G. Del Bino

Bio: G. Del Bino is an academic researcher from New York Medical College. The author has contributed to research in topics: Topoisomerase & Cell cycle. The author has an hindex of 14, co-authored 16 publications receiving 2887 citations. Previous affiliations of G. Del Bino include Free University of Brussels & Memorial Sloan Kettering Cancer Center.

Features of apoptotic cells measured by flow cytometry

Abstract: The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Comparison of methods based on annexin-V binding, DNA content or TUNEL for evaluating cell death in HL-60 and adherent MCF-7 cells.

Abstract: HL-60 and MCF-7 cells were treated with 0.15 microM camptothecin (CPT) or with the solvent dimethylsulfoxide (DMSO) for the controls, for 2, 3 and 4 h or for 24, 48 and 72 h, respectively. The apoptotic index (AI) was then evaluated in parallel by the following flow cytometric methods: (1) double staining of unfixed cells with fluoresceinated annexin V and propidium iodide (PI), this after detachment by trypsinization in the case of MCF-7 cultures; (2) prefixation in 70% ethanol, extraction of degraded, low molecular weight DNA with 0.2 M phosphatecitrate buffer and analysis of the DNA content stained with PI; (3) TUNEL, i.e. labelling of DNA strand breaks with biotin-dUTP, followed by staining with streptavidin-fluorescein and counterstaining with PI. In HL-60 cells, the three methods gave similar results for the AI (3-4% in the controls and at 2 h of CPT treatment, and 35-43% at 3 and 4 h after CPT). This indicates that CPT-induced membrane alteration and DNA fragmentation occurred concomitantly in those cells. For MCF-7 cells, CPT-induced apoptosis developed more slowly, the AI, whether based on annexin V or on DNA content, remained unchanged at 24 h, then was increasing to 8% at 48 h and to 25% at 72 h of treatment. In these cells, the TUNEL index did not increase prior to 72 h, and the increase was minor (up to 9% vs. 2-3% in the controls) at 72 h of the treatment. This indicates that in MCF-7 cells DNA strand breaks cannot be effectively labelled, which may be due to inaccessibility of 3'-OH ends in the breaks to exogenous terminal deoxynucleotidyl transferase. The mechanism of endonucleolytic DNA fragmentation thus may be different, depending on the cell type.

Diverse Effects of Camptothecin, an Inhibitor of Topoisomerase I, on the Cell Cycle of Lymphocytic (L1210, MOLT-4) and Myelogenous (HL-60, KG1) Leukemic Cells

Abstract: Exposure of mouse lymphocytic L1210 cells to 0.02-0.5 micrograms/ml of camptothecin (CAM) causes a slowdown in the rate of cell progression through S and G2 phases of the cell cycle; the "terminal" point of CAM action is about 1 h prior to mitosis. Some cells also enter higher DNA ploidy and progress through the cycle at that ploidy. CAM exerts similar effects (S- and G2-phase arrest, entrance to higher DNA ploidy, low initial cytotoxicity) on human lymphocytic MOLT-4 leukemia cells. In contrast, treatment of human promyelocytic HL-60 cells with CAM results in the immediate (occurring as early as 2 h after treatment) death of S- and G2+M-phase cells; the dead cells exhibit decreased DNA stainability with intercalating dyes, suggestive of DNA degradation. Although CAM is less cytotoxic to another human myelogenous leukemic cell line, KG1, the latter cells also respond like HL-60, namely by selective death in S and G2. The data indicate that there may be a tissue (leukemia type) specificity in the response of cells to camptothecin and suggest that myelogenous leukemias, especially those characterized by high proliferation rates, may be especially sensitive to the cytotoxic action of this and perhaps other topoisomerase I inhibitors.

Inhibitors of proteases prevent endonucleolysis accompanying apoptotic death of HL-60 leukemic cells and normal thymocytes.

Abstract: Exposure of human promyelocytic leukemic HL-60 cells to the topoisomerase I inhibitor camptothecin (CAM) triggers endonucleolytic activity and apoptotic death of these cells. The nucleolytic effect is seen 2-4 h after drug addition and is highly selective to cells progressing through S phase. Concomitant with degradation of DNA, which is preferential to the nucleosomal DNA linker sections, extensive proteolysis takes place in these cells. Cellular RNA, however, is initially degraded to a much lesser degree than DNA or protein. Both endonucleolysis and proteolysis triggered by CAM in S-phase HL-60 cells can be prevented by the protease inhibitors N-tosyl-L-phenylalanylchloromethyl ketone (TPCK), N-tosyl-L-lysylchloromethyl ketone (TLCK) or partly by N-tosyl-L-arginine methyl ester (TAME), added simultaneously with CAM, or up to 30 min after exposure to CAM, at their respective concentrations known to inhibit proteases. The protective effect of these protease inhibitors on DNA degradation cannot be due to the suppression of cell progression through S phase because cells still replicate DNA in their presence, albeit at a reduced rate. Furthermore, TPCK and TLCK protect rat thymocytes against endonucleolysis induced by prednisolone. In the latter cell system, (considered a classic model of apoptosis), endonucleolysis, which primarily affects G0/G1 cells, is unrelated to cell progression through S phase. The present data suggest that the endonucleolysis and proteolysis which accompany apoptotic cell death are coupled, and the proteolytic step is needed for DNA degradation to occur.

Early redistribution of plasma membrane phosphatidylserine is a general feature of apoptosis regardless of the initiating stimulus: inhibition by overexpression of Bcl-2 and Abl.

Abstract: A critical event during programmed cell death (PCD) appears to be the acquisition of plasma membrane (PM) changes that allows phagocytes to recognize and engulf these cells before they rupture. The majority of PCD seen in higher organisms exhibits strikingly similar morphological features, and this form of PCD has been termed apoptosis. The nature of the PM changes that occur on apoptotic cells remains poorly defined. In this study, we have used a phosphatidylserine (PS)-binding protein (annexin V) as a specific probe to detect redistribution of this phospholipid, which is normally confined to the inner PM leaflet, during apoptosis. Here we show that PS externalization is an early and widespread event during apoptosis of a variety of murine and human cell types, regardless of the initiating stimulus, and precedes several other events normally associated with this mode of cell death. We also report that, under conditions in which the morphological features of apoptosis were prevented (macromolecular synthesis inhibition, overexpression of Bcl-2 or Abl), the appearance of PS on the external leaflet of the PM was similarly prevented. These data are compatible with the notion that activation of an inside-outside PS translocase is an early and widespread event during apoptosis.

Features of apoptotic cells measured by flow cytometry

Abstract: The present review describes several methods to characterize and differentiate between two different mechanisms of cell death, apoptosis and necrosis. Most of these methods were applied to studies of apoptosis triggered in the human leukemic HL-60 cell line by DNA topoisomerase I or II inhibitors, and in rat thymocytes by either topoisomerase inhibitors or prednisolone. In most cases, apoptosis was selective to cells in a particular phase of the cell cycle: only S-phase HL-60 cells and G0 thymocytes were mainly affected. Necrosis was induced by excessively high concentrations of these drugs. The following cell features were found useful to characterize the mode of cell death: a) Activation of an endonuclease in apoptocic cells resulted in extraction of the low molecular weight DNA following cell permeabilization, which, in turn, led to their decreased stainability with DNA-specific fluorochromes. Measurements of DNA content made it possible to identify apoptotic cells and to recognize the cell cycle phase specificity of the apoptotic process. b) Plasma membrane integrity, which is lost in necrotic but not apoptotic cells, was probed by the exclusion of propidium iodide (PI). The combination of PI followed by Hoechst 33342 proved to be an excellent probe to distinguish live, necrotic, early- and late-apoptotic cells. c) Mitochondrial transmembrane potential, assayed by retention of rhodamine 123 was preserved in apoptotic but not necrotic cells. d) The ATP-dependent lysosomal proton pump, tested by the supravital uptake of acridine orange (AO) was also preserved in apoptotic but not necrotic cells. e) Bivariate analysis of cells stained for DNA and protein revealed markedly diminished protein content in apoptotic cells, most likely due to activation of endogenous proteases. Necrotic cells, having leaky membranes, had minimal protein content. f) Staining of RNA allowed for the discrimination of G0 from G1 cells and thus made it possible to reveal that apoptosis was selective to G0 thymocytes. g) The decrease in forward light scatter, paralleled either by no change (HL-60 cells) or an increase (thymocytes) of right angle scatter, were early changes during apoptosis. h) The sensitivity of DNA in situ to denaturation, was increased in apoptotic and necrotic cells. This feature, probed by staining with AO at low pH, provided a sensitive and early assay to discriminate between live, apoptotic and necrotic cells, and to evaluate the cell cycle phase specificity of these processes. i) The in situ nick translation assay employing labeled triphosphonucleotides can be used to reveal DNA strand breaks, to detect the very early stages of apoptosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Membrane blebbing during apoptosis results from caspase-mediated activation of ROCK I

Abstract: The execution phase of apoptosis is characterized by marked changes in cell morphology that include contraction and membrane blebbing. The actin-myosin system has been proposed to be the source of contractile force that drives bleb formation, although the biochemical pathway that promotes actin-myosin contractility during apoptosis has not been identified. Here we show that the Rho effector protein ROCK I, which contributes to phosphorylation of myosin light-chains, myosin ATPase activity and coupling of actin-myosin filaments to the plasma membrane, is cleaved during apoptosis to generate a truncated active form. The activity of ROCK proteins is both necessary and sufficient for formation of membrane blebs and for re-localization of fragmented DNA into blebs and apoptotic bodies.