Mechanisms of O6-methylguanine induced senescence and transcriptional repression in glioblastoma cells

O6-甲基鸟嘌呤诱导胶质母细胞瘤细胞衰老和转录抑制的机制

• 批准号: 398037827
• 负责人: Professor Dr. Markus Christmann
• 金额: --
• 依托单位: Institut für Toxikologie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2018
• 资助国家: 德国
• 起止时间: 2017-12-31 至 2022-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/398037827?language=en
• 关键词: Mechanisms; O6; methylguanine; induced; senescence

An important group of genotoxic agents acts via methylation at the O6-position of guanine, giving rise to the formation of O6-methylguanine (O6MeG) in the DNA. Among these agents is the anticancer drug temozolomide (TMZ), which is used in the 1st line therapy of high-grade gliomas, notably glioblastomas. In our previous work we showed that TMZ induces in glioblastoma cells not only apoptosis and autophagy, but also senescence, which was triggered by O6MeG. In extended (unpublished) background studies we also show that senescence occurs in G2 phase cells, which is dependent on p21 activity. Since the success of glioblastoma therapy is related to the DNA repair capacity of the tumor, we addressed the question whether TMZ-induced senescence leads to alterations in DNA repair capacity. These studies showed that TMZ-induced senescence is strongly associated with transcriptional repression of the DNA repair proteins EXO1, MSH2, MSH6 und RAD51, with p21 playing a role also in this repression process. In this grant proposal we wish to analyze in two intertwined subprojects (TP1, Kaina; TP2, Christmann) the molecular mechanisms underlying TMZ-induced senescence, focusing on the role of persistent DNA damage and sustained DDR activation. Specifically we want to address the questions of a) which signaling mechanisms activated by O6MeG trigger senescence, b) what makes activation of DDR following primary DNA damage in a fraction of cells permanent, c) can sustained DDR be alleviated, d) are senescent cells prone to DNA damaging agents, e) can senescent cells regain propliferation capacity, f) is DNA repair capacity and drug sensitivity of recovered senescent cells comparable or different from parental cells that have not undergone senescence? Further we wish to study g) the molecular mechanism underlying senescence associated p21 mediated transcriptional repression of repair genes using ChiP-Seq and mass-spectrometry to find p21 interaction partners, h) whether transcriptional repression of DNA repair genes EXO1, MSH2, MSH6 und RAD51 is affected by epigenetic mechanisms, i.e. alterations in the chromatin structure or promoter hypermethylation. The experimental data will be assessed as to whether the DNA repair capacity in senescent cells can be harnessed in order to prevent their escape from the senescent state, eliminating them selectively by anticancer drug treatment.

一组重要的遗传毒性物质通过鸟嘌呤O 6位的甲基化作用，导致DNA中O 6-甲基鸟嘌呤（O 6 MeG）的形成。在这些药物中，有抗癌药物替莫唑胺（TMZ），用于高级别胶质瘤（尤其是胶质母细胞瘤）的一线治疗。在我们以前的工作中，我们发现TMZ不仅诱导胶质母细胞瘤细胞凋亡和自噬，而且诱导衰老，这是由O 6 MeG触发的。在扩展的（未发表的）背景研究中，我们还表明，衰老发生在G2期细胞，这是依赖于p21活性。由于胶质母细胞瘤治疗的成功与肿瘤的DNA修复能力有关，我们提出了TMZ诱导的衰老是否会导致DNA修复能力改变的问题。这些研究表明TMZ诱导的衰老与DNA修复蛋白EXO 1、MSH 2、MSH 6和RAD 51的转录抑制密切相关，p21也在该抑制过程中发挥作用。在这项资助提案中，我们希望在两个相互交织的子项目（TP 1，Kaina; TP 2，Christmann）中分析TMZ诱导衰老的分子机制，重点关注持续DNA损伤和持续DDR激活的作用。具体地说，我们想解决以下问题：a）由O 6 MeG激活的哪些信号传导机制触发衰老，B）是什么使得在一部分细胞中原发性DNA损伤后DDR的激活永久，c）持续的DDR能否被缓解，d）衰老细胞是否易于受到DNA损伤剂的影响，e）衰老细胞能否恢复增殖能力，f）恢复的衰老细胞的DNA修复能力和药物敏感性与未经历衰老的亲本细胞相当还是不同？此外，我们希望研究g）衰老相关的p21介导的修复基因的转录抑制的分子机制，使用ChiP-Seq和质谱法来寻找p21相互作用伴侣，h）DNA修复基因EXO 1、MSH 2、MSH 6和RAD 51的转录抑制是否受到表观遗传机制的影响，即染色质结构或启动子超甲基化的改变。实验数据将评估衰老细胞中的DNA修复能力是否可以被利用，以防止它们逃离衰老状态，通过抗癌药物治疗选择性地消除它们。