Dendritic spine loss and neurodegeneration is rescued by Rab11 in models of Huntington's disease.

Huntington's disease (HD) is a fatal neurodegenerative disorder caused by expansion of a polyglutamine tract in the huntingtin protein (htt) that mediates formation of intracellular protein aggregates. In the brains of HD patients and HD transgenic mice, accumulation of protein aggregates has been causally linked to lesions in axo-dendritic and synaptic compartments. Here we show that dendritic spines - sites of synaptogenesis - are lost in the proximity of htt aggregates because of functional defects in local endosomal recycling mediated by the Rab11 protein. Impaired exit from recycling endosomes (RE) and association of endocytosed protein with intracellular structures containing htt aggregates was demonstrated in cultured hippocampal neurons cells expressing a mutant htt fragment. Dendrites in hippocampal neurons became dystrophic around enlarged amphisome-like structures positive for Rab11, LC3 and mutant htt aggregates. Furthermore, Rab11 overexpression rescues neurodegeneration and dramatically extends lifespan in a Drosophila model of HD. Our findings are consistent with the model that mutant htt aggregation increases local autophagic activity, thereby sequestering Rab11 and diverting spine-forming cargo from RE into enlarged amphisomes. This mechanism may contribute to the toxicity caused by protein misfolding found in a number of neurodegenerative diseases.

Percutaneous imaging-guided ablation therapies in the treatment of symptomatic bone metastases: preliminary experience.

PURPOSE: The treatment of pain in bone metastases is currently multidisciplinary. Among the various therapies, local radiotherapy is the gold standard for pain palliation from single metastasis, even though the maximum benefit is obtained between 12 and 20 weeks from initiation. In carefully selected patients, several ablation therapies achieve this objective in 4 weeks. The purpose of this study was to assess the technical success, effectiveness and possible complications of percutaneous ablation therapies in patients with symptomatic bone metastases. MATERIALS AND METHODS: From November 2003 to May 2008, ten ablation treatments were performed in ten patients with acute pain from metastatic bone lesions. Patient selection and choice of the most appropriate ablation treatment was made based on lesion characteristics. Three patients were treated with radiofrequency, one with plasma-mediated radiofrequency, two with plasma-mediated radiofrequency and cementoplasty, three with radiofrequency and cementoplasty and one with microwave. RESULTS: Assessments were based not only on imaging but also on the visual analogue scale (VAS) score for determining pain and on changes in morphine-equivalent doses. In both cases, 3-month follow-up showed a statistically significant reduction of pain. In no case did local complications occur either during or after treatment. Only one patient treated with radiofrequency (1/9, 11%) developed low-grade fever and general malaise during the 6 days following the procedure, compatible with a post-radiofrequency syndrome, which was treated with acetaminophen (paracetamol) only and resolved on day 7. CONCLUSIONS: Percutaneous ablation therapies represent a safe and valuable alternative for treating localised pain from single bone metastasis, providing rapid (4-week) relief of symptoms and a significant reduction in morphine doses. This contributes to improving the quality of life of patients with metastatic disease.

Image-guided percutaneous treatment of abdominal-pelvic abscesses: a 5-year experience.

PURPOSE: This study was undertaken to evaluate the efficacy of image-guided percutaneous drainage in treating abdominal and pelvic abscesses. MATERIALS AND METHODS: From August 2001 to August 2006, 95 patients (49 men and 46 women; mean age 61 years, range 25-92) with 107 abscesses underwent image-guided percutaneous drainage. Thirty-one abscesses were retroperitoneal (9 peripancreatic, 17 perirenal, 5 pararenal), 37 intraperitoneal (2 in communication with the small bowel), 8 intrahepatic (2 in communication with the extrahepatic biliary system and 2 with the intrahepatic biliary system), 4 perisplenic and 27 pelvic (4 in communication with the large bowel). Seventy-one of 107 procedures were performed with ultrasonographic (US) guidance and 36/107 with computed tomography (CT) guidance. All procedures were carried out with 8-to 14-Fr pigtail drainage catheters. RESULTS: Immediate technical success was achieved in 107/107 fluid collections. No major complications occurred. In 98/107 abscesses, we obtained progressive shrinkage of the collection (>50%) with consequent clinical success. In 9/107 cases, percutaneous drainage was unable to resolve the fluid collection. There were 12 cases of catheter displacement and six of obstruction. CONCLUSIONS: Percutaneous drainage is feasible and effective in treating abdominal and pelvic abscesses. It may be considered both as a preparatory step for surgery and a valuable alternative to open surgery. Failure of the procedure does not, however, preclude a subsequent surgical operation.

A novel paradigm for rapid ABT-737-induced apoptosis involving outer mitochondrial membrane rupture in primary leukemia and lymphoma cells.

Primary chronic lymphocytic leukemia (CLL) cells are exquisitely sensitive to ABT-737, a small molecule BCL2-antagonist, which induces many of the classical biochemical and ultrastructural features of apoptosis, including BAX/BAK oligomerization, cytochrome c release, caspase activation and chromatin condensation. Surprisingly, ABT-737 also induces mitochondrial inner membrane permeabilization (MIMP) resulting in mitochondrial matrix swelling and rupture of the outer mitochondrial membrane (OMM), so permitting the rapid efflux of cytochrome c from mitochondrial cristae and facilitating rapid caspase activation and apoptosis. BAX and BAK appear to be involved in the OMM discontinuities as they localize to the OMM break points. Notably, ABT-737 induced mitochondrial matrix swelling and OMM discontinuities in other primary B-cell malignancies, including mantle cell, follicular and marginal zone lymphoma cells but not in several cell lines studied. Thus, we describe a new paradigm of apoptosis in primary B-cell malignancies, whereby targeting of BCL2 results in all the classical features of apoptosis together with OMM rupture independent of caspase activation. This mechanism may be far more prevalent than hitherto recognized due to the failure of most methods, used to measure apoptosis, to recognize such a mechanism.

The plasma membrane Na+/Ca2+ exchanger is cleaved by distinct protease families in neuronal cell death.

Neurodegenerative conditions commonly involve loss of neuronal connectivity, synaptic dysfunction with excessive pruning, and ionic imbalances. These often serve as a prelude to cell death either through the activation of apoptotic or necrotic death routines or excess autophagy. In many instances, a local or generalized Ca2+ deregulation is involved in signaling or executing cell death. We have recently shown that in brain ischemia, and during excitotoxicity triggered by excess glutamate, the irreversible Ca2+ deregulation leading to necrosis is due to calpain-mediated modulation of the plasma membrane Na+/Ca2+ exchanger (NCX). Here we show that the NCX can also be cleaved by caspases in neurons undergoing apoptosis, which suggests that cleavage of the main Ca2+ extrusion pathway is a lethal event in multiple forms of cell death.

Cleavage of plasma membrane calcium pumps by caspases: a link between apoptosis and necrosis.

Neuronal death, which follows ischemic injury or is triggered by excitotoxins, can occur by both apoptosis and necrosis. Caspases, which are not directly required for necrotic cell death, are central mediators of the apoptotic program. Here we demonstrate that caspases cleave and inactivate the plasma membrane Ca(2+) pump (PMCA) in neurons and non-neuronal cells undergoing apoptosis. PMCA cleavage impairs intracellular Ca(2+) handling, which results in Ca(2+) overload. Expression of non-cleavable PMCA mutants prevents the disturbance in Ca(2+) handling, slows down the kinetics of apoptosis, and markedly delays secondary cell lysis (necrosis). These findings suggest that caspase-mediated cleavage and inactivation of PMCAs can lead to necrosis, an event that is reduced by caspase inhibitors in brain ischemia.

Apoptosis in caspase-inhibited neurons.

BACKGROUND: There is growing evidence of apoptosis in neurodegenerative disease. However, it is still unclear whether the pathological manifestations observed in slow neurodegenerative diseases are due to neuronal loss or whether they are related to independent degenerative events in the axodendritic network. It also remains elusive whether a single, caspase-based executing system involving caspases is responsible for neuronal loss by apoptosis. MATERIALS AND METHODS: Long-term exposure to the microtubule-disassembling agent, colchicine, was used to disrupt the axodendritic network and eventually trigger caspase-3-mediated apoptosis in cultures of cerebellar granule cells. For this model, we investigated the role of Bcl-2 and caspases in neurite degeneration and death of neuronal somata. RESULTS: Early degeneration of the axodendritic network occurred by a Bcl-2 and caspase-independent mechanism. Conversely, apoptosis of the cell body was delayed by Bcl-2 and initially blocked by caspase inhibition. However, when caspase activity was entirely blocked by zVAD-fmk, colchicine-exposed neurons still underwent delayed cell death characterized by cytochrome c release, chromatin condensation to irregularly shaped clumps, DNA-fragmentation, and exposure of phosphatidylserine. Inhibitors of the proteasome reduced these caspase-independent apoptotic-like features of the neuronal soma. CONCLUSION: Our data suggest that Bcl-2-dependent and caspase-mediated death programs account only partially for neurodegenerative changes in injured neurons. Blockage of the caspase execution machinery may only temporarily rescue damaged neurons and classical apoptotic features can still appear in caspase-inhibited neurons.

Caspases mediate nucleoporin cleavage, but not early redistribution of nuclear transport factors and modulation of nuclear permeability in apoptosis.

In eukaryotic cells, both soluble transport factors and components of the nuclear pore complex mediate protein and RNA trafficking between the nucleus and the cytoplasm. Here, we investigated whether caspases, the major execution system in apoptosis, target the nuclear pore or components of the nuclear transport machinery. Four nucleoporins, Nup153, RanBP2, Nup214 and Tpr are cleaved by caspases during apoptosis. In contrast, the nuclear transport factors, Ran, importin alpha and importin beta are not proteolytically processed, but redistribute across the nuclear envelope independently and prior to caspase activation. Also, mRNA accumulates into the nucleus before caspases become active. Microinjection experiments further revealed that early in apoptosis, the nucleus becomes permeable to dextran molecules of 70 kD molecular weight. Redistribution of import factors and mRNA, as well as nuclear permeabilisation, occur prior to caspase-mediated nucleoporin cleavage. Our findings suggest that the apoptotic programme includes modifications in the machinery responsible for nucleocytoplasmic transport, which are independent from caspase-mediated degradation of nuclear proteins.

Cascade of caspase activation in potassium-deprived cerebellar granule neurons: targets for treatment with peptide and protein inhibitors of apoptosis.

Cerebellar granule neurons (CGN) cultured in the presence of serum and depolarizing potassium concentrations undergo apoptosis when switched to serum-free medium containing physiological potassium concentrations. Here we show that processing of the key protease, caspase-3, depends on the activation of caspase-9, but not of caspase-8. Selective peptide inhibitors of caspase-9 block processing of caspase-3 and caspase-8 and inhibit apoptosis, whereas a selective inhibitor of caspase-8 blocks neither processing of caspase-3 nor cell death. The data obtained with peptide inhibitors were confirmed by adenovirally mediated ectopic expression of the cytokine response modifier A (crmA), the baculovirus protein p35, and the X chromosome-linked inhibitor of apoptosis (XIAP). Further, caspase-8-activating death receptors do not mediate apoptosis in CGN and potassium withdrawal-induced apoptosis evolves unaltered in gld or lpr mice, which harbor mutations in the CD95/CD95 ligand system. Thus, neuronal apoptosis triggered by potassium deprivation is death receptor-independent but involves the mitochondrial pathway of caspase activation.

Differential effects of bcl-2 on cell death triggered under ATP-depleting conditions.

The intracellular ATP concentration decides on the onset of either apoptosis or necrosis in Jurkat cells exposed to death stimuli. Bcl-2 can block apoptotic demise, which occurs preferably under conditions of high cellular ATP levels. Here, we investigated the effects of Bcl-2 on the necrotic type of cell demise that prevails under conditions of energy loss. ATP levels were modulated by using mitochondrial inhibitors, such as rotenone or S-nitrosoglutathione, in medium either lacking glucose or supplemented with glucose to stimulate glycolytic ATP generation. Under conditions of ATP depletion, staurosporine (STS) induced >90% necrosis in vector control-transfected cells, whereas bcl-2-transfected cells were protected. Thus, the antiapoptotic protein Bcl-2 can reduce the overall amount of cell death in ATP-depleted cells regardless whether it occurs by apoptosis or necrosis. Cytochrome c release, normally preceding STS-induced necrosis, was also inhibited by Bcl-2. However, Bcl-2 did not prevent an initial STS-induced drop of the mitochondrial membrane potential (DeltaPsi(m)). Therefore, the mechanisms whereby Bcl-2 prevents cell death and favors retention of cytochrome c in the mitochondria require neither the maintenance of mitochondrial DeltaPsi nor the maintenance of normal ATP levels.

Additive effects of caspase inhibitor and lazaroid on the survival of transplanted rat and human embryonic dopamine neurons.

Major practical constraints on neural grafting in Parkinson's disease are the shortage of human donor tissue and the great loss of dopamine neurons during the grafting procedure. The vast majority of implanted embryonic dopamine neurons are believed to die within a few days of transplantation surgery, at least in part through apoptosis. We have previously found that survival of nigral grafts in rodents can be significantly augmented by pretreatment with the caspase inhibitor Ac-YVAD-cmk or by lazaroids (lipid peroxidation inhibitors). We now report that pretreatment with the caspase inhibitor Ac-DEVD-cmk, but not z-VAD-fmk, results in a significantly improved survival of transplanted dopamine neurons of similar magnitude to that achieved in this study using Ac-YVAD-cmk (both 220-230% of control). In addition, we found that treatment of the graft tissue with tirilazad mesylate (a lazaroid allowed for clinical use) almost doubled the survival of grafted dopamine neurons. When Ac-YVAD-cmk and tirilazad mesylate treatments were combined, the number of surviving dopamine neurons increased significantly further to 280% of control. Importantly, the same combination of neuroprotectants enhanced the survival of human dopamine neurons xenotransplanted to immunosuppressed rats (to 240% of control). In conclusion, these results suggest that combining treatments that counteract oxidative stress and caspase activation is a valuable strategy to enhance nigral graft survival that should be considered for clinical application.

Apoptotic cleavage of scaffold attachment factor A (SAF-A) by caspase-3 occurs at a noncanonical cleavage site.

Members of the caspase family of cysteine proteases play essential roles in the disintegration of cellular architecture during apoptosis. Caspases have been grouped into subfamilies according to their preferred cleavage sites, with the "apoptotic executioner" caspase-3 as the prototype of DEXD-dependent proteases. We show here that caspase-3 is more tolerant to variations of the cleavage site than previously anticipated and present an example of a noncanonical recognition site that is efficiently cleaved by caspase-3 in vitro and in vivo. The new cleavage site was identified in human scaffold attachment factor A, one of the major scaffold attachment region DNA-binding proteins of human cells thought to be involved in nuclear architecture by fastening chromatin loops to a proteinaceous nuclear skeleton, the so-called nuclear matrix or scaffold. Using an amino-terminal recombinant construct of scaffold attachment factor A and recombinant caspase-3, we have mapped the cleavage site by matrix-assisted laser desorption ionization/time of flight mass spectrometry and Edman sequencing. We find that cleavage occurs after Asp-100 in a sequence context (SALD) that does not conform to the hitherto accepted DEXD consensus sequence of caspase-3. A point mutation, D100A, abrogates cleavage by recombinant caspase-3 in vitro and during apoptosis in vivo, confirming SALD as a novel caspase-3 cleavage site.

Calcium transients in single fibers of low-frequency stimulated fast-twitch muscle of rat.

Ca(2+) transients were investigated in single fibers isolated from rat extensor digitorum longus muscles exposed to chronic low-frequency stimulation for different time periods up to 10 days. Approximately 2.5-fold increases in resting Ca(2+) concentration ([Ca(2+)]) were observed 2 h after stimulation onset and persisted throughout the stimulation period. The elevated [Ca(2+)] levels were in the range characteristic of slow-twitch fibers from soleus muscle. In addition, we noticed a transitory elevation of the integral [Ca(2+)] per pulse with a maximum ( approximately 5-fold) after 1 day. Steep decreases in rate constant of [Ca(2+)] decay could be explained by an immediate impairment of Ca(2+) uptake and, with longer stimulation periods, by an additional loss of cytosolic Ca(2+) binding capacity resulting from a decay in parvalbumin content. A partial recovery of the rate constant of [Ca(2+)] decay in 10-day stimulated muscle could be explained by an increasing mitochondrial contribution to Ca(2+) sequestration.

Tributyltin-induced apoptosis requires glycolytic adenosine trisphosphate production.

The toxicity of tributyltin chloride (TBT) involves Ca(2+) overload, cytoskeletal damage, and mitochondrial failure leading to cell death by apoptosis or necrosis. Here, we examined whether the intracellular ATP level modulates the mode of cell death after exposure to TBT. When Jurkat cells were energized by the mitochondrial substrate, pyruvate, low concentrations of TBT (1-2 microM) triggered an immediate depletion of intracellular ATP followed by necrotic death. When ATP levels were maintained by the addition of glucose, the mode of cell death was typically apoptotic. Glycolytic ATP production was required for apoptosis at two distinct steps. First, maintenance of adequate ATP levels accelerated the decrease of mitochondrial membrane potential, and the release of the intermembrane proteins adenylate kinase and cytochrome c from mitochondria. A possible role of the adenine nucleotide exchanger in this first ATP-dependent step is suggested by experiments performed with the specific inhibitor, bongkrekic acid. This substance delayed cytochrome c release in a manner similar to that caused by ATP depletion. Second, caspase activation following cytochrome c release was only observed in ATP-containing cells. Bcl-2 had only a minor effect on TBT-triggered caspase activation or cell death. We conclude that intracellular ATP concentrations control the mode of cell death in TBT-treated Jurkat cells at both the mitochondrial and caspase activation levels.

Differential effects of Bcl-2 overexpression on fibre outgrowth and survival of embryonic dopaminergic neurons in intracerebral transplants.

The causes of death of transplanted neurons are not known in detail, but apoptotic mechanisms involving caspase activation are likely to play a role. We examined whether overexpression of the anti-apoptotic protein Bcl-2 may enhance the survival of dopaminergic [tyrosine hydroxylase (TH)-immunoreactive] grafted neurons. For this purpose, we prepared cells from embryonic day 13 ventral mesencephalon (VM) of mice overexpressing human Bcl-2, or from their wild-type littermates. The bcl-2 transgene was strongly expressed in these cells, and resulted in protection of neuronal cultures from death triggered by serum deprivation or exposure to staurosporine. To model pretransplantation stress more closely in vitro, we stored dissociated embryonic mesencephalic cells for 8 h in the same type of medium used for intracerebral transplantation. This resulted in massive cell death as quantified by lactate dehydrogenase (LDH) release, and increased DNA fragmentation. Although this cell loss was strongly reduced by a caspase inhibitor, Bcl-2 had no significant protective effect. Finally, mesencephalic cell suspensions were xenografted into the striatum of immunosuppressed hemiparkinsonian rats. Neither the survival of TH-immunopositive transplanted neurons nor the functional recovery of the rats was improved by Bcl-2, although the Bcl-2 protein was strongly expressed in transgenic grafts 5 weeks after implantation, and dopaminergic fibre outgrowth from the grafts was significantly improved. These data suggest that cell death in neuronal transplants involves apoptotic mechanisms that can bypass negative regulation by Bcl-2.

Execution of apoptosis: converging or diverging pathways?

There is increasing evidence that apoptosis and necrosis represent only two of several possible ways for cells to die. These two types of demise can occur simultaneously in tissues or cell cultures exposed to the same stimulus, and often local metabolic conditions and the intensity of the same initial insult decide the prevalence of either apoptosis or necrosis. Recent work has shown that execution of the apoptotic programme involves a relatively limited number of pathways. According to a general view, these would converge to activate the caspase family of proteases. However, there is increasing evidence that apoptotic-like features can be observed also in cells where caspases are inhibited by cell-permeable tripeptides, such as z-VaD-Ala-Asp-fluoromethyl ketone (z-VAD-fmk), or analogous compounds. This has posed the question as to whether apoptosis may or may not occur in a caspase independent way, and whether caspase inhibitors may be effective in the treatment of disease. Also relevant is the understanding that low intracellular energy levels during apoptosis can preclude caspase activation, and consequently decide the occurrence and mode of demise in damaged cells. In vivo, incomplete execution of damaged cells by apoptosis may have profound implications, as their persistence within a tissue, followed by delayed lysis, may elicit delayed pro-inflammatory reactions. In this minireview, we discuss some recent findings suggesting that cells may use diverging execution pathways, with different implications in pathology and therapy.

Prevention of endotoxin-induced lethality, but not of liver apoptosis in poly(ADP-ribose) polymerase-deficient mice.

Activation of poly-(ADP-ribose) polymerase (PARP) is often associated with cytotoxicity, but its precise role in shock-induced lethality and in different modes of tissue injury is still unknown. We took advantage of the existence of mice with a targeted deletion of the PARP gene (PARP-/-) to examine the differential sensitivity of wild-type (wt) and PARP-/- mice toward endotoxin (LPS)-induced lethality and different forms of liver damage. All PARP-/- animals survived high-dose (20 mg/kg) LPS-mediated shock, which killed 60% of wt animals. Moreover, LPS-induced necrotic liver damage was significantly reduced. In contrast, when apoptotic liver damage was induced via injection of low concentrations of LPS (30 microgram/kg) into D-galactosamine-sensitized mice, or via activation of hepatic cell death receptors, PARP-/- animals were not protected. We conclude that PARP is involved in systemic LPS toxicity, while it plays a minor role in apoptotic liver damage mediated by TNF or CD95.

ATP controls neuronal apoptosis triggered by microtubule breakdown or potassium deprivation.

BACKGROUND: Early loss of neurites followed by delayed damage of neuronal somata is a feature of several neurodegenerative diseases. Death by apoptosis would ensure the rapid removal of injured neurons, whereas conditions that prevent apoptosis may facilitate the persistence of damaged cells and favor inflammation and disease progression. MATERIALS AND METHODS: Cultures of cerebellar granule cells (CGC) were treated with microtubule disrupting agents. These compounds induced an early degeneration of neurites followed by apoptotic destruction of neuronal somata. The fate of injured neurons was followed after co-exposure to caspase inhibitors or agents that decrease intracellular ATP (deoxyglucose, S-nitrosoglutathione, 1-methyl-4-phenylpyridinium). We examined the implications of energy loss for caspase activation, exposure of phagocytosis markers, and long-term persistence of damaged cells. RESULTS: In CGC exposed to colchicine or nocodazole, axodendritic degeneration preceded caspase activation and apoptosis. ATP-depleting agents or protein synthesis inhibition prevented caspase activation, translocation of the phagocytosis marker, phosphatidylserine, and apoptotic death. However, they did not affect the primary neurite loss. Repletion of ATP by enhanced glycolysis restored all apoptotic features. Peptide inhibitors of caspases also prevented the apoptotic changes in the cell bodies, although the axodendritic net was lost. Under this condition cell demise still occurred 48 hr later in a caspase-independent manner and involved plasma membrane lysis at the latest stage. CONCLUSIONS: Inhibition of the apoptotic machinery by drugs, energy deprivation, or endogenous mediators may result in the persistence and subsequent lysis of injured neurons. In vivo, this may favor the onset of inflammatory processes and perpetuate neurodegeneration.

Inhibition of mitochondrial ATP generation by nitric oxide switches apoptosis to necrosis.

Under pathological conditions, the mode of cell death, apoptosis or necrosis, is relevant for the subsequent fate of the tissue. Cell demise may be shaped by endogenous mediators such as nitric oxide (NO) which interfere with subroutines of the death program. Here we show that apoptosis of Jurkat cells elicited by either staurosporine (STS) or anti-CD95 antibodies in glucose-free medium is converted to necrosis by NO donors. In the presence of NO, release of mitochondrial cytochrome c was delayed and activation of execution caspases was prevented. Stimulated cells died nonetheless. The switch in the mode of cell death was due to NO-dependent failure of mitochondrial energy production. Restoration of intracellular ATP by glucose supplementation recovered the cells' ability to activate caspases and undergo apoptosis. In this system, the apoptosis/necrosis conversion promoted by NO was not mediated by cyclic guanosine monophosphate-dependent mechanisms, poly-(ADP-ribose)-polymerase (PARP) activation, or inhibition of caspases due to S-nitrosylation and glutathione depletion. In contrast, depleting intracellular ATP with rotenone, an inhibitor of mitochondrial complex I mimicked the effect of NO. The findings presented here suggest that NO can decide the shape of cell death by lowering intracellular ATP below the level required to allow the coordinated execution of apoptosis.

Nitric oxide inhibits execution of apoptosis at two distinct ATP-dependent steps upstream and downstream of mitochondrial cytochrome c release.

The endogenous mediator nitric oxide (NO) blocked apoptosis of Jurkat cells elicited by staurosporine, anti-CD95 or chemotherapeutics, and switched death to necrosis. The switch in the mode of cell death was dependent on the ATP loss elicited by NO. This affected two distinct steps of the apoptotic cascade. First, the release of cytochrome c from mitochondria was delayed by NO. Second, processing of procaspases-3/7 to the active proteases was prevented even after cytochrome c had been released. Thus, NO interferes with execution steps of apoptosis both upstream and downstream of cytochrome c release.