cytochalasin-b and Necrosis

BteA is one of the effectors secreted from the Bordetella bronchiseptica type III secretion system. It has been reported that BteA induces necrosis in mammalian cells; however, the roles of BteA during the infection process are largely unknown. In order to investigate the BteA functions, morphological changes of the cells infected with the wild-type B. bronchiseptica were examined by time-lapse microscopy. L2 cells, a rat lung epithelial cell line, spread at 1.6 hours after B. bronchiseptica infection. Membrane ruffles were observed at peripheral parts of infected cells during the cell spreading. BteA-dependent cytotoxicity and cell detachment were inhibited by addition of cytochalasin D, an actin polymerization inhibitor. Domain analyses of BteA suggested that two separate amino acid regions, 200-312 and 400-658, were required for the necrosis induction. In order to examine the intra/intermolecular interactions of BteA, the amino- and the carboxyl-terminal moieties were purified as recombinant proteins from Escherichia coli. The amino-terminal moiety of BteA appeared to interact with the carboxyl-terminal moiety in the pull-down assay in vitro. When we measured the amounts of bacteria phagocytosed by J774A.1, a macrophage-like cell line, the phagocytosed amounts of B. bronchiseptica strains that deliver BteA into the host cell cytoplasm were significantly lower than those of strains that lost the ability to translocate BteA into the host cell cytoplasm. These results suggest that B. bronchiseptica induce necrosis by exploiting the actin polymerization signaling pathway and inhibit macrophage phagocytosis.

Topics: Actin Cytoskeleton; Amino Acids; Animals; Bacterial Proteins; Bacterial Secretion Systems; Bordetella bronchiseptica; Cell Shape; Chlorocebus aethiops; COS Cells; Cytochalasin B; Endocytosis; Gentamicins; L-Lactate Dehydrogenase; Macrophages; Mice; Mutant Proteins; Necrosis; Phagocytes; Phagocytosis; Protein Multimerization; Protein Structure, Tertiary; Rats; Signal Transduction; Time-Lapse Imaging

The cytokinesis-block micronucleus cytome (CBMN) assay, introduced by Fenech, was used to demonstrate different types of DNA damage in MOLT-3 human lymphoblastoid cells exposed to 10 μM zidovudine (AZT). In addition, we explored the cytoprotective potential of two antioxidants, WR-1065 and Tempol, to decrease AZT-induced genotoxicity. Binucleated cells, arrested by Cytochalasin B (Cyt B), were evaluated for micronuclei (MN), caused by DNA damage or chromosomal loss, and chromatin nucleoplasmic bridges (NPBs), caused by telomere attrition. Additionally, nuclear buds (NBUDs), caused by amplified DNA, and apoptotic and necrotic (A/N) cells were scored. We hypothesized that AZT exposure would increase the frequency of genotoxic end points, and that the antioxidants Tempol and WR-1065 would protect against AZT-induced genotoxicity. MOLT-3 cells were exposed to 0 or 10 µM AZT for a total of 76 hr. After the first 24 hr, 0 or 5 µM WR-1065 and/or 0 or 200 µM Tempol were added for the remainder of the experiment. For the last 28 hr (of 76 hr), Cyt B was added to arrest replication after one cell division, leaving a predominance of binucleated cells. The nuclear division index (NDI) was similar for all treatment groups, indicating that the exposures did not alter cell viability. MOLT-3 cells exposed to AZT alone had significant (P < 0.05) increases in MN and NBs, compared to unexposed cells. Both Tempol and WR-1065 protected against AZT-induced MN formation (P < 0.003 for both), and WR-1065, but not Tempol, reduced the levels of A/N (P = 0.041). In cells exposed to AZT/Tempol there were significantly reduced levels of NBUDs, compared to cells exposed to AZT alone (P = 0.015). Cells exposed to AZT/WR-1065 showed reduced levels of NPBs, compared to cells exposed to AZT alone (P = 0.037). Thus WR-1065 and Tempol protected MOLT-3 cells against specific types of AZT-induced DNA damage.

Topics: Antioxidants; Apoptosis; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Cell Survival; Chromatin; Chromosomes; Cyclic N-Oxides; Cytochalasin B; DNA Damage; Humans; Mercaptoethylamines; Micronucleus Tests; Mutagens; Necrosis; Radiation-Protective Agents; Spin Labels; Zidovudine

The cytokinesis-block micronucleus cytome (CBMN cyt) assay is a new and comprehensive technique for measuring DNA damage, cytostasis, and cytotoxicity in different tissue types, including lymphocytes. DNA damage events are scored specifically in once-divided binucleated cells. These events include; (a) micro-nuclei (MNi), a biomarker of chromosome breakage and/or whole chromosome loss; (b) nucleoplasmic bridges (NPBs), a biomarker of DNA misrepair and/or telomere end-fusions; and (c) nuclear buds (NBUDs), a biomarker of elimination of amplified DNA and/or DNA repair complexes. Cytostatic effects are measured via the proportion of mono-, bi-, and multinucleated cells and cytotoxicity via necrotic and/or apoptotic cell ratios. The assay has been applied to the biomonitoring of in vivo exposure to genotoxins, in vitro genotoxicity testing and in diverse research fields, such as nutrigenomics and pharmacogenomics. It has also been shown to be important as a predictor of normal tissue and tumor radiation sensitivity and cancer risk. This protocol also describes the current established methods for culturing lymphocytes, slide preparation, cellular and nuclear staining, scoring criteria, data recording, and analyses.

Topics: Apoptosis; Cell Nucleus; Cell Separation; Cells, Cultured; Centrifugation; Cytochalasin B; Cytokinesis; Humans; Lymphocyte Count; Lymphocytes; Micronucleus Tests; Necrosis; Staining and Labeling; Tissue Fixation

Apoptosis and necrosis need to be differentiated in order to distinguish drug-induced cell death from spontaneous cell death due to hypoxia. The ability to differentiate between these two modes of cell death, especially at an early stage in the process, could have a significant impact on accessing the outcome of anticancer drug therapy in the clinic. Nuclear magnetic resonance spectroscopy was used to distinguish apoptosis from necrosis in human cervical carcinoma (HeLa) cells. Apoptosis was induced by treatment with the topoisomerase II inhibitor etoposide, whereas necrosis was induced by the use of ethacrynic acid or cytochalasin B. We found that the intensity of the methylene resonance increases significantly as early as 6 h after the onset of apoptosis, but that no such changes occur during necrosis. The spectral intensity ratio of the methylene to methyl resonances also shows a high correlation with the percentage of apoptotic cells in the sample (r2=0.965, P<0.003).

Topics: Apoptosis; Cytochalasin B; Ethacrynic Acid; Female; HeLa Cells; Humans; Magnetic Resonance Spectroscopy; Necrosis; Protons; Uterine Cervical Neoplasms

Homocysteine (Hcy), an immediate precursor of methionine (Met), is considered a risk factor for cardiovascular disease, Alzheimer's disease and neural tube defects. Hcy concentration is also reported to correlate positively with the micronucleus index in lymphocytes in vivo, a marker of chromosome damage. However, it is unclear whether Hcy is genotoxic or simply a biomarker of folate deficiency, a known cause of chromosome damage. We investigated whether high concentrations of Hcy are genotoxic to human lymphocytes in vitro using the cytokinesis-block micronucleus assay. Eighteen lymphocyte cultures were initiated in Met-free and serum-free RPMI 1640 medium for each of four male volunteers aged 22-23 years. At 0, 24, 44 and 72 h, cultures were spiked with L-Hcy or L-Met to achieve concentrations ranging between 50 and 400 microM. The concentration of Hcy at 96 h ranged from 19.45 +/- 2.34 to 149.02 +/- 28.16 microM in Hcy cultures and 0.91 +/- 0.17 to 2.15 +/- 0.9 microM in Met cultures spiked with 50 and 400 microM of metabolite, respectively. Forty-four hours after mitogen stimulation, cytokinesis was inhibited with cytochalasin B. After 96 h, cells were transferred to microscope slides and the frequency of micronucleated-binucleate and necrotic cells was scored. Neither Hcy (P = 0.24) nor Met (P = 0.93) had an apparent dose effect on micronucleus frequency. However, when data were pooled, micronucleus frequency was moderately higher (50.1%) in Hcy- than in Met-spiked cultures (P = 0.04; paired t-test). Hcy concentration was positively correlated with necrosis (P < 0.0005; r(2)= 0.276), however, when data were pooled, levels of necrosis were higher in Met- than in Hcy-spiked cultures (P= 0.01; paired t-test). Further research is required to define more clearly the genotoxic and cytotoxic potential of homocysteine and its metabolites.

Topics: Adult; Analysis of Variance; Cells, Cultured; Cytochalasin B; Dose-Response Relationship, Drug; Homocysteine; Humans; Lymphocytes; Male; Methionine; Micronucleus Tests; Models, Chemical; Mutagens; Necrosis; Time Factors

Mutations in the Wiskott-Aldrich syndrome protein (WASP) have been hypothesized to cause defective actin cytoskeletal function. This resultant dysfunction of the actin cytoskeleton has been implicated in the pathogenesis of Wiskott-Aldrich syndrome (WAS). In contrast, it was found that stimulated actin polymerization is kinetically normal in the hematopoietic lineages affected in WAS. It was also found that the actin cytoskeleton in WAS platelets is capable of producing the hallmark cytoarchitectural features associated with activation. Further analysis revealed accelerated cell death in WAS lymphocytes as evidenced by increased caspase-3 activity. This increased activity resulted in accelerated apoptosis of these cells. CD95 expression was also increased in these cells, suggesting an up-regulation in the FAS pathway in WAS lymphocytes. Additionally, inhibition of actin polymerization in lymphocytes using cytochalasin B did not accelerate apoptosis in these cells. This suggests that the accelerated apoptosis observed in WAS lymphocytes was not secondary to an underlying defect in actin polymerization caused by mutation of the WAS gene. These data indicate that WASP does not play a universal role in signaling actin polymerization, but does play a role in delaying cell death. Therefore, the principal consequence of mutations in the WAS gene is to accelerate lymphocyte apoptosis, potentially through up-regulation of the FAS-mediated cell death pathway. This accelerated apoptosis may ultimately give rise to the clinical manifestations observed in WAS. (Blood. 2000;95:1283-1292)

Topics: Actins; Adenosine Diphosphate; Apoptosis; Blood Platelets; Cell Survival; Cytochalasin B; Cytoskeleton; Dinoprost; fas Receptor; Humans; In Vitro Techniques; Kinetics; Leukocytes; Lymphocytes; Mutation, Missense; N-Formylmethionine Leucyl-Phenylalanine; Necrosis; Point Mutation; Proteins; Reference Values; src Homology Domains; Tetradecanoylphorbol Acetate; Thrombocytopenia; Wiskott-Aldrich Syndrome; Wiskott-Aldrich Syndrome Protein

A method is described for the inclusion of apoptotic and necrotic cells in the cell counts obtained in the cytokinesis-block micronucleus (CBMN) assay, which is conventionally used solely for the assessment of chromosome breakage, chromosome loss and frequency of dividing cells. The morphological criteria for the recognition and discrimination between necrotic, apoptotic and viable cells are described. Using this comprehensive method we have evaluated the cytotoxic and genotoxic effects of hydrogen peroxide (0-100 microM) in lymphocytes exposed in RPMI 1640 medium. The results obtained indicated significant (P < 0.05) correlations between hydrogen peroxide concentration and the frequency of micronucleated cells (r = 0.39), necrotic cells (r = 0.73), apoptotic cells (r = -0.26) and binucleated cells (r = -0.55). Almost similar results were obtained using the cytosine arabinoside modification of the CBMN assay, which enables excision-repaired sites to be converted to micronuclei. Some of the above end-points were significantly (P < 0.05) correlated with each other (necrosis and apoptosis, R = -0.39; necrosis and micronucleated cell frequency, R = 0.46; necrosis and binucleated cells, R = -0.78; apoptosis and binucleated cells, R = 0.32). It was therefore necessary to use multiple regression analysis to identify the main event induced by hydrogen peroxide, which was necrosis (beta = 0.57, P = 0.0001) and not micronucleus formation (beta = 0. 15, P = 0.1332). Using an ELISA assay we showed that hydrogen peroxide did not induce 8-hydroxydeoxyguanosine. Our data show that the proposed comprehensive test system may provide a better procedure for classifying potential toxic chemicals and enable discrimination between agents that primarily induce cytotoxic effects as opposed to genotoxic effects. The integration of apoptosis and necrosis into the micronucleus assay may also be of practical use in radiosensitivity studies.

Topics: Adult; Apoptosis; Cell Division; Cells, Cultured; Cytochalasin B; DNA; DNA Damage; Dose-Response Relationship, Drug; Humans; Hydrogen Peroxide; Lymphocytes; Male; Micronucleus Tests; Models, Biological; Necrosis; Time Factors

Using the Ehrlich ascite carcinoma cells, exposed to oxidative stress, heat shock, cytochalasin B, vinblastine, Triton X-100 and energy starvation, morphological changes, DNA degradation, and form of cell death were investigated. A rather clear specificity of cell morphology, characteristic of apoptosis, was revealed for the most of treatments, including membrane blebs, chromatin condensation and apoptotic body formation. Karyorhexis was not common for the examined cells. After energy starvation, oxidative stress, heat shock and Triton X-100 treatment, the apoptotic cells were seen to undergo secondary necrosis rather soon; this was not the case after cytochalasin B or vinblastine treatments. In cells exposed to heat shock, cellular pyknosis was frequently developed. Such cells did not undergo secondary necrosis for a long time. The DNA degradation in cells occurred in chaotic manner (without ladder pattern). The fraction of degraded DNA corresponded to that of cells undergoing secondary necrosis.

Topics: Animals; Antineoplastic Agents, Phytogenic; Apoptosis; Carcinoma, Ehrlich Tumor; Cytochalasin B; DNA Damage; DNA, Neoplasm; Electrophoresis, Polyacrylamide Gel; Energy Metabolism; Hot Temperature; Mice; Mice, Inbred C57BL; Microscopy, Fluorescence; Necrosis; Octoxynol; Oxidative Stress; Staining and Labeling; Surface-Active Agents; Trypan Blue; Tumor Cells, Cultured; Vinblastine

Human promyelomonocytic leukemia cells HL-60 were treated with etoposide or cytochalasin B to induce apoptosis or necrosis, respectively. We report here that during necrosis, the DNA-repair associated nuclear enzyme poly(ADP-ribose) polymerase (PARP) was degraded differently from that observed during apoptosis. While apoptotic HL-60 cells exhibit only the signature 89 kDa fragment of PARP, necrosis of these cells was accompanied by formation of major fragments at MWr approximately 89 and 50 kDa and minor fragments at approximately 40 and 35 kDa. The necrosis-specific degradation of PARP was coincident with other changes detected by flow cytometric analysis, but earlier than the extensive degradation of DNA. Therefore, the unique necrotic degradation of PARP could be used as a sensitive indicator for necrotic death of cells.

Topics: Apoptosis; Cytochalasin B; DNA Repair; HL-60 Cells; Humans; Necrosis; Poly(ADP-ribose) Polymerases

Topics: Animals; Cell Count; Chick Embryo; Cytochalasin B; Indoles; Mitosporic Fungi; Necrosis; Phagocytes; Toes