apoptosis induced byinhibitors oftheplasma membrane nadh-oxidase...

Vol. 7, 1315-1325, October 1996 Cell Growth & Differentiation 1315

Apoptosis Induced by Inhibitors of the Plasma MembraneNADH-oxidase Involves BcI-2 and Calcineurin

Ernst J. Wolvetang,1 Jan A. Larm,2Penny Moutsoulas, and Alfons Lawen3

Department of Biochemistry and Molecular Biology [J. A. L, P. M.,A. L] and Centre for Molecular Biology and Medicine [E. J. W.], MonashUniversity, Wellington Road, Clayton, Melbourne, Victoria 3168,Australia

AbstractActivation of the plasma membrane NADH-oxidoreductase (PMOR) system by addition of growthfactors or extracellular electron acceptors stimulatescellular proliferation. We now show that the vanilloidscapsaicin, dihydrocapsaicin, and resiniferatoxin areinhibitors of the NADH-oxidase activity of the PMORsystem and that both these and two previouslyidentified PMOR inhibitors (chloroquine and retinoicacid) induce apoptosis in human B-cell and mousemyeloid cell lines. At the optimal concentration, PMORinhibitors can induce between 50 and 70% of apoptosisin mouse myeloid and human B-cell lines within 8-12h, provided these cell lines do not express Bcl-2. Theimmunosuppressants cyclosporin A and fujimycin(tacrolimus) inhibit PMOR inhibitor-induced apoptosis.By using combinations of these immunosuppressantsand excess amounts of their nonimmunosuppressiveanalogues, we demonstrate that in human B-cell linesthe BcI-2-sensitive apoptotic pathway triggered byPMOR inhibitors involves signaling through the proteinphosphatase calcineurin. We suggest that the PMORsystem is a redox sensor that can, depending on theambient redox environment and the availability ofgrowth factors, regulate plasma membrane calciumfluxes and signal for apoptosis through calcineurin.BcI-2, a protein that is thought to inhibit apoptosis byregulating reactive oxygen species and calcium fluxesin the cell, inhibits this apoptotic pathway.

IntroductionThe PMOR4 system is found in all eukaryotic cells analyzedthus far (1). It transfers electrons from cytoplasmic NADH via

Received 5/30/96; revised 7/12/96; accepted 7/31/96.The costs of publication of this article were defrayed in part by thepayment of page charges. This article must therefore be hereby markedadvertisement in accordance with 18 U.S.C. Section 1734 solely to mdi-cate this fact.1 Present address: Centre for Early Human Development, Institute ofReproduction and Development, Monash Medical Centre, Level 5, 246Clayton Road, Clayton, Melbourne, Victoria 3i68, Australia. Phone: 6i-3-9550-5480; Fax: 61 -3-9550-5568; E-mail: [email protected] Present address: Department of Pharmacology, Monash University,Wellington Road, Clayton, Melbourne, Victoria 3i 68, Australia.3 To whom requests for reprints should be addressed. Phone: 61 -3-9905-371 1 ; Fax: 61 -3-9905-4699; E-mail: [email protected] The abbreviations used are: PMOR, plasma membrane NADH-oxi-

Q-i 0 to extracellular electron acceptors (2-4). The PMORsystem displays two separate enzymic activities, NADH-fer-ricyanide reductase activity that can use extracellular fern-cyanide as a terminal electron acceptor (5, 6) and NADH-

oxidase activity for which oxygen is the terminal electronacceptor (7, 8). This relatively unknown multifunctional en-zyme has been suggested to contain flavin (9), Q-iO (iO),nonheme iron (i 1), and cytochrome b (1 2) and is importantfor cell proliferation. This is exemplified by several observa-tions: (a) the PMOR system is up-regulated by growth factorsand hormones (8, 13, 14); (b) the expression of Fos and Mycincreases after PMOR stimulation (1 5, 16), accompanied byan increased binding of GTP to p2i’� (i7 i 8); and (c) thePMOR system is associated with tyrosine kinases (i 9), pro-ton efflux (3), and calcium influx (20-22). The PMOR systemis up-regulated in cells of neoplastic liver nodules comparedwith normal liver (7) and is, due to its localization, easilyaccessible to metabolic inhibitors. Therefore, the PMOR sys-tem is an attractive target for the development of anticancerdrugs that would not be subject to multidrug resistance. ThePMOR system plays an important role in cellular redox ho-meostasis and appears in this aspect to function as a backupsystem to the mitochondrial respiratory chain. Cells that aredepleted of a functional mitochondrial respiratory chain (p�cells) through a 4-week incubation with ethidium bromidebecome totally dependent on the addition of pyruvate fortheir survival (23). Such p#{176}cells react by gradually up-regu-lating the PM NADH-ferricyanide reductase activity 5-fold(24, 25). This up-regulation allows the p#{176}cells to proliferatewithout pyruvate, provided that PMOR substrates, such asdichloroindophenol, ferricyanide, and Q-i 0, are added to themedium instead (24, 26). Insulin, ferric transferrmn, and Q-iOare known to directly stimulate the PM NADH-oxidoreduc-tase system. Indeed, HeLa cells can be grown without serumwhen insulin and Q-iO are added to the growth medium (6).This growth-stimulating effect of Q-iO and insulin is pre-vented by the presence of 200 �M capsaicin, suggesting thatcapsaicin can act as a Q-i 0 antagonist in the PMOR systemof intact cells (6). Capsaicin was subsequently reported to

inhibit not only the growth of HeLa cells but also HL-60,ovarian carcinoma, and mammary adenocarcinoma cells(27). Therefore, we decided to investigate in more detail theeffects of inhibition of the PM NADH-oxidase activity bycapsaicin and two other vanilloids, dihydrocapsaicin andresiniferatoxin, on human and mouse blood cell lines. Reti-noic acid (8, 28) and chloroquine (29) were previously shownto be inhibitors of PM NADH-oxidase activity. All five com-

doreductase; Cy, cyclosporin; DAPI, 4,6-diamidino-2-phenylindole;FDCP, factor-dependent cell Paterson’s; FK-506, fujimycin (tacrolimus);PM, plasma membrane; PPlase, peptidyl-prolyl-cis/trans-isomerase;Q-iO, ccenzyme Q-1O.

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Fig. 1. Effect of capsaicin, dihydrocapsaicmn, and resiniferatoxin on theNADH-oxidase activity from purified rat liver PMs. PMs were isolated fromfresh rat liver as described (8). The purity of the PM preparations (morethan 90% pure on a protein basis) was determined by marker enzymeanalysis and electron microscopy. The measurement of NADH-oxidaseactivity was performed as described (8) in the presence of the indicatedconcentrations of capsaicin (R), dihydrocapsaicin (#{149}),and resiniferatoxin(A). The results are the means of three experiments carried out with threeseparate PM preparations. Bars, SD.

Inhibitor [jiM]

1316 Apoptosis Induced by Plasma Membrane Oxidase Inhibitors

pounds were tested for their ability to trigger apoptosis inmouse and human blood cell lines. Apoptosis is a regulatedform of cellular death, distinct from necrosis, and is impor-tant in the selective removal of cells during development(30-32), in which case it is referred to as programmed celldeath. During apoptosis, cells shrink, and the nuclei con-dense and fragment (33-36). In the final stages of apoptosis,cells break up in small vesicles containing condensed nu-clear fragments and intact organelles. A deregulation or ab-errant induction of apoptosis can lead to cancer (37, 38),autoimmune diseases (39), or the destruction of the immunesystem after HIV infection (40, 41). Apoptosis can be inducedby a spectrum of stresses ranging from growth factor re-moval and glucocorticoids to calcium ionophores and UVirradiation. Irrespective of the insult, however, in most casesapoptosis is inhibited by Bcl-2. Bcl-2 is the prototype of agrowing family of proteins that homodimerize and het-erodimenze to inhibit apoptosis; its exact mode of action,

however, is still unclear. Bcl-2 was found to be associatedwith the outer mitochondrial endoplasmic reticulum and PMs(42, 43). There is evidence to suggest that Bcl-2 may beregulating reactive oxygen species fluxes (44, 45) and/orcalcium release (46) at these cellular locations. We now showthat inhibitors of the PMOR system induce apoptosis, whichcan be inhibited by Bcl-2 and involves calcineurin activation.

ResultsInhibitors of PM NADH-oxidase Induce Apoptosis in Bcl-2-negative Cells. Capsaicin, which is a 0-i 0 analogue anda vanilloid, was previously reported to act as an inhibitor ofthe PM NADH-oxidase (6, 14, 42). We have now analyzed indetail the effect of three vanilloids, capsaicin, dihydrocapsa-icin, and resiniferatoxin, on the NADH-oxidase activity ofhighly (90%) purified PMs from rat liver. All three com-pounds, but in particular resiniferatoxin, were found to beeffective inhibitors of the NADH-oxidase function of thePMOR system (Fig. i), whereas they do not inhibit its reduc-tase activity (47). These three vanilloids and two PM NADH-oxidase inhibitors described earlier, chloroquine (28) andretinoic acid (8, 27), have been tested for their capacity toinduce apoptosis in vitro. On incubation of the human Daudiand BL-29 lymphoblastoid cell lines with these compounds,at concentrations that inhibit PM NADH-oxidase activity byat least 50%, approximately one-half of the treated cellsunderwent apoptosis within 8 h. Fig. 2 shows the typical

condensed and fragmented nuclear morphology of Daudicells undergoing apoptosis after an 8-h treatment with 200�M capsaicin (Fig. 2, 1B), 100 �M dihydrocapsaicin (Fig. 2,1C), iO �M resiniferatoxin (Fig. 2, 1D), 250 p.M chloroquine(Fig. 2, 1E), and 40 p.�i retinoic acid (Fig. 2, iF). Interestingly,at these concentrations, none of the NADH-oxidase inhibi-tors is able to induce apoptosis in Namalwa cells (Fig. 2,2B-2F). Because the proto-oncogene product BcI-2 isknown to prevent or strongly inhibit apoptosis in many ex-perimental systems studied thus far (36, 48, 49), we studiedBcI-2 expression in Daudi, BL-29, and Namalwa cells. West-em blot analysis shows that BL-29 and Daudi cells expressBcl-2 minimally if at all (Fig. 3, Lanes 1 and 3), in contrast toNamalwa cells (Fig. 3, Lane 2), suggesting that the induction

of apoptosis by PM NADH-oxidase inhibitors is inhibited byBcI-2 expression. To verify this, we examined a mouse cellmodel system consisting of FDCP-i cells, which do notexpress human Bcl-2 (Fig. 3, Lane 4), and FDBCL-2 cells,which only differ from FDCP-i cells in that they constitutivelyexpress human Bcl-2 (Fig. 3, Lane 5). When these cells were

cultured in the presence of the set of PMOR inhibitors, wefound that, as for the results obtained with the lymphoblas-toid cells, only FDCP-i cells (not expressing Bcl-2) wereinduced to undergo apoptosis (Fig. 2, 3B-3F). FDBCL-2 cellsshowed no signs of apoptosis after incubation with thePMOR inhibitors (Fig. 2, 4B-4F). The morphological data ofall human lymphoblastoid and mouse myeloid cell lines thatwere treated with the five PMOR inhibitors studied are sum-marized in Fig. 4. In every case, cell lines expressing Bcl-2were totally resistant to induction of apoptosis by PM NADH-oxidase inhibitors, whereas all Bcl-2-negative cell lines weresensitive. To demonstrate that the Bcl-2-expressing cell linestested were capable of undergoing apoptotic cell death, weincubated these cell lines with 2 p.g/ml rotenone, an inhibitorof mitochondrial complex I. As reported previously (50), ro-tenone induces apoptosis, irrespective of whether cells ex-press BcI-2. Indeed, all cell lines examined in this studyundergo apoptosis after treatment with rotenone (Figs. 2Gand 4).

In addition to the morphological assessment of inductionof apoptosis, we also analyzed the formation of DNA frag-ments, both by detection of the typical DNA-laddering pat-tern and by the use of a commercial DNA fragmentation kit.Cell lines that do not express BcI-2 (FDCP-i and Daudi) and

Cell line Bcl-2 Experimental conditions

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Cell Growth & Differentiation 1317

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Fig. 2. Effect of PMOR inhibitors on the nuclear morphology of blood cells differing in expression of Bcl-2. Human lymphoblastoid Daudi (Bcl-2 negative:row 1), Namalwa cells (Bcl-2 positive; row 2), and mouse myeloid cell lines FDCP-1 (BcI-2 negative; row 3) and FDBCL-2 (BcI-2 positive; row 4) wereincubated with 200 �M capsaicin (B), 1 00 jiM dihydrocapsaicin (C), 1 0 �M resiniferatoxin (D), 250 MM chloroquine (F), 40 �tM retinoic acid (F) or 2 �tg/mlrotenone (G). Control incubations (A) contained only ethanol or Me2SO. Cells were cytospun onto a glass slide and fixed, and nuclear DNA was stained withDAPI and subsequently analyzed under a fluorescence microscope.

26 kDa .4

Fig. 3. Western blot analysis of BcI-2 expression in various cultured celllines. Lymphoblastoid cells (1 o� cells) were harvested and lysed, and thelysate supernatant was boiled in sample buffer as described in ‘Materialsand Methods.” One hundred .tg protein were loaded in each well of a 15%SDS-polyacrylamide gel, and the proteins were separated and electro-transferred to a polyvinylidene difluoride membrane, which was subse-quently incubated with a monoclonal antibody against BcI-2. Cross-re-acting antibodies were visualized with a sheep antimouse alkalinephosphatase conjugate. Lanes 1-5 contain cell extracts from BL-29,Namalwa, Daudi, FDCP-i , and FDBCL-2 cells, respectively.

that showed morphological characteristics of apoptosis after

incubation with our set of PM NADH-oxidase inhibitors also

displayed an increased amount of fragmented DNA (Fig. 5A)

and the characteristic 200-bp laddering pattern in agarose

gel electrophoresis (Fig. SB). In both assays, none of these

inhibitors increased DNA fragmentation in cells that express

Bcl-2 (FDBCL-2 and Namalwa).

Induction of apoptosis is dependent on the concentration

of PM NADH-oxidase inhibitor used, as exemplified for the

induction of apoptosis by the three vanilloids capsaicin (Fig.

6A), dihydrocapsaicin (Fig. 6B), and resiniferatoxin (Fig. 6C)

in Daudi cells. The concentration dependence of apoptosisinduced by PM NADH-oxidase inhibitors capsaicin and dihy-

drocapsaicin closely follows the inhibition ofthe NADH-oxidase

activity from isolated rat liver PMs by these vanilloid corn-

pounds (Fig. 1). At the concentrations tested none of the

PMOR-inhibitors induced apoptosis in Narnalwa cells that ex-

press Bcl-2 (Fig. 6, A-C). The same holds true for the vehicles

in which the inhibitors were added, i.e. , 0.2% Me2SO in the

case of resiniferatoxin and 0.2% ethanol for the other inhibitors.

In all cell lines tested, apoptosis induced by capsaicin

proceeded much faster than apoptosis induced by rotenone,

as exemplified in Fig. 7 for the FDCP-1 cells. Although this

rapid induction of apoptosis in FDCP-1 cells by vanilloids

such as capsaicin was totally abolished in the Bcl-2-express-

ing FDBCL-2 cells, the kinetics of apoptosis induced by

rnitochondrial respiratory chain inhibitors such as rotenone

remained unaffected (Fig. 7). Identical results were found

previously using the human lymphoblastoid cell line BM

13674, which does not express Bcl-2, and BMX 13674 cells,

a subclone of this cell line that expresses Bcl-2 (25).

Specificity of PM NADH-oxidase Inhibitors. Because

the PM NADH-oxidase has not yet been cloned, purified to

homogeneity, or reconstituted in a cell-free system, specific

inhibitors have not yet been identified. Previously, chloro-

quine and retinoic acid, both compounds with multiple cel-

lular targets, were found to inhibit NADH-oxidase activity in

isolated PMs from rat liver and to inhibit growth of cultured

human cell lines. Using a similar approach, we have now

identified capsaicin, dihydrocapsaicin, and resiniferatoxin as

inhibitors of the PM NADH-oxidase. Similarly to chloroquine

or retinoic acid, however, vanilloids also cannot be consid-

ered specific inhibitors of the PM NADH-oxidase. Vanilloids

have been reported to destroy a specific subset of neurons

by binding to a specific vanilloid receptor (51), to inhibit

complex I activity in isolated mitochondria (52-55), to inhibit

retrograde vesicle transport in neurons (56), and to change

the fluidity properties of membranes (57). To address these

issues, we performed the following experiments. First, we

investigated the possible involvement of a vanilloid receptor

100

Daudi BL-29 Namalwa FDCP-l FDBCL-2


5 E. J. Wolvetang, A. Ngo, and M. Degli Esposti, unpublished results.

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Fig. 4. Effect of PMOR inhibitors on the amount ofmorphological apoptosis in cell lines that differ in ex-pression of Bcl-2. Bcl-2-positive (Namalwa and FD-BCL-2) and BcI-2-negative (Daudi, BL-29, andFDCP-i)cells were incubated with 200 � capsaicin or100 � dihydrocapsaicin, 40 �M retinoic acid, 250 j�i

chloroquine, 1 0 �M resiniferatoxin, or 2 �g/ml rotenonefor 8 h. The percentage of morphologically detectableapoptosis was determined from the number of cellsdisplaying the typical condensed nuclear appearanceof apoptotic cells after staining with the DNA-stainingdye DAPI. The results are the means of at least threeseparate experiments. Bars, SD.

in vanilloid-induced apoptosis. In cultured neurons, capsaz-

epine can act as an antagonist for the binding of vanilloids totheir receptor (58). In Table 1 we show that preincubation ofDaudi cells with increasing capsazepine concentrations upto 100 p.M did not inhibit apoptosis induced by the subse-quent addition of capsaicin, resiniferatoxin, or retinoic acid.These results suggest that a vanilloid receptor is not involvedin vanilloid-induced apoptosis in cultured human blood celllines. One striking feature of the PMOR system from rat liverPMs is the ability of coenzyme 0-10 to reverse enzymeinhibition. This is thought to reflect the central role of acoenzyme Q-1O-binding site for PMOR activity. Table 2shows that preincubation of Daudi cells with 20 �M coen-zyme Q-1O can prevent about 50% of capsaicin- and res-iniferatoxin-induced apoptosis. These results suggest thatcapsaicin and resiniferatoxin induce apoptosis through theirinteraction with the coenzyme Q-1O-binding site of thePMOR system and not by some unspecific effect on mem-brane fluidity. Capsaicin can, however, also inhibit mitochon-drial complex I activity by acting as a coenzyme Q-l 0 an-tagonist (52-55). Indeed, like capsaicin, dihydrocapsaicinand resiniferatoxin are also able to inhibit mitochondrial com-plex I activity in submitochondrial particles from bovineheart.5 Although these compounds clearly induce apoptosisthrough a Bcl-2-sensitive pathway, in contrast to the BcI-2-insensitive one stimulated by rotenone, the question re-mained as to whether capsaicin and its analogues weretriggering apoptosis through inhibition of mitochondrial com-plex I activity, inhibition of PM NADH-oxidase activity, orboth. Therefore, we analyzed the effect of preincubation ofDaudi cells with ferric transferrmn, a compound that is knownto stimulate the PM NADH-oxidase activity but that does notaffect mitochondrial complex I activity in intact cells. Table 3shows that preincubation with increasing transferrmn concen-

trations prevented up to 50% of capsaicin- and resinifera-toxin-induced apoptosis in Daudi cells. Transferrin had noeffect on rotenone-induced apoptosis. Subsequently, theeffect of preincubation of Daudi cells with antisera raisedagainst partially purified preparations of the PMOR systemfrom rat liver was investigated (antisera kindly provided byD. J. Morr#{233},Purdue University, West Lafayette, IN). Table 4shows that a polyclonal antiserum raised against the rat liverPM NADH-oxidase prevented 50% of capsaicin-, resinifera-toxin-, and retinoic acid-induced apoptosis. Preincubationwith the same concentration of preimmune serum had noeffect on apoptosis induced by these compounds.

Apoptosis Triggered by PM NADH-oxidase InhibitorsInvolves Signaling through Calcineurin. In activated T

cells, apoptosis can be inhibited by the immunosuppres-sants CyA and FK-506 (59-62). The mechanism by whichthese immunosuppressants inhibit T-cell apoptosis appearsto involve the inhibition of the calcium- and calmodulin-dependent serine and threonine protein phosphatase cal-cineurin (63-65). CyA and FK-506 first bind to their cognatebinding proteins, cyclophilin and FK-506 binding protein,respectively. Subsequently, these immunosuppressant-im-munophilin complexes bind to calcineurin and inhibit its pro-tein phosphatase activity, thereby preventing the nuclearformation of several active transcription factors (66). Be-cause both the PMOR system and mitochondria are knownto be important for cellular calcium homeostasis, we ascer-tamed whether the calcium-dependent activation of cal-cineurin was involved in the apoptotic pathways triggered bythe PM NADH-oxidase inhibitor capsaicin and/or the mito-chondrial respiratory chain inhibitor rotenone. We found that0.3 �t�i CyA or FK-506 inhibited capsaicin-induced apoptosisin the human lymphoblastoid cell lines Daudi and BL-29 byabout 50% (Fig. 8). The newly described immunosuppres-sant SDZ 21 4-1 03 (67, 68) has an effect on capsaicin-in-duced apoptosis similar to that of CyA (Fig. 8). Rotenone-induced apoptosis, on the other hand, remained unaffected

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Fig. 5. The effect of the PMOR inhibitors on the amount (A) and sizedistribution (B) of DNA fragments in cell lines that differ in expression ofBcl-2. Bcl-2-positive (Namalwa and FDBCL-2) and BcI-2-negative (Daudiand FDCP-i) cell lines were incubated with 200 �.tM capsaicin or 10 �.LM

resiniferatoxin for 1 6 h. A, the amount of fragmented DNA produced wasdetermined with an ELISA kit from Boehnnger Mannheim. The results arethe means of two separate experiments. Bars, SD; AU. , arbitrary unit. B,the cells were harvested and the DNA was isolated as described in“Materials and Methods.” An aliquot of the DNA was separated on a 1.8%agarose gel and visualized as described (44). Cont, control; Cap, capsa-cm; RFT, resiniferatoxin.


6 E. J. Wolvetang, K. L. Johnson, and A. Lawen, unpublished results.

by the presence of CyA or FK-506 (Fig. 8). Because both

immunosuppressants inhibit the PPlase activity (69) of their

cognate binding proteins, as well as calcineurin, the effect of

the immunosuppressants on capsaicin-induced apoptosis

could also be explained by an overall lowered cellular PPlase

activity. To exclude this possibility, we used several analoguesof the immunosuppressants with different biological activities.

Two CyA analogues that still inhibit the PPlase activity of cy-

clophilin but form a Cy-cyclophilin complex that can no longer

bind to and inhibit calcineurin were tested, [MeVal4]CyA (70)

and [Melle4]CyA (71). Both compounds are no longer able to

inhibit capsaicin-induced apoptosis (Fig. 8). The FK-506 ana-

logue rapamycin has similar effects on its cognate immunophi-

lin and calcineunn as the above-mentioned CyA analogues.

Rapamycin differs from these compounds, however, in that it

retains its immunosuppressive activity through a different

mechanism of action not involving calcineunn (63, 72, 73).

Again, the inhibitory effect of FK-506 on capsaicin-induced

apoptosis was not observed when FK-506 in the medium was

replaced by rapamycin. Although the inhibition of the PPlase

activity is not sufficient for the inhibition of apoptosis, it is

needed, similar to the situation in immunosuppression. This is

shown by the use of analogues that no longer bind to their

respective immunophilins, CyH (74) and SDZ 282-320 [9,15-(5-methoxy-3-methyl-2,6-pyridylen; synthesized according to the

methods of Nussbaumer eta!. (75)]. Both compounds exhibited

no effect on the apoptotic response to inhibitors of the PM

oxidase (Fig. 8). Furthermore, excess amounts (1 0 �LM) of either

[MeVal4]CyA or [Melle4]CyA, but not CyH or rapamycin, cancompete for CyA-cyclophilin binding. In a similar way, rapamy-

cm, but not SDZ 282-320 or [MeIle4]CyA, can compete for

FK-506-FK-506-binding protein binding (Fig. 8). These results

clearly indicate the need for a calcineurin-binding (and, there-

fore, inhibiting) immunosuppressant-immunophilin complex for

inhibition of apoptosis that is capsaicin induced and Bcl-2

sensitive. In accordance with these results, apoptosis induced

by other PM NADH-oxidase inhibitors such as dihydrocapsa-

icin (100 j.LM), resiniferatoxin (10 .LM), retinoic acid (40 MM), and

chloroquine (200 jiM) was also inhibited by CyA and FK-506

(results not shown).

DiscussionWe have shown that the vanilloids capsaicin, dihydrocapsaicin,

and resiniferatoxin are potent inhibitors of the NADH-oxidase

activity of the PMOR system from rat liver. We have used these

three inhibitors together with two known rat liver PM NADH-

oxidase inhibitors, retinoic acid and chloroquine, to show thatPM NADH-oxidase inhibitors induce apoptosis in a time- andconcentration-dependent manner, as shown by the con-

densed, fragmented nuclear morphology (Fig. 2), the formationof DNA fragments, and the 200-bp laddering patterns of iso-

lated DNA (Fig. 5). The exact signal that triggers apoptosis after

PMOR inhibition is not yet known. Inhibition of the PMORsystem through interference with the coenzyme 0-binding site

may, in analogy to inhibition of the mitochondrial respiratory

chain by 0 antagonists, redirect the “normal” electron flow in

the complex, generating reactive oxygen species and a pro-

oxidant environment at the PM level. Oxidative stress is known

to trigger calcium influx over the PM. Preliminary observations

indicate that complexing extracellular calcium with EGTA or

intracellular calcium with bis-(O-aminophenoxy)-ethane-

N,N,N’,N’-tetraacetic acid tetra-(acetoxymethyl)-ester inhibits

PMOR inhibitor-induced apoptosis of B-cell lines.6 Previously,the PMOR system was found to regulate trans-PM calciumfluxes in synaptosomes (20-22). We hypothesize that inhibitorsof PM NADH-oxidase generate oxidative stress at the PM,

which subsequently triggers calcium influx and apoptosis. This

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would be consistent with the ability of Bcl-2 to prevent apop-tosis induced by PM NADH-oxidase inhibitors, because Bcl-2has been reported to regulate transmembrane calcium fluxes(43, 76) and to function in an antioxidant pathway (46, 77). Tworecent publications (78, 79) claim that their findings that BcI-2protects cells from hypoxia-induced apoptosis dispute the pro-miss that Bcl-2 works in an antioxidant manner. Our studies,however, suggest that BcI-2 may act to protect cells fromrenegade electrons when there is no electron sink available. Ifone assumes that oxygen is the final in vWo acceptor of rene-gade electrons (which must not necessarily be produced by thePMOR system), hypoxia may well inhibit the oxidase activity.Thus, BcI-2 may still work via an antioxidant mechanism, al-though in the electrochemical rather than the oxygen sense.

Because we recognized that neither vanilloids nor retinoic

acid and chloroquine are very specific inhibitors of PMNADH-oxidase, it was important to ascertain that the capa-bility of these compounds to induce apoptosis was causedby their inhibitory action on PM NADH-oxidase activity. Ourobservation that capsazepine, a compound that competes

with vanilloids for binding to the neuronal vanilloid receptor,does not inhibit vanilloid-induced apoptosis suggests that

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Fig. 7. Kinetics of induction of apoptosis by capsaicin and rotenone incells that differ in expression of BcI-2. FDCP-1 (#{149}and � or FDBCL-2 (Aand Y) cells were incubated with 200 �a,i capsaicin � and Y), 2 �g/mlrotenone (#{149}and A), or 0.2% ethanol (#{149})for various periods. At theindicated time intervals, aliquots were drawn from the cell suspensions,and the amount of morphological apoptosis was determined as describedin “Materials and Methods.” The results are the averages of two separateexperiments. The ethanol incubations represent the averages of both celllines, which did not significantly differ.

Fig. 6. Effect of the capsaicin (A), dihydrocapsaicin (B), and resinifera-toxin (C) concentration on the amount of morphological apoptosis inhuman Iyrnphoblastoid Daudi and Namalwa cells. Daudi (#{149})and Namalwa(�) cells were incubated for 8 h with increasing concentrations of capsa-icin, dihydrocapsaicin, or resiniferatoxin. Control incubations receivedonly the vehicle ethanol or Me2SO. The amount of apoptosis was deter-mined from the number of nuclei displaying the typical condensed andfragmented morphology of apoptotic cells. The results are the averages ofat least two separate experiments. Bais, SD.


Table 1 The effect of capsazepine, a vanilloid receptor antagonist, on apoptosis induced by PMOR inhibitors

Daudi cells were incubated for 8 h with 200 �u.i capsaicin, 20 pi� resiniferatoxin, or 40 p�i retinoic acid in the presence of increasing concentrations ofcapsazepine of up to 100 p.M. Capsazepine was added 10-15 mm before induction of apoptosis with the PMOR inhibitors. The amount of apoptosis wasdetermined from the number of nuclei displaying the typical condensed and fragmented morphology of apoptotic cells. The results are the averages of twoseparate experiments.

Apoptosis (% of total cell number after 8 h)(capsazepine (AM)

0 5 10 20 50 100

Ethanol (0.2%) 3 5 6 9 i5 20Capsaicin (200 MM) 55 53 52 61 69 72Resiniferatoxin (20 AM) 42 37 39 36 44 48Retinoic acid (40 AM) 40 41 37 47 45 51

Table 2 Prevention of vanilloid-induced apoptosis by coenzyrne 0-10

Cells were incubated for iO mm with the concentrations of 0-10 mdi-cated. After this, apoptosis was induced by either 200 AM capsaicin or 20

AM resiniferatoxin. Apoptosis was analyzed after 8 h by morphologicalassessment. The means of several experiments are given as percentagesapoptosis of total cell number ± SD; the numbers of experiments per-formed are given in parentheses.

+0.3% Ethanol +20 AM 0-10

Control 7 ± 3 (5) iO ± 1 (2)

Capsaicin 63 ± 8 (5) 39 ± 8 (5)Resiniferatoxin 56 ± 5 (3) 20 ± 9 (3)

binding to such a putative receptor is not involved in va-nilloid-induced apoptosis, at least in human blood cell lines.It remains to be established whether the selective destruc-

tion of nociceptive neurons after administration of capsaicinor resiniferatoxin during embryonal development and theanalgesic properties of these compounds in adult tissues isrelated to their ability to induce apoptosis. Preincubation withcoenzyme Q-1O can partly prevent vanilloid-induced apop-tosis, strengthening the idea that these compounds triggerapoptosis by acting as coenzyme Q-1O antagonists. Be-cause this property of capsaicin also underlies the ability ofthis compound to inhibit mitochondrial complex I activity, wenext investigated the effect of transferrmn, a known stimulatorof the PMOR system which does not affect mitochondrial

complex I, on capsaicin-induced apoptosis. As expected,transferrin was able to prevent partially vanilloid-inducedapoptosis. The strongest evidence for the involvement of thePMOR system in vanilloid- and retinoic acid-induced apop-tosis was obtained, however, by preincubating the cells witha polyclonal antiserum against partially purified PM NADH-oxidase preparations from rat liver. These antibodies, but notpreimmune serum, prevented about 50% of vanilloid- andretinoic acid-induced apoptosis. These data, together withthe different BcI-2 sensitivity, time dependence, and cal-cineurin involvement of apoptosis induced by PM NADH-oxidase compared with apoptosis induced by mitochondrialinhibitors, strongly support the idea that PM NADH-oxidaseinhibition underlies the induction of apoptosis by vanilloids,

retinoic acid, and chloroquine in cultured mammalian bloodcell lines.

Capsaicin-induced apoptosis can be inhibited by CyA andFK-506. In contrast, apoptosis induced by the mitochondrialrespiratory chain inhibitors rotenone, antimycin A, and oligo-

mycin remained unaffected by the presence of these immu-nosuppressants (Fig. 8 and results not shown). The use ofcombinations of these immunosuppressants with analoguesthat either no longer bind to their cognate immunophilin orbind immunophilin but no longer inhibit calcineurin activitydemonstrates that the inhibition of PMOR inhibitor-inducedapoptosis by immunosuppressants can be ascribed to inhi-bition of calcineurin. In this respect, apoptosis triggered byPM NADH-oxidase inhibition resembles apoptosis triggeredby the calcium ionophore ionomycin or surface 1gM ligationin mature B cells (80, 81) and activation-induced apoptosis ofT-ceII hybridomas (62), which can also be inhibited by im-munosuppressants that inhibit calcineurin. Calcineurin has

also been reported to be involved in calcium (ionomycin)-induced apoptosis in baby hamster kidney cells (82). On theother hand, in immature B cells, the same immunosuppres-sants can induce apoptosis (83) and can increase the son-sitivity to apoptotic stimuli in immature T cells (84) andthereby lead to a stimulation of calcineurin. At the concen-trations used in this study, neither CyA nor FK-506 had anydirect effect on ferricyanide reductase or NADH-oxidase ac-tivity of the PMOR system from isolated rat liver.7 Our ob-servation that the newly described CyA analogue SDZ 214-1 03 inhibits capsaicin-induced apoptosis as effectively as

CyA indicates that its mechanism of action also involvesbinding to cyclophilin and inhibition of calcineurin. Themechanism through which calcineurin inhibition preventsPMOR inhibitor-induced B-cell apoptosis is currently underinvestigation. It is possible that an increase in the expressionof Bcl-2 (85) and tumor necrosis factor a (86), which ismediated by calcineurin and inhibits calcium-induced apop-tosis in B cells (82), may also underlie the inhibition of ap-optosis induced by PM NADH-oxidase inhibitors by cal-cineurin-inhibiting immunosuppressants reported here.

Tyrosine kinase activity has been reported to be associatedwith the PMOR system (19). We have shown here that inhibitionofthe PM NADH-oxidase leads to apoptosis through a pathwayinvolving calcineurin. The classic pathway of calcineurin acti-vation involves the activation of phospholipase C, which in turnis activated through activation of tyrosine kineses (64). There-fore, we expect tyrosine kinase activation to be involved in the

7 J. A. Larm, F. Valllant, E. J. Wolvetang, and A. Lawen, unpublishedresults.


8 K. L Johnson and A. Lawen, unpublished results.

Table 3 The effect of ferric transfemn, an activator of the PMOR system, on apoptosis induced by PMOR inhibitors

Daudi cells were incubated for 8 h with 200 AM capsaicin, 20 AM resiniferatoxin, or 40 AM retinoic acid in the presence of increasing concentrations offemc transferrmn of up to 500 AM. Femc transferrmn was added 30 mm before induction of apoptosis with the PMOR inhibitors. The amount of apoptosis wasdetermined from the number of nuclei displaying the typical condensed and fragmented morphology of apoptotic cells. The results are the averages of twoseparate experiments.

Apoptosis (% of total cell number after 8 h)Ferric transferrin (�g/ml)

0 10 20 50 100 500

Ethanol (0.2%) 4 6 7 6 9 10

Capsaicin (200 AM) 51 55 43 37 30 33

Resiniferatoxin (20 AM) 41 44 38 29 18 23

Retinoic acid (40 AM) 39 42 36 25 20 26

Table 4 Prevention of apoptosis indu ced by PM NADH-o xidase inhibit ors by antiserum raised against the plasma me mbrane oxidase

Cells were incubated for 10 mm with 100 �g protein of either preimmune serum, antiplasma membrane reductase, or antiplasma membrane oxidaseserum. After this, apoptosis was induced by either 200 AM capsaicin, 20 AM resiniferatoxin, or 40 AM retinoic acid. Apoptosis was analyzed after 8 h bymorphological assessment. The means of several experiments are given as percentages of apoptosis of total cell numbers ± SD; the numbers ofexperiments performed are given in parentheses.

+0.2% Ethanol Preimmune Antireductase Antioxidase

Control 6 ± 4 (4) 6 ± 3 (2) n.d.a nd.Capsaicin 58 ± 6 (5) 55 ± 6 (2) 47 ± 4 (2) 30 ± 3 (2)Resiniferatoxin 57 ± 7 (3) 63 ± ii (3) 43 ± 12 (3) 32 ± 10(3)Retinoic acid 39 ± 2 (3) 40 ± 1 (2) 36 ± 2 (2) 23 ± 2 (2)

a nd., not done.

early signaling processes of PM NADH-oxidase inhibitor-in-duced apoptosis. Preliminary data from our laboratory� usingvarious inhibitors of protein tyrosine kinases suggest that thisindeed holds true. Further studies on this aspect are currentlyunder way in our laboratory.

Materials and MethodsCell Lines and Culture Conditions. The human lymphoblastoid cell linesDaudi, Namaiwa, and BL-29 were maintained in Iogarithmical growthusing RPMI 1640 (Life Technologies, Inc., Melbourne, Australia) supple-mented with 100 units/mI penicillin, 100 �g/ml streptomycin, iO% (v/v)

fetal calf serum, 5 mM 3-(N-morpholino)propanesulfonic acid-KOH (pH6.8), 50 �g/ml uridine, 1 mM sodium pyruvate, and 2 mr�i L-glutamine.Cultures in 75- or 25-mi culture flasks were incubated at 37#{176}Cin ahumidified gas mixture containing 5% CO2 balanced with air.

The interleukin 3-dependent mouse myeloid cell line FDCP-i and theFDBCL-2 cell line were kindly provided by D. Vaux (The Walter and Eliza

Hall Institute for Medical Research, Melbourne, Australia). The FDBCL-2

cell line was derived from FDCP-i cells by transfection with a vectorconstitutively expressing human BcI-2 (87). The RPMI 1640 of these cellsadditionally contained 5% interleukin 3-conditioned medium derived fromWEHI-3B cells (88).

Morphological Determination of Apoptosis. To 2 ml cells (2-5 x io�cells/mI) was added capsaicin, dihydrocapsaicin, chloroquine, retinoicacid, or rotenone, each dissolved in ethanol, or resiniferatoxin, dissolvedin Me2SO, to the final concentrations indicated. Immunosuppressants, atthe final concentrations indicated, were always added 1 0-1 5 mm prior toany further additions to allow for entry into the cells and association with

their respective binding proteins. Cells were cultured in 16- or 24-wellculture dishes. After 8 or 1 6 h, a 400-A’ aliquot was drawn from the

suspension and transferred to slides, which had been coated with 5 A1poly-L-lysine (10 �g/ml), by using a Shandon cytospmn (5 mm at 2000 rpm).The cells were fixed for 10 mm in a mixture of ethanol:acetic acid (3:1),

washed for 1 mm in distilled water, air dried, and stained with a drop of 0.1

pg/mI DAPI for 10 mm. The adhered cells were washed twice with distilled

water, air dried, and mounted with antifading medium [10 mg/mI p-phenyldiamine in 90% glycerol (pH 9.0)]. The slides were observed undera fluorescence microscope (Nikon Microphot-FX) at an excitation wave-length of 280 nm. The percentage of apoptotic nuclei was determined by

counting more than 500 cells from at least three separate determinations.The data are presented as averages ± SD.

Gel Electrophoresis of DNA Fragments. Cells were grown in 50-mIculture flasks at a similar density and treated with inhibitors as describedabove. About io� cells were harvested by centrifugation (5 mm, 150 x g).The pellet was immediately lysed in medium containing 1 % (w/v) SDS, 0.5

mg/mI proteinase K(Boehringer Mannheim, Mannheim, Germany), and iOmM Tris-HCI (pH 8.0) and incubated for 12 h at 37#{176}C.Protein was sub-

sequently precipitated by addition of 5 volume saturated NaCI solution.

The DNA in the supernatant was precipitated with 3 volume ethanol at-20#{176}C,taken up in 10 m� Tris-HCI (pH 8.5), and stored at -70”C. The

amount of DNA was determined in each sample with the fluorescent dye

Hoechst 33258 (89). Subsequently, 0.5-i .0 �g DNA was examined forDNA fragmentation as described (90). An EcoRl-HindIll digest of A phageDNA was used as a standard.

Quantitation of DNA Fragmentation. The quantity of DNA fragments

generated was determined with a standard cellular DNA fragmentationELISA kit from Boehringer Mannheim (No. 1585 045) according to the

recommendations of the supplier.Immunoblotting. About 1 o7 cells, grown as described above, were

suspended in lysis buffer [i % NP4O, 1 m�i phenylmethylsulfonyl fluoride,2% aprotinin, 2 mM EDTA, and 10 m�i Tris-HCI (pH 8.0)], left on ice for 15

mm, and subsequently centrifuged for 10 mm in an Eppendorf centrifuge.An aliquot of the supematant containing 50-i 00 mg protein was boiled in

sample buffer for 5 mm. The proteins were separated on a 1 5% SDS-

polyacrylamide gel and electrotransferred (Sartoblot; Sartorius, Mel-

bourne, Australia) at 30 V for 1-2 h to a polyvinylidene difluoride mem-brane (Bio-Rad, Hercules, CA). The filters were blocked overnight at 4#{176}C

with blocking buffer [5% nonfat milk powder plus 2% fetal calf serum inPBS (pH 7.4)]. The filters were incubated for 1 h with a 1 :1 dilution of the

supernatant of hybridoma cell line Bcl-2/i 00 (kindly provided by David

Mason, John Radcliffe Hospital, Oxford, United Kingdom; Ref. 91) in

blocking buffer. Subsequently, the filters were incubated with a 1:2000

dilution of sheep antimouse IgG alkaline phosphatase conjugate (Silenus,

Percentage Apoptosis after 8 Hours Percentage Apoptosis after 8 Hours

0 10 20 30 40 50 60 70� I � � �F �

-

� II H

-----H

BL-29


Contr�

Cape

Caps + CyA

Caps + CyA + CyH

Caps + CyA + (Me\�&)CyA

Cape + CyA + (Mefle’)CyA

Caps + CyA + Rapamycin

Caps + CyH

Cape + (MO�I$�)CyA

Cape + CMeRO’)CYA

Caps + Fj(�506

Cape + FK-506 + 50Z 280�629

Cape + FK-506 + (MeVd’EYA

Caps + FK-506 + lMelte’)CyA

Cape + FK-506 + Rapamycin

Cape #{247}Rapamydn

Cape + SDZ 280�629

Rotenone

Rotenone + CyA

Rotenone + FK�506

0 10 20 30 40 50 60 70(_� � - �

I

�

�II �

Daudi

Fig. 8. Inhibitory effect of immunosuppressants on capsaicin-induced apoptosis correlates with inhibition of calcineurin. Daudi (left panel) or BL-29 (rightpanel) cells were preincubated with 0.3 AM immunosuppressant for 1 0-i 5 mm before induction of apoptosis with either 200 �i capsaicmn (Caps) or 2 A�/mlrotenone. To compete for immunophilin binding, a 10 AM concentration of a second immunosuppressant was added 1 0 mm before the first immunosup-pressant. The amount of apoptosis was determined from the number of nuclei displaying the typical condensed and fragmented morphology of apoptoticcells. The results are the averages of at least three separate experiments. Bais, SD.

Hawthorn, Australia) in blocking buffer for 1 h and stained for alkalinephosphatase activity using p-nitrophenylphosphate as a substrate. Be-tween each step, the filters were washed three times for 10 mm with PBS

containing 0.05% Tween 20.Isolation of PM5 from Rat Uver and PM NADH-oxldoreductase

Activity. PMs were isolated from fresh rat liver using the aqueous two-phase partitioning procedure as described (92). The purity of the PMpreparations obtained was determined by marker enzyme analysis and

electron microscopy. Only PMs that were found to be more than 90% pureon a protein basis have been used for the measurement of NADH-oxidaseactivity as described (8).

Mitochondrial Complex I Assays. Submitochondrial particles, elec-tron transport particles (ETPH), were prepared from bovine heart as de-

scribed (93). NADH-oxidase and NADH-Q-i reductase activities were

determined as described elsewhere (94).

AcknowledgmentsThe present work succeeded studies on mitochondrial function and PMoxidoreductase activities initiated by Professor A. W. Linnane (Centre forMolecular Biology and Medicine, Monash University), whom we thank forhis initial generous financial support and interest. Valuable discussionswith Profs. F. L Crane and T. M. Buttke and Drs. I. R. Mackay and F.Vaillant are gratefully acknowledged. We appreciate the kind donations of

FDCP-i and FDBCL-2 cell lines by Dr. D. Vaux, immunosuppressants by

Dr. Ren#{233}Traber (Sandoz Pharma Ltd., Basel, Switzeriand), SAN 547 A byDr. L Hagmann (Sandoz Agro Ltd., Basel, Switzeriand), and SDZ 282-320by Dr. M. Grassberger (Sandoz Research Institute, Vienna, Austria). We

acknowledge the skillful assistance of Dr. K. Krauer with Western blotting,K. L. Johnson for cytometry, and B. Veitch for photomicrography.

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