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1.
Oncogene ; 33(3): 300-7, 2014 Jan 16.
Article in English | MEDLINE | ID: mdl-23318441

ABSTRACT

ARF is a multifunctional tumor suppressor that acts as both a sensor of oncogenic stimuli and as a key regulator of ribosome biogenesis. Recently, our group established the DEAD-box RNA helicase and microRNA (miRNA) microprocessor accessory subunit, DDX5, as a critical target of basal ARF function. To identify other molecular targets of ARF, we focused on known interacting proteins of DDX5 in the microprocessor complex. Drosha, the catalytic core of the microprocessor complex, has a critical role in the maturation of specific non-coding RNAs, including miRNAs and ribosomal RNAs (rRNAs). Here, we report that chronic or acute loss of Arf enhanced Drosha protein expression. This induction did not involve Drosha mRNA transcription or protein stability but rather relied on the increased translation of existing Drosha mRNAs. Enhanced Drosha expression did not alter global miRNA production but rather modified expression of a subset of miRNAs in the absence of Arf. Elevated Drosha protein levels were required to maintain the increased rRNA synthesis and cellular proliferation observed in the absence of Arf. Arf-deficient cells transformed by oncogenic Ras(V12) were dependent on increased Drosha expression as Drosha knockdown was sufficient to inhibit Ras-dependent cellular transformation. Thus, we propose that ARF regulates Drosha mRNA translation to prevent aberrant cell proliferation and Ras-dependent transformation.


Subject(s)
Cell Transformation, Neoplastic/genetics , Cyclin-Dependent Kinase Inhibitor p16/genetics , Ribonuclease III/genetics , ras Proteins/genetics , Animals , Apoptosis/genetics , Blotting, Western , Cell Cycle/genetics , Cell Proliferation , Cell Transformation, Neoplastic/metabolism , Cells, Cultured , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Embryo, Mammalian/cytology , Fibroblasts/cytology , Fibroblasts/metabolism , Gene Expression , Mice , Mice, Knockout , MicroRNAs/genetics , Protein Biosynthesis , RNA Interference , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Ribonuclease III/metabolism , ras Proteins/metabolism
2.
Oncogene ; 30(1): 77-86, 2011 Jan 06.
Article in English | MEDLINE | ID: mdl-20802533

ABSTRACT

Nucleophosmin (NPM/B23) is a multifunctional oncoprotein whose protein expression levels dictate cellular growth and proliferation rates. NPM is translationally responsive to hyperactive mammalian target of rapamycin (mTOR) signals, but the mechanism of this regulation is not understood. Using chimeric translational reporters, we found that the 3' untranslated region (UTR) of the NPM messenger (m)RNA is sufficient to mediate its translational modulation by mTOR signalling. We show that far upstream element (FUSE)-binding protein 1 (FBP1) interacts specifically with the 3' UTR of NPM to repress translation. Overexpression of FBP1 resulted in translational repression of NPM mRNAs, whereas depletion of FBP1 caused a dramatic increase in NPM translation and resulted in enhanced overall cell proliferation. Thus, we propose that FBP1 is a key regulator of cell growth and proliferation through its ability to selectively bind the NPM 3' UTR and repress NPM translation.


Subject(s)
DNA Helicases/metabolism , DNA-Binding Proteins/metabolism , Nuclear Proteins/biosynthesis , 5' Untranslated Regions , Cell Growth Processes/genetics , DNA Helicases/genetics , DNA-Binding Proteins/genetics , Humans , Nuclear Proteins/antagonists & inhibitors , Nuclear Proteins/genetics , Nucleophosmin , Protein Binding , Protein Biosynthesis , RNA, Messenger/genetics , RNA, Messenger/metabolism , RNA-Binding Proteins , Signal Transduction , Sirolimus/metabolism
3.
Oncogene ; 28(36): 3209-20, 2009 Sep 10.
Article in English | MEDLINE | ID: mdl-19561638

ABSTRACT

Nucleophosmin (NPM), an oligomeric phosphoprotein and nucleolar target of the ARF tumor suppressor, contributes to several critical cellular processes. Previous studies have shown that the human NPM's phosphorylation by cyclin E-cyclin-dependent kinase 2 (cdk2) on threonine (Thr) 199 regulates its translocation from the centrosome during cell cycle progression. Given our previous finding that ARF directly binds NPM, impeding its transit to the cytoplasm and arresting cells before S-phase entry, we hypothesized that ARF might also inhibit NPM phosphorylation. However, ARF induction did not impair phosphorylation of the cdk2 target residue in murine NPM, Thr198. Furthermore, phosphorylation of Thr198 occurred throughout the cell cycle and was concomitant with increases in overall NPM expression. To investigate the cell's presumed requirement for NPM-Thr198 phosphorylation in promoting the processes of growth and proliferation, we examined the effects of a non-phosphorylatable NPM mutant, T198A, in a clean cell system in which endogenous NPM had been removed by RNA interference. Here, we show that the T198A mutant is fully capable of executing NPM's described roles in nucleocytoplasmic shuttling, ribosome export and cell cycle progression. Moreover, the proliferative defects observed with stable NPM knockdown were restored by mutant NPM-T198A expression. Thus, we demonstrate that the reduction in NPM protein expression blocks cellular growth and proliferation, whereas phosphorylation of NPM-Thr198 is not essential for NPM's capacity to drive cell cycle progression and proliferation.


Subject(s)
Cell Proliferation , Mutation , Nuclear Proteins/metabolism , Threonine/metabolism , Animals , Blotting, Western , Breast Neoplasms/metabolism , Breast Neoplasms/pathology , Cell Cycle , Colonic Neoplasms/metabolism , Colonic Neoplasms/pathology , Cyclin-Dependent Kinase Inhibitor p16/genetics , Cyclin-Dependent Kinase Inhibitor p16/metabolism , Female , Green Fluorescent Proteins/genetics , Green Fluorescent Proteins/metabolism , HeLa Cells , Humans , Immunohistochemistry , Male , Mice , Mice, Knockout , NIH 3T3 Cells , Nuclear Proteins/genetics , Nucleophosmin , Phosphorylation , Prostatic Neoplasms/metabolism , Prostatic Neoplasms/pathology , Proto-Oncogene Proteins c-mdm2/genetics , Proto-Oncogene Proteins c-mdm2/metabolism , RNA Interference , Threonine/genetics , Tumor Suppressor Protein p14ARF/genetics , Tumor Suppressor Protein p14ARF/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism
4.
Histol Histopathol ; 22(8): 895-903, 2007 08.
Article in English | MEDLINE | ID: mdl-17503347

ABSTRACT

The mammalian target of rapamycin (mTOR) is a highly conserved serine/threonine protein kinase that regulates a number of diverse biologic processes important for cell growth and proliferation, including ribosomal biogenesis and protein translation. In this regard, hyperactivation of the mTOR signaling pathway has been demonstrated in numerous human cancers, including a number of inherited cancer syndromes in which individuals have an increased risk of developing benign and malignant tumors. Three of these inherited cancer syndromes (Lhermitte-Duclos disease, neurofibromatosis type 1, and tuberous sclerosis complex) are characterized by significant central nervous system dysfunction and brain tumor formation. Each of these disorders is caused by a genetic mutation that disrupts the expression of proteins which negatively regulate mTOR signaling, indicating that the mTOR signaling pathway is critical for appropriate brain development and function. In this review, we discuss our current understanding of the mTOR signaling pathway and its role in promoting ribosome biogenesis and cell growth. We suggest that studies of this pathway may prove useful in identifying molecular targets for biologically-based therapies of brain tumors associated with these inherited cancer syndromes as well as sporadic central nervous system tumors.


Subject(s)
Brain Neoplasms/metabolism , Cell Proliferation , Central Nervous System/metabolism , Protein Kinases/metabolism , Signal Transduction , Animals , Antibiotics, Antineoplastic/pharmacology , Antibiotics, Antineoplastic/therapeutic use , Brain Neoplasms/drug therapy , Brain Neoplasms/genetics , Brain Neoplasms/pathology , Cell Proliferation/drug effects , Central Nervous System/drug effects , Central Nervous System/pathology , Humans , Mutation , Protein Kinase Inhibitors/pharmacology , Protein Kinase Inhibitors/therapeutic use , Protein Kinases/genetics , Ribosomes/metabolism , Signal Transduction/drug effects , Sirolimus/pharmacology , Sirolimus/therapeutic use , TOR Serine-Threonine Kinases
5.
Gynecol Oncol ; 104(3): 727-31, 2007 Mar.
Article in English | MEDLINE | ID: mdl-17126894

ABSTRACT

OBJECTIVE: The primary objective of this study was to determine the rate of response to matuzumab in patients with recurrent, EGFR-positive ovarian, or primary peritoneal cancer. Secondary end points included safety and tolerability, time to tumor progression, duration of response, and overall survival. METHODS: A multi-institutional single arm phase II trial. RESULTS: Of 75 women screened for the study, 37 were enrolled and treated. Median age of the treated patient population was 58 years, and most patients had more than four prior lines of chemotherapy. Therapy was well tolerated, the most common toxicities being a constellation of skin toxicities, including rash, acne, dry skin, and paronychia, as well as headache, fatigue, and diarrhea. Serious adverse events were very rare but included a single episode of pancreatitis that may have been drug related. All patients completed therapy, receiving 1 to 30 infusions of matuzumab. There were no formal responses (RR=0%, 95% CI: 0-9.5%), although 7 patients (21%) were on therapy for more than 3 months with stable disease. CONCLUSIONS: Matuzumab at the dose and schedule selected is well tolerated. In this population of very heavily pretreated patients with epithelial ovarian and primary peritoneal malignancies, there was no evidence of significant clinical activity when matuzumab was administered as monotherapy.


Subject(s)
Antibodies, Monoclonal/therapeutic use , Antineoplastic Agents/therapeutic use , Ovarian Neoplasms/drug therapy , Peritoneal Neoplasms/drug therapy , Adult , Aged , Aged, 80 and over , Antibodies, Monoclonal, Humanized , Cetuximab , Drug Resistance, Neoplasm , ErbB Receptors/biosynthesis , ErbB Receptors/immunology , Female , Humans , Middle Aged , Organoplatinum Compounds/pharmacology , Ovarian Neoplasms/enzymology , Ovarian Neoplasms/immunology , Peritoneal Neoplasms/immunology
7.
J Mol Biol ; 314(2): 263-77, 2001 Nov 23.
Article in English | MEDLINE | ID: mdl-11718560

ABSTRACT

Understanding the interaction of Arf and Hdm2 has recently become a central issue in cancer biology. In response to hyperproliferative signals, p14(Arf) stabilizes p53 by binding to Hdm2 and inhibits the ubiquitination and subsequent proteosome-dependent degradation of p53. The medical importance of the Arf-Hdm2-p53 regulatory system is highlighted by the finding that either p53 or p14(Arf) are lost or modified in virtually all human cancers. Isolated Arf and Hdm2 domains are dynamically disordered in solution, yet they retain the ability to interact in vitro and in cellular assays. Upon binding, domains of both Arf and Hdm2 undergo a dramatic transition from disordered conformations to extended structures comprised of beta-strands. The presence of domains from both proteins are necessary and sufficient for the formation of the highly stable extended beta structures. We have mapped sites within Arf and Hdm2 that interact at a resolution of five amino acid residues using surface plasmon resonance. Surface plasmon resonance and circular dichroism spectropolarimetry confirm the presence of multiple interaction domains within each protein. Both p14(Arf) (human) and p19(Arf) (mouse) interact with Hdm2 through two short motifs present in their N termini. The Arf interacting region of Hdm2 is also composed of two short sequences located in the central acidic domain, between residues 235-264 and 270-289. The binding-induced structural transition is also induced by short peptides, 15 amino acids in length, that contain the binding motifs. Micro-injection and live cell imaging of proteins tagged with fluorescent labels was used to confirm the in vivo function of the interaction domains. Arf and Hdm2 thus appear to interact through a novel mechanism that exerts control over the cell division cycle. The novel molecular mechanism of interaction and the limited size of the protein domains involved provide opportunities for the development of anticancer therapeutics.


Subject(s)
Nuclear Proteins , Proto-Oncogene Proteins/chemistry , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p14ARF/chemistry , Tumor Suppressor Protein p14ARF/metabolism , 3T3 Cells , Amino Acid Motifs , Amino Acid Sequence , Animals , Binding Sites , Cell Nucleolus/chemistry , Cell Nucleolus/metabolism , Circular Dichroism , Cyclin-Dependent Kinase Inhibitor p16 , Gene Deletion , Humans , Magnetic Resonance Spectroscopy , Mice , Molecular Sequence Data , Peptide Fragments/chemistry , Peptide Fragments/metabolism , Protein Binding , Protein Interaction Mapping , Protein Structure, Secondary , Protein Structure, Tertiary , Proto-Oncogene Proteins c-mdm2 , Sequence Alignment , Surface Plasmon Resonance , Tumor Suppressor Protein p14ARF/genetics , Tumor Suppressor Protein p53/metabolism
8.
Biochemistry ; 40(8): 2379-86, 2001 Feb 27.
Article in English | MEDLINE | ID: mdl-11327858

ABSTRACT

Arf is a tumor suppressor that regulates p53 function and is a frequent target for loss in human cancers. Through two novel mechanisms, Arf inhibits the oncoprotein Hdm2, a negative regulator of p53. (1) Arf inhibits the E3 ubiquitin ligase activity of Hdm2 that leads to p53 degradation, and (2) Arf sequesters Hdm2 within nucleoli. These activities of Arf promote p53-mediated cell cycle arrest and apoptosis. Fundamental to these processes are interactions between Arf and Hdm2. Here we show that a peptide containing the 37 N-terminal amino acids of mouse Arf (mArfN37) localizes to nucleoli, sequesters Hdm2 within nucleoli, and causes cell cycle arrest. Circular dichroism and NMR spectroscopy show that mArfN37 is largely unstructured under aqueous conditions; however, the peptide adopts two alpha-helices (helix 1, residues 4-14; and helix 2, residues 20-29) in 2,2,2-trifluoroethanol (TFE). Each helix contains an amino acid motif that is repeated twice in mArfN37, once in each helix. The two helices, however, do not interact but are connected by an apparently flexible linker. The repeated motif contains Arg residues spaced by a hydrophobic segment that may be involved in Hdm2 recognition and binding. The RRPR nucleolar localization signal, contained within residues 31-34, appears to be disordered under all conditions. The identification of two Arf structural modules suggests that short peptides containing the repeated motif may function as Arf mimics and may allow the design of small molecule Arf mimics in the future.


Subject(s)
Peptide Fragments/chemistry , Peptide Fragments/physiology , Proteins/chemistry , Proteins/physiology , Tumor Suppressor Protein p53/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Cell Line , Circular Dichroism , Genes, Tumor Suppressor , Mice , Mice, Knockout , Molecular Sequence Data , Nuclear Magnetic Resonance, Biomolecular , Peptide Fragments/genetics , Protein Structure, Tertiary/genetics , Proteins/genetics , Recombinant Proteins/biosynthesis , Recombinant Proteins/chemistry , Solutions , Solvents , Trifluoroethanol , Tumor Suppressor Protein p14ARF
9.
Genes Dev ; 14(18): 2358-65, 2000 Sep 15.
Article in English | MEDLINE | ID: mdl-10995391

ABSTRACT

The p19(ARF) tumor suppressor antagonizes Mdm2 to induce p53-dependent cell cycle arrest. Individual TKO (triple knock out) mice nullizygous for ARF, p53, and Mdm2 develop multiple tumors at a frequency greater than those observed in animals lacking both p53 and Mdm2 or p53 alone, demonstrating that p19(ARF) can act independently of the Mdm2-p53 axis in tumor surveillance. Reintroduction of ARF into TKO mouse embryo fibroblasts (MEFs), but not into those lacking both p53 and ARF, arrested the cell division cycle in the G1 phase. Inhibition of the retinoblastoma protein had no effect on the ability of ARF to arrest TKO MEFs. Thus, in the absence of Mdm2, p19(ARF) interacts with other targets to inhibit cell proliferation.


Subject(s)
Genes, Tumor Suppressor , Nuclear Proteins , Proteins/metabolism , Tumor Suppressor Protein p53/physiology , Animals , Cells, Cultured , Fluorescent Antibody Technique , Immunoblotting , Mice , Mice, Inbred C57BL , Mice, Knockout , Neoplasms/genetics , Neoplasms/pathology , Polymerase Chain Reaction , Proteins/genetics , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-mdm2 , Tumor Suppressor Protein p14ARF , Tumor Suppressor Protein p53/genetics
10.
J Biol Chem ; 275(35): 27473-80, 2000 Sep 01.
Article in English | MEDLINE | ID: mdl-10842176

ABSTRACT

Oncogenic Ras induces two products of the INK4a/ARF tumor suppressor locus (p16(INK4a) and p19(ARF)) in primary human and rodent fibroblasts, ultimately leading to a permanent state of cell cycle arrest resembling replicative senescence. Whereas p16(INK4a) antagonizes the activities of cyclin D-dependent kinases, p19(ARF) activates the p53 transcription factor. Immortalized rodent fibroblast cell lines that lack INK4a/ARF function, ARF alone, or p53 are resistant to the growth inhibitory effects of oncogenic Ras and instead continue to proliferate and undergo morphological transformation. Primary mouse embryo fibroblasts lacking Cip1 and Kip1 genes encoding inhibitors of cyclin-dependent kinase-2 were used to further explore the effects of oncogenic Ras on arrest of the cell division cycle. Although early passage primary fibroblast strains that lack both p21(Cip1) and p27(Kip1) fail to assemble cyclin D-dependent kinases, oncogenic Ras retained its ability to induce p19(ARF), but not p16(INK4a), protecting Cip/Kip-null cells from proliferating and undergoing transformation. Under these conditions, Ras did not induce G(1) phase arrest but instead triggered DNA synthesis, abnormal nuclear divisions, failure of cytokinesis, and emergence of polyploid cells. Therefore, in the absence of p16(INK4a), p21(Cip1), and p27(Kip1), oncogenic Ras affects the functions of genes required for completion of the cell cycle.


Subject(s)
Cell Cycle Proteins , Cell Division/physiology , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , Embryo, Mammalian/cytology , Microtubule-Associated Proteins/metabolism , Oncogene Protein p21(ras)/physiology , Protein Biosynthesis , Tumor Suppressor Proteins , 3T3 Cells , Animals , Cell Cycle , Cyclin D1/biosynthesis , Cyclin D1/metabolism , Cyclin-Dependent Kinase Inhibitor p21 , Cyclin-Dependent Kinase Inhibitor p27 , Embryo, Mammalian/metabolism , Enzyme Activation , Fibroblasts/cytology , Fibroblasts/metabolism , Humans , Mice , Tumor Suppressor Protein p14ARF
11.
Mol Cell Biol ; 20(7): 2517-28, 2000 Apr.
Article in English | MEDLINE | ID: mdl-10713175

ABSTRACT

The ARF tumor suppressor protein stabilizes p53 by antagonizing its negative regulator, Mdm2 (Hdm2 in humans). Both mouse p19(ARF) and human p14(ARF) bind to the central region of Mdm2 (residues 210 to 304), a segment that does not overlap with its N-terminal p53-binding domain, nuclear import or export signals, or C-terminal RING domain required for Mdm2 E3 ubiquitin ligase activity. The N-terminal 37 amino acids of mouse p19(ARF) are necessary and sufficient for binding to Mdm2, localization of Mdm2 to nucleoli, and p53-dependent cell cycle arrest. Although a nucleolar localization signal (NrLS) maps within a different segment (residues 82 to 101) of the human p14(ARF) protein, binding to Mdm2 and nucleolar import of ARF-Mdm2 complexes are both required for cell cycle arrest induced by either the mouse or human ARF proteins. Because many codons of mouse ARF mRNA are not recognized by the most abundant bacterial tRNAs, we synthesized ARF minigenes containing preferred bacterial codons. Using bacterially produced ARF polypeptides and chemically synthesized peptides conjugated to Sepharose, residues 1 to 14 and 26 to 37 of mouse p19(ARF) were found to interact independently and cooperatively with Mdm2, while residues 15 to 25 were dispensable for binding. Paradoxically, residues 26 to 37 of mouse p19(ARF) are also essential for ARF nucleolar localization in the absence of Mdm2. However, the mobilization of the p19(ARF)-Mdm2 complex into nucleoli also requires a cryptic NrLS within the Mdm2 C-terminal RING domain. The Mdm2 NrLS is unmasked upon ARF binding, and its deletion prevents import of the ARF-Mdm2 complex into nucleoli. Collectively, the results suggest that ARF binding to Mdm2 induces a conformational change that facilitates nucleolar import of the ARF-Mdm2 complex and p53-dependent cell cycle arrest. Hence, the ARF-Mdm2 interaction can be viewed as bidirectional, with each protein being capable of regulating the subnuclear localization of the other.


Subject(s)
Cell Nucleolus/metabolism , Nuclear Proteins , Proteins/metabolism , Proto-Oncogene Proteins/metabolism , 3T3 Cells , Amino Acid Sequence , Animals , Cell Cycle/genetics , Cell Division/genetics , Fluorescent Antibody Technique , Humans , Mice , Molecular Sequence Data , Mutation , Peptide Fragments/metabolism , Protein Binding/genetics , Protein Conformation , Proteins/genetics , Proto-Oncogene Proteins c-mdm2 , Recombinant Proteins , Retroviridae/genetics , Transfection , Tumor Suppressor Protein p14ARF , Tumor Suppressor Protein p53/metabolism
12.
Curr Opin Genet Dev ; 10(1): 94-9, 2000 Feb.
Article in English | MEDLINE | ID: mdl-10679383

ABSTRACT

The ARF tumor suppressor connects pathways regulated by the retinoblastoma protein and p53. ARF inactivation reduces p53-dependent apoptosis induced by oncogenic signals. Nucleolar relocalization of Mdm2 by ARF connotes a novel mechanism for preventing p53 turnover and provides a framework for understanding how stress signals cooperate to regulate p53 function.


Subject(s)
ADP-Ribosylation Factors/physiology , Genes, Tumor Suppressor , Nuclear Proteins , Tumor Suppressor Protein p53/physiology , Animals , Carrier Proteins/physiology , Cyclin-Dependent Kinase Inhibitor p16 , Cyclin-Dependent Kinases/antagonists & inhibitors , Humans , Mice , Oncogenes , Proto-Oncogene Proteins/physiology , Proto-Oncogene Proteins c-mdm2
13.
Nat Cell Biol ; 1(1): 20-6, 1999 May.
Article in English | MEDLINE | ID: mdl-10559859

ABSTRACT

The Ink4/Arf locus encodes two tumour-suppressor proteins, p16Ink4a and p19Arf, that govern the antiproliferative functions of the retinoblastoma and p53 proteins, respectively. Here we show that Arf binds to the product of the Mdm2 gene and sequesters it into the nucleolus, thereby preventing negative-feedback regulation of p53 by Mdm2 and leading to the activation of p53 in the nucleoplasm. Arf and Mdm2 co-localize in the nucleolus in response to activation of the oncoprotein Myc and as mouse fibroblasts undergo replicative senescence. These topological interactions of Arf and Mdm2 point towards a new mechanism for p53 activation.


Subject(s)
ADP-Ribosylation Factors/metabolism , Cell Nucleolus/metabolism , Nuclear Proteins , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/metabolism , 3T3 Cells , ADP-Ribosylation Factors/genetics , Animals , Cell Nucleolus/ultrastructure , Cellular Senescence , Feedback , Fibroblasts/cytology , Fibroblasts/physiology , Gene Expression Regulation , Genes, myc , Genes, p53 , Mice , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins c-mdm2 , Recombinant Proteins/metabolism , Transfection
14.
Genes Dev ; 13(20): 2658-69, 1999 Oct 15.
Article in English | MEDLINE | ID: mdl-10541552

ABSTRACT

Transgenic mice expressing the c-Myc oncogene driven by the immunoglobulin heavy chain enhancer (Emu) 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 Emu-myc transgenic mice. Many Emu-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. Emu-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 Emu-myc mice died of lymphoma within a few weeks of birth. About half of the tumors arising in ARF hemizygous or ARF nullizygous Emu-myc transgenic mice also overexpressed Mdm2. Therefore, Myc activation strongly selects for spontaneous inactivation of the ARF-Mdm2-p53 pathway in vivo, cancelling its protective checkpoint function and accelerating progression to malignancy.


Subject(s)
Genes, myc , Genes, p53 , Lymphoma, B-Cell/etiology , Lymphoma, B-Cell/genetics , Nuclear Proteins , Proteins/genetics , Proto-Oncogene Proteins/genetics , Animals , Apoptosis/genetics , B-Lymphocytes/cytology , Cells, Cultured , Enhancer Elements, Genetic , Female , Hematopoietic Stem Cells/cytology , Immunoglobulin Heavy Chains/genetics , Lymphoma, B-Cell/pathology , Male , Mice , Mice, Inbred C57BL , Mice, Knockout , Mice, Transgenic , Mutation , Proto-Oncogene Proteins c-mdm2 , Tumor Suppressor Protein p14ARF
15.
Proc Natl Acad Sci U S A ; 95(14): 8292-7, 1998 Jul 07.
Article in English | MEDLINE | ID: mdl-9653180

ABSTRACT

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 approximately 75 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 p53-dependent function through an additional mechanism.


Subject(s)
Gene Expression Regulation, Neoplastic , Nuclear Proteins , Proteins/genetics , Proteins/metabolism , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Tumor Suppressor Protein p53/genetics , Tumor Suppressor Protein p53/metabolism , 3T3 Cells , Animals , Genes, Tumor Suppressor , Humans , Mice , Neoplasm Proteins/genetics , Neoplasm Proteins/metabolism , Proto-Oncogene Proteins c-mdm2 , Retroviridae , Transfection , Tumor Suppressor Protein p14ARF
16.
Am J Physiol ; 273(4): G804-11, 1997 10.
Article in English | MEDLINE | ID: mdl-9357821

ABSTRACT

Cytokines, growth factors, and alterations in the extracellular matrix composition may play a role in maintaining hepatic stellate cells (HSC) in the activated state that is responsible for hepatic fibrogenesis. However, the signal transduction pathways that are stimulated by these factors in HSC remain to be fully elucidated. Recent evidence indicates that the mitogen-activated protein kinase (MAPK) family, including c-Jun NH2-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK), plays an important role in the cellular response to stress. The aims of this study were to investigate whether fibronectin (FN) or the inflammatory cytokines interleukin-1alpha (IL-1alpha) and tumor necrosis factor-alpha (TNF-alpha) activate JNK, ERK, and AP-1 activity in HSC and induce the gene expression of the matrix metalloproteinase transin. Treatment of HSC with FN resulted in an up to 4.5-fold increase in ERK activity and a 2.1-fold increase in JNK activity. IL-1alpha and TNF-alpha produced up to a fourfold increase in JNK activity and a twofold increase in ERK activity. We then compared the effects of FN, IL-1alpha, and TNF-alpha on AP-1 activity and metalloproteinase mRNA induction. All three compounds increased AP-1 binding and promoter activity, and transin mRNA levels were increased 1.8-fold by FN, 2.2-fold by IL-1alpha, and 2.8-fold by TNF-alpha. Therefore, FN and inflammatory cytokines increase MAPK activity, stimulate AP-1 activity, and increase transin gene expression in HSC. Signal transduction pathways involving the MAPK family may play an important role in the regulation of matrix metalloproteinase expression by cytokines and FN in HSC.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Fibronectins/pharmacology , Interleukin-1/pharmacology , Liver/metabolism , Matrix Metalloproteinase 3/biosynthesis , Mitogen-Activated Protein Kinases , Transcription Factor AP-1/metabolism , Transcription, Genetic/drug effects , Tumor Necrosis Factor-alpha/pharmacology , Animals , Cells, Cultured , Chloramphenicol O-Acetyltransferase/biosynthesis , Genes, Reporter , JNK Mitogen-Activated Protein Kinases , Liver/cytology , Liver/drug effects , Male , Rats , Rats, Sprague-Dawley , Tetradecanoylphorbol Acetate/pharmacology , Transfection
17.
Biochem J ; 326 ( Pt 1): 61-8, 1997 Aug 15.
Article in English | MEDLINE | ID: mdl-9337851

ABSTRACT

In Chinese hamster embryo fibroblasts (IIC9 cells), platelet-derived growth factor (PDGF) stimulated mitogen-activated protein kinase/extracellular-signal-regulated kinase (MAP kinase/ERK) activity, but not that of c-jun N-terminal kinase (JNK), and induced G1 phase progression. ERK1 activation was biphasic and was sustained throughout the G1 phase of the cell cycle. PDGF induced cyclin D1 protein and mRNA levels in a time-dependent manner. Inhibition of PDGF-induced ERK1 activity by the addition of a selective inhibitor of MEK1 (MAP kinase kinase/ERK kinase 1) activation, PD98059, or transfection with a dominant-negative ERK1 (dnERK-) was correlated with growth arrest. In contrast, growth was unaffected by expression of dominant-negative JNK (dnJNK-). Interestingly, addition of PD98059 or dnERK-, but not dnJNK-, resulted in a dramatic decrease in cyclin D1 protein and mRNA levels, concomitant with a decrease in cyclin D1-cyclin-dependent kinase activity. To investigate the importance of sustained ERK1 activation, ERK1 activity was blocked by the addition of PD98059 throughout G1. Addition of PD98059 up to 4 h after PDGF treatment decreased ERK1 activity to the levels found in growth-arrested IIC9 cells. Loss of cyclin D1 mRNA and protein expression was observed within 1 h after inhibition of the second sustained phase of ERK1 activity. Disruption of sustained ERK1 activity also resulted in G1 growth arrest. These data provide evidence for a role for sustained ERK activity in controlling G1 progression through positive regulation of the continued expression of cyclin D1, a protein known to positively regulate G1 progression.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cyclin D1/biosynthesis , G1 Phase , Mitogen-Activated Protein Kinases , Animals , Calcium-Calmodulin-Dependent Protein Kinases/antagonists & inhibitors , Calcium-Calmodulin-Dependent Protein Kinases/physiology , Cell Division/drug effects , Cell Line , Cricetinae , Cricetulus , Enzyme Activation/drug effects , Fibroblasts , Flavonoids/pharmacology , G1 Phase/drug effects , JNK Mitogen-Activated Protein Kinases , Mitogen-Activated Protein Kinase 3 , Platelet-Derived Growth Factor/pharmacology , Time Factors , Up-Regulation/drug effects
18.
J Biol Chem ; 272(28): 17312-9, 1997 Jul 11.
Article in English | MEDLINE | ID: mdl-9211868

ABSTRACT

Modulation of the components involved in mitogenic signaling cascades is critical to the regulation of cell growth. GTP-binding proteins and the stimulation of phosphatidylcholine (PC) hydrolysis have been shown to play major roles in these cascades. One of the enzymes involved in PC hydrolysis, a PC-specific phospholipase C (PC-PLC) has received relatively little attention. In this paper we examined the role of a particular heterotrimeric GTP-binding protein, Go, in the regulation of cell growth and PC-PLC-mediated hydrolysis of PC in IIC9 fibroblasts. The Go alpha-subunit was ablated in IIC9 cells by stable expression of antisense RNA. These stably transfected cells acquired a transformed phenotype as indicated by: (a) the formation of multiple foci in monolayer cultures, (b) the acquisition of anchorage-independent growth in soft agar; and (c) an increased level of thymidine incorporation in the absence of added mitogens. These data implicate Goalpha as a novel tumor suppressor. Interestingly, PC-PLC activity was constitutively active in the Goalpha-ablated cells as evidenced by the chronically elevated levels of diacylglycerol and phosphorylcholine in the absence of growth factors. In contrast, basal activities of PC-phospholipase D, phospholipase A2, or phosphoinositol-PLC were not affected. These data demonstrate, for the first time, a role for Go in regulating cell growth and provide definitive evidence for the existence of a PC-PLC in eukaryotic cells. The data further indicate that a subunit of Go, is involved in regulating this enzyme.


Subject(s)
Cell Transformation, Neoplastic , GTP-Binding Proteins/physiology , Type C Phospholipases/metabolism , Animals , Cell Division , Choline Kinase/metabolism , Cricetinae , Cricetulus , Diglycerides/metabolism , Enzyme Activation , GTP-Binding Protein alpha Subunits, Gi-Go , GTP-Binding Proteins/genetics , Humans , Oligonucleotides, Antisense/metabolism , Phenotype , Phosphatidate Phosphatase/metabolism , Phosphatidylcholines/metabolism , Phosphatidylinositols/metabolism , Phospholipase D/metabolism , Phospholipases A/metabolism , Phospholipases A2 , Rats , Transducin/pharmacology
19.
J Biol Chem ; 272(28): 17320-6, 1997 Jul 11.
Article in English | MEDLINE | ID: mdl-9211869

ABSTRACT

We have generated stable IIC9 cell lines, Goa1 and Goa2, that overexpress full-length antisense Goalpha RNA. As shown previously, expression of antisense Goalpha RNA ablated the alpha subunit of the heterotrimeric G protein, Go, resulting in growth in the absence of mitogen. To better understand this change in IIC9 phenotype, we have characterized the signaling pathway and cell cycle events previously shown to be important in control of IIC9 G1/S phase progression. In this paper we clearly demonstrate that ablation of Goalpha results in growth, constitutively active Ras/ERK, elevated expression of cyclin D1, and constitutively active cyclin D1-CDK complexes, all in the absence of mitogen. Furthermore, these characteristics were abolished by the transient overexpression of the transducin heterotrimeric G protein alpha subunit strongly suggesting the transformation of Goalpha-ablated cells involves Gobetagamma subunits. This is the first study to implicate a heterotrimeric G protein in tumor suppression.


Subject(s)
Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cyclin-Dependent Kinases/metabolism , Cyclins/metabolism , GTP-Binding Proteins/physiology , Oncogene Proteins/metabolism , Proto-Oncogene Proteins , ras Proteins/metabolism , Animals , Cell Line , Cell Transformation, Neoplastic , Cricetinae , Cyclin D1 , Cyclin-Dependent Kinase 4 , Enzyme Activation , G1 Phase , GTP-Binding Protein alpha Subunits, Gi-Go , Platelet-Derived Growth Factor/metabolism
20.
J Biol Chem ; 272(52): 32966-71, 1997 Dec 26.
Article in English | MEDLINE | ID: mdl-9407076

ABSTRACT

Platelet-derived growth factor (PDGF)-induced Ras activation is required for G1 progression in Chinese hamster embryo fibroblasts (IIC9 cells). Ras stimulates both extracellular signal-related kinase (ERK) activation and RhoA activation in response to PDGF stimulation. Inhibition of either of these Ras-stimulated pathways results in growth arrest. We have shown previously that Ras-stimulated ERK activation is essential for the induction and continued G1 expression of cyclin D1. In this study we examine the role of Ras-induced RhoA activity in G1 progression. Unstimulated IIC9 cells expressed high levels of the G1 cyclin-dependent kinase inhibitor p27(KIP1). Stimulation with PDGF resulted in a dramatic decrease in p27(KIP1) protein expression. This decrease was attributed to increased p27(KIP1) protein degradation. Overexpression of dominant-negative forms of Ras or RhoA completely blocked PDGF-induced p27(KIP1) degradation, but only dominant-negative Ras inhibited cyclin D1 protein expression. C3 transferase also inhibited PDGF-induced p27(KIP1) degradation, thus further implicating RhoA in p27(KIP1) regulation. Overexpression of dominant-negative ERK resulted in inhibition of PDGF-induced cyclin D1 expression but had no effect on PDGF-induced p27(KIP1) degradation. These data suggest that Ras coordinates the independent regulation of cyclin D1 and p27(KIP1) expression by the respective activation of ERK and RhoA and that these pathways converge to determine the activation state of complexes of cyclin D1 and cyclin-dependent kinase in response to mitogen.


Subject(s)
Botulinum Toxins , Calcium-Calmodulin-Dependent Protein Kinases/metabolism , Cell Cycle Proteins , Cyclin D1/metabolism , Enzyme Inhibitors/metabolism , GTP-Binding Proteins/metabolism , Microtubule-Associated Proteins/metabolism , Mitogen-Activated Protein Kinases , Platelet-Derived Growth Factor/pharmacology , Tumor Suppressor Proteins , ras Proteins/metabolism , ADP Ribose Transferases/metabolism , Animals , Cricetinae , Cyclin-Dependent Kinase Inhibitor p27 , Cyclin-Dependent Kinases/antagonists & inhibitors , Cytoskeleton/metabolism , G1 Phase , Humans , Macromolecular Substances , Mitogen-Activated Protein Kinase 3 , rhoA GTP-Binding Protein
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