Signaling through the antigen receptor of B lymphocytes activates a ...

Oncogene (1999) 18, 4091 ± 4098 ã 1999 Stockton Press All rights reserved 0950 ± 9232/99 $12.00 http://www.stockton-press.co.uk/onc

Signaling through the antigen receptor of B lymphocytes activates a p53-independent pathway of c-Myc-induced apoptosis Hiroyuki Hagiyama1,2, Takahiro Adachi1, Tsutomu Yoshida1, Takashi Nomura3, Nobuyuki Miyasaka2, Tasuku Honjo3 and Takeshi Tsubata*,1 1

Department of Immunology, Medical Research Institute, Tokyo Medical and Dental University, 1-5-45 Yushima, Bunkyo-ku, Tokyo 113-8510, Japan; 2First Department of Internal Medicine, Faculty of Medicine, Tokyo Medical and Dental University, 1-545 Yushima, Bunkyo-ku, Tokyo 113-8519, Japan; 3Department of Medical Chemistry, Kyoto University Graduate School of Medicine, Kyoto 606-8501, Japan

Deregulated expression of c-Myc has been shown to induce or enhance apoptosis in various dierent cell types. c-Myc requires p53 for apoptosis in some but not all the cell types, indicating heterogeneous mechanisms for c-Myc-induced apoptosis. In B lymphoma line WEHI-231, stable expression of c-Myc has been demonstrated to protect cells from BCR-mediated apoptosis. However, stable expression of c-Myc carrying pro-apoptotic functions may generate variant cells resistant to apoptosis. By utilizing an inducible system for c-Myc, we demonstrated here that deregulated expression of c-Myc induced apoptosis of WEHI-231 by itself, indicating that c-Myc induces apoptosis in WEHI-231 as is the case for other cell types. When transactivation of p53 was inactivated, WEHI-231 cells overexpressing c-Myc no longer underwent apoptosis in the absence of other stimuli, but showed markedly enhanced apoptosis in the presence of BCR ligation. These results indicate that deregulated c-Myc expression enhances apoptosis by a p53-independent pathway in the presence of BCR signaling but requires p53 for apoptosis in the absence of BCR crosslinking in WEHI-231. BCR ligation may thus activate a p53-independent pathway of c-Myc-induced apoptosis. Keywords: apoptosis; B lymphocyte; c-Myc; p53

Introduction The c-Myc protein plays an important role in cell proliferation by transactivating target genes such as Cdc25 phosphatase essential for cell cycle progression (Galaktionov et al., 1996). Presumably due to the activity for cell proliferation, deregulated expression of c-Myc induces oncogenesis (Adams et al., 1985; DallaFavera et al., 1982). In contrast, overexpression of cMyc induces apoptosis of ®broblasts upon serum deprivation or treatment with cytokines or chemicals carrying anti-proliferative activity (Evan et al., 1992). Moreover, c-Myc enhances apoptosis of various cell types induced by various stimuli, such as irradiation (Chen et al., 1994), tumor necrosis factor (TNF) (Klefstrom et al., 1994), growth factor deprivation (Askew et al., 1991) and anti-cancer drugs (Dong et al.,

*Correspondence: T Tsubata Received 20 October 1998; revised 2 February 1999; accepted 26 February 1999

1997). Apoptosis by deregulated c-Myc expression may be involved in maintenance of homeostasis by counteracting its oncogenic activity. Indeed, expression of Bcl2 carrying anti-apoptotic function markedly enhances the oncogenesis by deregulated c-Myc expression (Strasser et al., 1990). Overexpression of c-Myc enhances expression of the tumor suppressor gene p53 (Hermeking and Eick, 1994; Reisman et al., 1993). In embryonic ®broblasts obtained from p53-de®cient mice, deregulated c-Myc expression fails to induce apoptosis (Hermeking and Eick, 1994; Wagner et al., 1994), indicating that p53 is involved in c-Myc-mediated apoptosis. In contrast, lines of evidence suggest that a p53-independent pathway is also involved in c-Myc-induced apoptosis. Indeed, renal tubular epithelial cells of p53-de®cient mice undergo apoptosis in the presence of deregulated c-Myc expression (Trudel et al., 1997), indicating that c-Myc-mediated apoptosis does not require p53 in renal tubular cells. Moreover, deregulated c-Myc induces apoptosis in epithelial cells and Rat1 fibroblasts when p53-mediated transactivation is almost completely abrogated by a dominant negative form of p53 or the SV-40 T antigen that inactivates p53 by physical association (Klefstrom et al., 1994; Lenahan and Ozer, 1996; Sakamuro et al., 1995). In epithelial cells and Rat1 cells, p53-mediated transactivation appears dispensable in c-Myc-induced apoptosis, although involvement of a transactivation-independent pathway (Caelles et al., 1994; Haupt et al., 1995; Wang et al., 1996) is not yet ruled out. Regardless of whether c-Myc-induced apoptosis is induced by serum deprivation or treatment with TNF, Rat1 cells do not require p53-mediated transactivation and embryonic fibroblasts require p53 for apoptosis (Hermeking and Eick, 1994; Klefstrom et al., 1997; Wagner et al., 1994). Thus, requirement of p53 or its transactivation for c-Myc-induced apoptosis may depend on cell types but not types of apoptotic stimulation. However, little is known about what induces pathways independent of p53 or its transactivation for c-Myc-induced apoptosis in certain cell types. Interaction with antigens appears to trigger activation and clonal expansion of B cells. However, antigen receptor of B cells (BCR) is also capable of transmitting a negative signal, resulting in functional inactivation or apoptosis of B cells (Goodnow, 1992). Such negative signaling via BCR may play a role in maintaining the normal immune system by inactivating or deleting self-reactive B cells upon interaction with self-antigens. B cell lines such as WEHI-231 undergo

c-Myc-induced apoptosis of B lymphocytes H Hagiyama et al

4092

apoptosis when BCR is ligated (Benhamou et al., 1990; Hasbold and Klaus, 1990). In WEHI-231 cells, BCR ligation markedly enhances the level of c-Myc within 1 h (McCormack et al., 1984). Subsequently, the level decreases and becomes undetectable after 8 h. Recently, Wu et al. (1996a) demonstrated that WEHI-231 transfectants stably expressing c-Myc fail to undergo apoptosis by BCR ligation, suggesting that overexpression of c-Myc fails to enhance but rather blocks BCRmediated apoptosis. Down-modulation of c-Myc by BCR ligation may thus be responsible for BCRmediated apoptosis. This conclusion is also supported by the ®nding that treatment with TGF-b or inhibitor of I-kB phosphorylation down-modulates the c-Myc level, followed by apoptosis in WEHI-231 cells (Fischer et al., 1994; Warner et al., 1992; Wu et al., 1996b). Thus, apoptosis is induced by down-modulation of cMyc in WEHI-231 but by deregulated c-Myc expression in many other cell types such as ®broblasts and epithelial cells. Therefore, c-Myc is suggested to play a distinct role in WEHI-231 from other cell types. Although the ®nding on WEHI-231 transfectants constitutively expressing c-Myc is the key evidence suggesting that deregulated c-Myc expression induces survival in WEHI-231 cells, this transfection may select variant cells resistant to apoptosis. Granted that c-Myc carries pro-apoptotic function in B cells as well, WEHI-231 transfected with the expression plasmid may undergo apoptosis by the expression of c-Myc. Only variant cells carrying defects in apoptosis may survive the selection. Those variant cells may highly express c-Myc but fail to undergo apoptosis as described by Wu et al. (1996a). To exclude the generation of variant cells resistant to apoptosis, we transfect WEHI-231 cells with the plasmid coding for a chimera consisting of c-Myc and hormone binding domain of the estrogen receptor (ER) (Eilers et al., 1989). The MycER chimera is transcriptionally inactive in the absence of estrogen, but is able to transactivate the target genes for c-Myc in the presence of estradiol. By using this induction system, we demonstrate that deregulated c-Myc expression induces apoptosis by itself in a p53-dependent manner in WEHI-231 cells. In contrast, c-Myc markedly enhances apoptosis upon BCR crosslinking even when p53-mediated transactivation is completely inactivated by a dominant negative form of p53. These results strongly suggest that BCR signaling activates a p53-independent pathway for cMyc-induced apoptosis. Results Expression of c-Myc induces apoptosis of WEHI-231 in the absence of BCR crosslinking and enhances BCR-mediated apoptosis To ask whether expression of c-Myc induces apoptosis, we transfected WEHI-231.5 cells with an expression plasmid coding for the MycER chimeric molecule. When the expression of MycER was assessed by Western blotting using anti-ER antibody, the level of MycER was very low in nuclear extracts from the MycER transfectants in the absence of b-estradiol, whereas the treatment with 0.1 mM b-estradiol markedly increased the amount of nuclear MycER (Figure

1a). Since the treatment with b-estradiol did not increase the amount of MycER protein in total cell lysates from the MycER transfectants (data not shown), MycER is most likely translocated to the nuclei in the presence of b-estradiol, resulting in transactivation of the target genes of c-Myc. In the absence of b-estradiol, a small number of dead cells was observed in WEHI-231.5 MycER transfectants (W-MycER) but not parent WEHI-231.5 cells. When MycER is activated by the treatment with 0.1 mM bestradiol, a sizable fraction of the W-MycER cells died in 48 h, whereas almost all the parent WEHI-231 cells survived the same treatment (Figure 1b). This indicates that induction of c-Myc alone induces death of WEHI231 cells. A few W-MycER cells died in the absence of estradiol presumably due to leaky transactivation activity of MycER without association with estrogen or a small amount of estrogen activity contained in the serum added to the culture. To test whether c-Mycinduced death of WEHI-231 is due to apoptosis, we measured the DNA contents of nuclei prepared from W-MycER cells by ¯ow cytometry. Treatment of WMycER cells with b-estradiol markedly increased the percentage of hypodiploid nuclei characteristic for

Figure 1 c-Myc induces death of WEHI-231 cells. (a) Treatment with b-estradiol induces nuclear translocation of MycER in WEHI-231.5 transfectants. WEHI-231.5 cells (3.06106) and the same number of cells of two independently obtained WEHI-231.5 MycER transfectants (W-MycER2 and W-MycER4) were cultured in 10 ml of phenol red-free medium with or without 0.1 mM b-estradiol (E2). After 6 h, cells were collected. Nuclei were then prepared and lysed in 30 ml of phospholysis buer. Nuclear extracts (100 mg) were separated by SDS ± PAGE under reducing conditions. Expression of MycER was detected by Western blotting using anti-human estrogen receptor a chain Ab. The band for MycER is indicated by an arrow. (b) Treatment with b-estradiol induces death of WEHI-231.5 MycER transfectants. WEHI-231.5 cells (1.06104) and the same number of WMycER2 and W-MycER4 were cultured in 0.2 ml of phenol redfree medium with or without 0.1 mM b-estradiol (E2). After 48 h, numbers of alive and dead cells were counted by trypan blue exclusion, and percentage of dead cells were calculated. Representative data of three experiments are shown


4093

Figure 2 Apoptosis of WEHI-231 cells. W-MycER2 cells (5.06104) were cultured in 1.0 ml of phenol red-free medium with or without 0.1 mM b-estradiol (E2) for 48 h. Alternatively, the same number of W-MycER2 cells were cultured in 1.0 ml of medium with 0.1 mM b-estradiol (E2) for 24 h, followed by addition of anti-IgM Ab to the ®nal concentration of 10 mg/ml and culturing for another 24 h. Cells were then harvested, lysed in 0.2 ml of 0.15% Triton-X in PBS and incubated with 5 mg/ml of RNase A and 50 mg/ml of propidium iodide (PI). DNA contents of the nuclei were analysed by FACSCalibur1. Percentages of hypodiploid nuclei are indicated. Representative data of three experiments are shown

apoptosis (Figure 2). Taken together, induction of cMyc alone induces apoptosis in WEHI-231 cells. To assess the eect of c-Myc on BCR-mediated apoptosis, we pretreated both parent WEHI-231.5 cells and W-MycER cells with 0.1 mM b-estradiol for 24 h. We then crosslinked BCR by adding anti-IgM Ab to the ®nal concentration of 10 mg/ml and cultured for additional 24 h. This treatment failed to kill parent WEHI-231.5 cells regardless of the presence of antiIgM, in agreement with the previous result showing that more than 24 h are required for BCR-mediated death of WEHI-231 cells (Hasbold and Klaus, 1990). In contrast, the combination of anti-IgM and bestradiol killed almost all the W-MycER transfectants, whereas around half of W-MycER cells died by the treatment with b-estradiol alone (Figure 3a). This indicates that BCR signaling induces death of WEHI231 in 24 h in the presence of deregulated c-Myc expression. To exclude the possibility that b-estradiol is degraded during the culture, resulting in the downregulation of c-Myc activity, we added b-estradiol every 12 h. However, this treatment together with antiIgM treatment for 24 h also killed W-MycER cells almost completely (Figure 3b). Moreover, ¯ow cytometry revealed that treatment with anti-IgM enhances the generation of hypodiploid nuclei characteristic for apoptosis in W-MycER cells in the presence of b-estradiol (Figure 2). Taken together, deregulated expression of c-Myc most likely enhances BCR-mediated apoptosis of WEHI-231.

Figure 3 c-Myc enhances BCR-mediated apoptosis of WEHI231. (a) WEHI-231.5 cells (1.06104) and the same number of cells of two independently obtained WEHI-231.5 MycER transfectants (W-MycER2 and W-MycER4) were cultured in 0.2 ml of phenol red-free medium with 0.1 mM b-estradiol for 24 h. Subsequently, anti-IgM Ab were added to the ®nal concentration of 10 mg/ml and cells were cultured for another 24 h. As a control, the same number of WEHI-231.5, W-MycER2 and W-MycER4 cells were cultured with 0.1 mM b-estradiol in the absence of anti-IgM Ab for 48 h. Cells were then collected and the numbers of alive and dead cells were counted by trypan blue exclusion. Representative data of three experiments are shown. (b) W-MycER2 cells (1.06104) were cultured in 0.2 ml of phenol redfree medium in 96 well plates, and 20 pmole of b-estradiol (E2) (0.1 mM at the ®nal concentration) was added either once at the initiation of the culture (61) or every 12 h (64). After 48 h, cells were collected and the number of dead and alive cells were counted by trypan blue exclusion. In some wells, cells were treated with 10 mg/ml of anti-IgM Ab for the last 24 h of culture. Representative data of three experiments are shown

WEHI-231 requires p53 for apoptosis induced by c-Myc alone but not for apoptosis induced by c-Myc together with BCR ligation To ask whether p53 is essential for apoptosis of WEHI-231 induced by c-Myc, we transfected WMycER cells with the expression plasmid for the dominant negative form of p53 (p53DD) containing the oligomerization domain but lacking the DNA binding domain (Shaulian et al., 1992). We chose two transfectants W-MycER-p53DD11 and W-MycERp53DD21 for further analysis. When we examined the level of p53 in WEHI-231 transfectants by


4094

Figure 4 The protein level of p53 in WEHI-231 cells. Cells (2.06105) of WEHI-231 (lane 1), the same number of untreated (lane 2) or X-ray (1000 R)-treated (lane 3) W-MycER2 cells, or 1.06105 cells of a W-MycER transfectant expressing a dominant negative form of p53 (W-MycER-p53DD11) (lane 4) were lysed in 10 ml of phospholysis buer (a). Alternatively, either 2.06105 W-MycER2 cells (b) or 1.06105 W-MycER-p53DD11 cells (c) were precultured with 0.1 mM b-estradiol (E2) alone for 24 h and then treated with 10 mg/ml of anti-IgM Ab (a-Ig) together with 0.1 mM b-estradiol for indicated duration. Cells were then collected and lysed in 10 ml of phospholysis buer. As a control, the same number of untreated W-MycER2 (b) or W-MycER-p53DD11 cells (c) were lysed in phospholysis buer. Proteins (40 mg (a, b) or 30 mg (c)) were separated by SDS ± PAGE under reducing conditions, followed by Western blotting using anti-p53 Ab. The bands for p53 and p53DD are indicated by arrows

Western blotting, W-MycER-p53DD11 cells showed a much higher level of endogenous p53 comparing to W-MycER cells (Figure 4a), in agreement with previous ®ndings (Shaulian et al., 1992). Although the protein level of p53 was markedly increased by X-irradiation of 1000 R in W-MycER cells (Figure 4a), treatment with anti-IgM together with b-estradiol failed to increase the p53 level both in W-MycER cells and W-MycER-p53DD11 cells (Figure 4b and c). Another transfectant W-MycER-p53DD21 cells showed essentially the same results in the changes of the protein level of p53 by irradiation or treatment with anti-IgM (data not shown). These results indicate that BCR signaling together with deregulated c-Myc expression does not up-regulate the level of p53 either in the presence or absence of p53DD. Although deregulated c-Myc expression has been shown to up-regulate p53 expression in ®broblasts (Hermeking and Eick, 1994; Reisman et al., 1993), cMyc may not signi®cantly transactivate p53 in B cells. To ask whether p53DD blocks transactivation by p53, we tested the DNA binding capacity of p53 in W-MycER cells and W-MycER-p53DD cells either untreated or treated with X-irradiation of 1000 R. The DNA binding capacity of p53 was almost undetectable in W-MycER-p53DD11 even when the level of endogenous p53 is up-regulated by Xirradiation (Figure 5a). In W-MycER-p53DD21, DNA binding of p53 was markedly reduced. Transactivation by p53 thus appeared to be markedly inhibited in W-MycER-p53DD cells. This conclusion was also supported by Western blotting for the p21Cip1 protein, the product of a p53-inducible gene. Indeed, X-irradiation induced expression of

p21Cip1 in W-MycER cells but not W-MycERp53DD11 nor W-MycER-p53DD21 cells (Figure 5b), indicating that p53DD blocks expression of the p53-inducible gene. Taken together, DNA binding and transactivation by p53 are almost completely suppressed in W-MycER-p53DD cells even when p53 is up-regulated by X-irradiation. Since BCR crosslinking does not up-regulate p53, the transcriptional activity of p53 appears to be abrogated almost completely in W-MycER-p53DD cells even in the presence of BCR signaling. As is the case for W-MycER cells, both W-MycERp53DD11 and W-MycER-p53DD21 cells showed a small number of dead cells in the absence of b-estradiol (Figure 6a). When treated with b-estradiol, the number of dead cells did not change in both of the W-MycERp53DD transfectants, whereas around a half of WMycER cells died by the same treatment. This indicates that WEHI-231 cells require p53 for apoptosis induced by c-Myc alone. In contrast, almost all the W-MycERp53DD cells died by the treatment with anti-IgM and b-estradiol, as is the case for W-MycER cells, whereas neither parent WEHI-231.5 cells nor WEHI-231.5 transfected with p53DD alone (W-p53DD) died by the same treatment (Figure 6b). This strongly suggests that p53-mediated transactivation is not essential for rapid apoptosis of WEHI-231 induced by deregulated c-Myc expression in the presence of BCR signaling. Taken together, deregulated expression of c-Myc may require p53-mediated transactivation for apoptosis in the absence of BCR signaling but not in the presence of BCR signaling. BCR signaling is thus likely to activate a p53-independent pathway of c-Myc-induced apoptosis.


4095

Figure 5 Dominant negative activity of p53DD (a) p53DD inhibits sequence-speci®c DNA binding of p53 in WEHI-231. WMycER4 cells (5.06107) and the same number of two independently obtained W-MycER transfectants expressing a dominant negative form of p53 (W-MycER-p53DD11 and WMycER-p53DD21) were exposed to X-ray (1000 R). Either irradiated or untreated cells (5.06107) were then homogenized and nuclei were incubated with extraction buer. The nuclear extracts were mixed with a 32P-labeled target DNA of p53, followed by immunoprecipitation with goat anti-p53 Ab. Labeled DNA in the precipitates were extracted and electrophoresed on a 5% native polyacrylamide gel. The radioactivity of the labeled DNA was quanti®ed by an image analyzer. Representative data of two experiments are shown. (b) p53DD inhibits expression of p21Cip1 induced by X-irradiation. W-MycER4 cells (3.06106) and the same number of W-MycER-p53DD11 and W-MycERp53DD21 cells were exposed to 3000 R X-ray. Irradiated or untreated cells were cultured in medium for 12 h, and then lysed in 50 ml of phospholysis buer. Proteins (80 mg) were separated by SDS ± PAGE under reducing conditions, followed by Western blotting using anti-p21Cip1 Ab. The blot was reprobed with antitubulin Ab to ensure equal loading of the immunoblot. The band for p21 is indicated by an arrow. Representative data of three experiments are shown

Discussion By utilizing the inducible system for c-Myc, we demonstrate here that deregulated expression of cMyc induces apoptosis by itself in WEHI-231. Moreover, BCR-mediated apoptosis is enhanced in WEHI-231 overexpressing c-Myc, although a sizable fraction of c-Myc-expressing WEHI-231 cells undergo apoptosis in the absence of BCR ligation, resulting in high background death. Since the dominant negative form of p53 (p53DD) blocks apoptosis induced by cMyc alone, enhancement of BCR-mediated apoptosis by c-Myc is more clearly observed in WEHI-231 cells expressing p53DD together with c-Myc. When c-Myc is activated, they die almost completely by BCR ligation for 24 h, but show only a marginal death in the absence of BCR crosslinking. In contrast, only

Figure 6 Eect of a dominant negative form of p53 on cell death of WEHI-231 induced by c-Myc. WEHI-231.5 cells (1.06104) and the same number of WEHI-231.5 transfectants expressing p53DD alone (W-p53DD), MycER alone (W-MycER2 and 4) and those expressing both MycER and p53DD (W-MycER-p53DD11 and 21) were cultured in 0.2 ml of phenol red-free medium with or without 0.1 mM b-estradiol (E2) (a). Alternatively, cells were cultured in 0.2 ml of phenol red-free medium with 0.1 mM bestradiol in the presence or absence of anti-IgM Ab as in the legend to Figure 3a (b). After 48 h, numbers of alive and dead cells were counted by trypan blue exclusion, and percentages of dead cells were calculated. The data represent mean+s.d. of triplicate cultures. Representative data of three experiments are shown

few of them die by BCR ligation for 24 h in the absence of deregulated c-Myc activation as is the case for parent WEHI-231 cells and WEHI-231 cells expressing p53DD alone. Thus, deregulated expression of c-Myc clearly induces marked apoptosis in the presence of BCR ligation in WEHI-231 expressing p53DD. Since DNA binding capacity is almost undetectable in a WEHI-231 transfectant expressing p53DD even when p53 is up-regulated by Xirradiation, p53DD may be able to block p53mediated transactivation almost completely in the transfectant. Therefore, deregulated c-Myc expression most likely induces rapid apoptosis in BCR-ligated WEHI-231 by a pathway independent of p53-induced transactivation. Recent studies revealed that p53 carries a pro-apoptotic function even in the absence


4096

of its transactivation capacity (Caelles et al., 1994; Haupt et al., 1995; Wang et al., 1996). However, overexpression of particular alleles of transactivationde®cient p53 is required for apoptosis and the apoptosis is much reduced than that induced by wild type p53 (Haupt et al., 1995). We show that BCR ligation fails to up-regulate the level of p53 in WEHI-231 p53DD transfectants and that BCRcrosslinked WEHI-231 transfectants eciently undergo c-Myc-induced apoptosis even in the absence of p53-mediated transactivation. It is thus unlikely that the transactivation-independent as well as -dependent apoptotic pathway of p53 is involved in c-Mycinduced apoptosis of BCR-crosslinked WEHI-231. Taken together, p53 may not be involved in c-Mycinduced apoptosis of WEHI-231 in the presence of BCR signaling. In contrast, c-Myc-induced apoptosis of WEHI-231 in the absence of BCR signaling requires p53, as deregulated expression of c-Myc alone induces apoptosis in untransfected WEHI-231 but not in WEHI-231 p53DD transfectants. BCR ligation thus most likely activates a p53-independent pathway of c-Myc-induced apoptosis. Two lines of evidence have suggested that upregulation of c-Myc blocks the BCR-mediated apoptosis, which is inconsistent with our results showing that overexpression of c-Myc enhances apoptosis of WEHI-231. First, Fischer et al. (1994) have demonstrated that treatment of WEHI-231 with antisense oligonucleotides unexpectedly blocks both down-modulation of c-Myc and apoptosis of BCRcrosslinked WEHI-231. Second, Wu et al. (1996a) have demonstrated that WEHI-231 transfectants constitutively expressing c-Myc fail to undergo apoptosis upon BCR crosslinking. However, oligonucleotides carrying unmethylated CpG nucleotides have been shown to activate B cells (Krieg et al., 1995) and block BCRmediated apoptosis of WEHI-231 (Yi et al., 1996). Since antisense oligonucleotides that Fischer et al. (1994) have utilized contain unmethylated CpG, the oligonucleotides abrogate BCR-mediated negative signaling presumably due to stimulation by unmethylated CpG sequences, resulting in restoration of the cMyc level and abrogation of apoptosis. Furthermore, we demonstrate here that apoptosis is induced by deregulated c-Myc expression alone in WEHI-231. Thus, transfection with a plasmid conferring stable expression of c-Myc may induce apoptosis by itself, and only variant cells resistant to apoptosis may survive. This could be the reason why the c-Myc transfectants obtained by Wu et al. (1996a) show resistance to BCR-mediated apoptosis. To avoid obtaining variant cells resistant to apoptosis, we have kept exogenous c-Myc inactive during transfection and selection, and activated c-Myc in the transfectants by b-estradiol using the MycER chimera. Moreover, we have excluded the possibility that c-Myc activity is down-modulated due to degradation or inactivation of estradiol during the culture, resulting in apoptosis. Indeed, addition of estradiol every 12 h also markedly enhances BCR-mediated apoptosis (Figure 3b). Thus, deregulated expression of c-Myc most likely enhances but not abrogates BCR-mediated apoptosis of WEHI231 cells. BCR crosslinking induces a biphasic change in cMyc expression in WEHI-231 cells. The level of c-

Myc is markedly enhanced within 1 h and then gradually decreases (McCormack et al., 1984). After 8 h, the level becomes far below that of unstimulated cells. Since overexpression of c-Myc enhances BCRmediated apoptosis of WEHI-231, transient upregulation of c-Myc by BCR-ligation could be involved in BCR-mediated apoptosis. This is also supported by the ®nding by Scott et al. (1996) that treatment with antisense oligonucleotides for c-myc reduces the c-Myc level and blocks BCR-mediated apoptosis in primary B cells. c-Myc may thus induce transcription of the gene(s) involved in apoptosis, although cells die after the c-Myc level is downmodulated. The c-Myc induced gene(s) may trigger an apoptotic cascade taking more than 10 h or require for apoptosis co-existence of some other genes expressed after c-Myc is down-modulated. In contrast, apoptosis of WEHI-231 induced by TGF-b or an inhibitor of I-kB phosphorylation also associates with down-modulation of c-Myc (Fischer et al., 1994; Warner et al., 1992; Wu et al., 1996b). Thus, down-modulation of c-Myc in the late phase of BCR-mediated apoptosis might also play a role in the induction of apoptosis. However, the downmodulation of c-Myc is not essential for BCRmediated apoptosis, as deregulated c-Myc expression fails to block but rather enhances BCR-mediated apoptosis. Embryonic ®broblasts with deregulated c-Myc expression from wild type but not p53-de®cient mice have been shown to undergo apoptosis regardless of whether apoptosis is induced by serum deprivation or treatment with TNF together with deregulated c-Myc expression (Hermeking and Eick, 1994; Klefstrom et al., 1997; Wagner et al., 1994), indicating that c-Mycinduced apoptosis requires p53 in embryonic fibroblasts. In contrast, transgenic renal tubular cells undergo apoptosis by deregulated c-Myc in p53de®cient mice clearly indicates that a p53-independent pathway is involved in c-Myc-induced apoptosis (Trudel et al., 1997). Moreover, Rat1 ®broblasts overexpressing c-Myc undergo apoptosis in the absence of p53-mediated transactivation upon serum deprivation or treatment with TNF (Klefstrom et al., 1997; Lenahan and Ozer, 1996). These ®ndings suggest that requirement of p53 for c-Myc-induced apoptosis depends on cell types or cell lines but not the stimuli inducing apoptosis together with deregulated c-Myc expression such as serum deprivation or TNF. However, we show here that c-Myc-induced apoptosis requires p53 in WEHI-231 in the absence but not presence of BCR signaling, strongly suggesting that the p53-independent pathway can be activated by transmembrane signals such as a signal via BCR. Rat1 cells might have acquired such a pathway by yet unknown signals during their dierentiation or establishment. Future studies will elucidate the molecular mechanisms for activation of the p53independent pathway of c-Myc-induced apoptosis. Since c-Myc-induced apoptosis is suggested to counteract to its oncogenic activities (Evan and Littlewood, 1993), the elucidation of the mechanisms for c-Myc-induced apoptosis may enhance our understanding of the mechanisms for oncogenesis and the development of new therapeutic strategies for cancers in which c-Myc expression is enhanced.


Materials and methods Cell culture WEHI-231.5 is a subclone of WEHI-231 cells obtained in our laboratory. WEHI-231.5 and its transfectants were cultured in RPMI-1640 medium supplemented with 10% FCS, 50 mM 2-mercaptoethanol and 2 mM L-glutamine. For induction of apoptosis, either 1.0 or 5.06104 cells were cultured in 0.2 or 1.0 ml of phenol red-free medium, respectively, with 0.1 mM b-estradiol, 10 mg/ml of F (ab')2 fragments of anity puri®ed goat anti-mouse IgM Ab (Cappel, Durham, NC, USA) or both. After 24 to 48 h, cells were collected, and numbers of alive and dead cells were counted by trypan blue exclusion. For detection of cells containing hypodiploid nuclei characteristic for apoptosis, cells were suspended in 0.2 ml of 0.15% Triton-X in PBS, and mixed with 0.2 ml of 10 mg/ ml RNase A and 20 ml of 1 mg/ml propidium iodide. Cells were then analysed by FACSCalibur1 (Becton Dickinson, San Jose, CA, USA). Western blotting Cells (1 ± 306105) were lysed in 10 ± 50 ml of phospholysis buer (1% Triton X-100, 0.1% SDS, 0.5% sodium deoxycholate, 10 mM sodium phosphate pH 7.5, 0.1 M NaCl). Alternatively, 36106 nuclei were prepared using a homogenizer, and lysed in 30 ml of phospholysis buer. Proteins were separated by SDS ± PAGE under reducing conditions. Western blotting was done using antibodies speci®c for c-Myc (9E10), p53 (M-19, Santa Cruz Biotechnology, Santa Cruz, CA, USA), p21Cip1 (C-19, Santa Cruz Biotechnology), tubulin (TUB 2.1, Seikagaku Co., Tokyo, Japan) or the human estrogen receptor a chain (HC-20, Santa Cruz Biotechnology). Plasmids and transfection The expression plasmid pMIK-MycER was constructed by cloning the EcoRI fragment of pMV7MycER (a gift from Dr M Bishop) (Eilers et al., 1989) into EcoRI-digested pMIKNeo (a gift from Dr K Maruyama). For constructing an expression plasmid for a dominant negative form of p53 (pMIK-p53DD), we generated mutant p53 cDNA containing the oligomerization domain but not the DNA binding domain as described previously (Shaulian et al., 1992) using pLSV-Nc51 (a gift from Dr K Seno) (Eliyahu et al., 1988). p53DD has been shown to carry a strong dominant negative eect (Shaulian et al., 1992), as oligomerization of p53DD with wild type p53 disturbs proper oligomerization of the DNA binding domain required for transactivation of the target genes (Wang et al., 1995). The mutant p53 was then inserted in pMIKNeo opened by EcoRI and XhoI. WEHI231.5 cells were transfected with pMIK-MycER, pMIKp53DD or both by electroporation and transfectants were selected by 1.2 mg/ml of Geneticin (Wako Pure Chemical, Osaka, Japan). MycER transfectants were cultured in phenol red-free medium. Expression of MycER and p53DD in the transfectants were con®rmed by Western blotting. DNA binding assay Nuclear extracts were prepared as described by Wu et al. (1984) with slight modi®cation. Brie¯y, 5.06107 cells treated

with or without X-irradiation (1000 R) were harvested, washed with PBS and resuspended in 500 ml solution I (10 mM HEPES (pH 7.9), 10 mM KCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM DTT, 0.5 mM PMSF). After 5 min, cells were homogenized and centrifuged at 600 g for 5 min. Supernatants were then removed and pellets were resuspended in 2 ml of extraction buer (solution II) (10 mM HEPES (pH 7.9), 0.4 M NaCl, 1.5 mM MgCl2, 0.1 mM EGTA, 0.5 mM DTT, 0.5 mM PMSF). The crude nuclei were extracted for 30 min with continuous rotation, followed by centrifugation at 100 000 g for 50 min. The supernatants were dialyzed against solution III (10 mM HEPES (pH 7.9), 50 mM NaCl, 0.1 mM EDTA, 0.5 mM DTT, 0.5 mM PMSF) overnight. Nuclear extracts were then cleared by centrifugation at 10 000 g for 10 min. DNA binding of p53 was determined as described by Shaulian et al. (1992) with slight modi®cation. A synthetic oligonucleotide containing a palindromic high-anity binding sequence for p53 (5'-p-TCGACGGACATGCCCGGGCATGTCCG-3') (Funk et al., 1992) was self-annealed, oligomerized by a ligation kit (Takara, Kusatsu, Japan), and inserted in PicaGene basic vector (Nippon Gene, Tokyo, Japan), resulting in pPB-p53bs which contains two tandem copies of the binding sequence for p53. After restriction digestion of pPB-p53bs by NheI and HindIII, the insert containing two copies of the binding sequence was isolated, labeled by Klenow fragment of DNA polymerase I (Takara, Kusatsu, Japan) in the presence of a-[32P]dCTP (Amersham, Buckinghamshire, England). Nuclear extracts (50 mg) were pre-absorbed with protein G sepharose (Pharmacia Biotech, Uppsala, Sweden) and then mixed with 40 ng of the radiolabeled target DNA in 200 ml of binding buer (20 mM Tris-HCl (pH 7.2), 75 mM NaCl, 10% glycerol, 1% Nonidet P-40, 5 mM EDTA, 30 mg/ml poly(dI-dC), 1 mM DTT). Mixture was incubated at 48C for 90 min, and subsequently 2 mg of goat anti-p53 Ab M-19 was added. After an additional 30-min incubation at 48C, immune complexes were collected by protein G sepharose. Beads were washed twice at 48C with binding buer containing 2% glycerol and resuspended in a solution containing 1% SDS, 10 mM Tris-HCl (pH 8.0) and 2 mM EDTA. After a 10-min incubation at 378C, the beads were removed by centrifugation and the supernatants were extracted by phenol/chloroform. DNA was ethanol precipitated in the presence of 20 mg of glycogen. The labeled DNA was electrophoresed on 5% native polyacrylamide gel and visualized by autoradiography. Radioactivity of labeled DNA was quanti®ed by an image analyzer BAS25001 (Fuji Film, Tokyo, Japan).

Acknowledgements We thank Drs M Bishop, K Seno, S Miyatake, K Maruyama for plasmids. This work was supported in part by grants from the Ministry of Education, Science, Sports and Culture of Japan, the Science and Technology Agency of Japan, the Yamanouchi Foundation for Research on Metabolic Disorders, Ciba-Geigy Foundation (Japan) for the Promotion of Science, and Uehara Memorial Foundation, and the Program for Promotion of Fundamental Studies in Health Sciences of the Organization for Drug ADR Relief, R&D Promotion and Product Review of Japan.

References Adams JM, Harris AW, Pinkert CA, Corcoran LM, Alexander WS, Cory S, Palmiter RD and Brinster RL. (1985). Nature, 318, 533 ± 538. Askew DS, Ashmun RA, Simmons BC and Cleveland JL. (1991). Oncogene, 6, 1915 ± 1922.

Benhamou LE, Cazenave PA and Sarthou P. (1990). Eur. J. Immunol., 20, 1405 ± 1407. Caelles C, Helmberg A and Karin M. (1994). Nature, 370, 220 ± 223.

4097


4098

Chen CH, Zhang J and Ling CC. (1994). Radiat. Res., 139, 307 ± 315. Dalla-Favera R, Bregni M, Erikson J, Patterson D, Gallo RC and Croce CM. (1982). Proc. Natl. Acad. Sci. USA, 79, 7824 ± 7827. Dong J, Naito M and Tsuruo T. (1997). Oncogene, 15, 639 ± 647. Eilers M, Picard D, Yamamoto KR and Bishop JM. (1989). Nature, 340, 66 ± 68. Eliyahu D, Gold®nger N, Pinhasi-Kimhi O, Shaulsky G, Skurnik Y, Arai N, Rotter V and Oren M. (1988). Oncogene, 3, 313 ± 321. Evan GI and Littlewood TD. (1993). Curr. Opin. Genet. Dev., 3, 44 ± 49. Evan GI, Wyllie AH, Gilbert CS, Littlewood TD, Land H, Brooks M, Waters CM, Penn LZ and Hancock DC. (1992). Cell, 69, 119 ± 128. Fischer G, Kent SC, Joseph L, Green DR and Scott DW. (1994). J. Exp. Med., 179, 221 ± 228. Funk WD, Pak DT, Karas RH, Wright WE and Shay JW. (1992). Mol. Cell. Biol., 12, 2866 ± 2871. Galaktionov K, Chen X and Beach D. (1996). Nature, 382, 511 ± 517. Goodnow CC. (1992). Annu. Rev. Immunol., 10, 489 ± 518. Hasbold J and Klaus GG. (1990). Eur. J. Immunol., 20, 1685 ± 1690. Haupt Y, Rowan S, Shaulian E, Vousden KH and Oren M. (1995). Genes Dev., 9, 2170 ± 2183. Hermeking H and Eick D. (1994). Science, 265, 2091 ± 2093. Klefstrom J, Arighi E, Littlewood T, JaÈaÈttelaÈ M, Saksela E, Evan GI and Alitalo K. (1997). EMBO J., 16, 7382 ± 7392. Klefstrom J, Vastrik I, Saksela E, Valle J, Eilers M and Alitalo K. (1994). EMBO J., 13, 5442 ± 5450. Krieg AM, Yi AK, Matson S, Waldschmidt TJ, Bishop GA, Teasdale R, Koretzky GA and Klinman DM. (1995). Nature, 374, 546 ± 549. Lenahan MK and Ozer HL. (1996). Oncogene, 12, 1847 ± 1854.

McCormack JE, Pepe VH, Kent RB, Dean M, MarshakRothstein A and Sonenshein GE. (1984). Proc. Natl. Acad. Sci. USA, 81, 5546 ± 5550. Reisman D, Elkind NB, Roy B, Beamon J and Rotter V. (1993). Cell Growth Dier., 4, 57 ± 65. Sakamuro D, Eviner V, Elliott KJ, Showe L, White E and Prendergast GC. (1995). Oncogene, 11, 2411 ± 2418. Scott DW, Lamers M, Kohler G, Sidman CL, Maddox B and Carsetti R. (1996). Int. Immunol., 8, 1375 ± 1385. Shaulian E, Zauberman A, Ginsberg D and Oren M. (1992). Mol. Cell. Biol., 12, 5581 ± 5592. Strasser A, Harris AW, Bath ML and Cory S. (1990). Nature, 348, 331 ± 333. Trudel M, Lanoix J, Barisoni L, Blouin MJ, Desforges M, L'Italien C and D'Agati V. (1997). J. Exp. Med., 186, 1873 ± 1884. Wagner AJ, Kokontis JM and Hay N. (1994). Genes Dev., 8, 2817 ± 2830. Wang Y, Schwedes JF, Parks D, Mann K and Tegtmeyer P. (1995). Mol. Cell. Biol., 15, 2157 ± 2165. Wang XW, Vermeulen W, Coursen JD, Gibson M, Lupold SE, Forrester K, Xu G, Elmore L, Yeh H, Hoeijmakers JH and Harris CC. (1996). Genes Dev., 10, 1219 ± 1232. Warner GL, Ludlow JW, Nelson DA, Gaur A and Scott DW. (1992). Cell Growth Dier., 3, 175 ± 181. Wu C. (1984). Nature, 311, 81 ± 84. Wu M, Arsura M, Bellas RE, FitzGerald MJ, Lee H, Schauer SL, Sherr DH and Sonenshein GE. (1996a). Mol. Cell. Biol., 16, 5015 ± 5025. Wu M, Lee H, Bellas RE, Schauer SL, Arsura M, Katz D, FitzGerald MJ, Rothstein TL, Sherr DH and Sonenshein GE. (1996b). EMBO J., 15, 4682 ± 4690. Yi A-K, Hornbeck P, Lafrenz DE and Krieg AM. (1996). J. Immunol., 157, 4918 ± 4925.