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

一组重要的基因毒性物质通过甲基化作用于鸟嘌呤的o6位点，在DNA中形成o6 -甲基鸟嘌呤（O6MeG）。这些药物中有抗癌药物替莫唑胺（TMZ），用于一线治疗高级别胶质瘤，特别是胶质母细胞瘤。我们在前期研究中发现，TMZ不仅诱导胶质母细胞瘤细胞凋亡和自噬，而且诱导衰老，而衰老是由O6MeG引发的。在扩展的（未发表的）背景研究中，我们还表明衰老发生在G2期细胞中，这取决于p21的活性。由于胶质母细胞瘤治疗的成功与肿瘤的DNA修复能力有关，我们研究了tmz诱导的衰老是否会导致DNA修复能力的改变。这些研究表明，tmz诱导的衰老与DNA修复蛋白EXO1、MSH2、MSH6和RAD51的转录抑制密切相关，p21也参与了这一抑制过程。在这项拨款申请中，我们希望在两个相互关联的子项目（TP1, Kaina; TP2, Christmann）中分析tmz诱导衰老的分子机制，重点关注持续性DNA损伤和持续性DDR激活的作用。具体来说，我们想要解决的问题是：a) O6MeG激活的信号机制触发衰老，b)是什么使得部分细胞原发性DNA损伤后的DDR激活成为永久性的，c)能否缓解持续的DDR， d)衰老细胞是否容易受到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是否与未经历衰老的亲本细胞相当或不同？我们希望进一步研究g)衰老相关的p21介导的修复基因转录抑制的分子机制，使用ChiP-Seq和质谱技术寻找p21的相互作用伙伴，h) DNA修复基因EXO1、MSH2、MSH6和RAD51的转录抑制是否受到表观遗传机制的影响，即染色质结构的改变或启动子的超甲基化。实验数据将评估是否可以利用衰老细胞的DNA修复能力，以防止它们从衰老状态中逃脱，并通过抗癌药物治疗选择性地消除它们。

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 In extended (unpublished) background studies we also show that senescence occurs in G2 phase cells, which is dependent on p21 activity.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.