Prion Protein Prevents Human Breast Carcinoma Cell Line from Tumor Necrosis Factor ␣-Induced Cell Death
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[CANCER RESEARCH 64, 719–727, January 15, 2004] Prion Protein Prevents Human Breast Carcinoma Cell Line from Tumor Necrosis Factor ␣-Induced Cell Death Maryam Diarra-Mehrpour,1 Samuel Arrabal,2,5 Abdelali Jalil,1 Xavier Pinson,1 Catherine Gaudin,1 Genevie`ve Pie´tu,3 Amandine Pitaval,3 Hugues Ripoche,4 Marc Eloit,2 Dominique Dormont,5 and Salem Chouaib1 1Laboratoire de Cytokines et Immunologie des Tumeurs Humaines, Institut National de la Sante´ et de la Recherche Me´dicale U-487, Institut Gustave Roussy Pavillon de Recherche 1 and Institut Fe´de´ratif de Recherche, Villejuif, France; 2Unite´Mixte de Recherche 1161 de Virologie Institut National de Recherche Agronomique Agence Franc¸aise de Se´curite´Sanitaire des Aliments E´ cole National Ve´te´rinaire d’Alfort, Maisons Alfort, France; 3Commisariat a`l’E´ nergie Atomique, Service de Ge´nomique Fonctionnelle, Evry, France; 4Unite´Mixte de Recherche 8125 Centre National de la Recherche Scientifique, Institut Gustave Roussy Pavillon de Recherche 2, Villejuif, France; and 5Commisariat a` l’E´ nergie Atomique, Service de Neurovirologie, Centre de Recherche du Service Sante´ des Arme´s, E´ cole Pratique des Hautes E´ tudes, Institut Paris sud sur les Cytokines, Universite´Paris XI, Fontenay-aux-Roses, France ABSTRACT lopathies (TSE), which are fatal neurodegenerative diseases. TSEs are characterized by vacuolation of neurons, astroglyosis, and the accu- To define genetic determinants of tumor cell resistance to the cytotoxic mulation of PrPres, an abnormal protease-resistant form of the host- action of tumor necrosis factor ␣ (TNF), we have applied cDNA microar- encoded PrPc, in the central nervous system. Although the physiolog- rays to a human breast carcinoma TNF-sensitive MCF7 cell line and its ical function of the normal cellular form of PrPc is not known, the established TNF-resistant clone. Of a total of 5760 samples of cDNA c res examined, 3.6% were found to be differentially expressed in TNF-resist- protease-resistant form of PrP (PrP ) plays an essential role in the ant 1001 cells as compared with TNF-sensitive MCF7 cells. On the basis transmission and propagation of TSE, and it is widely admitted in c of available literature data, the striking finding is the association of some the scientific community that the protease-resistant form of PrP differentially expressed genes involved in the phosphatidylinositol-3- (PrPres) is a major component, if not a unique component, of the kinase/Akt signaling pathway. More notably, we found that the PRNP infectious particle (for reviews see Refs. 10–12). Recently, the pos- gene coding for the cellular prion protein (PrPc), was 17-fold overex- sibility that PrPc may participate in cell death regulation has been pressed in the 1001 cell line as compared with the MCF7 cell line. This raised. Such activity might be correlated with the fact that an inter- differential expression was confirmed at the cell surface by immuno- action between PrPc and the Bcl-2 oncoprotein has been found in the staining that indicated that PrPc is overexpressed at both mRNA and yeast two-hybrid system (13). Some studies have, indeed, described protein levels in the TNF-resistant derivative. Using recombinant adeno- c viruses expressing the human PrPc, our data demonstrate that PrPc how PrP displays an antiapoptotic action that is similar to Bcl-2 overexpression converted TNF-sensitive MCF7 cells into TNF-resistant activity (14, 15). Other authors reported that stress-induced protein 1 c cells, at least in part, by a mechanism involving alteration of cytochrome is a cell surface ligand for PrP that transduces neuroprotective signals c c release from mitochondria and nuclear condensation. (16, 17). Some other models suggest, however, that PrP has a proapoptotic action. O’Donovan et al. (18) have reported that prion protein fragment 106–126 induced apoptosis via mitochondrial dis- INTRODUCTION ruption in a human neuroblastoma cell line. More recent data have c Tumor necrosis factor ␣ (TNF) is a cytokine with powerful direct also pointed out a proapoptotic function of the PrP , involving an tumor-killing capacity (1). Although considerable progress has been overexpression of the p53 tumor suppression factor (19, 20). made in identifying gene products that regulate TNF-induced cell In the present work, we studied the molecular mechanisms of tumor death, the understanding of the mechanism of cell resistance to the cell resistance to the cytotoxic action of TNF in a human breast cytotoxic action of TNF observed in several tumor cells remains carcinoma model. For this purpose, we have used the cDNA microar- limited. Therefore, knowledge of the molecular and biochemical ray technique and adenovirus-mediated gene transfer to extend pre- vious observations. We provide, for the first time, evidence that the mechanisms of tumor cell resistance to the cytotoxic action of TNF c may ultimately provide new approaches to enhance its therapeutic PrP protects human breast carcinoma against apoptosis mediated by efficiency against human malignancies. TNF. We have earlier shown (2–5) that cell surface expression of TNF receptors in a TNF-sensitive human breast carcinoma cell line model MATERIALS AND METHODS was necessary but not sufficient to mediate an apoptotic response, and that postreceptor mechanisms are important in controlling cell sus- Cell Lines and Viruses. TNF-resistant 1001 cells were established from ceptibility to the cytotoxic action of TNF. Overexpression of several TNF-resistant RA-1 cells transfected by p55 TNF receptor cDNA. Parental proteins, such as Bcl-2, c-myc, MnSOD, heat-shock protein 70, and a RA-1 cells were derived from TNF-sensitive human breast carcinoma MCF7 20 zinc finger protein, has also been associated with TNF resistance cells after continuous exposure to increasing dose of recombinant TNF as (6–9). described previously (21). All of the cells were maintained and propagated in RPMI 1640 containing 10% FCS. Replication-defective AxCMNtTA and The cellular prion protein (PrPc) is an ubiquitous host protein AdTRMet viruses were described previously (22). Both viruses were produced expressed by all known mammals, predominantly in the brain. It is and amplified in HEK293 cells. well known for its implication in transmissible spongiform encepha- Production of the cDNA Microarrays. Microarrays were prepared with a set of 5760 cDNA clones selected from a normalized infant brain library. The Received 6/13/03; revised 10/23/03; accepted 11/10/03. 3Ј and/or 5Ј ends of these cDNA clones have been sequenced previously (23, Grant support: Grants from INSERM and Association pour la Recherche sur le 24). Each cDNA clone insert was amplified by PCR and was spotted onto glass Cancer [Grants 4255 (to M. D-M.) and 5129 (to S. C.)] and Ligue Nationale Contre le PolySilane slides (CMT GAPS II; Corning) with a robot Microgrid II (BIO- Cancer (Val de Marne 2002). The costs of publication of this article were defrayed in part by the payment of page Robotics) under constant humidity and temperature. charges. This article must therefore be hereby marked advertisement in accordance with cDNA Microarrays Analysis. Total RNA was extracted from tumor cell 18 U.S.C. Section 1734 solely to indicate this fact. lines using the RNeasy Midi kit (Qiagen, S.A., Courtaboeuf, France) according Requests for reprints: Maryam Diarra-Mehrpour, INSERM, Unite´U487 Laboratoire to the manufacturer’s protocol. Twenty-five g of total RNA and 1 ng of 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: mRNA luciferase were incubated in a cocktail containing Cy3 or Cy5-dUTP [email protected]. (MEN) and SuperScript II reverse transcriptase (Life Technologies, Inc.). 719 Downloaded from cancerres.aacrjournals.org on September 29, 2021. © 2004 American Association for Cancer Research. PrPc PREVENTS BREAST CARCINOMA CELLS FROM APOPTOSIS Labeled targets were hybridized to microarray slides at 42°C for 16 h. After Terminal Deoxynucleotidyl Transferase-Mediated Nick End Labeling washing, slides were scanned with an Axon 4000B fluorescence laser-scanning Assay. DNA fragmentation was detected in fixed cells, by terminal de- instrument with a resolution of 10 m (Axon Instruments, Foster City, CA). To oxynucleotidyl transferase-mediated nick end labeling staining with the in situ discard systematic errors because of dye incorporation, the same sample was cell death detection kit, fluorescein (Roche), following the standard protocol. labeled either with Cy-5 or with Cy-3, and appropriate targets were combined Confocal Scanning Immunofluorescence Microscopy. After appropriate ϫ 4 (Dye-swap). Image analyses were performed using the GenePix Pro 3.0 transduction and treatment with TNF, 5 10 cells were washed once with software (Axon). Cy5:Cy3 intensity ratios from each gene were calculated and PBS and were fixed with 4% paraformaldehyde in PBS for 60 min. Cells were were globally normalized to make the median value of the log2 ratio equal to 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 zero. This corrects for dye bias, photo multiplier tube (PMT) voltage imbal- blocked with FCS 10% in PBS for 20 min. Cells were then incubated with a ance, and variations between channels in amounts of samples hybridized. monoclonal antibody against cytochrome c (BD PharMingen) for 60 min. Cells Genes were considered as significantly modulated according to the following were washed three times with PBS, and were incubated with Alexa 488 criteria in more than four among six experiments: (a) signal/noise ratio Ͼ3; (b) conjugated goat antimouse IgG (Molecular Probes) for 30 min in the dark. 2-fold or greater change in expression level; (c) similar value in Dye-swap After three washings with PBS, nuclei were stained with DAPI for 5 min and ␦ ϭ assay; and (d) significance analysis of microarrys (SAM) test, 2.1; median were examined under LSM 510 confocal microscope (Zeiss) as described ϭ ϭ false significant number 0.03676; false discovery rate 0.018%.