Fluorescein Isothiocyanate-annexin Research Articles

Interrelated Roles for Mcl-1 and BIM in Regulation of TRAIL-mediated Mitochondrial Apoptosis

The current study demonstrates a novel cross-talk mechanism between the TRAIL receptor death signaling pathway and the mitochondria. This newly identified pathway is regulated at the mitochondrial outer membrane by a complex between the prosurvival Bcl-2 member, Mcl-1 and the BH3-only protein, Bim. Under non-apoptotic conditions, Bim is sequestered by Mcl-1. Direct degradation of Mcl-1 by TRAIL-activated caspase-8 or caspase-3 produces Mcl-1-free Bim that mediates a Bax-dependent apoptotic cascade. Using Mcl-1 or Bim RNAi, we demonstrate that a loss in Mcl-1 expression significantly enhances the mitochondrial apoptotic response to TRAIL that is now mediated by freed Bim. Whereas overexpression of Mcl-1 contributes to the preservation of the mitochondrial membrane potential, Mcl-1 knockdown facilitates the Bim-mediated dissipation of this potential. Loss of Mcl-1 contributes to an increased level of caspase activity downstream of the mitochondrial response to TRAIL. Furthermore, the Mcl-1 expression level at the mitochondrial outer membrane determines the release efficiency for the apoptogenic proteins cytochrome c, Smac, and HtrA2 in response to Bim. These are the first findings to demonstrate the involvement of Bim in the TRAIL-mediated mitochondrial cascade. They also suggest that Mcl-1 may serve as a direct substrate for TRAIL-activated caspases implying the existence of a novel TRAIL/caspase-8/Mcl-1/Bim communication mechanism between the extrinsic and the intrinsic apoptotic pathways.

Thiopental-induced Apoptosis in Lymphocytes Is Independent of CD95 Activation

Barbiturate coma is used in patients with traumatic brain injury whenever increases in intracranial pressure remain unresponsive to less aggressive therapeutic regimens. However, barbiturate-mediated neuroprotection correlates with lymphopenia, which increases the risk of infection. The mechanisms by which barbiturates lead to lymphopenia remain to be determined. Freshly isolated human lymphocytes and Jurkat cells were incubated with the barbiturate thiopental for 24 and 48 h. Apoptosis was measured by fluorescein isothiocyanate-Annexin and propidium iodide staining, rhodamine 123 staining, and the terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling method. Caspase-3 activity was detected by Western blot and substrate cleavage assay. Thiopental dose-dependently (5-500 microg/ml) increased apoptosis in Jurkat cells from basal levels (4.4 +/- 1.9%) to 29.7 +/- 2.8% after 24 h and 39.7 +/- 3.2% after 48 h, whereas in lymphocytes, thiopental-induced necrosis was observed. Parallel to apoptosis, thiopental dose-dependently increased caspase-3-like activity in Jurkat cells. However, the pan-caspase inhibitor z-VAD-fmk (20 microm) only marginally reduced thiopental-induced (250 microg/ml) apoptosis in Jurkat cells (20.2 +/- 2.5 to 17.2 +/- 2.5%) and necrosis in lymphocytes (39.2 +/- 7.5 to 30.7 +/- 14%). In contrast, anti-CD95-induced apoptosis in Jurkat cells (27.0 +/- 2.0%) was completely blocked by z-VAD-fmk (8.1 +/- 1.8%). Neither expression of CD95 on Jurkat cells nor pretreatment with a neutralizing anti-CD95 antibody influenced thiopental-induced apoptosis, indicating that thiopental induces apoptosis independently of the CD95 system. The nuclear factor kappaB inhibitor gliotoxin accelerated both thiopental- and CD95-mediated apoptosis, indicating a mutual control mechanism of thiopental- and CD95-induced apoptosis. Thiopental directly induces cell death in lymphocytes and Jurkat cells by a CD95-independent mechanism.

Experimental validation of deuterium oxide-mediated antitumoral activity as it relates to apoptosis in murine malignant astrocytoma cells.

Deuterium oxide (D2O), or heavy water, affects a variety of biological activities different from those of water. The authors examined the antitumoral effect of D2O on brain neoplasms and demonstrated D2O-mediated cytotoxicity by using a Rous sarcoma virus-induced murine malignant astrocytoma cell line, RSVM. The mechanism of the observed cytotoxicity may involve D2O-induced apoptosis and cell-cycle modulation. The authors performed an assay with methylthiazol tetrazolium bromide and a trypan blue dye exclusion test to confirm in vitro D2O-mediated cytotoxicity for RSVM cells. At D2O concentrations of 10 to 50%, the cytotoxic effect was dose and time dependent. Flow cytometry analysis revealed programmed cell death (apoptosis) and the accumulation of RSVM cells during the G2/M phase. By applying the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling method, fluorescein isothiocyanate-annexin V and propidium iodide double staining, and caspase-family protease activity analysis, the authors demonstrated both DNA fragmentation and enhancement of caspase activity after a 48-hour treatment with D2O, thus indicating that D2O induces apoptosis in RSVM cells. Apoptotic DNA fragmentation was completely abolished by the caspase inhibitor Z-VAD-FMK (benzyloxycarbonil-Val-Ala-Aps-fluoromethylketone). The findings indicate that the caspase activation pathway may be involved in D2Oinduced apoptosis. The authors found that D2O is cytotoxic to malignant astrocytoma cells. The mechanism of D2O-mediated cytotoxicity involved the induction of apoptosis and cell accumulation during the G2/M phase. This D2O-induced apoptosis is modulated through the caspase activation pathway.

Complement activation by apoptotic endothelial cells following hypoxia/reoxygenation.

Reperfusion of ischaemic tissue initiates an inflammatory reaction that increases tissue injury. Complement activation at the endothelium contributes to this inflammation. This study investigated the mechanism of complement activation following reoxygenation of hypoxic human umbilical vein endothelial cells (HUVEC) as a model for complement activation observed on endothelium in reperfused ischaemic tissue. HUVEC cultured in 1% oxygen followed by reoxygenation activated the classical complement pathway resulting in C3 deposition. There was an increase in apoptotic cells in these cultures that was demonstrated by binding of fluorescein isothiocyanate-Annexin V and staining for hypodiploid nuclei. To determine if apoptotic HUVEC activate complement, uniformly apoptotic cells were produced by serum and growth factor deprivation. These cells, but not the control HUVEC, activated the classical complement pathway in the absence of antibody or other serum factors. To determine if apoptotic cells in the reoxygenated cultures were activating complement, fluorescent analysis was done. Annexin V binding and C3d deposition on cells from reoxygenated cultures showed complete concordance on the subpopulation of apoptotic cells. In addition, complement activation following reoxygenation of HUVEC was eliminated by treatment of the cultures with a caspase inhibitor during reoxygenation. These results suggest that oxidative damage to endothelial cells during reoxygenation initiates apoptosis with exposure of phosphatidylserine. Apoptotic cells directly activate the classical pathway of complement by binding C1. Activation of complement at the endothelium may contribute to the inflammatory response as well as clearance and repair.

Impaired survival of bone marrow hematopoietic progenitor cells in cyclic neutropenia

AbstractCyclic neutropenia (CN) is a congenital hematopoietic disorder characterized by remarkably regular oscillations of blood neutrophils from near normal to extremely low levels at 21-day intervals. Recurring episodes of severe neutropenia lead to repetitive and sometimes life-threatening infections. To investigate the cellular mechanism of CN, the ultrastructure and the proliferative and survival characteristics of bone marrow–derived CD34+ early progenitors, CD33+/CD34− myeloid progenitors, and CD15+ neutrophil precursors from CN patients and healthy volunteers were studied. The ultrastructural studies showed profound apoptotic features in bone marrow progenitor cells in CN. Colony-forming assays demonstrated a 75% decrease in the number of early myeloid-committed colonies compared with controls. Long-term culture-initiating cell assays demonstrated a 6-fold increase in production of primitive progenitor cells in CN. To determine whether accelerated apoptosis might account for the underproduction of myeloid progenitors, the hematopoietic subpopulations were labeled with fluorescein isothiocyanate–annexin V and analyzed by flow cytometry. Short-term culture of CN cells resulted in apoptosis of approximately 65% of CD34+ cells, 80% of CD33+/CD34− cells, and more than 70% of CD15+ cells, as compared with 20%, 7%, and 15% apoptosis in respective control subpopulations. Evidence of accelerated apoptosis of bone marrow progenitor cells was observed in all 8 patients participating in the study, regardless of the stage in the CN cycle in which bone marrow aspirations were obtained. Granulocyte colony-stimulating factor therapy of CN patients significantly improved survival of bone marrow progenitor cells. These data indicate that ineffective production of neutrophils is due to accelerated apoptosis of bone marrow myeloid progenitor cells in CN.

Externalization of phosphatidylserine may not be an early signal of apoptosis in neuronal cells, but only the phosphatidylserine-displaying apoptotic cells are phagocytosed by microglia.

Earlier reports on nonneural cells have shown that the normally inner plasma membrane lipid, phosphatidylserine (PS), flip-flops out during the early stages of apoptosis, whereas DNA laddering and plasma membrane permeabilization occur during the late stages. In this study, the applicability of these parameters to CNS-derived neuronal cells was tested using hippocampal HN2-5, cells that undergo apoptosis under anoxia. Because such insults on unsynchronized cells, e.g., undifferentiated HN2-5 cells, result in both early and late apoptotic cells, we mechanically separated these cells into three fractions containing (a) cells that had completely detached during anoxia, (b) cells that remained weakly attached to the tissue culture dish and, once detached by trituration in serum-containing medium, did not reattach, and (c) cells that reattached in 2-3 h. Fractions a and b contained cells that showed pronounced DNA laddering, whereas cells in fraction c did not show any DNA laddering. Double staining with fluorescein isothiocyanate-annexin V (which binds to PS) and propidium iodide (which stains the DNA in cells with a permeable cell membrane) revealed that all cells in fraction a had a permeable cell membrane (propidium iodide-positive) and PS molecules in the outer leaflet of the plasma membrane (fluorescein isothiocyanate-annexin V-positive). By contrast, fractions b and c contained cells with no externalized PS molecules. Cells in fractions a-c also showed, respectively, 50-, 21-, and 5.5-fold higher caspase-3 (CPP32) activity than that in healthy control cells. All these results show that fraction a contained late apoptotic cells, which also had the highest CPP32 activity; cells in fraction b were at an intermediate stage, when DNA laddering had already occurred; and fraction c contained very early apoptotic cells, in which no DNA laddering had yet occurred. Therefore, in the neuronal HN2-5 cells, externalization of PS occurs only during the final stages of apoptosis when the cells have completely lost their adhesion properties. Further experiments showed that ameboid microglial cells isolated from neonatal mouse brain phagocytosed only the cells in fraction a. These results show that in CNS-derived HN2-5 cells, (a) PS externalization is a late apoptotic event and is concomitant with a complete loss of surface adhesion of the apoptotic cells and (b) PS externalization is crucial for microglial recognition and phagocytosis of the apoptotic HN2-5 cells. Thus, PS externalization could be causally linked to the final detachment of apoptotic neuronal cells, which in turn prepares them for rapid phagocytosis by microglia.