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p53-independent apoptosis is induced by the p19ARF tumor suppressor
BBRC Biochemical and Biophysical Research Communications 295 (2002) 621–629 www.academicpress.com
p53-independent apoptosis is induced by the p19ARF tumor suppressor Keitaro Tsuji,a,b Kiyohisa Mizumoto,b Haruka Sudo,a Keisuke Kouyama,a Etsuro Ogata,c and Masaaki Matsuokaa,* b
a Department of Pharmacology, KEIO University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Shirokane, Minato-ku, Tokyo 108-8641, Japan c Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo 170-8455, Japan
Received 1 June 2002
Abstract p19ARF is a potent tumor suppressor. By inactivating Mdm2, p19ARF upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19ARF in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19ARF using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19ARF . In agreement, p19ARF -mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53independent pathway for p19ARF -induced apoptosis that is insensitive to inhibition by Mdm2. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: p19ARF ; p53; Mdm2; Apoptosis; Cell cycle arrest
Two tumor suppressor proteins, p19ARF and p16INK4a , are generated from the INK4a/ARF locus [1,2]. Knockout experiments have indicated that both genes antagonize the occurrence of various tumors [3–5]. p16INK4a upregulates the function of the retinoblastoma tumor suppressor protein by inhibiting cyclinD/cdk activities and induces cell cycle arrest [6]. p19ARF upregulates function of the p53 tumor suppressor protein by inhibiting Mdm2 activity [7–10]. In response to various genotoxic stress and oncogenic signals, the p53 homotetramer is activated [11]. As a transcriptional factor, p53 induces two major distinct cellular phenotypes, cell cycle arrest mediated by p21Cip1 [12] and cell death. Among broad-range transcriptional targets for p53, putative mediators for p53-induced cell death include Bax [13], Noxa [14], p53AIP [15], PUMA [16], and so on. Universal and essential mediators have not yet been identified. Mdm2, another transcriptional target for p53, is a major negative regulator for p53.
*
Corresponding author. Fax: +81-3-3359-8889. E-mail address: [email protected] (M. Matsuoka).
Mdm2 inhibits p53 activity by directly binding to it to antagonize its transcriptional activity and by enhancing its degradation. Mdm2 itself is an E3 ubiquitin ligase for p53 [17] and potentiates shuttling of p53 to cyctoplasmic proteosomes [18]. p19ARF antagonizes Mdm2 activity in two ways: it binds to and relocalizes Mdm2 in the nucleolus where Mdm2 is not able to inhibit p53 activity [19,20] and act itself as an inhibitor for Mdm2 ubiquitin ligase [21,22]. In some situations, p14ARF (the human ARF) or p19ARF upregulates p53 activity without sequestering Mdm2 in the nucleolus [23,24]. Several oncogenic signals induce expression of p19ARF , indicating that p19ARF acts as a safeguard for tumor development [25–28]. Mice nullizygous for ARF, p53, and Mdm2 develop tumors at a higher frequency than mice lacking both p53 and Mdm2 or p53 alone [29], indicating that the ARF suppresses tumor development not only by the Mdm2– p53 axis. In this relation, two studies have reported opposite conclusions. Primary mouse embryonic fibroblasts (MEFs) deficient in p53, Mdm2, and ARF genes are slowly arrested in the G1 phase by retrovirus-mediated overexpression of p19ARF while cell cycle arrest is
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not induced by reintroduction of p19ARF into cells deficient in p53 and ARF [29]. This indicated that Mdm2 has a negative effect on p53-independent cell cycle arrest induced by p19ARF . Another study demonstrated that p19ARF might act independently of p53 using MEF immortalization assays [30]. In both normal MEFs and MEFs lacking p53, expression of p19ARF antisense constructs inhibits the ability of endogenous p19ARF to induce replicative senescence, and results in cell immortalization, whereas subsequent excision of the integrated antisense vector with cre-recombinase restored the growth suppression by p19ARF . They concluded that p53-independent growth suppression by p19ARF occurred only in the presence of Mdm2. Currently, however, the precise molecular mechanism of p53-independent tumor-suppressing activity mediated by the ARF gene remains to be clarified. The ARF gene sensitizes cells to apoptosis induced by appropriate collateral signals [26,28,31–33]. Regarding p53-mediated antiproliferative activities, several studies have indicated that the regions of p53 responsible for induction of apoptosis are distinct from those responsible for cell cycle arrest [34–37]. Similarly, cell death and cell cycle arrest may be regulated by p19ARF in quite distinct manner. Here, we developed an adenovirus-mediated p19ARF expression system to analyze ARF-induced cell death. In this expression system, cells begin to die without additional cell deathinducing signals. Materials and methods Cell culture and transfection. U2OS cells (p53-intact), Saos-2 cells (p53-deficient), and HEK293 cells were obtained from ATCC. 10(1) mouse fibroblasts (p53-deficient) were provided by Dr. G.P. Zambetti (St. Jude Children’s Res. Hosp., TN). All these cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine/ml. Primary p53/ Mdm2/ARF-null mouse embryonal fibroblasts (MEFs) as well as MEFs nullizygous for ARF and p53, were and gifts from Dr. C.J. Sherr (St. Jude Children’s Res. Hosp., TN). Plasmids and adenoviral vectors. The p19ARF cDNA and the Mdm2 plasmid were provided by Dr. C.J. Sherr and Dr. M. Oren (Weizmann Institute, Israel). The system of a replication-deficient adenoviral vector, pAxCAwt, described in detail [39], was purchased from TaKaRa (Shiga, Japan). Structures of adenoviral expression cosmids for p53 and LacZ were described [38]. To express p19ARF , we used an adenovirus cre/loxP-regulated expression vector (TaKaRa, Shiga, Japan) [40]. In this vector, pAxCALNLw, a stuffer DNA fragment sandwiched by two loxP sequences, is located just upstream of p19ARF cDNA and interferes with expression of p19ARF . If an adenovirus expressing cre-recombinase (AxCANCre) is introduced into cells together, the stuffer is cut out and p19ARF begins to be expressed. To exclude the possibility that recombinant p19ARF viruses have nucleotide mutations, we PCR-amplified p19ARF DNA with pfu DNA polymerase (Strategene) and sequenced it. As a template for PCR amplification, DNA was isolated from purified viruses. To construct a adenoviral vector for Mdm2, the full-length cDNA was inserted into the SwaI site of pAxCAwt. A control cre/loxP-regulated LacZ adenovirus was purchased from TaKaRa.
Adenoviral vector-mediated expression. Recombinant adenoviruses were generated by the method described by Miyake et al. [38]. All viruses were grown in HEK293 cells and purified by double CsCl2 gradient ultracentrifugation. Concentrated viruses were stored at )80 °C. Contamination or regeneration of the wild-type adenovirus in HEK293 cells was examined by amplifying a 0.4 kb fragment from E1A region with PCR (forward-ATGAGACATATTATCTGCC ACGGAGGTGTTATTAC, reverse-CCTCTTCATCCTCGTCGTCACTGGGTGGAAAGCCA) using purified virus-derived DNAs as templates [41]. Virus titers were determined with two independent methods as described [39]. The particle numbers of purified adenoviruses were examined with a spectrophotometer at 260 nm (one absorbance unit at 260 nm is equivalent to 1:1 1012 ). Bioactive virus titers were estimated as 1/10 of these numbers, based on previous reports [42,43]. Actual bioactive virus titers were determined with the plaque assay method [38]. To correct titer variation derived from manipulation of the plaque assays and condition of HEK293 cells, we simultaneously determined titers of LacZ viruses as references, whose titers had been beforehand determined, and adjusted each virus titer accordingly. Virus titers determined by calculation of the virus particle number with a spectrophotometer at 260 nm were mostly very similar to those determined by examination of the infective virus number using the plaque assay method, except for p73 viruses, as described [39]. Infection was carried out by adding recombinant adenoviruses to serum-containing media as described [38]. Unless specified, cells (5 104 ) seeded on six-well plates were incubated with virus-containing media at the indicated multiplicity of infection (moi) at 37 °C for 60 min with constant agitation. Cell death assays and cell cycle analysis. At 48 h after infection, cells were harvested. Cell cycle determination was performed by FACS analysis with FACS Calibure from Becton–Dickinson (Franklin Lakes, NJ) as described previously [39]. Numbers of cells belonging to subG1 DNA content were determined with a MODFIT program. Apoptosis was visualized with TUNEL staining using the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics, Basel, Switzerland), that detected DNA strand breaks by terminal transferase-mediated dUTP nick-end labeling. Briefly, U2OS cells (5 104 ), seeded onto six-well plates and infected with indicated viruses, were harvested at 48 h after infection. After being fixed with 2% paraformaldehyde and permeabilized in 0.1% Triton X-100, TUNEL reaction was performed at 37 °C for 60 min. TUNEL-positive cells were visualized by FACS analysis with FACS Calibure from Becton– Dickinson (Franklin Lakes, NJ) according to manufacturer’s instructions. Measurement of cell growth. Cells (2:5 104 ) were seeded onto sixwell plates and infected with indicated viruses. Plating efficiency for Saos-2 cells is about 70–80%. At 24, 48, and 72 h after infection, cells were counted. Dead cells were determined by trypan blue exclusion. Cell fragments were not counted as trypan blue-positive cells. Numbers of total dead cells were therefore underestimated. Antibodies. A rabbit polyclonal antibody to p53 (FL-393) and a mouse monoclonal antibody to Hdm2 (SMP14) were purchased from Santa Cruz Biotech (Santa Cruz, CA). The anti-Hdm2 antibody (SMP14) cross-reacts with Mdm2 but its potency to recognize Mdm2 is much weaker. A rabbit polyclonal antibody to p19ARF (Ab80) was purchased from Abcam (Cambridge, UK). Immunoprecipitation and immunoblotting. Immunoprecipitation and immunoblotting procedures were described previously [44]. Cells were suspended at 5 106 /ml in NP40 lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.5% Nonidet P-40) containing 2.5 lg/ml of leupeptin, 5 lg/ml of aprotinin, 0.2 mM PMSF (phenylmethylsulfonyl fluoride), and 0.1 mM orthovanadate and sonicated at 4 °C. The cleared supernatants were then incubated for 2 h with indicated antibodies and precipitated for 1 h with 20 ll of 1:1 slurry of protein G–Sepharose FF/ml at 4 °C. Immunoblotted signals were visualized with an ECL detection kit from Amersham Pharmacia Biotech (Uppsala, Sweden).
K. Tsuji et al. / Biochemical and Biophysical Research Communications 295 (2002) 621–629 Immunocytochemistry. U2OS cells (5 104 ), seeded onto six-well plates and infected with adenoviruses, were fixed at 24 h after infection with 100% ethanol for 30 min at room temperature, rinsed for 45 min with PBS (phosphate-buffered saline), and then stained with the antip19ARF antibody for 60 min at 37 °C. Cells were stained with FITCconjugated goat anti-rabbit IgG (Vector Laboratory, CA) for 60 min at 37 °C. Before detection, they were washed for 45 min with PBS. Fluorescence signals were detected with a laser scanning, confocal microscope LSM (Carl Zeiss, Germany).
Results Adenovirus-mediated expression of p19ARF induces cell death in p53-intact U2OS cells
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was recognized with immunostaining procedures (Fig. 1A). Immunoblotting demonstrated that p19ARF was properly expressed and, furthermore, induced expression of endogenous p53 (Fig. 1B), indicating that ectopically expressed p19ARF is biologically active, even in human cells such as U2OS cells. Immunostaining showed that p19ARF was expressed in about 80% of U2OS cells if cells are infected with cre-ARF at a moi of 100 and cre at a moi of 80. In such a condition, cells began to die around 36 h after infection. By FACS analysis at 48 h, fraction of cells belonging to subG1 population was determined to be 21% (Fig. 2A-3). On the other hand, cells did not die if they were coinfected
To tightly regulate ectopic expression levels of p19ARF and to increase ectopic expression efficiency up to 100% of cells, the adenovirus-mediated expression system was used. With an ordinary adenovirus expression system, however, p19ARF recombinant viruses were not successfully generated, possibly because of its overwhelming toxicity to HEK293 host cells during expansion of viruses. With an adenovirus cre/loxP-regulated expression system, a salvage method, we successfully obtained a cre/ loxP-regulated p19ARF recombinant virus (designated creARF). By sequencing the full p19ARF DNA PCR-amplified from recombinant cre-ARF viruses-derived DNA, we confirmed that there were no mutated nucleotides. To examine whether the adenovirus cre/loxP system works properly for expression of p19ARF , we coinfected U2OS cells, ARF-deficient cells, with the cre-ARF viruses and the cre-recombinase-expressing viruses (designated cre viruses) and monitored expression of p19ARF by two methods (Fig. 1). Nucleolar localization of p19ARF , the well-known unique subcellular localization,
Fig. 1. Adenovirus cre/loxP-regulated expression of p19ARF in U2OS cells. (A) U2OS cells (5 104 ), seeded onto six-well plates and infected with cre-ARF viruses at a moi of 100 and cre viruses at moi of 80, were fixed at 24 h after infection. Subcellular localization of p19ARF was examined with a laser scanning, confocal microscope LSM. (B) U2OS cells (5 104 ), seeded onto six-well plates and infected with cre-ARF viruses at a moi of 100 and cre viruses at a moi of 80, or control viruses at indicated mois, were harvested at 24 h for immunoblotting with antip19ARF antibody (upper) and anti-p53 antibody (lower). LZ indicates the ordinary LacZ viruses.
Fig. 2. p19ARF -induced cell death (apoptosis) in U2OS cells. (A) U2OS cells, coinfected with cre-ARF viruses at a moi of 100 and cre viruses at a moi of 80 (panel 3), or control viruses at indicated mois (panels 1 and 2), were trypsinized and stained with PI for FACS analysis at 48 h. (B) U2OS cells, not infected (panel 1), or coinfected with cre-ARF viruses at a moi of 100 or 50, and cre viruses at moi of 80 (panels 2 and 3), or control viruses at indicated mois (panels 4 and 5), were harvested for TUNEL assays. The vertical and the horizontal axes indicate FSC and FL1-H, respectively. Dots shifted rightward to higher FL1-H indicated cells stained with TUNEL.
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with cre-ARF together with LacZ viruses in place of cre viruses (Fig. 2A-1). This was also the case if LacZ (or creLacZ) and cre viruses were used for coinfection (Fig. 2A2), indicating that cells were dead because p19ARF was toxic, but not because virus infection itself nor overexpression itself of a gene was toxic. Furthermore, TUNEL assays demonstrated that p19ARF -induced cell death is apoptosis (Fig. 2B). Because the p53 gene is intact in U2OS cells, it could be assumed that induction of p53 expression contributes to the death of U2OS cells. Adenovirus-mediated expression of p19ARF induces cell death in p53-deficient Saos-2 cells To test for p53-independent pathways in p19ARF -mediated cell death, we used Saos-2 cells for adenoviral infection in which p53 is deleted. We coinfected Saos-2 cells with cre-ARF and cre viruses and confirmed that p19ARF could also be expressed in Saos-2 cells by immunostaining (data not shown) and immunoblotting (Fig. 3A). Because Saos-2 cells were relatively insensitive to adenoviral infection as compared with U2OS cells, four times more viruses than those used for U2OS cells, were needed to express p19ARF in 80% of Saos-2 cells (data not shown). A few Saos-2 cells tend to die naturally. Without any insult, 3–5% of cells is considered to be dead by estimation of the subG1 fraction with FACS analysis (Fig. 3B). As observed in U2OS cells, death of Saos-2 cells was induced in a p19ARF -dependent manner (Figs. 3B and C). At 48 h after coinfection of Saos-2 cells with creARF at a moi of 400 and cre at a moi of 160, the percentage of cells belonging to subG1 area was increased up to 18.6% (Fig. 3B). Death of Saos-2 cells induced by p19ARF was increased proportionally to expression level of p19ARF (data not shown). p19ARF -induced cell death of Saos-2 cells was considered to be apoptosis and 12.4% of total cells was TUNEL-positive whereas fewer than 4% of control cells were TUNEL-positive (Fig. 4A). In addition to these cell death assays, cell growth as well as cell death was monitored by counting live cells and trypan blue-positive cells at 24, 48, and 72 h after coinfection of Saos-2 cells with cre-ARF and cre viruses, or control viruses (Fig. 4B). At 24 h, both live cell numbers and trypan blue-positive cell numbers are very similar among the three types of cells. At 48 h, however, increase of trypan blue-positive cells and decrease of live cells were observed only for cells coinfected with cre-ARF and cre viruses. From 48 to 72 h, a remarkable change of live cell numbers and trypan blue-positive cell numbers was not observed for cells coinfected with cre-ARF and cre viruses. Apparently, however, numbers of cell fragments increased (data not shown). Cell fragments were not counted as trypan blue-positive cells in this assay. Based on these findings, we concluded that death of Saos-2 cells was induced by p19ARF . Meanwhile, we were not sure whether cell cycle arrest was induced by p19ARF .
Fig. 3. p19ARF -induced cell death in Saos-2 cells. (A) Saos-2 cells (5 104 ), seeded on six-well plates, were coinfected with indicated mois of cre-ARF and cre viruses, or control viruses. At 48 h after infection, cells were harvested for immunoblotting with anti-p19ARF antibody. (B and C) Saos-2 cells, coinfected with cre-ARF viruses at a moi of 400 and cre viruses at moi of 160 (3), or control viruses at indicated mois (1, 2), were trypsinized and stained with PI for FACS analysis at 48 h. DNA histograms are shown for each preparation in (B). The sub-G1 cells were considered dead. In (C), percentages of dead cells were determined by another experiment with N ¼ 3. c-ARF and c-LZ indicate cre-ARF and cre-LacZ viruses.
To rule out that this p19ARF -induced p53-independent cell death is particular to Saos-2 cells, we next examined the 10(1) mouse fibroblasts, another p53-deficient cell line [28] with the same assays. We found that cell death was induced in a p19ARF -dependent manner even in 10(1) cells, although the magnitude is relatively small (data not shown). p19ARF induces cell death in MEFs nullizygous for ARF and p53 To examine whether p19ARF -induced p53-independent cell death occurs in the presence or absence of
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Fig. 4. p19ARF -induced cell death in Saos-2 cells is apoptosis. (A) Saos-2 cells (5:0 104 ), seeded on six-well plates, were infected with indicated mois of cre-ARF, cre, LacZ, or cre-LacZ viruses. At 48 h after infection, cells were analyzed with TUNEL reaction using FACS. The vertical and the horizontal axes indicate cell numbers and FL1-H, respectively. Cells shifted to higher FLI-H represent TUNEL-positive cells. (B) Saos-2 cells (2:5 104 ), seeded on six-well plates, were coinfected with indicated mois of cre-ARF, cre, LacZ, or cre-LacZ viruses. At 24, 48, and 72 h, number of live cells and trypan blue-positive cells were counted in 0.4% trypan blue-containing PBS. This experiment was performed at 24 h with N ¼ 1 and at 48 h as well as at 72 h with N ¼ 3. Cell fragments were not counted as trypan blue-positive cells in this assay.
Mdm2, we used MEFs, derived from mice nullizygous for ARF and p53, for the adenovirus-mediated expression of p19ARF . Immunostaining has indicated that we needed cre-ARF viruses at a moi of 800 and cre viruses at a moi of 320 to express p19ARF in 80% of MEFs (data not shown). It has been well known that the original hosts of adenovirus used for this expression system are human cells and mouse-derived cells are usually resistant to infection. At 48 h after coinfection of MEFs with cre-ARF viruses and cre viruses, or control viruses, the percentage of TUNEL-positive cells was examined. If p19ARF was expressed, the percentage of TUNEL-positive cells increased to 12.6% (Fig. 5A, 1) while without expression of p19ARF , it was fewer than 5% (Fig. 5A, 2 and 3). Considering that fewer than 0.5% of MEFs not infected with adenoviruses are TUNEL-positive (data not shown), we concluded that adenoviral infection itself is a little toxic to MEFs (Fig. 5A, 2 and 3). However, expression of p19ARF significantly increased the percentage of TUNEL-positive cells. FACS analysis also indicated that cell number belonging to subG1 fraction significantly increased when p19ARF was expressed (data not shown). These findings indicated that cell death was induced in a p19ARF -dependent manner, even in MEFs deficient in both ARF and p53. In a similar fashion, expression of p19ARF induced apoptosis of MEFs nullizygous for ARF, Mdm2, and p53 (Fig. 5B). Thus, we concluded that p19ARF -induced p53-independent apoptosis occurs, irrespective of Mdm2 status with the adenovirus-mediated expression system.
Coexpression of Mdm2 inhibits p19ARF -induced apoptosis in U2OS cells Mdm2 inhibits p53 activities [19–22]. We then asked how ectopic expression of Mdm2 alters p19ARF -induced apoptosis in both p53-intact and p53-deficient cells. To address this question, we developed an adenovirus vector to express Mdm2. Adenovirus-mediated coexpression of Mdm2 remarkably, but not completely, inhibits p19ARF -induced apoptosis in U2OS cells (Fig. 6A). If we observed that p53-induced apoptosis was almost completely inhibited by coexpression of Mdm2 (Fig. 6B), we could speculate that there is a p53-independent pathway insensitive to inhibition by Mdm2 in U2OS cells. Note that expression of endogenous Hdm2 is upregulated with expression of the ARF genes, as previously observed (Fig. 6C) [10,23]. Ectopic expression of Mdm2 suppressed expression of endogenous Hdm2 through inactivation of p53, a transcriptional activator for endogenous Hdm2 (Fig. 6C). In contrast, ectopic expression of Mdm2 did not inhibit p19ARF -induced apoptosis in Saos-2 (Fig. 6D), suggesting that the p53-independent pathways for p19ARF -induced apoptosis is insensitive to inhibition by Mdm2.
Discussion Cell cycle arrest and cell death have been examined as two major phenotypes of the tumor-suppressing genes. Induction of cell cycle arrest has been generally moni-
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Fig. 5. p19ARF -induced cell death in p53/ARF-null MEFs (A) and p19ARF -induced cell death in p53/Mdm2/ARF-null MEFs (B). MEFs (5 104 ) in passage 5–8, seeded on six-well plates, were coinfected with indicated mois of cre-ARF, cre, LacZ, or cre-LacZ viruses. At 48 h after infection, cells were harvested for TUNEL assays. The vertical and the horizontal axes indicate FSC and FL1-H, respectively. Dots shifted rightward to higher FL1H indicated TUNEL-positive cells.
tored as a tumor-suppressing phenotype of the ARF. On the contrary, induction of cell death by p19ARF has been rarely monitored because p19ARF -induced cell death needs associated cell death-inducing insults [26,28,31– 33]. Extensive works have established that the functional domains of the p53 tumor suppressor responsible for cell cycle arrest and cell death are not identical [34–37], urging us to examine both tumor-suppressing phenotypes to know whole activities of the ARF tumor suppressor. By adenovirus-mediated expression of p19ARF , apoptosis can be induced without additional signals, indicating that p19ARF -induced cell death can be easily examined with this system. Adenoviral infection itself seems to be a little toxic to cells [45]. This explains why we could easily see cell death with the adenovirus-mediated expression of tumor suppressor genes. However, infection at higher mois, up to 1600, itself never induced death of established cell lines such as U2OS, Saos-2, and 10(1) at least within 48 h [39]. Furthermore, coexpression of Mdm2 inhibited p19ARF -induced cell death in p53-intact U2OS cells (Fig. 6). This findings strongly support the idea that p19ARF -induced cell death is not an
artificial outcome evoked by the adenovirus-mediated overexpression. Emerging evidence has indicated that there are p53independent tumor-suppressing pathways mediated by the ARF tumor suppressor [24,29,30]. Slowly occurring cell cycle arrest was induced by overexpression of p19ARF in MEFs lacking ARF, p53, and Mdm2 [29]. Cell cycle arrest was not induced by reintroduction of p19ARF in MEFs deficient in ARF and p53, indicating that Mdm2 has a negative effect on p53-independent cell cycle arrest induced by p19ARF . In another study [30], p19ARF inhibited immortalization of p53-deficient MEFs only in the presence of Mdm2. They claimed that ARF exerts p53-independent tumor-suppressing activity through Mdm2 and Rb. With adenovirus-mediated expression system, p19ARF -induced p53-independent apoptosis was not inhibited by coexpression of Mdm2 (Fig. 6), indicating that the p53-independent p19ARF induced pathway is insensitive to inhibition by Mdm2. Downstream molecular mediators for p19ARF -induced apoptosis may be different from those for p19ARF induced cell cycle arrest. p21Cip1 , a transcriptional target
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tion, are the ones of possible downstream mediators. p19ARF sequestered E2Fs in the nucleolus and predisposes them to degradation, inhibiting the function of E2Fs for the G1-S transition [49]. From a standpoint of apoptosis, however, E2Fs are not considered to be downstream mediators for p19ARF because increase, but not decrease, of expression of E2Fs induces apoptosis. To totally understand the role of the ARF tumor suppressor, we need to identify the actual downstream mediators of p19ARF for both apoptosis and cell cycle, other than Mdm2 and p53.
Acknowledgments Fig. 6. p19ARF -induced cell death is partially inhibited by coexpression of Mdm2. (A) U2OS cells, not infected (lane 1), or coinfected with indicated viruses at indicated mois, were trypsinized and stained with PI for FACS analysis at 48 h. ARF () indicates coinfection with creARF viruses at a moi of 200 and cre-viruses at a moi of 80. Mdm2 and LZ indicate the Mdm2 viruses and the ordinary LacZ viruses. Numbers 2 and 4 indicate mois of 200 and 400. (B) U2OS cells, not infected, or coinfected with indicated viruses at indicated mois, were trypsinized and stained with PI for FACS analysis at 48 h. p53 () indicates infection with p53 viruses at a moi of 50. Mdm2 and LZ indicates the Mdm2 viruses and the ordinary LacZ viruses. Numbers 2 and 4 indicate mois of 200 and 400. (C) U2OS cells, not infected, or coinfected with indicated viruses at indicated mois, were harvested at 24 h for immunoblotting with anti-p19ARF antibody (upper), anti-p53 antibody (middle), and anti-Mdm2 antibody (lower). ARF () indicate coinfection with creARF viruses at a moi of 100 and cre-viruses at a moi of 80. Mdm2 and LZ indicates the Mdm2 viruses and the ordinary LacZ viruses. Numbers 1 and 2 indicate mois of 100 and 200. (D) Saos2 cells, not infected, or coinfected with indicated viruses at indicated mois, were trypsinized and stained with PI for FACS analysis at 48 h. ARF () indicates coinfection with creARF viruses at a moi of 400 and cre-viruses at a moi of 160. Mdm2 and LZ indicates the Mdm2 viruses and the ordinary LacZ viruses. Numbers 4, 8, and 12 indicate mois of 400, 800, and 1200.
for p53, mediates p19ARF -induced cell cycle arrest in p53-intact cells [12]. However, p19ARF -induced p21Cip1 independent cell cycle arrest has also been observed [24,29]. Many candidates for p53-mediated apoptosis have been identified [13–16], although universal mediators for p53 have not identified. Currently, we do not know what the downstream mediators are for the p53independent apoptosis mediated by p19ARF . p73 is not a main mediator, at least for the p53-independent pathways in Saos-2 cells because coexpression of p73DD, a dominant-negative form for p73, does not inhibit p19ARF -induced apoptosis (unpublished observation by K.T. and M.M.). Several putative functional interactors with p19ARF have been characterized. They include some E2Fs [46], DNA topoisomerase [47], pex19p [48], Spinophilin/ Neurabin II as a type 1 protein-phosphatase-binding protein [49], and hypoxia-inducible factor-1a [50]. So far, E2Fs, trancription factors involved in G1-S transi-
We are indebted to Tomo Yoshida, Kazumi Nishihara, Kouichi Tsuchiya, and Dovie Wylie for expert technical assistance. This work is supported in part by the charitable trust ARAKI grant. We are grateful to Dr. Kyoji Ikeda for helpful advice, to Dr. Charles J. Sherr, Dr. Martine Roussel, and Dr. Moshe Oren for the p19ARF plasmid, TKO MEFs, and DKO MEFs, and to Dr. G.P. Zambetti for 10(1) cells. This work has been completed with extensive help and beneficial criticism by Dr. Ikuo Nishimoto.
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p53-independent apoptosis is induced by the p19ARF tumor suppressor Keitaro Tsuji,a,b Kiyohisa Mizumoto,b Haruka Sudo,a Keisuke Kouyama,a Etsuro Ogata,c and Masaaki Matsuokaa,* b
a Department of Pharmacology, KEIO University School of Medicine, 35 Shinanomachi, Shinjuku-ku, Tokyo 160-8582, Japan Department of Biochemistry, School of Pharmaceutical Sciences, Kitasato University, Shirokane, Minato-ku, Tokyo 108-8641, Japan c Cancer Institute Hospital, Japanese Foundation for Cancer Research, Tokyo 170-8455, Japan
Received 1 June 2002
Abstract p19ARF is a potent tumor suppressor. By inactivating Mdm2, p19ARF upregulates p53 activities to induce cell cycle arrest and sensitize cells to apoptosis in the presence of collateral signals. It has also been demonstrated that cell cycle arrest is induced by overexpressed p19ARF in p53-deficient mouse embryonic fibroblasts, only in the absence of the Mdm2 gene. Here, we show that apoptosis can be induced without additional apoptosis signals by expression of p19ARF using an adenovirus-mediated expression system in p53-intact cell lines as well as p53-deficient cell lines. Also, in primary mouse embryonic fibroblasts (MEFs) lacking p53/ ARF, p53-independent apoptosis is induced irrespective of Mdm2 status by expression of p19ARF . In agreement, p19ARF -mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53independent pathway for p19ARF -induced apoptosis that is insensitive to inhibition by Mdm2. Ó 2002 Elsevier Science (USA). All rights reserved. Keywords: p19ARF ; p53; Mdm2; Apoptosis; Cell cycle arrest
Two tumor suppressor proteins, p19ARF and p16INK4a , are generated from the INK4a/ARF locus [1,2]. Knockout experiments have indicated that both genes antagonize the occurrence of various tumors [3–5]. p16INK4a upregulates the function of the retinoblastoma tumor suppressor protein by inhibiting cyclinD/cdk activities and induces cell cycle arrest [6]. p19ARF upregulates function of the p53 tumor suppressor protein by inhibiting Mdm2 activity [7–10]. In response to various genotoxic stress and oncogenic signals, the p53 homotetramer is activated [11]. As a transcriptional factor, p53 induces two major distinct cellular phenotypes, cell cycle arrest mediated by p21Cip1 [12] and cell death. Among broad-range transcriptional targets for p53, putative mediators for p53-induced cell death include Bax [13], Noxa [14], p53AIP [15], PUMA [16], and so on. Universal and essential mediators have not yet been identified. Mdm2, another transcriptional target for p53, is a major negative regulator for p53.
*
Corresponding author. Fax: +81-3-3359-8889. E-mail address: [email protected] (M. Matsuoka).
Mdm2 inhibits p53 activity by directly binding to it to antagonize its transcriptional activity and by enhancing its degradation. Mdm2 itself is an E3 ubiquitin ligase for p53 [17] and potentiates shuttling of p53 to cyctoplasmic proteosomes [18]. p19ARF antagonizes Mdm2 activity in two ways: it binds to and relocalizes Mdm2 in the nucleolus where Mdm2 is not able to inhibit p53 activity [19,20] and act itself as an inhibitor for Mdm2 ubiquitin ligase [21,22]. In some situations, p14ARF (the human ARF) or p19ARF upregulates p53 activity without sequestering Mdm2 in the nucleolus [23,24]. Several oncogenic signals induce expression of p19ARF , indicating that p19ARF acts as a safeguard for tumor development [25–28]. Mice nullizygous for ARF, p53, and Mdm2 develop tumors at a higher frequency than mice lacking both p53 and Mdm2 or p53 alone [29], indicating that the ARF suppresses tumor development not only by the Mdm2– p53 axis. In this relation, two studies have reported opposite conclusions. Primary mouse embryonic fibroblasts (MEFs) deficient in p53, Mdm2, and ARF genes are slowly arrested in the G1 phase by retrovirus-mediated overexpression of p19ARF while cell cycle arrest is
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not induced by reintroduction of p19ARF into cells deficient in p53 and ARF [29]. This indicated that Mdm2 has a negative effect on p53-independent cell cycle arrest induced by p19ARF . Another study demonstrated that p19ARF might act independently of p53 using MEF immortalization assays [30]. In both normal MEFs and MEFs lacking p53, expression of p19ARF antisense constructs inhibits the ability of endogenous p19ARF to induce replicative senescence, and results in cell immortalization, whereas subsequent excision of the integrated antisense vector with cre-recombinase restored the growth suppression by p19ARF . They concluded that p53-independent growth suppression by p19ARF occurred only in the presence of Mdm2. Currently, however, the precise molecular mechanism of p53-independent tumor-suppressing activity mediated by the ARF gene remains to be clarified. The ARF gene sensitizes cells to apoptosis induced by appropriate collateral signals [26,28,31–33]. Regarding p53-mediated antiproliferative activities, several studies have indicated that the regions of p53 responsible for induction of apoptosis are distinct from those responsible for cell cycle arrest [34–37]. Similarly, cell death and cell cycle arrest may be regulated by p19ARF in quite distinct manner. Here, we developed an adenovirus-mediated p19ARF expression system to analyze ARF-induced cell death. In this expression system, cells begin to die without additional cell deathinducing signals. Materials and methods Cell culture and transfection. U2OS cells (p53-intact), Saos-2 cells (p53-deficient), and HEK293 cells were obtained from ATCC. 10(1) mouse fibroblasts (p53-deficient) were provided by Dr. G.P. Zambetti (St. Jude Children’s Res. Hosp., TN). All these cells were grown in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 2 mM glutamine/ml. Primary p53/ Mdm2/ARF-null mouse embryonal fibroblasts (MEFs) as well as MEFs nullizygous for ARF and p53, were and gifts from Dr. C.J. Sherr (St. Jude Children’s Res. Hosp., TN). Plasmids and adenoviral vectors. The p19ARF cDNA and the Mdm2 plasmid were provided by Dr. C.J. Sherr and Dr. M. Oren (Weizmann Institute, Israel). The system of a replication-deficient adenoviral vector, pAxCAwt, described in detail [39], was purchased from TaKaRa (Shiga, Japan). Structures of adenoviral expression cosmids for p53 and LacZ were described [38]. To express p19ARF , we used an adenovirus cre/loxP-regulated expression vector (TaKaRa, Shiga, Japan) [40]. In this vector, pAxCALNLw, a stuffer DNA fragment sandwiched by two loxP sequences, is located just upstream of p19ARF cDNA and interferes with expression of p19ARF . If an adenovirus expressing cre-recombinase (AxCANCre) is introduced into cells together, the stuffer is cut out and p19ARF begins to be expressed. To exclude the possibility that recombinant p19ARF viruses have nucleotide mutations, we PCR-amplified p19ARF DNA with pfu DNA polymerase (Strategene) and sequenced it. As a template for PCR amplification, DNA was isolated from purified viruses. To construct a adenoviral vector for Mdm2, the full-length cDNA was inserted into the SwaI site of pAxCAwt. A control cre/loxP-regulated LacZ adenovirus was purchased from TaKaRa.
Adenoviral vector-mediated expression. Recombinant adenoviruses were generated by the method described by Miyake et al. [38]. All viruses were grown in HEK293 cells and purified by double CsCl2 gradient ultracentrifugation. Concentrated viruses were stored at )80 °C. Contamination or regeneration of the wild-type adenovirus in HEK293 cells was examined by amplifying a 0.4 kb fragment from E1A region with PCR (forward-ATGAGACATATTATCTGCC ACGGAGGTGTTATTAC, reverse-CCTCTTCATCCTCGTCGTCACTGGGTGGAAAGCCA) using purified virus-derived DNAs as templates [41]. Virus titers were determined with two independent methods as described [39]. The particle numbers of purified adenoviruses were examined with a spectrophotometer at 260 nm (one absorbance unit at 260 nm is equivalent to 1:1 1012 ). Bioactive virus titers were estimated as 1/10 of these numbers, based on previous reports [42,43]. Actual bioactive virus titers were determined with the plaque assay method [38]. To correct titer variation derived from manipulation of the plaque assays and condition of HEK293 cells, we simultaneously determined titers of LacZ viruses as references, whose titers had been beforehand determined, and adjusted each virus titer accordingly. Virus titers determined by calculation of the virus particle number with a spectrophotometer at 260 nm were mostly very similar to those determined by examination of the infective virus number using the plaque assay method, except for p73 viruses, as described [39]. Infection was carried out by adding recombinant adenoviruses to serum-containing media as described [38]. Unless specified, cells (5 104 ) seeded on six-well plates were incubated with virus-containing media at the indicated multiplicity of infection (moi) at 37 °C for 60 min with constant agitation. Cell death assays and cell cycle analysis. At 48 h after infection, cells were harvested. Cell cycle determination was performed by FACS analysis with FACS Calibure from Becton–Dickinson (Franklin Lakes, NJ) as described previously [39]. Numbers of cells belonging to subG1 DNA content were determined with a MODFIT program. Apoptosis was visualized with TUNEL staining using the In Situ Cell Death Detection Kit, Fluorescein (Roche Diagnostics, Basel, Switzerland), that detected DNA strand breaks by terminal transferase-mediated dUTP nick-end labeling. Briefly, U2OS cells (5 104 ), seeded onto six-well plates and infected with indicated viruses, were harvested at 48 h after infection. After being fixed with 2% paraformaldehyde and permeabilized in 0.1% Triton X-100, TUNEL reaction was performed at 37 °C for 60 min. TUNEL-positive cells were visualized by FACS analysis with FACS Calibure from Becton– Dickinson (Franklin Lakes, NJ) according to manufacturer’s instructions. Measurement of cell growth. Cells (2:5 104 ) were seeded onto sixwell plates and infected with indicated viruses. Plating efficiency for Saos-2 cells is about 70–80%. At 24, 48, and 72 h after infection, cells were counted. Dead cells were determined by trypan blue exclusion. Cell fragments were not counted as trypan blue-positive cells. Numbers of total dead cells were therefore underestimated. Antibodies. A rabbit polyclonal antibody to p53 (FL-393) and a mouse monoclonal antibody to Hdm2 (SMP14) were purchased from Santa Cruz Biotech (Santa Cruz, CA). The anti-Hdm2 antibody (SMP14) cross-reacts with Mdm2 but its potency to recognize Mdm2 is much weaker. A rabbit polyclonal antibody to p19ARF (Ab80) was purchased from Abcam (Cambridge, UK). Immunoprecipitation and immunoblotting. Immunoprecipitation and immunoblotting procedures were described previously [44]. Cells were suspended at 5 106 /ml in NP40 lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 1 mM DTT, and 0.5% Nonidet P-40) containing 2.5 lg/ml of leupeptin, 5 lg/ml of aprotinin, 0.2 mM PMSF (phenylmethylsulfonyl fluoride), and 0.1 mM orthovanadate and sonicated at 4 °C. The cleared supernatants were then incubated for 2 h with indicated antibodies and precipitated for 1 h with 20 ll of 1:1 slurry of protein G–Sepharose FF/ml at 4 °C. Immunoblotted signals were visualized with an ECL detection kit from Amersham Pharmacia Biotech (Uppsala, Sweden).
K. Tsuji et al. / Biochemical and Biophysical Research Communications 295 (2002) 621–629 Immunocytochemistry. U2OS cells (5 104 ), seeded onto six-well plates and infected with adenoviruses, were fixed at 24 h after infection with 100% ethanol for 30 min at room temperature, rinsed for 45 min with PBS (phosphate-buffered saline), and then stained with the antip19ARF antibody for 60 min at 37 °C. Cells were stained with FITCconjugated goat anti-rabbit IgG (Vector Laboratory, CA) for 60 min at 37 °C. Before detection, they were washed for 45 min with PBS. Fluorescence signals were detected with a laser scanning, confocal microscope LSM (Carl Zeiss, Germany).
Results Adenovirus-mediated expression of p19ARF induces cell death in p53-intact U2OS cells
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was recognized with immunostaining procedures (Fig. 1A). Immunoblotting demonstrated that p19ARF was properly expressed and, furthermore, induced expression of endogenous p53 (Fig. 1B), indicating that ectopically expressed p19ARF is biologically active, even in human cells such as U2OS cells. Immunostaining showed that p19ARF was expressed in about 80% of U2OS cells if cells are infected with cre-ARF at a moi of 100 and cre at a moi of 80. In such a condition, cells began to die around 36 h after infection. By FACS analysis at 48 h, fraction of cells belonging to subG1 population was determined to be 21% (Fig. 2A-3). On the other hand, cells did not die if they were coinfected
To tightly regulate ectopic expression levels of p19ARF and to increase ectopic expression efficiency up to 100% of cells, the adenovirus-mediated expression system was used. With an ordinary adenovirus expression system, however, p19ARF recombinant viruses were not successfully generated, possibly because of its overwhelming toxicity to HEK293 host cells during expansion of viruses. With an adenovirus cre/loxP-regulated expression system, a salvage method, we successfully obtained a cre/ loxP-regulated p19ARF recombinant virus (designated creARF). By sequencing the full p19ARF DNA PCR-amplified from recombinant cre-ARF viruses-derived DNA, we confirmed that there were no mutated nucleotides. To examine whether the adenovirus cre/loxP system works properly for expression of p19ARF , we coinfected U2OS cells, ARF-deficient cells, with the cre-ARF viruses and the cre-recombinase-expressing viruses (designated cre viruses) and monitored expression of p19ARF by two methods (Fig. 1). Nucleolar localization of p19ARF , the well-known unique subcellular localization,
Fig. 1. Adenovirus cre/loxP-regulated expression of p19ARF in U2OS cells. (A) U2OS cells (5 104 ), seeded onto six-well plates and infected with cre-ARF viruses at a moi of 100 and cre viruses at moi of 80, were fixed at 24 h after infection. Subcellular localization of p19ARF was examined with a laser scanning, confocal microscope LSM. (B) U2OS cells (5 104 ), seeded onto six-well plates and infected with cre-ARF viruses at a moi of 100 and cre viruses at a moi of 80, or control viruses at indicated mois, were harvested at 24 h for immunoblotting with antip19ARF antibody (upper) and anti-p53 antibody (lower). LZ indicates the ordinary LacZ viruses.
Fig. 2. p19ARF -induced cell death (apoptosis) in U2OS cells. (A) U2OS cells, coinfected with cre-ARF viruses at a moi of 100 and cre viruses at a moi of 80 (panel 3), or control viruses at indicated mois (panels 1 and 2), were trypsinized and stained with PI for FACS analysis at 48 h. (B) U2OS cells, not infected (panel 1), or coinfected with cre-ARF viruses at a moi of 100 or 50, and cre viruses at moi of 80 (panels 2 and 3), or control viruses at indicated mois (panels 4 and 5), were harvested for TUNEL assays. The vertical and the horizontal axes indicate FSC and FL1-H, respectively. Dots shifted rightward to higher FL1-H indicated cells stained with TUNEL.
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with cre-ARF together with LacZ viruses in place of cre viruses (Fig. 2A-1). This was also the case if LacZ (or creLacZ) and cre viruses were used for coinfection (Fig. 2A2), indicating that cells were dead because p19ARF was toxic, but not because virus infection itself nor overexpression itself of a gene was toxic. Furthermore, TUNEL assays demonstrated that p19ARF -induced cell death is apoptosis (Fig. 2B). Because the p53 gene is intact in U2OS cells, it could be assumed that induction of p53 expression contributes to the death of U2OS cells. Adenovirus-mediated expression of p19ARF induces cell death in p53-deficient Saos-2 cells To test for p53-independent pathways in p19ARF -mediated cell death, we used Saos-2 cells for adenoviral infection in which p53 is deleted. We coinfected Saos-2 cells with cre-ARF and cre viruses and confirmed that p19ARF could also be expressed in Saos-2 cells by immunostaining (data not shown) and immunoblotting (Fig. 3A). Because Saos-2 cells were relatively insensitive to adenoviral infection as compared with U2OS cells, four times more viruses than those used for U2OS cells, were needed to express p19ARF in 80% of Saos-2 cells (data not shown). A few Saos-2 cells tend to die naturally. Without any insult, 3–5% of cells is considered to be dead by estimation of the subG1 fraction with FACS analysis (Fig. 3B). As observed in U2OS cells, death of Saos-2 cells was induced in a p19ARF -dependent manner (Figs. 3B and C). At 48 h after coinfection of Saos-2 cells with creARF at a moi of 400 and cre at a moi of 160, the percentage of cells belonging to subG1 area was increased up to 18.6% (Fig. 3B). Death of Saos-2 cells induced by p19ARF was increased proportionally to expression level of p19ARF (data not shown). p19ARF -induced cell death of Saos-2 cells was considered to be apoptosis and 12.4% of total cells was TUNEL-positive whereas fewer than 4% of control cells were TUNEL-positive (Fig. 4A). In addition to these cell death assays, cell growth as well as cell death was monitored by counting live cells and trypan blue-positive cells at 24, 48, and 72 h after coinfection of Saos-2 cells with cre-ARF and cre viruses, or control viruses (Fig. 4B). At 24 h, both live cell numbers and trypan blue-positive cell numbers are very similar among the three types of cells. At 48 h, however, increase of trypan blue-positive cells and decrease of live cells were observed only for cells coinfected with cre-ARF and cre viruses. From 48 to 72 h, a remarkable change of live cell numbers and trypan blue-positive cell numbers was not observed for cells coinfected with cre-ARF and cre viruses. Apparently, however, numbers of cell fragments increased (data not shown). Cell fragments were not counted as trypan blue-positive cells in this assay. Based on these findings, we concluded that death of Saos-2 cells was induced by p19ARF . Meanwhile, we were not sure whether cell cycle arrest was induced by p19ARF .
Fig. 3. p19ARF -induced cell death in Saos-2 cells. (A) Saos-2 cells (5 104 ), seeded on six-well plates, were coinfected with indicated mois of cre-ARF and cre viruses, or control viruses. At 48 h after infection, cells were harvested for immunoblotting with anti-p19ARF antibody. (B and C) Saos-2 cells, coinfected with cre-ARF viruses at a moi of 400 and cre viruses at moi of 160 (3), or control viruses at indicated mois (1, 2), were trypsinized and stained with PI for FACS analysis at 48 h. DNA histograms are shown for each preparation in (B). The sub-G1 cells were considered dead. In (C), percentages of dead cells were determined by another experiment with N ¼ 3. c-ARF and c-LZ indicate cre-ARF and cre-LacZ viruses.
To rule out that this p19ARF -induced p53-independent cell death is particular to Saos-2 cells, we next examined the 10(1) mouse fibroblasts, another p53-deficient cell line [28] with the same assays. We found that cell death was induced in a p19ARF -dependent manner even in 10(1) cells, although the magnitude is relatively small (data not shown). p19ARF induces cell death in MEFs nullizygous for ARF and p53 To examine whether p19ARF -induced p53-independent cell death occurs in the presence or absence of
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Fig. 4. p19ARF -induced cell death in Saos-2 cells is apoptosis. (A) Saos-2 cells (5:0 104 ), seeded on six-well plates, were infected with indicated mois of cre-ARF, cre, LacZ, or cre-LacZ viruses. At 48 h after infection, cells were analyzed with TUNEL reaction using FACS. The vertical and the horizontal axes indicate cell numbers and FL1-H, respectively. Cells shifted to higher FLI-H represent TUNEL-positive cells. (B) Saos-2 cells (2:5 104 ), seeded on six-well plates, were coinfected with indicated mois of cre-ARF, cre, LacZ, or cre-LacZ viruses. At 24, 48, and 72 h, number of live cells and trypan blue-positive cells were counted in 0.4% trypan blue-containing PBS. This experiment was performed at 24 h with N ¼ 1 and at 48 h as well as at 72 h with N ¼ 3. Cell fragments were not counted as trypan blue-positive cells in this assay.
Mdm2, we used MEFs, derived from mice nullizygous for ARF and p53, for the adenovirus-mediated expression of p19ARF . Immunostaining has indicated that we needed cre-ARF viruses at a moi of 800 and cre viruses at a moi of 320 to express p19ARF in 80% of MEFs (data not shown). It has been well known that the original hosts of adenovirus used for this expression system are human cells and mouse-derived cells are usually resistant to infection. At 48 h after coinfection of MEFs with cre-ARF viruses and cre viruses, or control viruses, the percentage of TUNEL-positive cells was examined. If p19ARF was expressed, the percentage of TUNEL-positive cells increased to 12.6% (Fig. 5A, 1) while without expression of p19ARF , it was fewer than 5% (Fig. 5A, 2 and 3). Considering that fewer than 0.5% of MEFs not infected with adenoviruses are TUNEL-positive (data not shown), we concluded that adenoviral infection itself is a little toxic to MEFs (Fig. 5A, 2 and 3). However, expression of p19ARF significantly increased the percentage of TUNEL-positive cells. FACS analysis also indicated that cell number belonging to subG1 fraction significantly increased when p19ARF was expressed (data not shown). These findings indicated that cell death was induced in a p19ARF -dependent manner, even in MEFs deficient in both ARF and p53. In a similar fashion, expression of p19ARF induced apoptosis of MEFs nullizygous for ARF, Mdm2, and p53 (Fig. 5B). Thus, we concluded that p19ARF -induced p53-independent apoptosis occurs, irrespective of Mdm2 status with the adenovirus-mediated expression system.
Coexpression of Mdm2 inhibits p19ARF -induced apoptosis in U2OS cells Mdm2 inhibits p53 activities [19–22]. We then asked how ectopic expression of Mdm2 alters p19ARF -induced apoptosis in both p53-intact and p53-deficient cells. To address this question, we developed an adenovirus vector to express Mdm2. Adenovirus-mediated coexpression of Mdm2 remarkably, but not completely, inhibits p19ARF -induced apoptosis in U2OS cells (Fig. 6A). If we observed that p53-induced apoptosis was almost completely inhibited by coexpression of Mdm2 (Fig. 6B), we could speculate that there is a p53-independent pathway insensitive to inhibition by Mdm2 in U2OS cells. Note that expression of endogenous Hdm2 is upregulated with expression of the ARF genes, as previously observed (Fig. 6C) [10,23]. Ectopic expression of Mdm2 suppressed expression of endogenous Hdm2 through inactivation of p53, a transcriptional activator for endogenous Hdm2 (Fig. 6C). In contrast, ectopic expression of Mdm2 did not inhibit p19ARF -induced apoptosis in Saos-2 (Fig. 6D), suggesting that the p53-independent pathways for p19ARF -induced apoptosis is insensitive to inhibition by Mdm2.
Discussion Cell cycle arrest and cell death have been examined as two major phenotypes of the tumor-suppressing genes. Induction of cell cycle arrest has been generally moni-
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Fig. 5. p19ARF -induced cell death in p53/ARF-null MEFs (A) and p19ARF -induced cell death in p53/Mdm2/ARF-null MEFs (B). MEFs (5 104 ) in passage 5–8, seeded on six-well plates, were coinfected with indicated mois of cre-ARF, cre, LacZ, or cre-LacZ viruses. At 48 h after infection, cells were harvested for TUNEL assays. The vertical and the horizontal axes indicate FSC and FL1-H, respectively. Dots shifted rightward to higher FL1H indicated TUNEL-positive cells.
tored as a tumor-suppressing phenotype of the ARF. On the contrary, induction of cell death by p19ARF has been rarely monitored because p19ARF -induced cell death needs associated cell death-inducing insults [26,28,31– 33]. Extensive works have established that the functional domains of the p53 tumor suppressor responsible for cell cycle arrest and cell death are not identical [34–37], urging us to examine both tumor-suppressing phenotypes to know whole activities of the ARF tumor suppressor. By adenovirus-mediated expression of p19ARF , apoptosis can be induced without additional signals, indicating that p19ARF -induced cell death can be easily examined with this system. Adenoviral infection itself seems to be a little toxic to cells [45]. This explains why we could easily see cell death with the adenovirus-mediated expression of tumor suppressor genes. However, infection at higher mois, up to 1600, itself never induced death of established cell lines such as U2OS, Saos-2, and 10(1) at least within 48 h [39]. Furthermore, coexpression of Mdm2 inhibited p19ARF -induced cell death in p53-intact U2OS cells (Fig. 6). This findings strongly support the idea that p19ARF -induced cell death is not an
artificial outcome evoked by the adenovirus-mediated overexpression. Emerging evidence has indicated that there are p53independent tumor-suppressing pathways mediated by the ARF tumor suppressor [24,29,30]. Slowly occurring cell cycle arrest was induced by overexpression of p19ARF in MEFs lacking ARF, p53, and Mdm2 [29]. Cell cycle arrest was not induced by reintroduction of p19ARF in MEFs deficient in ARF and p53, indicating that Mdm2 has a negative effect on p53-independent cell cycle arrest induced by p19ARF . In another study [30], p19ARF inhibited immortalization of p53-deficient MEFs only in the presence of Mdm2. They claimed that ARF exerts p53-independent tumor-suppressing activity through Mdm2 and Rb. With adenovirus-mediated expression system, p19ARF -induced p53-independent apoptosis was not inhibited by coexpression of Mdm2 (Fig. 6), indicating that the p53-independent p19ARF induced pathway is insensitive to inhibition by Mdm2. Downstream molecular mediators for p19ARF -induced apoptosis may be different from those for p19ARF induced cell cycle arrest. p21Cip1 , a transcriptional target
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tion, are the ones of possible downstream mediators. p19ARF sequestered E2Fs in the nucleolus and predisposes them to degradation, inhibiting the function of E2Fs for the G1-S transition [49]. From a standpoint of apoptosis, however, E2Fs are not considered to be downstream mediators for p19ARF because increase, but not decrease, of expression of E2Fs induces apoptosis. To totally understand the role of the ARF tumor suppressor, we need to identify the actual downstream mediators of p19ARF for both apoptosis and cell cycle, other than Mdm2 and p53.
Acknowledgments Fig. 6. p19ARF -induced cell death is partially inhibited by coexpression of Mdm2. (A) U2OS cells, not infected (lane 1), or coinfected with indicated viruses at indicated mois, were trypsinized and stained with PI for FACS analysis at 48 h. ARF () indicates coinfection with creARF viruses at a moi of 200 and cre-viruses at a moi of 80. Mdm2 and LZ indicate the Mdm2 viruses and the ordinary LacZ viruses. Numbers 2 and 4 indicate mois of 200 and 400. (B) U2OS cells, not infected, or coinfected with indicated viruses at indicated mois, were trypsinized and stained with PI for FACS analysis at 48 h. p53 () indicates infection with p53 viruses at a moi of 50. Mdm2 and LZ indicates the Mdm2 viruses and the ordinary LacZ viruses. Numbers 2 and 4 indicate mois of 200 and 400. (C) U2OS cells, not infected, or coinfected with indicated viruses at indicated mois, were harvested at 24 h for immunoblotting with anti-p19ARF antibody (upper), anti-p53 antibody (middle), and anti-Mdm2 antibody (lower). ARF () indicate coinfection with creARF viruses at a moi of 100 and cre-viruses at a moi of 80. Mdm2 and LZ indicates the Mdm2 viruses and the ordinary LacZ viruses. Numbers 1 and 2 indicate mois of 100 and 200. (D) Saos2 cells, not infected, or coinfected with indicated viruses at indicated mois, were trypsinized and stained with PI for FACS analysis at 48 h. ARF () indicates coinfection with creARF viruses at a moi of 400 and cre-viruses at a moi of 160. Mdm2 and LZ indicates the Mdm2 viruses and the ordinary LacZ viruses. Numbers 4, 8, and 12 indicate mois of 400, 800, and 1200.
for p53, mediates p19ARF -induced cell cycle arrest in p53-intact cells [12]. However, p19ARF -induced p21Cip1 independent cell cycle arrest has also been observed [24,29]. Many candidates for p53-mediated apoptosis have been identified [13–16], although universal mediators for p53 have not identified. Currently, we do not know what the downstream mediators are for the p53independent apoptosis mediated by p19ARF . p73 is not a main mediator, at least for the p53-independent pathways in Saos-2 cells because coexpression of p73DD, a dominant-negative form for p73, does not inhibit p19ARF -induced apoptosis (unpublished observation by K.T. and M.M.). Several putative functional interactors with p19ARF have been characterized. They include some E2Fs [46], DNA topoisomerase [47], pex19p [48], Spinophilin/ Neurabin II as a type 1 protein-phosphatase-binding protein [49], and hypoxia-inducible factor-1a [50]. So far, E2Fs, trancription factors involved in G1-S transi-
We are indebted to Tomo Yoshida, Kazumi Nishihara, Kouichi Tsuchiya, and Dovie Wylie for expert technical assistance. This work is supported in part by the charitable trust ARAKI grant. We are grateful to Dr. Kyoji Ikeda for helpful advice, to Dr. Charles J. Sherr, Dr. Martine Roussel, and Dr. Moshe Oren for the p19ARF plasmid, TKO MEFs, and DKO MEFs, and to Dr. G.P. Zambetti for 10(1) cells. This work has been completed with extensive help and beneficial criticism by Dr. Ikuo Nishimoto.
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