c-myc protooncogene positively regulates cell proliferation and overexpression of c-myc is found in many solid tumors and leukemias. In the present study we used the K562 human myeloid leukemia cell line as a model to study the functional interaction between c-Myc and p53. Using two different methods, we generated K562 transfectant cell lines with conditional expression of either c-Myc or p53. The cells expressed the p53Vall35 mutant, which adopts a wild-type conformation at 32°C, while c-Myc induction was achieved with a zinc-inducible expression vector. We found that p53 in wild-type conformation induces growth arrest and apoptosis of K562. Expression of c-Myc significantly attenuated apoptosis and impaired the transcriptional activity of p53 on p21WAF1, Bax and cytomegalovirus promoters. The impairment of p21WAF1 transactivation by c-Myc was confirmed by transfection of a c-Myc-estrogen receptor fusion protein and by induction of c-myc by zinc in transfected cells. Also, p53-mediated up-regulation of p21WAF1 mRNA protein were significantly reduced by c-Myc, while Bax levels were unaffected. Consistently, c-Myc increased cyclin-dependent kinase 2 activity in K562 cells expressing p53 in wild-type conformation. These results suggest that c-Myc overexpression may antagonize the pro-apoptotic function of p53, thus providing a molecular mechanism for the frequently observed deregulation of c-myc in human cancer.
p53 is a transcription factor which is activated upon DNA damage and promotes cell growth arrest and apoptosis (Gottlieb and Oren, 1996; Ko and Prives, 1996; Levine, 1997). Mutations and deletions of the p53 tumor suppressor gene is a common alteration in human cancer. In many neoplasias, and particularly those of hematopoietic origin, loss of normal p53 function occur during tumor progression, and tumors without wild-type p53 are more aggressive and have worse prognosis (Wallace-Brodeur and Lowe, 1999; Kirsch and Kastan, 1999). Under genotoxic stress, p53 binds to specific DNA sequences and activates transcription of different genes. Some p53 target genes are involved in apoptosis control, e.g. bax, fas, and IGF-BP3, while others mediate cell cycle arrest, e.g. cyclin G, gadd45, 14-3-3σ and the cyclin-dependent kinase inhibitor p21WAF1. However, the mechanisms by which p53 induces apoptosis are not completely understood (Hansen and Oren, 1997; Amundson et al., 1998; Bates and Vousden, 1999).
c-Myc is a transcription factor involved in the control of cell proliferation, differentiation and apoptosis. Mitogenic stimulation induces c-myc expression whereas growth arrest is usually accompanied by suppression of c-myc (Henriksson and Lüscher, 1996; Amati et al., 1998; Bouchard et al., 1998) c-Myc heterodimerizes with the protein Max and the dimer binds to regulatory regions of target genes. These include genes involved in cell cycle control and DNA and RNA metabolism (Grandori and Eisenman, 1997; Dang 1999). Consistent with its positive effect on cell growth, deregulated expression of c-myc is a common finding in human cancer (Garte, 1993; Henriksson and Lüscher, 1996). Paradoxically, c-Myc overexpression also mediates apoptosis in different cell types subjected to sub-optimal growth conditions, e.g. deprivation of growth factors or hypoxia (for recent reviews see Hoffman and Lieberman, 1998; Hueber and Evan, 1998; Thompson, 1998). c-Myc-mediated apoptosis requires wild-type p53 in fibroblasts (Hermeking and Eick, 1994; Wagner et al., 1994; Rupnow et al., 1998), although there are examples where the apoptosis induced by c-Myc is p53-independent (Selvakumaran et al., 1994; Hsu et al., 1995; Sakamuro et al., 1995; Trudel et al., 1997; Stähler and Roemer 1998; Hagiyama et al., 1999). Besides, there are other data revealing a functional interaction between c-Myc and p53: (i) wild-type p53 represses c-myc promoter (Moberg et al., 1992; Ragimov et al., 1993) and mutated p53 activates c-myc promoter (Frazier et al. 1998); (ii) c-Myc induces p53 expression (Hermeking and Eick, 1994; Reisman et al., 1993; Roy et al., 1994; Kirch et al., 1999) (iii) c-myc and loss of wild-type of p53 function cooperate in the development of leukemia and lymphoma (Blyth et al., 1995; Lotem and Sachs, 1995; Eischen et al., 1999). They also cooperate in the transformation of mouse fibroblasts (Taylor et al., 1992), the immortalization of hematopoietic precursor cells (Metz et al., 1995) and the promotion of genomic instability (Yin et al., 1999).
Chronic myeloid leukemia (CML) is usually diagnosed in a benign chronic phase, characterized by a clonal granulocytosis. The chronic phase lasts 1–3 years and progresses to a fatal blast crisis. The molecular hallmark of all CMLs is the expression of the Bcr-Abl kinase, a fusion protein arising from a translocation (Melo, 1996). The disease evolution suggests that secondary gene alterations are required for progression from chronic CML phase to blast crisis, but there is no evidence, to date, that a single molecular change is responsible for this progression. Alterations of p53 are very rare in the chronic phase of CML, and they occur with frequencies ranging 10–25% of the blast crisis cases (Ahuja et al., 1991; Prokocimer and Rotter, 1994; Nakai and Misawas, 1995; Stuppia et al., 1997) although this frequency may be much lower, depending on the population analysed (Peller et al., 1998). Interestingly, c-myc expression is higher in cells derived from blast crisis CML than from the chronic phase (Preisler et al., 1990; Handa et al., 1997), and recent reports indicate a positive correlation of c-myc amplification with progression to blast crisis (Beck et al., 1998; Jennings and Mills, 1998). Moreover, the presence of an extra allele of c-myc (chromosome 8 trisomy) is the most common karyotypic alteration in CML blast crisis (46% of cases) (Alimena et al., 1987).
K562 cells derive from CML in blast crisis and are deficient in p53, due to the deletion of one allele and a frameshift mutation in the other (Bi et al., 1993; Law et al., 1993). We have used the K562 system to study the role of c-Myc on p53 function. We show here that c-Myc counteracts the apoptosis mediated by wild-type p53 and the up-regulation of p21WAF1 expression.
Wild-type p53 induces apoptosis in K562 cells
Kp53A1 cells are K562 transfected with a p53Vall35 mutant, which were fully characterized previously (Ehinger et al., 1997). This protein adopts a wild-type conformation at 32°C, whereas it behaves like mutated p53 at 37°C (Michalovitz et al., 1990; Milner and Medcalf, 1990). As described (Law et al., 1993), no p53 was detected by immunoblot in parental K562 cells, but significant amounts of p53 protein were detected at 37 and 32°C in Kp53A1 and Kp53C1 cells (Figure 1a). To confirm the presence of p53 in wild-type conformation at 32°C we analysed the expression of bax and p21WAF1, two well-known transcriptional targets of p53. Upon transfer to 32°C, the mRNA expression of both genes was clearly induced in Kp53A1, but not in parental K562 (Figure 1b) or vector-transfected KCMV cells (not shown). These results demonstrate that p53 is transcriptionally active at 32°C in Kp53A1. Transfer of cells of 32°C resulted in growth arrest, predominantly in G1 phase (not shown), as well as progressive decrease in cell viability and appearance of apoptotic cells. The morphological analysis of cells revealed a dramatic increase of cells showing typical features of apoptosis: membrane bebbling, cell shrinkage, chromatin condensation and fragmentation and formation of apoptotic bodies (Figure 1c). Similar result was observed for Kp53C1 cells (not shown). The apoptotic nature of Kp53A1 cell death at 32°C was also confirmed by the internucleosomal fragmentation of genomic DNA (Figure 1d). At 32°C the cells also exhibited other markers of apoptosis such as abnormal chromatin condensation revealed by DAPI staining and annexin V binding to the cell surface (see below). Thus, cell death induced by p53Vall35 at 32°C in K562 cells had the properties of typical apoptosis. This apoptosis showed a slow kinetics, as few apoptotic cells were detected after 24 h at 32°C.
Construction of transfectant cell lines with conditional expression of c-Myc and p53 in wild-type conformation
To study the effect of c-Myc on p53-mediated apoptosis of K562 we generated double transfectants with conditional expression of p53 in the wild-type conformation and c-Myc. Kp53A1 cells were transfected with pHeBO-MT-myc, a zinc-inducible c-Myc expression vector (Grignani et al., 1990). Individual clones resistant to G418 (as Kp53A1 cells) and hygromycin (transfected with c-myc) were isolated. One cell clone was selected for further experiments and termed Kp53A1/myc. The construction of this line and its expected expression pattern is schematized in Figure 2a. The expression of ectopic c-myc mRNA is markedly induced after 24 h of treatment with 75 μM ZnSO4 (Figure 2b). The transfected gene lacks the first non-coding c-myc exon and therefore it produces a smaller mRNA than the endogenous gene (Grignani et al., 1990). Kp53A1/myc cells also overexpressed c-Myc protein after zinc addition, as shown by immunoblot (Figure 2c). To rule out the possibility that the results were due to the particular cell clone selected, we generated another double transfectant cell line by the inverse pathway to that followed to generate Kp53A1/myc cells. Thus, we infected KmycB cells (with zinc-inducible expression of c-myc) (Delgado et al., 1995), with a retrovirus expressing the p53Vall35 mutant. Cells resistant to hygromycin (as KmycB cells) and puromycin (infected with the p53 retroviral vector) were selected and dubbed KmycB/p53 (Figure 2a). The expression of exogenous c-myc mRNA (Figure 2b) and protein (Figure 2c) were dramatically induced after 24 h of culture in the presence of 75 μM ZnSO4 in KmycB/p53 cells. The expression of endogenous c-myc was reduced at 32°C in Kp53A1/myc and KmycB/p53 cells (Figure 2b and data not shown). This result agrees with the reported induction of c-myc by mutated p53 and repression by wild-type p53 in other systems (see Introduction).
c-Myc protects K562 cells from p53-mediated apoptosis
We then analysed the effect of c-Myc induction on p53-mediated apoptosis. As shown in Figure 3a, when cells were shifted to 32°C in the presence of 75 μM ZnSO4, the loss in cell viability was attenuated. Likewise, the fraction of cells with apoptotic morphology was significantly reduced in the presence of 75 μM ZnSO4 (60 versus 20% apoptotic cells after 4 days with zinc). No significant changes in cell morphology were observed in those cells not undergoing apoptosis in the zinc-treated cultures (not shown). The extent of apoptosis protection was similar for Kp53A1/myc and KmycB/p53 cell lines (Figure 3a). No changes in viability were detected among the different cell lines when the experiments were performed at 37°C (not shown). The abrogation of apoptosis mediated by c-Myc was also demonstrated by determining the metabolic rate, using the WST-1 tetrazolium salt reduction test (not shown). Altogether, the results confirmed that c-Myc overexpression protects K562 cells from p53-mediated apoptosis. However, the induction of c-Myc at 32°C (i.e., with p53 in wild-type conformation) in Kp53A1/myc and KmycB/p53 cells did not restore full growth ability, and did not change the cell cycle phase distribution. Cell counts in the presence of zinc increased about twofold after 24 h at 32°C but did not grow further, and no changes were detected in the extent of G1 arrest with respect to untreated cells (not shown). The protection from apoptosis by c-Myc was also determined by the translocation of phosphatidylserine to the outer layer of cell membranes, as determined by annexin V binding. Our results showed that the shift-down to 32°C resulted in a dramatic increase in the number of cells positive for annexin V. However, the presence of 75 μM ZnSO4 resulted in a decrease of about 50% in the fraction of Kp53A1/myc and KmycB/p53 cells bound to annexin (Figure 3b).
Next we analysed the effect of c-Myc on p53-mediated apoptosis by determining the internucleosomal DNA fragmentation. DNA laddering was clearly reduced in both Kp53A1/myc and KmycB/p53 cell lines incubated at 32°C for 72 h in the presence of 75 μM ZnSO4, with respect to cells incubated without zinc (Figure 3c). In contrast, extensive DNA fragmentation was observed in Kp53A1 cells either in the presence or absence of ZnSO4. We also determined the effect of c-Myc expression on apoptosis by analysing the extent of chromatin condensation by DAPI staining. KmycB/p53 and Kp53A1/myc cells were incubated for 72 h at 32°C in the presence or absence of 75 μM ZnSO4, and the fraction of apoptotic cells was dramatically reduced in zinc-treated cells (Figure 3d). We conclude that c-Myc inhibits the apoptosis mediated by wild-type p53 in K562. As p53 may be expressed at different levels among individual cells, it is formally possible that c-Myc overexpression allows the growth of those cells with lower p53 expression. Thus, a population of p53-negative and apoptosis-resistant cells could be selected during zinc treatment at 32°C of Kp53A1/myc or KmycB/p53 cells. However, the expression of p53, as assessed by immunoblot, did not decrease significantly after 96 h at 32°C in the presence of zinc (not shown). Also, p53 is detected by immunofluorescence in the nuclei of Kp53A1/myc cells incubated at 32°C in the presence of zinc (not shown), ruling out a c-Myc-mediated impairment of p53 translocation to the nucleus.
c-Myc antagonizes the transcriptional activities of p53 in K562
To investigate possible mechanisms by which c-Myc could exert its protective effect on p53-mediated apoptosis, we studied the transactivation activity of p53 on the promoters of bax and p21WAF1, two p53 target genes involved in apoptosis and cell cycle arrest. We first confirmed that co-transfection of wild-type p53 expression vector (pLPC-hp53) into the parental K562 cells resulted in a dramatic activation of the p21WAF1 promoter (58-fold, mean of four experiments). The induction was similar at 32°C and 37°C (not shown). The effect of c-Myc on p53-dependent transcription was studied by transfecting into K562, pLPC-hp53 with and without an expression vector encoding c-Myc (LMycSN), together with the p53-responsive bax, mdmd2 and p21WAF1 promoter-luciferase reporters. Interestingly, c-Myc reduced the transactivation of the three promoters by 40–60% depending on the promoter analysed (Figure 4a). We also tested whether c-Myc impaired the p53-mediated transcriptional repression of cytomegalovirus (CMV) immediate early promoter (Subler et al., 1992; Jackson et al., 1993). The results showed that this repression was clearly attenuated by co-expression of c-Myc (Figure 4a).
We investigated the c-Myc effect in Kp53A1 and Kp53C1 cells. First, we confirmed that the shift to 32°C of Kp53A1 cells resulted in a notable increase in the activity of p21WAF1 promoter, in agreement with the presence of p53 in wild-type conformation (Figure 4b). This increase was abolished by co-transfection of the papilloma virus E6 gene expression vector (Figure 4b), or by deleting the p53-responsive elements of the p21WAF1 promoter (not shown), demonstrating that p53 was responsible for this transactivation. Next, Kp53A1 and Kp53C1 cells were co-transfected with the different reporters together with a c-Myc expression vector (LMycSN) and incubated at 32 and 37°C. Transcriptional activation of bax and p21WAF1 promoters was dramatically induced at 32°C (Figure 4c), but it was reduced by 40–60% upon co-transfection of c-Myc expression vector. Likewise, p53-mediated repression of CMV promoter was reduced by c-Myc expression (Figure 4c). Thus, c-Myc impaired both the transcriptional activation and repression activities of p53 in the K562 model. In contrast, the p53-independent up-regulation of p21WAF1 induced by phorbol esters or okadaic acid in K562 (Zeng and El-Deiry, 1996) was only marginally inhibited by c-Myc (5–20%) (not shown).
As p21WAF1 transactivation correlated to the changes at protein level (see below), we sought to confirm the c-Myc effect on p53-induced transactivation of p21WAF1 promoter by three approaches. First, we transfected the p21WAF1-luciferase construct along with increasing amounts of c-myc expression vector (6, 10 and 14 μg of LMycSN) into Kp53A1. The results revealed an increased blocking effect of the p53-induced transactivation of p21WAF1 with higher c-myc doses. With the highest amount of transfected c-myc expression, the activity of p21WAF1 promoter fell to 20% of control activity (Figure 5a). Second, we transfected the p21WAF1-luciferase reporter, along with an expression vector for c-MycERTM. This is a fusion protein of c-Myc and a mutant hormone binding site of estrogen receptor. The c-Myc activity of this protein is activated upon addition of 4-hydroxytamoxifen (Littlewood et al., 1995). The results indicated that p21WAF1 promoter activity was reduced by 4-hydroxytamoxifen in a concentration-dependent manner, reaching 60% of inhibition in cells treated with 500 nM 4-hydroxytamoxifen (Figure 5b). No effect of the hormone on promoter activity was detected in cells transfected with the empty vector.
Finally, we tested p21WAF1 promoter activity in Kp53A1/myc or KmycB/p53 cells at 32°C in the absence and presence of zinc, i.e., under conditions in which c-Myc induction was concomitant with apoptosis protection. When the cells were shifted to 32°C, the expression of luciferase was induced by about 10-fold (Figure 5c, white bars), in accordance with the change to a wild-type conformation of p53. When the experiment was conducted in the presence of zinc the activity of the p21WAF1 promoter was reduced by 40% compared to the cells without zinc (Figure 5c). Essentially, the same results were obtained with KmycB/p53 and Kp53A1/myc cells. The reduction in p21WAF1 promoter activity upon zinc addition was reproduced in 10 independent experiments among the two cell lines. Similar results were observed with 75 and 100 μM ZnSO4 (not shown). Zinc addition also reduced the activity of Bax promoter in these cell lines (not shown). As expected, zinc addition did not decrease p53-mediated p21WAF1 transactivation of Kp53A1 cells shifted to 32°C (Figure 5c) or K562 transfected with a wild-type p53 expression vector (not shown). To assess that zinc induced active c-Myc, we used a luciferase reporter carrying a Myc-responsive promoter (pGL2M4-Luc). The results confirmed that c-Myc transcriptional activity is elevated when protecting cells from apoptosis (Figure 5c).
c-Myc antagonizes the up-regulation of p21WAF1 mediated by p53 in K562
We investigated the levels of mRNA and protein of Bax, Mdm2 and p21WAF1 in Kp53A1/myc and KmycB/p53 cells. As expected from the presence of p53 in wild-type conformation, the shift to 32°C resulted in the induction of bax and p21WAF1 mRNA expression (Figure 6a, lanes 4–7). After addition of 75 μM ZnSO4 a dramatic induction of exogenous c-myc mRNA was observed. The endogenous c-myc expression declined at 32°C, in consistency with the reported effect of mutant and wild-type p53. When c-Myc expression was induced by zinc addition, no significant changes in bax mRNA levels (Figure 6a) and protein (not shown) were observed. In contrast, levels of p21WAF1 mRNA were clearly decreased in both cell lines after 24 h of incubation at 32°C in the presence of zinc (Figure 6a, lanes 4 and 7) compared to parallel cultures without zinc (Figure 6a, lanes 5 and 6). The effect of c-Myc on mRNA was also observed at the level of p21WAF1 protein, as shown by immunoblot (Figure 6b). The correlation between p21WAF1 protein down-regulation and inhibition of cyclin-dependent kinase 2 (CDK2) was studied in Kp53A1/myc and KmycB/p53 cells in the presence and absence of c-Myc induction. The result showed that incubation of the cells at 32°C caused a marked repression of CDK2 activity and that c-Myc counteracted this repression. This result was obtained assaying both the CDK2- and the cyclin E-associated kinase activity (Figure 6c). Altogether, our results show that c-Myc impairs the induction of p21WAF1 expression mediated by wild-type p53 in K562 cells and that this effect occurs at the transcriptional level.
The present study provides evidence that c-Myc antagonizes both the apoptosis and the up-regulation of p21WAF1 mediated by p53. Using the p53Val135 mutant we first showed that p53 in wild-type form induces growth arrest followed by cell death of K562. The cell death induced by wild-type p53 in K562 transfectants has the morphological and biochemical features of typical apoptosis. p53-induced K562 cell death has been reported (Kremenetskaya et al., 1997; Trepel et al., 1997). In contrast, other studies indicate that K562 expressing wild-type p53 are able to grow, albeit at a slower rate and accompanied by erythroid differentiation (Ehinger et al., 1995; Feinsten et al., 1992). The most obvious explanation for these differences is that higher levels of p53 in wild-type conformation are easier to obtain in stable transfectants with a termosensitive mutant, since the transfection with constitutive wild-type p53 will select against those cell clones overexpressing the pro-apoptotic protein.
Interestingly, ectopic expression of c-Myc counteracts the apoptosis induced by wild-type p53 in K562 cells. This result seems paradoxical as c-Myc induce apoptosis in different cell types under conditions of growth factor deprivation. However, c-Myc overexpression induces little apoptosis of K562 cells in the absence of serum (Cañelles et al., 1997). This is consistent with the lack of expression of p53 (Law et al., 1993), p19ARF (Delgado et al., 2000) and CD95/FAS (McGahon et al., 1995) in K562, as the three proteins are required for c-Myc-mediated apoptosis in fibroblasts (Hermeking and Eick, 1994; Hueber et al., 1997; Wagner et al., 1994; Zindy et al., 1998). However, c-Myc does not confer an unspecific anti-apoptotic effect on K562 cells, as it does not protect from other apoptotic stimuli as okadaic acid or ceramides (Cañelles et al., 1997; our unpublished results). The antiapoptotic effect of c-Myc has also been described in lymphoid cells (Sonenshein, 1997; Felsher and Bishop, 1999) and keratinocytes (Waikel et al., 1999), but its relationship with p53 function in these processes is unknown.
How does c-Myc impair the p53-mediated apoptosis in K562 cells? One obvious possibility is that c-Myc abrogates the transcriptional activation of p53 target genes. We tested bax and p21WAF1 genes, and the p53-mediated transactivation of both promoters was attenuated by c-Myc overexpression. Moreover, c-Myc attenuates the p53-mediated repression of CMV immediate early promoter. Thus c-Myc interferes with both the transcriptional activation and repression mediated by p53. Bax mRNA and protein levels were unchanged by c-Myc expression, ruling out the Bax down-regulation as a mechanism for apoptosis protection. In contrast, the expression of p21WAF1 mRNA and protein was dramatically reduced. Inhibition of p53-dependent induction of p21WAF1 is a newly described activity of c-Myc. In NIH3T3 fibroblasts the ectopic expression of c-Myc abrogates the p21WAF1 inhibitory activity on cyclin dependent kinases but it does not modify its expression (Hermeking et al., 1995). During the preparation of this manuscript, it has been reported that c-Myc inhibits phorbol ester-induced p21WAF1 up-regulation in prostate and breast cancer cell lines (Mitchell and El-Deiry, 1999). Thus, c-Myc-mediated inhibition of p21WAF1 transactivation described herein for K562 may occur widely among tumor cells. Other recent studies show that loss of c-Myc in rat Rat-1 cells results in a reduction of p21WAF1 (Mateyak et al., 1999). This reinforces the idea that c-Myc does not directly interact with p21WAF1 or Bax promoters and that the effect on p53 transcriptional activity depends on the cell type.
Does the c-Myc-mediated down-regulation of p21WAF1 explain the abrogation of p53-dependent apoptosis?. Given that the mechanism of p53-induced apoptosis is not well understood, we can only speculate on the possible mechanisms that are responsible for c-Myc-mediated supression of apoptosis. p21WAF1 appears to be necessary for growth arrest but not for apoptosis induced by wild-type p53 (Bates and Vousden, 1999; Hansen and Oren, 1997). It is formally possible that p21WAF1 accumulation eventually results in apoptosis of K562 cells, as has already been described in other systems as neuroblastoma (Poluha et al., 1996), lung carcinoma (Adachi et al., 1996), breast carcinoma (Sheikh et al., 1995), colon carcinoma (Chinery et al., 1997; Polyak et al., 1996), and lymphoid cells (Wu et al., 1998). However, it has been reported that expression of another p53 mutant (p53Val143) in K562 results in p21WAF1 up-regulation without concomitant apoptosis (Kobayashi et al., 1995). Also, co-transfection of an antisense-p21WAF1 expression vector did not alleviate the apoptosis due to p53 in K562 (our unpublished results). Thus it seems unlikely that p21WAF1 decline by itself is responsible for the protective effect of c-Myc. Our results rather indicate that c-Myc interferes with the ability of p53 to act as transcription factor in K562, as it not only antagonizes the activation of Bax and p21WAF1 promoters but also the repression of CMV promoter. It must be noted that there might be many p53-target genes involved in apoptosis, and therefore it is possible that c-Myc interferes with p53-dependent induction of other pro-apoptotic proteins.
Independently of the mechanisms involved, our results offer a new clue for the role of c-Myc in the development of leukemia. On one hand, c-myc overexpression has been implicated in the malignant progression of human tumors (Garte, 1993; Henriksson and Lüscher, 1996; Berns et al., 1992). On the other hand, apoptosis is a well-documented consequence of c-Myc ectopic expression in cells subjected to growth factor deprivation or to hypoxia. Moreover, a correlation between high c-myc expression and apoptosis has been reported for some solid tumors (Hoffman and Liebermann, 1998; Alarcon et al., 1996; Donzelli et al., 1999). However, the pro-apoptotic effect of c-Myc is at odds with the frequently found c-myc overexpression in human cancer. This is most intriguing in those tumors retaining p53 function, which occurs in a majority of the cases in some hematological neoplasias, e.g. CML blast crisis (Ahuja et al., 1991; Prokocimer and Rotter 1994; Nakai and Misawa, 1995; Stuppia et al., 1997), Burkitt lymphomas (Newcomb, 1995) and mouse plasmacytomas (Gutierrez et al., 1992). Thus, c-Myc overexpression must confer some selective advantage which counteracts its apoptotic effect. It has been suggested that tumors produce survival factors able to block the apoptotic effect of c-Myc, leaving the proliferative pathway unrestrained. Although our data do not contradict this hypothesis, they indicate that, at least in some tumors, an additional mechanism may be operating which can explain c-myc overexpression during tumor progression. Given the fact that p53 plays a pivotal role in mediating drug- or radiation-induced apoptosis, we propose that c-Myc overexpression could protect tumor cells from apoptosis mediated by p53.
Materials and methods
Cell lines, transfection and retroviral transduction
All cell lines were grown in RPMI 1640 medium (Gibco-Life Sciences) supplemented with 8% fetal calf serum and gentamycin (80 μg/ml). Kp53A1 cells (originally termed K562/ptsp53/A1) are K562 cells expressing a mousep53Val 135 mutant (Ehinger et al., 1997). The KCMV cell lines are K562 transfected with neo-resistant vector (Ehinger et al., 1997). KmycB are K562 cells transfected with a zinc-inducible c-myc gene (Delgado et al., 1995). Kp53C1 cells were generated by infecting K562 cells with retrovirus containing the vector pBabePuro-p53Val135 (provided by M Oren, Weizmann Institute, Revohot, Israel). The vector was electroporated into Phoenix-A cells and the supernatant used to infect K562 cells in the presence of 6 μg/ml polybrene. Infected cells were selected with 2 μg/ml puromycin and the resulting culture of puromycin-resistant cells was termed Kp53C1. To generate the Kp53A1/myc cell line, Kp53A1 cells were electroporated at 260 v and 1 mFa with pHeBO-MT-myc vector which carries a c-myc gene under the control of metallothionein promoter (Grignani et al., 1990). Cells and selected with 0.5 mg/ml of G418 and 0.1 mg/ml of hygromycin. Cell clones were isolated by limiting dilution. KmycB/p53 cells were generated by infecting KmycB cells with pBabePuro-p53Val135 retroviruses as described above. The p53 transfectant cell lines tended to lose the apoptotic phenotype after prolonged cell culture, and fresh stocks were used for most experiments.
When indicated, exponentially growing cells at a concentration of 2×105 cells/ml were transferred to 32°C. Cell morphology was analysed by May-Grünwald-Giemsa or 4,6-diamino-2-phenylindole (DAPI) staining of cytocentrifuge preparations. At least 200 cells were scored for each determination of apoptotic or metaphasic cells. Cell growth and viability were assayed with hemocytometer and the trypan blue exclusion test and by colorimetry based on the reduction of the tetrazolium salt WST-1 (Roche Molecular Biochemicals). To determine the presence on internucleosomal fragmentation (DNA laddering), cellular DNA was prepared and analysed by agarose gel electrophoresis as described (Cañelles et al., 1997). Binding of annexin V to cell surface was carried out by flow cytometry with annexin V-FITC kit following the manufacturer's instructions (Genzyme Diagnostics).
RNA, protein and kinase activity analysis
Total RNA was isolated from cells by the acid guanidine thiocyanate method and Northern blots were prepared and hybridized according to standard procedures. Probe for human c-myc was as described (Delgado et al., 1995); for p53, a 2.1 kb XhoI–BamHI fragment from pLTRp53cG corresponding to mouse cDNA (provided by M Oren); for p21WAF1 a 0.6 kb NotI fragment from pBSK-p21 corresponding to human cDNA (provided by M Serrano, Centro Nacional de Biotecnología, Madrid); for bax, a 0.5 kb EcoRI fragment form pSSFV-Bax corresponding to human cDNA (provided by S Korsmeyer, Washington University Medical School, St. Louis, USA). For immunoblots, cell lysates were prepared and immunoblotted as described (Cañelles et al., 1997). c-Myc and p21WAF1 were detected with rabbit polyclonal antibodies, p53 with the monoclonal antibody PAb240 (Santa Cruz Biotech.), and α-tubulin with a rabbit polyclonal antibody (provided by N Cowan, New York University, NY, USA). Immunocomplexes were detected with peroxidase-conjugated secondary antibodies (Cappel) and chemiluminescent method (ECL, Amersham). For kinase assays, cyclin E and CDK2 immunoprecipitations were performed essentially as described (Mateyak et al., 1999), using anti-cyclin E (M-20, Santa Cruz) and anti-CDK2 (M2, Santa Cruz) antibodies. Kinase activity of the immunoprecipitates was assayed using histone H1 as substrate.
Cells were transfected by electroporation at 260 v and 1 mFa in a Bio-Rad electroporator. Cells (4–8×106) were co-transfected with the appropriate plasmids and after 14 h of incubation at 37°C, cultures were split into aliquots and further incubated for 24 h at 32 or 37°C in the absence or presence of 100 μM ZnSO4 or 50–500 nM 4-hydroxytamoxifen. Cells were lysed and the luciferase activity was measured by a dual-luciferase reporter gene assay system (Promega). Plasmids used were: pGL3-Waf1-Luc (provided by M Oren) which contains a 2.3-kb of p21WAF1 promoter upstream of the firefly luciferase cDNA, pGL3-Bax-Luc (provided by M Oren), CMV-Luc (Bellon et al., 1994), pLPC-hp53 vector expressing wild-type p53 (provided by M Serrano) and its corresponding empty vector pLPCX (Serrano et al., 1997); pGL2M4-Luc (provided by R Eisenman, Fred Hutchinson Cancer Research Center, Seattle, USA), LMycSN (provided by R Eisenman) and the empty vector LXSN (Miller et al., 1993), pCMV16-E6 which carries the human papilloma virus E6 gene (provided by K Cho, Johns Hopkins University, Baltimore, USA), and pBpuro-c-mycERTM (provided by T Littlewood, Imperial Cancer Research Fund, London, UK) and the vector pBabePuro (Littlewood et al., 1995). In each transfection experiment, the same amount of total DNA was transfected for all samples, using the empty vectors as carrier DNA. One μg of the pRL-TK plasmid encoding for Renilla luciferase (Promega) was co-transfected in each case. Promoter activity was defined as the ratio between light units generated by the firefly and Renilla luciferases.
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We thank Pilar Frade and Rosa Blanco for technical assistance, and Kathleen Cho, Nicholas Cowan, Riccardo Dalla Favera, Robert Eisenman, Abelardo López-Rivas, Stanley Korsmeyer, Trevor Littlewood, Moshe Oren and Manuel Serrano for plasmids and antibodies. We are gratefully indebted to Dirk Eick and Moshe Oren for helpful comments on the manuscript. This work has been supported by grant PM98-0109, Spanish Ministry of Education and Culture, and Biomed 96-3532 from European Community.
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