Dominant-negative cAMP-responsive element-binding protein inhibits proliferating cell nuclear antigen and DNA repair, leading to increased cellular radiosensitivity
Dominant-negative cAMP-responsive element-binding protein inhibits proliferating cell nuclear antigen and DNA repair, leading to increased cellular radiosensitivity
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DOI：
10.1074/jbc.m304012200
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发表时间：
2003-08-08
影响因子：
4.8
通讯作者：
Schmidt-Ullrich, RK
中科院分区：
文献类型：
--
作者：
Amorino, GP;Mikkelsen, RB;Schmidt-Ullrich, RK
选择性抑制表皮生长因子受体或丝裂原活化蛋白激酶（MAPK）导致癌细胞的放射增敏。一种潜在的机制涉及cAMP反应元件结合蛋白，其通过表皮生长因子受体/MAPK途径被辐射激活，并调节增殖细胞核抗原（PCNA）的合成，PCNA是一种参与电离辐射诱导的DNA损伤修复的蛋白质。为了测试CREB在细胞放射敏感性中的作用，用表达显性阴性CREB突变体（CR 133或KCREB）的质粒转染CHO细胞，并在48小时后测量各种终点。基础水平的PCNACAT报告构建体的活性分别减少了60和40%以下的CR 133和KCREB的表达，观察到类似的减少PCNA蛋白水平。脉冲场凝胶电泳测量结果表明，CR 133抑制辐射诱导的DNA双链断裂的修复，这种效果被逆转过表达的PCNA;显性负CREB也显着抑制分裂剂量恢复。克隆形成试验用于确定存活分数; CR 133和KCREB的显性阴性CREB表达细胞与对照（单独载体）相比的剂量增强比分别为1.5和1.3。重要的是，突变CREB和组成型表达PCNA蛋白的构建体的共转染将CHO细胞的放射敏感性恢复到野生型水平。此外，表达CREB突变体的细胞没有表现出显著的细胞周期再分布。这些数据表明，CREB的遗传破坏导致放射增敏，并且这种效应可以通过涉及PCNA表达降低和DNA修复抑制的机制来解释。
Selective inhibition of the epidermal growth factor receptor or mitogen-activated protein kinase ( MAPK) results in radiosensitization of cancer cells. One potential mechanism involves cAMP-responsive element-binding protein, which is activated by radiation via the epidermal growth factor receptor/MAPK pathway and which regulates synthesis of proliferating cell nuclear antigen ( PCNA), a protein involved in repair of ionizing radiation-induced DNA damage. To test for a role of CREB in cellular radiosensitivity, CHO cells were transfected with plasmids expressing dominant-negative CREB mutants (CR133 or KCREB), and various endpoints were measured 48 h later. Basal levels of PCNACAT reporter construct activity were reduced by 60 and 40% following expression of CR133 and KCREB, respectively; similar decreases were observed in PCNA protein levels. Pulsed-field gel electrophoresis measurements showed that CR133 inhibited the repair of radiation-induced DNA double-strand breaks, and this effect was reversed by over-expression of PCNA; dominant-negative CREB also significantly inhibited split-dose recovery. Clonogenic assays were used to determine surviving fraction; the dose enhancement ratios for dominant-negative CREB-expressing cells compared with control ( vector alone) were 1.5 and 1.3 for CR133 and KCREB, respectively. Importantly, co-transfection of mutant CREB and a construct constitutively expressing PCNA protein restored radiosensitivity of CHO cells back to wild-type levels. Moreover, cells expressing either CREB mutant showed no significant cell cycle redistribution. These data demonstrate that genetic disruption of CREB results in radiosensitization, and that this effect can be explained by a mechanism involving decreased PCNA expression and inhibition of DNA repair.