Apoptosome-independent activation of the lysosomal cell death pathway by caspase-9.

The apoptosome, a heptameric complex of Apaf-1, cytochrome c, and caspase-9, has been considered indispensable for the activation of caspase-9 during apoptosis. By using a large panel of genetically modified murine embryonic fibroblasts, we show here that, in response to tumor necrosis factor (TNF), caspase-8 cleaves and activates caspase-9 in an apoptosome-independent manner. Interestingly, caspase-8-cleaved caspase-9 induced lysosomal membrane permeabilization but failed to activate the effector caspases whereas apoptosome-dependent activation of caspase-9 could trigger both events. Consistent with the ability of TNF to activate the intrinsic apoptosis pathway and the caspase-9-dependent lysosomal cell death pathway in parallel, their individual inhibition conferred only a modest delay in TNF-induced cell death whereas simultaneous inhibition of both pathways was required to achieve protection comparable to that observed in caspase-9-deficient cells. Taken together, the findings indicate that caspase-9 plays a dual role in cell death signaling, as an activator of effector caspases and lysosomal membrane permeabilization.

Oxidation reduces the fibrillation but not the neurotoxicity of the prion peptide PrP106-126.

There is increasing evidence that soluble oligomers of misfolded protein may play a role in the pathogenesis of protein misfolding diseases including the transmissible spongiform encephalopathies (TSE) where the protein involved is the prion protein, PrP. The effect of oxidation on fibrillation tendency and neurotoxicity of different molecular variants of the prion peptide PrP106-126 was investigated. It was found that methionine oxidation significantly reduced amyloid fibril formation and proteinase K resistance, but it did not reduce (but rather increase slightly) the neurotoxicity of the peptides in vivo (electroretinography after intraocular injections in mice) and in vitro (in primary neuronal cultures). We furthermore found that the bovine variant of PrP106-126, containing only one methionine residue, showed both reduced fibril forming capacity and in vivo and in vitro neurotoxicity. The findings imply (I) that there is not a simple relation between the formation of amyloid fibrils and neurotoxicity of PrP106-126 derived peptides, (II) that putative, soluble, non-amyloid protofibrils, presumed to be present in increased proportions in oxidized PrP106-126, could play a role in the pathogenesis of TSE and III) that the number of methionine residues in the PrP106-126 peptide seems to have a pivotal role in determining the physical and biological properties of PrP106-126.

Ischemia leads to apoptosis--and necrosis-like neuron death in the ischemic rat hippocampus.

Morphological evidence of apoptosis in transient forebrain ischemia is controversial. We therefore investigated the time sequence of apoptosis-related antigens by immunohistochemistry and correlated it with emerging nuclear patterns of cell death in a model of transient forebrain ischemia in CA1 pyramidal cells of the rat hippocampus. The earliest ischemic changes were found on day 2 and 3, reflected by an upregulation of phospho-c-Jun in a proportion of morphologically intact CA1 neurons, which matched the number of neurons that succumbed to ischemia at later time points. At day 3 and later 3 ischemic cell death morphologies became apparent: pyknosis, apoptosis-like cell death and necrosis-like cell death, which were confirmed by electron microscopy. Activated caspase-3 was present in the vast majority of cells with apoptosis-like morphology as well as in a small subset of cells undergoing necrosis; its expression peaked on days 3 to 4. Silver staining for nucleoli, which are a substrate for caspase-3, revealed a profound loss of nucleoli in cells with apoptosis-like morphology, whereas cells with necrosis-like morphology showed intact nucleoli. Overall, cells with apoptosis-like morphology and/or caspase-3 expression represented a minor fraction (<10%) of ischemic neurons, while the vast majority followed a necrosis-like pathway. Our studies suggest that CA1 pyramidal cell death following transient forebrain ischemia may be initiated through c-Jun N-terminal kinase (JNK) pathway activation, which then either follows an apoptosis-like cell death pathway or leads to secondary necrosis.

Functionally induced changes in water transport in the proximal tubule segment of rat kidneys.

To eliminate freezing artifacts in the proximal tubule cells, two cryotechniques were applied to normal rat kidneys, ie, freeze substitution and special freeze drying. In addition, salt depletion and salt loading were applied to groups of rats to evaluate whether the segmental structure of the proximal tubule could be altered. In the superficial part of the renal cortex of normal kidneys, the typical first segment structure in the proximal tubule was generally present in the early postglomerular fraction of the tubule. However, in the second segment, a special cellular phenomenon was constantly present, comprising a significant intercellular space that was easily identified using a light microscope. In the third segment, in which the presence of basolateral interdigitations is minimal, the small lateral space, which was found to be present in cryopreparations between neighboring cells from the normal kidney, was found to be enlarged by heavy salt loading of short duration. It is concluded that these cryotechniques demonstrate quantitative structural variations between superficial and deep nephrons, as well as the presence of extracellular areas between the cells of the second and the third segment, representing a structural background for the essential transport of water from the proximal tubules to the peritubular capillaries.

Sensitization to the lysosomal cell death pathway by oncogene-induced down-regulation of lysosome-associated membrane proteins 1 and 2.

Expression and activity of lysosomal cysteine cathepsins correlate with the metastatic capacity and aggressiveness of tumors. Here, we show that transformation of murine embryonic fibroblasts with v-H-ras or c-src(Y527F) changes the distribution, density, and ultrastructure of the lysosomes, decreases the levels of lysosome-associated membrane proteins (LAMP-1 and LAMP-2) in an extracellular signal-regulated kinase (ERK)- and cathepsin-dependent manner, and sensitizes the cells to lysosomal cell death pathways induced by various anticancer drugs (i.e., cisplatin, etoposide, doxorubicin, and siramesine). Importantly, K-ras and erbb2 elicit a similar ERK-mediated activation of cysteine cathepsins, cathepsin-dependent down-regulation of LAMPs, and increased drug sensitivity in human colon and breast carcinoma cells, respectively. Notably, reconstitution of LAMP levels by ectopic expression or by cathepsin inhibitors protects transformed cells against the lysosomal cell death pathway. Furthermore, knockdown of either lamp1 or lamp2 is sufficient to sensitize the cells to siramesine-induced cell death and photo-oxidation-induced lysosomal destabilization. Thus, the transformation-associated ERK-mediated up-regulation of cysteine cathepsin expression and activity leads to a decrease in the levels of LAMPs, which in turn contributes to the enhanced sensitivity of transformed cells to drugs that trigger lysosomal membrane permeabilization. These data indicate that aggressive cancers with high cysteine cathepsin levels are especially sensitive to lysosomal cell death pathways and encourage the further development of lysosome-targeting compounds for cancer therapy.

Anti-cancer agent siramesine is a lysosomotropic detergent that induces cytoprotective autophagosome accumulation.

A sigma-2 receptor ligand siramesine induces lysosomal leakage and cathepsin-dependent death of cancer cells in vitro and displays potent anti-cancer activity in vivo. The mechanism by which siramesine destabilizes lysosomes is, however, unknown. Here, we show that siramesine induces a rapid rise in the lysosomal pH that is followed by lysosomal leakage and dysfunction. The rapid accumulation of siramesine into cancer cell lysosomes, its ability to destabilize isolated lysosomes, and its chemical structure as an amphiphilic amine indicate that it is a lysosomotropic detergent. Notably, siramesine triggers also a substantial Atg6- and Atg7-dependent accumulation of autophagosomes that is associated with a rapid and sustained inhibition of mammalian target of rapamycin complex 1 (mTORC1; an inhibitor of autophagy). Siramesine fails, however, to increase the degradation rate of long-lived proteins. Thus, the massive accumulation of autophagosomes is likely to be due to a combined effect of activation of autophagy signaling and decreased autophagosome turnover. Importantly, pharmacological and RNA interference-based inhibition of autophagosome formation further sensitizes cancer cells to siramesine-induced cytotoxicity. These data identify siramesine as a lysosomotropic detergent that triggers cell death via a direct destabilization of lysosomes and cytoprotection by inducing the accumulation of autophagosomes. Threrefore, the combination of siramesine with inhibitors of autophagosome formation appears as a promising approach for future cancer therapy.

Control of macroautophagy by calcium, calmodulin-dependent kinase kinase-beta, and Bcl-2.

Macroautophagy is an evolutionary conserved lysosomal pathway involved in the turnover of cellular macromolecules and organelles. In spite of its essential role in tissue homeostasis, the molecular mechanisms regulating mammalian macroautophagy are poorly understood. Here, we demonstrate that a rise in the free cytosolic calcium ([Ca(2+)](c)) is a potent inducer of macroautophagy. Various Ca(2+) mobilizing agents (vitamin D(3) compounds, ionomycin, ATP, and thapsigargin) inhibit the activity of mammalian target of rapamycin, a negative regulator of macroautophagy, and induce massive accumulation of autophagosomes in a Beclin 1- and Atg7-dependent manner. This process is mediated by Ca(2+)/calmodulin-dependent kinase kinase-beta and AMP-activated protein kinase and inhibited by ectopic Bcl-2 located in the endoplasmatic reticulum (ER), where it lowers the [Ca(2+)](ER) and attenuates agonist-induced Ca(2+) fluxes. Thus, an increase in the [Ca(2+)](c) serves as a potent inducer of macroautophagy and as a target for the antiautophagy action of ER-located Bcl-2.

No difference in early cellular response of the pseudo-synovial membrane after total hip arthroplasty: comparison of 3 combinations of bearing materials.

BACKGROUND: Wear-resistant bearing materials may hypothetically reduce chronic inflammation in the pseudosynovial membrane as compared to less wear-resistant bearing materials such as polyethylene. We assessed the foreign body response in the pseudosynovial membrane in vivo after total hip replacement. METHODS: 37 patients from a larger prospective randomized trial of 225 patients had biopsies taken arthroscopically from the artificial hip joint (i.e. the pseudosynovial membrane) 1 year after insertion of the implant. All patients had an identical hip prosthesis (Bimetric-RingLoc) except for the bearing materials, which consisted of polyethylene on zirconia, CoCr on CoCr, or alumina on alumina. Histological quantification was performed on 2-mum-thick semi-thin plastic sections or paraffin sections by point counting technique to compare the volume fraction of macrophages, granulomas and endothelial cells in biopsies of the pseudosynovial membrane. RESULTS: The median macrophage volume fractions for polyethylene-on-zirconia bearing material (n = 15), CoCr-on-CoCr (n = 9), and alumina-on-alumina (n = 11) were 0.02, 0.04, and 0.004, respectively. The median granuloma volume fractions for polyethylene-on-zirconia (n = 13), CoCr-on-CoCr (n = 9), and alumina-on-alumina (n = 13) were 0.02, 0.04, and 0.02, respectively. The median endothelial cell volume fractions for polyethylene-on-zirconia (n = 15), CoCr-on-CoCr (n = 9), and alumina-on-alumina (n = 11) were 0.03, 0.02, and 0.05, respectively. Statistical analysis showed no significant differences between the three groups with the different bearings with respect to volume fraction of macrophages, granulomas and endothelial cells. INTERPRETATION: Our study demonstrated that a granulomatous inflammation is a common finding in non-loose implants as early as 1 year after the operation not demonstrating a difference in macrophages and granuloma formation with the various bearing materials. Thus a high volume fraction of macrophages was found in the osteoarthritis control group compared to the operated group.

Epidermal growth factor receptor mutation type III transfected into a small cell lung cancer cell line is predominantly localized at the cell surface and enhances the malignant phenotype.

In the present study we transfected the epidermal growth factor receptor (EGFR)-negative small cell lung cancer cell line, GLC3, with the type III EGFR mutation (EGFRvIII). The EGFRvIII protein could be detected by Western blot analysis as a 145-kDa protein, which by immunohistochemistry appeared to be localized at the cell surface. Ultrastructurally EGFRvIII was expressed mainly at the cell surface with clusters at cell-cell contacts. In the in vitro invasion assay, GLC3-EGFRvIII cells had a approximately 5-fold increased invasion compared with uninduced GLC3-EGFRvIII, GLC3-Tet-On and the parental cell line. GLC3-Tet-On appeared uniform in size with adherence junctions at cell-cell contacts. In uninduced GLC3-EGFRvIII cells adherence junctions were also present but less distinct. In doxycycline-pretreated GLC3-EGFRvIII cells, adherence junctions were absent. We conclude that the expression of EGFRvIII results in a more malignant phenotype. This effect appears to involve the disruption of adherence junctions.

Calcium and calpain as key mediators of apoptosis-like death induced by vitamin D compounds in breast cancer cells.

The active form of vitamin D(3) (1,25(OH)(2)D(3)) induces an increase in the intracellular free calcium ([Ca(2+)](i)) and caspase-independent cell death in human breast cancer cells. Here we show that the treatment of MCF-7 breast cancer cells with 1,25(OH)(2)D(3) or its chemotherapeutic analog, EB 1089, releases Ca(2+) from the endoplasmic reticulum. The increase in [Ca(2+)](i) was associated with the activation of a calcium-dependent cysteine protease, mu-calpain. Interestingly, ectopic expression of a calcium-binding protein, calbindin-D(28k), in MCF-7 cells not only attenuated the elevation in [Ca(2+)](i) and calpain activation, but also reduced death triggered by vitamin D compounds. Similarly, the inhibition of calpain activity by structurally unrelated chemical inhibitors increased the survival of the cells and reduces the amount of annexin V-positive cells. Despite the complete absence of effector caspase activation, transmission electron microscopy of MCF-7 cells treated with 1,25(OH)(2)D(3) or EB 1089 revealed apoptosis-like morphology characterized by the condensed cytoplasm, nuclei, and chromatin. Overall, these results suggest that calpain may take over the role of the major execution protease in apoptosis-like death induced by vitamin D compounds. Thus, these compounds may prove useful in the treatment of tumors resistant to therapeutic agents dependent on the classical caspase cascade.