Effects of trichostatin A, a histone deacetylase inhibitor, on the regulation of apoptosis in H-ras-transformed breast epithelial cells

This study examined the mechanism for the anti-cancer effects of histone deacetylase (HDAC) inhibitor trichostatin A (TsA) in H-ras-transformed human breast epithelial (MCF10A-ras) cells. The effects of TsA on anti-cancer effects of MCF10A-ras cells were determined by measuring the level of cell cycle regulator expression and apoptotic cell death using Western blotting and flow cytometry analysis, respectively. TsA induced morphological changes, apoptotic cell death and modulation of the cell cycle regulatory proteins in the MCF10A-ras cells. TsA increased the levels of acetylated histone H3 and H4 in MCF10A-ras cells. In addition, TsA markedly down-regulated the expression of cyclin D1 and CDK4, up-regulated the expression of p21WAF1 and p53 and induced cell cycle arrest at the G1 phase in MCF10A-ras cells. The levels of hyperphosphorylation of the Rb protein were lower in MCF10A-ras cells after the TsA treatment. Furthermore, the up-regulation of p53 promoted Bax expression, which led to the activation of pro-caspase-3 and eventually to apoptosis in MCF10A-ras cells. TsA significantly increased the levels of ERK1/2 phosphorylation in MCF10A-ras cells. Overall, the TsA-activated ERK pathway plays an important role in cell cycle arrest and apoptosis through the ERK-dependent induction of p21 in Ras-related human cancer cells.     

叠氮胸苷对人胶质母细胞瘤细胞株端粒酶活性、增殖和凋亡的影响

目的 探讨叠氮胸苷(AZT)对TJ905 人胶质母细胞瘤细胞株端粒酶活性、增殖和凋亡的影响及其机制.方法 体外培养TJ905细胞分别经50、100及200 μmol/L叠氮胸苷处理24 h后,用端粒重复扩增法检测端粒酶活性及集落形成效率;继续培养并维持相应叠氮胸苷浓度,取第1、3和6代细胞,利用Western blot检测cyclin A表达水平,用流式细胞术检测细胞周期分布并结合单细胞凝胶电泳检测细胞凋亡,用Ki-67免疫细胞化学染色检测细胞增殖活性.结果 叠氮胸苷可抑制TJ905细胞的端粒酶活性.50和100 μmol/L组cyclin A表达水平均显著低于对照组(P<0.01),并均呈时间和剂量依赖性降低.三个用药组的Go/G1期细胞均显著少于对照组(P<0.05～0.01),S期细胞均显著多于对照组(P<0.05～0.01);在各用药组第1代GO/G1期细胞减少和S期细胞增加均呈剂量依赖性;各用药组S期细胞增加均呈显著的时间依赖性,而Go/G1期细胞减少仅在50 μmol/L组呈时间依赖性.各用药组第1和6代的凋亡细胞数均显著多于对照组(P<0.05～0.01);各用药组第6代的凋亡细胞数随用药浓度升高而相应增加(P<0.05～0.01),且均多于同浓度组的第1和第3代(P<0.05～0.01).各用药组集落形成效率及Ki-67阳性细胞数均显著低于对照组(P<0.01),二者还随用药浓度增加和(或)作用时间延长相应减少.对照组各代间以上各指标的差异均无统计学意义(P>0.05).结论 叠氮胸苷可通过抑制端粒酶活性抑制TJ905细胞cyclin A表达,阻止该细胞从S期向G2期过渡,发挥其抑制增殖和诱导凋亡的作用.     

Pim-1激酶抑制剂SMI-4a抑制U937细胞增殖并诱导凋亡

目的:研究丝/苏氨酸蛋白激酶Pim-1抑制剂SMI-4a对人类急性髓系白血病细胞株U937的生长抑制、促凋亡作用及其可能机制。方法:CCK-8法检测不同浓度SMI-4a作用不同时间对U937细胞的生长抑制率;Annexin V-PI及Hoechst 33342染色法检测SMI-4a作用前后细胞凋亡情况,集落形成实验检测SMI-4a对U937细胞集落形成能力的影响;Western blot法检测SMI-4a对U937细胞核及细胞浆内β-catenin表达变化及细胞内凋亡相关蛋白的表达变化;免疫荧光法检测β-catenin在细胞内的表达变化。结果:CCK-8结果显示SMI-4a可以抑制U937细胞的活力,并呈时间和剂量依赖性;Annexin V-PI及Hoechst 33342染色结果显示SMI-4a可以促进U937细胞凋亡;集落形成实验证实SMI-4a可以抑制U937细胞的集落形成能力;Western blot实验结果显示SMI-4a作用于U937细胞48 h后细胞浆内的β-catenin表达增加,细胞核内的β-catenin表达减少,细胞内促凋亡蛋白Bax和PARP表达增强,抑凋亡蛋白Bcl-2表达明显减弱;免疫荧光进一步验证了SMI-4a作用后的U937细胞核内的β-catenin表达量明显减少。结论:SMI-4a诱导U937细胞凋亡是通过上调促凋亡基因的表达、下调凋亡抑制基因的表达来实现的。     

神经酰胺介导粒细胞凋亡机理初探

目的 观察神经酰胺对ＨＬ－６０及中性粒细胞（ＰＭＮ）凋亡产生的影响,探讨其在粒细胞发生过程中可能的调节作用。方法：采用ＤＮＡ凝胶电泳,流式细胞术,光镜及ＲＴ－ＰＣＲ等技术对细胞进行凋亡鉴定及相关基因ｂｃｌ－２,ｃ－ｍｙｃ,ｍＲＮＡ表达分析。结果 神经酰胺（ｃ２－ｃｅｒａｍｉｄｅ）肿瘤坏死因子（ＴＮＦα）及长春新碱（Ｖｉｎｃｒｉｓｔｉｎｅ）均可不同程度地诱导ＨＬ－６０及成熟ＰＭＮ产生凋亡,ＣＰＫ激活     

三氧化二砷对K562细胞端粒酶活性及P53蛋白表达水平的调节

目的：研究三氧化二砷(As2O3)诱导急性白血病K562细胞的凋亡机制。方法：以不同浓度的As2O3作用于体外培养的K562细胞，检测细胞生长抑制率，观察细胞凋亡时的形态学变化，对细胞端粒酶活性及P53蛋白的表达水平进行检测。结果：As2O3可显著地降低K562细胞端粒酶活性，升高P53蛋白的表达水平，抑制细胞的生长，诱导细胞发生凋亡。并呈现出明显的量-效与时-效关系。结论：As2O3能抑制K562细胞的生长并诱导细胞发生凋亡，降低细胞端粒酶活性及升高P53蛋白的表达水平是其重要作用机制之一。     

A novel histone deacetylase inhibitor, Scriptaid, induces growth inhibition, cell cycle arrest and apoptosis in human endometrial cancer and ovarian cancer cells

Histone deacetylase inhibitors (HDACIs) can inhibit cell proliferation, induce cell cycle arrest, and stimulate the apoptosis of cancer cells. We investigated the effects of a novel HDACI, Scriptaid, on the Ishikawa endometrial cancer cell line, SK-OV-3 ovarian cancer cell line, and normal human endometrial epithelial cells. Endometrial and ovarian cancer cells were treated with various concentrations of Scriptaid, and its effect on cell growth, cell cycle, apoptosis, and related measurements was investigated. 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays showed that all endometrial and ovarian cancer cell lines were sensitive to the growth inhibitory effect of Scriptaid, although normal endometrial epithelial cells were viable after treatment with the same doses of Scriptaid that induced the growth inhibition of endometrial and ovarian cancer cells. Cell cycle analysis indicated that their exposure to Scriptaid decreased the proportion of cells in the S phase and increased the proportion in the G0/G1 and/or G2/M phases of the cell cycle. Induction of apoptosis was confirmed by annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with the altered expression of genes related to cell growth, malignant phenotype, and apoptosis. Furthermore, Scriptaid treatment of these cell lines increased acetylation of H3 and H4 histone tails. These results raise the possibility that Scriptaid may prove particularly effective in the treatment of endometrial and ovarian cancers.     

顺铂对葡萄膜黑色素瘤细胞端粒酶活性的抑制

目的 研究顺铂对人葡萄膜黑色素瘤细胞端粒酶活性的抑制作用,为临床治疗人葡萄膜黑色素瘤提供理论依据.方法 采用特定时间下不同浓度以及特定浓度下不同作用时间的端粒酶抑制剂顺铂作用于体外培养的人黑色素瘤细胞.采用多聚酶链反应--酶联免疫吸附测定(PCR-ELISA)及聚丙烯酰胺凝胶电泳法(PCR-PAGE)测定细胞中端粒酶活性的变化.结果 作用72 h,端粒酶活性在顺铂浓度达到0.10mg/L后开始下降,当浓度达到1.00mg/L后其活性下降至(0.173±0.007).当顺铂浓度固定10.00 mg/L,顺铂作用时间达到24 h后开始出现抑制作用,48 h时达到抑制高峰,端粒酶活性下降至(0.276±0.024).随着顺铂浓度的增加及作用时间的延长,端粒酶活性逐渐下降(P＜0.05).PCR-PAGE显示顺铂浓度增加及作用时间延长,端粒酶活性的显色条带越来越少.结论 顺铂可有效降低体外培养的人眼葡萄膜黑色素瘤细胞端粒酶活性,并呈浓度和时间依赖性.     

Differential expression of bcl-2 and bax genes during the E.coli-induced apoptosis of human U937 cells

Theimportantroleofapoptoticcelldeathindif ferentdiseasesandphysiologicalconditionhas beendemonstratedinmanystudies,andmany factorsinvolvedinthedeathsignalingpathways havebeendefined[1],inwhichbcl2geneand othermembersofthisgenefamilywereprovedto playanimportantroleinembryogenesis,tissue remodelingandimmuneresponsesbytheiractions servingaseitherinhibitorsorpromotersofapopto sis[2].Usually,Bcl2processesanti apoptotic effectandBaxisalsoonememberoftheBcl2family[3].Previousworkshaddocumentedthe piv…     

替米沙坦抑制U937细胞增殖及诱导凋亡

目的:探讨替米沙坦对U937细胞株的生长抑制及凋亡诱导作用。方法:分别以不同浓度的替米沙坦处理人类急性髓系白血病细胞U937;以CCK-8法检测不同浓度替米沙坦对U937细胞的生长抑制作用;以集落形成实验观察不同浓度替米沙坦对U937细胞集落形成能力的影响;以Annexin V-PI双染法及Hoechst 33342染色法检测不同浓度替米沙坦作用前后U937细胞凋亡程度的变化;以流式细胞术检测U937细胞表面抗原CD11b的阳性表达率,瑞氏染色后倒置显微镜进行细胞形态学观察,了解U937细胞的分化情况;以Western blot法检测不同浓度替米沙坦作用U937细胞后凋亡相关蛋白表达量的改变。结果:CCK-8实验结果证实替米沙坦呈时间和剂量依赖性抑制U937细胞的生长;集落形成实验显示低剂量替米沙坦可以完全抑制U937细胞的集落形成能力;Annexin V-PI双染法及Hoechst 33342法结果证实替米沙坦可以诱导U937细胞凋亡;细胞表面抗原流式检测术及瑞氏染色结果证实替米沙坦可以促进部分U937细胞分化;Western blot实验结果证实替米沙坦作用于U937细胞72 h后,促凋亡相关蛋白cleaved PARP及cleaved caspase-3蛋白的水平明显增高。结论:替米沙坦可以抑制细胞增殖以及诱导U937细胞部分分化,并通过caspase依赖的凋亡途径触发U937细胞凋亡。     

Epigallocatechin gallate inhibits nitric oxide-induced apoptosis in rat PC12 cells

Nitric oxide (NO) is associated with many pathophysiology of the central nervous system including brain ischemia, neurodegeneration and inflammation. Epigallocatechin gallate (EGCG) is a major compound of green tea polyphenol that has shown the protective activity against neuronal diseases. This study examined the effect of EGCG on NO-induced cell death in PC12 cells. The administration of sodium nitroprusside (SNP), a NO donor, decreased the cell viability and induced apoptosis showing characterization such as cell shrinkage and chromatin condensation as well as subG1 fraction of cell cycles. EGCG inhibited the cytotoxicity and apoptotic morphogenic changes induced by SNP. EGCG attenuated the production of reactive oxygen species (ROS) by SNP, and ameliorated the SNP-induced Bax to Bcl-2 expression ratio leading to apoptosis. In addition, EGCG prevented the release of cytochrome c from the mitochondria into the cytosol as well as the upregulation of the voltage-dependent anion channel (VDAC), a cytochrome c releasing channel, in the mitochondria of SNP-treated cells. EGCG abrogated the activation of caspase-9, caspase-8 and caspase-3 induced by SNP. These results demonstrate that EGCG has a protective effect against SNP-induced apoptosis in PC12 cells by scavenging ROS and modulating the signal molecules associated with cytochrome c, caspases, VDAC and the Bcl-2 family. These findings suggest that EGCG might be a natural neuroprotective substance.     

Apicidin, a novel histone deacetylase inhibitor, has profound anti-growth activity in human endometrial and ovarian cancer cells

Histone deacetylase inhibitors (HDACIs) can inhibit proliferation, induce cell cycle arrest and stimulate apoptosis of cancer cells. Our purpose was to investigate the antiproliferative effects of a novel HDACI, apicidin, on the Ishikawa endometrial cancer cell line, the SK-OV-3 ovarian cancer cell line and normal human endometrial epithelial cells. Endometrial and ovarian cancer cells were treated with various concentrations of apicidin, and the effects on cell growth, cell cycle, apoptosis and related measurements were investigated. MTT assays showed that all endometrial and ovarian cancer cell lines were sensitive to the growth inhibitory effect of apicidin, although normal endometrial epithelial cells were viable after the treatment with the same doses of apicidin that induced the growth inhibition of endometrial and ovarian cancer cells. Cell cycle analysis indicated that their exposure to apicidin decreased the proportion of cells in S-phase and increased the proportion in G0/G1 and/or G2/M phases of the cell cycle. Induction of apoptosis was confirmed by Annexin V staining of externalized phosphatidylserine and loss of the transmembrane potential of mitochondria. This induction occurred in concert with the altered expression of p21WAF1, p27KIP1, p16, cyclin A, and E-cadherin. Furthermore, apicidin treatment of these cell lines increased acetylation of H3 and H4 histone tails. These results suggest that apicidin exhibits the antiproliferative effects through selective induction of genes related to cell growth, malignant phenotype, and apoptosis. The findings raise the possibility that apicidin may prove particularly effective in the treatment of endometrial and ovarian cancers.     

Enforced expression of the apoptosis inhibitor Bcl-2 ablates tolerance induction in DNA-reactive B cells through a novel mechanism

How self tolerance is maintained during B cell development in the bone marrow has been a focal area of study in immunology. Receptor editing, anergy and clonal deletion all play important roles in the regulation of autoimmunity in the immature population. The mechanisms of tolerance induction in the periphery, however, are less well characterized. Overexpression of the apoptosis inhibitor Bcl-2 rescues autoreactive B cells from deletion and can contribute to the development of autoimmune disease in certain genetic backgrounds. Using a peptide-induced autoimmunity model, we recently identified a peripheral tolerance checkpoint in antigen-activated B cells that have undergone class switching and somatic hypermutation. At this checkpoint, receptor editing, induced by antigen engagement, dampened the autoantibody response. In this study, we show that receptor editing fails to be induced in antigen-activated DNA-reactive B cells that overexpress Bcl-2 (Bcl-2 Tg). The failure to induce RAG and receptor editing is likely due, at least partially, to the lack of self antigen. First, the levels of circulating DNA and of apoptotic bodies in the spleen of Bcl-2 Tg mice are significantly lower than in control mice. Second, in Bcl-2 Tg mice, RAG can be induced in a population of antigen-activated B cells by providing exogenous soluble antigen. These data suggest that, in addition to its anti-apoptotic activity, Bcl-2 may indirectly inhibit tolerance induction in B cells acquiring anti-nuclear antigen reactivity after peripheral activation by limiting the availability of self antigen.     

淫羊藿甙抑制肿瘤细胞端粒酶活性及其调节机制的研究

目的 :探讨中药单体淫羊藿甙 (ICA)抑制肿瘤HL 6 0细胞端粒酶活性及作用机制。方法 :采用MTT法 ,NBT染色法 ,TRAP PCR ,RT PCR法和流式细胞术。结果 :ICA显著抑制HL 6 0细胞端粒酶活性 ,且端粒酶活性下降与细胞表面粒细胞分化抗原CD11b表达率呈负相关 ;诱导HL 6 0细胞向粒细胞方向分化 ;改变HL 6 0细胞周期各时相的分布 ,表现为G0 /G1期细胞逐渐增多 ,S期细胞逐渐减少 ;上调分化相关基因p2 1、下调增殖相关基因c mycmRNA和蛋白表达水平。结论 :阐明了ICA显著抑制HL 6 0细胞端粒酶活性 ,并从基因 蛋白 细胞效应水平揭示了其调节端粒酶活性的可能机制。     

A role of inhibitor of apoptosis (IAP) proteins in increased lymphocyte apoptosis in aged humans

Lymphocytes from aged humans show increased death-receptor-mediated apoptosis, which is associated with an increased and early activation of caspases. Inhibitor of apoptosis (IAP) proteins inhibit apoptosis by inhibiting activation and activity of caspases. Therefore, we examine the expression of two of the IAPs, the cIAP-2 and XIAP in lymphocytes from young and aged subjects by Western blotting. Lymphocytes from aged expressed significantly less cIAP2 whereas no difference was observed in XIAP expression between young and aged subjects. These data may suggest that decreased cIAP2 may play a role in increased apoptosis in aged humans. Possible mechanisms for the regulation of IAPs in aging are discussed.     

Detection of telomerase activity in human cells and tumors by a telomeric repeat amplification protocol (TRAP)

The association of human telomerase activity with an indefinite replicative capacity of cells in vitro and advanced tumors in vivo is gaining wide support. The increasing interest in studying various aspects of telomerase expression in cancer required the development of a sensitive and reliable protocol for the extraction and detection of telomerase activity in cell culture material, and from small tissue samples obtained from biopsy, surgical reaction of tumors, and autopsy. Recently a novel procedure for the extraction and detection of telomerase activity was developed (Science 1994; 266: 2011–2015) which resulted in an estimated 10⁴ fold improvement in detectability compared with previous methods. The described procedures not only dramatically increased sensitivity but also allowed fast and efficient detection of telomerase activity in a large number of samples. A number of technical aspects which are of eritical importance for reproducibility and reliability of this assay using clinical material are addressed in this report. In addition, new methods to perform telomerase assays without the use of radioisotopes are described.Abbreviations AEBSF 4-(2-aminoethyl)-benzenesulfonyl fluoride hydrochlorine - BCA bieinchoninic acid - CCD camera charged coupled device camera - CHAPS 3-[(3-cholamidopropyl)-dimethyl-ammonio]-l-propanesulfonate - DEPC diethyl pyrocarbonate - DPBS Dulbecco's phosphate buffered saline - EGTA ethylene glycol-bis(ß-aminoethyl ether)-N,N,N',N'-tetraacetic acid - HPLC high pressure liquid chromatography - HPV human papillomavirus - PDA piperazine diacrylamide - PCR polymerase chain reaction - T4g32 protein T4 gene 32 protein - TRAP telomeric repeat amplification protocol - TRF terminal restriction fragment     

Expression and localization of inhibitor of apoptosis proteins in normal human tissues

The family of inhibitor of apoptosis (IAP) proteins can suppress apoptosis induced by a variety of triggers. Among the IAPs, cIAP1, cIAP2, and XIAP have been characterized as inhibitors of specific caspases, and their expression, together with that of survivin, has been shown in several studies to play a role as tumor marker and prognostic factor for the survival of patients with cancer. Although survivin is usually not expressed in normal adult tissues, cIAP1, cIAP2, and XIAP have been found broadly expressed at messenger RNA level within normal cells. Here, we report an immunohistochemical study in a comprehensive panel of normal human tissues, and we confirm at the protein level the wide expression of IAPs. These results are consistent with a physiological role of IAPs in normal cells. Moreover, we show that IAPs' expression levels and localization patterns differ depending on the cell lineage. The variable subcellular localization of the IAPs within different cell types suggests that compartmentalization may contribute to regulate their function. The physiological role of these proteins should be further investigated to help tailor IAP-targeted therapeutic strategies for patients with cancer and circumvent possible side effects.     

槲皮素对U937细胞系抑制增殖和诱导凋亡作用的研究

目的 探讨黄酮类化合物槲皮素（Que）对人类单核细胞白血病U937细胞系的抑制增殖和诱导凋亡的作用。方法 应用MTT法检测不同浓度槲皮素对U937细胞的增殖抑制作用；AO／PI荧光染色后倒置荧光显微镜下观察细胞形态学变化；琼脂糖凝胶电泳测定细胞DNA的片段化；应用流式细胞仪检测细胞凋亡率及细胞周期分布。结果槲皮素能明显抑制U937细胞增殖，并存在剂量-效应关系和时间-效应关系；诱导U937细胞出现凋亡所具有的形态学和生化特征；随着槲皮素浓度升高，凋亡细胞和坏死细胞比例增加；将细胞特异性地阻滞在S期，出现凋亡峰。结论 槲皮素能抑制U937细胞增殖，诱导细胞凋亡，并具有细胞周期特异性。