p14ARF induces G2 cell cycle arrest in p53- and p21-deficient cells by down-regulating p34cdc2 kinase activity

The human INK4a gene locus encodes two structurally unrelated tumor suppressor proteins, p16(INK4a) and p14(ARF). Although primarily proposed to require a functional p53.Mdm-2 signaling axis, recently p14(ARF) has been implicated in p53-independent cell cycle regulation. Here we show that p14(ARF) preferentially induces a G(2) arrest in tumor cells lacking functional p53 and/or p21. Expression of p14(ARF) impaired mitotic entry and enforced a primarily cytoplasmic localization of p34(cdc2) that was associated with a decrease in p34(cdc2) kinase activity and reduced p34(cdc2) protein expression. A direct physical interaction between p14(ARF) and p34(cdc2) was, nevertheless, ruled out by lack of co-immunoprecipitation. The p14(ARF)-induced depletion of p34(cdc2) was associated with impaired cdc25C phosphatase expression and a prominent shift to inhibitory Tyr-15-phosphorylation in G(2)-arrested cells lacking either p53, p21, or both. Finally, reconstitution of p34(cdc2) using a constitutively active, phosphorylation-deficient p34(cdc2AF) mutant alleviated this p14(ARF)-induced G(2) arrest, thereby allowing cell cycle progression. Taken together, these data indicate that p14(ARF) arrests cells lacking functional p53/p21 in the G(2) phase of the cell cycle by targeting p34(cdc2) kinase. This may represent an important fail-safe mechanism by which p14(ARF) protects p53/p21-deficient cells from unrestrained proliferation.     

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.     

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.     

ARF——一种新的抑癌因子的研究进展

INK4a/ARF基因位于人染色体9p21,是人类肿瘤中最常见的基因失活位点之一.INK4a/ARF基因有两套各自独立的启动子,通过可变阅读框,能够编码两种蛋白质：p16^INK4a和p14^ARF（ARF在鼠细胞中为p19^ARF）.p16作为CDK4/6的抑制因子,能够阻断pRb磷酸化,将细胞周期阻断在G1期;而ARF可结合原癌蛋白MDM2,稳定p53,将细胞周期阻断在G1期和G2/M转换期,或诱导细胞凋亡.因此ARF蛋白和p16一样也是一种肿瘤抑制因子.     

p53-independent apoptosis is induced by the p19ARF tumor suppressor

p19(ARF) is a potent tumor suppressor. By inactivating Mdm2, p19(ARF) 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 p19(ARF) 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 p19(ARF) 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 p19(ARF). In agreement, p19(ARF)-mediated apoptosis in U2OS cells, but not in Saos2 cells, was attenuated by coexpression of Mdm2. We thus conclude that there is a p53-independent pathway for p19(ARF)-induced apoptosis that is insensitive to inhibition by Mdm2.     

p14ARF activates a Tip60-dependent and p53-independent ATM/ATR/CHK pathway in response to genotoxic stress

p14ARF is a tumor suppressor that controls a well-described p53/Mdm2-dependent checkpoint in response to oncogenic signals. Here, new insights into the tumor-suppressive function of p14ARF are provided. We previously showed that p14ARF can induce a p53-independent G2 cell cycle arrest. In this study, we demonstrate that the activation of ATM/ATR/CHK signaling pathways contributes to this G2 checkpoint and highlight the interrelated roles of p14ARF and the Tip60 protein in the initiation of this DNA damage-signaling cascade. We show that Tip60 is a new direct p14ARF binding partner and that its expression is upregulated and required for ATM/CHK2 activation in response to p14ARF. Strikingly, both p14ARF and Tip60 products accumulate following a cell treatment with alkylating agents and are absolutely required for ATM/CHK2 activation in this setting. Moreover, and consistent with p14ARF being a determinant of CHK2 phosphorylation in lung carcinogenesis, a strong correlation between p14ARF and phospho-CHK2 (Thr68) protein expression is observed in human lung tumors (P < 0.00006). Overall, these data point to a novel regulatory pathway that mediates the p53-independent negative-cell-growth control of p14ARF. Inactivation of this pathway is likely to contribute to lung carcinogenesis.     

Knockdown of CDK2AP1 in Primary Human Fibroblasts Induces p53 Dependent Senescence

Cyclin Dependent Kinase-2 Associated Protein-1 (CDK2AP1) is known to be a tumor suppressor that plays a role in cell cycle regulation by sequestering monomeric CDK2, and targeting it for proteolysis. A reduction of CDK2AP1 expression is considered to be a negative prognostic indicator in patients with oral squamous cell carcinoma and also associated with increased invasion in human gastric cancer tissue. CDK2AP1 overexpression was shown to inhibit growth, reduce invasion and increase apoptosis in prostate cancer cell lines. In this study, we investigated the effect of CDK2AP1 downregulation in primary human dermal fibroblasts. Using a short-hairpin RNA to reduce its expression, we found that knockdown of CDK2AP1in primary human fibroblasts resulted in reduced proliferation and in the induction of senescence associated beta-galactosidase activity. CDK2AP1 knockdown also resulted in a significant reduction in the percentage of cells in the S phase and an accumulation of cells in the G1 phase of the cell cycle. Immunocytochemical analysis also revealed that the CDK2AP1 knockdown significantly increased the percentage of cells that exhibited γ-H2AX foci, which could indicate presence of DNA damage. CDK2AP1 knockdown also resulted in increased mRNA levels of p53, p21, BAX and PUMA and p53 protein levels. In primary human fibroblasts in which p53 and CDK2AP1 were simultaneously downregulated, there was: (a) no increase in senescence associated beta-galactosidase activity, (b) decrease in the number of cells in the G1-phase and increase in number of cells in the S-phase of the cell cycle, and (c) decrease in the mRNA levels of p21, BAX and PUMA when compared with CDK2AP1 knockdown only fibroblasts. Taken together, this suggests that the observed phenotype is p53 dependent. We also observed a prominent increase in the levels of ARF protein in the CDK2AP1 knockdown cells, which suggests a possible role of ARF in p53 stabilization following CDK2AP1 knockdown. Altogether, our results show that knockdown of CDK2AP1 in primary human fibroblasts reduced proliferation and induced premature senescence, with the observed phenotype being p53 dependent.     

Regulation of p14ARF Through Subnuclear Compartmentalization

The p53-mediated pathway cell cycle arrest and apoptosis is central to cancer and an important point of focus for therapeutics development. The p14ARF ("ARF") tumor suppressor induces the p53 pathway in response to oncogene activation or DNA damage. However, ARF is predominantly nucleolar in localization and engages in several interactions with nucleolar proteins, whereas p53 is nucleoplasmic. This raises the question as to how ARF initiates its involvement in the p53 pathway. We have found that UV irradiation of cells disrupts the interaction of ARF with two of its nucleolar binding partners, B23(NPM, nucleophosmin, NO38, numatrin) and topoisomerase I, and promotes an immediate and transient subnuclear redistribution of ARF to the nucleoplasm, where it can engage the p53 pathway (Lee et al, Cancer Research 65:9834-42; 2005). The results support a model in which the nucleolus serves as a p53 upstream sensor of cellular stress, and add to a growing body of evidence that nucleolar sequestration of ARF prevents activation of p53. The results also have therapeutic implications for therapies based on exploiting p53 and other cellular stress response pathways to suppress cancer.     

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.     

p14(ARF)-induced apoptosis in p53 protein-deficient cells is mediated by BH3-only protein-independent derepression of Bak protein through down-regulation of Mcl-1 and Bcl-xL proteins

The p14(ARF) tumor suppressor plays a central role in regulating cell cycle arrest and apoptosis. We reported previously that p14(ARF) is capable of triggering apoptosis in a p53-independent manner. However, the mechanism remained unclear. Here we demonstrate that the p53-independent activation of the mitochondrial apoptosis pathway by p14(ARF) is primarily mediated by the pro-apoptotic Bax-homolog Bak. Expression of p14(ARF) exclusively triggers a N-terminal conformational switch of Bak, but not Bax, which allows for mitochondrial permeability shift, release of cytochrome c, activation of caspases, and subsequent fragmentation of genomic DNA. Although forced expression of Bak markedly sensitizes toward p14(ARF)-induced apoptosis, re-expression of Bax has no effect. Vice versa, knockdown of Bak by RNA interference attenuates p14(ARF)-induced apoptosis, whereas down-regulation of Bax has no effect. Bak activation coincides with a prominent, caspase-independent deprivation of the endogenous Bak inhibitors Mcl-1 and Bcl-x(L). In turn, mitochondrial apoptosis is fully blocked by overexpression of either Mcl-1 or Bcl-x(L). Taken together, these data indicate that in the absence of functional p53 and Bax, p14(ARF) triggers mitochondrial apoptosis signaling by activating Bak, which is facilitated by down-regulating anti-apoptotic Mcl-1 and Bcl-x(L). Moreover, our data suggest that the simultaneous inhibition of two central endogenous Bak inhibitors, i.e. Mcl-1 and Bcl-x(L), may be sufficient to activate mitochondrial apoptosis in the absence of BH3-only protein regulation.     

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.     

Recombinant adenovirus-mediated p14(ARF) overexpression sensitizes human breast cancer cells to cisplatin

p14(ARF), the alternative product from the human INK4a/ARF locus, is one of the major targets for alterations in the development of human cancers. Overexpression of p14(ARF) results in cell cycle arrest and apoptosis. To examine the potential therapeutic role of re-expressing p14(ARF) gene product in human breast cancer, a recombinant adenovirus expressing the human p14(ARF) cDNA (Adp14(ARF)) was constructed and used to infect breast cancer cells. Five days after infection, Adp14(ARF) had considerable cytotoxicity on p53-wild-type MCF-7 cells. A time-course study showed that Adp14(ARF) infection of MCF-7 cells at 100pfu/cell increased the number of cells in G0/G1 phase and decreased that in S and G2/M phases. The presence of apoptotic cells was confirmed using the TUNEL assay. Adp14(ARF)-mediated expression of p14(ARF) also resulted in a considerable increase in the amounts of p53 and its target proteins, p21(WAF1) and MDM2. Furthermore, the combination treatment of MCF-7 cells with Adp14(ARF) and cisplatin resulted in a significantly greater cell death. Together, we conclude that p14(ARF) plays an important role in the induction of cell cycle arrest and apoptosis in breast cancer cells and recombinant adenovirus-mediated p14(ARF) expression greatly increases the sensitivity of these cells to cisplatin. These results demonstrate that the proper combination of Adp14(ARF) with conventional chemotherapeutic drug(s) could have potential benefits in treating breast cancer that carries wild-type p53 gene.     

p14ARF Post-Transcriptional Regulation of Nuclear Cyclin D1 in MCF-7 Breast Cancer Cells: Discrimination between a Good and Bad Prognosis?

As part of a cell's inherent protection against carcinogenesis, p14ARF is upregulated in response to hyperproliferative signalling to induce cell cycle arrest. This property makes p14ARF a leading candidate for cancer therapy. This study explores the consequences of reactivating p14ARF in breast cancer and the potential of targeting p14ARF in breast cancer treatment. Our results show that activation of the p14ARF-p53-p21-Rb pathway in the estrogen sensitive MCF-7 breast cancer cells induces many hallmarks of senescence including a large flat cell morphology, multinucleation, senescence-associated-β-gal staining, and rapid G1 and G2/M phase cell cycle arrest. P14ARF also induces the expression of the proto-oncogene cyclin D1, which is most often associated with a transition from G1-S phase and is highly expressed in breast cancers with poor clinical prognosis. In this study, siRNA knockdown of cyclin D1, p21 and p53 show p21 plays a pivotal role in the maintenance of high cyclin D1 expression, cell cycle and growth arrest post-p14ARF induction. High p53 and p14ARF expression and low p21/cyclin D1 did not cause cell-cycle arrest. Knockdown of cyclin D1 stops proliferation but does not reverse senescence-associated cell growth. Furthermore, cyclin D1 accumulation in the nucleus post-p14ARF activation correlated with a rapid loss of nucleolar Ki-67 protein and inhibition of DNA synthesis. Latent effects of the p14ARF-induced cellular processes resulting from high nuclear cyclin D1 accumulation included a redistribution of Ki-67 into the nucleoli, aberrant nuclear growth (multinucleation), and cell proliferation. Lastly, downregulation of cyclin D1 through inhibition of ER abrogated latent recurrence. The mediation of these latent effects by continuous expression of p14ARF further suggests a novel mechanism whereby dysregulation of cyclin D1 could have a double-edged effect. Our results suggest that p14ARF induced-senescence is related to late-onset breast cancer in estrogen responsive breast cancers and/or the recurrence of more aggressive breast cancer post-therapy.