Contents lists available at SciVerse ScienceDirect
Cell Calcium
jo u rn al hom epa ge: www.elsev ier .com/ locate /ceca
alcium ionophore A23187 reveals calcium related cellular stress as “I-Bodies”:n old actor in a new role
mit Verma1, Anant Narayan Bhatt ∗,1, Abdullah Farooque, Suchit Khanna,aurabh Singh, Bilikere S. Dwarakanath ∗
etabolic and Cell Signaling Group, Division of Radiation Biosciences, Institute of Nuclear Medicine and Allied Sciences, Brig. S. K. Mazumdar Marg, Delhi 110054, India
r t i c l e i n f o
rticle history:eceived 22 June 2011eceived in revised form 28 August 2011ccepted 29 August 2011vailable online 28 September 2011
eywords:23187alcium
a b s t r a c t
Calcimycin (A23187) is an ionophore widely used in studies related to calcium dynamics in cells,but its fluorometric potential to reveal intracellular physiology has not been explored. Exploiting themicroenvironment-induced changes in its fluorescence, we show that a brief exposure of cells to non-toxic concentrations (≤3 �M) of the ionophore results in the characteristic organization of the ionophoreforming brightly fluorescent cytoplasmic bodies termed “I-Bodies”, which are closely related to stresslinked disturbances/changes in calcium homeostasis. “I-Bodies” appear to be Ca2+ rich intracellular sitesformed during stress-induced release of intracellular Ca2+, causing dysfunction and aggregation of mito-chondria, providing scaffold for high density packing of A23187. Formation of “I-Bodies” in cells exposed
ancer biomarkeritochondrial aggregation
to ionizing radiation and certain anticancer drugs suggest their potential in revealing alterations in cal-cium signaling and mitochondrial function during (related to) macromolecular damage-induced celldeath. The absence of “I-Bodies” in non-malignant cells and their varying numbers in malignant cells with5 fold increase in fluorescence imply that they can be potential biomarkers of cancer. Thus, “I-Bodies”are novel indicators of endogenous and induced stress linked to disturbances in calcium homeostasis in
erve
cells, with a potential to s
. Introduction
Calcium ionophore A23187 (calcimycin), isolated from Strepto-yces chartreuses is a hydrophobic (carboxylic polyether) molecule
hat selectively binds to calcium, a metal ion, enabling it to pene-rate the hydrophobic interior of the lipid bilayer thereby increasingellular permeability of calcium ion [1–3]. Available evidences sug-est that two monovalent units of A23187 associate with the cation4] forming a 2:1 dimer complex [5]. Further, the ionophore haslso been found to be intrinsically fluorescent with emission in thelue region upon UV excitation that leads to higher quantum yieldollowing association with phospholipids and membranes as com-ared to aqueous phase [6,7]. A23187 has been extensively used tolucidate mechanisms underlying the transport of calcium acrosshospholipid bilayers as well as other divalent cations [8,9]. It haslso been used to understand the role of calcium ion in several cel-
ular phenomena like proliferation, differentiation, and apoptosisesides its antibacterial activity [10,11].
Several studies have shown a strong relationship betweencalcium and mitochondria both during physiological and patho-physiological conditions [12]. Stress induced efflux of calciumsequestered in the endoplasmic reticulum (ER) generally relocatesto mitochondria in the cytoplasm that act as a crucial regulatorsof intracellular Ca2+ homeostasis [13–15]. Mitochondria lie in theclose proximity of plasma membrane and ER, where they playan important role in buffering the increased intracellular calcium[Ca2+]i after stress induced influx from extra-cellular milieu orrelease from intracellular stores (mainly ER) [16]. When mitochon-drial Ca2+ load exceeds the buffering capacity of the matrix, [Ca2+]m
rises steeply, leading to irreversible swelling, uncoupling and lossof soluble mitochondrial content that in turn results in openingof a large nonselective pore encompassing both the mitochon-drial membranes. This process is referred to as the mitochondrialpermeability transition (MPT) [17]. The functional impairmentof calcium and mitochondrial dynamics are now understood toplay a role in the pathogenesis of many unrelated disorders suchas dementia, Alzheimer’s, epilepsy, Parkinson’s, cardiomyopathy,coronary artery disease, chronic fatigue syndrome, retinitis pig-mentosa, diabetes and most importantly cancer [18]. Despite the
renewed interest in the role of mitochondria in calcium signaling,the molecular mechanisms that allow these organelles to rapidlyaccumulate and release large Ca2+ loads are still poorly under-stood.
The present study demonstrates that alterations in calciumomeostasis and mitochondrial dysfunction can be probed using23187, as its fluorescence enhances significantly under theseonditions due to a characteristic organization of the ionophoren to discrete, punctuated and brightly fluorescing bodies calledI-Bodies”. We show that stress induced release of Ca2+ from intra-ellular sites, which accumulates in the energized mitochondriaeading to mitochondrial dysfunction and aggregation, are the keyteps in “I-Bodies” formation. Further, the mitophagic vacuolesncircling these calcium rich clusters of dysfunctional mitochon-ria appear to provide scaffold for A23187 aggregation leading toI-Bodies” formation. Our results also show the potential of endoge-ous “I-Bodies” as a biomarker of cancer.
cals Co. (St Louis, USA), where as Fluo-3-AM, Mitotracker greennd 3,3′-dihexyloxacarbocyaniniodode (DiOC6), were procuredrom Molecular Probes (Eugene, USA). Etoposide, camptothecin,aclitaxel, cisplatin and doxorubicin were purchased from Daburharmaceuticals. Bhabhatron-II, a teletherapy machine fromanacea, Medical Technologies Pvt Ltd (Bangalore, India) was useds a source for �-ray irradiation.
.2. Sources of cell lines
The cerebral glioma cell line (BMG-1; diploid, wild type p53)stablished by us earlier [14], were cultured in DMEM (con-aining 1 g Glucose/l) with 5% foetal bovine serum containingntibiotics, i.e. penicillin (100 units/ml), streptomycin (50 units/ml)nd nystatin (2 �g/ml) in humidified 5% CO2 incubator at 37 ◦C.uman head and neck squamous carcinoma cell lines (KB), mouse
eukemic monocyte macrophage (J774.1), mouse embryonic fibro-last (NIH3T3), human embryonic kidney cells (HEK293), human
ung epithelial adenocarcinoma cells (A549), human embryonicormal lung fibroblast (MRC-5) and mouse normal monocyteacrophage (Raw 264.7) were obtained from NCCS, Pune, India and
ultured in DMEM (4.5 g Glucose/l) containing 10% foetal bovineerum and antibiotics. Stock culture was maintained in the expo-ential growth phase by passaging them every 3 days with theirespective growth medium in 25 cm2 plastic flask (BD, Mountain-iew, USA).
.3. Drug treatment and A23187 staining
Cells were seeded on sterile microscopic cover glass and incu-ated overnight at 37 ◦C in 5% CO2. Following overnight incubation,ttached cells were treated with either anticancer drugs such astoposide (100 �M), paclitaxel (100 �M), cisplatin (100 �M), dox-rubicin (100 �M) and camptothecin (5 �M) for 4 h or irradiated10 Gy; 4 h) with Co-60 gamma-rays. After the treatment, cells
ere incubated with 3 �M of A23187 for 20 min at 37 ◦C in growthedia for enumerating “I-Bodies” and 50 �M monodansylcadaver-
nes (MDC) for 30 min at 37 ◦C in growth media to stain mitophagicacuoles. Cells were then washed with PBS and visualized using UV
50 (2011) 510– 522 511
excitation in a fluorescence microscope (Olympus BX 60 fluores-cence microscope, Japan; fitted with Grundig FA87 monochromeCCD camera, Place) or analyzed on FACS LSRII flow cytometer (Bec-ton Dickinson, Mountainview, CA, USA) using 355 nm laser forexcitation with appropriate filters for collecting blue fluorescence.All the images were captured using 40× objective.
2.4. Determination of mitochondrial membrane potential (�� m)
Flow cytomeric analysis of mitochondrial membrane potentialwas carried out using DiOC6 staining. Briefly after treatment, cellsin situ were incubated with DiOC6 (40 nM for 30 min) in growthmedia at 37 ◦C, washed, scrapped and re-suspended in PBS beforemeasurements. The green fluorescence of DiOC6 was analyzed ina flow cytometer (LSR II, BD, Mountainview, CA, USA) followingexcitation using 488 nm laser.
2.5. Co-localization of JC-1 and mitotracker green with “I-Bodies”
Cells were grown on sterile cover glass for microscopic exam-ination of mitochondria by JC-1 and mitotracker green. Followingincubation overnight, cells were treated with etoposide (100 �M;4 h) and washed with cold PBS (pH 7.4). Subsequently, cells wereco-incubated in culture medium containing either JC-1 (2 �g/mL;30 min) with A23187 (3 �M; 20 min) or mitotracker green (100 nM;30 min) with A23187 (3 �M; 20 min) at 37 ◦C in 5% CO2. Following awash with cold PBS, cells were examined under the microscope andimages were captured using 40× objective. Pseudo coloring wasused for merged images to reveal “I-Bodies” formed by A23187.
2.6. Measurement of intracellular calcium
The qualitative and quantitative analyses of intracellular cal-cium were carried out using Fluo-3-AM by fluorescence microscopyand flow cytometry, respectively. For microscopy, attached cellstreated with etoposide (100 �M; 4 h), on sterile cover glass, werestained with Fluo-3-AM (3 �M; 30 min), washed once with cold PBSbefore examination. For flow cytometry, suspension of treated cellswere prepared and loaded with Fluo-3-AM (3 �M; 30 min), washedwith cold PBS, and resuspended in PBS for measurements with LSRII flow cytometer (BD, Mountainview, CA, USA).
2.7. Metabolic viability by MTT assay
The cells were plated in 96 well plates (5000 cells/200 �l/well).Following A23187 (3 �M; 20 min) treatment, stock MTT solution(20 �l) was added and cells were incubated at 37 ◦C in the darkfor 2 h. The medium was removed and formazan crystals formedby the cells were dissolved using 150 �l of DMSO. The absorbancewas read at 570 nm using 630 nm as reference wavelength on anELISA reader (Biotech Instruments, USA).
2.8. Implantation of tumors in mice
The inbred Swiss albino strain ‘A’ and Balb/c female mice (10–12weeks) used in these studies were obtained from the Institute’scentral animal facility and weighed 20–25 g at the time of tumorimplantation. EAT and DL cells were maintained in the peritonealcavity of strain A and Balb/c female mice, respectively. Tumors wereimplanted according to the protocol reported earlier [19]. In brief,
tumors in the form of ascites were developed by injecting 25 × 106
cells intra-peritoneally and solid tumors were implanted subcuta-neously by injecting 15 × 106 cells (in 0.1–0.15 ml volume) into theright hind leg of the mouse.
5 alcium
2
dwbocHa
2
laebtcE
3
3i
clWhlA(ttflp“iptibicsso(wavBNWfw(Baonf
12 A. Verma et al. / Cell C
.9. Isolation of murine tumor and peripheral blood cells
Ascites tumor cells were drawn from peritoneal cavity after 7ays of implantation using 28 gauge needle. Further, cells wereashed twice with PBS, examined viability (using trypan blue),
efore staining with A23187. Blood was collected from mouserbital plexus in the heaparinized vials. Peripheral blood mononu-lear cells were separated from density gradient method usingistopaque. PBMCs in the form of buffy coat were carefully sep-rated after centrifugation and washed twice with PBS before use.
.10. Ethics statement
All animal experiments were conducted according to the guide-ines established by CPCSEA (Committee for the Purpose of Controlnd Supervision on Experiments on Animals), Indian National Sci-nce Academy (INSA) and European Society for handling tumorearing animals [20]. Institute’s Animal Ethics Committee registra-ion number is 8/1999/CPCSEA; 09.03.1999. All animal experimentsonducted for this study were duly approved by Institute’s Animalthics Committee.
. Results
.1. “I-Bodies” are aggregates of highly fluorescent calciumonophore A23187 in cells
Although, the ionophore A23187 (calcimycin) is widely used inell biology studies related to calcium dynamics [21], the intracel-ular status of its fluorescence has not received much attention.
e report here for the first time the formation of discrete andighly fluorescent punctuated bodies observed nearly 20 min fol-
owing incubation of cancer cell lines (human glioma; BMG-1) with23187 (Fig. 1a). Interestingly, cells incubated with Br-A23187
non fluorescent analogue of A23187) under similar experimen-al conditions did not show any fluorescence (Fig. 1a), suggestinghat it was specific to A23187 (data not shown). These punctuateduorescent bodies that are distributed throughout the cytoplasm,redominantly in the perinuclear region have been designated asI-Bodies”. The number of “I-Bodies” enhanced multi-folds follow-ng treatment of cells with etoposide (Fig. 1a), a topoisomerase IIoison, which also induces oxidative stress and apoptosis. Forma-ion of “I-Bodies” was accompanied by approximately sevenfoldncrease in the intensity of blue fluorescence from cells that coulde rapidly and objectively analyzed by flow cytometry (Fig. 1b). An
ncrease in the number of “I-Bodies” and enhanced cellular fluores-ence was also observed with other DNA damaging and oxidativetress inducing agents like camptothecin and ionizing radiation,imilar to etoposide (Fig. 1c). These results suggest that A23187rganizes itself in cells in a characteristic punctuated structurecalled here as “I-Bodies”) and the number of “I-Bodies” increasesith an increase in the degree of stress induced by DNA damaging
nd other oxidative stress causing agents. Generality of this obser-ation was examined by using few well established malignant (KB,MG-1, J774, A549) as well as non-malignant (MRC-5, HEK293,IH3T3, Raw 264.7) cell lines treated with etoposide (Fig. 1d).hile the numbers of endogenous “I-Bodies” varied among dif-
erent malignant cell lines (KB, BMG-1, J774.1, A549), “I-Bodies”ere nearly absent in all the non-malignant cell lines investigated
MRC-5, NIH3T3, HEK293, Raw 264.7, Fig. 1d), suggesting that “I-odies” are related to subtle differences in the physiological status
nd endogenous stress levels that may be related to calcium home-stasis in these two groups of cells. However, induction of largeumbers of “I-Bodies” in both malignant and non-malignant cells
ollowing treatment with etoposide (Fig. 1d) lends support to the
50 (2011) 510– 522
proposition that “I-Bodies” formation is indeed related to the stresslevel. Since concentrations (or doses) of stress inducing agents viz.etoposide, ionizing radiation, camptothecin used here also resultsin profound cell death, these observations raises the hope that “I-Bodies” of A23187 can be used as an indicator of cellular responsesto induced stress, besides serving as a unique signature related toendogenous physiological status. In order to enhance its biolog-ical importance, we systematically investigated the potential ofvarious stress conditions in “I-Bodies” formation and underlyingmechanisms.
3.2. High intracellular calcium [Ca2+]i is required for “I-Bodies”formation
To understand the genesis of A23187 mediated “I-Bodies” for-mation, we first examined the role of intracellular Ca2+, as theionophore predominantly interacts with calcium and thereforeis likely to critically influence its organization. Probing the rela-tionship between intracellular Ca2+ concentration and cellularfluorescence due to “I-Bodies”, we compared the fluorescence ofFluo-3 (calcium indicator) and A23187 in cells treated with increas-ing concentration of etoposide (Fig. 2a). Results clearly show thatetoposide induced increase in A23187 fluorescence closely followsthe rise of [Ca2+]i under these conditions, with a positive corre-lation between the two (Fig. 2a inset), strongly suggesting therole of calcium in “I-Bodies” formation. To verify this suggestion,we disturbed the calcium homeostasis with BAPTA (an intracel-lular calcium chelator) for 1 h prior to etoposide treatment andobserved the formation of “I-Bodies”. Results obtained in BMG-1cells clearly showed that BAPTA reduced the “I-Bodies” formationunder these conditions (Fig. 2b), strengthening the proposition thatintracellular calcium strongly influences the “I-Bodies” formation.Flow cytometric analysis complemented microscopic observa-tions, where BAPTA reduced the A23187 fluorescence in etoposidetreated cells by nearly 70% (Fig. 2c). To further substantiate therole of intracellular calcium, we mobilized endoplasmic reticulumstored calcium using thapsigargin (SERCA inhibitor) and observedthe “I-Bodies” formation. Interestingly, thapsigargin induced “I-Bodies” formation, suggesting that “I-Bodies” formation is linkedto the release of calcium from intracellular stores (Fig. 2d). On theother hand, half an hour prior incubation of cells with EGTA (extracellular Ca2+ chelator) before exposing to etoposide did not alterthe formation of “I-Bodies” implying that extracellular Ca2+ hadnegligible role in “I-Bodies” formation (Fig. 2e).
3.3. “I-Bodies” are calcium rich intracellular site
To examine the correlation between calcium and “I-Bodies”, wefirst examined their localization in cells by microscopy in etopo-side treated cells after staining with Fluo-3 (an intracellular calciumindicator). The green fluorescence of Fluo-3 with vesicles like clus-tering primarily around perinuclear area enhanced significantlyfollowing etoposide treatment in both the cell lines (Fig. 3a) thatwere similar to the “I-Bodies”. The resemblance in the pattern ofFluo-3 fluorescence with “I-Bodies” suggested that “I-Bodies” arecalcium rich cytoplasmic organelle or bodies where A23187 accu-mulate with self organization capability. The persistence of A23187fluorescence (I-Bodies) for extended period of time suggests thatthese bodies retain Ca2+ even after incubation with the ionophorebecause A23187 may be acting like Ca2+/H+ exchanger and notallowing the Ca2+ to move out, which could be due to the acidic pHof these bodies [22]. However, when etoposide treated cells were
co-incubated with A23187 and Fluo-3, the blue fluorescence from“I-Bodies” did not co-localize with green fluorescence from Fluo-3(Fig. 3b), possibly due to either higher affinity of A23187 to Ca2+ orlow esterase activity in these cells [23].
alcium
3“
fopas
F(wiBi
A. Verma et al. / Cell C
.4. Dysfunctional mitochondria are the intracellular sites for theI-Bodies”
In our efforts to identify the intracellular site(s) of “I-Bodies”ormation, we primarily considered mitochondria as possible
rganelles, as they have a buffering capacity for increased cyto-lasmic Ca2+ level during stress, thereby maintaining homeostasisnd normal cellular physiological functions [24]. Moreover, etopo-ide is known to induce perinuclear clustering of mitochondria
ig. 1. “I-Bodies” of calcium ionophore A23187 in cells. (a) Photo-micrographs of punct100 �M etoposide; 4 h) human glioma cells (BMG-1). Cells grown on sterile cover glass waith UV excitation. (b) Flow cytometric analysis of blue fluorescence (from A23187) in c
s presented. (c) Photo-micrographs of BMG-1 cells treated with camptothecin (5 �M;
odies”. (d) “I-Bodies” in malignant (A549, BMG-1, J774.1, KB) and non-malignant (HEK29nterpretation of the references to colour in this figure legend, the reader is referred to th
50 (2011) 510– 522 513
[25], which is very similar to the intracellular location of “I-Bodies”observed here; although “I-Bodies” were also seen in the otherregions of cytoplasm; albeit to a lesser extent. We first exam-ined the consequences of disturbing mitochondrial uniporter onetoposide-induced “I-Bodies” formation using Ruthenium red (RR),
2+
which attenuates the influx of cytoplasmic Ca into the mito-chondria [26–28]. RR delays the increased Ca2+ clearance fromthe cytoplasm, although it does not inhibit the mitochondrialCa2+ import completely. Exposure of cells to RR for 30 min prior
uated bodies of A23187 fluorescence (I-Bodies) observed in untreated and treateds stained with A23187 (3 �M for 20 min) and viewed under fluorescence microscopeells. Fold change in the relative fluorescence intensity in comparison with control4 h) and ionizing radiation (10 Gy; 4 h post exposure) for the enumeration of “I-3, MRC-5, NIH3T3, Raw 264.7) cell lines treated with etoposide, (100 �M; 4 h). (Fore web version of the article.)
514 A. Verma et al. / Cell Calcium 50 (2011) 510– 522
(Cont
tB(AFaCi
tt[t
Fig. 1.
o etoposide treatment significantly reduced the number of “I-odies”, particularly their aggregation in the perinuclear regionFig. 4a), which was accompanied by a threefold reduction in the23187 fluorescence measured flow cytometrically (Supportingig. S1). However, RR added 2 h after exposure to etoposide did notffect the “I-Bodies” formation (Fig. 4a), suggesting that influx ofa2+ into the mitochondria (released from ER by etoposide; Fig. 2)
s a mandatory requirement for “I-Bodies” formation.Since etoposide induced mitochondrial aggregation and forma-
ion of “I-Bodies” appears to be linked with the release of Ca2+ fromhe ER [25], majority of which accumulates in the mitochondria29] causing further damage to the organelle [17], we hypothesizedhat “I-Bodies” are indeed functionally compromised mitochon-
inued)
dria (diminished membrane potential due to Ca2+ overloading) thatallow high density packing of A23187. To verify this hypothesis, weexamined the co-localization of A23187 fluorescence with a poten-tiometric cationic dye JC-1, which accumulates in the mitochondriagiving green/red fluorescence depending on the membrane poten-tial. As expected, the blue fluorescence (from “I-Bodies”) anddiffused green fluorescence were co-localized in etoposide treatedcells (Fig. 4b), suggesting that “I-Bodies” are perhaps damagedmitochondria that had lost the membrane potential essentially
because of irreversible damage due to massive loading of Ca2+.To substantiate these findings, we examined the co-localizationof A23187 fluorescence with mitotracker green, which binds tothe mitochondria regardless of its membrane potential [30]. The
A. Verma et al. / Cell Calcium 50 (2011) 510– 522 515
Fig. 2. Intracellular calcium release induces “I-Bodies” formation. (a) The relationship between intracellular Ca2+ concentration (measured by Fluo-3) and A23187 fluorescencemeasured by flow cytometry in etoposide treated cells (10–100 �M; 4 h). Fold change in relative fluorescence is presented. Inset shows correlation between A23187 andFluo-3 fluorescence. (b) Photo-micrographs of etoposide treated cells pre-incubated with intracellular calcium chelator BAPTA (20 �M; 1 h) showing the absence of “I-Bodies”.( rimeni thapi g “I-D
mflso
c) Flow cytometeric analysis of A23187 fluorescence carried out under similar expes presented. (d) Photomicrographs showing “I-Bodies” in BMG-1 cells treated withncubated with EGTA (10 mM; for 30 min) prior to etoposide treatment for revealin
itotracker green fluorescence co-localized very well with A23187uorescence and was prominent in fixed cells (Fig. 4c), lendingupport to the proposition that mitochondria are indeed the sitesf “I-Bodies” formation. Interestingly, not all the mitochondria
tal condition as described for (b); fold changes in the relative fluorescence intensitysigargin (1 �M; 1 h) before A23187 staining. (e) Photomicrographs of BMG-1 cellsots”.
stained by mitotracker green showed A23187 fluorescence, sug-gesting that only few mitochondria or few clusters of mitochondria(that are probably damaged) act as sites for A23187 organizationand involved in the formation of “I-Bodies”.
516 A. Verma et al. / Cell Calcium 50 (2011) 510– 522
F rescenH ed BM( erred
3e
cdCwmItnAtrCwbiccrm“m
ig. 3. “I-Bodies” are calcium rich bodies. (a) Photomicrograph showing Fluo-3 fluoEK cells. (b) The figure shows co-staining of Fluo-3 and A23187 in etoposide treat
For interpretation of the references to colour in this figure legend, the reader is ref
.5. Maintenance of mitochondrial membrane potential isssential for “I-Bodies” formation
Available evidences suggest that uptake of Ca2+ into mito-hondria involve electrophoretic transport through Ca2+ uniporterriven by the mitochondrial membrane potential, ��m [31,32].isplatin, paclitaxel and doxorubicin are known to disrupt ��m,hich prevents mitochondria from buffering (accumulation in theitochondria) the elevated cytosolic Ca2+ in the cells [33–35].
nterestingly, cisplatin, doxorubicin and paclitaxel that also alterhe Ca2+ homeostasis and elevate intracellular Ca2+ [36] didot induce “I-Bodies” formation but showed only a diffused23187 fluorescence (Fig. 5a). To determine if imbalance in
he redox status (induced by these drugs) leading to the dis-uption of ��m is associated with compromised mitochondriala2+ influx inhibiting “I-Bodies” formation, we incubated cellsith N-acetyl cysteine (NAC) and pyruvate (known antioxidants)
efore the addition of paclitaxel and cisplatin. Interestingly,ncubation of cells with pyruvate and NAC before exposure toisplatin and paclitaxel significantly increased both the mito-hondrial membrane potential and “I-Bodies” (Fig. 5b, c). These
esults reiterated the role of redox balance linked mitochondrialembrane potential and mitochondrial Ca2+ sequestration in the
I-Bodies” formation. To confirm this further, we disrupted theitochondrial membrane potential using FCCP (carbonyl cyanide
ce (3 �M; for 30 min) and “I-Bodies” in etoposide treated (100 �M; 4 h) BMG-1 andG-1 cells. A23187 was given pseudo color (red) when merged with Fluo-3 (green).to the web version of the article.)
p-trifluoromethoxyphenylhydrazone) before the exposure of cellsto etoposide and analyzed the A23187 fluorescence. As expected,cells with depolarized mitochondria showed a significant reduc-tion in A23187 fluorescence (Fig. 5d), confirming the requirementof ��m and redox balance for “I-Bodies” formation.
3.6. “I-Bodies” as a potential biomarker of cancer
It is well known that cancer cells are associated with an alteredlevel of oxidative and metabolic stress with disturbed Ca2+ home-ostasis, linked to the oncogenic transformation and physiologicalstatus [37,38]. We also observed varying levels of “I-Bodies” in allmalignant cell lines, while “I-Bodies” were nearly absent in the non-malignant cells (Fig. 1d). Flow cytometric measurements of bluefluorescence related to “I-Bodies” showed nearly fivefold differ-ence between the malignant and non-malignant cells suggestingthat perhaps “I-Bodies” can be used as a signature of cancer cells.To strengthen the potential of “I-Bodies” as a biomarker of cancer,we examined “I-Bodies” fluorescence of normal (peripheral bloodmononuclear cells; PBMCs) and tumor cells under in vivo condi-tions. Large numbers of “I-Bodies” could be seen in both the in vivo
tumors investigated (Ehrlich Ascites Tumor; EAT and Dalton’s lym-phoma; DL) grown as ascitic fluid (Fig. 6b). Interestingly, significantnumbers of “I-Bodies” were found in the PBMC’s of tumor bearingmice, while they were totally absent in the PBMC’s of normal mice
A. Verma et al. / Cell Calcium 50 (2011) 510– 522 517
Fig. 4. Mitochondria are targets of A23187 mediated “I-Bodies” formation. (a) Photomicrographs showing effects of Ruthenium red (10 �M; 1 h) on etoposide (100 �M; 4 h)induced “I-Bodies” in BMG-1 cells. Ruthenium red was added either before (pre-treatment) or 3 h after the addition of etoposide (post-treatment) and before staining withA23187 to observe “I-Bodies” formation. (b) Photomicrographs showing co-localization of “I-Bodies” (pseudo red color) with JC-1 fluorescence in etoposide treated BMG-1cells. JC-1 (1.5 �M; 30 min) and A23187 was added after etoposide treatment, respectively. The arrow in green panel indicates the monomeric form of JC-1 dye, whereas thearrow in red panel indicates the “I-Bodies”. (c) Co-localization of mitotracker green fluorescence and “I-Bodies” in etoposide treated BMG-1 cells, showing effects of ethanolfixation. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of the article.)
518 A. Verma et al. / Cell Calcium 50 (2011) 510– 522
Fig. 5. Maintained mitochondrial membrane potential is a pre-requisite for “I-Bodies” formation. (a) Photomicrographs showing the lack of “I-Bodies” formation in BMG-1cells treated with cisplatin (100 �M), paclitaxel (100 �M) and doxorubicin (100 �M) for 4 h. (b) Cells are pre-treated with antioxidants sodium pyruvate (1 mM) and N-acetylc or 4 hfl as alsw 00 �Mm
(sst[
ysteine (1 mM) for 1 h followed by cisplatin (100 �M) and paclitaxel (100 �M) fuorescence microscope. (c) A23187 and DiOC6 (40 nM for 30 min) fluorescence were treated with carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP; 1easured by flow cytometer is presented as fold change.
Fig. 6c). These observations suggest that “I-Bodies” are perhaps
ensitive enough to reveal even small alterations in Ca2+ relatedtress levels of cells, caused by the circulating tumor secreted fac-ors, which target all body cells including the hematopoietic cells39].
and than cells was stained with A23187 before “I-Bodies” were observed usingo observed under similar experimental conditions using flow cytometer. (d) Cells
for 15 min) followed by etoposide (100 �M for 4 h). Mean fluorescence intensity
4. Discussion
The calcium ionophore A23187 has been widely used in studiesrelated to calcium dynamics, while its intrinsic fluorescence prop-erty has been exploited to elucidate mechanisms underlying the
A. Verma et al. / Cell Calcium
Fig. 6. (a) Comparison of A23187 fluorescence in non-malignant (HEK 293, MRC-5, Raw 264.7 and NIH3T3) and malignantly transformed (A549, BMG-1, KB, J774.1and U87) cell lines. Data presented here is the ratio of A23187 fluorescence andauto-fluorescence in respective cell lines. (b) Formation of “I-Bodies” in isolatedmurine tumor cells (Ehrlich ascites tumor cells; EAT and Dalton’s lymphoma; DL)from ascitic fluid. The figure shows photomicrograph of cells stained with A23187.(f
tisetitabtssghtn(t
all the above mentioned conditions. Therefore, it is pertinent
c) Photomicrograph of A23187 stained PBMCs (peripheral blood mononuclear cells)rom control and tumor bearing animals.
ransport of divalent cations across phospholipid bilayers [7]. Thenterference of its fluorescence with other fluorochromes havingimilar spectral characteristics while studying calcium dynamics isither reduced using very low amounts of A23187 or replaced withhe non-fluorescent analogue Br-A23187. Contrary to these lim-tations, present studies investigate the application of ionophorehat is related to its ability to reveal stress induced mitochondrialggregation, associated with Ca2+ overload, which is presented asrightly fluorescing “I-Bodies” under various physiological condi-ions. It is well known that A23187 molecules aggregate as a dimerictack in phospholipid vesicles and the dimensions of which areufficient to span the bilayer thickness [4,40]. Although, A23187et partitioned in most of the membranous cellular organelles, itsigh density packing in specific membranous bodies of malignantlyransformed cells (as compared to diffused A23187 fluorescence in
on-malignant cells) as well as under conditions of induced stressetoposide, etc.) clearly indicate the differential intracellular dis-ribution of A23187 in stressed and unstressed cells. Therefore,
50 (2011) 510– 522 519
we propose that “I-Bodies” are either hypo-polarized, damagedmitochondria or mitophagic vacuole encircling dysfunctional mito-chondria [41] formed during stress, scaffolding A23187 with highdensity. Protracted exposure (several hours) of cells to various con-centrations of A23187 is known to be cytotoxic, inducing apoptosis[42]. However, the conditions employed here to reveal “I-Bodies”are completely non-toxic as revealed by cell viability assays (pro-pidium iodide (PI) uptake and MTT assay; Supporting Fig. S2), whichis due to very short (20 min) exposure time and the low concentra-tion (3 �M) of A23187.
Disturbances in the calcium homeostasis related to alterationsin cell signaling brought about by malignant transformation areperhaps one of the early events in carcinogenesis, suggestive of analtered physiological state [43]. While this low to moderate level ofchronic stress is not detrimental and may in fact drive the pro-cess of carcinogenesis, excessive stress (transient or protracted)induced by extraneous agents (like radiation, drugs, and heat) dis-turbs the functional signaling networks and physiological state thatmay either rescue cells from stress or commit them to death. Since“I-Bodies” seen at any instant is a snap-shot picture of a dynamicprocess involving events in cellular responses to stress, variations inthe abundance of endogenous and induced “I-Bodies” among cellsmay represent the rate of occurrence of these events. “I-Bodies”found mainly in malignantly transformed or cancer cell lines (BMG-1, KB, A549, and J774.1) but not in the normal cells (MRC-5, NIH3T3,HEK293, and Raw 264.7) suggest that it can be considered as a rapidbiomarker of cancer.
Since the “I-Bodies” are formed due to disturbed calcium home-ostasis (Fig. 2) and altered mitochondrial status (Fig. 4), its presencein cancer cells is in line with the well known altered calcium sig-naling and mitochondrial function [44]. One of the reasons for theformation of “I-Bodies” in cancer cells could be oxidized mem-brane fatty acid due to persistent oxidative stress, which servesas permanent endogenous ionophore [45] for the release of Ca2+
from ER stores that accumulate in the mitochondria, destroyingits structure and function, thereby forming scaffold for “I-Bodies”formation. Although the numbers of “I-Bodies” are significantlyincreased when the malignantly transformed cells are incubatedwith stress inducing agents (viz. etoposide; Fig. 1d), appearance of“I-Bodies” even in etoposide treated untransformed cells (Fig. 1d)suggests that intracellular dissemination of calcium related stressleading to “I-Bodies” formation is a general phenomenon not onlyrestricted to malignant cells. The macromolecular insult by agentslike radiation and camptothecin also formed “I-Bodies” indicatingthat stress induced changes in the physiological state is a prereq-uisite for this phenomenon to occur. Etoposide induced cell deathis orchestrated by irreversible release of intracellular calcium fromER stores into the cytoplasm [46]. A direct relationship with etopo-side induced release of Ca2+ from intracellular stores and “I-Bodies”observed in BMG-1 cells (Fig. 2), strongly suggests that this is theprimary step in the process of “I-Bodies” formation. These resultsalso demonstrate that “I-Bodies” are intracellular Ca2+ rich mem-branous organelles where A23187 aggregate to form “I-Bodies”(Figs. 2 and 3).
Mitochondrion with their origin as a cellular symbiont witheukaryotic cytoplasm is the primary organelle acting as bufferfor the Ca2+ transitions during stress [24]. One of the commonfeatures of mitochondrial damage is its aggregation as observedin a variety of conditions like Parkinson’s disease, cancer [47],viral infection [48,49] and exposure of cells to TNF-� [50] aswell as anticancer drugs like etoposide [25]. Interestingly, highcytoplasmic and mitochondrial Ca2+ is a common feature among
to consider mitochondria as sites of “I-Bodies” formation. Mito-chondrial participation in “I-Bodies” formation was revealed byJC-1 and mitotracker green (Fig. 4c, d) suggesting that “I-Bodies”
520 A. Verma et al. / Cell Calcium 50 (2011) 510– 522
Fig. 7. (a) Photomicrographs showing MDC-labeled vesicles in etoposide treated BMG-1 cells. Similar pattern of MDC labeling in stressed cells suggest that “I-Bodies” aremitophagic vacuoles. (b) The figure shows the sequential occurrence of events during the process of stress induced “I-Bodies” formation. The first step is release of Ca2+
from ER, which accumulates in energized mitochondria with well maintained ��m (second step). In the third step, Ca2+ overloading causes swelling and formation ofn ion ofm
asmmripBc
on-functional damaged mitochondria. The fourth and final step is the accumulatitochondria (4).
re indeed Ca2+ rich mitochondrial aggregates formed as a con-equence of stress (viz. etoposide induced) showing disturbedembrane potential and high Ca2+ accumulation. Moreover, treat-ent with etoposide and many other stress conditions leading to
elease of Ca2+ from intracellular stores has been recently shown to
nduce perinuclear clustering of mitochondria [25], which is com-arable to the perinuclear “I-Bodies” formation in etoposide treatedMG-1 cells observed here (Fig. 1a). Further, the lack of perinu-lear “I-Bodies” formation when the uniporter was inhibited using
A23187 in the membranous structure of mitophagic vacuole encircling damaged
Ruthenium red (Fig. 4a), confirmed that “I-Bodies” are indeed irre-versibly damaged mitochondria.
Etoposide enhances mitochondrial potential (hyper-polarization) and activates anti-oxidant defence as initial responseto the stress [25]. These energized mitochondria with intact
membrane potential then clear elevated Ca2+ in the cytoplasm.However, excessive stress results in mitochondrial overloadingof Ca2+, exceeding its buffering capacity, and leading to irre-versible damage. This results in clustering of functionally impaired
alcium
mcrimriiprftaa
acmcdlaarcficicgpmflwem
iCdrcvsBrtf“mtatcMocmcitgiB“
[
[
[
[
[
[
[
[
[
[
A. Verma et al. / Cell C
itochondria, leading to aggregation primarily in the perinu-lear region [25]. Interestingly, FCCP (an uncoupler) significantlyeduced the “I-Bodies” in etoposide treated cells (Fig. 5d andnset images) suggesting that balanced redox status linked to
aintained mitochondrial membrane potential is an obligatoryequirement for “I-Bodies” formation. Lack of “I-Bodies” formationn cells treated with cisplatin, paclitaxel and doxorubicin, whichnduces ROS dependent reduction in mitochondrial membraneotential along with elevated cytoplasmic calcium levels (Fig. 5a),eiterated that mitochondrial membrane potential is a criticalactor in “I-Bodies” formation. This was substantiated further byhe reappearance of “I-Bodies” when cells were pre-treated withntioxidants like pyruvate and NAC before exposure to thesegents (Fig. 5b, c).
Mitochondrial autophagy (mitophagy or mitoptosis) [51] is andaptive metabolic response, by which damaged mitochondriaan be catabolized either to remove defective structures or as aeans of providing macromolecules for energy generation under
onditions of nutrient starvation and hypoxia, which is a processependent on hypoxia inducible factor (HIF-1�) [52]. This is also in
ine with the observations that most cancer cells are associated with strong HIF signaling linked to reduced respiratory metabolismnd enhanced glycolysis, the seventh hallmark of cancer [53]. Ouresults demonstrating endogenous “I-Bodies” formation in all theancer cells investigated provide a link between mitochondrial dys-unction and cancer. It also suggest that the presence of “I-Bodies”n cancer cells could be due to the snapshot view of ongoing mito-hondrial autophagy process at any given time or genetic defectn autophagic clearance of dysfunctional mitochondria in cancerells similar to Parkinson’s disease [41]. These observations sug-est that there is a strong possibility that the bilayer membraneroviding scaffold to A23187 in “I-Bodies” formation could be theitophagic vacuole encircling the dysfunctional mitochondria and
uorescent labeling of similar perinuclear clusters on incubationith MDC (probe for staining autophagosomes) (Fig. 7a) [54] in
toposide treated cells validated the hypothesis that “I-Bodies” areitophagic vacuoles encircling damaged mitochondria.The sequence of events of “I-Bodies” formation is summarized
n Fig. 7b. Endogenous or induced stress stimulates the release ofa2+ from intracellular stores, energized and polarized mitochon-ria sequesters the released Ca2+ leading to excess Ca2+ overloadingesults in mitochondrial swelling and loss of membrane potentialausing irreversible damage to the mitochondria. The mitophagicacuole may then envelop these damaged mitochondria providingcaffold for the high density packing of A23187 that appear as “I-odies”. Because “I-Bodies” represent the fraction of mitochondriaendered dysfunctional due to stress induced calcium accumula-ion and overload, “I-Bodies” (or A23187) can also be used as a probeor identifying the presence of dead mitochondria in cells. Further,I-Bodies” status can also be used to predict the Ca2+ mediateditochondrial dependent cell death under various stress condi-
ions. A strong correlation between the abundance of “I-Bodies”nd flow cytometrically measured blue fluorescence suggests thathis can be used as a rapid assay for enumerating the physiologi-al status of cells, complementing other biochemical parameters.oreover, our results not only elucidate the complete mechanism
f endogenous and exogenous stress induced perinuclear mito-hondrial aggregation in cells, but also prompt us to propose thatitochondrial aggregation observed in various patho-physiological
onditions viz. cancer, and Parkinson’s disease is due to stressnduced persistent Ca2+ release/leak from intracellular stores. Dis-urbances in calcium homeostasis and mitochondrial dysfunction
enerated during the process of carcinogenesis appear to favoronophore aggregation in cells forming “I-Bodies”. Therefore, “I-odies” can also be a potential biomarker of cancer. Moreover,I-Bodies” formation and enhanced A23187 fluorescence observed
[
50 (2011) 510– 522 521
in PBMCs of tumor bearing mice can be very useful as it canbe exploited to quickly identify the presence of occult malignantlesions with a simple blood analysis, which may then be followedby detailed investigations using appropriate imaging modalities(Fig. 6).
Conflict of interest
None declared.
Acknowledgements
This work was supported by grants (INM 301, 311/1.4) fundedby Defence Research and Development Organisation, Governmentof India. A.V. was supported with a fellowship from Indian Coun-cil of Medical Research. We acknowledge Dr. Sudhir Chandna forextending the fluorescence microscope facility, Dr Seema Gupta foruseful discussions and Dr. R.P. Tripathi, Director INMAS for constantsupport and encouragement.
Appendix A. Supplementary data
Supplementary data associated with this article can be found, inthe online version, at doi:10.1016/j.ceca.2011.08.007.
References
[1] P.W. Reed, H.A. Lardy, A23187: a divalent cation ionophore, J. Biol. Chem. 247(1972) 6970–6977.
[2] M.J. Zmijewski Jr., R. Wong, J.W. Paschal, D.E. Dorman, The biosynthesis ofantibiotic A23187, Tetrahedron 39 (1983) 1255–1263.
[3] M.O. Chaney, P.V. Demarco, N.D. Jones, J.L. Occolowitz, Letter: the structure ofA23187, a divalent cation ionophore, J. Am. Chem. Soc. 96 (1974) 1932–1933.
[4] S.V. Balasubramanian, K.R.K. Easwaran, Aggregation of calcium ionophore(A23187) in phospholipid vesicles, Biochem. Biophys. Res. Commun. 158 (1989)891–897.
[5] M.O. Chaney, N.D. Jones, M. Debono, The structure of the calcium complex ofA23187, a divalent cation ionophore antibiotic, J. Antibiot. 29 (1976) 424–427.
[6] A.H. Caswell, B.C. Pressman, Kinetics of transport of divalent cations acrosssarcoplasmic reticulum vesicles induced by ionophores, Biochem. Biophys. Res.Commun. 49 (1972) 292–298.
[7] M.A. Kolber, D.H. Haynes, Fluorescence study of the divalent cation-transportmechanism of ionophore A23187 in phospholipid membranes, J. Biophys. 36(1981) 369–391.
[8] G.D. Case, J.M. Vanderkooi, A. Scarpa, Physical properties of biological mem-branes determined by the fluorescence of the calcium ionophore A23187, Arch.Biochem. Biophys. 162 (1974) 174–185.
[9] J.S. Puskin, A.I. Vistnes, M.T. Coene, A fluorescence study of A23187 interactionwith phospholipid vesicles, Arch. Biochem. Biophys. 206 (1981) 164–172.
10] J.D. Bartlett, J.D. Luethy, S.G. Carlson, S.J. Sollott, N.J. Holbrook, Calciumionophore A23187 induces expression of the growth arrest and DNA dam-age inducible CCAAT/enhancer-binding protein (C/EBP)-related gene, gadd153.Ca2+ increases transcriptional activity and mRNA stability, J. Biol. Chem. 267(1992) 20465–20470.
11] J.E. Westhead, Semiautomated turbidimetric bioassay for the ionophoreA23187, Antimicrob. Agents Chemother. 11 (1977) 916–918.
12] P.S. Brookes, Y. Yoon, J.L. Robotham, M.W. Anders, S.S. Sheu, Calcium, ATP, andROS: a mitochondrial love–hate triangle, Am. J. Physiol. Cell Physiol. 287 (2004)C817–C833.
13] E. Carafoli, Calcium signaling: a tale for all seasons, Proc. Natl. Acad. Sci. U.S.A.99 (2002) 1115–1122.
14] T.E. Gunter, L. Buntinas, G. Sparagna, R. Eliseev, K. Gunter, Mitochondrial cal-cium transport: mechanisms and functions, Cell Calcium 28 (2000) 285–296.
15] T.E. Gunter, D.I. Yule, K.K. Gunter, R.I. Eliseev, J.D. Salter, Calcium and mitochon-dria, FEBS Lett. 567 (2004) 96–102.
17] G. Szabadkai, M.R. Duchen, Mitochondria: the hub of cellular Ca2+ signalling,Physiology Bethesda 23 (2008) 84–94.
18] I.P. De Castro, L.M. Martins, R. Tufi, Mitochondrial quality control and neu-rological disease: an emerging connection, Expert Rev. Mol. Med. 12 (2010)1–20.
19] P. Workman, P. Twentymen, F. Balkwill, et al., United Kingdom Co-ordinating
Committee on Cancer Research (UKCCCR) guidelines for the welfare of animalsin experimental neoplasia (ed. 2), Br. J. Cancer 77 (1998) 1–10.
20] S. Gupta, R. Mathur, B.S. Dwarakanath, The glycolytic inhibitor 2-deoxy-d-glucose enhances the efficacy of etoposide in Ehrlich ascites tumor-bearingmice, Cancer Biol. Ther. 4 (2005) 87–94.
21] J.O. Hensold, G. Dubyak, D.E. Housman, Calcium ionophore, A23187, inducescommitment to differentiation but inhibits the subsequent expression of ery-throid genes in murine erythroleukemia cells, Blood 77 (1991) 1362–1370.
23] P.L. Rockwell, B.T. Storey, Determination of the intracellular dissociation con-stant, Kd, of the Fluo-3–Ca2+ complex in mouse sperm for use in estimatingintracellular Ca2+ concentrations, Mol. Reprod. Dev. 54 (1999) 418–428.
24] D.F. Babcock, J. Herrington, P.C. Goodwin, Y.B. Par, B. Hille, Mitochondrial par-ticipation in the intracellular Ca2+ network, J. Cell Biol. 136 (1997) 833–844.
25] N. Haga, N. Fujita, T. Tsuruo, Mitochondrial aggregation precedes cytochrome crelease from mitochondria during apoptosis, Oncogene 22 (2003) 5579–5585.
26] C.L. Moore, Specific inhibition of mitochondrial Ca2+ transport by rutheniumred, Biochem. Biophys. Res. Commun. 42 (1971) 298–305.
27] K.C. Reed, F.L.A. Bygrave, A low molecular weight ruthenium complex inhibitoryto mitochondrial Ca2+ transport, FEBS Lett. 46 (1974) 109–114.
28] W.L. Ying, J. Emerson, M.J. Clarke, D.R. Sanadi, Inhibition of mitochondrial cal-cium ion transport by an oxo-bridged dinuclear ruthenium ammine complex,Biochemistry 30 (1991) 4949–4952.
29] J. Herrington, Y.B. Park, D.F. Babcock, B. Hille, Dominant role of mitochondriain clearance of large Ca2+ loads from rat adrenal chromaffin cells, Neuron 16(1996) 219–228.
30] H. Oubrahim, E.R. Stadtman, P.B. Chock, Mitochondria play no roles in Mn(II)-induced apoptosis in HeLa cells, Proc. Natl. Acad. Sci. U.S.A. 98 (2001)9505–9510.
31] T.E. Gunter, D.R. Pfeiffer, Mechanisms by which mitochondria transport cal-cium, Am. J. Physiol. 258 (1990) C755–C786.
32] K.K. Gunter, T.E. Gunter, Transport of calcium by mitochondria, J. Bioenerg.Biomembr. 26 (1994) 471–485.
33] J.F. Kidd, M.F. Pilkington, M.J. Schell, et al., Paclitaxel affects cytosolic cal-cium signals by opening the mitochondrial permeability transition pore, J. Biol.Chem. 277 (2002) 6504–6510.
34] F. Splettstoesser, A.M. Florea, D. Busselberg, IP (3) receptor antagonist, 2-APB,attenuates cisplatin induced Ca2+ influx in HeLa-S3 cells and prevents acti-vation of calpain and induction of apoptosis, Br. J. Pharmacol. 151 (2007)1176–1186.
35] K.V. Norren, V.A. Helvoort, J.M. Argiles, et al., Direct effects of doxorubicin onskeletal muscle contribute to fatigue, Br. J. Cancer 100 (2009) 311–314.
36] N.A. Santos, C.S. Catao, N.M. Martins, et al., Cisplatin-induced nephrotoxicity isassociated with oxidative stress, redox state unbalance, impairment of ener-getic metabolism and apoptosis in rat kidney mitochondria, Arch. Toxicol. 81(2007) 495–504.
37] T.N. Seyfried, L.M. Shelton, Cancer as a metabolic disease, NutrMetab (Lond). 7(2010) 7.
38] G. Amuthan, G. Biswas, K.H. Ananadatheerthavarada, et al., Mitochondrialstress-induced calcium signaling, phenotypic changes and invasive behaviorin human lung carcinoma A549 cells, Oncogene 21 (2002) 7839–7849.
[
[
50 (2011) 510– 522
39] R. Roy, S.M. Singh, A. Shanker, Mechanism of thymocyte apoptosis induced byserum of tumor-bearing host: the molecular events involved and their inhibi-tion by thymosin alpha-1, Int. J. Immunopharmacol. 22 (4) (2000) 309–321.
40] G. Jyothi, A. Surolia, K.R.K. Easwaran, A23187—channel behaviour: fluorescencestudy, J. Biosci. 19 (1994) 277–282.
41] S. Geisler, K.M. Holmnstrom, D. Skujat, et al., PINK1/Parkin-mediatedmitophagy is dependent on VDAC1 and p62/SQSTM1, Nat. Cell Biol. 12 (2010)119–131.
42] N. Kajitani, H. Kobuchi, H. Fujita, et al., Mechanism of A23187-inducedapoptosis in HL-60 cells: dependency on mitochondrial permeability tran-sition but not on NADPH oxidase, Biosci. Biotechnol. Biochem. 71 (2007)2701–2711.
43] A. Bergner, J. Kellner, A. Tufman, R.M. Huber, Endoplasmic reticulum Ca2+ home-ostasis is altered in small and non-small cell lung cancer cell lines, J. Exp. Clin.Cancer Res. 28 (2009) 1–7.
44] I. Kaddour-Djebbar, V. Choudhary, C. Brooks, et al., Specific mitochondrial cal-cium overload induces mitochondrial fission in prostate cancer cells, Int. J.Oncol. 36 (2010) 1437–1444.
45] C. Serhan, P. Anderson, E. Goodman, P. Dunham, G. Weissmann, Phosphatidateand oxidized fatty acids are calcium ionophores. Studies employing arsenazoIII in liposomes, J. Biol. Chem. 256 (1981) 2736–2741.
46] J.B.A. Custodio, C.M.P. Cardosoa, L.M. Almeida, Thiol protecting agents andantioxidants inhibit the mitochondrial permeability transition promoted byetoposide: implications in the prevention of etoposide-induced apoptosis,Chem. Biol. Interact. 140 (2002) 169–184.
47] J. Neustadt, S.R. Pieczenik, Medication-induced mitochondrial damage and dis-ease, Mol. Nutr. Food Res. 52 (7) (2008) 780–788.
48] S. Desagher, J.C. Martinou, Mitochondria as the central control point of apopto-sis, Trends Cell Biol. 10 (9) (2000) 369–377.
49] S. Kim, H.Y. Kim, S. Lee, al. et, Hepatitis B virus X protein induces perinuclearmitochondrial clustering in microtubule- and dynein-dependent manners, J.Virol. 81 (4) (2007) 1714–1726.
50] K. De Vos, E. Gossens, E. Boone, et al., The 55-kDa tumor necrosis factor receptorinduces clustering of mitochondria through its membrane-proximal region, J.Biol. Chem. 273 (1998) 9673–9680.
51] K.G. Lyamzaeva, O.K. Nepryakhina, V.B. Saprunova, et al., Novel mechanism ofelimination of malfunctioning mitochondria (mitoptosis): formation of mitop-totic bodies and extrusion of mitochondrial material from the cell, Biochim.Biophys. Acta 1777 (7-8) (2008) 817–825.
52] H. Zhang, M. Bosh-Marse, L.A. Shimoda, et al., Mitochondrial autophagy is anHIF-1 dependent adaptive metabolic response to hypoxia, J. Biol. Chem. 283(16) (2008) 10892–10903.
53] G. Kroemer, J. Pouyssegur, Tumor cell metabolism: cancer’ Achilles’ heel, CancerCell. 13 (2008) 472–482.
54] C. Roberta, J. Davies, S. Beaucourt, C. Isidoro, Murphy, Autophagy-dependentcell survival and cell death in an autosomal dominant familial neurohypophy-seal diabetes insipidus in vitro model, FASEB 19 (2005) 1021–1023.
