Efficiency of homocysteine plus copper in inducing apoptosis is inversely proportional to gamma-glutamyl transpeptidase activity.

Hyperhomocysteinemia represents an independent risk factor for atherosclerosis, but the mechanisms leading to cellular dysfunctions remain unknown. Using ECV304 cells, we found that homocysteine (Hcy) plus copper (Cu2+) induced cytotoxic effects: loss of cell adhesion, increased permeability to PI, and the occurrence of morphologically apoptotic cells. This form of apoptosis, inhibited by Z-VAD-fmk, was associated with a loss of mitochondrial potential, a cytosolic release of cytochrome c, activation of caspase-3, degradation of poly(ADP-ribose)polymerase, and internucleosomal DNA fragmentation. However, the ability of Hcy plus Cu2+ to induce apoptosis decreased when the pretreatment culture time increased. As a positive correlation was found between the length of time of culture before treatment and the enhancement of gamma-glutamyl transpeptidase (gamma-GT) activity, we asked whether gamma-GT was involved in the control of Hcy plus Cu2+-induced apoptosis. Therefore, ECV304 cells were treated with either acivicin or dexamethasone, inhibiting and stimulating gamma-GT, respectively. In ECV304 cells and human umbilical venous endothelial cells, acivicin favored Hcy plus Cu2+-induced apoptosis whereas dexamethasone counteracted the apoptotic process. As acivicin and dexamethasone were also capable of modulating cell death in ECV304 cells treated with antitumoral drugs, our data emphasize that the involvement of gamma-GT in the control of apoptosis is not restricted to Hcy but also concerns other chemical compounds.

Mitochondria-targeting drugs arsenic trioxide and lonidamine bypass the resistance of TPA-differentiated leukemic cells to apoptosis.

Exposure of U937 human leukemic cells to the phorbol ester 12-O-tetradecanoylphorbol 13-acetate (TPA) induces their differentiation into monocyte/macrophage-like cells. This terminal differentiation is associated with a resistant phenotype to apoptosis induced by the topoisomerase II inhibitor etoposide. The inhibition occurs upstream of the mitochondrial release of cytochrome c and the activation of procaspase-2, -3, -6, -7, -8, and -9. By using cell-free systems, it was demonstrated that the mitochondrial pathway to cell death that involves mitochondrial membrane depolarization, cytochrome c release and cytosolic activation of procaspases by cytochrome c/dATP remains functional in TPA-differentiated U937 cells. Accordingly, 2 drugs recently shown to target the mitochondria, namely lonidamine and arsenic trioxide, bypass the resistance of TPA-differentiated U937 cells to classical anticancer drugs. Cell death induced by the 2 compounds is associated with mitochondrial membrane depolarization, release of cytochrome c and Smac/Diablo from the mitochondria, activation of caspases, poly(ADP-ribose) polymerase cleavage and internucleosomal DNA fragmentation. Moreover, the decreased glutathione content associated with the differentiation process amplifies the ability of arsenic trioxide to activate the mitochondrial pathway to cell death. Similar results were obtained by comparing undifferentiated and TPA-differentiated human HL60 leukemic cells. These data demonstrate that mitochondria-targeting agents bypass the resistance to classical anticancer drugs induced by TPA-mediated leukemic cell differentiation. (Blood. 2001;97:3931-3940)

Ceramide generation occurring during 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis is caspase independent and is not required to trigger cell death.

Biological activities of oxysterols seem tightly regulated. Therefore, the ability to induce cell death of structurally related oxysterols, such as those oxidized at C7(7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), was investigated on U937 cells at different times of treatment in a concentration range of 5-80 microg/ml. Whereas all oxysterols accumulate inside the cells, strong inhibition of cell growth and increased permeability to propidium iodide were observed only with 7beta-hydroxycholesterol and 7-ketocholesterol, which trigger an apoptotic process characterized by the occurrence of cells with fragmented and/or condensed nuclei, and by various cellular dysfunctions: loss of mitochondrial transmembrane potential, cytosolic release of cytochrome c, activation of caspase-9 and -3 with subsequent enhanced activity of caspase-3, degradation of poly(ADP-ribose) polymerase, and increased accumulation of cellular C16 : 0 and C24 : 1 ceramide species. This ceramide generation is not attributed to caspase activation since inhibition of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis by Z-VAD-fmk (100 microM), a broad spectrum caspase inhibitor, did not reduce C16 : 0 and C24 : 1 ceramide species accumulation. Conversely, when U937 cells were treated with 7beta-hydroxycholesterol and 7-ketocholesterol in the presence of fumonisin B1 (100 microM), a specific inhibitor of ceramide synthase, C16 : 0 and C24 : 1 ceramide species production was completely abrogated whereas apoptosis was not prevented. Noteworthy, 7alpha-hydroxycholesterol induced only a slight inhibition of cell growth. Collectively, these results are consistent with the notion that the alpha or beta hydroxyl radical position of oxysterols oxidized at C7 plays a key role in the induction of the apoptotic process. In addition, our findings demonstrate that 7beta-hydroxycholesterol- and 7-ketocholesterol-induced apoptosis involve the mitochondrial signal transduction pathway and they suggest that C16 : 0 and C24 : 1 ceramide species generated through ceramide synthase play a minor role in the commitment of 7beta-hydroxycholesterol- and 7-ketocholesterol-induced cell death.

Impairment with various antioxidants of the loss of mitochondrial transmembrane potential and of the cytosolic release of cytochrome c occuring during 7-ketocholesterol-induced apoptosis.

Previous investigations of our laboratory have shown that 7-ketocholesterol was a potent inducer of apoptosis involving a release of cytochrome c into the cytosol, and a lipid peroxidation process that could be the consequence of a production of radical oxygen species. According to these considerations, we asked whether some antioxidants were able to counteract 7-ketocholesterol-induced apoptosis, and whether prevention of cell death was associated with the impairment of mitochondrial events implied in the commitment to apoptosis, i.e., opening of the mitochondrial megachannels leading to the loss of the mitochondrial transmembrane potential (DeltaPsim), and release of cytochrome c from mitochondria into the cytosol. To this end, we studied the effects of glutathione (15 mM), N-acetylcysteine (15 mM), vitamin E (100 microM), vitamin C (50 microM) and melatonin (1 mM) on U937 cells treated with 7-ketocholesterol (40 microg/ml). Only glutathione, N-acetylcysteine, and vitamin E prevented apoptosis measured by the occurrence of cells with condensed and/or fragmented nuclei, as well as the loss of DeltaPsim, and the release of cytochrome c. However, all the antioxidants used were potent inhibitors of the production of O(2)(*) occuring under treatment with 7-ketocholesterol. Collectively, our data demonstrate that impairment of apoptosis by glutathione, N-acetylcysteine, and vitamin E correlates with the prevention of mitochondrial dysfunctions, and they underline that the ability of antioxidants to counteract 7-ketocholesterol-induced apoptosis does not only depend on their capability to inhibit the production of O(2)(*).

Glutathione is implied in the control of 7-ketocholesterol-induced apoptosis, which is associated with radical oxygen species production.

In a number of experimental systems, inhibition of apoptosis by antioxidants has led to the production of radical oxygen species (ROS) in certain apoptotic forms of cell death. Since antioxidant therapies can reduce vascular dysfunctions in hypercholesterolemic patients who frequently have increased plasma levels of oxysterols constituting potent inducers of apoptosis, we speculate that oxysterol-induced apoptosis could involve oxidative stress. Here, we tested the protective effects of the aminothiols glutathione (GSH) and N-acetylcysteine (NAC), which are two potent antioxidants, on apoptosis induced by 7-ketocholesterol in U937 cells, and we present evidence indicating that oxidative processes are involved in 7-ketocholesterol-induced cell death. Thus, GSH and NAC prevented phenomenona linked to apoptosis such as reduction of cell growth, increase cellular permeability to propidium iodide, and occurrence of nuclear condensation and/or fragmentation, and they delayed internucleosomal DNA fragmentation. In addition, cell treatment with GSH impaired cytochrome c release into the cytosol and degradation of caspase-8 occurring during cell death. During 7-ketocholesterol-induced apoptosis, we also observed a rapid decrease in cellular GSH content, oxidation of polyunsaturated fatty acids, and a production of ROS by flow cytometry with the use of the dye 2', 7'-dichlorofluorescin-diacetate; both phenomena were inhibited by GSH. Prevention of cell death by GSH and NAC does not seem to be a general rule since these antioxidants impaired etoposide (but not cycloheximide) -induced apoptosis. Taken together, our data demonstrate that GSH is implied in the control of 7-ketocholesterol-induced apoptosis associated with the production of ROS.

Different patterns of IL-1beta secretion, adhesion molecule expression and apoptosis induction in human endothelial cells treated with 7alpha-, 7beta-hydroxycholesterol, or 7-ketocholesterol.

Among oxysterols oxidized at C7 (7alpha-, 7beta-hydroxycholesterol, and 7-ketocholesterol), 7beta-hydroxycholesterol and 7-ketocholesterol involved in the cytotoxicity of oxidized low density lipoproteins (LDL) are potent inducers of apoptosis. Here, we asked whether all oxysterols oxidized at C7 were able to trigger apoptosis, to stimulate interleukin (IL)-Ibeta and/or tumor necrosis factor (TNF)-alpha secretion, and to enhance adhesion molecule expression (intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin) on human umbilical venous endothelial cells (HUVECs). Only 7beta-hydroxycholesterol and 7-ketocholesterol were potent inducers of apoptosis and of IL-1beta secretion. TNF-alpha secretion was never detected. Depending on the oxysterol considered, various levels of ICAM-1, VCAM-1 and E-selectin expression were observed. So, oxysterols oxidized at C7 differently injure and activate HUVECs, and the alpha- or beta-hydroxyl radical position plays a key role in apoptosis and IL-1beta secretion.

[Flow cytometry measurement of DNA fragmentation in the course of cell death via apoptosis. New techniques for evaluation of DNA status for the pathologist]. / Mise en évidence de la fragmentation d'ADN par cytométrie en flux au cours de la mort cellulaire par apoptose. Nouvelles techniques d'évaluation de l'état de l'ADN pour le pathologiste.

Cell death by apoptosis is characterized by DNA fragmentation in 200-250 and/or 30-50 kb followed or not by internucleosomal DNA fragmentation in 180-200 pb. Such characteristics have been used to distinguish between necrotic and apoptotic cells, and also to identify and quantify apoptotic cells by flow cytometry. In the case of internucleosomal DNA fragmentation, the analysis of DNA content constitutes the easiest method to identify apoptotic cells giving an hypoploid cell population commonly called "Sub G1". The identification of the "Sub G1" does not depend on the dyes used; however according to the method of cell fixation and permeabilization, of the divalent cations (Ca2+, Mg2+) present in the staining buffers and of the use of trypsin, the "Sub G1" population may be more or less difficult to identify. To detect apoptotic cells whatever the pattern of DNA fragmentation, the most commonly used methods are either in situ nick-translation or TUNEL (TdT dUTP Nick End Labelling). Thus, flow cytometry offers a wide range of attractive techniques to characterize apoptotic cells but it requires the use of methodological controls for validating results.