Catherine Brenner

Guidelines for the use and interpretation of assays for monitoring cell death in higher eukaryotes

Cell death is essential for a plethora of physiological processes, and its deregulation characterizes numerous human diseases. Thus, the in-depth investigation of cell death and its mechanisms constitutes a formidable challenge for fundamental and applied biomedical research, and has tremendous implications for the development of novel therapeutic strategies. It is, therefore, of utmost importance to standardize the experimental procedures that identify dying and dead cells in cell cultures and/or in tissues, from model organisms and/or humans, in healthy and/or pathological scenarios. Thus far, dozens of methods have been proposed to quantify cell death-related parameters. However, no guidelines exist regarding their use and interpretation, and nobody has thoroughly annotated the experimental settings for which each of these techniques is most appropriate. Here, we provide a nonexhaustive comparison of methods to detect cell death with apoptotic or nonapoptotic morphologies, their advantages and pitfalls. These guidelines are intended for investigators who study cell death, as well as for reviewers who need to constructively critique scientific reports that deal with cellular demise. Given the difficulties in determining the exact number of cells that have passed the point-of-no-return of the signaling cascades leading to cell death, we emphasize the importance of performing multiple, methodologically unrelated assays to quantify dying and dead cells.

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, ΔΨm, 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.

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.

Oxidation of a critical thiol residue of the adenine nucleotide translocator enforces Bcl-2 independent permeability transition pore opening and apoptosis.

Mitochondrial membrane permeabilization is a critical event in the process leading to physiological or chemotherapy-induced apoptosis. This permeabilization event is at least in part under the control of the permeability transition pore complex (PTPC), which interacts with oncoproteins from the Bcl-2 family as well as with tumor suppressor proteins from the Bax family, which inhibit or facilitate membrane permeabilization, respectively. Here we show that thiol crosslinking agents including diazenedicarboxylic acid bis 5N,N-dimethylamide (diamide), dithiodipyridine (DTDP), or bis-maleimido-hexane (BMH) can act on the adenine nucleotide translocator (ANT), one of the proteins within the PTPC. ANT alone reconstituted into artificial lipid bilayers suffices to confer a membrane permeabilization response to thiol crosslinking agents. Diamide, DTDP, and BMH but not tert-butylhydroperoxide or arsenite cause the oxidation of a critical cysteine residue (Cys 56) of ANT. Thiol modification within ANT is observed in intact cells, isolated mitochondria, and purified ANT. Recombinant Bcl-2 fails to prevent thiol modification of ANT. Concomitantly, a series of different thiol crosslinking agents (diamide, DTDP, and BMH, phenylarsine oxide) but not tert-butylhydroperoxide or arsenite induce mitochondrial membrane permeabilization and cell death irrespective of the expression level of Bcl-2. These data indicate that thiol crosslinkers cause a covalent modification of ANT which, beyond any control by Bcl-2, leads to mitochondrial membrane permeabilization and cell death.

Viral Control of Mitochondrial Apoptosis

Throughout the process of pathogen–host co-evolution, viruses have developed a battery of distinct strategies to overcome biochemical and immunological defenses of the host. Thus, viruses have acquired the capacity to subvert host cell apoptosis, control inflammatory responses, and evade immune reactions. Since the elimination of infected cells via programmed cell death is one of the most ancestral defense mechanisms against infection, disabling host cell apoptosis might represent an almost obligate step in the viral life cycle. Conversely, viruses may take advantage of stimulating apoptosis, either to kill uninfected cells from the immune system, or to induce the breakdown of infected cells, thereby favoring viral dissemination. Several viral polypeptides are homologs of host-derived apoptosis-regulatory proteins, such as members of the Bcl-2 family. Moreover, viral factors with no homology to host proteins specifically target key components of the apoptotic machinery. Here, we summarize the current knowledge on the viral modulation of mitochondrial apoptosis, by focusing in particular on the mechanisms by which viral proteins control the host cell death apparatus.

The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite, and 4-hydroxynonenal

Nitric oxide (NO), peroxynitrite, and 4-hydroxynonenal (HNE) may be involved in the pathological demise of cells via apoptosis. Apoptosis induced by these agents is inhibited by Bcl-2, suggesting the involvement of mitochondria in the death pathway. In vitro, NO, peroxynitrite and HNE can cause direct permeabilization of mitochondrial membranes, and this effect is inhibited by cyclosporin A, indicating involvement of the permeability transition pore complex (PTPC) in the permeabilization event. NO, peroxynitrite and HNE also permeabilize proteoliposomes containing the adenine nucleotide translocator (ANT), one of the key components of the PTPC, yet have no or little effects on protein-free control liposomes. ANT-dependent, NO-, peroxynitrite- or HNE-induced permeabilization is at least partially inhibited by recombinant Bcl-2 protein, as well as the antioxidants trolox and butylated hydroxytoluene. In vitro, none of the tested agents (NO, peroxynitrite, HNE, and tert-butylhydroperoxide) causes preferential carbonylation HNE adduction, or nitrotyrosylation of ANT. However, all these agents induced ANT to undergo thiol oxidation/derivatization. Peroxynitrite and HNE also caused significant lipid peroxidation, which was antagonized by butylated hydroxytoluene but not by recombinant Bcl-2. Transfection-enforced expression of vMIA, a viral apoptosis inhibitor specifically targeted to ANT, largely reduces the mitochondrial and nuclear signs of apoptosis induced by NO, peroxynitrite and HNE in intact cells. Taken together these data suggest that NO, peroxynitrite, and HNE may directly act on ANT to induce mitochondrial membrane permeabilization and apoptosis.

GAPDH, a novel regulator of the pro-apoptotic mitochondrial membrane permeabilization.

Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) is a pleiotropic enzyme that is overexpressed in apoptosis and in several human chronic pathologies. Here, we report that the protein accumulates in mitochondria during apoptosis, and induces the pro-apoptotic mitochondrial membrane permeabilization, a decisive event of the intrinsic pathway of apoptosis. GAPDH was localized by immunogold labeling and identified by matrix-assisted laser desorption/ionization-time of flight and nano liquid chromatography mass spectroscopy/mass spectroscopy in the mitochondrion of various tissues and origins. In isolated mitochondria, GAPDH can be imported and interact with the voltage-dependent anion channel (VDAC1), but not the adenine nucleotide translocase (ANT). The protein mediates a cyclosporin A-inhibitable permeability transition, characterized by a loss of the inner transmembrane potential, matrix swelling, permeabilization of the inner mitochondrial membrane and the release of two pro-apoptotic proteins, cytochrome c and apoptosis-inducing factor (AIF). This novel function of GAPDH might have implications for the understanding of mitochondrial biology, oncogenesis and apoptosis.

Permeabilization of the mitochondrial inner membrane during apoptosis: impact of the adenine nucleotide translocator.

Mitochondrial membrane permeabilization can be a rate limiting step of apoptotic as well as necrotic cell death. Permeabilization of the outer mitochondrial membrane (OM) and/or inner membrane (IM) is, at least in part, mediated by the permeability transition pore complex (PTPC). The PTPC is formed in the IM/OM contact site and contains the two most abundant IM and OM proteins, adenine nucleotide translocator (ANT, in the IM) and voltage-dependent anion channel (VDAC, in the OM), the matrix protein cyclophilin D, which can interact with ANT, as well as apoptosis-regulatory proteins from the Bax/Bcl-2 family. Here we discuss that ANT has two opposite functions. On the one hand, ANT is a vital, specific antiporter which accounts for the exchange of ATP and ADP on IM. On the other hand, ANT can form a non-specific pore, as this has been shown by electrophysiological characterization of purified ANT reconstituted into synthetic lipid bilayers or by measuring the permeabilization of proteoliposomes containing ANT. Pore formation by ANT is induced by a variety of different agents (e.g. Ca2+, atractyloside, thiol oxidation, the pro-apoptotic HIV-1 protein Vpr, etc.) and is enhanced by Bax and inhibited by Bcl-2, as well as by ADP. In isolated mitochondria, pore formation by ANT leads to an increase in IM permeability to solutes up to 1500 Da, swelling of the mitochondrial matrix, and OM permeabilization, presumably due to physical rupture of OM. Although alternative mechanisms of mitochondrial membrane permeabilization may exist, ANT emerges as a major player in the regulation of cell death. Cell Death and Differentiation (2000) 7, 1146–1154

Systems biology of cisplatin resistance: past, present and future

The platinum derivative cis-diamminedichloroplatinum(II), best known as cisplatin, is currently employed for the clinical management of patients affected by testicular, ovarian, head and neck, colorectal, bladder and lung cancers. For a long time, the antineoplastic effects of cisplatin have been fully ascribed to its ability to generate unrepairable DNA lesions, hence inducing either a permanent proliferative arrest known as cellular senescence or the mitochondrial pathway of apoptosis. Accumulating evidence now suggests that the cytostatic and cytotoxic activity of cisplatin involves both a nuclear and a cytoplasmic component. Despite the unresolved issues regarding its mechanism of action, the administration of cisplatin is generally associated with high rates of clinical responses. However, in the vast majority of cases, malignant cells exposed to cisplatin activate a multipronged adaptive response that renders them less susceptible to the antiproliferative and cytotoxic effects of the drug, and eventually resume proliferation. Thus, a large fraction of cisplatin-treated patients is destined to experience therapeutic failure and tumor recurrence. Throughout the last four decades great efforts have been devoted to the characterization of the molecular mechanisms whereby neoplastic cells progressively lose their sensitivity to cisplatin. The advent of high-content and high-throughput screening technologies has accelerated the discovery of cell-intrinsic and cell-extrinsic pathways that may be targeted to prevent or reverse cisplatin resistance in cancer patients. Still, the multifactorial and redundant nature of this phenomenon poses a significant barrier against the identification of effective chemosensitization strategies. Here, we discuss recent systems biology studies aimed at deconvoluting the complex circuitries that underpin cisplatin resistance, and how their findings might drive the development of rational approaches to tackle this clinically relevant problem.

Propionibacteria induce apoptosis of colorectal carcinoma cells via short-chain fatty acids acting on mitochondria.

The genus Propionibacterium is composed of dairy and cutaneous bacteria which produce short-chain fatty acids (SCFA), mainly propionate and acetate, by fermentation. Here, we show that P. acidipropionici and freudenreichii, two species which can survive in the human intestine, can kill two human colorectal carcinoma cell lines by apoptosis. Propionate and acetate were identified as the major cytotoxic components secreted by the bacteria. Bacterial culture supernatants as well as pure SCFA induced typical signs of apoptosis including a loss of mitochondrial transmembrane potential, the generation of reactive oxygen species, caspase-3 processing, and nuclear chromatin condensation. The oncoprotein Bcl-2, which is known to prevent apoptosis via mitochondrial effects, and the cytomegalovirus-encoded protein vMIA, which inhibits apoptosis and interacts with the mitochondrial adenine nucleotide translocator (ANT), both inhibited cell death induced by propionibacterial SCFA, suggesting that mitochondria and ANT are involved in the cell death pathway. Accordingly, propionate and acetate induced mitochondrial swelling when added to purified mitochondria in vitro. Moreover, they specifically permeabi-lize proteoliposomes containing ANT, indicating that ANT can be a critical target in SCFA-induced apoptosis. We suggest that propionibacteria could constitute probiotics efficient in digestive cancer prophylaxis via their ability to produce apoptosis-inducing SCFA.