Cell Death Subroutines Research Articles

Vital dyes and virtual deaths

Dear Editor, In 2009 and 2012, Cell Death & Differentiation has published recommendations of the Nomenclature Committee on Cell Death (NCCD) for the Classification of cell death and Molecular definitions of cell death subroutines, respectively. Under the subtitle When is a cell ‘dead'?, and in an accompanying table entitled Molecular or morphological criteria to define dead cells, the 2009 paper featured a set of three criteria; the fulfillment of any single one is considered to indicate cell death.1 The first criterion in this list is Loss of plasma membrane integrity, and parameters commonly used to assess integrity of the plasma membrane were mentioned, namely exclusion from cells of vital dyes like trypan blue or propidium iodide. It should be noted that injuries of the plasma membrane may be transient. This is now widely accepted, and here some key publications related to this issue are reviewed. For instance, mechanical stress can cause lesions of the plasma membrane spanning several micrometers, but they can be rapidly repaired by calcium influx-dependent mechanisms.2 Similarly, plasma membrane perforation by various pore forming proteins may be reversible.3 Some pore-forming proteins, for example, streptolysin O, render cells permeable for vital dyes and even for macromolecules, whereas small pore-forming proteins like α-toxin of S. aureus form lesions too small to permit flux of these molecules. However, all kinds of membrane lesions cause a drop of potassium and cellular ATP concentrations, which however, may be reversible if reconstitution of membrane integrity is achieved in time. Transient plasma membrane damage may not only trigger transcriptional responses, and release of mediators,4 but also cell cycle progression.5 Therefore, loss of membrane integrity is not even a reliable predictor of imminent death. Cellular tools for mending perforated membranes are diverse and comprise calcium influx-dependent or calcium influx-independent mechanisms. However, vesicle traffic emerges as a common denominator of resealing processes,6, 7, 8 which appear to be evolutionarily conserved and operate in vivo.8 As all definitions of cell death are necessarily ‘package deals' with the tacit assumption that the observed changes are irreversible, investigators are facing the question for how long they have to observe an indicator of a presumed, irreversible process before they may actually regard it as such. As membrane repair depends on the energy consuming task of vesicle traffic, irreversibility of loss of membrane integrity may be ascertained when cellular ATP is depleted. Although more work is required to fully understand and appreciate the mechanisms and relevance of membrane repair, the available evidence warrants a reassessment of Loss of plasma membrane integrity in itself as a bona fide criterion to define cell death.

Molecular Determinants of Bendamustine (BDM) Toxicity towards Hodgkin (H) and Reed-Sternberg (RS) Cell Lines From Hodgkin Lymphoma (HL).

AbstractAbstract 2763Patients with HL recurring after stem cell transplantation (SCT) are mostly incurable. Therefore development of new agents and strategies is an impellent medical need in this setting. BDM is a hybrid purine analogue/bi-functional alkylator active in B-cell tumors. Despite preliminary evidences of efficacy in refractory HL, the molecular mechanisms underlying the potential activity of BDM towards malignant H-RS cells were never explored.We investigated patterns of BDM cytotoxicity (12.5 to 100 mmol/L) in a panel of HL-derived cell lines (L1236, L428, KMH2, HDLM2, L540) and its time-dependent (8, 24, 48, 72 hrs) effects on genes involved in DNA-damage stress and repair response, apoptosis and cell cycle checkpoints by Q-RT-PCR. BDM induced a significant time- and dose-dependent inhibition of growth and survival in all cell types. L1236 cells displayed the highest sensitivity to the agent with an IC50, at 48 hrs, of 10.7 mmol/L, as opposed to KMH2, L428, L540 and HDLM2 cells with IC50 of 11.1, 12.4, 14.8 and 16.2 mmol/L, respectively. BDM elicited a dose-dependent increase of apoptosis (30% to 50% at 72 hrs) in all cell lines, as shown by Annexin-V/propidium iodide staining. The exquisite sensitivity to BDM of L1236 cells was confirmed by clonogenic assays, since these cells, after a 24 hr exposure, showed the lowest IC50 for secondary colony formation (0.7± 0.06 mmol/L) as compared to all other cell lines (IC50 range: 3.1 ± 0.28 to 15.0 ± 1.27 mmol/L).Most notably, however, BDM, within the same concentration range, activated different cell death subroutines among the various cell lines. Q-RT-PCR disclosed that BDM-induced cell death in L1236 was mainly dependent on triggering of apoptosis, as shown by the early (8–24 hrs) up regulation of the proapoptotic genes NOXA and p21, but not p27, without appreciable changes in expression levels of genes related to activation of the mitotic catastrophe process, i.e. PLK1, Aurora A kinase (AAK), and cyclin B1. In contrast, induction of mitotic catastrophe was a main determinant of BDM action on KMH2, L428 and L540 cells, as shown by early (8 hrs) and sustained (48 hrs) down-regulation of PLK1, AAK and cyclin B1 genes, without early changes in NOXA and p21 expression levels. This was confirmed by highly aberrant mitosis and further multinucleation. Interestingly, BDM induced the sequential activation of both these cell death pathways in HDLM2 cells only. In this cells, the early (8–24 hrs) down regulation of PLK1, AAK and cyclin B1 genes, was later (48–72 hrs) followed by a significant induction of NOXA, p21 and p27 genes. The highest sensitivity of L1236 cells to BDM correlated with the delayed (72 hrs) induction of the DNA-repair genes EXO-1 and ATR, but not ATM, as opposed to the earlier (24 hrs) and sustained up regulation of these DNA-repair genes, and of ATM, in all other cell types. Accordingly, only after 72 hrs from exposure to BDM, the proliferation-related genes C-MYC, E2F2 and cyclins (D1, D2) were up regulated in surviving L1236 cells, along with a >70% reduction in G0/G1 cells, a significant (48%) increase of cells in the G2 phase of the cell cycle and a 20% increment of those in S phase. Conversely, in the other cell lines, S phase accumulation (35% to 60.5% increase) and up regulation of proliferation-related genes in surviving cells occurred, as early as 24 hrs after exposure to BDM.Overall, these results indicate that BDM affects different cell death pathways among HL-derived cell lines. Specifically, in L1236 cells the agent induces the early up regulation of proapototic genes and G2 arrest, accompanied by a delayed activation of DNA repair genes. This changes may lead to cell death through apoptosis rather than mitotic catastrophe which, conversely, appeared the predominant cell death pathway activated by BDM in the other cell lines. Notably, while determinants of BDM toxicity in ‘bona fide' HL cell lines consistently overlap with those described for tumor B-cells of non-Hodgkin lymphomas (mitotic catastrophe), L1236 cells display a different response pattern to the agent, more reminiscent of BDM action on myeloma tumor cells. Since only L1236 cells are clonotypically related to primary H-RS cells from which were derived, our results suggest that optimal BDM dosing and scheduling in HL may be different from those adopted for other B-cell tumors. In this sense, a lower BDM dosing through a weekly schedule is currently being tested at our institution in refractory HL. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Transient receptor potential melastatin 4 and cell death

Cell death proceeds by way of a variety of “cell death subroutines,” including several types of “apoptosis,” “regulated necrosis,” and others. “Accidental necrosis” due to profound adenosine triphosphate (ATP) depletion or oxidative stress is distinguished from regulated necrosis by the absence of death receptor signaling. However, both accidental and regulated necrosis have in common the process of “oncosis,” a physiological process characterized by Na+ influx and cell volume increase that, in necrotic cell death, is required to produce the characteristic features of membrane blebbing and membrane rupture. Here, we review emerging evidence that the monovalent cation channel, transient receptor potential melastatin 4 (TRPM4), is involved in the cell death process of oncosis. Potential involvement of TRPM4 in oncosis is suggested by the fact that the two principal regulators of TRPM4, intracellular ATP and Ca2+, are both altered during necrosis in the direction that causes TRPM4 channel opening. Under physiological conditions, activation of TRPM4 promotes Na+ influx and cell depolarization. Under pathological conditions, unchecked activation of TRPM4 leads to Na+ overload, cell volume increase, blebbing and cell membrane rupture, the latter constituting the irreversible end stage of necrosis. Emerging data indicate that TRPM4 plays a crucial role as end executioner in the accidental necrotic death of ATP-depleted or redox-challenged endothelial and epithelial cells, both in vitro and in vivo. Future studies will be needed to determine whether TRPM4 also plays a role in regulated necrosis and apoptosis.

MiRDeathDB: a database bridging microRNAs and the programmed cell death

Dear Editor, MicroRNAs (miRNAs) are a class of small non-coding RNAs, which negatively regulate protein-coding genes and have been associated to almost all known physiological and pathological processes.1 Apoptosis, autophagy and programmed necrosis are three distinct yet intimately connected functional modules of programmed cell death (PCD). In the past few years, increasing evidences suggested that aberrant expression of miRNAs regulate different subroutines of PCD.2, 3 Exploring the roles of miRNAs in PCD network has attracted interests of researchers from a wide range of areas. Therefore, we developed miRDeathDB (http://rna-world.org/mirdeathdb/), a knowledge depository that integrates information for experimentally identified miRNAs and their targets in PCD network. In total, 210 records covering 86 miRNAs and 95 protein-coding genes were collected in five model species. Users can search the PCD-associated miRNAs and their targets based on gene principal symbols. In addition, users can retrieve plenty of genomic, proteomic and disease-associated data via links to external resources. Detailed information on database construction and usage can be found in Supplementary File. miRDeathDB provides a valuable resource for archiving PCD knowledge and can be used to understand general features of miRNAs in cell death network. For example, we found that miR-21 regulated eight cell death-related protein, while BCL2 was regulated by 16 miRNAs (Supplementary Figure 1). Similarly, autophagic protein BECN1 and MAP1LC3B were also targeted by multiple miRNAs (Supplementary Figure 1). These examples indicated a single miRNA could target multiple cell death-related proteins and one cell death-related protein could be regulated by multiple miRNAs, highlighting the plasticity and complexity of the regulatory relationships between miRNAs and cell death-related proteins. Cross-talk among different modules of PCD is intricate and fine tuned. Accumulating evidences suggested cell death subroutines share several protein-coding regulators that are essential for their respective executions.2 It seems also true for miRNA regulators. As shown in Supplementary Figure 1, miR-101, miR-204 and miR-221 have dual roles in apoptosis and autophagy. On the basis of these observations, we recently proposed that miRNAs may work as molecular switches between apoptosis and autophagy.3 miRNAs can target common protein-coding regulators of these two cell death modules, or simultaneously modulating multiple targets, which function either in autophagy or apoptosis. Although the roles of miRNAs in programmed necrosis still remain elusive at present, recent studies have elucidated several shared protein-coding components of the machineries regulating necrosis, autophagy and apoptosis.4 Therefore, it would be interesting to examine whether or not miRNAs stand at the cross-road of necrosis and other subroutines in PCD network. Several cell death-related databases centered on protein-coding genes have been built recently.5, 6 Complemented with these useful databases, we constructed miRDeathDB and established a novel link between miRNAs and PCD. To keep up with these two rapidly growing research fields, we will continuously curate the literature data and update miRDeathDB. We hope this resource will become a bridge between the miRNAs and cell death research and stimulate further interest on the role of miRNAs in PCD network.

Caspase-3 and prostaglandins signal for tumor regrowth in cancer therapy

Chemo- and radio-therapeutic regimens frequently kill cancer cells by inducing apoptosis, a cell-death subroutine that involves the activation of a particular class of proteases called caspases. In a recent issue of Nature Medicine, Huang et al. (2011) show that caspase activation in dying tumor cells causes the release of soluble lipid messengers, notably prostaglandin E(2), that stimulate tumor cell proliferation. In this short review, we will discuss the clinical and therapeutic implications of these findings.

FADD: an endogenous inhibitor of RIP3-driven regulated necrosis

FADD: an endogenous inhibitor of RIP3-driven regulated necrosis

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.

Apoptotic and necrotic death mechanisms are concomitantly activated in the same cell after cerebral ischemia.

Both necrotic and apoptotic cell death mechanisms are activated after cerebral ischemia. However, whether they are concomitantly active in the same cell or in discrete cell populations is not known. We investigated activation of both pathways at the cellular level in mice brains subjected to transient or permanent focal ischemia. Four hours after ischemia, diffuse cathepsin-B spillage into cytoplasm, suggesting lysosomal leakage, was observed within neurons immunoreactive for the active form of caspase-3 (p20). Ischemic neurons with a leaky plasma membrane (positive for propidium iodide) were colabeled with caspase-cleaved actin fragment and exhibited TUNEL-positive nuclei having apoptotic morphology. At 72 hours, up to 27% of cells with caspase activity displayed morphological features suggestive of secondary necrosis. These data, demonstrating an early and concurrent increase in caspase-3 and cathepsin-B activities followed by appearance of caspase-cleavage products, DNA fragmentation, and membrane disintegration, suggest that subroutines of necrotic and apoptotic cell death are concomitantly activated in ischemic neurons and that the dominant cell death phenotype is determined by the relative speed of each process.

Molecular switches deciding the death of injured neurons.

The endpoints used to evaluate neurotoxicity of drugs and chemicals are multiple and reflect the complexity of the nervous system. In many instances, loss of function can result from a temporary impairment of synaptic activity. However, exposure to some neurotoxic conditions may eventually lead to neuronal loss and be fatal to the organism. Execution of the apoptotic program seems to be the mechanism involved in loss of neurons in human neurodegenerative conditions. Apoptosis is a conserved mode of cell death, prominent in developmental conditions, whose main execution pathway converges on the activation of the caspase family of proteases. However, there is increasing evidence that cell death in post developmental conditions is more complex. Other routines or subroutines of cell death can be activated under toxic or pathological conditions, and several protease families may contribute to produce apoptotic-like features or other phenotypically different forms of cells death. This has posed the question as to whether classical apoptosis is a valid endpoint to test the effect of neurotoxic agents and whether inhibitors of the caspase subroutine to cell death may then be used to treat diseases characterized by an excess of apoptosis. The recognition of the molecular switches that toggle between cell death subroutines becomes, therefore, of central importance in biomedicine and toxicology.