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-Induced Cell Death
1 Laboratoire de Cytokines et Immunologie des Tumeurs Humaines, Institut National de la Santé et de la Recherche Médicale U-487, Institut Gustave Roussy Pavillon de Recherche 1 and Institut Fédératif de Recherche, Villejuif, France;
2 Unité Mixte de Recherche 1161 de Virologie Institut National de Recherche Agronomique Agence Française de Sécurité Sanitaire des Aliments École National Vétérinaire d’Alfort, Maisons Alfort, France;
3 Commisariat à l’Énergie Atomique, Service de Génomique Fonctionnelle, Evry, France;
4 Unité Mixte de Recherche 8125 Centre National de la Recherche Scientifique, Institut Gustave Roussy Pavillon de Recherche 2, Villejuif, France; and
5 Commisariat à l’Énergie Atomique, Service de Neurovirologie, Centre de Recherche du Service Santé des Armés, École Pratique des Hautes Études, Institut Paris sud sur les Cytokines, Université Paris XI, Fontenay-aux-Roses, France
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF), we have applied cDNA microarrays to a human breast carcinoma TNF-sensitive MCF7 cell line and its established TNF-resistant clone. Of a total of 5760 samples of cDNA examined, 3.6% were found to be differentially expressed in TNF-resistant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis of available literature data, the striking finding is the association of some differentially expressed genes involved in the phosphatidylinositol-3-kinase/Akt signaling pathway. More notably, we found that the PRNP gene coding for the cellular prion protein (PrPc), was 17-fold overexpressed in the 1001 cell line as compared with the MCF7 cell line. This differential expression was confirmed at the cell surface by immunostaining that indicated that PrPc is overexpressed at both mRNA and protein levels in the TNF-resistant derivative. Using recombinant adenoviruses expressing the human PrPc, our data demonstrate that PrPc overexpression converted TNF-sensitive MCF7 cells into TNF-resistant cells, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondria and nuclear condensation. |
INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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(TNF) is a cytokine with powerful direct tumor-killing capacity (1)
. Although considerable progress has been made in identifying gene products that regulate TNF-induced cell death, the understanding of the mechanism of cell resistance to the cytotoxic action of TNF observed in several tumor cells remains limited. Therefore, knowledge of the molecular and biochemical mechanisms of tumor cell resistance to the cytotoxic action of TNF may ultimately provide new approaches to enhance its therapeutic efficiency against human malignancies. We have earlier shown (2, 3, 4, 5) that cell surface expression of TNF receptors in a TNF-sensitive human breast carcinoma cell line model was necessary but not sufficient to mediate an apoptotic response, and that postreceptor mechanisms are important in controlling cell susceptibility to the cytotoxic action of TNF. Overexpression of several proteins, such as Bcl-2, c-myc, MnSOD, heat-shock protein 70, and a 20 zinc finger protein, has also been associated with TNF resistance (6, 7, 8, 9) .
The cellular prion protein (PrPc) is an ubiquitous host protein expressed by all known mammals, predominantly in the brain. It is well known for its implication in transmissible spongiform encephalopathies (TSE), which are fatal neurodegenerative diseases. TSEs are characterized by vacuolation of neurons, astroglyosis, and the accumulation of PrPres, an abnormal protease-resistant form of the host-encoded PrPc, in the central nervous system. Although the physiological function of the normal cellular form of PrPc is not known, the protease-resistant form of PrPc (PrPres) plays an essential role in the transmission and propagation of TSE, and it is widely admitted in the scientific community that the protease-resistant form of PrPc (PrPres) is a major component, if not a unique component, of the infectious particle (for reviews see Refs. 10, 11, 12 ). Recently, the possibility that PrPc may participate in cell death regulation has been raised. Such activity might be correlated with the fact that an interaction between PrPc and the Bcl-2 oncoprotein has been found in the yeast two-hybrid system (13) . Some studies have, indeed, described how PrPc displays an antiapoptotic action that is similar to Bcl-2 activity (14 , 15) . Other authors reported that stress-induced protein 1 is a cell surface ligand for PrPc that transduces neuroprotective signals (16 , 17) . Some other models suggest, however, that PrPc has a proapoptotic action. O’Donovan et al. (18) have reported that prion protein fragment 106–126 induced apoptosis via mitochondrial disruption in a human neuroblastoma cell line. More recent data have also pointed out a proapoptotic function of the PrPc, involving an overexpression of the p53 tumor suppression factor (19 , 20) .
In the present work, we studied the molecular mechanisms of tumor cell resistance to the cytotoxic action of TNF in a human breast carcinoma model. For this purpose, we have used the cDNA microarray technique and adenovirus-mediated gene transfer to extend previous observations. We provide, for the first time, evidence that the PrPc protects human breast carcinoma against apoptosis mediated by TNF.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Production of the cDNA Microarrays.
Microarrays were prepared with a set of 5760 cDNA clones selected from a normalized infant brain library. The 3' and/or 5' ends of these cDNA clones have been sequenced previously (23
, 24)
. Each cDNA clone insert was amplified by PCR and was spotted onto glass PolySilane slides (CMT GAPS II; Corning) with a robot Microgrid II (BIORobotics) under constant humidity and temperature.
cDNA Microarrays Analysis.
Total RNA was extracted from tumor cell lines using the RNeasy Midi kit (Qiagen, S.A., Courtaboeuf, France) according to the manufacturer’s protocol. Twenty-five µg of total RNA and 1 ng of mRNA luciferase were incubated in a cocktail containing Cy3 or Cy5-dUTP (MEN) and SuperScript II reverse transcriptase (Life Technologies, Inc.). Labeled targets were hybridized to microarray slides at 42°C for 16 h. After washing, slides were scanned with an Axon 4000B fluorescence laser-scanning instrument with a resolution of 10 µm (Axon Instruments, Foster City, CA). To discard systematic errors because of dye incorporation, the same sample was labeled either with Cy-5 or with Cy-3, and appropriate targets were combined (Dye-swap). Image analyses were performed using the GenePix Pro 3.0 software (Axon). Cy5:Cy3 intensity ratios from each gene were calculated and were globally normalized to make the median value of the log2 ratio equal to zero. This corrects for dye bias, photo multiplier tube (PMT) voltage imbalance, and variations between channels in amounts of samples hybridized. Genes were considered as significantly modulated according to the following criteria in more than four among six experiments: (a) signal/noise ratio >3; (b) 2-fold or greater change in expression level; (c) similar value in Dye-swap assay; and (d) significance analysis of microarrys (SAM) test, 
= 2.1; median false significant number = 0.03676; false discovery rate = 0.018%. For clustering analysis, we used Cluster and Tree initialization of Genesis software developed at Graz University of Technology.
TaqMan (Applied Biosystems) Real-Time Quantitative Reverse Transcription-PCR Analysis.
One µg of RNA was reversed transcribed using random hexamers according to the manufacturer’s recommendations (Applied Biosystems). Quantitative real-time PCR was performed using 5 µl of diluted cDNA (1 µl in 19 µl of water) in a final volume of 25 µl according to the manufacturer’s recommendations (Applied Biosystems). PCR primers and probe for the PRNP gene target were designed by Applied Biosystems and used as the manufacturer’s recommendations. The amount of sample RNA was normalized by the amplification of an endogenous control (18S). The relative quantification of the transcripts was derived using the standard curve method (Applied Biosystems User Bulletin 2, ABI PRISM 7700 Sequence Detection system).
PrPc Protein Expression Analysis.
The cells were seeded at 1 x 106 cells/well in a 6-well plate for overnight incubation. Indirect immunofluorescence experiment was performed using Pri 308 antihuman PrPc monoclonal antibody and was analyzed by fluorescence-activated cell sorting (FACS) as described previously (22)
.
To release cell surface PrPc, 5 x 104 cells/well were seeded on sterile slides in a 6-well plate for overnight incubation. The cell were then treated with 1 unit/ml phosphoinositol phospholipase C (PIPLC; Sigma) in opti-MEM at 30°C for 1 h. Cells were washed once with PBS and were fixed with 4% paraformaldehyde in PBS for 60 min. After three washings with PBS, nonspecific sites were blocked with PBS-BSA (1 mg/ml) for 20 min. Cells were then incubated with Pri 308 antihuman PrPc monoclonal antibody (which recognize a synthetic peptide encompassing residues 106–126 of human PrPc) or Pri908 antihuman PrPc monoclonal antibody (which recognize a synthetic peptide encompassing residues 214–230 of human PrPc) diluted to 1:100 in PBS-BSA (1 mg/ml) for 60 min. Cells were washed three times with PBS and were incubated with Alexa 488 conjugated goat antimouse IgG (Molecular Probes) diluted to 1:100 in PBS-BSA (1 mg/ml) for 30 min in the dark. After three washings with PBS, the slides were examined under LSM 510 confocal microscope (Zeiss).
MCF7 and 1001 Cell Transduction and TNF Treatment.
Cells were seeded at 104 cells/well in a 24-well plate [for methylthiazol tetrazolium (MTT) assay], or at 5 x 105 cells/well in a 6-well plate [for terminal deoxynucleotidyl transferase-mediated nick end labeling (TUNEL) assay], or at 5 x 104 cells/well on sterile slides in a 6 well plate (confocal scanning immunofluorescence microscopy) for overnight. Cells were transduced with AxCMNtTA, AdTRMet, or a combination of these two viruses at a multiplicity of infection of 10 for each virus. After 48-h incubation at 37°C, the medium was removed and was replaced by fresh medium in the absence or presence of TNF (100 ng/ml).
Determination of Cell Viability by MTT Assay.
Viability was assessed by the conversion of MTT (Sigma) to a formazan product. After appropriate incubation of cells with TNF, MTT (0.25 mg/ml) was added and was incubated for 4 h at 37°C. After the addition of 100 µl of Me2SO to each well, the plates were read at 620 nm on a micro ELISA plate reader. Cell viability (%) = 100 x (A1/A0), where A1 and A0 were the absorbance (A) obtained from TNF-treated and -untreated cells, respectively.
Terminal Deoxynucleotidyl Transferase-Mediated Nick End Labeling Assay.
DNA fragmentation was detected in fixed cells, by terminal deoxynucleotidyl transferase-mediated nick end labeling staining with the in situ cell death detection kit, fluorescein (Roche), following the standard protocol.
Confocal Scanning Immunofluorescence Microscopy.
After appropriate transduction and treatment with TNF, 5x 104 cells were washed once with PBS and were fixed with 4% paraformaldehyde in PBS for 60 min. Cells were then rinsed three times with PBS-SDS (0.1% in PBS) was used to permeabilize the cells for 10 min. After three washings with PBS, nonspecific sites were blocked with FCS 10% in PBS for 20 min. Cells were then incubated with a monoclonal antibody against cytochrome c (BD PharMingen) for 60 min. Cells were washed three times with PBS, and were incubated with Alexa 488 conjugated goat antimouse IgG (Molecular Probes) for 30 min in the dark. After three washings with PBS, nuclei were stained with DAPI for 5 min and were examined under LSM 510 confocal microscope (Zeiss) as described previously (25)
.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Real-time PCR analysis with two sample genes (v-erb-b2 and PLAUR) confirmed the pattern of differential gene expression identified by cDNA microarray (data not shown). To better understand the potential importance of each gene and to define genes most likely to be associated with TNF-resistance acquisition by tumor cells, we clustered differentially expressed genes into different categories, using National Center for Biotechnology Information Clusters of Orthologous Gene Classification" and Kyoto Encyclopedia of Genes and Genomes (KEGG). Interestingly, whereas the genes overexpressed in TNF-resistant 1001 cells were found to be associated with translation, immune response, cell adhesion/motility, matrix interaction-communication, and human diseases, those overexpressed in TNF-sensitive MCF7 cells appeared to be involved in replication repair, posttranslational modification, and protein turnover (Table 1)
. Although it is beyond the scope of this report to describe in detail all of the genes that were differentially expressed, some of these are subject to a higher level of scrutiny. Three genes (PARG1, RAB31, and ARFGAP1) associated with Rho GTPase activator, six genes (DSP6, PTPRM, PTPRE, moesin, myosin X, and BPAG1) associated with phosphatidylinositol 3-kinase (PI3K)/Akt signaling pathway (26, 27, 28)
, and, surprisingly, one gene (PRNP) associated with prion disease were overexpressed in TNF-resistant 1001 cells. An overexpression of IGFBP5 in MCF7 cell line was also found. Our results are fully in line with available data that have already been demonstrated (see "Discussion"; for review see Ref. 29
), suggesting that the PI3K/Akt signaling pathway is involved in tumor cell resistance to the cytotoxic action of TNF.
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. We further investigated the implication of the PrPc in cell resistance to TNF. Immunofluorescence analysis, performed using Pri 308 antihuman PrP monoclonal antibody, indicated that PrPc was highly expressed at the surface of 1001 resistant cells (mean fluorescence intensity = 94) and at a lower level at the surface of MCF7 cells (mean fluorescence intensity = 10; Fig. 2B
). The data showed that the PrPc protein expression in 1001 was 10 times higher than in MCF7 cells. PrPc is normally attached to the cell membrane via a glycosylphosphatidylinositol anchor. The confocal scanning fluorescence analysis also showed that PrPc was highly expressed at the surface of 1001 resistant cells. It has been shown that the enzyme phosphoinositol phospholipase C releases PrPc from the cells into medium. Treatment with phosphoinositol phospholipase C decreased the amount of PrPc at the surface of 1001 cells. PrPc expression at the surface of MCF 7 is low before and after phosphoinositol phospholipase C treatment (Fig. 2C)
. These results confirmed the differential PRNP gene expression identified by TaqMan reverse transcription-PCR analysis and demonstrated that, in the TNF-resistant 1001 cell line, PrPc is overexpressed at both mRNA and protein levels as compared with MCF7 cell line.
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100% of cell transduction and 25-fold overexpression of the PrPc in MCF7 cells (Fig. 3
A, left panel); and 80% of cell transduction and 4.5-fold overexpression of the PrPc in 1001 cells (Fig. 3
A, right panel). We have tested whether the overexpression of this protein can protect TNF-sensitive MCF7 cells to the cytotoxic action from TNF. Whereas only 30% of viability was observed in untransduced MCF7 cells, AxCMNtTA- and AdTRMet- cotransduced cells exhibited more than 80% cell viability. It should be noted that transduction with control adenoviruses (AxCMNtTA or AdTRMet) used alone or in combination with TNF, had no effect on cell viability (30% viability; Fig. 3
A, left panel). The same TNF treatment and viral transduction of 1001 cells, which naturally overexpress PrPc or follow PrPc induction, did not seem to modify the cell viability and confirmed the total resistance (100% viability) of these cells to TNF-mediated cytotoxicity (Fig. 3
A, right panel).
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B, left panel), AxCMNtTA- and AdTRMet-cotransduced cells exhibited only 0.3% of apoptotic cells (Fig. 3
B, right panel). It should be noted that transduction with control adenoviruses (AxCMNtTA or AdTRMet) used alone or in combination with TNF, had no effect on cell death (results not shown). The same TNF treatment and viral transduction of 1001 cells did not seem to modify the cell death and confirmed the absolute resistance (0.001 % cell death) of these cells to TNF-mediated cytotoxicity (Fig. 3C)
. These results showed unambiguously that overexpression of PrPc in MCF7 cells resulted in the acquisition of cell resistance to TNF-mediated cytotoxicity and indicated that PrPc protects MCF7 cells against the proapoptotic action of TNF. Furthermore, using a panel of human breast carcinoma cell lines, we have demonstrated a correlation between susceptibility to cytotoxic action of TNF and expression for PrPc. In this context, the PrPc ectopic expression in the BT20 TNF-sensitive cells was found efficient to confer resistance to TNF-induced cell death (data not shown). These observations suggest that the PrPc antiapoptotic role is applicable to other human breast carcinoma cells besides the MCF7 cell line.
PrPc Overexpression Abrogates Cytochrome c Release and Nuclear Condensation in TNF-Sensitive MCF7 Cells.
Our previous data indicated the involvement of mitochondrial pathway in the acquisition of tumor resistance to TNF-induced cell death (25)
. We, therefore, asked whether the PrPc effect involves this pathway. As shown in Fig. 4A
, cytochrome c was found to be localized to mitochondria (control medium) that was released from mitochondria when apoptosis was induced after TNF treatment (control with TNF; Fig. 4
A, MCF7). Nuclear condensation confirmed cell death induction, as evaluated by DNA staining with DAPI (Fig. 4B
, MCF7). Neither event was influenced by transduction with control adenoviruses AxCMNtTA or AdTRMet used alone or in the presence of TNF. However, when MCF7 cells were cotransduced with AxCMNtTA and AdTRMet, the cytochrome c release in response to TNF was no longer observed (Fig. 4A)
. Moreover, transduction of MCF7 cells with recombinant adenoviruses expressing PrPc protects from nuclear condensation after TNF treatment (Fig. 4B)
. As expected, neither cytochrome c release to cytosol, nor nuclear condensation, were observed in 1001 cells after TNF treatment. The transduction with the adenoviruses described above in TNF-treated and -untreated cells had no effect on such translocation (Fig. 4A)
and nuclear condensation (Fig. 4B)
. These results showed that PrPc overexpression converts TNF-sensitive cells to TNF-resistant cells by a mechanism, at least in part, involving the mitochondrial pathway.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Among the multitude of genes identified on our microarray, the striking finding is that a great number of differentially expressed genes is implicated in the PI3K/Akt signaling pathway. For example, DSP6, PTPRM, and PTPRE genes that were overexpressed in 1001 compared with MCF7 cell lines have been identified in the Kyoto Encyclopedia of Genes and Genomes as presenting a similar PTEN function in the PI3K signaling pathway (26
, 28)
. Weng et al. (27)
demonstrated that overexpression of wild-type PTEN in MCF7 leads to the suppression of cell growth through the blockade of cell cycle progression and the inhibition of Akt phosphorylation. In contrast, expression of the phosphatase-mutated dead isoform promoted cell growth and had an opposite effect on the phosphorylation of Akt. Our in vitro cell proliferation assay coupled to in vivo xenograft studies indicated that cell growth of 1001 cells was more important than that of MCF7 cells (data not shown). These results suggest that DSP6, PTPRM, and PTPRE genes would behave in the same way as the mutated PTEN gene and, thus, presumably, promote cell growth in the 1001 cell line. We also found an up-regulation of members of the ezrin/radixin/moesin protein family, Moesin, myosin X, and BPAG1. Ezrin, a plasma membrane-actin cytoskeleton linker, interacts with p85, the regulatory subunit of PI3K and plays a role in cell survival signaling (28)
. As another example, IGFBP-5, is overexpressed in MCF7 cells (Table 1B)
. Examination of signaling pathways involved in apoptosis revealed that phosphorylation of PI3K and the forkhead transcription factor was blocked by IGFBP-5 (30)
. This may, in part, further explain the sensitivity of MCF7 to TNF and confirm that tumor resistance to the cytotoxic action of TNF is complex and multiparametric. Surprisingly, extensive studies of our model also indicated that PrPc was overexpressed at both mRNA and protein levels in TNF-resistant derivative. At present, little is known about the exact physiological function of this protein, which is more widely studied for its implication in TSE. However, recent data have pointed to a potential role of PrPc in the regulation of apoptosis (14 , 16 , 17) . This could be only one of several functions of the protein, but it might be of particular interest, because the pathological processes of TSE may include a loss of function of PrPc when converted to the protease-resistant form of PrPc (PrPres). The role of PrPc in apoptosis remains a subject of debate. Whereas some studies suggest that it protects cells from proapoptotic agents, others tend to demonstrate that PrPc sensitizes cells to apoptotic stimuli. However, the exact mechanisms by which PrPc may induce susceptibility to apoptosis are not fully described. We have, therefore, investigated a possible role of PrPc in the acquisition of cell resistance to TNF. The results of the protein overexpression in MCF7 cells using recombinant adenoviruses unambiguously demonstrated that PrPc was implicated in the acquisition of a TNF-resistant phenotype of this cell line. Taken together with gene expression profiling, these data suggest that PrPc may induce resistance to TNF by involving the PI3K/Akt pathway. Interestingly, a recent study has already reported that PrPc activates this transduction signaling pathway in primary murine neurons, but as an external ligand to an unknown cellular receptor, leading to an enhanced neuronal survival (31) .
Other reports have shown that the murine PrPc may protect rat and murine retinal explants from anisomycine-mediated cell death. This phenomenon requires PrPc activation via an external ligand, which seems to be the stress-inducible protein 1, through a cAMP/PKA-dependent pathway (16
, 17)
. It is conceivable that, in our model, PrPc is also activated by a ligand that could be the laminin 2, which is overexpressed in 1001 cells (Table 1A)
. In fact, PrPc has been previously shown to interact with the laminin receptor precursor and the laminin receptor at the cell surface (32
, 33)
. The possible interaction of this complex with laminin 2 could lead to the transduction of a protective signal. It would not be the first time that laminin is related to PrPc, because the laminin-induced PC12 neuronal cell differentiation implicates PrPc (16
, 34)
.
PrPc has also been related to apoptotic cascades involving Bcl-2 or p53. Two different human and murine neuronal cell models were described to be protected, respectively, from Bax and serum deprivation mediated apoptosis by either PrPc or Bcl-2 expression (14, 15) . Our data indicate that PrPc overexpression converts TNF-sensitive cells to TNF-resistant cells by a mechanism involving, at least in part, the alteration of cytochrome c release from mitochondria to the cytosol and nuclear condensation. Although it is clearly established that Bcl-2 is an important regulator of mitochondrial apoptotic cascades, a previous report indicates that overexpression of Bcl-2 in MCF7 did not reduce the cell sensitivity to TNF (6) . This suggests that, in the MCF7 cell line, PrPc-induced protection from the cytotoxic action of TNF is independent from the Bcl-2 pathway. Recently, it has been reported that the murine PrPc may sensitize human HEK293 epithelial and murine TSM1 neuronal cell lines to staurosporine-induced cell death (19 , 20) . The proapoptotic action of PrPc observed in these models, which is in apparent contradiction to our observations, is related to a p53-apoptosis pathway. In this regard, we have previously reported that, whereas MCF7 cells express wild-type p53, this protein was found mutated in the TNF-resistant 1001 derivative clone (5) . We have also shown that wild-type p53 overexpression converts TNF-resistant 1001 into TNF-sensitive 1001 by a mechanism involving mitochondrial pathway (6 , 25) . In this context, it will be interesting to evaluate the effect of p53 inactivation on PrPc overexpression in the sensitivity of MCF7 cells to TNF cytotoxicity.
Our data strongly indicate that PrPc exhibits an antiapoptotic action on MCF7 cells in response to the cytotoxic action of TNF, at least in part, by a mechanism involving alteration of cytochrome c release from mitochondrial and nuclear condensation, which may be linked to the PI3K/Akt pathway. Taken together with previously published studies, these observations are in agreement with PrPc playing a key role in apoptosis. Whether it might be a pro- or antiapoptotic action seems to depend on a combination of several factors: the cell phenotype (neuronal versus epithelial, normal versus cancer); the PrPc species (human, murine versus ovine); and the apoptotic agent (TNF, staurosporine, Bax or anisomycin). To our knowledge, the present work links, for the first time, PrPc to the acquisition of a resistance phenotype by tumor cells.
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ACKNOWLEDGMENTS |
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We thank O. Brison (Unité Mixte Recherche 8125, Villejuif), J. Soudon (PHARMACELL, Paris), J. C. Ahomadeghe (Institut Gustave Roussy, Villejuif), J. Bassam (Institut curie, Paris), and N. Sarafan-Vasseur (Equipe Mixte INSERM 99-06, Rouen) for providing human breast carcinoma cell lines; J-R. Bertrand for help with the computer graphics; Philippe Dessen for continuous help with the microarray data analysis and the reading of the manuscript; J. Grassi for providing us the Pri 308 and Pri 908 antihuman PrPc monoclonal antibodies (service de pharmacologie et d’immunologie, Commisariat à l’énergie atomique, Saclay); Rodica Stancou for technical assistance; and Yan Lecluse for fluorescence-activated cell sorting (FACS) analysis.
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FOOTNOTES |
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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.
Requests for reprints: Maryam Diarra-Mehrpour, INSERM, Unité U487 Laboratoire Cytokines et Immunologie des Tumeurs Humaines, Institut Gustave Roussy, PR1, F-94805 Villejuif cedex, France. Phone: 331-42-11-46-50; Fax: 331-42-11-52-88; E-mail: mehrpour{at}igr.fr
Received 6/13/03. Revised 10/23/03. Accepted 11/10/03.
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). Cells were then treated or not with TNF (100 ng/ml) for 72 h, and the cell viability was measured by a methylthiazol tetrazolium (MTT) assay, performed in replicate of three samples (
). TNF-sensitive MCF7 (B) and TNF-resistant 1001 (C) cell lines were transduced and then treated (solid histogram) or not (open histogram) with TNF (100 ng/ml) for 24 h. Apoptosis was measured by terminal deoxynucleotidyl transferase-mediated nick end labeling assay. Percentages, apoptotic cells in TNF-treated cells. M1, apoptotic cell region.