Martin Francis L, Williamson Sally J M, Paleologou Katerina E, Hewitt Rebecca, El-Agnaf Omar M A, Allsop David
Department of Biological Sciences, I.E.N.S., Lancaster University, Lancaster, UK.
J Neurochem. 2003 Nov;87(3):620-30. doi: 10.1046/j.1471-4159.2003.02013.x.
Lewy bodies in the brains of patients with Parkinson's disease (PD) contain aggregates of alpha-synuclein (alpha-syn). Missense mutations (A53T or A30P) in the gene encoding alpha-syn are responsible for rare, inherited forms of PD. In this study, we explored the susceptibility of untransfected human dopaminergic BE(2)-M17 neuroblastoma cells, cells transfected with vector only, or cells transfected with wild-type alpha-syn, A30P alpha-syn or A53T alpha-syn to Fe(II)-induced DNA damage in the form of single-strand breaks (SSBs). DNA SSBs were detected following 2-h treatments with various concentrations of Fe(II) (0.01-100.0 microm), using the alkaline single cell-gel electrophoresis ('Comet') assay and quantified by measuring comet tail length (CTL) microm). Fe(II) treatment induced significant increases in CTL in cells transfected with A30P alpha-syn or A53T alpha-syn, even at the lowest concentrations of Fe(II) tested. In comparison, untransfected cells, vector control cells or cells transfected with wild-type alpha-syn exhibited increases in SSBs only when exposed to concentrations of 1.0 microm Fe(II) and above. Even when exposed to higher concentrations (10.0-100.0 microm) of Fe(II), untransfected cells, vector control cells or cells transfected with wild-type alpha-syn were less susceptible to DNA-damage induction than cells transfected with A30P alpha-syn or A53T alpha-syn. Incorporation of DNA-repair inhibitors, hydroxyurea and cytosine arabinoside, enhanced the sensitivity of DNA damage detection. Susceptibility to Fe(II)-induced DNA damage appeared to be dependent on alpha-syn status because cells transfected with wild-type alpha-syn or A53T alpha-syn were equally susceptible to the damaging effects of the mitochondrial respiratory chain inhibitor rotenone. Overall, our data are suggestive of an enhanced susceptibility to the toxic effects of Fe(II) in neuroblastoma cells transfected with mutant alpha-syn associated with inherited forms of PD.
帕金森病（PD）患者大脑中的路易小体包含α-突触核蛋白（α-syn）聚集体。编码α-syn的基因中的错义突变（A53T或A30P）导致罕见的遗传性PD形式。在本研究中，我们探究了未转染的人多巴胺能BE(2)-M17神经母细胞瘤细胞、仅用载体转染的细胞，或用野生型α-syn、A30P α-syn或A53T α-syn转染的细胞对Fe(II)诱导的单链断裂（SSB）形式的DNA损伤的易感性。在用各种浓度的Fe(II)（0.01 - 100.0微摩尔）处理2小时后，使用碱性单细胞凝胶电泳（“彗星”）试验检测DNA SSB，并通过测量彗星尾长（CTL，微摩尔）进行定量。即使在测试的最低Fe(II)浓度下，Fe(II)处理也会使转染了A30P α-syn或A53T α-syn的细胞中的CTL显著增加。相比之下，未转染的细胞、载体对照细胞或转染了野生型α-syn的细胞仅在暴露于1.0微摩尔及以上浓度的Fe(II)时才会出现SSB增加。即使暴露于更高浓度（10.0 - 100.0微摩尔）的Fe(II)，未转染的细胞、载体对照细胞或转染了野生型α-syn的细胞对DNA损伤诱导的敏感性也低于转染了A30P α-syn或A53T α-syn的细胞。加入DNA修复抑制剂羟基脲和阿糖胞苷可增强DNA损伤检测的敏感性。对Fe(II)诱导的DNA损伤的易感性似乎取决于α-syn状态，因为转染了野生型α-syn或A53T α-syn的细胞对线粒体呼吸链抑制剂鱼藤酮的损伤作用同样敏感。总体而言，我们的数据表明，转染了与遗传性PD形式相关的突变α-syn的神经母细胞瘤细胞对Fe(II)的毒性作用的易感性增强。
