O6 -methylguanine methyltransferase promoter methylation status of glioblastoma cell line clonal population.

IF 1.3 4区 医学 Q4 CLINICAL NEUROLOGY
Neuropathology Pub Date : 2024-02-01 Epub Date: 2023-06-29 DOI:10.1111/neup.12931
Mitsuhiro Anan, Rolando Fausto Del Maestro, Nobuhiro Hata, Minoru Fujiki
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Abstract

Glioblastoma (GBM) remains a treatment-resistant malignant brain tumor in large part because of its genetic heterogeneity and epigenetic plasticity. In this study, we investigated the epigenetic heterogeneity of GBM by evaluating the methylation status of the O6 -methylguanine methyltransferase (MGMT) promoter in individual clones of a single cell derived from GBM cell lines. The U251 and U373 GBM cell lines, from the Brain Tumour Research Centre of the Montreal Neurological Institute, were used for the experiments. To evaluate the methylation status of the MGMT promoter, pyrosequencing and methylation-specific PCR (MSP) were used. Moreover, mRNA and protein expression levels of MGMT in the individual GBM clones were evaluated. The HeLa cell line, which hyper-expresses MGMT, was used as control. A total of 12 U251 and 12 U373 clones were isolated. The methylation status of 83 of 97 CpG sites in the MGMT promoter were evaluated by pyrosequencing, and 11 methylated CpG sites and 13 unmethylated CpG sites were evaluated by MSP. The methylation status by pyrosequencing was relatively high at CpG sites 3-8, 20-35, and 7-83, in both the U251 and U373 clones. Neither MGMT mRNA nor protein was detected in any clone. These findings demonstrate tumor heterogeneity among individual clones derived from a single GBM cell. MGMT expression may be regulated, not only by methylation of the MGMT promoter but by other factors as well. Further studies are needed to clarify the mechanisms underlying the epigenetic heterogeneity and plasticity of GBM.

胶质母细胞瘤细胞系克隆群体的 O6 -甲基鸟嘌呤甲基转移酶启动子甲基化状况。
胶质母细胞瘤(GBM)仍然是一种耐药的恶性脑肿瘤,这在很大程度上是因为它具有遗传异质性和表观遗传可塑性。在这项研究中,我们通过评估源自 GBM 细胞系的单个细胞克隆中 O6 -甲基鸟嘌呤甲基转移酶(MGMT)启动子的甲基化状态,研究了 GBM 的表观遗传异质性。实验使用了蒙特利尔神经研究所脑肿瘤研究中心的 U251 和 U373 GBM 细胞系。为了评估 MGMT 启动子的甲基化状态,使用了热测序和甲基化特异性 PCR (MSP)。此外,还评估了各个 GBM 克隆中 MGMT 的 mRNA 和蛋白表达水平。MGMT高表达的HeLa细胞系被用作对照。共分离出 12 个 U251 和 12 个 U373 克隆。通过热测序评估了 MGMT 启动子 97 个 CpG 位点中 83 个位点的甲基化状态,通过 MSP 评估了 11 个甲基化 CpG 位点和 13 个未甲基化 CpG 位点。在 U251 和 U373 克隆中,热释光测序法对 CpG 位点 3-8、20-35 和 7-83 的甲基化状态进行了评估。在任何克隆中都没有检测到 MGMT mRNA 或蛋白质。这些发现表明,来自单个 GBM 细胞的克隆之间存在肿瘤异质性。MGMT 的表达可能不仅受 MGMT 启动子甲基化的调控,还受其他因素的调控。要弄清 GBM 表观遗传异质性和可塑性的内在机制,还需要进一步的研究。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
Neuropathology
Neuropathology 医学-病理学
CiteScore
4.10
自引率
4.30%
发文量
105
审稿时长
6-12 weeks
期刊介绍: Neuropathology is an international journal sponsored by the Japanese Society of Neuropathology and publishes peer-reviewed original papers dealing with all aspects of human and experimental neuropathology and related fields of research. The Journal aims to promote the international exchange of results and encourages authors from all countries to submit papers in the following categories: Original Articles, Case Reports, Short Communications, Occasional Reviews, Editorials and Letters to the Editor. All articles are peer-reviewed by at least two researchers expert in the field of the submitted paper.
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