IL——3和羟基脲协同诱导K562细胞凋亡

The effect of IL-3 and hydroxyurea on human erythroleukemia cell line (K562 cells) was demonstrated by using the electro-microscopy and flow cytometry. Our data showed that neither IL-3 nor hydroxyurea could induce the apoptosis of K562 cells alone. However, the IL-3 and hydroxyurea could induce the apoptosis of K562 cells cooperatively. Analysis with flow cytometry showed that the percentage of apoptotic cells was about 31.90% after K562 cells were induced by IL-3 and hydroxyurea cooperatively for 5 days, and the sub-G1 peak (apoptotic peak) was detected in the induced K562 cells. Meanwhile, the percentage of S-phase in the IL-3 and hydroxyurea induced K562 cells was increased, and the proliferation of the induced K562 cells was inhibited significantly. Furthermore, the IL-3 and hydroxyurea induced K562 cells showed chromatin condensation with regular crescents at the nuclear edges and apoptotic bodies. It suggested that IL-3 could enhance the sensitivity of K562 cells to hydroxyurea and the apoptosis of K562 cells could be induced by IL-3 and hydroxyurea cooperatively.     

IL-3和羟基脲协同诱导K562细胞凋亡

本文应用流式细胞分选仪和电子显微镜研究了IL-3和羟基脲对人红白血病细胞株(K562细胞)凋亡的影响.结果显示IL-3和羟基脲分别诱导K562细胞,不能引起细胞凋亡;而IL-3和羟基脲协同诱导K562细胞,可以引起细胞凋亡.用流式细胞仪检测到IL-3和羟基脲协同诱导K562细胞后,DNA含量低于二倍体的细胞数达31.90%,并产生明显的凋亡小峰.同时,IL-3和羟基脲协同诱导K562细胞,可抑制细胞周期中的S期,阻止细胞从S期进入G2/M期,使细胞周期延长,对K562细胞的生长和增殖具有抑制作用.在电镜下可观察到IL-3和羟基脲协同诱导的K562细胞,出现典型的凋亡细胞形态,细胞核内染色质浓缩、凝聚,紧靠在核膜边沿,形成新月形或环状的染色质结构,产生凋亡小体.提示IL-3和羟基脲具有协同效应,IL-3可提高K562细胞对羟基脲的敏感性,并可协同羟基脲诱导K562细胞凋亡.     

Gamma irradiation induced apoptotic changes in the chromatin structure of human erythroleukemia K562 cells

Exponentially growing human erythroleukemia K562 cells were synchronized by centrifugal elutriation prior to and after Co⁶⁰ γ-irradiation (4 Gy). Forward scatter flow cytometry used for size analysis revealed the increase of an early apoptotic cell population ranging from lower (0.05 C-value) to higher DNA content (∼1 C) as the cells progressed through the S phase. The increase in cellular DNA content expressed in C-values correlated with apoptotic chromatin changes manifested as many small apoptotic bodies in early S phase and larger but less numerous disintegrated apoptotic bodies in late S phase. Most significant changes after exposure to γ-irradiation took place in early S phase resulting in an increase of nuclear size by more than 50%. Cell fractions containing irradiated cells showed enhanced growth arrest at 2.4 C-value, which was accompanied by apoptosis. Apoptotic cell cycle arrest near to the G₁/G₀ checkpoint and apoptotic changes indicate that the radiation resistance of K562 cells is related to the bypass of the early stage of the p53 apoptotic pathway. Apoptotic changes in chromatin structure induced by γ-irradiation indicate that these injury-specific changes can be identified and distinguished from chromatin changes induced by UV radiation or heavy metals.     

羟基脲诱导前后HEL细胞内GATA转录因子与人β—类珠蛋白基因调控元件 …

HEL cells, a human erythroleukemia cell line, expressing mainly the gamma-globin genes, small amount of epsilon-globin gene, but not beta-globin gene. Our previous studies demonstrated that beta-globin gene could be expressed in HEL cells induced by hydroxyurea. However, the molecular mechenism is still unknown. Here the binding patterns of GATA factors (GATA-1 and GATA-2) to the regulatory elements of human beta-globin gene were examined with the nuclear extracts from hydroxyurea-induced and uninduced HEL cells. Our results showed in EMSA assay that GATA factors could bind to the core sequence of HS2(-10681 to -10971 bp), the 3' flanking sequence of HS2 core(-10323 to -10680 bp) and the promoter of human beta-globin gene(+20 to -112 bp). However, the binding patterns between hydroxyurea-induced and uninduced HEL cells were different. Furthermore, by using Western-blot analysis, our data showed that the amount of GATA-2 was decreased in hydroxyurea-induced HEL cells. In contrast to GATA-2, the amount of GATA-1 was increased in hydroxyurea-induced HEL cells. These results showed that the different members of GATA family might play different roles during the differentiation of erythrocytes. GATA-1 may stimulate the differentiation of HEL cells, while GATA-2 can probably inhibit the differentiation of HEL cells.     

羟基脲对人红白血病细胞株中球蛋白基因表达和细胞分化的影响

人红白血病细胞株（ＨＥＬ细胞）中珠蛋白基因表达具有自己的特点。即只表达胚胎型的γ－珠蛋白基因而不表达成人型的β－珠蛋白基因。羟基脲是一种抑制ＤＮＡ的小分子有机化合物。它在临床上被用来治疗地中海盆血和镰刀型贫血。我们的实验结果表明：胡羟基脲浓度增加ＨＥＬ细胞增殖速度减慢。用常规ＲＴ－ＰＣＲ方法和定量ＰＣＲ分析证明,用羟基脲诱导ＨＥＬ细胞后,β－珠蛋白基因表达增加,α－珠蛋白基因表达减少,而γ－珠蛋白     

维甲酸诱导的人大肠癌细胞凋亡

本研究应用光镜、电镜技术、DNA凝胶电泳、流式细胞术及末端脱氧核苷酰转移酶原位标记(TUNEL法),观察全反式维甲酸ATRA诱导的人大肠癌CCL229细胞凋亡特征。RA诱导CCL229细胞凋亡,光、电镜下观察到凋亡小体形成等典型的形态学改变,琼脂糖凝胶电泳上呈现特征性的DNA ladder,DNA直方图上显示亚二倍体峰。10-8mol/L-105mol/L范围内,RA诱导CCL229细胞凋亡表现出时间和剂量依赖性。     

Chromatin condensation during apoptosis is accompanied by degradation of lamin A+B, without enhanced activation of cdc2 kinase

Chromatin condensation paralleled by DNA fragmentation is one of the most important criteria which are used to identify apoptotic cells. However, comparable changes are also observed in interphase nuclei which have been treated with cell extracts from mitotic cells. In this respect it is known that in mitosis, the lamina structure is broken down as a result of lamin solubilization and it is possible that a similar process is happening in apoptotic cells. The experiments described in this study have used confluent cultures of an embryonic fibroblast cell line which can be induced to undergo either apoptosis at low serum conditions or mitosis. Solubilization of lamin A+B was analyzed by immunoblotting and indirect immunofluorescence. These studies showed that in mitotic cells lamina breakdown is accompanied by lamin solubilization. In apoptotic cells, a small amount of lamin is solubilized before the onset of apoptosis, thereafter, chromatin condensation is accompanied by degradation of lamin A+B to a 46-kD fragment. Analysis of cellular lysates by probing blots with anti- PSTAIR followed by anti-phosphotyrosine showed that in contrast to mitosis, dephosphorylation on tyrosine residues did not occur in apoptotic cells. At all timepoints after the onset of apoptosis there was no significant increase in the activation of p34cdc2 as determined in the histone H1 kinase assay. Coinduction of apoptosis and mitosis after release of cells from aphidicolin block showed that apoptosis could be induced in parallel with S-phase. The sudden breakdown of chromatin structure may be the result of detachment of the chromatin loops from their anchorage at the nuclear matrix, as bands of 50 kbp and corresponding multimers were detectable by field inversion gel electrophoresis (FIGE). In apoptotic cells all of the DNA was fragmented, but only 14% of the DNA was smaller than 50 kbp. DNA strand breaks were detected at the periphery of the condensed chromatin by in situ tailing (ISTAIL). Chromatin condensation during apoptosis appears to be due to a rapid proteolysis of nuclear matrix proteins which does not involve the p34cdc2 kinase.     

三尖杉酯碱诱导HL-60细胞程序性死亡过程中的细胞质和细胞核出泡与染色质凝集

本文利用视频显微影像反差增强技术(VideoEnhancement Contrast,VEC)对三尖杉酯碱诱导的单个HL-60活细胞程序死亡(Apo-ptosis,Apo)全过程进行了观察,结果表明每个Apo细胞在染色质凝集前都要发生细胞核的出泡,而每一个核出泡又都是由相应的质出泡所诱导的,但并不是每个质出泡都能诱导核出泡,质出泡的次数远远高于核出泡,提示核、质出泡可能与染色质凝集有关,并且核、质出泡是程序死亡细胞形成Apo小体所必需的。进一步研究则说明核、质出泡与微丝解聚和重组有关。核、质出泡虽可加速细胞程序死亡过程中的染色质凝集,但并不是程序死亡细胞染色质凝集所必需的,提示HL-60细胞程序死亡过程中的核变化和质变化可能是相对独立的。     

Induction of apoptosis by all-trans retinoic acid in the human myeloma cell line RPMI 8226 and negative regulation of some of its typical morphological features by dexamethasone.

We investigated the effects of all-trans retinoic acid (RA) and dexamethasone (Dex) on the in vitro growth of the human myeloma cell line RPMI 8226. RA inhibited RPMI 8226 cell growth by both antiproliferative effect and induction of apoptosis. Typical morphological and biochemical characteristics of apoptosis including chromatin condensation, apoptotic bodies formation and internucleosomal DNA cleavage were detected after 4 days of treatment with 1 microM RA. In situ TUNEL assay demonstrated that DNA cleavage preceded chromatin condensation. The expression of tissue transglutaminase (tTG), an enzyme proposed to play a role in apoptosis was induced with RA, as shown by both enzymatic assay and in situ immunofluorescence detection. Dex, when used alone, had no effect on cell growth and apoptosis. When combined to RA, Dex did not interfere with the RA-dependent inhibition of cell proliferation, but unexpectedly inhibited both quantitatively and qualitatively several morphological and biochemical features of the apoptosis induced by RA. Dex did not affect RA-induced DNA breaks formation but impeded the progression of chromatin condensation and the formation of apoptotic bodies. Interestingly, Dex also inhibited the RA-dependent induction of tTG. RU486, a glucocorticoid antagonist, counteracted all Dex effects. Taken together these data demonstrate that key cytoplasmic and nuclear events occurring during apoptosis are differentially regulated by RA and Dex in myeloma cell line RPMI 8226.     

Caspase-3 activity and carbonyl cyanide m-chlorophenylhydrazone-induced apoptosis in HL-60

The role of caspase proteases in carbonyl cyanide m-chlorophenylhydrazone (CCCP)-induced apoptosis of human promyelocytic HL-60 cells was examined. Treatment of HL-60 cells with micromolar concentrations of CCCP resulted in cell death, with typical apoptotic features such as chromatin condensation, formation of apoptotic bodies, nucleosomal fragmentation of DNA and a distinct increase in caspase-3 activity. The results, however, indicated that full caspase-3 inhibition by the selective inhibitor N-benzyloxycarbonyl-Asp-Glu-Val-Asp fluoromethyl ketone (Z-DEVD-FMK) did not prevent cell death, nor did it affect the manifestation of apoptotic hallmarks, including apoptotic bodies formation and nucleosomal DNA fragmentation. The only distinct effect that Z-DEVD-FMK exhibited was to retard the disruption of the plasma membrane. We therefore assume that caspase-3 activity itself is not essential for the manifestation of apoptotic features mentioned above. Similarly, the pan-specific caspase inhibitor N-benzyloxycarbonyl-Val-Ala-Asp fluoromethyl ketone (Z-VAD-FMK) did not prevent cell death. On the contrary, Z-VAD-FMK completely prevented DNA cleavage and apoptotic body formation, but it failed to completely counteract chromatin condensation. Thus, in the presence of Z-VAD-FMK, application of CCCP concentrations that otherwise induced apoptosis, resulted in the appearance of two morphologically different groups of dead cells with intact DNA. The first group included cells with necrotic-like nuclear morphology, and therefore could be taken as being "truly" necrotic in nature, because they had intact DNA. The cells of the second group formed small single-spherical nuclei with condensed chromatin. In spite of having intact DNA, they could not be taken as "truly" necrotic cells. It is evident that in the experimental system, caspase proteases play an essential role in the formation of apoptotic bodies and in the cleavage of nucleosomal DNA, but not in the condensation of chromatin. Therefore, it is likely that the choice between cell death modalities is not solely a matter of the caspase proteases present.     

双歧杆菌诱发实验性大肠癌凋亡的电镜观察

本文以大肠癌裸鼠移植瘤为动物模型,用透射电镜观察了青春型双歧杆菌对移植瘤超微结构的影响。结果表明：双歧杆菌经荷瘤鼠腹腔注射后能诱导多量大肠癌细胞凋亡,凋亡早期的细胞主要表现为核染色质浓缩边聚;中期为核膜破裂,胞核崩解,凋亡小体形成;晚期主要为邻近活细胞吞噬凋亡小体。提示双歧杆菌诱导大肠癌细胞程序化死亡可能是其抑瘤机制之一。     

鸭呼肠孤病毒强毒株致鸭胚原代成纤维细胞凋亡的研究

通过光镜、电镜、DNA Ladder法、流式细胞术、荧光染色对鸭呼肠孤病毒(DRV)诱导鸭胚原代成纤维细胞(DEF)凋亡情况进行检测.结果显示,光镜可见细胞形态学上出现细胞皱缩,染色质浓染边移;电镜观察到细胞胞浆浓缩,细胞核染色质凝聚、部分形成凋亡小体;荧光染色结果显示,在感染后24h有激发绿色荧光的凋亡细胞出现,随着时间的推移,激发红色荧光的死亡细胞数量增多;DNA Ladder检测到感染后24～144h的DNA样品呈梯形条带;流式细胞术于感染后24h检测到凋亡细胞,其数量在72～96h达到高峰,144h开始下降.研究结果表明,DRV在DEF增殖的过程中具有诱导宿主细胞凋亡的作用.     

Postovulatory follicle: a model for experimental studies of programmed cell death or apoptosis in teleosts

This is the first evidence of programmed cell death, or apoptosis, occurring in the postovulatory follicle (POF) of teleost fish. Females of Astyanax bimaculatus lacustris were submitted to induced ovulation through injecting pituitary extract. Ultrastructural analyses of POFs at time intervals varying from zero to four days postspawning showed several characteristic events of the apoptosis. Typical apoptotic figures, such as nucleus with chromatin condensation underlying the nuclear envelope in a crescent pattern and apoptotic bodies at different stages of formation and reabsorption, were observed in the follicular cells a few days after the onset of the postovulatory period. The results indicated that apoptosis is the major mechanism responsible for the elimination of the follicular cells in the POFs of A. bimaculatus lacustris during ovarian recovery postspawning. It is suggested that POFs might be used as an experimental model in dynamic studies involving cell death in teleosts.     

Expression of human cytomegalovirus immediate early antigens is responsible for a novel chromatin structure of infected human embryonic lung cells

Whereas human embryonic lung (HEL) cells displayed chromatin fibers composed of a repeat of conventional nucleosomes of 15 nm in diameter, human cytomegalovirus (HCMV) infection induced transient appearance of a novel chromatin structure composed of a repeat of large ellipsoids of 45-65 nm X 15-30 nm with linkers of 50-60 nm long and 6-7 nm thick. Essentially the same change in chromatin structure could be induced when uninfected HEL cell nuclei were incubated in vitro with a 0.4 M NaCl nuclear extract from HCMV-infected HEL cells expressing immediate early antigens (IEA's) or with a similar nuclear extract from NIH/3T3 cells constitutively expressing HCMV IEA's. The latter cell line was established by transformation of the mouse cells with a plasmid carrying the HCMV major immediate early and immediate early 2 genes. These results together with those of control experiments suggest that the expression of IEA's is directly or indirectly responsible for the appearance of the novel chromatin structure in HCMV-infected HEL cells.     

褐飞虱若虫中肠凋亡细胞的检测

为揭示褐飞虱Niloparvata lugens Stl若虫在发育过程中中肠的凋亡细胞,使用末端脱氧核苷酸转移酶介导的dUTP-生物素断端标记法(TUNEL)进行中肠组织切片检测,结果表明,1～5龄若虫中肠分别存在2%～5%的凋亡细胞。利用4′,6-二脒基-2-苯基吲哚二盐酸(DAPI)染色法检测表明,存在Ⅰ,Ⅱa和Ⅱb期凋亡的细胞核,其特征包括染色体浓缩、边缘化及细胞核碎裂。透射电子显微镜检测结果表明,早期凋亡的细胞呈现染色质浓缩、边缘化特征,晚期凋亡的细胞出现细胞核碎裂、形成凋亡小体及细胞质空泡化等。本研究揭示了在正常发育过程中褐飞虱若虫中肠有少量的细胞发生了凋亡。通过人工干预的方式调控中肠细胞的凋亡进程有可能使之成为防治该水稻害虫的新靶标。     

The effect of prostacyclin agonists on the differentiation of phorbol ester treated human erythroleukemia cells

Phorbol-12-myristate-13-acetate (PMA) induces megakaryocytopoeisis in human erythroleukemia (HEL) cells which is characterized by the increase in cell size, increase in nuclear polyploidization and expression of megakaryocyte marker, CD41. However, upon treatment with 100 nM of selective prostacyclin (IP) agonist beraprost inhibits the induced differentiation. Moreover, selective non-prostanoid IP agonist, BMY 45778 prevents PMA induced megakaryocytopoeisis in HEL cells similarly, while prostaglandin E(2) and specific EP(3) agonist sulprostone have no effect. Thus, IP receptor is involved. Furthermore, adenylate cyclase activator forskolin and cAMP analog dibutyryl-cAMP also prevented PMA induced megakaryocytopoeisis in HEL cells. Thus, IP agonists inhibition of PMA induced megakaryocytopoeisis in HEL cells may involve a cAMP dependent pathway.     

A proteomic approach to the identification of molecular targets in subsequent apoptosis of HEL cells after diosgenin‐induced megakaryocytic differentiation

Diosgenin is a plant steroid which is able to induce megakaryocytic differentiation of human erythroleukemia (HEL) cells followed by apoptosis at a later stage. Apoptosis markers and phospho‐kinases involved during the subsequent apoptosis of megakaryocytes after diosgenin‐induced differentiation in these cells were detected using a proteomic approach. In mature megakaryocytes undergoing apoptosis, we observed increased expression of intrinsic apoptosis markers such as Bax/Bcl‐2 ratio and cleaved caspase‐9 as well as extrinsic apoptosis markers including cell death receptors and cleaved caspase‐8. Furthermore, we demonstrated the link between both apoptotic pathways by Bid cleavage and confirmed the executive phase of apoptosis by caspase‐3 cleavage. For the first time, we examined kinase activation and showed that kinases including Src, Tor, Akt, CREB, RSK and Chk2 may be implicated in signalling of subsequent apoptosis of mature megakaryocytes after diosgenin‐induced differentiation of HEL cells. J. Cell. Biochem. 107: 785–796, 2009. © 2009 Wiley‐Liss, Inc.     

Three distinct stages of apoptotic nuclear condensation revealed by time-lapse imaging, biochemical and electron microscopy analysis of cell-free apoptosis

During apoptotic execution, chromatin undergoes a phase change from a heterogeneous, genetically active network to an inert highly condensed form that is fragmented and packaged into apoptotic bodies. We have previously used a cell-free system to examine the roles of caspases or other proteases in apoptotic chromatin condensation and nuclear disassembly. But so far, the role of DNase activity or ATP hydrolysis in this system has not yet been elucidated. Here, in order to better define the stages of nuclear disassembly in apoptosis, we have characterized the apoptotic condensation using a cell-free system and time-lapse imaging. We demonstrated that the population of nuclei undergoing apoptosis in vitro appears to follow a reproducible program of nuclear condensation, suggesting the existence of an ordered biochemical pathway. This enabled us to define three stages of apoptotic chromatin condensation: stage 1 ring condensation; stage 2 necklace condensation; and stage 3 nuclear collapse/disassembly. Electron microscopy revealed that neither chromatin nor detectable subnuclear structures were present inside the stage 1 ring-condensed structures. DNase activity was not essential for stage 1 ring condensation, which could occur in apoptotic extracts depleted of all detectable DNase activity. However, DNase(s) were required for stage 2 necklace condensation. Finally, we demonstrated that hydrolyzable ATP is required for stage 3 nuclear collapse/disassembly. This requirement for ATP hydrolysis further distinguished stage 2 from stage 3. Together, these experiments provide the first steps towards a systematic biochemical characterization of chromatin condensation during apoptosis.