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Introduction
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Defects in signaling pathways leading to the classic mitochondrion- and caspase-mediated apoptosis are frequent in primary tumors (1, 2). As a result, alternative cell death programs are gaining increasing interest among cancer researchers. Until recently, lysosomes have been considered "suicide bags" that, through the release of unspecific enzymes, cause autolysis and damage neighboring cells during uncontrolled tissue damage. However, accumulating data now show that lysosomes also function as death signal integrators in many controlled death paradigms (3, 4). Lysosomal proteases and cathepsins translocate from the lysosomal lumen to the cytosol in response to a wide variety of death stimuli such as TNF (5, 6), Fas (7), p53 activation (8), microtubule stabilizing agents (9), oxidative stress (7, 10), staurosporine (11), growth factor deprivation (7), and lysosomotropic agents (10, 12). Once released to the cytosol, cathepsins, especially cysteine cathepsins B and L and aspartyl cathepsin D, may trigger the mitochondrial outer membrane permeabilization followed by caspase- or apoptosis-inducing factormediated apoptosis (5, 8, 1113) or mediate caspase- and/or apoptosis-inducing factorindependent programmed cell death (PCD) with apoptosis- or necrosis-like morphology (6, 14). Because the latter pathway can circumvent most of the known resistance mechanisms occurring in tumor cells, lysosomal membrane permeabilization (LMP) appears to be a promising target for novel cancer drugs.
The heat shock protein 70 (Hsp70) family of proteins consists of both constitutively expressed and stress-inducible molecular chaperones that are localized to different intracellular compartments (15). The major stress-inducible Hsp70 (also called Hsp72) is highly expressed in the cytosol and plasma membrane of primary tumors of various origins, whereas its expression in unstressed normal cells is very low and restricted to the cytosol (1517). The role of Hsp70 in tumorigenesis is supported by experimental data showing that its high expression is required for the growth of human tumor xenografts in immunodeficient mice (18), and that it enhances the tumorigenic potential of rodent cells in syngenic animals (19). Furthermore, its high expression correlates with poor therapeutic outcome in human breast cancer (20). The molecular mechanism underlying the tumorigenic potential of Hsp70 is as yet unclear, but it may be explained by its ability to confer resistance to both apoptosis- and necrosis-like PCD induced by diverse stimuli (21, 22). In vitro studies have suggested that Hsp70 may directly interfere with the apoptosis signaling machinery by binding to the apoptotic protease-activating factor-1 (22) or apoptosis-inducing factor (23) and thereby inhibit the apoptosome-mediated activation of caspases or apoptosis-inducing factorinduced nuclear changes, respectively. However, most studies using cellular death models suggest that Hsp70-mediated inhibition of caspase-mediated PCD occurs upstream of mitochondrial outer membrane permeabilization and apoptosome formation (2426). Furthermore, Hsp70 can effectively rescue cells from caspase-independent PCD induced by TNF, heat shock, serum starvation, or oxidative stress (6, 23, 24, 27, 28), and the depletion of Hsp70 induces caspase-independent apoptosis-like PCD in various human tumor cell lines (18, 29). Here, we aim at determining the mechanism through which Hsp70 regulates tumor cell survival with special focus on LMP and caspase-independent cell death.
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Materials and Methods
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Cell Culture.
WEHI-V2 and WEHI-V3 are vector-transfected and WEHI-hsp-2 and WEHI-hsp-18 Hsp70-transfected single cell clones of WEHI-S murine fibrosarcoma cells (28). ME-180v and ME-180as are single cell clones of ME-180 human cervix carcinoma cells transfected with vector and Hsp70 antisense (as) cDNA, respectively (28). HeLa human cervix carcinoma cell line was provided by J. Lukas (Danish Cancer Society, Copenhagen, Denmark). CX+ (>90% of cell positive) and CX (<20% of cells positive) cells are stable sublines of CX2 human colon carcinoma cells sorted according to the Hsp70 surface expression (17). HBL-100 is an immortalized breast epithelial cell line provided by P. Briand (Danish Cancer Society, Copenhagen, Denmark). Murine embryonic fibroblasts (MEFs) explanted from days 1416 wild type or homozygote Hsp70 transgenic CBA x C57Bl/6 mice (provided by G. Kollias, Hellenic Pasteur Institute, Athens, Greece, and G. Pagoulatos, University of Ioannina, Ioannina, Greece; reference 30) embryos were passaged once a week (68,000 cells/cm2) with a change of medium on day three until they reached senescence. Senescent cells were maintained by medium change twice a week, and passaging was restarted upon immortalization. Immortalized MEFs (iMEFs) were used at passages 1520. DMEM (Invitrogen) supplemented with 10% heat-inactivated calf serum (Biological Industries), 0.1 mM of nonessential amino-acids (Invitrogen), 110 mg/ml Na-pyruvate (Merck) and antibiotics were used as growth medium for MEFs. Other cells were propagated in RPMI 1640 (Invitrogen) supplemented with 6% heat-inactivated calf serum and antibiotics (complete medium) at 37°C in a humidified air atmosphere with 5% CO2. All cells were repeatedly tested and found negative for mycoplasma by Hoechst (H-33342; Molecular Probes) staining and immunofluorescence microscopy.
Cell Death Induction and Determination.
Ad.asHsp70 and Ad.ß-Gal are adenoviral (Ad.) shuttle vectors carrying bases 475796 of the published human Hsp70 sequence in as orientation and ß-galactosidase (ß-Gal) cDNA, respectively (29). The infections were performed as described previously using the lowest multiplicity of infection resulting in a 100% infection (29). Recombinant human TNF was provided by A. Cerami (Kenneth S. Warren Laboratories, Tarrytown, NY), recombinant murine TNF was obtained from R&D Systems, and etoposide and hydrogen peroxide were obtained from Sigma-Aldrich. To obtain Fas ligandcontaining supernatant, confluent Neuro2 cells were provided with fresh serum-free medium, and after 24 h at 37°C, the supernatant was collected, centrifuged at 600 g for 10 min, and stored in aliquots at 80°C. The blue light was delivered to CX cells by a custom-made lamp house consisting of a 100-W Hg gas discharge lamp, a bp filter (450500 nm), and a mirror system (Leica) and to iMEFs by the 488-nm laser of the Axiovert 100M confocal microscope (Carl Zeiss MicroImaging, Inc.).
-irradiation was delivered to exponentially growing cells by a 137Cs source at a dose rate of 1 Gy/10 s.
z-Val-Ala-DL-Asp-CH2F (zVAD-fmk) obtained from Bachem, PD150606 obtained from Calbiochem-Novabiochem, CA-074-Me obtained from Peptides International, z-Phe-Ala-CH2F (zFA-fmk) obtained from Enzyme System Products, Mu-Leu-HphVSPh (LHVS; provided by J. Palmer, Celera, San Francisco, CA; reference 31), and Ac-Asp-Glu-Val-Asp-aldehyde (DEVD-CHO) obtained from BIOMOL Research Laboratories, Inc. were dissolved in DMSO. When using the protease inhibitors, DMSO concentration of all samples was adjusted to 0.2%. The experiments with ME-180 transfectants were performed in RPMI 1640 supplemented with 0.5% FCS after a preincubation of cells in this medium for 1824 h. Experiments with other cells were performed in complete medium.
3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT) reduction and lactate dehydrogenase (LDH) release assays (cytotoxicity detection kit; Roche) were used to analyze the survival of the cells as described previously (6). Staining with annexin V conjugated with fluorescein isothiocyanate (Bender Medsystems) and flow cytometry (FACSCaliburTM; Becton Dickinson) analysis were used to detect apoptotic cells exposing phosphatidylserine on the outer leaflet of the plasma membrane.
Measurement of Enzyme Activities.
To measure cytosolic enzyme activities, subconfluent cells were treated with an extraction buffer (250 mM sucrose, 20 mM Hepes, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, and 1 mM pefabloc, pH 7.5) containing 15 µg/ml digitonin for 1215 min on ice. The digitonin concentration and treatment times were optimized to result in the total release of the cytosolic LDH activity without disruption of lysosomes. To measure the total cellular cysteine cathepsin activity, cells were treated with the aforementioned extraction buffer containing 200 µg/ml digitonin for 1215 min on ice and, when indicated, incubated with the indicated protease inhibitors or recombinant human Hsp70 (rhHsp70; StressGen Biotechnologies) for 30 min on ice before the measurement. The effector caspase and cysteine cathepsin activities were estimated by adding one volume of 20 µM Ac-DEVD7-amino-trifluoromethylcoumarin (AFC) (BIOMOL Research Laboratories, Inc.) in caspase reaction buffer (100 mM Hepes, 20% glycerol, 0.5 mM EDTA, 0.1% CHAPS, 5 mM dithiothreitol (DTT), and 1 mM pefabloc, pH 7.5) or 20 µM zFR-AFC (Enzyme System Products) in cathepsin reaction buffer (50 mM sodium acetate, 4 mM EDTA, 8 mM DTT, and 1 mM pefabloc, pH 6.0), respectively. The Vmax of the liberation of AFC (excitation, 400 nm; emission, 489 nm) was measured for 20 min at 30°C with a Spectramax Gemini fluorometer (Molecular Devices). ß-N-acetyl-glucosaminidase (NAG) activity was estimated by adding three volumes of 0.2 M sodium citrate buffer, pH 4.5, containing 300 µg/ml 4-methylumbelliferyl-2-acetamido-2-deoxy-ß-D-glucopyranoside (Sigma-Aldrich). The Vmax of the liberation of methylumbelliferyl (excitation, 356 nm; emission, 444 nm) was measured for 20 min at 30°C with a Spectramax Gemini fluorometer. LDH activity of the cytosol determined by a cytotoxicity detection kit (Roche) was used as an internal standard with which protease activities were normalized.
In Vitro Apoptosome Assay.
Subconfluent cultures of HeLa cells were harvested by scraping on ice, washed in ice-cold PBS, and resuspended in equal volume of ice-cold isotonic lysis buffer (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 1 mM DTT, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 100 µg/ml pefabloc). After a 30-min incubation on ice, the cells were lysed by 30 strokes of a Dounce homogenizer and centrifuged at 750 g for 10 min. The obtained supernatant was further centrifuged at 10,000 g for 10 min and at 20,000 g for 30 min. The clarified supernatant was removed carefully, stored in aliquots at 80°C, and used at protein concentrations ranging from 5 to 10 mg/ml. The apoptosome was activated by the addition of 1 mM dATP (dissolved in ddH20 and adjusted to pH 7.0; ICN Biomedicals) and 1 µM horse heart cytochrome c (Sigma-Aldrich) to the cytosolic HeLa cell extract (protein concentration; 510 mg/ml) containing 100 µM DEVD-AFC (BIOMOL Research Laboratories, Inc.). When indicated, 2 µM recombinant human Hsp70 (StressGen Biotechnologies) was added to the cytosolic extract before the addition of cytochrome c and dATP. After a 30-min incubation at 37°C, the Vmax of the liberation of AFC was measured as mentioned before. After 2 h at 37°C, the samples from the in vitro apoptosome assay were mixed with 0.25 volumes of 4 x Laemmli sample buffer and the immunodetection of proteins separated by SDS-PAGE was performed as described in the next paragraph.
Immunoblot Analysis, Immunocytochemistry, and Electron Microscopy.
The primary antibodies used included murine monoclonal antibodies against human Hsp70 (Multimmune GmbH), cathepsin B (Oncogene Research Products), cathepsin L and DFF45/ICAD (Transduction Laboratories), caspase-3 and -9 (BD Biosciences), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Biogenesis), as well as polyclonal goat antibodies against rat cathepsin B (32), rabbit antibodies against human cathepsin D (no. 06-467; Upstate Biotechnology), and rabbit antiserum against Hsp70 raised (Neosystem) by immunizing a rabbit with an ovalbumin-coupled peptide (YTKDNNLLGRFELSG) corresponding to amino acids 450463 in the human Hsp70 sequence (P08107). Immunodetection of proteins (20 µg/lane) separated by 810% SDS-PAGE and transferred to nitrocellulose was performed using ECL Western blotting reagents (Amersham Biosciences), and the indicated primary antibodies and appropriate secondary antibodies were obtained from DakoCytomation.
To visualize cathepsins and cytochrome c, cells were fixed in 20° methanol for 10 min at 25°C and 4% formaldehyde for 30 min followed by 0.1% Triton for 10 min at 25°C, respectively. After blocking for 30 min in 10% FCS, slides were stained with the indicated primary antibodies and biotinylated (cathepsin B; DakoCytomation) or Alexa Fluor488conjugated antimouse IgG (Molecular Probes). The indicated nuclei were stained in the end with 2 µl/ml ethidium bromide for 12 min at 25°C preceded by the 10-min treatment with 0.5 mg/ml RNase A at 37°C. Biotinylated IgG was visualized by peroxidase-conjugated streptavidinbiotin complex and diaminobenzidine/H2O2 according to the manufacturer's instructions (SteptABC; Vector Laboratories) and images were taken with a digital camera mounted on a BX60 microscope (Olympus). Confocal images were obtained with an Axiovert 100M microscope equipped with LSM 510 system (Carl Zeiss MicroImaging, Inc.).
For immunoelectron microscopy, CX+ and CX tumor cells were fixed in 8% paraformaldehyde in 250 mM Hepes buffer for 1 h. After two washes, free aldehyde groups were quenched with 50 mM NH4Cl for 10 min. For cryoprotection, the cell pellets were incubated in 2.1 M sucrose in 17% polyvinylpyrrolodone at 20°C for 30 min before freezing in liquid nitrogen. Ultrathin sections (70 nm) were cut at 100°C on an Ultracut E microtome (FC4E; Reichart-Jung) using a glass knife and mounted on 150-mesh Parlodion (Mallinckrodt Specialty Chemicals)coated nickel grids. Immunogold labeling of Hsp70 was performed with 10-nm gold particles and of cathepsin D with 5-nM gold particles. In brief, the grids were rinsed and blocked in 0.1% acetylated BSA buffer before incubation with murine anti-Hsp70 antibody (1:100) and/or rabbit anti-cathepsin D antibody (1:200) for 18 h at 4°C. After washing, the grids were incubated individually (single staining) or subsequently (double staining) for 3 h at 25°C in 1% BSA buffer containing 1:75 dilutions of Aurion goat antimouse IgG/IgM (10 nm GP) and goat antirabbit IgG (5 nm GP; both from Amersham Biosciences). Nonspecific binding was blocked by extensive washing in 0.1% acetylated BSA buffer. An additional fixation in 2% glutaraldehyde in PBS was performed after immunostaining. The sections were stained in uranyl acetate/methyl cellulose and viewed in an EM 10CR electron microscope (Carl Zeiss MicroImaging, Inc.). This method is optimized to the localization of antigens that are localized in both intracellular and extracellular compartments.
Fractionation of iMEFs.
Subconfluent cultures of iMEF-Hsp-4 cells were harvested by scraping on ice, washed in ice-cold PBS, and resuspended (20 x 106 cells/ml) in ice-cold SCA buffer (20 mM Hepes-KOH, pH 7.5, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 250 mM sucrose, 1 mM DTT, 10 µg/ml aprotinin, 1 µg/ml leupeptin, 1 µg/ml pepstatin A, and 1 mM pefabloc). After a 30-min incubation on ice, cells were lysed by 3040 strokes in a Dounce homogenizer and centrifuged at 750 g for 5 min. The pellet was collected as the heavy membrane fraction (intact cells and nuclei). The supernatant obtained was centrifuged at 750 g for 5 min, the pellet was discarded, and the new supernatant was divided into two tubes and further centrifuged at 20,000 g for 20 min. The resulting supernatants were collected as the cytosolic fraction. The pellets were resuspended in 450 µl SCA buffer with or without 800 µg/ml digitonin, incubated for 30 min on ice, and centrifuged at 20,000 g for 20 min. The pellets were collected as the light membrane fractions (cellular organelles including endosomes and lysosomes) and the supernatants as the supernatants of the light membrane fraction (without digitonin; washing buffer and with digitonin; the contents of permeabilized cellular organelles). All fractions were mixed with 1:3 volumes of 3x Laemmli sample buffer and subjected to immunoblot analysis.
Lysosomal Volume and Permeability Assessment.
To label lysosomes, cells were incubated with 5 µM acridine orange (Molecular Probes) for 15 min at 37°C and washed twice with PBS before the indicated treatments or measurements. Acridine orange is a metachromatic fluorochrome and a weak base that exhibits red fluorescence when highly concentrated in acidic lysosomes and green fluorescence when outside the lysosomes. Total lysosomal volume and lysosomal integrity were evaluated by assessing the red fluorescence (FL2) and green fluorescence (FL1), respectively, by flow cytometry using a flow cytometer (Becton Dickinson) or confocal microscopy (Axiovert 100M microscope equipped with LSM 510 system; Carl Zeiss MicroImaging, Inc.).
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Results
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Hsp70 Depletion Triggers LMP Followed by Cathepsin-mediated PCD.
We have shown recently that the depletion of Hsp70 by adenoviral transfer of antisense Hsp70 cDNA (Ad.asHsp70) induces massive caspase-independent PCD in diverse tumor cell lines, but not in nontumorigenic epithelial cells or fibroblasts (18, 29). To find clues to the tumor-specific death pathway suppressed by Hsp70, we analyzed the ability of various protease inhibitors to rescue tumor cells from the death induced by Hsp70 depletion. Remarkably, the inhibition of cysteine cathepsin activity by broad spectrum cysteine cathepsin inhibitors zFA-fmk (6) and LHVS (31) or high concentrations of zVAD-fmk (6) conferred significant protection against Ad.asHsp70-induced death in MCF-7 and MDA-MB-468 breast cancer cells (Fig. 1, A and B). A more specific inhibition of cathepsin B by CA-074-Me conferred smaller, but significant, protection in MCF-7 cells. Due to an unspecific toxicity CA-074-Me and LHVS in the long-term assay, they could not be applied to MDA-MB-486 and MCF-7 cells, respectively. As we have shown earlier, selective inhibition of caspases by DEVD-CHO at concentrations up to 200 µM (not depicted) or low concentrations of zVAD-fmk (<50 µM) did not increase the survival of Ad.asHsp70-treated MCF-7 cells (Fig. 1 A), whereas such treatments conferred complete protection against TNF-induced apoptosis in MCF-7 cells (33). Also, 50 µM PD150606 capable of inhibiting calpain activation in MCF-7 cells was unable to rescue cells from Ad.asHsp70 (unpublished data).
The authors have no conflicting financial interests.