A platform for interrogating cancer-associated p53 alleles.

p53 is the most frequently mutated gene in human cancer. Compelling evidence argues that full transformation involves loss of growth suppression encoded by wild-type p53 together with poorly understood oncogenic activity encoded by missense mutations. Furthermore, distinguishing disease alleles from natural polymorphisms is an important clinical challenge. To interrogate the genetic activity of human p53 variants, we leveraged the Drosophila model as an in vivo platform. We engineered strains that replace the fly p53 gene with human alleles, producing a collection of stocks that are, in effect, 'humanized' for p53 variants. Like the fly counterpart, human p53 transcriptionally activated a biosensor and induced apoptosis after DNA damage. However, all humanized strains representing common alleles found in cancer patients failed to complement in these assays. Surprisingly, stimulus-dependent activation of hp53 occurred without stabilization, demonstrating that these two processes can be uncoupled. Like its fly counterpart, hp53 formed prominent nuclear foci in germline cells but cancer-associated p53 variants did not. Moreover, these same mutant alleles disrupted hp53 foci and inhibited biosensor activity, suggesting that these properties are functionally linked. Together these findings establish a functional platform for interrogating human p53 alleles and suggest that simple phenotypes could be used to stratify disease variants.

Essential versus accessory aspects of cell death: recommendations of the NCCD 2015.

Cells exposed to extreme physicochemical or mechanical stimuli die in an uncontrollable manner, as a result of their immediate structural breakdown. Such an unavoidable variant of cellular demise is generally referred to as 'accidental cell death' (ACD). In most settings, however, cell death is initiated by a genetically encoded apparatus, correlating with the fact that its course can be altered by pharmacologic or genetic interventions. 'Regulated cell death' (RCD) can occur as part of physiologic programs or can be activated once adaptive responses to perturbations of the extracellular or intracellular microenvironment fail. The biochemical phenomena that accompany RCD may be harnessed to classify it into a few subtypes, which often (but not always) exhibit stereotyped morphologic features. Nonetheless, efficiently inhibiting the processes that are commonly thought to cause RCD, such as the activation of executioner caspases in the course of apoptosis, does not exert true cytoprotective effects in the mammalian system, but simply alters the kinetics of cellular demise as it shifts its morphologic and biochemical correlates. Conversely, bona fide cytoprotection can be achieved by inhibiting the transduction of lethal signals in the early phases of the process, when adaptive responses are still operational. Thus, the mechanisms that truly execute RCD may be less understood, less inhibitable and perhaps more homogeneous than previously thought. Here, the Nomenclature Committee on Cell Death formulates a set of recommendations to help scientists and researchers to discriminate between essential and accessory aspects of cell death.

Effectors of alcohol-induced cell killing in Drosophila.

Heavy alcohol consumption provokes an array of degenerative pathologies but the signals that couple alcohol exposure to regulated forms of cell death are poorly understood. Using Drosophila as a model, we genetically establish that the severity of ethanol challenge dictates the type of death that occurs. In contrast to responses seen under acute exposure, cytotoxic responses to milder challenges required gene encoding components of the apoptosome, Dronc and Dark. We conducted a genome-wide RNAi screen to capture targets that specifically mediate ethanol-induced cell death. One effector, Drat, encodes a novel protein that contains an ADH domain but lacks essential residues in the catalytic site. In cultured cells and neurons in vivo, depletion of Drat conferred protection from alcohol-induced apoptosis. Adults mutated for Drat showed both improved survival and enhanced propensities toward sedation after alcohol challenge. Together, these findings highlight novel effectors that support regulated cell death incited by alcohol stress in vitro and in vivo.

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.

No death without life: vital functions of apoptotic effectors.

As a result of the genetic experiments performed in Caenorhabditis elegans, it has been tacitly assumed that the core proteins of the 'apoptotic machinery' (CED-3, -4, -9 and EGL-1) would be solely involved in cell death regulation/execution and would not exert any functions outside of the cell death realm. However, multiple studies indicate that the mammalian orthologs of these C. elegans proteins (i.e. caspases, Apaf-1 and multidomain proteins of the Bcl-2 family) participate in cell death-unrelated processes. Similarly, loss-of-function mutations of ced-4 compromise the mitotic arrest of DNA-damaged germline cells from adult nematodes, even in a context in which the apoptotic machinery is inoperative (for instance due to mutations of egl-1 or ced-3). Moreover, EGL-1 is required for the activation of autophagy in starved nematodes. Finally, the depletion of caspase-independent death effectors, such as apoptosis-inducing factor (AIF) and endonuclease G, provokes cell death-independent consequences, both in mammals and in yeast (Saccharomyces cerevisiae). These results corroborate the conjecture that any kind of protein that has previously been specifically implicated in apoptosis might have a phylogenetically conserved apoptosis-unrelated function, most likely as part of an adaptive response to cellular stress.

The molecular archaeology of a mitochondrial death effector: AIF in Drosophila.

Apoptosis-inducing factor (AIF) is a phylogenetically conserved redox-active flavoprotein that contributes to cell death and oxidative phosphorylation in Saccharomyces cerevisiae, Caenorhabditis elegans, mouse and humans. AIF has been characterized as a caspase-independent death effector that is activated by its translocation from mitochondria to the cytosol and nucleus. Here, we report the molecular characterization of AIF in Drosophila melanogaster, a species in which most cell deaths occur in a caspase-dependent manner. Interestingly, knockout of zygotic D. melanogaster AIF (DmAIF) expression using gene targeting resulted in decreased embryonic cell death and the persistence of differentiated neuronal cells at late embryonic stages. Although knockout embryos hatch, they undergo growth arrest at early larval stages, accompanied by mitochondrial respiratory dysfunction. Transgenic expression of DmAIF misdirected to the extramitochondrial compartment (DeltaN-DmAIF), but not wild-type DmAIF, triggered ectopic caspase activation and cell death. DeltaN-DmAIF-induced death was not blocked by removal of caspase activator Dark or transgenic expression of baculoviral caspase inhibitor p35, but was partially inhibited by Diap1 overexpression. Knockdown studies revealed that DeltaN-DmAIF interacts genetically with the redox protein thioredoxin-2. In conclusion, we show that Drosophila AIF is a mitochondrial effector of cell death that plays roles in developmentally regulated cell death and normal mitochondrial function.

p53 directs focused genomic responses in Drosophila.

p53 is a fundamental determinant of cancer susceptibility and other age-related pathologies. Similar to mammalian counterparts, Drosophila p53 integrates stress signals and elicits apoptotic responses that maintain genomic stability. To illuminate core-adaptive functions controlled by this gene family, we examined the Drosophila p53 regulatory network at a genomic scale. In development, the absence of p53 impacted constitutive expression for a surprisingly broad scope of genes. By contrast, stimulus-dependent responses governed by Drosophila p53 were limited in scope. The vast majority of stress responders were induced and p53 dependent (RIPD) genes. The signature set of 29 'high stringency' RIPD genes identified here were enriched for intronless loci, with a non-uniform distribution that includes a recently evolved cluster unique to Drosophila melanogaster. Two RIPD genes, with known and unknown biochemical activities, were functionally examined. One RIPD gene, designated XRP1, maintains genome stability after genotoxic challenge and prevents cell proliferation upon induced expression. A second gene, RnrL, is an apoptogenic effector required for caspase activation in a model of p53-dependent killing. Together, these studies identify ancient and convergent features of the p53 regulatory network.

Bifunctional killing activity encoded by conserved reaper proteins.

Drosophila activators of apoptosis mapping to the Reaper region function, in part, by antagonizing IAP proteins through a shared RHG motif. We isolated Reaper from the Blowfly L. cuprina, which triggered extensive apoptosis in Drosophila cells. Conserved regions of Reaper were tested in the context of GFP fusions and a second killing activity, distinct from the RHG, was identified. A 20 amino-acid peptide, designated R3, conferred targeting to a focal compartment and promoted membrane blebbing. Killing by the R3 fragment did not correlate with translational suppression or with reduced DIAP1 levels. Likewise, R3-induced cell deaths were only modestly suppressed by silencing of Dronc and involved no detectable association with DIAP1. Instead, a second IAP-binding domain, distinct from the R3, was identified at the C terminus of Reaper that bound to DIAP1 but failed to trigger apoptosis. Collectively, these findings are inconsistent with single effector models for cell killing by Reaper and suggest, instead, that Reaper encodes conserved bifunctional death activities that propagate through distinct effector pathways.

Morpholino antisense oligonucleotides: tools for investigating vertebrate development.

Antisense oligonucleotides provide a promising approach to investigating gene function in vivo, but their ability to offer unambiguous insights into phenotypes has been debated. The recent use of morpholino antisense oligonucleotides in zebrafish embryos may prove a major advance, but rigorous controls are essential.

Drosophila p53 binds a damage response element at the reaper locus.

The tumor suppressor gene p53 regulates multiple cellular responses to DNA damage, but the transcriptional targets that specify these responses are incompletely understood. We describe a Drosophila p53 homolog and demonstrate that it can activate transcription from a promoter containing binding sites for human p53. Dominant-negative forms of Drosophila p53 inhibit both transactivation in cultured cells and radiation-induced apoptosis in developing tissues. The cis-regulatory region of the proapoptotic gene reaper contains a radiation-inducible enhancer that includes a consensus p53 binding site. Drosophila p53 can activate transcription from this site in yeast and a multimer of this site is sufficient for radiation induction in vivo. These results indicate that reaper is a direct transcriptional target of Drosophila p53 following DNA damage.

Guardian ancestry: fly p53 and damage-inducible apoptosis.

The tumor suppressor, p53, is among the most commonly mutated genes in human cancers. Recent reports describe shared and divergent properties of a Drosophila p53 homolog Dmp53. Like its mammalian counterpart, Dmp53 also functions in damage-induced cell death. In this model system, the apoptosis activator reaper has emerged as an important target gene. Together with the wealth of genomic data available in Drosophila, continued studies on Dmp53 promise new insights into the regulation and function of this important gene family.

The Drosophila caspase Dredd is required to resist gram-negative bacterial infection.

The Drosophila innate immune system discriminates between pathogens and responds by inducing the expression of specific antimicrobial peptide-encoding genes through distinct signaling cascades. Fungal infection activates NF-kappaB-like transcription factors via the Toll pathway, which also regulates innate immune responses in mammals. The pathways that mediate antibacterial defenses, however, are less defined. We have isolated loss-of-function mutations in the caspase encoding gene dredd, which block the expression of all genes that code for peptides with antibacterial activity. These mutations also render flies highly susceptible to infection by gram-negative bacteria. Our results demonstrate that Dredd regulates antibacterial peptide gene expression, and we propose that Dredd, Immune Deficiency and the P105-like rel protein Relish define a pathway that is required to resist gram-negative bacterial infections.

Dark is a Drosophila homologue of Apaf-1/CED-4 and functions in an evolutionarily conserved death pathway.

Here we identify a new gene, dark, which encodes a Drosophila homologue of mammalian Apaf-1 and Caenorhabditis elegans CED-4, cell-death proteins. Like Apaf-1, but in contrast to CED-4, Dark contains a carboxy-terminal WD-repeat domain necessary for interactions with the mitochondrial protein cytochrome c. Dark selectively associates with another protein involved in apoptosis, the fly apical caspase, Dredd. Dark-induced cell killing is suppressed by caspase-inhibitory peptides and by a dominant-negative mutant Dredd protein, and enhanced by removal of the WD domain. Loss-of-function mutations in dark attenuate programmed cell deaths during development, causing hyperplasia of the central nervous system, and other abnormalities including ectopic melanotic tumours and defective wings. Moreover, ectopic cell killing by the Drosophila cell-death activators, Reaper, Grim and Hid, is substantially suppressed in dark mutants. These findings establish dark as an important apoptosis effector in Drosophila and raise profound evolutionary considerations concerning the relationship between mitochondrial components and the apoptosis-promoting machinery.

An emerging blueprint for apoptosis in Drosophila.

Apoptosis research demonstrates that, even though the multitude of regulatory circuits controlling programmed cell death might diverge, core elements of the 'apoptotic engine' are widely conserved. Therefore, studies in less complex model systems, such as the nematode and the fly, should continue to have a profound impact on our understanding of the process. This review explores genes and molecules that control apoptosis in Drosophila. The death inducers Reaper, Grim and Hid relay signals, possibly through IAPs (inhibitor of apoptosis proteins) and Dark (an Apaf-1/Ced-4 homologue), to trigger caspase function. This animal model promises continued insights into the determinants of cell death in 'naturally occurring' and pathological contexts.