组蛋白
抗辐射性
九氟化硫
乙酰化
DNA损伤
组蛋白脱乙酰基酶
彗星试验
生物
分子生物学
生物化学
组蛋白H2A
DNA
化学
遗传学
细胞培养
重组DNA
基因
夜蛾
作者
Kanupriya Sharma,Ashish Kumar,Sudhir Chandna
标识
DOI:10.1016/j.bbagen.2016.03.004
摘要
Lepidopteran insect cells withstand multifold higher radiation doses and suffer far less DNA damage despite carrying numerous structural/functional homologies with mammalian cells. Since DNA-histone interactions significantly influence radiation-induced DNA damage, we investigated the role of histones in insect cell radioresistance.Modified comet assay was used to assess the γ-radiation-induced DNA damage following serial histone depletion by varied salt concentrations. Acid-Urea-Triton (AUT) gel analysis combined with in silico predictions was used to compare mammalian and insect histones and acetylation status while HDAC activity was assessed/modified for studying the latter's role in radioresistance. Cell death was measured by morphological analysis and flow cytometry.High-salt extraction pattern from Sf9 nuclei suggested stronger DNA-histone affinity as the two core histones H2A/H2B could be extracted at much higher (2M) concentration as compared to 1.2M NaCl in mammalian (AA8) cells. Electrophoretic mobility of unirradiated Sf9 cells remained unaltered at all salt concentrations (0.14M-2M NaCl), and radiation-induced DNA damage increased only by 2M-NaCl pre-treatment. In silico analysis confirmed excellent conservation of Lepidopteran H2A/H2B sequence with human histones including comparable N-terminal lysine residues, yet these had ~60% lower acetylation. Importantly, insect cells showed ~70% higher histone deacetylase activity whose inhibition by Trichostatin-A reversed hypo-acetylation state and caused significant radiosensitization, thereby confirming the protective contribution of reduced acetylation.Our study reveals that the hypo-acetylated state of well-conserved core histones, maintained by considerable HDAC activity, contributes significantly in Lepidopteran radioresistance.This investigation shows constitutively high activity of HDACs as a potential radioprotective mechanism existing in insect cells.
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