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NEOPLASIA
From the Faculty of Medicine and Pharmacy, INSERM U517
and INSERM U498, IFR 100, Dijon, France.
Exposure of U937 human leukemic cells to the phorbol ester
12-O-tetradecanoylphorbol 13-acetate (TPA) induces their
differentiation into monocyte/macrophage-like cells. This terminal
differentiation is associated with a resistant phenotype to
apoptosis induced by the topoisomerase II inhibitor etoposide.
The inhibition occurs upstream of the mitochondrial release of
cytochrome c and the activation of procaspase-2, -3, -6, -7, -8, and
-9. By using cell-free systems, it was demonstrated that the
mitochondrial pathway to cell death that involves mitochondrial
membrane depolarization, cytochrome c release and cytosolic activation
of procaspases by cytochrome c/dATP remains functional in
TPA-differentiated U937 cells. Accordingly, 2 drugs recently shown to
target the mitochondria, namely lonidamine and arsenic trioxide, bypass
the resistance of TPA-differentiated U937 cells to classical anticancer
drugs. Cell death induced by the 2 compounds is associated with
mitochondrial membrane depolarization, release of cytochrome c and
Smac/Diablo from the mitochondria, activation of caspases,
poly(ADP-ribose) polymerase cleavage and internucleosomal DNA
fragmentation. Moreover, the decreased glutathione content associated
with the differentiation process amplifies the ability of arsenic
trioxide to activate the mitochondrial pathway to cell death. Similar
results were obtained by comparing undifferentiated and
TPA-differentiated human HL60 leukemic cells. These data demonstrate
that mitochondria-targeting agents bypass the resistance to classical
anticancer drugs induced by TPA-mediated leukemic cell differentiation.
(Blood. 2001;97:3931-3940) Chemotherapeutic drugs can kill cultured leukemic
cell lines by inducing apoptosis.1 This mode of cell death
may be clinically relevant because apoptotic blast cells have been
detected in the peripheral blood of patients with acute leukemia who
received chemotherapeutic drugs.2 Hallmarks of apoptosis
in leukemic cells include characteristic morphologic changes such as
cell shrinkage, membrane blebbing, and cell fragmentation into
apoptotic bodies, internucleosomal DNA fragmentation, and limited
proteolytic cleavage of selective intracellular proteins such as the
nuclear enzyme poly(ADP-ribose)polymerase (PARP). This cell death
phenotype is induced by activation of a constitutively expressed
apoptotic machinery that involves a family of cysteine proteases known
as caspases, organized in a branched proteolytic cascade. These enzymes are synthesized as inactive proenzymes (procaspases) whose proteolysis at internal aspartate residues generates large and small
subunits.3 Heterodimerization of these subunits is
required to form the active enzyme (caspase) that cleaves
intracellular substrates, either a downstream procaspase or other
cellular proteins.4
Activation of the caspase cascade in response to specific damage
induced by chemotherapeutic drugs is the consequence of a disruption of
the mitochondrial membrane barrier function.5 Although the
exact mechanism remains controversial, mitochondrial changes lead to
the release of soluble apoptogenic proteins such as cytochrome
c,6 apoptosis-inducing factor (AIF),7 and Smac/Diablo8,9 from the mitochondrial intermembrane space to the cytosol. Once in the cytosol, cytochrome c, in the presence of
adenosine triphosphate (ATP), induces conformational changes of an
adaptor molecule designated Apaf-1 that recruits and activates procaspase-9 molecules.10,11 In turn, caspase-9 activates
the downstream caspase cascade that involves caspase-3 and other
effector enzymes.12 Smac/Diablo was recently shown to
suppress the inhibition of caspases by proteins known as inhibitor of
apoptosis proteins (IAPs), thereby increasing their sensitivity
to the cytochrome c/ATP pathway of activation.8,9 Once
activated, effector caspases cleave a limited set of essential
cellular proteins, leading to cell
dismantling.13-15 Mitochondrial function during apoptosis is controlled by the Bcl-2 family of proteins that could prevent the opening of the permeability transition pore or stabilize the barrier function of the outer mitochondrial membrane and prevent the release of apoptogenic molecules.5,16
How the specific damage induced by chemotherapeutic drugs leads
to mitochondrial changes and activation of the caspase cascade under
the control of Bcl-2-related proteins remains poorly understood. We
have shown previously that differentiation of human myeloid leukemia
cell lines along a macrophagic pathway induced by exposure to the
phorbol ester 12-O-tetradecanoylphorbol 13-acetate
(TPA) correlated with a resistance phenotype to apoptosis induced by a
series of anticancer drugs with distinct primary intracellular targets.17,18 This inhibition occurs downstream of
specific damage, measured as cleavage complexes, induced by
topoisomerase poisons.17 In addition, TPA-differentiated
cells are resistant to apoptosis induced by exposure to Fas-ligand, a
specific pathway that was proposed as an amplifier system in
drug-induced cell death.19 A resistance to drug-induced
and TRAIL-mediated apoptosis was also reported in vitamin
D3- and dimethyl sulfoxide (DMSO)-differentiated HL60
cells, respectively.20,21
Identification of the molecular mechanisms that modulate cell
sensitivity to chemotherapeutic drugs in differentiated cells could
help to define strategies combining differentiating and cytotoxic
agents for treating acute leukemias. The present study further explores
the level of inhibition of drug-induced apoptosis in TPA-differentiated
cells. By using cell-free systems, we demonstrate that the pathway
involving mitochondria and the downstream caspase cascade remains
functional in terminally differentiated cells. Based on this
observation, we tested the ability of 2 chemotherapeutic drugs recently
shown to target the mitochondria, namely the indazole-3-carboxylic acid
derivative lonidamine and the metalloid arsenic trioxide, to induce
apoptosis in these cells.
Drugs and chemical reagents
Antibodies
Cell culture and differentiation The human leukemic cell lines U937 and HL60 (American Type Culture Collection, Rockville, MD) were grown in suspension in RPMI 1640 medium with glutamax-I (Gibco BRL, Life Technologies, Cergy Pontoise, France) supplemented with 10% (vol/vol) fetal bovine serum (BioWhittaker, Fontenay-sous-bois, France) in an atmosphere of 95% air and 5% CO2 at 37°C. Cell viability was determined by using a trypan blue dye exclusion assay. To ensure exponential growth, cells were resuspended at a density of 0.5 × 106 cells/mL in fresh medium 24 hours before each treatment. To induce differentiation, cells were culture in the presence of 20 nM TPA for 24 hours (HL60) or 72 hours (U937) as previously described.17,18 After treatment, adherent cells were harvested using a cell scraper. U937 and HL60 cell clones containing the full-length human bcl-2 cDNA were kindly provided by Jacqueline Bréard (INSERM 461, Chatenay-Malabry, France) and Michele Allouche (CNRS-UPCM, CHU Purpan, Toulouse, France), respectively.DNA fragmentation DNA fragmentation was quantified by using a previously reported filter elution assay.22 Briefly, exponentially growing cells were incubated with 0.02 µCi/mL [2-14C]-thymidine and cultured at 37°C for 2 days. Then, cells were chased in isotope-free medium overnight, resuspended in fresh medium, and treated with different agents before or after TPA treatment. Approximately 1.0 × 106 treated or untreated labeled cells were loaded onto a protein-absorbing filter (polyvinylidene difluoride filters, 0.65 µm pore size, 25 mm diameter; Durapore membrane, Millipore, Saint-Quentin en Yvelines, France). Cells were then washed once with ice-cold phosphate-buffered saline (PBS) and subsequently lysed in 0.2% sodium sarkosyl, 2 M NaCl, 0.04 M EDTA, pH 10.0. Filters were washed with 0.02 M EDTA, pH 10.0. DNA was depurinated by incubation of filters in 1 M HCl at 65°C for 45 minutes, then released from the filters with 0.4 M NaOH for 45 minutes at room temperature. Radioactivity was counted by liquid scintillation spectrometry in each fraction (wash, lysis, EDTA wash, and filter). DNA fragmentation was measured as the fraction of disintegrations per minute (dpm) in the lysis fraction plus EDTA wash relative to the total intracellular dpm. For analyzing DNA fragmentation by agarose gel electrophoresis, cellular DNA was extracted by a previously described salting-out procedure,23 and electrophoresis was performed in 1.8% agarose gel in Tris-borate-EDTA buffer (pH 8.0) at 20 V for 14 hours. After electrophoresis, DNA was visualized by ethidium bromide staining.Western blot analysis After treatment, whole cell lysates were prepared by lysing the cells in boiling buffer (1% SDS, 1 mM Na-vanadate, 10 mM Tris pH 7.4) in the presence of protease inhibitors (0.1 mM phenylmethylsulfonyl fluoride [PMSF, 2.5 µg/mL pepstatin, 10 µg/mL aprotinin, 5 µg/mL leupeptin). Viscosity of the samples was reduced by several passages through a 26-gauge needle. Protein concentration was measured by the Bio-Rad DC protein assay kit (Ivry sur Seine, France). Thirty micrograms protein was incubated in loading buffer (125 mM Tris-HCl, pH 6.8, 10% -mercaptoethanol, 4.6% SDS, 20% glycerol, and 0.003%
bromophenol blue), separated by sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), and electroblotted to polyvinylidene difluoride membrane (Bio-Rad). After blocking nonspecific binding sites overnight by 5% nonfat milk in TPBS (0.1% PBS-Tween 20), the membrane was incubated for 2 hours at room temperature with primary antibody. After
2 washes in TPBS, the membrane was incubated with horseradish peroxidase-conjugated goat antimouse or antirabbit antibodies (Jackson
ImmunoResearch Laboratories, West Grove, PA) for 30 minutes at room
temperature, then washed twice in TPBS. Immunoblot was revealed using
enhanced chemiluminescence detection kit (Amersham) by autoradiography.
Cell fractionation Cytosolic fractions were prepared by resuspending the cells in ice-cold buffer A (250 mM sucrose, 20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 17 µg/mL PMSF, 8 µg/mL aprotinin, 2 µg/mL leupeptin [pH 7.4]) before passing them through an ice-cold cell homogenizer. Unlysed cells and nuclei were pelleted by a 10-minute, 750g spin. This step was repeated twice. The supernatant was centrifuged at 10 000g for 25 minutes, and the resultant supernatant was further centrifuged at 100 000g for 1 hour. The supernatant (cytosolic fraction) was frozen at 80°C. Nuclei-free,
mitochondria-free cytosolic extracts (cell-free extracts) were
generated as previously described.24 Briefly, cells
(1-2 × 108) were pelleted and washed twice with PBS, pH
7.2, followed by a single wash with 4 mL ice-cold cell extract buffer
(20 mM HEPES, 10 mM KCl, 1.5 mM MgCl2, 1 mM EDTA, 1 mM
EGTA, 1 mM dithiothreitol, 100 µM PMSF, 2 µg/mL aprotinin, 10 µg/mL leupeptin [pH 7.4]). Two volumes ice-cold extract buffer were
added to 1 vol packed cell pellet before the cellular suspension was
transferred to a 2-mL Dounce homogenizer. Cells were allowed to swell
under the hypotonic condition for 20 minutes, then were disrupted with
30 strokes of a B-type. Lysis was confirmed with the trypan
blue dye exclusion test before centrifugation of lysates at 16 000g for 15 minutes at 4°C. Caspase activation by the
cytochrome c/dATP combination was tested in these clarified supernatants.
Purification of mitochondria Purified mitochondria were isolated as previously described.24 Briefly, cells (1 × 108) were resuspended in buffer H (300 mM saccharose, 5 mM N-tris(hydroxymethyl)methyl-2-amino-ethanesulfonic acid, 200 µM EGTA, pH 6.9), homogenized in a Potter-Thomas homogenizer, then centrifuged for 10 minutes at 760g. The supernatant was recovered while the pellet was resuspended in H buffer and centrifuged to recover the supernatant as above. Both supernatants were mixed and centrifuged for 10 minutes at 8740g before the mitochondria pellet was resuspended in buffer H.Activation of cell-free apoptosis Cell-free extracts (10 µL, 5-10 mg/mL protein) were incubated at 37°C with 5 µM horse heart cytochrome c (Sigma) and 1 mM dATP (Pharmacia). Then caspase activation was determined by Western blot analysis. For cell-free reaction activated by atractyloside, purified mitochondria were exposed to 5 mM atractyloside for 30 minutes and pelleted by a 10 000g spin for 10 minutes before cytochrome c release was determined by Western blot in both the supernatant and the mitochondria pellet.Analysis of mitochondrial membrane potential Mitochondrial membrane depolarization was analyzed using the DePsipher kit (R&D Systems, Abington, United Kingdom) according to the manufacturer's instructions. Briefly, 106 cells were incubated for 20 minutes at 37°C in the presence of 5 µg/mL DepSipher (R&D Systems) solution and then washed 2 times in PBS. In some cases, cells were treated for 30 minutes with the uncoupling agent carbonyl cyanide m-chlorophenylhydrazone (100 µM; Sigma-Aldrich). Analysis was performed by the use of a FACscan cytometer (Becton Dickinson, Le Pont de Claix, France).Quantification of intracellular glutathione and reactive oxygen species content by flow cytometry The level of cellular glutathione (GSH) was determined by flow cytometry after staining with monochlorobimane (Molecular Probes) as previously described.25 Briefly, 200 µM monochlorobimane [syn-(ClCH2, CH3)-1,5-diazabicyclo-[3.3.0]-octa-3,6-dione-2,8-dione)] was added to cell suspensions for 30 minutes at 37°C. After 2 washes in PBS, cells were resuspended in PBS and analyzed on a Bio-Rad flow cytometer (Hercules, CA). For reactive oxygen species (ROS) measurements, cells were incubated for 15 minutes at 37°C in the presence of 6.6 µM dihydro-ethidium (Sigma-Aldrich) and analyzed by flow cytometry.
TPA-induced differentiation of U937 human leukemic cells prevents etoposide-induced apoptosis U937 leukemic cells were induced to differentiate along a macrophagic pathway on exposure to 20 nM TPA. After a 48- to 72-hour exposure to TPA, more than 90% cells demonstrate a differentiated phenotype including adhesion to the culture flask and increased expression of the glycoprotein CD11b at the cell surface. On continuous exposure to 50 µM etoposide, most of the parental U937 cells undergo apoptosis in 6 hours, as demonstrated by staining nuclear chromatin with Hoechst 33342, whereas TPA-differentiated cells strongly resist drug-induced cell death (Figure 1).
To link the resistance phenotype to the differentiation process, we
exposed the cells for 4.5 hours to 50 µM etoposide at various times
after the beginning of TPA treatment (20 nM) (Figure 2). Apoptosis was identified by Hoechst
staining of nuclear chromatin (Figure 2A) and quantified by measuring
nuclear DNA fragmentation (Figure 2B). Exposure to TPA provoked a
time-dependent decrease of etoposide-induced morphologic changes and
DNA fragmentation that were almost completely inhibited 48 to 72 hours
after the beginning of TPA exposure. Accordingly, by using
agarose gel electrophoresis, the characteristic nucleosome-sized
ladder fragmentation of nuclear DNA observed in parental
undifferentiated cells exposed for 4.5 hours to the drug was not
detected in TPA-differentiated cells treated in the same conditions
(Figure 2B, insert).
Etoposide treatment of parental cells also induced a decrease of procaspase-2, -3, -6, -7, -8, and -9 expression, as determined by Western blot analysis (Figure 2C). By using antibodies reacting with the cleavage fragments that characterize active enzymes, decreased expression of the 48-kd procaspase-2, the 32-kd procaspase-3, the 35-kd procaspase-7, and the 48-kd procaspase-9 correlated with the appearance of a p35, a p19/p17, a p10, and a p37 active fragment, respectively. The 30-kd fragment identified with the anti-caspase-9 antibody might correspond to the previously described caspase-9b isoform.26 The significance of the 18-kd band remains unknown. All these etoposide-induced events were progressively inhibited as U937 cells progressed toward the differentiation phenotype on TPA exposure. This inhibition became identifiable between 6 and 12 hours after the beginning of TPA exposure, depending on the experiment. In addition, TPA-induced differentiation was associated with a progressive inhibition of the 116-kd DNA repair enzyme PARP cleavage into an 85-kd N-terminal fragment (Figure 2C). Caspases remain able to be activated in cell-free extracts from TPA-induced differentiated cells Exposure of undifferentiated U937 cells to 50 µM etoposide for 4.5 hours triggered the appearance of cytochrome c in the cytosol. This event was prevented by TPA-induced differentiation (Figure 3A). To determine whether the cytochrome c-mediated activation of the caspase cascade was influenced by the differentiation process, we used a previously described cell-free system.24 Exposure of cell-free extracts from undifferentiated U937 cells to 5 µM cytochrome c and 1 mM dATP induced a time-dependent decrease of the expression of procaspase-2, -3, -6, -7, and -9, suggesting their cleavage into active fragments. Accordingly, immunoblot experiments detected the time-dependent appearance of the 35-kd active fragment of caspase-2, the 19- and 17-kd fragments of caspase-3, the 11-kd fragment of caspase-6, the 30-kd fragment of caspase-7, and the 37-kd fragment of caspase-9 (Figure 3B). In this time-course, procaspase-8 expression remained unchanged. TPA-mediated differentiation of U937 cells induced some changes in the subcellular localization of procaspases, mainly the disappearance of procaspase-2 from the cytosol (O.S. et al, manuscript in preparation). The other procaspases remained sensitive to activation by the cytochrome c-dATP combination with kinetics similar to that observed in cytosolic extracts from undifferentiated cells (Figure 3B). Thus, TPA-induced differentiation does not significantly influence procaspase activation by cytochrome c/dATP.
Mitochondria from TPA-induced differentiated U937 cells remain reactive The mechanisms that account for the mitochondrial release of cytochrome c are still controversial. One of the proposed mechanisms involves a decrease of the mitochondrial membrane potential  m.5 To determine whether TPA-induced differentiation
was associated with a decreased reactivity of mitochondria to
uncoupling agents, we exposed undifferentiated and TPA-differentiated
U937 cells for 30 minutes to 100 µM of the uncoupling agent carbonyl
cyanide m-chlorophenylhydrazone (mCICCP). This treatment induced a
switch from the mitochondrial aggregate (FL2-H) to the cytosolic
monomer (FL1-H) that was similar in mitochondria from both
undifferentiated and TPA-differentiated cells indicating mitochondrial
depolarization (Figure 4A). A mechanism
proposed to account for the mitochondrial release of cytochrome c was
the opening of the megachannel that can be induced by exposing purified
mitochondria to atractyloside (5 mM, 30 minutes). This compound induced
the release of cytochrome c from mitochondria of undifferentiated and
differentiated U937 cells with a similar efficacy (Figure 4B). In the
presence of cell-free extracts from undifferentiated U937 cells, the
effect of atractyloside on purified mitochondria resulted in cleavage of the DEVD-AFC substrate, suggesting caspase activation (data not
shown). Together these results suggested that the mitochondrial pathway
to cell death may remain functional in TPA-differentiated U937
cells.
Mitochondria-targeting agent lonidamine induced apoptosis of TPA-differentiated cells The previous observations suggested that drugs shown to directly target the mitochondria might overcome the resistance of TPA-differentiated cells to etoposide and various other classical anticancer drugs.18 We first tested the mitochondria-targeting drug lonidamine.27 The ability of this compound to induce apoptosis of TPA-differentiated cells was only slightly delayed in TPA-differentiated compared to undifferentiated U937 cells, as demonstrated by quantifying DNA fragmentation (Figure 5A) and by counting the percentage of cells demonstrating nuclear chromatin condensation after a 24- to 48-hour exposure to 200 µM lonidamine (Table 1). Lonidamine-induced apoptotic DNA fragmentation and chromatin condensation were completely inhibited by stable overexpression of Bcl-2 in U937 cells (Figure 5A; Table 1). In undifferentiated and TPA-differentiated cells, lonidamine induced mitochondrial depolarization (Figure 5C), cytochrome c release in the cytosol (Figure 5B), procaspase-3 cleavage in its active fragments, and PARP cleavage (Figure 5D). All these events were prevented by Bcl-2 overexpression. The recently described Smac/Diablo protein8,9 was only slightly released in the cytosol on lonidamine exposure, an effect that was not prevented by Bcl-2 overexpression (Figure 5B).
TPA-induced differentiation sensitizes U937 cells to arsenic trioxide-induced apoptosis As2O3 is another member of this novel class of chemotherapeutic agents that induce apoptosis, at least in part, by acting on the mitochondria.28,29 As2O3 induced apoptosis of undifferentiated U937 cells in a dose- and time-dependent manner (Figure 6A; Table 1). TPA-induced differentiation did not prevent, and even sensitized, U937 cells to As2O3-induced apoptosis. The differentiation process also increased the ability of As2O3 to induce mitochondrial depolarization (Figure 6C), cytochrome c release from the mitochondria to the cytosol (Figure 6B), procaspase-3 cleavage in its active fragments and PARP cleavage (Figure 6D). All these events were prevented by Bcl-2 overexpression in U937 cells (Figure 6A-D; Table 1). Smac/Diablo protein was released in the cytosol on As2O3 treatment more efficiently than on lonidamine exposure. Again, this effect was not prevented by Bcl-2 overexpression (Figure 6B). Because As2O3-induced apoptosis has been shown to be modulated by the cellular GSH redox system, with increased intracellular levels of reduced GSH having an inhibitory effect,30 we measured the influence of TPA-induced differentiation on cellular GSH and ROS content in untreated and As2O3-treated cells. By using flow cytometry methods, we observed that the differentiation process was associated with a decrease in the basal level of GSH (Figure 7A) and an increase in the basal ROS content in the cells (Figure 7B). Arsenic exposure decreased GSH content and increased ROS level in parental cells. These effects were prevented by Bcl-2 overexpression. In differentiated cells, As2O3 treatment further decreased GSH content without increasing the cellular ROS content, as measured by using dihydro-ethidium (Figure 7A-B).
Lonidamine and arsenic trioxide also bypass the resistance of TPA-differentiated HL60 cells to classical chemotherapeutic drugs The results obtained in U937 cells were reproduced in HL60 cells whose exposure to TPA also induces a macrophagic-like differentiation process with a resistant phenotype to classical anticancer drugs.17 TPA-differentiated HL60 cells, which were resistant to etoposide-induced apoptosis, demonstrated a sensitivity to lonidamine that was only slightly delayed compared to that of parental cells, whereas their sensitivity to As2O3-induced cell death was greatly increased (Figure 8; Table 1).
Classical chemotherapeutic drugs induce apoptosis by compromising
mitochondrial function in an indirect fashion TPA-mediated inhibition of etoposide-induced apoptosis increased as leukemic cells progressed toward the macrophagic differentiation phenotype. Short-term effects of TPA are mainly the activation of several protein kinase C (PKC) isoenzymes.34-36 The differentiation process involves several events downstream of the transient activation of PKCs.37 Thus, the differentiation-related resistance of leukemic cells to etoposide-induced cell death might be distinguished from the direct consequences of PKC activation. A differentiation-related resistance to apoptosis induced by various stimuli has been described in leukemic cells exposed to either 1,25-dihydroxyvitamin D338,39 or DMSO.21 A resistance to growth factor deprivation-induced cell death was also identified in differentiated myocytes.40 Thus, several differentiation processes in different cell types were associated with a decreased sensitivity to apoptosis induced by a series of distinct stimuli. Most pathways to apoptosis converge on the mitochondrial release of
apoptogenic molecules to the cytosol and the activation of the caspase
cascade.1,4,5 The current study confirms that etoposide
activates a variety of caspases while inducing apoptosis of
undifferentiated U937 cells. Various mechanisms negatively interfere
with this final pathway, including overexpression of antiapoptotic
proteins of the Bcl-2 family that prevents the cytosolic release of
mitochondrial soluble molecules7,41 and overexpression of
heat-shock proteins such as HSP27 and HSP70 that prevent cytochrome c-mediated activation of the caspase cascade by interacting with cytochrome c24 and the Apaf-1 adaptor
molecule,42 respectively. Despite the fact that the
TPA-induced differentiation process induces some changes in the
subcellular localization of procaspases (O.S. et al, manuscript in
preparation) and modulates the expression of inhibitory proteins Lonidamine (1-[(2,4-dichlorophenylmethyl) methyl]-1H-indazole-3-carboxylic acid]) is an antineoplastic drug derived from indazole-3-carboxylic acid. This compound, which was shown to exacerbate the response of human tumor cells to cisplatin, irradiation, doxorubicin, or cyclophosphamide, has been tested in phase II and III trials of metastatic breast cancer and ovarian cancer.44-48 Although several distinct mechanisms were proposed to account for lonidamine cytotoxic effects,49,50 recent data suggest that mitochondria could be the main subcellular target of this compound. Lonidamine directly acts on the permeability transition pore complex when tested on purified organelles, an effect inhibited by Bcl-2.27 Since we have shown that the mitochondrial pathway to cell death remains unchanged in TPA-differentiated cells, lonidamine may have demonstrated a similar efficacy on undifferentiated and TPA-differentiated leukemic cells. Actually, TPA-induced differentiation slightly, but reproducibly, delays lonidamine-induced apoptosis in U937 and HL60 cells, suggesting a negative effect of the differentiation process on some other potential consequences of lonidamine treatment, such as cytosolic calcium increase and intracellular lactate accumulation. This effect of the differentiation process on lonidamine-induced cell death remains limited compared with the strong inhibition of apoptosis triggered by classical anticancer drugs. As2O3 is another therapeutic compound that was
suggested to directly target the mitochondria.28,29
Although As2O3-induced apoptosis was initially
described in acute promyelocytic cell lines,51,52 this
compound induces the death of many other leukemia cell
types.53-56 The hypothesis that
As2O3 directly targets the mitochondria is
based on the observation that exposure of purified organelles to
arsenic triggers opening of the permeability transition pore and the
release of soluble intermembrane proteins.28 In whole
cells, As2O3 induces disruption of the
mitochondrial transmembrane potential In conclusion, we have shown that the TPA-mediated differentiation of
human myeloid leukemic cell lines induces a resistance phenotype to
classical anticancer drugs such as etoposide without inhibiting the
mitochondrial pathway to cell death. The differentiation process has a
limited influence on lonidamine-induced cell death and sensitizes
leukemic cells to As2O3-mediated apoptosis
(Figure 9). Although the
As2O3 doses required for inducing
apoptosis in differentiated cells remain high compared with
clinically achieved concentrations,68 these observations
suggest that chemotherapeutic drugs that directly or rapidly target the
mitochondria in vitro warrant further clinical investigation in the
treatment of acute leukemias. These drugs may represent an effective
strategy to bypass the resistance of leukemic cells to classical
anticancer drugs, or they may be used in combination with
differentiating agents.
We thank M. F. Poupon, X. Wang, and D. G. Chen for providing lonidamine, anti-Smac antibody, and As2O3, respectively.
Submitted November 14, 2000; accepted February 27, 2001.
Supported by grants from the Ligue National Contre le Cancer (Equipe labelisée "la ligue") and the Association Régionale pour l'Enseignement et la Recherche Scientifique. O.S. is the recipient of a grant from the Fondation pour la Recherche Médicale.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Eric Solary, Faculty of Medicine and Pharmacy, INSERM U517, 7 boulevard Jeanne d'Arc, 21033 Dijon, France; e-mail: esolary{at}u-bourgogne.fr.
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Top
Abstract
Introduction
) and TPA-differentiated (
) U937 cells were
treated with 50 µM etoposide for indicated times. Percentages of
apoptotic cells were determined by fluorescent microscopy after Hoechst
staining. Each value is the mean ± SD of triplicate
determinations.
, U-BCL2) were treated for
indicated times with 200 µM lonidamine. (A) Apoptotic DNA
fragmentation was quantified using a filter elution assay. Values from
1 of 2 independent experiments are shown (mean ± SD of triplicate
samples). (B) Western blot analysis of cytochrome c and Smac/Diablo
expression in cytosolic fractions from U937, U-TPA, and U-BCL2 cells
treated (+) or not treated (
for example, by inducing
DNA damage that triggers p53 activation and interferes with cell cycle
progression
chimeric proteins to nuclear bodies,
but not 
in human interleukin-3-dependent cells suppresses apoptosis and induces bcl-2 expression.
Blood.
1998;91:823-829