Lysosome-mediated apoptosis is associated with cathepsin D-specific processing of bid at Phe24, Trp48, and Phe183.

Bax-mediated permeabilization of the outer mitochondrial membrane and release of apoptogenic factors into the cytosol are key events that occur during apoptosis. Likewise, apoptosis is associated with permeabilization of the lysosomal membrane and release of lysosomal cathepsins into the cytosol. This report identifies proteolytically active cathepsin D as an important component of apoptotic signaling following lysosomal membrane permeabilization in fibroblasts. Lysosome-mediated cell death is associated with degradation of Bax sequestering 14-3-3 proteins, cleavage of the Bax activator Bid, and translocation of Bax to mitochondria, all of which were cathepsin D-dependent. Processing of Bid could be reproduced by enforced lysosomal membrane permeabilization, using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride (MSDH). We identified three cathepsin D-specific cleavage sites in Bid, Phe24, Trp48, and Phe183. Cathepsin D-cleaved Bid induced Bax-mediated release of cytochrome c from purified mitochondria, indicating that the fragments generated are functionally active. Moreover, apoptosis was associated with cytosolic acidification, thereby providing a more favorable environment for the cathepsin D-mediated cleavage of Bid. Our study suggests that cytosolic cathepsin D triggers Bax-mediated cytochrome c release by proteolytic activation of Bid.

Lysosome-targeted stress reveals increased stability of lipofuscin-containing lysosomes.

Cellular ageing is associated with accumulation of undegradable intralysosomal material, called lipofuscin. In order to accelerate the lipofuscin accumulation, confluent, growth-arrested human fibroblasts were cultured under hyperoxic conditions. To provide a better insight into the effects of lipofuscin on cellular functions, we compared lysosomal stability in control and lipofuscin-loaded human fibroblasts under conditions of lysosome-targeted stress induced by exposure to either the lysosomotropic detergent MSDH or the redox-cycling quinone naphthazarin. We show that lysosomal damage, assessed by acridine-orange relocation, translocation of cathepsin D to the cytosol, and alkalinization of lysosomes, is more pronounced in control than in lipofuscin-loaded fibroblasts. Finding that lysosomal integrity was less affected or even preserved in case of lipofuscin-loaded cells enables us to suggest that lipofuscin exerts lysosome-stabilizing properties.

Evaluation of electron spin resonance for studies of superoxide anion production by human neutrophils interacting with Staphylococcus aureus and Staphylococcus epidermidis.

The present study evaluates electron spin resonance (ESR) and the spin trapper 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO) for analysis of superoxide radical production by human neutrophils interacting with viable Staphylococcus aureus and Staphylococcus epidermidis bacteria. To avoid auto-activation due to interaction with glass surfaces, neutrophils were preincubated in plastic tubes until the peak response was reached, and then transferred to a quartz flat cell to record the ESR spectra. The time point for peak response was identified by parallel analysis of the bacteria-neutrophil interaction using luminol amplified chemiluminescence. We found detectable ESR spectra from neutrophils interacting with as few as five bacteria of the weak activating S. epidermidis per neutrophil. Addition of the NADPH oxidase inhibitor diphenylene iodonium totally abolished spectra. Catalase, DMSO or an iron chelator had no impact on the produced spectra and ionomycin, a selective activator of intracellular NADPH oxidase, gave significant ESR spectra. Taken together, our results indicate that DEPMPO is cell permeable and detects NADPH oxidase derived superoxide anions formed in phagosomes or released by human neutrophils phagocytosing viable S. aureus and S. epidermidis. The technique may be used as a sensitive tool to evaluate superoxide anion production in human neutrophils.

Increased resistance of lipofuscin-loaded prematurely senescent fibroblasts to starvation-induced programmed cell death.

Alterations of cellular structures often found in ageing cells is mainly the result of production of reactive oxygen species and a consequence of aerobic life. Both oxidative stress and decreased degradative capacity of lysosomal system cause accumulation of intralysosomal age-related pigment called lipofuscin. To investigate the influence of lipofuscin on cell function, we compared survival of lipofuscin-loaded and control human fibroblasts following complete starvation induced by exposure to phosphate-buffered saline (PBS). Starving of control fibroblasts resulted in lysosomal alkalinisation, relocation of cathepsin D to the cytosol, caspase-3 activation and, finally, cell death, which became evident 72 h after the start of exposure to PBS. Increase of lysosomal pH was significantly less prominent in lipofuscin-loaded cells than in controls and was accompanied neither by leakage of cathepsin D nor by caspase-3 activation even 96 h after the initiation of starvation. Suppression of autophagy by 3-methyladenine (3-MA) accelerated cell death, while inhibition of cathepsin D delayed it, implying an important role of autophagy in cell survival during starvation and showing the involvement of lysosomes in starvation-induced cell death. Disturbed apoptotic response found in lipofuscin-loaded cells can be interpreted as an example of hormesis--an adaptation to low doses of otherwise harmful agents, in this case of lipofuscin, which has a protective effect at moderate amounts but becomes toxic at large quantities.

Cytosolic acidification and lysosomal alkalinization during TNF-alpha induced apoptosis in U937 cells.

Apoptosis is often associated with acidification of the cytosol and since loss of lysosomal proton gradient and release of lysosomal content are early events during apoptosis, we investigated if the lysosomal compartment could contribute to cytosolic acidification. After exposure of U937 cells to tumor necrosis factor-alpha, three populations; healthy, pre-apoptotic, and apoptotic cells, were identified by flow cytometry. These populations were investigated regarding intra-cellular pH and apoptosis-associated events. There was a drop in cytosolic pH from 7.2 +/- 0.1 in healthy cells to 6.8 +/- 0.1 in pre-apoptotic, caspase-negative cells. In apoptotic, caspase-positive cells, the pH was further decreased to 5.7 +/- 0.04. The cytosolic acidification was not affected by addition of specific inhibitors towards caspases or the mitochondrial F(0)F(1)-ATPase. In parallel to the cytosolic acidification, a rise in lysosomal pH from 4.3 +/- 0.3, in the healthy population, to 4.8 +/- 0.3 and 5.5 +/- 0.3 in the pre-apoptotic- and apoptotic populations, respectively, was detected. In addition, lysosomal membrane permeability increased as detected as release of cathepsin D from lysosomes to the cytosol in pre-apoptotic and apoptotic cells. We, thus, suggest that lysosomal proton release is the cause of the cytosolic acidification of U937 cells exposed to TNF-alpha.

Lysosomal membrane permeabilization during apoptosis--involvement of Bax?

Bcl-2 family members have long been known to control permeabilization of the mitochondrial membrane during apoptosis, but involvement of these proteins in lysosomal membrane permeabilization (LMP) was not considered until recently. The aim of this study was to investigate the mechanism underlying the release of lysosomal proteases to the cytosol seen during apoptosis, with special emphasis on the role of Bax. In human fibroblasts, exposed to the apoptosis-inducing drug staurosporine (STS), the release of the lysosomal protease cathepsin D to the cytosol was observed by immunocytochemistry. In response to STS treatment, there was a shift in Bax immunostaining from a diffuse to a punctate pattern. Confocal microscopy showed co-localization of Bax with both lysosomes and mitochondria in dying cells. Presence of Bax at the lysosomal membrane was confirmed by immuno-electron microscopy. Furthermore, when recombinant Bax was incubated with pure lysosomal fractions, Bax inserted into the lysosomal membrane and induced the release of lysosomal enzymes. Thus, we suggest that Bax is a mediator of LMP, possibly promoting the release of lysosomal enzymes to the cytosol during apoptosis.

Ultraviolet A and B affect human melanocytes and keratinocytes differently. A study of oxidative alterations and apoptosis.

Ultraviolet (UV) radiation is an etiologic agent for malignant melanoma and non-melanoma skin cancer, but the spectral range responsible for tumor induction is still to be elucidated. In this study, we compared effects of UVA and UVB irradiation on normal human melanocytes (MCs) and keratinocytes (KCs) in vitro. We demonstrate that UVA irradiation induces immediate loss of reduced glutathione (GSH) in both MCs and KCs. Exposure to UVA also causes reduced plasma membrane stability, in both cell types, as estimated by fluorescein diacetate retention and flow cytometry. Furthermore, we noted reduction in proliferation and higher apoptosis frequency 24 h after UVA irradiation. UVB irradiation of KCs caused instant reduction of reduced GSH and impaired plasma membrane stability. We also found decline in proliferation and increased apoptosis after 24 h. In MCs, on the other hand, UVB had no effect on GSH level or plasma membrane stability, although increased apoptotic cell death and reduced proliferation was detected. In summary, MCs and KCs showed similar response towards UVA, while UVB had more pronounced effects on KCs as compared to MCs. These results might have implications for the induction of malignant melanoma and non-melanoma skin cancer.

Analysis of cytosolic and lysosomal pH in apoptotic cells by flow cytometry.

Several reports indicate that the cytosol is acidified during apoptosis although the mechanism is not yet fully elucidated. The most acidic organelle found in the cell is the lysosome, raising the possibility that lysosomal proton release may contribute to the cytosolic acidification. We here describe methods for measurement of the cytosolic and lysosomal pH in U937 cells by a dual-emission ratiometric technique suitable for flow cytometry. Cytosolic pH was analysed in cells loaded with the fluorescent probe BCECF, while lysosomal pH was determined after endocytosis of FITC-dextran. Standard curves were obtained by incubating cells in buffers with different pH in the presence of the proton ionophore nigericin. Apoptosis was induced by exposure of cells to 10 ng/ml TNF-alpha for 4 h, and apoptotic cells were identified using a fluorescent marker for active caspases. By gating of control and apoptotic cells, the cytosolic and lysosomal pH were calculated in each population. The cytosolic pH was found to decrease from 7.2+/-0.1 to 5.8+/-0.1 and the lysosomal increased from 4.3+/-0.4 to 5.2+/-0.3. These methods will be useful in future attempts to evaluate the involvement of lysosomes in the acidification of the cytosol during apoptosis.