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Hoechst Staining (hoechst + staining)
Selected AbstractsCharacteristics of okadaic acid,induced cytotoxic effects in CHO K1 cellsENVIRONMENTAL TOXICOLOGY, Issue 6 2003C. Huynh-Delerme Abstract This article reports the results of investigations into the process of cell death induced in the Chinese hamster ovary cell K1 subclone (CHO K1) by okadaic acid (OA), a hydrophobic polyether produced by marine dinoflagellates. The IC50 was about 13 nM OA after 24 h of treatment, as determined using neutral red. With the MTT assay, the IC50 was 25 nM, although in this case 25% of the initial staining was still observed at 100 nM. Hoechst staining showed that mitotic figures accumulated at 12 nM OA after a 24- or 48-h treatment. In experiments limited to a 3-day treatment without changing the medium, CHO K1 cells were engaged in the death process at 50 nM OA after about 20 h and at 10 nM OA after 48 h. In many cells nuclear fragmentation that resulted in the apparent appearance of vesicles correlated with increasing cellular volume. But additional cell fragmentation was not observed with any treatment, and the chromatin material seemed to progressively disappear inside the cells. DNA fragmentation was analyzed by electrophoresis and with the TUNEL technique. With both techniques, the DNA was fragmented by 48 h in both 25 and 50 nM OA. Electrophoresis showed that both adherent and nonadherent cells were affected. Annexin-positive/ propidium iodide (PI),negative cells were rarely observed after OA treatment. Some were seen under the scanning cytometer after 20 h at 50 nM OA or after 48 h at 10 nM OA, but they were never detected by flow cytometry. Most of the time scanning cytometry showed either unstained cells or PI-positive (annexin-positive or -negative) cells (48 h, 50 nM, or 72 h, 10 nM). Flow cytometry cytograms showed two cell subpopulations: one composed of a majority of smaller cells, the other of larger cells. The larger cells markedly decreased with time and OA treatment (50 and 100 nM). Stained-cell counting showed that all cells that stained were both annexin- and PI positive and that most PI-positive cells were smaller. Ki67 antigen labeling showed the proliferative activity of CHO K1 cultures but also demonstrated the loss of this activity in smaller cells treated with 50 nM OA for 48 h. We concluded that in our culture conditions the main OA target within CHO K1 cultures was dividing cells. Our results suggest that cells with disturbed metaphase,anaphase enter apoptosis, leading to necrotic daughter cells. © 2003 Wiley Periodicals, Inc. Environ Toxicol 18: 383,394, 2003 [source] Gambogic acid induces apoptosis by regulating the expression of Bax and Bcl-2 and enhancing caspase-3 activity in human malignant melanoma A375 cellsINTERNATIONAL JOURNAL OF DERMATOLOGY, Issue 2 2009Xiaoyuan Xu Objectives, To investigate the effect of a Chinese traditional medicine, gambogic acid (GA), on human malignant melanoma (MM) A375 cells and to study the mechanism of apoptosis induced by GA. Methods, A375 cells were treated with GA at different doses and for different times, and their proliferation and viability were detected by 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay. Apoptosis induced by GA in A375 cells was observed by annexin-V/propidium iodide doubling staining flow cytometry assay and Hoechst staining. To further determine the molecular mechanism of apoptosis induced by GA, the changes in expression of Bcl-2 and Bax were detected by real-time reverse transcriptase-polymerase chain reaction (RT-PCR) and Western blot, and caspase-3 activity was measured by fluorescence resonance energy transfer (FRET) probe. Results, After incubation with GA, A375 cell proliferation was dramatically inhibited in a dose-dependent manner. After these cells had been exposed to GA for 24, 36 and 48 h, the IC50 values were 1.57 ± 0.05, 1.31 ± 0.20, and 1.12 ± 0.19 µg/mL, respectively. Treatment of A375 cells with GA (2.5,7.5 µg/mL) for 36 h resulted in an increased number of early apoptotic cells, which ranged from 27.6% to 41.9%, in a dose-dependent manner, compared with only 3.5% apoptotic cells in the non-GA-treated group. An increase in Bax and decrease in Bcl-2 expression were found by real-time RT-PCR and Western blot. Caspase-3 activity was increased in a dose-dependent manner, observed by FRET probe. Conclusion, GA can inhibit the proliferation of A375 cells and induce their apoptosis, which may be related to the up-regulation of the Bax/Bcl-2 ratio and caspase-3 activity. [source] Non-apoptogenic killing of hela cervical carcinoma cells after short exposure to the alkylating agent N -methyl- N, -nitro- N -nitrosoguanidine (MNNG)JOURNAL OF CELLULAR BIOCHEMISTRY, Issue 6 2003Józefa W, sierska-G Abstract We examined the action of N -methyl- N, -nitro- N -nitrosoguanidine (MNNG) on HeLa cells and compared it with that of cisplatin (CP). MNNG directly killed a substantial number of cells within 1 hour and resulted in strong DNA-damage as evidenced by Comet measurements. Despite appearance of DNA lesions, p53 protein was not activated. Analysis of HeLa cells treated with MNNG for 1h, 3h and 6h by flow cytometry and by Hoechst staining did not reveal any sub-G1 cell population and chromatin condensation/fragmentation characteristic for apoptosis, respectively. Also, no biochemical changes typical for apoptosis such as activation of caspase-3 or release of cytochrome C from mitochondria were detected. Inactivation of PARP-1 reduced the direct cytotoxicity exerted by MNNG. Our results showing that despite appearance of severe DNA lesions after short exposure of HeLa cells to MNNG neither activation of p53 response nor induction of apoptosis occurred implicate that generation of strong DNA damage is not sufficient to stabilize p53 protein in HeLa cells. Our data unequivocally show that the conscientious determination of the type of cell death induced by genotoxic agents is necessary. The assessment of the changes based on at least a few independent criteria is required to discriminate between apoptosis and necrosis. Since the alkylating agents generate DNA strand breaks, the recruitment of methods based on determination of DNA cleavage such as DNA ladder or TUNEL assay for evaluation of apoptosis is not adequate. © 2003 Wiley-Liss, Inc. [source] Alteration of argyrophilic nucleolar organizer region associated (Ag-NOR) proteins in apoptosis-induced human salivary gland cells and human oral squamous carcinoma cellsJOURNAL OF ORAL PATHOLOGY & MEDICINE, Issue 4 2001Yasuhiro Morimoto Abstract: The level of argyrophilic nucleolar organizer regions (AgNORs) and AgNOR-associated proteins (Ag-NOR proteins) varies with cell activity, including ribosomal biogenesis occurring in proliferating cells. Proteins associated with some AgNORs are detected by a specific silver staining. To investigate a possible relationship between apoptosis and the AgNORs or Ag-NOR proteins, we examined the changes of AgNORs and Ag-NOR proteins during apoptosis in a human salivary gland cell line, HSG cells, and a human oral squamous carcinoma cell line, SCC-25 cells. Apoptosis was induced by treatment of HSG and SCC-25 cells with okadaic acid. Proteins prepared from HSG and SCC-25 cells treated with varying concentrations of okadaic acid (OA) were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by transferring to transfer membranes and staining for Ag-NOR proteins by modified Western blot analysis. Four major bands (110 kDa, 43 kDa, 39kDa, and 37 kDa) were detected in the proteins obtained from the control cells. The level of the 110-kDa protein decreased in the proteins prepared from OA-induced apoptotic cells; however, the reaction intensity of the other three bands was changed in apoptotic cells. An additional band of an 80-kDa Ag-NOR protein appeared and increased in the apoptotic cells. Cellular fractionation of HSG cells and SCC-25 cells was done with or without apoptotic induction. An 80-kDa Ag-NOR protein was detected in the nuclear fraction prepared from the apoptotic cells, while the 110-kDa protein decreased in the nuclear fraction of these cells. The 110-kDa Ag-NOR protein may be nucleolin (C23) as deduced from its AgNOR staining features, including molecular weight. The 80-kDa protein may be the cleavage product of the 110-kDa protein. In the cell-free apoptotic system, in which intact nuclei of HSG cells were incubated with the cytosol fraction of apoptotic HSG and SCC-25 cells, the 80-kDa Ag-NOR protein was detected in nuclei incubated with the cytosol fraction of apoptotic cells, while the level of the 110-kDa protein decreased. The changes of Ag-NOR proteins in nuclei prepared from SCC-25 cells incubated with cytosol fractions prepared from HSG and SCC-25 cells were identical to those of the HSG cells. The alternation of AgNORs in apoptosis-induced HSG cells was also examined using double staining with Hoechst 33342 and silver nitrate. Hoechst staining revealed typical apoptotic nuclei, which exhibited highly fluorescent condensed chromatin in OA-treated HSG cells. Silver grains representing AgNORs were not detected in the cells undergoing apoptosis. The dual-imposition view confirmed that AgNORs, which are visible as dots in nucleoli in the control cells, disappeared from the apoptotic nuclei of HSG cells. Our results indicate that the 110-kDa nucleolar Ag-NOR protein is associated with apoptosis and is cleaved during apoptosis. [source] Extracts of various species of Epilobium inhibit proliferation of human prostate cellsJOURNAL OF PHARMACY AND PHARMACOLOGY: AN INTERNATI ONAL JOURNAL OF PHARMACEUTICAL SCIENCE, Issue 5 2003Annabella Vitalone This study examined whether various species of Epilobium, a phytotherapeutic agent used in folk medicine as a treatment for benign prostatic hyperplasia, may have an antiproliferative effect in PZ-HPV-7 human prostatic epithelial cells in-vitro. The MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl-tetrazolium bromide) test, [methyl- 3H]thymidine incorporation into DNA and flow cytometry analysis were used to evaluate cell proliferation. Ethanolic extracts of E. spicatum, E. rosmarinifolium and E. tetragonum inhibited DNA synthesis in PZ-HPV-7 cells. While at high concentrations all extracts were cytotoxic, DNA synthesis was also decreased at levels that caused no or little cytotoxicity. Treatment of cells with Epilobium extracts did not result in a formation of DNA fragments (evaluated by the TUNEL assay) or chromatin condensation (assessed by Hoechst staining). Flow cytometry analysis indicated that Epilobium extracts inhibit the progression of the cell cycle from the G0/G1 phase. These results suggest that extracts of Epilobium inhibit proliferation of human PZ-HPV-7 cells in-vitro by affecting progression of the cell cycle. This study provides some initial biological plausibility for the use of Epilobium extracts in benign prostatic hyperplasia. [source] Viral ssRNA Induces First Trimester Trophoblast Apoptosis through an Inflammatory MechanismAMERICAN JOURNAL OF REPRODUCTIVE IMMUNOLOGY, Issue 1 2010Paulomi B. Aldo Citation Aldo PB, Mulla MJ, Romero R, Mor G, Abrahams VM. Viral ssRNA induces first-trimester trophoblast apoptosis through an inflammatory mechanism. Am J Reprod Immunol 2010; 64: 27,37 Problem, Infection during pregnancy represents a significant cause of mobility and mortality. While viruses pose a major threat, little is known about their effect on early pregnancy, or the mechanisms involved. The objective of this study was to characterize the trophoblast response following exposure to viral ssRNA. Method of study, First trimester trophoblast cells were treated with or without viral ssRNA. Cytokine production was measured using multiplex analysis and ELISA. Apoptosis was determined using Hoechst staining, cell viability, and caspase activity assays. Results, Treatment of trophoblasts with viral ssRNA increased their secretion of IL-8, IL-6, and IFN,. However, the ssRNA also induced trophoblast apoptosis. To test whether the viral ssRNA-induced inflammatory response was responsible for this induction of apoptosis, conditioned media (CM) from trophoblasts were added to a fresh culture of cells. The CM from viral ssRNA-treated induced higher levels of trophoblast apoptosis than the control CM. Moreover, recombinant IFN, induced trophoblast apoptosis. Conclusion, We demonstrate that viral ssRNA induces a pro-inflammatory and type I interferon response in the trophoblast and this inflammatory process may indirectly induce trophoblast apoptosis. These results provide a novel mechanism by which certain viral infections might compromise placental integrity and function, and therefore, pregnancy outcome. [source] The novel ruthenium,, -linolenic complex [Ru2(aGLA)4Cl] inhibits C6 rat glioma cell proliferation and induces changes in mitochondrial membrane potential, increased reactive oxygen species generation and apoptosis in vitroCELL BIOCHEMISTRY AND FUNCTION, Issue 1 2010Geise Ribeiro Abstract The present study reports the synthesis of a novel compound with the formula [Ru2(aGLA)4Cl] according to elemental analyses data, referred to as Ru2GLA. The electronic spectra of Ru2GLA is typical of a mixed valent diruthenium(II,III) carboxylate. Ru2GLA was synthesized with the aim of combining and possibly improving the anti-tumour properties of the two active components ruthenium and , -linolenic acid (GLA). The properties of Ru2GLA were tested in C6 rat glioma cells by analysing cell number, viability, lipid droplet formation, apoptosis, cell cycle distribution, mitochondrial membrane potential and reactive oxygen species. Ru2GLA inhibited cell proliferation in a time and concentration dependent manner. Nile Red staining suggested that Ru2GLA enters the cells and ICP-AES elemental analysis found an increase in ruthenium from <0.02 to 425,mg/Kg in treated cells. The sub-G1 apoptotic cell population was increased by Ru2GLA (22,±,5.2%) when analysed by FACS and this was confirmed by Hoechst staining of nuclei. Mitochondrial membrane potential was decreased in the presence of Ru2GLA (44,±,2.3%). In contrast, the cells which maintained a high mitochondrial membrane potential had an increase (18,±,1.5%) in reactive oxygen species generation. Both decreased mitochondrial membrane potential and increased reactive oxygen species generation may be involved in triggering apoptosis in Ru2GLA exposed cells. The EC50 for Ru2GLA decreased with increasing time of exposure from 285,µM at 24,h, 211,µM at 48,h to 81,µM at 72,h. In conclusion, Ru2GLA is a novel drug with antiproliferative properties in C6 glioma cells and is a potential candidate for novel therapies in gliomas. Copyright © 2009 John Wiley & Sons, Ltd. [source] 2125: High glucose sensitizes human retinal endothelial cells for IFN-g-mediated apoptosisACTA OPHTHALMOLOGICA, Issue 2010R NAGARAJ Purpose The biochemical mechanisms by which inflammatory cytokines cause damage in the diabetic retina are poorly understood. Indoelamine 2, 3-dioxygenase (IDO) is an inducible by IFN-, enzyme and is the first enzyme of the kynurenine pathway, which produces cytotoxic kynurenines. In this study we have investigated the role of IDO in apoptosis of human retinal capillary endothelial cells (HREC) under hyperglycemic conditions. Methods HREC were cultured in medium containing high glucose (25 mM) or low glucose (7.5 mM) and incubated with 1-100 U/ml of IFN-,. IDO activity was measured by an HPLC assay. Expression of IFN-, receptor 1, and activation of the JAK-STAT signaling pathway along with activation of PKC-, was assessed by Western blotting. HREC apoptosis was measured by Hoechst staining. The role of IDO in HREC apoptosis was evaluated in the presence specific chemical inhibitors of the kynurenine pathway. Results IFN-, dose-dependently activated JAK-STAT signaling and PKC-,, and upregulated IDO. The IDO-mediated tryptophan oxidation led to formation of kynurenines, which was followed by chemical modification of proteins by kynurenines in HREC. These changes were accompanied by production of reactive oxygen species (ROS) and depletion of protein-free thiols. IFN-, inhibited cell cycle at low concentrations and caused caspase-3-mediated apoptosis and at higher concentrations, and those effects were amplified in the presence of high glucose in HREC. We found that IFN-, mediated cytotoxicity in HREC was primarily due to ROS generated by 3-hydroxykynurenine. Conclusion Our results suggest that high glucose sensitizes HREC to deleterious effects IFN-, and provide a novel mechanistic pathway for retinal capillary endothelial cell death in diabetes. [source] | |||||||||||||