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Myc is an enigma wrapped in a mystery. Attempts to identify Myc target genes, particularly in cancer, have been fraught with dead ends and context-specific functions. Lin et al. and Nie et al. address this conundrum by showing that Myc acts to amplify the output of existing transcriptionally active genes.

Deregulated expression of c-MYC occurs in a broad range of human cancers and is often associated with poor prognosis, indicating a key role for this oncogene in tumour progression. However, as established human tumours often bear multiple genetic lesions, it is difficult to determine whether c-MYC is instrumental in the initiation/progression of the tumour, or indeed whether inactivating c-MYC would lead to tumour regression. Regulatable transgenic mouse models of oncogenesis have shed light on these issues and provide hope for effective cancer therapies.

c-Myc promotes apoptosis by destabilizing mitochondrial integrity, leading to the release of proapoptotic effectors including holocytochrome c. Candidate mediators of c-Myc in this process are the proapoptotic members of the Bcl-2 family. We show here that fibroblasts lacking Bak remain susceptible to c-Myc-induced apoptosis whereas bax-deficient fibroblasts are resistant. However, despite this requirement for Bax, c-Myc activation exerts no detectable effects on Bax expression, localization, or conformation. Moreover, susceptibility to c-Myc-induced apoptosis can be restored in bax-deficient cells by ectopic expression of Bax or by microinjection of a peptide comprising a minimal BH3 domain. Microinjection of BH3 peptide also restores sensitivity to c-Myc-induced apoptosis in p53-deficient primary fibroblasts that are otherwise resistant. By contrast, there is no synergy between BH3 peptide and c-Myc in fibroblasts deficient in both Bax and Bak. We conclude that c-Myc triggers a proapoptotic mitochondrial destabilizing activity that cooperates with proapoptotic members of the Bcl-2 family.

We propose that deregulation of proliferation, together with a reduction in apoptosis, creates a platform that is both necessary and can be sufficient for cancer. The secondary traits of diverse neoplasms are a consequence of cell proliferation, tissue expansion, and other outcomes of this platform.

Cellular senescence, a form of cell-cycle arrest, is a tumor-suppressor mechanism triggered by multiple tumor-promoting insults, including oncogenic stress and DNA damage. The role of cyclin-dependent kinase 2 (CDK2) regulation has been evaluated in models of replicative senescence, but little is known regarding its role in other senescence settings. Using in vitro and in vivo models of DNA damage–and oncogene-induced cellular senescence, it was determined that activation of the tumor-suppressor protein p53 (TP53) resulted in repression of the CDK2 transcript that was dependent on intact RB. Ectopic CDK2 expression was sufficient to bypass p53-dependent senescence, and CDK2-specific inhibition, either pharmacologically (CVT313) or by use of a dominant-negative CDK2, was sufficient to induce early senescence. Pharmacologic inhibition of CDK2 in an in vivo model of pineal tumor decreased proliferation and promoted early senescence, and it also decreased tumor penetrance and prolonged time to tumor formation in animals lacking p53. In conclusion, for both oncogene- and DNA damage–induced cellular senescence, CDK2 transcript and protein are decreased in a p53- and RB-dependent manner, and this repression is necessary for cell-cycle exit during senescence. Implications: These data show that CDK2 inhibition may be useful for cancer prevention in premalignant hyperproliferative lesions, as well as established tumors. Mol Cancer Res; 13(1); 29–40. ©2014 AACR.

Gliomas are the most common primary tumours affecting the adult central nervous system and respond poorly to standard therapy. Myc is causally implicated in most human tumours and the majority of glioblastomas have elevated Myc levels. Using the Myc dominant negative Omomyc, we previously showed that Myc inhibition is a promising strategy for cancer therapy. Here, we preclinically validate Myc inhibition as a therapeutic strategy in mouse and human glioma, using a mouse model of spontaneous multifocal invasive astrocytoma and its derived neuroprogenitors, human glioblastoma cell lines, and patient-derived tumours both in vitro and in orthotopic xenografts. Across all these experimental models we find that Myc inhibition reduces proliferation, increases apoptosis and remarkably, elicits the formation of multinucleated cells that then arrest or die by mitotic catastrophe, revealing a new role for Myc in the proficient division of glioma cells., Myc has been implicated in the development of multiple types of cancer. Here, the authors explore the therapeutic potential and mechanism of action of Myc inhibition in mouse and human models of glioblastoma, an aggressive type of tumour that is often resistant to conventional therapy.

Beneath the complexity and idiopathy of every cancer lies a limited number of 'mission critical' events that have propelled the tumour cell and its progeny into uncontrolled expansion and invasion. One of these is deregulated cell proliferation, which, together with the obligate compensatory suppression of apoptosis needed to support it, provides a minimal 'platform' necessary to support further neoplastic progression. Adroit targeting of these critical events should have potent and specific therapeutic consequences.

Caspase-8 is a member of the cysteine proteases, which are implicated in apoptosis and cytokine processing. Like all caspases, caspase-8 is synthesized as an inactive single polypeptide chain zymogen procaspase and is activated by proteolytic cleavage, through either autoactivation after recruitment into a multimeric complex or trans-cleavage by other caspases. Thus, ligand binding-induced trimerization of death receptors results in recruitment of the receptor-specific adapter protein Fas-associated death domain (FADD), which then recruits caspase-8. Activated caspase-8 is known to propagate the apoptotic signal either by directly cleaving and activating downstream caspases or by cleaving the BH3 Bcl2-interacting protein, which leads to the release of cytochrome c from mitochondria, triggering activation of caspase-9 in a complex with dATP and Apaf-1. Activated caspase-9 then activates further "downstream caspases," including caspase-8. Knockout data indicate that caspase-8 is required for killing induced by the death receptors Fas, tumor necrosis factor receptor 1, and death receptor 3. Moreover, caspase-8-/- mice die in utero as a result of defective development of heart muscle and display fewer hematopoietic progenitor cells, suggesting that the FADD/caspase-8 pathway is absolutely required for growth and development of specific cell types.

In order to study the effect of c-Myc activation in T lymphocytes in vivo, we generated transgenic mice that express a 4-hydroxytamoxifen (4-OHT)-dependent switchable c-myc oncoprotein under the control of the proximal lck promoter. Activation of c-MycER causes no obvious alteration in T cell ontogeny. However, using MHC class I restricted H-Y-TCR transgenic mice, we found that c-Myc activation in vivo enhances the efficiency of positive selection. Moreover, splenic T cells derived from lck-c-mycER transgenic mice in which c-Myc had been activated exhibited increased proliferation in vitro in response to activation with anti-CD3/CD28 antibody. Activation of c-MycER also promotes apoptosis in thymocytes in vitro.

Caspase-3 is an effector of apoptosis in experimental models of Parkinson's disease (PD). However, its potential role in the human pathology remains to be demonstrated. Using caspase-3 immunohistochemistry on the postmortem human brain, we observed a positive correlation between the degree of neuronal loss in dopaminergic (DA) cell groups affected in the mesencephalon of PD patients and the percentage of caspase-3-positive neurons in these cell groups in control subjects and a significant decrease of caspase-3-positive pigmented neurons in the substantia nigra pars compacta of PD patients compared with controls that also could be observed in an animal model of PD. This suggests that neurons expressing caspase-3 are more sensitive to the pathological process than those that do not express the protein. In addition, using an antibody raised against activated caspase-3, the percentage of active caspase-3-positive neurons among DA neurons was significantly higher in PD patients than in controls. Finally, electron microscopy analysis in the human brain and in vitro data suggest that caspase-3 activation precedes and is not a consequence of apoptotic cell death in PD.

Release of cytochrome c from mitochondria triggers activation of caspase proteases and death of a cell by apoptosis. However, the mechanism and kinetics of cytochrome c release remain unknown. Here we study this event by using green fluorescent protein (GFP)-tagged cytochrome c, and find that the release of cytochrome-c-GFP always precedes exposure of phosphatidylserine and the loss of plasma-membrane integrity - characteristics of apoptotic cells. Once initiated, the release of cytochrome- c-GFP continues until all of the protein is released from all mitochondria in individual cells, within about 5 minutes, regardless of the type or strength of stimulus or the time elapsed since the stimulus was applied. Temperatures ranging from 24 degrees C to 37 degrees C do not change the duration of release, and nor does the addition of caspase inhibitors. Further, we find that the electron-transport chain can maintain the mitochondrial transmembrane potential even after cytochrome c has been released.

The protooncogene c- myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. However, the consequences of activating c-Myc in an adult tissue, in which these cellular processes are part of normal homeostasis, remain unknown. In order to achieve this, we have targeted expression of a switchable form of the c-Myc protein to the skin epidermis, a well characterized homeostatic tissue. We show that activation of c-MycER TM in adult suprabasal epidermis rapidly triggers proliferation and disrupts differentiation of postmitotic keratinocytes. Sustained activation of c-Myc is sufficient to induce papillomatosis together with angiogenesis—changes that resemble hyperplastic actinic keratosis, a commonly observed human precancerous epithelial lesion. All these premalignant changes spontaneously regress upon deactivation of c-MycER TM .

p53 is a pivotal bulwark against cancer, but exactly how it suppresses tumors remains elusive, in part because it modulates such diverse biological processes via so many downstream pathways. In a recent issue of Cell, Godar et al. (2008) now identify another string to p53's anticancer bow-repression of the CD44 cell-surface glycoproteins that coordinate many attributes of tumor progression.

Both tumors and normal tissues need a blood supply for oxygen, nutrients, and waste removal. However, whereas normal vasculature is hierarchically assembled into efficient networks of arteries, capillaries, and veins, the blood vessels of tumors are a mess—chaotic, leaky, inefficient, and barely making do. Why the difference? Do tumor vessels lack the signals to mature or, instead, is their maturation actively suppressed? What triggers and maintains tumor vasculature? In a recent study using a switchable Myc-driven mouse tumor model, we addressed these fundamental questions. We identified the inflammatory cytokine interleukin-1β as an essential initiating trigger of vascular endothelial growth factor–dependent angiogenesis. Here, we consider how kinetic studies using regulatable forms of Myc or other oncogenes can shed new light on the way tumors initiate and maintain their aberrant blood supplies. [Cancer Res 2007;67(15):7059–61]

Gerard Evan reviews an inspiring, at times frustrating, chronicle of the war on cancer by one of its generals.

Two papers in this issue of Cell (3xBergmann, A, Agapite, J, McCall, K, and Steller, H. Cell. 1998; 95Abstract | Full Text | Full Text PDF | PubMed | Scopus (335)See all References, 13xKaruda, P and White, K. Cell. 1998; 95See all References) indicate that a critical target of this pathway is the death-inducing protein Hid. Karuda and White used a genetic screen for mutations that ameliorate the lethality of the proapoptotic Reaper protein transgenically targeted to the Drosophila eye using the glass multimer reporter (GMR). Mutations that interfere with any component of the DER-Ras-MAPK pathway exacerbate Reaper-mediated killing whilst corresponding gain-of-function mutations suppress it. The anti-apoptotic effect of the Ras/MAPK pathway is not confined to the eye: disseminated expression of a constitutively active Ras mutant (Q13) greatly suppresses the widespread cell death that accompanies normal embryonic development in the fly. This developmental death requires genes within the 75C1-2 region of Drosophila chromosome 3 that contains the apoptotic effectors reaper, hid, and grim. Thus, the product of one or more of these genes is the initiator of cell death triggered by interfering with Ras/MAPK signaling.Subsequent analysis showed that expression of hid, but not reaper or grim, is directly modulated by Ras/MAPK signaling. Activating mutations of either Dras1 or Draf suppress hid expression, an effect mimicked by ectopic expression of Pointed P2, an Ets domain transcription factor that is functionally activated by MAPK. Conversely, both a dominant interfering mutant of the DER receptor and ectopic expression of Yan, a transcription factor that is inactivated by MAPK and which antagonizes Pointed P2, induce accumulation of hid mRNA and promote apoptosis. An important role for hid as a target for Ras/MAPK signaling is confirmed by the fact that such ectopic apoptosis is, at least partially, inhibited in hidnull mutants. hidnull mutants are also resistant to killing by ectopically expressed Reaper, which might suggest that Hid is a necessary effector of Reaper action. Curiously, however, hidnull mutants also exhibit resistance to killing by ectopically expressed hid, an observation that may indicate that Hid is lethal only above a critical threshold level, one never reached in the absence of expression of the endogenous gene.An alternative explanation might be that Hid action is also modulated by posttranscriptional mechanisms. This notion is validated by the study of Bergmann et al., who used a genetic screen designed to look directly for modifiers of eye ablation induced by GMR-directed expression of hid. Again, the initial trawl exposed a clutch of genes that implicate the DER/Ras/MAPK pathway in regulating Hid-mediated cell death: gap1, encoding a GTPase-activating protein, and sprouty and argos, which encode secreted inhibitors of DER activation. Again, loss-of-function alleles of DER, Dras1, Draf, or rolled exacerbate Hid killing, whereas the corresponding gain-of-function mutations inhibit it. In contrast to Karuda and White, Bergmann et al. find no evidence for suppression of Reaper-induced apoptosis by Ras, although both agree that Grim-induced cell death is independent of Ras signaling.In all organisms, Ras has multiple downstream effectors whose relative contribution to any Ras-dependent process may be dissected using Ras effector mutants that activate only one effector pathway at a time (Downward 1998xDownward, J. Curr. Opin. Genet. Dev. 1998; 8: 49–54Crossref | PubMed | Scopus (458)See all ReferencesDownward 1998). Bergmann et al. use effector mutants to confirm that the Raf/MAPK pathway is the major antiapoptotic pathway in the fly eye, although a weaker ancillary survival signal is mediated by the phosphatidylinositol 3-kinase (PI3-K)/Dakt pathway; the arcane Ral GDS pathway does not appear to be involved. Bergmann et al. acknowledge that the Ras/MAPK pathway affects hid expression, but concentrate on the possibility that MAPK inhibits Hid by directly phosphorylating the Hid protein. They confirm this genetically by showing that mutation of key MAPK target residues in Hid generates a protein with enhanced lethality over the wild type and that is unaffected by Ras/MAPK signaling. Thus, the DER/Ras/MAPK pathway is a critical pathway promoting cell survival that has the Hid protein as its principal target.Does this discovery have wider implications for our understanding of the regulation of apoptosis? In principle, the answer should be yes. However, there are two potential “flies in this ointment.” The first has already been alluded to—no counterpart of Hid is known in the nematode or vertebrate. There are two possibilities: either Hid is present in these organisms but has yet to reveal itself in any genetic or biochemical analysis, or Hid (and maybe Grim and Reaper too) are arthropod inventions. The remarkable overall similarities in metazoan biology makes the latter possibility unpalatable, but without any understanding of Hid function at the molecular level, it is difficult to search for analogous functions in other species. In order to place Hid, and the pathways that regulate it, into a larger context, elucidation of Hid functions is essential.The second problem lies in any interpretation of signal transduction as a linear sequence of affectors and effectors. Although initial genetic studies in the fly eye indicated a comfortingly linear Ras→Raf→MAPKK→MAPK signaling sequence, an increasing amount of data indicates that all is not so simple. Both the receptors at the apex of this pathway and Ras itself are linked to a variety of other signaling pathways, and the same may well be true of the MAPKK Dsor and the MAPK Rolled. Furthermore, flow down the Ras→Raf→MAPK pathway is subject to extensive modulation by other intersecting signaling pathways within each cell type and through control of the intensity and timing of activation (Freeman 1997xFreeman, M. Development. 1997; 124: 261–270PubMedSee all ReferencesFreeman 1997).As in the fly, the well-characterized mammalian survival signaling pathways activated by the high affinity NGF receptor TrkA and by IGF-IR also utilize Ras as a key nexus. However, although the Raf/MAPK signaling pathway suppresses apoptosis in some neuronal and myeloid cells, studies with Ras effector mutants (identical to those used in the study of Bergmann et al.) indicate that the predominant survival signal appears to route through the Ras effector PI3-K and the serine/threonine protein kinase Akt (PKB) (Downward 1998xDownward, J. Curr. Opin. Genet. Dev. 1998; 8: 49–54Crossref | PubMed | Scopus (458)See all ReferencesDownward 1998). One critical target of Akt is Bad, a BH3 protein and antagonist of Bcl2 that is inactivated by Akt phosphorylation (Franke and Cantley 1997xFranke, T.F and Cantley, L.C. Nature. 1997; 390: 116–117Crossref | PubMed | Scopus (150)See all ReferencesFranke and Cantley 1997). Although in Drosophila, Akt exerts appreciable antiapoptotic activity (Staveley et al. 1998xStaveley, B.E, Ruel, L, Jin, J, Stambolic, V, Mastronardi, F.G, Heitzler, P, Woodgett, J.R, and Manoukian, A.S. Curr. Biol. 1998; 8: 599–602Abstract | Full Text | Full Text PDF | PubMedSee all ReferencesStaveley et al. 1998), Bergmann et al. find that it provides only a weak ancillary survival signal in the fly eye. So does this indicate a different use of pathways in fly and vertebrate? Probably not. It is clear that Ras sits at the apex of multiple effectors, each of which presides over its own suite of diverging and multifunctional signaling pathways. In such a network of pleiotropic effectors, no single cell fate is ever really controlled by any single effector (Figure 1Figure 1). Rather, the predominant outcome of Ras signaling is a result of a network of interactions between differing signaling pathways wherein some signals are reinforced while others are gated. Fate, in cells as in man, is a tangled web.Figure 1The Ras/MAP Kinase Signaling Pathway: The Legend and the RealitySee text for details.View Large Image | View Hi-Res Image | Download PowerPoint Slide

The MYC proto-oncogene has long been implicated in the control of normal cell growth and its deregulation is associated with the development of neoplasia. The MYC protein has a well-established role as a component of signal-transduction pathways promoting both proliferation and apoptosis. Because signalling pathways that drive cell death and cell proliferation are so tightly coupled, a synergy between genetic lesions leading to suppression of cell death and those promoting cell proliferation is observed during carcinogenesis. We discuss such synergy with respect to the cooperating oncogenes MYC, RAS and BCL2.

In multicellular organisms, mutations in somatic cells affecting critical genes that regulate cell proliferation and survival cause fatal cancers. Repair of the damage is one obvious option, although the relative inconsequence of individual cells in metazoans means that it is often a “safer” strategy to ablate the offending cell. Not surprisingly, corruption of the machinery that senses or implements DNA damage greatly predisposes to cancer. Nonetheless, even when oncogenic mutations do occur, there exist potent mechanisms that limit the expansion of affected cells by suppressing their proliferation or triggering their suicide. Growing understanding of these innate mechanisms is suggesting novel therapeutic strategies for cancer.

Members of the tumour necrosis factor (TNF) receptor family exert pleiotropic effects and can trigger both apoptosis and proliferation [1]. In their cytoplasmic region, some of these receptors share a conserved sequence motif – the ‘death domain' – which is required for transduction of the apoptotic signal by recruiting other death-domain-containing adaptor molecules like the Fas-associated protein FADD/MORT1 or the TNF receptor-associated protein TRADD [2–4]. FADD links the receptor signal to the activation of the caspase family of cysteine proteases [5,6]. Functional inactivation of individual receptor family members often fails to exhibit a distinctive phenotype, probably because of redundancy [7–9]. To circumvent this problem, we used a dominant-negative mutant of FADD (FADD-DN) which should block all TNF receptor family members that use FADD as an adaptor. We established transgenic mice expressing FADD-DN under the influence of the lck promoter and investigated the consequences of its expression in T cells. As expected, FADD-DN thymocytes were protected from death induced by CD95 (Fas/Apo1), whereas apoptosis induced by ultraviolet (UV) irradiation, anti-CD3 antibody treatment or dexamethasone was unaffected, as was spontaneous cell death. Surprisingly, however, we also observed profound inhibition of thymocyte proliferation in vivo and of activation-induced proliferation of thymocytes and mature T cells in vitro . This inhibition of proliferation was not due to increased cell death and appeared to be p53 dependent.

Normal fibroblasts are resistant to the cytotoxic action of tumor necrosis factor (TNF), but are rendered TNF-sensitive upon deregulation of c-Myc. To assess if oncoproteins induce the cytotoxic TNF activity by modulating TNF signaling, we investigated the TNF-elicited signaling responses in fibroblasts containing a conditionally active c-Myc protein. In association with cell death, c-Myc impaired TNF-induced activation of phospholipase A2, JNK protein kinase and cell survival-signaling-associated NF-kappaB transcription factor complex. The TNF-induced death of mouse primary fibroblasts expressing deregulated c-Myc was inhibited by transient overexpression of the p65 subunit of NF-kappaB, which increased NF-kappaB activity in the cells. Unlike other TNF-induced signals, TNF-induced accumulation of the wild-type p53 mRNA and protein was not inhibited by c-Myc. TNF, with c-Myc, induced apoptosis in mouse primary fibroblasts but only weakly in p53-deficient primary fibroblasts. The C-terminal domain of p53, which is a transacting dominant inhibitor of wild-type p53, failed to inhibit apoptosis by c-Myc and TNF, suggesting that the cell death was not dependent on the transcription-activating function of p53. Taken together, the present findings show that the cytotoxic activity of TNF towards oncoprotein-expressing cells involves p53 and an impaired signaling for survival in such cells.

Induction of apoptosis by oncogenes like c-mycmay be important in restraining the emergence of neoplasia. However, the mechanism by which c-mycinduces apoptosis is unknown. CD95 (also termed Fas or APO-1) is a cell surface transmembrane receptor of the tumor necrosis factor receptor family that activates an intrinsic apoptotic suicide program in cells upon binding either its ligand CD95L or antibody. c-myc–induced apoptosis was shown to require interaction on the cell surface between CD95 and its ligand. c-Myc acts downstream of the CD95 receptor by sensitizing cells to the CD95 death signal. Moreover, IGF-I signaling and Bcl-2 suppress c-myc–induced apoptosis by also acting downstream of CD95. These findings link two apoptotic pathways previously thought to be independent and establish the dependency of Myc on CD95 signaling for its killing activity.

Cysteine proteases of the ICE/CED‐3 family (caspases) are required for the execution of programmed cell death (PCD) in a wide range of multicellular organisms. Caspases are implicated in the execution of apoptosis in Drosophila melanogaster by the observation that expression of baculovirus p35, a caspase inhibitor, blocks cell death in vivo in Drosophila . We report here the identification and characterization of drICE, a D.melanogaster caspase. We show that overexpression of drICE sensitizes Drosophila cells to apoptotic stimuli and that expression of an N‐terminally truncated form of drICE rapidly induces apoptosis in Drosophila cells. Induction of apoptosis by rpr overexpression or by cycloheximide or etoposide treatment of Drosophila cells results in proteolytic processing of drICE. We further show that drICE is a cysteine protease that cleaves baculovirus p35 and Drosophila lamin DmO in vitro and that drICE is expressed at all the stages of Drosophila development at which PCD can be induced. Taken together, these results strongly argue that drICE is an apoptotic caspase that acts downstream of rpr . drICE is therefore the first unequivocal link between the molecular machinery of Drosophila cell death and the conserved machinery of Caenorhabditis elegans and vertebrates. Identification of drICE should facilitate the elucidation of upstream regulators and downstream targets of caspases by genetic screening.

Apoptosis as a form of programmed cell death (PCD) in multicellular organisms is a well-established genetically controlled process that leads to elimination of unnecessary or damaged cells. Recently, PCD has also been described for unicellular organisms as a process for the socially advantageous regulation of cell survival. The human Bcl-2 family member Bak induces apoptosis in mammalian cells which is counteracted by the Bcl-x(L) protein. We show that Bak also kills the unicellular fission yeast Schizosaccharomyces pombe and that this is inhibited by coexpression of human Bcl-x(L). Moreover, the same critical BH3 domain of Bak that is required for induction of apoptosis in mammalian cells is also required for inducing death in yeast. This suggests that Bak kills mammalian and yeast cells by similar mechanisms. The phenotype of the Bak-induced death in yeast involves condensation and fragmentation of the chromatin as well as dissolution of the nuclear envelope, all of which are features of mammalian apoptosis. These data suggest that the evolutionarily conserved metazoan PCD pathway is also present in unicellular yeast.

Background: Three principal genes are involved in developmental programmed cell death (PCD) in the nematode worm Caenorhabditis elegans . The ced-3 and ced-4 genes are both required for each PCD, whereas ced-9 acts to prevent the death-promoting actions of these genes in cells that are destined to survive. Vertebrate homologues of both ced-3 and ced-9 have been identified as the genes encoding the caspase cysteine proteases and the Bcl-2 family, respectively. In contrast, no vertebrate homologue of ced-4 is known. The CED-3/caspases are important effectors of apoptosis that are presumed to act by cleaving specific target substrates. However, the molecular functions of the CED-9/Bcl-2 and CED-4 proteins are unknown. The unicellular yeast Schizosaccharomyces pombe shares many general cellular properties with metazoa, but has no identified cell suicide machinery. We have therefore used S. pombe as a naive model cell system in which to examine the biological effects of cell-death proteins. Results: Induction of wild-type ced-4 expression in S. pombe resulted in rapid focal chromatin condensation and lethality. Mutation of the putative nucleotide-binding P-loop motif of CED-4 (K165Q) eliminated the lethal phenotype. Immunolocalization of CED-4 to the condensed chromatin suggested that the phenotype may result from an intrinsic activity of CED-4. Co-expression of ced-9 prevented CED-4-induced chromatin condensation and lethality, and caused the relocalization of CED-4 to endoplasmic reticulum and outer mitochondrial membranes. A direct interaction between CED-4 and CED-9 was confirmed by yeast two-hybrid analysis. Conclusions: Using S. pombe as a model system in which to assay CED-4 function, we have identified a potential direct role for CED-4 in chromatin condensation. Chromatin condensation is a ubiquitous feature of metazoan apoptosis that has yet to be linked to an effector. The CED-9-mediated rescue of CED-4-induced lethality in this system and the interaction of the two proteins in the yeast two-hybrid analysis suggest that CED-9 inhibits CED-4 action by direct physical association.

There is increasing evidence for a central role in mammalian apoptosis of the interleukin-1β– converting enzyme (ICE) family of cysteine proteases, homologues of the product of the nematode “death” gene, ced-3. Ced-3 is thought to act as an executor rather than a regulator of programmed cell death in the nematode. However, it is not known whether mammalian ICE-related proteases (IRPs) are involved in the execution or the regulation of mammalian apoptosis. Moreover, an absolute requirement for one or more IRPs for mammalian apoptosis has yet to be established. We have used two cell-permeable inhibitors of IRPs, Z-Val-Ala-Asp.fluoromethylketone (ZVAD.fmk) and t-butoxy carbonyl-Asp.fluoromethylketone (BD.fmk), to demonstrate a critical role for IRPs in mammalian apoptosis induced by several disparate mechanisms (deregulated oncogene expression, ectopic expression of the Bcl-2 relative Bak, and DNA damage–induced cell death). In all instances, ZVAD.fmk and BD.fmk treatment inhibits characteristic biochemical and morphological events associated with apoptosis, including cleavage of nuclear lamins and poly-(ADP-ribose) polymerase, chromatin condensation and nucleosome laddering, and external display of phosphatidylserine. However, neither ZVAD.fmk nor BD.fmk inhibits the onset of apoptosis, as characterized by the onset of surface blebbing; rather, both act to delay completion of the program once initiated. In complete contrast, IGF-I and Bcl-2 delay the onset of apoptosis but have no effect on the kinetics of the program once initiated. Our data indicate that IRPs constitute part of the execution machinery of mammalian apoptosis induced by deregulated oncogenes, DNA damage, or Bak but that they act after the point at which cells become committed to apoptosis or can be rescued by survival factors. Moreover, all such blocked cells have lost proliferative potential and all eventually die by a process involving cytoplasmic blebbing.

Using a series of insulin-like growth factor I (IGF-I) receptor mutants, we have attempted to define domains required for transmitting the antiapoptotic signal from the receptor and to compare these domains with those required for mitogenesis or transformation. In FL5.12 cells transfected with wild-type IGF-I receptors, IGF-I affords protection from interleukin 3 withdrawal but is not mitogenic. An IGF-I receptor lacking a functional ATP binding site provided no protection from apoptosis. However, receptors mutated at tyrosine residue 950 or in the tyrosine cluster (1131, 1135, and 1136) within the kinase domain remained capable of suppressing apoptosis, although such mutations are known to inactivate transforming and mitogenic functions. In the C terminus of the IGF-I receptor, two mutations, one at tyrosine 1251 and one which replaced residues histidine 1293 and lysine 1294, abolished the antiapoptotic function, whereas mutation of the four serines at 1280 to 1283 did not. Interestingly, receptors truncated at the C terminus had enhanced antiapoptotic function. In Rat-1/ c-MycER fibroblasts, the Y950F mutant and the tyrosine cluster mutant could still provide protection from c-Myc-induced apoptosis, whereas mutant Y1250/1251F could not. These studies demonstrate that the domains of the IGF-I receptor required for its antiapoptotic function are distinct from those required for its proliferation or transformation functions and suggest that domains of the receptor required for inhibition of apoptosis are necessary but not sufficient for transformation.

mir-17-92, a potent polycistronic oncomir, encodes six mature miRNAs with complex modes of interactions. In the Eμ-myc Burkitt’s lymphoma model, mir-17-92 exhibits potent oncogenic activity by repressing c-Myc-induced apoptosis, primarily through its miR-19 components. Surprisingly, mir-17-92 also encodes the miR-92 component that negatively regulates its oncogenic cooperation with c-Myc. This miR-92 effect is, at least in part, mediated by its direct repression of Fbw7, which promotes the proteosomal degradation of c-Myc. Thus, overexpressing miR-92 leads to aberrant c-Myc increase, imposing a strong coupling between excessive proliferation and p53-dependent apoptosis. Interestingly, miR-92 antagonizes the oncogenic miR-19 miRNAs; and such functional interaction coordinates proliferation and apoptosis during c-Myc-induced oncogenesis. This miR-19:miR-92 antagonism is disrupted in B-lymphoma cells that favor a greater increase of miR-19 over miR-92. Altogether, we suggest a new paradigm whereby the unique gene structure of a polycistronic oncomir confers an intricate balance between oncogene and tumor suppressor crosstalk. DOI: http://dx.doi.org/10.7554/eLife.00822.001, eLife digest The role of genes, in very simple terms, is to be transcribed into messenger RNA molecules, which are then translated into strings of amino acids that fold into proteins. Each of these steps is extremely complex, and a wide range of other molecules can speed up, slow down, stop or otherwise disrupt the expression of genes as protein products. Genes can also code for nucleic acids that are not translated into proteins, such as microRNAs. These are small RNA molecules that can reduce the production of proteins by repressing the translation step and/or by partially degrading the messenger RNA molecules. mir-17-92 is a gene that exemplifies much of this complexity. It codes for six different microRNAs in a single primary transcript, and has been implicated in a number of cancers, including lung cancer, Burkitt’s lymphoma and other forms of lymphomas and leukemia. One of six microRNAs has a longer evolutionary history than the remaining five: mir-92 is found in vertebrates, chordates and invertebrates, whereas the other five are only found in vertebrates. However, it is not known how or why the mir-17-92 gene evolved to code for multiple different microRNAs. Olive et al. have studied how these mir-17-92 microRNAs functionally interact in mice with Burkitt’s lymphoma, a form of cancer that is associated with a gene called c-Myc being over-activated. Mutations in this gene promote the proliferation of cells, and in cooperation with other genetic lesions, this ultimately leads to cancer. mir-17-92 is implicated in this cancer because it represses the process of programmed cell death (which is induced by the protein c-Myc) that the body employs to stop tumors growing. Olive et al. found that deleting one of the six microRNAs, miR-92, increased the tendency of the mir-17-92 gene to promote Burkitt’s lymphoma. By repressing an enzyme called Fbw7, miR-92 causes high levels of c-Myc to be produced. While this leads to the uncontrolled proliferation of cells that promotes cancer, it also increases programmed cell death, at least in part, by activating the p53 pathway, a well-known tumor suppression pathway. The experiments also revealed that the action of miR-92 and that of one of the other microRNAs, miR-19, were often opposed to each other. These findings have revealed an unexpected interaction among different components within a single microRNA gene, which acts to maintain an intricate balance between pathways that promote and suppress cancer. DOI: http://dx.doi.org/10.7554/eLife.00822.002

The origin recognition complex (ORC) binds to origins of replication in budding yeast. We have cloned a Xenopus homolog of the largest ORC polypeptide (XORC1). Immunodepletion of XOrc1 from Xenopus egg extracts blocks the initiation of DNA replication. We have purified Xenopus ORC, consisting of a protein complex similar to yeast ORC. In Xenopus egg extracts, ORC associates with chromatin throughout G1 and S phases. RLF-M, a component of the replication licensing system, also associates with chromatin early in the cell cycle but dissociates during S phase. We show that the assembly of RLF-M onto chromatin is dependent on the presence of chromatin-bound ORC, leading to sequential assembly of initiation proteins onto replication origins during the cell cycle.

Apoptosis is a common feature of the nervous system, occurring physiologically during development and pathologically in several diseases. In view of the latter, pathways that regulate apoptosis in neurones are of particular interest, and recent advances in this field implicate several signalling pathways in the induction of apoptosis, after withdrawal of nerve growth factor. Recent information drawn from both mammalian and invertebrate models enables us to assign components of the apoptotic pathway as either regulators or effectors.

Synthetic peptides play an important role in many areas of biological research. Advances in synthetic chemistry and automation over the past few years have resulted in increasingly reliable and rapid syntheses. As a result, peptides are now frequently employed in immunological studies, structural studies, as enzyme substrates, in ligand/receptor studies, and as probes for a range of molecular interactions. This review describes solid-phase peptide synthesis and the applications of synthetic peptides in molecular biology and biochemistry.

Early tumor evolution through sustained oncogene activity selectively bypasses the engagement of cell-intrinsic tumor-suppressor signaling and cell-extrinsic microenvironmental restrictions. How the immune system may be involved is virtually unknown. We describe here the contributions of a conditional and reversible low-level expression of Myc in a mouse model of KrasG12D-driven non-small cell lung cancer. Deregulated Myc activity results in highly expansive tumors that appear embedded in inflamed regions and leads to a rapid reduction of mouse survival. Myc activation imposes an immediate switch to an immunosuppressive and pro-angiogenic microenvironment, facilitated through IL23- and CCL9-associated recruitment of PD-L1 loaded macrophages and local exclusion of T-lymphocytes. Reversibly, blocking the activity of Myc-driven IL23 and CCL9 expression or withdrawal of deregulated Myc activity in tumors established by oncogene cooperation results in tumor cell death and regression, associated with a collapse of the established microenvironmental changes and re-engagement of cytotoxic T-cells. During oncogene cooperation with Ras, deregulated Myc directs a sufficient and necessary switch to a microenvironment that shields tumor growth and expansion from immune suppression. Citation Format: Roderik M. Kortlever, Nicole M. Sodir, Catherine H. Wilson, Deborah L. Burkhart, Lamorna Swigart, Trevor D. Littlewood, Gerard I. Evan. Deregulated Myc is an immunosuppressive switch. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 535.

A number of proteins have been rendered functionally oestrogen-dependent by fusion with the hormone-binding domain of the oestrogen receptor. There are, however, several significant disadvantages with such fusion proteins. First, their use in cells in vitro requires phenol red-free medium and laborious stripping of steroid hormones from serum in order to avoid constitutive activation. Secondly, control of oestrogen receptor fusion proteins in vivo is precluded by high endogenous levels of circulating oestrogens. Thirdly, the hormone-binding domain of the oestrogen receptor functions as a hormone-dependent transcriptional activation domain making interpretation of fusions with transcription factors problematical. In order to overcome these drawbacks we have used a transcriptionally inactive mutant of the murine oestrogen receptor which is unable to bind oestrogen yet retains normal affinity for the synthetic ligand, 4-hydroxytamoxifen. When the hormone-binding domain of this mutant oestrogen receptor is fused to the C-terminus of the c-Myc protein, Myc-induced proliferation and apoptosis in fibroblasts becomes dependent on 4-hydroxytamoxifen, but remains refractory to 17 beta-oestradiol.

Progression of premalignant lesions is restrained by oncogene-induced senescence. Oncogenic Ras triggers senescence in many organs, including the lung, which exhibits high levels of the angiogenesis inhibitor thrombospondin-1 (TSP-1). The contribution of TSP-1 upregulation to the modulation of tumorigenesis in the lung is unclear. Using a mouse model of lung cancer, we have shown that TSP-1 plays a critical and cell-autonomous role in suppressing Kras-induced lung tumorigenesis independent of its antiangiogenic function. Overall survival was decreased in a Kras-driven mouse model of lung cancer on a Tsp-1–/– background. We found that oncogenic Kras–induced TSP-1 upregulation in a p53-dependent manner. TSP-1 functioned in a positive feedback loop to stabilize p53 by interacting directly with activated ERK. TSP-1 tethering of ERK in the cytoplasm promoted a level of MAPK signaling that was sufficient to sustain p53 expression and a senescence response. Our data identify TSP-1 as a p53 target that contributes to maintaining Ras-induced senescence in the lung.

Despite their apparent success in pre-clinical trials, metalloproteinase (MMP) inhibitors proved to be inefficacious in clinical settings. In an effort to understand the underlying causes of this unanticipated outcome, we modeled the consequences of long-term MMP inhibition by removing one of the major players in tumorigenesis, MMP9, in two complimentary mouse models of pancreatic neuroendocrine carcinogenesis: Myc;BclXl and RIP1-Tag2. By employing gel zymography and a fluoregenic solution assay, we first established that MMP9 is expressed and activated in Myc;BclXl tumors in an interleukin-1β-dependent manner. The genetic deletion of MMP9 in Myc;BclXl mice impairs tumor angiogenesis and growth analogous to its absence in the RIP1-Tag2 model. Notably, tumors that developed in the context of MMP9-deficient backgrounds in both models were markedly more invasive than their typical wild-type counterparts, and expressed elevated levels of pro-invasive cysteine cathepsin B. The increased invasion of MMP9-deficient tumors was associated with a switch in the spectrum of inflammatory cells at the tumor margins, involving homing of previously undetected, cathepsin-B expressing CD11b;Gr1-positive cells to the invasive fronts. Thus, plasticity in the tumor inflammatory compartment is partially responsible for changes in the expression pattern of tumor-associated proteases, and may contribute to the compensatory effects observed on MMP inhibition, hence accounting for the heightened tumor progression described in late stage clinical trials.

Apoptosis of mammalian cell is under the control of a wide range of intracellular and extracellular factors-amongst them proteases, protein kinases, cytokines and the protein products of oncogenes and tumour suppressor genes. The c-myc proto-oncogene encodes an essential component of the cell's proliferative machinery and its deregulated expression is implicated in many cancers. Under certain conditions, c-Myc also acts as a potent inducer of apoptosis. We have developed a 'switchable' chimaeric c-Myc protein whose activity is dependent on the synthetic ligand, 4-hydroxytamoxifen. In cells expressing this switchable c-Myc, proliferation and apoptosis in cultured fibroblasts can be regulated by addition of 4-hydroxytamoxifen. We have further demonstrated the utility of a switchable gene transcription system for the induction of proteins with pro-apoptotic effect. Myc-induced apoptosis is inhibited by the action of certain cytokines or by expresson of exogenous proteins with anti-apoptotic potential such as Bcl-2. We show that inhibition of p53 using dominant negative molecules inhibits apoptosis induced by DNA damage but has little effect on Myc-induced apoptosis. Finally, we have also been able to modulate a relatively late stage in apoptosis using inhibitors of cysteine proteases. Our data suggest a model in which the integrated activities of several proteins with diverse molecular functions may determine whether a particular cell undergoes apoptosis but that, once the actual catalytic machinery is engaged, the apoptotic process is irreversible.

Vascular smooth muscle (VSM) cell proliferation contributes to the pathogenesis of atherosclerosis, restenosis after angioplasty and vein graft disease. The regulation of genes involved in VSM cell proliferation, particularly by naturally occurring inhibitors, is therefore of some importance. We have investigated the role of the c-myc proto-oncogene in growth arrest of exponentially proliferating rat VSM cells, following mitogen withdrawal, treatment with heparin (50 micrograms/ml), interferon-gamma (IFN-gamma) (100 i.u./ml), or the cyclic nucleotide analogues, 8-bromo-adenosine-3′5′-cyclic monophosphate (8-Br-cAMP; 0.1 mM) and 8-bromoguanosine-3′5′-cyclic monophosphate (8-Br-cGMP; 0.1 mM). Growth arrest was accompanied by down-regulation of c-Myc protein and mRNA following treatment with all inhibitors. Serum withdrawal or IFN-gamma treatment suppressed c-myc expression by more than 50% within 2 h, and this occurred throughout the cell cycle. Platelet-derived growth factor, epidermal growth factor and basic fibroblast growth factor all contributed independently to the maintenance of c-myc expression. Heparin, 8-Br-cAMP or 8-Br-cGMP also suppressed c-myc, but this occurred later, after 24-48 h, and was also observed following arrest by metabolic block. We conclude that c-myc expression is linked to VSM cell growth arrest in response to endogenous regulators and metabolic block. Down-regulation of c-myc expression may thus be an essential part of the arrest programme in VSM cells induced by many pharmacological agents.

Structural and functional similarities have been discovered between two mammalian proteins, Bcl-2 and interleukin 1 β -converting enzyme, and proteins encoded by nematode cell-death genes.

Several recent studies have implicated oncogenes and tumour suppressor genes in the regulation of programmed cell death (apoptosis). Lesions in the cell death pathway appear to be important in both carcinogenesis and the evolution of drug resistance in tumours. They include deregulated expression of genes such as bcl-2, loss of p53, and autocrine activation of anti-apoptotic signal transduction pathways. Paradoxically, a number of dominant oncogenes appear to act as potent inducers of apoptosis. This suggests that the pathways of cell proliferation and cell death may be tightly coupled, an idea that may have dramatic implications for models of oncogene co-operation and carcinogenesis.

Restenosis after angioplasty is due predominantly to accumulation of vascular smooth muscle cells (VSMCs). The resistance of restenosis to pharmacological treatment has prompted investigation of genes involved in VSMC proliferation. We have examined the effect on VSMC proliferation of blocking expression of the c-myc proto-oncogene with antisense oligodeoxynucleotides, both in vitro and in a rat carotid artery injury model of angioplasty restenosis. Antisense c-myc oligodeoxynucleotides reduced average cell levels of c-myc mRNA and protein by 50-55% and inhibited proliferation of VSMCs when mitogenically stimulated from quiescence or when proliferating logarithmically (IC50 = 10 micrograms/ml). Corresponding sense c-myc, two-base-pair mismatch antisense c-myc, antisense alpha-actin or glyceraldehyde phosphate dehydrogenase oligodeoxynucleotides did not suppress c-myc expression or inhibit VSMC proliferation. Antisense c-myc inhibition was relieved by overexpression of an exogenous c-myc gene. After balloon catheter injury, peak c-myc mRNA expression occurred at 2 h. Antisense c-myc applied in a pluronic gel to the arterial adventitia reduced peak c-myc expression by 75% and significantly reduced neointimal formation at 14 d, compared with sense c-myc and gel application alone. We conclude that c-myc expression is required for VSMC proliferation in vitro and in the vessel wall. C-myc is a therefore a potential target for adjunctive therapy to reduce angioplasty restenosis.

A new paper implicates the Myc oncoprotein in the direct induction of DNA damage and consequent genome instability in cultured cells. However, it is less clear whether Myc induces the same genetic pandemonium in vivo.

MdmX, also known as Mdm4, is a critical negative regulator of p53, and its overexpression serves to block p53 tumor suppressor function in many cancers. Consequently, inhibiting MdmX has emerged as an attractive approach to restoring p53 function in those cancers that retain functional p53. However, the consequences of acute systemic MdmX inhibition in normal adult tissues remain unknown. To determine directly the effects of systemic MdmX inhibition in normal tissues and in tumors, we crossed mdmX−/− mice into the p53ERTAM knockin background. In place of wild-type p53, p53ERTAM knockin mice express a variant of p53, p53ERTAM, that is completely dependent on 4-hydroxy-tamoxifen for its activity. MdmX inhibition was then modeled by restoring p53 function in these MdmX-deficient mice. We show that MdmX is continuously required to buffer p53 activity in adult normal tissues and their stem cells. Importantly, the effects of transient p53 restoration in the absence of MdmX are nonlethal and reversible, unlike transient p53 restoration in the absence of Mdm2, which is ineluctably lethal. We also show that the therapeutic impact of restoring p53 in a tumor model is enhanced in the absence of MdmX, affording a significant extension of life span over p53 restoration in the presence of MdmX. Hence, systemic inhibition of MdmX is both a feasible therapeutic strategy for restoring p53 function in tumors that retain wild-type p53 and likely to be significantly safer than inhibition of Mdm2.

We have identified a protein motif, related to the zinc finger, which defines a newly discovered family of proteins. The motif was found in the sequence of the human RING1 gene, which is proximal to the major histocompatibility complex region on chromosome six. We propose naming this motif the "RING finger" and it is found in 27 proteins, all of which have putative DNA binding functions. We have synthesized a peptide corresponding to the RING1 motif and examined a number of properties, including metal and DNA binding. We provide evidence to support the suggestion that the RING finger motif is the DNA binding domain of this newly defined family of proteins.

c-Myc (Myc) and Max proteins dimerize and bind DNA through basic-helix-loop-helix-leucine zipper motifs (b-HLH-LZ). Using a genetic approach, we demonstrate that binding to Max is essential for Myc transforming activity and that Myc homodimers are inactive. Mutants of Myc and Max that bind efficiently to each other but not to their wild-type partners were generated by either exchanging the HLH-LZ domains or reciprocally modifying LZ dimerization specificities. While transformation defective on their own, complementary mutants restore Myc transforming activity when coexpressed in cells. The HLH-LZ exchange mutants also have dominant negative activity on wild-type Myc function. In addition, wild-type max antagonizes myc function in a dose-dependent manner, presumably through competition of Max-Max and Myc-Max dimers for common target DNA sites. Therefore, Max can function as both suppressor and activator of Myc. A general model for the role of Myc and Max in growth control is discussed.

Summary Non-small cell lung carcinoma (NSCLC) is the leading cause of cancer-related death worldwide, with an overall 5-year survival rate of only 10–15% 1. Deregulation of the Ras pathway is a frequent hallmark of NSCLC, often through mutations that directly activate Kras 2. p53 is also frequently inactivated in NSCLC and, since oncogenic Ras can be a potent trigger of p53 3, it seems likely that oncogenic Ras signalling plays a major and persistent part in driving the selection against p53. Hence, pharmacological restoration of p53 is an appealing therapeutic strategy for treating this disease 4. Here, we model the likely therapeutic impact of p53 restoration in a spontaneously evolving mouse model of NSCLC initiated by sporadic oncogenic activation of endogenous Kras 5. Surprisingly, p53 restoration failed to induce significant regression of established tumours although it did result in a significant decrease in the relative proportion of tumours classed as high grade. This is due to selective activation of p53 only in the more aggressive tumour cells within each tumour. Such selective activation of p53 correlates with marked up regulation in Ras signal intensity and induction of the oncogenic signalling sensor p19ARF 6. Our data indicate that p53-mediated tumour suppression is triggered only when oncogenic Ras signal flux exceeds a critical threshold. Importantly, the failure of low-level oncogenic Kras to engage p53 reveals inherent limits in the capacity of p53 to restrain early tumour evolution and to the efficacy of therapeutic p53 restoration to eradicate cancers.

The role of cell surface galactosyltransferase in mediating isoproterenol-induced parotid gland hypertrophy and hyperplasia was examined in rat parotid gland acinar cells. Introduction of the transferase modifier, α-lactalbumin, or galactosyltransferase-associated kinase inhibitor trifluoperazine, into β-agonist-treated rats prevented acinar cell proliferation as determined by [ 3 H]thymidine incorporation after 96 h of treatment. However, [ 3 H]thymidine incorporation into DNA after 24 h of treatment, with injection of a combination of isoproterenol/α-lactalbumin or isoproterenol/trifluoperazine, was similar to injections of isoproterenol alone; suggesting that acinar cells could be stimuated to undergo a single round of DNA synthesis. Northern blot analysis of myc and fos expression followed a similar pattern of down-regulation to control levels after 96 h but not after 24 h. Hybridization with erb B showed little change with proliferation, confirming previous observations on protein levels of the EGF-receptor in acinar cells. Western blot analysis of nuclear protein expression of myc revealed that isoproterenol caused an increase in a 62-kDa protein which was again down-regulated with inhibition of cell proliferation. Analysis of protein levels of Rb 110 protein showed no change in protein level in the nucleus with cell proliferation, but did show an associated increase in protein phosphorylation in response to growth stimulation.