Inhibition of DNA-dependent Protein Kinase Catalytic Subunit by Small Molecule Inhibitor NU7026 Sensitizes Human Leukemic K562 Cells to Benzene Metabolite-induced Apoptosis

Benzene is an established leukotoxin and leukemogen in humans. We have previously re- ported that exposure of workers to benzene and to benzene metabolite hydroquinone in cultured cells induced DNA-dependent protein kinase catalytic subunit （DNA-PKcs） to mediate the cellular response to DNA double strand break （DSB） caused by DNA-damaging metabolites. In this study, we used a new, small molecule, a selective inhibitor of DNA-PKcs, 2-（morpholin-4-yl）-benzo[h]chomen-4-one （NU7026）, as a probe to analyze the molecular events and pathways in hydroquinone-induced DNA DSB repair and apoptosis. Inhibition of DNA-PKcs by NU7026 markedly potentiated the apoptotic and growth inhibitory effects of hydroquinone in proerythroid leukemic K562 cells in a dose-dependent manner. Treatment with NU7026 did not alter the production of reactive oxygen species and oxidative stress by hydroquinone but repressed the protein level of DNA-PKcs and blocked the induction of the kinase mRNA and protein expression by hydroquinone. Moreover, hydroquinone increased the phos- phorylation of Akt to activate Akt, whereas co-treatment with NU7026 prevented the activation of Akt by hydroquinone. Lastly, hydroquinone and NU7026 exhibited synergistic effects on promoting apop- tosis by increasing the protein levels of pro-apoptotic proteins Bax and caspase-3 but decreasing the protein expression of anti-apoptotic protein Bcl-2. Taken together, the findings reveal a central role of DNA-PKcs in hydroquinone-induced hematotoxicity in which it coordinates DNA DSB repair, cell cycle progression, and apoptosis to regulate the response to hydroquinone-induced DNA damage.

气态甲醛致雌性小鼠生殖细胞DNA-蛋白质交联的研究

为了研究气态甲醛对雌性小鼠生殖细胞的影响,以昆明雌性小鼠卵巢为实验材料,采取动态吸入式染毒方式,应用KCl-SDS沉淀法检测了气态甲醛染毒后所引起的小鼠卵巢生殖细胞DNA-蛋白质交联(DPC)效应.结果表明,较低浓度的甲醛(0.5mg·m-3),即可导致明显的DPC效应(与对照相比,p<0.05),并且随着染毒浓度的升高(0.5、1.0、3.0mg·m-3),DPC系数也逐渐升高.上述结果表明在实验所设浓度范围内,甲醛对小鼠卵巢生殖细胞的DNA损伤可以引起DNA-蛋白质的交联,并且DPC系数随着染毒浓度的增大而增大.DNA-蛋白质交联是DNA分子的一种严重损伤,气态甲醛对雌性小鼠生殖细胞具有一定的影响.

氟致体外培养牛脾淋巴细胞DNA-蛋白质交联作用的研究

为探讨氟致牛脾淋巴细胞DNA-蛋白质交联作用的机理,以牛脾淋巴细胞为材料,用终浓度0.2、0.4、0.6和0.8 mmol/L氟化钠(NaF)溶液染毒,培养24 h,检测牛脾淋巴细胞内谷胱甘肽过氧化物酶(GSH-Px)和超氧化物歧化酶(SOD)活性、丙二醛(MDA)含量及DNA-蛋白质交联(DPC)效应。结果显示,与对照组比较,淋巴细胞内GSH-Px、SOD活性呈不同程度的降低,而MDA含量、DPC系数不同程度增大,且存在着剂量-效应关系。证实,氟能够通过诱导体外培养牛脾淋巴细胞发生氧化应激导致其发生DNA-蛋白质交联。

苯致模拟失重大鼠肝细胞DNA-蛋白质交联的研究

目的:探讨苯吸入染毒对尾吊模拟失重大鼠肝细胞DNA-蛋白质交联(DNA-protein crosslinks,DPC)作用。方法:将30只雄性SD大鼠随机分为3组:对照组和2个染毒组。对照组吸入新鲜空气,染毒组分别给予浓度为10 mg/m3和50 mg/m3的苯气体进行吸入染毒,每天24 h,连续7 d。结果:与对照组相比,低浓度的气态苯(10 mg/m3)可以诱导显著的DNA-蛋白质交联作用(P<0.05),高浓度的气态苯(50 mg/m3)可以诱导极显著的DNA-蛋白质交联作用(P<0.01)。结论:高低浓度苯吸入染毒均可致肝细胞内的DPC系数显著上升,且随苯浓度上升,细胞中的DPC含量出现极显著上升。

Long non-coding RNA DPP10-AS1 represses the proliferation and invasiveness of glioblastoma by regulating miR-24-3p/CHD5 signaling pathway

This investigation aimed to unveil new prospective diagnosis-related biomarkers together with treatment targets against glioblastoma.Methods:The expression levels of long non-coding RNA(lncRNA)DPP10-AS1 were assessed using real-time quantitative polymerase chain reaction(RT-qPCR)within both the patient tissue specimens and glioblastoma cell lines.The relationship between lncRNA DPP10-AS1 expression in glioblastoma and patient prognosis was investigated.Cell Counting Kit-8(CCK-8),transwell,and clonogenic experiments were utilized to assess tumor cells’proliferation,invasiveness,and migratory potentials after lncRNA DPP10-AS1 expression was up or down-regulated.Using an online bioinformatics prediction tool,the intracellular localization of lncRNA DPP10-AS1 and its target miRNA were predicted,and RNA-FISH verified results.A dual-luciferase reporter experiment validated the relationship across miR-24-3p together with lncRNA DPP10-AS1.MiR-24-3p expression within glioblastoma was identified through RT-qPCR,and potential link across miR-24-3p and lncRNA DPP10-AS1 was assessed using Pearson correlation analysis.Moreover,influence from lncRNA DPP10-AS1/miR-24-3p axis upon glioblastoma cell progression was assessed in vivo via a subcutaneous xenograft tumor model.Results:The expression of lncRNA DPP10-AS1 was notably reduced in both surgical specimens of glioblastoma and the equivalent cell lines.Low level of lncRNA DPP10-AS1 in glioblastoma is following poor prognosis.The downregulation of lncRNA DPP10-AS1 in glioblastoma cells resulted in enhanced cellular proliferation,migration,and invasion capabilities,accompanied by downregulated E-cadherin and upregulated vimentin and N-cadherin.Additionally,the observed upregulation of lncRNA DPP10-AS1 demonstrated a substantial inhibitory function upon proliferation,invasion,and migratory capabilities of LN229 cells.Subcellular localization disclosed that lncRNA DPP10-AS1 had a binding site that interacted with miR-24-3p.Upregulated miR-24-3p was detected in glioblastomas,displaying an inverse correlation with lncRNA DPP10-AS1 expression.MiR-24-3p downstream target has been determined as chromodomain helicase DNA binding protein 5(CHD5).LncRNA DPP10-AS1 affected the invasion and proliferation of glioblastoma by controlling the miR-24-3p/CHD5 axis.Conclusion:The present study demonstrated that lncRNA DPP10-AS1 can inhibit the invasive,migratory,and proliferative properties of glioblastoma by regulating the miR-24-3p/CHD5 signaling pathway.Consequently,lncRNA DPP10-AS1 has potential as a tumor suppressor and might be utilized for accurate diagnosis and targeted treatments of glioblastomas.

氯氰菊酯对小鼠肝细胞的氧化损伤和维生素E的抗氧化作用

目的研究氯氰菊酯对小鼠肝组织的氧化损伤。方法昆明小鼠随机分为5组,包括1个阴性对照组(花生油)、3个氯氰菊酯染毒组(10、20和40 mg/kg)和1个高剂量氯氰菊酯(40 mg/kg)+维生素E(100 mg/kg)组,灌胃7 d,测定肝组织匀浆活性氧(ROS)、还原型谷胱甘肽(GSH)、丙二醛(MDA)、8-羟基脱氧鸟苷(8-OHdG)的含量和肝组织细胞DNA-蛋白质交联(DPC)系数;并观察病理损伤。结果随着氯氰菊酯染毒剂量的升高,肝组织ROS、MDA、8-OHdG含量和DPC系数逐渐上升,GSH含量逐渐降低,各指标呈一定的剂量-效应关系,40 mg/kg组的上述指标与对照组差异均有统计学意义(P<0.05,P<0.01)。病理学观察可见40 mg/kg组肝细胞水肿严重,体积明显增大,胞浆疏松化甚至气球样变,肝索排列较紊乱。与单独氯氰菊酯染毒组相比较,40 mg/kg+VE组肝组织的ROS、MDA、8-OHdG含量和DPC系数均有下降,GSH含量上升(P<0.05,P<0.01)。结论 40mg/kg的氯氰菊酯能造成小鼠肝组织的氧化损伤和病理损伤,并可被维生素E所拮抗。