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M F Roussel

Publications and source records attributed to M F Roussel.

At least 55 records · Page 3Linked to original sources
PubMed1998

Assembly of cyclin D-dependent kinase and titration of p27Kip1 regulated by mitogen-activated protein kinase kinase (MEK1).

A constitutively active form of mitogen-activated protein kinase kinase (MEK1) was synthesized under control of a zinc-inducible promoter in NIH 3T3 fibroblasts. Zinc treatment of serum-starved cells activated extracellular signal-regulated protein kinases (ERKs) and induced expression of cyclin D1. Newly synthesized cyclin D1 assembled with cyclin-dependent kinase-4 (CDK4) to form holoenzyme complexes that phosphorylated the retinoblastoma protein inefficiently. Activation of the MEK1/ERK pathway neither triggered degradation of the CDK inhibitor kinase inhibitory protein-1 (p27(Kip1)) nor led to activation of cyclin E- and A-dependent CDK2, and such cells did not enter the DNA synthetic (S) phase of the cell division cycle. In contrast, zinc induction of active MEK1 in cells also engineered to ectopically overexpress cyclin D1 and CDK4 subunits generated levels of cyclin D-dependent retinoblastoma protein kinase activity approximating those achieved in cells stimulated by serum. In this setting, p27(Kip1) was mobilized into complexes containing cyclin D1; cyclin E- and A-dependent CDK2 complexes were activated; and serum-starved cells entered S phase. Thus, although the activity of p27(Kip1) normally is canceled through a serum-dependent degradative process, overexpressed cyclin D1-CDK complexes sequestered p27(Kip1) and reduced the effective inhibitory threshold through a stoichiometric mechanism. A fraction of these cells completed S phase and divided, but they were unable to continuously proliferate, indicating that other serum-responsive factors ultimately became rate limiting for cell cycle progression. Therefore, the MEK/ERK pathway not only acts transcriptionally to induce the cyclin D1 gene but functions posttranslationally to regulate cyclin D1 assembly with CDK4 and to thereby help cancel p27(Kip1)-mediated inhibition.

3T3 Cells↗
PubMed1997

Tumor suppression at the mouse INK4a locus mediated by the alternative reading frame product p19ARF.

The INK4a tumor suppressor locus encodes p16INK4a, an inhibitor of cyclin D-dependent kinases, and p19ARF, an alternative reading frame protein that also blocks cell proliferation. Surprisingly, mice lacking p19ARF but expressing functional p16INK4a develop tumors early in life. Their embryo fibroblasts (MEFs) do not senesce and are transformed by oncogenic Ha-ras alone. Conversion of ARF+/+ or ARF+/- MEF strains to continuously proliferating cell lines involves loss of either p19ARF or p53. p53-mediated checkpoint control is unperturbed in ARF-null fibroblast strains, whereas p53-negative cell lines are resistant to p19ARF-induced growth arrest. Therefore, INK4a encodes growth inhibitory proteins that act upstream of the retinoblastoma protein and p53. Mutations and deletions targeting this locus in cancer cells are unlikely to be functionally equivalent.

3T3 Cells↗
PubMed1997

Expression of the p16INK4a tumor suppressor versus other INK4 family members during mouse development and aging.

Four INK4 proteins can prevent cell proliferation by specifically inhibiting cyclin D-dependent kinases. Both p18INK4c and p19INK4d were widely expressed during mouse embryogenesis, but p16INK4a and p15INK4b were not readily detected prenatally. Although p15INK4b, p18INK4c and p19INK4d were demonstrated in many tissues by 4 weeks after birth, p16INK4a protein expression was restricted to the lung and spleen of older mice, with increased, more widespread mRNA expression during aging. Transcripts encoding the INK4a alternative reading frame product p19ARF were not detected before birth but were ubiquitous postnatally. Expression of p16INK4a and p15INK4b was induced when mouse embryos were disrupted and cultured as mouse embryo 'fibroblasts' (MEFs). The levels of p16INK4a and p18INK4c, but not p15INK4b or p19INK4d, further increased as MEFs approached senescence. Following crisis and establishment, three of four independently-derived cell lines became polyploid and expressed higher levels of functional p16INK4a. In contrast, one MEF line that sustained bi-allelic deletions of INK4a initially remained diploid. Therefore, loss of p16INK4a and other events predisposing to polyploidy may represent alternative processes contributing to cell immortalization. Whereas p18INK4c and p19INK4d may regulate pre- and postnatal development, p16INK4a more likely plays a checkpoint function during cell senescence that underscores its selective role as a tumor suppressor.

Aging↗
PubMed1997

Features of macrophage differentiation induced by p19INK4d, a specific inhibitor of cyclin D-dependent kinases.

The mitogen-dependent induction of cyclin D-dependent kinase activity is required for cells to enter the DNA synthetic (S) phase of their division cycle. Immature 32Dcl3 myeloid cells (32D) proliferating in the presence of interleukin-3 (IL-3) normally express cyclins D2 and D3, which assemble into binary holoenzyme complexes with their catalytic subunits, CDK4 and CDK6. When 32D cells are switched to medium containing granulocyte colony-stimulating factor (G-CSF) instead of IL-3, D-type cyclins are degraded and, in the absence of their associated kinase activity, the cells arrest in the first gap phase (G1) of the cell cycle and differentiate to neutrophils. We derived 32D cells in which the expression of p19INK4d, a specific polypeptide inhibitor of CDK4 and CDK6, is regulated by the heavy metal-inducible sheep metallothionein promoter. Induction of p19INK4d in response to zinc prolonged cell survival in the absence of growth factor treatment. When maintained in medium containing both IL-3 and zinc, these cells lost cyclin D-dependent kinase activity, underwent G1 phase arrest, and acquired certain morphologic, antigenic, and functional properties of mononuclear phagocytes. Cells induced to express p19INK4d did not synthesize receptors for macrophage colony-stimulating factor (M-CSF/CSF-1) and reverted to an immature myeloid phenotype when shifted back into medium containing IL-3 alone. These cells exhibited accelerated differentiation to neutrophils in response to G-CSF but also gave rise to macrophage-like cells when maintained in medium containing both G-CSF and zinc. Therefore, the acquisition of macrophage properties in response to zinc treatment neither depended upon IL-3 nor upon G1 phase arrest per se and instead reflects some ability of p19INK4d, and presumably cyclin D-dependent kinases, to affect myeloid differentiation.

Carrier Proteins↗
PubMed1997

Regulation of cell cycle entry and G1 progression by CSF-1.

Proliferation, differentiation, and survival of monocytes, macrophages, and their immediate progenitors is regulated by the macrophage colony-stimulating factor (CSF-1). CSF-1 initiates a mitogenic response by binding to its receptor (CSF-1R), thereby activating the receptor's intrinsic tyrosine kinase activity and initiating signaling via multiple effector-mediated pathways. CSF-1 is required throughout G1 to ensure entry of bone marrow-derived macrophages into S phase, and persistent CSF-1R kinase activity is necessary to the expression of both immediate early (e.g., c-fos, c-jun, and c-myc) and delayed early (e.g., D-type cyclins) response genes. Ectopic expression of human CSF-1R in different mouse cell lines, including fibroblasts, IL-3-dependent myeloid cells, and early pre-B cells, confers CSF-1 responsiveness by replacing the cells' requirements for other mitogenic growth factors. NIH-3T3 fibroblasts engineered to express a human CSF-1 receptor point mutant (CSF-1R [Y809F]) fail to proliferate in response to CSF-1 and remain arrested in the early G1 phase of the cell cycle. Despite CSF-1-dependent transcription of fos and jun family members, c-myc, D-type, and E-type G1 cyclin mRNAs are not expressed in the latter cells in response to growth factor stimulation. However, enforced expression of c-myc or D-type cyclins, but not cyclin E, resensitizes cells bearing CSF-1R (Y809F) to the mitogenic effects of CSF-1, enabling them to proliferate continuously in liquid culture and to form colonies in agar in response to the growth factor. D-type cyclin mutants defective in binding to the retinoblastoma protein (pRB) were unable to rescue mutant receptor signaling, suggesting that the ability of D-type cyclin-dependent kinases to cancel pRB's growth-suppressive function is necessary for CSF-1-induced G1 exit. By contrast, cyclin E must function in a different pathway. Cells rescued by c-myc were prevented from entering S phase by microinjection of antibodies to cyclin D1. Conversely, cyclin D1-rescued cells were inhibited from forming CSF-1-dependent colonies in agar when challenged with either a dominant-negative c-myc mutant or mad, a transcription factor which competes with myc for max, its requisite heterodimeric partner. Thus, although the expression of c-myc and D-type cyclins is rate limiting for G1 phase progression, their functions are interdependent, with both activities being required for mitogenicity.

3T3 Cells↗
PubMed1997

Complementation of growth factor receptor-dependent mitogenic signaling by a truncated type I phosphatidylinositol 4-phosphate 5-kinase.

Substitution of phenylalanine for tyrosine at codon 809 (Y809F) of the human colony-stimulating factor 1 (CSF-1) receptor (CSF-1R) impairs ligand-stimulated tyrosine kinase activity, prevents induction of c-MYC and cyclin D1 genes, and blocks CSF-1-dependent progression through the G1 phase of the cell cycle. We devised an unbiased genetic screen to isolate genes that restore the ability of CSF-1 to stimulate growth in cells that express mutant CSF-1R (Y809F). This screen led us to identify a truncated form of the murine type Ibeta phosphatidylinositol 4-phosphate 5-kinase (mPIP5K-Ibeta). This truncated protein lacks residues 1 to 238 of mPIP5K-Ibeta and is catalytically inactive. When we transfected cells expressing CSF-1R (Y809F) with mPIP5K-Ibeta (delta1-238), CSF-1-dependent induction of c-MYC and cyclin D1 was restored and ligand-dependent cell proliferation was sustained. CSF-1 normally triggers the rapid disappearance of CSF-1R (Y809F) from the cell surface; however, transfection of cells with mPIP5K-Ibeta (delta1-238) stabilized CSF-1R (Y809F) expression on the cell surface, resulting in elevated levels of ligand-activated CSF-1R (Y809F). These results suggest a role for PIP5K-Ibeta in receptor endocytosis and that the truncated enzyme compensated for a mitogenically defective CSF-1R by interfering with this process.

Amino Acid Sequence↗
PubMed1997

Expression of INK4 inhibitors of cyclin D-dependent kinases during mouse brain development.

In situ hybridization of mouse embryo sections demonstrated expression of mRNAs encoding two polypeptide inhibitors (p18INK4c and p19INK4d) of cyclin D-dependent kinase (CDK) 4 and CDK6 in the central nervous system. No expression of two other INK4 members, p16INK4a and p15INK4b, was observed. The p19INK4d and p18INK4c proteins formed complexes with either CDK4 or CDK6 in a temporal pattern consistent with the results of in situ hybridization. Expression of INK4c was observed at embryonic day 13.5 in neuroepithelial zones of the developing brain, being restricted to dividing neuroblasts but absent from differentiating postmitotic neurons. In the neocortex, p18INK4c was expressed precisely at those developmental stages when neuroblasts switch from a symmetric to an asymmetric pattern of cell division with concomitant increases in their G1 interval. INK4d RNA was detected from embryonic day 11.5 onward, at higher levels than INK4c and with a distinctly different spatial and temporal pattern. Marked INK4d expression was seen in dorsal root ganglia, spinal cord, and focally throughout the brain, but primarily in postmitotic neurons. Neural expression of INK4d continued postnatally into adulthood in postmitotic cells of the dentate gyrus, the pyramidal layer of the hippocampus, and in discrete regions of the cerebral cortex, cerebellum, thalamus, and brainstem. Downregulation of p19INK4d in the dentate gyrus after kainic acid-induced seizures indicated that its expression could also be modified in nondividing cells by excitotoxic stress. Therefore, p19INK4d may contribute to maintaining the quiescent state, acting as a buffer to prevent reactivation of cyclin D-dependent kinases in terminally differentiated cells.

Animals↗
PubMed1996

Phosphorylation of Ets1 regulates the complementation of a CSF-1 receptor impaired in mitogenesis.

Ets1, the founder member of the Ets transcription factor family, is involved in a variety of developmental and cellular processes. Previous studies have shown that serine phosphorylation of Ets1 inhibits its DNA binding activity, suggesting that phosphorylation is important in the regulation of Ets1 function. To further examine Ets1 phosphorylation, we ectopically expressed Ets1 in fibroblasts and stimulated these cells with serum. Using two-dimensional tryptic phosphopeptide analysis and site-directed mutagenesis, we found that Ets1 was phosphorylated on threonine 38, a residue conserved in several Ets proteins. Substitution of this residue with alanine enhanced CSF-1-dependent colony formation in semi-solid medium of NIH3T3 cells expressing a mitogenically defective CSF-1 receptor [Y809F]. Threonine 38 is part of a consensus amino-acid sequence frequently recognized and targeted by members of the MAP kinase family. Moreover, this residue is phosphorylated in vitro by recombinant ERK2, which suggests that the kinase which phosphorylates threonine 38 in vivo is a member of the MAP kinase family. In addition, phosphorylation on threonine 38 seems to negatively regulate Ets1 activity in response to growth-factor stimulation.

3T3 Cells↗
PubMed1996

Cloning and expression of the murine Elf-1 cDNA.

The Ets family of transcription factors has been implicated in the etiology of several types of cancer. We cloned and characterized the gene encoding the murine homologue of one Ets family member, Elf-1 (mElf-1), in order to gain insight into its cellular physiology. We examined mElf-1 mRNA expression in normal mouse tissues and in several murine and human cell lines. Expression of mElf-1, although highest in lymphocytes, was observed in a number of hematopoietic cell lineages, including the myeloid, macrophages and erythroid lineages, and was lowest in the murine fibroblast cell line, NIH3T3. Analysis of human and fetal tissue mRNAs confirmed that mElf-1 is expressed in a variety of cell lineages, principally in hematopoietic cells. Western blot analysis using antiserum generated to a synthetic C-terminal peptide of mElf-1, and extracts prepared from cell lines that expressed the mElf-1 mRNA identified multiple mElf-1 species that migrated near the 97-kDa molecular weight marker. Site selection analysis indicated that the binding site preferred by mElf-1 is very similar to that of the Drosophila melanogaster homologue, E74, and to that of Fli-1, another Ets family member. We conclude that the expression of mElf-1 is not restricted to the lymphoid lineage, and suggest that Elf-1 may regulate the transcription of a broad spectrum of genes.

Amino Acid Sequence↗
PubMed1996

A potential role for Elf-1 in terminal transferase gene regulation.

The terminal deoxynucleotidyltransferase (TdT) gene represents an attractive model for the analysis of gene regulation during an early phase of lymphocyte development. In previous studies, we identified a DNA element, termed D', which is essential for TdT promoter activity in immature lymphocytes, and two classes of D'-binding factors, Ikaros proteins and Ets proteins. Here, we report a detailed mutant analysis of the D' element which suggests that an Ets protein, rather than an Ikaros protein, activates TdT transcription. Since multiple Ets proteins are expressed in developing lymphocytes and are capable of binding to the D' element, DNA affinity chromatography was used to determine if one of the Ets proteins might bind to the D' element with a uniquely high affinity, thereby implicating that protein as a potential TdT activator. Indeed, one binding activity was greatly enriched in the high-salt eluates from a D' affinity column. Peptide microsequencing revealed that the enriched protein was Elf-1. Immunoblot analyses confirmed that in nuclear extracts, Elf-1 has a significantly higher affinity for the D' sequence than does another Ets protein, Ets-1. Transactivation and expression studies support the hypothesis that Elf-1 activates TdT transcription in immature T and B cells. Finally, a D' mutation which selectively reduces Elf-1 binding, but not the binding of other Ets proteins, was found to greatly reduce TdT promoter activity. Although Elf-1 previously had been implicated in the inducible activation of genes in mature T and B cells, our results suggest that it also plays an important role in regulating genes during an early phase of lymphocyte development.

Amino Acid Sequence↗
PubMed1996

Inhibition of cell proliferation by the Mad1 transcriptional repressor.

Mad1 is a basic helix-loop-helix-leucine zipper protein that is induced upon differentiation of a number of distinct cell types. Mad1 dimerizes with Max and recognizes the same DNA sequences as do Myc:Max dimers. However, Mad1 and Myc appear to have opposing functions. Myc:Max heterodimers activate transcription while Mad:Max heterodimers repress transcription from the same promoter. In addition Mad1 has been shown to block the oncogenic activity of Myc. Here we show that ectopic expression of Mad1 inhibits the proliferative response of 3T3 cells to signaling through the colony-stimulating factor-1 (CSF-1) receptor. The ability of over-expressed Myc and cyclin D1 to complement the mutant CSF-1 receptor Y809F (containing a Y-to-F mutation at position 809) is also inhibited by Mad1. Cell cycle analysis of proliferating 3T3 cells transfected with Mad1 demonstrates a significant decrease in the fraction of cells in the S and G2/M phases and a concomitant increase in the fraction of G1 phase cells, indicating that Mad1 negatively influences cell cycle progression from the G1 to the S phase. Mutations in Mad1 which inhibit its activity as a transcription repressor also result in loss of Mad1 cell cycle inhibitory activity. Thus, the ability of Mad1 to inhibit cell cycle progression is tightly coupled to its function as a transcriptional repressor.

3T3 Cells↗
PubMed1995

Signaling by ABL oncogenes through cyclin D1.

Oncogenic signals induce cellular proliferation by deregulating the cell division cycle. Cyclin D1, a regulator of G1-phase progression, acts synergistically with ABL oncogenes in transforming fibroblasts and hematopoietic cells in culture. Synergy with v-Abl depended on a motif in cyclin D1 that mediates its binding to the retinoblastoma protein, suggesting that ABL oncogenes in part mediate their mitogenic effects via a retinoblastoma protein-dependent pathway. Overexpression of cyclin D1, but not cyclin E, rescued a signaling-defective src-homology 2 (SH2) domain mutant of BCR-ABL for transformation of cells in culture and malignant tumor formation in vivo. These results demonstrate that cyclin D1 can provide essential signals for malignant transformation in concert with an activated tyrosine kinase.

Animals↗
PubMed1995

Rescue of defective mitogenic signaling by D-type cyclins.

Three gene products, including Myc and the D- and E-type G1 cyclins, are rate limiting for G1 progression in mammalian fibroblasts. Quiescent mouse NIH 3T3 fibroblasts engineered to express a mutant colony-stimulating factor (CSF-1) receptor (CSF-1R 809F) fail to synthesize c-myc and cyclin D1 mRNAs upon CSF-1 stimulation and remain arrested in early G1 phase. Ectopic expression of c-myc or either of three D-type cyclin genes, but not cyclin E, resensitized these cells to the mitogenic effects of CSF-1, enabling them to proliferate continuously in liquid culture and to form colonies in agar in response to the growth factor. Rescue by cyclin D1 was enhanced by c-myc but not by cyclin E and was reversed by infecting cyclin D1-reconstituted cells with a retroviral vector encoding catalytically inactive cyclin-dependent kinase 4. Induction of cyclin D1 mRNA by CSF-1 was restored in cells forced to express c-myc, and vice versa, suggesting that expression of the two genes is interdependent. Cells reconstituted with c-myc were prevented from entering S phase when microinjected with a monoclonal antibody to cyclin D1, and conversely, those rescued by cyclin D1 were inhibited from forming CSF-1-dependent colonies when challenged with a dominant-negative c-myc mutant. Cyclin D mutants defective in binding to the retinoblastoma protein were impaired in rescuing mitogenic signaling. Therefore, Myc and D-type cyclins collaborate during the mitogenic response to CSF-1, whereas cyclin E functions in a separate pathway.

3T3 Cells↗
PubMed1995

A variant Ewing's sarcoma translocation (7;22) fuses the EWS gene to the ETS gene ETV1.

Most Ewing's sarcomas or related primitive neuroectodermal tumors have the (11;22)(q24;q12) or less frequently the (21;22)(q22;q12) translocation. These rearrangements fuse the EWS gene on chromosome 22q12 to either the FLI1 or ERG genes, both members of the ETS family of transcription factors. Simple variant chromosomal translocations have been occasionally described in these tumors. We have identified a third Ewing's sarcoma translocation, the t(7;22)(p22;q12), that fuses EWS to the human homologue of the murine ETS gene ER81. This gene, designated ETV1 (for ETS Translocation Variant), is located on chromosome band 7p22. Identical EWS nucleotide sequences found in the majority of EWS-FLI1 and EWS-ERG chimeric transcripts are fused to a portion of ETV1 encoding an ETS domain with sequence specific DNA-binding activity. These findings confirm that the fusion of EWS to different ETS family members can result in a similar tumor phenotype.

Adult↗
PubMed1995

DNA synthesis induced by some but not all growth factors requires Src family protein tyrosine kinases.

The Src family of protein tyrosine kinases have been implicated in the response of cells to several ligands. These include platelet-derived growth factor (PDGF), epidermal growth factor (EGF), and colony stimulating factor type 1 (CSF-1, in macrophages and in fibroblasts engineered to express the receptor). We recently described a microinjection approach which we used to demonstrate that Src family kinases are required for PDGF-induced S phase entry of fibroblasts. We now use this approach to ask whether other ligands also require Src kinases to stimulate cells to replicate DNA. An antibody specific for the carboxy terminus of Src, Fyn, and Yes (anti-cst.1) inhibited Src kinase activity in vitro and caused morphological reversion of Src transformed cells in vivo. Microinjection of this antibody was used to demonstrate that Src kinases were required for both CSF-1 and EGF to drive cells into the S phase. Expression of a kinase-inactive form of Src family kinases also prevented EGF- and CSF-1-stimulated DNA synthesis. However, even though the Src family kinases were necessary for both PDGF- and EGF-induced DNA synthesis in Swiss 3T3 cells, the responses to two other potent growth factors for these cells, lysophosphatidic acid and bombesin, were unaffected by the neutralizing antibodies. Therefore, some but not all growth factors required functional Src family kinases to transmit mitogenic responses.

3T3 Cells↗
PubMed1995

Novel INK4 proteins, p19 and p18, are specific inhibitors of the cyclin D-dependent kinases CDK4 and CDK6.

Cyclin D-dependent kinases act as mitogen-responsive, rate-limiting controllers of G1 phase progression in mammalian cells. Two novel members of the mouse INK4 gene family, p19 and p18, that specifically inhibit the kinase activities of CDK4 and CDK6, but do not affect those of cyclin E-CDK2, cyclin A-CDK2, or cyclin B-CDC2, were isolated. Like the previously described human INK4 polypeptides, p16INK4a/MTS1 and p15INK4b/MTS2, mouse p19 and p18 are primarily composed of tandemly repeated ankyrin motifs, each ca. 32 amino acids in length, p19 and p18 bind directly to CDK4 and CDK6, whether untethered or in complexes with D cyclins, and can inhibit the activity of cyclin D-bound cyclin-dependent kinases (CDKs). Although neither protein interacts with D cyclins or displaces them from preassembled cyclin D-CDK complexes in vitro, both form complexes with CDKs at the expense of cyclins in vivo, suggesting that they may also interfere with cyclin-CDK assembly. In proliferating macrophages, p19 mRNA and protein are periodically expressed with a nadir in G1 phase and maximal synthesis during S phase, consistent with the possibility that INK4 proteins limit the activities of CDKs once cells exit G1 phase. However, introduction of a vector encoding p19 into mouse NIH 3T3 cells leads to constitutive p19 synthesis, inhibits cyclin D1-CDK4 activity in vivo, and induces G1 phase arrest.

Amino Acid Sequence↗
PubMed1994

Monoclonal antibodies to mammalian D-type G1 cyclins.

D-type cyclins are necessary and rate-limiting for G1 progression during the mammalian cell cycle. Cyclins D1, D2, and D3 are encoded by distinct genes and are expressed in proliferating cells in a lineage-specific manner. Monoclonal antibodies (mAbs) generated to bacterially produced recombinant D-type cyclins were able to react with the native proteins expressed in mammalian cells. One mouse and three rat mAbs immunoprecipitated cyclin D1 from mouse macrophages. Only rat mAbs reacted with human cyclin D1 and cross-reacted with cyclin D2 expressed in proliferating T lymphocytes and human tumor cell lines. A single rat mAb to cyclin D2 exhibited a pattern of reactivity reciprocal to that of rat mAbs to D1. Three rat mAbs reacted specifically with mouse or human cyclin D3, but did not cross-react with cyclins D1 or D2 from either species. Representative mAbs were useful for immunoblotting and detected D-type cyclins coprecipitating in complexes recovered with antiserum to cyclin-dependent kinase-4 (CDK4). Because these mAbs detect D-type cyclins in the nuclei of fixed permeabilized cells, they should prove useful in documenting cyclin overexpression in those human tumors in which the genes are amplified or are targets of specific chromosomal rearrangements.

Animals↗