Down-regulation of DNMT3b in PC3 cells effects locus-specific DNA methylation, and represses cellular growth and migration

<p>Abstract</p> <p>Background</p> <p>Aberrations in DNA methylation patterns promote changes in gene expression patterns and are invariably associated with neoplasia. DNA methylation is carried out and maintained by several DNA methyltransferases (DNMTs) among which DNMT1 functions as a maintenance methylase while DNMT3a and 3b serve as de novo enzymes. Although DNMT3b has been shown to preferentially target the methylation of DNA sequences residing in pericentric heterochromatin whether it is involved in gene specific methylation remains an open question. To address this issue, we have silenced the expression of DNMT3b in the prostate-derived PC3 cells through RNA interference and subsequently studied the accompanied cellular changes as well as the expression profiles of selected genes.</p> <p>Results</p> <p>Our results demonstrate that DNMT3b depletion results in increased apoptosis and reduced migration of PC3 cells compared to the untransfected control cells. Reduced DNMT3b expression resulted in hypomethylation of retinoblastoma (Rb), retinoic-acid receptor β (RAR-β), and adenomatous polyposis coli (APC) gene promoters, and also culminated in increased expression of <it>CDKN3 </it>and <it>cytochrome b5</it>. Although DNMT3b silenced cells were found to have reduced growth and migratory potential, there was no apparent changes in their invasive ability compared to the parental PC3 cell line.</p> <p>Conclusion</p> <p>Our findings reveal that DNMT3b preferentially targets certain gene promoters in PC3 cells and that its depletion significantly reduces growth and migration of PC3 cells.</p

Silencing of MBD1 and MeCP2 in prostate-cancer-derived PC3 cells produces differential gene expression profiles and cellular phenotypes

Alterations in genomic CpG methylation patterns have been found to be associated with cell transformation and neoplasia. Although it is recognized that methylation of CpG residues negatively regulates gene expression, how the various MBPs (methyl-binding proteins) contribute to this process remains elusive. To determine whether the two well characterized proteins MeCP2 (methyl-CpG-binding protein 2) and MBD1 (methyl-CpG-binding domain 1) have distinct or redundant functions, we employed RNAi (RNA interference) to silence their expression in the prostate cancer-derived PC3cell line, and subsequently compared cell growth, invasion and migration properties of these cell lines in addition to their respective mRNA-expressionprofiles. Cells devoid of MeCP2 proliferated more poorly compared with MBD1-deficient cells and the parental PC3 cells. Enhanced apoptosis was observed in MeCP2-deficient cells, whereas apoptosis in parental and MBD1-deficient cells appeared to be equivalent. Boyden chamber invasion and wound-healing migration assays showed that MBD1-silenced cells were both more invasive and migratory compared with MeCP2-silenced cells. Finally, gene chip microarray analyses showed striking differences in the mRNA-expression profiles obtained from MeCP2- and MBD1-depleted cellsrelative to each other as well as when compared with control cells. The results of the present study suggest that MeCP2 and MBD1 silencing appear to affect cellular processes independently in vivo and that discrete sets of genes involved in cellular proliferation, apoptosis, invasion and migration are targeted by each protein

Antiproliferative Effects of DNA Methyltransferase 3B Depletion Are Not Associated with DNA Demethylation

Silencing of genes by hypermethylation contributes to cancer progression and has been shown to occur with increased frequency at specific genomic loci. However, the precise mechanisms underlying the establishment and maintenance of aberrant methylation marks are still elusive. The de novo DNA methyltransferase 3B (DNMT3B) has been suggested to play an important role in the generation of cancer-specific methylation patterns. Previous studies have shown that a reduction of DNMT3B protein levels induces antiproliferative effects in cancer cells that were attributed to the demethylation and reactivation of tumor suppressor genes. However, methylation changes have not been analyzed in detail yet. Using RNA interference we reduced DNMT3B protein levels in colon cancer cell lines. Our results confirm that depletion of DNMT3B specifically reduced the proliferation rate of DNMT3B-overexpressing colon cancer cell lines. However, genome-scale DNA methylation profiling failed to reveal methylation changes at putative DNMT3B target genes, even in the complete absence of DNMT3B. These results show that DNMT3B is dispensable for the maintenance of aberrant DNA methylation patterns in human colon cancer cells and they have important implications for the development of targeted DNA methyltransferase inhibitors as epigenetic cancer drugs

MicroRNA-mediated drug resistance in breast cancer

Chemoresistance is one of the major hurdles to overcome for the successful treatment of breast cancer. At present, there are several mechanisms proposed to explain drug resistance to chemotherapeutic agents, including decreased intracellular drug concentrations, mediated by drug transporters and metabolic enzymes; impaired cellular responses that affect cell cycle arrest, apoptosis, and DNA repair; the induction of signaling pathways that promote the progression of cancer cell populations; perturbations in DNA methylation and histone modifications; and alterations in the availability of drug targets. Both genetic and epigenetic theories have been put forward to explain the mechanisms of drug resistance. Recently, a small non-coding class of RNAs, known as microRNAs, has been identified as master regulators of key genes implicated in mechanisms of chemoresistance. This article reviews the role of microRNAs in regulating chemoresistance and highlights potential therapeutic targets for reversing miRNA-mediated drug resistance. In the future, microRNA-based treatments, in combination with traditional chemotherapy, may be a new strategy for the clinical management of drug-resistant breast cancers

DNA methylation profile of multiple genes involved in bladder cancer among Saudi population – A pilot study

Background: To identify bladder cancer specific methylated DNA sequences for the Saudi population in order to detect and predict bladder cancer progression.Methods: In this study, we analysed DNA methylation levels of 48 tumour suppressor genes loci in 24 bladder tissues (19 bladder cancer samples and 5 control samples taken from histologically normal bladders).  DNA Methylation analysis was done using Human Tumour Suppressor Genes EpiTect Methyl II Complete PCR Array from Qiagen TM.Results: We identified significant difference in DNA hypermethylation levels at APC, BRCA1, CDH1, CDH13, CDKN2A, DAPK1, ESR1, FHIT, MGMT, RASSF1, SOCS1, TIMP3, TP73, VHL, WIF1 between controls and cancerous samples. It was also observed that CADM1 and DKK3 were differentially methylated in non-muscle invasive versus muscle invasive bladder cancer samples. Additionally, DNA hypermethylation of ESR1 was notified as the novel tumour suppressor gene specific for the Saudi population in bladder cancer.Conclusion: Our findings suggest that these aberrant DNA methylation patterns in bladder cancer are disease and population specific and have a potential to develop as distinct DNA methylation-based biomarkers in future.Keywords: Bladder Cancer; DNA methylation; Epigenetics; Hypermethylation; Tumor Markers  

Utility of DNA methylation markers for diagnosing cancer.

DNA methylation occurs at the CpG residues and serves as a powerful epigenetic mechanism that negatively regulates gene expression. This process is catalyzed by DNA methyltransferases and occurs within CpG islands found in the promoter regions of \u3e70% of human genes. Given the important role of DNA methylation in regulating gene expression, un-programmed changes in methylation patterns are expected to either silence or activate transcription of tumor suppressor genes (via hypermethylation) or oncogenes (via demethylation), respectively, and by doing so promote a disease state. In light of the fact that a number of different cancers are frequently associated with hypermethylated tumor suppressor genes together with the observation that tumor derived genomic DNAs are present in various body fluids including serum/plasma, urine, sputum and bronchial lavage, methylated DNA has shown tremendous promise to serve as a robust biomarker for detecting cancer. Over the last several years protocols for capturing small amounts of DNA in circulation have been developed. Once captured, DNA methylation may be readily monitored by restriction enzyme digestion or bisulfite conversion followed by amplification of the desired genomic region with the polymerase chain reaction (PCR). New technologies which employ methyl-binding protein or antibodies that bind specifically to methylated-CpG residues have now enabled investigators to interrogate the status of entire DNA methyome of diseased tissue in an efficient and cost-effective manner. In this review, we describe the various tumor suppressor genes that are frequently hypermethylated in different cancers and how these and other methylated loci may be employed as clinically useful biomarkers for diagnosing cancer noninvasively using readily available body fluids