Multipolar mitosis of tetraploid cells: inhibition by p53 and dependency on Mos.

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.

Calreticulin exposure dictates the immunogenicity of cancer cell death.

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.

A novel dendritic cell subset involved in tumor immunosurveillance.

The interferon (IFN)-gamma-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-gamma 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-gamma 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.

Caspase-dependent immunogenicity of doxorubicin-induced tumor cell death.

Systemic anticancer chemotherapy is immunosuppressive and mostly induces nonimmunogenic tumor cell death. Here, we show that even in the absence of any adjuvant, tumor cells dying in response to anthracyclins can elicit an effective antitumor immune response that suppresses the growth of inoculated tumors or leads to the regression of established neoplasia. Although both antracyclins and mitomycin C induced apoptosis with caspase activation, only anthracyclin-induced immunogenic cell death was immunogenic. Caspase inhibition by Z-VAD-fmk or transfection with the baculovirus inhibitor p35 did not inhibit doxorubicin (DX)-induced cell death, yet suppressed the immunogenicity of dying tumor cells in several rodent models of neoplasia. Depletion of dendritic cells (DCs) or CD8+T cells abolished the immune response against DX-treated apoptotic tumor cells in vivo. Caspase inhibition suppressed the capacity of DX-killed cells to be phagocytosed by DCs, yet had no effect on their capacity to elicit DC maturation. Freshly excised tumors became immunogenic upon DX treatment in vitro, and intratumoral inoculation of DX could trigger the regression of established tumors in immunocompetent mice. These results delineate a procedure for the generation of cancer vaccines and the stimulation of anti-neoplastic immune responses in vivo.

Lysosomal membrane permeabilization induces cell death in a mitochondrion-dependent fashion.

A number of diseases are due to lysosomal destabilization, which results in damaging cell loss. To investigate the mechanisms of lysosomal cell death, we characterized the cytotoxic action of two widely used quinolone antibiotics: ciprofloxacin (CPX) or norfloxacin (NFX). CPX or NFX plus UV light (NFX*) induce lysosomal membrane permeabilization (LMP), as detected by the release of cathepsins from lysosomes. Inhibition of the lysosomal accumulation of CPX or NFX suppresses their capacity to induce LMP and to kill cells. CPX- or NFX-triggered LMP results in caspase-independent cell death, with hallmarks of apoptosis such as chromatin condensation and phosphatidylserine exposure on the plasma membrane. LMP triggers mitochondrial membrane permeabilization (MMP), as detected by the release of cytochrome c. Both CPX and NFX* cause Bax and Bak to adopt their apoptotic conformation and to insert into mitochondrial membranes. Bax-/- Bak-/- double knockout cells fail to undergo MMP and cell death in response to CPX- or NFX-induced LMP. The single knockout of Bax or Bak (but not Bid) or the transfection-enforced expression of mitochondrion-targeted (but not endoplasmic reticulum-targeted) Bcl-2 conferred protection against CPX (but not NFX*)-induced MMP and death. Altogether, our data indicate that mitochondria are indispensable for cell death initiated by lysosomal destabilization.

Radiation and inhibition of angiogenesis by canstatin synergize to induce HIF-1alpha-mediated tumor apoptotic switch.

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-1alpha increases the activity of the canstatin-induced alpha(v)beta(5) 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-1alpha 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.

Inhibition of macroautophagy triggers apoptosis.

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, or Atg12) 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.

Mutant MyoD lacking Cdc2 phosphorylation sites delays M-phase entry.

The transcription factors MyoD and Myf-5 control myoblast identity and differentiation. MyoD and Myf-5 manifest opposite cell cycle-specific expression patterns. Here, we provide evidence that MyoD plays a pivotal role at the G(2)/M transition by controlling the expression of p21(Waf1/Cip1) (p21), which is believed to regulate cyclin B-Cdc2 kinase activity in G(2). In growing myoblasts, MyoD reaccumulates during G(2) concomitantly with p21 before entry into mitosis; MyoD is phosphorylated on Ser5 and Ser200 by cyclin B-Cdc2, resulting in a decrease of its stability and down-regulation of both MyoD and p21. Inducible expression of a nonphosphorylable MyoD A5/A200 enhances the MyoD interaction with the coactivator P/CAF, thereby stimulating the transcriptional activation of a luciferase reporter gene placed under the control of the p21 promoter. MyoD A5/A200 causes sustained p21 expression, which inhibits cyclin B-Cdc2 kinase activity in G(2) and delays M-phase entry. This G(2) arrest is not observed in p21(-/-) cells. These results show that in cycling cells MyoD functions as a transcriptional activator of p21 and that MyoD phosphorylation is required for G(2)/M transition.

Metabolic effects of fasting on human and mouse blood in vivo.

Starvation is a strong physiological stimulus of macroautophagy/autophagy. In this study, we addressed the question as to whether it would be possible to measure autophagy in blood cells after nutrient deprivation. Fasting of mice for 48 h (which causes ∼20% weight loss) or starvation of human volunteers for up to 4 d (which causes <2% weight loss) provokes major changes in the plasma metabolome, yet induces only relatively minor alterations in the intracellular metabolome of circulating leukocytes. White blood cells from mice and human volunteers responded to fasting with a marked reduction in protein lysine acetylation, affecting both nuclear and cytoplasmic compartments. In circulating leukocytes from mice that underwent 48-h fasting, an increase in LC3B lipidation (as assessed by immunoblotting and immunofluorescence) only became detectable if the protease inhibitor leupeptin was injected 2 h before drawing blood. Consistently, measurement of an enhanced autophagic flux was only possible if white blood cells from starved human volunteers were cultured in the presence or absence of leupeptin. Whereas all murine leukocyte subpopulations significantly increased the number of LC3B+ puncta per cell in response to nutrient deprivation, only neutrophils from starved volunteers showed signs of activated autophagy (as determined by a combination of multi-color immunofluorescence, cytofluorometry and image analysis). Altogether, these results suggest that white blood cells are suitable for monitoring autophagic flux. In addition, we propose that the evaluation of protein acetylation in circulating leukocytes can be adopted as a biochemical marker of organismal energetic status.

Chemosensitization by erythropoietin through inhibition of the NF-kappaB rescue pathway.

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 IkappaBalpha, provoked the translocation of NF-kappaB p65/50 to the nucleus and stimulated the expression of an NF-kappaB-activatable reporter gene. All these signs of NF-kappaB 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-kappaB inhibition. EPO lost its death-facilitating effects in the presence of an NF-kappaB inhibitor, underscoring the cause-effect relationship between EPO-mediated chemosensitization and NF-kappaB inhibition. Altogether, these results suggest that, at least in a specific subset of tumors, EPO receptor agonists can prevent activation of the NF-kappaB pathway, thereby enhancing the propensity of EPO receptor-positive tumor cells to undergo apoptosis.

Enrichment of non-synchronized cells in the G1, S and G2 phases of the cell cycle for the study of apoptosis.

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-kappaB 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.

Dynamic evolution of the adenine nucleotide translocase interactome during chemotherapy-induced apoptosis.

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-immunoprecipitation 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 beta 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.

Wild-type p53 induced sensitization of mutant p53 TNF-resistant cells: role of caspase-8 and mitochondria.

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 p53-mutated 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 c 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 cross-talk 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.

Quantitation of mitochondrial alterations associated with apoptosis.

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 (DeltaPsi(m)) 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 DeltaPsi(m), based on the incubation of cells with cationic lipophilic fluorochromes, the uptake of which is driven by the DeltaPsi(m). Certain DeltaPsi(m)-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).

Mitochondrion-dependent caspase activation by the HIV-1 envelope.

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.

Cytofluorometric assessment of cell cycle progression.

One of the most prominent features of cellular senescence, a stress response that prevents the propagation of cells that have accumulated potentially oncogenic alterations, is a permanent loss of proliferative potential. Thus, at odds with quiescent cells, which resume proliferation when stimulated to do so, senescent cells cannot proceed through the cell cycle even in the presence of mitogenic factors. Here, we describe a set of cytofluorometric techniques for studying how chemical and/or physical stimuli alter the cell cycle in vitro, in both qualitative and quantitative terms. Taken together, these methods allow for the identification of bona fide cytostatic effects as well as for a refined characterization of cell cycle distributions, providing information on proliferation, DNA content as well as on the presence of cell cycle phase-specific markers. At the end of the chapter, a set of guidelines is offered to assist researchers that approach the study of the cell cycle with the interpretation of results.

Immunosurveillance against tetraploidization-induced colon tumorigenesis.

Circumstantial evidence suggests that colon carcinogenesis can ensue the transient tetraploidization of (pre-)malignant cells. In line with this notion, the tumor suppressors APC and TP53, both of which are frequently inactivated in colon cancer, inhibit tetraploidization in vitro and in vivo. Here, we show that-contrarily to their wild-type counterparts- Tp53 (-/-) colonocytes are susceptible to drug-induced or spontaneous tetraploidization in vitro. Colon organoids generated from tetraploid Tp53 (-/-) cells exhibit a close-to-normal morphology as compared to their diploid Tp53 (-/-) counterparts, yet the colonocytes constituting these organoids are characterized by an increased cell size and an elevated expression of the immunostimulatory protein calreticulin on the cell surface. The subcutaneous injection of tetraploid Tp53 (-/-) colon organoids led to the generation of proliferating tumors in immunodeficient, but not immunocompetent, mice. Thus, tetraploid Tp53 (-/-) colonocytes fail to survive in immunocompetent mice and develop neoplastic lesions in immunocompromised settings only. These results suggest that tetraploidy is particularly oncogenic in the context of deficient immunosurveillance.

Cytofluorometric purification of diploid and tetraploid cancer cells.

During malignant transformation, cells can increase their ploidy and hence become polyploid (mostly tetraploid). Frequently, however, tetraploid cells undergo asymmetric divisions, in turn entailing a reduction in ploidy and the acquisition of a pseudo-diploid, aneuploid state. To investigate such a stepwise aneuploidization process, we developed a cytofluorometric method (based on the heterogeneity in cell size and/or chromatin content) that allows for the cloning and subsequent functional analysis of cells with distinct ploidies. Here, we detail this methodology, which has been instrumental for investigating the functional link between ploidy status and oncogenesis.

A fluorescence-microscopic and cytofluorometric system for monitoring the turnover of the autophagic substrate p62/SQSTM1.

Autophagic flux can be measured by determining the declining abundance of autophagic substrates such as sequestosome 1 (SQSTM1, better known as p62), which is sequestered in autophagosomes upon its direct interaction with LC3. However, the total amount of p62 results from two opposed processes, namely its synthesis (which can be modulated by some cellular stressors including autophagy inducers) and its degradation. To avoid this problem, we generated a stable cell line expressing a chimeric protein composed by p62 and the HaloTag (®) protein, which serves as a receptor for fluorescent HaloTag (®) ligands. Upon labeling with HaloTag (®) ligands (which form covalent, near-to-undissociable bonds with the Halotag (®) receptor) and washing, the resulting fluorescent labeling is not influenced by de novo protein synthesis, therefore allowing for the specific monitoring of the fusion protein decline without any interference by protein synthesis. We demonstrate that a HaloTag (®) -p62 fusion protein stably expressed in suitable cell lines can be used to monitor autophagy by flow cytometry and automated fluorescence microscopy. We surmise that this system could be adapted to high-throughput applications.

Extracellular ATP acts on P2Y2 purinergic receptors to facilitate HIV-1 infection.

Extracellular adenosine triphosphate (ATP) can activate purinergic receptors of the plasma membrane and modulate multiple cellular functions. We report that ATP is released from HIV-1 target cells through pannexin-1 channels upon interaction between the HIV-1 envelope protein and specific target cell receptors. Extracellular ATP then acts on purinergic receptors, including P2Y2, to activate proline-rich tyrosine kinase 2 (Pyk2) kinase and transient plasma membrane depolarization, which in turn stimulate fusion between Env-expressing membranes and membranes containing CD4 plus appropriate chemokine co-receptors. Inhibition of any of the constituents of this cascade (pannexin-1, ATP, P2Y2, and Pyk2) impairs the replication of HIV-1 mutant viruses that are resistant to conventional antiretroviral agents. Altogether, our results reveal a novel signaling pathway involved in the early steps of HIV-1 infection that may be targeted with new therapeutic approaches.