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Stress granules (SG) are dynamic cytoplasmic foci in which stalled translation initiation complexes accumulate. In conditions of acute cellular redox, stress cells manipulated to lose the expression of apoptosis-inducing factor (AIF) nucleate SG signature proteins (e.g. TIA-1, PABP1) more efficiently than AIF-positive controls. AIF also inhibited SG formation induced by the RasGAP-associated endoribonuclease G3BP. Retransfection of mouse AIF into cells subjected to human AIF-specific siRNA revealed that only AIF imported into mitochondria could repress SGs and that redox-active domains of AIF, which are dispensable for its apoptogenic action, were required for SG inhibition. In response to oxidative stress, AIF-negative cells were found to deplete non-oxidized glutathione more rapidly than AIF-expressing cells. Exogenous supplementation of glutathione inhibited SG formation elicited by arsenate or G3BP. Together, these data suggest that the oxidoreductase function of AIF is required for the maintenance of glutathione levels in stress conditions and that glutathione is a major regulator of SG. [ABSTRACT FROM AUTHOR]

Tetraploidy can constitute a metastable intermediate between normal diploidy and oncogenic aneuploidy. Here, we show that the absence of p53 is not only permissive for the survival but also for multipolar asymmetric divisions of tetraploid cells, which lead to the generation of aneuploid cells with a near-to-diploid chromosome content. Multipolar mitoses (which reduce the tetraploid genome to a sub-tetraploid state) are more frequent when p53 is downregulated and the product of the Mos oncogene is upregulated. Mos inhibits the coalescence of supernumerary centrosomes that allow for normal bipolar mitoses of tetraploid cells. In the absence of p53, Mos knockdown prevents multipolar mitoses and exerts genome-stabilizing effects. These results elucidate the mechanisms through which asymmetric cell division drives chromosomal instability in tetraploid cells. [ABSTRACT FROM AUTHOR]

Tumor radioresponsiveness depends on endothelial cell death, which leads in turn to tumor hypoxia. Radiation-induced hypoxia was recently shown to trigger tumor radioresistance by activating angiogenesis through hypoxia-inducible factor 1—regulated (HIF-1—regulated) cytokines. We show here that combining targeted radioiodide therapy with angiogenic inhibitors, such as canstatin, enhances direct tumor cell apoptosis, thereby overcoming radio-induced HIF-1—dependent tumor survival pathways in vitro and in vivo. We found that following dual therapy, HIF-1α increases the activity of the canstatin-induced αvβ5 signaling tumor apoptotic pathway and concomitantly abrogates mitotic checkpoint and tetraploidy triggered by radiation. Apoptosis in conjunction with mitotic catastrophe leads to lethal tumor damage. We discovered that HIF-1 displays a radiosensitizing activity that is highly dependent on treatment modalities by regulating key apoptotic molecular pathways. Our findings therefore support a crucial role for angiogenesis inhibitors in shifting the fate of radiation-induced HIF-1α activity from hypoxia-induced tumor radioresistance to hypoxia-induced tumor apoptosis. This study provides a basis for developing new biology-based clinically relevant strategies to improve the efficacy of radiation oncology, using HIF-1 as an ally for cancer therapy. [ABSTRACT FROM AUTHOR]

Tumor radioresponsiveness depends on endothelial cell death, which leads in turn to tumor hypoxia. Radiation-induced hypoxia was recently shown to trigger tumor radioresistance by activating angiogenesis through hypoxia-inducible factor 1—regulated (HIF-1—regulated) cytokines. We show here that combining targeted radioiodide therapy with angiogenic inhibitors, such as canstatin, enhances direct tumor cell apoptosis, thereby overcoming radio-induced HIF-1—dependent tumor survival pathways in vitro and in vivo. We found that following dual therapy, HIF-1α increases the activity of the canstatin-induced αvβ5 signaling tumor apoptotic pathway and concomitantly abrogates mitotic checkpoint and tetraploidy triggered by radiation. Apoptosis in conjunction with mitotic catastrophe leads to lethal tumor damage. We discovered that HIF-1 displays a radiosensitizing activity that is highly dependent on treatment modalities by regulating key apoptotic molecular pathways. Our findings therefore support a crucial role for angiogenesis inhibitors in shifting the fate of radiation-induced HIF-1α activity from hypoxia-induced tumor radioresistance to hypoxia-induced tumor apoptosis. This study provides a basis for developing new biology-based clinically relevant strategies to improve the efficacy of radiation oncology, using HIF-1 as an ally for cancer therapy. [ABSTRACT FROM AUTHOR]

Anthracyclin-treated tumor cells are particularly effective in eliciting an anticancer immune response, whereas other DNA-damaging agents such as etoposide and mitomycin C do not induce immunogenic cell death. Here we show that anthracyclins induce the rapid, preapoptotic translocation of calreticulin (CRT) to the cell surface. Blockade or knockdown of CRT suppressed the phagocytosis of anthracyclin-treated tumor cells by dendritic cells and abolished their immunogenicity in mice. The anthracyclin-induced CRT translocation was mimicked by inhibition of the protein phosphatase 1/GADD34 complex. Administration of recombinant CRT or inhibitors of protein phosphatase 1/GADD34 restored the immunogenicity of cell death elicited by etoposide and mitomycin C, and enhanced their antitumor effects in vivo. These data identify CRT as a key feature determining anticancer immune responses and delineate a possible strategy for immunogenic chemotherapy. [ABSTRACT FROM AUTHOR]

Abstract: The susceptibility of cells to apoptosis induction is deeply influenced by their position in the cell cycle. Unfortunately, however, current methods for the enrichment of cells in defined phases of the cell cycle are mostly based on the synchronization of cells by agents or conditions that are intrinsically toxic and induce apoptosis on their own. We developed a novel procedure for the purification of cells in distinct phases of the cell cycle. This method is based on the stable transfection of cells with a chimeric protein made up by histone H2B and green fluorescent protein (GFP). Cytofluorometric purification of cells defined by their size and their H2B-GFP-dependent fluorescence (which reflects chromatin and hence DNA content) allowed for the efficient separation of diploid and tetraploid cells in the fluorescence-activated cell sorter (FACS). Moreover, when applied to diploid cells, this method allowed for the enrichment of live, functional cells in the G1, S and G2 phases of the cell cycle. FACS-purified cells were viable and readily resumed the cell cycle upon reculture. While staurosporine was equally toxic for cells in any phase of the cell cycle, camptothecin was particularly toxic for cells in the S phase. Moreover, BAY11-7082, a specific inhibitor of the IKK complex required for NF-κB activation, exhibited a particular cell cycle-specific profile of toxicity (G2>S>G1). These results delineate a novel procedure for studying the intersection between cell cycle regulation and cell death mechanisms. [Copyright &y& Elsevier]

The interferon (IFN)-γ–induced TRAIL effector mechanism is a vital component of cancer immunosurveillance by natural killer (NK) cells in mice. Here we show that the main source of IFN-γ is not the conventional NK cell but a subset of B220+Ly6C− dendritic cells, which are atypical insofar as they express NK cell-surface molecules. Upon contact with a variety of tumor cells that are poorly recognized by NK cells, B220+NK1.1+ dendritic cells secrete high levels of IFN-γ and mediate TRAIL-dependent lysis of tumor cells. Adoptive transfer of these IFN-producing killer dendritic cells (IKDCs) into tumor-bearing Rag2−/−Il2rg−/− mice prevented tumor outgrowth, whereas transfer of conventional NK cells did not. In conclusion, we identified IKDCs as pivotal sensors and effectors of the innate antitumor immune response. [ABSTRACT FROM AUTHOR]

Mammalian cells were observed to die under conditions in which nutrients were depleted and, simultaneously, macroautophagy was inhibited either genetically (by a small interfering RNA targeting Atg5, Atg6/ Beclin 1–1, Atg10, orAtg12) or pharmacologically (by 3-methyladenine, hydroxychloroquine, bafilomycin A1, or monensin). Cell death occurred through apoptosis (type 1 cell death), since it was reduced by stabilization of mitochondrial membranes (with Bcl-2 or vMIA, a cytomegalovirus-derived gene) or by caspase inhibition. Under conditions in which the fusion between lysosomes and autophagosomes was inhibited, the formation of autophagic vacuoles was enhanced at a preapoptotic stage, as indicated by accumulation of LC3-II protein, ultrastructural studies, and an increase in the acidic vacuolar compartment. Cells exhibiting a morphology reminiscent of (autophagic) type 2 cell death, however, recovered, and only cells with a disrupted mitochondrial transmembrane potential were beyond the point of no return and inexorably died even under optimal culture conditions. All together, these data indicate that autophagy may be cytoprotective, at least under conditions of nutrient depletion, and point to an important cross talk between type 1 and type 2 cell death pathways. [ABSTRACT FROM AUTHOR]

Two cell lines that exemplify erythropoietin (EPO) receptor-positive tumors, human renal carcinoma cell lines RCC and the myelomonocytic leukemia cell line U937, were investigated for the apoptosis-modulatory potential of EPO. Cells cultured in the presence of EPO exhibited an elevated apoptotic response to cancer chemotherapeutic agents such as daunorubicin (Dauno) and vinblastine (VBL). Chemosensitization by EPO did not involve an increase in p53 activation, yet correlated with enhanced Bax/Bak-dependent mitochondrial membrane perturbation and caspase maturation. In vitro monotherapy with Dauno or VBL induced the degradation of I?Ba, provoked the translocation of NF-?B p65/50 to the nucleus and stimulated the expression of an NF-?B-activatable reporter gene. All these signs of NF-?B activation were perturbed in the presence of EPO. Inhibition of JAK2, one of the receptor-proximal elements of EPO-mediated signal transduction, greatly diminished the EPO-mediated chemosensitization and NF-?B inhibition. EPO lost its death-facilitating effects in the presence of an NF-?B inhibitor, underscoring the cause-effect relationship between EPO-mediated chemosensitization and NF-?B inhibition. Altogether, these results suggest that, at least in a specific subset of tumors, EPO receptor agonists can prevent activation of the NF-?B pathway, thereby enhancing the propensity of EPO receptor-positive tumor cells to undergo apoptosis.Oncogene (2005) 24, 737-745. doi:10.1038/sj.onc.1208205 Published online 6 December 2004 [ABSTRACT FROM AUTHOR]

Cells expressing the human immunodeficiency virus (HIV-1) envelope glycoprotein complex (Env) can fuse with CD4+ cells. When the apoptotic pathway is initiated in Env+ cells (‘donor cells’), co-culture with a healthy CD4+ fusion partner (‘acceptor cells’) results in apoptosis of the syncytium and thus is ‘contagious’. The cell-to-cell transmission of the lethal signal was only observed when the nuclei from donor cells exhibited pre-apoptotic chromatin condensation (PACC), correlating with comet assay-detectable DNA strand breaks, which precede caspase activation, as well as the loss of the mitochondrial transmembrane potential. Transmission of the lethal signal resulted into mitochondrial alterations, and caspase-dependent nuclear pyknosis with chromatinolysis affecting both the donor and the acceptor nuclei. In the presence of caspase inhibitors, all nuclei of the syncytium formed by fusion of the pre-apoptotic and the healthy cell manifested PACC, exhibited DNA lesions and lost transcriptional activity. Transmission of the lethal signal did not require donor cells to contain a nucleus or mitochondrial DNA, yet was inhibited when two mitochondrion-stabilizing proteins, Bcl-2 or vMIA, were overexpressed. Contagious apoptosis could be induced in primary human T cells, as well as in vivo, in T cells exposed to dying Env-expressing cells. Altogether, these data point to a novel mechanism through which HIV-1 can induce bystander killing. [ABSTRACT FROM AUTHOR]

The mitochondrial permeability transition pore complex (PTPC) is involved in the control of the mitochondrial membrane permeabilization during apoptosis, necrosis and autophagy, indeed, the adenine nucleotide translocator (ANT) and the voltage-dependent anion channel (VDAC), two major components of PTPC, are the targets of a variety of proapoptotic inducers. Using co-immuno- precipitation and proteomic analysis, we identified some of the interacting partners of ANT in several normal tissues and human cancer cell lines. During chemotherapy- induced apoptosis, some of these interactions were constant (e.g. ANT-VDAC), whereas others changed strongly concomitantly with the dissipation of the mitochondrial transmembrane potential and until nuclear degradation occurred (e.g. Bax, Bcl-2, subunits of the respiratory chain, a subunit of the phosphatase PP2A, phospholipase PLC β 4 and IP3 receptor). in addition, a glutathione-S-transferase (GST) interacts with ANT in normal tissue, in colon carcinoma cells and in vitro. This interaction is lost during apoptosis induction, suggesting that GST behaves as an endogenous repressor of PTPC and ANT pore opening. Thus, ANT is connected to mitochondrial proteins as well as to proteins from other organelles such as the endoplasmic reticulum forming a dynamic polyprotein complex. Changes within this ANT interactome coordinate the lethal response of cells to apoptosis induction. [ABSTRACT FROM AUTHOR]

Cells expressing the envelope glycoprotein complex (Env) encoded by the human immunodeficiency virus can fuse with cells expressing Env receptors (CD4 and CXCR4). The resulting syncytia undergo apoptosis. We developed a cytofluorometric assay for the quantitation of syncytium formation and syncytial apoptosis. Using this methodology, we show that caspase activation in syncytia is inhibited by pharmacological or genetic intervention on cyclin-dependent kinase-1, p53, and mitochondrial membrane permeabilization (MMP). Thus, transfection of fusing cells with the viral mitochondrial inhibitor of apoptosis encoded by cytomegalovirus, a specific inhibitor of MMP, prevented the mitochondrial cytochrome c release and abolished simultaneously the activation of caspase-3. Conversely, inhibition of caspases did not prevent MMP. These results indicate that Env-elicited syncytial apoptosis involves the intrinsic (mitochondrial) pathway. [Copyright &y& Elsevier]

Mitochondria undergo two major changes during early apoptosis. On the one hand, the outer mitochondrial membrane becomes permeable to proteins, resulting in the release of soluble intermembrane proteins (SIMPs) from the mitochondrion. On the other hand, the inner mitochondrial membrane transmembrane potential (ΔΨm) is reduced. These changes occur in most, if not all, models of cell death and can be taken advantage of to detect apoptosis at an early stage. Here, we delineate methods for the detection of alterations in the ΔΨm, based on the incubation of cells with cationic lipophilic fluorochromes, the uptake of which is driven by the ΔΨm. Certain ΔΨm-sensitive dyes can be combined with other fluorochromes to detect simultaneously cellular viability, plasma membrane exposure of phosphatidylserine residues, or the mitochondrial production of reactive oxygen species (ROS). In addition, we describe an immunofluorescence method for the detection of two functionally important proteins translocating from mitochondria, namely, the caspase co-activator cytochrome c and the caspase-independent death effector apoptosis inducing factor (AIF). [Copyright &y& Elsevier]

In the present study, we have investigated the mechanisms by which the restoration of wild-type (wt) p53 functions in p53 mutant cells increases their susceptibility to the cytotoxic action of tumor necrosis factor (TNF). Our data indicate that the resistance of p53mutated cl.1001 cells to TNF-induced cell death was not due to a defect in the expression of TRADD and FADD, yet correlated with a reduced caspase-8 activation as well as a deficient mitochondrial membrane permeabilization. Moreover, cl.1001 cells failed to translocate the mitochondrial AIF and cytochrome to the nucleus and to the cytosol, respectively, in response to TNF. Sensitization of these cells, following infection with a recombinant adenovirus encoding wtp53, to TNF-induced cytotoxicity resulted in the restoration of caspase-8 cleavage and the reestablishment of mitochondrial signs of apoptosis. These findings suggest that the crosstalk between p53 and TNF-induced cell death depends on mitochondria and that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF-resistant tumors to the cytotoxic action of this cytokine. [ABSTRACT FROM AUTHOR]

An increasing number of experimental chemotherapeutic agents induce apoptosis by directly triggering mitochondrial membrane permeabilization (MMP). Here we examined MMP induced by lonidamine, arsenite, and the retinoid derivative CD437. Cells overexpressing the cytomegalovirus-encoded protein vMIA, a protein which interacts with the adenine nucleotide translocator, were strongly protected against the MMP-inducing and apoptogenic effects of lonidamine, arsenite, and CD437. In a cell-free system, lonidamine, arsenite, and CD437 induced the permeabilization of ANT proteoliposomes, yet had no effect on protein-free liposomes. The ANT-dependent membrane permeabilization was inhibited by the two ANT ligands ATP and ADP, as well as by recombinant Bcl-2 protein. Lonidamine, arsenite, and CD437, added to synthetic planar lipid bilayers containing ANT, elicited ANT channel activities with clearly distinct conductance levels of 20±7, 100±30, and 47±7 pS, respectively. Altering the ATP/ADP gradient built up on the inner mitochondrial membrane by inhibition of glycolysis and/or oxidative phosphorylation differentially modulated the cytocidal potential of lonidamine, arsenite, and CD437. Inhibition of F0F1ATPase without glycolysis inhibition sensitized to lonidamine-induced cell death. In contrast, only the combined inhibition of glycolysis plus F0F1ATPase sensitized to arsenite-induced cell death. No sensitization to cell death induction by CD437 was achieved by glucose depletion and/or oligomycin addition. These results indicate that ANT is a target of lonidamine, arsenite, and CD437 and unravel an unexpected heterogeneity in the mode of action of these three compounds. Oncogene (2001) 20, 7579–7587. [ABSTRACT FROM AUTHOR]