RÉSUMÉ
Crisis periods in development are critical periods of cell death that have long been suggested as "epigenetic crises" which are central to normal and abnormal embryogenesis. Under in-vitro culture conditions, there are similar crisis periods or Hayflick limits of culture senescence. Epigenetic modulations from CpG methylation coupled to DNA replication provide an alternate timing mechanism to the telomere/telomerase biological clock. Physiological cell death in both development and in in-vitro isolates is primarily apoptotic. Arguments of divergent apoptotic death commitments as caspase dependent and independent pathways seem to suggest that there is no possibility of a global life and death signal. However recent reports implicating CpG specific cleavage in apoptosis implies that the powerful imprint mark that silences genes, protects genes from nuclease restriction, and modulates chromatin conformations, could provide a common commitment pathway of convergence in the death cascade. If the imprint mark were central to the apoptotic commitment, then apoptosis is Lamarckian not Darwinian.
Sujet(s)
Apoptose , Réplication de l'ADNRÉSUMÉ
A CpG-specific commitment common to both caspase-dependent and -independent cell deaths implies critical gene activity from epigenetic modulation. Using a focused microarray (genechip) of 22 housekeeping genes, which have canonical CpG islands at 5'-promoter regions, here we show critical regulation of vital intermediary metabolism and cell structure that are common to both caspase-dependent fasL-mediated and caspase-independent etoposide-mediated cell deaths. Gene activity of at least twofold under or over control levels and common to both cell death pathways was considered to be significantly regulated in common. Seven genes controlling energy production in glycolysis, tricarboxylic acid cycle, and the respiratory electron transport chain were significantly downregulated in common. Energy depletion is lethal. Downregulated pyruvate dehydrogenase E1 gene, in addition, suggested primary metabolic acidification. Cell acidification is also lethal. Critical derangement of the cell structure was suggested by common downregulation of the basal histone gene H2A.X which is required for nucleosome assembly. Common upregulation of the alpha-tubulin gene suggested perturbation of vital microtubular dynamics. Gene-commanded cell suicide was suggested. We further show that a Bcl-2 overexpression of three- to fourfold above normal levels could not prevent the CpG-specific megabase DNA cleavages in the two cell death pathways, but abolished their low-molecular-weight 200-bp ladder cleavages. Together with incomplete suppression of the other apoptotic expressions, the Bcl-2 inhibition point appeared downstream from the CpG-cleavage commitment point.
Sujet(s)
Apoptose/génétique , Caspases/métabolisme , Mort cellulaire/génétique , Ilots CpG/génétique , Régulation de l'expression des gènes , Gènes bcl-2/génétique , Annexine A5/métabolisme , Apoptose/effets des médicaments et des substances chimiques , Caspases/génétique , Cycle cellulaire , Lignée cellulaire , Fragmentation de l'ADN , Électrophorèse en champ pulsé , Métabolisme énergétique/génétique , Étoposide/pharmacologie , Ligand de Fas , Humains , Glycoprotéines membranaires/pharmacologie , Séquençage par oligonucléotides en batterie , Cellules cancéreuses en cultureRÉSUMÉ
Cell death in mammals seems to have caspase-dependent and -independent pathways unlike that in Caenorhabditis elegans where CED-3 protease activation is the central command. A recent suggestion to define apoptosis as the caspase-dependent or caspase-committed cell death form and leave cell death committed by other pathways as just cell death was meant to categorize the apparent divergence in mammalian cell death pathways. However, we show CpG oligonucleotides (ODN) blocking caspase-dependent fas(CD95) ligand-mediated apoptosis as well as caspase-independent etoposide-mediated apoptosis and etoposide-zVAD-mediated necrosis. CpG specificity was demonstrated by reversing the CpG motif or replacing it with a methylated motif (mCpG) which failed to inhibit. CpG ODN blocked CpG-specific DNA cleavage by rare-cutting NotI restriction, which produced a megabase cleavage pattern similar to that in the fasL and etoposide cell death inductions. CpG ODN inhibition was similar to that by CpG-specific SssI methylase. A common CpG-specific commitment point preceding caspase-dependent and -independent cell death pathways was suggested. CpG-specific modulation is a key epigenetic mechanism in genomic imprinting, resisting nuclease restriction, and patterning of chromatin conformations. It is now shown to have a powerful effect modulating cell death.
Sujet(s)
Caspases/métabolisme , Ilots CpG , Oligonucléotides/métabolisme , Chlorométhyl cétones d'acides aminés/pharmacologie , Annexine A5/pharmacologie , Apoptose/effets des médicaments et des substances chimiques , Inhibiteurs des caspases , Lignée cellulaire , Inhibiteurs de la cystéine protéinase/pharmacologie , Fragmentation de l'ADN , Antienzymes/pharmacologie , Étoposide/pharmacologie , Cytométrie en flux , Fluorescéine-5-isothiocyanate/pharmacologie , Colorants fluorescents/pharmacologie , Humains , Nécrose , Inhibiteurs de la synthèse d'acide nucléique/pharmacologie , Phosphatidylsérine/pharmacologie , Facteurs temps , Cellules cancéreuses en culture , Antigènes CD95/métabolismeRÉSUMÉ
Cell growth in human Chang liver cells was arrested in late mitosis with 600 microM zVAD-fmk. Associated cell death manifested cell shrinkage and phagocytic marking shown by phosphatidylserine (PS) externalization, in a dose-dependent manner. While low molecular weight internucleosomal ladder cleavages were suppressed, there were however high molecular weight DNA cleavages extending up to megabase level in association with chromatin condensation that appeared more marked than the staurosporine-induced positive-apoptosis control. Caspase-3 activity was suppressed. Specific caspase-3 inhibitor DEVD-cho also produced cell growth suppression with late mitosis arrest, and cell shrinkage which was expressed concomitantly with phagocytic PS marking in similar dose-dependent manners at 50 to 100 microM concentrations. Cell shrinkage, PS externalization, and high molecular weight DNA cleavages associated with chromatin condensation without 200 bp ladder fragmentations, were dissociated from caspase-3 activity. Anti-caspases inducing late mitosis arrest provided fresh insights into late mitosis progression.
Sujet(s)
Chlorométhyl cétones d'acides aminés/pharmacologie , Apoptose , Inhibiteurs des caspases , Mitose/effets des médicaments et des substances chimiques , Oligopeptides/pharmacologie , Caspase-3 , Caspases/métabolisme , Numération cellulaire , Cellules cultivées , Fragmentation de l'ADN , Humains , Foie/cytologie , Foie/effets des médicaments et des substances chimiques , Phosphatidylsérine/métabolismeRÉSUMÉ
Previous suggestions of CpG-specific apoptotic commitment implied critical epigenetic modulation of housekeeping genes which have canonical CpG islands at 5' promoter regions. Differential housekeeping gene activity however has not been shown. Using a focussed microarray (genechip) of 22 housekeeping genes we show this in apoptosis induced in human Chang liver cells by DCNP (2,6-dichloro-4-nitrophenol), a non-genotoxic inhibitor of sulfate detoxification. 3-7 folds downregulation of 9 genes in glycolysis, tricarboxylic acid cycle and the respiratory electron transport chain suggested gene-directed energy depletion which was correlated with observed ATP depletion. 4 folds downregulation of the pyruvate dehydrogenease gene suggested gene-directed metabolic acidosis which was correlated with observed cell acidification. Other differential housekeeping gene activity, including 4 folds upregulation of microtubular alpha-tubulin gene, and 2 folds upregulation of ubiquitin, also had a bearing on apoptosis. Broadspectrum zVAD-fmk caspase inhibition abolished 200 bp DNA ladder fragmentations but not the CpG-specific megabase fragmentations and other hallmarks of cell destruction, suggesting a caspase-independent cell death. Death appeared committed at gene-level.
Sujet(s)
Apoptose/effets des médicaments et des substances chimiques , Métabolisme énergétique/effets des médicaments et des substances chimiques , Régulation de l'expression des gènes/effets des médicaments et des substances chimiques , Hépatocytes/effets des médicaments et des substances chimiques , Nitrophénols/pharmacologie , Séquençage par oligonucléotides en batterie , Adénosine triphosphate/métabolisme , Chlorométhyl cétones d'acides aminés/pharmacologie , Apoptose/physiologie , Inhibiteurs des caspases , Caspases/métabolisme , Lignée cellulaire , Inhibiteurs de la cystéine protéinase/pharmacologie , Fragmentation de l'ADN , Relation dose-effet des médicaments , Métabolisme énergétique/génétique , Métabolisme énergétique/physiologie , Activation enzymatique , Cytométrie en flux , Hépatocytes/cytologie , Humains , Concentration en ions d'hydrogène , Mitochondries/métabolismeRÉSUMÉ
Etoposide induced a megabase (Mb) fragmentation pattern identical with that from genomic digestion by NotI restriction endonuclease which specifically cleaves CpG islands in euchromatin domains. Redigestion by NotI produced no change, suggesting cleavage in the same or closely related sites in euchromatin domains. Preferential euchromatin cleavage was further suggested by harvested metaphase chromosomes showing self-inflicted resolution of light G-bandings (R-bandings), the euchromatin domains. Autodegeneration following Mb euchromatin fragmentations was shown by their degradation into 200 bp ladders, and expressions of apoptotic and "non-apoptotic" active death morphologies that were also seen conjointly in the same cell. The endstage further showed heterochromatin masses anchored to the nucleolus by novel ball-and-socket joints where dislocations occurred with nuclear leakage.
Sujet(s)
Apoptose , Chromatine/ultrastructure , Fragmentation de l'ADN , Lignée cellulaire , Chromatine/effets des médicaments et des substances chimiques , Chromosomes/effets des médicaments et des substances chimiques , DNA restriction enzymes/pharmacologie , Étoposide/pharmacologie , Euchromatine , Humains , Foie/ultrastructure , Microscopie électronique , Phase S , Facteurs tempsRÉSUMÉ
Apoptosis seems characterized by a cascade of megabase to 200-bp fragmentations and by a commitment to perish at the initial level. How that could be achieved seems unclear. Preferential cleavage of transcriptionally active chromatin by apoptotic nuclease activity has long been suggested. We show here the manifestation of self-inflicted G-banding patterns in mitotic chromosomes, or G-band expression, occurring concurrently with a pattern of megabase fragmentations in two apoptotic systems that we have established in human Chang liver cells using (a) staurosporine and (b) vanadyl(4) prepulsing. We further show that rare-cutting NotI and MluI restriction endonucleases with C-G dinucleotide sequence specificity had produced similar G-bandings and megabase fragmentations cascading down to the 200-bp ladder fragmentation that were also associated with the expression of characteristic apoptotic morphologies by the digested cells. CpG-specific methylation using the methylase SssI abolished the DNA fragmentation cascade, G-banding, and apoptotic expressions induced by NotI and MluI, implicating endonuclease cleavage of active chromatin, where CpG islands are concentrated, as the initiating event. Reproducing the G-bandings and megabase fragmentations by directly applying NotI and MluI endonucleases to fixed chromosomes and extracted genomic DNA, respectively, further confirmed the notion of endonucleolytic cleavage of active chromatin as the causation. Nuclease-digested light G-band regions of chromosomes appeared to be the chromosome sites providing the megabase fragments. Transcriptionally active genes of the genome are known to be preferentially cleaved by nuclease activity and are established as being concentrated in the light G-bandings that correspond to R-bandings, which are also known to be the sites of more frequent cytogenetic breakpoints. Manifestation of self-inflicted G-banding patterns (G-banding expression) in apoptosis would then imply cleavage of the transcriptionally active genes in every light G-band site of every chromosome in the genome. This must be suicidal.
Sujet(s)
Apoptose , Protéines bactériennes , Fragmentation de l'ADN , Cycle cellulaire , Lignée cellulaire , Chromosomes , Type II site-specific deoxyribonuclease/métabolisme , Cytométrie en flux , Expression des gènes , Humains , Mitose , Staurosporine/pharmacologie , Facteurs temps , Vanadates/pharmacologieRÉSUMÉ
Staurosporine is a protein kinase inhibitor that is known to block G1/S and G2/M cell cycle transitions, resulting in the accumulation of G1 or G2 interphase cells, depending on the applied dosage. It is, however, not known to block mitosis (M-phase) progression. We show here that a 15 min prepulse with 500 nM staurosporine had caused mitotic cell arrest in human Chang liver cells, accumulating telophase cells distinguished by their doublet nuclei and cytokinetic constrictions. The arrested telophase cells perished by apoptosis. Telophase-specific cell cycle arrest and apoptosis are novel. Staurosporine targeting cytokinesis could provide insights into late mitosis where control mechanisms remain largely unknown.
Sujet(s)
Apoptose , Antienzymes/pharmacologie , Foie/cytologie , Foie/effets des médicaments et des substances chimiques , Mitose/effets des médicaments et des substances chimiques , Staurosporine/pharmacologie , Télophase , Lignée cellulaire , ADN/métabolisme , Fragmentation de l'ADN , Phase G1/effets des médicaments et des substances chimiques , Phase G2/effets des médicaments et des substances chimiques , Humains , Inhibiteurs de protéines kinases , Phase S/effets des médicaments et des substances chimiquesRÉSUMÉ
Zinc (Zn) is a trace element in human cells and regarded as an essential nutrient with established deficiency states affecting multiple organs in the body. However, it has been reported that Zn uptake is associated with some serious harmful effects, such as inhibition of DNA synthesis and enhanced toxicity from reactive oxygen species. We have previously shown that in vivo administration of Zn2+ in C57/6J mice induces weight loss and massive hair loss where the normal course hair becomes replaced by fine vello hair, simulating the side effects from cancer chemotherapy where oxidative free radical damage is implicated in association with DNA fragmentation and programmed cell death (PCD). Here, in vitro flow cytometric studies on human Chang liver showed Zn2+ causing cell condensation with DNA fragmentation that occurred in a dose-dependent manner, an effect replicated by micrococcal nuclease digestion. Specific terminal deoxynucleotidyl transferase-(TdT) mediated labeling of 3'-OH ends of DNA nicks corroborated the flow cytometric profiles of propidium iodide-DNA binding where degradation of both 2 and 4 N genomic DNA resulted in a solitary 1N peak presentation. DNA degradation concomitant with cell condensation is seen as an established hallmark of PCD. We further showed that Zn2+ could enhance the generation of hydroxyl free radicals (OH.) by the transition metal vanadium. Glutathione, the cell's main reducing agent, underwent corresponding reduction. The results suggested that Zn supplementation could induce features resembling PCD.
Sujet(s)
Apoptose/effets des médicaments et des substances chimiques , Chlorures/pharmacologie , Fragmentation de l'ADN/effets des médicaments et des substances chimiques , Foie/effets des médicaments et des substances chimiques , Stress oxydatif/effets des médicaments et des substances chimiques , Composés du zinc/pharmacologie , Cycle cellulaire/effets des médicaments et des substances chimiques , Cellules cultivées , Milieux de culture sans sérum , ADN/analyse , DNA nucleotidylexotransferase/métabolisme , Nucléotides désoxyuridyliques/métabolisme , Cytométrie en flux , Glutathion/métabolisme , Humains , Radical hydroxyle/métabolisme , Foie/cytologie , Micrococcal nuclease/métabolisme , Microscopie de contraste de phase , Vanadates/pharmacologieRÉSUMÉ
BACKGROUND: Apoptosis is a morphologically distinctive form of programmed cell death/cell suicide in which genomic DNA degradation/fragmentation and variegated dense chromatin aggregates are characteristic hallmarks that have never been demonstrated in mitotic cells. Perceptions of mutual exclusivity between apoptosis and mitosis imply that M-phase cells cannot be apoptotic. However, in the present study we show apoptotic morphologies in M-phase cells after an acute oxidative stress and endonuclease digestion. METHODS: Degradation of genomic DNA in human Chang liver cells (American Type Culture Collection, ATCC CCL13) was demonstrated by flow cytometric cell-by-cell evaluation of (a) propidium iodide intercalative binding to DNA and (b) terminal deoxynucleotidyl transferase (TdT)-mediated 3'OH nick end labeling (TUNEL) of fragmented DNA. Oxidative stress was imposed by a 30-min prepulse with 200 microM vanadyl(4), which produces hydroxyl free radicals (OH*), the most reactive of the free radical species. Oxidative stress in the cells was demonstrated by evaluating glutathione-S-transferase (GST)-mediated monochlorobimane-glutathione adduct fluorescence for glutathione content, the main reducing agent of a cell, and methylene blue redox metachromasia, which is a deep color when oxidized and colorless when reduced. Cells with DNA fragmentation were highlighted by TUNEL. Apoptotic morphologies were visualized by staining with Giemsa and neutral red dyes and by DNA-propidium iodide binding to chromatin. Direct endonuclease induction of apoptotic morphologies in permeabilized M-phase cells was produced by 1 hr incubation (37 degrees C) with 16 units/ml of micrococcal nuclease. RESULTS: The genomic DNA of proliferative cells, namely in G2/M phase of the cell cycle, was degraded by vanadyl(4) prepulsing and by micrococcal nuclease digestion, concomitantly with DNA fragmentation shown by TUNEL. Cytological profiles showed GSH depletion and M-phase cells with particularly high oxidative reactivity indicated by methylene blue redox metachromasia. DNA fragmentation in M-phase cells was highlighted by TUNEL. Characteristic apoptotic condensations, ranging from single-ball condensations to "pulverized" aggregates of a mitotic catastrophe, buddings, and "apoptotic bodies," were found in prophase, metaphase, anaphase, and telophase mitotic cells. The observed separation of condensed chromatin aggregates from the main chromosome mass in prophase and metaphase cells could explain micronuclei, linking it with apoptosis. Direct endonuclease digestion readily produced apoptotic morphologies in interphase and in M-phase cells. CONCLUSION: Apoptotic morphologies in M-phase cells can be induced indirectly via oxidative stress or directly via endonuclease activity, which has long been established as a pervading hallmark of apoptosis.
Sujet(s)
Apoptose , Mitose/physiologie , Stress oxydatif/physiologie , Cellules cultivées , ADN/analyse , ADN/effets des médicaments et des substances chimiques , Fragmentation de l'ADN/effets des médicaments et des substances chimiques , Endonucleases/pharmacologie , Glutathion/analyse , Humains , Foie/cytologie , Microscopie de fluorescence , Vanadates/pharmacologieRÉSUMÉ
Vanadium has recently been reported to induce interphase and M-phase (mitotic) programmed cell death via the generation of hydroxyl free radicals (OH*). In this paper, the effects of antioxidants on: (a) vanadyl[IV]-generated OH* free radical levels; and (b) cellular glutathione in vanadyl [IV]-treated Chang liver cells were evaluated. The surface morphology of vanadyl-treated mitotic cells was studied by confocal and scanning microscopy. The free radical scavengers zinc chloride, glucose and thiourea reduced the levels of vanadyl-induced OH* free radicals and partially prevented the depletion of cellular glutathione. Concurrent with OH* free radical production, vanadyl-treated telophase cells exhibited excessive cell blebbing and cell shrinkage. The morphological features demonstrated in vanadyl-induced mitotic programmed cell death as a consequence of oxidative stress is novel.
Sujet(s)
Radical hydroxyle/toxicité , Foie/effets des médicaments et des substances chimiques , Vanadates/toxicité , Apoptose/effets des médicaments et des substances chimiques , Cellules cultivées , Chlorures/pharmacologie , Piégeurs de radicaux libres/pharmacologie , Glucose/pharmacologie , Glutathion/métabolisme , Humains , Foie/cytologie , Foie/ultrastructure , Microscopie confocale , Microscopie électronique à balayage , Mitose/effets des médicaments et des substances chimiques , Stress oxydatif , Spectrophotométrie UV , Télophase , Thiourée/pharmacologie , Vanadates/métabolisme , Composés du zinc/pharmacologieRÉSUMÉ
The relationship between cell cycling and apoptosis/programmed cell death has been perceived as either checkpoint arrests or mitotic aberration where common pathways between mitosis and apoptosis seem suggested. We show here evidence implicating both perceptions of cell cycle involvement. The process was initiated by hydroxyl free radicals (OH*) generated intracellularly from internalized vanadyl(4). Intranuclear sequestration of vanadyl(4) was verified by nuclear microscopy. Resultant high oxidative reactivity in the nucleus was shown by the redox indicator methylene blue, suggesting direct oxidative damage to genomic DNA. Oxidative stress was further enhanced by depletion of glutathione which is the main cellular reducing agent. Genomic degradation and fragmentation was confirmed by flow cytometric evaluation of terminal deoxynucleotidyl transferase (TdT)-mediated 3'OH end-labelling (TUNEL) of DNA nicks, and cell cycle DNA profiling demonstrating sub-G1 (sub-2N) accumulation. With DNA degradation, there was a G2M transient with hyperdiploid right-shifting, consistent with G2 arrest. G2 arrest was subsequently 'released' with abolition of G2M and all other cell cycle phases except for a solitary sub-G1 (apoptotic) peak. The cytological profile of this 'release' phenomenon was initially marked by the appearance of clusters of mitotic and apoptotic cells. At later stages, the cell population was composed exclusively of nuclear ghosts, apoptotic cells, mitotic cells, and mitotic cells with both chromosomes and apoptotic condensations. Concurrent and conjoint expression of cell death and cell division as the exclusive process of an entire cell population refuted the notion of mutual exclusivity between life and death. Zn2+, an endonuclease inhibitor, abolished all observed cytological and DNA profile changes.
RÉSUMÉ
Endonuclease activation causing genomic degradation is a pervasive hallmark of apoptosis and a suggested precipitating or commitment step in the suicidal process. Directly applied endonuclease activity has produced apoptotic-like effects in isolated nuclei, but not yet shown as an initiating apoptogen in whole cells. Mechanistically genomic damage inflicted by a variety of DNA-damaging agents is also known to produce mitotic catastrophe condensations characterizing cell cycle derangement. Morphological and molecular similarities between apoptosis and mitotic catastrophe have been noted, but their conjoint expressions from directly applied endonuclease activity has also not been shown. We show here micrococcal nuclease (MNase) initiating apoptosis in human Chang liver cells which expressed both apoptotic and mitotic catastrophe condensations. Genomic profiling showed (a) the two stage apoptotic sequence of large (50 kb) and small (200 bp) fragment cleavage demonstrated by pulse field and normal gel electrophoresis, respectively; (b) the sub-G1 ;apoptotic peak' with shrunken cells from flow cytometric evaluation of PI-DNA binding and laser forward scattering, (c) 3' OH termini typical of apoptotic DNA fragments labelled by terminal deoxynucleotidyl transferase (TdT)-mediated fluorescence tagging especially in the shrunken cells, and (d) positive comet assay of the apoptotic genome. Nuclear shrinkage evaluated by confocal image analysis was consistent with the apoptotic response, as was Zn2+ ion sensitivity, an established inhibitor of apoptotic expression. Endonuclease activity per se is apoptogenetic and mechanistically convergent with the mitotic catastrophe pathway in the proliferative cycle.
RÉSUMÉ
In a previous report, we have demonstrated that acute zinc administration reduced the hepatic cytochrome P450 content in female C57/6J mice. In this extended toxicological study, we investigated the effects of zinc administration on (a) the hepatic cytochrome P450 content of both male and female mice to evaluate whether the sex of the animal had any influence on the zinc effects and (b) the hepatocytes at the ultrastructural level. Two doses of zinc chloride at 28 micrograms g-1 body weight (equivalent to LD50 in chronic treatment) were administered intraperitoneally to male and female C57/6J mice at 24 h intervals. Significant reduction of hepatic cytochrome P450 content was observed to occur the next day in both acutely treated male and female mice. On examination under transmission electron microscopy, evidence was found of toxic injury to the hepatocytes of mice livers in the zinc-treated group. Glutathione-monochlorobimane adduct formation (which is specifically catalysed by glutathione transferase) was found to be depressed in Chang liver cells. The findings indicate that acute zinc administration reduced the hepatic cytochrome P450 content in C57/6J mice irrespective of gender.
Sujet(s)
Cytochrome P-450 enzyme system/métabolisme , Foie/enzymologie , Foie/ultrastructure , Zinc/toxicité , Animaux , Femelle , Glutathion/métabolisme , Injections péritoneales , Dose létale 50 , Foie/effets des médicaments et des substances chimiques , Mâle , Souris , Souris de lignée C57BL , Microscopie électronique , Zinc/sangRÉSUMÉ
Very little is known about the modulation of vanadium accumulation in cells, although this ultratrace element has long been seen as an essential nutrient in lower life forms, but not necessarily in humans where factors modulating cellular uptake of vanadium seem unclear. Using nuclear microscopy, which is capable of the direct evaluation of free and bound (total) elemental concentrations of single cells we show here that an NH4Cl acidification prepulse causes distinctive accumulation of vanadium (free and bound) in human Chang liver cells, concentrating particularly in the nucleus. Vanadium loaded with acidification but leaked away with realkalinization, suggests proton-dependent loading. Vanadyl(4), the oxidative state of intracellular vanadium ions, is known to be a potent source of hydroxyl free radicals (OH). The high oxidative state of nuclei after induction of vanadyl(4) loading was shown by the redox indicator methylene blue, suggesting direct oxidative damage to nuclear DNA. Flow cytometric evaluation of cell cycle phase-specific DNA composition showed degradation of both 2N and 4N DNA phases in G1, S and G2/M cell cycle profiles to a solitary IN DNA peak, in a dose-dependent manner, effective from micromolar vanadyl(4) levels. This trend was reproduced with microccocal nuclease digestion in a time response, supporting the notion of DNA fragmentation effects. Several other approaches confirmed fragmentation occurring in virtually all cells after 4mM V(4) loading. Ultrastructural profiles showed various stages of autophagic autodigestion and well defined plasma membrane outlines, consistent with programmed cell death but not with necrotic cell death. Direct intranuclear oxidative damage seemed associated with the induction of mass suicide in these human Chang liver cells following vanadium loading and nuclear sequestration.
Sujet(s)
Apoptose/effets des médicaments et des substances chimiques , Noyau de la cellule/métabolisme , Foie/cytologie , Vanadium/métabolisme , Vanadium/pharmacologie , Chlorure d'ammonium/métabolisme , Chlorure d'ammonium/pharmacologie , Lignée cellulaire , ADN/métabolisme , Altération de l'ADN , Cytométrie en flux , Fluorescéines/métabolisme , Colorants fluorescents/métabolisme , Humains , Concentration en ions d'hydrogène , Radical hydroxyle/métabolisme , Foie/effets des médicaments et des substances chimiques , Foie/métabolisme , Foie/ultrastructure , Bleu de méthylène/métabolisme , Micrococcal nuclease/métabolisme , Microscopie , Spectrométrie de fluorescence , Rayons XRÉSUMÉ
Zinc, an essential trace element, is important for normal cell growth. Growing children, especially at puberty, require increased zinc (2.8 mg/day for males and 2.65 mg/day for females). The DNA profile and cell cycle of human Chang liver cells grown in 0-900 mumol/L zinc chloride supplemented serum-free media for 24 h were analyzed using a Coulter flow cytometer. There was no significant difference in the G1, S and G2/M phases between zinc treated cells and control cultures except at 90 and 900 mumol/L zinc chloride. At these two higher dosages, fragmentation of genomic DNA into sub-2N DNA (sub-G1 DNA), generally considered a hallmark of programmed cell death (PCD), was noted. Results of the present study seem to suggest that growth regulation by zinc during growth spurts such as at puberty, could also be influenced by other factors besides its direct effect on DNA synthesis. In addition, high dosages of zinc could be cytotoxic.
Sujet(s)
Cycle cellulaire/effets des médicaments et des substances chimiques , ADN/biosynthèse , Foie/cytologie , Zinc/pharmacologie , Lignée cellulaire , ADN/effets des médicaments et des substances chimiques , Cytométrie en flux , HumainsRÉSUMÉ
Large channel endocytosis is considered to be the characteristic of specialized endocytic cells like macrophages and phagocytes while small pit endocytosis involving clathrin protein coatings are the membrane recycling macromolecular pathways for most eukaryotic cells. We show here that extracellular ATP induced cells to internalize their plasma membrane by large channel endocytosis. In the process of plasma membrane internalization, flat protracted cells round up and become easily detachable from the substrate. Scanning transmission ion microscopy (STIM) revealed an increase in cell density as the ATP treated cells assumed a rounded morphology. The increase in cell density could be attributed to endocytic internalization of cell membrane and debris.
Sujet(s)
Adénosine triphosphate/pharmacologie , Endocytose/effets des médicaments et des substances chimiques , Foie/cytologie , Numération cellulaire/effets des médicaments et des substances chimiques , Lignée cellulaire/cytologie , Lignée cellulaire/métabolisme , Lignée cellulaire/ultrastructure , Humains , Microscopie électronique , Microscopie électronique à balayageRÉSUMÉ
Intraperitoneal zinc chloride was administered at 7.5 micrograms/g body weight and 15 micrograms/g body weight to 10-12 weeks old Swiss albino mice for 5 consecutive days. Control animals were given normal saline. The testis and epididymis were dissected and examined under the light microscope. Micrographs of the testes appeared normal in both treated and nontreated animals. However the group of animals treated with the higher dosage of zinc chloride showed evidence of rupture and collapse of the epididymal epithelial lining. The testes were not affected probably because of (a) known higher testicular concentration of metallothioneins which can bind the zinc and consequently detoxify the metal and (b) "stratified" epithelium comprising of spermatogenic and Sertoli cells.
Sujet(s)
Épididyme/effets des médicaments et des substances chimiques , Zinc/pharmacologie , Animaux , Relation dose-effet des médicaments , Épididyme/cytologie , Épithélium/effets des médicaments et des substances chimiques , Mâle , Souris , Testicule/cytologie , Testicule/effets des médicaments et des substances chimiques , Testicule/ultrastructureRÉSUMÉ
The effect of staurosporine, an antimicrobial agent and inhibitor of protein kinase C (PKC) on programmed cell death/apoptosis (PCD) was investigated in the human oral cavity epidermoid carcinoma KB cell line. Staurosporine-treated oral KB carcinoma cells exhibited morphological features characteristic of apoptosis such as (a) cell shrinkage and increased nuclear fluorescence (quantitated by image analysis with laser scanning confocal microscopy), (b) nuclear condensation and fragmentation observed under fluorescence microscopy with propidium-iodide-DNA staining and (c) chromatin condensation seen under transmission electron microscopy. Specific terminal deoxynucleotidyl transferase (TdT)-mediated labeling of 3'OH ends of DNA breaks in staurosporine-treated cells confirmed DNA fragmentation. In addition, we show the concomitant existence of M-phase PCD with interphase PCD in staurosporine-treated KB cells. It would appear that staurosporine induces apoptosis regardless of the cell cycle phase and that mitosis and apoptosis are not necessarily mutually exclusive events.
Sujet(s)
Alcaloïdes/pharmacologie , Apoptose/effets des médicaments et des substances chimiques , Interphase/effets des médicaments et des substances chimiques , Mitose/effets des médicaments et des substances chimiques , Protéine kinase C/antagonistes et inhibiteurs , Humains , Cellules KB , Staurosporine , Cellules cancéreuses en cultureRÉSUMÉ
Recent studies using real time imaging demonstrated relative nuclear insulation for ion-size particles. We show here that acidification and recovery converted the insulated interphase nuclei of KB carcinoma and nontumorigenic Chang cells into intense nuclear accumulating states marked by sequestration of the exogenous supravital dyes neutral red, methylene blue, and brilliant cresyl blue. The phenomenon was not affected by Na(+)-free and HCO3(-)-free conditions nor by the presence of cationic and anionic antiport regulators of cytosolic pH. Cytological, microspectrophotometric, and flow cytometric evaluation of whole cell populations showed that the nuclear influx was abolished by omitting the pH recovery response, and by modulating the recovery response. The abolition of nuclear influx in the presence of the P-ATPase and Fzero-ATPase inhibitors, vanadyl(IV) ions and oligomycin, respectively, suggest that H(+)-translocating ATPase pumps are involved in regulating cytosolic acidification in Na(+)-free and HCO3-conditions vanadyl(IV) inhibited nuclear uptake of supravital dyes in a dose dependent manner. Nuclear uptake of dyes, however, was not affected by up to 1 mM of genistein even though tyrosine-specific phosphorylation and DNA synthesis were abolished. Upgradient nuclear influx involving proton pump is novel. KB cancer cells and nontumorigenic Chang cells had differential dye accumulations induced by acidification and recovery.