Detection of the apoptosis of Jurkat cell using an electrorotation chip

The apoptosis of cells is one of the fields that attract increasing attention in biology today.Usually,the cells are treated with chemicals when detecting apoptosis.It is highly desired to detect apoptosis in a real-time basis.Apoptosis of Jurkat cells was studied using a real-time electrorotation chip.This chip allows the detection of the cell membrane capacitance changes during the course of apoptosis and therefore facilitates the analysis of apoptosis in a real-time basis without involving any chemical treatment.     

Jurkat细胞凋亡的实时电旋转芯片检测

细胞凋亡是当今生物学中最受关注的领域之一。当前细胞凋亡的检测方法一般都需要对细胞进行化学处理,不能实现对凋亡的实时监测。为了研究细胞凋亡过程中细胞膜电容的变化规律,采用电旋转芯片测量凋亡细胞的电旋转频谱,进而推算出细胞的膜电容。结果显示,随着时间的增加,凋亡细胞的膜电容逐渐减小。在此基础上提出一种用电旋转芯片测定细胞膜电容的方法来检测细胞凋亡。该方法无需对细胞进行化学处理,可以实现对细胞凋亡的实时监测,为研究细胞凋亡提供了一种新的工具。     

Membrane dielectric responses of bufalin-induced apoptosis in HL-60 cells detected by an electrorotation chip

A non-invasive electrorotation (ROT) technique was used to monitor the apoptosis-induced changes in HL-60 cells. The specific membrane capacitance of the cells fell from 15.6 ± 0.9 mF/cm² to 6.4 ± 0.6 mF/cm² after 48 h treatment with 10 nM bufalin, a component of bufadienolides in traditional Chinese medicine, Chan Su. However, the average membrane conductance remained almost constant during the first 24 h of treatment and then increased afterwards. Apoptosis was verified by a DNA fragmentation assay and scanning electron microscopy. The results demonstrate that the ROT technique gives a quantitative analysis of the toxic damage by chemicals to cells and can be exploited in the testing and development of new pharmaceuticals and active cell agents. Chengjun Huang and Ailiang Chen contributed equally to this work.     

Analysis of parasites by electrorotation

AIMS: The application of the AC electrokinetic technique of electrorotation for studying eukaryotic parasite transmission stages is reviewed. Electrorotation is a noninvasive technique that utilizes electrically energized microelectrode structures within micro-fluidic chambers to probe the physiological structure of micro-organisms. Application of the technique to the transmission life cycle stages of three separate genera of protozoan parasites, Cryptosporidium, Giardia and Cyclospora, and one nematode genus Ascaris, each of significant public health importance, is described. METHODS AND RESULTS: Standard electrorotation apparatus, consisting of micro-fabricated electrodes in a fluidic chip, quadrature sinusoidal signal generator, microscope and image capture system, was used to study each organism. Spectra of cellular rotation rate were recorded as a function of applied electric field frequency and compared with standardized biological tests, where appropriate, to illustrate the effectiveness and versatility of the electrorotation technique. CONCLUSIONS: Electrorotational determination of the viability of individual G. intestinalis cysts, Cryptosporidium parvum and Cyclospora cayetanensis oocysts has been achieved. The sporulation state of Cyclospora cayetanensis oocysts was also readily determined, as was the fertilization state of A. suum ova. SIGNIFICANCE AND IMPACT OF THE STUDY: Electrorotation is a simple, noninvasive and versatile analytical technique suited to a wide range of particle types and capable of incorporation into integrated Lab-on-a-chip devices.     

神经酰胺与细胞凋亡

在多细胞有机体中,凋亡对正常组织的发育和稳态是必需的。神经酰胺（ceramide,Cer）作为一种脂类分子,不仅是细胞膜的重要组成部分,而且是重要的凋亡参与者。在细胞膜上,它能够自发地聚集,形成招募死亡受体和配体的结构域从而促进信号的传导和放大;在细胞质中,它作为信号通路的中介者,通过许多不同的分子机制去调控机体的凋亡通路;在细胞核膜和核内,通过其代谢变化,决定细胞的增殖或凋亡;而且它的浓度和位置以及与其它分子的比例及其相互作用都会影响到细胞和组织的命运。综述了近来关于Cer及其代谢物的凋亡作用。     

Oxidative stress signalling in the apoptosis of Jurkat T-lymphocytes

Within the first 24 h after castration of an adult male rat, the vascular system of the ventral prostate gland undergoes a degenerative process that drastically reduces blood flow to the tissue. Since the vascular degeneration precedes the loss of the prostatic epithelium (by apoptosis), we have proposed that the onset of epithelial cell apoptosis in this tissue is caused by an ischemic/hypoxic environment resulting from the loss of blood flow. In order to further evaluate the extent to which ischemia/hypoxia might be a factor in apoptosis of the prostate epithelium after castration, we analyzed for biomarkers of cellular hypoxia in rat ventral prostates during the first 3 days following castration. Ventral prostate tissues removed from hypoxyprobe-1-treated adult male rats (uncastrated controls; surgically castrated for 24, 48 or 72 h, or sham-castrated for equivalent times) were directly analyzed for evidence of hypoxia by in situ immunohistochemical evaluation of hypoxyprobe-1 adduct formation in the prostate cells. Protein extracts from these tissues were also tested for expression of the 120 kDa hypoxia-inducible factor-1-alpha (HIF-1-alpha) protein as well as for expression of mitogen-activated protein kinase (MAPK) and c-Jun N-terminal kinase (JNK) proteins using a Western blot assay. The tyrosine phosphorylation status of the latter signaling molecules was also evaluated by Western blotting using anti-tyrosine phosphate antibodies. Our results showed that epithelial cells of the rat ventral prostate stained positively for hypoxyprobe-1 adducts at all times after castration, whereas cells in control tissues were unstained by this procedure. In addition, the prostatic expression of HIF-1-alpha protein was increased approximately 20-fold at 48 h after castration compared to control tissues. Finally, although prostatic MAPK and JNK protein expression was unaltered during the early period after castration, phosphorylation of the JUN kinase protein was significantly elevated, indicating that this stress-activated cellular signaling pathway becomes more active subsequent to castration. These results support our proposal that early castration-induced degeneration and constriction of the vascular system of the rat ventral prostate gland leads to reduced oxygenation of prostatic epithelial cells and the activation of hypoxic cellular signaling in these cells through upregulation of HIF-1-alpha expression and stimulation of the JUN kinase signaling pathway.     

Dielectric properties of alginate beads and bound water relaxation studied by electrorotation

The electrical and dielectric properties of Ba²⁺ and Ca²⁺ cross‐linked alginate hydrogel beads were studied by means of single‐particle electrorotation. The use of microstructured electrodes allowed the measurements to be performed over a wide range of medium conductivity from about 5 mS/m to 1 S/m. Within a conductivity range, the beads exhibited measurable electrorotation response at frequencies above 0.2 MHz with two well‐resolved co‐ and antifield peaks. With increasing medium conductivity, both peaks shifted toward higher frequency and their magnitudes decreased greatly. The results were analyzed using various dielectric models that consider the beads as homogeneous spheres with conductive loss and allow the complex rotational behavior of beads to be explained in terms of conductivity and permittivity of the hydrogel. The rotation spectra could be fitted very accurately by assuming (a) a linear relationship between the internal hydrogel conductivity and the medium conductivity, and (b) a broad internal dispersion of the hydrogel centered between 20 and 40 MHz. We attribute this dispersion to the relaxation of water bound to the polysaccharide matrix of the beads. The dielectric characterization of alginate hydrogels is of enormous interest for biotechnology and medicine, where alginate beads are widely used for immobilization of cells and enzymes, for drug delivery, and as microcarriers for cell cultivation. © 1999 John Wiley & Sons, Inc. Biopoly 50: 227–237, 1999     

蛋白质芯片技术检测脑损伤大鼠血清差异蛋白

目的:研究大鼠脑损伤后血清中蛋白质表达谱的变化及其特点.方法:采用弱阳离子交换芯片(WCX2)结合表面增强激光解析电离飞行时间质谱技术分析大鼠闭合性脑损伤后4 h、8 h、12 h、24 h、48 h血清中蛋白质表达谱的改变.结果:与对照组相比,脑损伤后血清中有2个蛋白质的表达谱发生改变.其中差异蛋白5648Da,在4 h、8 h和12 h组表达降低(P＜0.01),24 h和48 h组表达恢复;差异蛋白9681Da在对照组、24 h和48 h组几乎不表达,而4 h、8 h和12 h组表达增加(P＜0.05).结论:脑损伤可引起血清中蛋白质表达谱发生变化.     

Mechanisms of B cell receptor induced apoptosis

B cell receptor (BCR)-mediated apoptosis plays a key role in the negative selection (deletion) of autoreactive B cells. Mechanisms of BCR-mediated apoptosis have been widely studied in cell lines representing both immature (bone marrow) and mature (germinal center) B cells. However, there is much inconsistency and controversy concerning the possible mechanisms of BCR-mediated apoptosis, which may reflect differences in the origin or the maturational stage of the cell line used. Based on recent studies, collapse of mitochondrial membrane potential (Delta Psi m) seems to be an essential event for BCR-mediated apoptosis in both mature and immature cells. The collapse of Delta Psi m is dependent on the synthesis of new proteins, which are involved in the permeability change of mitochondrial membranes. Mitochondrial dysfunction induces activation of caspases, cysteine proteases, which play a central role in apoptosis. However, instead of caspases, other effector proteases, such as cathepsins or calpains, may also be responsible for the organized destruction of cell components seen during BCR-mediated apoptosis.     

N-acetylphytosphingosine-induced apoptosis of Jurkat cells is mediated by the conformational change in Bak

N-acetylphytosphingosine (NAPS), a sphingolipid derivative, is one of the well-known signal molecules that mediates various cellular functions, including cell growth, differentiation, and apoptosis. In this study, we demonstrated that NAPS induces apoptosis of Jurkat cells by activating Bak, but not Bax, which are both members of a proapoptotic subfamily of the Bcl-2 proteins. NAPS activated caspase-8 in a FADD-independent manner, but the lack of caspase-8 did not suppress the activation of caspase-3 and -9 and cell death, indicating that caspase-8 activation does not play an important role in NAPS-induced cell death. The overexpression of Bcl-x_L, an anti-apoptotic protein, completely inhibited the activation of the caspases and apoptosis, assuming that NAPS-induced apoptosis was initiated by the mitochondria. The expression levels of pro- and anti-apoptotic Bcl-2 family members were not changed by the NAPS treatment. However, Bad was translocated from the cytosol into the mitochondria, where it bound to Bcl-x_L, and Bak was dissociated from Bcl-x_L and conformationally changed. Taken together, these findings indicate that NAPS induced apoptosis of Jurkat cells in a mitochondria-dependent manner that was controlled by the translocation of Bad and the conformational change in Bak. These authors contributed equally to this paper     

Hypotonically induced changes in the plasma membrane of cultured mammalian cells

Summary Cells from three cell lines were electrorotated in media of osmotic strengths from 330 mOsm to 60 mOsm. From the field-frequency dependence of the rotation speed, the passive electrical properties of the surfaces were deduced. In all cases, the area-specific membrane capacitance (C _m) decreased with osmolality. At 280 mOsm (iso-osmotic), SP2 (mouse myeloma) and G8 (hybridoma) cells had C _mvalues of 1.01 ± 0.04 F/cm² and 1.09 ± 0.03 F/cm², respectively, whereas dispase-treated L-cells (sarcoma fibroblasts) exhibited C _m=2.18±0.10/F/cm². As the osmolality was reduced, the C _mreached a well-defined minimum at 150 mOsm (SP2) or 180 mOsm (G8). Further reduction in osmolality gave a 7% increase in C _m, after which a plateau close to 0.80F/cm2²was reached. However, the whole-cell capacities increased about twofold from 200 mOsm to 60 mOsm. L-cells showed very little change in C _mbetween 280 mOsm and 150 mOsm, but below 150 mOsm the C _mdecreased rapidly. The changes in C _mcorrelate well with the swelling of the cells assessed by means of van't Hoff plots. The apparent membrane conductance (including the effect of surface conductance) decreased with C _m, but then increased again instead of exhibiting a plateau. The rotation speed of the cells increased as the osmolality was lowered, and eventually attained almost the theoretical value. All measurements indicate that hypo-osmotically stressed cells obtain the necessary membrane area by using material from microvilli. However, below about 200 mOsm the whole-cell capacities indicate the progressive incorporation of extra membrane into the cell surface.We thank Mr. B.G. Klarmann for his help with the measurements. This work was supported by grants of the DFG (SFB 176 B5 to U.Z. and W.M.A.) and of the BMFT (DARA 50 WB 9212 to U.Z.). We also thank the Umweltbundesamt, Berlin, for support enabling the construction of some of the rotation generators used in this work.     

Transglutaminase overexpression sensitizes neuronal cell lines to apoptosis by increasing mitochondrial membrane potential and cellular oxidative stress

'Tissue' transglutaminase (tTG) selectively accumulates in cells undergoing apoptosis both in vivo and in vitro. Considering the central role played by mitochondria in apoptosis, we investigated the relationships existing amongst tTG expression, apoptosis and mitochondrial function. To this aim we studied the mechanisms of apoptosis in a neuronal cell line (SK-N-BE (2)) in which the tTG-expression was driven by a constitutive promoter. Furthermore, a tet-off inducible promoter was also used in 3T3 fibroblastic cells used as control. Both cell lines, when expressing tTG, appeared 'sensitized' to apoptosis. Strikingly, we found major differences in the morphological features of mitochondria among cell lines in the absence of apoptotic stimuli. In addition, these ultrastructural characteristics were associated with specific functional features: (i) constitutively hyperpolarized mitochondria and (ii) increased reactive oxygen intermediates production. Importantly, after mitochondrial-mediated apoptosis by staurosporine, a rapid loss of mitochondrial membrane potential was found in tTG cells only. Taken together, these results seem to suggest that, via hyperpolarization, tTG might act as a 'sensitizer' towards apoptotic stimuli specifically targeted to mitochondria. These results could also be of pathogenetic relevance for those diseases that are characterized by increased tTG and apoptotic rate together with impaired mitochondrial function, e.g. in some neurodegenerative disease.     

Diallyl trisulfide induces apoptosis in Jurkat cells by the modification of cysteine residues in thioredoxin

We reported the regulation of protein function by oxidative modification of the specific cysteine residue(s) by diallyl trisulfide (DATS). In this study, we examined if DATS modifies the cysteine residue of thioredoxin (Trx) by urea-polyacryl amide gel electrophoresis. DATS modified two specific cysteine residues in Trx and this oxidative modification of cysteine residues would be sole causative of the apoptosis induced by DATS in leukemic cells.     

Cepharanthine activates caspases and induces apoptosis in Jurkat and K562 human leukemia cell lines.

Cepharanthine (CEP) is a known membrane stabilizer that has been widely used in Japan for the treatment of several disorders such as anticancer therapy-provoked leukopenia. We here report that apoptosis was induced by low concentrations (1-5 microM) of CEP in a human leukemia T cell line, Jurkat, and by slightly higher concentrations (5-10 microM) in a human chronic myelogenous leukemia (CML) cell line K562, which expresses a p210 antiapoptotic Bcr-Abl fusion protein. Induction of apoptosis was confirmed in both Jurkat and K562 cells by DNA fragmentation and typical apoptotic nuclear change, which were preceded by disruption of mitochondrial membrane potential and were induced through a Fas-independent pathway. CEP treatment induced activation of caspase-9 and -3 accompanied by cleavage of PARP, Bid, lamin B1, and DFF45/ICAD in both Jurkat and K562 cells, whereas caspase-8 activation and Akt cleavage were observed only in Jurkat cells. The CEP-induced apoptosis was completely blocked by zVAD-fmk, a broad caspase inhibitor. Interestingly, CEP treatment induced remarkable degradation of the Bcr-Abl protein in K562 cells, and this degradation was prevented partially by zVAD-fmk. When used in combination with a nontoxic concentration of herbimycin A, lower concentrations (2-5 microM) of CEP induced obvious apoptosis in K562 cells with rapid degradation or decrease in the amount of Bcr-Abl and Akt proteins. Our results suggest that CEP, which does not have bone marrow toxicity, may possess therapeutic potential against human leukemias, including CML, which is resistant to anticancer drugs and radiotherapy.     

Minerval induces apoptosis in Jurkat and other cancer cells

Minerval is an oleic acid synthetic analogue that impairs lung cancer (A549) cell proliferation upon modulation of the plasma membrane lipid structure and subsequent regulation of protein kinase C localization and activity. However, this mechanism does not fully explain the regression of tumours induced by this drug in animal models of cancer. Here we show that Minerval also induced apoptosis in Jurkat T‐lymphoblastic leukaemia and other cancer cells. Minerval inhibited proliferation of Jurkat cells, concomitant with a decrease of cyclin D3 and cdk2 (cyclin‐dependent kinase2). In addition, the changes that induced on Jurkat cell membrane organization caused clustering (capping) of the death receptor Fas (CD95), caspase‐8 activation and initiation of the extrinsic apoptosis pathway, which finally resulted in programmed cell death. The present results suggest that the intrinsic pathway (associated with caspase‐9 function) was activated downstream by caspase‐8. In a xenograft model of human leukaemia, Minerval also inhibited tumour progression and induced tumour cell death. Studies carried out in a wide variety of cancer cell types demonstrated that apoptosis was the main molecular mechanism triggered by Minerval. This is the first report on the pro‐apoptotic activity of Minerval, and in part explains the effectiveness of this non‐toxic anticancer drug and its wide spectrum against different types of cancer.     

Carotenoids induce apoptosis in the T-lymphoblast cell line Jurkat E6.1

Epidemiologically, a high-carotenoid intake via a fruit- and vegetable-rich diet is associated with a decreased risk of various forms of cancer. The mechanisms by which carotenoids exert this protective effect are controversial. In this study, we examined the potency of a range of carotenoids commonly found in human plasma to induce apoptosis in Jurkat E6.1 malignant T-lymphoblast cells. At a concentration of 20 microM, the order of potency to induce apoptosis after 24 h was: beta-carotene > lycopene > lutein > beta-cryptoxanthin = zeaxanthin. Canthaxanthin failed to induce apoptosis under these conditions. beta-Carotene induced apoptosis in a time- and concentration-dependent manner with a lowest effective concentration of about 3 microM. Pre-conditioning of beta-carotene for 72 h destroyed its pro-apoptotic activity almost completely, whereas degradation for 6 h or less did not, indicating that either beta-carotene itself and/or an early degradation product of beta-carotene are the death-inducing compounds. Apoptosis induced by beta-carotene was characterized by chromatin condensation and nuclear fragmentation, DNA degradation, PARP cleavage and caspase-3 activation. The antioxidant BO-653 inhibited the degradation of beta-carotene in vitro and significantly increased its cytotoxicity, indicating that a pro-oxidant effect of beta-carotene is unlikely to cause its pro-apoptotic activity. The induction of apoptosis in transformed cells by carotenoids may explain their protective effect against cancer formation in humans. Possible pathways for induction of apoptosis by carotenoids are discussed.     

Effect of the microbial lipopeptide on tumor cell lines: apoptosis induced by disturbing the fatty acid composition of cell membrane

Microbial lipopeptides play an important role in apoptosis induction of tumor cells. However, there is little knowledge about the relationship between apoptosis induction and membrane fatty acids. The present study focused on the effects of lipopeptides produced by Bacillus subtilis HSO121 on Bcap-37 cell lines. 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl (MTT) colorimetric assay and surface tension measurements, showed that the critical micelle concentration (CMC) was a critical level for the inhibitory activity of lipopeptides on the growth of Bcap-37 cells. Under the CMC, the order of least to greatest cytotoxicity effect on cancer cell lines by lipopeptides is C₁₃-lipopeptide<C₁₄-lipopepitde<C₁₅-lipopeptide. Above CMC, all lipopeptides directly exert cytolytic activity. The flow cytometric analysis and Hoechst33258 staining experiments confirmed the apoptosis of Bcap-37 cell lines induced by lipopeptides in a dose-dependent manner. This apoptosis was associated with a significant decrease of the unsaturated degree of the cellular fatty acids of Bcap-37 cell lines due to the changes in the cellular fatty acids composition induced by the lipopeptide treatment. These results indicated that disturbance of the cellular fatty acid composition of breast cancer cell lines were related to in the cell apoptosis. Furthermore, significant difference in IC₅₀ values of tumor cells and normal cell showed that the lipopeptide exerted selective cytotoxicity on the cancer cells. Thus HSO121 lipopeptides may have potential applications as an anticancer leads.     

Construction of novel single-cell screening system using a yeast cell chip for nano-sized modified-protein-displaying libraries

A novel screening system using a microchamber array chip was developed for construction of combinatorial nano-sized protein libraries in combination with yeast cell surface engineering. It is possible to place a single yeast cell into each microchamber, to observe its behavior, and to pick up the target cell. The microchamber array chip is referred to as a “yeast cell chip.” A single EGFP-displaying yeast cell could be detected, picked up by a micro-manipulator, and cultivated on agar medium. Furthermore, a catalytic reaction, the hydrolysis of fluorescein dioctanate, by a single yeast cell displaying Rhizopus oryzae lipase (ROL) was carried out in one microchamber. The ROL-encoding gene in a single ROL-displaying cell was amplified by PCR. These results demonstrate that this yeast cell chip in combination with cell surface engineering could be used as a tool in a high-throughput screening system not only for a single living cell and a whole-cell catalyst with a nano-sized protein cluster but also for modified nano-sized and functional protein molecules from protein libraries on the cell surface.     

Decrease in cell surface sialic acid in etoposide-treated Jurkat cells and the role of cell surface sialidase

The present study investigated the mechanism underlying alterations of cell surface sugar chains of Jurkat cells by inducing apoptosis with etoposide, an inhibitor of topoisomerase II. Within 3[emsp4 ]h of etoposide treatment, flowcytometric analysis revealed a decrease in Maackia amurensis agglutinin recognized 2,3-linked sialic acid moieties and an increase in Ricinus communis agglutinin recognized galactose. The results suggested that asialo-sugar chains on glycoconjugates were rapidly induced on the etoposide-treated cell surface. To clarify the desialylation mechanism, we studied 2,3-sialyltransferase mRNA expression and the activity of sialidase on the cell surface during etoposide-induced apoptosis. The expression of hST3Gal III and hST3Gal IV mRNAs were down-regulated and sialidase activity on the cell surface increased threefold within 2[emsp4 ]h of etoposide treatment. Moreover, the decrease in 2,3-linked sialic acid levels was significantly suppressed in the presence of 2,3-dehydro-2-deoxy-N-acetylneuraminic acid, an inhibitor of sialidase. These results suggested that activation or exposure of sialidase on the cell surface was induced by etoposide treatment and was the main cause of the decrease in sialic acids.     

Influence of expressed TRAIL on biophysical properties of the human leukemic cell line Jurkat

The cDNA fragment of human TRAIL (TNF-related apoptosis inducing ligand) was cloned into RevTet-On, a Tetregulated and high-level gene expression system. The gene expression system was constructed in a human leukemic cell line: Jurkat. By using RevTet-On TRAIL gene expression system in Jurkat as a cell model, we studied the influence of TRAIL gene on the changes of cellular apoptosis before and after the TRAIL gene expression, which was induced by adding tetracycline derivative doxycycline (Dox). The results indicated that the cellular apoptosis ratio was largely dependent on the TRAIL gene expression level. Moreover, it was found that the apoptosis-inducing TRAIL could cause significant changes in the biophysical properties of Jurkat cells. The cell surface charge density decreased, the membrane fluidity declined, the elastic coefficients K1 increased, and the proportion of α-helix in membrane protein secondary structure decreased. Thus, the apoptosis-inducing TRAIL gene caused significant changes on the biomechanic properties of Jurkat cells.