The Drosophila caspases Strica and Dronc function redundantly in programmed cell death during oogenesis

Programmed cell death (PCD) in the Drosophila ovary occurs either during mid-oogenesis, resulting in degeneration of the entire egg chamber or during late oogenesis, to facilitate the development of the oocyte. PCD during oogenesis is regulated by mechanisms different from those that control cell death in other Drosophila tissues. We have analyzed the role of caspases in PCD of the female germline by examining caspase mutants and overexpressing caspase inhibitors. Imprecise P-element excision was used to generate mutants of the initiator caspase strica. While null mutants of strica or another initiator caspase, dronc, display no ovary phenotype, we find that strica exhibits redundancy with dronc, during both mid- and late oogenesis. Ovaries of double mutants contain defective mid-stage egg chambers similar to those reported previously in dcp-1 mutants, and mature egg chambers with persisting nurse cell nuclei. In addition, the effector caspases drice and dcp-1 also display redundant functions during late oogenesis, resulting in persisting nurse cell nuclei. These findings indicate that caspases are required for nurse cell death during mid-oogenesis, and participate in developmental nurse cell death during late oogenesis. This reveals a novel pathway of cell death in the ovary that utilizes strica, dronc, dcp-1 and drice, and importantly illustrates strong redundancy among the caspases.     

Anti-viral strategies of cytotoxic T lymphocytes are manifested through a variety of granule-bound pathways of apoptosis induction

Cytotoxic T cells and natural killer cells together constitute a major defence against virus infection, through their ability to induce apoptotic death in infected cells. These cytolytic lymphocytes kill their targets through two principal mechanisms, and one of these, granule exocytosis, is essential for an effective in vivo immune response against many viruses. In recent years, the authors and other investigators have identified several distinct mechanisms that can induce death in a targeted cell. In the present article, it is postulated that the reason for this redundancy of lethal mechanisms is to deal with the array of anti-apoptotic molecules elaborated by viruses to extend the life of infected cells. The fate of such a cell therefore reflects the balance of pro-apoptotic (immune) and anti-apoptotic (viral) strategies that have developed over eons of evolutionary time.     

Lysosomal cathepsins in embryonic programmed cell death

During limb development, expression of cathepsin D and B genes prefigure the pattern of interdigital apoptosis including the differences between the chick and the webbed digits of the duck. Expression of cathepsin L is associated with advanced stages of degeneration. Analysis of Gremlin-/- and Dkk-/- mouse mutants and local treatments with BMP proteins reveal that the expression of cathepsin B and D genes is regulated by BMP signaling, a pathway responsible for triggering cell death. Further cathepsin D protein is upregulated in the preapoptotic mesenchyme before being released into the cytosol, and overexpression of cathepsin D induces cell death in embryonic tissues by a mechanism including mitochondrial permeabilization and nuclear translocation of AIF. Combined inhibition of cathepsin and caspases suggests a redundancy in the apoptotic molecular machinery, providing evidence for compensatory activation mechanisms in the cathepsin pathway when caspases are blocked. It is concluded that lysosomal enzymes are functionally implicated in embryonic programmed cell death.     

Molecular analysis of functional redundancy among anti-apoptotic Bcl-2 proteins and its role in cancer cell survival

Bcl-2 family proteins act as essential regulators and mediators of intrinsic apoptosis. Several lines of evidence suggest that the anti-apoptotic members of the family, including Bcl-2, Bcl-xL and Mcl-1, exhibit functional redundancy. However, the current evidence is largely indirect, and based mainly on pharmacological data using small-molecule inhibitors. In order to study compensation and redundancy of anti-apoptotic Bcl-2 proteins at the molecular level, we used a combined knockdown/overexpression strategy to essentially replace the function of one member with another. The results show that HeLa cells are strictly dependent on Mcl-1 for survival and correspondingly refractory to the Bcl-2/Bcl-xL inhibitor ABT-263, and remain resistant to ABT-263 in the context of Bcl-xL overexpression because endogenous Mcl-1 continues to provide the primary guardian role. However, if Mcl-1 is knocked down in the context of Bcl-xL overexpression, the cells become Bcl-xL-dependent and sensitive to ABT-263. We also show that Bcl-xL compensates for loss of Mcl-1 by sequestration of two key pro-apoptotic Bcl-2 family members, Bak and Bim, normally bound to Mcl-1, and that Bim is essential for cell death induced by Mcl-1 knockdown. To our knowledge, this is the first example where cell death induced by loss of Mcl-1 was rescued by the silencing of a single BH3-only Bcl-2 family member. In colon carcinoma cell lines, Bcl-xL and Mcl-1 also play compensatory roles, and Mcl-1 knockdown sensitizes cells to ABT-263. The results, obtained employing a novel strategy of combining knockdown and overexpression, provide unique molecular insight into the mechanisms of compensation by pro-survival Bcl-2 family proteins.     

Genetic control of programmed cell death (apoptosis) in Drosophila

Programmed cell death, or apoptosis, is a highly conserved cellular process that has been intensively investigated in nematodes, flies, and mammals. The genetic conservation, the low redundancy, the feasibility for high-throughput genetic screens and the identification of temporally and spatially regulated apoptotic responses make Drosophila melanogaster a great model for the study of apoptosis. Here, we review the key players of the cell death pathway in Drosophila and discuss their roles in apoptotic and non-apoptotic processes.     

Activation of intrinsic and extrinsic pathways in apoptotic signaling during UV-C-induced death of Jurkat cells: the role of caspase inhibition

We have examined UV irradiation-induced cell death in Jurkat cells and evaluated the relationships that exist between inhibition of caspase activity and the signaling mechanisms and pathways of apoptosis. Jurkat cells were irradiated with UV-C light, either with or without pretreatment with the pan-caspase inhibitor, z-VAD-fmk (ZVAD), or the more selective caspase inhibitors z-IETD-fmk (IETD), z-LEHD-fmk (LEHD), and z-DEVD-fmk (DEVD). Flow cytometry was used to examine alterations in viability, cell size, plasma membrane potential (PMP), mitochondrial membrane potential (DeltaPsi(mito)), intracellular Na(+) and K(+) concentrations, and DNA degradation. Processing of pro-caspases 3, 8, and 9 and the pro-apoptotic protein Bid was determined by Western blotting. UV-C irradiation of Jurkat cells resulted in characteristic apoptosis within 6 h after treatment and pretreatment of cells with ZVAD blocked these features. In contrast, pretreatment of the cells with the more selective caspase inhibitors under conditions that effectively blocked DNA degradation and inhibited caspase 3 and 8 processing as well as Bid cleavage had little protective effect on the other apoptotic characteristics examined. Thus, both intrinsic and extrinsic pathways are activated during UV-induced apoptosis in Jurkat cells and this redundancy appears to assure cell death during selective caspase inhibition.     

Caspase-independent programmed cell death with necrotic morphology.

Cell death is generally classified into two large categories: apoptosis represents active, programmed cell death, while necrosis represents passive cell death without underlying regulatory mechanisms. Recent progress revealed that caspases, a family of cysteine proteases, play a central role in the regulation of apoptosis. Unexpectedly, however, caspase inhibition occasionally turns the morphology of programmed cell death from apoptotic into necrotic without inhibiting death itself. In this article, we review different models of caspase-independent programmed cell death showing necrotic-like morphology, including our Ras-mediated caspase-independent cell death. Based on these findings, we suggest the existence of a necrotic-like cell death regulated by cellular intrinsic death programs distinct from that of apoptosis. Even though type 2 physiological cell death, or autophagic degeneration, has been recognized as a necrotic-like programmed cell death for a long time, the underlying molecular mechanisms have not been identified despite its physiological significance. This has been in part due to the previous absence of adequate caspase-independent cellular models to study, recent efforts may now help to elucidate these mechanisms.     

New concepts in radiation-induced apoptosis: 'premitotic apoptosis' and 'postmitotic apoptosis'

Formerly, the mechanisms responsible for the killing of cells by ionizing radiation were regarded as being divided into two distinct forms, interphase death and reproductive death. Since they were defined based on the classical radiobiological concepts using a clonogenic cell survival assay, biochemical and molecular biological mechanisms involved in the induction of radiation-induced cell death were not fully understood in relation to the modes of cell death. Recent multidisciplinary approaches to cell death mechanism have revealed that radiation-induced cell death is divided into several distinct pathways by the time course and cell-cycle position, and that apoptotic cell death plays a key role in almost every mode of cell death. This review discusses the mechanisms of radiation-induced apoptosis in relation to cellcycle progression and highlights a new concept of the mode of cell death: 'premitotic apoptosis' and 'postmitotic apoptosis'. The former is a rapid apoptotic cell death associated with a prompt activation of caspase-3, a key enzyme of intracellular signaling of apoptosis. Arapid execution of cell killing in premitotic apoptosis is presumably due to the prompt activation of a set of pre-existed molecules following DNA damages. In contrast, the latter is a delayed apoptotic cell death after cell division, and unlike premitotic apoptosis, it neither requires a rapid activation of caspase-3 nor is inhibited by a specific inhibitor, Ac-DEVD-CHO. A downregulation of anti-apoptotic genes such as MAPK and Bcl-2 may play a key role in this mode of cell death. Characterization of these two types of apoptotic cell death regarding the cell cycle regulation and intrcellular signaling will greatly help to understand the mechanisms of radiation-induced apoptosis.     

Expansion and evolution of cell death programmes

Cell death has historically been subdivided into regulated and unregulated mechanisms. Apoptosis, a form of regulated cell death, reflects a cell's decision to die in response to cues and is executed by intrinsic cellular machinery. Unregulated cell death (often called necrosis) is caused by overwhelming stress that is incompatible with cell survival. Emerging evidence, however, suggests that these two processes do not adequately explain the various cell death mechanisms. Recent data point to the existence of multiple non-apoptotic, regulated cell death mechanisms, some of which overlap or are mutually exclusive with apoptosis. Here we examine how and why these different cell death programmes have evolved, with an eye towards new cytoprotective therapeutic opportunities.     

Viruses associated with human cancer

It is estimated that viral infections contribute to 15-20% of all human cancers. As obligatory intracellular parasites, viruses encode proteins that reprogram host cellular signaling pathways that control proliferation, differentiation, cell death, genomic integrity, and recognition by the immune system. These cellular processes are governed by complex and redundant regulatory networks and are surveyed by sentinel mechanisms that ensure that aberrant cells are removed from the proliferative pool. Given that the genome size of a virus is highly restricted to ensure packaging within an infectious structure, viruses must target cellular regulatory nodes with limited redundancy and need to inactivate surveillance mechanisms that would normally recognize and extinguish such abnormal cells. In many cases, key proteins in these same regulatory networks are subject to mutation in non-virally associated diseases and cancers. Oncogenic viruses have thus served as important experimental models to identify and molecularly investigate such cellular networks. These include the discovery of oncogenes and tumor suppressors, identification of regulatory networks that are critical for maintenance of genomic integrity, and processes that govern immune surveillance.     

Flexibility exists in the region of [A6-A11, A7-B7] disulfide bonds during insulin precursor folding

Programmed cell death or apoptosis is the regulatory mechanism for removing unneeded cells during animal development and in tissue homeostasis. Perturbation of the cell death mechanisms leads to various disorders, including neurodegenerative diseases, immunodeficiency diseases, and tumors. c-Jun N-terminal kinase (JNK) has crucial roles in the regulation of cell death in response to many stimuli. Since JNK is highly conserved from yeast to mammals, genetic studies using model animals are helpful in understanding the principal cell death mechanisms regulated by JNK. For example, loss-of-function studies using the targeted disruption of murine genes have established the genetic framework of the mechanisms of the cell death induced by UV radiation. Also, in Drosophila, many cell death-related genes have been identified by genetics. Genetic studies of JNK-dependent cell death mechanisms should shed light on the regulation of both physiological and pathological cell death.     

What makes the mitochondria a killer? Can we condition them to be less destructive?

Cardioprotection, such as preconditioning and postconditioning, has been shown to result in a significant reduction in cell death. Many of the signaling pathways activated by cardioprotection have been elucidated, but there is still a lack of understanding of the mechanisms by which these signaling pathways reduce cell death. Mitochondria have been reported to be an important player in many types of apoptotic and necrotic cell death. If mitochondria play an important role in cell death, then it seems reasonable to consider that cardioprotective mechanisms might act, at least in part, by opposing mitochondrial cell death pathways. One of the major mechanisms of cell death in ischemia-reperfusion is suggested to be the opening of a large conductance pore in the inner mitochondrial membrane, known as the mitochondrial permeability transition pore. Inhibition of this mitochondrial pore appears to be one of the major mechanisms by which cardioprotection reduces cell death. Cardioprotection activates a number of signaling pathways that reduce the level of triggers (reactive oxygen species and calcium) or enhances inhibitors of the mitochondrial permeability transition pore at the start of reperfusion. This article is part of a Special Issue entitled: Mitochondria and Cardioprotection.     

It's a noisy business! Genetic regulation at the nanomolar scale

Many molecules that control genetic regulatory circuits act at extremely low intracellular concentrations. Resultant fluctuations (noise) in reaction rates cause large random variation in rates of development, morphology and the instantaneous concentration of each molecular species in each cell. To achieve regulatory reliability in spite of this noise, cells use redundancy in genes as well as redundancy and extensive feedback in regulatory pathways. However, some regulatory mechanisms exploit this noise to randomize outcomes where variability is advantageous.     

Mitochondrial mechanisms of apoptosis in response to DNA damage

Genome integrity is essential for cell viability, while damage to the DNA structure is a key factor inducing cell death. Among all cell death programs, those involving mitochondrial proteins are of particular importance. Activation of various protective epigenetic mechanisms in response to DNA damage prevents cell death. The outcome of genotoxic stress—cell death versus survival—depends on the balance of proapoptotic and antiapoptotic signaling. This concept provides a rational basis for improving the efficacy of anticancer therapy by combining DNA-damaging exposures with inhibition of antiapoptotic mechanisms.     

Surveying the landscape of emerging and understudied cell death mechanisms

Cell death can be a highly regulated process. A large and growing number of mammalian cell death mechanisms have been described over the past few decades. Major pathways with established roles in normal or disease biology include apoptosis, necroptosis, pyroptosis and ferroptosis. However, additional non-apoptotic cell death mechanisms with unique morphological, genetic, and biochemical features have also been described. These mechanisms may play highly specialized physiological roles or only become activated in response to specific lethal stimuli or conditions. Understanding the nature of these emerging and understudied mechanisms may provide new insight into cell death biology and suggest new treatments for diseases such as cancer and neurodegeneration.     

Prion protein function and the disturbance of early embryonic development in zebrafish

Transmissible Spongiform Encephalopathies (TSE) or prion diseases are a threat to food safety and to human and animal health. The molecular mechanisms responsible for prion diseases share similarities with a wider group of neurodegenerative disorders including Alzheimer disease and Parkinson disease and the central pathological event is a disturbance of protein folding of a normal cellular protein that is eventually accompanied by neuronal cell death and the death of the host. Prion protein (PrP) is a constituent of most normal mammalian cells and its presence is essential in the pathogenesis of TSE. However, the function of this normal cellular protein remains unclear. The prevention of PRNP gene expression in mammalian species has been undramatic, implying a functional redundancy. Yet PrP is conserved from mammals to fish. Recent studies of PrP in zebrafish have yielded novel findings showing that PrP has essential roles in early embryonic development. The amenability of zebrafish to global technologies has generated data indicating the existence of “anchorless” splice variants of PrP in the early embryo. This paper will discuss the possibility that the experimentalist’s view of PrP functions might be clearer at a greater phylogenetic distance.     

Retrograde transport of neurotrophins: fact and function

Retrograde signals generated by nerve growth factor (NGF) and other neurotrophins promote the survival of appropriately connected neurons during development, and failure to obtain sufficient retrograde signals may contribute to neuronal death occurring in many neurodegenerative diseases. The discovery over 25 years ago that NGF supplied to the axon terminals is retrogradely transported to the cell bodies suggested that NGF must reach the cell body to promote neuronal survival. Research during the intervening decades has produced a refinement of this hypothesis. The current hypothesis is that NGF bound to TrkA at the axon terminal is internalized into signaling endosomes, with NGF in their lumens bound to phosphorylated TrkA in their membranes, which are retrogradely transported to the cell bodies, where TrkA activates downstream signaling molecules that promote neuronal survival and regulate many aspects of neuronal gene expression. This model has been extrapolated to retrograde signaling by all neurotrophins. We consider the evidence for this model, focusing on results of experiments with neurons in compartmented cultures. Results to date indicate that while the transport of signaling endosomes containing NGF bound to TrkA may carry retrograde signals, retrograde survival signals can be carried by another mechanism that is activated by NGF at the axon terminal surface and travels to the cell body unaccompanied by the NGF that initiated it. It is hypothesized that multiple mechanisms of retrograde signaling exist and function under different circumstances. The newly discovered potential for redundancy in retrograde signaling mechanisms can complicate the interpretation of experimental results.     

The relative contribution of pro-apoptotic p53-target genes in the triggering of apoptosis following DNA damage in vitro and in vivo

The p53 pathway displays a large degree of redundancy in the expression of a number of pro-apoptotic mechanisms following DNA damage that, among others, involves increased expression of several pro-apoptotic genes through transactivation. Spatial and temporal cellular contexts contribute to the complexity of the regulation of apoptosis, hence different genes may show a cell- and tissue-dependent specificity with regard to the regulation of cell death and act in concert or show redundancy with one and another. We used siRNA technology to assess the effect of multiple ablations of documented pro-apoptotic p53 target genes (PPG) in the colorectal cancer cell line HCT116 and generated mice deficient in both of the extrinsic and intrinsic PPGs genes Dr5 and Puma following treatment with chemotherapeutics and ionizing radiation. DR5, Fas, Bax, Bad, Puma and Bnip3L were induced by 5-FU and adriamycin (ADR) in HCT116 cells in a p53-dependent manner. The resulting caspase 3/7 activity in HCT116 cells following treatment were suppressed by ablated expression of the PPGs in the extrinsic as well as the intrinsic pathway. To our surprise, knocking-down any of the PPGs concomitantly with DR5 did not further inhibit caspase 3/7 activity whereas inhibiting DR5-expression in HCT116Bax knockdown (kd) and HCT116Fas kd did, suggesting that these genes act downstream or in synergy with DR5. This was supported by our in vivo observations, since Puma and Dr5 were equally efficient in protecting cells of the spleen from sub-lethal radiation-induced apoptosis but less effective compared with irradiated p53^-/- mice. To our surprise, Dr5^-/-; Puma^-/- mice did not show additive protection from radiation-induced apoptosis in any of the investigated organs. Our data indicates that the intrinsic pathway may rely on extrinsic signals to promote cell death in a cell- and tissue-dependent manner following DNA damage. Furthermore, p53 must rely on mechanisms independent of DR5 and PUMA to initiate apoptosis following γ-radiation in the spleen and thymus in vivo.