The importance of epigenetic aberrations in the pathogenesis of leukemias has been revealed by recurrent gene mutations that highlight epigenetic pathways as well as by the clinical success of ...therapies like 5-azacytidine and decitabine that work through epigenetic mechanisms. However, precise mechanisms of how gene mutations lead to leukemias and how epigenetic therapies induce clinical remissions are elusive. Current scientific inquiries that take advantage of techniques that can distinguish among the various covalent cytosine modifications at single base resolution are likely to shed light on the ways epigenetic pathways drive leukemogenesis as well as how the hypomethylating drugs induce clinical remissions. The hope is that these studies will also reveal which patients are likely to respond to epigenetic therapies. Thus, the future is likely to bring a new wave of diagnostic and prognostic tools that probe the epigenomics of leukemia to help clinicians in their management of patients.
Taking advantage of their ability to integrate their genomes into the host genome, lentiviruses have been used to rapidly produce transgenic mice in biomedical research. In most cases, transgenes ...delivered by lentiviral vectors have resisted silencing mediated by epigenetic modifications in mice. However, some studies revealed that methylation caused decreased transgene expression in mice. Therefore, there is conflicting evidence regarding the methylation-induced silencing of transgenes delivered by lentiviral transduction in mice. In this study, we present evidence that the human
TTR
transgene was silenced by DNA methylation in the liver of a transgenic mouse model generated by lentiviral transduction. The density of methylation on the transgene was increased during reproduction, and the expression of the transgene was completely silenced in mice of the F2 generation. Interestingly, 5-azacytidine (5-AzaC), a methyltransferase inhibitor, potently reactivated the silenced genes in neonatal mice whose hepatocytes were actively proliferating and led to stable transgene expression during development. However, 5-AzaC did not rescue liver transgene expression when administered to adult mice. Moreover, 5-AzaC at the given dose had low developmental toxicity in the newborn mice. In summary, we demonstrate the methylation-induced silencing of an exogenous gene in the liver of a mouse model generated by lentiviral transduction and show that the silenced transgene can be safely and efficiently reactivated by 5-AzaC treatment, providing an alternative way to obtain progeny with stable transgene expression in the case of the methylation of exogenous genes in transgenic mice generated by lentiviral transduction.
Cisplatin resistance is one of the major reasons leading to the high death rate of ovarian cancer. Methyl-Capture sequencing (MethylCap-seq), which combines precipitation of methylated DNA by ...recombinant methyl-CpG binding domain of MBD2 protein with NGS, global and unbiased analysis of global DNA methylation patterns. We applied MethylCap-seq to analyze genome-wide DNA methylation profile of cisplatin sensitive ovarian cancer cell line A2780 and its isogenic derivative resistant line A2780CP. We obtained 21,763,035 raw reads for the drug resistant cell line A2780CP and 18,821,061reads for the sensitive cell line A2780. We identified 1224 hyper-methylated and 1216 hypomethylated DMRs (differentially methylated region) in A2780CP compared to A2780. Our MethylCap-seq data on this ovarian cancer cisplatin resistant model provided a good resource for the research community. We also found that A2780CP, compared to A2780, has lower observed to expected methylated CpG ratios, suggesting a lower global CpG methylation in A2780CP cells. Methylation specific PCR and bisulfite sequencing confirmed hypermethylation of PTK6, PRKCE and BCL2L1 in A2780 compared with A2780CP. Furthermore, treatment with the demethylation reagent 5-aza-dC in A2780 cells demethylated the promoters and restored the expression of PTK6, PRKCE and BCL2L1.
Several studies have demonstrated the possibility to revert differentiation process, reactivating hypermethylated genes and facilitating cell transition to a different lineage. Beside the epigenetic ...mechanisms driving cell conversion processes, growing evidences highlight the importance of mechanical forces in supporting cell plasticity and boosting differentiation. Here, we describe epigenetic erasing and conversion of dermal fibroblasts into insulin-producing cells (EpiCC), and demonstrate that the use of a low-stiffness substrate positively influences these processes. Our results show a higher expression of pluripotency genes and a significant bigger decrease of DNA methylation levels in 5-azacytidine (5-aza-CR) treated cells plated on soft matrix, compared to those cultured on plastic dishes. Furthermore, the use of low-stiffness also induces a significant increased up-regulation of ten-eleven translocation 2 (
Tet2)
and histone acetyltransferase 1 (
Hat1)
genes, and more decreased histone deacetylase enzyme1 (
Hdac1)
transcription levels. The soft substrate also encourages morphological changes, actin cytoskeleton re-organization, and the activation of the Hippo signaling pathway, leading to yes-associated protein (YAP) phosphorylation and its cytoplasmic translocation. Altogether, this results in increased epigenetic conversion efficiency and in EpiCC acquisition of a mono-hormonal phenotype. Our findings indicate that mechano-transduction related responsed influence cell plasticity induced by 5-aza-CR and improve fibroblast differentiation toward the pancreatic lineage.
Porcine circovirus type 2 (PCV2) is the main pathogen causing post-weaning multisystemic wasting syndrome (PMWS), which mainly targets the body's immune system and poses a serious threat to the ...global pig industry. 5-Azacytidine is a potent inhibitor of DNA methylation, which can participate in many important physiological and pathological processes, including virus-related processes, by inhibiting gene expression. However, the impact of 5-Aza on PCV2 replication in cells is not yet clear. We explored the impact of 5-Aza on PCV2 infection utilizing PK15 cells as a cellular model. Our objective was to gain insights that could potentially offer novel therapeutic strategies for PCV2. Our results showed that 5-Aza significantly enhanced the infectivity of PCV2 in PK15 cells. Transcriptome analysis revealed that PCV2 infection activated various immune-related signaling pathways. 5-Aza may activate the MAPK signaling pathway to exacerbate PCV2 infection and upregulate the expression of inflammatory and apoptotic factors.
Using direct conversion technology, normal adult somatic cells can be routinely switched from their original cell type into specific differentiated cell types by inducing the expression of ...differentiation‐related transcription factors. In this study, normal human dermal fibroblasts (NHDFs) are directly converted into cardiomyocyte‐like cells by drug and gene delivery using carboxymethylcellulose (CMC) nanoparticles (CiCMC‐NPs). CMC‐based multifunctional nanogels containing specific cardiomyocyte‐related genes are designed and fabricated, including GATA4, MEF2C, and TBX5 (GMT). However, GMT alone is insufficient, at least in vitro, in human fibroblasts. Hence, to inhibit proliferation and to induce differentiation, 5‐azacytidine (5‐AZA) is conjugated to the hydroxyl group of CMC in CiCMC‐NPs containing GMT; in addition, the CMC is coated with polyethylenimine. It is confirmed that the CiCMC‐NPs have nanogel properties, and that they exhibit the characteristic effects of 5‐AZA and GMT. When CiCMC‐NPs‐containing 5‐AZA and GMT are introduced into NHDFs, cardiomyocyte differentiation is initiated. In the reprogrammed cells, the mature cardiac‐specific markers cardiac troponin I and α‐actinin are expressed at twofold to threefold higher levels than in NHDFs. Engineered cells transplanted into live hearts exhibit active pumping ability within 1 day. Histology and immunohistology of heart tissue confirm the presence of transplanted engineered NHDF cells at injection sites.
Cardiomyocyte‐inducing carboxymethylcellulose nanoparticles are biocompatible nanogel‐type nanoparticles used as a dual delivery system in normal human dermal fibroblasts.
This study aimed to screen osteosarcoma (OS) prognosis relevant genes for methylation dysregulation, and the functional mechanisms of FES overexpression in OS cells were investigated.
The OS ...prognosis relevant genes with differentially methylated positions (DMPs) identified from the GSE36001 and GSE36002 datasets, and the UCSC database, were used as a training set to construct a risk model, while the GSE21257 dataset was used as validation set. The expression levels of several key genes in OS cells after 5-Aza-2'-deoxycytidine treatment were detected by qPCR. The effects of FES overexpression on cell proliferation, cell cycle, migration, and invasion of MNNG/HOS were analyzed by CCK8, flow cytometry, and Transwell assays.
A total of 31 candidate genes, corresponding to 36 DMPs, were identified as OS prognosis relevant genes; from these, the top 10 genes were used to construct a risk model. Following validation of the risk model, FES, LYL1, MAP4K1, RIPK3, SLC15A3, and STAT3 showed expression changes between the OS and control samples. qPCR results showed that the expression of FES was significantly downregulated in three OS cell lines and increased after 5-Aza-DC treatment. The proliferation, cell cycle progression, migration, and invasion of MNNG/HOS cells were significantly inhibited after transfection with FES overexpression plasmid, and the protein expression of FYN and β catenin were decreased in MNNG/HOS cells by FES overexpression.
The decrease in FES by hypermethylation was associated with OS prognosis, and might contribute to the proliferation, migration, and invasion of OS cells. FES, and its upstream FYN and β catenin, might coordinately exert a tumor suppressor effect in OS cells.
In this study, importance of Wnt-β-catenin pathway in the development of uterine cervical carcinoma was evaluated. For this purpose, the profiles (expression/methylation/deletion) of β-catenin, ...p-β-catenin (Y654), Wnt3a, and APC were studied in disease free normal cervical epithelium (
n
= 9), adjacent normal cervical epithelium of primary tumors (
n
= 70), CIN (
n
= 28), CACX (
n
= 102) samples, and two CACX cell lines (HeLa and SiHa). Immunohistochemical analysis revealed high/medium (74–95%) expression of β-catenin/p-β-catenin (Y654) and Wnt3a and low expression (23–26%) of APC in proliferating basal–parabasal layers contrary to differentiated spinous layer in normal cervix irrespective of HPV16 infection. The expression profile of the genes in the basal–parabasal layers did not change significantly during development of CACX. High (66%) promoter methylation of APC was seen in basal–parabasal layers and the cervical lesions (42–69%), unlike in spinous layers (25%). The promoter methylation status of APC was validated by in vitro demethylation experiments using 5-aza-dC in CACX cell lines. However, additional deletion of APC was significantly increased from CIN (12%) to stage I/II (40%) and became comparable in stage III/IV (48%) of the tumor. Patients with alterations (deletion/methylation) of APC and high/medium expression of Wnt3a/β-catenin/p-β-catenin (Y654) showed significantly poor survival. Thus our data indicate that cumulative effect of Wnt3a overexpression and APC inactivation are needed for overexpression of β-catenin during the development of CACX.
Epstein-Barr virus (EBV), a member of the
family, maintains a lifelong latent infection in human B cells. Switching from the latent to the lytic phase of its lifecycle allows the virus to replicate ...and spread. The viral lytic cycle is induced in infected cultured cells by drugs such as sodium butyrate and azacytidine. Lytic reactivation can be inhibited by natural products and pharmaceuticals. The anticonvulsant drugs valproic acid and valpromide inhibit EBV in Burkitt lymphoma cells. Therefore, other drugs that treat neurological and psychological disorders were investigated for effects on EBV lytic reactivation. Clozapine, an atypical antipsychotic drug used to treat schizophrenia and bipolar disorder, was found to inhibit the reactivation of the EBV lytic cycle. Levels of the viral lytic genes BZLF1, BRLF1, and BMLF1 were decreased by treatment with clozapine in induced Burkitt lymphoma cells. The effects on viral gene expression were dependent on the dose of clozapine, yet cells were viable at an inhibitory concentration of clozapine. One metabolite of clozapine-desmethylclozapine-also inhibited EBV lytic reactivation, while another metabolite-clozapine-N-oxide-had no effect. These drugs may be used to study cellular pathways that control the viral lytic switch in order to develop treatments for diseases caused by EBV.
In recent years, recurrent somatic mutations in epigenetic regulators have been identified in patients with hematological malignancies. Furthermore, chromosomal translocations in which the fusion ...protein partners are themselves epigenetic regulators or where epigenetic regulators are recruited/targeted by oncogenic fusion proteins have also been described. Evidence has accumulated showing that "epigenetic drugs" are likely to provide clinical benefits in several hematological malignancies, granting their approval for the treatment of myelodysplastic syndromes and cutaneous T-cell lymphomas. A large number of pre-clinical and clinical trials evaluating epigenetic drugs alone or in combination therapies are ongoing. The aim of this review is to provide a comprehensive summary of known epigenetic alterations and of the current use of epigenetic drugs for the treatment of hematological malignancies.
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