Department of Biochemistry and Molecular Biology, Cancer Research Center, Seoul National University College of Medicine, Seoul 110-799, Republic of Korea.
Biochem Biophys Res Commun. 2012 Jun 1;422(2):256-62. doi: 10.1016/j.bbrc.2012.04.139. Epub 2012 May 1.
Cyclic AMP is involved in the regulation of metabolism, gene expression, cellular growth and proliferation. Recently, the cAMP signaling system was found to modulate DNA-damaging agent-induced apoptosis by regulating the expression of Bcl-2 family proteins and inhibitors of apoptosis. Thus, we hypothesized that the cAMP signaling may modulate DNA repair activity, and we investigated the effects of the cAMP signaling system on γ-ray-induced DNA damage repair in lung cancer cells. Transient expression of a constitutively active mutant of stimulatory G protein (GαsQL) or treatment with forskolin, an adenylyl cyclase activator, augmented radiation-induced DNA damage and inhibited repair of the damage in H1299 lung cancer cells. Expression of GαsQL or treatment with forskolin or isoproterenol inhibited the radiation-induced expression of the XRCC1 protein, and exogenous expression of XRCC1 abolished the DNA repair-inhibiting effect of forskolin. Forskolin treatment promoted the ubiquitin and proteasome-dependent degradation of the XRCC1 protein, resulting in a significant decrease in the half-life of the protein after γ-ray irradiation. The effect of forskolin on XRCC1 expression was not inhibited by PKA inhibitor, but 8-pCPT-2'-O-Me-cAMP, an Epac-selective cAMP analog, increased ubiquitination of XRCC1 protein and decreased XRCC1 expression. Knockdown of Epac1 abolished the effect of 8-pCPT-2'-O-Me-cAMP and restored XRCC1 protein level following γ-ray irradiation. From these results, we conclude that the cAMP signaling system inhibits the repair of γ-ray-induced DNA damage by promoting the ubiquitin-proteasome dependent degradation of XRCC1 in an Epac-dependent pathway in lung cancer cells.
环磷酸腺苷（cAMP）参与代谢、基因表达、细胞生长和增殖的调节。最近发现，cAMP 信号系统通过调节 Bcl-2 家族蛋白和凋亡抑制剂的表达来调节 DNA 损伤剂诱导的细胞凋亡。因此，我们假设 cAMP 信号可能调节 DNA 修复活性，并研究了 cAMP 信号系统对肺癌细胞中γ射线诱导的 DNA 损伤修复的影响。刺激型 G 蛋白（GαsQL）的组成型活性突变的瞬时表达或使用 forskolin（一种腺苷酸环化酶激活剂）处理，增强了辐射诱导的 DNA 损伤，并抑制了 H1299 肺癌细胞中损伤的修复。GαsQL 的表达或使用 forskolin 或异丙肾上腺素处理抑制了 XRCC1 蛋白的辐射诱导表达，并且外源性表达 XRCC1 消除了 forskolin 的 DNA 修复抑制作用。福司柯林处理促进了 XRCC1 蛋白的泛素化和蛋白酶体依赖性降解，导致蛋白在γ射线照射后半衰期显著缩短。福司柯林对 XRCC1 表达的影响不受 PKA 抑制剂的抑制，但 Epac 选择性 cAMP 类似物 8-pCPT-2'-O-Me-cAMP 增加了 XRCC1 蛋白的泛素化并降低了 XRCC1 表达。Epac1 的敲低消除了 8-pCPT-2'-O-Me-cAMP 的作用，并在 γ 射线照射后恢复了 XRCC1 蛋白水平。从这些结果中，我们得出结论，cAMP 信号系统通过促进 Epac 依赖性途径中 XRCC1 的泛素化蛋白酶体依赖性降解，抑制了肺癌细胞中 γ 射线诱导的 DNA 损伤的修复。
