Stabilization of apoptotic cells: generation of zombie cells.

Apoptosis is characterized by degradation of cell components but plasma membrane remains intact. Apoptotic microtubule network (AMN) is organized during apoptosis forming a cortical structure beneath plasma membrane that maintains plasma membrane integrity. Apoptotic cells are also characterized by high reactive oxygen species (ROS) production that can be potentially harmful for the cell. The aim of this study was to develop a method that allows stabilizing apoptotic cells for diagnostic and therapeutic applications. By using a cocktail composed of taxol (a microtubule stabilizer), Zn(2+) (a caspase inhibitor) and coenzyme Q10 (a lipid antioxidant), we were able to stabilize H460 apoptotic cells in cell cultures for at least 72 h, preventing secondary necrosis. Stabilized apoptotic cells maintain many apoptotic cell characteristics such as the presence of apoptotic microtubules, plasma membrane integrity, low intracellular calcium levels and mitochondrial polarization. Apoptotic cell stabilization may open new avenues in apoptosis detection and therapy.

Apoptotic microtubules delimit an active caspase free area in the cellular cortex during the execution phase of apoptosis.

Apoptotic microtubule network (AMN) is organized during apoptosis, forming a cortical structure beneath plasma membrane, which has an important role in preserving cell morphology and plasma membrane permeability. The aim of this study was to examine the role of AMN in maintaining plasma membrane integrity during the execution phase of apoptosis. We demonstrated in camptothecin-induced apoptosis in H460 cells that AMN delimits an active caspase free area beneath plasma membrane that permits the preservation of cellular cortex and transmembrane proteins. AMN depolymerization in apoptotic cells by a short exposure to colchicine allowed active caspases to reach the cellular cortex and cleave many key proteins involved in plasma membrane structural support, cell adhesion and ionic homeostasis. Cleavage of cellular cortex and plasma membrane proteins, such as α-spectrin, paxilin, focal adhesion kinase (FAK), E-cadherin and integrin subunit ß4 was associated with cell collapse and cell detachment. Otherwise, cleavage-mediated inactivation of calcium ATPase pump (PMCA-4) and Na(+)/Ca(2+) exchanger (NCX) involved in cell calcium extrusion resulted in calcium overload. Furthermore, cleavage of Na(+)/K(+) pump subunit ß was associated with altered sodium homeostasis. Cleavage of cell cortex and plasma membrane proteins in apoptotic cells after AMN depolymerization increased plasma permeability, ionic imbalance and bioenergetic collapse, leading apoptotic cells to secondary necrosis. The essential role of caspase-mediated cleavage in this process was demonstrated because the concomitant addition of colchicine that induces AMN depolymerization and the pan-caspase inhibitor z-VAD avoided the cleavage of cortical and plasma membrane proteins and prevented apoptotic cells to undergo secondary necrosis. Furthermore, the presence of AMN was also critical for proper phosphatidylserine externalization and apoptotic cell clearance by macrophages. These results indicate that AMN is essential to preserve an active caspase free area in the cellular cortex of apoptotic cells that allows plasma membrane integrity during the execution phase of apoptosis.

Coenzyme Q10 distribution in blood is altered in patients with fibromyalgia.

OBJECTIVE: Coenzyme Q10 (CoQ(10)) is an essential electron carrier in the mitochondrial respiratory chain and a strong antioxidant. Signs and symptoms associated with muscular alteration and mitochondrial dysfunction, including oxidative stress, have been observed in patients with fibromyalgia (FM). The aim was to study CoQ(10) levels in plasma and mononuclear cells, and oxidative stress in FM patients. METHODS: We studied CoQ(10) level by HPLC in plasma and peripheral mononuclear cells obtained from patients with FM and healthy control subjects. Oxidative stress markers were analyzed in both plasma and mononuclear cells from FM patients. RESULTS: Higher level of oxidative stress markers in plasma was observed respect to control subjects. CoQ(10) level in plasma samples from FM patients was doubled compared to healthy controls and in blood mononuclear cells isolated from 37 FM patients was found to be about 40% lower. Higher levels of ROS production was observed in mononuclear cells from FM patients compared to control, and a significant decrease was induced by the presence of CoQ(10). CONCLUSION: The distribution of CoQ(10) in blood components was altered in FM patients. Also, our results confirm the oxidative stress background of this disease probably due to a defect on the distribution and metabolism of CoQ(10) in cells and tissues. The protection caused in mononuclear cells by CoQ(10) would indicate the benefit of its supplementation in FM patients.

Cytotoxic effects of amitriptyline in human fibroblasts.

Amitriptyline is a tricyclic antidepressant widely used in the treatment of chronic pain. The objective of the present study was to investigate the potential cytotoxic effects of amitriptyline in human fibroblasts primary culture. Human fibroblast cells were cultured from healthy subjects and incubated with 50 microM and 100 microM amitriptyline. Cell counting was performed to study dose-dependency of toxicity. Lipid peroxidation analysis and western blotting for antioxidants catalase and mitochondrial superoxide dismutase (MnSOD) were carried out in order to evaluate oxidative stress. To investigate mitochondria damage the following determinations were made: cytochrome c, citrate synthase, and mitochondrial membrane potential (DeltaPsi(m)). Amitriptyline reduced significantly the number of cultured cells, resulting in a decrease of 45.2%, 65.0% and 94.9% when treated with 20 microM, 50 microM and 100 microM amitriptyline, respectively. This drug enhanced the production of oxidized products during lipid peroxidation, inverting the reduced/oxidized ratio to 25% reduction and 75% oxidation after 24h of amitriptyline administration. A decreased in catalase protein levels has been also observed. Moreover, amitriptyline treatment induced a significant decrease of cytochrome c, DeltaPsi(m), and citrate synthase activity; revealing mitochondrial damage. These findings suggest that amitriptyline has a strong cytotoxic effect in human fibroblasts, decreasing growth rate and mitochondrial activity, and increasing oxidative stress.

Nuclear caspase-3 and caspase-7 activation, and poly(ADP-ribose) polymerase cleavage are early events in camptothecin-induced apoptosis.

Chemotherapy-induced apoptosis by DNA-damaging drugs is thought to be generally dependent on the release of cytochrome c and the subsequent activation of caspase-9 and -3. However, the molecular mechanism of how damaged DNA triggers the apoptotic process is not clear. To better understand the mechanisms underlying this process, we examined drug-induced apoptosis in cultured H-460 cells. Using cell fractionation, western blotting, and immunofluorescence assays, we show that the activation of nuclear caspases-7 and -3, and poly(ADP-ribose) polymerase (PARP) cleavage, are early events in camptothecin-induced apoptosis. Moreover, we demonstrate that these events precede the release of cytochrome c and apoptotic inducing factor, and the activation of caspases 2, 8, 9 and 12. Together our results suggest that drugs acting at the DNA level can initiate apoptosis via nuclear caspase activation.

Muscle coenzyme Q10 concentrations in patients with probable and definite diagnosis of respiratory chain disorders.

Coenzyme Q(10) (CoQ) deficiency syndrome is a disorder of unknown ethiology that may cause different forms of mitochondrial encephalomyopathy. In the present study our aim was to analyse CoQ concentration and mitochondrial respiratory chain (MRC) enzyme activities in muscle biopsies of patients with clinical suspicion and/or biochemical-molecular diagnosis of a mitochondrial disorder. We studied 36 patients classified into 3 groups: 1) 14 patients without a definitive diagnosis of mitochondrial disease, 2) 13 patients with decreased CI + III and II + III activities of the MRC, and 3) 9 patients with definitive diagnosis of mitochondrial disease. Only 1 of the 14 patients of group 1 showed slightly reduced CoQ values in muscle. Six of the 13 patients from group 2 showed partial CoQ deficiency in muscle and 1 of the 9 cases from group 3 presented a slight CoQ deficiency. Significantly positive correlation was observed between CI + III and CII + III activities with CoQ concentrations in the 36 muscle homogenates from patients (r = 0.555; p = 0.001; and r = 0.460; p = 0.005, respectively). In conclusion, measurement of MRC enzyme activities is a useful tool for the detection of CoQ deficiency, which should be confirmed by CoQ quantification.

Camptothecin-induced apoptosis in non-small cell lung cancer is independent of cyclooxygenase expression.

Recent observations show a positive correlation between the expression of cyclooxygenase (COX), especially COX-2), and cancer development. Here we tested the hypothesis that expression of COX-2 could influence apoptosis in lung cancer cell lines. To address this question, we determined the effects of camptothecin-induced apoptosis on three lung cancer cell lines which over express COX-1 (CORL23), COX-2 (MOR-P) and neither isoform (H-460), and determine if these effects were prostaglandin mediated. We also compared the effects of non-selective and isoenzyme selective COX-2 inhibitors on camptothecin-induced apoptosis in these three cell lines. Camptothecin induced apoptosis in all three cell lines independently of COX-1 or COX-2 expression. Indomethacin, a non-selective COX inhibitor and NS398, a selective COX-2 inhibitor had no effect on camptothecin-induced apoptosis at concentrations that abolished prostaglandin production. In conclusion, these finding suggest that the COX pathway is not involved in camptothecin-induced apoptosis of non-small cell lung cancer cell lines.

Anticancer drugs induce increased mitochondrial cytochrome c expression that precedes cell death.

Recent studies have demonstrated that cytochrome c plays an important role in cell death. In the present study, we report that teniposide and various other chemotherapeutic agents induced a dose-dependent increase in the expression of the mitochondrial respiratory chain proteins cytochrome c, subunits I and IV of cytochrome c oxidase, and the free radical scavenging enzyme manganous superoxide dismutase. The teniposide-induced increase of cytochrome c was inhibited by cycloheximide, indicating new protein synthesis. Elevated cytochrome c levels were associated with enhanced cytochrome c oxidase-dependent oxygen uptake using TMPD/ascorbate as the electron donor, suggesting that the newly synthesized proteins were functional. Cytochrome c was released into the cytoplasm only after maximal levels had been reached in the mitochondria, but there was no concomitant decrease in mitochondrial membrane potential or caspase activation. Our results suggest that the increase in mitochondrial protein expression may play a role in the early cellular defense against anticancer drugs.

Tumor necrosis factor-alpha increases the steady-state reduction of cytochrome b of the mitochondrial respiratory chain in metabolically inhibited L929 cells.

The mechanism of tumor necrosis factor alpha (TNFalpha)-induced cytotoxicity in metabolically inhibited cells is unclear, although some studies have suggested that mitochondrial dysfunction and generation of reactive oxygen species may be involved. Here we studied the effect of TNFalpha on the redox state of mitochondrial cytochromes and its involvement in the generation of reactive oxygen species in metabolically inhibited L929 cells. Treatment with TNFalpha and cycloheximide (TNFalpha/CHX) induced mitochondrial cytochrome c release, increased the steady-state reduction of cytochrome b, and decreased the steady-state reduction of cytochromes cc(1) and aa(3). TNFalpha/CHX treatment also induced lipid peroxidation, intracellular generation of reactive oxygen species, and cell death. Furthermore, as the cells died mitochondrial morphology changed from an orthodox to a hyperdense and condensed and finally to a swollen conformation. Antimycin A, a mitochondrial respiratory chain complex III inhibitor that binds to cytochrome b, blocked the formation of reactive oxygen species, suggesting that the free radicals are generated at the level of cytochrome b. Moreover, antimycin A, when added after 3 h of TNFalpha/CHX treatment, arrested the further release of cytochrome c and the cytotoxic response. We propose that the reduced cytochrome b promotes the formation of reactive oxygen species, lipid peroxidation of the cell membrane, and cell death.

Increased mitochondrial cytochrome c levels and mitochondrial hyperpolarization precede camptothecin-induced apoptosis in Jurkat cells.

Mitochondria play a central role in apoptosis through release of cytochrome c and activation of caspases. In the present study, we showed that, in Jurkat human T cells, camptothecin-induced apoptosis is preceded by (i) an increase in cytochrome c and subunit IV of cytochrome c oxidase (COX IV) levels in mitochondria; and (ii) an elevation of the mitochondrial membrane potential (Delta(Psi)m). These events are followed by cytochrome c release into the cytosol, cytochrome c and COX IV depletion from mitochondria, externalization of phosphatidylserine (PS), disruption of Delta(Psi)m, caspase activation, poly(ADP-ribose)polymerase cleavage and DNA fragmentation. The pan-caspase inhibitor z-VAD.fmk blocked camptothecin-induced PS externalization, disruption of Delta(Psi)m and DNA fragmentation, suggesting that these events are mediated by caspase activation. In contrast, z-VAD did not prevent cytochrome c release, despite preventing cytochrome c and COX IV depletion from mitochondria. Together, these data suggest that mitochondrial cytochrome c and COX IV enrichment are early events preceding the onset of apoptosis and that cytochrome c release is upstream of caspase activation and loss of Delta(Psi)m. Furthermore, prevention by z-VAD of cytochrome c and COX IV depletion in mitochondria suggests the possibility that a caspase-like activity in mitochondria is involved in the proteolytic depletion of respiratory chain proteins. Activation of this activity may play an important role in drug-induced apoptosis.

Tumor necrosis factor-alpha increases ATP content in metabolically inhibited L929 cells preceding cell death.

The effects of tumor necrosis factor-alpha (TNF) on ATP levels were studied in metabolically inhibited L929 cells. Treatment of these cells with TNF in the presence of actinomycin D or cycloheximide induces cyclic changes in the intracellular ATP content preceding cell death. After 3 h of incubation, the intracellular ATP content increased by 48 +/- 6% (p < 0.001), but at 4 h, it decreased to the control level. Two hours later, it increased again by 23 +/- 5% over the control level (p < 0.001). Coinciding with cell death, ATP content decreased progressively until almost complete depletion. These changes in ATP content were associated with parallel alterations in the respiratory coupling and with increased generation of reactive oxygen species. The mechanism by which TNF/actinomycin D or TNF/cycloheximide increased cellular ATP seemed to be dependent on the mitochondrial ATP synthesis and related to the cytotoxic effect of TNF, since blockade of mitochondrial electron transport prevented the increase in cellular ATP, the formation of reactive oxygen species, and the apoptotic cell death caused by TNF. We suggest that the TNF/actinomycin D- or TNF/cycloheximide-induced changes in intracellular ATP levels may be involved in the cytotoxic effect of TNF in metabolically inhibited L929 cells.

Tumor necrosis factor alpha inhibits collagen alpha 1(I) gene expression in rat hepatic stellate cells through a G protein.

BACKGROUND & AIMS: Tumor necrosis factor alpha (TNF-alpha) inhibits collagen gene expression in cultured fibroblasts. By binding to cell surface receptors, TNF-alpha promotes signals within the cells. The purpose of this study was to investigate the role played by G proteins in TNF-alpha-induced inhibition of collagen gene expression. METHODS: Effect of TNF-alpha on collagen alpha 1(I) messenger RNA (mRNA) level was measured in cultured hepatic stellate cells in basal condition and after inhibiting or activating G proteins or the major intracellular signal transduction pathways. RESULTS: TNF-alpha significantly decreased the level of alpha 1(I) collagen mRNA. Treatment of cells with pertussis toxin inhibited this effect, whereas blocking adenylate cyclase or protein kinase A had no effect. Likewise, blocking phospholipase A2, phospholipase C1 calcium channels, calmodulin, or protein kinase C did not eliminate the inhibitory effect of TNF-alpha on collagen mRNA. On the other hand, C2-ceramide and sphingomyelinase reproduced the effect of TNF-alpha on collagen gene expression, and TNF-alpha did not increase the effect of sphingomyelinase. CONCLUSIONS: TNF-alpha-induced inhibition of alpha 1(I) collagen gene expression in a hepatic stellate cell line may be mediated by a pertussis toxin-sensitive G protein. TNF-alpha may inhibit this gene by using sphingomyelin/ceramide as an intracellular signal transduction pathway.

Down-regulation of tumor necrosis factor receptors by blockade of mitochondrial respiration.

We have studied the effect of blockade of mitochondrial respiration on the binding of human 125I-TNF alpha to L929 cell receptors. Specific TNF alpha binding was decreased to about 20-40% of controls by blocking mitochondrial respiration. This effect was dose- and time-related and was observed independently of the level at which the respiration was blocked (respiratory chain, proton backflow, ATPase, anaerobiosis). This blockade had no effect on the half-life of the specific TNF alpha binding, the internalization or degradation of TNF alpha-receptor complexes, or the number of TNF alpha-binding sites. Scatchard analysis of TNF alpha binding data indicated a 2-4-fold decrease in the affinity of these binding sites. These effects did not appear to be related to the protein kinase C activity or to reactive oxygen radicals, since they were not antagonized by pretreatment of cells with oxygen radical scavengers, deferoxamine, or inhibitors of protein kinase C. Decrease in TNF alpha binding capacity correlated significantly with cellular ATP content (r = 0.94; p < 0.01) and with the cytocidal activity of TNF alpha against L929 cells. These findings suggest that blockade of mitochondrial respiration down-regulates the binding of TNF alpha to cells, most likely by changing the affinity of receptors for this cytokine. This down-regulation may increase the resistance of cells to TNF alpha cytotoxicity.

Toxic oil stimulates collagen synthesis acting at a pretranslational level in cultured fat-storing cells.

BACKGROUND/AIMS: The toxic oil syndrome appeared in Spain in 1981 as a result of ingestion of rapeseed oil denatured with aniline. Some patients developed scleroderma-like skin lesions and liver cirrhosis. Mechanisms of these fibrotic lesions are not known. The present study was designed to investigate the effect of toxic oils on collagen metabolism. METHODS: We measured the relative rate of collagen production, absolute rate of collagen synthesis, production, secretion, and degradation, proline transport, steady-state levels of procollagen alpha 1(l)-messenger RNA (mRNA) in cultured fat-storing cells, and chloramphenicol acetyltransferase activity in transfected cells. RESULTS: Toxic oils increased collagen synthesis, procollagen alpha 1(l)-mRNA levels, and chloramphenicol acetyltransferase activity in cultured fat-storing cells. Effect on collagen production correlated with lipid peroxide content in oils. Cycloheximide, alpha-tocopherol, and methylene blue prevented the increase in procollagen alpha 1(l)-mRNA. Oleylanilide and linoleylanilide, markers for toxic oils, reproduced the stimulatory effects of toxic oils on collagen production and procollagen alpha 1(l)-mRNA. CONCLUSIONS: Toxic oils increased collagen synthesis by acting on the promoter of procollagen alpha 1(l) gene, probably through lipid peroxides derived from acylanilides. We suggest that toxic oil may have stimulated procollagen gene expression through the formation of adducts of aldehydes with some transcription factor.