and silencing of multiple genes, including tumor suppressor genes (TSGs) that regulate critical processes such as cell cycle control, DNA repair and angiogenesis. Tissue inhibitor of metalloproteinase-3 (TIMP3) is an extracellular matrix-bound protein which regulates matrix composition and affects tumor growth, invasion and angiogenesis. It mediates vascular endothelial growth factor (VEGF) by blocking the binding of VEGF to VEGF receptor-2 and inhibits downstream signaling. This study focused on the hypermethylation status of the TIMP3 gene with clinical parameters in invasive breast ductal carcinoma (IDC) samples. Materials and methods. - DNA extraction and methylation specific PCR (MSP) was performed on 173 patients with invasive breast carcinoma. Both specific methylated and unmethylated primers for each gene were used for PCR and the products were visualized on agarose gel. The methylation status of TIMP3 was then compared with corresponding patients' clinicopathologic characteristics. Results. - Methylation frequencies of TIMP3 in the breast cancer samples were 20.81 %. Among the hypermethylated cancers, 50% were tumor grade II-III, 44.44% were positive in lymph node involvement (LN), 36.11% were positive in lymphovascular permeation (LVP); 44.44%, 22.22% and 47.22% for the overexpressions in estrogen receptor (ER), progesterone receptor(PR) and c-erbB2, respectively. Conclusion. - The result demonstrated that hypermethylation of TIMP3 in IDC might be associated with high tumor grading and lymph nodes metastasis, and overexpression of ER, PR and c-erbB2, respectively.