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Loss of neurons in chronic neurodegenerative diseases may occur over a period of many years. Once initiated, neuronal cell death is accompanied by distinct phenotypic changes including cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing and phosphatidylserine (PS) exposure at the plasma membrane. It is still poorly understood which events mark the point of no return for dying neurons. Here we analyzed the neuronal cell line SH-SY5Y expressing cytochrome C (Cyto.C)-GFP. Cells were exposed temporarily to ethanol (EtOH) and tracked longitudinally in time by light and fluorescent microscopy. Exposure to EtOH induced elevation of intracellular Ca 2+ and reactive oxygen species, cell shrinkage, neurite retraction, mitochondrial fragmentation, nuclear condensation, membrane blebbing, PS exposure and Cyto.C release into the cytosol. Removing EtOH at predetermined time points revealed that all phenomena except Cyto.C release occurred in a phase of neuronal cell death in which full recovery to a neurite-bearing cell was still possible. Our findings underscore a strategy of treating chronic neurodegenerative diseases by removing stressors from neurons and harnessing intracellular targets that delay or prevent trespassing the point of no return.

Cytochromes c are ubiquitous proteins, essential for life in most organisms. Their distinctive characteristic is the covalent attachment of heme to their polypeptide chain. This post-translational modification is performed by a dedicated protein system, which in many Gram-negative bacteria and plant mitochondria is a nine-protein apparatus (CcmA-I) called System I. Despite decades of study, mechanistic understanding of the protein-protein interactions in this highly complex maturation machinery is still lacking. Here, we focused on the interaction of CcmC, the protein that sources the heme cofactor, with CcmE, the pivotal component of System I responsible for the transfer of the heme to the apocytochrome. Using in silico analyses, we identified a putative interaction site between these two proteins (residues Asp 47 , Gln 50 , and Arg 55 on CcmC; Arg 73 , Asp 101 , and Glu 105 on CcmE), and we validated our findings by in vivo experiments in Escherichia coli Moreover, employing NMR spectroscopy, we examined whether a heme-binding site on CcmE contributes to this interaction and found that CcmC and CcmE associate via protein-protein rather than protein-heme contacts. The combination of in vivo site-directed mutagenesis studies and high-resolution structural techniques enabled us to determine at the residue level the mechanism for the formation of one of the key protein complexes for cytochrome c maturation by System I.

et al., [Objective]: To establish the role of the transcription factor Pax4 in pancreatic islet expansion and survival in response to physiological stress and its impact on glucose metabolism, we generated transgenic mice conditionally and selectively overexpressing Pax4 or a diabetes-linked mutant variant (Pax4R129W) in β-cells. [Research and Methods]: Glucose homeostasis and β-cell death and proliferation were assessed in Pax4- or Pax4R129W-overexpressing transgenic animals challenged with or without streptozotocin. Isolated transgenic islets were also exposed to cytokines, and apoptosis was evaluated by DNA fragmentation or cytochrome C release. The expression profiles of proliferation and apoptotic genes and β-cell markers were studied by immunohistochemistry and quantitative RT-PCR. [Results]: Pax4 but not Pax4R129W protected animals against streptozotocin-induced hyperglycemia and isolated islets from cytokine-mediated β-cell apoptosis. Cytochrome C release was abrogated in Pax4 islets treated with cytokines. Interleukin-1β transcript levels were suppressed in Pax4 islets, whereas they were increased along with NOS2 in Pax4R129W islets. Bcl-2, Cdk4, and c-myc expression levels were increased in Pax4 islets while MafA, insulin, and GLUT2 transcript levels were suppressed in both animal models. Long-term Pax4 expression promoted proliferation of a Pdx1-positive cell subpopulation while impeding insulin secretion. Suppression of Pax4 rescued this defect with a concomitant increase in pancreatic insulin content. [Conclusions]: Pax4 protects adult islets from stress-induced apoptosis by suppressing selective nuclear factor-κB target genes while increasing Bcl-2 levels. Furthermore, it promotes dedifferentiation and proliferation of β-cells through MafA repression, with a concomitant increase in Cdk4 and c-myc expression., This work was funded by the Swiss National Science Foundation (grants 310030-119763 to B.R.G.; 310000-116750/1 to C.B.W. and B.R.G.; 310000-109402, 310000-122423, and CR32I3-129987 to P.M.; and 3100A0103867 NCCR “Frontiers-in-Genetics” to P.L.H.), the Juvenile Diabetes Research Foundation (grants 7-2005-1158 to C.B.W. and B.R.G.; 40-2011-11 to P.M.; and 26-2007-928 and 26-2008-640 to P.L.H.), the EU (FP-7, BETAIMAGE 222980, IMI IMIDIA, and C2008-T7 to P.M. and FP-6, β Cell Therapy Consortium to P.L.H.), the National Institutes of Health/National Institutes of Diabetes and Digestive and Kidney Diseases (the Beta Cell Biology Consortium to P.L.H.), and the Foundation Progreso y Salud, Junta de Andalucia (to B.R.G.). K.H.H.H. was supported by a GeMet/Novo Nordisk fellowship. No other potential conflicts of interest relevant to this article were reported.

During apoptosis, mitochondria lose their membrane potential and undergo fragmentation around the time of release of cytochrome c. Apoptotic fission is at least in part sustained by the translocation of dynamin-related protein 1 (Drp1), normally located in the cytosol, to mitochondria. This process depends on dephosphorylation of Drp1 by the phosphatase calcineurin. Here, we report the identification of a novel inhibitor of this process. A polypeptide (PPD1) from the immunophilin FKBP52 inhibits calcineurin activation triggered by mitochondrial dysfunction. PPD1 blocks Drp1 translocation to mitochondria and fragmentation of the organelle. PPD1 delays apoptosis by intrinsic stimuli by preventing fragmentation and release of cytochrome c. Cells expressing PPD1 display enhanced clonogenic ability after exposure to staurosporine. A genetic analysis revealed that the activity of PPD1 is independent of the BH3-only protein BAD, another target of calcineurin during apoptosis, and is not additive to inhibition of Drp1. Thus, PPD1 is a novel inhibitor of apoptosis that elucidates the function of calcineurin-dependent mitochondrial fragmentation in the amplification of cell death.

Mastoparan (MP), a potent facilitator of mitochondrial permeability transition (PT), could be used as an antitumor agent, if it were encapsulated in a tumor selective delivery system. We recently developed transferrin-modified liposomes (Tf-L) with a pH-sensitive fusogenic peptide (GALA), which delivers an encapsulated fluorescent marker into cytosol efficiently. In this study, we encapsulated MP into Tf-L with GALA for the selective delivery to mitochondria of tumor cells. The MP showed potent PT activity at concentrations above 25 microM in a homogenate of K 562 cells as well as in isolated mitochondria in the presence of phosphate. Tf-L equipped with cholesteryl GALA can release encapsulated sulforhodamine B, while Tf-L failed, as evidenced by confocal laser scanning microscopy. The MP, which was delivered with Tf-L with GALA, released cytochrome c (cyt c) from mitochondria to the cytosol, while free MP released cyt c not only to the cytosol but also extracellulary. These results demonstrate the utility of MP in Tf-L with GALA for cancer therapy.

Uncoupling protein 2 (UCP2) is suggested to be a regulator of reactive oxygen species production in mitochondria. We performed a detailed study of brain injury, including regional and cellular distribution of UCP2 mRNA, as well as measures of oxidative stress markers following permanent middle cerebral artery occlusion in UCP2 knockout (KO) and wild-type (WT) mice. Three days post ischemia, there was a massive induction of UCP2 mRNA confined to microglia in the peri-infarct area of WT mice. KO mice were less sensitive to ischemia as assessed by reduced brain infarct size, decreased densities of deoxyuridine triphosphate nick end-labelling (TUNEL)-labelled cells in the peri-infact area and lower levels of lipid peroxidation compared with WT mice. This resistance may be related to the substantial increase of basal manganese superoxide dismutase levels in neurons of KO mice. Importantly, we found a specific decrease of mitochondrial glutathione (GSH) levels in UCP2 expressing microglia of WT, but not in KO mice after ischemia. This specific association between UCP2 and mitochondrial GSH levels regulation was further confirmed using lipopolysaccharide models of peripheral inflammation, and in purified peritoneal macrophages. Moreover, our data imply that UCP2 is not directly involved in the regulation of ROS production but acts by regulating mitochondrial GSH levels in microglia.

Hyperoxia is known to induce extensive alveolar cell death by still poorly defined mechanisms. In this study, the mitochondria-dependent cell death pathway was explored during hyperoxia-induced lung injury in mice. We observed a progressive release of cytochrome c from the mitochondria into the cytosol of alveolar cells. This release was accompanied by the translocation of the proapoptotic protein Bax from cytosol to mitochondria without detectable activation of caspase-3. As cytochrome c release can be induced by mitochondrial membrane alteration and permeability transition (MPT), mice were treated with cyclosporin A, which specifically inhibits MPT. Cyclosporin A treatment prevented mitochondrial release of cytochrome c during hyperoxia and concomitantly preserved mitochondria from extensive swelling and crista disorganization, as assessed by electron microscopy analysis of alveolar epithelial cells. These morphological and biochemical observations correlated with decreased lung tissue damage, as evaluated by morphological score and lung weight. In conclusion, mitochondrial damage and cytochrome c release are important linked events in hyperoxia-induced lung injury and can be efficiently blocked by cyclosporin A.

A generic optical biosensing strategy was developed that relies on the absorbance enhancement phenomenon occurring in a multiple scattering matrix. Experimentally, inserts made of glass fiber membrane were placed into microplate wells in order to significantly lengthen the trajectory of the incident light through the sample and therefore increase the corresponding absorbance. Enhancement factor was calculated by comparing the absorbance values measured for a given amount of dye with and without the absorbance-enhancing inserts in the wells. Moreover, the dilution of dye in solutions with different refractive indices (RI) clearly revealed that the enhancement factor increased with the ΔRI between the membrane and the surrounding medium, reaching a maximum value (EF>25) when the membranes were dried. On this basis, two H2O2-biosensing systems were developed based on the biofunctionalization of the glass fiber inserts either with cytochrome c or horseradish peroxidase (HRP) and the analytical performances were systematically compared with the corresponding bioassay in solution. The efficiency of the absorbance-enhancement approach was particularly clear in the case of the cytochrome c-based biosensor with a sensitivity gain of 40 folds and wider dynamic range. Therefore, the developed strategy represents a promising way to convert standard colorimetric bioassays into optical biosensors with improved sensitivity.

Reporting in Molecular Cell, Sheridan et al. (2008) and Breckenridge et al. (2008) show that mitochondrial fragmentation is not required to induce cell death. Meanwhile, Yamaguchi et al. show that proapoptotic Bcl-2 family members promote cytochrome c mobilization through Opa1-mediated cristae remodeling. Therefore, the connection between mitochondrial structure and apoptosis is more complex than previously imagined.

Metformin is used as an anti-diabetic drug. Metformin ameliorates insulin resistance by improving insulin sensitivity in liver and skeletal muscle. Reduced mitochondrial content has been reported in type 2 diabetic muscles and it may contribute to decreased insulin sensitivity characteristic for diabetic muscles. The molecular mechanism behind the effect of metformin is not fully clarified but inhibition of complex I in the mitochondria and also activation of the 5′AMP activated protein kinase (AMPK) has been reported in muscle. Furthermore, both AMPK activation and metformin treatment have been associated with stimulation of mitochondrial function and biogenesis. However, a causal relationship in skeletal muscle has not been investigated. We hypothesized that potential effects of in vivo metformin treatment on mitochondrial function and protein expressions in skeletal muscle are dependent upon AMPK signaling. We investigated this by two weeks of oral metformin treatment of muscle specific kinase dead α 2 (KD) AMPK mice and wild type (WT) littermates. We measured mitochondrial respiration and protein activity and expressions of key enzymes involved in mitochondrial carbohydrate and fat metabolism and oxidative phosphorylation. Mitochondrial respiration, HAD and CS activity, PDH and complex I-V and cytochrome c protein expression were all reduced in AMPK KD compared to WT tibialis anterior muscles. Surprisingly, metformin treatment only enhanced respiration in AMPK KD mice and thereby rescued the respiration defect compared to the WT mice. Metformin did not influence protein activities or expressions in either WT or AMPK KD mice. We conclude that two weeks of in vivo metformin treatment enhances mitochondrial respiration in the mitochondrial deficient AMPK KD but not WT mice. The improvement seems to be unrelated to AMPK, and does not involve changes in key mitochondrial proteins.

The vacuolating cytotoxin (VacA) is an important virulence factor of Helicobacter pylori with pleiotropic effects on mammalian cells, including the ability to trigger mitochondria-dependent apoptosis. However, the mechanism by which VacA exerts its apoptotic function is unclear. Using a genetic approach, in this study we show that killing by VacA requires the proapoptotic Bcl-2 family members BAX and BAK at the mitochondrial level, but not adequate endoplasmic reticulum Ca(2)(+) levels, similarly controlled by BAX and BAK. A combination of subcellular fractionation and imaging shows that wild-type VacA, but not mutants in its channel-forming region, induces the accumulation of BAX on endosomes and endosome-mitochondria juxtaposition that precedes the retrieval of active BAX on mitochondria. It is noteworthy that in Bax- and Bak-deficient cells, VacA is unable to cause endosome-mitochondria juxtaposition and is not retrieved in mitochondria. Thus, VacA causes BAX/BAK-dependent juxtaposition of endosomes and mitochondria early in the process of cell death, revealing a new function for these proapoptotic proteins in the regulation of relative position of organelles.

Aims Here we investigated the mechanisms by which cardiovascular CB1 cannabinoid receptors may modulate the cardiac dysfunction, oxidative stress, and interrelated cell death pathways associated with acute/chronic cardiomyopathy induced by the widely used anti-tumour compound doxorubicin (DOX). Methods and results Both load-dependent and -independent indices of left-ventricular function were measured by the Millar pressure–volume conductance system. Mitogen-activated protein kinase (MAPK) activation, cell-death markers, and oxidative/nitrosative stress were measured by molecular biology/biochemical methods and flow cytometry. DOX induced left-ventricular dysfunction, oxidative/nitrosative stress coupled with impaired antioxidant defense, activation of MAPK (p38 and JNK), and cell death and/or fibrosis in hearts of wide-type mice (CB1+/+), and these effects were markedly attenuated in CB1 knockouts (CB1−/−). In human primary cardiomyocytes expressing CB1 receptors (demonstrated by RT–PCR, western immunoblot, and flow cytometry) DOX, likewise the CB1 receptor agonist HU210 and the endocannabinoid anandamide (AEA), induced MAPK activation and cell death. The DOX-induced MAPK activation and cell death were significantly enhanced when DOX was co-administered with CB1 agonists AEA or HU210. Remarkably, cell death and MAPK activation induced by AEA, HU210, and DOX ± AEA/HU210 were largely attenuated by either CB1 antagonists (rimonabant and AM281) or by inhibitors of p38 and JNK MAPKs. Furthermore, AEA or HU210 in primary human cardiomyocytes triggered increased reactive oxygen species generation. Conclusion CB1 activation in cardiomyocytes may amplify the reactive oxygen/nitrogen species-MAPK activation-cell death pathway in pathological conditions when the endocannabinoid synthetic or metabolic pathways are dysregulated by excessive inflammation and/or oxidative/nitrosative stress, which may contribute to the pathophysiology of various cardiovascular diseases.

International audience; PIDD (p53-induced protein with a death domain [DD]), together with the bipartite adapter protein RAIDD (receptor-interacting protein-associated ICH-1/CED-3 homologous protein with a DD), is implicated in the activation of pro–caspase-2 in a high molecular weight complex called the PIDDosome during apoptosis induction after DNA damage. To investigate the role of PIDD in cell death initiation, we generated PIDD-deficient mice. Processing of caspase-2 is readily detected in the absence of PIDDosome formation in primary lymphocytes. Although caspase-2 processing is delayed in simian virus 40–immortalized pidd−/− mouse embryonic fibroblasts, it still depends on loss of mitochondrial integrity and effector caspase activation. Consistently, apoptosis occurs normally in all cell types analyzed, suggesting alternative biological roles for caspase-2 after DNA damage. Because loss of either PIDD or its adapter molecule RAIDD did not affect subcellular localization, nuclear translocation, or caspase-2 activation in high molecular weight complexes, we suggest that at least one alternative PIDDosome-independent mechanism of caspase-2 activation exists in mammals in response to DNA damage.

The pancreatic beta cell dysfunction is critical cycle in the pathogenesis of diabetes. Hyperglycemia is one of factors that induce pancreatic beta cell dysfunction, but the underlying mechanisms have not been well elucidated. In this study, we reported that a mitochondrial fission modulator, Dynamin-related protein 1 (Drp-1), plays an important role in high glucose induced beta cell apoptosis. Drp-1 expressed in islet beta cells was increased drastically under hyperglycemia conditions. Induction of Drp-1 expression significantly promoted high glucose induced apoptosis in Drp-1WT (Drp-1 wild type) inducible beta cell line, but not in Drp-1K38A (a dominant negative mutant of Drp1) inducible beta cell line. We further demonstrated that mitochondrial fission, cytochrome C release, mitochondrial membrane potential decreased, caspase-3 activation and generation of reactive oxygen species were enhanced by induction of Drp-1WT, but prevented by Drp-1K38A in pancreatic beta cells under high glucose condition. These results indicated that Drp-1 mediates high glucose induced pancreatic beta cell apoptosis.

Apoptosis represents a physiological form of cell death, the perturbation of which may contribute to the development of several diseases connected with accumulation of unwanted cells or excessive cell loss. We have previously shown that the continuous presence of low concentrations of H2O2 (generated by the action of glucose oxidase) was able to inhibit caspase-mediated apoptosis in Jurkat cells. The main purpose of the present study was to elucidate the exact molecular mechanism(s) underlying this inhibitory action of H2O2. The results presented show that events like outer mitochondrial membrane permeabilization, release of cytochrome c from mitochondria, oligomerization of Apaf-1, and recruitment of procaspase-9 to apoptosomes were taking place normally, but further advancement toward activation of the execution caspases was interrupted when H2O2 was present during the apoptotic process. From the results presented in this work, it emerges that the inhibition of procaspase-9 autoactivation was probably due to the reversible oxidation of sensitive cysteine residues in this molecule. Remarkably, caspase-9 activation and the ensuing caspase cascade proceeded normally in the presence of H2O2 under conditions of iron deprivation, indicating that the inhibition of procaspase-9 activation was an iron-dependent process. Collectively, these results highlighted the potential role of available intracellular iron ions in signaling mechanisms related to apoptotic cell death. Free Radic Biol Med

Bcl-2 is an antiapoptotic molecule that prevents oxidative stress damage and cell death. We investigated the possible protective mechanisms mediated by Bcl-2 during hyperoxia-induced cell death in L929 cells. In these cells, hyperoxia promoted apoptosis without DNA fragmentation. Overexpression of Bcl-2 significantly protected cells from oxygen-induced apoptosis, as shown by measurement of lactate dehydrogenase release, quantification of apoptotic nuclei, and detection of Annexin-V-positive cells. Bcl-2 partially prevented mitochondrial damage and interfered with the mitochondrial proapoptotic signaling pathway: it reduced Bax translocation to mitochondria, decreased the release of cytochrome c, and inhibited caspase 3 activation. However, treatment with the caspase inhibitor Z-VAD.fmk failed to rescue the cells from death, indicating that protection provided by Bcl-2 was due not only to caspase inhibition. Bcl-2 also prevented the release of mitochondrial apoptotic inducing factor, a mediator of caspase-independent apoptosis, correlating with the absence of oligonucleosomal DNA fragmentation. In addition, Bcl-2-overexpressing cells showed significantly higher intracellular amounts of glutathione after 72 h of oxygen exposure. In conclusion, our results demonstrate that the overexpression of Bcl-2 is able to prevent hyperoxia-induced cell death, by affecting mitochondria-dependent apoptotic pathways and increasing intracellular antioxidant compounds.

The mitochondrial outer membrane contains protein import machineries, the translocase of the outer membrane (TOM) and the sorting and assembly machinery (SAM). It has been speculated that TOM or SAM are required for Bax-induced release of intermembrane space (IMS) proteins; however, experimental evidence has been scarce. We used isolated yeast mitochondria as a model system and report that Bax promoted an efficient release of soluble IMS proteins while preproteins were still imported, excluding an unspecific damage of mitochondria. Removal of import receptors by protease treatment did not inhibit the release of IMS proteins by Bax. Yeast mutants of each Tom receptor and the Tom40 channel were not impaired in Bax-induced protein release. We analyzed a large collection of mutants of mitochondrial outer membrane proteins, including SAM, fusion and fission components, but none of these components was required for Bax-induced protein release. The released proteins included complexes up to a size of 230 kDa. We conclude that Bax promotes efficient release of IMS proteins through the outer membrane of yeast mitochondria while the inner membrane remains intact. Inactivation of the known protein import and sorting machineries of the outer membrane does not impair the function of Bax at the mitochondria.

Mitochondria are complex organelles whose internal structure and cytosolic organization is controlled by a growing number of 'mitochondria-shaping' proteins. These include mitochondrial proteins such as the large dynamin-related GTPases Mitofusin (Mfn) 1 and 2, Optic Atrophy 1 (Opa1); as well as the cytosolic dynamin-related protein 1 (Drp1) and its receptor on the outer mitochondrial membrane Fis1. These proteins influence not only the shape of mitochondria, but also the function of the organelle and eventually integrated cellular signalling cascades, including apoptosis. We undertook a genetic approach to elucidate the function and regulation of these proteins. Opa1 is involved in the regulation of mitochondrial fusion, by co-operating with Mfn1. Moreover, Opa1 independently from mitochondrial fusion regulates the crista remodelling pathway of apoptosis. Oligomers of a membrane bound and a soluble form of Opa1, produced by Parl, an inner membrane rhomboid protease, are disrupted early during apoptosis, leading to remodelling of the mitochondrial cristae and redistribution of the mitochondrial cytochrome c. The importance of this pathway is substantiated by the phenotype of the Parl-/- mouse, which displays excess apoptosis in multiple tissues. Cells lacking Parl are more susceptible to apoptotic stimuli and the reintroduction of a soluble form of Opa1 rescues their phenotype.

The mitochondrial pathway of apoptosis in vertebrates is dependent on the process of mitochondrial outer membrane permeabilization (MOMP), which leads to the release of proteins from the mitochondrial intermembrane space into the cytosol. “Upstairs” of this event are the Bcl-2 family proteins that regulate and mediate MOMP; “downstairs” is the activation of caspases that orchestrate the dismantling of the cell. In the Connections Map database at Science 's Signal Transduction Knowledge Environment (STKE), the pathways that define the mitochondrial pathway of apotosis are illustrated, with the bulk of control occurring “upstairs” of MOMP.

c-Jun N-terminal kinase (JNK), a member of the mitogen-activated protein kinase family, is activated in response to a number of extracellular stimuli, including inflammatory cytokines, UV irradiation and ischaemia. A large body of evidence supports a role for JNK signalling in stress-induced apoptosis. It has been hypothesized that JNK may contribute to the apoptotic response by regulating the intrinsic cell death pathway involving the mitochondria. Here, we examined the role of the JNK signalling pathway in hippocampal CA1 apoptotic neurones following transient ischaemia in gerbils. We showed early activation of death receptor-dependent apoptosis (caspase-8 activation 2 days after ischaemia) and a biphasic activation of caspase-3 and caspase-9 after ischaemia. Activation of the mitochondrial pathway, as measured by cytochrome c release, appeared as a late event (5-7 days after ischaemia). AS601245, a novel JNK inhibitor, antagonized activation of both pathways and significantly protected CA1 neurones from cell death. Our results suggest a key role of JNK in the control of death receptor and mitochondrial-dependent apoptosis after transient ischaemia.

Proteasome inhibitors exhibit antitumor activity against malignancies of different histology. Yet, the mechanisms underlying this effect are poorly understood. Recent evidence indicates that antiapoptotic factors may also accumulate as a consequence of exposure to these drugs, possibly reducing their cytotoxicity. These include the Bcl-2 family member Mcl-1, whose down-regulation has been proposed to initiate apoptosis in response to genotoxic stimuli. In this study, we found that proteasome inhibitors release cyotochrome c and second mitochondria-derived activator of caspase (SMAC)/Diablo and trigger the subsequent apoptotic cascade in spite of concomitant Mcl-1 increase. However, our data indicate that subtraction of Mcl-1 during apoptosis, although not required for early release of proapoptotic factors, is probably relevant in speeding up cell demise, since RNA interference-mediated Mcl-1 silencing is lethal in lymphoma cells. Consistent with this, the cytotoxic effects of proteasome inhibitors are enhanced when Mcl-1 increase is impeded. Thus, this study identifies Mcl-1 accumulation as an unwanted molecular consequence of exposure to proteasome inhibitors, which slows down their proapoptotic effects. Pharmacologic or genetic approaches targeting Mcl-1, including therapeutic RNAi, may increase the effectiveness of these compounds. (Blood. 2005;105:3255-3262)