Loss of one allele of ARF rescues Mdm2 haploinsufficiency effects on apoptosis and lymphoma development (original) (raw)
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Disruption of the ARF-Mdm2-p53 tumor suppressor pathway in Myc-induced lymphomagenesis
Genes & Development, 1999
Transgenic mice expressing the c-Myc oncogene driven by the immunoglobulin heavy chain enhancer (Eµ) develop B-cell lymphoma and exhibit a mean survival time of approximately 6 months. The protracted latent period before the onset of frank disease likely reflects the ability of c-Myc to induce a p53-dependent apoptotic program that initially protects animals against tumor formation but is disabled when overtly malignant cells emerge. In cultured primary mouse embryo fibroblasts, c-Myc activates the p19 ARF -Mdm2-p53 tumor suppressor pathway, enhancing p53-dependent apoptosis but ultimately selecting for surviving immortalized cells that have sustained either p53 mutation or biallelic ARF deletion. Here we report that p53 and ARF also potentiate Myc-induced apoptosis in primary pre-B-cell cultures, and that spontaneous inactivation of the ARF-Mdm2-p53 pathway occurs frequently in tumors arising in Eµ-myc transgenic mice. Many Eµ-myc lymphomas sustained either p53 (28%) or ARF (24%) loss of function, whereas Mdm2 levels were elevated in others. Its overexpression in some tumors lacking p53 function raises the possibility that Mdm2 can contribute to lymphomagenesis by interacting with other targets. Eµ-myc transgenic mice hemizygous for ARF displayed accelerated disease (11-week mean survival), and 80% of these tumors lost the wild-type ARF allele. All ARF-null Eµ-myc mice died of lymphoma within a few weeks of birth. About half of the tumors arising in ARF hemizygous or ARF nullizygous Eµ-myc transgenic mice also overexpressed Mdm2. Therefore, Myc activation strongly selects for spontaneous inactivation of the ARF-Mdm2-p53 pathway in vivo, canceling its protective checkpoint function and accelerating progression to malignancy. Corresponding author. E-MAIL john.cleveland@stjude.org; FAX (901) 525-8025. 2658 GENES & DEVELOPMENT 13:2658-2669 © 1999 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/99 $5.00; www.genesdev.org
Arf induces p53-dependent and -independent antiproliferative genes
Cancer research, 2003
The tumor suppressor p19(Arf) (p14(ARF) in humans), encoded by the Ink4a/Arf locus, is mutated, deleted, or silenced in many forms of cancer. p19(Arf) induces growth arrest by antagonizing the activity of the p53-negative regulator, Mdm2, thereby inducing a p53 transcriptional response. p19(Arf) can also inhibit cell cycle progression of mouse embryo fibroblasts lacking Cip1 or lacking both Mdm2 and p53, although in the absence of p53, arrest occurs more slowly. Profiling with high-density oligonucleotide GeneChips and cDNA microarrays was used to interrogate mouse genes, the expression of which was induced or suppressed by a conditionally regulated Arf gene. Cluster analysis of temporal gene expression patterns and validation of the results by RNA analysis identified Arf-responsive genes whose induction was both p53-dependent and -independent. The latter included four members of the B-cell translocation gene family (Btg1, Btg2, Btg3, and Tob1) that were demonstrated to inhibit cell...
Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2
Proceedings of the National Academy of Sciences, 1998
The INK4a-ARF locus encodes two proteins, p16 INK4a and p19 ARF , that restrain cell growth by affecting the functions of the retinoblastoma protein and p53, respectively. Disruption of this locus by deletions or point mutations is a common event in human cancer, perhaps second only to the loss of p53. Using insect cells infected with baculovirus vectors and NIH 3T3 fibroblasts infected with ARF retrovirus, we determined that mouse p19 ARF can interact directly with p53, as well as with the p53 regulator mdm2. ARF can bind p53-DNA complexes, and it depends upon functional p53 to transcriptionally induce mdm2 and the cyclin-dependent kinase inhibitor p21 Cip1 , and to arrest cell proliferation. Binding of p19 ARF to p53 requires the ARF N-terminal domain (amino acids 1-62) that is necessary and sufficient to induce cell cycle arrest. Overexpression of p19 ARF in wild type or ARF-null mouse embryo fibroblasts increases the half-life of p53 from 15 to Ϸ75 min, correlating with an increased p53-dependent transcriptional response and growth arrest. Surprisingly, when overexpressed at supra-physiologic levels after introduction into ARF-null NIH 3T3 cells or mouse embryo fibroblasts, the p53 protein is handicapped in inducing this checkpoint response. In this setting, reintroduction of p19 ARF restores p53's ability to induce p21 Cip1 and mdm2, implying that, in addition to stabilizing p53, ARF modulates p53dependent function through an additional mechanism. MATERIALS AND METHODS Cell Culture. Mammalian cells were maintained in DMEM plus 10% fetal bovine serum, 2 mM glutamine, and 100 units͞ml penicillin and streptomycin (GIBCO). Balb-3T3 (10)1 cells (ARF wild-type, p53-deleted) were a gift of Arnold Levine (Princeton University, Princeton, NJ). MEFs at passages 6-9 were established as described (14); those lacking p53 came from embryos of p53-null mice (The Jackson Laboratory). Charles Sawyers (University of California, Los Angeles) provided helper and vector retrovirus plasmids. Virus production and infection were performed as previously described, and growth arrest was determined by measurements of DNA The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked ''advertisement'' in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Mdm2 Regulates p53 Independently of p19ARF in Homeostatic Tissues
Molecular and Cellular Biology, 2004
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.
p53-Dependent and -Independent Functions of the Arf Tumor Suppressor
Cold Spring Harbor Symposia on Quantitative Biology, 2005
The Ink4a-Arf locus encodes two closely wedded tumor suppressor proteins (p16 Ink4a and p19 Arf) that inhibit cell proliferation by activating Rb and p53, respectively. With few exceptions, the Arf gene is repressed during mouse embryonic development, thereby helping to limit p53 expression during organogenesis. However, in adult mice, sustained hyperproliferative signals conveyed by somatically activated oncogenes can induce Arf gene expression and trigger a p53 response that eliminates incipient cancer cells. Disruption of this tumor surveillance pathway predisposes to cancer, and inactivation of INK4a-ARF by deletion, silencing, or mutation has been frequently observed in many forms of human cancer. Although it is accepted that much of Arf's tumor-suppressive activity is mediated by p53, more recent genetic evidence has pointed to additional p53independent functions of Arf, including its ability to inhibit gene expression by a number of other transcription factors. Surprisingly, the enforced expression of Arf in mammalian cells promotes the sumoylation of several Arf-interacting proteins, implying that Arf has an associated catalytic activity. We speculate that transcriptional down-regulation in response to Arfinduced sumoylation may account for Arf's p53-independent functions.
International Journal of Molecular Sciences, 2012
Transcriptional activation of MYC is a hallmark of many B cell lineage neoplasms. MYC provides a constitutive proliferative signal but can also initiate ARF-dependent activation of p53 and apoptosis. The E3 ubiquitin ligase, ARF-BP1, encoded by HUWE1, modulates the activity of both the MYC and the ARF-p53 signaling pathways, prompting us to determine if it is involved in the pathogenesis of MYC-driven B cell lymphomas. ARF-BP1 was expressed at high levels in cell lines from lymphomas with either wild type or mutated p53 but not in ARF-deficient cells. Downregulation of
Proceedings of the National Academy of Sciences
Cancer development is driven by activated oncogenes and loss of tumor suppressors. While oncogene inhibitors have entered routine clinical practice, tumor suppressor reactivation therapy remains to be established. For the most frequently inactivated tumor suppressor p53, genetic mouse models have demonstrated regression of p53-null tumors upon p53 reactivation. While this was shown in tumor models driven by p53 loss as the initiating lesion, many human tumors initially develop in the presence of wild-type p53, acquire aberrations in the p53 pathway to bypass p53-mediated tumor suppression, and inactivate p53 itself only at later stages during metastatic progression or therapy. To explore the efficacy of p53 reactivation in this scenario, we used a reversibly switchable p53 (p53ERTAM) mouse allele to generate Eµ-Myc–driven lymphomas in the presence of active p53 and, after full lymphoma establishment, switched off p53 to model late-stage p53 inactivation. Although these lymphomas had...
Molecular and Cellular Biology, 2002
Recent studies have shown the p19 ARF tumor suppressor to be involved in the response to oncogenic stress by regulating the activity of p53. This response is mediated by antagonizing the function of Mdm2, a negative regulator of p53, indicating a pathway for tumor suppression that involves numerous genes altered in human tumors. We previously described a transgenic mouse brain tumor model in which oncogenic stress, provided by cell-specific inactivation of the pRb pathway, triggers a p53-dependent apoptotic response. This response suppresses the growth of developing tumors and thus represents a bona fide in vivo tumor suppressor activity. We further showed that E2F1, a transcription factor known to induce p19 ARF expression, was required for the response. Here, we use a genetic approach to test whether p19 ARF functions to transduce the signal from E2F1 to p53 in this tumor suppression pathway. Contrary to the currently accepted hypothesis, we show that a deficiency in p19 ARF has no impact on p53-mediated apoptosis or tumor suppression in this system. All measures of p53 function, including the level of apoptosis induced by pRb inactivation, the expression of p21 (a p53-responsive gene), and the rate of tumor growth, were comparable in mice with and without a functional p19 ARF gene. Thus, although p19 ARF is required in some cell types to transmit an oncogenic response signal to p53, it is dispensable for this function in an in vivo epithelial system. These results underscore the complexity of p53 tumor suppression and further indicate the existence of distinct cell-specific pathways that respond to similar stimuli.
Arf Induces p 53-dependent and-independent Antiproliferative Genes 1 , 2
2003
The tumor suppressor p19 (p14 in humans), encoded by the Ink4a/Arf locus, is mutated, deleted, or silenced in many forms of cancer. p19 induces growth arrest by antagonizing the activity of the p53negative regulator, Mdm2, thereby inducing a p53 transcriptional response. p19 can also inhibit cell cycle progression of mouse embryo fibroblasts lacking Cip1 or lacking both Mdm2 and p53, although in the absence of p53, arrest occurs more slowly. Profiling with high-density oligonucleotide GeneChips and cDNA microarrays was used to interrogate mouse genes, the expression of which was induced or suppressed by a conditionally regulated Arf gene. Cluster analysis of temporal gene expression patterns and validation of the results by RNA analysis identified Arf-responsive genes whose induction was both p53-dependent and -independent. The latter included four members of the B-cell translocation gene family (Btg1, Btg2, Btg3, and Tob1) that were demonstrated to inhibit cell proliferation in pri...
p19Arf induces p53-dependent apoptosis during Abelson virus-mediated pre-B cell transformation
Proceedings of the National Academy of Sciences, 1998
The Ink4a͞Arf locus encodes p16 Ink4a and p19 Arf and is among the most frequently mutated tumor suppressor loci in human cancer. In mice, many of these effects appear to be mediated by interactions between p19 Arf and the p53 tumor-suppressor protein. Because Tp53 mutations are a common feature of the multistep pre-B cell transformation process mediated by Abelson murine leukemia virus (Ab-MLV), we examined the possibility that proteins encoded by the Ink4a͞Arf locus also play a role in Abelson virus transformation. Analyses of primary transformants revealed that both p16 Ink4a and p19 Arf are expressed in many of the cells as they emerge from the apoptotic crisis that characterizes the transformation process. Analyses of primary transformants from Ink4a͞Arf null mice revealed that these cells bypassed crisis. Because expression of p19 Arf but not p16 Ink4a induced apoptosis in Ab-MLV-transformed pre-B cells, p19 Arf appears to be responsible for these events. Consistent with the link between p19 Arf and p53, Ink4a͞Arf expression correlates with or precedes the emergence of cells expressing mutant p53. These data demonstrate that p19 Arf is an important part of the cellular defense mounted against transforming signals from the Abl oncoprotein and provide direct evidence that the p19 Arf -p53 regulatory loop plays an important role in lymphoma induction.