Electrical effects on the proliferation of living HeLa cells cultured on optically transparent electrode surface.

Low d.c. potential application induced changes of cellular morphology and growth of living cells on a potential-controlled electrode. At a potential range higher than +0.7 V (vs. Ag/AgCl), serious electric effects on cell viability, membrane permeability, and cytoskeletal morphology of HeLa cells were observed. On the other hand, at lower than +0.5 V no effect was observed. At the boundary potential range between +0.5 V to +0.7 V, where HeLa cells were cultured on the potential-controlled optically transparent In2O3 electrode (OTE) surface, intriguing effects on HeLa cells appeared. At this potential range, where HeLa cells cultured on a potential-applied OTE, all the cells were alive accompanying morphological change. The morphology of HeLa cells returned to their normal spindle shape, when potential application to the electrode was cut off. At a potential of +0.65 V, cell proliferation ratio of cultured cell on an electrode was about one-fifth of that on a non-controlled electrode. These results suggest that low d.c. electrical effects induce significant change in cellular morphology and function.     

伦敦白胶和茶：一种既快又安全的用于透射电子显微镜的细菌样品制备方法

【目的】检测乌龙茶提取物是否可作为电子染色剂取代醋酸双氧铀用于细菌细胞染色,使其能在透射电子显微镜下进行观察。【方法】利用伦敦白胶对细菌样品(大肠杆菌和金黄色葡萄球菌)进行胶块的制备,再在复染铅与不复染铅这两种情况下对超薄切片样品进行3种不同染色剂的电子染色,之后在透射电子显微镜下观察比较其不同之处。这3种不同的染色剂分别是醋酸双氧铀、0.05%乌龙茶提取物以及0.1%乌龙茶提取物。首先将带有超薄切片样品的铜网悬浮于不同的待比较染液中10?15 min,若需进一步用柠檬酸铅复染,则将经3次蒸馏水冲洗过后的铜网再次悬浮于柠檬酸铅染液中8?10 min。【结果】复染铅的情况下,在透射电子显微镜下无论是大肠杆菌还是金黄色葡萄球菌,利用3种电子染色剂进行染色的结果均非常相似。【结论】实验结果表明,在观察细菌结构中,乌龙茶提取物可以替代醋酸双氧铀进行透射电子显微镜样品的电子染色。     

The role of phosphorylation and limited proteolytic cleavage of talin and vinculin in the disruption of focal adhesion integrity

Chemical agents which activate specific kinases were employed to disrupt the stress fiber and focal adhesion organization of cells spread on a substratum. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, an activator of protein kinase C, promoted a rapid loss of stress fibers and focal adhesions from African green monkey kidney (BSC-1) cells. This was paralleled by an increase in the level of talin phosphorylation suggesting that this may play a role in the removal of talin from focal adhesions. Similar morphological changes were produced in the rat embryo fibroblast line (REF 52) by dibutyryl-cAMP, which stimulates protein kinase A. In contrast, however, the phosphorylation of talin was reduced in REF 52 cells when treated with dibutyryl cAMP. In untreated cells we found that the levels of vinculin phosphorylation were very low relative to the levels of talin phosphorylation and did not change following drug treatment in either cell line. Although limited proteolytic cleavage of cytoskeletal proteins represents a potential mechanism for focal adhesion disruption, we observed no proteolysis of talin or vinculin in response to either drug treatment.     

Operculina turpethum attenuates N-nitrosodimethylamine induced toxic liver injury and clastogenicity in rats

The root extract of Operculina turpethum (OTE) has been used as an anti-inflammatory, purgative, and hepato-protective agent. N-Nitrosodimethylamine (NDMA) is a potent hepatotoxin that induces fibrosis of the liver. In the present study, we examined the therapeutic effects of OTE root extract against NDMA-induced hepatotoxicity and clastogenicity in rats. Hepatic fibrosis was induced in adult male albino rats through serial intraperitoneal administrations of NDMA at a concentration of 10 mg/kg body weight on three consecutive days of each week over a period of three weeks. A group of rats received OTE orally in doses of 75, 150 and 200 mg/kg body weight at 5 h after the administration of NDMA. The controls and treated animals were sacrificed on days-7, 14 and 21 after the start of the administration of NDMA. The progression of hepatic fibrosis as well as the amelioration effect of OTE was evaluated through histopathologically as well as by immunohistochemical staining for the activation of hepatic stellate cells. Alterations in serum and liver biochemical parameters and LDH isoenzymes were also studied. Serial administration of NDMA resulted in well formed fibrosis in the liver and induction of micronuclei in the bone marrow cells. Staining of α-SMA demonstrated activated stellate cells from day-7 onwards which was dramatically increased on day-21. An elevation of micronuclei count, liver function enzymes, serum hydroxyproline levels and LDH isoenzymes 4 and 5 were also observed. All these changes were remarkably reduced in OTE administered animals and fibrogenesis was completely absent. Our results suggest that OTE has hepatoprotective and anti-clastogenic effects against NDMA-induced hepatic fibrosis. Therefore OTE may be used as a hepatoprotective agent against various liver diseases including toxic liver injury.     

Effects of the inhibitors of dynamics of cytoskeletal structures on the development of apoptosis induced by the tumor necrosis factor

Changes in cytoskeletal structures have been investigated during apoptosis of epithelial HeLa cells induced by tumor necrosis factor- (TNF-). Shape and surface cell activity were investigated by time-lapse video microscopy, and changes of the cytoskeletal structure were studied by immune fluorescent microscopy. Addition of TNF- to HeLa cell culture caused early disruption of the actin cytoskeleton and vinculin-containing focal contacts, keratin filaments, and microtubules. Rounding of cells, general blebbing, and nuclear fragmentation were observed at the terminal apoptotic stages. Actomyosin complex inhibitors, H7 and HA1077, suppressed blebbing (but not cell rounding) and activated the development of apoptosis. The latter suggests that in contrast to blebbing the general rounding does not depend on increased contractility of actomyosin cortex. These cytoskeletal inhibitors accelerated the development of apoptosis of HeLa cells and increased sensitivity of HeLa-Bcl-2 cells (transfected with DNA encoding antiapoptotic protein Bcl-2) to TNF-induced apoptosis. Damage of cytoskeletal structures significantly attenuated antiapoptotic activity of Bcl-2 in the HeLa-Bcl-2 cells. It is suggested that the stimulation of apoptosis by cytoskeletal inhibitors may be attributed to the altered distribution of cell organelles, especially, mitochondria.     

AFM-detected apoptotic changes in morphology and biophysical property caused by paclitaxel in Ishikawa and HeLa cells

The apoptosis of cancer cells is associated with changes in the important cell properties including morphology, surface roughness and stiffness. Therefore, the changes in morphology and biophysical properties can be a good way of evaluating the anticancer activity of a drug. This study examined the effect of paclitaxel on the properties of Ishikawa and HeLa cells using atomic force microscopy (AFM), and the relationship between the changes in morphology and the biophysical properties and apoptosis was discussed. The viability and proliferation of the cells were analyzed using the methylthiazol tetrazolium (MTT) method and a TUNEL assay to confirm cellular apoptosis due to a paclitaxel treatment. AFM observations clearly showed the apoptotic morphological and biophysical changes in Ishikawa and HeLa cells. After the paclitaxel treatment, the cell membrane was torn and holed, the surface roughness was increased, and the stiffness was decreased. These changes were observed more apparently after a 24 h treatment and in Ishikawa cells compared to HeLa cells. The MTT and TUNEL assays results revealed the Ishikawa cells to be more sensitive to paclitaxel than HeLa cells and definite apoptosis occurred after a 24 h treatment. These results showed good agreement with the AFM results. Therefore, research on the morphological and biophysical changes by AFM in cancer cells will help to evaluate the anticancer activities of the drugs.     

The fate of E- and P-cadherin during the early stages of apoptosis.

Caspases are responsible for the proteolysis of many cytoskeletal proteins in apoptotic cells. It has been demonstrated here that during cisplatin-induced apoptosis of human embryo retinoblasts both E- and P-cadherin were degraded by caspases, giving initially major polypeptide products of apparent molecular weights 48 K and 104 K respectively. This proteolysis occurred over a similar time-scale to the observed degradation of PARP and to the onset of DNA fragmentation but appreciably later than p53 induction and cleavage of Mdm2 and p21. Addition of caspase inhibitors such as Z-VAD-FMK inhibited apoptosis and cadherin degradation. Co-immunoprecipitation studies carried out on viable cells confirmed previously observed complexes between cadherins and alpha and beta catenin and between the catenins themselves. These interactions were sustained in apoptotic cells as long as the protein components remained intact. Using confocal microscopy it has been shown that cytoskeletal changes associated with apoptosis precede degradation of catenins and cadherins by several hours. In particular, after addition of cisplatin relatively rapid (within 3 h) re-localization of adherens junction proteins from the cell periphery to the cytoplasm was observed whereas little cadherin or catenin degradation occurred until 10 h. We conclude that neither caspase-mediated degradation of cytoskeletal components nor disruption of adherens junction protein-protein interactions is required for morphological change.     

RacB regulates cytoskeletal function in Dictyostelium spp

Thus far, 14 homologues of mammalian Rac proteins have been identified in Dictyostelium. It is unclear whether each of these genes has a unique function or to what extent they play redundant roles in actin cytoskeletal organization. To investigate the specific function of RacB, we have conditionally expressed wild-type (WT-RacB), dominant negative (N17-RacB), and constitutively activated (V12-RacB) versions of the protein. On induction, cells expressing V12-RacB stopped growing, detached from the surface, and formed numerous spherical surface protrusions while cells overexpressing WT-RacB became flattened on the surface. In contrast, cells overexpressing N17-RacB did not show any significant morphological abnormalities. The surface protrusions seen in V12-RacB cells appear to be actin-driven protrusions because they were enriched in F-actin and were inhibitable by cytochalasin A treatment. The protrusions in V12-RacB cells did not require myosin II activity, which distinguishes them from blebs formed by wild-type cells under stress. Finally, we examined the functional consequences of expression of wild-type and mutant RacB. Phagocytosis, endocytosis, and fluid phase efflux rates were reduced in all cell lines expressing RacB proteins but the greatest decrease was observed for cells expressing V12-RacB. From these results, we conclude that like other members of the Rho family, RacB induces polymerization of actin but the consequences of activation appear to be different from other Dictyostelium Rac proteins so far investigated, resulting in different morphological and functional changes in cells.     

Network analysis and proteomic identification of vimentin as a key regulator associated with invasion and metastasis in human hepatocellular carcinoma cells

Poor prognoses have long been associated with the high relapse and metastasis of human hepatocellular carcinoma (HCC). To achieve long-term survival, it is necessary to identify new HCC biomarkers and investigate their roles in cell mobility and invasiveness. Of note, overexpression of vimentin (Vim) was significantly correlated with tumor nuclear grade (p=0.01) and the invasive potential, indicating that Vim may be a promising candidate in regulating HCC metastasis. RNA interference-mediated silencing of Vim (siVim) suppressed the invasive and migratory propensity, and matrix metalloproteinase (MMP)-9 activity, and elicited morphological changes in poorly differentiated SK-Hep-1 cells. Moreover, we performed a comprehensive proteomic analysis to survey global protein changes mediated by siVim in SK-Hep-1 cells. Significant changes in cytoskeleton protein but not messenger RNA levels encoding these targeted proteins were observed. All of the data in the current study and a network analysis implied that abolition of Vim may disturb the expression and stability of various cytoskeletal proteins through promoting the ubiquitin system, resulting in impaired cell adhesion and motility. Collectively, an integrated approach represents a modality to explore novel relationships in a proteome complex and highlights the functional roles of Vim in HCC metastasis. This article is part of a Special Issue entitled: Translational Proteomics.     

Changes in cell-substratum adhesion and nuclear-cytoskeletal anchorage during cytodifferentiation of BeWo choriocarcinoma cells

Changes in the substratum anchorage of cells and nuclei were examined during methotrexate (MTX)-induced cytodifferentiation of BeWo human choriocarcinoma cells. During this process cytotrophoblast-like cells (CTLs) transform into giant mono- and multinucleated syncytiotrophoblast-like cells (STLs). Cells treated with MTX for 24 h exhibited significantly faster rates of substratum detachment by EDTA, trypsin-EDTA, EDTA-glycine, and DMSO than did uninduced controls. The decrease in cell-substratum adhesiveness occurred prior to the onset of morphological transformation. By 48 h, when morphological transformation was first observed, there had occurred a marked change in nuclear-cytoskeletal anchorage to the substratum, as evidenced by a difference in sensitivity of Triton-extracted STL and CTL monolayers to detachment by KI. STL monolayers were completely detached within 5 min of exposure to 0.3 M KI, while CTL monolayers remained firmly attached to the substratum for at least 3 h. KI-extracted residues were examined by electron microscopy and found to consist of nuclear shells attached to intermediate filaments. When cytoskeletal residues and KI-extracted proteins of STL and CTL cells were compared by two-dimensional polyacrylamide gel electrophoresis (PAGE), qualitative and quantitative differences were seen in a number of minor components. Thus the sensitivity of STL nuclear-cytoskeletal monolayers to removal by KI, an effective actin depolymerizing agent, may involve changes in the organization, stability, or interactions of actin with other components of the cytoskeletal framework.     

Label-free monitoring of apoptosis by surface plasmon resonance detection of morphological changes

Apoptosis can be routinely characterized using biomolecular markers such as in the TUNEL and the annexin V assays or by using fluorescent caspase substrates. Apoptosis can also be semi-quantitatively characterized using microscopy, which targets morphological features such as cell rounding, nuclear condensation and fragmentation as well as cell membrane blebbing. This label-free approach provides a limited resolution for the evolution of these events in time and relies heavily on subjective identification of the morphological features. Here we propose a label-free assay based on surface plasmon resonance (SPR) detection of minute morphology changes occurring as a result of apoptosis induction in an endothelial cell model (EA.hy926). At first, annexin V assays confirmed that our cellular model was responsive to TRAIL over a 12-hour period. Then, we show that SPR allows accurate monitoring of apoptosis by measuring (1) the duration of the latency period during which the apoptotic signal is integrated by the initiator caspases and transmitted to the executioner caspases, (2) the rate of the execution phase in which death substrates are cleaved and morphological changes occur, and (3) the total extent of apoptosis. Using these parameters, we characterized the responses obtained with TRAIL (EA.hy926, HeLa, AD-293) and the anti-Fas antibody (HeLa) for the extrinsic pathways and UV exposure (HeLa) for the intrinsic pathways. By comparing the SPR time-course of apoptosis with phase contrast micrographs, we demonstrate that the cell morphological hallmarks of apoptosis are the major contributors to the SPR signal. Altogether, our results validate the use of SPR as an accurate label-free assay for the real-time monitoring of apoptosis-triggered cell morphological changes.     

Microtubule rearrangements during mitosis in multinucleate cells

The peroxidase-antiperoxidase (PAP) method for the detection of polymerized tubulin has been used to study the microtubule rearrangements during mitosis in PtK1 and HeLa multinucleate cells obtained by polyethyleneglycol (PEG)-mediated fusion. We demonstrate here that the transition of the microtubular cytoskeleton from interphase to mitosis is an inducible event and independent of the factor(s) responsible for chromatin condensation and nuclear envelope breakdown. However, for the induction of the microtubule rearrangements nuclear envelope breakdown is required. At midprophase, cytoskeletal microtubule rearrangements start for multinucleate PtK1 cells, whereas in HeLa cells such changes are delayed, and a more abrupt transition is observed here. After complete nuclear envelope breakdown (prometaphase) mitotic asters and spindles but no cytoplasmic (interphase) microtubuli can be observed in both systems. Metaphase is characterized by an interaction between the different mitotic poles which show the form of bipolar spindles, but individual separated mitotic poles far removed from the chromatin can also be seen.     

Identification of DISE-inducing shRNAs by monitoring cellular responses

Off-target effects (OTE) are an undesired side effect of RNA interference (RNAi) caused by partial complementarity between the targeting siRNA and mRNAs other than the gene to be silenced. The death receptor CD95 and its ligand CD95L contain multiple sequences that when expressed as either si- or shRNAs kill cancer cells through a defined OTE that targets critical survival genes. Death induced by survival gene elimination (DISE) is characterized by specific morphological changes such as elongated cell shapes, senescence-like enlarged cells, appearance of large intracellular vesicles, release of mitochondrial ROS followed by activation of caspase-2, and induction of a necrotic form of mitotic catastrophe. Using genome-wide shRNA lethality screens with eight different cancer cell lines, we recently identified 651 genes as critical for the survival of cancer cells. To determine whether the toxic shRNAs targeting these 651 genes contained shRNAs that kill cancer cell through DISE rather than by silencing their respective target genes, we tested all shRNAs in the TRC library derived from a subset of these genes targeting tumor suppressors (TS). We now report that only by monitoring the responses of cancer cells following expression of shRNAs derived from these putative TS it was possible to identify DISE-inducing shRNAs in five of the genes. These data indicate that DISE in general is not an undefined toxic response of cells caused by a random OTE but rather a specific cellular response with shared features that points at a specific biological function involving multiple genes in the genome.     

TMEM166, a novel transmembrane protein,regulates cell autophagy and apoptosis

Programmed cell death can be divided into apoptosis and autophagic cell death. We describe the biological activities of TMEM166 (transmembrane protein 166, also known as FLJ13391), which is a novel lysosome and endoplasmic reticulum-associated membrane protein containing a putative TM domain. Overexpression of TMEM166 markedly inhibited colony formation in HeLa cells. Simultaneously, typical morphological characteristics consistent with autophagy were observed by transmission electron microscopy, including extensive autophagic vacuolization and enclosure of cell organelles by double-membrane structures. Further experiments confirmed that the overexpression of TMEM166 increased the punctate distribution of MDC staining and GFP-LC3 in HeLa cells, as well as the LC3-II/LC3-I proportion. On the other hand, TMEM166-transfected HeLa and 293T cells succumbed to cell death with hallmarks of apoptosis including phosphatidylserine externalization, loss of mitochondrial transmembrane potential, caspase activation and chromatin condensation. Kinetic analysis revealed that the appearance of autophagy-related biochemical parameters preceded the nuclear changes typical of apoptosis in TMEM166-transfected HeLa cells. Suppression of TMEM166 expression by small interference RNA inhibited starvation-induced autophagy in HeLa cells. These findings show for the first time that TMEM166 is a novel regulator involved in both autophagy and apoptosis.     

Pineal peptide with an inhibiting effect on growth of HeLa S3 tumor cells

The aim of the present study was to assess whether a peptide fraction isolated from calf pineal glands has an effect on proliferation and morphology of HeLa S3 tumor cells. Under the experimental conditions adopted, the results showed that the peptide has a marked inhibitory effect on proliferation of HeLa S3 cells and that permeabilization with calcium phosphate of the plasmatic membrane increases this effect. Moreover, the pineal peptide affects the cytoskeletal morphology of HeLa cells by modifying the distribution of actin. The peptide is probably internalized by the cells and irreversibly modifies the cytoskeletal morphology with consequent inhibition of cellular proliferation.     

Change of Morphology and Cytoskeletal Protein Gene Expression during Dibutyryl cAMP-induced Differentiation in C6 Glioma Cells

Elevation of the intracellular cAMP level induces morphological changes of astrocyte-like differentiation in C6 glioma cells. Such changes may be accompanied with expression of cytoskeletal protein genes. We therefore analyzed morphological changes after a treatment with dibutyryl cAMP (dbcAMP) and then assessed the expression of cytoskeletal protein genes by a quantitative real-time polymerase chain reaction. The cell number remained unaltered upon incubation with 1 mM dbcAMP in medium supplemented with 0.1% fetal bovine serum (FBS), whereas the number and lengths of processes increased, when compared with those of cells incubated in medium supplemented with 0.1% or 10% FBS only. The amounts of β-actin, γ-actin, and β-tubulin mRNAs in C6 cells, but not α-tubulin mRNA, increased during the early proliferation in DMEM containing 10% FBS. The expression of cytoskeletal protein genes decreased when incubated with 0.1% FBS or 1 mM dbcAMP in 0.1% FBS, compared with those of cells cultured in 10% FBS. These results indicated that, during the early proliferation in normal culture condition, the expression of cytoskeletal protein genes in C6 cells, except α-tubulin, increased, while in differentiating or differentiated C6 glioma cells, cAMP-induced morphological changes were not accompanied with elevation of gene expression for cytoskeletal proteins, such as actin and tubulin.     

Differences in virus-induced cell morphology and in virus maturation between MVA and other strains (WR,Ankara, and NYCBH) of vaccinia virus in infected human cells

Live recombinants based on attenuated modified vaccinia virus Ankara (MVA) are potential vaccine candidates against a broad spectrum of diseases and tumors. To better understand the efficacy of MVA as a human vaccine, we analyzed by confocal and electron microscopy approaches MVA-induced morphological changes and morphogenetic stages during infection of human HeLa cells in comparison to other strains of vaccinia virus (VV): the wild-type Western Reserve (WR), Ankara, and the New York City Board of Health (NYCBH) strains. Confocal microscopy studies revealed that MVA infection alters the cytoskeleton producing elongated cells (bipolar), which do not form the characteristic actin tails. Few virions are detected in the projections connecting neighboring cells. In contrast, cells infected with the WR, Ankara, and NYCBH strains exhibit a stellated (multipolar) or rounded morphology with actin tails. A detailed transmission electron microscopy analysis of HeLa cells infected with MVA showed important differences in fine ultrastructure and amounts of the viral intermediates compared to cells infected with the other VV strains. In HeLa cells infected with MVA, the most abundant viral forms are intracellular immature virus, with few intermediates reaching the intracellular mature virus (IMV) form, at various stages of maturation, which exhibit a more rounded shape than IMVs from cells infected with the other VV strains. The "IMVs" from MVA-infected cells have an abnormal internal structure ("atypical" viruses) with potential alterations in the core-envelope interactions and are unable to significantly acquire the additional double envelope to render intracellular envelope virus. The presence of potential cell-associated envelope virus is very scarce. Our findings revealed that MVA in human cells promotes characteristic morphological changes to the cells and is able to reach the IMV stage, but these virions were not structurally normal and the subsequent steps in the morphogenetic pathway are blocked.     

Generation of ROS in cells on exposure to CW and pulsed near-infrared laser tweezers.

We report the results of a study on generation of reactive oxygen species (ROS) and changes in the membrane potential of mitochondria of carcinoma of cervix (HeLa) and Chinese hamster ovary (CHO) cells following exposure to continuous wave (cw) or pulsed Nd: YAG laser (1064 nm). For a given laser irradiation, the generation of ROS and induced changes in the membrane potential of mitochondria were more pronounced for HeLa cells as compared to CHO cells. However, in both the cells the laser dose required to elicit a given change was much lower with pulsed laser exposure compared to that required with a cw laser exposure. This suggests involvement of photothermal effects in the laser irradiation induced changes. Mechanistic studies using quenchers for ROS suggest that laser irradiation leads to generation of hydroxyl radicals.     

HeLa cells apoptosis induced by 1,7-dimethyl-1,4,7,10-tetraazacyclododecane

Preliminary pharmacological tests showed that 1,7-dimethyl-1,4,7,10-tetraazacyclododecane (DMC) had antitumor activity against HeLa and A549 cell lines in vitro. The HeLa cells apoptosis induced by DMC was examined by flow cytometric meter, and further confirmed by observing the morphological changes and DNA fragmentation. No observation of A549 cells induced apoptosis was observed by DMC.