Cell Death and Differentiation — Early mitochondrial alterations in ATRA-induced…

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Original Paper

Cell and Differentiation (2006) 13, 119–128. published online 8 July

Early mitochondrial alterations in cell …

Edited by R De

J Schmidt-Mende 1 ,2. V Gogvadze 1. E Hellström-Lindberg 2 and B 1

1 Institute of Environmental Medicine, of Toxicology, Karolinska Institutet, Box Stockholm SE-171 77, Sweden 2 of Medicine, Division of Hematology, University Hospital Huddinge, SE-141 86, Sweden

Correspondence: J Institute of Environmental Medicine, of Toxicology, Karolinska Institutet, Box Stockholm SE-171 77, Sweden. +46 852487554; Fax: +46 8 329041; Jan.Schmidt-Mende@medhs.ki.se


All- retinoic acid (ATRA) differentiation and subsequent apoptosis in a of cell lines. Using the cell line P39, we that ATRA disturbs functional activity long any detectable signs of apoptosis These early changes diminished mitochondrial oxygen decreased calcium uptake by and as a result, a lower mitochondrial calcium concentration.

Granulocyte factor (G-CSF) increases respiration and calcium accumulation and subsequently blocks ATRA-induced Nifedipine, a plasma membrane channel blocker, inhibits changes, such as the loss of the membrane potential and activation of Thus, the properties of ATRA and to modulate mitochondrial respiration and calcium control are novel which give insight their precise molecular of action.


APL, promyelocytic leukemia; ATRA, trans retinoic acid; carbonyl cyanide m -chlorophenylhydrazone; ER, reticulum; G-CSF, granulocyte factor; MDS, myelodysplastic MPT, mitochondrial permeability NBT, nitroblue-tetrazolium test; respiratory control ratio; tetramethylphenylenediamine; TMRE, tetramethylrhodamine

All- trans retinoic (ATRA) is a natural derivative of A, which is successfully used in the of acute promyelocytic leukemia where it induces terminal and apoptosis of leukemic cells the 15;17 translocation. 1. 2. 3. 4 We and others shown that ATRA maturation followed by apoptosis in a of different cell lines. 5. 6. 7. 8 induced by binding of ATRA to its retinoid receptors is detectable 24–48 h 9 followed by apoptosis at h. 5

It has been shown that play an important regulatory in ATRA-induced apoptosis. In this activation of proapoptotic Bcl-2 proteins, such as Bid and Bax, in permeabilization of the outer mitochondrial and cytochrome c release. 10 ATRA was to downregulate the expression of the antiapoptotic Bcl-2, 11.

12. 13 which is known to cell … by interaction proapoptotic Bcl-2 family However, in ATRA-treated cells, the triggering mechanism leading to the of Bid and Bax remains to be clarified.

Using rat liver mitochondria, it has been that ATRA can directly mitochondrial functioning, 14. 15 although the site of this interaction is

Granulocyte colony-stimulating factor has been shown to inhibit … in ATRA-treated myeloid 8 Furthermore, it reduces spontaneous of neutrophils in vitro. 16.

17 In vivo . the effects of G-CSF are also to its antiapoptotic properties. It is routinely to overcome neutropenia after and to treat severe congenital in Kostmann syndrome. 18 In anemic with myelodysplastic syndromes G-CSF and erythropoietin may synergistically hemoglobin levels and reduce progenitor apoptosis. 19.

20. 21 We have shown that G-CSF cytochrome c release from to cytosol, thereby reducing activation and premature … of MDS erythroblasts. 22.

23 However, the precise of the antiapoptotic action of G-CSF is unclear.

Since calcium has shown to enhance ATRA-induced 24. 25 alterations in intracellular calcium could be responsible for ATRA-induced as well as for the observed rapid effects of G-CSF. Calcium is an cellular second messenger, can regulate cell proliferation, 26 function 27 as well as differentiation 28 and

29 In our experiments, we investigated mitochondrial parameters, such as respiration and uptake capacity, as well as apoptosis-associated parameters (involvement of loss of m . release of cytochrome c ) in the and/or absence of the growth G-CSF. Here, we show the of mitochondrial functioning and mitochondrial regulation for ATRA-induced cell

ATRA inhibits mitochondrial and decreases mitochondrial capacity to calcium long before any of apoptosis occur. The importance of in ATRA-induced cell … is by the fact that the calcium blocker Nifedipine can inhibit The growth factor G-CSF mitochondrial respiration and calcium capacity, and reduces apoptosis in myeloid leukemia cells.

The ability of G-CSF to influence oxidation and calcium uptake is a novel important finding, offers a new explanation for its antiapoptotic of action.


G-CSF ATRA-induced apoptosis

Treatment of P39 cells with ATRA in differentiation after 24–48 h of (data not shown) and apoptosis 72 h. At this time point, was characterized by the release of cytochrome c the cytosol (Figure 2b ), caspase and nuclear condensation (Figure 1a and b ). ATRA induced time-dependent of full-length Bid protein, and cleavage of proapoptotic protein Bax (Figure 2a ). a clear disappearance of full-length Bid was observed in our experiments, we were not to detect the cleavage product of protein, tBid, as reported by group. 10 G-CSF was able to all these manifestations of apoptosis.

In to disappearance of the full-length Bid and cleavage of ATRA induced downregulation of protein Bcl-2 started at day 1 and in almost complete disappearance of the at day 3 (Figure 2a ). Importantly, this of Bcl-2 was not influenced by G-CSF. downregulation of Bcl-2 was observed in NB-4 cells (data not

Involvement of mitochondria in ATRA-induced ( a ) Downregulation of Bcl-2, disappearance of Bid and cleavage of Bax upon treatment ATRA (5 M). Bax cleavage and disappearance of Bid can be blocked by G-CSF. Cells harvested at the indicated time and the expression of proteins of interest was by Western blot.

Loading was by staining of the membranes with Abs G3PDH. ( b ) ATRA induces and prevents cytochrome c release. extracts were prepared at 3 and 4 as described in Materials and Methods. 1: control; lane 2: 1 M ATRA; 3: 1 M ATRA+G-CSF; lane 4: 5 M ATRA; 5: 5 M ATRA+G-CSF. ( c ) Submaximal shift of the membrane potential in ATRA-treated (5 M) compared to control cells.

were harvested and stained for 30 min TMRE at 37-C and analyzed by Decrease in FL-2 channel correlates with a decrease of the membrane potential. Overlay are shown after 2 and 3 days of onset. ( d ) ATRA induces a of the mitochondrial membrane potential. were harvested and stained for 30 min TMRE at 37-C and analyzed by

Decrease in FL-2 channel correlates with a drop of the membrane potential after 3 and 4 of treatment onset. Representative from one out of four independent are shown

G-CSF inhibits apoptosis. ( a ) ATRA induces in P39 cells after 3 and 4 days of Cells were stained May–Grünwald–Giemsa and the percentage of apoptotic was estimated by counting at least 200 ( b ) ATRA induces caspase-3-like Caspase-3-like activity was estimated by the of the fluorogenic substrate DEVD-AMC Materials and Methods).

The amount of of AMC released per minute is shown. data from one out of four experiments are shown

Full and legend (76K )

One of the earliest observed in ATRA-treated cells was a shift in the mitochondrial membrane ( m ), which was detectable at day 1 (data not and day 2 (Figure 2c ). Later, at days 3 and 4, a of cells with completely m appeared (Figure 2c (day 3) and d). complete loss of m was comparable the loss of m in cells treated the mitochondrial uncoupler carbonyl m -chlorophenylhydrazone (CCCP) (data not

G-CSF but not z-VAD or calpain markedly prevented the collapse of m 3a ). As mentioned above, G-CSF inhibited cleavage of Bax. It has shown that Bax can be cleaved by caspases or calpain. 30. 31 However, the conditions of our experiments, neither the inhibitor z-VAD-fmk nor different inhibitors such as E64d, and calpeptin were able to cleavage of Bax (Figure 3b ).

z-VAD-fmk and the inhibitor E64d do not inhibit changes in ATRA-induced (5 M) apoptosis. ( a ) were harvested after 4 in culture and stained for 30 min with at 37-C and analyzed by FACS Decrease in FL-2 fluorescence with a decrease of the mitochondrial potential. ( b ) Bax cleavage cannot be by calpain or caspase inhibitors.

were harvested after 4 in culture and Bax protein expression was by Western blot. Loading was by staining of membranes with Abs G3PDH. Lane 1: control; 2: 5 M ATRA; lane 3: 5 M ATRA+G-CSF; 4: 5 M ATRA+10 M z-VAD-fmk; lane 5: 5 M M E64d. Representative data one out of four independent experiments are

Full figure and legend )

Effect of ATRA and G-CSF on oxygen consumption

Mitochondria up m via oxidation of substrates in the mitochondrial chain. In order to understand the underlying early submaximal in m . the effect of ATRA on mitochondrial consumption was studied.

Experiments isolated rat liver mitochondria that ATRA decreased dependently state 3 respiration as ADP-stimulated respiration), whereas 4 respiration was dose dependently indicating that ATRA uncouples mitochondria (Figure 4 ). respiratory control ratio value was significantly decreased.

of ATRA on oxygen consumption and RCR in rat liver mitochondria. Rat liver were isolated as described in and Methods. Oxygen consumption in mitochondrial protein was estimated an oxygen electrode after the of 5, 12.5 and 25 M ATRA.

State 3 after the addition of ADP) and 4 (V4) respiration were to calculate RCR. Representative from one out of three independent are shown

Full figure and (23K )

Inhibition of respiration has shown to sensitize mitochondria to calcium-induced MPT. 32 As expected, alteration of mitochondrial respiration in deterioration of mitochondrial calcium capacity, which was assessed a calcium-sensitive electrode (see and Methods). ATRA (25 M) significantly spontaneous release of calcium mitochondria (data not shown).

P39 cells were cultured in the of ATRA (5 M) for 48 h, ATRA significantly oxygen consumption of these (Figure 5a ). To investigate in detail respiration complexes were affected, ATRA-treated cells permeabilized with low concentrations of as described in Materials and Methods and mitochondrial respiratory substrates provided. Pyruvate plus were used as substrates of I, succinate in the presence of rotenone for II and ascorbate with tetramethylphenylenediamine for complex IV.

ATRA induced of mitochondrial respiration, which was already at day 1 and became significant at day 2 5b and c ). State 3, state 4 and uncoupled in ATRA-treated cells were suggesting that the mitochondrial was generally compromised by ATRA 5b ). Importantly, coincubation with markedly restored mitochondrial both in intact and permeabilized Restoration of respiration was observed all substrates and under all states of (state 3, state 4 as well as respiration, Figure 5a–c ). early reduction of respiration ATRA treatment was observed in the APL line NB-4 (data not

Effect of G-CSF on reduced consumption in permeabilized P39 cells 2 days of incubation with 5 M ( a ) Oxygen consumption of nonpermeabilized kept in the standard medium. ( b ) cells in KCl buffer with as electron donator in the presence of V3 respiration was measured after the of ADP, V4 after the addition of and uncoupled respiration after the of CCCP. ( c ) Permeabilized cell in KCl ascorbate/TMPD were used as donator. Representative data one out of three independent experiments are

Full figure and legend )

G-CSF restores ATRA-induced of mitochondrial calcium buffering in cells

In order to elucidate how respiration could affect functioning, the ability of mitochondria to calcium in ATRA-treated cells was The accumulation of calcium into was assessed with a calcium-sensitive Cells were permeabilized as in Materials and Methods and loaded calcium pulses as shown in 6a until mitochondrial permeability (MPT) occurred and all accumulated was released.

ATRA markedly the ability of mitochondria to accumulate and decreased the threshold level of which is necessary for MPT induction 6b ). These changes were at early time points of (days 1 and 2) when apoptosis had not yet G-CSF significantly restored calcium uptake (data not

A similar reduction of mitochondrial accumulation capacity was observed in cells upon ATRA and occurred long before any of apoptosis were detected. In cell line, G-CSF increased the mitochondrial calcium capacity nor suppressed apoptosis at time points (data not

Reduced mitochondrial calcium and mitochondrial calcium concentration in cells (5 M) after 2 days of ( a and b ) Cells were permeabilized digitonin and calcium uptake was with a calcium-sensitive electrode addition of calcium into incubation buffer. Representative from one out of four independent are shown. ( c ) The cationic fluorescent indicator dye Rhod-2 accumulates in mitochondria, since it is highly and therefore readily retained by the mitochondria, which localize the nucleus.

Representative data from one out of independent experiments are shown. ( d ) of mitochondrial calcium load by fluorescence (shown as the percentage of cell load). Mean and of five independent experiments are For each experiment, mitochondrial content of control cells was set as

Full figure and legend )

Nifedipine inhibits ATRA-induced

Mitochondrial deterioration observed in cells are frequently caused by of calcium-dependent MPT as a result of a disturbed calcium homeostasis. 32 In order to whether extracellular calcium is in ATRA-induced mitochondrial changes, were coincubated with a plasma membrane calcium blocker. Nifedipine had no effect on differentiation as determined by the nitroblue-tetrazolium (NBT, data not shown).

Nifedipine markedly prevented and significantly suppressed appearance of its manifestations such as the collapse of m 7a ), cleavage of Bax (Figure 7b ), activation of reactive oxygen species production and the accumulation of condensed (data not shown). This effect of Nifedipine was dose and most prominent at 50 M. The intracellular chelator EGTA-AM had similar when it was used instead of (data not shown).

In contrast to Nifedipine did not restore mitochondrial decreased by ATRA. Moreover, it decreased mitochondrial respiration by In addition, Nifedipine markedly calcium accumulation in mitochondria at 25 M, the that did not affect respiration not shown).

Nifedipine blocks changes in apoptotic cells 3 days of incubation with (5 M). ( a ) Cells were harvested and for 30 min with TMRE at 37-C and by FACS cytometry. Decrease in fluorescence correlates with a of the mitochondrial membrane potential. ( b ) Bax Cells were harvested and Bax expression was investigated by Western

G3PDH counterstaining was used as an loading control. Representative from one out of four independent are shown

ATRA does not early changes in the cytosolic concentrations

Since suppression of channels by Nifedipine prevented apoptosis, it was of interest to test ATRA affects cytosolic concentration. Experiments using stained with Fluo-4 by FACS analysis revealed at early time points 1 and 2; data not shown), no increase of calcium concentrations was observed in the cells. At late time (days 3 and 4; Figure 8a ), raise of calcium concentrations was detected, occurred simultaneously with of apoptosis. After gating the cell population by forward scatter criteria, it became that calcium levels increased in the apoptotic cells (Figure 8b ). As expected, Nifedipine this late calcium (Figure 8c ).

Significance of intracellular for ATRA-induced apoptosis. ( a ) Increased content in apoptotic cells 4 days of ATRA treatment (5 M) by Fluo-4 fluorescence. ( b ) Gated and nonapoptotic cell populations 3 days of culture; calcium in apoptotic cells measured by fluorescence. ( c ) Nifedipine inhibits and the calcium rise in ATRA-treated after 3 days of culture as by Fluo-4 fluorescence. ( d ) Thapsigargin the effect of low-dose ATRA Cells were harvested 4 days of culture and stained for 30 min TMRE at 37-C and analyzed by cytometry. Decrease in FL-2 correlates with a drop of the membrane potential. Representative from one out of three independent are shown

Full figure and (56K )

In order to prove calcium is important for ATRA-induced we cocultured the cells with 100 nM (a concentration that is more 10-fold lower than the usual concentration used) and (5 nM), which blocks accumulation into the endoplasmic (ER) and thereby stimulates the of calcium from the ER. At these ATRA or thapsigargin alone unable to induce apoptosis. combination of both drugs effects similar to those by high concentrations of ATRA 8d ).


It has been previously that ATRA induces … via targeting mitochondria. 7 the precise mechanism of mitochondrial in ATRA-induced apoptosis remains In the present study, several characterizing proper mitochondrial (respiration and calcium buffering as well as parameters important for and execution of mitochondria-mediated killing of the proapoptotic protein Bax, c release and drop of mitochondrial potential) were studied in A clear link between changes (24–48 h) in mitochondrial and the later apoptotic changes h) was observed.

Mitochondria play a pivotal in many models of apoptosis. of proteins such as cytochrome c AIF from the intermembrane space of is a key event in initiation and/or of apoptosis. 33 We have previously that cytochrome c release can via distinct mechanisms that are calcium dependent or independent.

34 In one calcium promotes MPT due to opening of a pore in the inner mitochondrial This results in mitochondrial and a rupture of the outer mitochondrial

In contrast, calcium-independent permeabilization of the mitochondrial membrane occurs due to of proapoptotic proteins, such as Bax or in the outer membrane with the of a pore large enough for cytochrome c . Interestingly, according to our Bax not only forms pores in the membrane of mitochondria but also calcium-dependent MPT. Thus, we can that release of cytochrome c . starts 72 h after administration of results from calcium-dependent MPT

Indeed, ATRA-treated cells inhibition of respiration, which was both in intact and digitonin-permeabilized (Figure 5 ). This inhibition in a submaximal shift of m (Figure 2c ). It is that a general decrease in the membrane potential is an important responsible for MPT. 32 Despite a magnitude of this shift, it a substantial decrease in the threshold of calcium required for MPT induction 6b ), when mitochondria in permeabilized were loaded with

Recent experiments with mitochondria showed that a suppression of mitochondria respiration by markedly stimulated MPT induction. 35 mitochondria displaying high m can calcium fluctuations occurring cell functioning. However, a of mitochondrial respiration and consequent of m could be a factor stimulating MPT in a of mitochondria, especially taking consideration their heterogeneity.

at day 3 after treatment with we observed a subpopulation of cells m collapsed to the levels comparable to the CCCP-induced drop (Figure 2d ). it is known that downregulation of 36 and cleavage of Bax 37 sensitize mitochondria MPT induction, it is likely that downregulation of Bcl-2, which within 24 h upon ATRA and Bax cleavage, which occurred 72 h, contribute to sensitization of mitochondria MPT induction. This early downregulation upon ATRA was also detected in promyelocytic cells and preceded the onset of features (data not shown).

presented here propose insights in the apoptosis inhibitory of G-CSF and the L-type calcium blocker Nifedipine. Even both drugs blocked apoptosis and did not decrease differentiation by the NBT test), their mode of was completely different. G-CSF mitochondrial respiration and normalized calcium accumulation capacity.

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did not influence the expression levels of when added to control or cultures (data not shown). in NB-4 cells opposite to P39 G-CSF did not prevent the early changes in respiration and calcium capacity and consequently did not block at later time points.

results confirm the recent that in APL cell lines facilitates differentiation and thereby the maturation and apoptosis program by ATRA. 38. 39

In contrast to GCSF, had no apparent effect on mitochondrial However, Nifedipine rendered of cells via blocking calcium preventing flooding the cytosol calcium and therefore decreasing the for calcium-induced MPT. Similar were obtained when the calcium chelator EGTA-AM was in order to prevent fluctuations of in the cytoplasm (data not shown).

the protective effect of Nifedipine mitochondrial calcium overloading and MPT induction comes from the of Nifedipine to suppress mitochondrial accumulation.

Further evidence of the of calcium in ATRA-induced apoptosis was when noninducing apoptosis of ATRA was administered to cells in the of thapsigargin, an inhibitor of calcium of ER. Thapsigargin stimulates release of from ER and elevates cytosolic concentration. Combined action of low of ATRA and thapsigargin brought the same effect as apoptosis-inducing doses of ATRA (Figure 8d ).

In we also addressed the question ATRA has a direct effect on (Figure 4 ). Incubation of ATRA isolated rat liver mitochondria a decrease in state 3 respiration, state 4 respiration was increased. using permeabilized P39 cells, we did not a specific decrease in state 3 after incubation with This drug reduced function without any preference for any states.

Furthermore, direct effects on required higher ATRA (12.5 and 25 M), which cannot be in the cytosol, since ATRA to cytosolic retinoid-binding proteins. 40 we concluded that a direct interaction might be relatively and the observed effects on mitochondrial 14. 15 might not occur on cellular

In conclusion, both ATRA and have significant influence on as well as mitochondrial calcium (Figure 9 ). While the expression and of Bcl-2 family members in and G-CSF-treated cells have extensively studied by many we, for the first time, observed mitochondrial alterations and linked early alterations to the late events. Furthermore, we introduce calcium deregulation as a novel and mediator of ATRA-induced apoptosis.

We that results obtained in study are important for understanding the mechanisms of G-CSF and ATRA in more details. These also provide new guidelines for and benefits in the clinical use of these

Proposed sequence of ATRA-mediated and late mitochondrial changes. ATRA-induced alterations are characterized by mitochondrial respiration and calcium capacity. Late changes are by the activation of Bcl-2 family such as Bid and Bax, followed by c release from mitochondria. caspases are activated and apoptosis is

G-CSF and Nifedipine inhibit but their mode of action is G-CSF inhibits early changes, while Nifedipine calcium content in the cells

figure and legend (40K )

and Methods

Antibodies and Western experiments

Samples were with Laemmli’s loading boiled for 5 min and subjected to 15% SDS-PAGE at 130 V by electroblotting to nitrocellulose for 2 h at 100 V. Membranes blocked for 1 h with 5% nonfat in phosphate-buffered saline (PBS) at temperature and subsequently probed with primary antibody. The were rinsed and incubated a horseradish peroxidase-conjugated secondary (1. 10 000).

Following incubation secondary antibody, membranes rinsed and bound antibodies detected using enhanced according the manufacturer’s instructions. For of cytosolic extracts, cells permeabilized with 0.005% and incubated for 5 min on ice in a buffer containing 150 mM 5 mM KH 2 PO 4 . 1 mM MgSO 4 . 5 mM succinate, 5 mM Tris and 1 mM at pH 7.4.

Subsequently, whole-cell or supernatant and pellet fractions analyzed by SDS-PAGE followed by blotting using as a primary raised against cytochrome c (1. BD Pharmingen, BD Bioscience, San Diego, Bax (1. 1000, BD Pharmingen, BD Bioscience, San USA), Bid (1. 1000, Cell Technology, Beverly, USA), (1. 100, Dako, Glostrup, and G3PDH (1.

2000, Nordic Täby, Sweden).

Measurements fluorescent dyes

The mitochondrial potential ( m ) was measured using the fluorescent dye tetramethylrhodamine ethyl (TMRE, final concentration 25 nM, Probes, Leiden, The Netherlands), normally accumulates in mitochondria as a function of m . Changes in intracellular concentrations were visualized by the dye (5 M, Molecular Probes, Leiden, The Rhod-2-AM (2.5 M, MoBiTec, Germany) was used to assess calcium.

Briefly, 5 10 5 (FACS or 4 10 6 (plate reader measurements) were collected, spun and resuspended in 0.5 and 2 ml, respectively, of phenol-red-free medium supplemented with 2% FCS the dyes in the indicated concentrations. 30 min incubation at 37-C in the dark, the were stored on ice and analyzed on a Flow cytometer (Becton San Jose, CA, USA). Necrotic were excluded based on and side scatter criteria and were analyzed using the software (Becton Dickinson).

stained with the calcium-sensitive dye were washed and reincubated for 30 min in without the dye at 37-C to reduce background of the dye. The multivalent Rhod-2-AM is accumulated by energized according to the mitochondrial membrane hydrolyzed and trapped in the mitochondrial

Measurements of the Rhod-2 fluorescence performed using a plate (Labsystems, Stockholm, Sweden) at 544 nm and 590 emission wavelengths. To obtain fluorescence ( f max ) cells were lysed by a detergent, Trition Minimum fluorescence value ( f min ) was upon addition of the calcium EGTA (5 mM, final concentration).

calcium concentrations were using the formula [Ca] m = k d ( f f min )/( f max — f ), where k d (the constant)=570 nM and f =fluorescence. Fluorescence was used to confirm that the majority of Rhod-2 fluorescence was with mitochondria.

Caspase assay

The measurement of (Peptide Institute, Osaka, cleavage was performed using a assay. One million cells sedimented and washed once PBS. After centrifugation, were resuspended in 25 l PBS, to a microtiter plate and combined the appropriate peptide substrate in a standard reaction buffer mM HEPES, 10% sucrose, 5 mM dithiothreitol and 3-[(3-cholamidopropyl) dimethylammonio]-1-propanesulfonate (CHAPS), pH

Cleavage of the fluorogenic peptide was monitored by AMC liberation in a Fluoroscan II reader (Labsystems, Stockholm, using 355 nm excitation and 460 nm emission Fluorescence units were into the amount of AMC (pmol) a standard curve generated free AMC. Data duplicate samples were analyzed by linear regression.

of rat liver mitochondria

The liver of Sprague–Dawley rats was minced on resuspended in 50 ml of MSH buffer (210 mM 70 mM sucrose, 5 mM HEPES, pH 7.5), with 1 mM EDTA and homogenized a glass homogenizer and Teflon Homogenates were centrifuged at 600 g for 8 min at The supernatant was decanted and recentrifuged at g for 15 min to form a mitochondrial pellet was resuspended in MSH buffer without and centrifuged again at 5500 g for 15

The final mitochondrial pellet was in MSH buffer at a protein concentration of mg/ml.

Measurement of mitochondrial

Isolated rat liver mitochondria (1 mg) or P39 (5 10 6 ) were resuspended in 1 or 0.3 ml of buffer mM KCl, 5 mM KH 2 PO 4 . 1 mM MgSO 4 . 5 mM Tris, pH respectively. Measurement of respiration was at 30-C. Cells were with 0.005% digitonin. (5 mM) in the presence of rotenone (2 M), malate+pyruvate (5 mM and TMPD (0.5 mM)+ascorbate (1 mM) used as mitochondrial substrates.

in the oxygen concentration were with an oxygen electrode Instruments, Norfolk, UK) and analyzed the OxygraphPlus software (Hansatech Norfolk, UK). ADP and CCCP used in concentrations of 75 and 0.2 M for the experiments cells or 200 and 1 M for the experiments with mitochondria, respectively.

Isolated with an RCR (defined as the rate of in the presence of ADP divided by the rate following the expenditure of ADP) 3.5 were used for all experiments. mitochondria were prepared for experiment and used within 4 h. V4 was measured in mitochondria as the steady-state after expenditure of ADP. In basal V4 respiration was estimated in the of 1 M atractyloside, which blocks ADP into mitochondria.

For measurement of calcium loading, 1.5 10 6 cells suspended in 400 l of buffer (150 mM 5 mM KH 2 PO 4 . 5 mM succinate, 1 mM MgSO 4 . 5 mM Tris, pH Cells were permeabilized 0.005% digitonin and 2 M rotenone was in order to maintain pyridine in a reduced form. Mitochondrial uptake was induced by sequential of calcium to the cells.

Calcium changes were registered a calcium-sensitive electrode (Thermo Beverly, USA) and visualized a chart recorder.

Analysis of

Differentiation was determined with NBT stimulation with phorbol-myristate-acetate as elsewhere. 8 Briefly, 1 10 6 cells spun down at 240 g for 5 min. were resuspended in 20% FBS in RPMI to 37-C.

The cell suspension was with equal volume of NBT solution (NBT, 1.5 mg/ml and 400 ng/ml in PBS) prewarmed to and incubated for 30 min. Cytospins were air-dried, fixed in and counterstained with May–Grünwald–Giemsa. The of cell containing dark formazan deposits was counted in a of 200 cells.

Apoptotic morphology

was assessed by estimating morphologic as fragmented nuclei and condensed in May–Grünwald–Giemsa staining on cytospined The number of apoptotic cells was in percentage of a total (minimum cells counted per slide.


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