Regulation of DNA repair and drug sensitivity by cytokines in immunocompetent cells

细胞因子是包括免疫反应在内的生物过程的重要细胞间介质。我们研究了细胞因子是否对DNA修复有影响，使用新鲜分离的人单核细胞作为模型系统，这些单核细胞经GM-CFS/IL-4成熟为巨噬细胞和树突状细胞（dc）。我们观察到，人类单核细胞不表达可检测量的DNA修复蛋白XRCC1，连接酶III， PARP-1和DNA- pkcs。在细胞因子刺激单核细胞向巨噬细胞或dc分化的过程中，这些修复蛋白的表达上调。XRCC1、PARP-1和DNA-PKCS在基因/mRNA水平上发生调控。缺乏修复蛋白的表达会导致碱基切除和DNA双链断裂修复缺陷，以及单核细胞对单功能烷基化剂（包括替莫唑胺、电离辐射和氧化剂）的超敏反应。我们的初步数据显示，小鼠单核细胞也缺乏XRCC-1、连接酶III、PARP-1和DNA-PKCS，这表明这是一种普遍的生物学现象。-在这项拨款申请中，我们希望在人类和小鼠系统中扩展先前的研究。我们希望阐明在CD34+细胞成熟为单核细胞前体的过程中，XRCC1、PARP-1和DNA-PKCS何时以及如何下调，修复蛋白是否在已建立的单核细胞样人类细胞中也缺乏，它们是否在分化过程中上调，以及相应的修复基因如何被调节。在小鼠中，我们希望确定骨髓中发生XRCC1、连接酶III、PARP-1和DNA-PKCS下调的单核细胞前体。此外，我们想要评估小鼠单核细胞是否也对包括ROS在内的基因毒物过敏，其他基因是否在单核细胞受到细胞因子刺激后受到差异调节和上调，最后，单核细胞的过敏是否导致单核细胞的消耗和巨噬细胞的减少，在全身照射和烷基化剂（包括抗癌药物替莫唑胺）治疗后。

Cytokines are important intercellular mediators of biological processes including the immune response. Whether cytokines have an impact on DNA repair has been studied by us using as a model system freshly isolated human monocytes, which were matured by GM-CFS/IL-4 into macrophages and dendritic cells (DCs). We observed that human monocytes do not express detectable amounts of the DNA repair proteins XRCC1, Ligase III, PARP-1 and DNA-PKCS. During cytokine-stimulated differentiation of monocytes into macrophages or DCs the expression of these repair proteins is upregulated. The regulation occurs for XRCC1, PARP-1 and DNA-PKCS on gene/mRNA level. Lack of expression of the repair proteins results in a defect in base excision and DNA doble-strand break repair and hypersensitivity of monocytes to monofunctional alkylating agents, including temozolomide, ionising radiation and oxidative agents. Our preliminary data revealed that also mouse monocytes lack XRCC-1, Ligase III, PARP-1 and DNA-PKCS, indicating this is a general biological phenomenon. - In this grant proposal we wish to extend the previous studies in both human and mouse system. We wish to elucidate when and how during maturation of CD34+ cells into monocyte precursors XRCC1, PARP-1 and DNA-PKCS become downregulated, whether the repair proteins are also lacking in established monocyte-like human cells, whether they become upregulated durig differentiation and how the corresponding repair genes are regulated. In mice, we wish to determine the monocyte precursor in the bone marrow in which downregulation of XRCC1, Ligase III, PARP-1 and DNA-PKCS occurs. Further, we want to assess whether mouse monocytes are also hypersensitive to genotoxicants, including ROS, whether other genes are differentially regulated and subject to upregulation following cytokine stimulation of monocytes and, finally, whether hypersensitivity of monocytes results in depletion of monocytes and the descending population, i.e. macrophages, following whole body irradiation and treatment with alkylating agents, including the anticancer drug temozolomide.