Apoptosis in factor-dependent haematopoietic cells is linked to calcium-sensitive mitochondrial...

British Journal of Haematology 2000, 109, 221±234

Apoptosis in factor-dependent haematopoietic cells is linked to

calcium-sensitive mitochondrial rearrangements and

cytoskeletal modulation

JOHN GARLAND,1 GILLIAN BROW N,2 JULIAN BEASLEY2 and RO BIN BROW N

3 1Institute of Clinical Science, Exeter

University, Noy Scott House, Wonford, Exeter EX2 5EQ, UK, 2Biomedical Research Laboratories, and 3Cell Biology Unit,

Glaxo-Wellcome Medicines Research Centre, Gunnels Wood Road, Stevenage, Herts SG1 2NY, UK

Received 12 October 1999; accepted for publication 1 December 1999

Summary. Apoptosis in murine haematopoietic interleukin(IL)3-dependent cell lines is induced within 6±8 h by IL-3withdrawal. Direct introduction of cytochrome c by electro-poration induces apoptosis within 2 h and was inhibited bycaspase inhibitors, such as Z-VADfmk and Z-Dfmk. Wereport here that apoptosis induced by IL-3 withdrawal wasrefractory to these inhibitors but was accompanied bystriking redistribution of mitochondria, which aggregatedinto an area associated with centrioles without loss of Dcm.Both mitochondrial redistribution and apoptosis wereinhibited by the calcium ionophore, ionomycin. Nocodozole,an inhibitor of microtubule assembly, also induced apopto-sis, which was unaffected by caspase inhibitors. Althoughnocodozole did not alter mitochondrial distribution, itsignificantly reduced Dcm, and both reduction of Dcm andapoptosis were inhibited by ionomycin. Oligomycin, whichinhibits the mitochondrial FoF1 ATPase, similarly induced

apoptosis, which was unaffected by caspase inhibitors butwas inhibited by ionomycin. Further, oligomycin stimulatedthe novel formation and release of surface membrane-derived vesicles containing mitochondria with intact Dcm;ionomycin also inhibited their production. In all theseconditions, Bcl-2 protected cells from apoptosis. Our studiesshow that apoptosis induced by three very different agentsshares insensitivity to caspase inhibitors, suppression byionomycin and effects on mitochondria, which all appearto be linked to cytoskeletal/microtubule activity. Theysuggest that microtubules and the cytoskeleton play animportant role in apoptosis through mechanisms affectingmitochondria but which are independent of cytochrome crelease.

Keywords: apoptosis, mitochondrial movement, cytoskele-ton, calcium.

Apoptosis (programmed cell death) is effected by proteolyticactivation of a cascade of cysteine±aspartate proteases(caspases) (Salvesen & Dixit, 1997; Thornberry & Lazebnik,1998). Caspases have numerous substrates, such as PARPand nuclear lamin, and can be grouped into threefunctional families on the basis of sequence homology andsubstrate preferences. Caspase 3 plays a central effector role,for example in apoptotic DNA fragmentation throughactivating caspase-activated DNase (CAD) by proteolyticremoval of an inhibitory subunit, I-CAD (Enari et al, 1998;Janicker et al, 1998). Death-inducing signalling complex(DISC) receptors, such as TNFr-1, activate procaspase 3through activation of procaspase 9 via receptor interaction

with adaptor proteins, such as TRADD and FAD (Nagata,1997; Ashkenazi & Dixit, 1998). Alternatively, apoptogenicfactors are released from mitochondria. These includecytochrome c (Kluck et al, 1997a; Yang et al, 1997),apoptosis inducing factor (AIF; Lorenzo et al, 1999) andcaspases (Krajewski et al, 1999; Susin et al, 1999).Cytochrome c binds to APAF 1 (ced 4-like), which recruitsand activates caspase 9 in the presence of ATP, leading toprocaspase 3 activation (Li et al, 1997; Reed, 1997). Themechanisms of cytochrome c release from mitochondria arestill unclear, but have been linked to changes in mitochon-drial transmembrane potential (Dcm) (Zamzami et al,1995a,b) and to the mitochondrial pore-inducing functionof pro-apoptotic proteins, such as Bax, BAD and BID, all ofwhich cause loss in Dcm and stimulate cytochrome c releasewhen directed to the mitochondrial outer membrane (Zhaet al, 1996; Gross et al, 1997, 1999). Cytochrome c release

221 q 2000 Blackwell Science Ltd

Correspondence: John Garland, Laboratory of Vascular Cell Biology,Department of Pathology, Manchester University Medical School,

Oxford Road, Manchester M13 9PT, UK.

222 J. Garland et al

q 2000 Blackwell Science Ltd, British Journal of Haematology 109: 221±234

is inhibited by antiapoptotic proteins, such as bcl-2 and bcl-XL (Kluck et al, 1997b; Yang et al, 1997; Ashkenazi & Dixit,1998) but, under some circumstances, Bcl-2 may alsoinhibit Bax-induced apoptosis even in the presence ofcytochrome c release (Rosse et al, 1998).

Murine IL-3-dependent cells, such as Bo and Baf-3,undergo rapid apoptosis on IL-3 withdrawal, which issuppressed by Bcl-2 (Vaux et al, 1988; Hockenbery et al,1993; Rosse et al, 1998). Apoptosis by IL-3 withdrawal isnot, however, accompanied by changes in mitochondrialDcm (Garland & Halestrap, 1997), and cells protected fromapoptosis by bcl-2 or activated MAP-KK (Perkins et al,1996) survive in a metabolically arrested state for manydays without any change in intracellular ATP (Garland et al,1998). However, electroporation of exogenous cytochromec induces apoptosis within 2 h regardless of IL-3signalling, expression of bcl-2, MAP-KK or proliferativestatus and is inhibited by the known caspase inhibitorsZ-VADfmk and Z-Ddcbmk (Garland & Rudin, 1998). Wewished to confirm that apoptosis induced by IL-3 with-drawal was similarly related to cytochrome c release frommitochondria despite the lack of change in Dcm. Wetherefore investigated Dcm and mitochondrial distributionduring IL-3 withdrawal and apoptosis induced by twoother pharmacologically completely distinct agents: oligo-mycin, which inhibits the mitochondrial FoF1 ATPsynthase; and nocodozole, which disrupts the microtubulecytoskeleton.

MATERIALS AND METHODS

Cell lines and reagents. The cell lines Bo, Baf3, B15(overexpressing human Bcl-2) and A15 (overexpressing Bcl-2and activated MAP-KK) have been described previously(Perkins et al, 1996) and were cultured in RPMI-1640medium supplemented with 10% fetal calf serum (FCS) and10% WEHI-3b-conditioned medium as a source of IL-3.Ionomycin, oligomycin, atractyloside (Sigma) were firsttitrated and subsequently used at 5±10� concentrationproducing an observable effect in 50% of cells. z-VADfmkand Ac-DEVD-fmk were obtained from Calbiochem, andz-Dfmk from the Alexis Corporation (Switzerland). All werekept as 100 mmol/l solutions in dimethyl sulphoxide(DMSO) at 2808C and used at 50 mmol/l or as indicated.Nocodozole was from Sigma.

Histochemistry and flow cytometry. For JC1 staining, cellswere adjusted to 2±5 � 105/ml in complete medium.JC1 (5 ml; kindly provided by Dr Bo Chen, Dana FarberInstitute, Boston, USA; stock solution 2 mg/ml inDMSO) was deposited on the wall of a tube containing1 ml of cell suspension, mixed rapidly by flicking thetube and incubated for 15 min at 378C. Cytospinpreparations were then made. Slides were examined undera Nikon fluorescence microscope equipped with a triplebandpass cube for red and green fluorescence. Slides wereviewed and photographed using a 100� oil immersionobjective.

For DNA histochemistry, cytospin preparations werepermeabilized with 70% methanol and stained with

DAPI. For flow cytometric analysis of DNA fragmentation,1±2 � 106 cells were spun, washed once in RPMI withoutserum, spun and fixed for 2±6 h at 48C in 70% methanol.After spinning, the pellets were resuspended and stained for30 min in 250 ml of DNA Prep (Coulter) at 378C andanalysed on a Coulter Epics Elite flow cytometer. Forwardand side-scatter gates were set to exclude necrotic material.Flow cytometry gates A, C, D and E represented preG1, G1, Sand G2/M phases. (Note: apoptosis causes loss in S±G2/M,and the pre-G1 DNA peak also underestimates apoptosis;Garland & Rudin, 1998.) All cytometer samples were alsoviewed under direct phase microscopy before fixing andstaining and apoptotic cells counted.

For electron microscopy, cells were fixed in 4% glutaral-dehyde before osmium fixation and embedding. Sectionswere stained with uranyl acetate.

Electroporation. For electroporation, cells were adjusted to107/ml in complete medium. Bovine cytochrome c (Sigma)to 80 mg/ml final concentration or control protein (inactiveyeast cytochrome c) was added to 250 ml of cell suspensionin 40-mm cuvettes and held on ice for 15 min. Cells wereelectroporated using 250 V, 900 mF (Biorad Genepulser),returned to ice for 15 min, then washed twice in completemedium to remove dead cells and cytochrome c beforereculture.

RESULTS

IL-3 withdrawal causes mitochondrial redistribution withoutalteration in Dcm

JC1 is a carbocyanine fluorescent dye (excitation 490 nm,emission 527 nm, green), which is concentrated withinmitochondria. In the presence of a net negative charge, Jaggregates form with a change in emission to 590 nm(orange), which can be used to measure Dcm quantitatively(Reers et al, 1991, 1995). We found that JC1 is considerablymore reliable as an indicator of Dcm in IL-3-dependent cellsthan Di-O-(C6)3 or Rh123 (Salvioli et al, 1997). Both ofthese generate high backgrounds, and the discrimination ofspecific mitochondrial fluorescence is difficult. An addedadvantage of JC1 is that, under direct white light micro-scopy, mitochondria containing orange-fluorescent J aggre-gates appear blue in colour and can be imaged directly (seeFig 1). To eliminate changes induced by IL-3 withdrawalthat may be masked by apoptosis, we first used B15 andA15 cells, which survive for many days without IL-3. In allgrowing cells stained with JC1, mitochondria showed atypical dispersed perinuclear distribution (Fig 1). However,after 24 h of IL-3 deprivation, mitochondria were re-arranged to form striking, greatly extended linear arrays(Fig 1). Mitochondria were all stained bright orange,showing retention of Dcm. Electron microscopy (Fig 2)showed that the mitochondria were drawn to a fan-shapedarea between nuclear lobes associated with centrioles, thusimplicating microtubules in the rearrangement. When IL-3-starved parental Bo cells were examined, similar earlyredistribution of mitochondria was seen (not shown), butthe cells apoptosed before its completion.

Mitochondrial Rearrangements in Apoptotic IL-3-dependent Cells 223


Inhibition of apoptosis and reversal of mitochondrialredistribution by calcium ionophoreBecause the calcium ionophore ionomycin has been reportedto inhibit apoptosis induced by IL-3 withdrawal in a calcium-dependent fashion (Rodriguez-Tarduchy et al, 1997), weinvestigated its effect on both apoptosis and the mitochon-drial rearrangements after IL-3 withdrawal (Fig 3). Iono-mycin completely inhibited apoptosis in IL-3-deprivedparental Bo cells as reported previously (Fig 3). However,it had no effect on apoptosis induced directly by electro-poration of cytochrome c (Fig 4) or on growth or the cellcycle (not shown), suggesting that it could not inhibit eventsdownstream of cytochrome c. We therefore asked whetherionomycin also altered the mitochondrial rearrangements seenafter long-term IL-3 deprivation. In A15 cells quiesced withoutIL-3 for 3 days, IL-3 reversed the mitochondrial aggregationwithin 1±2 h, returning them to a dispersed perinuclearlocation (Fig 5). Surprisingly, ionomycin (200 nmol/l21mmol/l) also completely reversed mitochondrial aggregationafter only 1 h exposure (Fig 5) and, in fact, was as effective asIL-3. Similar results were observed in B15 cells and Bo cells

quiesced for shorter times. These experiments thereforesuggested that both apoptosis and mitochondrial rearrange-ments induced by IL-3 withdrawal were dependent on calcium,possibly linked to rapid changes in the cytoskeleton.

Caspase inhibitors do not block apoptosis induced by IL-3withdrawalIf ionomycin and bcl-2/MAPKK inhibited apoptosis bypreventing cytochrome c release and thereby caspase 3activation during IL-3 withdrawal, such apoptosis should beinhibited by z-Dfmk and z-VADfmk (Garland & Rudin,1998). However, neither prevented or delayed apoptosis(Fig 6) even when used at 200 mmol/l. This suggested thatcytochrome c-dependent activation of caspases was notinvolved in apoptosis induced by IL-3 withdrawal and thatprotection by ionomycin was not therefore through theinhibition of cytochrome c release.

The microtubule disrupting agent, nocodozole, induces apoptosiswithout altering mitochondrial distribution, but reduces Dcm

The above results on mitochondrial aggregation suggested

Fig 1. Mitochondrial rearrangements in A15 cells induced by IL-3 withdrawal. Upper row, brightfield; lower row, fluorescence. Cells werestained with JC1. In brightfield, mitochondria appear as blue. Left: control cells (C) growing in IL-3. Right: cells quiesced for 3 days (Q). In

control cells, mitochondria are dispersed; in quiescent cells, they are aggregated and form extended linear arrays. Note that all mitochondria

are orange under fluorescence, showing retention of Dcm.



that apoptosis and IL-3 signalling were both linked tomicrotubule activity. Nocodozole prevents microtubulereassembly by binding to tubulin, but does not affect stablemicrotubule arrays. We therefore expected that nocodozolemight duplicate the effects of IL-3 withdrawal on thecytoskeleton and therefore induce apoptosis and mitochon-drial rearrangements. However, although 50±100 mmol/lnocodozole induced both chromatin condensation andaccelerated apoptosis in Bo cells, mitochondria were notrearranged. Unexpectedly, many mitochondria in nocodo-zole-treated cells showed reduced Dcm (Fig 7), with mostcells having a 50% reduction in JC1 orange-fluorescentmitochondria. Because nocodozole inhibits only dynamicmicrotubules, these results suggested, first, that mitochon-dria are associated with the stable cytoskeleton and,secondly, that tubulin or nocodozole itself may in someway regulate mitochondrial membrane potential. In apop-tosis-resistant B15 and A15 cells, nocodozole similarlyinduced chromosome condensation and reduced Dcm, butapoptosis was significantly delayed. Nocodozole-inducedapoptosis was completely insensitive to caspase inhibitors(Fig 8) but, like IL-3 withdrawal, was inhibited byionomycin (Fig 7). Ionomycin also inhibited the chromatincondensation and reduction in Dcm. These results suggestedthat nocodozole-induced apoptosis was linked to a calcium-dependent pathway primarily involving the cytoskeletonand not to the cytochrome c/APAF-1 pathway, despite thereductions in Dcm. Thus, although nocodozole-induced

apoptosis did not duplicate the mitochondrial rearrange-ments seen with IL-3 withdrawal, it shared with it the keyfeatures of inhibition by calcium ionophore and insensitivityto caspase inhibitors (Fig 8).

Mitochondrial rearrangements are induced by the mitochondrialpoison oligomycinOligomycin binds to the Fo complex of the mitochondrialFoF1 ATPase and therefore inhibits ATP synthesis depen-dent on oxidative phosphorylation, and hyperpolarizes theinner mitochondrial membrane. In yeasts and mammaliancells, oligomycin has been shown to inhibit Bax-induced celldeath and has been used to demonstrate that the cellularchoice between apoptotic or necrotic cell death may bedetermined by intracellular ATP levels (Eguchi et al, 1997;Leist et al, 1997; Matzuyama et al, 1998). As IL-3-dependent cells do not generate ATP by oxidative phosphor-ylation, nor do their ATP levels change during apoptosisinduction (Garland & Halestrap, 1997), we reasoned thathyperpolarization of the inner mitochondrial membranewould inhibit any cytochrome c release dependent onreduced Dcm (Matzuyama et al, 1998), if this was indeedinduced but not detected during IL-3 withdrawal. Oligomy-cin should therefore inhibit apoptosis. However, contrary toprediction, oligomycin at 200±500 nmol/l induced apopto-sis in all Bo cells supplemented with IL-3 in 24±48 h andaccelerated apoptosis in its absence. Oligomycin alsoinduced apoptosis in IL-3-supplemented B15 and A15

Fig 2. Electron micrograph of quiesced A15

cells. Mitochondria (M) are drawn into a fan-shaped, electron-lucent area between nuclear

lobes (N) centred on the centriole (C).



cells. However, < 40% of cells survived. These thencontinued to grow in oligomycin with IL-3, albeit with anextended generation time (24 h compared with 10 h), andproduced a fully viable culture with indefinite survival.Oligomycin-resistant cells died in the absence of IL-3 within12 h (data not shown). In growing oligomycin-resistantcultures, we noticed that all cells exhibited extensive surfacemembrane blebbing (Figs 9 and 10). These blebs containedJC1 orange-fluorescent material; mitochondria appeared tocollect opposite to, and tracked into, them through stalksattaching the blebs to the cell (Fig 9). It was thus possibleto construct a sequence whereby mitochondria weresequestered into the blebs, which were then released

from the cell. In support of this, spun supernates of culturesof oligomycin-resistant B15 or A15 cells contained manysmall JC1 orange-fluorescent vesicles. The presence ofmitochondria-derived material in membrane blebs wasconfirmed by electron microscopy (Fig 10). However,nuclear morphology was completely unaffected (Fig 9).This indicated that oligomycin specifically affected thecytoskeleton associated with surface membrane mobilityand mitochondrial movement and not the mitotic appara-tus. We excluded the possibility that oligomycin-inducedmitochondrial migration was coupled to a possible inhibi-tion of mitochondrial ATP production because atractyloside(which inhibits the mitochondrial adenine nucleotidetransporter and disrupts mitochondrial ATP synthesis)had no effect on growth and survival (Fig 11), apoptosis orcell morphology or blebbing at any concentration up to1 mmol/l. As with both IL-3 withdrawal and nocodozole,oligomycin-induced apoptosis was not suppressed bycaspase inhibitors, but all its effects were prevented byionomycin (Fig 11). Further, cells growing in oligomycinremained fully susceptible to cytochrome c-induced apop-tosis, which was suppressed by caspase inhibitors (Fig 12).This again suggested that oligomycin-induced apoptosiswas separate and mechanistically different from thatinduced by cytochrome c activation of caspases.

DISCUSSION

We expected that apoptosis induced by IL-3 withdrawalinvolved the release of cytochrome c and activation ofcaspases via APAF 1. Instead, we found that it wasinsensitive to caspase inhibitors, but confirmed that it wasinhibited by the calcium ionophore ionomycin (Baffy et al,1993; Rodriguez-Tarduchy et al, 1997). Although we againfailed to find any reduction in Dcm (Garland & Halestrap,1997), we observed massive mitochondrial rearrangementsthat could also be rapidly reversed by ionomycin. Oligomy-cin and nocodozole, two very different agents, similarlyinduced apoptosis that was not inhibited by caspaseinhibitors, but was inhibited by ionomycin. Further,nocodozole, a specific microtubule-disrupting agent, unex-pectedly but significantly reduced Dcm., and oligomycin, aninhibitor of mitochondrial FoF1 ATP synthase, inducednovel continuous production of surface vesicles into whichmitochondria migrated. Because apoptosis induced bycytochrome c is inhibited by caspase inhibitors but not byionomycin, the present results indicate that apoptosisinduced by IL-3 withdrawal, nocodozole and oligomycin isnot dependent on primary mitochondrial cytochrome crelease, but rather on calcium-sensitive cytoskeletalmodulation, which nevertheless affects mitochondria.

It is known that outer mitochondrial membrane perme-ability and cytochrome c release is regulated by the bcl-2family of proteins (e.g. bcl-2, Bcl-XL, Bax, BAD; Jacobson,1997). However, in many cells, cytochrome c release is alsocoupled to reduction in membrane Dcm and changes in thepermeability transition pore (PTP), both with inner mem-brane location. Association between inner and outermembrane function is therefore necessary for the release

Fig 3. Inhibition of apoptosis by ionomycin. Bo cells were cultured

with IL-3 (1 C), without IL-3 (C) or without IL-3 but with

500 nmol/l ionomycin (I). Cells were examined by flow cytometry

after 20 h. Visible apoptosis was: with IL-3, , 1%; without IL-3,40%; without IL-3 with ionomycin, 8%.



of intermembrane space proteins, such as cytochrome c,dependent on inner membrane Dcm, and PTP. Physical`megachannels' (Zoratti & Szabo, 1995) and physiologicalmitochondrial disruption dependent on PTP opening and

Ca21 have been proposed. Recent studies indicate that theouter membrane protein Bax and the voltage-dependentanion channel (VDAC)-forming protein, porin, can associatewith isolated PTP (Beutner et al, 1998; Marzo et al, 1998;

Fig 5. Reversal of mitochondrial redistribution by 500 nmol/l ionomycin in quiescent B15 cells. Conditions as described in the legend to Fig 1.B15 cells were quiesced by IL-3 withdrawal for 24 h (left), then recultured with IL-3 (middle) or 500 nmol/l ionomycin (right). Cells were

stained with JC1 after 1 h and deposited on slides with a cytocentrifuge. Note reversal of mitochondrial rearrangement with both IL-3 and

ionomycin.

Fig 4. Ionomycin does not inhibit cytochrome c-induced apoptosis. Bo cells were electroporated with cytochrome c as described in Materialsand methods and recultured in IL-3 with or without 500 nmol/l ionomycin. Cells were fixed, stained with propidium iodide (PI) and examined

by flow cytometry after 4 h. Left: control cells (cont) without ionomycin; right: with ionomycin (iono). Note equal induction of apoptosis.

Arrow, preG1 apoptotic DNA. Percentage of visible apoptosis was 66% in controls and 61% with ionomycin. Similar results were obtained

when ionomycin was added before electroporation. c, control cells; cyt c, electroporated with bovine cytochrome c.



Narita et al, 1998). Moreover, cyclophilin, another compo-nent of the PTP (Woodfield et al, 1998), is inhibited bycyclosporin A, which closes PTP and inhibits apoptosis andcytochrome c release in many cells. Thus, evidence for a

potential complex formed from outer and inner membraneproteins appears to exist.

Could such a complex be regulated by the cytoskeletonand calcium? Both the cytoskeleton and actin can modulate

Fig 6. Caspase inhibitors suppress apoptosis induced by cytochrome c but not IL-3 withdrawal. Visible apoptotic fractions given in brackets.

Left (1): Baf 3 cells electroporated with cytochrome c and cultured with IL-3 for 4 h; 1 C: control, without cytochrome c; Cyt C: electroporated

with bovine cytochrome c (53%); Cyt C/VAD: electroporated with bovine cytochrome c and cultured with 50 mmol/l z-VADfmk (9%). Right (±):cells cultured for 22 h without IL-3; C: control (72%); VAD: with 50 mmol/l z-VADfmk (65%); DEVD: with 50 mmol/l Ac-DEVDfmk (70%).



porin activity (Kay et al, 1997; Rappaport et al, 1998; Xuet al, 1999). Mitochondria attach to microtubules (Hegge-ness et al, 1978; Jung et al, 1993; Periera et al, 1997) andare moved via a specific microtubule motor (KIF-1B) duringFas-induced apoptosis (Nangaku et al, 1994; De Vost et al,1998). With regard to calcium, localized calcium `micro-domains' have been described, which delineate sites ofcontact between the endoplasmic reticulum (ER), andtherefore possibly ER microtubules, to the mitochondrialouter membrane (Rizzuto et al, 1998). Intracellular Ca21

determines both mitochondrial Ca21 and PTP conformation(Davidson & Halestrap, 1990; Denton & McCormack, 1990;Halestrap & Davidson, 1990) and can be modulated by GTPrequired for tubulin polymerization and by cytoskeletalintegrity (Hajnoczky et al, 1994). Ichas & Mazat (1998)described how cytosolic Ca21 increases can lead to PTPopening, collapse of Dcm, swelling and release of inter-membrane/matrix components. Interestingly, the modelinvolves localized calcium `microdomains' close to mito-chondria. Consistent with such a model, ionomycin, which

Fig 7. Inhibition by ionomycin (I) of cytoskeletal effects and loss of Dcm induced by nocodozole (NOC) in Bo cells. Cells were incubated without

IL-3 with or without 50 mmol/l nocodozole with or without 500 nmol/l ionomycin for 6 h. (A) With nocodozole, stained with DAPI (centre)

and JC1 (lower); (B) with nocodozole and ionomycin, stained with DAPI (centre) and JC1 (lower); (C) flow cytometry of PI-stained cells. C:

control; I: with ionomycin; NOC: with nocodozole; NOC 1 I: with nocodozole and ionomycin. Note that nocodozole reduced Dcm, shown byreduction in orange fluorescence (A). Ionomycin suppressed changes in both Dcm and chromatin condensation (B). An apoptotic cell is shown

(arrow). Nocodozole induces significant apoptosis (C, arrow), which is inhibited by ionomycin, and cells accumulate in G2/M phase.



principally increases cytosolic Ca21 through release frombound stores (Gwag et al, 1999), usually induces apopto-sis (Naganuma et al, 1999). However, although IL-3withdrawal has been shown to redistribute Ca21 fromcytosol to mitochondria (Baffy et al, 1993), the inhibition ofapoptosis by ionomycin under all the conditions describedhere cannot readily be explained by such models.

In our cells also, both Bcl-2 and MAP-KK exertantiapoptosis effects. Phosphorylation of Bcl-2 on serine70 appears to be required for its antiapoptotic activity(Ruvulo et al, 1999), which can be stimulated by micro-tubule-associated protein kinase (MAP-K) activation (Vranaet al, 1999) via MAP-K kinase (MAP-KK). Thus, expressionof MAP-KK in A15 cells is normally protective via Bcl-2phosphorylation However, microtubule damage induceshyperphosphorylation of Bcl-2, which renders it inactive

Fig 7. Continued

Fig 8. Apoptosis induced by nocodozole is not inhibited by caspase

inhibitors. Bo cells were cultured without IL-3 with or withoutnocodozole or 50 mmol/l caspase inhibitors (see Fig 6). C, control;

N, with nocodozole; N/VAD, with nocodozole and z-VADfmk; N/

DEVD:, with nocodozole and Ac-DEVDfmk. Cells were fixed and

stained with PI for flow cytometry after 6 h. Note extensiveapoptosis, which is unaffected by caspase inhibitors. Similar results

were obtained with IL-3 present (data not shown).



and can induce apoptosis (Haldar et al, 1997; Srivastavaet al, 1998). Microtubule disruption and inactivation of Bcl-2 by nocodozole may thus explain the loss of Dcm, or it maybe related to direct suppression of PTP closure byunpolymerized tubulin (Evtodienko et al, 1996). Ca21

changes could also affect calcium-dependent phosphatases,such as calcineurin, thereby altering, for example, BADphosphorylation and its interaction with Bcl-2, as well asthe phosphorylation of bcl-2 itself (Zha et al, 1996; Shiasakiet al, 1997). With regard to oligomycin, low levels of ATPcan induce actin-dependent membrane blebbing (Gabai et al,1992), and oligomycin may reduce intracellular [ATP] viathe mitochondrial FoF1 ATP synthase acting as an ATPasewhen Fo is inhibited. However, this does not explain whymitochondria specifically migrate into vesicles. Also, as cellsappear to be fully viable, blebbing seems unlikely to berelated to cell demise. Further work is needed to clarify thiseffect.

We hypothesize that the cytoskeleton associates with

outer mitochondrial membrane complexes that requireCa21 to maintain complex integrity. Changes in thecytoskeleton or Ca21 cause complex discontinuity andpotential release of matrix and/or intermembrane spaceproteins. Our study therefore raises the intriguing issue that,while cells appear to be fully primed to activate apoptosisthrough cytochrome c-APAF 1, other mechanisms forapoptosis induction appear to exist that are highly dependenton Ca21 and the cytoskeleton, but not necessarily invoking aprimary mitochondrial cytochrome c-dependent pathway.The activity and location of defined caspases in these cellsduring apoptosis under these conditions is thus important todefine and is currently under investigation.

ACKNOWLEDGMENTS

This work was supported by the Northcott-Devon MedicalFoundation and The Paul Janssen Research Foundation. We

Fig 9. Mitochondrial migration into membrane blebs induced by 300 nmol/l oligomycin. B15 cells were selected for resistance to oligomycin

over 3 weeks. Left: brightfield (upper) and fluorescence (lower) micrographs of JC1-stained cells showing: (a) early extension of cell; (b) cell with

early membrane blebbing showing two polar blebs with mitochondria lining up before migration (arrows); (c) late cell with large membranebleb about to be released; (d) released bleb containing mitochondria. Middle: membrane bleb just before detachment. Note apposition of

mitochondria opposite bleb and stalk of attachment and retention of Dcm. Right: blebbing cell stained with DAPI. Note prophase nuclear

morphology is completely unaffected by oligomycin.


Fig 10. Electron micrograph showing migra-

tion of mitochondria into a bleb. Note that the

mitochondrial membranes (arrow) appearatypical, however.

Fig 11. Inhibition of oligomycin-induced apoptosis by ionomycin.

Bo cells were incubated in 300 nmol/l oligomycin (OM) or

250 nmol/l atractyloside (AT) with IL-3 with or without

500 nmol/l ionomycin (I) and examined by flow cytometry after22 h. Note that atractyloside had no effect. Ionomycin almost

completely inhibited apoptosis.



thank Dr M. Collins (ICRF, London) for the provision of Bo,B15 and A15 cells.

NOTE ADDED IN PROOF

A recent article by Zhu et al (Nature 403, 98; 2000)describes the induction of apoptosis by endoplasmicreticulum-associated caspase 12 activated by endoplasmicreticulum stress. Our findings linking apoptosis withcytoskeletal rearrangement would be entirely compatiblewith such a mechanism.

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