The ARF tumor suppressor is a crucial component of the cellular response to hyperproliferative signals, including oncogene activation, and functions by inducing a p53-dependent cell growth arrest and apoptosis program. It has recently been reported that the ARF mRNA can produce a smARF isoform that lacks the NH2-terminal region required for p53 activation. Overexpression of this isoform can induce autophagy, a cellular process characterized by the formation of cytoplasmic vesicles and the digestion of cellular content, independently of p53. However, the level of this isoform is extremely low in cells, and it remains unclear whether the predominant form of ARF, the full-length protein, is able to activate autophagy. Here, we show that full-length ARF can induce autophagy in 293T cells where p53 is inactivated by viral proteins, and, notably, expression of the NH2-terminal region alone, which is required for nucleolar localization, is sufficient for autophagy activation, independently of p53. Given the reported ability of p53 to induce autophagy, we also investigated the role of p53 in ARF-mediated autophagy induction. We found that full-length ARF expression induces p53 activation and promotes autophagy in a p53-positive cell line, and that ARF-mediated autophagy can be abrogated, at least in part, by RNAi-mediated knockdown of p53 in this cellular context. Thus, our findings modify the current view regarding the mechanism of autophagy induction by ARF and suggest an important role for autophagy in tumor suppression via full-length ARF in both p53-dependent and p53-independent manners, depending on cellular context. [Cancer Res 2008;68(2):352–7]

The ARF tumor suppressor (p14ARF in humans and p19ARF in mice) is a nucleolar protein that is a crucial component of the cellular response to aberrant oncogene activation (1). ARF is transcriptionally induced in response to the overexpression or mutational activation of growth-promoting genes, including MYC and RAS, and responds in turn by inhibiting the p53-specific ubiquitin ligases MDM2 (24) and ARF-BP1 (5), leading to the initiation of a p53-dependent cell growth arrest and apoptosis program. ARF has also been shown to have p53-independent functions (1). Mice that lack Arf, Trp53, and Mdm2 develop a more severe tumor phenotype than mice that lack Trp53 alone, and the overexpression of ARF can induce cell cycle arrest and apoptosis in p53-null cell lines.

It has recently been shown that the p19ARF mRNA can produce a short isoform of the ARF protein by internal initiation of translation at methionine 45 (6). This isoform, dubbed short mitochondrial ARF or smARF, lacks the ARF NH2-terminal region that contains the MDM2 and ARF-BP1 binding domains required for ubiquitin ligase inhibition and, consequently, for p53-dependent ARF function. smARF also lacks the p19ARF nucleolar localization signal and is therefore excluded from the nucleolar compartment, localizing to mitochondria instead. Reef et al. (6) suggest that this isoform induces cell death by autophagy, a cellular process associated with type II programmed cell death and characterized by the formation of cytosolic double-membrane vesicles, called autophagosomes, that engulf cellular content and fuse with lysosomes to digest it (7). Autophagy has been implicated in tumor suppression (8). In particular, the autophagy gene beclin 1 was shown to be monoallelically deleted in a high percentage of breast, ovarian, and prostate tumors, and mice that are heterozygous for beclin 1 display an increased incidence of tumor formation (9, 10). It has also recently been shown that autophagy can be activated by the p53 tumor suppressor (11, 12). Although smARF can activate autophagy, this isoform is very unstable and present at low or undetectable endogenous levels (6), and the role of the predominant full-length form of ARF in autophagy induction is still unknown. We report that nucleolar full-length ARF can indeed induce autophagy in HEK 293T cells. We also show that nucleolar p14ARF can induce autophagy in U2-OS cells and that this induction is p53-dependent.

Cells and plasmids. H1299, 293T, and U2-OS cells were grown in DMEM (Cellgro) supplemented with 10% fetal bovine serum (Sigma). NARF cells were grown as described (13). p19ARF cDNA was obtained by cloning from a mouse spleen cDNA library and inserted into the pIRESneo vector (Clontech) with a FLAG tag at the COOH terminus. N62p19ARF and smARF were amplified from p19ARF and inserted into pIRESneo also with COOH-terminus FLAG tags. pIRESneo/p19ARF(M45A)-FLAG was generated using the QuikChange Site-Directed Mutagenesis kit (Stratagene). pEGFP-light chain 3 (LC3) was kindly provided by S. Jin (University of Medicine and Dentistry of New Jersey, Piscataway, NJ).

Antibodies and Western blots. Cell lysis for Western blots was carried out in FLAG lysis buffer containing protease inhibitor cocktail (Sigma) and 1 mmol/L DTT (Acros). The antibodies used to detect FLAG, β-actin, β-tubulin, green fluorescent protein (GFP), p14ARF, p53, p21, poly(ADP)ribose polymerase (PARP)-1, and p53 up-regulated modulator of apoptosis (PUMA) are M2, AC-15 (Sigma), H-235 (Santa Cruz Biotechnology), JL-8 (Clontech), Ab-4 (Lab Vision), DO-1, C-19, F-2 (Santa Cruz), and Ab-1 (Calbiochem), respectively. The MDM2 antibody was generated in rabbit against residues 1 to 110 of human MDM2.

Transfection of H1299 and 293T cells. H1299 cells were plated on glass coverslips in a 12-well plate and transfected the following day with 0.8 μg of the appropriate ARF expression plasmid using Lipofectamine 2000 (Invitrogen). Cells were treated for 30 min with 25 nmol/L MitoTracker dye (Invitrogen) 24 h posttransfection, fixed for 20 min in 4% paraformaldehyde, permeabilized with 0.2% Triton-X-100, stained with FLAG primary and Alexa Fluor 488 anti-mouse secondary (Invitrogen) antibodies, counterstained with 4′,6′-diamidino-2-phenylindole to visualize nuclei, and mounted on glass slides. Cells were visualized under the ×40 objective of a Nikon Eclipse 80i fluorescence microscope, and images were captured using a Photometrics CoolSNAP HQ2 camera.

HEK 293T cells were plated at 2 × 106 cells per 100-mm plate and transfected the following day with 1 μg of GFP or GFP-LC3 plasmid and 19 μg of empty vector or of the appropriate ARF expression plasmid. Cell and plasmid amount were scaled accordingly for smaller plate sizes. For the cell death assay, images were captured using the ×10 objective of the microscope described above. For the autophagy assay, cells were plated on glass coverslips and images were captured using the ×60 objective of a Nikon Eclipse E400 fluorescence microscope equipped with a Photometrics CoolSNAP EZ camera.

Autophagy and RNAi in U2-OS cells. U2-OS cells and the NARF derivative (14) were infected for 2 h with adenovirus expressing GFP-LC3 (15) and induced with 1 mmol/L isopropyl-l-thio-B-d-galactopyranoside (IPTG; Sigma) for 48 h. For the RNAi experiment, cells were transfected with control siRNA or siGENOME SMARTpool against human TP53 (Dharmacon) using Lipofectamine 2000 (Invitrogen) then infected and induced as above. All images were captured using the ×40 objective of the Eclipse 80i microscope described above.

Cellular localization of p19ARF fragments. The NH2 terminus of p19ARF was previously shown to be necessary and sufficient for p53-dependent cell cycle arrest (16). Residues 1 to 37 of p19ARF harbor a nucleolar localization signal as well as the Mdm2 binding sites of the protein (Fig. 1A) that are required for the inhibition of Mdm2 function and the activation of p53. Mouse ARF protein lacking the NH2 terminus was shown to localize to the nucleoplasm and cytoplasm and to be defective in the induction of growth arrest (16). A reported short mitochondrial isoform of p19ARF (smARF) is a product of internal initiation of translation at methionine 45, and, as a result, it lacks the domains required for nucleolar localization and Mdm2 inhibition (6). To determine the localization of the p19ARF fragments, we generated COOH-terminus FLAG-tagged constructs expressing the full-length p19ARF protein, the NH2 terminus of p19ARF (amino acids 1–62; N62p19ARF), and the short isoform of p19ARF (amino acids 45–169; smARF; Fig. 1A). We also generated a construct expressing full-length p19ARF that harbors a methionine 45 to alanine substitution [p19ARF(M45A)], and that is therefore unable to express the short mitochondrial isoform. The expression of these constructs is shown in Fig. 1B. As expected, when expressed in H1299 cells, full-length p19ARF and N62p19ARF localize to nucleoli, whereas smARF localizes to mitochondria (Fig. 1C).

Full-length p19ARF and the ARF NH2-terminal domain induce cell death when overexpressed in HEK 293T cells. It has been reported that the short mitochondrial form of p19ARF can induce cell death when overexpressed in HEK 293T cells (6). As shown in Fig. 2A, transient transfection of the smARF construct in 293T cells, where p53 is inactivated by the expression of viral transforming proteins, causes widespread cell death after 48 h, characterized by the occurrence of many rounded and floating cells in accordance with the previously published data. Surprisingly, we found that transient transfection of the full-length p19ARF construct, the M45A mutant and the NH2-terminal domain, all of which have nucleolar localization, can also induce cell death, similarly to the short mitochondrial form (Fig. 2A and B). The cell death observed in cells transfected with the M45A mutant indicates that this phenotype is not due to the expression of a low or undetectable amount of the smARF isoform from the p19ARF construct, but that it is caused by the full-length ARF protein itself. All cells were cotransfected with GFP expression plasmid with high transfection efficiency (Fig. 2A,, right). Cells were trypsinized 48 h posttransfection, and live cells were quantitated as shown in Fig. 2C. To determine whether the observed cell death is due to apoptosis, we detected cleavage of the apoptosis marker PARP-1 by Western blot. As show in Fig. 2D, and consistent with the previously published result for smARF, transfection of ARF constructs does not cause PARP cleavage, whereas transfection of Adenine Nucleotide Translocase-1 (ANT-1), which was previously reported to induce apoptosis (17), results in significant PARP cleavage.

Overexpression of full-length ARF and the ARF NH2 terminus induces autophagy. Reef et al. (6) showed that smARF causes cell death in 293T cells by activating autophagy. Given the similar cell death phenotype observed after transfection of the nucleolar forms of p19ARF, we tested the ability of the full-length ARF protein and the NH2-terminal fragment to induce autophagy in 293T cells. Microtubule-associated protein LC3 is commonly used as a specific marker for autophagy. Upon induction of autophagy, the predominantly cytoplasmic LC3 protein, LC3I, is conjugated to phosphatidylethanolamine to yield an LC3II form that associates with autophagosomal membranes (18). When viewed under the microscope, the localization of LC3 protein to cytoplasmic dots is an indicator of autophagic process (15). As shown in Fig. 3A, cotransfection of p19ARF, p19ARF(M45A), and N62p19ARF with GFP-tagged LC3 (GFP-LC3) induces the formation of GFP puncta in 293T cells, as previously observed with the transfection of the smARF isoform (Fig. 3A,, bottom). Quantitation of the percentage of GFP-LC3–positive cells displaying cytoplasmic punctate staining is shown in Fig. 3B. Moreover, overexpression of p19ARF and N62p19ARF induces a significant increase in the level of the autophagic LC3II form, which can be detected by Western blot as the faster migrating LC3 protein (Fig. 3C). Overexpression of smARF, on the other hand, does not cause a detectable increase in LC3II level in the same experiment. Reef et al. (6) showed that knockdown of Beclin-1, an essential autophagy gene, protects against smARF-induced cell death in 293T cells. We sought to investigate whether knockdown of Beclin-1 could also rescue cell death induced by full-length ARF. For this purpose, we treated 293T cells with control siRNA or siRNA against Beclin-1, then transfected treated cells with control vector or expression plasmids for p19ARF(M45A) and smARF. As shown in Supplementary Fig. S1, knockdown of Beclin-1 attenuates cell death induced by full-length ARF, indicating that autophagy contributes to ARF-mediated cell death. Additionally, we sought to determine whether induction of autophagy by full-length ARF is p53-dependent in 293T cells, where p53 is generally considered to be inactivated by the overexpression of SV40 large T antigen. We show in Supplementary Fig. S2 that RNAi knockdown of p53 in 293T cells does not inhibit p19ARF-mediated autophagy, indicating that although nucleolar full-length ARF can induce autophagy in this cell line, it does so independently of p53.

To determine whether full-length ARF can induce autophagy in other cell lines, we used a U2-OS derivative in which the expression of p14ARF is under control of an IPTG-inducible promoter (NARF; ref. 14). The full-length p14ARF protein induced in these cells was previously shown to localize to nucleoli as expected (13). NARF cells were infected with adenovirus expressing GFP-LC3 then treated for 48 h to induce p14ARF expression. As shown in Fig. 3D (left), treatment of NARF cells with IPTG results in a significant increase of LC3 puncta compared with untreated cells, whereas the treatment of parental ARF-null U2-OS cells with IPTG had no effect on the induction of autophagy. We verified the levels of ARF and p53 after IPTG treatment of NARF cells by Western blot (Fig. 3D , right). As expected, IPTG treatment results in significant induction of p14ARF and in an increase in the level of p53 and of the p53 transcriptional targets MDM2 and p21.

Autophagy induced by full-length ARF is p53-dependent in U2-OS cells. p53 was previously shown to activate autophagy when overexpressed in the Saos-2 osteosarcoma cell line (12). We showed in Fig. 3 that ARF overexpression activates autophagy in the p53-positive cell line U2-OS, and that p53 is stabilized in response to ARF induction by IPTG. We therefore sought to investigate whether autophagy induced by ARF in U2-OS cells is p53-dependent. We transfected NARF cells with control siRNA or siRNA against p53 then infected the cells with GFP-LC3 adenovirus and induced p14ARF expression with IPTG as described previously. Treatment of control siRNA-transfected cells with IPTG induced significant GFP-LC3 puncta in ∼40% of all cells, whereas <10% of treated cells showed GFP-LC3 puncta after p53 knockdown (Fig. 4A and B). When analyzed by Western blot, IPTG treatment of cells transfected with control siRNA induces significant ARF expression, with a corresponding increase in the level of p53 and of the p53 transcriptional targets MDM2, p21, and PUMA. Treatment of control siRNA cells with IPTG also results in an increase in the level of LC3-II, confirming the activation of autophagy in these cells. On the other hand, expression of p53 and the p53 transcriptional targets is undetectable in cells transfected with p53 siRNA, even after ARF induction with IPTG, and there is almost no detectable increase in the level of LC3II (Fig. 4C).

We show in this report that p19ARF and its NH2-terminal domain, both of which localize to the nucleolar compartment, can induce autophagy when overexpressed in 293T cells, where p53 is inactive. We also show that induction of p14ARF in U2-OS cells can activate autophagy in a p53-dependent manner. It has previously been reported that a short isoform of p19ARF that lacks the ARF NH2 terminus can induce autophagy (6). This isoform localizes to mitochondria, and lacks the domains required for MDM2 binding and for p53-dependent ARF function. Our data suggest that the predominant ARF isoform can induce autophagy by a different mechanism, independent of smARF, both in p53-dependent and p53-independent manners depending on the cellular context. In particular, the p53 dependence of autophagy induced by p14ARF indicates that the ARF NH2 terminus, which is required for its p53-dependent function, is crucial for the activation of autophagy by ARF under some conditions. Furthermore, although p53 was already shown to activate autophagy by inhibition of the mammalian target of rapamycin pathway (11) and through the transcriptional target DRAM (12), the ability of ARF to modulate this activation was not known.

The ARF protein is crucial for the cellular response to oncogene activation, and tumor cells that harbor mutations in growth-promoting genes frequently eliminate ARF or p53 to maintain their hyperproliferative state (19). Although autophagy is primarily known to be involved in the response to nutrient starvation and other environmental cues, our data suggest that it may also be involved in the cellular response to oncogene activation via the ARF tumor suppressor. The role of autophagy in cancer development is complex, as it has been implicated both in tumor survival and in tumor cell death, depending on the context (20). Our results support the notion that autophagy is involved in tumor suppression, particularly as an effector of the ARF oncogene response pathway.

Note: Supplementary data for this article are available at Cancer Research Online (http://cancerres.aacrjournals.org/).

Grant support: NIH and the National Cancer Institute (W. Gu).

The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

We thank S. Jin for providing us with the GFP-LC3 expression plasmid, A. Tolkovsky for the adenovirus expressing GFP-LC3, G. Peters for the NARF cell line, and K. Ryan for helpful discussions.

1
Sherr CJ. Divorcing ARF and p53: an unsettled case.
Nat Rev Cancer
2006
;
6
:
663
–73.
2
Zhang Y, Xiong Y, Yarbrough WG. ARF promotes MDM2 degradation and stabilizes p53: ARF-INK4a locus deletion impairs both the Rb and p53 tumor suppression pathways.
Cell
1998
;
92
:
725
–34.
3
Pomerantz J, Schreiber-Agus N, Liegeois NJ, et al. The Ink4a tumor suppressor gene product, p19Arf, interacts with MDM2 and neutralizes MDM2's inhibition of p53.
Cell
1998
;
92
:
713
–23.
4
Korgaonkar C, Zhao L, Modestou M, Quelle DE. ARF function does not require p53 stabilization or Mdm2 relocalization.
Mol Cell Biol
2002
;
22
:
196
–206.
5
Chen D, Kon N, Li M, Zhang W, Qin J, Gu W. ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor.
Cell
2005
;
121
:
1071
–83.
6
Reef S, Zalckvar E, Shifman O, et al. A short mitochondrial form of p19ARF induces autophagy and caspase-independent cell death.
Mol Cell
2006
;
22
:
463
–75.
7
Levine B, Klionsky DJ. Development by self-digestion: molecular mechanisms and biological functions of autophagy.
Dev Cell
2004
;
6
:
463
–77.
8
Jin S. p53, Autophagy and tumor suppression.
Autophagy
2005
;
1
:
171
–3.
9
Qu X, Yu J, Bhagat G, et al. Promotion of tumorigenesis by heterozygous disruption of the beclin 1 autophagy gene.
J Clin Invest
2003
;
112
:
1809
–20.
10
Yue Z, Jin S, Yang C, Levine AJ, Heintz N. Beclin 1, an autophagy gene essential for early embryonic development, is a haploinsufficient tumor suppressor.
Proc Natl Acad Sci U S A
2003
;
100
:
15077
–82.
11
Feng Z, Zhang H, Levine AJ, Jin S. The coordinate regulation of the p53 and mTOR pathways in cells.
Proc Natl Acad Sci U S A
2005
;
102
:
8204
–9.
12
Crighton D, Wilkinson S, O'Prey J, et al. DRAM, a p53-induced modulator of autophagy, is critical for apoptosis.
Cell
2006
;
126
:
121
–34.
13
Llanos S, Clark PA, Rowe J, Peters G. Stabilization of p53 by p14ARF without relocation of MDM2 to the nucleolus.
Nat Cell Biol
2001
;
3
:
445
–52.
14
Stott FJ, Bates S, James MC, et al. The alternative product from the human CDKN2A locus, p14(ARF), participates in a regulatory feedback loop with p53 and MDM2.
EMBO J
1998
;
17
:
5001
–14.
15
Bampton ET, Goemans CG, Niranjan D, Mizushima N, Tolkovsky AM. The dynamics of autophagy visualized in live cells: from autophagosome formation to fusion with endo/lysosomes.
Autophagy
2005
;
1
:
23
–36.
16
Weber JD, Kuo ML, Bothner B, et al. Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex.
Mol Cell Biol
2000
;
20
:
2517
–28.
17
Bauer MK, Schubert A, Rocks O, Grimm S. Adenine nucleotide translocase-1, a component of the permeability transition pore, can dominantly induce apoptosis.
J Cell Biol
1999
;
147
:
1493
–502.
18
Kabeya Y, Mizushima N, Ueno T, et al. LC3, a mammalian homologue of yeast Apg8p, is localized in autophagosome membranes after processing.
EMBO J
2000
;
19
:
5720
–8.
19
Zindy F, Eischen CM, Randle DH, et al. Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization.
Genes Dev
1998
;
12
:
2424
–33.
20
Hippert MM, O'Toole P S, Thorburn A. Autophagy in cancer: good, bad, or both?
Cancer Res
2006
;
66
:
9349
–51.

Supplementary data