Interferon-gamma Sensitizes Human Myeloid Leukemia Cells to Death Receptor-mediated Apoptosis by a Pleiotropic Mechanism*

Nieves VarelaDagger §, Cristina Muñoz-Pinedo||, Carmen Ruiz-Ruiz||, Gema Robledo, Miriam PedrosoDagger , and Abelardo López-Rivas**

From the  Instituto de Parasitología y Biomedicina, Consejo Superior de Investigaciones Científicas, calle Ventanilla 11, 18001 Granada, Spain and the Dagger  Centro Nacional de Sanidad Agropecuaria, carretera de Tapaste y Autopista Nacional, San José de las Lajas, La Habana, Cuba

Received for publication, January 29, 2001


    ABSTRACT
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The role of interferon (IFN)-gamma as a sensitizing agent in apoptosis induced by ligation of death receptors has been evaluated in human myeloid leukemia cells. Incubation of U937 cells with IFN-gamma sensitized these cells to apoptosis induced by tumor necrosis factor-alpha , agonistic CD95 antibody, and tumor necrosis factor-related apoptosis-inducing ligand. Other human myeloid leukemic cells were also sensitized by IFN-gamma to death receptor-mediated apoptosis. Treatment of U937 cells with IFN-gamma up-regulated the expression of caspase-8 and potently synergized with death receptor ligation in the processing of caspase-8 and BID cleavage. Concomitantly, a marked down-regulation of BCL-2 protein was also observed in cells incubated with IFN-gamma . Furthermore, the caspase-dependent generation of a 23-kDa fragment of BCL-2 protein, the release of cytochrome c from mitochondria and the activation of caspase-9 were also enhanced upon death receptor ligation in IFN-gamma -treated cells. Ectopically expressed Bcl-2 protein inhibited IFN-gamma -induced sensitization to apoptosis. In summary, these results indicate that IFN-gamma sensitizes human myeloid leukemic cells to a death receptor-induced, mitochondria-mediated pathway of apoptosis.


    INTRODUCTION
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Interferons (IFNs)1 are a family of natural glycoproteins that share antiviral, immunomodulatory, and anti-proliferative effects (1). In mice, a defect in the transcriptional regulation of IFN-dependent genes causes a marked increase in the number of myeloid cells and hematologic alterations similar to chronic human myelogenous leukemia (2). In both chronic and acute human myeloid leukemias, a decrease of IFN-modulated transcriptional activity has been reported (3, 4). On the other hand, the anti-tumor properties of IFNs against a variety of tumor cells such as lymphomas, melanomas, and multiple myeloma has also been demonstrated (5, 6). Furthermore, clinically and experimentally, IFN-gamma has been shown to enhance the anti-tumor effects of anti-metabolite on cancer cells (5, 7). Positive anti-tumor effects have also been obtained by immunotherapy with natural IFNs and interleukins, particularly in combination strategies (8). In tumor cell lines, IFN-gamma can induce or modulate cell death either as a single agent or in combination with chemotherapeutic drugs (9).

Apoptosis is an active form of cell death that plays a fundamental role in normal development, tissue homeostasis, and pathological situations (10-12). CD95 (Fas/Apo-1) receptor, a member of TNF/nerve growth factor receptor family (13, 14), is a potent inducer of apoptosis in the immune system upon interaction with its natural ligand CD95L, a type II integral membrane protein homologous to TNF (15). TRAIL, a recently identified member of the TNF family with homology to CD95L (16), induces apoptosis (17) upon binding to death-domain containing receptors, TRAIL-R1 and TRAIL-R2 (also known as DR4 and DR5, respectively) (18-21). Although the expression of CD95L seems to be more restricted to lymphoid cells (15), TRAIL transcripts are detectable in many normal organs and tissues (16), suggesting that this ligand may be non-toxic to normal cells.

Death receptors are expressed in many tumor cells that can therefore be killed by the appropriate ligands (22). However, expression of death receptors is not always sufficient to allow an apoptotic response since there are examples of tumor cells, including myeloid leukemic cells, that express significant levels of death receptors in the plasma membrane but are resistant to death ligands (22, 23). Understanding the mechanisms that sensitize tumor cells to death ligand-induced apoptosis could therefore be an important objective in the development of therapies to treat malignancies like human myelogenous leukemias. In this respect, IFN-gamma and IFN-alpha can up-regulate the expression of a number of apoptosis-related proteins in different types of cells (9, 24, 25). In certain cancer cells, including U937 myeloid leukemic cells, it has been reported that IFN-gamma induces sensitization to CD95-mediated apoptosis by up-regulating the expression of ICE/caspase-1 (26, 27). However, more recent data have demonstrated that caspase-1/ICE is not involved in the proteolytic cascade activated upon CD95 cross-linking at the cell surface by CD95L or CD95 antibody (28-30).

The above data prompted us to investigate the effects of IFN-gamma on death receptor-induced apoptosis in the human U937 myeloid leukemic cell line. We were particularly interested to ascertain whether IFN-gamma could enhance the sensitivity of these cells to TRAIL-induced apoptosis, in view of the importance of TRAIL as a rather selective anti-tumor protein (31). In this report we show that IFN-gamma sensitizes U937 cells and other human myeloid leukemic cell lines to CD95-, TNF-R-, and TRAIL-R-mediated apoptosis. Following treatment of U937 cells with IFN-gamma , we have observed a significant up-regulation of caspase-8 and a marked down-regulation of BCL-2 protein. Furthermore, we demonstrate that in U937 cells, IFN-gamma facilitates several biochemical events involved in death receptor-induced mitochondria-mediated apoptosis.

    EXPERIMENTAL PROCEDURES
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Materials-- RPMI 1640 medium and fetal bovine serum were obtained from Life Technologies, Inc. CH-11 monoclonal antibody (mAb) reacting with CD95 was from Upstate Biotechnology Inc. (Lake Placid, NY). Human IFN-gamma , human TNF-alpha and recombinant human TRAIL were obtained from PreproTech EC Ltd (London, United Kingdom). Mouse anti-BAX mAb, mouse anti-BAD mAb, and mouse anti-cytochrome c mAb were obtained from PharMingen (San Diego, CA). Mouse anti-BCL-2 mAb was from Dako (Glostrup, Denmark). Mouse anti-human caspase-8 mAb and rabbit anti-caspase-9 polyclonal antibodies were purchased from Cell Diagnostica (Münster, Germany) and StressGen Biotechnologies Corp. (Victoria, Canada), respectively. Goat polyclonal anti-caspase-3 antibody was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). Rabbit polyclonal antiserum against PARP was purchased from Roche Molecular Biochemicals (Mannheim, Germany). Mouse anti-FADD mAb was from Transduction Laboratories (Lexington, KY). Rabbit anti-BID polyclonal antibody was generously provided by Dr. X. Wang (Howard Hughes Medical Institute, Dallas, TX). Rabbit polyclonal antibodies to caspase-9 p37 fragment and caspase-3 p17 subunit were obtained from New England Biolabs (Beverly, MA). Monoclonal antibody to alpha-tubulin was purchased from Sigma (Poole, United Kingdom). Benzyloxycarbonyl-Val-Ala-Asp- (OMe)fluoromethyl ketone (Z-VAD-fmk) was from Enzyme System Inc. (Dublin, CA).

Cell Culture-- The various human myeloid leukemic cell lines used in this study were maintained in culture in RPMI 1640 medium containing 10% fetal calf serum and 1 mM L-glutamine, at 37 °C in a humidified 5% CO2, 95% air incubator. Cell viability was determined by the trypan blue dye exclusion method.

Determination of Apoptotic Cells-- Analysis by flow cytometry of hypodiploid apoptotic cells was performed on a FACScan cytometer using the Cell Quest software (Becton Dickinson, Mountain View, CA), after extraction of the degraded DNA from apoptotic cells following a recently described method (32).

Phosphatidylserine exposure on the surface of apoptotic cells was detected by flow cytometry after staining with Annexin-V-FLUOS (Roche Molecular Biochemicals).

Analysis of DNA cleavage into oligonucleosome-length fragments was performed following a method described previously (33).

Cytochrome c Release from Mitochondria-- For measurements of cytochrome c release from mitochondria, cells were lysed and cytosolic fractions were separated from mitochondria as described (34). Cytosolic proteins (40 µg of protein) were mixed with Laemmli buffer and resolved on 12% SDS-polyacrylamide minigels. Cytochrome c was determined by Western blot analysis as described below.

Cell Extracts and Western Blot Analysis of Proteins-- Cells were pelleted and lysed in Laemmli buffer. After sonication, proteins were resolved on 7.5% SDS-polyacrylamide minigels for determination of PARP cleavage or 12% SDS for analysis of other proteins, and electrophoretically transferred onto Immobilon (Millipore). Membranes were blocked with 5% milk powder in PBS plus 0.1% Tween 20 (PBS/Tween) for 1 h and washed with PBS/Tween. For protein detection, immunoblots were probed with alpha -tubulin mAb (1:40000), BAX mAb (1 µg/ml), BAD mAb (1:500), cytochrome c mAb (0.5 µg/ml), polyclonal antiserum against PARP (1:2000), caspase-3 antibody (1:1000), caspase-8 mAb (1:200), caspase-9 antibody (1:1000), caspase-9 (37-kDa fragment, 1:500), caspase-3 (17-kDa subunit, 1:500), BID antibody (1:2000), or BCL-2 mAb (1:1000). After washing, membranes were incubated with horseradish peroxidase-conjugated anti-rabbit IgG (1:2000; Dako) or horseradish peroxidase-conjugated anti-mouse Ig (1:2000, Dako). Bound antibody was visualized by enhanced chemiluminescence (ECL, Amersham Pharmacia Biotech), according to manufacturer's instructions.

Reverse Transcriptase-Polymerase Chain Reaction (RT-PCR)-- Total RNA was isolated from cells with Trizol reagent (Life Technologies, Inc.) as recommended by the supplier. cDNAs were synthesized from 2 µg of total RNA using a RT-PCR kit (PerkinElmer Life Sciences) with the supplied oligo(dT) primer under conditions described by the manufacturer. PCR reactions were performed using the following primers: TRAIL-R1 sense, 5'-CTGAGCAACGCAGACTCGCTGTCCAC-3'; TRAIL-R1 antisense, 5'-TCCAAGGACACGGCAGAGCCTGTGCCAT-3'; TRAIL-R2 sense, 5'-GCCTCATGGACAATGAGATAAAGGTGGCT-3'; TRAIL-R2 antisense, 5'-CCAAATCTCAAAGTACGCACAAACGG-3'; BAK sense, 5'-CCTGTTTGAGAGTGGCATC-3'; BAK antisense, 5'-TCGTACCACAAACTGGCCCA-3'; IRF-1 sense, 5'-CTTAAGAACCAGGCAACCTCTGCCTTC-3'; IRF-1 antisense, 5'-GATATCTGGCAGGGAGTTCATG-3'; BCL-2 sense, 5'-AGATGTCCAGCCAGCTGC ACCTGAC-3'; BCL-2 antisense, 5'AGATAGGCACCAGGGTGAGCAAGCT-3'; beta -actin sense, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3'; and beta -actin antisense, 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3', giving products of 506, 502, 266, 406, 367, and 661 base pairs, respectively. Expression of beta -actin was used as a control of RNA integrity and equal gel loading. Cycle conditions for all PCR reactions were 1 min at 95 °C, 1 min at 55 °C, and 1 min at 72 °C for 30 cycles. FLIP was analyzed by RT-PCR as described recently (35).

    RESULTS
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ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

IFN-gamma Sensitizes U937 Human Myeloid Leukemic Cells to Death Receptor-mediated Apoptosis-- Activation of death receptors in tumor cells by appropriate ligands or agonistic antibodies results in the death of target cells by apoptosis (22). However, some tumor cells are not very sensitive to death receptor-mediated apoptosis unless protein synthesis is inhibited (36). This is also the case for the human promyelocytic cell line U937 (Fig. 1). As shown in Fig. 1, U937 cells were markedly sensitized to CD95-mediated death by co-treatment with the protein synthesis inhibitor cycloheximide. In order to find a physiologically relevant sensitizing agent, in this work we have examined the ability of IFN-gamma to modulate the apoptotic response of human U937 myeloid leukemic cells upon death receptor ligation in the cell surface. IFN-gamma can induce an anti-proliferative response in a variety of tumor cells (1). It could also activate an apoptotic program or sensitize cells to apoptosis induced by other stimuli (27, 37, 38), although the mechanism underlying the sensitization process remains unclear. Results shown in Fig. 2 indicate that pre-incubation of U937 cells with IFN-gamma (10 units/ml) for 24 h markedly sensitized these leukemic cells to apoptosis upon death receptor activation. In these experiments we observed that IFN-gamma facilitated the generation of hypodiploid apoptotic cells by a subsequent treatment with TNF-alpha , CD95 agonistic antibody, or TRAIL (Fig. 2a). This effect was paralleled by the externalization of phosphatidylserine in the plasma membrane of U937 cells (data not shown). Other apoptotic features like activation of caspase-3 (Fig. 2b), DNA fragmentation in a ladder pattern (Fig. 2c), and PARP cleavage (Fig. 2d) were also markedly enhanced by IFN-gamma . In the experiments involving CD95 IgM, an irrelevant IgM antibody did not cooperate with IFN-gamma in the induction of apoptosis (results not shown).


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Fig. 1.   Incubation of U937 cells with cycloheximide sensitizes these leukemic cells to CD95-mediated death. U937 cells were treated for 24 h with different doses of CD95 antibody CH-11 in the absence or presence of cycloheximide (0.25 µg/ml). After this incubation, cell viability was assessed by the trypan blue dye exclusion method. Data shown are representative of three separate experiments.


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Fig. 2.   IFN-gamma sensitizes U937 cells to death receptor-mediated apoptosis. Cells were pre-incubated for 24 h with 10 units/ml IFN-gamma and treated for an additional 24-h period with CD95 mAb (20 ng/ml), TNF-alpha (1 ng/ml), or TRAIL (10 ng/ml). Apoptotic features were determined as described under "Experimental Procedures." a, percentage of apoptotic cells as determined by cytofluorimetric analysis of DNA content. b, caspase-3 activation, measured by the generation of 17-kDa subunit. c, DNA fragmentation into oligonucleosome-length fragments. d, proteolytic cleavage of poly(ADP-ribose) polymerase. In a, error bars represent S.D. from three independent experiments. In b-d, the results illustrate a representative experiment from at least three different experiments.

Sensitization by IFN-gamma to death receptor-induced apoptosis was also observed in two other human myeloid leukemic cell lines examined. Results shown in Fig. 3 demonstrate that incubation of either HL-60 (Fig. 3a) or THP-1 (Fig. 3b) cells with IFN-gamma sensitized them to a subsequent treatment with TNF-alpha or CD95 antibody.


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Fig. 3.   Other myeloid leukemic cells are also sensitized by IFN-gamma to death receptor-induced apoptosis. HL-60 (a) or THP-1 (b) cells were incubated for 24 h with 10 units/ml IFN-gamma and treated for an additional 36-h period with either CD95 mAb (20 ng/ml) or TNF-alpha (1 ng/ml). Apoptotic cells were determined by flow cytometry as described under "Experimental Procedures." Percentage of hypodiploid apoptotic cells is shown. The results illustrate a representative experiment from at least three different experiments.

It has been reported that IFN-gamma can elevate the expression of CD95 in several acute myelogenous leukemic cell lines, including U937 cells (39, 40) and that this effect could explain the enhanced sensitivity of IFN-gamma -treated cells to apoptosis mediated by CD95 receptors. However, U937 cells express a high number of CD95 molecules in their surface, and in our experiments we have observed only a slight increase in CD95 expression in IFN-gamma -treated cells (data not shown). Furthermore, we have determined by RT-PCR the expression of pro-apoptotic TRAIL receptors (DR4 and DR5) in U937 cells treated with IFN-gamma (Fig. 4). As a control of IFN-gamma action, we determined the expression of the transcription factor IRF-1, an IFN-gamma -regulated gene (Fig. 4). Expression of TRAIL receptors was not significantly elevated by IFN-gamma in U937 cells after 24 h (Fig. 4) or 48 h (data not shown). We also examined by RT-PCR the levels of TRAIL decoy receptors DcR1 and DcR2 in cells incubated in the presence of IFN-gamma for 24 h. Results not shown indicated that the cellular levels of these anti-apoptotic receptors did not change upon IFN-gamma treatment. Although we have not investigated the expression of TNF-R in U937 cells, the above results suggested that the sensitization to apoptosis observed in IFN-gamma -treated cells should be probably related to changes in the intracellular levels of apoptosis regulators rather than to an increase in the expression of death receptors.


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Fig. 4.   Expression of TRAIL receptors upon treatment with IFN-gamma . U937 cells were incubated for 24 h with 10 units/ml IFN-gamma . RT-PCR of TRAIL-R1, TRAIL-R2, IRF-1, and beta -actin were performed as described under "Experimental Procedures."

Up-regulation of Caspase-8 and Down-regulation of BCL-2 in U937 Cells Treated with IFN-gamma -- Regulation of the expression and/or activity of the death receptor-inducing signaling complex (DISC) components could be a strategy used by tumor cells to escape from the host immune system (41-43). Caspase-8, the most apical caspase required in death receptor-mediated apoptosis (44), is also a cellular target of oncogenic viruses, to protect transformed cells from death receptor-induced apoptosis (45). The intracellular signaling mechanism involved in the activation of apoptosis by death receptors comprises different activities (46). The adapter protein FADD is responsible for coupling death receptors to the initiator caspase-8 (47). We have examined the levels of both FADD and caspase-8 in U937 cells following treatment with IFN-gamma . Results in Fig. 5 indicate that procaspase-8, but not FADD, was up-regulated in these leukemic cells after 24 h of incubation in the presence of IFN-gamma . This could be relevant in the mechanism of IFN-gamma -induced sensitization of U937 cells to death receptor-mediated apoptosis as overexpression of procaspase-8 by transfection has been reported to facilitate apoptosis (48). On the contrary, the cellular levels of procaspase-9, which plays an important role in the mitochondria-mediated pathway of apoptosis, and procaspase-3, responsible for many of the nuclear changes during apoptosis, did not change in U937 cells treated with IFN-gamma for up to 48 h (Fig. 5).


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Fig. 5.   Effect of IFN-gamma on the expression of FADD and caspases. Cells were incubated for the indicated times with 10 units/ml IFN-gamma . Expression levels of FADD and caspase-8, -3, and -9 were determined by Western blot. Results are representative of at least three independent experiments.

To further characterize the cellular modulators of apoptosis that may be responsible for the sensitizing action of IFN-gamma in U937 cells, we analyzed the expression of the apoptosis inhibitor FLIP and several pro-apoptotic members of the BCL-2 family. As shown in Fig. 6 (a and b), IFN-gamma treatment did not modify the cellular levels of FLIP, BAK, BAX, BAD, or BID, determined either by RT-PCR or Western blot analysis. In these experiments, the cellular expression of IRF-1 mRNA was up-regulated by IFN-gamma , serving as an internal control of IFN-gamma activity.


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Fig. 6.   Effect of IFN-gamma on the expression of FLIP and pro-apoptotic members of the BCL-2 family. a, levels of FLIP and BAK mRNA as detected by RT-PCR, after treatment with 10 units/ml IFN-gamma . IRF-1 mRNA was amplified as a control for IFN-gamma -mediated gene regulation. b, protein levels of BAX, BAD, and BID, as detected by Western blotting, after incubation of cells with 10 units/ml IFN-gamma for the indicated times. Results are representative of at least three independent experiments.

Interestingly, when we determined the expression of anti-apoptotic BCL-2 protein, a marked decline in the cellular levels of this anti-apoptotic protein was observed following IFN-gamma treatment (Fig. 7). A decreased level of BCL-2 was clearly observed after 24 h of IFN-gamma treatment and remained low for at least 48 h. This effect was also observed at the mRNA level (Fig. 7). Although we cannot exclude an IFN-gamma -induced decrease of BCL-2 mRNA stability, these results may suggest a negative regulation of BCL-2 gene transcription by IFN-gamma as described recently (49). Reduction in the levels of BCL-2 could be an important event in the regulation of cellular sensitivity to stress treatments, which operate through a mitochondrial pathway of apoptosis (50). A decrease in cellular BCL-2 protein levels could also sensitize type II cells to CD95-mediated apoptosis (51). CD95 type II cells are also characterized by a markedly increased sensitivity to death receptor-induced apoptosis upon inhibition of protein synthesis (52). Sensitivity of U937 cells to death receptor-induced apoptosis is considerably enhanced by cycloheximide treatment (Fig. 1) (53), which suggests that U937 cells are probably type II cells (52).


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Fig. 7.   Down-regulation of BCL-2 following IFN-gamma treatment. U937 cells were incubated for the indicated times with 10 units/ml IFN-gamma and the level of BCL-2 protein was determined by Western blot analysis (upper panel). In the lower panel, cells were treated for 24 h in absence or presence of IFN-gamma (10 units/ml) and the expression of BCL-2 mRNA was determined by RT-PCR.

Facilitation by IFN-gamma of a Death Receptor-mediated Mitochondrial Pathway of Apoptosis in U937 Cells-- In order to ascertain the steps in death receptor-mediated apoptosis that are affected by IFN-gamma treatment, we first examined the activation of caspase-8 by analyzing the processing of this caspase into various specific proteolytic fragments. As shown in Fig. 8a, activation of either death receptor clearly synergized with IFN-gamma in the stimulation of procaspase-8 processing. By immunoblot analysis of caspase-8 in treated cells, we detected both the 55- and 53-kDa inactive proforms corresponding to caspase-8a and -8b as well as the 43- and 41-kDa intermediate products corresponding to cleavage of both caspase-8a and -8b between the large and small subunits. We also detected the presence of the large 18-kDa subunit, which would lead upon combination with the small 10-kDa subunit to the assembly of the active caspase (54). Caspase-8 is the most upstream caspase required in death receptor-mediated apoptosis (44), although it could also be activated downstream of mitochondria through an amplification pathway regulated by this organelle during apoptosis (52). One of the consequences of the activation of caspase-8 at the DISC upon CD95 ligation is the cleavage of BID, a BH3 domain-containing member of the BCL-2 family, to generate a 15-kDa fragment that translocates to mitochondria (55). Insertion of cleaved BID into the mitochondrial membrane causes the release of cytochrome c from mitochondria (55). We have examined the effect of IFN-gamma treatment on the cleavage of BID following death receptor activation. Results shown in Fig. 8b indicate that IFN-gamma treatment did not affect the cellular level of BID, as observed previously (Fig. 6b), but it markedly facilitated the cleavage of BID upon death receptor ligation, as determined by the decrease in the level of intact BID in the cells. Results presented in Fig. 8 (a and b) also indicated that there was a good correlation between the degree of caspase-8 activation and the extent of intact BID protein loss, suggesting a possible cause-effect relationship between both events.


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Fig. 8.   IFN-gamma -mediated sensitization of U937 cells to death receptor-induced caspase-8 activation, BID cleavage, and generation of 23-kDa BCL-2 fragment. Cells were incubated with 10 units/ml IFN-gamma for 24 h and subsequently treated for 24 h with CD95 mAb (20 ng/ml), TNF-alpha (1 ng/ml), or TRAIL (10 ng/ml). After this treatment, procaspase-8 processing was assayed by Western blot analysis (a). Bands corresponding to intact proforms (55 and 53 kDa), cleaved intermediate products (43 and 41 kDa), and large subunit (18 kDa) of caspase-8 are marked with arrows. Results are representative of three independent experiments. b, the cellular level of BID was determined by Western blot following treatment with IFN-gamma and death receptor activator. beta -Tubulin expression was used as a control of equal gel loading. c, following IFN-gamma treatment, Z-VAD-fmk (100 µM) was added to some cultures 1 h before death receptor activation. BCL-2 was detected by Western blotting. Panels show two different exposures of the same immunoblot membrane to demonstrate both the formation of 23-kDa fragment (upper panel) and the down-regulation of BCL-2 (lower panel). The results shown are representatives of four independent experiments.

When analyzing BCL-2 protein levels in cells incubated in the presence of IFN-gamma and subsequently treated with TRAIL, TNF-alpha , or CD95 antibody, we observed the formation of a 23-kDa fragment of BCL-2 protein (Fig. 8c). In the presence of Z-VAD-fmk, an inhibitor of multiple caspases, generation of the 23-kDa fragment of BCL-2 was markedly abrogated (Fig. 8c). Processing by caspases of the 26-kDa anti-apoptotic BCL-2 protein to produce a pro-apoptotic 23-kDa fragment has been observed previously in different cell types upon treatment with various apoptotic stimuli (56, 57). This caspase-dependent cleavage of BCL-2 appears to promote further caspase activation as part of a positive feedback loop for executing cell death. This mechanism involves the localization of the 23-kDa fragment to mitochondria to induce cytochrome c release from this organelle (57).

The above results indicated that, in U937 cells, IFN-gamma promoted the generation of two different cytochrome c-releasing factors upon death receptor activation. To evaluate the possible involvement of this mitochondria-derived factor in IFN-gamma -induced sensitization of U937 cells to death receptor-mediated apoptosis, we determined the release of cytochrome c into the cytosol of cells incubated with IFN-gamma and death receptor activators. In Fig. 9a, we show that cytosolic cytochrome c was elevated following treatment of cells with the various combinations of IFN-gamma and death receptor activators. Once released from mitochondria, cytochrome c will bind to Apaf-1, an event that triggers oligomerization of Apaf-1/cytochrome c in complexes that activate procaspase-9 (58). Fig. 9b illustrates the activation of procaspase-9 in cells treated with IFN-gamma and subsequently incubated with death receptor activators. Formation of 37-kDa fragment of caspase-9 was only observed in those cultures subjected to combined treatments (Fig. 9b). These results suggested that IFN-gamma facilitated the activation by death receptors of a mitochondria-operated pathway of apoptosis in human myeloid leukemic U937 cells. It has been demonstrated that Bcl-2/Bcl-XL can block cell death by preventing the activation of the mitochondria-regulated pathway of apoptosis (59-61). The importance of this pathway in IFN-gamma -induced sensitization was further examined in U937 cells transfected with a cDNA encoding anti-apoptotic Bcl-2. Results shown in Fig. 10 demonstrate that U937Bcl-2 cells were markedly protected from apoptotic cell death induced by the combination of IFN-gamma and the various death receptor agonists.


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Fig. 9.   Death receptor-induced cytochrome c release into the cytosol and caspase-9 processing are enhanced by pre-incubation of U937 cells with IFN-gamma . Cells were pre-incubated with 10 units/ml IFN-gamma for 24 h and treated with CD95 mAb (20 ng/ml), TNF-alpha (1 ng/ml), or TRAIL (10 ng/ml) for an additional 24-h period. a, cytosolic fractions obtained as described under "Experimental Procedures" were subjected to electrophoresis, and cytochrome c was detected by Western blot analysis. The mitochondrial Bcl-2 protein was undetectable in the cytosolic samples. b, whole extracts of U937 cells, treated as described previously, were analyzed for the formation of cleaved caspase-9 (37-kDa fragment) by Western blot. beta -Tubulin levels were used as control of equal gel loading. Results are representative of two independent experiments.


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Fig. 10.   U937 cells ectopically expressing Bcl-2 protein are resistant to IFN-gamma -induced sensitization to death receptor-mediated apoptosis. Cells were incubated for 24 h with 10 units/ml IFN-gamma and treated for another 24-h period with either CD95 mAb (20 ng/ml), TNF-alpha (1 ng/ml), or TRAIL (10 ng/ml). Hypodiploid apoptotic cells were determined as described under "Experimental Procedures." Percentage of apoptotic cells is shown. Results show a representative experiment from at least three different experiments.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we have demonstrated that treatment of different human myeloid leukemic cell lines with IFN-gamma markedly enhances the sensitivity of these cells to death receptor-mediated apoptosis. In our report, we have also shown that IFN-gamma treatment up-regulates the expression of caspase-8 protein in U937 cells. Regulation by IFN-gamma of caspase expression and activation has been described in human erythroid progenitor cells as well as in colon and breast tumor cells (9, 26, 62, 63). In myeloid leukemic cells, IFN-gamma caused an increased expression of caspase-1/ICE gene, following strong induction of the IRF-1 gene (27), the product of which is a transcriptional activator of the caspase-1 gene (64). However, recent data have demonstrated that caspase-1/ICE is not involved in the proteolytic cascade activated upon death receptor cross-linking at the cell surface (30). Our results are the first demonstration of the induction of caspase-8 protein in human myeloid leukemic cells upon IFN-gamma treatment. In this report we have also shown that IRF-1 is clearly induced by IFN-gamma in U937 cells. At present, we can only speculate with the possibility of similarities between caspase-1 and -8 in terms of the mechanism regulating their expression by IFN-gamma (27, 65).

Caspase-8 is recruited in zymogen form to the DISC upon ligation of CD95 at the cell surface, by either CD95L or agonistic CD95 antibodies (66). This caspase also participates in the initial signaling complex during TNF-alpha (44). Despite some recent controversy, several reports have demonstrated the involvement of FADD and caspase-8 in TRAIL-induced apoptosis of tumor cells (67-70). After recruitment to the DISC, caspase-8 is autoprocessed to generate the active form that can cleave other substrates, including executioner caspases. According to the induced-proximity model for caspase-8 activation, a locally high concentration of this caspase zymogen would promote its autoprocessing and the release of the active caspase (71). It is therefore possible that the increased expression of caspase-8 found in IFN-gamma -treated U937 cells might facilitate formation of the DISC upon death receptor activation and subsequently activate an apoptotic program. This can be especially important in CD95 type II cells, like the U937 cell line (72), which show a reduced DISC formation upon CD95 engagement at the cell surface (51). In these cells, mitochondria may function as amplifiers of apoptosis activating caspase-9 and executioner caspases, through the release of cytochrome c (52). This proposition is in agreement with our results showing the cleavage of BID, an increased release of cytochrome c from mitochondria, and the activation of caspase-9 upon death receptor activation in IFN-gamma -treated U937 cells. Regarding the role of IFN-gamma -induced caspase-8 elevation in the sensitization of U937 cells to TRAIL-induced apoptosis, a similar hypothesis could be proposed for TRAIL-R-mediated cell death (70). However, at present we can not exclude the possibility of the processing of caspase-8 observed in our studies being the result of both the formation of the DISC and its activation downstream of mitochondria.

In addition to the up-regulation of caspase-8, IFN-gamma treatment caused a marked decrease in the cellular levels of anti-apoptotic BCL-2 mRNA and protein in U937 cells. This could play a potentially important role in the sensitization process induced by IFN-gamma , as a major mechanism for the anti-apoptotic action of BCL-2 is to prevent the release of cytochrome c from mitochondria (59, 61). To our knowledge, our results are the first indication of a down-regulation of BCL-2 in human myeloid leukemic cells by IFN-gamma . In agreement with our results, it has been recently demonstrated that expression of Bcl-X(L), another member of the BCL-2 family of anti-apoptotic proteins, is inhibited in U937 cells overexpressing the interferon consensus sequence-binding protein (2). It has been reported previously that IFN-gamma induced apoptosis in colon carcinoma cells, which was correlated with the down-regulation of BCL-2 and the up-regulation of BAX (73). On the other hand, the importance of BCL-2 in regulating IFN-gamma -induced apoptosis has been reported in HeLa cells (74). In these studies, overexpression of BCL-2 blocked interferon-induced double-stranded RNA-dependent protein kinase-activated apoptosis.

Besides its effect in the expression of BCL-2 in U937 cells, we have also demonstrated here that IFN-gamma promoted the caspase-dependent cleavage of remaining BCL-2 protein to generate a 23-kDa fragment, upon death receptor activation. A similar proteolytic fragment of BCL-2 has been shown to be produced by caspase-3 during staurosporine-induced apoptosis in breast tumor cells (57). This caspase-generated BCL-2 fragment localizes to mitochondria and promotes the release of cytochrome c (57), which contributes to amplification of the caspase cascade (56). Therefore, in human leukemic U937 cells, IFN-gamma not only diminished the levels of anti-apoptotic mitochondrial membrane-operating BCL-2 protein but, by promoting the generation of pro-apoptotic fragments of BID and BCL-2 upon death receptor activation, it could also favor mitochondria-mediated apoptosis. This apoptotic pathway was prevented in U937 cells that expressed ectopic Bcl-2, further supporting the role of mitochondria in IFN-gamma -induced sensitization to death receptor-mediated apoptosis.

Although the above observations open the possibility of using death ligands as anti-leukemic agents in combination with IFN-gamma , severe toxicity has been observed in systemic anti-tumor treatments with TNF-alpha or CD95 agonistic antibody. TNF-alpha causes a lethal inflammatory response (75), and CD95 mAb produces lethal liver damage (76). However, repeated systemic exposure of non-human primates to elevated doses of TRAIL did not cause significant changes in clinical parameters (31, 77), although it may affect normal human hepatocytes (78). Interestingly, in contrast to myeloid leukemic cells, treatment of normal human monocytes with IFN-gamma or -alpha induces the down-regulation of pro-apoptotic TRAIL receptors, which renders these cells resistant to TRAIL-mediated apoptosis (79). Altogether, these data suggest that sensitizing regimes like IFN-gamma may be useful after cautious studies, in therapeutic strategies with non-toxic death receptor ligands or drugs for the treatment of certain human myeloid malignancies.

    ACKNOWLEDGEMENTS

We thank Dr. Faustino Mollinedo (Consejo Superior de Investigaciones Científicas, Salamanca, Spain) for THP-1 cells. U937 cells transfected with full-length human Bcl-2 (U937Bcl-2) were kindly donated by Dr. Carmen Garrido (Faculty of Medicine and Pharmacy, Dijon, France).

    FOOTNOTES

* This work was supported in part by Ministerio de Educación y Cultura Grants 1FD97-0514-C02-01 and SAF2000-0118-C03-01 and by Consejo Superior de Investigaciones Científicas/Ministerio de Ciencia, Technología y Medio Ambiente Grant 99CU0004 (to A.L.-R.).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.

§ Recipient of a fellowship from Junta de Andalucia.

|| Both authors contributed equally to this work.

** To whom correspondence should be addressed. Tel.: 34-958-80-51-88; Fax: 34-958-20-33-23; E-mail: alrivas@ipb.csic.es.

Published, JBC Papers in Press, February 14, 2001, DOI 10.1074/jbc.M100815200

    ABBREVIATIONS

The abbreviations used are: IFN, interferon; TNF, tumor necrosis factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; ICE, interleukin-1-beta -converting enzyme; DISC, death-inducing signaling complex; PARP, poly(ADP-ribose) polymerase; Z-VAD-fmk, benzyloxycarbonyl-Val-Ala-Asp-(OMe)fluoromethyl ketone; FLIP, FLICE-inhibitory protein; IRF-1, interferon regulatory factor-1; mAb, monoclonal antibody; PCR, polymerase chain reaction; RT, reverse transcription; PBS, phosphate-buffered saline; FADD, fas-associated death domain.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

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