Loss of mitochondrial membrane potential and caspase activation enhance apoptosis in irradiated K562 cells treated with herbimycin A

Purpose: We previously reported that herbimycin A (HMA) alters the mode of cell death of K562 cells induced by radiation and enhanced their radiosensitivity. In the present study, we explored the apoptosis-inducing activity of HMA and the fundamental mechanism via which it regulates radiation-induced cell death.

Materials and methods: Chronic myelogenous leukemia (CML) cell line K562 was used. For X-irradiation and drug treatment, cells were plated at approximately 2 × 10⁵ cells/ml. Exponentially growing cells were treated with 10 Gy of X-ray using a 6-MeV X-ray machine at a dose rate of 200 – 300 cGy/min. The cells were treated with 0.25 μM HMA immediately after irradiation and HMA remained for the entire culture period. The modes of cell death were discriminated by morphological changes, analysis of cell cycle, analysis of the mitochondrial events, and the expression of apoptosis-related proteins.

Results: Our data demonstrates that radiation induced a significant time-dependent increase of cell death and failed to sustain a prolonged G2 arrest in K562 cells. Radiation-induced cell death caused the accumulation of cyclinB1 and weak nuclear fragmentation, suggesting a mitotic catastrophe. This mitotic catastrophe was dependent upon the mitochondrial permeability transition pore (PTP) opening and was independent of caspase-3. In contrast, K562 cells treated with radiation and HMA had an accelerated cell death and induced a p53-independent apoptosis. This apoptotic pathway was dependent upon an initial hyperpolarization of the mitochondrial inner membrane, following the release of cytochrome c and subsequent caspase-3 activation.

Conclusions: Two mechanisms of radiation-induced cell death in K562 cells, mitotic catastrophe and apoptosis, are regulated through distinct pathways, mitochondria and caspase-independent and -dependent, respectively. The findings of this study may provide new insights into improving the efficiency of radiotherapy in CML patients.     

PB231诱导K562细胞凋亡的初步研究

 目的研究PB231诱导K 562细胞凋亡作用,探讨PB231的抗肿瘤机制.方法采用荧光显微镜观察细胞形态学变化,MTT法测定PB231对K 562细胞生长抑制作用,琼脂糖凝胶电泳检测细胞DNA裂解.结果K562细胞经PB31处理后,在倒置光显微镜下可见细胞变形和凋亡小体形成;荧光显微镜下可见染色质凝集.MTT法检测其IC50值为5×10-5M.琼脂糖凝胶电泳呈典型的"DNA Ladder".结论PB231可诱导K562细胞发生凋亡.这可能是其抑制K562细胞生长的作用机理之一.     

高压氧对一氧化碳中毒大鼠海马神经细胞线粒体膜电位与细胞凋亡的影响

目的 观察一氧化碳中毒（COP）后脑损伤时线粒体膜电位及细胞凋亡数量的变化以及高压氧（HBO）对其影响。方法Wistar大鼠共128只,采用配伍组设计,按随机抽样原则将动物分成4组16小组。正常对照组8只;CO组COP后第1,5,10,15,20天各8只;CO＋HP组高气压处理后第1,5,10,15,20天各8只：CO＋HBO组HBO处理后第1,5,10,15,20天各8只。用流式细胞仪测定COP大鼠在不同时间段海马神经细胞线粒体膜电位及海马神经细胞凋亡细胞相对百分比（％）的变化和HBO治疗后的改变。结果CO组在COP后大鼠海马神经细胞线粒体膜电位在第1,5,10天降低（P＜0．01－0．05）;CO＋HBO组在第1,5天降低（P＜0．01－0．05）。CO组COP后大鼠海马神经细胞凋亡细胞相对百分比在第1,5,10,15,20天高于对照组（P＜0．01）;而CO＋HBO组只在第1,5,10天高于对照组（P＜0．01）。结论COP后大鼠海马神经细胞线粒体膜电位降低,凋亡细胞数增加,并持续很长时间,而HBO处理可缩短线粒体膜电位降低的减少凋亡细胞数百分比。     

辐射后造血细胞表面IL-11受体表达的变化

目的 探讨体外照射后造血细胞表面IL-11受体(IL-11R)蛋白和基因表达水平的变化,为临床更合理有效地应用rhIL-11治疗急性放射病提供理论和实验依据。方法 采用流式细胞术及RT-PCR法检测了不同照射剂量和照射后不同时间K562细胞表面IL-11R蛋白和基因表达水平的变化。结果 随着照射剂量的升高,K562细胞表面IL-11Rα蛋白的表达量逐渐升高,其中5Gy和10Gy照射组照射后24h IL-11Rα蛋白表达水平较正常显著增高。结论 照射后24h IL-llRα蛋白及基因表达水平升高可能是机体自我保护的一种适应性反应,为早期应用IL-11提供了物质基础。     

瑞香狼香水提物小鼠药物血清诱导K562细胞凋亡

目的从细胞凋亡角度探讨瑞香狼毒(SCL)抗肿瘤作用机理.方法用SCL小鼠药物血清处理培养的人白血病K562细胞,MTT比色法观察对细胞生长的抑制作用,荧光显微镜观察凋亡细胞的形态变化,流式细胞仪观察DNA改变.结果SCL(5～20)g*kg-1小鼠药物血清显著抑制K562细胞增殖,明显诱导K562细胞凋亡的形态学改变和DNA变化.凋亡细胞率与小鼠服用SCL水提物的剂量呈正相关.结论SCL水提物可诱导肿瘤细胞凋亡.     

替米沙坦对糖尿病大鼠心肌线粒体膜电位及心肌细胞凋亡的作用

目的 探讨替米沙坦对1型糖尿病大鼠氧化应激水平的影响.方法 成年雄性Wistar大鼠一次性腹腔注射链脲佐菌素(STZ)建立1型糖尿病大鼠模型.16只成模大鼠随机分为糖尿病(DM)组和替米沙坦(T)组,每组8只;另选健康大鼠8只作为正常对照(Con)组.12周后测量大鼠体重(BW)和心脏重量(HW),计算HW/BW;可见分光光度计分别测定血清还原型谷胱甘肽(GSH)、丙二醛(MDA)含量及超氧化物歧化酶(SOD)活力;透射电镜观察心肌细胞超微结构.结果 STZ注射后7d及12周DM组与T组大鼠血糖差异均无统计学意义,但均显著高于Con组(P<0.01).12周时,与Con组比较,DM组血清SOD活力降低,MDA含量增高,GSH含量下降(P<0.01),T组血清SOD活力降低(P<0.01),MDA含量增高(P<0.05),而GSH含量无明显改变(P>0.05).12周时,与DM组比较,T组血清SOD活力升高,MDA含量降低,GSH含量升高(P<0.01).Con组心肌纤维排列整齐,线粒体呈圆形或椭圆形,结构清晰,线粒体膜完整,嵴排列整齐,未见肿胀及空泡变性.DM组心肌细胞线粒体肿胀,线粒体膜模糊、增厚,嵴不规则或断裂、溶解,可见线粒体空泡变性.T组心肌细胞超微结构损伤较DM组明显减轻.结论 替米沙坦可抑制1型糖尿病大鼠的氧化应激水平,对糖尿病大鼠心肌发挥保护作用.     

JTV1对K562细胞增殖和凋亡的影响及其机制

目的观察上调JTV1基因的表达对人白血病K562细胞系增殖与凋亡的影响并探讨其机制。方法将携带有JTV1基因的pcDNA3.1-JTV1载体转染人K562细胞作为阳性转染组,转染pcDNA3.1空载体作为空载体转染组,未转染的K562细胞为未转染组。通过集落形成实验检测各组K562细胞的增殖能力,采用流式细胞术与Annexin-PI双染色结合分析细胞周期和细胞凋亡率,同时采用RT-PCR法检测JTV1基因及凋亡相关基因Bcl-2、Bax、C-myc mRNA水平的变化,采用Western blotting检测基因JTV1及凋亡相关基因Bcl-2、Bax、C-myc蛋白水平的变化。结果集落形成实验结果显示上调JTV1基因表达后K562细胞的增殖能力明显降低。流式细胞术与Annexin-PI双染色检测结果显示,与空载体转染组及未转染组相比,阳性转染组K562细胞的G期细胞比例明显升高(P<0.05);阳性转染组中Bax基因的mRNA水平和蛋白水平较空载体转染组及未转染组均明显上升,而Bcl-2基因和C-myc基因的mRNA水平和蛋白水平则明显下调(P<0.05)。结论 JTV1基因过表达可能通过抑制基因Bcl-2和C-myc的表达增强基因Bax的表达,从而抑制K562细胞系的增殖并促进其凋亡。     

激光激发5-氨基乙酰丙酸己酯对人红白血病耐药/非耐药细胞株的杀伤效应研究

目的探讨5-氨基乙酰丙酸己酯-光动力疗法(He-ALA-PDT)对人红白血病细胞株K562及其耐阿霉素细胞株K562/ADM的杀伤效应。方法实验设4组:PDT组(加光敏剂并接受光照)、激光组(不加光敏剂只接受光照)、暗毒性组(只加光敏剂而不接受光照)、正常对照组(不加光敏剂且不接受光照)。用不同浓度(0.025、0.1、0.4、1.6mmol/L)的He-ALA避光孵育细胞,给予不同功率密度(4.5、9、18、36J/cm2)的630nm波长激光照射后,继续培养12h,观察细胞形态学变化,以CCK8法测定细胞存活率,采用甲基纤维素培养体系分析白血病细胞克隆形成能力的变化。结果暗毒性组、激光组均未对细胞产生明显杀伤作用,而PDT组能明显杀伤白血病细胞,且细胞存活率随He-ALA浓度和激光功率密度的增加而降低,且在相同条件下,K562细胞的存活率显著低于K562/ADM细胞(P<0.05)。PDT组的集落形成率明显低于正常对照组(P<0.001),且与K562/ADM细胞相比,K562细胞的集落形成率降低程度更为显著(P<0.001)。结论He-ALA-PDT可杀伤K562、K562/ADM细胞,并显著降低白血病细胞的克隆形成能力;K562/ADM细胞对PDT的反应不如K562细胞敏感。     

bcl—2在足叶乙甙诱导的K562细胞凋亡中的作用

目的研究ｂｃｌ－２及其表达产物在足叶乙甙诱导的Ｋ５６２细胞凋亡中的作用。方法采用细胞形态观察,ＤＮＡ电泳,ＤＮＡ缺口的原位检测观察Ｋ５６２细胞的凋亡,用原位杂交法检测ｂｃｌ－２ｍＲＮＡ的表达,用免疫荧光法检测ｂｃｌ－２蛋白的表达。结果２０μｇ／ｍｌ的足叶乙甙能诱导Ｋ５６２细胞发生凋亡,在此过程中,ｂｃｌ－２ｍＲＮＡ及ｂｃｌ－２蛋白的表达均下降。结论ｂｃｌ－２及其表达产物的下调在足叶乙甙诱导的Ｋ５６２细胞凋亡中起重要作用。     

过氧化氢对肠上皮细胞线粒体膜电位及细胞凋亡的影响

目的 :探讨过氧化氢在体外对肠上皮细胞线粒体膜电位的影响及其与细胞凋亡的关系。方法 :肠上皮细胞株 (SW -4 80 ) ,采用过氧化氢 (H2 O2 )损伤模型 ,用 5 0、10 0、2 0 0、4 0 0 μmol·L-1和 4mmol·L-1作用 1、3、6、12及 2 4h。线粒体功能采用噻唑蓝法 (MTT法 ) ;用罗丹明 (Rhodamine- 12 3)荧光探针检查活细胞线粒体膜电位变化 ;用Annexin -Ⅴ荧光探针 ,流式细胞仪检测早期凋亡细胞 ;同时观察细胞形态学和线粒体超微结构的改变。结果 :H2 O2 在低浓度 (5 0、10 0、2 0 0 μmol·L-1)时线粒体功能未见明显改变 ,在高浓度 (4 0 0 μmol·L-1、4mmol·L-1)时可导致线粒体功能不可逆的进行性下降 ,线粒体内嵴减少、肿胀 ;同时使线粒体膜电位显著降低 ,细胞凋亡率显著增加。结论 :线粒体参与了H2 O2 诱导肠上皮细胞的早期程序性死亡及随后的细胞死亡 ,这种损伤作用与线粒体结构、功能改变和膜电位下降密切相关。     

糖尿病大鼠小肠平滑肌细胞线粒体膜电位变化及其意义

目的　研究糖尿病大鼠小肠平滑肌细胞线粒体膜电位变化与细胞凋亡的关系。方法　Wistar大鼠 70只 ,随机分为 :对照组(n =30 ) ,腹腔注射等量的生理盐水。糖尿病模型组(n=4 0 ) ,以链脲菌素 6 0mg/kg腹腔注射 ;造模 2个月后行大鼠胃排空和肠道传输速度测定 ,应用激光共聚焦显微镜检测小肠平滑肌线粒体膜电位 ,脱氧核糖核苷酸末端转移酶介导的缺口末端标记技术 (TUNEL)和流式细胞仪检测细胞凋亡指数 ,Westernblot免疫印迹检测细胞色素C含量。结果　模型组大鼠小肠平滑肌细胞线粒体膜电位 (36 8.8±5 6 4 )显著低于对照组 (96 0 5± 12 6 3,P <0 0 1) ;TUNEL染色显示实验组小肠平滑肌细胞凋亡指数 (9 6 3%± 2 4 7% )显著高于对照组(3 2 3%± 1 2 8% ,P <0 0 1) ;流式细胞仪检测提示实验组小肠平滑肌细胞凋亡率为 15 0 % ,明显高于对照组 (3 0 % ,P <0 0 1)。Westernblot免疫印迹检测实验组细胞色素C含量较对照组明显增高(P <0 0 1)。结论　糖尿病大鼠的胃肠道动力改变和线粒体功能改变与细胞凋亡有关。     

瑞香狼毒小鼠药物血清增敏化疗药物对K562／VCR耐药细胞的抗癌活性

目的研究瑞香狼毒(SC)小鼠药物血清与细胞毒化疗药物联合应用对K562/VCR多药耐药细胞的协同抗肿瘤效应.方法小鼠口服SC水提物6g*kg-1,2h后采集血清.SC药物血清与长春新碱(VCR)、阿霉素(Dox)或足叶乙甙(VP-16)联合处理K562/VCR多药耐药细胞,检测细胞MTT转化率,克隆形成和DNA合成能力.结果5%SC药物血清显著增加细胞毒化疗药物对K562/VCR耐药细胞的敏感性;VCR、Dox、VP-16对K562/VCR的IC50较单独化疗药物组分别减小6.3、15.0和18.5倍;细胞克隆数分别减少45.1%、66.6%和58.6%;[3H]TdR掺入K562/VCR细胞DNA也显著减少.增敏作用随SC药物血清比例的增高而增强.结论瑞香狼毒可增加化疗药物对多药耐药肿瘤细胞的敏感性.     

Treatment of breast tumor cells in vitro with the mitochondrial membrane potential dissipater valinomycin increases 18F-FDG incorporation.

Mitochondrial membrane potential is essential for adenosine triphosphate (ATP) synthesis by oxidative phosphorylation, and its abolition is an early event during apoptosis, a type of cell death commonly exhibited by tumor cells responding to treatment. Dissipation of mitochondrial membrane potential can be specifically induced using the K+ ion channel-opening agent valinomycin and has been used in this study to determine how the loss of mitochondrial membrane potential could influence 18F-FDG incorporation. METHODS: MCF-7 cells were treated with valinomycin for 30 min, inducing loss of mitochondrial membrane potential as determined using flow cytometry with the JC-1 probe. 18F-FDG incorporation, the initial rate of O-methyl-D-glucose incorporation (a measure of glucose transport), hexokinase activity and subcellular distribution, ATP content using bioluminescence, and lactate production were determined on control and valinomycin-treated cells. RESULTS: A 30-min treatment of MCF-7 cells with 1 micromol of valinomycin per liter resulted in absence of red fluorescence from JC-1, indicative of dissipation of mitochondrial membrane potential. 18F-FDG incorporation was significantly increased by 30 min of treatment with valinomycin and was still apparent after 3.5 h of incubation. Hexokinase activity and subcellular distribution were not significantly different between control cells and cells treated for 30 min with valinomycin. Glucose transport was moderately though significantly increased, and lactate production was also increased. CONCLUSION: Loss of mitochondrial membrane potential is associated with increased 18F-FDG incorporation, glucose transport, and lactate production.     

PTD-OD-HA融合蛋白对K562细胞中Bcr-Ab1的抑制作用观察

目的探讨PTD-OD-HA融合蛋白转导K562细胞的动力学、定位及其与Bcr-Abl定位的关系,以及对Bcr-Abl寡聚化和Bcr-Abl酪氨酸激酶活性的影响。方法采用FITC标记PTD-OD-HA融合蛋白,观察蛋白转导K562细胞的效率与剂量和时间的关系,激光共聚焦显微镜检测融合蛋白在细胞内的定位,免疫共沉淀法检测融合蛋白与Bcr-Abl的相互作用,Western blotting检测Bcr-Abl的磷酸化水平。结果 PTD-OD-HA转导K562细胞呈剂量和时间依赖性。PTD-OD-HA定位于K562细胞胞质,与Bcr-Abl共定位,且能与Bcr-Abl蛋白相互作用,进而干扰Bcr-Abl同源寡聚化,并可降低Bcr-Abl的磷酸化水平。结论在K562细胞中,PTD-OD-HA融合蛋白能有效抑制Bcr-Abl同源寡聚化及磷酸化水平。     

Radio- and chemo-sensitization of human erythroleukemic K562 cells by the histone deacetylase inhibitor Trichostatin A

Histone deacetylase inhibitors are emerging therapeutic agents for cancer. In addition to effecting hyperacetylation of core histones, they have been shown to induce biologic effects such as cell cycle redistribution, cytostasis and in certain cases apoptosis in a variety of cell lines. In this study, the purpose was to investigate the effects of Trichostatin A (TSA) - the most potent histone deacetylase inhibitor identified to date - in human erythroleukemic K562 cells. Further aims were to examine the effect of TSA pre-treatment on the chemosensitivity of the cells to the anthracycline, doxorubicin, and on radiosensitivity. In all experiments the cells were treated with 0.2, 0.5 and 2 mM TSA for 24 h prior to analysis for histone acetylation status, cell growth and survival. In parallel assays, TSA treated cells were exposed to doxorubicin or g-radiation and subsequently analyzed for clonogenic survival. The findings indicated that TSA exhibits potent histone deacetylase inhibitor activity in K562 cells, resulting in hyperacetylation of histones 3 and 4 at the concentrations tested. Furthermore, treatment of cells with TSA resulted in dose-dependent inhibition of proliferation, reduction in clonogenic survival and induction of apoptosis. Moreover, the findings of clonogenic survival assays indicated that pre-treatment of K562 cells with TSA augmented the cytotoxic potency of doxorubicin. The magnitude of sensitization to 10 mM doxorubicin-mediated cell death was approximately 2-fold in cells that were treated with 0.2 mM TSA and 5-fold in cells exposed to 0.5 and 2 mM TSA, compared to cells that had not been pre-treated with the histone deacetylase inhibitor. Similarly, exposure of K562 cells to TSA prior to irradiation resulted in dose-dependent radiosensitization. The dose modification factors at D(37) were calculated to be 1.3, 1.6 and 2.5 for cells treated with 0.2, 0.5 and 2 mM TSA, respectively. These findings provide additional evidence that histone deacetylase inhibitors can increase the cytotoxic efficiency of chemotherapeutic drugs, particularly those which target DNA, and can enhance the sensitivity of cells to g-radiation. More generally, the results support the development of histone deacetylase inhibitors as potential clinical chemo- and radio-sensitizers.     

N-ras基因在K562细胞中的表达研究

目的探讨N-ras基因突变及表达水平与慢性粒细胞白血病发病的关系。方法以慢粒细胞株K562细胞为研究对象，采用RT-PCR和直接测序的方法对N-ras的表达水平及突变进行检测。结果RT-PCR结果示N-ras基因在K562细胞中表达升高，直接测序结果表明N-ras基因在K562细胞中不存在突变。结论N-ras基因在K562细胞中高表达，而其高表达机制与突变无关。     

89Sr诱发K562癌细胞凋亡及其相关基因表达研究

目的 探讨^89Sr诱发K562癌细胞凋亡放射毒理效应的分子机理。方法 采用分子病理和定量病理技术研究^89Sr内照射诱导的K562癌细胞凋亡，剂量效应关系和重要相关基因bcl-和bax在其中的表达。结果 K562细胞经^89Sr内照射6和24h，提取DNA，琼脂糖凝胶电泳图谱上出现典型的“阶梯状”区带，伴有“拖尾”，而对照组未见“阶梯状”区带，荧光双标记流式细胞仪法定量检测，^89Sr内照射6，9，12，24和48h后，K562细胞凋亡率和坏死率均较对照组有明显升高（P＜0．01），随着^89Sr内照射时间的延长，累积剂量的提高，均无显著变化（P＞0．05），免疫组织化学染色显示，对照组K562细胞中bcl-2和bax蛋白均有表达，bcl-2蛋白表达阳性细胞率82．8％，bax为44.4%,bcl-2/bax比值1.86;^89Sr内照射24h后的K562细胞中，bcl-2蛋白表达阳性细胞率31.4%,bax为38.2%,bcl-2/bax比值0.82，与对照组相比，差异显著（P＜0．05）；RT－PCR显示bcl-2 mRNA在对照组K56细胞中高表达；^89Sr内照射12h后已有明显的下调，24h后则非常显著。结论 ^89Sr内照射K562细胞后，细胞凋亡与细胞坏死并存，且^89Sr诱导K5622细胞的凋亡可能是通过bcl-2 mRNA及其蛋白表达降低，bcl-2/bax比值下降而调控的。     

As2O3诱导HL-60,K562及NB4细胞的凋亡

目的 :研究三氧化二砷 (As2 O3)对慢性粒细胞K5 6 2 (CML) ,急性粒细胞白血病细胞株HL - 6 0 (AMLM2 )的作用并与细胞株NB4(APLM3)作比较。方法 :采用形态学和流式细胞术观察不同浓度As2 O3 对HL - 6 0 ,K5 6 2及NB4细胞的作用。结果 :As2 O3不仅能诱导NB4的凋亡 ,提高其浓度至 2 .7μM和 8.1μM ,也能诱导K5 6 2 ,HL - 6 0的凋亡。As2 O3 不能诱导三种细胞分化。结论 :As2 O3 对细胞诱导的凋亡是非选择性的且不能诱导分化。     

PTD-OD-HA融合蛋白对K562细胞中Bcr-Ab1的抑制作用观察

目的 探讨PTD-OD-HA融合蛋白转导K562细胞的动力学、定位及其与Bcr-Ab1定位的关系,以及对Bcr-Ab1寡聚化和Bcr-Ab1酪氨酸激酶活性的影响.方法 采用FITC标记PTD-OD-HA融合蛋白,观察蛋白转导K562细胞的效率与剂量和时间的关系,激光共聚焦显微镜检测融合蛋白在细胞内的定位,免疫共沉淀法检测融合蛋白与Bcr-Ab1的相互作用,Western blotting检测Bcr-Ab1的磷酸化水平.结果 PTD-OD-HA转导K562细胞呈剂量和时间依赖性.PTD-OD-HA定位于K562细胞胞质,与Bcr-Ab1共定位,且能与Bcr-Ab1蛋白相互作用,进而干扰Bcr-Ab1同源寡聚化,并可降低Bcr-Ab1的磷酸化水平.结论 在K562细胞中,PTD-OD-HA融合蛋白能有效抑制Bcr-Ab1同源寡聚化及磷酸化水平.