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[Cancer Research 60, 5673-5680, October 15, 2000]
© 2000 American Association for Cancer Research

Biochemistry and Biophysics

Interferon-{gamma} Treatment Elevates Caspase-8 Expression and Sensitizes Human Breast Tumor Cells to a Death Receptor-induced Mitochondria-operated Apoptotic Program1

Carmen Ruiz-Ruiz, Cristina Muñoz-Pinedo and Abelardo López-Rivas2

Instituto de Parasitología y Biomedicina Consejo Superior de Investigaciones Centificas, 18001 Granada, Spain


   ABSTRACT
Top
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In this report, we have assessed the role of IFN-{gamma} as a sensitizing agent in apoptosis mediated by activation of death receptor CD95 in breast tumor cells. Treatment of the tumor cell lines MCF-7 and MDA-MB231 with IFN-{gamma} significantly facilitated apoptosis induced by CD95 receptor ligation at the plasma membrane, independently of p53 status. In contrast, IFN-{gamma} treatment did not enhance the apoptotic effect of the DNA-damaging drug, doxorubicin. Analysis of apoptosis regulators indicated that caspase-8 mRNA and protein levels were up-regulated in both of the cell lines after treatment with IFN-{gamma}. Furthermore, IFN-{gamma} sensitized MCF-7 and MDA-MB231 cells to CD95-mediated activation of caspase-8, induction of cytochrome c release from mitochondria, and processing of caspase-9. Release of cytochrome c, caspases activation, and apoptosis were prevented in MCF-7 cells overexpressing Bcl-2. Altogether these results indicate that IFN-{gamma}, maybe through the elevation of caspase-8 levels, sensitizes human breast tumor cells to a death receptor-mediated, mitochondria-operated pathway of apoptosis.


   INTRODUCTION
Top
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Apoptotic cell death plays a fundamental role in normal development, tissue homeostasis, and pathological situations (1, 2, 3) . The CD95 (Fas/Apo-1) receptor, a member of the TNF3 /nerve growth factor receptor family (4 , 5) , is a potent inducer of apoptosis in the immune system on interaction with its natural ligand CD95L, a type II integral-membrane protein homologous to TNF-{alpha} (6) . Whereas the expression of CD95L seems to be more restricted to lymphoid cells (6) , CD95 antigen is also expressed outside the immune system in many nontransformed cells, including mammary epithelial cells (7) . In contrast, breast tumor cells express low levels of CD95 mRNA and protein and are usually not killed by CD95 antibodies (8) .

IFNs are a family of natural glycoproteins that share antiviral, immunomodulatory, and antiproliferative effects (9) . Their antitumor activity against a variety of tumor cells such as lymphomas, melanomas, and multiple myeloma has also been reported (10 , 11) . Clinically and experimentally, it has been demonstrated that IFN-{gamma} can enhance the antitumor effects of antimetabolite on cancer cells (11 , 12) . In vitro, IFN-{gamma} can induce or modulate apoptosis either as a single agent or in combination with other chemotherapeutic agents (13) . In breast cancer cells, positive results have been obtained by immunotherapy with natural IFNs and interleukins, particularly in combination strategies (14) . However, the mechanism of IFNs-mediated modulation of cellular susceptibility to apoptosis has not been elucidated. IFN-{gamma} and IFN-{alpha} can up-regulate the expression of a number of apoptosis-related proteins including TNF-R, CD95 and other death receptors as well as their respective ligands, different members of the Bcl-2 family, and caspases in different types of cells (13 , 15 , 16) . Moreover, the tumor suppressor IRF-1 has been proposed to play a role in apoptosis and to be a transcriptional activator of the ICE/caspase-1 gene (17) . In breast cancer cells, it has been reported that IFN-{gamma} induces sensitization to CD95-mediated apoptosis by up-regulating the expression of ICE/caspase-1 (8) . However, recent results have indicated that caspase-1/ICE does not play a crucial role in apoptosis on death-receptor cross-linking by ligands (18, 19, 20) .

We have recently reported that DNA-damaging drugs sensitize breast tumor cells to CD95-mediated apoptosis by inducing the expression of cell membrane CD95 receptor (21) . The accumulation of p53 in drug-treated cells is required for the up-regulation of CD95. However, inactivating p53 mutations are frequently observed in breast cancer cells that abrogate p53-dependent gene transcription (22, 23, 24) . The above data prompted us to investigate the effects of IFN-{gamma} on CD95 receptor-induced apoptosis in both p53wild-type and p53 mutated breast tumor cells. In this report, we show that IFN-{gamma} sensitizes breast tumor cells to CD95 receptor-mediated apoptosis in a p53-independent way. Although IFN-{gamma} induces the expression of CD95 in the membrane of breast cancer cells, it does not seem to be the sole mechanism by which IFN-{gamma} regulates apoptosis induced by death receptor ligation. We have observed a marked up-regulation of caspase-8, which plays a pivotal role in the proteolytic cascade leading to apoptosis on activation of death receptors (25, 26, 27) . In this report, we also show that IFN-{gamma} facilitates the induction, on CD95 ligation, of biochemical events such as the activation of initiator caspase-8, the release of cytochrome c from mitochondria, and the processing of caspase-9, which are important events in CD95-mediated apoptosis of certain cells (28) . Finally, we demonstrate that apoptosis induced by the combination of IFN-{gamma} and a CD95 agonistic antibody probably involves mitochondrial events, inasmuch as it is inhibited in cells overexpressing Bcl-2.


   MATERIALS AND METHODS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents and Antibodies.
RPMI 1640 and fetal bovine serum were obtained from Life Technologies-Europe. CH11 mAb (IgM) reacting with CD95 was from Upstate Biotechnology (Lake Placid, NY). Human IFN-{gamma} was obtained from PreproTech EC LTD (London, England). Anti-CD95 rabbit polyclonal IgG antibody (C-20) was purchased from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Anti-CD95 DX2 mAb (IgG1), mouse anti-Bax mAb, mouse anti-Bad mAb, and mouse anti-cytochrome c mAb were obtained from PharMingen (San Diego, CA). Mouse anti-Bcl2 mAb was from DAKO (Denmark). Mouse antihuman caspase-8 mAb was purchased from Cell Diagnostica (Münster, Germany). Rabbit anti-caspase-9 polyclonal antibody was from StressGen Biotechnologies Corp. (Victoria, Canada). Rabbit anticleaved caspase-9 polyclonal antibody was obtained from New England BioLabs Inc. (Beverly, MA). Mouse anti-{alpha}-tubulin mAb, calcium ionophore A23187 and phorbol-12,13-dibutyrate were from Sigma Immunochemicals (St. Louis, MO). Rabbit polyclonal antiserum against PARP was obtained 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, Texas). We are also grateful to Dr. Michael Hahne (University of Lausanne, Lausanne, Switzerland) for the gift of recombinant human CD95 ligand and CD95 ligand cross-linker. pcDNA3-bcl-2 plasmid was kindly provided by Dr. Jacint Boix (University of Lleida, Lleida, Spain).

Cell Lines.
The human breast tumor cell lines MCF-7 and MDA-MB231 were kindly provided by Dr. M. Ruiz de Almodovar (Department of Radiology, University of Granada, Granada, Spain). They were maintained in culture in RPMI 1640 containing 10% fetal bovine serum, 1 mM L-glutamine and gentamicin, at 37°C in a humidified 5% CO2/95% air incubator. Stable cell lines overexpressing human Bcl-2 protein were generated by transfection of MCF-7 cells with either pcDNA3 or pcDNA3-hbcl-2 DNA, using FUGENE reagent (Roche Molecular Biochemicals) according to the manufacturer’s instructions. Resistant clones were selected in culture medium with 2 mg/ml G418 sulfate (Sigma Chemical Co.) and analyzed for the overexpression of hBcl-2 by Western blot.

Analysis of Cell Viability and Apoptosis.
Cell viability was determined by the crystal violet method as described previously (29) . PS exposure on the surface of apoptotic cells was detected by flow cytometry after staining with Anexin-V-FLUOS (Roche Molecular Biochemicals). Flow cytometry was performed on a FACScan cytometer using the Cell Quest software (Becton Dickinson, Mountain View, CA).

Determination of Cell Surface CD95 Expression.
Cells were detached from the culture flask with RPMI medium containing 3 mM EDTA, and cytofluorimetric analysis of CD95 was performed with CD95 mouse monoclonal IgG antibody DX2 (2 µg/ml; Ref. 30 ).

Immunoblot Detection of Proteins.
After detachment with RPMI/EDTA, cells (5 x 105) were washed with PBS and lysed in 20 µl of sample buffer [50 mM Tris-HCl (pH 6.8), 6 M urea, 6% 2-mercaptoethanol, 3% SDS, and 0.003% bromphenol blue]. Cell samples were sonicated, and proteins were resolved on SDS-polyacrylamide minigels and detected as described previously (21) .

For measurements of cytochrome c release, cells were lysed and cytosolic fractions were separated from mitochondria as described previously (31) . Cytosolic proteins (40 µg of protein) were mixed with Laemmli buffer and resolved on SDS-12% PAGE minigels. Cytochrome c was determined by Western blot analysis as described above.

RT-PCR.
Total RNA was isolated from cells with Trizol reagent (Life Technologies, Inc. Grand Island, NY) as recommended by the supplier. cDNAs were synthesized from 2 µg of total RNA using a RNA PCR kit (Perkin-Elmer) with the supplied oligo d(T) primer under conditions described by the manufacturer. PCR reactions were performed using the following primers: human CD95L sense, 5'-CAGGACTGAGAAGAAGTAAAACCG-3', and human CD95L antisense, 5'-CTCCAAAGATGATGCTGTG-3'; human Bak sense, 5'-CCTGTTTGAGAGTGGCATC-3', and human Bak antisense, 5'-TCGTACCACAAACTGGCCCA-3'; human IRF-1 sense, 5'-CTTAAGAACCAGGCAACCTCTGCCTTC-3', and human IRF-1 antisense, 5'-GATATCTGGCAGGGAGTTCATG-3'; and human ß-actin sense, 5'-TGACGGGGTCACCCACACTGTGCCCATCTA-3', and human ß-actin antisense, 5'-CTAGAAGCATTTGCGGTGGACGATGGAGGG-3'—giving products of 440, 266, 406, and 661 bp, respectively. Cycle conditions for all of the PCR reactions were 1 min at 95°C, 1 min at 55°C, and 1 min at 72°C for 30 cycles with the exception of hCD95L. For hCD95L, PCR was carried out under the same conditions for 40 cycles.

Northern Blot Analysis of Caspase-8 mRNA.
Total RNA (20 µg) was run on 1% agarose/formaldehyde gel and transferred to nylon membranes (Hybond-N, Amersham Pharmacia Biotech, Buckinghamshire, England). Membranes were hybridized to a cDNA probe for caspase-8 labeled with [{alpha}-32P]dCTP (Amersham Pharmacia Biotech), using a random primer labeling kit (Roche Molecular Biochemicals). Caspase-8 cDNA probe was generated by RT-PCR, as described above, using the following primers for PCR amplification: sense 5'-GATATTGGGGAACAACTGGAC-3' and antisense 5'-CATGTCATCATCCAGTTTGCA-3'.


   RESULTS
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Sensitization by IFN-{gamma} of Breast Tumor Cells to Death Receptor-mediated Apoptosis Is Independent of p53 Status.
We reported recently that breast tumor cells expressing wild type p53 can be sensitized to CD95-mediated apoptosis by treatments that caused DNA damage (21) . These treatments up-regulated the cell membrane expression of CD95 through a p53-dependent mechanism. However, inactivating p53 mutations are frequently observed in breast tumor cells (24) and can abrogate p53-dependent elevation of cell cycle inhibitors and apoptosis-inducing molecules (32 , 33) . To circumvent this problem, we have analyzed the ability of IFN-{gamma} treatment to sensitize breast tumor cells to death receptor-mediated apoptosis in both p53 wild-type and p53 mutated breast cancer cell lines. Treatment of both MCF-7 (p53 wt) and MDA-MB231 (mutant p53) breast tumor cells with the combination of IFN-{gamma} and CD95 antibody caused an important decrease in cell viability as determined by the crystal violet method (Fig. 1, A and B)Citation . In both cell lines, treatment with either IFN-{gamma} or CD95 antibody had only a slight effect on the number of viable cells at the end of the incubation period. Similar results were obtained in EVSA-T cells, another breast tumor cell line harboring a mutant p53 (results not shown). Analysis of PS exposure in the extracellular side of the plasma membrane, a marker of apoptosis, indicated that the synergistic loss of cell viability observed (Fig. 1A)Citation was in fact attributable to facilitation of this type of cell death (Fig. 1, C and D)Citation . Fig. 1DCitation also shows that synergism with IFN-{gamma} was observed not only with CD95 antibody but also with soluble recombinant CD95 ligand. Results not shown demonstrated that IFN-{gamma} also sensitized both of the human breast tumor cell lines to apoptosis induced by TRAIL, another member of the TNF-{alpha} family of death-inducing ligands that preferentially kills tumor cells (34 , 35) . However, IFN-{gamma} did not enhance the effect of doxorubicin, a DNA-damaging drug that at certain concentrations may cause apoptosis in breast tumor cells (Fig. 2Citation ; Ref. 21 ).



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  		Fig. 1. IFN-{gamma} sensitized breast tumor cells to CD95-mediated cell death. Cell viability was assessed by crystal violet staining after 48 h treatment of MCF-7 cells (A) and 72 h treatment of MDA-MB231 cells (B), without or with 10 ng/ml IFN-{gamma} in the presence or in the absence of 500ng/ml CD95 mAb, CH11. PS externalization in MCF-7 cells was determined after 48-h incubation without or with 10 ng/ml IFN-{gamma} in the presence or in the absence of either 500 ng/ml CH11 (C) or 100 ng/ml recombinant human CD95 ligand and 200 ng/ml CD95 ligand cross-linker (D). Bars, SD from at least two independent experiments.

 


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  		Fig. 2. IFN-{gamma} did not sensitize breast tumor cells to doxorubicin-induced cell death. Cell viability was assessed by the crystal violet method after 48-h incubation of MCF-7 cells with different concentrations of doxorubicin (Doxo) or CD95 antibody (500 ng/ml) in the presence of IFN-{gamma} (10 ng/ml). Bars, SD from two independent experiments.

 
Up-Regulation of CD95 Receptors by IFN-{gamma} Is Not Sufficient to Sensitize Breast Tumor Cells to CD95-mediated Apoptosis.
Our findings (Figs. 1Citation and 2)Citation indicated that IFN-{gamma} enhanced the sensitivity of breast tumor cells to CD95 receptor-induced apoptosis, only in the presence of exogenous CD95 antibody or ligand. These observations suggested that IFN-{gamma} treatment was not able to induce the expression of endogenous CD95L. This hypothesis was confirmed by RT-PCR analysis of CD95L mRNA. Treatment with IFN-{gamma} for up to 48 h did not induce CD95L mRNA expression in MCF-7 cells (Fig. 3)Citation . As a control of CD95L expression, human Jurkat T-lymphocytes, treated in parallel for 5 h with calcium ionophore and phorbol ester, showed a marked up-regulation of CD95L mRNA. To investigate the mechanism underlying the sensitization of breast tumor cells by IFN-{gamma} to apoptosis-mediated-by-death-receptor activation, we first analyzed the expression of CD95 receptors in cells treated with IFN-{gamma} (Fig. 4)Citation . Total CD95 protein (Fig. 4A)Citation and CD95 receptor expression at the cell membrane (Fig. 4B)Citation significantly increased after treatment of MCF-7 cells with IFN-{gamma}, as previously reported by other investigators who analyzed levels of mRNA for CD95 (8) . In contrast, untreated MDA-MB231 cells expressed a significant amount of total CD95 protein and CD95 membrane receptors and IFN-{gamma} treatment only slightly enhanced these levels (Fig. 4, A and B)Citation but markedly sensitized these cells to CD95-mediated cell death (Fig. 1B)Citation . To further assess the importance of the increase in CD95 levels in the facilitation by IFN-{gamma} of CD95-mediated cell death, we carried out several experiments in MCF-7 cells with the DNA-damaging drug, doxorubicin. We have previously shown that treatment of MCF-7 cells with doxorubicin concentrations of 100 ng/ml or higher caused p53 accumulation that was followed by a marked elevation of CD95 expression and that synergized with CD95 agonistic antibody in the induction of apoptosis (21) . At a lower concentration (10 ng/ml), doxorubicin produced an effect on CD95 expression (Fig. 4C)Citation that was similar to the effect observed in cells incubated in the presence of IFN-{gamma} (Fig. 4A)Citation . However, in contrast to the sensitization to apoptosis induced by IFN-{gamma} (Fig. 1)Citation , doxorubicin did not facilitate CD95-mediated cell death (Fig. 4D)Citation . Although we cannot completely exclude a certain role of increased CD95 in IFN-{gamma}-induced sensitization to apoptosis, these observations and the results obtained in MDA-MB231 cells suggested that elevation of CD95 expression by IFN-{gamma} is not sufficient to explain the synergism found in CD95-mediated apoptosis. The observations and results also suggested that changes in intracellular apoptotic mediators should be involved in the proapoptotic effect of IFN-{gamma} in breast tumor cells.



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  		Fig. 3. Expression of CD95L is not induced by IFN-{gamma} in breast tumor cells. MCF-7 cells were treated with IFN-{gamma} (10 ng/ml) for the times indicated. As a control of CD95L expression, Jurkat cells were treated with calcium ionophore A23418 and phorbol-12,13-dibutyrate (I+P) for 5 h. After treatment, mRNA was extracted from cells and analyzed by RT-PCR as indicated in "Materials and Methods."

 


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  		Fig. 4. Treatment of breast tumor cells with either IFN-{gamma} or doxorubicin induced similar up-regulation of CD95 expression but differentially sensitized MCF-7 cells to CD95-induced apoptosis. CD95 protein expression was determined by Western blotting (A) and by flow cytometry (B) in MCF-7 and MDA-MB231 cells, after treatment without or with 10 ng/ml IFN-{gamma} for either 24 h (A) or 48 h (B). (C) CD95 receptors expression was analyzed by flow cytometry in MCF-7 cells after incubation for 48 h without or with 10 ng/ml doxorubicin. (D) MCF-7 cells were treated without or with 10 ng/ml doxorubicin in the presence or in the absence of 500 ng/ml CD95 antibody CH11. Cell viability was assessed after 48 h by crystal violet staining. Bars, SD from two different experiments.

 
Up-Regulation of Caspase-8/FLICE in Breast Tumor Cells after Treatment with IFN-{gamma}.
It has been reported that IFN-{gamma} modulates apoptosis in colon cancer cells by sensitizing the cells to killing by apoptotic stimuli (13) . In these cells, IFN-{gamma} induced changes in an array of genes of the apoptotic pathway, including death receptors, caspases, and the proapoptotic member of the bcl-2 family, Bak. To further investigate the mechanism of IFN-{gamma}-mediated facilitation of apoptosis in breast tumor cells, we carried out the analysis of the expression of different apoptosis-related molecules on treatment with a sensitizing concentration of IFN-{gamma}. In these experiments, we did not observe any changes in the levels of the bcl-2 family proteins Bax, Bad, Bcl-2, or Bid after treatment with IFN-{gamma}, as determined by Western blot analysis (Fig. 5A)Citation . We also determined by RT-PCR the mRNA expression for proapoptotic Bak, the levels of which are elevated in human colon adenocarcinoma cells after IFN-{gamma} treatment. As shown in Fig. 5ACitation , Bak mRNA levels remained unchanged after 36 h of incubation in the presence of IFN-{gamma}. In these cells, IFN-{gamma} treatment caused an induction of mRNA for the transcriptional activator IRF-1, an IFN inducible gene that served as a control for IFN-{gamma} action (36) .



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  		Fig. 5. IFN-{gamma} up-regulated the expression of caspase-8 in breast cancer cells. MCF-7 and MDA-MB231 cells were treated without or with 10 ng/ml IFN-{gamma} for the indicated periods of time. Expression of Bax, Bad, Bcl-2, and Bid (A), and of caspase-8, FADD, and caspase-9 (B) were determined by Western blotting. In (A) the expression of Bak mRNA was analyzed by RT-PCR. RT-PCR products of ß-actin and IRF-1 were used as controls of RNA input and IFN-{gamma} action, respectively. C, expression levels of caspase-8 mRNA were detected by Northern blot analysis in MCF-7 and MDA-MB231 cells after treatment without or with IFN-{gamma} (10 ng/ml) for 15 h or 36 h, respectively; lower panel, ethidium bromide staining of loaded RNA.

 
Caspases, cysteine proteases of the CED3/ICE family, are essential elements of the death receptor-initiated pathway of apoptosis. They play a role in both the initiation and the execution phases of apoptosis induced on death receptor cross-linking by specific ligands (37, 38, 39) . It has been shown that IFN-{gamma} can up-regulate the expression of several members of this family in different cells (13 , 40) . In breast tumor cells, it was reported that IFN-{gamma} treatment increased the expression of caspase-1/ICE (8) . It was suggested that this effect could have a role in the IFN-{gamma}-induced sensitization of these tumor cells to CD95-mediated apoptosis. However, it is now clear that caspase-1/ICE is not involved in the proteolytic cascade leading to apoptosis on death-receptor cross-linking by ligands (18, 19, 20) . Instead, ligation of death receptors results in the formation of the DISC, a complex that comprises the adapter molecule FADD/MORT1 and caspase-8 (41) . Formation of the DISC results in the release of active caspase-8 and the induction of apoptosis through either a caspases cascade (CD95 type I cells) or a mitochondria-mediated pathway (CD95 type II cells; 28 ). We, therefore, decided to determine the levels of both FADD/MORT1 and caspase-8 in breast tumor cells treated with IFN-{gamma}. Results shown in Fig. 5BCitation indicate that there was a clear up-regulation of caspase-8 protein in both MCF-7 and MDA-MB231 cells treated with IFN-{gamma}. In agreement with these data, analysis of mRNA levels for caspase-8 during IFN-{gamma} treatment revealed a marked increase of this mRNA in both of the tumor cell lines (Fig. 5C)Citation . Interestingly, it has been reported that overexpression of caspase-8 is sufficient to sensitize cells to apoptosis (25) . We also analyzed the expression of FADD protein, the adapter molecule that recruits caspase-8 to the DISC in death receptor-mediated apoptosis. As shown in Fig. 5BCitation , FADD levels did not change in MCF-7 cells incubated in the presence of IFN-{gamma}. Recruitment and activation of caspase-8 at the DISC can be sometimes prevented by the presence of the endogenous inhibitor c-FLIP, a determinant of susceptibility to death receptor-mediated apoptosis (42) . This protein could be highly expressed in tumor cells (43) . To ascertain whether IFN-{gamma} was reducing the expression of such an inhibitor in breast tumor cells, we examined by RT-PCR analysis the mRNA expression of c-FLIP in MCF-7 and MDA-MB231 cells. Results not shown indicated that these tumor cells did not express detectable levels of c-FLIP.

Caspase-9 forms a multiprotein complex with Apaf-1 and cytochrome c and is a key element in mitochondria-mediated caspase activation (44 , 45) . In the breast tumor cell lines MCF-7 and MDA-MB231, caspase-9 protein was expressed, but we did not observe any significant change in the cellular levels of this caspase on IFN-{gamma} treatment (Fig. 5B)Citation .

IFN-{gamma} Promotes, in Breast Tumor Cells, the Activation of an CD95-induced Mitochondria-operated Apoptotic Pathway That Is Inhibited by Bcl-2 Overexpression.
Mitochondria can play a pivotal role in apoptosis induced by different apoptotic inducers, particularly DNA-damaging agents (46 , 47) . In CD95-expressing type II cells, release of apoptotic factors from mitochondria on CD95 activation in the plasma membrane is a necessary step in CD95-induced apoptosis (28) . MCF-7 cells have been ascribed to the type II group of cells based on the fact that Bcl-xL blocked apoptosis in cells transfected with CD95 (28 , 48) . To get further insight into the mechanism of IFN-{gamma}-induced sensitization of breast tumor cells to death receptor-mediated apoptosis, we analyzed several biochemical events which are known to be elicited on CD95 ligation in the plasma membrane. In this respect, CD95-mediated caspase-8 activation, which was not observed in cells treated with CD95 antibody alone, was clearly induced by CD95 antibody in both breast tumor cell lines when IFN-{gamma} was present in the culture medium (Fig. 6A and B)Citation . Cytochrome c release from mitochondria is a crucial step in the formation of the apoptosome, during activation of caspase-9 (49 , 50) . Data shown in Fig. 6, A and BCitation , indicate that IFN-{gamma} treatment facilitated CD95-induced release of cytochrome c in breast tumor cells. As a consequence probably of cytochrome c elevation in the cytosol, there was an activation of caspase-9 processing, measured as formation of the Mr 32,000 fragment, in cells incubated in the presence of both CD95 antibody and IFN-{gamma} (Fig. 6, A and B)Citation . It is interesting that both the activation of caspase-8 and the release of cytochrome c can be observed as early as 6 h after the addition of CD95 antibody, whereas caspase-9 processing is only observed at later times (Fig. 6, A and B)Citation , which suggests an ordered relationship between these events. Finally, the triggering of this apoptotic pathway by the combination of CD95 antibody and IFN-{gamma} resulted in the activation of executioner caspases as determined by the proteolytic cleavage of the nuclear substrate PARP (Fig. 6C)Citation . Altogether, these results supported the hypothesis that in breast tumor cells, IFN-{gamma}-promoted sensitization to death receptor-induced apoptosis was associated with the activation of a mitochondria-operated apoptotic pathway.



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  		Fig. 6. IFN-{gamma} facilitated CD95-induced activation of a mitochondria-operated apoptotic pathway in breast cancer cells. MCF-7 (A, C) or MDA-MB231 (B, C) cells were preincubated for 24 h in the presence or absence of 10 ng/ml IFN-{gamma} before treatment without or with 500 ng/ml CD95 antibody CH11 for the indicated times. In B, middle and lower panels, IFN-{gamma}-treated MDA-MB231 cells were incubated with CD95 antibody for 36 h. Activation of caspase-8, release of cytochrome c from mitochondria, activation of caspase-9, and cleavage of PARP were determined by Western blot. Arrows, the cleaved intermediate forms of caspase-8 (41–43 kDa), caspase-9 (32 kDa) and the proteolytic fragment of PARP (85 kDa). In B, {alpha}-tubulin was used as control of loaded protein. kDa, Mr in thousands.

 
The importance of mitochondria-regulated events in IFN-{gamma}-induced sensitization was further analyzed in MCF-7 cells transfected with a cDNA encoding antiapoptotic human Bcl-2. Several clones were selected that overexpressed Bcl-2 protein. Results obtained with a representative clone are shown in Fig. 7Citation . In MCF-7bcl-2 cells, Bcl-2 protein expression was markedly elevated as compared with cells transfected with an empty vector (Fig. 7A)Citation . Interestingly, MCF-7bcl-2 cells were completely protected from cell death induced by the combination of IFN-{gamma} and agonistic CD95 antibody (Fig. 7B)Citation . Antiapoptotic Bcl-2 inhibits the activation of executioner caspases and apoptosis by preventing the release of cytochrome c from mitochondria (51) . As shown in Fig. 7CCitation , both the release of cytochrome c from mitochondria and the activation of PARP cleavage were clearly prevented in MCF-7 cells overexpressing Bcl-2. It has been demonstrated that in MCF-7 cells—stably expressing CD95—caspase-8 is recruited to the DISC after the triggering of CD95 (48) . In these cells, overexpression of Bcl-xL did not interfere with activation of caspase-8, but it blocked CD95-mediated apoptosis (28 , 52 , 53) . We have shown (Fig. 7, B and C)Citation that overexpression of Bcl-2 prevented apoptosis as well as cytochrome c release and the activation of executioner caspases. However, when examining caspase-8 activation, we found no differences between control-transfected and Bcl-2-overexpressing MCF-7 cells on treatment with IFN-{gamma} and CD95 antibody (Fig. 7D)Citation . Taken together, our data suggest that, in IFN-{gamma} sensitized cells, Bcl-2 blocks the CD95 pathway downstream of caspase-8 activation and upstream of cytochrome c release and executioner caspases. These results also indicate that sensitization by IFN-{gamma} to CD95-induced apoptosis in MCF-7 breast tumor cells occurs through a mitochondria-mediated pathway. It is interesting that in Bcl-2-overexpressing MCF-7 cells, apoptosis was inhibited despite activation of caspase-8 (Fig. 7Citation , B, C, and D), which, in principle, could lead to activation of a caspases cascade (type I CD95 cells). However, MCF-7 cells are deficient in caspase-3 expression, and this caspase is required to activate the type I pathway in MCF-7 cells that overexpress CD95 (28) . On the other hand, it is possible that the activation of caspase-8 that is observed in our experiments on treatment with IFN-{gamma} and CD95 antibody (Fig. 6, ACitation and B, and Fig. 7DCitation ) is not sufficient to activate the CD95 type I pathway, in contrast to other studies using CD95-overexpressing cells (28 , 52 , 53) , in which the recruitment of FADD and caspase-8 to the DISC and the activation of caspase-8 were markedly stimulated on CD95 ligation.



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  		Fig. 7. Overexpression of Bcl-2 prevented IFN-{gamma}-promoted CD95-mediated apoptosis in breast tumor cells. MCF-7 cells were transfected with either pcDNA3-neo vector or pcDNA3-bcl-2 vector, and Bcl-2 expression levels were determined by Western blot analysis (A). In B, pcDNA3-neo (control) or pcDNA3-bcl-2 (Bcl-2) cells were treated with or without IFN-{gamma} (10 ng/ml) in the presence or absence of CD95 antibody (500 ng/ml) for 48 h, and cell viability was assessed by crystal violet staining. Bars, SD from two different experiments. Release of cytochrome c from mitochondria and PARP cleavage (C) and caspase-8 activation (D) were determined by Western blot analysis in both MCF-7 and MCF-7Bcl-2 cells incubated for 24 h with or without IFN-{gamma} (10 ng/ml) and subsequently treated in the presence or absence of CD95 antibody (500 ng/ml) for 24 h.

 

   DISCUSSION
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Different mechanisms seem to be involved in the resistance of tumor cells to CD95L-induced apoptosis. Loss of CD95 receptor expression has been observed in different tumor cells, including hepatocellular and breast carcinomas (7 , 8 , 54) . However, in other cases, the resistance to death receptor-mediated apoptosis is found in tumor cells expressing a significant number of CD95 receptors, e.g., in certain colon carcinoma cells (55) . In the breast, the presence of CD95 protein in the cell surface of normal mammary epithelial cells has been reported (7) . Although a role has not been yet ascribed to CD95 in apoptosis of these cells in vivo, normal breast epithelial cells are sensitive to CD95-mediated apoptosis in vitro (8) . In contrast, malignant breast cell lines express low levels of CD95 protein and are resistant to CD95-mediated apoptosis (8) . We have previously confirmed these results and demonstrated that genotoxic agents up-regulated the expression of CD95 in breast tumor cells by a p53-dependent mechanism and sensitized these cells to CD95-induced apoptosis (21) . However, p53 is frequently mutated and inactivated in breast tumor cells (56) ; and, therefore, it could be important to find alternative treatments to sensitize malignant breast epithelial cells to death receptor-mediated cell death. In our study, we have found that IFN-{gamma} sensitizes breast tumor cells to death receptor-mediated apoptosis but not to doxorubicin-induced apoptosis. In this respect, breast tumor cells seem to behave differently from colon cancer cells, in which IFN-{gamma} treatment sensitizes these tumor cells to apoptosis that is induced by CD95 receptor activation, irradiation, and antitumor agents (13) . Our results also indicate that, besides its effect on death receptor expression, IFN-{gamma} might be regulating the intracellular apoptotic machinery (46) . In this respect, it has been reported that IFN-{gamma} modulates a p53-independent apoptotic pathway through the regulation of several apoptosis-related genes, in a human colon adenocarcinoma cell line (13) . These genes included CD95 and TNFR1, several members of the caspase family, and two members of the bcl-2 family: bak and Mcl-1. However, the relative contribution of each of these genes to IFN-{gamma}-mediated promotion of apoptosis was not established in these studies.

Apart from caspase-8, no other changes were observed in the levels of the apoptosis-related mRNAs and proteins analyzed in our study, in contrast to colon adenocarcinoma cells (13) . In our report, we have provided evidence for the up-regulation of caspase-8 mRNA and protein on IFN-{gamma} treatment in both wild-type and mutant p53-expressing human breast cancer cells. This caspase is the first caspase required in death receptor-mediated apoptosis (57) , although it could be also activated downstream of mitochondria through an amplification pathway regulated by this organelle (58) . Caspase-8 is recruited in zymogen form to the DISC on ligation of CD95 at the cell surface, by either CD95L or agonistic CD95 antibodies (25) . After recruitment, 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 the autoprocessing and the release of the active caspase (39) . It is possible that the increased expression of caspase-8 found in IFN-{gamma}-treated breast tumor cells, might facilitate formation of the DISC triggered on CD95-receptor activation and thus subsequently activate an apoptotic program. In this respect, it is interesting to mention that IFN-{gamma} did not increase the apoptotic effect of the DNA-damaging drug, doxorubicin, in breast tumor cells. DNA-damaging treatments usually activate apoptosis through a mitochondrial pathway that shares several elements with death receptor-induced apoptotic mechanism in CD95 type II cells (59) . However, an important difference between both mechanisms is the absolute requirement for caspase-8 recruitment to the DISC in death receptor-mediated apoptosis but not in DNA damage-induced cell death (57 , 59) . The fact that IFN-{gamma} sensitizes breast tumor cells to death receptor-mediated apoptosis but not to doxorubicin-induced death, suggests that IFN-{gamma} must be acting at an early step in the apoptotic process, such as the activation of initiator caspase-8 at the DISC. In CD95 type II cells like the MCF-7 cell line, mitochondria may function as amplifiers activating caspase-9 and executioner caspases (58) . This proposition is in agreement with our results indicating the activation by CD95 agonistic antibody of a mitochondria-regulated pathway of apoptosis in IFN-{gamma}-treated cells. The importance of this IFN-{gamma}-promoted mitochondrial pathway was confirmed by experiments in breast tumor cells that overexpressed Bcl-2. In these cells, the release of cytochrome c from mitochondria, the activation of executioner caspases, and apoptosis were markedly inhibited.

Previous data (8) have indicated that caspase-1/ICE is up-regulated in some breast cancer cell lines on treatment with IFN-{gamma}. This study also showed that overexpression of caspase-1/ICE sensitized these cells to CD95-mediated apoptosis. However, more recent data have demonstrated that caspase-1/ICE is not involved in the proteolytic cascade activated on CD95 cross-linking at the cell surface by CD95L or CD95 antibody (18, 19, 20) . An explanation for the observed increase in CD95-mediated apoptosis in caspase-1/ICE-transfected MCF-7 cells (8) is that overexpression of this caspase somehow replaced caspase-8 in the activation of the apoptotic machinery (60) . Alternatively, overexpressed caspase-1/ICE could be a substrate of CD95-activated caspase-8 and provoke an amplification of caspase signaling (61) . How IFN-{gamma} can regulate the expression of the caspase-8 gene is not known. IFN-{gamma} activates the signal transducer and activator of transcription (STAT) signaling pathway, which can play important roles in cell proliferation, differentiation, and apoptosis (9 , 62) . Furthermore, it was demonstrated that IFN-{gamma} activated STAT1 and induced apoptosis in various cell types (63) . Activation of apoptosis by IFN-{gamma} correlated with the induction of ICE/caspase-1 (63) . IFN-{gamma}-induced activation of the caspase-1 promoter was dependent on the binding of IRF-1 (64) . Although the elements in the caspase-8 gene that are responsible for activating caspase-8 expression have not been identified, one can speculate with the possibility of similarities between caspase-1 and -8 in terms of the mechanism regulating their expression by IFN-{gamma}.

Regulation of the expression and/or activity of the DISC components could be a strategy used by virally infected or tumor cells to escape from the host immune system. Protection of virus-infected cells against death-receptor-induced apoptosis may lead to higher virus production and contribute to the persistence and oncogenicity of several FLIP-encoding viruses (65) . In this respect, caspase-8 inhibitors like CrmA and v-FLIP are present in cells infected by different viruses (43 , 66) . Down-regulation of CD95 expression is also observed in adenovirus-infected cells (67) . Human melanomas express elevated levels of FLIP and are resistant to death receptor-mediated apoptosis (27) . The gene for caspase-8 is frequently inactivated in neuroblastoma, a tumor of the peripheral nervous system (68) . Caspase-8 is a cellular target of the Mr 14,700 protein of adenovirus type 5, that protected cells from death receptor-induced apoptosis (69) . Therefore, down-regulation of caspase-8 levels or activity in tumor cells may be an important mechanism in the evasion of the immune response. In this respect, our data indicate that sensitizing regimens like IFN-{gamma} may be used in combination strategies with nontoxic death-receptor ligands, for instance TRAIL, in the treatment of human breast cancer.


   FOOTNOTES
 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

1 Supported by grants from Fundación Ramón Areces and Ministerio de Educación y Cultura (1FD97-0514-C02-01) to A. L-R. C. M-P. is recipient of a fellowship from Ministerio de Educación y Cultura. Back

2 To whom requests for reprints should be addressed, at Instituto de Parasitología y Biomedicina, CSIC, calle Ventanilla 11, 18001 Granada, Spain. Phone: 34-958-80-51-88; Fax: 34-958-20-33-23; E-mail: alrivas1{at}ipb.csic.es Back

3 The abbreviations used are: TNF, tumor necrosis factor; TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; FLIP, FLICE-inhibitory protein; DISC, death-inducing signaling complex; IRF, IFN regulatory factor; mAb, monoclonal antibody; PS, phosphatidylserine; RT-PCR, reverse transcription-PCR; PARP, poly(ADP-ribose-polymerase; CD95L, CD95 ligand. Back

Received 12/10/99. Accepted 8/ 9/00.


   REFERENCES
Top
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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