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J Biol Chem, Vol. 275, Issue 13, 9244-9250, March 31, 2000


Caspase-8-mediated Intracellular Acidification Precedes Mitochondrial Dysfunction in Somatostatin-induced Apoptosis*

Danni LiuDagger , Giovanni MartinoDagger , Muthusamy ThangarajuDagger , Monika SharmaDagger , Fawaz Halwani§, Shi-Hsiang Shen, Yogesh C. PatelDagger , and Coimbatore B. SrikantDagger ||

From the Dagger  Fraser Laboratories, Department of Medicine, and the § Department of Pathology, McGill University and Royal Victoria Hospital, Montreal, Quebec H3A 1A1 and the  Pharmaceutical Sector, N.R.C. Biotechnology Research Institute, Montreal, Quebec H4P 2R2, Canada

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Activation of initiator and effector caspases, mitochondrial changes involving a reduction in its membrane potential and release of cytochrome c (cyt c) into the cytosol, are characteristic features of apoptosis. These changes are associated with cell acidification in some models of apoptosis. The hierarchical relationship between these events has, however, not been deciphered. We have shown that somatostatin (SST), acting via the Src homology 2 bearing tyrosine phosphatase SHP-1, exerts cytotoxic action in MCF-7 cells, and triggers cell acidification and apoptosis. We investigated the temporal sequence of apoptotic events linking caspase activation, acidification, and mitochondrial dysfunction in this system and report here that (i) SHP-1-mediated caspase-8 activation is required for SST-induced decrease in pHi. (ii) Effector caspases are induced only when there is concomitant acidification. (iii) Decrease in pHi is necessary to induce reduction in mitochondrial membrane potential, cyt c release and caspase-9 activation and (iv) depletion of ATP ablates SST-induced cyt c release and caspase-9 activation, but not its ability to induce effector caspases and apoptosis. These data reveal that SHP-1-/caspase-8-mediated acidification occurs at a site other than the mitochondrion and that SST-induced apoptosis is not dependent on disruption of mitochondrial function and caspase-9 activation.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Apoptosis is a physiological process of cell death indispensable for the maintenance of multicellular organisms. This process drives the cell into self-destruction via a common execution pathway. The cellular machinery utilized for this process creates distinct apoptotic features of cell shrinkage, cytoplasmic and nuclear condensation, membrane blebbing, chromatin compaction, and fragmentation of chromosomal DNA into 180-base pair multimers. A central event in the process of apoptosis is the activation of cysteine aspartate proteases (caspases) (1). Active caspases consist of dimeric complexes of ~20- and 10-kDa fragments derived from the procaspases that exist as inactive zymogens by internal proteolytic cleavage at cysteine-aspartate sites (2). Mammalian caspases can be divided into initiator (e.g. caspases 2, 8, 9, 10) and effector (caspases 3, 4, 5, 6, 7, 11, 12, and 13) enzymes. A feature of apoptosis that impinges on caspases is altered mitochondrial function characterized by a reduction in the electrochemical gradient across the mitochondrial membrane (Delta psi m)1 and release of mitochondrial cyt c into the cytoplasm (3-15). Cyt c is necessary for caspase-9 activation (16, 17). Caspase-9 can function as an initiator caspase when mitochondrial dysfunction is the primary event in apoptosis, whereas it serves to amplify the apoptotic signaling of other initiator caspases under conditions in which disruption of mitochondria is a late event (16-19).

In some models of apoptosis activation of caspases is associated with intracellular acidification (20-23). The question of whether intracellular acidification is necessary for inducing caspases or occurs merely as a consequence of caspase activation has remained an issue of debate (24-32). For instance, contradictory reports suggest that the pan-caspase inhibitor z-VAD-fmk prevents decrease in pHi, whereas acidification per se was found to activate z-VAD-fmk-sensitive caspases (27, 32). Since z-VAD-fmk inhibits both initiator and effector caspases, its use does not allow differentiation between caspases that may be activated in a pHi-sensitive and -insensitive manner during apoptosis associated with acidification. The temporal relationship between mitochondrial dysfunction and acidification has not been definitively established, although it was reported recently that loss of Delta psi m may trigger a decrease in intracellular pH (pHi) in hematopoietic cells (27, 32).

Somatostatin (SST) receptor (SSTR)-mediated cytotoxic signaling triggers acidification and apoptosis in MCF-7 and T47D breast cancer cells: prevention of acidification by pH clamping inhibited its ability to induce apoptosis (33-35). Translocation of the tyrosine phosphatase SHP-1 from the cytosol to the membrane is an early and essential event in SST-induced cell acidification and apoptosis (34-36). When ectopically expressed, SHP-1 lowered the resting pHi of MCF-7 cells (pHi = 7.07 versus 7.25 in cells transfected with the empty vector), and amplified not only the cytotoxic action of SST, but also acidification-induced apoptosis. Moreover, agonist-induced acidification and acidification-induced apoptosis were both inhibited by the dominant negative suppressive effect SHP-1C455S (35). In the present study we undertook to delineate the temporal sequence of activation of different caspases in relation to cellular acidification and mitochondrial dysfunction during SST-induced apoptosis in MCF-7 cells. We present evidence demonstrating that caspase 8 activation is necessary for intracellular acidification to occur during SST-induced apoptosis and that the effector caspases are induced only as a consequence of the decrease in pHi. Moreover, the reduction in Delta psi m and the release of cyt c into the cytosol from the mitochondria also occur distal to acidification. Depletion of ATP prevented the activation of caspase-9 but only partially inhibited its ability to activate the terminal caspases and induce apoptosis. These data suggest that SST-induced, acidification-dependent, apoptosis is not dependent on mitochondrial dysfunction.

    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The MCF-7 cell line (clone HTB22) was obtained from ATCC. Special reagents were obtained from the following sources: [D-Trp8]SST-14 (Bachem, Torrance, CA); annexin-V labeling kit (Roche Diagnostics, Montreal, CA); nigericin (ICN, Costa Mesa, CA). Carboxy-SNARF-1 acetoxymethyl ester and 3,3'-dihexyloxacarbocyanine iodide (DiOC6(3)) (Molecular Probes, Eugene, OR). Aminomethylcoumarin derivatives (caspase substrates) and aldehyde derivatives (caspase inhibitors) of tetrapeptide sequences that are recognized by distinct caspases: IETD (caspase-8), LEHD (caspase-9), and DEVD (caspases-3/-7) (BioMol Research Laboratories, Plymouth Meeting, PA). Antibodies against the different caspases and cyt c were purchased from Pharmingen (San Diego, CA). All other reagents used were of analytical grade and were obtained from regular commercial sources.

Cell Culture and Incubation Conditions-- Cells were plated in 75-cm2 culture flasks and grown in minimal essential medium containing non-essential amino acids and supplemented with 10% fetal bovine serum. Cells were incubated in the presence or absence of 100 nM [D-Trp8]SST-14 for different time periods as indicated. To examine the effect of direct acidification, cells were incubated in medium supplemented with 140 mM K+ and 10 nM nigericin. Caspase inhibitors were dissolved in dimethyl sulfoxide and used at 1:1000 dilution to yield a final concentration of 50 mg/ml. Depletion of intracellular ATP was achieved in glucose-deprived cells by inhibiting F0/F1-ATPase with oligomycin (37). Briefly, cells were incubated with 10 mM oligomycin in glucose-free Dulbecco's modified Eagle's medium (Canadian Life Technologies, Guelph, Ontario) supplemented with 50 mM malic acid, 2 mM glutamate, 1 mM sodium pyruvate, 10 mM HEPES/Na+ (pH 7.4), 0.05 mM beta -mercaptoethanol, and 10% dialyzed fetal bovine serum as described by Eguchi et al. (38) prior to peptide treatment. Cellular ATP was measured using a commercial luciferase luminescence assay kit (Sigma). The ATP concentration decreased by >83 ± 6% (n = 4) following oligomycin treatment (data not shown).

Detection of Apoptosis-- Apoptosis was determined by annexin-V positivity using the annexin-V-FLUOS kit (Roche Diagnostics, Montreal, Canada) or by the presence of oligonucleosomal DNA fragments as described previously (34, 35, 39). Cells labeled with fluorescein isothiocyanate-conjugated annexin-V and propidium iodide were analyzed by flow cytometry in a Becton Dickinson Vantage Plus flow cytometer. A 5-watt argon laser generating light at 351-363 nm was used as the excitation source and fluorescein isothiocyanate fluorescence was detected with a 560-nm short pass dichroic filter while propidium iodide fluorescence was detected using a 610-nm long pass filter. At least 10,000 gated events were recorded for each sample and the data analyzed by Winlist software (Verity Software House, ME). To assess DNA fragmentation, DNA was extracted twice with phenol/chloroform and once with chloroform from cells incubated in lysis buffer (500 mM Tris-HCl (pH 9) containing 2 mM EDTA, 10 mM NaCl, 1% SDS, and 1 mg/ml proteinase K) at 48 °C for 30 h. DNA extracts were incubated with 300 µg/ml bovine pancreatic RNase A at 37 °C for 1 h and 10-µg aliquots of DNA samples containing 10 µg/ml ethidium bromide were subjected to electrophoresis on 1.2% (w/v) agarose gels using the Hoefer SwitchbackTM pulse controller and visualized under UV light.

Measurement of Intracellular pH-- For measuring intracellular pH, cells were loaded with 10 µM acetoxymethylester derivative of SNARF-1 for the final hour of incubation in the absence or presence of 100 nM [D-Trp8]SST-14 at 37 °C (39). The cells were then scraped, washed, and maintained at 37 °C. Intracellular carboxy SNARF-1 was excited at 488 nm and emission was recorded at both 580 and 640 nm with 5-nm band pass filters with linear amplifiers in a Becton-Dickinson FACStar Vantage cytometer. The ratio of the emissions at these wavelengths was electronically calculated and used as a parameter indicative of pHi. The intracellular pH values were estimated by comparison of the mean ratios of the samples to a calibration curve of intracellular pH generated by incubation of carboxy-SNARF-1 loaded cells in buffers ranging in pH from 8.0 to 6.25 and containing the proton ionophore nigericin (33). Cells with fluorescence of <50 units were excluded in the calculation of the ratio of the emissions at 580 and 640 nm.

Measurement of Mitochondrial Membrane Potential-- DiOC6(3) (50 nM final concentration) was added to the cells 15 min prior to the completion of incubation. The cells were then washed to remove excess fluorochrome, scraped, and maintained at 37 °C. DiOC6(3) fluorescence was measured in a EPICS 750 series Flow Cytometer (Coulter Electronics, Hialeah, FL) with the excitation and emission wavelengths set at 488 and 520 nm, respectively. At least 10,000 events were recorded for each sample and the data analyzed by WinList Program (Verity Software House, Topsham, ME).

Subcellular Fractionation and Western Blotting-- Cells were washed in phosphate-buffered saline and resuspended in 500 ml of a buffer containing 25 mM Hepes-KOH buffer (pH 7.4) containing 10 mM KCl, 1.5 mM MgCl2, 5 mM EDTA, 1 m M EGTA, 2 mM dithiothreitol, 250 mM sucrose, 0.2% Triton X-100, and protease inhibitor mixture (Roche Diagnostics, Montreal, CA). The cells were homogenized in a Pyrex homogenizer using a type B pestle. Cell debris and nuclei were removed by centrifugation at 1,000 × g for 10 min at 4 °C. Mitochondrial fraction was then pelleted by centrifugation at 10,000 × g for 20 min. The supernatant obtained at this stage was re-centrifuged at 40,000 × g for 1 h to obtain cytosolic fraction.

Thirty micrograms of cytosolic fractions prepared from cells incubated under different experimental conditions were subjected to SDS-polyacrylamide gel electrophoresis. The separated proteins were blotted onto nitrocellulose membranes and subjected to immunoblot analysis for cyt c, or caspases-8, -9, -3, and -7.

Measurement of Caspase Activity-- Activities of caspases were measured in the lysates measuring the in vitro hydrolysis of DEVD-AMC (caspases-3 and -7), IETD-AMC (caspase-8), and LEHD-AMC (caspase-9) (40, 41). The fluorescence of the aminomethylcoumarin released from the substrates was measured in a Perkin-Elmer spectrofluorimeter with the excitation and emission wavelengths set at 380 and 460 nm, respectively. Enzyme activity was quantitated against a standard fluorescence curve generated using aminomethylcoumarin over a concentration range of 0-1000 nM.

    RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In order to determine the hierarchy of caspase activation during acidification-dependent apoptosis we measured the time course of [D-Trp8]SST-14-induced changes in enzyme activities using substrates that display specificity for initiator and effector caspases in vitro: IETD-AMC (caspase-8) and DEVD-AMC (caspases-3/-7) respectively. In cells incubated with 100 nM [D-Trp8]SST-14 a concentration which induced maximal apoptosis (35, 39), the IETD-AMC hydrolyzing activity was maximal by 3 h (6-fold increase over the basal value of 0.5 nmol/mg protein, Fig. 1), but had fallen to basal levels by 24 h. By contrast, DEVD-specific caspase activity increased by <3-fold during SST treatment but continued to increase and remained elevated even at 24 h (1.21 ± 0.14 and 3.61 ± 0.7, respectively, compared with 0.45 ± 0.05 nmol/mg protein in untreated control cells). When acidification was prevented by pH clamping by the inclusion of nigericin, SST-induced increase in IETDase was unaffected whereas its ability to induce DEVDase activity was completely inhibited (Fig. 2). We next examined the effect of selective inhibitors of these caspases on SST-induced acidification and apoptosis. IETD-CHO (the tetrapeptide aldehyde inhibitor of caspase-8) prevented the decrease pHi in SST-treated cells whereas DEVD-CHO (the caspase-3/-7 inhibitor) was without effect (Fig. 3A). By contrast, the ability of SST to induce apoptosis was suppressed by both inhibitors as confirmed by DNA fragmentation analysis (Fig. 3B) and by annexin-V positivity (not shown). The temporal sequence of activation of the different caspases during [D-Trp8]SST-14-induced apoptosis was confirmed by measuring the effect of each of the caspase inhibitors on the activities of other caspases (Fig. 4). IETD-specific caspase activation by SST was unaffected by DEVD-CHO (Fig. 4A) but the inductive effect of SST on DEVD-specific caspase activity was totally inhibited by IETD-CHO (Fig. 4B).


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  		Fig. 1.   . [D-Trp8]SST-14-induced activation of caspase-8 (IETDase) precedes that of caspases-3/-7 (DEVDase) in MCF-7 cells. Enzyme activities were measured using the aminomethylcoumarin derivatives of the tetrapeptide substrates in extracts of cells incubated with 100 nM peptide at the indicated times. IETDase activity was maximal at 3 h, and declined to basal level by 24 h (top panel). By contrast, DEVDase activity was maximal at 24 h (bottom panel). Values represent nanomole of aminomethylcoumarin liberated from the substrates during 30 min incubation with cell extracts in vitro and was quantitated against the fluorescence readings of serially diluted aminomethylcoumarin as described under "Materials and Methods" (mean ± S.E., n = 6).


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  		Fig. 2.   Effect of pH clamping on caspase activation. [D-Trp8]SST-14 induced increase in caspase-8 (IETDase) activity was not affected by the prevention of acidification by nigericin (top panel) whereas pH clamping prevented the increase in caspase-3/-7 (DEVDase) activity (bottom panel). Enzyme activities were measured after 4 h (IETDase) or 24 h (DEVDase) treatment (mean ± S.E., n = 6).


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  		Fig. 3.   . Differential effects of caspase-inhibitors on [D-Trp8]SST-14-induced acidification, but not on apoptosis in MCF-7 cells. A, the decrease in pHi in cells incubated with 100 nM peptide for 24 h was prevented by the caspase-8 inhibitor IETD-CHO, but not by the caspase-3/-7 inhibitor, DEVD-CHO (mean ± S.E., n = 6). B, oligonucleosomal DNA fragmentation in peptide-treated cells was completely inhibited by both IETD-CHO and DEVD-CHO (figure representative of four different experiments).


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  		Fig. 4.   Inhibition of DEVDase does not prevent [D-Trp8]SST-14-induced activation of IETDase, whereas inhibition of IETDase abrogates induction of DEVDase. A, extracts of cells incubated with 100 nM peptide ± DEVD-CHO for 4 h were assayed for IETDase activity or for 24 h ± IETD-CHO for DEVDase assay (mean ± S.E., n = 6).

Mitochondrial dysfunction characterized by a reduction in its transmembrane potential (Delta Psi m) and release of cyt c into the cytosol, are characteristic features of apoptosis (6, 9, 10, 42, 43). An important arm of apoptotic signaling involves cyt c-dependent activation of caspase-9. Cyt c released from the mitochondria complexes with APAF-1 (the mammalian homolog of the pro-apoptotic protein CED-4 of Caenorhabditis elegans) and procaspase-9. Such activation of caspase-9 has been reported to be necessary for the full expression of nuclear apoptotic events (44). To determine whether mitochondrial dysfunction precedes or follows acidification, we compared the effects of pH clamping and different caspase inhibitors on Delta psi m, cyt c release, and caspase-9 activation in SST-treated cells. SST induced a decrease in Delta psi m in MCF-7 cells (Fig. 5A). Inhibition of acidification by pH clamping totally abrogated the ability of SST to decrease Delta psi m. Maximal effect was seen at 6 h when 48 ± 7% of SST-treated cells displayed a significant reduction in Delta psi m compared with the untreated control (Fig. 5B). Additionally, loss of Delta psi m during SST treatment was prevented almost completely by IETD-CHO but was decreased only by 23 ± 3% by DEVD-CHO (Fig. 5B). A marked increase in cytosolic cyt c content was seen in SST-treated cells (Fig. 6). Such an increase did not occur when acidification was prevented by pH clamping. SST-induced increase in cytosolic cyt c was completely suppressed by IETD-CHO but was not inhibited by inhibition of effector caspases by DEVD-CHO. We measured the caspase-9 activity in extracts of cells incubated with SST using the tetrapeptide substrate LEHD-AMC, a substrate with reported caspase-9 selectivity (41). LEHD-specific caspase activity was induced by SST in MCF-7 cells in an acidification-dependent manner (Fig. 7). Inhibition of caspase-9 activity with LEHD-CHO did not affect SST-induced loss of Delta psi m or the release of cyt c into the cytosol (not shown).


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  		Fig. 5.   [D-Trp8]SST-14-induced reduction in Delta psi m is acidification-dependent. A, cells were incubated for 6 h in the absence (panel 1) or presence of 100 nM peptide (panel 2) in regular medium or with the peptide in nigericin containing medium (panel 3), labeled with DiOC6(3) and analyzed by flow cytometry. Representative recordings of six separate measurements are shown. The reduction in Delta psi m in peptide- treated cells is evident from the decrease in the number of cells with the resting potential as well as from the appearance of the distinct peak of cells with lower DiOC6(3) fluorescence (panel 2). Inhibition of acidification prevented the ability of [D-Trp8]SST-14 to decrease Delta psi m (compare panels 3 and 1). B, quantitation of the effect of pH clamping, IETD-CHO and DEVD-CHO on D-Trp8-induced reduction in Delta psi m (mean ± S.E., n = 6). The effect of the peptide was only partially inhibited by DEVD-CHO, whereas it was completely abolished in the presence of IETD-CHO similar to that seen in cells clamped at physiological pH in the presence of nigericin. *, p < 0.001; **, p < 0.01.


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  		Fig. 6.   [D-Trp8]SST-14-induced increase in cytosolic cyt c precedes the activation of DEVDase. 30-mg protein aliquots from cytosolic extracts of cells incubated in the absence and presence of 100 nM [D-Trp8]SST-14 alone or with the indicated inhibitors were subjected to immunoblot analysis following electrophoresis and membrane transfer. Nigericin and IETD-CHO, but not DEVD-CHO, prevented [D-Trp8]SST-14-induced increase in cyt c.


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  		Fig. 7.   . Activation of caspase-9 (LEHDase) by [D-Trp8]SST-14 is attenuated by inhibition of acidification. Cells were incubated as described in the legend for Fig. 2B (mean ± S.E., n = 6).

Cyt c- and APAF-1-mediated activation of caspase-9 is an energy-dependent process requiring ATP (45, 46). In order to establish the extent to which SST-signaled apoptosis is mediated via ATP-dependent caspase-9 activation, we tested the effect of depleting intracellular ATP on the cytotoxic signaling of SST. In ATP-depleted cells, SST failed to activate LEHDase (Fig. 8A). ATP depletion also inhibited SST-induced increase in cytosolic cyt c (Fig. 8B). By contrast, ATP depletion decreased the inductive effect of SST on DEVDase activity only by 9 ± 1% (Fig. 8C). Likewise ATP depletion had minimal effect on the extent of apoptosis. Following 4 h treatment with SST, the number of annexin-V positive cells was 8.8 ± 1.5% in ATP-depleted and 12.1 ± 1.1% in ATP-replete MCF-7 cells. Precise quantitation of the long term effect of ATP depletion on SST-induced apoptosis was not possible because of significant necrosis (as determined by the presence of cells labeled with both propidium iodide and annexin V (data not shown)).


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  		Fig. 8.   Effect of ATP depletion on [D-Trp8]SST-14-induced cytotoxic signaling. ATP-depleted cells were prepared by incubating with oligomycin in glucose-free medium for 1 h. Control and ATP-depleted cells were incubated for 4 h in the absence (lanes 1 and 3) or presence of 100 nM peptide (lanes 2 and 4). A, LEHDase activation by the peptide seen in control cells was completely abolished by ATP-depletion (compare lanes 2 and 4). B, [D-Trp8]SST-14-induced increase in cytosolic cyt c was also inhibited by ATP depletion.

Immunoblot analysis confirmed the pH-independent generation of active caspase-8 (IETD-AMC-specific) by the formation of the 20-kDa caspase-8 fragment from the 50-kDa procaspase-8 in [D-Trp8]SST-14-treated cells. Inhibition of acidification by pH clamping did not prevent activation of caspase-8 (Fig. 9). By contrast, generation of the 20-kDa fragments of caspase-3 and caspase-7 (DEVD-AMC-specific proteases) from procaspase-3 (32 kDa) and procaspase-7 (35 kDa) and of caspase-9 (LEHD-AMC-specific) from procaspase-9 (48 kDa) occurred only if acidification was present.


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  		Fig. 9.   Immunoblot analysis demonstrating differential pH sensitivity of caspase activation. Formation of the 20-kDa active caspase from the inactive procaspase-8 induced by [D-Trp8]SST-14-mediated cytotoxic signaling was pH-independent. By contrast, the formation of the 20-kDa fragments from procaspases-9, -7, and -3 in peptide-treated cells was prevented by inhibition of acidification by nigericin (data representative of four independent experiments).

The present finding that caspase-8 precedes the onset of acidification prompted us to assess the importance of SHP-1 in the activation of IETDase and DEVDase by [D-Trp8]SST-14 and by direct acidification. IETDase activity was higher in [D-Trp8]SST-14 SST-treated cells expressing SHP-1 compared with the empty vector (3.75 ± 0.5 ± 3.1 ± 0.4 nmol/mg protein, Fig. 10, top panel). Likewise, agonist-induced increase in DEVDase activity was also higher in cells expressing SHP-1 (3.72 ± 0.43 versus 2.45 ± 0.36 nmol/mg protein in control vector-transfected cells, Fig. 10, bottom panel). As shown in this figure, SHP-1C455S suppressed the ability of [D-Trp8]SST-14 SST to activate both enzymes. Interestingly, basal activities of IETDase and DEVDase (0.85 ± 0.07 and 0.93 ± 0.1 nmol/mg protein, respectively) were slightly higher in cells overexpressing the wild type SHP-1. When subjected to direct acidification, IETDase activity was minimal in all three cell types (Fig. 11). In SHP-1 expressing cells, IETDase activity was lower than that seen under basal conditions at pH 7.2 (0.63 ± 0.07 versus 1.12 ± 0.14 nmol/mg of protein, respectively, compare Figs. 10 and 11). By contrast, DEVDase activity was higher in both vector control and SHP-1-transfected cells (2.45 ± 0.3 and 3.57 ± 0.5 nmol/mg of protein, respectively). The dominant negative effect of SHP-1C455S completely abolished the acidification-induced increase in DEVDase activity. Finally we assessed the role of SHP-1 in [D-Trp8]SST-14-induced reduction in Delta psi m. While the maximum number of cells that displayed decreased Delta psi m was the same in both control vector and SHP-1 transfected cells following incubation with [D-Trp8]SST-14, the rate of reduction in Delta psi was increased by ectopically expressed SHP-1 reaching maximal level by 90 min, a time point at which only 12% of the control vector-transfected cells displayed decrease in Delta psi m (Fig. 12). In SHP-1C455S-transfected cells no decrease in Delta psi m occurred even after 24 h treatment with [D-Trp8]SST-14.


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  		Fig. 10.   SHP-1 dependence of caspase activation by [D-Trp8]SST-14 in MCF-7 cells. The ability of the peptide to induce both IETDase (top panel) and DEVDase (bottom panel) during the 4-h incubation was higher in SHP-1 expressing cells compared with the empty vector control (VC) cells, and was abolished by the dominant negative effect of SHP-1C455S. Mean ± S.E., n = 3).


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  		Fig. 11.   Acidification-induced activation of DEVDase (speckled bars) in SHP-1-expressing MCF-7 cells was higher than that in control vector (VC)-transfected cells. SHP-1C455S suppressed acidification-induced increase in DEVDase activity. Acidification had no effect on IETDase activity (solid bars) in all three cell types.


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  		Fig. 12.   . SHP-1 dependence of [D-Trp8]SST-14-induced reduction in Delta psi m. Cells were incubated with 100 nM peptide for 90 min prior to measurement of Delta psi m. [D-Trp8]SST induced reduction in in Delta psi m was seen in 53 ± 4% of SHP-1 expressing cells whereas only 12.8 ± 1% of vector control (VC) cells displayed a reduction mitochondrial membrane potential. In cells expressing SHP-1C455S, the peptide failed to trigger the loss of mitochondrial membrane potential (mean ± S.E., n = 4).


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

In this study we demonstrated that the cytotoxic signaling of SST in MCF-7 cells activates multiple caspases and that SHP-1-dependent activation of caspase-8 precedes the decrease in pHi whereas acidification is necessary for the induction of the effector caspases. In accordance with this was the finding that inhibition of SST-induced acidification by pH clamping with nigericin did not affect SST-induced activation of caspase-8 while it completely abrogated the induction of the other caspases. Likewise, inhibition of caspase-8 by IETD-CHO prevented SST-induced acidification and activation of terminal caspases. By contrast, LEHD-CHO and DEVD-CHO did not prevent a SST-induced increase in caspase-8 activity and the decrease in pHi. Moreover, SST-induced increase in caspase-8 activity peaked by 3 h and declined thereafter paralleling the previously reported time course of acidification (35). The distal caspases, in contrast, displayed sustained increase in activity. These data demonstrate that caspase-8 activation is required for SST-induced acidification and, additionally, that its activity is pHi sensitive. This is supported by the finding that the 20-kDa fragment derived from procaspase-8 was present in cells with acidic pHi during SST treatment. By contrast, the generation of caspases-9, -3, and -7 from the respective procaspases occurred only when there was acidification. The detection of caspase-3 in the HTB22 clone of MCF-7 cells used in the present study contrasts with its reported absence in other clones of this cell line due to a 47-base pair deletion within the exon 3 of the caspase-3 gene (47, 48).2 We found that acidification per se was sufficient to activate the effector caspases in the absence of a detectable increase in caspase-8 activity. While this suggests that acidification may trigger the activation of the effector caspases directly, the possibility that transient activation of caspase-8 during rapid acidification may suffice to induce these caspases cannot be ruled out. The finding that SHP-1 is required not only for the induction of IETDase by SST but also of DEVDase by cell acidification reinforces the idea that SHP-1 modulates the apoptotic events both before and after cell acidification (34). The phosphatase-dependent processes that lead to caspase activation remain to be delineated.

Caspase-8 can activate caspases-3 and -7 directly and/or through induction of caspase-9 (17, 18, 45, 49). In order to assess the relative importance of caspase-9 in the cytotoxic signaling of SST, we compared the effect of SST in control and ATP-depleted MCF-7 cells. SST was unable to activate caspase-9 in ATP-depleted cells, but was still capable of activating DEVDase and inducing apoptosis. Thus, SST-induced apoptosis in MCF-7 cells involves caspase-8-mediated direct activation of terminal caspases as well as an amplifying effect mediated through mitochondrial dysfunction and consequent activation of caspase-9. These data support the concept that caspase-8 can activate apoptotic pathways involving effector caspases through both mitochondria-dependent and -independent pathways (17, 44, 50-54). The extent of SST-induced apoptosis was 34 ± 5% lower in ATP-depleted cells, an effect that could be accounted for by the loss of caspase-9-mediated activation of the terminal caspases and/or the loss of effector caspase-mediated activation of caspase-9. We found that DEVD-CHO only partially suppressed the effect of SST on Delta psi m and cyt c release suggesting that mitchondrial dysfunction may be caused to some extent by the action of the effector caspases as demonstrated previously in an in vitro model (10).

We showed that intracellular acidification precedes the onset of reduction in Delta psi m in MCF-7 cells exposed to the cytotoxic action of SST. Likewise, release of cyt c from the mitochondria and LEHDase activation was observed in cells subjected to direct acidification (details not shown). This is in contrast to the report that mitochondrial permeability transition causes acidification during valinomycin-induced apoptosis in hematopoetic cells (55). It is possible that the cause and effect relationship between mitochondrial dysfunction and cell acidification may be cell type-dependent. Indeed the existence of two cell types in which caspase-8 can trigger apoptosis without invoking mitochondrial dysfunction (type I cells) and those in which apoptosis is induced predominantly in a mitochondria-dependent manner (type II cells) has been described (51). The fact that mitochondrial dysfunction occurs late and is inconsequential in SST-signaled apoptosis adds credence to this idea. This is supported by the recently reported finding that ATP-dependent steps in Fas-mediated apoptosis in Type I cells are located downstream of caspase-3 (56).

The mechanism of SHP-1-/caspase-8-mediated inhibition of pH homeostasis remains to be elucidated. We have previously shown that amiloride and bafilomycin-1, which inhibit Na+/H+ exchanger (NHE) and H+-ATPase, respectively, trigger acidification and apoptosis in MCF-7 cells. Inhibition of NHE lowered the pHi to a greater extent than inhibition of H+-ATPase. (34). This raises the possibility that SHP-1 and caspase-8 mediated signaling may generate or unmask molecule(s) that may disrupt proton extrusion pathways involving these channels. The finding that SST-induced acidification does not occur at the mitochondria suggests that it inhibits the regulation of proton transport through NHE and H+-ATPase either at the cell membrane or some other subcellular locus. The existence of multiple NHE isoforms and their differential localization at the cell membrane (e.g. NHE-1 and NHE-2) or at the endoplasmic reticulum-nuclear envelope and endosomes (e.g. NHE-3) (57) raises the possibility that SST may inhibit some or all of the NHEs. Our present findings suggest that SHP-1- and caspase-8-mediated disruption of pH homeostasis may target these proton extrusion pathway(s). Studies are currently in progress to identify the subcellular site(s) and the underlying mechanism involved in SST-induced acidification.

In summary, these findings help define the temporal sequence of events that link the initiator and effector caspases with inhibition of pH homeostasis and mitochondrial dysfunction in acidification-dependent apoptosis. We demonstrated that (i) SHP-1-dependent activation of caspase-8 is required for SST-induced decrease in pHi while SHP-1-dependent activation of effector caspases is necessary for acidification-induced apoptosis. (ii) Mitochondrial dysfunction and activation of effector caspases occur distal to acidification and (iii) caspase-9 is not essential for SST-induced apoptosis to occur but, when induced, can amplify the cytotoxic signaling of SST.

    ACKNOWLEDGEMENT

We thank Dr. J. J. Lebrun for the use of the luminometer.

    FOOTNOTES

* This work was supported by Canadian Medical Research Council Grant MT-12603 and the U. S. Department of Defense Breast Cancer Program.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

|| To whom correspondence should be addressed: M3.15, Royal Victoria Hospital, 687 Pine Ave. W., Montreal, Quebec H3A 1A1, Canada. Tel.: 514-842-1231 (ext. 5359); Fax: 514-849-3681; E-mail: mdcs@musica.mcgill.ca.

2 R. U. Janicke, personal communication.

    ABBREVIATIONS

The abbreviations used are: Delta psi m, mitochondrial membrane potential; cyt c, cytochrome c; DiOC6(3), 3,3'-dihexyloxacarbocyanine iodide; NHE, Na+/H+ exchanger; pHi, intracellular pH; SST, somatostatin.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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