Succinate dehydrogenase (SDH) is an inner mitochondrial membrane protein complex that links the Krebs cycle to the electron transport system. It can produce significant amounts of superoxide ([Formula: see text]) and hydrogen peroxide (H2O2); however, the precise mechanisms are unknown. This fact hinders the development of next-generation antioxidant therapies targeting mitochondria. To help address this problem, we developed a computational model to analyze and identify the kinetic mechanism of [Formula: see text] and H2O2 production by SDH. Our model includes the major redox centers in the complex, namely FAD, three iron-sulfur clusters, and a transiently bound semiquinone. Oxidation state transitions involve a one- or two-electron redox reaction, each being thermodynamically constrained. Model parameters were simultaneously fit to many data sets using a variety of succinate oxidation and free radical production data. In the absence of respiratory chain inhibitors, model analysis revealed the 3Fe-4S iron-sulfur cluster as the primary [Formula: see text] source. However, when the quinone reductase site is inhibited or the quinone pool is highly reduced, [Formula: see text] is generated primarily by the FAD. In addition, H2O2 production is only significant when the enzyme is fully reduced, and fumarate is absent. Our simulations also reveal that the redox state of the quinone pool is the primary determinant of free radical production by SDH. In this study, we showed the importance of analyzing enzyme kinetics and associated side reactions in a consistent, quantitative, and biophysically detailed manner using a diverse set of experimental data to interpret and explain experimental observations from a unified perspective.

A 10-year-old Andalusian mare was presented for evaluation of weight loss, increasing periods of recumbency, and swelling of the lower limbs. Radiographs revealed severe palisading to solid periosteal new bone formation in numerous locations. Necropsy revealed a metastatic malignant adenocarcinoma of ovarian origin with secondary hypertrophic osteopathy.

Acute heat shock can induce apoptosis through a canonical pathway involving the upstream activation of caspase-2, followed by BID cleavage and stimulation of the intrinsic pathway. Herein, we report that the BH3-only protein BIM, rather than BID, is essential to heat shock-induced cell death. We observed that BIM-deficient cells were highly resistant to heat shock, exhibiting short and long-term survival equivalent to Bax(-/-)Bak(-/-) cells and better than either Bid(-/-) or dominant-negative caspase-9-expressing cells. Only Bim(-/-) and Bax(-/-)Bak(-/-) cells exhibited resistance to mitochondrial outer membrane permeabilization and loss of mitochondrial inner membrane potential. Moreover, while dimerized caspase-2 failed to induce apoptosis in Bid(-/-) cells, it readily did so in Bim(-/-) cells, implying that caspase-2 kills exclusively through BID, not BIM. Finally, BIM reportedly associates with MCL-1 following heat shock, and Mcl-1(-/-) cells were indeed sensitized to heat shock-induced apoptosis. However, pharmacological inhibition of BCL-2 and BCL-X(L) with ABT-737 also sensitized cells to heat shock, most likely through liberation of BIM. Thus, BIM mediates heat shock-induced apoptosis through a BAX/BAK-dependent pathway that is antagonized by antiapoptotic BCL-2 family members.

Bortezomib is a highly selective inhibitor of the 26S proteasome and has been approved for clinical use in the treatment of relapsing and refractory multiple myeloma and mantle cell lymphoma. Clinical trials are also underway to assess the role of bortezomib in several other human malignancies, including leukemia. However, the mechanism(s) by which bortezomib acts remain to be fully understood. Here, we studied the molecular requirements of bortezomib-induced apoptosis using the human T-cell leukemic Jurkat cells stably transfected with or without shRNA against apoptotic protease-activating factor-1 (Apaf-1). The Apaf-1-deficient Jurkat T cells were resistant to bortezomib-induced apoptosis, as assessed by caspase-3 activity, poly(ADP-ribose) polymerase cleavage, phosphatidylserine externalization, and hypodiploid DNA content. In contrast, Apaf-1-deficient cells were sensitive to Fas-induced apoptosis. Bortezomib induced an upregulation of the pro-apoptotic protein Noxa, loss of mitochondrial transmembrane potential, and release of cytochrome c in cells expressing or not expressing Apaf-1. Transient silencing of Apaf-1 expression in RPMI 8402 T-cell leukemic cells also diminished bortezomib-induced apoptosis. Fas-associated death domain (FADD)–deficient Jurkat cells were resistant to Fas-mediated apoptosis yet remained sensitive to bortezomib. Our results show that bortezomib induces apoptosis by regulating pathways that are mechanistically different from those activated upon death receptor ligation. Furthermore, in silico analyses of public transcriptomics databases indicated elevated Apaf-1 expression in several hematologic malignancies, including acute lymphoblastic and myeloid leukemia. We also noted variable Apaf-1 expression in a panel of samples from patients with acute lymphoblastic leukemia. Our results suggest that the expression of Apaf-1 may be predictive of the response to proteasome inhibition.

ADAM17 is a membrane-associated metalloprotease that cleaves proteins from the surface of neutrophils and modulates the density of various receptors and adhesion molecules. The protease activity of ADAM17 is highly inducible and occurs upon neutrophil activation as well as apoptosis. At this time, little is known about the signal transduction pathway that promotes ADAM17 activity in neutrophils upon the induction of apoptosis. We show that caspase-8 activation, Bid cleavage, and the release of mitochondrial reactive oxygen species are sequential transduction components of the Fas signaling cascade that induces ADAM17. This is different from ADAM17 stimulation upon overt neutrophil activation, which requires MAPK p38 or ERK, but not caspases and reactive oxygen species. ADAM17 activity in apoptotic neutrophils may serve to inactivate select effector molecules that promote the pro-inflammatory activity of recruited neutrophils. For instance, TNFα receptors TNF-RI and TNF-RII are substrates of ADAM17, and we show that they are shed during apoptosis, decreasing neutrophil sensitivity to TNFα. Altogether, our findings provide significant new insights into the signal transduction pathway that stimulates ADAM17 during induced neutrophil apoptosis. ADAM17 induction during apoptosis may rapidly diminish neutrophil sensitivity to the inflammatory environment, complementing other anti-inflammatory activities by these cells during inflammation resolution.

Exposure of cells to hyperthermia is known to induce apoptosis, although the underlying mechanisms are only partially understood. Here, we examine the molecular requirements necessary for heat-induced apoptosis using genetically modified Jurkat T-lymphocytes. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were completely resistant to heat-induced apoptosis, implicating the involvement of the mitochondria-mediated pathway. Pretreatment of wild-type cells with the cell-permeable biotinylated general caspase inhibitor b-VAD-fmk (biotin-Val-Ala-Asp(OMe)-CH(2)F) both inhibited heat-induced apoptosis and affinity-labeled activated initiator caspase-2, -8, and -9. Despite this finding, however, cells engineered to be deficient in caspase-8, caspase-2, or the caspase-2 adaptor protein RAIDD (receptor-interacting protein (RIP)-associated Ich-1/CED homologous protein with death domain) remained susceptible to heat-induced apoptosis. Additionally, b-VAD-fmk failed to label any activated initiator caspase in Apaf-1-deficient cells exposed to hyperthermia. Cells lacking Apaf-1 or the pro-apoptotic BH3-only protein Bid exhibited lower levels of heat-induced Bak activation, cytochrome c release, and loss of mitochondrial membrane potential, although cleavage of Bid to truncated Bid (tBid) occurred downstream of caspase-9 activation. Combined, the data suggest that caspase-9 is the critical initiator caspase activated during heat-induced apoptosis and that tBid may function to promote cytochrome c release during this process as part of a feed-forward amplification loop.

Free radicals are naturally produced in vivo, both by normal cellular metabolism and as a result of disease process or through xenobiotic activities. Free radicals have the potential to elicit many of the tissue changes associated with toxicities and disease processes, but are also a consequence of such damage. Thus, the cause-effect relationship between radicals and disease or toxicities has been difficult to establish. In recent years, improvements in analytical methodologies, as well as the realization that subtle effects induced by free radicals at the molecular level are important in modulating cellular signaling, have greatly improved our understanding of the roles of these reactive species in toxic mechanisms and disease processes. This chapter provides an overview of free radical processes as related to mechanisms of cell and tissue injury.

The extent to which the BH3-only protein Bid is important for intrinsic (mitochondria-mediated) apoptotic cell death induced by genotoxic stress remains controversial. In the present study, we examine this issue using a panel of gene-manipulated Bax-deficient Jurkat T-lymphocytes. Cells stably depleted of Bid were far less sensitive than control-transfected cells to etoposide-induced apoptosis. In particular, drug-induced Bak activation, cytochrome c release, loss of mitochondrial membrane potential, and caspase activation were all decreased in cells lacking Bid. Reconstitution experiments using recombinant proteins and permeabilized Bid-deficient cells demonstrated that truncated Bid (tBid), but not full-length Bid, potently induced Bak activation and the release of cytochrome c. Further, caspase-8-deficient Jurkat cells efficiently cleaved Bid and were sensitive to drug-induced apoptosis. By comparison, Apaf-1-deficient cells, as well as cells overexpressing full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP, failed to cleave Bid in response to genotoxic stress. These data suggest that tBid plays an important regulatory role in the execution of DNA damage-induced cytochrome c release and apoptosis. However, the fact that cleavage of Bid to tBid is mediated by executioner caspases suggests that a self-amplifying feed forward loop involving caspases, Bid, and mitochondria may help determine irreversible commitment to apoptosis.

The 90-kDa heat shock protein (Hsp90) assists in the proper folding of numerous mutated or overexpressed signal transduction proteins that are involved in cancer. Consequently, there is considerable interest in developing chemotherapeutic drugs that specifically disrupt the function of Hsp90. Here, we investigated the extent to which a novel novobiocin-derived C-terminal Hsp90 inhibitor, designated KU135, induced antiproliferative effects in Jurkat T-lymphocytes. The results indicated that KU135 bound directly to Hsp90, caused the degradation of known Hsp90 client proteins, and induced more potent antiproliferative effects than the established N-terminal Hsp90 inhibitor 17-allylamino-demethoxygeldanamycin (17-AAG). Closer examination of the cellular response to KU135 and 17-AAG revealed that only 17-AAG induced a strong up-regulation of Hsp70 and Hsp90. In addition, KU135 caused wild-type cells to undergo G(2)/M arrest, whereas cells treated with 17-AAG accumulated in G(1). Furthermore, KU135 but not 17-AAG was found to be a potent inducer of mitochondria-mediated apoptosis as evidenced, in part, by the fact that cell death was inhibited to a similar extent by Bcl-2/Bcl-x(L) overexpression or the depletion of apoptotic protease-activating factor-1 (Apaf-1). Together, these data suggest that KU135 inhibits cell proliferation by regulating signaling pathways that are mechanistically different from those targeted by 17-AAG and as such represents a novel opportunity for Hsp90 inhibition.

Activation of executioner caspases during receptor-mediated apoptosis in type II cells requires the engagement of the mitochondrial apoptotic pathway. Although it is well established that recruitment of mitochondria in this context involves the cleavage of Bid to truncated Bid (tBid), the precise post-mitochondrial signaling responsible for executioner caspase activation is controversial. Here, we used distinct clones of type II Jurkat T-lymphocytes in which the mitochondrial apoptotic pathway had been inhibited to investigate the molecular requirements necessary for Fas-induced apoptosis. Cells overexpressing either Bcl-2 or Bcl-xL were protected from apoptosis induced by agonistic anti-Fas antibody. By comparison, Apaf-1-deficient Jurkat cells were sensitive to anti-Fas, exhibiting Bid cleavage, Bak activation, the release of cytochrome c and Smac, and activation of executioner caspase-3. Inhibiting downstream caspase activation with the pharmacological inhibitor Z-DEVD-fmk or by expressing the BIR1/BIR2 domains of X-linked inhibitor of apoptosis protein (XIAP) decreased all anti-Fas-induced apoptotic changes. Additionally, pretreatment of Bcl-xL-overexpressing cells with a Smac mimetic sensitized these cells to Fas-induced apoptosis. Combined, our findings strongly suggest that Fas-mediated activation of executioner caspases and induction of apoptosis do not depend on apoptosome-mediated caspase-9 activation in prototypical type II cells.

Department of Pharmacology, Toxicology & Therapeutics (S.N.S., M.E.S., J.D.R.) and Department of Urology (S.B.C., G.A.V.), University of Kansas Medical Center, Kansas City, KS 66160; Department of Medicinal Chemistry (Y.L., A.C.D., B.S.J.B.) and Biotechnology Innovation & Optimization Center (M.T., R.A.R.), The University of Kansas, Lawrence, KS 66047; University of Kansas Cancer Center, Kansas City, KS 66160 (G.A.V., R.A.R., B.S.J.B., J.D.R.); and Department of Biochemistry and Molecular Biology, Oklahoma State University, Stillwater, OK 74078 (K.S., R.L.M.) Molecular Pharmacology Fast Forward. Published on September 9, 2009 as doi:10.1124/mol.109.058545

Mitochondrial outer membrane permeabilization and the release of intermembrane space proteins, such as cytochrome c, are early events during intrinsic (mitochondria-mediated) apoptotic signaling. Although this process is generally accepted to require the activation of Bak or Bax, the underlying mechanism responsible for their activation during true intrinsic apoptosis is not well understood. In the current study, we investigated the molecular requirements necessary for Bak activation using distinct clones of Bax-deficient Jurkat T-lymphocytes in which the intrinsic pathway had been inhibited. Cells stably overexpressing Bcl-2/Bcl-x(L) or stably depleted of Apaf-1 were equally resistant to apoptosis induced by the DNA-damaging anticancer drug etoposide as determined by phosphatidylserine externalization and caspase activation. Strikingly, characterization of mitochondrial apoptotic events in all three drug-resistant cell lines revealed that, without exception, resistance to apoptosis was associated with an absence of Bak activation, cytochrome c release, and mitochondrial membrane depolarization. Furthermore, we found that etoposide-induced apoptosis and mitochondrial events were inhibited in cells stably overexpressing either full-length X-linked inhibitor of apoptosis protein (XIAP) or the BIR1/BIR2 domains of XIAP. Combined, our findings suggest that caspase-mediated positive amplification of initial mitochondrial changes can determine the threshold for irreversible activation of the intrinsic apoptotic pathway.

Sequential activation of caspases is critical for the execution of apoptosis. Recent evidence suggests caspase 2 is a significant upstream caspase capable of initiating mitochondrial events, such as the release of cytochrome c. In particular, in vitro studies using recombinant proteins have shown that cleaved caspase 2 can induce mitochondrial outer membrane permeabilization directly or by cleaving the BH3-only protein BID (BH3 interacting domain death agonist). However, whether interchain cleavage or activation of procaspase 2 occurs prior to Apaf-1-mediated procaspase 9 activation under more natural conditions remains unresolved. In the present study, we show that Apaf-1-deficient Jurkat T-lymphocytes and mouse embryonic fibroblasts were highly resistant to DNA-damage-induced apoptosis and failed to cleave or activate any apoptotic procaspase, including caspase 2. Significantly, drug-induced cytochrome c release and loss of mitochondrial membrane potential were inhibited in cells lacking Apaf-1. By comparison, procaspase proteolysis and apoptosis were only delayed slightly in Apaf-1-deficient Jurkat cells upon treatment with anti-Fas antibody. Our data support a model in which Apaf-1 is necessary for the cleavage or activation of all procaspases and the promotion of mitochondrial apoptotic events induced by genotoxic drugs.

Caspases are cysteine proteases that play a central role in the execution of apoptosis. Recent evidence indicates that caspase-2 is activated early in response to genotoxic stress and can function as an upstream modulator of the mitochondrial apoptotic pathway. In particular, we have shown previously that fully processed caspase-2 can permeabilize the outer mitochondrial membrane and cause cytochrome c and Smac/DIABLO release from these organelles. Using permeabilized cells, isolated mitochondria, and protein-free liposomes, we now report that this effect is direct and depends neither on the presence or cleavage of other proteins nor on a specific phospholipid composition of the liposomal membrane. Interestingly, caspase-2 was also shown to disrupt the interaction of cytochrome c with anionic phospholipids, notably cardiolipin, and thereby enhance the release of the hemoprotein caused by treatment of mitochondria with digitonin or the proapoptotic protein Bax. Combined, our data suggest that caspase-2 possesses an unparalleled ability to engage the mitochondrial apoptotic pathway by permeabilizing the outer mitochondrial membrane and/or by breaching the association of cytochrome c with the inner mitochondrial membrane.

The mechanisms regulating mitochondrial outer-membrane permeabilization and the release of cytochrome c during apoptosis remain controversial. In the present study, we show in an in vitro model system that the release of cytochrome c may occur via moderate modulation of mitochondrial volume, irrespective of the mechanism leading to the mitochondrial swelling. In contrast with mitochondrial permeability transition-dependent release of cytochrome c, in the present study mitochondria remain intact and functionally active.

Despite an increasing interest in neural stem cell (NSC) research, relatively little is known about the biochemical regulation of cell death pathways in these cells. We demonstrate here, using murine-derived multipotent C17.2 NSCs, that cells undergo mitochondria-mediated cell death in response to apoptotic stimuli such as oxidative stress induced by 2,3-dimethoxy-1,4-naphthoquinone (DMNQ). In particular, treated cells exhibited apoptotic features, including Bax translocation, cytochrome c release, activation of caspase-9 and -3, chromatin condensation and DNA fragmentation. Although C17.2 cells possess the Fas receptor and express procaspase-8, agonistic Fas mAb treatment failed to induce apoptosis. Fas treatment activated the extracellular signal-regulated protein kinase (ERK) pathway, which may have an antiapoptotic as well as a growth stimulating role. Combined, our findings indicate that while NSCs are sensitive to cytotoxic stimuli that involve an engagement of mitochondria, Fas treatment does not induce death and may have an alternative role.

The mechanism by which caspase-2 executes apoptosis remains obscure. Recent findings indicate that caspase-2 is activated early in response to DNA-damaging antineoplastic agents and may be important for the engagement of the mitochondrial apoptotic pathway. We demonstrate here that fully processed caspase-2 stimulates mitochondrial release of cytochrome c and Smac/DIABLO, but not apoptosis-inducing factor (AIF). This event occurs independently of several Bcl-2 family proteins, including Bax, Bak and Bcl-2, and inactivation experiments reveal that the proteolytic activity of caspase-2 is not required for the effect. Further, functional studies of mitochondria indicate that processed caspase-2 stimulates state 4 respiration and decreases the respiratory control ratio as a result of, in large part, an uncoupling effect. Combined, our data suggest that caspase-2 retains a unique ability to engage directly the mitochondrial apoptotic pathway, an effect that requires processing of the zymogen but not the associated catalytic activity.