Adenovirus-mediated transfer of wild-type p53 gene sensitizes TNF resistant MCF7 derivatives to the cytotoxic eect of this cytokine: relationship with c-myc and Rb Maya Ameyar 1 , Vladimir Shatrov 1 , CeÂline Bouquet 2 , Corinne Capoulade 3 , Zhenzi Cai 1 , Rodica Stancou 1 , Christophe Badie 4 , Hedi Haddada 2 and Salem Chouaib* ,1 1 INSERM U487 `Cytokines et Immunologie des Tumeurs Humaines', Institut Gustave Roussy, 94805 Villejuif, France; 2 INSERM U362, Institut Gustave Roussy, 94805 Villejuif, France; 3 UMR 1598, Institut Gustave Roussy, 94805 Villejuif, France; 4 UMR CNRS 1772, Institut Gustave Roussy, 94805 Villejuif, France Tumor suppressor p53 is a nuclear transcription factor that blocks cell cycle progression and induces apoptosis. We have previously shown that the MCF7 resistance to the cytotoxic action of TNF correlates with p53 mutations. In the present study, we used a recombinant adenovirus carrying a wild-type p53 gene (Adwtp53) in order to investigate the eect of wt p53 transfer on modulation of cell resistance to the cytotoxic action of TNF. Our data indicate that infection of TNF resistant MCF7 cells (1001 and MCF7/Adr) with Adwtp53 resulted in the restoration of wt p53 expression and function as respectively revealed by the yeast assay and the induction of p53 inducible genes MDM2 and p21. Furthermore, the restoration of p53 function signi®cantly sensitized TNF resistant cells to TNF cytotoxic action. This correlated with a signi®cant down-regulation of c- myc in both TNF-resistant cell lines and a decrease of Retinoblastoma protein (Rb) in 1001 clone. In contrast, the eect of p53 seems to be independent from Bcl-2 and Bax protein level regulation. The present study suggests that the combination of TNF and Adwtp53 may be a potential strategy to sensitize mutant p53 TNF-resistant tumors to the cytotoxic action of this cytokine. Keywords: TNF; p53; c-myc; Rb Introduction Tumor necrosis factor (TNF) is a multifunctional cytokine that initiates a wide variety of biological responses. This factor acts as a host defence factor in immunological responses and plays a major role in tumor cell destruction mediated by cytotoxic T cells (Chouaib et al., 1991). However, despite the antitumor eects of TNF, its eective application is strongly limited by acquisition or innate resistance of certain tumor cells to its cytotoxic action. Although loco- regional administration of TNF partially resolved its systemic and toxic side eects (Lejeune et al., 1994), resistance of some tumor cells to the cytotoxic action of TNF remains a drawback to its eective application. Therefore, understanding the molecular and biochem- ical mechanisms of tumor cell resistance to the cytocidal action of TNF may ultimately provide new approaches to enhance its therapeutic ecacy against human malignancies. Several mechanisms have been reported to contri- bute to the cellular resistance to TNF-induced cell killing (Cai et al., 1997a; JaÈattela et al., 1992; Liston et al., 1996; Opipari et al., 1992; Rothe et al., 1995; Wong and Goeddel, 1988; Zyad et al., 1994). Recently, we have provided evidence for the existence of an association between the resistance of human breast adenocarcinoma cell line MCF7 to the cytotoxic action of TNF and the loss of p53 function (Cai et al., 1997b). p53 is the most frequently mutated gene in human cancers (Hainaut et al., 1998). Its mutations may interfere with some pathways responsible for apoptosis, including the transcription of some genes involved in the regulation of programmed cell death (PCD). It is clearly established that the nuclear phosphoprotein p53 is involved in the mediation of cell cycle checkpoints (Kastan et al., 1992). Its induction after DNA damage and certain stress signals (Gottlieb and Oren, 1996) permits cell cycle control, by induction of apoptosis or growth arrest, depending on the cell type. p53 is a transcriptional factor which activity can be abrogated by mutations, almost all aecting the DNA-binding domain, abolishing or reducing the ability of p53 to bind DNA (Gottlieb and Oren, 1996). This tumor suppressor gene probably induces a G1/S arrest through the combined inhibition of cyclin-dependent kinases and of DNA replication. The molecular mechanisms of p53 induced-apoptosis and its interac- tion with TNF cytotoxic pathways are less well characterized. Among target genes transactivated by p53 are GADD45 gene, which is involved in cell cycle arrest (Kastan et al., 1992) and MDM2 gene which forms a regulatory feedback loop with p53 (Barak et al., 1993; Chen et al., 1994; Momand et al., 1992; Wu et al., 1993). Another gene induced after exposure to DNA damaging agent and associated to wild-type p53 is p21 WAF1/CIP1 (El-Deiry et al., 1994; Harper et al., 1993) which encodes for an inhibitor of cyclin dependent kinase (Gu et al., 1993; Harper et al., 1993; Xiong et al., 1993). Bcl-2 and Bax are known to be regulators of the apoptotic cascade and may serve as pores of unknown speci®city in the mitochondrial membrane. (Miyashita and Reed, 1995; Williams and Smith, 1993; Antonsson et al., 1997; Schendel et al., 1997; Zamzami et al., 1998). While bax is activated by p53, bcl-2 gene is down- *Correspondence: S Chouaib Received 9 November 1998; revised 16 April 1999; accepted 20 April 1999 Oncogene (1999) 18, 5464 ± 5472 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $15.00 http://www.stockton-press.co.uk/onc