p53-independent apoptosis in myoblasts - Semantic Scholar

2397

Journal of Cell Science 112, 2397-2407 (1999) Printed in Great Britain © The Company of Biologists Limited 1999 JCS0411

p53-independent apoptosis induced by muscle differentiation stimuli in polyomavirus large T-expressing myoblasts Vanesa Gottifredi*, Angelo Peschiaroli, Gian Maria Fimia‡ and Rossella Maione§ Isituto Pasteur-Fondazione Cenci Bolognetti, Dipartimento di Biotecnologie Cellulari ed Ematologia, Sezione di Genetica Molecolare, Università di Roma La Sapienza, Viale Regina Elena 324, 00161 Roma, Italy *Present address: Department of Biological Sciences, 818 Fairchild Center, Columbia University, MC2424 1212 Amsterdam Avenue New York, NY 10027 USA ‡Present address: Institut de Genetique et de Biologie Moleculaire et Cellulaire, BP 163, 67404 Illkirch Cedex, C.U. de Strasbourg, France §Author for correspondence (e-mail: [email protected])

Accepted 12 May; published on WWW 24 June 1999

SUMMARY Abnormal proliferation signals, driven by cellular or viral oncogenes, can result in the induction of apoptosis under sub-optimal cell growth conditions. The tumor suppressor p53 plays a central role in mediating oncogene-induced apoptosis, therefore transformed cells lacking p53 are generally resistant to apoptosis-promoting treatments. In a previous work we have reported that the expression of polyomavirus large T antigen causes apoptosis in differentiating myoblasts and that this phenomenon is dependent on the onset of muscle differentiation in the absence of a correct cell cycle arrest. Here we report that polyomavirus large T increases the levels and activity of p53, but these alterations are not involved in the apoptotic

INTRODUCTION Increasing evidence has been accumulated, showing that the mechanisms underlying several processes of programmed cell death by apoptosis are linked to the regulation of proliferation/differentiation pathways (King and Cidlowski, 1995). Apoptosis has also been found to be associated with oncogenic activation. For example, the expression of viral oncoproteins, such as adenovirus E1A (Lowe and Ruley, 1993; Debbas and White, 1993), papillomavirus E7 (Pan and Griep, 1994) or polyomavirus large T (PyLT) (Fimia et al., 1998), and the deregulated expression of the proto-oncogene c-myc (Evan et al., 1992) or of the cell cycle-regulatory transcription factor E2F (Shan and Lee, 1994; Qin et al., 1994) in some conditions can induce apoptotic cell death instead of increased proliferation. The outcome of oncogene activity is generally influenced by several kinds of stress conditions, including lack of growth factors (Evan et al., 1992), cell density (Brezden and Rauth, 1996), treatment with DNA damaging drugs (Lowe et al., 1993), hypoxia (Graeber et al., 1996) or differentiation stimuli (Fimia et al., 1998), all conditions that lead normal cells to growth arrest. Since the apoptotic activity of these oncogenes require functional domains involved in growth deregulation (White et al., 1991;

mechanism. Apoptosis in polyomavirus large T-expressing myoblasts is not prevented by the expression of a p53 dominant-negative mutant nor it is increased by p53 overexpression. Moreover, forced differentiation induced through the over-expression of the muscle regulatory factor MyoD, leads to apoptosis without altering p53 function and, more significantly, even in a p53-null background. Our results indicate that apoptosis induced by the activation of muscle differentiation pathways in oncogene-expressing cells can occur in a p53-independent manner. Key words: p53, Polyomavirus large T, Apoptosis, Muscle differentiation, MyoD

Rudolph et al., 1996; Querido et al., 1997), it is commonly believed that apoptosis results from an unbalanced cell cycle progression occurring in the presence of a contrasting signal to growth arrest. However, the molecular mechanisms by which defects in cell proliferation result in the induction of cell death have not been elucidated and, to further complicate the picture, in some cases the induction of apoptosis and the occurrence of cell cycle progression have been dissociated (Packham et al., 1996; Rudolph et al., 1996; Phillips et al.,1997), thus suggesting that these two processes interact through a more complex relationship. So far, the only link well established between oncogenes and apoptosis is the tumor suppressor p53 (Choisy-Rossi and Yonish-Rouach, 1998). In addition to inducing growth arrest, p53 is believed to counteract tumorigenesis also by inducing apoptosis. The mechanisms of these effects are not yet fully understood and, although it has been well established that p53 can activate the expression of both inhibitors of proliferation and cell deathregulatory genes, several indications support the existence of both transactivation-dependent and independent pathways (Ko and Prives, 1996; Hansen and Oren, 1997). The levels of p53 are increased, as a result of protein stabilization, in response not only to stress conditions, such as DNA damage, starvation, hypoxia, or ribonucleotide depletion (Levine, 1997), but also

2398 V. Gottifredi and others to the activity of oncogenes such as adenovirus E1A (Lowe and Ruley 1993), papillomavirus E7 (Demers et al., 1994) SV40T (Farmer et al., 1992), myc (Hermecking and Eick, 1994; Wagner et al., 1994), ras (Serrano et al., 1997) and E2F1 (Hiebert et al., 1995). Although the mechanisms leading to p53 stabilization are multiple and not completely defined, several recent lines of evidence have involved the Ink4a tumor suppressor gene product ARF in the pathway by which both viral and cellular oncogenes induce p53 (Sherr, 1998, and references therein). Regardless of protein levels and transcriptional activity, p53 function is anyhow required in many cases of oncogene-induced apoptosis, as deduced by the survival effects of p53 dominant-negative mutants or by the inability of several oncogenes to induce apoptosis in p53-null cells (Debbas and White, 1993; Lowe et al., 1994; Hermecking and Eick, 1994). Once activated, p53 can induce growth arrest, differentiation, senescence or apoptosis, depending on a variety of factors, including the cell type, the accumulation level and the genetic background (Ko and Prives, 1996; Hansen and Oren, 1997; Levine, 1997; Almog and Rotter, 1997). One of the conditions, that in several circumstances influences the decision between cell cycle arrest or cell death is the functionality of the pathway involving the retinoblastoma (RB) tumor suppressor (White, 1994). In fact, RB inactivation by gene mutation or by oncoprotein binding results in an increased sensitivity to the induction of apoptosis both in vivo and in vitro (Howes et al., 1994; Morgenbesser et al., 1994; Lee et al., 1994; Almasan et al., 1995; Haas-Kogan et al., 1995; Tsai et al., 1998). Conversely, RB over-expression, as well as the activity of cyclin-dependent-kinase (cdk) inhibitors, which is expected to support RB function, prevent the induction of apoptosis in several circumstances (Haupt et al., 1995; Haas Kogan et al., 1995; Wang and Walsh, 1996; Park et al., 1996). E2F overexpression, a functional equivalent of RB inactivation, also causes cell death and, in most cases, it has been found that apoptosis involving defects in the RB pathway are p53dependent (Howes et al., 1994; Pan and Griep, 1994; Morgenbesser et al., 1994; Wu and Levine, 1994; Haupt et al., 1995). We have recently found that C2 myoblast cells, in which RB function is inactivated by the expression of PyLT, undergo apoptosis when subjected to muscle differentiation stimuli and, in particular, following the over-expression of the muscleregulatory factor MyoD (Fimia et al., 1998). From this study we have found that myoblast apoptosis is related to the simultaneous activation of proliferation and differentiation pathways, both processes appearing not correctly accomplished. The importance of functional RB for myoblast cell survival is demonstrated also in other reports (Wang and Walsh, 1996; Zacksenhaus et al., 1996; Wang et al., 1997), but the possible role of p53 in muscle cell apoptosis has not yet been investigated. Recent studies indicate that p53 transcriptional activity is modulated during differentiation of in vitro myoblast cell lines (Soddu et al., 1996; Tamir and Bengal, 1998). Although p53-null mice show a normal muscle development (Donehower et al., 1992) and fibroblast cells from these mice can be converted in vitro to the myogenic lineage (Halevy et al.,1995), nevertheless the interference with endogenous p53 function impairs the ability of C2 myoblasts to undergo myogenesis (Soddu et al., 1996). This observation

suggests that p53 can play a role, although still controversial, in myoblast differentiation. Therefore, on the basis of the finding that p53 function changes during muscle differentiation and of the well stated connection between RB- and p53dependent pathways, we investigated the role of p53 in the apoptosis induced by the activation of muscle differentiation pathways in PyLT-expressing cells. In this work we report that PyLT increases the levels of functional p53 in myoblast cells, but this alteration is not involved in the mechanism leading to apoptosis. Moreover, we show that the attempt to induce myogenesis in PyLT-expressing fibroblasts, through the ectopic expression of the muscle regulatory factor MyoD, results in cell death even in a p53-null background.

MATERIALS AND METHODS Cell cultures Mouse myoblast C2 cells, clone 7 (Yaffe and Saxel, 1977), the myoblast clones LT.N2 (Maione et al., 1994) and LT.N2DD, p53−/− mouse embryo fibroblasts, kindly provided by Dr T. Jacks (MIT, Cambridge, USA) and the PyLT-derived subclone LTp53−/− were maintained in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10%-20% foetal calf serum (FCS) (and under constant selection of 400 µg/ml geneticin in the case of LT.N2, 2.5 µg/ml puromycin plus geneticin in the case of LT.N2DD and 200 µg/ml hygromycin B in the case of LTp53−/− (all antibiotics from Sigma Chemicals) in a humidified 5% carbon dioxide atmosphere. To induce differentiation cells were grown to confluence and then shifted to DMEM supplemented with 0.5% FCS. Cell viability was assessed by microscopic observation and Trypan blue exclusion. Colony forming efficiency was determined by Giemsa staining. Staining of apoptotic nuclei Apoptotic nuclei were visualized following the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method developed by Gavrieli et al. (1992), with some minor modifications. Briefly, cells were fixed with 4% paraformaldehyde in PBS for 20 minutes, washed with Tris buffered saline (1× TBS: 20 mM Tris-HCl, pH 7.6, 140 mM NaCl) and incubated 5 minutes in 20 µg/ml proteinase K to strip nuclei from proteins. After three washes with TBS, samples were preincubated in TdT (terminal deoxynucleotidyl transferase) reaction buffer (30 mM Tris base, 140 mM sodium cacodylate pH 7.2, 1 mM cobalt chloride) for 15 minutes and then with the labelling mixture containing 40 µM biotin-16-dUTP and 0.3 U/µl TdT (both from Boehringer Mannheim) for 1 hour at 37°C in humid atmosphere. After washing, the reaction was terminated by incubating 5 minutes in 0.5 M EDTA, pH 8. To stain labeled nuclei samples were blocked with 4% BSA in PBS and then incubated in 20 µg/ml streptavidin-FITC for 30 minutes at room temperature. After three washes, a final stain with 1 µg/ml of the DNA binding fluorochrome DAPI (Boehringer Mannheim) was carried out to visualize total nuclei. Indirect immunofluorescence staining Cells grown on glass coverslips were fixed by immersion in methanol/acetone (3:7, v/v) for 15 minutes at −20°C and then air dried. Coverslips were then incubated for one hour at room temperature with a 1:10 dilution of either the broadly reactive pAb 421 or the conformation-specific pAb1620 (both from Oncogene Research), as primary antibodies against p53. After three washes with PBS, the coverslips were incubated with the secondary rhodamineconjugated antibody to mouse IgG (Cappel Immunochemicals) diluted 1:100 in PBS plus 3% bovine serum albumin, washed repeatedly with PBS, and mounted with 70% glycerol in PBS. The

p53-independent apoptosis in myoblasts 2399 samples were analysed under phase contrast and appropriate fluorescent light. Cell transfections and chloramphenicol acetyl transferase (CAT) assay Cells were transfected by the calcium phosphate precipitation method, with approximately 1×106 cells/100 mm diameter dish or 3×105 cells/60 mm diameter dish. LT.N2 subclones stably expressing the p53 dominant negative mutant were selected by cotransfecting each 100 mm dish with the following amounts of DNA: 2 µg of the pCMVDD plasmid, coding for the dimerization domain of p53 (p53DD), able to dominantly inactivate wild-type p53 through the formation of non-functional oligomers (Shaulian et al., 1992), kindly provided by Dr M. Oren (The Weizmann Institute of Science, Rehovot, Israel); 0.2 µg of pBABEpuro, containing the puromycin resistance gene under the control of the Moloney murine leukemia virus LTR (Morgenstern and Land, 1990), used as a selectable marker, made available by Dr B. Amati (Swiss Institute for Cancer Research, Epalinges, Switzerland); 8 µg of mouse genomic DNA as the carrier. Thirty-six hours after transfection, cells were diluted 1:7 into selective medium containing 2.5 µg/ml of puromycin (Sigma). The medium was changed every 23 days and, after 15 days, the surviving colonies were isolated and separately amplified. p53−/− fibroblasts stably expressing PyLT were obtained following the same protocol after co-transfection of the plasmids pLBE, coding for wild-type PyLT (Maione et al., 1994), and pSV2hygro, containing the hygromycin B resistance under the control of the SV40 promoter-enhancer region. Cell clones were selected in 200 µg/ml hygromycin. CAT assays were performed in duplicate by cotransfecting cells (60 mm dishes) with different combinations of the following plasmids: pG13CAT, containing multiple copies of a p53 DNA binding sequence (Kern et al., 1992), and made available by Dr L. Banks (International Centre for Genetics and Biotechnology, Trieste, Italy); pLBE coding for PyLT and previously described (Maione et al., 1994); pCMVDD coding for a p53 dominant negative mutant, as already mentioned above; pCMVβ-gal coding for β-galactosidase and used to monitor transfection efficiency. The DNA precipitates contained in all cases 1 µg of pG13CAT and 1 µg of pCMVβ-gal and, where indicated, 5 µg of pLBE, 5 µg of pCMVDD or 5 µg of each plasmid. The DNA concentrations were adjusted to 12 µg/precipitate, by adding, when necessary, pBR322 plasmid as a carrier. Forty-eight hours after transfection cells were disrupted by freezing and thawing. Cell extracts, normalized for the transfection efficiency, were assayed for CAT activity by the method of Gorman et al. (1982). Conversion percentage was determined using a Packard Instant Imager. Production of recombinant retroviruses and retroviral infections The pBabe retroviral vector coding for wild-type MyoD has been previously described (Fimia et al.,1998). The pBabe reroviral vector pES29, coding for wild-type human p53 was kindly provided by Dr R. Iggo (ISREC, Epalinges, Switzerland). BOSC 23 ecotropic packaging cells (Pear et al., 1993) were maintained in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS in a humidified 5% carbon dioxide atmosphere. To obtain recombinant retroviruses BOSC 23 cells were transfected as described (Pear et al., 1993). Briefly, 6×106 cells were seeded onto 100 mm tissue culture dishes in DMEM supplemented with 10% FCS and grown for 24 hours. Just before transfection 25 µM chloroquine was added to the culture medium and 20 µg of plasmid/100 mm dish were transfected with the calcium phosphate precipitation method. After 10 hours the medium was changed and cells were incubated for additional 16 hours in DMEM-10% FCS. Medium was again replaced with a smaller volume. The retroviral supernatant was harvested 24 hours later and, after removal of cell debris, frozen at −80°C for later use.

For retroviral infection, 3×105 cells were plated onto 60 mm dishes 24 hours before infection. Cells were then incubated with 1 ml of BOSC 23 retroviral supernatant supplemented with 4 µg/ml polybrene for 10 hours and then re-fed with fresh medium. Forty eight hours later cells were collected for analysis or further incubated in DMEM supplemented with 0.5% FCS or diluted into selective medium. Immunoprecipitation and western blots Cells were rinsed twice with phosphate-buffered saline and lysed on ice in 50 mM Tris-HCl, pH 8.0, 150 mM NaCl, 1 mM EGTA, pH 8.0, 100 mM NaF, pH 8.0, 10% glycerol, 1 mM MgCl2, 1% (v/v) Triton X-100 containing freshly added protease and phosphatase inhibitors (1 mM phenylmethylsulfonyl fluoride, 10 µg/ml leupeptin, 5 µg/ml aprotinin, 1 mM sodium orthovanadate). Lysates were subjected to ultrasonic treatment for 15 seconds and then clarified by centrifugation at 4°C. Protein concentrations were determined by BioRad protein assay reagent (Bio-Rad Laboratories, Hercules, CA). To perform p53 immunoprecipitation, 1 µg/sample of the monoclonal antibody pAb421 (Oncogene Research Products) was incubated with pre-cleaned cell lysates for 90 minutes at 4°C. Immunocomplexes were washed 3 times with cold NET-gel buffer (50 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, pH 8.0, 0.1% (v/v) Nonidet P-40, 0.25% gelatin, 1 mM sodium orthovanadate), eluted and denaturated in Laemmli buffer at room temperature to avoid IgG co-migration with the protein of interest. Proteins from either immunoprecipitations or cell lysates were resolved in 12% SDS-PAGE and transferred to nitrocellulose filters (Bio-Rad Laboratories, Hercules, CA). Blots were stopped and probed with specific antibodies after blocking nonspecific reactivity. Primary antibodies were diluted in TBS-T (20 mM Tris-HCl, pH 7.8, 150 mM NaCl, 0.05% Tween-20 containing 0.2% gelatin). After extensive washing, immunocomplexes were detected with horseradish peroxidase-conjugated species-specific secondary antiserum (BioRad Laboratories, Hercules, CA) followed by enhanced chemiluminiscence reaction (Amersham International plc, UK). The following primary antibodies and dilutions were used: (a) for p53 a 1:10 dilution of the pAb421 monoclonal antibody (Oncogene Research Products); (b) for PyLT, a 1:500 dilution of the 440 rabbit polyclonal serum, kindly provided by Dr B. Schaffhausen (Tufts University School of Medicine, Boston); (c) for MyoD, a 1:500 dilution of the rabbit polyclonal anti-MyoD antibody M-318 (sc-760 Santa Cruz); (d) for myogenin, the monoclonal anti-myogenin antibody IF5D (Wright et al., 1991) as undiluted hybridomasupernatant; (e) for bax, a 1:1000 dilution of the rabbit polyclonal antibody P-19 (sc-526 Santa Cruz).

RESULTS Py LT increases p53 levels and activity in myoblast cells Py LT-expressing myoblasts undergo apoptotic cell death after the shift to low serum-containing differentiation medium, a condition that otherwise supports differentiation in the parental C2 cells (Fimia et al., 1998). Deregulation of the cell cycle by several oncogenes can lead to p53-dependent apoptosis, in many cases accompanied by the stabilization of the protein. Therefore, to explore the possible involvement of p53 in this cell death, we first analysed the protein levels in PyLTexpressing myoblasts, with respect to the onset of apoptosis. Fig. 1A shows the morphological appearance and TUNEL staining of LT.N2 cells, a representative clone expressing PyLT (Maione et al., 1994), compared to the parental C2 cells, at different times after serum removal. When LT.N2 cells are shifted to differentiation medium, in the space of a few hours

2400 V. Gottifredi and others

A

Fig. 1. Expression of p53 in PyLTexpressing myoblasts during apoptosis. (A) Fluorescence micrographs of TUNEL-stained C2-7 and LT.N2 cells at different times after the shift to differentiation medium. The same fields were also photographed under phase contrast illumination to reveal morphological differentiation in C2.7 cells and cell death in LT.N2 cells. The numbers 0, 12 and 48 indicate the hours after the shift. The percentages ± s.e.m. of TUNEL-positive cells, calculated with respect to DAPIstained total nuclei were: C2.7 (0): 0.5±0.1; C2.7 (12): 2.8±0.4; C2.7 (48): 0.8±0.5; LT.N2 (0): 1.1±0.8; LT.N2 (12): 12.3±1.5; LT.N2 (48): 26.3±2.9. (B) Analysis of p53 levels in C2.7 and LT.N2 cells grown in the same conditions as in A. p53 protein was immunoprecipitated as described in Materials and Methods and then analysed by SDS-PAGE followed by western blot. SV3T3 (NIH 3T3 stably transfected with SV40 large T), expressing high levels of p53, were used as a positive control.

B

p53-independent apoptosis in myoblasts 2401

Fig. 2. Expression of wild-type p53 in C2.7 and LT.N2 cells. Fluorescence micrographs of C2.7 and LT.N2 cells, grown in 10% FCS-containing medium after immunostaining with either pAb421 or pAb1620, as indicated.

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CAT ACTIVITY

a phenomenon of apoptosis takes place, which parallels the onset of myotube formation in the C2 cultures. Some TUNELpositive, apoptotic nuclei are also observed in C2 cells, in some experiments, in agreement with a previous report (Wang and Walsh, 1996), but this phenomenon can be minimized by using low-passage, well differentiating cultures. To determine the protein levels of p53 in the same conditions, cell extracts from parallel cultures were analyzed by immunoprecipitation with the broadly reactive antibody pAb421. As shown in Fig. 1B, p53 levels are somewhat increased in LT.N2 with respect to C2 cells, but do not change with respect to survival or apoptosis conditions. A transient increase of the protein levels is rather observed in C2 cells at early times of differentiation, in agreement with previous reports showing that p53 expression and activity are up-regulated during C2 cell differentiation (Halevy et al., 1995; Soddu et al., 1996). To exclude the possibility that the increased stability of the protein in the PyLT-expressing clone was associated with an altered conformation, we also tested the reactivity of the conformationspecific pAb1620 antibody. Immunofluorescence staining with this antibody (Fig. 2), which does not recognise most mutant or denatured p53, suggests that LT.N2 express the wild-type form of p53, as previously reported for the parental C2 cells. Transient transfection of PyLT into C2 cells also leads to increased levels of p53 as we determined by immunofluorescence staining (data not shown), thus confirming that PyLT expression is responsible for the accumulation observed in LT.N2 cells. To test whether PyLT could modify in some way the p53-mediated transcriptional activity, we analysed the effects of the viral oncoprotein on the activity of a p53-responsive promoter. C2 cells were transiently co-transfected with a PyLT-expressing plasmid and the p53responsive construct pG13CAT, containing 13 repeats of the p53 binding site (Kern et al., 1992). Fig. 3 shows that PyLT up-regulates the reporter’s activity and that this increase is

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PG13CAT LARGE T p53 DD

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Fig. 3. Effect of PyLT on p53-mediated transcriptional activity. CAT assay was performed on C2 cell extracts after transient transfection with the p53 responsive construct (G13CAT) and different combinations of plasmids encoding PyLT (Large T) and the p53 dominant negative mutant (DD); symbol + indicates the presence of each plasmid. CAT activity was calculated as the percentage of conversion of chloramphenicol to the acetylated forms. Results represent the average values ± s.e.m. from three separate experiments performed in duplicate.

dependent on p53 since it is completely reverted by the cotransfection of the pCMVDD plasmid, coding for the p53dominant negative mutant p53DD (Shaulian et al., 1992). All these data indicate that PyLT increases both the levels and the activity of p53 in C2 myoblasts. This finding is in contrast with the ability of other viral oncoproteins, in particular SV40 large T, adenovirus E1B and also adenovirus E1A, to increase p53 levels but to inhibit its transactivation function (Blagosklonny, 1997). Apoptosis in PyLT-expressing myoblasts is not affected by the down- or up-regulation of p53 function To determine the possible role of the observed p53 alterations in this apoptosis, we analysed the effects of either inhibiting or further increasing the activity of p53 in LT.N2 myoblasts. In order to interfere with the activity of the endogenous protein we used the dominant negative mutant p53DD, which hinders the PyLT-dependent up-regulation of p53 activity (see Fig. 3) and which has been demonstrated to act as an effective antagonist of wild-type p53 in several biological functions, including the induction of apoptosis (Gottlieb et al., 1994; Bowman et al., 1996). LT.N2 subclones expressing p53DD were obtained following stable co-transfection of CMVDD plasmid and a selectable marker into LT.N2 cells (see Materials and Methods). Fig. 4A shows the abundant expression of p53DD in two representative subclones as determined by western blot with pAb421 antibody. The increased levels of the endogenous wild-type p53 in these sub-clones can be ascribed to an increased stabilization of the protein due to its binding in non-functional oligomers with DD miniprotein, as suggested in other cases (Shaulian et al., 1992; Gottlieb et al., 1994). The same figure also shows the levels of exogenously overexpressed p53, in LT.N2 cells transiently infected with a pBabe retroviral vector coding for the wild-type protein. As shown in

2402 V. Gottifredi and others does not affect at all LT.N2 cell proliferation (data not shown), in agreement with another report showing that PyLT interferes with the growth arrest activity of p53 in fibroblasts cells (Doherty and Freund, 1997).

Fig. 4. The over-expression of either a dominant negative mutant or wild-type p53 does not affect the viability of LT.N2 cells. (A) Western blot analysis showing the levels of both mutant (p53DD) and wild-type (p53) forms of p53 in LT.N2DD subclones (LT.N2DD11 and LT.N2DD15) and in pBabep53 retrovirus-infected LT.N2 cells (LT.N2p53), compared to LT.N2 and pBabe puro retrovirus-infected LT.N2 (LT.N2Babe). Cell extracts were prepared at the time called 0 in all the experiments, 48 hours after infection in the case of LT.N2p53 and LT.N2Babe. (B) Kinetics of cell death for LT.N2, LT.N2DD11 and LT.N2p53, after the shift to differentiation medium (DM). The percentage of non-viable cells was determined by Trypan blue exclusion, at different times indicated by the number of hours. Each point represents the means ± s.e.m. of three separate experiments.

Fig. 4B, however, neither the expression of p53DD, nor the over-expression of the wild-type protein affected at all LT.N2 cell viability. In fact, LT.N2DD clones did not show an increased survival nor a delayed kinetics of cell death with respect to LT.N2 control cells. Moreover, although p53 overexpression in oncogene-expressing cells, even more after serum deprivation, is expected to induce apoptosis, we were unable to detect any increased cell death in this system upon p53 retroviral infection. As expected, while p53 overexpression causes a slightly reduced growth rate in C2 cells, it

The over-expression of myogenic factors does not increase p53 function Apoptosis in LT.N2 myoblasts is dependent on the activation of early differentiation steps by myogenic factors and takes place to a greater extent and even in the presence of high serum, upon forced activation of differentiation pathways by overexpressed myogenic factors (Fimia et al., 1998). In light of previous reports and our own observations that p53 levels and activity are transiently increased at the onset of differentiation in C2 cells, we asked whether any change in p53 function could be revealed during apoptosis induced by forced differentiation. C2 and LT.N2 cells were either mock-infected or infected with a high titer retroviral vector coding for the muscle regulatory factor MyoD. The over-expression of this factor accelerates the onset of myogenesis in both C2 and LT.N2 cells as determined by the expression of the early differentiation marker myogenin, even in the presence of high-serum medium. This treatment, however, while in C2 cells induces a faster accomplishment of terminal differentiation, in LT.N2 cells causes an increased kinetics of cell death (Fimia et al., 1998). As shown in Fig. 5A, a consistent phenomenon of apoptosis can be visualised by TUNEL staining of LT.N2 but not C2 cells, at 48 hours after MyoD retrovirus infection. The analysis of p53 levels, however, as determined by western blot (Fig. 5B) shows that also in this case, like after serum removal, the amount of the protein does not increase in parallel with cell death; rather, it appears to be reduced in concomitance with the up-regulation of myogenin in LT.N2 cells. We did not observe any increase in p53 levels even in MyoD infected C2 cells, thus suggesting that the transient up-regulation observed when C2 cells are shifted to differentiation medium, does not depend in a simple manner on the activation of myogenic factors or, if it does, it could be related to a narrow temporal window of this process. Since the possibility cannot be excluded that p53 function can be up-regulated with no increase in the protein level, we also analysed the expression of the transcriptional target bax gene that, in addition to being indicative of p53 function, also encodes a pro-apoptotic protein involved in the mechanism by which p53 induces transcription-dependent apoptosis (Miyashita and Reed, 1995). This analysis shows that bax levels do not increase at all during apoptosis induced by forced differentiation, rather, to some extent, it is down-regulated. The over-expression of myogenic factors induces apoptosis in p53-null fibroblasts In order to confirm the lack of involvement of p53 in apoptosis induced by myogenic factors in the absence of functional RB, we reconstructed a similar system in a p53-null background. For this purpose we used fibroblast cells from mice knock-out for p53, taking advantage of the ability of most fibroblast cell lines to undergo the myogenic conversion and the consequent activation of muscle-specific pathways, in response to the ectopic expression of myogenic factors (Weintraub et al., 1989). p53−/− fibroblast clones were selected after stable transfection of PyLT and a representative one, expressing high levels of the viral oncoprotein, was analysed for the response

p53-independent apoptosis in myoblasts 2403

A

A Fig. 5. Analysis of p53 function during apoptosis induced by MyoD overexpression. C2.7 and LT.N2 cells were either infected with pBabeMyoD (C2.7 MyoD and LT.N2 MyoD) or mock infected with the empty retrovirus (C2.7 and LT.N2), kept in serum-containing medium and analysed 48 hours post-infection. (A) Fluorescence micrographs after TUNEL or DAPI staining. The percentages ± s.e.m. of TUNEL-positive cells, calculated with respect to DAPI-stained nuclei, were: C2.7: 0.2±0.1; C2.7 MyoD: 0.5±0.1; LT.N2: 1.3±0.8; LT.N2 MyoD: 11.3±1.4. (B) Western blot with antibodies specific for the indicated proteins.

2404 V. Gottifredi and others

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Fig. 6. Effects of MyoD expression in LTp53−/− fibroblasts. p53−/− and LT p53−/− fibroblasts were infected with pBabe puro or pBabe MyoD retrovirus and, after 48 hours, either shifted to differentiation medium or diluted into puromycin-containing selective medium. The phase contrast micrographs show the morphological appearance of the cell cultures 24 hours after the shift to differentiation medium (A) or one week after the incubation in selective medium (B).

to exogenous MyoD. Transient infection of the p53−/− parental cell line and of the PyLT-derived clone (LTp53−/−) with pBabe MyoD leads to the same phenomena observed in C2 and LT.N2 myoblasts, that is: full activation of the myogenic program in the former in contrast to abnormal differentiation and induction of cell death in the latter (Fig. 6A). As a further control, we also infected NIH3T3 fibroblasts (p53+/+) and a PyLTexpressing subclone. Also in this case, we observed MyoDinduced apoptosis in the presence of PyLT, without significant differences related to the presence or absence of p53. Rather, apoptosis in LTp53−/− cells is still more pronounced than that occurring in both LT.N2 and LT.NIH3T3 cells, probably as a consequence of the high tendency of these cells to undergo cell death upon exposure to a variety of sub-optimal growth conditions, even in the absence of MyoD infection (data not shown). The deleterious effect of MyoD on the survival of p53−/− fibroblasts expressing PyLT was still much more evident when we attempted to stably express the muscle regulatory factor in these cells (Fig. 6B; Table 1). Both p53−/− and LTp53−/− cells were infected either with the empty vector pBabePuro or with pBabeMyoD and, 48 hours later, diluted into selective medium. As shown in Table 1, in the space of about one week, many colonies grew up from each infection of p53−/− cultures, although MyoD reduced to some extent the

colony forming efficiency, likely due to its ability to induce terminal differentiation and growth arrest in a fraction of infected cells, even in high serum. A more dramatic effect was observed after MyoD infection of the PyLT-derived clone. In this case, in fact, not only the colony forming efficiency was much more strongly reduced (Table 1), but also, microscopic inspection of the colonies showed that most of them contained dying cells (Fig. 6B). A few clones co-expressing low levels of both MyoD and PyLT could be isolated at later times (approximately three weeks), but they grew very slowly, likely as result of a balance between cell proliferation and cell death. Table 1. MyoD expression strongly reduces the colony forming efficiency of LT p53−/− fibroblasts pBabe puro PBabe MyoD

p53−/−

LT p53−/−

71±7 37±6

62±10 6±3

p53−/− and LT p53−/− fibroblasts were infected with either pBabe puro or pBabe MyoD retrovirus and, 48 hours later, diluted into DMEM supplemented with 20% FCS and 2.5 µg/ml puromycin at the density of 200 cells/60 mm dish. The average number of colonies/dish was determined 1 week later, from 8 dishes obtained in two experiments performed with independent retroviral supernatants.

p53-independent apoptosis in myoblasts 2405 These cells, after confluence, expressed early differentiation markers and died by apoptosis, just like LT.N2 cells (data not shown). These results demonstrate that apoptosis induced by the activation of the myogenic program is independent of p53. DISCUSSION The mechanisms regulating apoptosis in oncogene-expressing cells are of considerable interest in light of the well recognised role of this form of cell death in limiting the expansion of tumor cells. A general picture can be drawn in which suboptimal growth conditions selectively induce apoptosis in cells forced to proliferate by oncogenes, in many cases through p53-dependent pathways. However, the outcome of oncogene activity is not so obvious, due to the existence of several distinct mechanisms acting in different cellular contexts. We have previously reported that C2 myoblast cells show an increased sensitivity to the induction of apoptosis upon expression of PyLT and that this phenomenon is dependent on the activation of the myogenic program in the absence of a correct RB function (Fimia et al., 1998). In this paper we report that, although the viral oncoprotein causes some changes in p53 function, apoptosis induced by forcing muscle differentiation in PyLT-expressing cells is p53-independent. There is little information about possible functional interactions between PyLT and p53, apart from the clear exclusion of a physical interaction between the two proteins (Manfredi and Prives, 1990). In line with previous observations that p53 accumulates in response to viral oncoproteins, we have found that PyLT-expressing myoblasts contain higher levels of p53 with respect to the parental cells. The same increase has been observed also in fibroblast cell lines stably transfected with the viral oncoprotein (data not shown). In both cases, however, the increased level of p53 is not so much significant as that observed upon expression of SV40 large T or adenovirus E1A (Deppert et al., 1987; Lowe and Ruley, 1993). The accumulation of p53 in response to viral oncoproteins has been recently explained by the findings that the over-expression of E2F1 or the lack of RB induce the expression of the tumor suppressor proteins ARF which, in turn, stabilize p53 by inhibiting MDM2-mediated degradation (Sherr, 1998, and references therein). This proposed mechanism does not exclude that an efficient p53 stabilization by oncogenic stimuli could be related to its loss of function and to the consequent impairment of negative feedback controls (Blagosklonny, 1997). In agreement with this view, both SV40 large T and AdE1A have been shown to inhibit the p53 transactivation function (Bargonetti et al., 1992; Jiang et al., 1993; Steegenga et al., 1996; Somasundaran et al., 1997), whereas PyLT leads to the up-regulation of a p53-responsive promoter. Therefore, a possible explanation is that the lower accumulation of p53 induced by PyLT with respect to other oncoproteins is related to the different effects on the activity of p53 itself. However, although PyLT increases transcriptional activation by p53 in transient cotransfection assays, there is no increase of the endogenous bax expression in the PyLT stablyexpressing clone. This discrepancy might reflect the different sensitivity of the two experimental approaches in revealing changes in p53 function. In any case, p53 does not appear to be inactivated by PyLT.

Several lines of evidence indicate that cell cycle, differentiation and apoptosis are coordinated in myoblast cells and that RB function plays a critical role in regulating the complex interplay between these processes (Maione and Amati, 1997). Myoblast cells, in which RB has been inactivated by gene mutation or by PyLT binding, show defects in differentiation and undergo apoptosis at high frequency in the absence of mitogens (Wang et al., 1997; Fimia al., 1998). Some apoptotic cell death is normally observed also in a fraction of C2 cells that have been shown to fail in the upregulation of the cdk inhibitor p21 (Wang and Walsh, 1996). Since p21 is thought to be important to support RB function during myoblast differentiation, it is likely that apoptosis in wild-type myoblasts occurs through the same mechanisms as in the PyLT-derivative, with the difference that RB inactivation by the viral oncoprotein strongly increases the frequency of the apoptotic phenomenon. We have recently suggested that the activation of the differentiation program, and not simply the removal of growth factors, is determinant in triggering cell death in myoblasts defective in RB function (Fimia et al., 1998). Several features of this system would have suggested an involvement of p53 in myoblast apoptosis. First, a functional connection between apoptosis, p53 and deregulation of the RB pathway is supported by numerous observations (as discussed in the Introduction). Second, the expression of PyLT in C2 cells leads to both an increased level of functional p53 and an increased sensitivity to the induction of apoptosis. Finally, p53 expression and activity have been shown to be activated at the onset of several differentiative processes, including myogenesis (Soddu et al., 1996; Tamir and Bengal, 1998). In contrast to our expectations, however, we obtained strong evidence indicating the lack of involvement of p53 in apoptosis induced by differentiation in PyLT-expressing myoblasts. Although the up-regulation of p53 occurs in an early phase of wild-type C2 myoblast differentiation, just at the same time as the apoptosis occurring in a fraction of these cells, we did not observe a correlation between the activation of myogenic factors and an increased p53 function in the PyLT-derivative. LT.N2 cells do not up-regulate p53 after the shift to differentiation medium. Rather, upon MyoD over-expression, both C2 and LT.N2 cells show a decreased p53 function, as determined by the levels of the well known transcriptional target bax. A more direct demonstration of the p53independence of this apoptosis comes from the observation that it is not affected by a p53 dominant negative mutant and, more significantly, it is very marked even in fibroblasts derived from p53-null mice which, in contrast, have been shown to be resistent to the induction of apoptosis by E2F or E1A (Kowalik et al., 1995; Lowe et al., 1994). Apoptosis in myoblast cells is characterized by the concomitant activation of differentiation and proliferation pathways (Fimia et al., 1998). Both processes, however, show evident abnormalities, probably due to the simultaneous activation of these two mutually exclusive mechanisms. PyLTexpressing myoblasts, as well as PyLT-expressing fibroblasts converted to the myogenic lineage, fail to down-regulate several cyclins and cdks, probably as a consequence of E2F activity, due to RB inactivation. Therefore, the induction of cell death in this system could involve the aberrant activity of some of these cell cycle regulatory factors, frequently invoked to

2406 V. Gottifredi and others play a role in apoptosis (Kasten and Giordano, 1998). On the other hand we have to consider that early events of myogenesis, in addition to muscle-specific gene activation, also include complex modifications of the expression pattern of several other factors such as those involved in cell-cell or cellextracellular matrix interactions (McDonald et al., 1995), which in turn, play critical roles in regulating cell proliferation, gene expression and cell survival (Sastry et al., 1996). More work is required to determine whether apoptosis is directly triggered by aberrant events of the differentiation program, due to the concomitant occurrence of cell cycle progression, or by some aberrant event of proliferation, due to the concomitant activation of differentiation pathways. Whatever is the triggering mechanism, we have found that differentiationinduced apoptosis acts through p53-independent pathways. The lack of involvement of p53 in an apoptotic mechanism involving RB defects or E2F excess would be in line with recent results showing that RB inactivation or E2F overexpression can induce apoptosis in the myocardium of p53−/− mice (Agah et al., 1997) and in p53-null human tumor cells (Hsieh et al., 1997; Phillips et al., 1997). Nevertheless, p53 and E2F simultaneously over-expressed in these cells cooperate in the induction of apoptosis, suggesting that the impairment of the RB/E2F pathway can utilize both p53-dependent and independent mechanisms. However, we did not find such a cooperation, since increased levels of p53 does not increase at all the cell death of PyLT-expressing myoblasts. This could be probably ascribed to differences between the cell systems and, in particular, to the involvement of muscle-specific functions in the determination of the apoptotic pathway. Whereas several mechanisms have been shown to mediate p53-dependent apoptosis (Hansen and Oren, 1997), little or nothing is yet known about the p53-independent pathways, an issue which deserves much attention in the field of tumor biology due to the generally low sensitivity of p53 lacking tumor cells to apoptosis-inducing treatments. Therefore, the study of alternative pathways activated by forced differentiation could be relevant to rescue an apoptotic response in oncogenetransformed cells lacking p53. We gratefully acknowledge Prof. Paolo Amati for continuous support, helpful discussions and critical reading of this manuscript. We also thank Luana Coltella for help in some experiments and Nazzareno Falcone for technical assistance. We are indebted to Drs L. Banks, R. Iggo, T. Jacks, M. Oren and B. Schaffhausen for the generous gift of cell lines, plasmids and antibodies. This work was supported by grants from the Associazione Italiana Ricerche sul Cancro (AIRC).

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