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Istituto di Farmacologia e Farmacognosia (O.C., A.G., L.P.)
Istituto di Chimica Biologica "Giorgio Fornaini" (M.F.), Universite?Degli Studi di Urbino "Carlo Bo", Urbino, Italy
Abstract
Exposure of U937 cells to an otherwise nontoxic concentration of peroxynitrite promotes a rapid necrotic response in the presence of pharmacological inhibitors of phospholipase A2. A 12-fold higher concentration of the oxidant, in the absence of additional treatments, caused remarkably greater DNA single-strand breakage, delayed formation of H2O2, and depletion of reduced glutathione but an identical level of toxicity. Cell death was prevented in both circumstances by nanomolar levels of arachidonic acid or by cyclosporin A via mechanisms unrelated to elimination of the above effects and was causally linked to prevention of mitochondrial permeability transition. Treatment with a high dose of peroxynitrite for 30 min caused an approximately 40% decline in ATP, both in the absence and presence of arachidonate, whereas only a small, arachidonic acid-sensitive reduction of the ATP pool was detected in cells treated with the low dose of peroxynitrite and the phospholipase A2 inhibitor. ATP-predepleted cells, however, were hypersensitive to peroxynitrite, and under these conditions, toxicity was not prevented by arachidonate. The above findings were reproduced in another promonocytic cell line, THP-1 cells. We concluded that the rapid necrotic response triggered by peroxynitrite in monocytes is mediated by a regulated process, not by ATP depletion, associated with reduced arachidonate availability. Supplementation of exogenous arachidonic acid always rescued cells via an ATP-dependent survival pathway.
Cell death, a process of paramount importance in a variety of physiological and pathological processes, is mediated by different mechanisms in which apoptosis and necrosis represent the two extremes on a continuum (Leist and Nicotera, 1997). Because apoptosis requires energy-dependent events, the choice among these different modes of cell death may be determined on the basis of the ATP availability (Nicotera and Melino, 2004). In the presence of ATP, a toxic treatment leads to apoptosis, whereas in its absence, cell death would take place via a passive mechanism (i.e., necrosis). In support of this notion are the following observations: 1) toxicity paradigms that cause necrosis are associated with depletion of the ATP pool (Lelli et al., 1998), which is, however, well-preserved using lower concentrations of the toxic agent under conditions in which apoptosis, but not necrosis, is concomitantly induced; 2) depletion of ATP, early in the apoptotic process, switches the predominant form of cell death from apoptosis to necrosis (Leist et al., 1997); and 3) activation of poly(ADP-ribose)polymerase leads to ATP depletion and necrosis, whereas inhibition of its activity prevents both ATP depletion and necrosis and eventually switches cells into apoptosis (Ha and Snyder, 1999). Thus, the above results define an important role for ATP in the regulation of cell death and imply that necrosis takes place in damaged cells unable to perform the highly energy-demanding processes involved in apoptosis.
However, necrosis may also represent the primary mode of cell death induced by an otherwise apoptotic treatment when parameters other than ATP are affected (e.g., when caspases are inhibited) (Hirsch et al., 1997; Nicotera and Melino, 2004). Furthermore, it is becoming increasingly clear that necrosis may also represent a primary mode of cell death in a variety of physiopathological conditions (Proskuyakov et al., 2003), and several early events described in the apoptotic process are also critical in the regulation of necrosis (Leist and Nicotera, 1997; Proskuyakov et al., 2003). A good example is given by mitochondrial permeability transition (MPT), which may trigger both apoptosis and necrosis (Kroemer et al., 1998) via mechanisms that are similarly regulated by proteins of the Bcl-2 family (Single et al., 2001; Proskuyakov et al., 2003). Although severe depletion of ATP was described in toxicity paradigms resulting in primary necrosis (Barros et al., 2001; Proskuyakov et al., 2003), whether ATP depletion does always occur and is in fact the most prominent cause of this lethal response remains to be established. This is an important question, because a general requirement for ATP depletion would demonstrate that necrosis is always a passive response, regardless of whether it is primarily induced or it takes place as a consequence of the inability of the cells to perform the apoptotic process.
The present study was designed to investigate whether depletion of ATP is needed in primary necrosis resulting from a toxicity paradigm that has been extensively characterized in our laboratory. We reported that exposure of U937 cells to peroxynitrite promotes an MPT-dependent necrosis within minutes, followed by immediate cell lysis (Sestili et al., 2001). An initial event triggered by peroxynitrite (i.e., inhibition of complex III of the mitochondrial respiratory chain), was responsible for the time-dependent formation of H2O2 that was essential for the occurrence of cell death (Tommasini et al., 2002a). We also showed that otherwise nontoxic concentrations of peroxynitrite nevertheless commit cells to MPT-dependent necrosis, which is, however, prevented by a cytoprotective signaling driven by arachidonic acid released by the cytosolic phospholipase A2 isoform (cPLA2) (Tommasini et al., 2002b, 2004a). We do not know whether arachidonate itself or some downstream product of the cyclooxygenase or lipoxygenase pathways is responsible for the survival signaling.
Thus, although this toxicity paradigm represents a model of "severe primary necrosis" in which extensive ATP depletion may occur, because both peroxynitrite and H2O2 potently inhibit the respiratory chain and glycolysis (Hyslop et al., 1988; Souza and Radi, 1998), it nevertheless presents some features of a highly regulated event.
The present study demonstrates a requirement for ATP in the above cytoprotective signaling and indicates that the ATP pool is well-preserved when the necrotic response, which takes place via a highly regulated mechanism independent of the damage accumulated, is elicited by suppression of the protective signaling.
Materials and Methods
Chemicals. 5,8,11,14-Eicosatetraynoic acid (ETYA), arachidonic acid, 2-deoxy-D-glucose, catalase, N-acetyl-L-cysteine, and most of the reagent grade chemicals were obtained from Sigma-Aldrich (Milan, Italy). Arachidonyl trifluoromethyl ketone (AACOCF3), cyclosporin A, and dihydrorhodamine-123 (DHR) were purchased from Calbiochem (San Diego, CA), Novartis (Bern, Switzerland), and Molecular Probes Europe (Leiden, The Netherlands), respectively.
Cell Culture and Treatment Conditions. U937 or THP-1 cells were cultured in suspension in RPMI 1640 medium (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum (Biological Industries, Kibbutz Beit Haemek, Israel), penicillin (50 U/ml), and streptomycin (50 e蘥/ml; SeraLab Ltd., Crawley Down, UK), at 37°C in T-75 tissue culture flasks (Corning Glassworks, Corning, NY) gassed with an atmosphere of 95% air/5% CO2.
Peroxynitrite was synthesized by the reaction of nitrite with acidified H2O2 as described by Radi et al. (1991), with minor modifications (Tommasini et al., 2002a). Treatments were performed in 2 ml of prewarmed saline A (8.182 g/l NaCl, 0.372 g/l KCl, 0.336 g/l NaHCO3, and 0.9 g/l glucose) containing 5 x 105 cells.
Viability Assay. Cytotoxicity was determined with the trypan blue exclusion assay. In brief, an aliquot of the cell suspension was diluted 1:1 (v/v) with 0.4% trypan blue, and the viable cells were counted with a hemocytometer.
Alkaline Halo Assay. DNA single-strand breakage was determined using the alkaline halo assay as described previously (Sestili and Cantoni, 1999). After treatments, the cells were resuspended at 2.0 x 104 cells/100 e蘬 in 1.5% low-melting agarose in phosphate-buffered saline (8 g/l NaCl, 1.15 g/l Na2HPO4, 0.2 g/l KH2PO4, and 0.2 g/l KCl), containing 5 mM EDTA and immediately sandwiched between an agarose-coated slide and a coverslip. After complete gelling, the coverslips were removed, and the slides were immersed in an alkaline buffer (0.1 M NaOH/1 mM EDTA, pH 12.5), washed, and stained for 5 min with 10 e蘥/ml ethidium bromide.
The ethidium bromide-labeled DNA was visualized using a confocal laser microscope (DVC 250; Bio-Rad, Hercules, CA), and the resulting images were taken and processed with a chilled charge-coupled device camera (5985; Hamamatsu Italy S.P.A., Milan, Italy) coupled with a Macintosh computer (Apple Computer, Cupertino, CA) using the NIH Image program (developed at the U.S. National Institutes of Health and available at http://rsb.info.nih.gov/nihimage/).
The level of DNA single-strand breakage was quantified by calculating the nuclear spreading factor values, which represent the ratio between the area of the halo (obtained by subtracting the area of the nucleus from the total area, nucleus + halo) and that of the nucleus, from 50 to 75 randomly selected cells per experiment per treatment condition.
DHR Oxidation and Imaging. Peroxynitrite-pretreated cells (3 min) were postincubated in fresh saline A containing 10 e DHR (7 min). After treatments, the cells were washed three times, resuspended in 20 e蘬 of saline A, and stratified on a slide. Fluorescence images were captured with a BX-51 microscope (Olympus, Tokyo, Japan) equipped with a SPOT-RT camera unit (Diagnostic Products, Los Angeles, CA). The excitation and emission wavelengths were 488 and 515 nm, respectively, with a 5-nm slit width for both emission and excitation. Images were collected with exposure times of 100 to 400 ms, digitally acquired, and processed for fluorescence determination at the single-cell level on a personal computer using Scion Image software (Scion Corporation, Frederick, MD). Mean fluorescence values were determined by averaging the fluorescence values of at least 50 cells per treatment condition per experiment.
ATP Determination. Ice-cold 2.5% perchloric acid was added to the cells (106 cells/ml). After a 20-min incubation in an ice bath, the solution was removed. Cell extracts were neutralized with K2CO3, and the precipitate was removed by centrifugation. The nucleotide-containing supernatants were filtered through 0.22 e pore micro-filters and analyzed for ATP content by reversed-phase high-performance liquid chromatography (Stocchi et al., 1985) using a Supelcosil LC-18 column (Supelco, Bellefonte, PA).
Biochemical Assays. The assay described by Beutler (1984) was used to measure nonprotein sulfydryl levels. Because GSH represents more than 90% of the nonprotein-SH, the latter will be referred to as GSH. In brief, cells (4 x 106) were washed three times with saline A and centrifuged; the pellet was then resuspended with 150 e蘬 of metaphosphoric acid solution [1.67% (v/v) metaphosphoric acid/0.2% EDTA/30% (w/v) NaCl], kept on ice for 5 min, and centrifuged at 10,000g for 5 min. The nonprotein thiol content was measured spectrophotometrically in the supernatant at 412 nm by using 5,5'-dithiobis(2-nitrobenzoic acid) (412 = 13,600 MeC1cmeC1). Protein content was assayed as described by Bradford (1976), with bovine serum albumin as standard. Phosphofructokinase activity was measured as described by Beutler (1984).
Statistical Analysis. Results are expressed as mean ± S.E.M. For comparison between two groups, the Student's unpaired t test was used.
Results
The results illustrated in Table 1 confirm our previous findings (Sestili et al., 2001) indicating that the loss of viable cells is detectable 15 min after the addition of 1.2 mM peroxynitrite, progressively increases for an additional 45 min, and is not paralleled by the appearance of nonviable cells (data not shown), an indication that cell lysis had indeed taken place. Also consistent with our previous findings (Tommasini et al., 2002b, 2004a) is the observation that an otherwise nontoxic concentration of peroxynitrite (100 e) causes, upon supplementation of a phospholipase A2 inhibitor (50 e AACOCF3), a lethal response identical with that obtained with 1.2 mM peroxynitrite. Once again, toxicity was associated with immediate cell lysis. The effects of AACOCF3 were mimicked by other phospholipase A2 inhibitors, including ETYA (50 e; data not shown).
Cells were exposed for 3 min to either 100 e or 1.2 mM peroxynitrite, centrifuged, and then postincubated for a further 57 min in saline A in the absence or presence (only for cells exposed to 100 e peroxynitrite) of AACOCF3 (50 e). In some experiments, arachidonic acid (0.1 e) or catalase (10 U/ml) was also added during the 57-min post-treatment incubation. In other experiments, cyclosporin A (0.5 e) was given to the cultures 5 min before peroxynitrite and freshly readded during the 57-min post-treatment incubation. The last experimental condition involved a 60-min exposure to N-acetyl-l-cysteine (5 mM) before treatment with peroxynitrite, as detailed above. After treatments, cells were analyzed for viability using the trypan blue exclusion assay. Results represent the mean ± S.E.M. from at least four separate experiments.
In the next set of experiments, the cells were treated as detailed above for 10 min, a time at which toxicity is not as yet apparent, and were analyzed for various biochemical parameters. The results illustrated in Fig. 1 indicate that treatment with the high dose of peroxynitrite produced a remarkably greater DNA single-strand breakage (A), delayed formation of H2O2 (B), and loss of GSH (C) than treatment of phospholipase A2 inhibitor-supplemented cells with the otherwise nontoxic concentration of peroxynitrite or with the low concentration of peroxynitrite alone. The latter two conditions failed to elicit a detectable DHR fluorescence response and produced identical effects on DNA strand scission. Depletion of GSH, however, was more pronounced in cells supplemented with the phospholipase A2 inhibitor. ETYA was used as a phospholipase A2 inhibitor because AACOCF3 is intrinsically fluorescent and interferes with the assays measuring DNA cleavage and oxidation of DHR. Taken together, the above results indicate that the same level of cell death mediated by two different toxicity paradigms is associated with an early accumulation of remarkably different levels of damage.
A very low concentration of exogenous arachidonic acid (100 nM) prevented toxicity induced in both conditions (Table 1). N-Acetyl-L-cysteine (5 mM) or catalase (10 U/ml), however, abolished the lethal response mediated by the intrinsically toxic concentration of peroxynitrite but was ineffective in cells exposed to the otherwise nontoxic concentration of peroxynitrite and AACOCF3. These results confirm our previous findings (Tommasini et al., 2004c) indicating that in both conditions, toxicity arises as a consequence of insufficient arachidonate release caused by either pharmacological inhibition of phospholipase A2 (treatment with the low concentration of peroxynitrite/AACOCF3)orH2O2-dependent inhibition of arachidonic acid release (treatment with the high concentration of peroxynitrite).
The cytoprotective effects of arachidonic acid were mimicked by the MPT inhibitor cyclosporin A (0.5 e) (Table 1). Neither of the two agents, however, affected the DNA single-strand breakage (A) and delayed formation of H2O2 (B) induced by the three different treatments. In contrast, arachidonic acid or cyclosporin A significantly reduced the extent of GSH depletion (C) observed in phospholipase A2-inhibited cells exposed to 100 e peroxynitrite but had hardly any effect in the two remaining conditions. Exposure to 1.2 mM peroxynitrite led to total depletion of cellular GSH. These results further establish the notion that toxicity is not a direct consequence of the overall cellular damage.
We next investigated the effect of the above treatments on the cellular ATP pool. Exposure to 100 e peroxynitrite did not affect ATP levels (Fig. 2A). Under the same conditions, however, the addition of AACOCF3 (Fig. 2B) caused a small reduction in cellular ATP after 15 min of incubation, a time at which many cells are committed to death and only a few dead cells are present because, as previously mentioned, necrotic cells lyse immediately. Identical levels of ATP were found after 30 min. Similar results were obtained by replacing AACOCF3 with ETYA (data not shown). When arachidonic acid or cyclosporin A prevented toxicity, the decline in ATP levels was also prevented. Exposure to 1.2 mM peroxynitrite caused a greater decline in ATP content insensitive to either arachidonic acid or cyclosporin A (Fig. 2C). This response was time-dependent, and after 30 min, a 40% reduction in the ATP content was detected. These results indicate that under the above treatment conditions, the ATP pool is very well preserved.
U937 cells, like most tumor cells, meet their high energy demand for cell proliferation by glycolysis (Brand and Hermfisse, 1997). Indeed a 60-min exposure to the complex I inhibitor rotenone (0.5 e) only reduced ATP content by 12%, suggesting a minor contribution of oxidative phosphorylation in the overall ATP synthesis. This is in contrast with the approximately 90% decrease mediated by 5 mM 2-deoxy-D-glucose, a glucose analog that competitively inhibits cellular uptake and use of glucose. Thus, the decline in ATP content mediated by 1.2 mM peroxynitrite is only partly caused by the inhibition of electron transport. Inhibition of glycolysis is therefore more likely to mediate the decline in ATP levels, an inference supported by the reduction in phosphofructokinase activity (30%, 0.046 U/mg protein in untreated cells) observed 10 min after the addition of peroxynitrite. Finally, accelerated ATP hydrolysis is expected to take place as a consequence of the extensive damage induced by peroxynitrite.
We next investigated the toxicity of peroxynitrite in ATP-predepleted cells and asked the question of whether arachidonic acid could promote survival also under these treatment conditions. For this purpose, the cells were incubated for 60 min in a glucose-free saline supplemented with 2-deoxy-D-glucose that, as previously mentioned, caused a 90% reduction in the ATP pool. We found that ATP depletion markedly sensitizes cells to peroxynitrite because a lethal response was observed after exposure to as little as 10 to 100 e peroxynitrite (Fig. 2D). It is interesting that exogenous arachidonic acid did not prevent toxicity in ATP-predepleted cells, indicating that the ability of the lipid messenger to promote survival is restricted to conditions associated with ATP availability.
The final set of experiments was performed with the aim of reproducing the most relevant findings in THP-1 cells, an additional promonocytic cell line. As illustrated in Fig. 3, the arachidonate-dependent cytoprotective signaling is not restricted to U937 cells (A), the ATP pool is well-preserved also in THP-1 cells (B), and predepletion of ATP in the latter cell type, as previously observed in U937 cells, abolishes the cytoprotective effects of arachidonic acid.
Discussion
The present study used two different toxicity paradigms (i.e., a high dose of peroxynitrite or an otherwise nontoxic concentration of peroxynitrite associated with a phospholipase A2 inhibitor) to show that the rapid necrotic response that in both conditions takes place as a consequence of the inhibition of a survival signaling is not dependent on ATP depletion.
We first provided evidence indicating that MPT-dependent necrosis is not a direct consequence of the overall cellular damage. Indeed, the two toxicity paradigms were associated with the accumulation of different levels of DNA single-strand breakage and H2O2 as well as with different depletion of GSH (Fig. 1). Furthermore, the addition of exogenous arachidonic acid or cyclosporin A was on the one hand invariably followed by prevention of toxicity, whereas on the other hand, survival was not always paralleled by a reduction of the induced damage. By comparing the results obtained using the low dose of peroxynitrite alone or associated with the phospholipase A2 inhibitor, the same conclusion can be reached. The nontoxic and toxic treatments produced identical levels of DNA strand scission and failed to promote detectable delayed formation of H2O2. The decrease in GSH, however, was greater in the second condition. The observation that arachidonic acid or cyclosporin A abolished the enhancing effects mediated by the phospholipase A2 inhibitor on GSH depletion, with no effect on GSH decline mediated by the nontoxic dose of peroxynitrite, strongly suggests a role for events downstream to MPT in causing a further decrease in the GSH pool. The high concentration of peroxynitrite led to total depletion of cellular GSH, insensitive to arachidonic acid or cyclosporin A. This indicates that oxidation of cellular thiols is directly mediated by peroxynitrite.
Consistent with the notion that toxicity is not a direct consequence of the overall cellular damage were also the results reported in a recent study from our laboratory (Tommasini et al., 2004c). We showed that the formation of H2O2, critical for cell-death induction by high concentrations of peroxynitrite (Tommasini et al., 2002a), was in fact a dispensable event under conditions in which cPLA2 was pharmacologically inhibited or genetically depleted. The effect of H2O2 was indeed upstream to arachidonate and was restricted to the inhibition of arachidonic acid release. Supplementation of exogenous arachidonate was therefore able to promote full survival even under conditions associated with extensive H2O2-dependent damage (e.g., in the presence of bona fide complex III inhibitors, which remarkably enhance formation of H2O2, or upon supplementation of high levels of exogenous H2O2). These observations therefore readily explain our findings that catalase or N-acetyl-L-cysteine prevented toxicity mediated by 1.2 mM peroxynitrite but not that mediated by 100 e peroxynitrite in phospholipase A2 inhibitor-supplemented cells (Table 1).
The fact that these two conditions produced identical levels of toxicity indicates that the specific lesion(s) committing cells to MPT is already induced by the low dose of peroxynitrite. An effect of peroxynitrite on the adenine nucleotide translocator was recently identified in Kroemer's laboratory (Vieira et al., 2001), and this event, in conjunction with alterations in the redox state (Costantini et al., 1996), may well be involved in the commitment to MPT, critically regulated by the arachidonate-dependent cytoprotective signaling. Indeed, using the calcein assay, we produced direct evidence of MPT induction prevented by cyclosporin A as well as by arachidonate (Tommasini et al., 2004a).
The above findings indicate that necrosis takes place as a consequence of a regulated process, an inference supported by the observation that the ATP pool was well preserved during drug exposure (Fig. 2, AeCC). Indeed, a 15- to 30-min treatment with 100 e peroxynitrite/AACOCF3 alone caused a very small reduction of cellular ATP. It is important to note that at 15 min, a significant proportion of the cells was committed to death, whereas at 30 min, the cell population was mainly represented by cells surviving treatment with peroxynitrite. The observation that the small decrease in ATP was prevented by arachidonic acid suggests that this response does not precede, but rather follows, MPT. MPT inhibition by cyclosporin A also consistently prevented the ATP decline. These results clearly demonstrate that cellular ATP is virtually identical in cells committed to death in survivors or in untreated cells. Exposure to 1.2 mM peroxynitrite caused a greater decrease in ATP content, and this response was insensitive to either arachidonic acid or cyclosporin A. This indicates that the severe nitrosative stress imposed by this treatment causes ATP depletion by impairing the machinery of ATP synthesis and/or via accelerated ATP hydrolysis. As a consequence, inhibition of MTP prevents toxicity but not the effects of peroxynitrite on cellular ATP. Hence, cells committed to death and surviving cells are equally affected in their machinery to produce/hydrolyze ATP.
We confirmed the notion that U937 cells, like most tumor cells, mainly produce their ATP by glycolysis. Indeed, 2-deoxy-D-glucose virtually depleted the ATP pool that was only marginally affected by the respiratory chain inhibitor rote-none. Thus, the significant decrease in ATP content mediated by the high dose of peroxynitrite is more likely to depend on inhibition of glycolysis than of electron transport. This conclusion is supported by the observed reduction in the activity of phosphofructokinase, a rate-limiting enzyme of glycolysis (Stryer, 1995). In addition, H2O2 may also contribute to the inhibition of glycolysis (Hyslop et al., 1988). Finally, as mentioned above, accelerated ATP hydrolysis is a very likely cause of ATP depletion because the cells are heavily injured by peroxynitrite. Although the ATP synthetic machinery seems to be partially compromised, this event is not expected to lead to toxicity, because identical ATP levels were found in cells rescued with arachidonic acid or cyclosporin A. Furthermore, we showed previously that prevention of MPT does not simply delay toxicity but rather promotes full survival and allows cells to proliferate as untreated cells (Sestili et al., 2001). Hence, the inhibition of ATP synthesis must be reversible.
These results are consistent with the notion that ATP depletion is not the cause of necrotic cell death. The execution of the arachidonate-dependent signaling, however, requires ATP. Important indications of this were provided by the observations that otherwise nontoxic levels of peroxynitrite kill cells previously depleted in ATP and that arachidonic acid fails to mediate cytoprotection under these conditions (Fig. 2D).
The arachidonate-dependent cytoprotective signaling is not restricted to U937 cells but is rather a general feature of cells belonging to the monocyte/macrophage lineage (Tommasini et al., 2004b). We confirmed the results obtained in U937 cells using an additional promonocytic cell line (i.e., THP-1 cells) (Fig. 3). Hence, these cells may cope with peroxynitrite using both endogenous and exogenous arachidonic acid to trigger an energy-dependent survival signaling pathway.
In conclusion, the results presented in this study further our knowledge of the mechanism whereby monocytic cells cope with peroxynitrite and provide important information on the role of ATP in primary necrosis in a selected toxicity paradigm. Previous studies have described necrosis as a sort of obligatory and passive response in which, regardless of the necrotic insult, the ATP levels rapidly decrease to 0 as a consequence of defective ATP synthesis associated with elevated ATP consumption (Proskuyakov et al., 2003). Cells unable to sustain the activity of ion pumps and other energy-demanding functions would therefore rapidly lose their membrane integrity and lyse. This is obviously not the case of U937 (or THP-1) cells challenged with peroxynitrite, which preserve their ATP levels and eventually die by necrosis via a highly regulated mechanism.
Thus, this lethal response may not just represent a cell failure to perform apoptosis. Monocytes sustain the inflammatory response by releasing an array of toxic molecules, including peroxynitrite, and survive in an environment in which other cells die by using stimuli that are detrimental for other cell types. The final physiological response of monocytes would then be a necrotic death as an extreme strategy to further support the inflammatory response. Thus, necrosis not only takes place via a highly regulated mechanism but might also represent a well-defined physiological event with important pathological implications (e.g., conditions in which inflammation is of pathological significance).
doi:10.1124/mol.104.009498.
References
Barros LF, Hermosilla J, and Castro J (2001) Necrotic volume increase and the early physiology of necrosis. Comp Biochem Physiol A Mol Integr Physiol 130: 401eC409.
Beutler E (1984) Red Cell Metabolism: A Manual of Biochemical Methods. Gree and Stratton, New York.
Bradford M (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248eC254.
Brand KA and Hermfisse U (1997) Aerobic glycolysis by proliferating cells: a protective strategy against reactive oxygen species. FASEB J 11: 388eC395.
Costantini P, Chernyak BV, Petronilli V, and Bernardi P (1996) Modulation of the mitochondrial permeability transition pore by pyridine nucleotides and dithiol oxidation at two separate sites. J Biol Chem 271: 6746eC6751.
Ha HC and Snyder SH (1999) Poly(ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion. Proc Natl Acad Sci USA 96: 13978eC13982.
Hirsch T, Marchetti P, Susin SA, Dallaporta B, Zamzami N, Marzo I, Geuskens M, and Kroemer G (1997) The apoptosis-necrosis paradox. Apoptogenic proteases activated after mitochondrial permeability transition determine the mode of cell death. Oncogene 15: 1573eC1582.
Hyslop PA, Hinshaw DB, Halsey WA, Schraufstatter IU, Sauerheber RD, Spragg RG, Jackson JH, and Cochrane GC (1988) Mechanisms of oxidant-mediated cell injury. The glycolytic and mitochondrial pathways of ADP phosphorylation are major intracellular targets inactivated by hydrogen peroxide. J Biol Chem 263: 1665eC1675.
Kroemer G, Dallaporta B, and Resche-Rigon M (1998) The mitochondrial death/life regulator in necrosis and apoptosis. Annu Rev Physiol 60: 619eC642.
Leist M and Nicotera P (1997) Breakthroughs and views. The shape of cell death. Biochim Biophys Res Commun 236: 1eC9.
Leist M, Single B, Castoldi AF, Kuhnle S, and Nicotera P (1997) Intracellular adenosine triphosphate (ATP) concentration: A switch in the decision between apoptosis and necrosis. J Exp Med 185: 1481eC1486.
Lelli JL Jr, Becks LL, Dabrowska MI, and Hinshaw DB (1998) ATP converts necrosis to apoptosis in oxidant-injured endothelial cells. Free Radic Biol Med 25: 694eC702.
Nicotera P and Melino G (2004) Regulation of the apoptosis-necrosis switch. Oncogene 23: 2757eC2765.
Proskuyakov SY, Konoplyannikov AG, and Gabai VL (2003) Necrosis: a specific form of programmed cell death Exp Cell Res 283: 1eC16.
Radi R, Beckman JS, Bush KM, and Freeman BA (1991) Peroxynitrite-induced membrane lipid peroxidation: the cytotoxic potential of superoxide and nitric oxide. Arch Biochem Biophys 288: 481eC487.
Sestili P and Cantoni O (1999) Osmotically driven radial diffusion of single-stranded DNA fragments on an agarose bed as a convenient measure of DNA strand scission. Free Radic Biol Med 26: 1019eC1026.
Sestili P, Tommasini I, and Cantoni O (2001) Peroxynitrite promotes mitochondrial permeability transition-dependent rapid U937 cell necrosis: survivors proliferate with kinetics superimposable on those of untreated cells. Free Radic Res 34: 513eC527.
Single B, Leist M, and Nicotera P (2001) Differential effects of Bcl-2 on cell death triggered under ATP-depleting conditions. Exp Cell Res 262: 8eC16.
Souza JM and Radi R (1998) Glyceraldehyde-3-phosphate dehydrogenase inactivation by peroxynitrite. Arch Biochem Biophys 360: 187eC194.
Stocchi V, Cucchiarini L, Magnani M, Chiarantini L, Palma P, and Crescentini G (1985) Simultaneous extraction and reverse-phase high-performance liquid chromatographic determination of adenine and pyridine nucleotides in human red blood cells. Anal Biochem 146: 118eC124.
Stryer L (1995) Biochemistry. Freeman and Co., New York.
Tommasini I, Guidarelli A, and Cantoni O (2004a) Non-toxic concentrations of peroxynitrite commit U937 cells to mitochondrial permeability transition-dependent necrosis that is however prevented by endogenous arachidonic acid. Biochem Pharmacol 67: 1077eC1087.
Tommasini I, Guidarelli A, Cerioni L, and Cantoni O (2004b) The arachidonate-dependent cytoprotective signaling evoked by peroxynitrite is a general response of the monocyte-macrophage lineage. Biochem Biophys Res Commun 31: 1191eC1195.
Tommasini I, Sestili P, and Cantoni O (2002a) Delayed formation of hydrogen peroxide mediates the lethal response evoked by peroxynitrite in U937 cells. Mol Pharmacol 6: 870eC878.
Tommasini I, Sestili P, Guidarelli A, and Cantoni O (2002b) Peroxynitrite stimulates the activity of cytosolic phospholipase A2 in U937 cells: the extent of arachidonic acid formation regulates the balance between cell survival or death. Cell Death Differ 9: 1368eC1376.
Tommasini I, Sestili P, Guidarelli A, and Cantoni O (2004c) Hydrogen peroxide generated at the level of mitochondria in response to peroxynitrite promotes U937 cell death via inhibition of the cytoprotective signalling mediated by cytosolic phospholipase A2. Cell Death Differ 11: 974eC984.
Vieira HL, Belzacq AS, Haouzi D, Bernassola F, Cohen I, Jacotot E, Ferri KF, El Hamel C, Bartle LM, Melino G, et al. (2001) The adenine nucleotide translocator: a target of nitric oxide, peroxynitrite and 4-hydroxynonenal. Oncogene 20: 4305eC4316.