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Induction of TNF-sensitive cellular phenotype by c-Myc involves p53 and impaired NF-kappa B activation

1997; Springer Nature; Volume: 16; Issue: 24 Linguagem: Inglês

10.1093/emboj/16.24.7382

1460-2075

Juha Klefström,

Cell death mechanisms and regulation

Article15 December 1997free access Induction of TNF-sensitive cellular phenotype by c-Myc involves p53 and impaired NF-κB activation Juha Klefstrom Juha Klefstrom Molecular/Cancer Biology Laboratory, Haartman Institute, PO Box 21, 00014 University of Helsinki, FinlandJ.Klefstrom and E.Arighi contributed equally to this work Search for more papers by this author Elena Arighi Elena Arighi Molecular/Cancer Biology Laboratory, Haartman Institute, PO Box 21, 00014 University of Helsinki, FinlandJ.Klefstrom and E.Arighi contributed equally to this work Search for more papers by this author Trevor Littlewood Trevor Littlewood Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Marja Jäättelä Marja Jäättelä Apoptosis Laboratory, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark Search for more papers by this author Eero Saksela Eero Saksela Molecular/Cancer Biology Laboratory, Haartman Institute, PO Box 21, 00014 University of Helsinki, Finland Search for more papers by this author Gerard I. Evan Gerard I. Evan Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Kari Alitalo Corresponding Author Kari Alitalo Molecular/Cancer Biology Laboratory, Haartman Institute, PO Box 21, 00014 University of Helsinki, Finland Search for more papers by this author Juha Klefstrom Juha Klefstrom Molecular/Cancer Biology Laboratory, Haartman Institute, PO Box 21, 00014 University of Helsinki, FinlandJ.Klefstrom and E.Arighi contributed equally to this work Search for more papers by this author Elena Arighi Elena Arighi Molecular/Cancer Biology Laboratory, Haartman Institute, PO Box 21, 00014 University of Helsinki, FinlandJ.Klefstrom and E.Arighi contributed equally to this work Search for more papers by this author Trevor Littlewood Trevor Littlewood Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Marja Jäättelä Marja Jäättelä Apoptosis Laboratory, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark Search for more papers by this author Eero Saksela Eero Saksela Molecular/Cancer Biology Laboratory, Haartman Institute, PO Box 21, 00014 University of Helsinki, Finland Search for more papers by this author Gerard I. Evan Gerard I. Evan Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK Search for more papers by this author Kari Alitalo Corresponding Author Kari Alitalo Molecular/Cancer Biology Laboratory, Haartman Institute, PO Box 21, 00014 University of Helsinki, Finland Search for more papers by this author Author Information Juha Klefstrom1, Elena Arighi1, Trevor Littlewood2, Marja Jäättelä3, Eero Saksela1, Gerard I. Evan2 and Kari Alitalo 1 1Molecular/Cancer Biology Laboratory, Haartman Institute, PO Box 21, 00014 University of Helsinki, Finland 2Biochemistry of the Cell Nucleus Laboratory, Imperial Cancer Research Fund, PO Box 123, 44 Lincoln's Inn Fields, London, WC2A 3PX UK 3Apoptosis Laboratory, Danish Cancer Society, Strandboulevarden 49, DK-2100 Copenhagen, Denmark *E-mail: [email protected] The EMBO Journal (1997)16:7382-7392https://doi.org/10.1093/emboj/16.24.7382 PDFDownload PDF of article text and main figures. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Normal fibroblasts are resistant to the cytotoxic action of tumor necrosis factor (TNF), but are rendered TNF-sensitive upon deregulation of c-Myc. To assess if oncoproteins induce the cytotoxic TNF activity by modulating TNF signaling, we investigated the TNF-elicited signaling responses in fibroblasts containing a conditionally active c-Myc protein. In association with cell death, c-Myc impaired TNF-induced activation of phospholipase A2, JNK protein kinase and cell survival-signaling-associated NF-κB transcription factor complex. The TNF-induced death of mouse primary fibroblasts expressing deregulated c-Myc was inhibited by transient overexpression of the p65 subunit of NF-κB, which increased NF-κB activity in the cells. Unlike other TNF-induced signals, TNF-induced accumulation of the wild-type p53 mRNA and protein was not inhibited by c-Myc. TNF, with c-Myc, induced apoptosis in mouse primary fibroblasts but only weakly in p53-deficient primary fibroblasts. The C-terminal domain of p53, which is a transacting dominant inhibitor of wild-type p53, failed to inhibit apoptosis by c–Myc and TNF, suggesting that the cell death was not dependent on the transcription-activating function of p53. Taken together, the present findings show that the cytotoxic activity of TNF towards oncoprotein-expressing cells involves p53 and an impaired signaling for survival in such cells. Introduction Tumor necrosis factor-α (TNF) is a cytokine capable of cell death induction. The ligand-bound, active p55 TNF receptor can interact with caspase proteases via cytoplasmic adaptor proteins TRADD and FADD (Boldin et al., 1996; Muzio et al., 1996). The action of caspases is thought to execute most if not all forms of apoptotic cell death. The active p55 kDa TNF receptor complexes also interact via TRADD with RIP and TRAF2 proteins, which are involved in the activation of c-Jun amino-terminal kinases (JNKs, also known as stress-activated protein kinases SAPKs) and NF-κB (Hsu et al., 1996; Liu et al., 1996b). The NF-κB transcription factor complex mediates part of the pleiotropic biological effects of TNF by binding to and activating promoter regions of genes encoding growth factors, chemokines and leukocyte adhesion molecules (reviewed in Verma et al., 1995). Various stress conditions, especially genotoxic stress, also activate JNKs and NF-κB by as yet uncharacterized mechanisms (Kyriakis et al., 1994; Liu et al., 1996a). In primary cell cultures, TNF initiates a wide variety of cellular responses other than death, whereas the induction of death occurs mainly in cells derived from tumors or infected with viruses (Carswell et al., 1975; Aderka et al., 1985). However, TNF sensitivity can be induced in virtually all types of cells by chemically blocking de novo protein synthesis. This effect may be due to an inhibition of the synthesis of proteins which protect cells against the cytotoxic activity of TNF. The pre-treatment of cells with TNF confers resistance to subsequent TNF cytotoxicity, which suggests that a subset of such proteins is TNF-inducible (Wallach, 1984). In this context, it has been shown that if the expression of the TNF-inducible manganese superoxide dismutase (MnSOD) is inhibited concomitantly with TNF stimulation, cells which otherwise are resistant to TNF die. Similarly, inactivation of the TNF-inducible NF-κB transcription factor complex potentiates the cytotoxic action of TNF or genotoxic agents and can render normal cells TNF-sensitive (Beg and Baltimore, 1996; Van Antwerp et al., 1996; Wang et al., 1996). Thus, some of the TNF signals emanating from the activated receptor apparently via the TRAF2 pathway increase cellular resistance against death, and these signals have a crucial role in the survival of cells exposed to TNF. Given that TNF exerts its cytotoxic effects towards cells derived from tumors or infected with viruses, the cellular sensitivity to TNF-induced death may be a consequence of the expression of viral or endogenous oncoproteins. We and others have shown that both deregulated c–Myc (Janicke et al., 1994; Klefstrom et al., 1994) and adenoviral E1A (Chen et al., 1987; Duerksen-Hughes et al., 1991) can render fibroblasts sensitive to the cytotoxic effects of TNF. It is not yet clear, however, how oncoproteins activate cell death signaling by TNF. In order to elucidate these mechanisms, we studied the TNF-induced signaling pathways in fibroblasts containing a conditionally active form of c-Myc (MycER, Eilers et al., 1989). While TNF stimulation increased the cellular activities of phospholipase A2 (PLA2), JNK and NF-κB transcription factor complex in fibroblasts, these responses were inhibited upon c-Myc activation. The c-Myc-induced TNF sensitivity was inhibited but not completely abrogated by the overexpression of the p65 subunit of NF-κB, which substantially increased cellular NF-κB activity. Thus, part of the c-Myc-induced TNF sensitivity can be attributed to an inhibition of signaling for cell survival. The TNF-induced accumulation of p53 mRNA and protein was not inhibited by c-Myc. The p53 protein was necessary for TNF-induced cell death, suggesting that, in addition to the impaired signaling for survival, the TNF-induced p53 dictates the cytotoxic effect of TNF towards oncogene-expressing cells. Results Induction of TNF sensitivity by c-Myc but not by the pro-apoptotic protein Bak The deregulated expression of c-Myc induces cellular TNF sensitivity, but it has been unclear whether this is a specific function of c-Myc or a result of c-Myc-induced secondary cellular changes. The activation of c-Myc increases the susceptibility of fibroblasts to undergo apoptosis (Evan et al., 1992), which makes it possible that TNF sensitivity is associated with a low cellular threshold to apoptosis. Serum and its component, insulin-like growth factor-I (IGF-I), counteract the apoptotic functions of c-Myc (Harrington et al., 1994). As shown in Figure 1A, the addition of IGF-I completely blocked Rat1 cell apoptosis upon c-Myc activation, as quantitated by the MTT assay. When both TNF and IGF-I were added to the cell cultures containing activated c-Myc, cell viability decreased in a TNF dose-dependent manner. Without c-Myc activation, TNF did not cause a decrease in cell survival. Similar results were obtained when the cells were grown in the presence of high concentrations of insulin or in 10% fetal calf serum (FCS) (data not shown). These experiments demonstrate that the cells expressing deregulated c-Myc are not protected against the cytotoxic effects of TNF by the presence of IGF-I or other serum survival factors. Figure 1.Survival of Rat1 cells following activation of c-Myc or Bak in the presence of various concentrations of TNF. (A) Induction of TNF sensitivity by c-Myc in the presence of IGF-I. Rat1-MycER cells serum starved for 2 days were incubated in serum-free medium containing 100 ng/ml IGF-I and equivalent amounts of either ethanol carrier, β-estradiol (β-e) or OHT, and various concentrations of TNF. After 2 days of incubation with the drugs, the viability of the cells was quantitated by the MTT assay. The results represent means ± SD of five independent experiments. (B) and (C) TNF sensitivity of apoptosis-prone cells. Rat1-MycER and Rat1-Bak cells were serum starved for 2 days, after which the medium was replaced with serum-free medium containing ethanol carrier, β-e or OHT, and TNF at the concentrations indicated. After incubation for 2 (Rat1–MycER) or 6 days (Rat1-Bak) with the drugs, the viability of the cells was quantitated as above. The results represent means ± SD of four to five independent experiments. Note that in serum-deprived cells, both c-Myc and Bak decrease cell survival but only cells with active c-Myc are killed by TNF. Download figure Download PowerPoint Bak is a protein of the Bcl2 family, which, like c-Myc, induces apoptosis in serum-deprived fibroblasts, but unlike c-Myc, Bak does not stimulate entry into the cell cycle (Chittenden et al., 1995). We tested whether the pro-apoptotic Bak induces TNF sensitivity in Rat1 cells. Experimental induction of Bak in serum-starved Rat1 cells decreased cell survival (Figure 1C). However, whereas TNF treatment in the serum-free conditions caused a further dose-dependent decrease in the survival of c-Myc-overexpressing cells (Figure 1B), it did not significantly change, in the same conditions, the survival of Bak-overexpressing cells (Figure 1C). The failure of Bak to induce TNF sensitivity was not due to an absence of TNF receptors, since a rapid cell death resulted if TNF was added to the cultures together with sublethal concentrations of the protein synthesis inhibitor cycloheximide (data not shown). These results show that the activation of the apoptotic machinery is not sufficient to induce TNF sensitivity. Thus, the induction of TNF sensitivity is a specific function of c-Myc. Regions of c-Myc required for the induction of TNF sensitivity c-Myc is a basic region helix–loop–helix leucine zipper (bHLHZip) transcription factor which can either stimulate or repress transcription. In a heterodimer with Max, c–Myc binds specific DNA sequences and activates transcription from promoters containing its target sequences. These functions of c-Myc are required for c-Myc-induced cell cycle progression, cell transformation and apoptosis (Amati et al., 1993). In contrast, c-Myc can also repress transcription without an absolute need for Max (Roy et al., 1993; Philipp et al., 1994). The integrity of a small amino-terminal region encompassing amino acids 92–106 of c–Myc is critical for repression of cyclin D1, but is dispensable for transformation. Conversely, the amino-terminal residues 128–143 of c-Myc are critical for transformation, but not for repression of cyclin D1. By using the amino-terminal mutant forms of c-Myc differing in their effects on cell transformation and cyclin D1 repression (Philipp et al., 1994), we wished to learn which functions of c–Myc are associated with the induction of TNF sensitivity. The mutant forms of c-Myc were introduced by retroviral infection into NIH-3T3 cells, followed by selection of drug-resistant clones, which were pooled. Equal expression of c-Myc mutants was confirmed in the pools by Western blot analysis (data not shown). In repeated experiments, equivalent numbers of cells from each clone were seeded on duplicate culture dishes. After overnight adherence, TNF was added to one of the duplicate cultures and cell death in the cultures was examined microscopically at various times thereafter. The results from these experiments are summarized in Table I. They indicate that only those c-Myc mutants having transforming activity enhance cellular TNF sensitivity as does the wild-type c-Myc. Thus, like other known c-Myc-mediated biological responses, the induction of TNF sensitivity also requires c-Myc interaction with Max (Klefstrom et al., 1994) and a competence of c-Myc for transcriptional activation. Table 1. N-terminal regions in c-Myc involved in the induction of TNF sensitivity Myc mutant Cyclin D1 repressiona Transformationa TNF sensitivity MYC + + + MYCΔ92–106 − + + MYCΔ128–143 + − − MYCΔ104–136 − − − a Philipp et al. (1994). Deregulated c-Myc inhibits TNF-induced stress signals To elucidate mechanisms whereby deregulated c-Myc induces TNF sensitivity, we examined whether c-Myc modulates TNF-induced signal transduction in the target cells. TNF-induced activation of PLA2, JNK and NF-κB was assessed in Rat1 cells containing the conditionally active form of c-Myc (MycER) or, as a control, in Rat1 cells expressing a similar form of c-MycΔ (MycΔER is devoid of transcriptional activity due to a deletion encompassing c-Myc residues 106–143). TNF increases PLA2 activity in association with its cytotoxic and mitogenic responses (Palombella and Vilcek, 1989; Jäättelä, 1993). This leads to the enzymatic release of arachidonic acid (AA) from membrane phospholipids. PLA2 activity in TNF-treated cells was examined by measuring the release of [3H]arachidonic acid into the cell culture supernatants (Jäättelä, 1993). TNF stimulation reproducibly caused the release of AA metabolites from the tested cells (Table II). AA release from the TNF-treated cells was ∼1.2- to 1.4-fold higher than the spontaneous release from cells incubated in medium alone. However, if c-Myc was activated with 4-hydroxytamoxifen (OHT) 6 h prior to the addition of TNF, the TNF-induced AA release was inhibited (Table II). The activation of c–Myc as such did not affect the release of AA metabolites in these conditions. The inhibition of the TNF-induced AA release was a specific effect of the transcription factor activity of c-Myc, since the TNF-induced AA release was not impaired by c-MycΔ. Table 2. Effect of c-Myc deregulation on the TNF-induced PLA2 acivity Cells Relative release of arachidonic acid −OHT/−TNF −OHT/+TNF +OHT/−TNF +OHT/+TNF Rat1 1.00 1.33 1.03 1.38 Rat1-MycER 1.00 1.22 1.01 1.08 Rat1-MycΔER 1.00 1.41 0.98 1.46 Cells were treated with 100 nM OHT to activate c-Myc and with 50 ng/ml TNF as indicated. The release of arachidonic acid after 20 h of treatment is reported relative to the spontaneous release from untreated cells (1.0). The values represent means of triplicate experiments repeated three times with similar results. We also determined whether c-Myc inhibits the early TNF-induced signals, such as JNK activation. TNF was added to cells containing either active or inactive c-Myc. Cells were lysed after 5 or 20 min TNF treatment, and the JNK isoforms were bound to an amino-terminal c-Jun fragment. The JNK–c-Jun complexes were subjected to kinase assays and the amount of phosphorylated c-Jun protein was determined using specific anti-phospho-c-Jun antibody. As shown in Figure 2, TNF treatment caused a transient activation of JNKs in Rat1 fibroblasts. Maximal JNK activity was observed variably at 5–20 min. However, if c-Myc was activated prior to TNF stimulation, no activation of JNKs was observed. In parallel assays, c–MycΔ did not impair the TNF-induced JNK activation. Figure 2.Deregulation of c-Myc inhibits TNF-induced JNK activity. Cells were pre-treated in normal growth medium with ethanol carrier or with OHT for 6 h before addition of 50 ng/ml TNF. TNF-stimulated cells were lysed at the indicated time points and the N-terminal fragment of c-Jun (1–89) was incubated with the lysates, isolated with the bound JNK isoforms and subjected to kinase assay. The amount of phosphorylation at the Ser63 residue of c-Jun was determined from immunoblots by using phospho-c-Jun-specific antibody. After antibody detection, the immunoblots were stained with colloidal gold to confirm equivalent amounts of N-terminal c-Jun in the samples (the lowermost panels of two immunoblots). The assay was repeated three times with similar results. Download figure Download PowerPoint The NF-κB transcription factor complex is implicated in counteracting the cytotoxic activity of TNF. We used a reporter construct assay to examine the transcriptional activity of NF-κB in the TNF-treated cells. This reporter contained binding sites for NF-κB in front of the minimal c-fos promoter and the luciferase gene. One or two days after transfection, TNF was added to the cultures and luciferase activity was measured from the cell lysates. As shown in Figure 3, TNF treatment stimulated the NF-κB activity in Rat1 fibroblasts. In comparison, c-Myc caused an ∼50% inhibition of the transcriptional activation by NF-κB (Figure 3A); c-MycΔ did not cause such an inhibition (Figure 3B). The effect of c-Myc on NF-κB activation by TNF was also analyzed in mouse embryonic fibroblasts (MEFs). The cells were infected by high-titer MycERtm retroviruses as described in Materials and methods. As with Rat1 cells, the activation of c-Myc also inhibited the TNF-induced NF-κB activation in early passage MEFs (Figure 3C). Figure 3.Deregulation of c-Myc inhibits TNF-induced transcriptional activity of NF-κB. Cells were transfected with 5 μg of CMV-βgal and pBIIX-Luc plasmid and assayed 1 or 2 days after transfection. Transfected cells were pre-incubated for 6 h with ethanol carrier or OHT prior to addition of 50 ng/ml TNF. After a further incubation for 6 or 18 h with TNF, the cells were lysed. The luciferase activities were measured from the lysates and normalized to β-galactosidase activities. The normalized values are shown in (A) and (B). In (C) the values are presented as fold induction relative to control (−OHT, −TNF). The results represent mean ± SD values of six (Rat1–MycER cells), two (Rat1-MycΔER cells) or four (MEF-MycERtm cells) separate transfection experiments. Download figure Download PowerPoint Overexpression of p65 increases NF-κB activity in cells containing deregulated c-Myc and inhibits TNF-induced cell death To test if the level of cellular NF-κB activity can affect the c-Myc-induced TNF sensitivity, the NF-κB subunits, p50 or p65, or the negative regulator of NF-κB activity, IκBα, were transiently overexpressed in the low passage MEF-MycERtm cells. To identify the transfected cells, p50, p65, IκBα or control vector was co-transfected into the cells with a β-galactosidase-encoding reporter vector. Aliquots of each transfected cell culture were seeded to duplicate wells. The cells in one well were treated with ethanol carrier (untreated cells) and in the other well with OHT (to activate c-Myc) and TNF. After 24 or 48 h incubation with the drugs, cells were fixed and stained with X-gal to identify the β-galactosidase-expressing cells. The total number of adherent blue cells in each well was scored by microscopy. The NF-κB activity in the transfected cells was monitored in two separate transfection experiments by including the NF-κB luciferase reporter in the co-transfection mixture. TNF treatment decreased the viability of the cells containing deregulated c-Myc and control vector (pGD); the number of blue cells in the wells treated for 24 h with OHT and TNF was ∼55% of the number in the untreated wells (Figure 4A). In comparison, 80% of the cells co-expressing deregulated c-Myc and transfected p65 survived a 24 h TNF treatment. The corresponding figures after 48 h were 30 and 50%, respectively (P <0.05, paired t-test). The NF-κB activity was ∼6-fold higher in the cells transfected with p65 than in the control cells (Figure 4B). Thus, the overexpression of p65 increased both the NF-κB activity and the viability of TNF-treated cells containing deregulated c-Myc. In contrast to p65, the overexpression of p50 did not significantly increase the NF-κB activity or the survival of treated cells (Figure 4A and B). The overexpression of IκBα caused a weak decrease of NF-κB activity and of cell viability (Figure 4A and B). Figure 4.Effect of NF-κB subunits on the viability and NF-κB activity of TNF-treated mouse embryonic fibroblasts with deregulated c-Myc. (A) Cells were co-transfected with 1 μg of CMV-βGal and 10 μg of one of the pGD expression vectors indicated in the figure. Aliquots of the transfected cell cultures were seeded on duplicate wells; one well was treated with ethanol carrier (untreated) and the other with OHT and 50 ng/ml TNF. After 24 or 48 h incubation with the drugs, cells were fixed and stained with X-gal. Each bar in the figure represents the ratio of transfected cells in the well treated with OHT and TNF to transfected cells in the untreated well. The results represent mean ± SD values of three separate transfection experiments. (B) Cells were co-transfected with 0.5 μg of CMV-βGal, 0.5 μg of pBIIX-Luc and 5 μg of one of the pGD expression vectors. Transfected cells were treated with OHT and 50 ng/ml TNF and, after 18 h, lysed and analyzed as in Figure 3. The results represent mean values of two separate transfection experiments. Download figure Download PowerPoint c-Myc does not inhibit the TNF-induced accumulation of p53 Cellular p53 protein accumulates when cells are subjected to genotoxic stress, but less is known about the effects of death-inducing cytokines on p53 expression. We observed that TNF stimulation of Rat1-MycER cells induced a marked increase in cellular p53 protein levels (Figure 5A, upper panel). In Rat1 cells with deregulated c-Myc, the p53 level gradually increased after 10 h incubation of cells with TNF (Figure 5A, lower panel). Furthermore, immunoprecipitation analyses using p53 conformation-specific antibodies showed that Rat1 cells expressed wild-type p53 (data not shown). In MEFs, TNF induced p53 protein only if c-Myc was activated prior to TNF-stimulation (Figure 5B). While TNF stimulation induced both p53 protein and mRNA, only p53 protein was induced by UV-C treatment (Figure 5A and C). It has been noted previously that genotoxic stress induces p53 by post-translational mechanisms (Maltzman and Czyzyk, 1984; Kastan et al., 1991). The present data indicate that p53 induction by TNF involved changes also at the steady-state mRNA level. Notably, in contrast to the other tested TNF responses, the TNF-induced p53 accumulation in fibroblasts was not inhibited by deregulated c-Myc. Figure 5.Analysis of p53 protein and mRNA levels in TNF-stimulated cells. (A) and (B) Induction of p53 protein by TNF or UV-C. Rat1–MycER cells were incubated for 0–12 h (12 h in the upper panel of A) and MEF-MycERtm cells for 24 h (B) in the presence or absence of 50 ng/ml TNF. OHT or an equivalent volume of ethanol carrier was added to cells 6 h prior to TNF. The last lane in the upper panel of (A) represents cells incubated for 6 h after exposure to 50 J/m2 UV-C light (Stratalinker, Stratagen). The cells were lysed, and equivalent amounts of total protein (60 μg) were analyzed by Western immunoblotting using anti-p53 Pab421 antibody. (C) Induction of p53 mRNA by TNF or UV-C. Northern blotting and hybridization analysis of p53 and GAPDH mRNAs in control Rat1 cells, cells treated for 12 h with 50 ng/ml TNF or incubated for 6 h after exposure to 50 J/m2 UV-C. The relative level of p53 induction in comparison with untreated Rat1 cells (1.0) was quantitated by phosphoimager (Fuji) analysis and normalized to gapdh signals. Download figure Download PowerPoint TNF, with c-Myc, induces apoptosis in mouse primary fibroblasts but only weakly in p53–deficient primary fibroblasts The main cellular responses to p53 accumulation are cell cycle inhibition and apoptosis, the latter occurring predominantly in cells expressing growth-deregulating oncoproteins. This prompted us to assess the role of TNF-induced p53 in the cell death. The conditionally active form of c-Myc was introduced into embryonic fibroblasts derived from normal mice (MEFs p53+/+) or from mice having a targeted disruption of the p53 gene (MEFs p53−/−). Since p53-deficient cells acquire genetic abnormalities during continuous culture (Harvey et al., 1993), MycERtm was introduced into MEFs by high-titer retroviruses. MEFs of passage 1–4 were infected and then selected in puromycin. An ∼90% efficiency of infection was obtained, which allowed us to harvest the MycERtm-expressing MEFs for assays within a week of the infection. An equivalent expression level of MycERtm protein in the retrovirally infected MEF p53+/+ and MEF p53−/− cells was confirmed by Western blot analysis (data not shown). For assays of TNF sensitivity, infected and uninfected MEF p53+/+ and MEF p53−/− cells of the same passage number were seeded on coverslips. c-Myc was activated with OHT, and 50 ng/ml of TNF was added to the cultures as indicated in Table III. Control MEFs were treated with TNF in parallel assays. At defined time points, cells on coverslips were scored for apoptotic nuclei. The results from these experiments are summarized in Table III. Both MEF p53+/+ and MEF p53−/− cells were resistant to the cytotoxic activity of TNF. However, if c-Myc was activated in the MEF p53+/+ cells, the rate of apoptosis increased substantially in the TNF-treated cultures. In contrast, when c-Myc was activated in the MEF p53−/− cells, the rate of apoptosis in the presence of TNF remained low. After a 3 day incubation with TNF, only ∼4% of MEF p53−/− cells with active c-Myc were undergoing apoptosis, in contrast to 12% of MEF p53+/+ cells with active c-Myc. We conclude that TNF-induced p53 is involved in the TNF-induced apoptosis. Table 3. c-Myc-induced TNF sensitivity in normal and p53-deficient mouse embryonic fibroblasts Day MEF p53+/+ MEF p53−/− −Myc +Myc −Myc +Myc −TNF +TNF −TNF +TNF −TNF +TNF −TNF +TNF 1 1.4 0.7 1.8 3.0 0.4 0.5 0.6 1.9 2 0.4 1.2 3.9 8.8 0.0 0.5 0.3 2.5 3 1.2 1.3 3.2 11.8 0.1 0.6 0.2 3.6 Cells were stained with propidium iodide and the condensed and fragmented nuclei were scored by immunofluorescence microscopic detection. The values represent means of two separate retrovirus infection experiments. In contrast to MEF p53+/+ cells, which enter a non-growing senescent phase after prolonged culture, the MEFp53−/− cells exhibit a high spontaneous immortalization rate (Harvey et al., 1993). This allowed us to establish from the MycERtm-expressing MEFp53−/− cells clonal cell lines whose TNF sensitivity was then assessed by the MTT assay. The viability of the five clones examined did not decrease when c-Myc was activated in the presence of TNF (data not shown). This was not due to a lack of functional TNF receptors, since the TNF treatment combined with sublethal amounts of the protein synthesis inhibitor cycloheximide caused a rapid cytotoxic response in these cells. The expression of the carboxy-terminal domain of p53 inhibits X-irradiation-induced apoptosis but not the c-Myc-mediated cell death About half of the tumor cells express transforming mutant p53 (Hollstein et al., 1996), which is devoid of its wild-type function as a transcription-activating sequence-specific DNA-