Patricia Boya

Mitochondrial membrane permeabilization is a critical step of lysosome-initiated apoptosis induced by hydroxychloroquine.

Hydroxychloroquine (HCQ) is a lysosomotropic amine with cytotoxic properties. Here, we show that HCQ induces signs of lysosomal membrane permeabilization (LMP), such as the decrease in the lysosomal pH gradient and the release of cathepsin B from the lysosomal lumen, followed by signs of apoptosis including caspase activation, phosphatidylserine exposure, and chromatin condensation with DNA loss. HCQ also induces mitochondrial membrane permeabilization (MMP), as indicated by the insertion of Bax into mitochondrial membranes, the conformational activation of Bax within mitochondria, the release of cytochrome c from mitochondria, and the loss of the mitochondrial transmembrane potential. To determine the molecular order among these events, we introduced inhibitors of LMP (bafilomycin A1), MMP (Bcl-XL, wild-type Bcl-2, mitochondrion-targeted Bcl-2, or viral mitochondrial inhibitor of apoptosis from cytomegalovirus), and caspases (Z-VAD.fmk) into the system. Our data indicate that caspase-independent MMP is rate-limiting for LMP-mediated caspase activation. Mouse embryonic fibroblasts lacking the expression of both Bax and Bak are resistant against hydroxychloroquine-induced apoptosis. Such Bax−/− Bak−/− cells manifest normal LMP, yet fail to undergo MMP and subsequent cell death. The data reported herein indicate that LMP does not suffice to trigger caspase activation and that Bax/Bak-dependent MMP is a critical step of LMP-induced cell death.

Endoplasmic reticulum stress-induced cell death requires mitochondrial membrane permeabilization

Accumulating evidence suggests that mitochondrial membrane permeabilization (MMP) is a rate-limiting step of programmed (developmental) cell death as well as stressinduced cell death, including in the context of anti-cancer chemotherapy or viral infection. This notion is re-enforced by the observation that the knock-out of proteins involved in the pathways leading to MMP or closely linked to MMP (Bax, Bak, Bim, AIF, cytochrome c, Apaf-1, caspase-9 etc.) results in a major phenotype. Recently, we have launched the working hypothesis that MMP may be a rate-limiting event of apoptosis induction even when cell death is initiated through a primary stimulus affecting other organelles than mitochondria such as nuclei (via p53 activation), lysosomes (via activation of cathepsins) or the endoplasmic reticulum (ER). The proapoptotic agents that specifically act on the ER include tunicamycin (TM, which inhibits N-linked glycosylation), brefeldin A (BFA, which inhibits ER-Golgi transport) and thapsigargin (TG, which inhibits the sarcoplasmic/endoplasmic Ca-ATPase SERCA). Whereas BFA and TG elicit a local unfolded protein response, TG depletes ER Ca and thus impinges on Ca signaling. Mouse embryonic fibroblasts lacking both Bax and Bak become resistant to apoptosis induction by TM and BFA, an observation that may be attributed to the obligatory participation of Bax and Bak in MMP induction, or alternatively, suggests an as yet poorly characterized function of Bax and Bak at the ER level. Indeed, Bax redistributes both to mitochondria and to ER upon apoptosis induction, and overexpression of Bax reportedly causes a loss of ER Ca content. To probe the importance of MMP for ER stress-induced cell death, we assessed the effects of two local MMP inhibitors, Bcl-XL and vMIA on cellular alterations provoked by TM, BFA, and TG (Figure 1). Bcl-XL is found inserted in intracellular membranes (in particular the outer mitochondrial membrane) and is known to stabilize the mitochondrial membrane barrier function by local interactions with pore forming proteins contained in the permeability transition pore complex, namely the voltage-dependent anion channel (VDAC), the adenine nucleotide translocase (ANT), and pro-apoptotic Bcl-2 family members. In contrast to Bcl-2, ± 14 no local ER effects have been described for Bcl-XL. 15 Viral mitochondrial inhibitor of apoptosis (vMIA) is encoded by the Cytomegalovirus UL37 gene, has no obvious structural similarity with Bcl-2-like molecules, and is exclusively found in mitochondrial membranes, where it specifically interacts with ANT but not with VDAC, as shown by mass spectroscopic identification of vMIAinteracting proteins and confirmed by co-immunoprecipitation assays. ± 18 TM, BFA, and TG induced signs of MMP in several different cell lines including B cell lymphoma BJAB (Figure 1a) and cervical carcinoma HeLa cells (Figure 1b). Such signs consist in the loss of the mitochondrial transmembrane potential (DCm), as determined by means of the DCm-sensitive fluorochrome, DiOC6 3 (Figure 1a), and the release of cytochrome c from mitochondria, as determined by immunofluorescence analysis (Figure 1b,c). Thus, both the permeability of the inner membrane (on which the DCm builds up) and that of the outer membrane (which retains cytochrome c) were compromised by the three different ER-targeted toxins. ER stress-induced MMP was an early event since it was detectable in a fraction of cells that still lack signs of chromatin condensation (not shown). Stable transfection with vMIA and Bcl-XL prevented ER stressinduced MMP (Figure 1a,b,c), both in BJAB and in HeLa cells. It has been reported that, in determined circumstances, for instance in type I cells stimulated by CD95 ligation, Bcl-2-mediated MMP inhibition is not sufficient for the prevention of apoptosis. Therefore, we assessed whether MMP inhibition would suffice to suppress apoptosis induction by TM, BFA, or TG. Clearly, vMIA and Bcl-XL overexpression did reduce the frequency of cells which manifest caspase activation (not shown), nuclear chromatin condensation (Figure 1b), phosphatidylserine exposure on the outer leaflet of the plasma membrane (as determined with an Annexin V-FITC conjugate, Figure 1d), and loss of viability (as determined by staining with propidium iodide, PI, Figure 1e). In conclusion, it appears that MMP inhibition by Bxl-XL or vMIA can protect cells against apoptosis induction by ERspecific toxins. Using a monoclonal antibody specific for the apoptogenic conformation of Bax (6A7), we determined the putative link between vMIA-induced (presumably ANTmediated ± 18 apoptosis inhibition and the Bax/Bak mediated MMP induced by ER stress. ER stress did induce an apoptosis-associated change in Bax conformation, linked to its aggregation in cytoplasmic spots (Figure 2f), some of which coincide with mitochondria (as determined by confocal microscopy, not shown). vMIA did prevent the apoptosis associated conformational change of Bax, as well as its aggregation, a finding that may link our previous observation that Bax and ANT can interact to induce MMP. In conclusion, it appears that ER stress can induce apoptosis through a reaction that depends on pro-apoptotic members of the Bcl-2 family (Bax, Bak) and which involves MMP as a critical step towards cellular demise. Rather than Cell Death and Differentiation (2002) 9, 465 ± 467 ã 2002 Nature Publishing Group All rights reserved 1350-9047/02

Viral and bacterial proteins regulating apoptosis at the mitochondrial level

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NF-κB and p53 Are the Dominant Apoptosis-inducing Transcription Factors Elicited by the HIV-1 Envelope

Mitochondrial membrane permeabilization (MMP) is a critical step of several apoptotic pathways. Some infectious intracellular pathogens can regulate (induce or inhibit) apoptosis of their host cells at the mitochondrial level, by targeting proteins to mitochondrial membranes that either induce or inhibit MMP. Pathogen‐encoded mitochondrion‐targeted proteins may or may not show amino acid sequence homology to Bcl‐2‐like proteins. Among the Bcl‐2‐unrelated, mitochondrion‐targeted proteins, several interact with the voltage‐dependent anion channel (VDAC) or with the adenine nucleotide translocator (ANT). While VDAC‐targeted proteins show homology to VDAC/porin, ANT‐targeted proteins possess relatively short cationic binding domains, which may facilitate insertion into the negatively charged inner mitochondrial membrane. It may be speculated that such proteins employ pre‐existing host‐intrinsic mechanisms of MMP control.

Cell permeable BH3-peptides overcome the cytoprotective effect of Bcl-2 and Bcl-X L

The coculture of cells expressing the HIV-1 envelope glycoprotein complex (Env) with cells expressing CD4 results into cell fusion, deregulated mitosis, and subsequent cell death. Here, we show that NF-κB, p53, and AP1 are activated in Env-elicited apoptosis. The nuclear factor κB (NF-κB) super repressor had an antimitotic and antiapoptotic effect and prevented the Env-elicited phosphorylation of p53 on serine 15 and 46, as well as the activation of AP1. Transfection with dominant-negative p53 abolished apoptosis and AP1 activation. Signs of NF-κB and p53 activation were also detected in lymph node biopsies from HIV-1–infected individuals. Microarrays revealed that most (85%) of the transcriptional effects of HIV-1 Env were blocked by the p53 inhibitor pifithrin-α. Macroarrays led to the identification of several Env-elicited, p53-dependent proapoptotic transcripts, in particular Puma, a proapoptotic “BH3-only” protein from the Bcl-2 family known to activate Bax/Bak. Down modulation of Puma by antisense oligonucleotides, as well as RNA interference of Bax and Bak, prevented Env-induced apoptosis. HIV-1–infected primary lymphoblasts up-regulated Puma in vitro. Moreover, circulating CD4+ lymphocytes from untreated, HIV-1–infected donors contained enhanced amounts of Puma protein, and these elevated Puma levels dropped upon antiretroviral therapy. Altogether, these data indicate that NF-κB and p53 cooperate as the dominant proapoptotic transcription factors participating in HIV-1 infection.

The chemopreventive agent N-(4-hydroxyphenyl)retinamide induces apoptosis through a mitochondrial pathway regulated by proteins from the Bcl-2 family

Peptides corresponding to the BH3 domains of Bax (BaxBH3) or Bcl-2 (Bcl2BH3) are potent inducers of apoptosis when fused to the Atennapedia plasma membrane translocation domain (Ant). BaxBH3Ant and Bcl2BH3Ant caused a mitochondrial membrane permeabilization (MMP) and apoptosis, via a mechanism that was not inhibited by overexpressed Bcl-2 or Bcl-XL, yet partially inhibited by cyclosporin A (CsA), an inhibitor of the mitochondrial permeability transition pore. When added to isolated mitochondria, BaxBH3 and Bcl2BH3 induced MMP, which was inhibited by CsA. However, Bcl-2 or Bcl-XL failed to inhibit MMP induced by BaxBH3 and Bc2BH3 in vitro, while they efficiently suppressed the induction of MMP by the Vpr protein (from human immunodeficiency virus-1), a ligand of the adenine nucleotide translocator (ANT). BaxBH3 but not Bcl2BH3 was found to interact with ANT, and only BaxBH3 (not Bcl2BH3) permeabilized ANT proteoliposomes and induced ANT to form non-specific channels in electrophysiological experiments. In contrast, both BaxBH3 and Bcl2BH3 were able to stimulate channel formation by recombinant Bax protein. Thus, BaxBH3 might induce MMP via an action on at least two targets, ANT and Bax-like proteins. In contrast, Bcl2BH3 would elicit MMP in an ANT-independent fashion. In purified mitochondria, two ligands of ANT, bongkrekic acid and the protein vMIA from cytomegalovirus, failed to prevent MMP induced by BaxBH3 or Bcl2BH3. In conclusion, BaxBH3 and Bcl2BH3 induce MMP and apoptosis through a mechanism which overcomes cytoprotection by Bcl-2 and Bcl-XL.

The C-terminal moiety of HIV-1 Vpr induces cell death via a caspase-independent mitochondrial pathway.

N-(4-hydroxyphenyl)retinamide (4-HPR, fenretinide) is a potent chemopreventive agent whose effect has been suggested to involve apoptosis induction. 4-HPR induces a loss of the mitochondrial transmembrane potential and the mitochondrial release of cytochrome c before caspase activation. Inhibition of mitochondrial membrane permeabilization (MMP) by transfection with Bcl-2 or the Cytomegalovirus UL37 gene product vMIA prevented caspase activation and cell death. In contrast to other retinoid derivatives, 4-HPR has no direct MMP-inducing effects when added to isolated mitochondria or when added to proteoliposomes containing the MMP-regulatory permeability transition pore complex (PTPC). Moreover, although reactive oxygen species (ROS) overproduction appears to be instrumental for 4-HPR-induced MMP and apoptosis, inhibition of the NF-κB or p53-mediated signal transduction pathways failed to modulate 4-HPR-induced apoptosis. 4-HPR was found to cause an antioxidant-inhibitable conformational change of both Bax and Bak, leading to the exposure of their N-termini and to the mitochondrial relocalization of Bax. Cells with a Bax−/− Bak−/− genotype were resistant against the 4-HPR-induced MMP, overproduction of ROS and cell death. Altogether, these data indicate that 4-HPR induces MMP through an ROS-mediated pathway that involves the obligatory contribution of the proapopotic Bcl-2 family members Bax and/or Bak.

Bcl-2 and CCND1/CDK4 expression levels predict the cellular effects of mTOR inhibitors in human ovarian carcinoma

Previous biochemical studies suggested that HIV-1-encoded Vpr may kill cells through an effect on the adenine nucleotide translocase (ANT), thereby causing mitochondrial membrane permeabilization (MMP). Here, we show that Vpr fails to activate caspases in conditions in which it induces cell killing. The knock-out of essential caspase-activators (Apaf-1 or caspase-9) or the knock-out of a mitochondrial caspase-independent death effector (AIF) does not abolish Vpr-mediated killing. In contrast, the cytotoxic effects of Vpr are reduced by transfection-enforced overexpression of two MMP-inhibitors, namely the endogenous protein Bcl-2 or the cytomegalovirus-encoded ANT-targeted protein vMIA. Vpr, which can elicit MMP through a direct effect on mitochondria, and HIV-1-Env, which causes MMP through an indirect pathway, exhibit additive (but not synergic) cytotoxic effects. In conclusion, it appears that Vpr induces apoptosis through a caspase-independent mitochondrial pathway.

Mitochondrial permeability transition as a novel principle of hepatorenal toxicity in vivo

Molecular markers enabling the prediction of sensitivity/resistance to rapamycin may facilitate further clinical development of rapamycin and its derivatives as anticancer agents. In this study, several human ovarian cancer cell lines (IGROV1, OVCAR-3, A2780, SK-OV-3) were evaluated for susceptibility to rapamycin-mediated growth inhibition. The differential expression profiles of genes coding for proteins known to be involved in the mTOR signaling pathway, cell cycle control and apoptosis were studied before and after drug exposure by RT-PCR. In cells exposed to rapamycin, we observed a dose-dependent downregulation of CCND1 (cyclin D1) and CDK4 gene expression and late G1 cell cycle arrest. Among these cell lines, SK-OV-3 cells resistant to both rapamycin and RAD001 were the sole to show the expression of the anti-apoptotic gene Bcl-2. Bcl-2/bclxL-specific antisense oligonucleotides restored the sensitivity of SK-OV-3 cells to apoptosis induction by rapamycin and RAD001. These results indicate that baseline Bcl-2 expression and therapy-induced downexpression of CCND1 and CDK4 may be regarded as molecular markers enabling the prediction and follow-up of the cellular effects on cell cycle and apoptosis induction of rapamycin in ovarian cancer. Furthermore, strategies to down regulate Bcl-2 in ovarian cancer may prove useful in combination with rapamycin or RAD001 for ovarian cancer.

Mitochondrion-targeted apoptosis regulators of viral origin ☆

Atractyloside (Atr) binds to the adenine nucleotide translocator (ANT) and inhibits ANT-mediated ATP/ADP exchange on the inner mitochondrial membrane. In addition, Atr can trigger opening of a non-specific ion channel, within the ANT-containing permeability transition pore complex (PTPC), which is subject to redox regulation and inhibited by cyclosporin A (CsA). Here we show that the cytotoxic effects of Atr, both in vivo and in vitro, are determined by its capacity to induce PTPC opening and consequent mitochondrial membrane permeabilization (MMP). Thus, the Atr-induced MMP and death of cultured liver cells are both inhibited by CsA as well as by glutathione (GSH) and enhanced by GSH depletion. Similarly, the hepatorenal toxicity of Atr, assessed in vivo, was reduced by treating mice with CsA or a diet rich in sulfur amino acids, a regime which enhances mitochondrial GSH levels. Atr injection induced MMP in hepatocytes and proximal renal tubular cells, and MMP was reduced by either CsA or GSH. Acetaminophen (paracetamol)-induced acute poisoning was also attenuated by CsA and GSH, both in vitro and in vivo. Altogether these data indicate that PTPC-mediated MMP may determine the hepatorenal toxicity of xenobiotics in vivo.