Influence of DNA DSB repair on cell sensitivity to chloroethylating cytostatics

本次更新申请的主要目的是继续研究DNA双链断裂（DSB）修复的作用，特别是同源重组在肿瘤（胶质母细胞瘤）细胞对氯乙基化抗癌药物（CENU）的抗性中的作用。在增殖细胞中，CENU诱导的DNA链间交联（ICL）修复是一个复杂的、多步骤的过程，是肿瘤细胞对这类化疗药物耐药的关键节点。参与DNA损伤信号和ICL修复的蛋白质的作用及其激活的时间顺序将被阐明。根据我们的模型，在ICL停止的复制分叉的修复过程中，为了响应CENU，形成了依赖复制的临界DSB。产生的DSB是高度细胞毒性病变，其修复是通过同源重组（HR）完成的。在提出的更新框架内，我们希望继续研究cenu诱导的信号传导，DSB修复和细胞死亡途径。我们计划将我们的研究扩展到下调或抑制HR蛋白的方法，并分析CENU用于治疗的胶质母细胞瘤细胞对细胞敏感性的影响。此外，我们将研究CENU慢性治疗对体外和小鼠异种移植胶质母细胞瘤细胞系中HR蛋白表达的影响。这些数据将有助于阐明重组修复在化疗期间对CENU的细胞耐药性发展中的作用。

The main objective of this renewal application is to continue research on the role of DNA double-strand break (DSB) repair and, specifically, the role of homologous recombination in the resistance of tumour (glioblastoma) cells to chloroethylating anticancer drugs (CENU). The repair of DNA inter strand cross links (ICL) induced by CENU in proliferating cells is a complex, multistep process, which is a key node in the tumour cell resistance to this group of chemotherapeutics. The role of proteins involved in DNA damage signaling and ICL repair and the chronological order of their activation will be elucidated. According to our model, in response to CENU critical DSB are formed replication dependently during the repair process at replication forks stalled by ICL. The generated DSB are highly cytotoxic lesions whose repair is accomplished by homologous recombination (HR). Within the frame of the proposed renewal, we wish to continue our research on CENU-induced signaling, DSB repair and cell death pathways. We plan to extend our investigations with methods for down-regulation or inhibition of HR proteins and analyze the effect on the cellular sensitivity in glioblastoma cells, for which CENU are applied in therapy. In addition, we will study the effect of chronic treatment with CENU on the expression of HR proteins in glioblastoma cell lines in vitro and in xenografts in mice. The data will help to clarify the involvement of recombinational repair in the development of cellular resistance to CENU during chemotherapy.