Mitochondrial release of caspase-2 and -9 during the apoptotic process.

The barrier function of mitochondrial membranes is perturbed early during the apoptotic process. Here we show that the mitochondria contain a caspase-like enzymatic activity cleaving the caspase substrate Z-VAD.afc, in addition to three biological activities previously suggested to participate in the apoptotic process: (a) cytochrome c; (b) an apoptosis-inducing factor (AIF) which causes isolated nuclei to undergo apoptosis in vitro; and (c) a DNAse activity. All of these factors, which are biochemically distinct, are released upon opening of the permeability transition (PT) pore in a coordinate, Bcl-2-inhibitable fashion. Caspase inhibitors fully neutralize the Z-VAD.afc-cleaving activity, have a limited effect on the AIF activity, and have no effect at all on the DNase activities. Purification of proteins reacting with the biotinylated caspase substrate Z-VAD, immunodetection, and immunodepletion experiments reveal the presence of procaspase-2 and -9 in mitochondria. Upon induction of PT pore opening, these procaspases are released from purified mitochondria and become activated. Similarly, upon induction of apoptosis, both procaspases redistribute from the mitochondrion to the cytosol and are processed to generate enzymatically active caspases. This redistribution is inhibited by Bcl-2. Recombinant caspase-2 and -9 suffice to provoke full-blown apoptosis upon microinjection into cells. Altogether, these data suggest that caspase-2 and -9 zymogens are essentially localized in mitochondria and that the disruption of the outer mitochondrial membrane occurring early during apoptosis may be critical for their subcellular redistribution and activation.

The permeability transition pore complex: a target for apoptosis regulation by caspases and bcl-2-related proteins.

Early in programmed cell death (apoptosis), mitochondrial membrane permeability increases. This is at least in part due to opening of the permeability transition (PT) pore, a multiprotein complex built up at the contact site between the inner and the outer mitochondrial membranes. The PT pore has been previously implicated in clinically relevant massive cell death induced by toxins, anoxia, reactive oxygen species, and calcium overload. Here we show that PT pore complexes reconstituted in liposomes exhibit a functional behavior comparable with that of the natural PT pore present in intact mitochondria. The PT pore complex is regulated by thiol-reactive agents, calcium, cyclophilin D ligands (cyclosporin A and a nonimmunosuppressive cyclosporin A derivative), ligands of the adenine nucleotide translocator, apoptosis-related endoproteases (caspases), and Bcl-2-like proteins. Although calcium, prooxidants, and several recombinant caspases (caspases 1, 2, 3, 4, and 6) enhance the permeability of PT pore-containing liposomes, recombinant Bcl-2 or Bcl-XL augment the resistance of the reconstituted PT pore complex to pore opening. Mutated Bcl-2 proteins that have lost their cytoprotective potential also lose their PT modulatory capacity. In conclusion, the PT pore complex may constitute a crossroad of apoptosis regulation by caspases and members of the Bcl-2 family.

Detection of Clostridium tyrobutyricum spores using polyclonal antibodies and flow cytometry.

AIMS: The present work investigates the feasibility of using flow cytometry (FCM) combined with fluorescent-labelled specific polyclonal antibodies for the detection and presumptive identification of Clostridium tyrobutyricum spores in bovine milk. METHODS AND RESULTS: Two fluorescent molecules (fluorescein isothiocyanate and Alexa Fluor 488) were conjugated to antispores polyclonal antibodies. Side scatter and forward scatter profiles of the Cl. tyrobutyricum spores marked with fluorescent antibodies permitted the detection of spores and differentiated them from other related microbial species. The detection limit of this method was 10(3) spores per 100 ml of milk, and results could be achieved in 2 h. CONCLUSIONS: FCM combined with fluorochrome-conjugated antibodies, especially Alexa Fluor, could be an efficacious means to detect and provide presumptive identification of Cl. tyrobutyricum spores, as well as differentiation from other Clostridium species that can also cause late blowing in cheese. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes the basis for the development of a method suitable for analysis of milk destined for cheese manufacture that would permit the detection of Cl. tyrobutyricum spores in a short period. This would enable the industry to use contaminated milk for dairy products other than cheese where Cl. tyrobutyricum does not cause a problem.

Bax and adenine nucleotide translocator cooperate in the mitochondrial control of apoptosis.

The proapoptotic Bax protein induces cell death by acting on mitochondria. Bax binds to the permeability transition pore complex (PTPC), a composite proteaceous channel that is involved in the regulation of mitochondrial membrane permeability. Immunodepletion of Bax from PTPC or purification of PTPC from Bax-deficient mice yielded a PTPC that could not permeabilize membranes in response to atractyloside, a proapoptotic ligand of the adenine nucleotide translocator (ANT). Bax and ANT coimmunoprecipitated and interacted in the yeast two-hybrid system. Ectopic expression of Bax induced cell death in wild-type but not in ANT-deficient yeast. Recombinant Bax and purified ANT, but neither of them alone, efficiently formed atractyloside-responsive channels in artificial membranes. Hence, the proapoptotic molecule Bax and the constitutive mitochondrial protein ANT cooperate within the PTPC to increase mitochondrial membrane permeability and to trigger cell death.

[Eating habits in physical therapy students]. / Hábitos dietéticos en estudiantes de fisioterapia.

OBJECTIVE: a) To know the eating and exercising habits of undergraduate Physical Therapy students; b) To promote awareness among these future health professionals and educators of the need to foster adequate eating habits among patients and the general population. METHODS: A dietary and physical activity questionnaire, regarding a seven-day period, distributed among students of the School of Physical Therapy (University of Valladolid) in the city of Soria, Spain (n = 131; sex (m/f): 38/93). RESULTS: Results show that a high percentage of the subjects follow the recommendations of the new Nutritional Pyramid of the Spanish Nutrition Society (SEN) regarding intake of meat, fish, milk, dairy products and exercise habits. This is not the case, however, for products such as pasta, bread and cereals, vegetables, fruits, legumes and olive oil. Furthermore, fast food is taken, on average, once a week despite the recommended sporadic monthly intakes. CONCLUSIONS: According to results, formative programmes should be carried out to enable these students to encourage healthy diets. Thus, diseases related to non-healthy eating habits could be prevented, and information on the issue could be spread among the population.

[Prevalence of osteoporosis assessed by quantitative ultrasound calcaneus measurements in institutionalized elderly population]. / Prevalencia de osteoporosis en ancianos institucionalizados, mediante ultrasonografía de calcáneo.

OBJECTIVES: To analyze using quantitative ultrasound of calcaneus (QUS) the prevalence of osteoporosis in institutionalized elderly people, in order to contribute to state reliable criteria (T-score units) for the diagnosis of osteoporosis which allow advances in bone fracture prevention. MATERIAL AND METHODS: Elderly people (n=171) were divided into separate groups according to sex and age criteria (three decades, from 70 to 90 years) and analyzed using QUS. RESULTS: Both globally and in the younger groups, women showed significantly lower values than men in all densitometry variables (p < 0.001). In the oldest group only T-score and BUA showed statistical differences (p = 0.039 y p = 0.025, respectively). The prevalence of osteoporosis in women was higher than in men whichever criteria were applied in all age groups. Applying the WHO criteria with QUS, the prevalence of osteoporosis in global population is close to that stated by DEXA using the same criteria. CONCLUSIONS: QUS could be useful to assess the bone mass evolution with age and for the diagnosis and monitoring of osteoporosis. In our elderly population, the WHO criteria for DEXA, are also the most suitable ones for QUS utilization.

Subcellular and submitochondrial mode of action of Bcl-2-like oncoproteins.

Bcl-2 is the prototype of a class of oncogenes which regulates apoptosis. Bcl-2-related gene products with either death-promoting and death-inhibitory activity are critically involved in numerous disease states and thus constitute prime targets for therapeutic interventions. The relative amount of death agonists and antagonists from the Bcl-2 family constitutes a regulatory rheostat whose function is determined, at least in part, by selective protein-protein interactions. Bcl-2 and its homologs insert into intracellular membranes including mitochondria, the endoplasmatic reticulum and the nuclear envelope. Many of the molecular genetic, ultrastructural, crystallographic and functional studies suggest that Bcl-2-related molecules exert their apoptosis-regulatory effects via regulating mitochondrial alterations preceding the activation of apoptogenic proteases and nucleases. Via a direct effect on mitochondrial membranes, Bcl-2 prevents all hallmarks of the early stage of apoptosis including disruption of the inner mitochondrial transmembrane potential and the release of apoptogenic protease activators from mitochondria. The mitochondrial permeability transition (PT) pore, also called mitochondrial megachannel or multiple conductance channel, is a multiprotein complex formed at the contact site between the mitochondrial inner and outer membranes, exactly at the same localization at which Bax, Bcl-2, and Bcl-XL are particularly abundant. The PT pore participates in the regulation of matrix Ca2+, pH, deltapsim, and volume and functions as a Ca2+-, voltage-, pH-, and redox-gated channel with several levels of conductance and little if any ion selectivity. Experiments involving the purified PT pore complex indicate that Bax, Bcl-2, and Bcl-XL exert at least part of their apoptosis-regulatory function by facilitating (Bax) or inhibiting (Bcl-2, Bcl-XL) PT pore opening. These findings clarify the principal (but not exclusive) mechanism of Bcl-2-mediated cytoprotection.

The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death.

Mitochondrial alterations including permeability transition (PT) constitute critical events of the apoptotic cascade and are under the control of Bcl-2 related gene products. Here we show that induction of PT is sufficient to activate CPP32-like proteases with DEVDase activity and the associated cleavage of the nuclear DEVDase substrate poly(ADP-ribose) polymerase (PARP). Thus, direct intervention on mitochondria using a ligand of the mitochondrial benzodiazepin receptor or a protonophore causes DEVDase activation. In addition, the DEVDase activation triggered by conventional apoptosis inducers (glucocorticoids or topoisomerase inhibitors) is prevented by inhibitors of PT. The protease inhibitor N-benzyloxycabonyl-Val-Ala-Asp-fluoromethylketone (Z-VAD.fmk) completely prevents the activation of DEVDase and PARP cleavage, as well as the manifestation of nuclear apoptosis (chromatin condensation, DNA fragmentation, hypoploidy). In addition, Z-VAD.fmk delays the manifestation of apoptosis-associated changes in cellular redox potentials (hypergeneration of superoxide anion, oxidation of compounds of the inner mitochondrial membrane, depletion of non-oxidized glutathione), as well as the exposure of phosphatidylserine residues in the outer plasma membrane leaflet. Although Z-VAD.fmk retards cytolysis, it is incapable of preventing disruption of the plasma membrane during protracted cell culture (12-24 h), even in conditions in which it completely blocks nuclear apoptosis (chromatin condensation and DNA fragmentation). Electron microscopic analysis confirms that cells treated with PT inducers alone undergo apoptosis, whereas cells kept in identical conditions in the presence of Z-VAD.fmk die from necrosis. These observations are compatible with the hypothesis that PT would be a rate limiting step in both the apoptotic and the necrotic modes of cell death. In contrast, it would be the availability of apoptogenic proteases that would determine the choice between the two death modalities.

The thiol crosslinking agent diamide overcomes the apoptosis-inhibitory effect of Bcl-2 by enforcing mitochondrial permeability transition.

In several different cell lines, Bcl-2 prevents the induction of apoptosis (DNA fragmentation, PARP cleavage, phosphatidylserine exposure) by the pro-oxidant ter-butylhydroperoxide (t-BHP) but has no cytoprotective effect when apoptosis is induced by the thiol crosslinking agent diazenedicarboxylic acid his 5N,N-dimethylamide (diamide). Both t-BHP and diamide cause a disruption of the mitochondrial transmembrane potential delta psi(m) that is not inhibited by the broad spectrum caspase inhibitor z-VAD.fmk, although z-VAD.fmk does prevent nuclear DNA fragmentation and poly(ADP-ribose) polymerase cleavage in these models. Bcl-2 stabilizes the delta psi(m) of t-BHP-treated cells but has no inhibitory effect on the delta psi(m) collapse induced by diamide. As compared to normal controls, isolated mitochondria from Bcl-2 overexpressing cells are relatively resistant to the induction of delta psi(m) disruption by t-BHP in vitro. Such Bcl-2 overexpressing mitochondria also fail to release apoptosis-inducing factor (AIF) and cytochrome c from the intermembrane space, whereas control mitochondria not overexpressing Bcl-2 do liberate AIF and cytochrome c in response to t-BHP. In contrast, Bcl-2 does not confer protection against diamide-triggered delta psi(m) collapse and the release of AIF and cytochrome c. This indicates that Bcl-2 suppresses the permeability transition (PT) and the associated release of intermembrane proteins induced by t-BHP but not by diamide. To further investigate the mode of action of Bcl-2, semi-purified PT pore complexes were reconstituted in liposomes in a cell-free, organelle-free system. Recombinant Bcl-2 or Bcl-X(L) proteins augment the resistance of reconstituted PT pore complexes to pore opening induced by t-BHP. In contrast, mutated Bcl-2 proteins which have lost their cytoprotective potential also lose their PT-modulatory capacity. Again, Bcl-2 fails to confer protection against diamide in this experimental system. The reconstituted PT pore complex itself cannot release cytochrome c encapsulated into liposomes. Altogether these data suggest that pro-oxidants, thiol-reactive agents, and Bcl-2 can regulate the PT pore complex in a direct fashion, independently from their effects on cytochrome c. Furthermore, our results suggest a strategy for inducing apoptosis in cells overexpressing apoptosis-inhibitory Bcl-2 analogs.

Bcl-2 and Bax regulate the channel activity of the mitochondrial adenine nucleotide translocator.

Bcl-2 family protein including anti-apoptotic (Bcl-2) or pro-apoptotic (Bax) members can form ion channels when incorporated into synthetic lipid bilayers. This contrasts with the observation that Bcl-2 stabilizes the mitochondrial membrane barrier function and inhibits the permeability transition pore complex (PTPC). Here we provide experimental data which may explain this apparent paradox. Bax and adenine nucleotide translocator (ANT), the most abundant inner mitochondrial membrane protein, can interact in artificial lipid bilayers to yield an efficient composite channel whose electrophysiological properties differ quantitatively and qualitatively from the channels formed by Bax or ANT alone. The formation of this composite channel can be observed in conditions in which Bax protein alone has no detectable channel activity. Cooperative channel formation by Bax and ANT is stimulated by the ANT ligand atractyloside (Atr) but inhibited by ATP, indicating that it depends on the conformation of ANT. In contrast to the combination of Bax and ANT, ANT does not form active channels when incorporated into membranes with Bcl-2. Rather, ANT and Bcl-2 exhibit mutual inhibition of channel formation. Bcl-2 prevents channel formation by Atr-treated ANT and neutralizes the cooperation between Bax and ANT. Our data are compatible with a ménage à trois model of mitochondrial apoptosis regulation in which ANT, the likely pore forming protein within the PTPC, interacts with Bax or Bcl-2 which influence its pore forming potential in opposing manners.

Lonidamine triggers apoptosis via a direct, Bcl-2-inhibited effect on the mitochondrial permeability transition pore.

The molecular mode of action of lonidamine, a therapeutic agent employed in cancer chemotherapy, has been elusive. Here we provide evidence that lonidamine (LND) acts on mitochondria to induce apoptosis. LND provokes a disruption of the mitochondrial transmembrane potential which precedes signs of nuclear apoptosis and cytolysis. The mitochondrial and cytocidal effects of LND are not prevented by inhibitors of caspases or of mRNA or protein synthesis. However, they are prevented by transfection-enforced overexpression of Bcl-2, an oncoprotein which inhibits apoptosis by stabilizing the mitochondrial membrane barrier function. Accordingly, the cell death-inducing effect of LND is amplified by simultaneous addition of PK11195, an isoquinoline ligand of the peripheral benzodiazepine receptor which antagonizes the cytoprotective effect of Bcl-2. When added to isolated nuclei, LND fails to provoke DNA degradation unless mitochondria are added simultaneously. In isolated mitochondria, LND causes the dissipation of the mitochondrial inner transmembrane potential and the release of apoptogenic factors capable of inducing nuclear apoptosis in vitro. Thus the mitochondrion is the subcellular target of LND. All effects of LND on isolated mitochondria are counteracted by cyclosporin A, an inhibitor of the mitochondrial PT pore. We therefore tested the effect of LND on the purified PT pore reconstituted into liposomes. LND permeabilizes liposomal membranes containing the PT pore. This effect is prevented by addition of recombinant Bcl-2 protein but not by a mutant Bcl-2 protein that has lost its apoptosis-inhibitory function. Altogether these data indicate that LND represents a novel type of anti-cancer agent which induces apoptosis via a direct effect on the mitochondrial PT pore.

Biosynthesis of docosahexaenoic acid in human cells: evidence that two different delta 6-desaturase activities may exist.

It has been proposed that synthesis of docosahexaenoic acid (22:6(n-3) in rat hepatocytes occurs by a route independent of delta 4-desaturase, which involves delta 6-desaturation and retroconversion (Voss A., Reinhart M., Sankarappa S. and Sprecher H. (1991) J. Biol. Chem. 266, 19995-20000). However, most cells exhibit these enzymatic activities and nevertheless synthesize low to undectectable amounts of 22:6(n-3). Moreover, there are few data on the occurrence of this pathway in human cells. In the present work, we have analysed the biosynthetic pathway of 22:6(n-3) in human Y-79 retinoblastoma and Jurkat T-cells. Y-79 cells were supplemented with 18:3(n-3) and 20:5(n-3) or incubated with [1-14C]18:3(n-3) and [1-14C]20:5(n-3) and lipids analysed by argentation TLC, reverse-phase TLC and GLC-mass spectrometry. Pulse-chase experiments revealed that synthesis of 22:6(n-3) from 20:5(n-3) in Y-79 cells occurred through two successive elongations, followed by a delta 6-desaturation of 24:5(n-3) to 24:6(n-3) and retroconversion to 22:6(n-3). Incubation of Y-79 cells with [1-14C]18:3(n-3) in medium containing 50 microM trans-9,12-18:2, a potent inhibitor of delta 6-desaturase, caused a reduction of 22:6(n-3) synthesis mainly by interfering with the desaturation of 18:3(n-3). However, when [1-14C]20:5(n-3) was used as precursor, synthesis of 22:6(n-3) was depressed to a lesser extent and mainly by reduction of 24:6(n-3) retroconversion. Neuronal differentiation of Y-79 cells caused a great increase in delta 6-desaturase activity on 18:3(n-3), though the amount of 22:6(n-3) synthesized did not change or diminish, suggesting the existence of a particular delta 6-desaturase involved in the synthesis of 22:6(n-3). The existence of a distinctive delta 6-desaturase activity could also explain why Jurkat cells growing in serum-free medium showed a near 3-fold increase in the synthesis of pentaenes from 18:3(n-3) and, at the same time, a large decrease in the synthesis of 22:6(n-3). The verification of the involvement of two delta 6-desaturase activities in 22:6(n-3) synthesis would have important implications for the formulation of the nutritional requirements of this fatty acid during development.

Biosynthesis of unsaturated fatty acids in the main cell lineages of human leukemia and lymphoma.

Unsaturated fatty acids are essential for the proliferation of many haematopoietic cells, but little is known about their biosynthetic pathways in these cells. We have studied the activity of the main desaturation-elongation enzymes in human B-(Reh-6, Raji, Ramos) and T-(CEM, Jurkat) lymphocytic, promonocytic (U937), promyelocytic (HL-60) and pluripotent myeloid (K562) cell lineages, as well as the changes induced by cell differentiation. Cells were incubated with 14C-labelled 18:0, 18:2(n - 6) and 18:3(n - 3) or supplemented with the corresponding unlabelled fatty acid and synthesis of polyunsaturated fatty acids (PUFA) was evaluated by argentation-TLC and GLC. The main activity present in most cells was delta 9-desaturase (range between 200-1000 pmol/24 h per 10(6) cells) that was regulated by the type of free fatty acids in culture media. A great variability in the activities of delta 6- and delta 5-desaturase was observed. They were virtually absent in B-cells and only one (Jurkat) T-cell line synthesized significant amounts of (n - 6) and (n - 3) PUFA. The main PUFA formed by Jurkat cells were 20:3 and 20:4(n - 6) (30 and 40%, respectively, of cell lipid radioactivity) and 20:5, 22:5 and 22:6(n - 3) (60, 20 and 10%, respectively, of cell radioactivity). Cell differentiation caused complex changes in desaturase activities. The activity of delta 9-desaturase increased with the degree of differentiation of B-cells. Differentiation of U937 cells to macrophages with PMA caused a 2-3-fold increase in the activity of (delta 6 + delta 5)- and delta 9-desaturases and no changes and a 2-fold decrease, respectively, if the inducer was DMSO. Differentiation of HL-60 cells to granulocytes with DMSO virtually abolished delta 9-desaturase activity and greatly reduced that of delta 6- and delta 5-desaturases. delta 9-Desaturase activity increased (2.5-fold) in myeloid K562 cells differentiated to erythroblasts with hemin. No induction of delta 6-desaturase, absent in K562 cells, occurred after differentiation to erythroblasts or megakaryoblasts and they synthesized alternative PUFA through sequential elongation and delta 5-desaturation of 18:2(n - 6) and 18:3(n - 3). The activities of delta 6- and delta 5-desaturase in HL-60 and U937 cells increased when differentiation also stimulated the synthesis of eicosanoids and extracellular release of PUFA.

Farnesyltransferase inhibitor BMS-214662 induces apoptosis in B-cell chronic lymphocytic leukemia cells.

B-cell chronic lymphocytic leukemia (B-CLL) cells develop resistance to nucleoside analogs over time. This chemoresistance may be caused by selection for B-CLL cells with defects in the particular apoptosis pathway triggered by these drugs. Therefore, anticancer agents that induce apoptosis through alternative pathways might be useful in treating chemoresistant B-CLL. Farnesyltransferase inhibitors (FTIs) are a class of synthetic drugs with definite molecular targets, which have demonstrated cytotoxicity against leukemic cell lines. We have studied the ex vivo effect of the FTI BMS-214662 on cells from 18 patients with B-CLL. Low concentrations (<1 microM) of BMS-214662 prevented farnesylation of the chaperone marker HDJ-2 and had no effect on Akt activation. BMS-214662 induced apoptosis in B-CLL cells from all patients studied, including those showing resistance to cladribine and fludarabine ex vivo and in vivo. Treatment with BMS-214662 induced loss of mitochondrial membrane potential (DeltaPsi(m)), phosphatidylserine exposure, proapoptotic conformational changes of Bax and Bak, reduction in Mcl-1 levels and activation of caspases 9 and 3. The general caspase inhibitor Z-VAD-fmk did not prevent BMS-214662-induced cell death. These results indicate that BMS-214662 may be a useful drug for treating B-CLL and, in particular, an alternative for the therapy of purine analog-resistant or relapsed B-CLL.

Role of caspases and apoptosis-inducing factor (AIF) in cladribine-induced apoptosis of B cell chronic lymphocytic leukemia.

We have evaluated the role of caspases and the mitochondrial apoptosis inducing-factor (AIF) in apoptosis induced by cladribine (2CdA), in vitro, in cells from patients of B-CLL and in peripheral blood lymphocytes from normal donors. In sensitive B-CLL cells, apoptosis was characterized by cell shrinking, loss of mitochondrial membrane potential (DeltaPsi(m)), phosphatidylserine exposure, activation of caspases 3, 7, 8 and 9, reduction of Mcl-1 levels, translocation of AIF from mitochondria to nucleus and chromatin condensation. No significant variations in the levels of Bcl-2, Bax and Bak proteins were noticed upon treatment with 2CdA. Co-treatment of cells with the pan-caspase inhibitor Z-VAD-fmk attenuated some morphological and biochemical characteristics of apoptosis and delayed 2CdA-induced DeltaPsi(m) loss, but did not prevent cell death. Z-VAD-fmk did not prevent 2CdA-induced AIF translocation but in this case apoptotic cells displayed only peripheral chromatin condensation, characteristic of AIF action. Reduced or negligible caspase 3 expression did not prevent 2CdA toxicity in cells from four patients. Cells from three patients that responded poorly to 2CdA lacked expression of caspases 9 or 3. Cells from another patient resistant to 2CdA expressed caspases 3, 7, 8 and 9 but they were not activated by treatment. These results indicate that execution of apoptosis is carried out independently by AIF and caspases, which are responsible for the development of apoptotic phenotype in response to 2CdA. Although caspases can also collaborate in DeltaPsi(m) loss, proapoptotic proteins from the Bcl-2 superfamily may be the key inducers of DeltaPsi(m) loss and apoptosis in B-CLL cells sensitive to 2CdA.

Two death pathways induced by sorafenib in myeloma cells: Puma-mediated apoptosis and necroptosis.

PURPOSE: Sorafenib is a multikinase inhibitor that targets the MAPK pathway and is currently used for the treatment of hepatocellular and renal carcinoma. Recently, it has been shown that sorafenib is also cytotoxic to multiple myeloma (MM) cells. Here, we have further analyzed the mechanism of sorafenib-induced death in MM cells. METHODS: Cell death induced by sorafenib in MM cell lines and in plasma cells from MM patients was evaluated by analysis of gene expression by RT-MLPA and quantitative PCR, protein levels and functionality by Western blot and flow cytometry and gene silencing with siRNA. RESULTS: Cell death was characterized by phosphatidylserine exposure, ΔΨm loss, cytochrome c release and caspase activation, hallmarks of apoptosis. DL50 at 24 h ranged from 6 to 10 µM. Ex vivo treatment with 20 µM sorafenib induced apoptosis in around 80 % myeloma cells from six multiple myeloma patients. Sorafenib induced caspase-dependent degradation of Bcl-xL and Mcl-1 proteins, destabilizing the mitochondria and speeding up the development of apoptosis. Sorafenib treatment increased levels of Puma at mRNA and protein level and gene silencing with siRNA confirmed a relevant role for Puma in the induction of apoptosis. Co-treatment with the pan-caspase inhibitor Z-VAD-fmk prevented cell death to a variable degree depending on the cell line. In RPMI 8226 cells, Z-VAD-fmk prevented most of sorafenib-induced death. However, death in MM.1S was only prevented by co-incubation with both Z-VAD-fmk and the RIP1K inhibitor necrostatin-1, indicating that under conditions of inefficient caspase activation, sorafenib induces death by necroptosis. CONCLUSION: Our results demonstrate a key role for Puma in the triggering of sorafenib-induced apoptosis and that this drug can also induce death by necroptosis in multiple myeloma cells.

Immunotherapy with liposome-bound TRAIL overcomes partial protection to soluble TRAIL-induced apoptosis offered by down-regulation of Bim in leukemic cells.

PURPOSE: Human Apo2-Ligand/TRAIL secreted by natural killer cells and cytotoxic T lymphocytes plays an important role immunosurveillance controlling tumor growth and metastasis. Moreover, the fact that Apo2L/TRAIL is capable of inducing cell death in tumor cells but not in normal cells makes this death ligand a promising anti-tumor agent. Previous data from our group demonstrated that Apo2L/TRAIL was physiologically released as transmembrane protein inserted in lipid vesicles, called exosomes. Recently, we demonstrated that artificial lipid nanoparticles coated with bioactive Apo2L/TRAIL (LUV-TRAIL) resembling the natural exosomes, greatly improved Apo2L/TRAIL activity and were able to induce apoptosis in hematological malignancies. In this study, we have deepened in the underlying mechanism of action of LUV-TRAIL in hematologic cells. METHODS/PATIENTS: Cytotoxic ability of LUV-TRAIL was assessed on Jurkat cells either over-expressing the anti-apoptotic protein Mcl1 or down-regulating the pro-apoptotic protein Bim previously generated in our laboratory. We also tested LUV-TRAIL cytotoxic ability against primary human leukemic cells from T-cell ALL patient. RESULTS: Silencing Bim but not Mcl-1 over-expression partially protects Jurkat cells from apoptosis induced by sTRAIL. LUV-TRAIL induced caspase-8 and caspase-3 activation and killed Jurkat-Mcl1 and Jurkat-shBim more efficiently than sTRAIL independently of the mitochondrial pathway. On the other hand, LUV-TRAIL were clearly more cytotoxic against primary leukemic cells from a T-cell ALL patient than sTRAIL. CONCLUSION: Tethering Apo2L/TRAIL to the surface of lipid nanoparticles greatly increases its bioactivity and could be of potential use in anti-tumor therapeutics.

CPP32 inhibition prevents Fas-induced ceramide generation and apoptosis in human cells.

Intracellular activation of sphingomyelinase, leading to ceramide generation, and ICE-like proteases have been implicated in TNF and Fas-induced apoptosis, but the links between these intracellular apoptotic mediators remain undefined. We show here that a specific peptide inhibitor of the ICE-like protease CPP32/Yama (DEVD-CHO) blocks anti-Fas-induced apoptosis in Jurkat and U937 cells, while having no effect on TNF-induced apoptosis in U937 cells. This peptide also prevents ceramide accumulation induced by Fas engagement. Jurkat and U937 cells, as well as their mtDNA-depleted derived lines (rho degree cells), were sensitive to ceramide toxicity, which was not prevented by ICE-like protease inhibitors. These results, taken together, suggest that ICE-like protease activation is a prerequisite for ceramide generation and subsequent apoptosis, at least in the case of Fas-induced cell death.

mtDNA-depleted U937 cells are sensitive to TNF and Fas-mediated cytotoxicity.

It has been proposed that TNF cytotoxicity is mediated by reactive oxygen intermediates generated by uncoupling of mitochondrial respiration. We have compared sensitive U937 cells and derived cell lines depleted of mtDNA for their ability to undergo TNF- and Fas-induced apoptosis. Cells lacking around 98% of mtDNA were still sensitive to TNF-induced apoptosis. U937 cells devoid of mtDNA (U937-rho degree) were resistant to TNF, but this was due to the loss of its 55 kDa receptor. U937-rho degree cells were also resistant to docosahexaenoic acid, which causes U937 cell death by lipid peroxidation. These cells were sensitive to anti-Fas toxicity. The results indicate that TNF and Fas-induced toxicity occurs by a mechanism mostly independent of mitochondrial free radical generation.

Nitric oxide induces apoptosis via triggering mitochondrial permeability transition.

Nitric oxide (NO) induces apoptosis in thymocytes, peripheral T cells, myeloid cells and neurons. Here we show that NO is highly efficient in inducing mitochondrial permeability transition, thereby causing the liberation of apoptogenic factors from mitochondria which can induce nuclear apoptosis (DNA condensation and DNA fragmentation) in isolated nuclei in vitro. In intact thymocytes, NO triggers disruption of the mitochondrial transmembrane potential, followed by hypergeneration of reactive oxygen species, exposure of phosphatidyl serine on the outer plasma membrane leaflet, and nuclear apoptosis. Inhibitors of mitochondrial permeability transition such as bongkrekic acid and a cyclophilin D-binding cyclosporin A derivative, N-methyl-Val-4-cyclosporin A, prevent the mitochondrial as well as all post-mitochondrial signs of apoptosis induced by NO including nuclear DNA fragmentation and exposure of phosphatidylserine residues on the cell surface. These findings indicate that NO can cause apoptosis via triggering of permeability transition.