Life Sciences, Vol. 49, pp. 1731-1737 Printed in the U.S.A.
TUMOR NECROSIS FACTOR INDUCES ACTIVATION OF MITOCHONDRIAL SUCCINATE DEHYDROGENASE
Christiane Levratt, James W. Larrick*, Susan C. Wright* *Palo Alto Institute for Molecular Medicine 2462 Wyandotte St. Mountain View, CA 94043 t Laboratoire de Biochimie, U.189, Faculte de Medecine, Lyon-Sud, France. (Received in final form September 26, 1991)
Summary We have studied TNF-induced changes in mitochondrial enzymes. One enzyme, succinate dehydrogenase (SDH), is specifically activated in TNF sensitive cells including U937 (human monocytic), WEHI-164 (murine fibrosarcoma), and ME-180 (human cervical carcinoma). SDH is activated by TNF concentrations which also cause cytolysis, however the enzyme activity is elevated several hours before maximum cytotoxicity is observed. In contrast, TNF does not activate SDH in TNF resistant variants derived from U937 and WEHI-164. The mechanism of TNF-mediated cytotoxicity is not well understood (1), and in fact may involve multiple intracellular pathways depending on the target cell studied (for review see 2). One approach to analyze these pathways has been to examine alterations in intracellular enzyme activities in response to TNF. Numerous studies have provided evidence for activation and/or induction of enzymes such as phospholipase A2 (3,4), manganese superoxide dismutatse (MnSOD) (5,6), and 2', 5' oligoadenylate synthetase (7). While some of these enzymes, such as PLA2, may be directly involved in the TNF lytic pathway, others (e.g. MnSOD), may be induced in the cell's attempt to repair TNFmediated injury. Recent studies suggesting that TNF inhibits mitochondrial respiration (8) prompted us to examine TNF-induced alterations in mitochondrial enzymes. We report that TNF rapidly activates SDH in cell lines sensitive to TNF-mediated cytolysis but not in TNF-resistant variants selected from the sensitive cell lines. Materials and Methods Bio(:hemical Reaaents were purchased from Sigma Chemical Co. [St. Louis, MO]. Human TNF~ [Sp. act. >107 U/mg] was from Amgen Inc. [Thousand Oak, CA]. Address correspondence to : James W. Larrick MD PhD, Palo Alto Institute for Molecular Medicine, 2462 Wyandotte Street, Mountain View, CA 94043.
0024-3205/91 $3.00 + .00 Copyright © 1991 Pergamon Press plc
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Isolation of TNF-resistant U937 and WEHI-164 cell lines is previously described (9). TNF resistant variants were selected from clones by increasing the concentration of TNF from 0.5 ng/ml to 10.0 ng/ml over a period of several months. Cytotoxicity assavs were performed as previously described (9). Briefly, cells (106/ml)in RPMI-1640 with 2.5% fetal bovine serum were exposed to TNF at 37°C for indicated times. Viable cells were assessed microscopically by trypan blue exclusion (0.4% wt/vol). Mitochondrial preparation. Dead cells were removed from U937 and U9TR cells by Ficoll Hypaque density gradient centrifugation in Ca + +/Mg + + free HBSS. ME-180 and WEHI164 monolayers were washed twice with PBS 2mM EDTA and harvested in Ca + + / M g + + free HBSS by scraping. Cells (3 X 106 cells/ml) were resuspended in 0.25 M sucrose, 10 mM Tris-HCI buffer pH 7.4, 1 mM EDTA and homogenized with a Dounce homogenizer (7ml size). The homogenate was spun 15 min. at 2000xg and supernatant stored on ice. The pellet was rehomogenized [same buffer] and spun 15 min at 2000xg. The supernatants were combined and spun 15 min. at 12000xg. The supernatant was discarded and the pellet (mitochondria) was washed once. Mitochondria were suspended in 0.25M sucrose 10mM Tris-HCI pH 7.4, and the protein content measured(10). Characterization of mitochondria isolated from U937 cells. Marker enzyme activities of mitochondrial compartments were determined in the cell homogenate, the isolated mitochondrial fraction and the microsomal fraction. The ratios of marker enzyme activities measured in mitochondria and in homogenate showed that the homogenization procedure lyses most of the cells. Marker enzyme activities detected in the microsome fraction were very low, inner membrane (cytochrome c oxidase and succinate dehydrogenase) and matrix (malate dehydrogenase) enzymes were not detectable. These results confirm that inner and outer mitochondrial membranes are not significantly damaged during the isolation of mitochondria. Enzyme assays. Rotenone-insensitive NADH cytochrome c reductase [EC 18.104.22.168] (11), hexokinase [EC 22.214.171.124 .] (12), adenylate kinase [EC 126.96.36.199.] (13), cytochrome c oxidase [EC 188.8.131.52.] (14), succinate dehydrogenase [EC 184.108.40.206.] (15) and malate dehydrogenase [EC 220.127.116.11.] (16) were assayed according to the methods described in the quoted articles. Activity_ of $DH [succinate:(2,6-dichloroindophenol) oxidoreductase] was determined by the method of Ackrell et al. (15). 100ug of mitochondria were added to 100mM sodium phosphate buffer pH 7.6 followed by the successive additions of 5mM EDTA, lmM KCN (freshly neutralized), 0.1mM phenazine metosulphate (PMS), 0.08mM cytochrome c and 25mM succinate. The absorbance change was measured at 550nm at RT. The reduction of cytochrome c in the absence of succinate was negligible and PMS was not reduced in both untreated and TNF-treated cells in the absence of succinate. Addition of phospholipase A2 did not improve the measurement [because mitochondria were incubated in the hypotonic medium]. Results Activities of the following enzymes: rotenone-insensitive NADH cytochrome c reductase and hexokinase (outer surface membrane), adenylate kinase (intermembrane space), cytochrome c oxidase and succinate dehydrogenase (inner membrane), and malate dehydrogenase (matrix) were measured in mitochondria isolated from TNF-treated U937 cells. Since it is not possible to obtain high quality mitochondrial preparations from cell populations containing a significant percentage of dead cells, the cells were treated with TNF for only 6 hr. At this time point, there is minimal cell death as shown by the cell
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viabilities reported in Table I. Any dead cells present were eliminated by Ficoll hypaque centrifugation prior to extracting the mitochondria. This rules out the possibility that any alterations in enzyme activity may be the result of cell death as opposed to an active cellular response to TNF. Of the enzymes tested, the only significant change was a 56% increase in SDH activity in mitochondria isolated from TNF-treated U937 cells (Table I). TNF also activated SDH in two other TNF-sensitive cell lines, ME-180 and WEHI-164 fibroblasts. These results suggest that TNF induced SDH activation is not an effect unique to U937 cells since it also occurs in other TNF-sensitive cell lines. TABLE 1 Effect of TNF on the succinate dehydrogenase activity in mitochondria isolated from several TNF-sensitive and TNF-resistant cell lines.
Succinate dehydrogenase activitya Cell lines
TNF Sensitive U937 ME-180 WEHI-164
0.160___0.010 0.195_+0.015 0.440_+0.010
0.250_0.025 0.370_+0.035 0.580-0.030
(+56) (+84) (+32)
89 73 86
TNF Resistant U9-TR WEHI-TR
aCells (10e ml) were TNF treated or untreated (control) with 10ng/ml of TNF for 6 hours. SDH activity is expressed as the absorbance change at 550nm per mg protein per min. Each value is the average _+ standard deviation of 4 experiments. Viability determined by trypan blue exclusion. The time course of TNF induced SDH activation was measured in mitochondria isolated from TNF-treated U937 cells. SDH activation increased with the length of TNF treatment (Fig.lA) reaching a maximum at 6 hr (55%) and declining at 12 hr. We compared the kinetics of the SDH effect with the kinetics of TNF-mediated cytolysis of U937 cultured under identical conditions. There is very little cell death until at least 12 hr of incubation, with maximal cytotoxicity occurring at 24 hr. Therefore, the peak of TNF-induced SDH activation occurs more than 6 hrs before maximal cell death occurs.
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Time Course of TNF-Induced Succinate Dehydrogenase (SDH) Activation in U937 Cells
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Dose Response of SDH Activation
'-r a 20 -
TNF Concentration (ng/ml)
FIG 1. Panel A:
Kinetics of TNF induced SDH activation.
U937 cells (106 cells/ml) were incubated with 10ng/ml of TNF and at different time points cells were assessed for SDH activity. The percent values for SDH activation are expressed as follows : [SDH specific activity assay/SDH specific activity control- 1] X 100 Each value is the average of 3 experiments. Panel B:
TNF-dose response for induction of SDH activation.
U937 cells (106 cells/ml) were incubated with different concentrations of TNF and SDH activity was measured at 6 hours (A) whereas cytotoxicity was assessed at 24 hours. The percent TNF-mediated SDH activation and cytotoxicity was determined as described in panel A. We then examined the TNF dose response on SDH activation using U937. Cells were incubated with various concentrations of TNF from I ng/ml/106 cells to 50ng/ml/106 cells. TNF induced SDH activation was directly proportional to the amount of TNF added to the cells (Fig.1B). U937 cells were cultured under identical conditions and their viability was determined by trypan blue exclusion after 24h. The results indicate that the dose response for TNF-induced cytotoxicty is similar to TNF activation of SDH in U937 cells (data not shown). The concentration of TNF which induced SDH activation by 50% after 6 hours of treatment, 10ng/ml/106 cells was adopted in the following experiments. These results show that the activation of SDH in mitochondria from sensitive cells is TNFdependent in a time and dose-response manner.
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We next investigated if TNF activated SDH in TNF resistant cell lines UgTR and WEHI-TR. These cells (selected from the parental cell lines U937 and WEHI-164) are >1000 fold more resistant to lysis by TNF in a 20-24h assay (9), although they still express a normal number of TNF receptors (unpublished observations). In the U937 variant, U9-TR, TNF induced a slight inhibition of SDH by 15% and no cells were killed (Table I). The activities of the other marker enzymes were not altered (data not shown). SDH activity did not increase in the TNF-resistant cells even after 24 hrs of exposure to TNF. Under these conditions, TNF did not change the activity of SDH in WEHI-TR cells. These results indicate that TNF-induced SDH activation correlates with the sensitivity of both of these tumor cell lines to TNF-mediated cytolysis. Discussion This study shows that TNF induces the activation of SDH, a mitochondrial enzyme which belongs to complex II of the respiratory chain. This response was observed in three distinct TNF-sensitive cell lines (U937, WEHI-164 and ME-180) but not in TNF-resistant variants (U9-TR and WEHI-TR). SDH activation is directly proportional to the concentration of TNF and to the length of exposure to TNF with maximal activation observed several hours prior to cytolysis. Similar findings have been reported in a non-cytotoxic model system examining the effects of TNF on preadipocytes. It was shown that TNF rapidly stimulates respiration as revealed by an increase in 02 consumption (17). We have also obtained preliminary data that TNF increases 02 consumption in U937 but not in the TNF resistant variant. Although both of these studies show TNF stimulates the respiratory chain, this has not been a universal finding for all cell lines. Our results differ from another study that has examined TNF-induced alterations on tumor cell respiration in vitro. Using digitonin permeabilization to assay mitochondrial electron transfer, Lancaster et al. (8) found that TNF treatment for 7-9 hours completely inhibited complex II (succinoxidase) and complex IV (cytochrome oxidase) of the respiratory chain in the TNF-sensitive LM murine fibroblasts. Based on these studies, it was postulated that bioenergetic dysfunction may be involved in the TNF lytic mechanism. It is likely that TNF activates different lytic pathways in different cell types. Thus, it has been shown that for the LM cell, TNF induces a necrotic form of cell death (18), similar to that induced by antibody and complement. In contrast, we have observed that both U937 and WEHI-164 undergo apoptosis in response to TNF as revealed by the characteristic morphological changes and DNA fragmentation which occur well before cell death. SDH activation may be involved in inducing apoptosis in U937 whereas inhibition of this enzyme may occur when TNF activates the necrotic pathway in LM cells. Several other observations are consistent with the hypothesis that activation of SDH is an essential step in the TNF lytic pathway in U937 cells. Both activation of SDH and tumor cell lysis exhibit a similar TNF dose response curve. Furthermore, SDH activity peaks at 6 hours after exposure to TNF which is hours before there is a maximal cytotoxic effect. Thus, elevation of SDH may be a transient intermediate step in the TNF lytic pathway. The lack of SDH activation in the TNF resistant variants provides additional support for a role of SDH in TNF cytolysis. Our unpublished studies have shown that the U9-TR cell line expresses normal numbers of functional TNF receptors that can mediate signal transduction since TNF can activate the nuclear regulatory molecule, NFkB, in these TNF resistant cells. Therefore, if our hypothesis is correct, the U9-TR variant may be TNF resistant due to a block in the TNF pathway that occurs following TNF-receptor
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interaction and prior to activation of SDH. However, our data still do not rule out the possibility that activation of SDH in normal U937 but not the variant is a coincidental phenomenon that is not related to the TNF lytic mechanism. Attempts to answer this question by adding inhibitors of SDH activation to TNF cytotoxicity assays yielded inconclusive results due to nonspecific toxic effects of the inhibitors. If SDH is involved in the TNF lytic mechanism, one could speculate that activation of this enzyme may contribute to cell injury through the generation of oxygen radicals. Previous studies have provided evidence for the involvement of oxygen radicals in TNF-mediated cytotoxicity (19,20), although their source was not identified. SDH is an electron donor for coenzyme Q which in the half-reduced form (ubisemiquinone) can react with molecular oxygen to generate oxygen radicals  (21,22). TNF activation of this reaction may be the source of membrane and DNA damaging free radicals. In summary, these studies demonstrate that activation of mitochondrial SDH is an early effect of TNF. It is possible that activation of SDH may contribute to tumor cell injury by production of free radicals. Antagonists of this process may provide novel strategies to prevent TNF-mediated cell injury in radiation damage, cachexia, sepsis, AIDS, malaria, etc. Potentiation ofthe process may facilitate the anti-proliferative/cytotoxic effects of TNF and related cytokines. Therefore, the present findings may provide the basis for a new approach in developing therapies to manipulate the cell's response to TNF. Acknowledgements This work was supported by CA 47669 awarded by the National Cancer Institute. References .
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