Mari Enoksson

Caspase-2 acts upstream of mitochondria to promote cytochrome c release during etoposide-induced apoptosis

DNA damage induced by the cancer chemotherapeutic drug etoposide triggers the onset of a series of intracellular events characteristic of apoptosis. Among the early changes observed is the release of cytochrome c from mitochondria, although the mechanism responsible for this effect is unclear. We demonstrate here a role for caspase-2 in etoposide-induced cytochrome crelease. In particular, Jurkat T-lymphocytes treated with an irreversible caspase-2 inhibitor, benzyloxycarbonyl-Val-Asp-Val-Ala-Asp-fluoromethyl ketone (z-VDVAD-fmk), or stably transfected with pro-caspase-2 antisense (Casp-2/AS) are refractory to cytochrome crelease stimulated by etoposide. Experiments performed using a reconstituted cell-free system indicate that etoposide-induced cytochrome c release by way of caspase-2 occurs independently of cytosolic factors, suggesting that the nuclear pool of pro-caspase-2 is critical to this process. Apart from inhibiting cytochrome c release, undermining caspase-2 activity results in an attenuation of downstream events, such as pro-caspase-9 and -3 activation, phosphatidylserine exposure on the plasma membrane, and DNA fragmentation. Taken together, our data indicate that caspase-2 provides an important link between etoposide-induced DNA damage and the engagement of the mitochondrial apoptotic pathway.

Caspase-2 Permeabilizes the Outer Mitochondrial Membrane and Disrupts the Binding of Cytochrome c to Anionic Phospholipids

Caspases are cysteine proteases that play a central role in the execution of apoptosis. Recent evidence indicates that caspase-2 is activated early in response to genotoxic stress and can function as an upstream modulator of the mitochondrial apoptotic pathway. In particular, we have shown previously that fully processed caspase-2 can permeabilize the outer mitochondrial membrane and cause cytochrome c and Smac/DIABLO release from these organelles. Using permeabilized cells, isolated mitochondria, and protein-free liposomes, we now report that this effect is direct and depends neither on the presence or cleavage of other proteins nor on a specific phospholipid composition of the liposomal membrane. Interestingly, caspase-2 was also shown to disrupt the interaction of cytochrome c with anionic phospholipids, notably cardiolipin, and thereby enhance the release of the hemoprotein caused by treatment of mitochondria with digitonin or the proapoptotic protein Bax. Combined, our data suggest that caspase-2 possesses an unparalleled ability to engage the mitochondrial apoptotic pathway by permeabilizing the outer mitochondrial membrane and/or by breaching the association of cytochrome c with the inner mitochondrial membrane.

Visualization of the compartmentalization of glutathione and protein-glutathione mixed disulfides in cultured cells

Fluorescence microscopy of A549 cells stained with a glutathione (L‐γ‐glutamyl‐Lcysteinylglycine, GSH)‐specific polyclonal antibody displayed uniform staining of the perinuclear cytosol, with the nuclear region apparently lacking GSH staining. This discontinuous staining was confirmed in other cell types and also corroborated in A549 cells stained with the thiol‐reactive dye mercury orange. The selectivity of antibody binding was confirmed by buthionine sulfoximine (BSO)‐dependent inhibition of GSH synthesis. However, confocal visualization of antibody‐stained A549 cells in the z‐plane revealed the majority of the perinuclear staining intensity in the upper half of the cell to be associated with mitochondria, as confirmed by double staining for cytochrome oxidase. Integration of the confocal signals from the nuclear and cytosolic regions halfway down the z‐plane showed that the GSH concentrations of these compartments are close to equilibrium. Confirmation of the relatively high levels of mitochondrial glutathione was provided in cells treated with BSO and visualized in z‐section, revealing the mitochondrial GSH content of these cells to be well preserved in apposition to near‐complete depletion of cytosolic/nuclear GSH. Localized gradients within the cytosolic compartment were also visible, particularly in the z‐plane. The antibody also provided initial visualization of the compartmentalization of protein‐GSH mixed disulfides formed in A549 cells exposed to diamide. Discontinuous staining was again evident, with heavy staining in membrane blebs and in the nuclear region. Using FACS analysis of anti‐GSH antibody‐stained Jurkat T lymhocytes, we also demonstrated population variations in the cellular compliment of GSH and protein‐GSH mixed disulfides, formed in response to diamide. In addition, we showed cell‐cycle variation in GSH content of the cells, with the highest levels of GSH associated with the G2/M mitotic phase of the cell cycle, using double staining with propidium iodide. Similar FACS analyses performed in isolated mitochondria presented a considerable variation in GSH content within mitochondria of uniform granularity from the same preparation.

Differential regulation of the mitochondrial and death receptor pathways in neural stem cells

Despite an increasing interest in neural stem cell (NSC) research, relatively little is known about the biochemical regulation of cell death pathways in these cells. We demonstrate here, using murine‐derived multipotent C17.2 NSCs, that cells undergo mitochondria‐mediated cell death in response to apoptotic stimuli such as oxidative stress induced by 2,3‐dimethoxy‐1,4‐naphthoquinone (DMNQ). In particular, treated cells exhibited apoptotic features, including Bax translocation, cytochrome c release, activation of caspase‐9 and ‐3, chromatin condensation and DNA fragmentation. Although C17.2 cells possess the Fas receptor and express procaspase‐8, agonistic Fas mAb treatment failed to induce apoptosis. Fas treatment activated the extracellular signal‐regulated protein kinase (ERK) pathway, which may have an antiapoptotic as well as a growth stimulating role. Combined, our findings indicate that while NSCs are sensitive to cytotoxic stimuli that involve an engagement of mitochondria, Fas treatment does not induce death and may have an alternative role.

Mitochondrial cytochrome c release may occur by volume-dependent mechanisms not involving permeability transition

The mechanisms regulating mitochondrial outer-membrane permeabilization and the release of cytochrome c during apoptosis remain controversial. In the present study, we show in an in vitro model system that the release of cytochrome c may occur via moderate modulation of mitochondrial volume, irrespective of the mechanism leading to the mitochondrial swelling. In contrast with mitochondrial permeability transition-dependent release of cytochrome c, in the present study mitochondria remain intact and functionally active.