抗三尖杉酯碱的HL60细胞抗粉防己碱诱导的细胞凋亡

目的：用粉防己碱（Ｔｅｔ）研究抗三尖杉酯碱（Ｈａｒ）的ＨＬ６０细胞抗细胞凋亡的机制。 方法：生长抑制，流式细胞术、ＤＮＡ凝胶电泳、蛋白质磷酸化和ＲＮＡ点杂交。 结果：抗性细胞对Ｔｅｔ没有交叉抗性，Ｔｅｔ诱导ＨＬ６０细胞发生凋亡，但不能诱导抗性细胞发生凋亡。Ｔｅｔ引起抗性细胞低于３０ｋＤａ的蛋白质高度磷酸化。Ｔｅｔ和Ｈａｒ诱导ＨＬ６０细胞ｃ－ｍｙｃ ｍＲＮＡ表达增高，而抗性细胞ｃ－ｍｙｃ ｍＲＮＡ表     

抗三尖杉酯碱的人白血病HL60细胞的特性

抗三尖杉酯碱的人白血病ＨＬ６０细胞的特性１何琪杨，周卫东，纪林，张鸿卿，何农高，薛绍白２（北京师范大学生物系，北京１００８７５，中国）关键词三尖杉酯碱类；白血病ＨＬ６０；多种抗药性；ｍｄｒ１基因；糖蛋白类；流式细胞术；聚合酶链反应目的：研究人ＨＬ６...     

粉防己碱和蝙蝠葛碱减低抗三尖杉酯碱的人白血病HL60细胞对阿…

研究粉防己碱和蝙蝠葛碱能否减抵抗三尖杉酯碱的人早幼粒白血病ＨＬ６０抗性株对阿霉素的抗性。细胞计数法和克隆形成法测定药物毒性，流式细胞光度术分析细胞周期变化，荧光法测定Ｄｏｘ含量。     

抗三尖杉酯碱HL－60细胞的抗程序性细胞死亡及其克服

三尖杉酯碱（ｈａｒｒｉｎｇｔｏｎｉｎ，ＨＴ）是中国产植物海南粗榧（ＣｅｐｈａｌｏｔａｘｕｓｈａｉｎａｎｅｎｓｉｓＬｉ）中提取的一种抗肿瘤药物，对急性粒细胞白血病、急性单核细胞白血病有较好疗效（１）。三尖杉酯碱可非常有效地诱导敏感ＨＬ－６０细胞程序性死亡（ａｐｏｐｔｏｓｉｓ，Ａｐｏ）（２，３）。但超过半致死剂量（ＩＣ５０）近百倍的ＨＴ却不能诱导抗三尖杉酯碱细胞ＨＴ１２程序性死亡。如用维拉帕米（ｖｅｒａｐａｍｉｌ，Ｖｐ）１０μｇ·ｍｌ－１逆转多药抗性后，ＨＴ虽可诱导ＨＴ１２细胞程序性死亡，但与敏感细胞相比，出现程序性细胞死亡的时间大大推迟，用药浓度也提高约１０倍。这些结果提示：程序性细胞死亡可能作为肿瘤细胞是否已形成抗药性的标志之一，同时也说明程序性细胞死亡相关因子可能参与肿瘤细胞抗药性的形成。     

抗三尖杉酯碱的人白血病HL60细胞的特性

研究人ＨＬ６０细胞抗三尖杉酯碱的机制。     

环孢菌素A阻断人HL－60抗药性细胞于G_1期而诱导敏感细胞凋亡

进一步研究了抗三尖杉酯碱的ＨＬ－６０细胞（ＨＲ２０）抗细胞凋亡的机制及该抗性和抗药性的关系。结果表明，环孢菌素Ａ（ＣｓＡ）２０，１０μｇ·ｍｌ￣（－１）诱导ＨＬ－６０细胞发生凋亡，而阻断ＨＲ２０细胞于Ｇ＿１期，就不能诱导细胞发生凋亡。低浓度的ＣｓＡ明显增加柔红霉素在ＨＲ２０细胞内的积聚，其逆转抗药性作用与阻断细胞周期运行无关。ＣｓＡ１０μｇ·ｍｌ￣（－１）处理ＨＲ２０细胞，可引起５０ｋＤａ的蛋白质高度磷酸化。结果提示：环孢菌素Ａ阻断抗三尖杉酯碱的ＨＬ－６０细胞于Ｇ＿１期，而诱导敏感的ＨＬ－６０细胞发生凋亡，其阻断作用与抗药性无关     

人白血病HL60细胞的分化状态对细胞凋亡的影响

用细胞培养和流式细胞术等方法，研究人白血病ＨＬ６０细胞诱导分化后，对三尖杉酯碱（Ｈａｒ）和喜树碱（Ｃａｍ）诱导细胞凋亡的影响。结果表明，１２豆蔻酰及１３乙酸佛波酯以１６ｎｍｏｌ·Ｌ－１浓度处理ＨＬ６０细胞２４ｈ，细胞向单核／巨噬细胞方向分化，阻断于Ｇ１期；分化细胞抗Ｈａｒ和Ｃａｍ诱导的细胞凋亡，但其ｃｍｙｃ基因的表达无变化。１４％二甲基亚砜处理ＨＬ６０细胞４８ｈ，细胞向粒细胞方向分化，阻断于Ｇ１期；分化细胞抗Ｃａｍ，而不抗Ｈａｒ诱导的细胞凋亡；分化细胞的ｃｍｙｃ基因表达明显下降。结果提示，人白血病ＨＬ６０细胞的分化状态，明显影响三尖杉酯碱和喜树碱诱导的细胞凋亡，但可能与ｃｍｙｃ基因的表达变化无关     

十四酰佛波乙酸酯抑制三尖杉酯碱诱导的人白血病HL—60细胞凋亡

目的：研究十四酰佛波乙酸酯（ＴＡ）预处理后，三尖杉酯碱（Ｈａｒ）和喜树碱（Ｃａｍ）诱导人白血病ＨＬ－６０细胞凋亡的变化。方法：染色质凝集观察，流式细胞术，ＤＮＡ琼脂糖凝胶电泳和点杂交。结果：ＴＡ２００ｎｍｏｌ·Ｌ＾－１预处理ＨＬ－６０细胞６ｈ，明显抑制Ｈａｒ０．１ｍｇ·Ｌ＾－１作用３ｈ诱导的细胞凋亡，但只部分抑制Ｃａｍ０．２ｍｇ·Ｌ＾－作用３ｈ诱导的细胞凋亡。ＴＡ预处理ＨＬ－６０细胞明显降低ｃ－ｍ     

尿,血,细胞匀浆中高三尖杉酯碱的高效液相色谱测定法

尿、血、细胞匀浆中高三尖杉酯碱的高效液相色谱测定法山东省劳动卫生职业病防治研究所济南２５０００１邵华三尖杉科三尖杉属植物为我国特有，高三尖杉酯碱（以下简称ＨＨＴ）是其中分离出来的－种抗癌药物，是治疗急性非淋巴细胞白血病的有效药物。目前，国内外对该药的...     

用改进逆流分配法分离三尖杉酯碱和高三尖杉酯碱

对三尖杉酯碱和高三尖杉酯碱混合物的分离利用经改进的逆流分配法，用经ｐＨ５缓冲液饱和的氯仿作流动相，经氯仿饱和的ｐＨ５磷酸氢二钠－枸橼酸作固定相，醋酸乙酯∶丙酮（６∶２．５）作展开剂，碱性硅胶板作薄层板，经对５批混合物进行分离，均分别获得三尖杉酯碱和高三尖杉酯碱的白色结晶。各项指标检查均符合标准。     

人白血病HL60细胞的分化状态对细胞凋亡的影响

用细胞培养和流式细胞术等方法,研究人白血病HL60细胞诱导分化后,对三尖杉酯碱(Har)和喜树碱(Cam)诱导细胞凋亡的影响。结果表明,12-豆蔻酰及13-乙酸佛波酯以16nmol·L^-1浓度处理HL60细胞24h,细胞向单核/巨噬细胞方向分化,阻断于G_{1期;分化细胞抗Har和Cam诱导的细胞凋亡,但其c-myc基因的表达无变化。1.4%二甲基亚砜处理HL60细胞48h,细胞向粒细胞方向分化,阻断于G1期;分化细胞抗Cam,而不抗Har诱导的细胞凋亡;分化细胞的c-myc基因表达明显下降。结果提示,人白血病HL60细胞的分化状态,明显影响三尖杉酯碱和喜树碱诱导的细胞凋亡,但可能与c-myc基因的表达变化无关。}     

粉防己碱和蝙蝠葛碱减低抗三尖杉酯碱的人白血病HL60细胞对阿霉素的抗性(英文)

目的:研究粉防己碱(Tet)和蝙蝠葛碱(Dau)能否减低抗三尖杉酯碱(Har)的人早幼粒白血病HL60抗性株对阿霉素(Dox)的抗性。方法:细胞计数法和克隆形成法测定药物毒性,流式细胞光度术分析细胞周期变化,荧光法测定Dox含量。结果:无细胞毒性的Tet和Dau明显地增强Dox对HL60抗性细胞的生长抑制作用,使克隆形成率从Dox单药的60％分别降低到0.2％,9.2％,使阻断在G_2M期的抗性细胞增多,但Tet和Dau不增强Dox对敏感的HL60细胞的毒性。Tet使胞内Dox积聚增加。结论:Tet和Dau减低抗Har的HL60细胞对Dox的抗性,其机制是增加Dox在细胞内积聚。     

人早幼粒白血病HL60细胞及其亚系中mdr－1基因和癌基因表达的关系

人早幼粒白血病ＨＬ６０细胞及其亚系中ｍｄｒ１基因和癌基因表达的关系周卫东，张鸿卿，方敏，薛绍白（北京师范大学生物系，北京１００８７５，中国）关键词癌基因；多种抗药性；流式细胞计量术；急性早幼粒白血病目的：研究人早幼粒白血病ＨＬ６０细胞系及其分别抗Ｈ...     

Anthraquinone antitumour agents, doxorubicin, pirarubicin and benzoperimidine BP1, trigger caspase-3/caspase-8-dependent apoptosis of leukaemia sensitive HL60 and resistant HL60/VINC and HL60/DOX cells

We examined the effect of selected anthraquinone antitumour agents - doxorubicin (DOX), pirarubicin (PIRA) and benzoperimidine BP1 - on inducing apoptosis of the sensitive leukaemia HL60 cell line and its multidrug resistance sublines overexpressing P-glycoprotein (HL60/VINC) and multidrug resistance-associated protein 1 (HL60/DOX). All agents used at IC50 and IC90 were able to influence the cell cycle of sensitive HL60 and resistant cells and induce apoptosis. Interestingly, it was seen that HL60/VINC cells were more susceptible to undergo caspase-3/caspase-8-dependent apoptosis induced by the studied anthraquinone compounds compared with HL60 and HL60/DOX cells. However, the examined agents did not change the expression of Fas receptors on the surface of HL60-sensitive and-resistant cells.     

Role of Ras‐related C3 botulinum toxin substrate 2 (Rac2), NADPH oxidase and reactive oxygen species in diallyl disulphide‐induced apoptosis of human leukaemia HL‐60 cells

1. Diallyl disulphide (DADS) has potential as a chemopreventive and therapeutic agent. Previous studies have reported that Ras‐related C3 botulinum toxin substrate 2 (Rac2), a regulatory subunit of the NADPH oxidase complex, is upregulated in DADS‐induced apoptosis in human leukaemia HL‐60 cells. The aim of the present study was to investigate the role of Rac2, NADPH oxidase and reactive oxygen species (ROS) in DADS‐induced apoptosis. 2. Expresssion of the Rac2 gene along with that of five other genes of NADPH oxidase subunits were in HL‐60 cells measured by Sybergreen quantitative real‐time polymerase chain reaction. RNA interference was used to test the effect of Rac2. Protein expression was evaluated using western blot analysis and ROS levels were measured by 2′,7′‐dichlorofluorescein diacetate (DCFH‐DA) fluorescence. DNA fragmentation and flow cytometry analysis were used to detect apoptotic cells. 3. Levels of Rac2 gene and protein were significantly upregulated and NADPH oxidase was activated in DADS‐induced apoptosis. Pretreatment of HL‐60 cells with small interfering (si) RNAs to inhibit Rac2 blocked DADS‐induced apoptosis. Diallyl disulphide‐induced intracellular ROS production was increased in phorbol myristate acetate‐stimulated cells, but decreased in Rac2 siRNA‐treated cells. In Rac2 siRNA‐treated cells, activator protein‐1 and caspase 3 levels decreased, c‐myc protein levels were increased and p38 protein levels were unchanged compared with Rac2‐competent, DADS‐treated cells. 4. These results demonstrate that NADPH oxidase is the main source of DADS‐induced ROS. In addition, Rac2 selectively activates the c‐Jun N‐terminal kinase pathway, but not the p38 pathway, in DADS‐induced apoptosis. So, Rac2, NADPH oxidase and ROS have a critical role in DADS‐induced apoptosis in human leukaemia HL‐60 cells.     

不同靶点的反义bcl—2寡核苷酸对白血病细胞足叶乙甙敏感性影响的研究

目的探讨针对bcl-2mRNA蛋白编码区的反义寡核苷酸对足叶乙甙诱导HL-60和K562细胞凋亡的影响。方法应用MTT法和流式细胞仪检测反义寡核苷酸与足叶乙甙联合作用的HL60和K562细胞中bcl-2蛋白表达和细胞凋亡及足叶乙甙IC50值。结果10μmol     

聚酯型儿茶素对HL-60细胞的诱导分化作用及其机制研究

目的　研究聚酯型儿茶素对人急性早幼粒白血病HL 6 0细胞的分化作用并探讨其作用机制。方法　应用电镜、NBT还原试验、同位素掺入试验、细胞内cAMP/cGMP浓度测定、原位杂交和免疫细胞化学等方法研究细胞分化及机制。结果　聚酯型儿茶素作用于HL 6 0细胞后 ,扫描电镜和透射电镜观察显示分化细胞的特征 ;NBT还原能力明显增强 ;[3H] TdR ,[3H] Leu掺入试验证明聚酯型儿茶素可明显抑制细胞DNA和蛋白质合成 ;聚酯型儿茶素作用后 ,细胞内cAMP浓度及cAMP/cGMP比值明显增加 ,HL 6 0细胞的c myc基因mRNA和蛋白产物的表达均明显降低 ,而c fos基因mRNA和蛋白产物的表达均明显升高。结论　聚酯型儿茶素可诱导HL 6 0细胞向成熟细胞分化 ,其分化作用可能与其升高细胞内cAMP/cGMP比值和抑制c myc基因表达、增强c fos基因表达有关。本研究为聚酯型儿茶素在肿瘤防治领域的开发应用提供了充分的理论依据     

Mechanisms of multidrug resistance in HL60 cells. Analysis of resistance associated membrane proteins and levels of mdr gene expression

HL60 cells isolated for resistance to Adriamycin do not contain P-glycoprotein, as determined with immunological probes. These cells, however, are multidrug resistant and defective in the cellular accumulation of drug. In view of these findings, we have examined in greater detail certain properties of the HL60/Adr cells and have compared these properties to an HL60 drug-resistant isolate (HL60/Vinc) which contains high levels of P-glycoprotein. The results of these studies demonstrated that verapamil induces a major increase in cellular drug accumulation in both HL60/Adr and HL60/Vinc isolates. An 125I-labeled photoaffinity analog of verapamil labeled P-glycoprotein contained in membranes of HL60/Vinc cells. In contrast, this agent did not label any protein selectively associated with drug resistance in membranes of the HL60/Adr isolate. The photoactive dihydropyridine calcium channel blocker [3H]azidopine and [125I]NASV, a photoaffinity analog of vinblastine, labelled P-glycoprotein in membranes from HL60/Vinc cells, whereas in experiments with the HL60/Adr isolate there was no detectable labeling of a drug resistance associated membrane protein. Additional studies have been carried out to analyze membrane proteins of HL60/Adr cells labeled with the photoaffinity agent 8-azido-alpha-[32P]ATP (AzATP32). The results demonstrate that this agent labeled a resistance associated membrane protein of 190 kilodaltons (P190). P190 is essentially absent in membranes of drug-sensitive cells. Labeling of P190 with AzATP32 in membranes of resistant cells was blocked completely when incubations were carried out in the presence of excess unlabeled ATP. Additional studies were carried out to analyze mdr gene amplification and expression in sensitive and resistant cells. Experiments carried out with human 5',mdr1 (1.1 kb) and mdr3 (1.0 kb) cDNAs demonstrate that both of these sequences were highly amplified in the HL60/Vinc isolate. Only the mrd1 gene sequence however, was overexpressed. In contrast, there was no detectable amplification or overexpression of mdr1 or mdr3 sequences in HL60/Adr cells. The results of this study thus identify a new nucleotide binding protein which is overexpressed in membranes of HL60 cells isolated for resistance to Adriamycin. P190, which exhibits properties distinct from P-glycoprotein, possibly functions in the energy-dependent drug efflux system contained in the HL60/Adr resistant isolate.     

Butein,a Plant Polyphenol,Induces Apoptosis Concomitant with Increased Caspase‐3 Activity,Decreased Bcl‐2 Expression and Increased Bax Expression in HL‐60 Cells

Abstract: In the present study we have investigated whether butein could induce apoptosis in human leukaemic HL‐60 cells. The treatment of HL‐60 cells with butein induced apoptotic cell death as determined by morphological and biochemical changes. Apoptotic DNA fragments in the butein‐treated HL‐60 cells were increased gradually as determined by flow cytometric analysis. The caspase‐3 activity was increased during butein‐induced apoptosis. However, caspase‐3 inhibitor abrogated the butein‐induced DNA fragmentation. Furthermore, the treatment of HL‐60 cells with butein decreased the expression of Bcl‐2 protein, but increased the expression of Bax protein. These results suggest that butein‐induced apoptosis is mediated through the activation of caspase‐3 and it is associated with changed expression of Bcl‐2 and Bax proteins.