Expression of histone H2AX phosphorylation and its potential to modulate adriamycin resistance in K562/A02 cell line

DNA repair processes play a role in the development of drug resistance which represents a huge obstacle to leukemia chemotherapy. Histone H2AX phosphorylation (ser139) (γH2AX) occurs rapidly at the onset of DNA double strand break (DSB) and is critical to the regulation of DSB repair. If DNA repair is successful, cells exposed to anti-neoplastic drugs will keep entering the cycle and develop resistance to the drugs. In this study, we investigated whether γH2AX can be used as an indicator of tumor chemosensitivity and a potential target for enhancing chemotherapy. K562 and multi-drug resistant cell line K562/A02 were exposed to adriamycin (ADR) and γH2AX formed. Flow cytometry revealed that percentage of cells expressing γH2AX was increased in a dose-dependent manner and the percentage of K562/A02 cells was lower than that of K562 cells when treated with the same concentration of ADR. In order to test the potential of γH2AX to reverse drug resistance, K562/A02 cells were treated with PI3K inhibitor LY294002. It was found that LY249002 decreased ADR-induced γH2AX expression and increased the sensitivity of K562/A02 cells to ADR. Additionally, the single-cell gel electrophoresis assay and the Western blotting showed that LY249002 enhanced DSBs and decreased the expression of repair factor BRCA1. These results illustrate chemosensitivity can partly be measured by detecting γH2AX and drug resistance can be reversed by inhibiting γH2AX.     

DNA损伤应答靶向抑制剂对卵巢癌细胞的化疗增敏作用

目的 观察DNA损伤应答抑制剂及其联合常规化疗药物（顺铂等）在卵巢癌耐药细胞株OVCAR-8中的作用,研究其化疗致敏效应。方法 利用针对DNA损伤应答关键信号蛋白质的抑制剂,与顺铂等连用处理卵巢癌细胞,分析这些药物处理方式对卵巢癌细胞的杀伤能力。MTT法检测不同药物作用后OVCAR-8的增殖抑制情况;免疫荧光法检测OVCAR-8中磷酸化组蛋白2A变体（γH2AX）和p53结合蛋白1（53BP1）的表达,观察二者在DNA损伤位点的募集和形成灶点的能力。结果 毛细血管扩张共济失调突变蛋白（ATM）/ATM和Rad 3相关蛋白（ATR）抑制剂与顺铂联用能抑制损伤修复机制的活化,明显减弱OVCAR-8细胞的增殖活力（P<0.01）,促进其凋亡;在羟基脲和Wortmannin联合处理时,OVCAR-8细胞的ATR转导信号（如γH2AX）减弱,细胞生存率明显降低（P<0.05）;多聚ADP-核糖聚合酶（PARP）抑制剂与顺铂联合处理OVCAR-8细胞未发现明显的增敏作用（P>0.05）。结论 适当的DNA损伤应答抑制剂有潜力提高常规化疗药物的抗肿瘤效果,以达到快速清除肿瘤细胞和防止产生耐药性的效果。     

ATR抑制剂VX-970对CPT诱导的结肠癌细胞生长的影响

目的:探讨ATR特异抑制剂VX-970对喜树碱抑制的结肠癌细胞HCT-116生长,促进结肠癌细胞HCT-116凋亡的影响。方法:采用克隆形成、MTS、流式细胞术等实验分别检测单独使用VX-970,CPT以及联合VX-970和CPT用药处理对细胞生长、细胞周期及凋亡等影响,蛋白免疫印迹检测DNA损伤关键蛋白p-ATR,p-Chk1,γH2AX等的表达。结果:VX-970增强CPT对HCT-116细胞生长和增殖的抑制作用,促进细胞凋亡和减弱细胞周期G2/M期的阻滞。结论:VX-970能显著增强CPT抑制HCT-116细胞生长,诱导细胞凋亡和提高细胞对CPT药物的敏感性。     

ABT737通过延迟宫颈癌细胞放射后DNA损伤修复及诱导凋亡而增敏放疗

【目的】 探讨类似BH-3的小分子物质ABT737诱导增敏放疗的作用及其分子机制?【方法】噻唑蓝法(MTT)检测不同浓度ABT737对HeLa细胞的生长抑制作用;细胞克隆形成实验检测ABT737联合放疗对放疗的增敏作用;免疫荧光法检测γ-H2AX观察DNA损伤修复情况;流式细胞术检测细胞凋亡;免疫印迹法检测Caspase-3?PARP的表达观察凋亡?【结果】与对照组相比,ABT737能显著抑制宫颈癌HeLa细胞的增殖(P < 0.05),并呈浓度依赖性,其IC50为15.7 μmol/L?体外培养克隆形成实验结果显示放疗+ABT737不同用药时间组的DEF值均大于1,放疗+8 μmol/L ABT737持续用药组为1.88,放疗+12 μmol/L ABT737用药72 h组为1.13,细胞存活分数(SF值)持续用药组为0.84,用药72 h组SF值为0.82;免疫荧光结果显示ABT737联合放疗处理HeLa细胞1 h后,放射线导致的γ-H2AX聚焦点数量及有γ-H2AX聚焦点生成的细胞数量均明显增加,上述处理24 h后,单纯放疗组γ-H2AX焦点消失,而联用ABT737处理组仍可观察到γ-H2AX焦点聚集?流式细胞术结果显示,单纯放疗组早期凋亡率(Annexin V+,PI-)为23.3%,ABT737联合放疗可以明显提高放射线诱导的细胞凋亡,早期凋亡率最高达50.3%?免疫印迹结果显示10 ?滋mol/L ABT737与2 Gy放射联合作用于Hela细胞后,凋亡蛋白cleaved Caspase-3与cleaved PARP的表达较单纯放疗组增加?【结论】 ABT737对宫颈癌Hela细胞具有放疗增敏作用,其机制与ABT737可延迟宫颈癌细胞放疗后DNA损伤修复及诱导凋亡有关?     

人参皂苷Rh2调控盘状结构域受体1对糖尿病大鼠肾纤维化和细胞凋亡的影响

目的 分析人参皂苷Rh2（G-Rh2）对糖尿病肾病（DN）大鼠肾纤维化和细胞凋亡的影响及其作用机制。方法 SPF级,雄性,SD大鼠30只,随机分为对照组（Control组）、DN组以及G-Rh2药物干预组。DN和G-Rh2药物干预组大鼠腹腔注射链脲佐菌素构建DN大鼠模型,Control组大鼠给予等量的柠檬酸缓冲溶液注射。待DN大鼠模型构建成功后,G-Rh2药物干预组给予G-Rh2（20 mg·kg-1 ·d-1）连续灌胃12周,Control和DN大鼠给予等量的生理盐水灌胃。HE染色和PAS染色观察大鼠肾组织形态,Masson染色观察大鼠肾组织纤维化程度,TUNEL染色观察肾组织细胞凋亡情况;Western blot检测大鼠肾组织CollagenI、CollagenIII、Cleaved-caspase-3、Bcl-2、DDR1表达水平;免疫组化定位检测肾组织DDR1表达情况。结果 与Control组相比,DN组肾组织纤维化程度加重,细胞凋亡指数升高（P<0.01）,CollagenI、CollagenIII、Cleaved-caspase-3、DDR1表达上调（P<0.01）,Bcl-2表达下调（P<0.01）;与DN组相比,G-Rh2药物干预组肾组织纤维化程度,细胞凋亡指数,CollagenI、CollagenIII、Cleaved-caspase-3、DDR1表达均显著降低（P<0.05或P<0.01）,Bcl-2表达显著升高（P<0.05）。结论 G-Rh2抑制糖DN肾纤维化和细胞凋亡可能与下调DDR1表达有关。     

卵巢透明细胞腺癌的DNA损伤应答反应

目的研究卵巢透明细胞腺癌的发生与DNA损伤应答之间的关系。方法对14例新鲜卵巢组织标本(正常卵巢组织3例,低分化卵巢肿瘤6例,透明细胞腺癌5例),采用X射线(2Gy)照射制备组织DNA损伤模型,分别对其进行组织冷冻切片,采用免疫荧光和Western免疫印迹方法检测DNA损伤应答反应。结果在X射线照射前,与正常卵巢组织细胞相比,透明细胞腺癌中存在大量内源性DNA损伤,X射线照射后组蛋白2A变体(H2AX)表现过度磷酸化,而损伤修复能力正常。p53结合蛋白1(53BP1)的激活与磷酸化的H2AX(γH2AX)不能共定位。结论卵巢透明细胞腺癌中DNA损伤异常激活,提示DNA损伤应答信号转导网络存在缺陷。     

上皮性卵巢癌细胞原代培养及放化疗相关的DNA损伤应答机制的研究

目的 通过上皮性卵巢癌组织细胞原代培养,检测DNA损伤应答过程中相关蛋白细胞定位的动态变化。方法 选取临床新鲜卵巢上皮性肿瘤标本28例（交界性浆液囊腺瘤6例,高分化浆液腺癌5例,中分化浆液腺癌6例,低分化浆液腺癌11例）,组织块经胶原酶A消化后培养,比较不同培养基（MCDB/M199培养基、原代细胞专用培养基、DMEM培养基）对原代肿瘤细胞正常形态维持、增殖潜力及纤维化的影响。离子射线（X光）、喜树碱（CPT）处理诱导细胞DNA损伤,间接免疫荧光法检测ATM蛋白激酶依赖性信号转导通路中DNA损伤（双链断裂）应答相关蛋白〔p53结合蛋白1（53BP1),磷酸化组蛋白H2AX(γ-H2AX)〕的应答特征。结果 MCDB/M199培养基有利于卵巢上皮性肿瘤细胞维持正常形态并减缓纤维化。未经处理前各级别卵巢肿瘤原代细胞中均存在不同程度的内源性损伤(53BP1、γ-H2AX灶点),且随着肿瘤细胞恶性程度的增加,53BP1、γ-H2AX灶点逐渐增多（P <0.05）。X光、CPT可以在卵巢癌原代细胞中诱导大量DNA损伤,提示原代培养的细胞有经典的DNA损伤应答反应。结论 成功建立了卵巢上皮性肿瘤直接分离培养方法,在卵巢上皮性肿瘤发展过程中,ATM转导通路检验点信号可被内源性损伤激活,CPT、X线处理会造成上皮性卵巢癌原代细胞中ATM通路的进一步激活来抑制肿瘤增殖,阻止肿瘤进展。     

H2AX参与白血病细胞诱导NK细胞凋亡的研究

目的 初步探讨H2AX磷酸化水平是否参与白血病细胞系诱导自然杀伤(NK)细胞凋亡.方法 体外培养 NK92细胞,利用Fas激活性抗体CH11作用于NK92细胞,用流式细胞术检测NK92细胞的凋亡情况;检测NK92细胞凋亡的同时用流式细胞学技术检测H2AX磷酸化的水平.结果 随着CH11浓度(0、2.5、5、10、20 μg/ml)的升高, NK92细胞凋亡现象明显增加,并呈剂量依赖性,表明NK细胞凋亡随着CH11浓度的升高而增加;同时H2AX的磷酸化水平也随CH11浓度(0、5、10、40、80 μg/ml)逐步增高,并呈剂量依赖性,提示H2AX的磷酸化作用参与细胞凋亡过程,并且随浓度的增加而增加.结论 H2AX的磷酸化作用参与白血病细胞诱导NK细胞的凋亡过程.     

丹酚酸B配伍丹皮酚对H_2O_2诱导的人脐静脉内皮细胞氧化损伤模型Bcl-2、Bax、caspase-3 mRNA及蛋白表达的影响

目的探讨丹酚酸B配伍丹皮酚对过氧化氢（H2O2）诱导的人脐静脉内皮细胞氧化损伤模型Bcl-2、Bax、caspase-3 mRNA及蛋白表达的影响。方法采用H2O2造成人脐静脉内皮细胞氧化损伤模型,利用SYBR GreenRT-PCR方法检测药物作用内皮细胞后凋亡相关基因Bcl-2、Bax、caspase-3 mRNA的表达变化,Western blot法检测内皮细胞凋亡相关蛋白Bcl-2、Bax、caspase-3的表达。结果丹酚酸B配伍丹皮酚能够使损伤内皮细胞中Bcl-2基因mRNA水平上升,而Bax、caspase-3基因的表达降低,与Western blot方法检测蛋白含量的结果较为一致,且数据比较差异有高度统计学意义（P〈0.01）。结论丹酚酸B配伍丹皮酚是通过调节Bcl-2、Bax基因及蛋白的表达,抑制下游分子caspase-3的活化而起到保护血管内皮细胞的作用。     

石英粉尘的DNA损伤作用研究

目的探讨石英粉尘对DNA的损伤作用。方法分析某矿山井下和井上,接触不同游离SiO2含量粉尘的工人尘肺发病率。用0、100、200、400μg/mL标准α石英刺激人胚肺成纤维细胞,采用免疫荧光技术检测H2AX磷酸化(γH2AX)水平作为DNA双链断裂损伤程度的指标。结果井下粉尘的游离SiO2含量高于井上,井下工人尘肺发病率(12.15%)高于井上(3.74%),差异有统计学意义(P﹤0.05)。γH2AX水平随石英剂量增高呈增高趋势,半定量结果分别为(521.9±233.1)、(823.3±201.5)、(1 375.5±311.5)、(1 545.6±145.7);差异均有统计学意义(P﹤0.05)。结论石英粉尘可引起DNA双链断裂,且存在剂量效应关系。     

甲醛致雌性小鼠卵巢细胞DNA损伤及Bcl-2和Bax表达的影响

目的探讨甲醛致小鼠卵巢细胞DNA损伤效应及对Bcl-2和Bax表达的影响与其相关作用的机制。方法采用单细胞凝胶电泳方法,检测甲醛染毒(剂量分别为0.3、1.2、4.8 mg/kg)后小鼠卵巢细胞DNA损伤情况,采用免疫组织化学法检测Bcl-2、Bax的表达。结果甲醛致卵巢组织结构损伤,染毒浓度越高,病理损害越明显;卵巢细胞Bcl-2下调,Bax表达上调,并有量-效关系,甲醛染毒各组Bcl-2和Bax表达的相对灰度值较对照组均有差异(P<0.01);试剂量范围内,小鼠卵巢细胞彗星率随甲醛染毒剂量增加而增高(P<0.05),其彗星尾长随甲醛染毒剂量增加而缩短,尤其高甲醛染毒组更短(P<0.01)。结论甲醛能致卵巢细胞DNA损伤效应,损伤小鼠卵巢的结构,使卵巢细胞凋亡增加,Bcl-2表达下调、Bax表达上调;Bcl-2和Bax表达的变化可能是诱导凋亡的机制。     

Expression and Significance of Bcl-2, Bax, Fas and Caspase-3 in Different Phases of Human Hemangioma

Summary： The relationship between Bcl-2, Bax, Fas, caspase-3 and development of hemangioma and the molecular mechanism was investigated. By using immunohistochemical S-P method, proliferating cell nuclear antigen was detected. According to the classification of Mulliken in combination with PCNA expression, 27 cases were identified as proliferating hemangioma and 22 cases as involutive hemangioma. Five normal skin tissues around the tumor tissue served as controls. By using immunohistochemical technique, the expression of Bcl-2, Bax, Fax and Caspase-3 was detected. The cells expressing Bcl-2, Bax, Fax and cappase-3 were identified as hemangioma endothelia by immunohistochemical staining of Ⅷ factor. The average absorbance （A） and average positive area rate of Bcl-2, Bax, Fas and caspase-3 expression were measured by using HPIAS-2000 imaging analysis system. The results showed that the expression of Bcl-2 in the endothelia of proliferating hemangioma was significantly higher that in involutive degenerative hemangioma endothelia and vascular endothelia of normal skin tissue （P〈0.01）. The expression of Bax, Fas and Caspase-3 in the endothelia of involutive hemangioma was obviously higher than in the endothelia of proliferating hemangioma and normal skin tissue （P〈0.01）. The expression of BAx and Fas in endothelia of proliferating hemangioma was higher than in those of normal skin tissue （P〈0.05）. It was suggested that Bcl-2, Bax, Fas and caspase-3 might be involved in the development and involution of hemangioma. Bcl-2 could promote the growth of hemangioma by inhibiting apoptosis of endothelia. Bax, Fas and caspase-3 promote the switch of hemangioma from proliferation to involution by inducing the apoptosis of hemangioma endothelia.     

应用蛋白质芯片检测复发鼻咽癌蛋白磷酸化表达

目的 比较复发鼻咽癌(rNPC)和初发鼻咽癌(pNPC)相关蛋白质磷酸化的差异.方法 提取在温州医学院附属第一医院2003年1月至2005年9月行放疗的4例复发鼻咽癌和4例初发鼻咽癌的总蛋白,应用磷酸化抗体蛋白质芯片与提取蛋白进行杂交,检测656种蛋白质磷酸化表达水平,筛选差异磷酸化蛋白并进行聚类.PhosphoSite plus在线磷酸化蛋白数据库分析磷酸化位点及其在信号通路中的作用.免疫组织化学验证差异磷酸化蛋白.结果 复发组鼻咽癌和初发组的蛋白质磷酸化表达不同,筛选出6个差异蛋白.KIT、ATP1A1、Synapsin、SEK1及Histone H2AX磷酸化水平在复发组上调(P=0.007 ～0.048),而c-Jun下调(P =0.030).复发组H2AX磷酸化表达0.390(0.175)高于初发组0.290(0.155),复发组c-Jun磷酸化表达0.625(0.145)低于初发组0.725(0.178),(均P＜0.05).其中c-Jun、H2AX、SEK1及KIT蛋白质磷酸化涉及DNA损伤修复能力增强、抑制凋亡及致瘤能力增强,与肿瘤复发有关.结论 蛋白质磷酸化水平的改变可能与鼻咽癌复发机制有关,为临床上放射治疗失败提供全新的策略.     

Synergistic effect of hyperthermia and neferine on reverse multidrug resistance in adriamycin-resistant SGC7901/ADM gastric cancer cells

Multidrug resistance(MDR) plays a major obstacle to successful gastric cancer chemotherapy.The purpose of this study was to investigate the MDR reversal effect and mechanisms of hyperthermia in combination with neferine(Nef) in adriamycin(ADM) resistant human SGC7901/ADM gastric cancer cells.The MDR cells were heated at 42℃ and 45℃ for 30 min alone or combined with 10 μg/mL Nef.The cytotoxic effect of ADM was evaluated by MTT assay.Cellular plasma membrane lipid fluidity was detected by fluorescence polarization technique.Intracellular accumulation of ADM was monitored with high performance liquid chromatography.Mdr-1 mRNA,P-glycoprotein(P-gp),γH2AX expression and γH2AX foci formation were determined by real-time PCR,Western blot and immunocytochemical staining respectively.It was found that different heating methods induced different cytotoxic effects.Water submerged hyperthermia had the strongest cytotoxicity of ADM and Nef combined with hyperthermia had a synergistic cytotoxicity of ADM in the MDR cells.The water submerged hyperthermia increased the cell membrane fluidity.Both water submerged hyperthermia and Nef increased the intracellular accumulation of ADM.The water submerged hyperthermia and Nef down-regulated the expression of mdr-1 mRNA and P-gp.The water submerged hyperthermia could damage DNA and increase the γH2AX expression of SGC7901/ADM cells.The higher temperature was,the worse effect was.Our results show that combined treatment of hyperthermia with Nef can synergistically reverse MDR in human SGC7901/ADM gastric cancer cells.     

电离辐射对沉默ATRX的HeLa细胞DNA损伤修复的影响

目的：靶向沉默宫颈癌HeLa细胞中α-地中海贫血/精神发育迟滞综合征X染色相关蛋白（ATRX）,检测电离辐射对ATRX、γH2AX和Rad51蛋白表达及γH2AX和Rad51焦点数的影响,探讨ATRX参与辐射后HeLa细胞DNA损伤修复的作用。方法： 3条ATRX-shRNA和阴性对照（Control-shRNA）的慢病毒载体转染293T细胞,收集慢病毒并感染HeLa细胞,利用puromycin筛选获得稳定沉默ATRX的细胞系,分别命名为shA₁-HeLa、shA₂-HeLa、shA₃-HeLa和shCon-HeLa,采用Western blotting法检测沉默ATRX效率以及电离辐射后ATRX、γH2AX和Rad51蛋白的表达,采用免疫荧光技术观察shCon-HeLa和shA₁-HeLa组中γH2AX和Rad51焦点并计数其数量。结果： shCon-HeLa细胞中可见ATRX蛋白表达,而shA₁-HeLa、shA₂-HeLa和shA₃-HeLa细胞中均无ATRX蛋白表达,表明沉默效率较高。在2和8 Gy剂量照射后1、6和24 h,shCon-HeLa组ATRX蛋白表达量逐渐升高,24 h时表达量最高,且8 Gy照射后1、6和24 h表达量均较高。4 Gy照射后0~6 h,与shCon-HeLa组比较,shA₁-HeLa组γH2AX焦点数在1 h明显升高（P<0.05）,而后逐渐降低,但在6 h焦点数仍明显高于shCon-HeLa组（P<0.01）;Rad51焦点数与γH2AX焦点数变化相一致,与shCon-HeLa组比较,shA₁-HeLa组Rad51焦点数在1 h明显升高（P<0.05）,在6 h时shA₁-HeLa焦点数仍明显高于shCon-HeLa组（P<0.01）。4 Gy照射后0~16 h,shA₁-HeLa组细胞中γH2AX和Rad51蛋白表达量均较shCon-HeLa组增加。结论：成功获得稳定沉默ATRX的HeLa细胞模型,电离辐射可诱导ATRX蛋白表达量增加,且沉默ATRX的HeLa细胞中γH2AX和Rad51焦点数及蛋白表达量均高于对照组,提示ATRX参与了辐射诱导的DNA损伤修复过程。     

同型半胱氨酸对C17.2小鼠神经干细胞的影响及作用机制

目的探讨同型半胱氨酸（Hcy）对C17.2小鼠神经干细胞（NSCs）的影响及可能的分子机制。方法培养C17.2小鼠NSCs,分为对照组、0.25mMHcy组、0.25mMHcy+5mMN-乙酰半胱氨酸（NAC）组;加入相应药物培养24h后,采用CCK-8法检测各组NSCs生长活力,流式细胞术和Caspase-3法检测Hcy对NSCs凋亡的影响,彗星实验检测Hcy对NSCsDNA的损伤程度,H2DCF-DA染色检测NSCs的氧自由基（ROS）水平。结果Hcy能诱导细胞ROS产生和DNA损伤,从而导致NSCs凋亡增加。相反,NAC可降低Hcy诱发的ROS产生,明显改善DNA损伤,提高细胞存活率。结论Hcy能诱导NSCsROS产生和DNA损伤,并导致NSCs凋亡;而减少Hcy引发的ROS产生可以明显改善DNA损伤并提高细胞存活率。     

糖皮质激素诱导特发性血小板减少性紫癜患儿外周血淋巴细胞凋亡的体外研究

为探讨糖皮质激素对儿童急性特发性血小板减少性紫癜(AITP)外周血淋巴细胞凋亡的诱导作用,对6例初诊儿童AITP外周血淋巴细胞进行糖皮质激素、糖皮质激素受体拮抗剂(RU486)等体外干预培养,用流式细胞术检测细胞凋亡,以凋亡率为定量指标,用DNA电泳对细胞凋亡进行定性分析.结果显示糖皮质激素组的淋巴细胞凋亡率明显高于对照组和经RU486预处理组,具有显著性差异;糖皮质激素组细胞的DNA电泳呈现清晰的"梯状"(1adder)条带,而对照组和RU486预处理组均未出现,提示糖皮质激素可诱导淋巴细胞凋亡,这一作用可被其受体拮抗剂阻断,说明糖皮质激素诱导AITP外周血淋巴细胞凋亡可能是通过糖皮质激素受体途径来实现的.     

Lower Phosphorylation of p38 MAPK Blocks the Oxidative Stress-induced Senescence in Myeloid Leukemic CD34~+CD38~- Cells

Leukemia seems to depend on a small population of "leukemia stem cells (LSCs)" for its growth and metastasis. However, the precise surviving mechanisms of LSCs remain obscure. Cellular senescence is an important obstacle for production and surviving of tumor cells. In this study we investigated the activated state of a pathway, in which reactive oxygen species (ROS) induces cellular senescence through DNA damage and phophorylation of p38 MAPK (p38), in myeloid leukemic CD34+CD38- cells. Bone marrow samples were obtained from patients with acute myeloid leukemia (AML, n=11) and chronic myeloid leukemia (CML, n=9). CD34+CD38- cells were isolated from mononuclear cells from these bone marrow samples, and K562 and KG1a cells (two kinds of myeloid leukemia cell lines) by mini-magnetic activated cell sorting. Hematopoietic stem cells (HSCs) from human cord blood served as controls. Intracellular ROS level was detected by flow cytometry. DNA damage defined as the γH2AX level was measured by immunofluorescence staining. Real-time RT-PCR was used to detect the expression of p21, a senescence-associated gene. Western blotting and immunofluo-rescence staining were employed to determine the p38 expression and activation. The proliferation and apoptosis of CD34+CD38- cells were detected by MTT assay and flow cytometry. Our results showed that ROS and DNA damage were substantially accumulated and p38 was less phosphorated in myeloid leukemic CD34+CD38- cells as compared with HSCs and H2O2-induced senescent HSCs. Furthermore, over-phosphorylation of p38 by anisomycin, a selective activator of p38, induced both the senescence-like growth arrest and apoptosis of CD34+CD38- cells from K562 and KG1a cell lines. These findings suggested that, although excessive accumulation of oxidative DNA damage was present in LSCs, the relatively decreased phosphorylation of p38 might help leukemic cells escape senescence and apoptosis.     

SET8 Inhibition Potentiates Radiotherapy by Suppressing DNA Damage Repair in Carcinomas

Objective SET8 is a member of the SET domain-containing family and the only known lysine methyltransferase(KMT) that monomethylates lysine 20 of histone H4(H4 K20 me1). SET8 has been implicated in many essential cellular processes, including cell cycle regulation, DNA replication, DNA damage response, and carcinogenesis. There is no conclusive evidence, however, regarding the effect of SET8 on radiotherapy. In the current study we determined the efficacy of SET8 inhibition on radiotherapy of tum...