Inactivation of the WNT5A Alternative Promoter B Is Associated with DNA Methylation and Histone Modification in Osteosarcoma Cell Lines U2OS and SaOS-2

Abstract Osteosarcoma is the most common primary malignant tumour of bone occurring in children and young adolescents, and is characterised by complex genetic and epigenetic changes. The miRNA miR-486-5p has been shown to be downregulated in osteosarcoma and in cancer in general. To investigate if the miR-486 locus is epigenetically regulated, we integrated DNA methylation and miR-486-5p expression data using cohorts of osteosarcoma cell lines and patient samples. An upstream CpG island of mir-486 was shown to be highly methylated in osteosarcoma cell lines as determined by methylation-specific PCR and direct bisulfite sequencing. High methylation levels were seen for osteosarcoma patient samples, cell xenografts and cell lines based on quantitative methylation-specific PCR. 5-Aza-2’-deoxycytidine treatment of osteosarcoma cell lines caused induction of miR-486-5p in osteosarcoma cell lines. When overexpressed, miR-486-5p affected cell morphology. miR-486-5p represents a highly cancer relevant, epigenetically regulated miRNA in osteosarcoma.

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Histone Modifications Associated with DNA Methylation and Transcriptional Repression of p15INK4b in Acute Myeloid Leukemia.

Blood ◽

10.1182/blood.v112.11.3354.3354 ◽

2008 ◽

Vol 112 (11) ◽

pp. 3354-3354

Author(s):

Thomas A. Paul ◽

Horatiu Muresan ◽

Emily Prentice ◽

Linda Wolff

Keyword(s):

Dna Methylation ◽

Histone Modification ◽

Cell Lines ◽

Histone Modifications ◽

Transcriptional Repression ◽

Myeloid Leukemia ◽

Methylation Status ◽

Hdac Inhibitors ◽

Chromosome 9 ◽

Repressive Histone Modification

Abstract p15INK4b is a cyclin-dependent kinase inhibitor known to regulate the G1-to-S transition of the cell cycle and to be involved in negatively regulating myeloid progenitor cell production. DNA hypermethylation leading to transcriptional silencing of p15INK4b has been reported in up to 70% of acute myeloid leukemia (AML) patient samples. In our study we sought to determine if p15INK4b DNA methylation in AML is accompanied by repressive histone modifications that contribute to the transcriptional repression of the gene at the chromatin level. Chromatin immunoprecipitation and DNA tiling microarrays (ChIP-on-chip) with 20bp resolution were utilized to assess the distribution of histone modifications over a 1.2 megabase region of human chromosome 9 including p15INK4b and adjacent tumor suppressor genes p14ARF and p16INK4a. We found that AML cell lines with p15INK4b hypermethylation (Kasumi-1, KG-1, and KG-1a) had high levels of the repressive histone modification trimethylation of lysine 27 of histone H3 (H3K27me3). Remarkably, this modification spanned the entire INK4b-ARF-INK4a region while little binding was observed in adjacent regions of chromosome 9. Binding of EZH2, the polycomb associated H3K27 histone methyltransferase, co-localized with H3K27me3 distribution over the INK locus. H3K27me3 was not identified at this region in AML cell lines without p15INK4b DNA methylation (U937 and HL-60). In contrast, histone modifications associated gene activation, trimethylation of lysine 4 of H3 (H3K4me3) and acetylation of lysine 9 (H3K9Ac), were found at the p15INK4b promoter in these cells. Enrichment of another repressive histone modification, trimethylation of histone H3 on lysine 9 (H3K9me3), did not correlate with the DNA methylation status of p15INK4b and appeared highest in exons 2 and 3 of p16INK4a in most cell lines. Since p15INK4b reactivation has been described as a component of a patient’s response to epigenetic therapies in AML treatment, we sought to determine the dynamics of histone modifications following treatment with the DNA methyltransferase (DNMT) inhibitor 5-aza-2’-deoxycytidine and histone deacetylase (HDAC) inhibitor tricostatin A. In KG-1 cells, a reduction in p15INK4b DNA methylation was observed following treatment with DNMT inhibitors. Unexpectedly, treatment with HDAC inhibitors alone was also capable of reducing p15INK4b DNA methylation suggesting that a repressive chromatin structure contributes to the DNA methylation in this cell line. Loss of DNA methylation was not sufficient for reactivation of p15INK4b expression as detectible expression was only observed following the combined treatment of DNMT and HDAC inhibitors. Reactivation was associated with an increase in the activation-associated histone modifications H3K4me3 and H3K9Ac at the promoter region and, unexpectedly, maintenance of the repressive modification H3K27me3. This “bivalent” histone modification pattern is characteristic of many developmentally poised genes in embryonic stem cells and correlates with the histone methylation status of p15INK4b we observed in CD34+ bone marrow progenitor/ stem cells. This data indicates that optimal epigenetic therapies targeted to reactivate p15INK4b expression should be designed to induce activating histone modifications in addition to reducing DNA methylation.

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DNA Methylation Inhibites ANXA1 Gene Expression in Nasopharyngeal Carcinoma Cell Lines*

PROGRESS IN BIOCHEMISTRY AND BIOPHYSICS ◽

10.3724/sp.j.1206.2009.00170 ◽

2009 ◽

Vol 36 (10) ◽

pp. 1319-1326 ◽

Cited By ~ 3

Author(s):

Shuang-Xiang TAN ◽

Rui-Cheng HU ◽

Ai-Guo DAI ◽

Cen-E TANG ◽

Hong YI ◽

...

Keyword(s):

Gene Expression ◽

Dna Methylation ◽

Nasopharyngeal Carcinoma ◽

Cell Lines ◽

Carcinoma Cell ◽

Nasopharyngeal Carcinoma Cell ◽

Carcinoma Cell Lines

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Epigenetic modifications in acute lymphoblastic leukemia: From cellular mechanisms to therapeutics

Current Gene Therapy ◽

10.2174/1566523220999201111194554 ◽

2020 ◽

Vol 20 ◽

Author(s):

Ezzatollah Fathi ◽

Raheleh Farahzadi ◽

Soheila Montazersaheb ◽

Yasin Bagheri

Keyword(s):

Dna Methylation ◽

Acute Lymphoblastic Leukemia ◽

Histone Modification ◽

Epigenetic Modification ◽

Lymphoblastic Leukemia ◽

Underlying Mechanism ◽

Methylation Pattern ◽

Epigenetic Alterations ◽

Cellular Mechanisms ◽

Novel Treatments

Background:: Epigenetic modification pattern is considered as a characteristic feature in blood malignancies. Modifications in the DNA methylation modulators are recurrent in lymphoma and leukemia, so that, the distinct methylation pattern defines different types of leukemia. Generally, the role of epigenetics is less understood and most investigations are focused on genetic abnormalities and cytogenic studies to develop novel treatments for patients with hematologic disorders. Recently, understanding the underlying mechanism of acute lymphoblastic leukemia (ALL), especially epigenetic altera-tions as a driving force in the development of ALL opens a new era of investigation for developing promising strategy, be-yond available conventional therapy. Objective:: This review will focus on a better understanding of the epigenetic mechanisms in cancer development and pro-gression, with an emphasis on epigenetic alterations in ALL including, DNA methylation, histone modification, and mi-croRNA alterations. Other topics that will be discussed include the use of epigenetic alterations as a promising therapeutic target in order to develop novel well-suited approaches against ALL. Conclusion:: According to the literature review, leukemogenesis of ALL is extensively influenced by epigenetic modifica-tions, particularly DNA hyper-methylation, histone modification, and miRNA alteration.

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Natural Antisense Transcript PEBP1P3 Regulates the RNA Expression, DNA Methylation and Histone Modification of CD45 Gene

Genes ◽

10.3390/genes12050759 ◽

2021 ◽

Vol 12 (5) ◽

pp. 759

Author(s):

Zhongjing Su ◽

Guangyu Liu ◽

Bin Zhang ◽

Ze Lin ◽

Dongyang Huang

Keyword(s):

Dna Methylation ◽

Alternative Splicing ◽

Histone Modification ◽

Antisense Rna ◽

Noncoding Rna ◽

Antisense Transcript ◽

Natural Antisense Transcript ◽

Regulation Of Expression ◽

Splicing Isoforms ◽

Intron 2

The leukocyte common antigen CD45 is a transmembrane phosphatase expressed on all nucleated hemopoietic cells, and the expression levels of its splicing isoforms are closely related to the development and function of lymphocytes. PEBP1P3 is a natural antisense transcript from the opposite strand of CD45 intron 2 and is predicted to be a noncoding RNA. The genotype-tissue expression and quantitative PCR data suggested that PEBP1P3 might be involved in the regulation of expression of CD45 splicing isoforms. To explore the regulatory mechanism of PEBP1P3 in CD45 expression, DNA methylation and histone modification were detected by bisulfate sequencing PCR and chromatin immunoprecipitation assays, respectively. The results showed that after the antisense RNA PEBP1P3 was knocked down by RNA interference, the DNA methylation of CD45 intron 2 was decreased and histone H3K9 and H3K36 trimethylation at the alternative splicing exons of CD45 DNA was increased. Knockdown of PEBP1P3 also increased the binding levels of chromatin conformation organizer CTCF at intron 2 and the alternative splicing exons of CD45. The present results indicate that the natural antisense RNA PEBP1P3 regulated the alternative splicing of CD45 RNA, and that might be correlated with the regulation of histone modification and DNA methylation.

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Anti-Metastatic and Anti-Angiogenic Effects of Curcumin Analog DK1 on Human Osteosarcoma Cells In Vitro

Pharmaceuticals ◽

10.3390/ph14060532 ◽

2021 ◽

Vol 14 (6) ◽

pp. 532

Author(s):

Muhammad Nazirul Mubin Aziz ◽

Nurul Fattin Che Rahim ◽

Yazmin Hussin ◽

Swee Keong Yeap ◽

Mas Jaffri Masarudin ◽

...

Keyword(s):

Cell Lines ◽

Safety Profile ◽

Quantitative Polymerase Chain Reaction ◽

Tube Formation ◽

Osteosarcoma Cell ◽

Curcumin Analog ◽

Human Osteosarcoma ◽

Human Osteosarcoma Cells ◽

Human Osteosarcoma Cell

Osteosarcoma (OS) is a life-threatening malignant bone tumor associated with poor prognosis among children. The survival rate of the patient is still arguably low even with intensive treatment provided, plus with the inherent side effects from the chemotherapy, which gives more unfavorable outcomes. Hence, the search for potent anti-osteosarcoma agent with promising safety profile is still on going. Natural occurring substance like curcumin has gained a lot of attention due to its splendid safety profile as well as it pharmacological advantages such as anti-metastasis and anti-angiogenesis. However, natural curcumin was widely known for its poor cellular uptake, which undermines all potential that it possesses. This prompted the development of synthetically synthesized curcuminoid analog, known as (Z)-3-hydroxy-1-(2-hydroxyphenyl)-3-phenylprop-2- en-1-one (DK1). In this present study, in vitro scratch assay, transwell migration/invasion assay, HUVEC tube formation assay, and ex vivo rat aortic ring assays were performed in order to investigate the anti-metastatic and anti-angiogenic potential of DK1. For further comprehension of DK1 mechanism on human osteosarcoma cell lines, microarray gene expression analysis, quantitative polymerase chain reaction (qPCR), and proteome profiler were adopted, providing valuable forecast from the expression of important genes and proteins related to metastasis and angiogenesis. Based on the data gathered from the bioassays, DK1 was able to inhibit the metastasis and angiogenesis of human osteosarcoma cell lines by significantly reducing the cell motility, number of migrated and invaded cells as well as the tube formation and micro-vessels sprouting. Additionally, DK1 also has significantly regulated several cancer pathways involved in OS proliferation, metastasis, and angiogenesis such as PI3K/Akt and NF-κB in both U-2 OS and MG-63. Regulation of PI3K/Akt caused up-regulation of genes related to metastasis inhibition, namely, PTEN, FOXO, PLK3, and GADD45A. Meanwhile, NF-κB pathway was regulated by mitigating the expression of NF-κB activator such as IKBKB and IKBKE in MG-63, whilst up-regulating the expression of NF-κB inhibitors such as NFKBIA and NFKBIE in U-2 OS. Finally, DK1 also has successfully hindered the metastatic and angiogenic capability of OS cell lines by down-regulating the expression of pro-metastatic genes and proteins like MMP3, COL11A1, FGF1, Endoglin, uPA, and IGFBP2 in U-2 OS. Whilst for MG-63, the significantly down-regulated oncogenes were Serpin E1, AKT2, VEGF, uPA, PD-ECGF, and Endoglin. These results suggest that curcumin analog DK1 may serve as a potential new anti-osteosarcoma agent due to its anti-metastatic and anti-angiogenic attributes.

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Association of expression of epigenetic molecular factors with DNA methylation and sensitivity to chemotherapeutic agents in cancer cell lines

Clinical Epigenetics ◽

10.1186/s13148-021-01026-4 ◽

2021 ◽

Vol 13 (1) ◽

Author(s):

Suleyman Vural ◽

Alida Palmisano ◽

William C. Reinhold ◽

Yves Pommier ◽

Beverly A. Teicher ◽

...

Keyword(s):

Dna Methylation ◽

Cell Line ◽

Cell Lines ◽

Cancer Cell ◽

Antitumor Agents ◽

Cancer Cell Lines ◽

Chemotherapeutic Agents ◽

Epigenetic Factors ◽

Expression Of Genes ◽

Genes Encoding

Abstract Background Altered DNA methylation patterns play important roles in cancer development and progression. We examined whether expression levels of genes directly or indirectly involved in DNA methylation and demethylation may be associated with response of cancer cell lines to chemotherapy treatment with a variety of antitumor agents. Results We analyzed 72 genes encoding epigenetic factors directly or indirectly involved in DNA methylation and demethylation processes. We examined association of their pretreatment expression levels with methylation beta-values of individual DNA methylation probes, DNA methylation averaged within gene regions, and average epigenome-wide methylation levels. We analyzed data from 645 cancer cell lines and 23 cancer types from the Cancer Cell Line Encyclopedia and Genomics of Drug Sensitivity in Cancer datasets. We observed numerous correlations between expression of genes encoding epigenetic factors and response to chemotherapeutic agents. Expression of genes encoding a variety of epigenetic factors, including KDM2B, DNMT1, EHMT2, SETDB1, EZH2, APOBEC3G, and other genes, was correlated with response to multiple agents. DNA methylation of numerous target probes and gene regions was associated with expression of multiple genes encoding epigenetic factors, underscoring complex regulation of epigenome methylation by multiple intersecting molecular pathways. The genes whose expression was associated with methylation of multiple epigenome targets encode DNA methyltransferases, TET DNA methylcytosine dioxygenases, the methylated DNA-binding protein ZBTB38, KDM2B, SETDB1, and other molecular factors which are involved in diverse epigenetic processes affecting DNA methylation. While baseline DNA methylation of numerous epigenome targets was correlated with cell line response to antitumor agents, the complex relationships between the overlapping effects of each epigenetic factor on methylation of specific targets and the importance of such influences in tumor response to individual agents require further investigation. Conclusions Expression of multiple genes encoding epigenetic factors is associated with drug response and with DNA methylation of numerous epigenome targets that may affect response to therapeutic agents. Our findings suggest complex and interconnected pathways regulating DNA methylation in the epigenome, which may both directly and indirectly affect response to chemotherapy.

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Correction to: Afatinib is active in osteosarcoma cell lines

Journal of Cancer Research and Clinical Oncology ◽

10.1007/s00432-020-03357-w ◽

2020 ◽

Vol 146 (10) ◽

pp. 2719-2719

Author(s):

Marlid Cruz-Ramos ◽

Yessica Zamudio-Cuevas ◽

Daniel Medina-Luna ◽

Karina Martínez-Flores ◽

Gabriela Martínez-Nava ◽

...

Keyword(s):

Cell Lines ◽

Osteosarcoma Cell

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