Emerging roles of caspase-3 in apoptosis.
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Caspases are crucial mediators of programmed cell death (apoptosis). Among them, caspase-3 is a frequently activated death protease, catalyzing the specific cleavage of many key cellular proteins. However, the specific requirements of this (or any other) caspase in apoptosis have remained largely unknown until now. Pathways to caspase-3 activation have been identified that are either dependent on or independent of mitochondrial cytochrome c release and caspase-9 function. Caspase-3 is essential for normal brain development and is important or essential in other apoptotic scenarios in a remarkable tissue-, cell type- or death stimulus-specific manner. Caspase-3 is also required for some typical hallmarks of apoptosis, and is indispensable for apoptotic chromatin condensation and DNA fragmentation in all cell types examined. Thus, caspase-3 is essential for certain processes associated with the dismantling of the cell and the formation of apoptotic bodies, but it may also function before or at the stage when commitment to loss of cell viability is made. (+info)
On the complexities of ceramide changes in cells undergoing apoptosis: lack of evidence for a second messenger function in apoptotic induction.
(50/8107)
The generation of cellular ceramides as a second messenger has been implicated as a regulatory and required step for the induction of apoptosis. In this study, we have applied a recently developed mass spectrometric technique to the determination of changes in physiological ceramide levels during apoptosis induced by tumor necrosis factor plus cycloheximide in U937 cells and the chemical agents anisomycin or geranylgeraniol in HL-60 cells. The mass spectrometric method has significant advantages over traditional methods for ceramide quantitation in that it determines the relative abundance of all ceramide species present in complex biological lipid mixtures individually and simultaneously. We quantitiated ceramides ranging from C14 to C26, finding that their basal levels and relative distribution varied significantly, both within and between different cell types. However, we were not able to detect any significant changes in either total ceramide content or species distribution until 1 h or more post-stimulation with any of these treatments, by which time the cells were in an advanced stage of apoptosis. Differences were also seen between all three treatments in the ceramide species distribution observed in these late stages of apoptosis. These data indicate that in vivo ceramide generation occurs as a consequence of apoptosis rather than as an essential second messenger involved in its induction. They also pose new questions about the potential roles that certain ceramide species may play in the late stages of apoptosis, and demonstrate a clear need to utilize the resolving power of mass spectrometry-based assays in any future investigations into the biological function of ceramides. (+info)
Inhibition of transforming growth factor beta1-induced hepatoma cell apoptosis by liver tumor promoters: characterization of primary signaling events and effects on CPP32-like caspase activity.
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The effects of the liver tumor promoters phenobarbital, clofibrate, dieldrin, and DDT on transforming growth factor-beta1 (TGFbeta)-induced apoptosis were studied in FTO-2B hepatoma cells. Inhibition of apoptosis by these compounds was strongly correlated with a decrease in CPP32-like caspase activity. Similar effects were obtained with insulin and dexamethasone. CPP32-like activity may thus provide a useful tool for quantiation of apoptosis under various treatment conditions. Diverse effects on apoptosis-associated cellular signaling proteins were observed: insulin led to an activation of the MAP kinases ERK1/2, of PKB/Akt and of NF-kappaB, phenobarbital and clofibrate enhanced NF-kappaB activity solely, while dexamethasone slightly enhanced NF-kappaB activity and increased the expression of Bcl-xL. Since inhibition of apoptosis was still detectable if the anti-apoptotic compounds were administered more than 10 h after TGFbeta, the diverse primary signals appear to converge at a presumably late stage of apoptosis, but upstream of activation of CPP32 or related caspases. (+info)
Differential involvement of DNases in HeLa cell apoptosis induced by etoposide and long term-culture.
(52/8107)
We have applied to human HeLa cells two different stimuli of apoptosis: the antitumoral drug etoposide, and a more 'physiological' death condition, obtained by growing cells in the same medium for long time periods, for up to 10 days. Analysis of different parameters demonstrated that in both experimental systems the same apoptotic features are visible. However, the DNA degradation pattern appeared to be different, suggesting the involvement of different DNases. In this view, we have analyzed the activity and expression of Ca2+-Mg2+-dependent and acid DNases. We have observed that DNase I is not modulated during apoptosis. In contrast, the acid L-DNase II (derived from Leukocyte Elastase Inhibitor by post-translational modification), recently identified in our laboratory, is mainly active in the apoptotic pathway induced by long term-culture. Furthermore, we have provided evidence that while caspase 3 is activated by both inducers, caspase 1 is essential only for the etoposide-induced apoptosis. (+info)
De novo expression of the class-A macrophage scavenger receptor conferring resistance to apoptosis in differentiated human THP-1 monocytic cells.
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The class-A macrophage scavenger receptor (MSR) is a trimeric multifunctional protein expressed selectively in differentiated monomyeloid phagocytes which mediates uptake of chemically modified lipoproteins and bacterial products. This study investigated whether MSR plays a role in the regulation of apoptosis, a model of genetically programmed cell death. De novo expression of MSR occurred in human THP-1 monocytic cells differentiated with phorbol esters, which activated a nuclear transcription factor binding to the Ap1/ets-like domain of the MSR promoter. The phorbol ester-stimulated THP-1 cells also expressed increased levels of the pro-apoptotic gene products, caspase-3 and Fas ligand, but the cells exhibited no change in apoptosis. Global activation of GTP-binding proteins with fluoride anions triggered apoptosis of THP-1 cells in a time- and concentration-dependent manner, demonstrated by nuclear shrinkage and fragmentation and internucleosomal DNA fragmentation. However, the MSR-expressing THP-1 macrophage-like cells showed a significant reduction in apoptosis compared to undifferentiated control THP-1 cells, which produce MSR at undetectable levels. Fluoride stimulation also triggered apoptosis of human Jurkat T cells. Stimulation with phorbol ester made no difference in apoptosis between treated and untreated Jurkat cells. Finally, Chinese hamster ovary (CHO) cells overexpressing the class-A MSR type I by cDNA transfection showed markedly increased resistance to G-protein-coupled apoptosis. Thus, de novo expression of MSR associated with monocyte maturation into macrophages appears to confer the resistance of macrophages to apoptotic stimulation by G-protein activation. (+info)
Activation of the CD95 (APO-1/Fas) pathway in drug- and gamma-irradiation-induced apoptosis of brain tumor cells.
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Chemotherapeutic agents and gamma-irradiation used in the treatment of brain tumors, the most common solid tumors of childhood, have been shown to act primarily by inducing apoptosis. Here, we report that activation of the CD95 pathway was involved in drug- and gamma-irradiation-induced apoptosis of medulloblastoma and glioblastoma cells. Upon treatment CD95 ligand (CD95-L) was induced that stimulated the CD95 pathway by crosslinking CD95 via an autocrine/paracrine loop. Blocking CD95-L/receptor interaction using F(ab')2 anti-CD95 antibody fragments strongly reduced apoptosis. Apoptosis depended on activation of caspases (interleukin 1beta-converting enzyme/Ced-3 like proteases) as it was almost completely abrograted by the broad range caspase inhibitor benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone. Apoptosis was mediated by cleavage of the receptor proximal caspase FLICE/MACH (caspase-8) and the downstream caspase CPP32 (caspase-3, Apopain) resulting in cleavage of the prototype caspase substrate PARP. Moreover, CD95 was upregulated in wild-type p53 cells thereby increasing responsiveness towards CD95 triggering. Since activation of the CD95 system upon treatment was also found in primary medulloblastoma cells ex vivo, these findings may have implications to define chemosensitivity and to develop novel therapeutic strategies in the management of malignant brain tumors. (+info)
Extended therapeutic window for caspase inhibition and synergy with MK-801 in the treatment of cerebral histotoxic hypoxia.
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In rats, striatal histotoxic hypoxic lesions produced by the mitochondrial toxin malonate resemble those of focal cerebral ischemia. Intrastriatal injections of malonate induced cleavage of caspase-2 beginning at 6 h, and caspase-3-like activity as identified by DEVD biotin affinity-labeling within 12 h. DEVD affinity-labeling was prevented and lesion volume reduced in transgenic mice overexpressing BCL-2 in neuronal cells. Intrastriatal injection of the tripeptide, N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (zVAD-fmk), a caspase inhibitor, at 3 h, 6 h, or 9 h after malonate injections reduced the lesion volume produced by malonate. A combination of pretreatment with the NMDA antagonist, dizocilpine (MK-801), and delayed treatment with zVAD-fmk provided synergistic protection compared with either treatment alone and extended the therapeutic window for caspase inhibition to 12 h. Treatment with cycloheximide and zVAD-fmk, but not with MK-801, blocked the malonate-induced cleavage of caspase-2. NMDA injections alone resulted in a weak caspase-2 cleavage. These results suggest that malonate toxicity induces neuronal death by more than one pathway. They strongly implicate early excitotoxicity and delayed caspase activation in neuronal loss after focal ischemic lesions and offer a new strategy for the treatment of stroke. (+info)
Crm-A, bcl-2 and NDGA inhibit CD95L-induced apoptosis of malignant glioma cells at the level of caspase 8 processing.
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Susceptibility to CD95 (Fas/APO-1)-mediated apoptosis in human glioma cells depends on CD95 expression and unknown factors that regulate signal transduction. Thus, LN-18 cells are highly sensitive to CD95 ligand (CD95L) whereas LN-229 cells require coexposure to inhibitors of RNA or protein synthesis for induction of apoptosis. Here, we report that caspase 8 and 3 activation, poly(ADP-ribose)polymerase cleavage and apoptosis are inhibited by the lipoxygenase inhibitor, nordihydroguaretic acid (NDGA), or ectopic expression of crm-A or bcl-2. CD95L-induced glioma cell apoptosis does not involve ceramide generation. Apoptosis induced by exogenous ceramide resembles CD95-mediated apoptosis in that bcl-2 is protective but differs in that NDGA and crm-A have no effect and in that cycloheximide (CHX) inhibits rather than potentiates ceramide-induced cell death. We conclude that caspase 8 and caspase 3 activation, but not ceramide generation, are required for CD95 ligand-induced apoptosis of glioma cells and that bcl-2, crm-A and NDGA all act upstream of caspases to inhibit apoptosis. (+info)