Intracellular triggering of Fas, independently of FasL, as a new mechanism of antitumor ether lipid-induced apoptosis.

Antitumor ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH(3); edelfosine) induces apoptosis in cancer cells, sparing normal cells. We have found that the apoptotic action of ET-18-OCH(3) required drug uptake and Fas in the target cell. Failure to accomplish one of these requirements prevents cell killing by the ether lipid. In human lymphoid leukemic cells, ET-18-OCH(3) does not promote Fas or FasL expression and ET-18-OCH(3)-induced apoptosis is not inhibited by pre-incubation with an anti-Fas blocking antibody that abrogates cell killing mediated by Fas/FasL interactions. ET-18-OCH(3)-resistant normal human Fas-positive fibroblasts do not incorporate ET-18-OCH(3), but undergo apoptosis upon ET-18-OCH(3) microinjection. Murine fibroblasts L929 and L929-Fas, stably transfected with human Fas cDNA, do not incorporate ET-18-OCH(3) and are resistant to its action when added exogenously. Microinjection of ET-18-OCH(3) induces apoptosis in L929-Fas cells, but not in wild-type L929 cells. Confocal laser scanning microscopy shows that ET-18-OCH(3) induces Fas clustering and capping during triggering of ET-18-OCH(3)-induced apoptosis. Microinjection-induced apoptosis and Fas clustering are specific for the molecular structure of ET-18-OCH(3). Our data indicate that ET-18-OCH(3) induces apoptosis via Fas after the ether lipid is inside the cell, and this Fas activation is independent of the interaction of Fas with its natural ligand FasL. This explains the selective action of ET-18-OCH(3) on tumors since only cancer cells incorporate sufficient amounts of the drug.

Induction of apoptosis in human mitogen-activated peripheral blood T-lymphocytes by the ether phospholipid ET-18-OCH3: involvement of the Fas receptor/ligand system.

1. Activated T-cells constitute a target for treatment of autoimmune diseases. We have found that the antitumour ether phospholipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3; edelfosine) induced dose- and time-dependent apoptosis in human mitogen-activated peripheral blood T-lymphocytes, but not in resting T-cells. T-lymphocytes were stimulated with phytohemagglutinin and interleukin-2 or with concanavalin A. Apoptosis was assessed by DNA fragmentation through cell cycle and TUNEL analyses, as well as through visualization of internucleosomal DNA fragmentation in agarose gels. 2. The ET-18-OCH3-mediated apoptotic response in activated T-lymphocytes was less intense than in human leukaemic T cell lines, such as Jurkat cells and Peer cells; namely about 25% apoptosis in activated T-cells versus about 46-61% apoptosis in T leukaemic cells after 24 h treatment with 10 microM ET-18-OCH3. 3. The ET-18-OCH3 thioether analogue BM 41.440 (ilmofosine) showed a similar apoptotic capacity to that found with ET-18-OCH3 in activated T-cells, whereas the phospholipid analogue hexadecylphosphocholine (miltefosine) failed to promote this response. 4. The uptake of [3H]-ET-18-OCH3 was much larger in activated T-cells than in resting lymphocytes. 5. Using a cytofluorimetric approach we have found that ET-18-OCH3 induced disruption of the mitochondrial transmembrane potential and production of reactive oxygen species in activated T-cells, but not in resting lymphocytes. 6. ET-18-OCH3 induced an increase in Fas (APO-1/CD95) ligand mRNA expression in activated T-cells, and incubation with a blocking anti-Fas (APO-1/CD95) antibody partially inhibited the ET-18-OCH3-induced apoptosis of activated T-lymphocytes. 7. These results demonstrate that mitogen-activated T-cells, unlike resting lymphocytes, are able to take up significant amounts of ET-18-OCH3, and are susceptible to undergo apoptosis by the ether lipid via, in part, the Fas (APO-1/CD95) receptor/ligand system. This ET-18-OCH3 apoptotic action can be of importance in the therapeutic action of this ether lipid in certain autoimmune diseases.

Biological effects of continuous exposure of embryos and young chickens to electromagnetic fields emitted by video display units.

The effects of continuous exposure of embryos and young chickens to electromagnetic fields (EMFs) emitted by video display units (VDUs) were investigated. Embryos and brood were continuously exposed during embryonic and postembryonic phases to EMFs emitted by two types of VDU (TV or computer). Embryonic mortality was evaluated in three independent experiments. Young chickens were immunized three times by porcine thyroglobulin (Tg). Blood samples were assayed after each immunization for specific anti-Tg antibodies (IgG), plasma corticosterone (CORT), and plasma melatonin (MLT). In the sham-exposed samples, embryonic death (10-33%) was restricted to the perinatal period and the IgG, CORT, and MLT responses of young chickens crested after the second immunization. Constant EMF exposure was accompanied by significantly increased fetal loss (47-68%) and markedly depressed levels of circulating anti-Tg IgG, CORT, and MLT. Collectively, these findings indicate that continuous exposure to EMFs, issuing from VDUs, adversely affects embryos and young chickens.

Chemiluminescence and nitrite determinations by the MALU macrophage cell line.

The MALU macrophage cell line is an in vitro coculture of mesothelial cells and alveolar macrophages from mice. During the culture, macrophages in a preactivated state can be collected in the supernatant (Lombard et al., 1988). We describe here two methods to measure reactive oxygen intermediate (ROI) and reactive nitrogen intermediate (RNI) production by the MALU macrophages. We measured ROI by the chemiluminescence assay with a luminometer and RNI was measured colorimetrically in a spectrophotometer with the Griess reagent. Different parameters (cell number, incubation time, temperature and activating substance) which can interfere with the cell response were analysed. Our results show that the MALU macrophage cell line produces large amounts of ROI and RNI after activation. This cell line is a good model for investigating the effect of pharmacological drugs on ROI and RNI production.