Kim Hyun-Suk, Guo Chunlu, Thompson Eric L, Jiang Yanlin, Kelley Mark R, Vasko Michael R, Lee Suk-Hee
Department of Biochemistry and Molecular Biology, Indianapolis, IN 46202, USA.
Department of Pharmacology and Toxicology, Indianapolis, IN 46202, USA.
Mutat Res. 2015 Sep;779:96-104. doi: 10.1016/j.mrfmmm.2015.06.010. Epub 2015 Jun 26.
Peripheral neuropathy is one of the major side effects of treatment with the anticancer drug, cisplatin. One proposed mechanism for this neurotoxicity is the formation of platinum adducts in sensory neurons that could contribute to DNA damage. Although this damage is largely repaired by nuclear excision repair (NER), our previous findings suggest that augmenting the base excision repair pathway (BER) by overexpressing the repair protein APE1 protects sensory neurons from cisplatin-induced neurotoxicity. The question remains whether APE1 contributes to the ability of the NER pathway to repair platinum-damage in neuronal cells. To examine this, we manipulated APE1 expression in sensory neuronal cultures and measured Pt-removal after exposure to cisplatin. When neuronal cultures were treated with increasing concentrations of cisplatin for two or three hours, there was a concentration-dependent increase in Pt-damage that peaked at four hours and returned to near baseline levels after 24h. In cultures where APE1 expression was reduced by ∼ 80% using siRNA directed at APE1, there was a significant inhibition of Pt-removal over eight hours which was reversed by overexpressing APE1 using a lentiviral construct for human wtAPE1. Overexpressing a mutant APE1 (C65 APE1), which only has DNA repair activity, but not its other significant redox-signaling function, mimicked the effects of wtAPE1. Overexpressing DNA repair activity mutant APE1 (226 + 177APE1), with only redox activity was ineffective suggesting it is the DNA repair function of APE1 and not its redox-signaling, that restores the Pt-damage removal. Together, these data provide the first evidence that a critical BER enzyme, APE1, helps regulate the NER pathway in the repair of cisplatin damage in sensory neurons.
周围神经病变是抗癌药物顺铂治疗的主要副作用之一。这种神经毒性的一种推测机制是感觉神经元中铂加合物的形成，这可能导致DNA损伤。尽管这种损伤在很大程度上通过核苷酸切除修复（NER）得以修复，但我们之前的研究结果表明，通过过表达修复蛋白APE1增强碱基切除修复途径（BER）可保护感觉神经元免受顺铂诱导的神经毒性。问题仍然是APE1是否有助于NER途径修复神经元细胞中铂损伤的能力。为了研究这一点，我们在感觉神经元培养物中操纵APE1的表达，并在暴露于顺铂后测量铂的去除情况。当用浓度不断增加的顺铂处理神经元培养物两到三个小时时，铂损伤呈浓度依赖性增加，在四小时达到峰值，并在24小时后恢复到接近基线水平。在使用针对APE1的小干扰RNA（siRNA）使APE1表达降低约80%的培养物中，八小时内铂的去除受到显著抑制，而通过使用人类野生型APE1的慢病毒构建体过表达APE1可逆转这种抑制。过表达仅具有DNA修复活性但不具有其他重要氧化还原信号功能的突变型APE1（C65 APE1），其效果与野生型APE1相似。过表达仅具有氧化还原活性的DNA修复活性突变型APE1（226 + 177APE1）无效，这表明是APE1的DNA修复功能而非其氧化还原信号功能恢复了铂损伤的去除。总之，这些数据首次证明了一种关键的BER酶APE1有助于调节NER途径以修复感觉神经元中的顺铂损伤。
