ABSTRACT
The roles of oxygen and reactive oxygen intermediates in apoptosis are unclear at present. Although oxygen and reactive oxygen intermediates are not required for the execution of apoptosis, oxygen may be involved in at least some forms of apoptosis. In this study we show that dexamethasone (Dex)-induced apoptosis of immature mouse thymocytes is completely inhibited by hypoxic culture. In contrast, anti-CD95 thymocyte apoptosis is unaffected by hypoxia, indicating the existence of two forms of thymocyte apoptosis: an oxygen-dependent pathway (Dex induced) and an oxygen-independent pathway (anti-CD95 induced). Furthermore, hypoxia inhibited mitochondrial permeability transition (PT) in Dex-treated, but not in anti-CD95-treated, thymocytes, suggesting that the oxygen-sensitive step is upstream of mitochondria. Both Dex- and anti-CD95-induced PT and apoptosis were dependent on activation of IL-converting enzyme-like protease, as PT and apoptosis were inhibited by preincubation with Cbz-Val-Ala-Asp-fluoromethyl ketone, an irreversible inhibitor of IL-converting enzyme-like proteases. In addition, hypoxia inhibited the activation by Dex of caspase-3 (CPP32)-like proteases. Our data show that the private signaling pathways of Dex (oxygen dependent) and anti-CD95 (oxygen independent) both converge upstream of mitochondrial changes. The oxygen-dependent step in Dex-induced apoptosis lies upstream of caspase-3-like protease activation. Our observations support a model of apoptosis signaling in which independent pathways (oxygen dependent and oxygen independent) particular to each stimuli converge at a central point in the apoptotic cascade.
Subject(s)
Apoptosis/drug effects , Apoptosis/immunology , Dexamethasone/pharmacology , Oxygen/physiology , T-Lymphocytes/cytology , T-Lymphocytes/drug effects , Thymus Gland/cytology , Thymus Gland/drug effects , Animals , Antibodies, Monoclonal/pharmacology , Cell Differentiation/drug effects , Cell Differentiation/immunology , Cell Hypoxia/drug effects , Cell Hypoxia/immunology , Cells, Cultured , Cyclic N-Oxides/pharmacology , Dexamethasone/antagonists & inhibitors , Endopeptidases/metabolism , Enzyme Inhibitors/pharmacology , Growth Inhibitors/antagonists & inhibitors , Growth Inhibitors/pharmacology , Immunosuppressive Agents/antagonists & inhibitors , Immunosuppressive Agents/pharmacology , Intracellular Membranes/drug effects , Intracellular Membranes/immunology , Intracellular Membranes/metabolism , Male , Mice , Mice, Inbred BALB C , Mitochondria/drug effects , Mitochondria/immunology , Mitochondria/metabolism , Nerve Tissue Proteins/antagonists & inhibitors , Nerve Tissue Proteins/metabolism , Permeability/drug effects , Rotenone/pharmacology , Spin Labels , T-Lymphocytes/enzymology , T-Lymphocytes/metabolism , Thymus Gland/enzymology , Thymus Gland/metabolism , Uncoupling Agents/pharmacology , fas Receptor/immunologyABSTRACT
Identification of biomolecules in complex biological mixtures represents a major challenge in biomedical, environmental, and chemical research today. Chemical separations with traditional detection schemes such as absorption, fluorescence, refractive index, conductivity, and electrochemistry have been the standards for definitive identifications of many compounds. In many instances, however, the complexity of the biomixture exceeds the resolution capability of chemical separations. Biosensors based on molecular recognition can dramatically improve the selectivity of and provide biologically relevant information about the components. This review describes how coupling chemical separations with online biosensors solves challenging problems in sample analysis by identifying components that would not normally be detectable by either technique alone. This review also presents examples and principles of combining chemical separations with biosensor detection that uses living systems, whole cells, membrane receptors, enzymes, and immunosensors.
Subject(s)
Biosensing Techniques , Chemistry/methods , Pharmaceutical Preparations/isolation & purification , Animals , Biophysics/methods , Chemistry/instrumentation , Enzymes, ImmobilizedABSTRACT
T cell apoptosis may play an important role in the depletion and functional defects of T cells in HIV disease. A number of investigators have shown that peripheral blood T cells in HIV disease undergo spontaneous and activation-induced apoptosis. We found recently that peripheral blood T cells from HIV+ individuals undergo apoptosis when stimulated through Fas. Also, a number of investigators have shown that Tat protein from HIV-1 can increase spontaneous and activation-induced apoptosis. In the present study we examined the effect of HIV type 1 Tat protein on spontaneous, activation-induced and Fas-induced apoptosis of peripheral blood T cells from HIV- individuals. We find that Tat protein has no effect on spontaneous apoptosis but does enhance activation-induced apoptosis of both CD4+ and CD8+ T cells. Tat, however, failed to enhance Fas-induced apoptosis of CD4+ and CD8+ T cells. Examining the mechanisms by which Tat induces apoptosis, we found that inhibitors of reactive oxygen intermediate (ROI) generation or neutralizers of ROI, such as rotenone, a potent inhibitor of mitochondrial complex I of the respiratory chain, and 3,3,5,5-tetramethylpyrroline N-oxide (TMPO), an electron spin trap, could both enhance the spontaneous apoptosis induced by Tat. This enhancement of Tat-induced apoptosis by rotenone and TMPO was independent of ICE activation as it could not be inhibited by the tripeptide z-VAD-fmk, an irreversible inhibitor of ICE/ced-3 protease homologs. These findings suggest that Tat induced enhancement of activation-induced cell death may involve complex mechanisms, some of which are ROI independent. These results indicate that a HIV-specific mechanism other than Tat is responsible for the previously observed increased susceptibility of peripheral blood T cells from HIV-infected individuals to undergo apoptosis in response to Fas stimulation.
