ARF-BP1/Mule is a critical mediator of the ARF tumor suppressor

Although the importance of the ARF tumor suppressor in p53 regulation is well established, numerous studies indicate that ARF also suppresses cell growth in a p53/Mdm2-independent manner. To understand the mechanism of ARF-mediated tumor suppression, we identified a ubiquitin ligase, ARF-BP1, as a key factor associated with ARF in vivo. ARF-BP1 harbors a signature HECT motif, and its ubiquitin ligase activity is inhibited by ARF. Notably, inactivation of ARF-BP1, but not Mdm2, suppresses the growth of p53 null cells in a manner reminiscent of ARF induction. Surprisingly, in p53 wild-type cells, ARF-BP1 directly binds and ubiquitinates p53, and inactivation of endogenous ARF-BP1 is crucial for ARF-mediated p53 stabilization. Thus, our study modifies the current view of ARF-mediated p53 activation and reveals that ARF-BP1 is a critical mediator of both the p53-independent and p53-dependent tumor suppressor functions of ARF. As such, ARF-BP1 may serve as a potential target for therapeutic intervention in tumors regardless of p53 status.     

Apoptosis induced by adenovirus-mediated p14ARF expression in U2OS osteosarcoma cells is associated with increased Fas expression

The INK4A/ARF locus on chromosome 9 is a tumor suppressor gene frequently mutated in human cancers. In order to study the effects of p14ARF expression in tumor cells, we constructed a recombinant adenovirus containing p14ARF cDNA (Adp14ARF). Adp14ARF infection of U2OS osteosarcoma cells which has wild type p53 and mutant p14ARF revealed high levels of p14 (ARF) expression within 24h. In addition, Adp14ARF-mediated expressing of p14 (ARF) was associated with increased levels of p53, p21, and mdm2 protein. Growth inhibition assays following Adp14ARF infection demonstrated that the growth of U2OS cells was inhibited relative to infection with control virus. Furthermore, TUNEL analysis as well as PARP cleavage assays demonstrated that Adp14ARF infection was associated with increased apoptosis in U2OS cell line and that it was associated with Adp14ARF induced overexpression of Fas and Fas-L. Addition of Fas-L neutralizing antibody NOK-1 decreased Adp14-mediated cell death, indicating that p14 (ARF) induction of the Fas pathway is associated with increased apoptosis. The finding that Adp14ARF infection did not induce Fas expression in U2OS/E6 and MCF/E6 cells suggests that wild type p53 expression may be necessary for Adp14ARF-mediated induction of Fas. The observation that overexpression of p53 by Adp53 infection in MCF-7 does not induce increased Fas protein levels nor apoptotic cell death suggests that p53 overexpression is required but not sufficient enough for apoptosis. These studies suggest there are other mechanisms other than induction of p53 in ARF-mediated apoptosis and gene therapy using Adp14ARF may be a promising treatment option for human cancers containing wild type p53 and mutant or deleted p14 expression.     

p14ARF interacts with DAXX: Effects on HDM2 and p53

The p14ARF (ARF) tumour suppressor plays an important role in the cellular response to oncogene activation. In this report, we demonstrate an interaction between ARF and DAXX, a highly conserved protein with identified roles in the regulation of gene expression. HDM2 was shown to interact with each of ARF and DAXX upon upregulation of expression as well as at lower expression levels following transfection of ARF and DAXX. Through immunofluorescence analysis, we observed that endogenous ARF and DAXX colocalize both to nucleoli and to nuclear bodies in cell lines that co-express both proteins. Similar results were obtained upon co-transfection of ARF and DAXX. Co-expression of ARF and DAXX was further found to inhibit ARF-mediated HDM2 sumoylation and to induce sumoylation and ubiquitination of DAXX itself, implicating DAXX as a substrate of ARF-mediated post-translational events. We also observed induction of p53 sumoylation in the presence of ARF and DAXX, an effect that was inhibited by upregulation of HDM2 expression. In summary, we have identified DAXX as a novel ARF binding partner and substrate of ARF-mediated sumoylation and suggest that DAXX acts as a modifier of both p53-dependent and p53-independent ARF function.     

Analysis of p53 tumor suppressor pathway genes in chronic lymphocytic leukemia

The p53 tumor suppressor gene plays an important role in preventing tumor development. The p53 protein interacts with other p53 signal pathway members to control cell proliferation. In this study, expression of the p53, Human homolog of murine Double Minute 2 (HDM2), p14Alternating Reading Frame (ARF), Zinc Finger and BTB domain containing 7A (ZBTB7A), and B-Cell Lymphoma 6 (BCL6) genes was quantitatively investigated by real-time polymerase chain reaction (PCR) in the peripheral blood of patients with chronic lymphocytic leukemia (CLL) and healthy controls. Plasma fibronectin levels were determined by enzyme-linked immunosorbent assay. Expression of the p53, p14, and HDM2 genes were significantly higher in the patients. However, ZBTB7A and BCL6 gene expression was not detectable in both groups. A positive correlation between p14ARF and HDM2 expression and a negative correlation between p53 and p14ARF expression was observed. Expression of the p14ARF and HDM2 genes were inversely correlated in the control group. Neither HDM2 nor p14ARF gene expression was correlated with p53 expression. The p53 gene was also analyzed for the presence of mutations. A splice-site mutation was found in a single patient. Our findings indicate that expression of the p53, p14ARF, and HDM2 genes are associated with CLL. Elucidation of the mutual interactions at the protein level warrants further studies.     

Mdm2 regulates p53 independently of p19(ARF) in homeostatic tissues

Tumor suppressor proteins must be exquisitely regulated since they can induce cell death while preventing cancer. For example, the p19(ARF) tumor suppressor (p14(ARF) in humans) appears to stimulate the apoptotic function of the p53 tumor suppressor to prevent lymphomagenesis and carcinogenesis induced by oncogene overexpression. Here we present a genetic approach to defining the role of p19(ARF) in regulating the apoptotic function of p53 in highly proliferating, homeostatic tissues. In contrast to our expectation, p19(ARF) did not activate the apoptotic function of p53 in lymphocytes or epithelial cells. These results demonstrate that the mechanisms that control p53 function during homeostasis differ from those that are critical for tumor suppression. Moreover, the Mdm2/p53/p19(ARF) pathway appears to exist only under very restricted conditions.     

Cyclin G1 has growth inhibitory activity linked to the ARF-Mdm2-p53 and pRb tumor suppressor pathways

Cyclin G1 is a p53-responsive gene that is induced in alternative reading frame (ARF)-arrested cells, yet its role in growth control is unclear. We tested its effects on growth and involvement in the ARF-Mdm2-p53 tumor suppressor pathway. We show that cyclin G1 interacts with ARF, Mdm2, and p53 in vitro and in vivo. At high levels, cyclin G1 induces a G(1)-phase arrest in mammalian cells that coincides with p53 activation. Conversely, lower levels of cyclin G1 lack intrinsic growth inhibitory effects yet potentiate ARF-mediated growth arrest. Notably, cyclin G1 is down-regulated by Mdm2 through proteasome-mediated degradation. These data suggest that cyclin G1 is a positive feedback regulator of p53 whose expression is restrained by Mdm2. Interestingly, growth inhibition by cyclin G1 does not require p53 but instead exhibits partial retinoblastoma protein (pRb) dependence. These findings reveal that cyclin G1 has growth inhibitory activity that is mechanistically linked to ARF-p53 and pRb tumor suppressor pathways.     

A novel nuclear interactor of ARF and MDM2 (NIAM) that maintains chromosomal stability

The ARF tumor suppressor signals through p53 and other poorly defined anti-proliferative pathways to block carcinogenesis. In a search for new regulators of ARF signaling, we discovered a novel nuclear protein that we named NIAM (nuclear interactor of ARF and MDM2) for its ability to bind both ARF and the p53 antagonist MDM2. NIAM protein is normally expressed at low to undetectable levels in cells because of, at least in part, MDM2-mediated ubiquitination and proteasomal degradation. When reintroduced into cells, NIAM activated p53, caused a G1 phase cell cycle arrest, and collaborated with ARF in an additive fashion to suppress proliferation. Notably, NIAM retains growth inhibitory activity in cells lacking ARF and/or p53, and knockdown experiments revealed that it is not essential for ARF-mediated growth inhibition. Thus, NIAM and ARF act in separate anti-proliferative pathways that intersect mechanistically and suppress growth more effectively when jointly activated. Intriguingly, silencing of NIAM accelerated chromosomal instability, and microarray analyses showed reduced NIAM mRNA expression in numerous primary human tumors. This study identifies a novel protein with tumor suppressor-like behaviors and functional links to ARF-MDM2-p53 signaling.     

P14ARF promotes accumulation of SUMO-1 conjugated (H)Mdm2

p14ARF tumour suppressor stabilises and activates p53 by directly interacting with (H)Mdm2 [(human) murine double minute 2 homologue] and inhibiting its E3 ubiquitin ligase activity. Here we demonstrate that p14ARF promotes accumulation of (H)Mdm2 conjugated to the small ubiquitin-like protein SUMO-1. Mutational analysis demonstrated that the N-terminus of Mdm2 is a target for p14ARF-mediated SUMO conjugation. SUMO modification requires residues 2-14 in p14ARF that interact with (H)Mdm2 and residues 82-101 in exon 2 involved in nucleolar localisation of p14ARF. These data suggest a novel role for p14ARF as a regulator of activity of (H)Mdm2, which could be related to its tumour suppressing activities.     

CARF is a novel protein that cooperates with mouse p19ARF (human p14ARF) in activating p53

The INK4a locus on chromosome 9p21 encodes two structurally distinct tumor suppressor proteins, p16(INK4a) and the alternative reading frame protein, ARF (p19(ARF) in mouse and p14(ARF) in human). Each of these proteins has a role in senescence of primary cells and activates pathways for cell cycle control and tumor suppression. The current prevailing model proposes that p19(ARF) activates p53 function by antagonizing its degradation by MDM2. It was, however, recently shown that stabilization of p53 by p14(ARF) occurs independent of the relocalization of MDM2 to the nucleolus. We have identified a novel collaborator of ARF, CARF. It co-localizes and interacts with ARF in the nucleolus. We demonstrate that CARF is co-regulated with ARF, cooperates with it in activating p53, and thus acts as a novel component of the ARF-p53-p21 pathway.     

The ARF tumour suppressor

The ARF tumour suppressor is a product of the INK4a/ARF locus; a sequence that is frequently altered in human cancer. ARF is upregulated by oncogenic stimuli and is a critical regulator of p53 stability through interactions with the mdm2 and ARF-BP1/Mule ubiquitin ligases. Cellular stress signals liberate ARF from the nucleolus where it is bound to B23/nucleophosmin. This nucleolar location of ARF may serve as a reservoir for the rapid induction of p53, but may also serve to co-ordinate effects on cell cycle, survival and growth. The biological functions of ARF interactions with other binding partners remain uncertain, but ARF-mediated sumoylation may represent a unifying effector pathway.     

A major functional difference between the mouse and human ARF tumor suppressor proteins

Suppression of tumorigenesis is considerably more stringent in the human than in the much shorter lived mouse species, and the reasons for this difference are poorly understood. We investigated functional differences in the control of the ARF (alternative reading frame) protein that acts upstream of p53 and is encoded along with p16(INK4a) at a major tumor suppressor locus in both the human and mouse genomes. The mouse and human ARF proteins are substantially divergent at their carboxyl termini. We have shown that the mouse ARF protein (p19ARF) interacts with Pex19p in the cell cytoplasm leading to its nuclear exclusion and repression of its p53 activation function. The human ARF protein (p14ARF) is substantially smaller than its mouse counterpart and is not subject to this functional inactivation by Pex19p. In an identical cellular background, ribozymes directed against Pex19p enhanced p19ARF- but not p14ARF-activated p53 function. This is the first demonstration of a functional difference between the mouse and human ARF proteins. In view of the major role of ARF in tumor suppression, this distinction may contribute to the different levels of tumor proneness of these species.     

Depletion of the nucleolar protein nucleostemin causes G1 cell cycle arrest via the p53 pathway

Nucleostemin (NS) is a nucleolar protein expressed in adult and embryo-derived stem cells, transformed cell lines, and tumors. NS decreases when proliferating cells exit the cell cycle, but it is unknown how NS is controlled, and how it participates in cell growth regulation. Here, we show that NS is down-regulated by the tumor suppressor p14(ARF) and that NS knockdown elevates the level of tumor suppressor p53. NS knockdown led to G1 cell cycle arrest in p53-positive cells but not in cells in which p53 was genetically deficient or depleted by small interfering RNA knockdown. These results demonstrate that, in the cells investigated, the level of NS is regulated by p14(ARF) and the control of the G1/S transition by NS operates in a p53-dependent manner.