Necroptosis is preceded by nuclear translocation of the signaling proteins that induce it

A signaling pathway that induces programmed necrotic cell death (necroptosis) was reported to be activated in cells by several cytokines and various pathogen components. The major proteins participating in that pathway are the protein kinases RIPK1 and RIPK3 and the pseudokinase mixed lineage kinase domain-like protein (MLKL). Recent studies have suggested that MLKL, once activated, mediates necroptosis by binding to cellular membranes, thereby triggering ion fluxes. However, our knowledge of both the sequence of molecular events leading to MLKL activation and the subcellular sites of these events is fragmentary. Here we report that the association of MLKL with the cell membrane in necroptotic death is preceded by the translocation of phosphorylated MLKL, along with RIPK1 and RIPK3, to the nucleus.Apart from the apoptotic cell death pathway that ligands of the tumor necrosis factor (TNF) family can activate, these ligands and various other inducers, including the interferons and various pathogen components, have in recent years been found also to trigger a signaling cascade that induces programmed necrotic death (necroptosis). This cascade encompasses sequential activation of the protein kinases RIPK1 and RIPK3 and the pseudokinase mixed lineage kinase domain-like protein (MLKL).^{1, 2, 3, 4, 5} RIPK3-mediated phosphorylation of MLKL triggers its oligomerization, which is necessary and sufficient for the induction of cell death,^{6, 7, 8} and can also trigger some non-deadly functions.⁹ MLKL was recently suggested to trigger cell death by binding to cellular membranes and initiating ion fluxes through them.^{6, 7, 8, 10} However, its exact molecular target in death induction is contentious.^{6, 8, 10, 11, 12} Current knowledge of the subcellular sites of MLKL action is based mainly on determination of the location of this protein close to the time of cell death. Here we present a detailed assessment of the cellular location of MLKL at different times following its activation. Our findings indicate that before cell death, MLKL translocates to the nucleus along with RIPK1 and RIPK3.     

Characterization of RIPK3-mediated phosphorylation of the activation loop of MLKL during necroptosis

Mixed lineage kinase domain-like pseudokinase (MLKL) mediates necroptosis by translocating to the plasma membrane and inducing its rupture. The activation of MLKL occurs in a multimolecular complex (the ‘necrosome''), which is comprised of MLKL, receptor-interacting serine/threonine kinase (RIPK)-3 (RIPK3) and, in some cases, RIPK1. Within this complex, RIPK3 phosphorylates the activation loop of MLKL, promoting conformational changes and allowing the formation of MLKL oligomers, which migrate to the plasma membrane. Previous studies suggested that RIPK3 could phosphorylate the murine MLKL activation loop at Ser345, Ser347 and Thr349. Moreover, substitution of the Ser345 for an aspartic acid creates a constitutively active MLKL, independent of RIPK3 function. Here we examine the role of each of these residues and found that the phosphorylation of Ser345 is critical for RIPK3-mediated necroptosis, Ser347 has a minor accessory role and Thr349 seems to be irrelevant. We generated a specific monoclonal antibody to detect phospho-Ser345 in murine cells. Using this antibody, a series of MLKL mutants and a novel RIPK3 inhibitor, we demonstrate that the phosphorylation of Ser345 is not required for the interaction between RIPK3 and MLKL in the necrosome, but is essential for MLKL translocation, accumulation in the plasma membrane, and consequent necroptosis.Regulated necrotic cell death, or ‘necroptosis,'' is mediated by the interaction of activated receptor-interacting kinase-3 (RIPK3) and mixed lineage kinase like (MLKL).^{1, 2, 3} The function of RIPK3 to promote necroptosis can be induced by the activity of receptor-interacting protein kinase-1 (RIPK1),⁴ and is antagonized by the proteolytic activity of a complex formed by RIPK1, FADD, caspase-8 and c-FLIP_L.^{5, 6, 7, 8, 9, 10} Inactive RIPK1 functions to inhibit RIPK3 activation, even under conditions in which RIPK3 is activated independently of RIPK1.^{11, 12, 13} These complex interactions help to account for the lethal effects of ablating FADD, caspase-8 or RIPK1.¹⁴MLKL is a substrate for RIPK3 kinase activity^{1, 2, 3} and appears to execute the process of necroptosis by targeting the plasma membrane.^{15, 16, 17} The phosphorylation of MLKL by RIPK3 has been proposed to promote necroptosis by inducing essential changes in the ‘latch'' of this pseudokinase, allowing the formation of oligomers, migration to plasma membrane^{15, 16, 17, 18} and binding to phosphatidylinositol lipids to directly disrupt membrane integrity.^{16, 19} Structurally, murine MLKL is composed of a pseudokinase domain (C-terminal region) and a four-helical bundle domain (4HBD) located in the N-terminal region.^{3, 20} The 4HBD domain is sufficient to oligomerize, bind to phosphatidylinositol lipids and trigger cell death.^{16, 19} However, the activation of full-length MLKL requires phosphorylation of residues in the activation loop in the pseudokinase domain. The residues Ser345, Ser347 and Thr349 within the murine MLKL activation loop are RIPK3 phosphorylation sites,³ corresponding to Thr357 and Ser358 in human MLKL.¹⁶ Upon RIPK3 phosphorylation, human MLKL shifts from its monomeric state to an active oligomeric state.¹⁶The residue Gln343 in the murine α-helix (residues Leu339 to Ser347) forms a hydrogen bond with Lys219 and the Ser345 and disruption of this coordinated state by phosphorylation of Ser345 has been proposed to destabilize the monomeric structure, promoting a conformational change in MLKL to an active state.^{3, 21} This hypothesis was supported by the specific mutations K219M, Q343A or S345D; all of which led to a form of MLKL form that promoted necroptosis independently of RIPK3.^{3, 16}In this study, we examine serine and threonine residues within the activation loop of MLKL for their roles in necroptosis. We have developed an antibody anti-phospho-Ser345 and explore its use as a marker for necroptosis in murine cell systems. Using this antibody, together with described and novel inhibitors of RIPK3, we more fully explore the role of modifications in the active loop of MLKL during the process of necroptosis.     

A cellular screen identifies ponatinib and pazopanib as inhibitors of necroptosis

Necroptosis is a form of regulated necrotic cell death mediated by receptor-interacting serine/threonine-protein kinase 1 (RIPK1) and RIPK3. Necroptotic cell death contributes to the pathophysiology of several disorders involving tissue damage, including myocardial infarction, stroke and ischemia-reperfusion injury. However, no inhibitors of necroptosis are currently in clinical use. Here we performed a phenotypic screen for small-molecule inhibitors of tumor necrosis factor-alpha (TNF-α)-induced necroptosis in Fas-associated protein with death domain (FADD)-deficient Jurkat cells using a representative panel of Food and Drug Administration (FDA)-approved drugs. We identified two anti-cancer agents, ponatinib and pazopanib, as submicromolar inhibitors of necroptosis. Both compounds inhibited necroptotic cell death induced by various cell death receptor ligands in human cells, while not protecting from apoptosis. Ponatinib and pazopanib abrogated phosphorylation of mixed lineage kinase domain-like protein (MLKL) upon TNF-α-induced necroptosis, indicating that both agents target a component upstream of MLKL. An unbiased chemical proteomic approach determined the cellular target spectrum of ponatinib, revealing key members of the necroptosis signaling pathway. We validated RIPK1, RIPK3 and transforming growth factor-β-activated kinase 1 (TAK1) as novel, direct targets of ponatinib by using competitive binding, cellular thermal shift and recombinant kinase assays. Ponatinib inhibited both RIPK1 and RIPK3, while pazopanib preferentially targeted RIPK1. The identification of the FDA-approved drugs ponatinib and pazopanib as cellular inhibitors of necroptosis highlights them as potentially interesting for the treatment of pathologies caused or aggravated by necroptotic cell death.Programmed cell death has a crucial role in a variety of biological processes ranging from normal tissue development to diverse pathological conditions.^{1, 2} Necroptosis is a form of regulated cell death that has been shown to occur during pathogen infection or sterile injury-induced inflammation in conditions where apoptosis signaling is compromised.^{3, 4, 5, 6} Given that many viruses have developed strategies to circumvent apoptotic cell death, necroptosis constitutes an important, pro-inflammatory back-up mechanism that limits viral spread in vivo.^{7, 8, 9} In contrast, in the context of sterile inflammation, necroptotic cell death contributes to disease pathology, outlining potential benefits of therapeutic intervention.¹⁰ Necroptosis can be initiated by death receptors of the tumor necrosis factor (TNF) superfamily,¹¹ Toll-like receptor 3 (TLR3),¹² TLR4,¹³ DNA-dependent activator of IFN-regulatory factors¹⁴ or interferon receptors.¹⁵ Downstream signaling is subsequently conveyed via RIPK1¹⁶ or TIR-domain-containing adapter-inducing interferon-β,^{8, 17} and converges on RIPK3-mediated^{13, 18, 19, 20} activation of MLKL.²¹ Phosphorylated MLKL triggers membrane rupture,^{22, 23, 24, 25, 26} releasing pro-inflammatory cellular contents to the extracellular space.²⁷ Studies using the RIPK1 inhibitor necrostatin-1 (Nec-1) ²⁸ or RIPK3-deficient mice have established a role for necroptosis in the pathophysiology of pancreatitis,¹⁹ artherosclerosis,²⁹ retinal cell death,³⁰ ischemic organ damage and ischemia-reperfusion injury in both the kidney³¹ and the heart.³² Moreover, allografts from RIPK3-deficient mice are better protected from rejection, suggesting necroptosis inhibition as a therapeutic option to improve transplant outcome.³³ Besides Nec-1, several tool compounds inhibiting different pathway members have been described,^{12, 16, 21, 34, 35} however, no inhibitors of necroptosis are available for clinical use so far.^{2, 10} In this study we screened a library of FDA approved drugs for the precise purpose of identifying already existing and generally safe chemical agents that could be used as necroptosis inhibitors. We identified the two structurally distinct kinase inhibitors pazopanib and ponatinib as potent blockers of necroptosis targeting the key enzymes RIPK1/3.     

TRAF2 is a biologically important necroptosis suppressor

Tumor necrosis factor α (TNFα) triggers necroptotic cell death through an intracellular signaling complex containing receptor-interacting protein kinase (RIPK) 1 and RIPK3, called the necrosome. RIPK1 phosphorylates RIPK3, which phosphorylates the pseudokinase mixed lineage kinase-domain-like (MLKL)—driving its oligomerization and membrane-disrupting necroptotic activity. Here, we show that TNF receptor-associated factor 2 (TRAF2)—previously implicated in apoptosis suppression—also inhibits necroptotic signaling by TNFα. TRAF2 disruption in mouse fibroblasts augmented TNFα–driven necrosome formation and RIPK3-MLKL association, promoting necroptosis. TRAF2 constitutively associated with MLKL, whereas TNFα reversed this via cylindromatosis-dependent TRAF2 deubiquitination. Ectopic interaction of TRAF2 and MLKL required the C-terminal portion but not the N-terminal, RING, or CIM region of TRAF2. Induced TRAF2 knockout (KO) in adult mice caused rapid lethality, in conjunction with increased hepatic necrosome assembly. By contrast, TRAF2 KO on a RIPK3 KO background caused delayed mortality, in concert with elevated intestinal caspase-8 protein and activity. Combined injection of TNFR1-Fc, Fas-Fc and DR5-Fc decoys prevented death upon TRAF2 KO. However, Fas-Fc and DR5-Fc were ineffective, whereas TNFR1-Fc and interferon α receptor (IFNAR1)-Fc were partially protective against lethality upon combined TRAF2 and RIPK3 KO. These results identify TRAF2 as an important biological suppressor of necroptosis in vitro and in vivo.Apoptotic cell death is mediated by caspases and has distinct morphological features, including membrane blebbing, cell shrinkage and nuclear fragmentation.^{1, 2, 3, 4} In contrast, necroptotic cell death is caspase-independent and is characterized by loss of membrane integrity, cell swelling and implosion.^{1, 2, 5} Nevertheless, necroptosis is a highly regulated process, requiring activation of RIPK1 and RIPK3, which form the core necrosome complex.^{1, 2, 5} Necrosome assembly can be induced via specific death receptors or toll-like receptors, among other modules.^{6, 7, 8, 9} The activated necrosome engages MLKL by RIPK3-mediated phosphorylation.^{6, 10, 11} MLKL then oligomerizes and binds to membrane phospholipids, forming pores that cause necroptotic cell death.^{10, 12, 13, 14, 15} Unchecked necroptosis disrupts embryonic development in mice and contributes to several human diseases.^{7, 8, 16, 17, 18, 19, 20, 21, 22}The apoptotic mediators FADD, caspase-8 and cFLIP suppress necroptosis.^{19, 20, 21, 23, 24} Elimination of any of these genes in mice causes embryonic lethality, subverted by additional deletion of RIPK3 or MLKL.^{19, 20, 21, 25} Necroptosis is also regulated at the level of RIPK1. Whereas TNFα engagement of TNFR1 leads to K63-linked ubiquitination of RIPK1 by cellular inhibitor of apoptosis proteins (cIAPs) to promote nuclear factor (NF)-κB activation,²⁶ necroptosis requires suppression or reversal of this modification to allow RIPK1 autophosphorylation and consequent RIPK3 activation.^{2, 23, 27, 28} CYLD promotes necroptotic signaling by deubiquitinating RIPK1, augmenting its interaction with RIPK3.²⁹ Conversely, caspase-8-mediated CYLD cleavage inhibits necroptosis.²⁴TRAF2 recruits cIAPs to the TNFα-TNFR1 signaling complex, facilitating NF-κB activation.^{30, 31, 32, 33} TRAF2 also supports K48-linked ubiquitination and proteasomal degradation of death-receptor-activated caspase-8, curbing apoptosis.³⁴ TRAF2 KO mice display embryonic lethality; some survive through birth but have severe developmental and immune deficiencies and die prematurely.^{35, 36} Conditional TRAF2 KO leads to rapid intestinal inflammation and mortality.³⁷ Furthermore, hepatic TRAF2 depletion augments apoptosis activation via Fas/CD95.³⁴ TRAF2 attenuates necroptosis induction in vitro by the death ligands Apo2L/TRAIL and Fas/CD95L.³⁸ However, it remains unclear whether TRAF2 regulates TNFα-induced necroptosis—and if so—how. Our present findings reveal that TRAF2 inhibits TNFα necroptotic signaling. Furthermore, our results establish TRAF2 as a biologically important necroptosis suppressor in vitro and in vivo and provide initial insight into the mechanisms underlying this function.     

Absence of RIPK3 predicts necroptosis resistance in malignant melanoma

Acquired or intrinsic resistance to apoptotic and necroptotic stimuli is considered a major hindrance of therapeutic success in malignant melanoma. Inhibitor of apoptosis proteins (IAPs) are important regulators of apoptotic and necroptotic cell death mediated by numerous cell death signalling platforms. In this report we investigated the impact of IAPs for cell death regulation in malignant melanoma. Suppression of IAPs strongly sensitized a panel of melanoma cells to death ligand-induced cell death, which, surprisingly, was largely mediated by apoptosis, as it was completely rescued by addition of caspase inhibitors. Interestingly, the absence of necroptosis signalling correlated with a lack of receptor-interacting protein kinase-3 (RIPK3) mRNA and protein expression in all cell lines, whereas primary melanocytes and cultured nevus cells strongly expressed RIPK3. Reconstitution of RIPK3, but not a RIPK3-kinase dead mutant in a set of melanoma cell lines overcame CD95L/IAP antagonist-induced necroptosis resistance independent of autocrine tumour necrosis factor secretion. Using specific inhibitors, functional studies revealed that RIPK3-mediated mixed-lineage kinase domain-like protein (MLKL) phosphorylation and necroptosis induction critically required receptor-interacting protein kinase-1 signalling. Furthermore, the inhibitor of mutant BRAF Dabrafenib, but not Vemurafenib, inhibited necroptosis in melanoma cells whenever RIPK3 is present. Our data suggest that loss of RIPK3 in melanoma and selective inhibition of the RIPK3/MLKL axis by BRAF inhibitor Dabrafenib, but not Vemurafenib, is critical to protect from necroptosis. Strategies that allow RIPK3 expression may allow unmasking the necroptotic signalling machinery in melanoma and points to reactivation of this pathway as a treatment option for metastatic melanoma.Over the past few years, necroptosis has been established as an alternative programmed form of cell death, contrasting caspase-dependent apoptosis. It is now evident that an ordered activation of the receptor-interacting protein kinases-1 and -3 (RIPK1 and RIPK3), and their downstream substrates is mandatory for the execution of necroptosis.^{1, 2, 3} Under caspase-limited conditions, the necroptotic cell signalling machinery is regulated by RIPK1, with the impact of scaffolding function as compared with kinase function still unclear.^{1, 4, 5, 6} RIPK1 interacts with and either autophosphorylates or transphosphorylates RIPK3 (for review, see Cho et al.,¹ Zhang et al.,² He et al.,³ and Vanden Berghe et al.⁷). When RIPK1 is active, RIPK3 phosphorylation and activation occurs within the assembled Necrosome (for review, see Remijsen et al.⁸) or Ripoptosome.^{4, 9, 10} RIPK3 then phosphorylates the pseudo kinase mixed-lineage kinase domain-like protein (MLKL).¹¹ MLKL in its active form allows its oligomerization, membrane accumulation, and complex formation within cellular membranes of the mitochondria¹² and cell membranes,¹³ and finally results in necroptosis.¹⁴The RIPK1/RIPK3/MLKL signalling network acts as a sensor for genotoxic stress⁹ and also has a key role in necroptosis regulation in keratinocyte skin cancer (SCC).⁴ In these epithelial cancers, cellular inhibitors of apoptosis proteins (cIAPs) block both apoptotic and necroptotic cell death.^{4, 5} Both apoptosis and necroptosis can be increasingly initiated by intrinsic or extrinsic stimuli when IAPs are suppressed by IAP antagonist. Extrinsic apoptosis mediated by activation of death receptors (DRs) such as cluster of differentiation 95 (CD95), TRAILR1/R2 or tumour necrosis factor receptor-1 (TNFR1) through ligation of respective death ligands (DLs) such as CD95L, TNF-related apoptosis-inducing ligand (TRAIL), and TNF initiates apoptosis either by direct activation of the caspase cascade (caspase-8/caspase-3) or via the intrinsic cell death signalling machinery regulated by pro-apoptotic members of the Bcl-2 family followed by caspase-3 activation.¹⁵ Inhibition of caspase-8 within the death-inducing signalling complex or complex II, or within the Ripoptosome can trigger CD95L-mediated,⁵ TRAIL-mediated¹⁶ or TNF-induced necroptosis.^{8, 17} A role for apoptosis resistance, cancer maintenance, and progression is widely assumed (for review, see Obexer et al.¹⁸), but the pathophysiological inhibitory or propagating function of necroptosis has not formally been demonstrated in cancer.Metastatic melanoma has an overall poor prognosis but novel therapeutics have revolutionized clinical practice for different subsets of patients. The use of inhibitors of the V600E- or V600K-mutated proto-oncogene serine/threonine protein kinase B-RAF (e.g., Dabrafenib or Vemurafenib) results in suppression of Ras/Raf/mitogen-activated protein kinase pathways and translate into unfortunately transient clinical responses (for review, see Spagnolo et al.¹⁹). The high recrudescence of metastatic melanoma following the treatment with BRAF inhibitors will potentially require combination therapies that activate additional tumour-inhibitory pathways. Combinations such as BRAF inhibitors with mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) inhibitors have already yielded impressive results²⁰ and other combination therapies may further improve clinical outcome.²¹ As BRAF inhibitors target the cell death pathway at best in an indirect manner, we reasoned that necroptosis induction could represent a novel option to improve melanoma therapy. Our investigations demonstrate for the first time that loss of RIPK3 during melanoma development is critical for necroptosis protection. Reactivation of the RIPK1/RIPK3/MLKL signalling machinery by RIPK3 reconstitution allows IAP antagonist/DL-mediated necroptosis in the presence of Vemurafenib, but not Dabrafenib. Here, Dabrafenib blocks necroptosis by interference with RIPK3-mediated MLKL phosphorylation. Therefore, strategies that increase RIPK3 expression in combination with Vemurafenib, but not Dabrafenib, likely represent an attractive strategy to overcome cell death resistance in melanoma.     

RIPK1- and RIPK3-induced cell death mode is determined by target availability

Both receptor-interacting protein kinase 1 (RIPK1) and RIPK3 can signal cell death following death receptor ligation. To study the requirements for RIPK-triggered cell death in the absence of death receptor signaling, we engineered inducible versions of RIPK1 and RIPK3 that can be activated by dimerization with the antibiotic coumermycin. In the absence of TNF or other death ligands, expression and dimerization of RIPK1 was sufficient to cause cell death by caspase- or RIPK3-dependent mechanisms. Dimerized RIPK3 induced cell death by an MLKL-dependent mechanism but, surprisingly, also induced death mediated by FADD, caspase 8 and RIPK1. Catalytically active RIPK3 kinase domains were essential for MLKL-dependent but not for caspase 8-dependent death. When RIPK1 or RIPK3 proteins were dimerized, the mode of cell death was determined by the availability of downstream molecules such as FADD, caspase 8 and MLKL. These observations imply that rather than a ‘switch'' operating between the two modes of cell death, the final mechanism depends on levels of the respective signaling and effector proteins.Mammalian cells can use a number of mechanisms to kill themselves. The best characterized depends on the Bcl-2 family members Bax and Bak that work via mitochondria to activate caspases.¹ Some caspases, notably caspase 8, can be activated independently of Bcl-2 family members, for example, after stimulation of members of the TNF receptor superfamily.² Recently, it has become apparent that some of these receptors, including TNFR1, can activate a third suicide mechanism that does not require caspases, and in which the morphology of the dying cell differs from classical apoptosis. This form of cell death, termed ‘necroptosis'', can often be blocked by necrostatin-1 (nec-1), an inhibitor of the kinase activity of receptor-interacting protein kinase 1 (RIPK1).^{3, 4} Accordingly, observations from several groups have shown that in some cell types, expression of RIPK1 can signal cell death by caspase-independent necroptosis.⁵It has previously been revealed that RIPK1 could function downstream of death receptors, but in those cases, cell death was usually blocked by coexpression of the viral inhibitor of caspases 1 and 8, CrmA,⁶ and typically exhibited a classical ‘apoptotic'' morphology. It was revealed that RIPK1 engages FADD via homotypic binding of their death domains (DDs), and FADD in turn activates caspase 8.^{6, 7}RIPK3, like RIPK1, bears a kinase domain and RIP homology interaction motif (RHIM), but unlike RIPK1 does not have a DD.^{8, 9, 10, 11} RIPK3 is required for necroptosis.^{12, 13} Furthermore, RIPK1 appears to activate RIPK3 in this pathway, as cell death could be blocked by nec-1.¹⁴ RIPK3 activates, by phosphorylation, MLKL, a pseudokinase essential for this death pathway.^{15, 16, 17} Once activated, MLKL forms multimers that trigger breaches of the plasma membrane.^{18, 19, 20}Although RIPK3 is necessary for necroptosis, it is unclear whether activation of RIPK3 is sufficient for cell death, because TNF activates signaling by many pathways in addition to those controlled by RIPK1.²¹ It is also unclear whether RIPK3 can contribute to apoptosis. Despite some reports to this effect,^{8, 9, 22} RIPK3 has been described as the necroptotic ‘switch'', implying its activity precipitates necroptosis to the exclusion of apoptosis.^{23, 24, 25}Here, we have directly activated RIP kinases without the confounding effects of multiple signals emanating from the target cell''s cytokine receptors, allowing us to define more precisely the functions of RIPK1 and RIPK3. We activated RIP kinases by dimerization using inducible lentiviral vectors, each encoding a chimera of a RIP kinase with subunit B of E. coli DNA gyrase.²⁶ We infected mouse embryonic fibroblasts (MEFs) that lack genes for, or expression of, various cell death proteins, induced expression of the RIPK chimera, caused its dimerization by addition of the divalent antibiotic coumermycin (C) and quantitated the resulting cell death.Our results reveal that each of RIPK1 and RIPK3 can contribute to both apoptosis and necroptosis depending on the biochemical context. Furthermore, necroptosis can occur in the absence of caspase 8 and FADD, which shows that the ripoptosome, with core components caspase 8, FADD and RIPK1,^{27, 28} is not required for necroptosis. Instead, we propose that dimers of RIPK1 and/or RIPK3 are the pivotal complexes from which both forms of cell death can progress.     

Differential roles of RIPK1 and RIPK3 in TNF-induced necroptosis and chemotherapeutic agent-induced cell death

Apoptosis is a key mechanism for metazoans to eliminate unwanted cells. Resistance to apoptosis is a hallmark of many cancer cells and a major roadblock to traditional chemotherapy. Recent evidence indicates that inhibition of caspase-dependent apoptosis sensitizes many cancer cells to a form of non-apoptotic cell death termed necroptosis. This has led to widespread interest in exploring necroptosis as an alternative strategy for anti-cancer therapy. Here we show that in human colon cancer tissues, the expression of the essential necroptosis adaptors receptor interacting protein kinase (RIPK)1 and RIPK3 is significantly decreased compared with adjacent normal colon tissues. The expression of RIPK1 and RIPK3 was suppressed by hypoxia, but not by epigenetic DNA modification. To explore the role of necroptosis in chemotherapy-induced cell death, we used inhibitors of RIPK1 or RIPK3 kinase activity, and modulated their expression in colon cancer cell lines using short hairpin RNAs. We found that RIPK1 and RIPK3 were largely dispensable for classical chemotherapy-induced cell death. Caspase inhibitor and/or second mitochondria-derived activator of caspase mimetic, which sensitize cells to RIPK1- and RIPK3-dependent necroptosis downstream of tumor necrosis factor receptor-like death receptors, also did not alter the response of cancer cells to chemotherapeutic agents. In contrast to the RIPKs, we found that cathepsins are partially responsible for doxorubicin or etoposide-induced cell death. Taken together, these results indicate that traditional chemotherapeutic agents are not efficient inducers of necroptosis and that more potent pathway-specific drugs are required to fully harness the power of necroptosis in anti-cancer therapy.Cell death by apoptosis is a natural barrier to cancer development, as it limits uncontrolled proliferation driven by oncogenes.¹ Chemotherapeutic agents that target apoptosis have been successful in anti-cancer therapy. However, cancer cells, especially cancer stem cells, often evolve multiple mechanisms to circumvent growth suppression by apoptosis.² This resistance to apoptosis is a major challenge for many chemotherapeutic agents. Targeting other non-apoptotic cell death pathways is an attractive therapeutic alternative.A growing number of recent studies show that there are distinct genetic programmed cell death modes other than apoptosis.³ Necroptosis is mediated by receptor interacting protein kinase 3 (RIPK3).⁴ In the presence of caspase inhibition and cellular inhibitor of apoptosis proteins (cIAPs) depletion, tumor necrosis factor (TNF) receptor 1 triggers a signaling reaction that culminates in binding of RIPK3 with its upstream activator RIPK1 through the RIP homotypic interaction motif (RHIM).⁴ RIPK1 and RIPK3 phosphorylation stabilizes this complex and promotes its conversion to an amyloid-like filamentous structure termed the necrosome.⁵ Once activated, RIPK3 recruits its substrate mixed lineage kinase domain-like (MLKL).⁶ Phosphorylated MLKL forms oligomers that translocate to intracellular membranes and the plasma membrane, which eventually leads to membrane rupture.^{7, 8, 9, 10}In addition to phosphorylation, RIPK1 and RIPK3 are also tightly regulated by ubiquitination, a process mediated by the E3 ligases cIAP1, cIAP2, and the linear ubiquitin chain assembly complex.¹¹ The ubiquitin chains on RIPK1 act as a scaffold to activate nuclear factor-κB (NF-κB) and mitogen-activated protein kinase pathways and inhibit formation of the necrosome. As such, depletion of cIAP1/2 by second mitochondria-derived activator of caspase (Smac) mimetics or removal of the ubiquitin chains by the de-ubiquitinating enzyme cylindromatosis (CYLD) promotes necroptosis.^{12, 13, 14, 15} In addition, RIPK1 and RIPK3 are cleaved and inactivated by caspase 8.^{16, 17, 18} Mice deficient for caspase 8 or FADD, an essential adaptor protein of caspase 8, suffer from embryonic lethality due to extensive RIPK1- or RIPK3-dependent necroptosis.^{19, 20, 21} Hence, caspase inhibition and IAP depletion are key priming signals for necroptosis.The physiological functions of RIPK1 and RIPK3 have been extensively investigated in infectious and sterile inflammatory diseases.^{4, 22} By contrast, their roles in cancer cells'' response to chemotherapeutics are poorly understood. Here we show that RIPK1 and RIPK3 expression is significantly decreased in human colon cancer tissues, suggesting that suppression of RIPK1 or RIPK3 expression is advantageous for cancer growth. However, the loss of RIPK1 and RIPK3 expression in colon cancer was not due to epigenetic DNA modification. Interestingly, RIPK1 and RIPK3 expression in colon cancer cells is reduced by hypoxia, a hallmark of solid tumor. We found that chemotherapeutic agents did not effectively elicit RIPK1/RIPK3-dependent necroptosis in colon cancer cells. Moreover, caspase inhibition and Smac mimetics, which are potent sensitizers for necroptosis, also did not enhance chemotherapeutic agent-induced cell death. These results show that traditional chemotherapeutic agents are not strong inducers of classical necroptosis in colon cancers and suggest that development of pathway-specific drugs is needed to harness the power of necroptosis in anti-cancer therapy.     

Distinct roles of RIP1–RIP3 hetero- and RIP3–RIP3 homo-interaction in mediating necroptosis

Necroptosis is mediated by a signaling complex called necrosome, containing receptor-interacting protein (RIP)1, RIP3, and mixed-lineage kinase domain-like (MLKL). It is known that RIP1 and RIP3 form heterodimeric filamentous scaffold in necrosomes through their RIP homotypic interaction motif (RHIM) domain-mediated oligomerization, but the signaling events based on this scaffold has not been fully addressed. By using inducible dimer systems we found that RIP1–RIP1 interaction is dispensable for necroptosis; RIP1–RIP3 interaction is required for necroptosis signaling, but there is no necroptosis if no additional RIP3 protein is recruited to the RIP1–RIP3 heterodimer, and the interaction with RIP1 promotes the RIP3 to recruit other RIP3; RIP3–RIP3 interaction is required for necroptosis and RIP3–RIP3 dimerization is sufficient to induce necroptosis; and RIP3 dimer-induced necroptosis requires MLKL. We further show that RIP3 oligomer is not more potent than RIP3 dimer in triggering necroptosis, suggesting that RIP3 homo-interaction in the complex, rather than whether RIP3 has formed homo polymer, is important for necroptosis. RIP3 dimerization leads to RIP3 intramolecule autophosphorylation, which is required for the recruitment of MLKL. Interestingly, phosphorylation of one of RIP3 in the dimer is sufficient to induce necroptosis. As RIP1–RIP3 heterodimer itself cannot induce necroptosis, the RIP1–RIP3 heterodimeric amyloid fibril is unlikely to directly propagate necroptosis. We propose that the signaling events after the RIP1–RIP3 amyloid complex assembly are the recruitment of free RIP3 by the RIP3 in the amyloid scaffold followed by autophosphorylation of RIP3 and subsequent recruitment of MLKL by RIP3 to execute necroptosis.Necroptosis is a type of programmed necrosis characterized by necrotic morphological changes, including cellular organelle swelling, cell membrane rupture,^{1, 2, 3} and dependence of receptor-interacting protein (RIP)1⁴ and RIP3.^{5, 6, 7} Physiological function of necroptosis has been illustrated in host defense,^{8, 9, 10, 11} inflammation,^{12, 13, 14, 15, 16} tissue injury,^{10, 17, 18} and development.^{19, 20, 21}Necroptosis can be induced by a number of different extracellular stimuli such as tumor necrosis factor (TNF). TNF stimulation leads to formation of TNF receptor 1 (TNFR1) signaling complex (named complex I), and complex II containing RIP1, TRADD, FAS-associated protein with a death domain (FADD), and caspase-8, of which the activation initiates apoptosis. If cells have high level of RIP3, RIP1 recruits RIP3 to form necrosome containing FADD,^{22, 23, 24} caspase-8, RIP1, and RIP3, and the cells undergo necroptosis.^{25, 26} Caspase-8 and FADD negatively regulates necroptosis,^{27, 28, 29, 30} because RIP1, RIP3, and CYLD are potential substrates of caspase-8.^{31, 32, 33, 34} Necrosome also suppresses apoptosis but the underlying mechanism has not been described yet. Mixed-lineage kinase domain-like (MLKL) is downstream of RIP3,^{35, 36} and phosphorylation of MLKL is required for necroptosis.^{37, 38, 39, 40, 41, 42}Apoptosis inducing complex (complex II) and necrosome are both supramolecular complexes.^{43, 44, 45} A recent study showed that RIP1 and RIP3 form amyloidal fibrils through their RIP homotypic interaction motif⁴⁶ (RHIM)-mediated polymerization, and suggested that amyloidal structure is essential for necroptosis signaling.⁴⁷ The RIP1–RIP3 heterodimeric amyloid complex is believed to function as a scaffold that brings signaling proteins into proximity to permit their activation. However, RIP1 and RIP3 also can each form fibrils on their own RHIM domains in vitro. It is unclear how the homo- and hetero-interactions are coordinated and organized on the amyloid scaffold to execute their functions in necroptosis. Here, we used inducible dimerization systems to study the roles of RIP1–RIP1, RIP1–RIP3, and RIP3–RIP3 interactions in necroptosis signaling. Our data suggested that it is the RIP1–RIP3 interaction in the RIP1–RIP3 heterodimeric amyloid complex that empowers to recruit other free RIP3; homodimerization of RIP3 triggers its autophosphorylation and only the phosphorylated RIP3 can recruit MLKL to execute necroptosis.     

Targeting heat-shock protein 90 with ganetespib for molecularly targeted therapy of gastric cancer

Gastric cancer (GC) remains the fifth most common cancer worldwide. Heat-shock protein 90 (HSP90) has become an attractive therapeutic target in treating cancers, because of its abnormally high expression in cancers. Several successful cases of HSP90 inhibitors capable of inhibiting GC inspired us to try ganetespib, a clinically promising and actively investigated second-generation HSP90 inhibitor in GC treatment. In our study, we show that ganetespib markedly reduced the growth of MGC-803 and also significantly inhibited the growth of SGC-7901 and MKN-28 in a dose-dependent manner. It induced G2/M cell-cycle arrest and apoptosis in all three cell lines, together with the related markers affected significantly. Mechanistically, ganetespib caused pronounced decrease of expression of classic HSP90 client proteins. Specifically, it greatly affected epidermal growth factor receptor (EGFR) signaling cascades by markedly decreasing the levels of total EGFR and EGFR on cell membranes. EGFR knockdown also induced cell-cycle arrest and apoptosis accompanied with a decrease of several EGFR downstream proteins. These results strongly support that EGFR signaling greatly contributes to the ganetespib inhibitory effects. Besides, we found that the responses of GC cell lines to ganetespib correlated well with their EGFR expression levels: MGC-803, as well as AGS and BGC-803, with higher EGFR expression responded to ganetespib better, whereas SGC-7901 and MKN-28 with lower EGFR levels were much less sensitive to ganetespib. Although SGC-7901 and MKN-28 were not very sensitive to ganetespib, ganetespib worked synergistically with radiation and cisplatin in killing them. Importantly, ganetespib significantly inhibited the growth of xenograft tumors in vivo as a single agent or in combination with cisplatin. Results of hematoxylin/eosin staining, TUNEL (terminal deoxynucleotidyl transferase dUTP nick-end labeling) assays, and immunohistochemistry staining of phosphorylated cyclin-dependent kinase 1 (pCDK1), EGFR and Ki-67 revealed significant differences in ganetespib-treated tumors. Collectively, our data suggest that ganetespib, as a new potent treatment option, can be used for the molecularly targeted therapy of GC patients according to their expression profiles of EGFR.Gastric cancer (GC) remains the fifth most common cancer worldwide, with an estimated 9 52 000 new cases (7% of total cancer incidence) and 7 23 000 deaths (9% of total cancer mortality) in 2012.¹ As a highly aggressive and lethal malignancy, the aggressive nature of GC is linked to mutations in tumor suppressor genes, oncogenes, growth factors and their receptors, and so on.² Till now, there are few effective treatment options for advanced patients with distant metastasis or recurrence.³ The detailed mechanisms that regulate GC are not yet fully understood; therefore, such situations underscore the persistent unmet need to identify therapeutics that target pathways involved in GC progression.Consequently, identification of key regulatory molecules in GC is of high priority for understanding the mechanism for tumor dissemination as well as the development of novel interventions. Aberrant expression and kinase activity of Src have been found in many different tumors, including GC.^{4, 5} Previous studies have shown that phosphorylated mammalian target of rapamycin (p-mTOR) was significantly overexpressed in advanced GC patients'' tumors and suggested that the PI3K/AKT/mTOR (phosphoinositide 3-kinase/AKT/mTOR) pathway is activated in GC with potential prognostic and predictive significance.^{6, 7} Aurora A overexpression has recently been reported in GC, and it was suggested to be associated with cancer progression and poor prognosis.^{8, 9, 10}In our previous work, we conducted data mining meta-analyses integrating results from multiple small interfering RNA (siRNA) screens to identify gene targets, which are necessary for the growth of different cancer cells. Among those genes, we found that heat-shock protein 90 (HSP90) was one of the most vital proteins for cancer cell survival.¹¹ As we know, HSP90 is involved in the regulation of numerous proteins important for GC pathogenesis, such as proteins important for cell adhesion (e.g., focal adhesion kinase), cell motility (e.g., epidermal growth factor receptor (EGFR), c-Src, phosphoinositide-dependent protein kinase 1 (PDK1)), and angiogenesis (e.g., hypoxia-inducible factor-1 (HIF-1), vascular endothelial growth factor receptor (VEGFR)).^{12, 13, 14, 15} For these reasons, HSP90 has been of considerable interest as a therapeutic target in GC.As an ATP-dependent molecular chaperone protein, HSP90 conducts the proper folding of myriad proteins.^{12, 14} Abnormally high expression of HSP90 has been found in GC and been greatly considered as an independent prognostic marker of GC progression.^{16, 17, 18} HSP90 remains an attractive therapeutic target in a variety of cancers,^{19, 20, 21, 22} and inhibition of HSP90 showed potent growth inhibitory effects on GC in cell cultures and in mouse models.^{23, 24, 25} Ganetespib is a particularly promising second-generation HSP90 inhibitor that does not suffer from the toxicity issues associated with earlier-generation HSP90 inhibitors and exhibits increased potency compared with first- and other second-generation agents.^{11, 26, 27, 28, 29}In this current study, using cell culture and xenograft mouse models, we sought to evaluate the effects of ganetespib treatments on GC cells, individually or in combination with other treatments. In addition, we searched for the possible mechanisms underlying the antitumor activities of ganetespib. And, our results suggested that, as a promising drug candidate, ganetespib has potent antitumor activities on GC, and it is worth being investigated further clinically for the molecularly targeted therapy of GC patients.     

New-Onset Diabetes Mellitus After Transplantation in a Cynomolgus Macaque (Macaca fasicularis)

A 5.5-y-old intact male cynomolgus macaque (Macaca fasicularis) presented with inappetence and weight loss 57 d after heterotopic heart and thymus transplantation while receiving an immunosuppressant regimen consisting of tacrolimus, mycophenolate mofetil, and methylprednisolone to prevent graft rejection. A serum chemistry panel, a glycated hemoglobin test, and urinalysis performed at presentation revealed elevated blood glucose and glycated hemoglobin (HbA1c) levels (727 mg/dL and 10.1%, respectively), glucosuria, and ketonuria. Diabetes mellitus was diagnosed, and insulin therapy was initiated immediately. The macaque was weaned off the immunosuppressive therapy as his clinical condition improved and stabilized. Approximately 74 d after discontinuation of the immunosuppressants, the blood glucose normalized, and the insulin therapy was stopped. The animal''s blood glucose and HbA1c values have remained within normal limits since this time. We suspect that our macaque experienced new-onset diabetes mellitus after transplantation, a condition that is commonly observed in human transplant patients but not well described in NHP. To our knowledge, this report represents the first documented case of new-onset diabetes mellitus after transplantation in a cynomolgus macaque.Abbreviations: NODAT, new-onset diabetes mellitus after transplantationNew-onset diabetes mellitus after transplantation (NODAT, formerly known as posttransplantation diabetes mellitus) is an important consequence of solid-organ transplantation in humans.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} A variety of risk factors have been identified including increased age, sex (male prevalence), elevated pretransplant fasting plasma glucose levels, and immunosuppressive therapy.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} The relationship between calcineurin inhibitors, such as tacrolimus and cyclosporin, and the development of NODAT is widely recognized in human medicine.^{7-10,15,17,19,21,25-28,31,33,34,37,38,42} Cynomolgus macaques (Macaca fasicularis) are a commonly used NHP model in organ transplantation research. Cases of natural and induced diabetes of cynomolgus monkeys have been described in the literature;^14,43,45 however, NODAT in a macaque model of solid-organ transplantation has not been reported previously to our knowledge.     

Plasma membrane poration by opioid neuropeptides: a possible mechanism of pathological signal transduction

Neuropeptides induce signal transduction across the plasma membrane by acting through cell-surface receptors. The dynorphins, endogenous ligands for opioid receptors, are an exception; they also produce non-receptor-mediated effects causing pain and neurodegeneration. To understand non-receptor mechanism(s), we examined interactions of dynorphins with plasma membrane. Using fluorescence correlation spectroscopy and patch-clamp electrophysiology, we demonstrate that dynorphins accumulate in the membrane and induce a continuum of transient increases in ionic conductance. This phenomenon is consistent with stochastic formation of giant (~2.7 nm estimated diameter) unstructured non-ion-selective membrane pores. The potency of dynorphins to porate the plasma membrane correlates with their pathogenic effects in cellular and animal models. Membrane poration by dynorphins may represent a mechanism of pathological signal transduction. Persistent neuronal excitation by this mechanism may lead to profound neuropathological alterations, including neurodegeneration and cell death.Neuropeptides are the largest and most diverse family of neurotransmitters. They are released from axon terminals and dendrites, diffuse to pre- or postsynaptic neuronal structures and activate membrane G-protein-coupled receptors. Prodynorphin (PDYN)-derived opioid peptides including dynorphin A (Dyn A), dynorphin B (Dyn B) and big dynorphin (Big Dyn) consisting of Dyn A and Dyn B are endogenous ligands for the κ-opioid receptor. Acting through this receptor, dynorphins regulate processing of pain and emotions, memory acquisition and modulate reward induced by addictive substances.^{1, 2, 3, 4} Furthermore, dynorphins may produce robust cellular and behavioral effects that are not mediated through opioid receptors.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29} As evident from pharmacological, morphological, genetic and human neuropathological studies, these effects are generally pathological, including cell death, neurodegeneration, neurological dysfunctions and chronic pain. Big Dyn is the most active pathogenic peptide, which is about 10- to 100-fold more potent than Dyn A, whereas Dyn B does not produce non-opioid effects.^{16, 17, 22, 25} Big Dyn enhances activity of acid-sensing ion channel-1a (ASIC1a) and potentiates ASIC1a-mediated cell death in nanomolar concentrations^{30, 31} and, when administered intrathecally, induces characteristic nociceptive behavior at femtomolar doses.^{17, 22} Inhibition of endogenous Big Dyn degradation results in pathological pain, whereas prodynorphin (Pdyn) knockout mice do not maintain neuropathic pain.^{22, 32} Big Dyn differs from its constituents Dyn A and Dyn B in its unique pattern of non-opioid memory-enhancing, locomotor- and anxiolytic-like effects.²⁵Pathological role of dynorphins is emphasized by the identification of PDYN missense mutations that cause profound neurodegeneration in the human brain underlying the SCA23 (spinocerebellar ataxia type 23), a very rare dominantly inherited neurodegenerative disorder.^{27, 33} Most PDYN mutations are located in the Big Dyn domain, demonstrating its critical role in neurodegeneration. PDYN mutations result in marked elevation in dynorphin levels and increase in its pathogenic non-opioid activity.^{27, 34} Dominant-negative pathogenic effects of dynorphins are not produced through opioid receptors.ASIC1a, glutamate NMDA (N-methyl-d-aspartate) and AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid)/kainate ion channels, and melanocortin and bradykinin B2 receptors have all been implicated as non-opioid dynorphin targets.^{5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 30, 31, 35, 36} Multiplicity of these targets and their association with the cellular membrane suggest that their activation is a secondary event triggered by a primary interaction of dynorphins with the membrane. Dynorphins are among the most basic neuropeptides.^{37, 38} The basic nature is also a general property of anti-microbial peptides (AMPs) and amyloid peptides that act by inducing membrane perturbations, altering membrane curvature and causing pore formation that disrupts membrane-associated processes including ion fluxes across the membrane.³⁹ The similarity between dynorphins and these two peptide groups in overall charge and size suggests a similar mode of their interactions with membranes.In this study, we dissect the interactions of dynorphins with the cell membrane, the primary event in their non-receptor actions. Using fluorescence imaging, correlation spectroscopy and patch-clamp techniques, we demonstrate that dynorphin peptides accumulate in the plasma membrane in live cells and cause a profound transient increase in cell membrane conductance. Membrane poration by endogenous neuropeptides may represent a novel mechanism of signal transduction in the brain. This mechanism may underlie effects of dynorphins under pathological conditions including chronic pain and tissue injury.     

Comparison of Biomarkers of Oxidative Stress and Cardiovascular Disease in Humans and Chimpanzees (Pan troglodytes)

In the oxidative stress hypothesis of aging, the aging process is the result of cumulative damage by reactive oxygen species. Humans and chimpanzees are remarkably similar; but humans live twice as long as chimpanzees and therefore are believed to age at a slower rate. The purpose of this study was to compare biomarkers for cardiovascular disease, oxidative stress, and aging between male chimpanzees and humans. Compared with men, male chimpanzees were at increased risk for cardiovascular disease because of their significantly higher levels of fibrinogen, IGF1, insulin, lipoprotein a, and large high-density lipoproteins. Chimpanzees showed increased oxidative stress, measured as significantly higher levels of 5-hydroxymethyl-2-deoxyuridine and 8-iso-prostaglandin F_2α, a higher peroxidizability index, and higher levels of the prooxidants ceruloplasmin and copper. In addition, chimpanzees had decreased levels of antioxidants, including α- and β-carotene, β-cryptoxanthin, lycopene, and tocopherols, as well as decreased levels of the cardiovascular protection factors albumin and bilirubin. As predicted by the oxidative stress hypothesis of aging, male chimpanzees exhibit higher levels of oxidative stress and a much higher risk for cardiovascular disease, particularly cardiomyopathy, compared with men of equivalent age. Given these results, we hypothesize that the longer lifespan of humans is at least in part the result of greater antioxidant capacity and lower risk of cardiovascular disease associated with lower oxidative stress.Abbreviations: 5OHmU, 5-hydroxymethyl-2-deoxyuridine; 8isoPGF_2α, 8-iso-prostaglandin F_2α; HDL, high-density lipoprotein; IGF1, insulin-like growth factor 1; LDL, low-density lipoprotein; ROS, reactive oxygen speciesAging is characterized as a progressive reduction in the capacity to withstand the stresses of everyday life and a corresponding increase in risk of mortality. According to the oxidative stress hypothesis of aging, much of the aging process can be accounted for as the result of cumulative damage produced by reactive oxygen species (ROS).^{6,21,28,41,97} Endogenous oxygen radicals (that is, ROS) are generated as a byproduct of normal metabolic reactions in the body and subsequently can cause extensive damage to proteins, lipids, and DNA.^6,41 Various prooxidant elements, in particular free transition metals, can catalyze these destructive reactions.⁶ The damage caused by ROS can be counteracted by antioxidant defense systems, but the imbalance between production of ROS and antioxidant defenses, over time, leads to oxidative stress and may contribute to the rate of aging.^28,97Oxidative stress has been linked to several age-related diseases including neurodegenerative diseases, ophthalmologic diseases, cancer, and cardiovascular disease.^21,28,97 Of these, cardiovascular disease remains the leading cause of adult death in the United States and Europe.⁷¹ In terms of cardiovascular disease, oxidative stress has been linked to atherosclerosis, hypertension, cardiomyopathy, and chronic heart failure in humans.^55,78,84 Increases in oxidant catalysts (prooxidants)—such as copper, iron, and cadmium—have been associated with hypertension, coronary artery disease, atherosclerosis, and sudden cardiac death.^98,102,106 Finally, both endogenous and exogenous antioxidants have been linked to decreased risk of cardiovascular disease, although the mechanisms behind this relationship are unclear.^11,52,53 However, the oxidative stress hypothesis of aging aims to explain not only the mechanism of aging and age-related diseases (such as cardiovascular disease) in humans but also the differences between aging rates and the manifestations of age-related diseases across species.The differences in antioxidant and ROS levels between animals and humans offer promise for increasing our understanding of human aging. Additional evidence supporting the oxidative stress hypothesis of aging has come from comparative studies linking differences in aging rates across taxa with both antioxidant and ROS levels.^{4,17-21,58,71,86,105} In mammals, maximum lifespan potential is positively correlated with both serum and tissue antioxidant levels.^{17,18,21,71,105} Research has consistently demonstrated that the rate of oxidative damage varies across species and is negatively correlated with maximum lifespan potential.^{4,19,20,58,71,86} However, few studies involved detailed comparisons of hypothesized biochemical indicators of aging and oxidative stress between humans and animals.⁶ This type of interspecies comparison has great potential for directly testing the oxidative stress hypothesis of aging.Much evolutionary and genetic evidence supports remarkable similarity between humans and chimpanzees.^95,100 Despite this similarity, humans have a lifespan of almost twice that of chimpanzees.^3,16,47 Most comparative primate aging research has focused on the use of a macaque model,^62,81,88 and several biochemical markers of age-related diseases have been identified in both humans and macaque monkeys.^{9,22,28,81,93,97} Several other species of monkeys have also been used in research addressing oxidative stress, antioxidant defenses, and maximum lifespan potential.^18,21,58,105 However, no study to date has examined biochemical indicators of oxidative stress and aging in chimpanzees and humans as a test of the oxidative stress hypothesis for aging. The purpose of this study is to compare biochemical markers for cardiovascular disease, oxidative stress, and aging directly between male chimpanzees and humans. Given the oxidative stress hypothesis for aging and the known role of oxidative stress in cardiovascular disease, we predict that chimpanzees will show higher levels of cardiovascular risk and oxidative stress than humans.