Walter Malorni

Classification of cell death: recommendations of the Nomenclature Committee on Cell Death

Different types of cell death are often defined by morphological criteria, without a clear reference to precise biochemical mechanisms. The Nomenclature Committee on Cell Death (NCCD) proposes unified criteria for the definition of cell death and of its different morphologies, while formulating several caveats against the misuse of words and concepts that slow down progress in the area of cell death research. Authors, reviewers and editors of scientific periodicals are invited to abandon expressions like ‘percentage apoptosis’ and to replace them with more accurate descriptions of the biochemical and cellular parameters that are actually measured. Moreover, at the present stage, it should be accepted that caspase-independent mechanisms can cooperate with (or substitute for) caspases in the execution of lethal signaling pathways and that ‘autophagic cell death’ is a type of cell death occurring together with (but not necessarily by) autophagic vacuolization. This study details the 2009 recommendations of the NCCD on the use of cell death-related terminology including ‘entosis’, ‘mitotic catastrophe’, ‘necrosis’, ‘necroptosis’ and ‘pyroptosis’.

Molecular definitions of cell death subroutines: recommendations of the Nomenclature Committee on Cell Death 2012

In 2009, the Nomenclature Committee on Cell Death (NCCD) proposed a set of recommendations for the definition of distinct cell death morphologies and for the appropriate use of cell death-related terminology, including ‘apoptosis’, ‘necrosis’ and ‘mitotic catastrophe’. In view of the substantial progress in the biochemical and genetic exploration of cell death, time has come to switch from morphological to molecular definitions of cell death modalities. Here we propose a functional classification of cell death subroutines that applies to both in vitro and in vivo settings and includes extrinsic apoptosis, caspase-dependent or -independent intrinsic apoptosis, regulated necrosis, autophagic cell death and mitotic catastrophe. Moreover, we discuss the utility of expressions indicating additional cell death modalities. On the basis of the new, revised NCCD classification, cell death subroutines are defined by a series of precise, measurable biochemical features.

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.

Galectin‐3 overexpression protects from apoptosis by improving cell adhesion properties

Galectin‐3 is a carbohydrate‐binding protein endowed with affinity for β‐galactosides. It plays a role in cell–cell and cell–matrix interactions. Furthermore, it has been hypothesized to be involved in tumor progression and metastasis. To address the role of galectin‐3 in the invasive and metastatic processes, we stably overexpressed galectin‐3 in human breast carcinoma cell lines, and we evaluated the influence of elevated galectin‐3 expression on several cell features, including cellular homotypic and heterotypic interactions and cell survival. No differences in various parameters related with cell growth features and proliferation were detected. By contrast, we found that galectin‐3 overexpressing cells, with respect to low galectin‐3 expressing cells, exerted: (1) a significantly enhanced adhesion to laminin, fibronectin and vitronectin exerted both directly or via increased expression of specific integrins, e.g., alpha‐4 and beta‐7; (2) a remodeling of those cytoskeletal elements associated with cell spreading, i.e., microfilaments; (3) an enhanced survival upon exposure to different apoptotic stimuli, such as cytokine and radiation. Collectively, our results indicate that overexpression of galectin‐3 may play a role in tumor cell invasion and metastasis by specifically influencing cell adhesion to the extracellular matrix. This may confer selective survival advantage and resistance to the particular homeless‐induced apoptosis called anoikia. Int. J. Cancer 85:545–554, 2000.

CD95 (APO‐1/Fas) linkage to the actin cytoskeleton through ezrin in human T lymphocytes: a novel regulatory mechanism of the CD95 apoptotic pathway

CD95 (APO‐1/Fas) is a member of the tumor necrosis factor receptor family, which can trigger apoptosis in a variety of cell types. However, little is known of the mechanisms underlying cell susceptibility to CD95‐mediated apoptosis. Here we show that human T cells that are susceptible to CD95‐mediated apoptosis, exhibit a constitutive polarized morphology, and that CD95 colocalizes with ezrin at the site of cellular polarization. In fact, CD95 co‐immunoprecipitates with ezrin exclusively in lymphoblastoid CD4+ T cells and primary long‐term activated T lymphocytes, which are prone to CD95‐mediated apoptosis, but not in short‐term activated T lymphocytes, which are refractory to the same stimuli, even expressing equal levels of CD95 on the cell membrane. Pre‐treatment with ezrin antisense oligonucleotides specifically protected from the CD95‐mediated apoptosis. Moreover, we show that the actin cytoskeleton integrity is essential for this function. These findings strongly suggest that the CD95 cell membrane polarization, through an ezrin‐mediated association with the actin cytoskeleton, is a key intracellular mechanism in rendering human T lymphocytes susceptible to the CD95‐mediated apoptosis.

Unravelling the Complexity of T Cell Abnormalities in Common Variable Immunodeficiency

We investigated several phenotypic and functional parameters of T cell-mediated immunity in a large series of common variable immunodeficiency (CVID) patients. We demonstrated that the vast majority of CVID patients presented multiple T cell abnormalities intimately related among them, the severity of which was reflected in a parallel loss of CD4+ naive T cells. A strong correlation between the number of CD4+ naive T cells and clinical features was observed, supporting the subgrouping of patients according to their number of naive CD4+ T lymphocytes. A reduced thymic output and disrupted CD4+ and CD8+ TCR repertoires paralleled the contraction of CD4+ naive T cell pools. The evaluation of activation markers and cytokine production indicated a strong T cell activation that was significantly related to the increased levels of T cell turnover and apoptosis. Finally, discrete genetic profiles could be demonstrated in groups of patients showing extremely diverse T cell subset composition and function. Naive CD4+ T cell levels were significantly associated with the switched memory B cell-based classification, although the concordance between the respective subgroups did not exceed 58.8%. In conclusion, our data highlight the key role played by the T cell compartment in the pathogenesis of CVID, pointing to the need to consider this aspect for classification of this disease.

Mitochondrial fission and cristae disruption increase the response of cell models of Huntington's disease to apoptotic stimuli

Huntingtons disease (HD), a genetic neurodegenerative disease caused by a polyglutamine expansion in the Huntingtin (Htt) protein, is accompanied by multiple mitochondrial alterations. Here, we show that mitochondrial fragmentation and cristae alterations characterize cellular models of HD and participate in their increased susceptibility to apoptosis. In HD cells, the increased basal activity of the phosphatase calcineurin dephosphorylates the pro‐fission dynamin related protein 1 (Drp1), increasing its mitochondrial translocation and activation, and ultimately leading to fragmentation of the organelle. The fragmented HD mitochondria are characterized by cristae alterations that are aggravated by apoptotic stimulation. A genetic analysis indicates that correction of mitochondrial elongation is not sufficient to rescue the increased cytochrome c release and cell death observed in HD cells. Conversely, the increased apoptosis can be corrected by manoeuvres that prevent fission and cristae remodelling. In conclusion, the cristae remodelling of the fragmented HD mitochondria contributes to their hypersensitivity to apoptosis.

N-Acetylcysteine inhibits apoptosis and decreases viral particles in HIV-chronically infected U937 cells

Apoptosis or programmed cell death (PCD) is a type of death occurring in various physiological processes. Several data suggest that: (1) apoptosis may play a critical role in AIDS pathogenesis; (2) an increase of endocellular free radical levels can be associated with activation of previously latent HIV virus. Tumor necrosis factor (TNF), a cytokine capable of inducing oxygen free radicals and apoptosis, appears also to be involved in HIV activation. The present findings, which elucidate a relationship between the percentage of apoptotic cells, reduced glutathione (GSH) depletion and an increase of p24 antigenemia, suggest that pretreatment with N‐acetylcysteine (NAC) is capable of decreasing the above‐mentioned phenomena in HIV‐infected U937 cells.

Galectin-1 sensitizes resting human T lymphocytes to Fas (CD95)-mediated cell death via mitochondrial hyperpolarization, budding, and fission

Galectins have emerged as a novel family of immunoregulatory proteins implicated in T cell homeostasis. Recent studies showed that galectin-1 (Gal-1) plays a key role in tumor-immune escape by killing antitumor effector T cells. Here we found that Gal-1 sensitizes human resting T cells to Fas (CD95)/caspase-8-mediated cell death. Furthermore, this protein triggers an apoptotic program involving an increase of mitochondrial membrane potential and participation of the ceramide pathway. In addition, Gal-1 induces mitochondrial coalescence, budding, and fission accompanied by an increase and/or redistribution of fission-associated molecules h-Fis and DRP-1. Importantly, these changes are detected in both resting and activated human T cells, suggesting that Gal-1-induced cell death might become an excellent model to analyze the morphogenetic changes of mitochondria during the execution of cell death. This is the first association among Gal-1, Fas/Fas ligand-mediated cell death, and the mitochondrial pathway, providing a rational basis for the immunoregulatory properties of Gal-1 in experimental models of chronic inflammation and cancer.

Galectin‐3 overexpression protects from cell damage and death by influencing mitochondrial homeostasis

Galectins are a family of proteins involved in several cell processes, including their survival and death. Galectin‐3 has in particular been described as an anti‐apoptotic molecule entangled with a number of subcellular activities including anoikis resistance. In this work we partially address the mechanisms underlying this activity pointing at two key factors in injury progression: the alteration of mitochondrial membrane potential and the formation of reactive oxygen species. Overexpression of galectin‐3 appears in fact to exert a protective effect towards both these events. On the basis of these data, we propose a reappraisal of the role of galectin‐3 as a regulator of mitochondrial homeostasis.