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M F Roussel

Publications and source records attributed to M F Roussel.

At least 37 records · Page 2Linked to original sources
PubMed2000

Cooperative signals governing ARF-mdm2 interaction and nucleolar localization of the complex.

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.

3T3 Cells↗
PubMed1999

Postnatal neuronal proliferation in mice lacking Ink4d and Kip1 inhibitors of cyclin-dependent kinases.

Development of the central nervous system requires proliferation of neuronal and glial cell precursors followed by their subsequent differentiation in a highly coordinated manner. The timing of neuronal cell cycle exit and differentiation is likely to be regulated in part by inhibitors of cyclin-dependent kinases. Overlapping and sustained patterns of expression of two cyclin-dependent kinases, p19(Ink4d) and p27(Kip1), in postmitotic brain cells suggested that these proteins may be important in actively repressing neuronal proliferation. Animals derived from crosses of Ink4d- null with Kip1-null mice exhibited bradykinesia, proprioceptive abnormalities, and seizures, and died at about 18 days after birth. Metabolic labeling of live animals with bromodeoxyuridine at postnatal days 14 and 18, combined with immunolabeling of neuronal markers, showed that subpopulations of central nervous system neurons were proliferating in all parts of the brain, including normally dormant cells of the hippocampus, cortex, hypothalamus, pons, and brainstem. These cells also expressed phosphorylated histone H3, a marker for late G(2) and M-phase progression, indicating that neurons were dividing after they had migrated to their final positions in the brain. Increased proliferation was balanced by cell death, resulting in no gross changes in the cytoarchitecture of the brains of these mice. Therefore, p19(Ink4d) and p27(Kip1) cooperate to maintain differentiated neurons in a quiescent state that is potentially reversible.

Animals↗
PubMed1999

Disruption of the ARF-Mdm2-p53 tumor suppressor pathway in Myc-induced lymphomagenesis.

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.

Animals↗
PubMed1999

Transforming activity of EWS/FLI is not strictly dependent upon DNA-binding activity.

In approximately 85% of Ewing sarcomas, chromosomal translocations give rise to the chimeric gene EWS/FLI, encoding the N-terminus of the RNA binding protein EWS fused to the DNA-binding domain of the ETS protein FLI-1. EWS/FLI is a stronger transcriptional activator than wild-type FLI-1, although both proteins bind to the same DNA sequences in vitro. In addition, EWS/FLI, but not FLI-1, is a transforming oncogene in NIH3T3 fibroblasts. EWS/FLI is thought to transform through its ability to deregulate the expression of target genes. We introduced several point mutations into the ETS domain of EWS/FLI that abolished DNA-binding activity. Although two of these mutations disrupted the transforming activity of EWS/FLI, one mutated protein containing a substitution of isoleucine 347 with glutamic acid (I347E) retained diminished transforming activity. In addition, EWS/FLI I347E did not activate expression of the endogenous EWS/FLI target gene manic fringe (MFNG). These studies demonstrate that a portion of the oncogenic activity of EWS/FLI is independent of FLI DNA-binding activity.

3T3 Cells↗
PubMed1999

Induction of ARF tumor suppressor gene expression and cell cycle arrest by transcription factor DMP1.

Expression of the DMP1 transcription factor, a cyclin D-binding Myb-like protein, induces growth arrest in mouse embryo fibroblast strains but is devoid of antiproliferative activity in primary diploid fibroblasts that lack the ARF tumor suppressor gene. DMP1 binds to a single canonical recognition site in the ARF promoter to activate gene expression, and in turn, p19(ARF) synthesis causes p53-dependent cell cycle arrest. Unlike genes such as Myc, adenovirus E1A, and E2F-1, which, when overexpressed, activate the ARF-p53 pathway and trigger apoptosis, DMP1, like ARF itself, does not induce programmed cell death. Therefore, apart from its recently recognized role in protecting cells from potentially oncogenic signals, ARF can be induced in response to antiproliferative stimuli that do not obligatorily lead to apoptosis.

3T3 Cells↗
PubMed1999

Cloning and chromosomal localization of the gene encoding human cyclin D-binding Myb-like protein (hDMP1).

The murine transcription factor murine cyclin D-binding Myb-like protein (mDmp1) arrests the cell cycle in G1 phase, through an activity that can be overridden by direct interaction with the D-type cyclins. Here, we describe the identification, sequence, chromosomal localization, and expression of the human cognate, hDMP1. The hDMP1 cDNA contains a 2280bp open reading frame that shares a high degree of identity with the mDmp1 coding region. The 4.4kb hDMP1 messenger RNA is ubiquitously expressed in normal human tissues, with highest levels in testis and substructures within the brain. By use of fluorescence in situ hybridization with a human genomic P1 probe, we assigned hDMP1 to chromosome 7, band q21. This chromosomal region is frequently deleted as part of the 7q-minus and monosomy 7 abnormalities of human acute myeloid leukemia (AML) and myelodysplastic syndrome (MDS). We analyzed hDMP1 copy number by fluorescence in situ hybridization in leukemic blasts from nine patients with abnormalities of the long arm of chromosome 7, and in each case one allele of the hDMP1 gene was deleted. Functional analysis of the mDmp1 protein has shown that it negatively regulates cell proliferation, which suggests that this gene is a candidate suppressor of malignant transformation. Further study will be needed to determine whether gene-specific mutations implicate hDMP1 as a tumor suppressor in acute leukemias with deletions of the long arm of chromosome 7 or in other types of human malignancy.

Amino Acid Sequence↗
PubMed1999

The p21(Cip1) and p27(Kip1) CDK 'inhibitors' are essential activators of cyclin D-dependent kinases in murine fibroblasts.

The widely prevailing view that the cyclin-dependent kinase inhibitors (CKIs) are solely negative regulators of cyclin-dependent kinases (CDKs) is challenged here by observations that normal up-regulation of cyclin D- CDK4 in mitogen-stimulated fibroblasts depends redundantly upon p21(Cip1) and p27(Kip1). Primary mouse embryonic fibroblasts that lack genes encoding both p21 and p27 fail to assemble detectable amounts of cyclin D-CDK complexes, express cyclin D proteins at much reduced levels, and are unable to efficiently direct cyclin D proteins to the cell nucleus. Restoration of CKI function reverses all three defects and thereby restores cyclin D activity to normal physiological levels. In the absence of both CKIs, the severe reduction in cyclin D-dependent kinase activity was well tolerated and had no overt effects on the cell cycle.

Animals↗
PubMed1999

Expression of c-Myc in response to colony-stimulating factor-1 requires mitogen-activated protein kinase kinase-1.

The mitogen-inducible gene c-myc is a key regulator of cell proliferation and transformation. Yet, the signaling pathway(s) that regulate its expression have remained largely unresolved. Using the mitogen-activated protein kinase kinase (MEK1/2) inhibitor PD98059 and dominant negative forms of Ras (N17) and ERK1 (K71R), we found that activation of Ras and extracellular signal-regulated kinase (ERK) is necessary for colony-stimulating factor-1 (CSF-1)-mediated c-Myc expression and DNA synthetic (S) phase entry. Quiescent NIH-3T3 cells expressing a partially defective CSF-1 receptor, CSF-1R (Y809F), exhibited impaired ERK1 activation and c-Myc expression and failed to enter the S phase of the cell division cycle in response to CSF-1 stimulation. Ectopic expression of a constitutively active form of MEK1 in cells expressing CSF-1R (Y809F) rescued c-Myc expression and S phase entry, but only in the presence of CSF-1-induced cooperating signals. Therefore, MEK1 participates in an obligate signaling pathway linking CSF-1R to c-Myc expression, but other signals from CSF-1R must cooperate with the MEK/ERK pathway to induce c-Myc expression and S phase entry in response to CSF-1 stimulation.

3T3 Cells↗
PubMed1999

A rate limiting function of cdc25A for S phase entry inversely correlates with tyrosine dephosphorylation of Cdk2.

The cdc25A phosphatase removes inhibitory phosphates from threonine-14 and tyrosine-15 of cyclin dependent kinase-2 (cdk2) in vitro, and it is therefore widely assumed that cdc25A positively regulates cyclin E- and A-associated cdk2 activity at the G1 to S phase transition of the mammalian cell division cycle. Human cdc25A was introduced into mouse NIH3T3 fibroblasts co-expressing a form of the colony-stimulating factor-1 (CSF-1) receptor that is partially defective in transducing mitogenic signals. Cdc25A enabled these cells to form colonies in semisolid medium containing serum plus human recombinant CSF-1 in a manner reminiscent of cells rescued by c-myc. However, cdc25A-rescued cells could not proliferate in chemically defined medium containing CSF-1 and continued to require c-myc function for S phase entry. When contact-inhibited cells overexpressing cdc25A were dispersed and stimulated to synchronously enter the cell division cycle, they entered S phase 2-3 h earlier than their parental untransfected counterparts. Shortening of G1 phase temporally correlated with more rapid degradation of the cdk inhibitor p27Kip1 and with premature activation of cyclin A-dependent cdk2. Paradoxically, tyrosine phosphorylation of cdk2 increased considerably as cells entered S phase, and cdc25A overexpression potentiated rather than diminished this effect. At face value, these results are inconsistent with the hypothesis that cdc25A acts directly on cdk2 to activate its S phase promoting function.

3T3 Cells↗
PubMed1999

Nucleolar Arf sequesters Mdm2 and activates p53.

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.

3T3 Cells↗
PubMed1999

Cyclin D- and E-dependent kinases and the p57(KIP2) inhibitor: cooperative interactions in vivo.

This study examines in vivo the role and functional interrelationships of components regulating exit from the G1 resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57(KIP2) inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57(KIP2) inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G1, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57(KIP2)-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57(KIP2) function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57(KIP2) to regulate the G1/S transition in a cell type highly dependent upon pRb.

Animals↗
PubMed1999

The oncogenic potential of the Pax3-FKHR fusion protein requires the Pax3 homeodomain recognition helix but not the Pax3 paired-box DNA binding domain.

The chimeric transcription factor Pax3-FKHR, produced by the t(2;13)(q35;q14) chromosomal translocation in alveolar rhabdomyosarcoma, consists of the two Pax3 DNA binding domains (paired box and homeodomain) fused to the C-terminal forkhead (FKHR) sequences that contain a potent transcriptional activation domain. To determine which of these domains are required for cellular transformation, Pax3, Pax3-FKHR, and selected mutants were retrovirally expressed in NIH 3T3 cells and evaluated for their capacity to promote anchorage-independent cell growth. Mutational analysis revealed that both the third alpha-helix of the homeodomain and a small region of the FKHR transactivation domain are absolutely required for efficient transformation by the Pax3-FKHR fusion protein. Surprisingly, point mutations in the paired domain that abrogate sequence-specific DNA binding retained transformation potential equivalent to that of the wild-type protein. This finding suggests that DNA binding mediated through the Pax3 paired box is not required for transformation. Our results demonstrate that the integrity of the Pax3 homeodomain recognition helix and the FKHR transactivation domain is necessary for efficient cellular transformation by the Pax3-FKHR fusion protein.

3T3 Cells↗
PubMed1998

Glycogen synthase kinase-3beta regulates cyclin D1 proteolysis and subcellular localization.

The activities of cyclin D-dependent kinases serve to integrate extracellular signaling during G1 phase with the cell-cycle engine that regulates DNA replication and mitosis. Induction of D-type cyclins and their assembly into holoenzyme complexes depend on mitogen stimulation. Conversely, the fact that D-type cyclins are labile proteins guarantees that the subunit pool shrinks rapidly when cells are deprived of mitogens. Phosphorylation of cyclin D1 on a single threonine residue near the carboxyl terminus (Thr-286) positively regulates proteasomal degradation of D1. Now, we demonstrate that glycogen synthase kinase-3beta (GSK-3beta) phosphorylates cyclin D1 specifically on Thr-286, thereby triggering rapid cyclin D1 turnover. Because the activity of GSK-3beta can be inhibited by signaling through a pathway that sequentially involves Ras, phosphatidylinositol-3-OH kinase (PI3K), and protein kinase B (Akt), the turnover of cyclin D1, like its assembly, is also Ras dependent and, hence, mitogen regulated. In contrast, Ras mutants defective in PI3K signaling, or constitutively active mitogen-activated protein kinase-kinase (MEK1) mutants that act downstream of Ras to activate extracellular signal-regulated protein kinases (ERKs), cannot stabilize cyclin D1. In direct contrast to cyclin D1, which accumulates in the nucleus during G1 phase and exits into the cytoplasm during S phase, GSK-3beta is predominantly cytoplasmic during G1 phase, but a significant fraction enters the nucleus during S phase. A highly stable D1 mutant in which an alanine is substituted for the threonine at position 286 and that is refractory to phosphorylation by GSK-3beta remained in the nucleus throughout the cell cycle. Overexpression of an active, but not a kinase-defective, form of GSK-3beta in mouse fibroblasts caused a redistribution of cyclin D1 from the cell nucleus to the cytoplasm. Therefore, phosphorylation and proteolytic turnover of cyclin D1 and its subcellular localization during the cell division cycle are linked through the action of GSK-3beta.

3T3 Cells↗
PubMed1998

The chimeric E2A-HLF transcription factor abrogates p53-induced apoptosis in myeloid leukemia cells.

Leukemic lymphoblasts expressing the E2A-HLF oncoprotein possess wild-type p53 genes, but do not undergo apoptosis in response to DNA damage. Experimentally, E2A-HLF prevents apoptosis due to growth factor deprivation or gamma-irradiation in interleukin-3 (IL-3)-dependent murine pro-B cells. To directly test the chimeric protein's ability to abrogate p53-mediated cell death, we used mouse myeloid leukemia cells (M1p53tsval) that constitutively express a temperature-sensitive (ts) mutant p53 gene and undergo apoptosis when p53 assumes an active wild-type configuration. This effect is blocked by treatment with IL-6, which allows the cells to survive in culture despite wild-type p53 activation. We introduced E2A-HLF into M1p53tsval cells and found that they were resistant to p53-mediated apoptosis and that E2A-HLF effectively substituted for the survival functions of IL-6. The expression of p53-responsive genes such as p21 and Bax was upregulated normally, suggesting that E2A-HLF acts downstream of p53 to block execution of the p53-induced apoptotic program. NFIL3, a growth factor-regulated bZIP protein that binds to the same DNA-consensus site as E2A-HLF, delays apoptosis in IL-3-dependent pro-B cells deprived of growth factor. By contrast, in the present study, enforced expression of NFIL3 failed to protect M1p53tsval cells from p53-dependent apoptosis and actively antagonized the ability of IL-6 to rescue cells from that fate, consistent with its role as either a transcriptional repressor or activator, depending on the cell type in which it is expressed. We conclude that the E2A-HLF chimera abrogates p53-induced apoptosis in leukemic cells, possibly through the transcriptional modulation of cell death pathways that are activated by p53 in response to DNA damage.

Animals↗
PubMed1998

Myc signaling via the ARF tumor suppressor regulates p53-dependent apoptosis and immortalization.

Establishment of primary mouse embryo fibroblasts (MEFs) as continuously growing cell lines is normally accompanied by loss of the p53 or p19(ARF) tumor suppressors, which act in a common biochemical pathway. myc rapidly activates ARF and p53 gene expression in primary MEFs and triggers replicative crisis by inducing apoptosis. MEFs that survive myc overexpression sustain p53 mutation or ARF loss during the process of establishment and become immortal. MEFs lacking ARF or p53 exhibit an attenuated apoptotic response to myc ab initio and rapidly give rise to cell lines that proliferate in chemically defined medium lacking serum. Therefore, ARF regulates a p53-dependent checkpoint that safeguards cells against hyperproliferative, oncogenic signals.

Animals↗
PubMed1998

E1A signaling to p53 involves the p19(ARF) tumor suppressor.

The adenovirus E1A oncogene activates p53 through a signaling pathway involving the retinoblastoma protein and the tumor suppressor p19(ARF). The ability of E1A to induce p53 and its transcriptional targets is severely compromised in ARF-null cells, which remain resistant to apoptosis following serum depletion or adriamycin treatment. Reintroduction of p19(ARF) restores p53 accumulation and resensitizes ARF-null cells to apoptotic signals. Therefore, p19(ARF) functions as part of a p53-dependent failsafe mechanism to counter uncontrolled proliferation. Synergistic effects between the p19(ARF) and DNA damage pathways in inducing p53 may contribute to E1A's ability to enhance radio- and chemosensitivity.

Adenovirus E1A Proteins↗
PubMed1998

Functional and physical interactions of the ARF tumor suppressor with p53 and Mdm2.

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.

3T3 Cells↗