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1.
Proc Natl Acad Sci U S A ; 110(14): 5689-94, 2013 Apr 02.
Artigo em Inglês | MEDLINE | ID: mdl-23493555

RESUMO

Inflammation and cancer, two therapeutic areas historically addressed by separate drug discovery efforts, are now coupled in treatment approaches by a growing understanding of the dynamic molecular dialogues between immune and cancer cells. Agents that target specific compartments of the immune system, therefore, not only bring new disease modifying modalities to inflammatory diseases, but also offer a new avenue to cancer therapy by disrupting immune components of the microenvironment that foster tumor growth, progression, immune evasion, and treatment resistance. McDonough feline sarcoma viral (v-fms) oncogene homolog (FMS) and v-kit Hardy-Zuckerman 4 feline sarcoma viral oncogene homolog (KIT) are two hematopoietic cell surface receptors that regulate the development and function of macrophages and mast cells, respectively. We disclose a highly specific dual FMS and KIT kinase inhibitor developed from a multifaceted chemical scaffold. As expected, this inhibitor blocks the activation of macrophages, osteoclasts, and mast cells controlled by these two receptors. More importantly, the dual FMS and KIT inhibition profile has translated into a combination of benefits in preclinical disease models of inflammation and cancer.


Assuntos
Aminopiridinas/farmacologia , Inflamação/tratamento farmacológico , Modelos Moleculares , Metástase Neoplásica/tratamento farmacológico , Proteína Oncogênica gp140(v-fms)/antagonistas & inibidores , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-kit/antagonistas & inibidores , Pirróis/farmacologia , Aminopiridinas/síntese química , Aminopiridinas/química , Animais , Sobrevivência Celular/efeitos dos fármacos , Cromatografia de Afinidade , Cristalização , Escherichia coli , Células Endoteliais da Veia Umbilical Humana , Humanos , Indóis , Macrófagos/efeitos dos fármacos , Mastócitos/efeitos dos fármacos , Estrutura Molecular , Mutação de Sentido Incorreto/genética , Proteína Oncogênica gp140(v-fms)/química , Proteína Oncogênica gp140(v-fms)/genética , Osteoclastos/efeitos dos fármacos , Conformação Proteica , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Proteínas Proto-Oncogênicas c-kit/química , Proteínas Proto-Oncogênicas c-kit/genética , Pirróis/síntese química , Pirróis/química , Células Sf9 , Spodoptera
2.
Lab Invest ; 84(3): 320-31, 2004 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-14767486

RESUMO

The fms oncogene encodes the macrophage colony-stimulating factor receptor (CSF1R), a transmembrane tyrosine kinase receptor, which is abnormally expressed in breast cancer. Transfection of wild-type CSF1R into HC11 mammary epithelial cells (HC11-CSF1R) renders the transfectants capable of in vitro local invasion and in vivo tumorigenesis. Transfection with CSF1R mutated to express phe at the tyr-721 autophosphorylation site (HC11-CSF1R-721) creates a phenotype that lacks metastastic competence but maintains local invasiveness. Conversely, HC11 cells transfected with CSF1R mutated at tyr-807 (HC11-CSF1R-807) retain their metastatic competence, but are not locally invasive. Our aims were to determine which genes were differentially expressed with transfection of HC11 with wild-type CSF1R, and to determine the effect of mutation at the autophosphorylation sites on gene expression, using 4.6 K cDNA microarrays. Complementary DNA from HC11, HC11-CSF1R-721 and HC11-CSF1R-807 were each hybridized together with HC11-CSF1R on individual arrays. A principal component spectral method combined with prenormalization procedures was used for sample clustering. Differentially expressed genes were identified by the analysis of variance. Confirmation by Northern blotting was performed for MAP kinase phosphatase-1, WDNM1 (extracellular proteinase inhibitor), Trop 2 (tumor-associated calcium signal transducer-2), procollagen type IV alpha, secretory leukoprotease inhibitor, prenylated snare protein Ykt6, ceruloplasmin and chaperonin 10. Many of these genes have not previously been associated with tumor invasion and metastasis. We have successfully identified genes that can be linked to the invasive phenotypes or to tumorigenesis. These genes provide a basis for further studies of metastatic progression and local invasiveness, and can be evaluated as therapeutic targets.


Assuntos
Genes fms , Neoplasias Mamárias Experimentais/genética , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/metabolismo , Linhagem Celular Tumoral , Feminino , Perfilação da Expressão Gênica , Humanos , Imuno-Histoquímica , Neoplasias Mamárias Experimentais/metabolismo , Neoplasias Mamárias Experimentais/secundário , Camundongos , Mutagênese Sítio-Dirigida , Invasividade Neoplásica/genética , Análise de Sequência com Séries de Oligonucleotídeos , Proteína Oncogênica gp140(v-fms)/genética , Proteína Oncogênica gp140(v-fms)/metabolismo , Fenótipo , Transfecção
3.
J Biol Chem ; 277(17): 14635-40, 2002 Apr 26.
Artigo em Inglês | MEDLINE | ID: mdl-11847211

RESUMO

The receptor for the macrophage colony-stimulating factor (CSF-1, also termed M-CSF), the tyrosine kinase c-Fms, was originally determined to be the oncogene product of the McDonough strain of feline sarcoma virus, v-Fms. The structural difference between c-Fms and v-Fms amounts to only five point mutations in the extracellular domain, two mutations in the cytoplasmic domain, and the replacement of 50 amino acids by 14 unrelated amino acids at the C-terminal tail. Here, we have identified c-Cbl as the direct binding partner for c-Fms. c-Cbl binds to phosphotyrosine residue 977 at the C-terminal end of feline c-Fms, which is absent in v-Fms. The replacement of the C-terminal end of v-Fms by the corresponding part of c-Fms (vc-Fms) restored the binding potential. As a result, vc-Fms reduced the transforming potency of v-Fms. The overexpression of Cbl did not influence the v-Fms-transformed phenotype, although c-Cbl forms a complex with v-Fms indirectly. In contrast, the expression of Cbl drastically reduced the vc-Fms-transformed phenotype and the activation of Erk and enhanced Fms ubiquitination via phosphotyrosine residue 977. Furthermore, the replacement of tyrosine 977 into phenylalanine in feline c-Fms and vc-Fms reduced the Cbl-dependent ubiquitination. These data suggest that an indirect association of c-Cbl via multimeric complex induced a different signaling pathway from the pathway induced by c-Cbl direct interaction.


Assuntos
Transformação Celular Neoplásica , Regulação para Baixo/fisiologia , Fator Estimulador de Colônias de Macrófagos/metabolismo , Proteína Oncogênica gp140(v-fms)/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Ubiquitina-Proteína Ligases , Células 3T3 , Animais , Humanos , Camundongos , Proteína Oncogênica gp140(v-fms)/genética , Fenótipo , Mutação Puntual , Ligação Proteica , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-cbl , Receptor de Fator Estimulador de Colônias de Macrófagos/fisiologia , Tirosina/metabolismo , Ubiquitina/metabolismo
4.
Oncogene ; 18(46): 6335-42, 1999 Nov 04.
Artigo em Inglês | MEDLINE | ID: mdl-10597233

RESUMO

We previously showed that Gi2 proteins interfere with the transduction of CSF-1 receptor (CSF-1R) proliferation signals (Corre and Hermouet, 1995). To identify CSF-1R pathways controlled by Gi2, we transfected v-fms, the oncogenic equivalent of CSF-1R, in NIH3T3 cells in which Gi2 proteins were inactivated by stably expressing a dominant negative mutant form of the alpha subunit of Gi2 (alpha i2-G204A). Expression of alpha i2-G204A resulted in decreased Src-kinase activity, delayed activation of p42 ERK-MAPK, decreased cyclin D1 expression and reduced proliferation in response to serum. In alpha i2-G204A cells transfected with v-fms, Src-kinase activity remained deficient but p42 MAPK activity and cyclin D1 expression were similar to those of vector/v-fms cells, suggesting that v-fms bypasses Src to activate the ERK-MAPK cascade. However, DNA synthesis and focus formation were inhibited by up to 80% in alpha i2-G204A/v-fms cells compared to vector/v-fms cells. We found that tyrosine phosphorylation of STAT3, also activated by CSF-1R/v-fms, was inhibited in alpha i2-G204A/v-fms cells; in addition, expression of an 85 kDa, C-terminal truncated form of STAT3 (STAT3 delta) was constitutively increased. Both the inhibition of v-fms-induced STAT3 tyrosine phosphorylation and the increased expression of STAT3 delta were reproduced by transfecting a dominant negative mutant of Src. Last, we show that expression of STAT3 delta 55C, a mutant form of STAT3 lacking the last 55 C-terminal amino acids, is sufficient to inhibit DNA synthesis and v-fms-induced transformation in NIH3T3 cells. In summary, adequate regulation by Gi2 proteins of the activity of both Src-kinase and STAT3 is required for optimal cell proliferation in response to CSF-1R/v-fms.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteínas Adaptadoras de Transporte Vesicular , Transformação Celular Viral/fisiologia , Proteínas de Ligação a DNA/fisiologia , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/fisiologia , Sistema de Sinalização das MAP Quinases/fisiologia , Proteína Oncogênica gp140(v-fms)/fisiologia , Proteínas Proto-Oncogênicas pp60(c-src)/fisiologia , Transativadores/fisiologia , Células 3T3 , Sequência de Aminoácidos , Animais , Divisão Celular , Transformação Celular Viral/genética , Ciclina D1/metabolismo , Replicação do DNA , Subunidades alfa Gi-Go de Proteínas de Ligação ao GTP/genética , Camundongos , Dados de Sequência Molecular , Fosforilação , Processamento de Proteína Pós-Traducional , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fyn , Proteínas Recombinantes de Fusão/fisiologia , Fator de Transcrição STAT3 , Deleção de Sequência , Proteínas Adaptadoras da Sinalização Shc , Proteína 1 de Transformação que Contém Domínio 2 de Homologia de Src , Transfecção
5.
Naunyn Schmiedebergs Arch Pharmacol ; 357(4): 378-84, 1998 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-9606022

RESUMO

We investigated the effects of the receptor-coupled protein tyrosine kinase (RTK) v-Fms on the membrane current properties of NIH3T3 mouse fibroblasts. We found that v-Fms, the oncogenic variant of the macrophage colony-stimulating factor receptor c-Fms, activates a K+ current that is absent in control cells. The activation of the K+ current was Ca2+-dependent, voltage-independent, and was completely blocked by the K+ channel blockers charybdotoxin, margatoxin and iberiotoxin with IC50 values of 3 nM, 18 nM and 76 nM, respectively. To identify signalling components that mediate the activation of this K+ current, NIH3T3 cells that express different mutants of the wild-type v-Fms receptor were examined. Mutation of the binding site for the Ras-GTPase-activating protein led to a complete abolishment of the K+ current. A reduction of 76% and 63%, respectively, was observed upon mutation of either of the two binding sites for the growth factor receptor binding protein 2. Mutation of the ATP binding lobe, which disrupts the protein tyrosine kinase activity of v-Fms, led to a 55% reduction of the K+ current. Treatment of wild-type v-Fms cells with Clostiridium sordellii lethal toxin or a farnesyl protein transferase inhibitor, both known to inhibit the biological function of Ras, reduced the K+ current amplitude to 17% and 6% of the control value, respectively. This is the first report showing that an oncogenic RTK can modulate K+ channel activity. Our results indicate that this effect is dependent on the binding of certain Ras-regulating proteins to the v-Fms receptor and is not abolished by disruption of its intrinsic protein tyrosine kinase activity. Furthermore, our data suggest that Ras plays a key role for K+ channel activation by the oncogenic RTK v-Fms.


Assuntos
Cálcio/fisiologia , Proteína Oncogênica gp140(v-fms)/fisiologia , Canais de Potássio/fisiologia , Células 3T3 , Animais , Toxinas Bacterianas/farmacologia , Camundongos , Técnicas de Patch-Clamp , Bloqueadores dos Canais de Potássio , Proteínas ras/fisiologia
6.
Cancer Res ; 58(8): 1650-3, 1998 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-9563477

RESUMO

Ribonucleotide reductase, which is composed of the two protein components R1 and R2, is a highly regulated enzyme activity that is essential for DNA synthesis and repair. Recent studies have shown that elevated expression of the rate-limiting R2 component increases Raf-1 protein activation and mitogen-activated protein kinase activity and acts as a novel malignancy determinant in cooperation with H-ras and rac-1. We show that R2 cooperation in cellular transformation extends to a variety of oncogenes with different functions and cellular locations. Anchorage-independent growth of cells transformed with v-fms, v-src, A-raf, v-fes, c-myc, and ornithine decarboxylase was markedly enhanced when the R2 component of ribonucleotide reductase was overexpressed. In addition, we observed that elevated R2 expression conferred on c-myc-transformed NIH 3T3 cells an increased tumorigenic potential in immunoincompetent mice. Taken together, these observations demonstrate that the R2 protein is not only a rate-limiting component for ribonucleotide reduction but that it is also capable of acting in cooperation with a variety of oncogenes to determine transformation and tumorigenic potential.


Assuntos
Transformação Celular Neoplásica , Ribonucleotídeo Redutases/fisiologia , Células 3T3 , Animais , Western Blotting , Linhagem Celular , Proteínas de Fusão gag-onc/fisiologia , Camundongos , Camundongos Nus , Transplante de Neoplasias , Proteína Oncogênica gp140(v-fms)/fisiologia , Proteína Oncogênica pp60(v-src)/fisiologia , Ornitina Descarboxilase/farmacologia , Proteínas Proto-Oncogênicas c-myc/fisiologia , Proteínas Proto-Oncogênicas c-raf/fisiologia , Ribonucleotídeo Redutases/metabolismo , Ensaio Tumoral de Célula-Tronco
7.
Oncogene ; 15(13): 1565-72, 1997 Sep 25.
Artigo em Inglês | MEDLINE | ID: mdl-9380408

RESUMO

Tyrosine autophosphorylation of the v-Fms oncogene product results in the formation of high-affinity binding sites for cellular proteins containing Src homology 2 (SH2) domains. These proteins transduce various mitogenic and morphogenic signals. As reported previously, Y696KNI in the kinase insert domain of v-Fms binds to the growth factor receptor bound protein 2 (Grb2), a stimulator of the Ras/Raf1 pathway. Here, we mapped Y921TNL within the C-terminal domain of Fms as a novel autophosphorylation site. We demonstrate that this site constitutes a second Grb2 binding site: a recombinant fusion protein (residues 904-944) containing phosphorylated Y921 bound Grb2 from FDCP-1Mac11 cell extracts significantly more efficiently than a corresponding protein (residues 617-759) containing Y696. A yeast two-hybrid system which allowed the formation of a functional Fms tyrosine kinase was employed to quantify binding of Grb2. Fms-protein containing either one of the two phosphorylation sites bound Grb2 equally well, binding was increased for proteins carrying both sites. In contrast, the simultaneous substitution of Y696 and Y921 by phenylalanines abolished Grb2 binding. Mouse NIH3T3 cells expressing the Y921F mutant Fms-protein showed a substantially higher content of fibronectin network than wild-type transformed cells and had largely lost their serum independent growth phenotype.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Proteína Oncogênica gp140(v-fms)/química , Proteínas/metabolismo , Células 3T3 , Animais , Sítios de Ligação , Linhagem Celular Transformada , Proteína Adaptadora GRB2 , Camundongos , Mutação , Mapeamento de Peptídeos , Fosforilação , Proteínas Tirosina Quinases/metabolismo , Proteínas Recombinantes/metabolismo , Domínios de Homologia de src
8.
C R Acad Sci III ; 320(2): 163-70, 1997 Feb.
Artigo em Francês | MEDLINE | ID: mdl-9181122

RESUMO

It is well known that the gp120-gp41 complex undergoes a conformational change after CD4 binding. It is likely that CD4 undergoes a conformational change as well. Recently, a calculation of the normal modes of the two N-terminal domains of CD4 has shown that a hinge-bending motion of one of these domains with respect to the other may occur. In the present study, results obtained previously are verified with two other normal mode calculations, starting from crystallographic structures of different origin. A scheme describing the first steps of the process leading to cell infection by human immunodeficiency virus (HIV) is then proposed. It rests upon the idea that CD4 and gp120-gp41 conformational changes allow for bringing the cell and virus membranes closer to each other.


Assuntos
Antígenos CD4/química , Fusão Celular/imunologia , HIV/fisiologia , Proteína gp120 do Envelope de HIV/química , Infecções por HIV/fisiopatologia , Técnicas In Vitro , Modelos Estruturais , Proteína Oncogênica gp140(v-fms)/química , Conformação Proteica , Estrutura Terciária de Proteína
9.
Br J Cancer ; 76(8): 977-82, 1997.
Artigo em Inglês | MEDLINE | ID: mdl-9376277

RESUMO

Co-expression of macrophage colony-stimulating factor (M-CSF) and its receptor (c-fms) is often found in ovarian epithelial carcinoma, suggesting the existence of autocrine regulation of cell growth by M-CSF. To block this autocrine loop, we have developed hammerhead ribozymes against c-fms mRNA. As target sites of the ribozyme, we chose the GUC sequence in codon 18 and codon 27 of c-fms mRNA. Two kinds of ribozymes were able to cleave an artificial c-fms RNA substrate in a cell-free system, although the ribozyme against codon 18 was much more efficient than that against codon 27. We next constructed an expression vector carrying a ribozyme sequence that targeted the GUC sequence in codon 18 of c-fms mRNA. It was introduced into TYK-nu cells that expressed M-CSF and its receptor. Its transfectant showed a reduced growth potential. The expression levels of c-fms protein and mRNA in the transfectant were clearly decreased with the expression of ribozyme RNA compared with that of an untransfected control or a transfectant with the vector without the ribozyme sequence. These results suggest that the ribozyme against GUC in codon 18 of c-fms mRNA is a promising tool for blocking the autocrine loop of M-CSF in ovarian epithelial carcinoma.


Assuntos
Carcinoma/genética , Carcinoma/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes fms , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/metabolismo , RNA Catalítico/metabolismo , Sequência de Bases , Sítios de Ligação , Divisão Celular/fisiologia , Coriocarcinoma/genética , Coriocarcinoma/metabolismo , Feminino , Humanos , Fator Estimulador de Colônias de Macrófagos/biossíntese , Fator Estimulador de Colônias de Macrófagos/genética , Dados de Sequência Molecular , Conformação de Ácido Nucleico , Proteína Oncogênica gp140(v-fms)/biossíntese , Proteína Oncogênica gp140(v-fms)/genética , Proto-Oncogene Mas , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transfecção , Células Tumorais Cultivadas
10.
J Biol Chem ; 271(40): 24476-81, 1996 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-8798707

RESUMO

Tyrosine autophosphorylation of the v-Fms oncogene product results in the formation of high affinity binding sites for cellular proteins with Src homology 2 (SH2) domains that are involved in various signal cascades. Tryptic digestion of the autophosphorylated v-Fms and of its cellular counterpart, the feline c-Fms polypeptide, gave rise to at least six common major phosphopeptides, four of which have been characterized previously. Employing site-directed mutagenesis and phosphopeptide mapping of in vitro phosphorylated glutathione S-transferase v-Fms fusion proteins as well as full-length v-Fms molecules expressed in various cells, we show here that Tyr543 of the juxtamembrane domain and Tyr696 of the kinase insert domain constitute major autophosphorylation sites. Recombinant fusion proteins containing the tyrosine-phosphorylated kinase insert domain bind the growth factor receptor bound protein 2 and the p85 and p110 subunits of phosphatidylinositol 3'-kinase. In contrast, fusion proteins containing the juxtamembrane domain phosphorylated on Tyr543 fail to bind any of the known SH2 domain-containing cellular proteins but associate specifically with an as yet undefined 55-kDa cellular protein that by itself is phosphorylated on tyrosine.


Assuntos
Proteína Oncogênica gp140(v-fms)/metabolismo , Proteínas/metabolismo , Tirosina/metabolismo , Células 3T3 , Animais , Gatos , Membrana Celular/metabolismo , Camundongos , Fosfatidilinositol 3-Quinases , Fosfopeptídeos/metabolismo , Fosforilação , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Ligação Proteica , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo
11.
Oncology ; 53(2): 99-103, 1996.
Artigo em Inglês | MEDLINE | ID: mdl-8604248

RESUMO

We studied the production of macrophage colony-stimulating factor (M-CSF) and the expression of c-fms mRNA, an M-CSF receptor, in four human ovarian cancer cell lines. All four cell lines expressed c-fms mRNA while three secreted M-CSF into the culture medium. The exogenous administration of M-CSF caused no significant enhancement of cellular proliferation in any cell line. Interestingly, the proliferation of KK cells was not affected by anti-M-CSF antibody. These results, taken together with the fact that ovarian cancer cells simultaneously produce M-CSF and c-fms, suggest that an autocrine mechanism may modulate cellular proliferation.


Assuntos
Adenocarcinoma/metabolismo , Fator Estimulador de Colônias de Macrófagos/biossíntese , Proteína Oncogênica gp140(v-fms)/biossíntese , Neoplasias Ovarianas/metabolismo , RNA Mensageiro/biossíntese , Adenocarcinoma/patologia , Anticorpos Anti-Idiotípicos/farmacologia , Divisão Celular/efeitos dos fármacos , Feminino , Humanos , Fator Estimulador de Colônias de Macrófagos/imunologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Neoplasias Ovarianas/patologia , Proto-Oncogene Mas , Células Tumorais Cultivadas
12.
J Virol ; 69(10): 6010-20, 1995 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-7666506

RESUMO

Expression of the v-fms oncogene of feline sarcoma virus in fibroblasts causes surface exposure of an activated receptor tyrosine kinase, v-Fms, that is autophosphorylated at multiple sites within its cytoplasmic domain. Cellular proteins interacting with this part of v-Fms modulate the mitogenic activity and morphology of the cells. We show here that the tyrosine residue in position 807 (Y-807) of the v-Fms molecule constitutes a major autophosphorylation site. The replacement of this residue by phenylalanine (Y807F mutation) allowed us to functionally dissect v-Fms-specific mitogenic and morphogenic cascades. Cells expressing the mutant v-Fms molecule resembled wild-type (wt) v-Fms-transformed (wt-v-Fms) cells in terms of [3H]thymidine uptake rates and activation of the Ras/Raf-1 mitogenic cascade. Such cells showed, however, a flat morphology and contained intact actin cables and fibronectin network. Our studies indicate that the v-Fms molecule controls cell morphology by a cascade that involves a direct interaction with p120RasGAP and p190RhoGAP: (i) in contrast to wt v-Fms molecules, the Y807F v-Fms protein failed to associate with and phosphorylate p120RasGAP; (ii) tight complexes between p120RasGAP and p190RhoGAP as well as detectable RhoGAP activity were present exclusively in wt-v-Fms cells; and (iii) p190RhoGAP was dispersed throughout the cytoplasm of wt-v-Fms cells, whereas its distribution was restricted to perinuclear regions of cells expressing the mutant v-Fms gene.


Assuntos
Transformação Celular Neoplásica , Genes fms , Proteína Oncogênica gp140(v-fms)/metabolismo , Oncogenes , Proteínas Tirosina Quinases/metabolismo , Proteínas/metabolismo , Vírus do Sarcoma Felino/fisiologia , Tirosina , Células 3T3 , Sequência de Aminoácidos , Animais , Replicação do DNA , Proteínas Ativadoras de GTPase , Glutationa Transferase/biossíntese , Cinética , Camundongos , Proteína Oncogênica gp140(v-fms)/biossíntese , Proteína Oncogênica gp140(v-fms)/isolamento & purificação , Fenilalanina , Plasmídeos , Mutação Puntual , Proteínas/isolamento & purificação , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/isolamento & purificação , Proteínas Recombinantes de Fusão/metabolismo , Vírus do Sarcoma Felino/genética , Timidina/metabolismo , Transfecção , Proteínas Ativadoras de ras GTPase
13.
Arch Virol ; 140(1): 179-86, 1995.
Artigo em Inglês | MEDLINE | ID: mdl-7646343

RESUMO

Cell transformation is characterized by overt changes in growth control and cell morphology. To study the role of tyrosine residues Y705 and Y807 of v-Fms of the McDonough strain of feline sarcoma virus in cell transformation we replaced them individually with phenylalanine residues. Cells expressing the mutant genes showed mitogenic properties similar to wild-type v-Fms transformed cells. However, the morphology of cells expressing the Y807F mutant remained the same as nontransformed cells. Four phosphoproteins of 190, 120, 55 and 50 kDa were detected in cells expressing the wild-type but were absent in cells expressing the mutant Y807F-v-fms gene.


Assuntos
Transformação Celular Viral , Proteína Oncogênica gp140(v-fms)/fisiologia , Vírus do Sarcoma Felino/fisiologia , Tirosina/fisiologia , Sequência de Bases , Linhagem Celular Transformada , Dados de Sequência Molecular , Oligodesoxirribonucleotídeos , Proteína Oncogênica gp140(v-fms)/química , Fosforilação , Vírus do Sarcoma Felino/química
14.
Int J Pept Protein Res ; 43(6): 520-8, 1994 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-7928082

RESUMO

The dipeptides N-carbomethoxycarbonyl-prolyl-phenylalanyl-benzyl esters (CPFs) play a significant role in the prevention of HIV-1 virus infection by interacting with the glycoprotein gp120, one of the envelope proteins of the HIV-1 virus. Of the four possible isomers of CPF, (L-pro-L-phe), (L-pro-D-phe), (D-pro-L-phe) and (D-pro-D-phe), herein denoted LL etc., the crystal structures of LL, stereoisomeric LD and the racemic mixture of LD/DL have been determined. All three peptides crystallize in the orthorhombic system and they all have similar cell dimensions: (i) LL, P2(1)2(1)2(1), a = 13.699(2), b = 25.893(5), c = 6.155(1) A, Z = 4, Dcale = 1.333 g cm-3, R = 0.070 for 1247 observed reflections; (ii) LD, P2(1)2(1)2(1), a = 11.663(2), b = 26.557(2), c = 7.281(1) A, Z = 4, Dcalc = 1.290 g cm-3, R = 0.054 for 1918 observed reflections; (iii) LD/DL, Pbc2(1), a = 11.953(2), b = 24.208(8), c = 7.782(2) A, Z = 4, Dcalc = 1.292 g cm-3, R = 0.080 for 894 observed reflections. Both the enantiomeric LD and the LD in racemic LD/DL have a similar conformation, an extended peptide chain with phi 1 = -76, -73 degrees; psi 1 = 160, 158 degrees, psi 2 = 123, 131 degrees and psi 2 = -172, -167 degrees, while peptide LL adopts a bent conformation at the Phe residue, phi 1 = -69 degrees, psi 1 = 158 degrees, phi 2 = -60 degrees and psi 2 = -34 degrees.(ABSTRACT TRUNCATED AT 250 WORDS)


Assuntos
Antivirais/química , Antivirais/farmacologia , Compostos de Benzil/química , Compostos de Benzil/farmacologia , Antígenos CD4/metabolismo , Dipeptídeos/química , Dipeptídeos/farmacologia , Proteína gp120 do Envelope de HIV/metabolismo , Proteína Oncogênica gp140(v-fms)/metabolismo , Cristalografia por Raios X , Ligação Proteica , Conformação Proteica , Estereoisomerismo , Relação Estrutura-Atividade
15.
Oncogene ; 9(5): 1429-35, 1994 May.
Artigo em Inglês | MEDLINE | ID: mdl-8152804

RESUMO

Chemotaxis, directed migration toward a gradient of a soluble substance, requires a cell to spatially distinguish the concentration of a chemoattractant at one end relative to its opposite end. Platelet-derived growth factor (PDGF) is a potent mitogen and chemoattractant. In the current study, we attempted to interfere with PDGF-BB mediated chemotaxis by abnormal expression of potential early components of the signaling cascade. We find that expression of the PDGF homolog v-Sis prevents cellular migration toward PDGF-BB, indicating that autocrine production of a PDGF receptor ligand will prevent the chemotactic response to exogenously added ligand. In addition, while it is known that PDGF receptor mutants incapable of activating tyrosine kinase activity cannot transduce a signal for mitogenesis or chemotaxis, the effects of excess tyrosine kinase activity on PDGF mediated chemotaxis have not been tested. We demonstrate that cells expressing constitutively active tyrosine kinase genes such as v-fms, v-fes, or v-src fail to migrate toward PDGF-BB whereas expression of the serine/threonine kinase v-mos does not block the chemotactic response. The results demonstrate that chemotaxis may be prevented by excess production of either ligand, receptor activity, or downstream signaling molecule. In addition, our results show that the signals that mediate chemotaxis are separable from those that regulate unstimulated random motility in the same cells.


Assuntos
Quimiotaxia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-mos/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Oncogênicas de Retroviridae/metabolismo , Células 3T3 , Animais , Proteína Tirosina Quinase CSK , Movimento Celular , Camundongos , Proteína Oncogênica gp140(v-fms)/metabolismo , Proteínas Oncogênicas v-sis , Proteínas Proto-Oncogênicas c-fes , Transdução de Sinais , Quinases da Família src
16.
Oncogene ; 9(3): 899-909, 1994 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-8108134

RESUMO

We wished to identify DNA sequences conferring suppression of proliferation and transformed phenotypes. Thus, we have transfected DNA from normal rat cells, covalently linked to neo DNA coding for neomycin resistance into a tumorigenic, HRAS transformed rat cell line. Phenotypic revertants were selected after the first cycle of transfection by enrichment procedures that served to eliminate transformed cells. The revertant clones continued to express the HRAS oncogene, but exhibited a lower tumorigenicity, loss of anchorage-independent proliferation, flat morphology, and retardation of growth in monolayer culture. The reverted phenotype could be transferred in a second cycle of transfection into the HRAS transformed rat cells. Neo DNA ligated to genomic donor DNA was used as a tagging sequence to molecularly clone the transferred DNA sequence in a recombinant phage. Fragments of the cloned DNA detect a 2.5 kb transcript in parental cells and revertants. Thus, the recombinant phage harbors a putative growth inhibitory gene, designated trg, that is expressed at a higher level in rat embryo fibroblasts and in the REF52 cell line. Introduction of recombinant phage DNA into established 208F and Rat-2 cells and into HRAS-, v-fgr-, v-fms- and v-raf-transformed rat cell lines resulted in inhibition of growth and induction of programmed cell death.


Assuntos
Apoptose/genética , Divisão Celular/genética , Animais , Linhagem Celular , Linhagem Celular Transformada , Clonagem Molecular , DNA/genética , Resistência a Medicamentos/genética , Genes ras , Neomicina/farmacologia , Proteína Oncogênica gp140(v-fms)/genética , Proteínas Oncogênicas v-raf , Oncogenes , Fenótipo , Proteínas Tirosina Quinases/genética , Ratos , Proteínas Oncogênicas de Retroviridae/genética , Transfecção
17.
J Virol ; 68(1): 411-24, 1994 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-8254751

RESUMO

The feline sarcoma virus oncogene v-fms has significantly contributed to the dissection of peptide growth factor action since it encodes the transmembrane tyrosine kinase gp140v-fms, a transforming version of colony-stimulating factor 1 receptor, a member of the growth factor receptor tyrosine kinase family. In this study, the functional significance of structural differences between distinct tyrosine kinase types, in particular between cellular receptors and viral transforming proteins of distinct structural types, has been further investigated, and their functional compatibility has been addressed. For this purpose, major functional domains of three structurally distinct tyrosine kinases were combined into two chimeric receptors. The cytoplasmic gp140v-fms kinase domain and the kinase domain of Rous sarcoma virus pp60v-src were each fused to the extracellular ligand-binding domain of the epidermal growth factor (EGF) receptor to create chimeras EFR and ESR, respectively, which were studied upon stable expression in NIH 3T3 fibroblasts. Both chimeras were faithfully synthesized and routed to the cell surface, where they displayed EGF-specific, low-affinity ligand-binding domains in contrast to the high- and low-affinity EGF-binding sites of normal EGF receptors. While the EFR kinase was EGF controlled for autophosphorylation and substrate phosphorylation in vitro, in vivo, and in digitonin-treated cells, the ESR kinase was not responsive to EGF. While ESR appeared to recycle to the cell surface upon endocytosis, EGF induced efficient EFR internalization and degradation, and phorbol esters stimulated protein kinase C-mediated downmodulation of EFR. Despite its ligand-inducible kinase activity, EFR was partly EGF independent in mediating mitogenesis and cell transformation, while ESR appeared biologically inactive.


Assuntos
Fator de Crescimento Epidérmico/farmacologia , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Regulação Viral da Expressão Gênica/efeitos dos fármacos , Proteína Oncogênica gp140(v-fms)/metabolismo , Proteínas Tirosina Quinases/metabolismo , Vírus do Sarcoma Felino/enzimologia , Células 3T3 , Animais , Sequência de Bases , Transporte Biológico , Adesão Celular , Divisão Celular/efeitos dos fármacos , Transformação Celular Neoplásica , Regulação para Baixo , Ativação Enzimática , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/metabolismo , Substâncias de Crescimento/farmacologia , Camundongos , Dados de Sequência Molecular , Proteína Oncogênica gp140(v-fms)/genética , Proteína Oncogênica pp60(v-src)/genética , Proteína Oncogênica pp60(v-src)/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção
18.
Mol Biol Cell ; 5(1): 81-95, 1994 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-7514458

RESUMO

Oncogenic activation of the macrophage colony stimulating factor (M-CSF) receptor (c-Fms) requires mutation or truncation of the carboxyl terminus and specific amino acid substitutions in or near the fourth immunoglobulin (Ig)-like loop in the extracellular domain. Using a murine c-Fms system, we investigated the effect of C-terminal truncation, substitutions at amino acids 301 and 374 in the fourth Ig-like loop of the extracellular domain, or the combined mutations on individual steps in receptor activation. The mutations at amino acids 301 and 374 were necessary, but not sufficient, for receptor dimerization in the absence of M-CSF. Only receptors with a truncated C-terminus as well as the extracellular domain mutations dimerized efficiently in the absence of M-CSF, suggesting that the C-terminus of c-Fms also regulates receptor oligomerization. Truncation of the C-terminus alone did not cause receptor dimerization and did not activate the kinase enzymatic activity. Thus, truncation of the C-terminus did not activate receptor monomers in cis. Receptors with both a truncated C-terminus and the extracellular domain mutations underwent ligand-independent aggregation, transphosphorylation, and phosphorylation of cellular proteins, followed by rapid internalization and degradation. These results suggest that M-CSF binding to c-Fms initiates activation by inducing conformational changes in both the cytoplasmic C-terminal domain and the fourth Ig-like loop of the extracellular domain, leading to the formation of stable receptor dimers.


Assuntos
Transformação Celular Neoplásica/genética , Endocitose , Mutação , Conformação Proteica , Processamento de Proteína Pós-Traducional , Proteínas Tirosina Quinases/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/genética , Animais , Células Cultivadas , Camundongos , Mutagênese Sítio-Dirigida , Proteína Oncogênica gp140(v-fms)/química , Fosforilação , Fosfotirosina , Coelhos , Ratos , Receptor de Fator Estimulador de Colônias de Macrófagos/química , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismo
19.
Mol Endocrinol ; 6(7): 1051-60, 1992 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-1324418

RESUMO

In order to precisely define the sequences that constitute the ras-responsive enhancers element present in the murine retrotransposon NVL3, point mutations were introduced into the previously defined minimal transcriptional enhancer DNA. Analyses of the effects of these point mutations in transient transfection experiments, in gel retention assays, and by methylation interference footprinting indicated that the enhancer element was composed of two binding sites for distinct nuclear factors. Both binding sites were required for activation of the enhancer by either ras or v-fms oncogenes, and the distinct nuclear factors were found in extracts from cells that contained either oncogene. UV cross-linking analysis revealed that the AP1-related binding site, TGACTCT, was recognized by a nuclear factor of apparent molecular size of 50 kilodaltons, that is probably c-jun. The other binding site, CAGGATAT, is very similar to sites recognized by the ets-family of transcription factors, and was recognized by the 120-kilodalton ras-responsive factor-1. Activation of the NVL3 element was reconstituted in an in vitro transcription assay. The ets-related binding site was necessary for this in vitro reconstitution of activity. Thus, the NVL3 enhancer is related to the previously described oncogene-responsive enhancer element present in polyoma virus and is also related to elements identified in several cellular genes known to be ras-responsive, including the transforming growth factor-beta 1 gene.


Assuntos
Proteínas de Ligação a DNA/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica , Proteína Oncogênica gp140(v-fms)/metabolismo , Proteína Oncogênica p21(ras)/metabolismo , Receptor de Fator Estimulador de Colônias de Macrófagos/metabolismo , Sequência de Bases , Sítios de Ligação , Elementos de DNA Transponíveis/genética , Genes fms , Genes ras , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Proteínas Proto-Oncogênicas c-jun/metabolismo , Sequências Reguladoras de Ácido Nucleico , Fatores de Transcrição/metabolismo
20.
Leuk Res ; 16(4): 403-10, 1992.
Artigo em Inglês | MEDLINE | ID: mdl-1314319

RESUMO

Agents which induce monocytic characteristics in HL-60 human acute promyelocytic leukemia cells induce mRNA for the fms proto-oncogene, which encodes the receptor for M-CSF. Previous studies of fms expression in HL-60 cells have characterized chiefly induction by phorbol esters of fms mRNA. Our studies of fms expression in HI-60 cells have characterized induction by vitamin D3 of the fms protein. We have used flow cytometry to correlate fms antigen with a monocyte-specific differentiation antigen recognized by antibody MO2 (CD14), with DNA content, and with the nuclear antigen Ki-67, a marker of cell cycling. HL-60 cells were cultured with or without 1 microM vitamin D for 7 days. fms antigen was found on 42 +/- 5.8% of the cells cultured without vitamin D, but on 63 +/- 4.3% of the cells cultured with vitamin D. MO2 binding was detected on only 2 +/- 0.5% of the cells without vitamin D, but on 59 +/- 9% with vitamin D. Cells cultured with vitamin D that were fms-positive were also predominantly (83%) MO2-positive. Analysis of DNA content, measured by propidium iodide staining, showed that 57 +/- 1.5% of cells cultured without vitamin D, but 93 +/- 0.5% of cells cultured with vitamin D, were in the G0/G1 cell cycle phase. Analysis of nuclear antigen Ki-67 revealed that, of the vitamin D-treated cells that were fms-positive, a significant proportion (37%) were still cycling. We conclude that (1) fms is demonstrable on some uninduced HL-60 cells, (2) when HL-60 cells are induced to develop monocytic characteristics by vitamin D, fms induction is part of the program for monocytic differentiation that includes MO2 expression, yet (3) some induced cells expressing fms are still cycling.


Assuntos
Colecalciferol/farmacologia , Regulação Leucêmica da Expressão Gênica/efeitos dos fármacos , Genes fms/efeitos dos fármacos , Leucemia Promielocítica Aguda/metabolismo , Proteína Oncogênica gp140(v-fms)/metabolismo , Antígenos de Diferenciação/metabolismo , Ciclo Celular/efeitos dos fármacos , DNA de Neoplasias/metabolismo , Citometria de Fluxo , Humanos , Antígeno Ki-67 , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/imunologia , Proteínas Nucleares/metabolismo , Proto-Oncogene Mas , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/imunologia , Células Tumorais Cultivadas/metabolismo
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