RESUMO
Treatment of metastatic renal cell carcinoma (mRCC) has improved significantly with the advent of agents targeting the mTOR pathway, such as temsirolimus and everolimus. However, their efficacy is thought to be limited by feedback loops and crosstalk with other pathways leading to the development of drug resistance. As CXCR4-CXCL12-CXCR7 axis has been described to have a crucial role in renal cancer; the crosstalk between the mTOR pathway and the CXCR4-CXCL12-CXCR7 chemokine receptor axis has been investigated in human renal cancer cells. In SN12C and A498, the common CXCR4-CXCR7 ligand, CXCL12, and the exclusive CXCR7 ligand, CXCL11, activated mTOR through P70S6K and 4EBP1 targets. The mTOR activation was specifically inhibited by CXCR4 antagonists (AMD3100, anti-CXCR4-12G5 and Peptide R, a newly developed CXCR4 antagonist) and CXCR7 antagonists (anti-CXCR7-12G8 and CCX771, CXCR7 inhibitor). To investigate the functional role of CXCR4, CXCR7 and mTOR in human renal cancer cells, both migration and wound healing were evaluated. SN12C and A498 cells migrated toward CXCL12 and CXCL11; CXCR4 and CXCR7 inhibitors impaired migration and treatment with mTOR inhibitor, RAD001, further inhibited it. Moreover, CXCL12 and CXCL11 induced wound healing while was impaired by AMD3100, the anti CXCR7 and RAD001. In SN12C and A498 cells, CXCL12 and CXCL11 promoted actin reorganization characterized by thin spikes at the cell periphery, whereas AMD3100 and anti-CXCR7 impaired CXCL12/CXCL11-induced actin polymerization, and RAD001 treatment further reduced it. In addition, when cell growth was evaluated in the presence of CXCL12, CXCL11 and mTOR inhibitors, an additive effect was demonstrated with the CXCR4, CXCR7 antagonists and RAD001. RAD001-resistant SN12C and A498 cells recovered RAD001 sensitivity in the presence of CXCR4 and CXCR7 antagonists. In conclusion, the entire axis CXCR4-CXCL12-CXCR7 regulates mTOR signaling in renal cancer cells offering new therapeutic opportunities and targets to overcome resistance to mTOR inhibitors.
Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Receptores CXCR4/metabolismo , Receptores CXCR/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Carcinoma de Células Renais/genética , Linhagem Celular Tumoral , Quimiocina CXCL12/genética , Quimiocina CXCL12/metabolismo , Humanos , Neoplasias Renais/genética , Receptores CXCR/genética , Receptores CXCR4/genética , Transdução de Sinais , Serina-Treonina Quinases TOR/genéticaRESUMO
We report that in breast cancer cells, tyrosine phosphorylation of the estradiol receptor alpha (ERalpha) by Src regulates cytoplasmic localization of the receptor and DNA synthesis. Inhibition of Src or use of a peptide mimicking the ERalpha p-Tyr537 sequence abolishes ERalpha tyrosine phosphorylation and traps the receptor in nuclei of estradiol-treated MCF-7 cells. An ERalpha mutant carrying a mutation of Tyr537 to phenylalanine (ER537F) persistently localizes in nuclei of various cell types. In contrast with ERalpha wt, ER537F does not associate with Ran and its interaction with Crm1 is insensitive to estradiol. Thus, independently of estradiol, ER537F is retained in nuclei, where it entangles FKHR-driving cell cycle arrest. Chromatin immunoprecipitation analysis reveals that overexpression of ER537F in breast cancer cells enhances FKHR interaction with cyclin D1 promoter. This mutant also counteracts cell transformation by the activated forms of Src or PI3-K. In conclusion, in addition to regulating receptor localization, ERalpha phosphorylation by Src is required for hormone responsiveness of DNA synthesis in breast cancer cells.
Assuntos
Neoplasias da Mama/metabolismo , Pontos de Checagem do Ciclo Celular/fisiologia , Estradiol/metabolismo , Receptor alfa de Estrogênio/metabolismo , Tirosina/metabolismo , Quinases da Família src/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Neoplasias da Mama/enzimologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células COS , Pontos de Checagem do Ciclo Celular/genética , Processos de Crescimento Celular/genética , Processos de Crescimento Celular/fisiologia , Linhagem Celular , Linhagem Celular Tumoral , Núcleo Celular/genética , Núcleo Celular/metabolismo , Chlorocebus aethiops , Ciclina D1/genética , Ciclina D1/metabolismo , Citoplasma/genética , Citoplasma/metabolismo , Receptor alfa de Estrogênio/genética , Feminino , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Humanos , Carioferinas/genética , Carioferinas/metabolismo , Células MCF-7 , Camundongos , Mutação , Células NIH 3T3 , Fenilalanina/genética , Fenilalanina/metabolismo , Fosfatidilinositol 3-Quinases/genética , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação , Regiões Promotoras Genéticas , Receptores Citoplasmáticos e Nucleares/genética , Receptores Citoplasmáticos e Nucleares/metabolismo , Fase S/genética , Transcrição Gênica , Tirosina/genética , Proteína ran de Ligação ao GTP/genética , Proteína ran de Ligação ao GTP/metabolismo , Quinases da Família src/genética , Proteína Exportina 1RESUMO
Celiac disease (CD) is characterized by abnormally high concentrations of certain peptides in the small bowel. These peptides can be grouped in 'toxic' and 'immunogenic' classes, which elicit an innate immune response and an HLA-mediated adaptive response, respectively. It is not clear on which molecular mechanisms responses to these different classes are based, but the 31-43 (P31-43) and the 56-68 (P56-68) A-gliadin fragments are usually adopted as sequence representatives of toxic and immunogenic peptides, respectively. Here we report fluorescence experiments aiming to mimic the interaction of these peptides with the cell membrane surface by using sodium dodecyl sulphate (SDS) as a membrane-mimetic medium. We show that P31-43 is able to bind SDS micelles in a way that resembles mixed micelle formation. On the other hand, no binding at all could be detected for P56-68. This different behaviour could be related to the paracellular or transcellular route through which gluten peptides may cross the intestinal epithelium, and open new insights into the pathogenetic mechanisms of CD.
Assuntos
Membrana Celular/metabolismo , Gliadina , Fragmentos de Peptídeos , Doença Celíaca/metabolismo , Membrana Celular/química , Corantes/química , Detergentes/química , Gliadina/química , Gliadina/imunologia , Gliadina/toxicidade , Humanos , Mucosa Intestinal/citologia , Mucosa Intestinal/metabolismo , Micelas , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/imunologia , Fragmentos de Peptídeos/toxicidade , Dodecilsulfato de Sódio/químicaRESUMO
In human mammary and prostate cancer cells, steroid hormones or epidermal growth factor (EGF) trigger association of the androgen receptor (AR)-estradiol receptor (ER) (alpha or beta) complex with Src. This interaction activates Src and affects the G1 to S cell cycle progression. In this report, we identify the sequence responsible for the AR/Src interaction and describe a 10 amino-acid peptide that inhibits this interaction. Treatment of the human prostate or mammary cancer cells (LNCaP or MCF-7, respectively) with nanomolar concentrations of this peptide inhibits the androgen- or estradiol-induced association between the AR or the ER and Src the Src/Erk pathway activation, cyclin D1 expression and DNA synthesis, without interfering in the receptor-dependent transcriptional activity. Similarly, the peptide prevents the S phase entry of LNCaP and MCF-7 cells treated with EGF as well as mouse embryo fibroblasts stimulated with androgen or EGF. Interestingly, the peptide does not inhibit the S phase entry and cytoskeletal changes induced by EGF or serum treatment of AR-negative prostate cancer cell lines. The peptide is the first example of a specific inhibitor of steroid receptor-dependent signal transducing activity. The importance of these results is highlighted by the finding that the peptide strongly inhibits the growth of LNCaP xenografts established in nude mice.
Assuntos
Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Receptores Androgênicos/metabolismo , Domínios de Homologia de src/fisiologia , Sequência de Aminoácidos , Antagonistas de Receptores de Andrógenos , Animais , Neoplasias da Mama/metabolismo , Humanos , Masculino , Camundongos , Peptídeos , Neoplasias da Próstata/metabolismo , Ligação Proteica , Receptores de Estradiol/antagonistas & inibidores , Receptores de Estradiol/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
The p85-associated phosphatidylinositol (PI) 3-kinase/Akt pathway mediates the oestradiol-induced S-phase entry and cyclin D1 promoter activity in MCF-7 cells. Experiments with Src, p85alpha and Akt dominant-negative forms indicate that in oestradiol-treated cells these signalling effectors target the cyclin D1 promoter. Oestradiol acutely increases PI3-kinase and Akt activities in MCF-7 cells. In NIH 3T3 cells expressing ERalpha, a dominant-negative p85 suppresses hormone stimulation of Akt. The Src inhibitor, PP1, prevents hormone stimulation of Akt and PI3-kinase activities in MCF-7 cells. In turn, stimulation of Src activity is abolished in ERalpha-expressing NIH 3T3 fibroblasts by co-transfection of the dominant-negative p85alpha and in MCF-7 cells by the PI3-kinase inhibitor, LY294002. These findings indicate a novel reciprocal cross-talk between PI3-kinase and Src. Hormone stimulation of MCF-7 cells rapidly triggers association of ERalpha with Src and p85. In vitro these proteins are assembled in a ternary complex with a stronger association than that of the binary complexes composed by the same partners. The ternary complex probably favours hormone activation of Src- and PI3-kinase-dependent pathways, which converge on cell cycle progression.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Fase S/fisiologia , Quinases da Família src/metabolismo , Divisão Celular/efeitos dos fármacos , Ciclina D1/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Receptor alfa de Estrogênio , Feminino , Humanos , Subunidades Proteicas , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-akt , Receptores de Estrogênio/metabolismo , Fase S/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Células Tumorais CultivadasRESUMO
Thyroid papillary carcinomas are characterized by RET/PTC rearrangements that cause the tyrosine kinase domain of the RET receptor to fuse with N-terminal sequences encoded by heterologous genes. This results in the aberrant expression of a ligand-independent and constitutively active RET kinase. We analysed actin reorganization induced by the RET/PTC1 oncogene in PC Cl 3 rat thyroid epithelial cells. Differently from oncogenes Src, Ras and Raf, RET/PTC1 caused actin filaments to form prominent stress fibers. Moreover, stress fibers were identified in human thyroid papillary carcinoma cell lines harboring RET/PTC1 rearrangements but not in thyroid carcinoma cells negative for RET/PTC rearrangements. RET/MEN 2A, a constitutively active but unrearranged membrane-bound RET oncoprotein, did not induce stress fibers in PC Cl 3 cells. Induction of stress fibers by RET/PTC1 was restricted to thyroid cells; it did not occur in NIH3T3 fibroblasts or MCF7 mammary cells. RET/PTC1-mediated stress fiber formation depended on Rho but not Rac small GTPase activity. In addition, inhibition of Rho, but not of Rac, caused apoptosis of RET/PTC1-expressing thyroid cells. We conclude that Rho is implicated in the actin reorganization and cell survival mediated by the chimeric RET/PTC1 oncogene in thyroid epithelial cells, both phenotypes being cell type- and oncogene type-specific.
Assuntos
Carcinoma Papilar/patologia , Proteínas de Drosophila , Proteínas de Fusão Oncogênica/fisiologia , Transdução de Sinais/fisiologia , Fibras de Estresse/fisiologia , Glândula Tireoide/citologia , Neoplasias da Glândula Tireoide/patologia , Proteínas rho de Ligação ao GTP/fisiologia , Células 3T3 , Actinas/metabolismo , Adenocarcinoma/patologia , Animais , Apoptose , Neoplasias da Mama/patologia , Linhagem Celular , Linhagem Celular Transformada , Sobrevivência Celular , Replicação do DNA , Dimerização , Feminino , Humanos , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Camundongos , Neoplasia Endócrina Múltipla Tipo 2a/genética , Neoplasia Endócrina Múltipla Tipo 2a/metabolismo , Invasividade Neoplásica , Proteínas de Neoplasias/metabolismo , Especificidade de Órgãos , Fenótipo , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases , Proteínas Recombinantes de Fusão/fisiologia , Transfecção , Células Tumorais CultivadasRESUMO
The RET/PTC3 oncogene arises from the fusion between the N-terminal encoding domain of the RFG gene and the tyrosine kinase encoding domain of RET receptor. RET/PTC3 is very frequent in papillary thyroid carcinomas, especially in children exposed to the Chernobyl accident. We have studied the functional consequences of the RFG-RET fusion. Here we show that the N-terminal coiled-coil domain of RGF mediates oligomerization and activation of the kinase and of the transforming capability of RET/PTC3. In addition, the RFG coiled-coil domain mediates a physical association between RET/PTC3 and RGF proteins, rendering RFG a bona fide substrate of RET/PTC3 kinase. Finally, we show that the coiled-coil domain of RGF is essential for the distribution of the RET/PTC3 protein at the membrane/particulate cell compartment level, where also most of the RFG protein is localized. We propose that fusion to the RFG coiled-coil domain provides RET kinase with a scaffold that mediates oligomerization and re-localization of the RET/PTC3 protein, a process that may be crucial for the signalling of this specific RET/PTC variant.
Assuntos
Proteínas de Drosophila , Proteínas de Fusão Oncogênica/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Células 3T3 , Animais , Linhagem Celular , Membrana Celular/enzimologia , Membrana Celular/metabolismo , Transformação Celular Neoplásica , Ativação Enzimática , Células Epiteliais/citologia , Humanos , Camundongos , Proteínas de Fusão Oncogênica/genética , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , Ratos , Receptores Proteína Tirosina Quinases/genética , Glândula Tireoide/citologia , TransfecçãoRESUMO
The proteins of the Ets family are transcription factors involved in signal transduction, cell cycle progression, and differentiation. In this study, we report that thyroid cell neoplastic transformation is associated with a dramatic increase in ETS transcriptional activity, which is dependent on the accumulation of Ets-1, Ets-2, and other Ets-related proteins. Inhibition of ETS transactivation activity by the Ets-dominant negative construct (Ets-Z) induced programmed cell death in human thyroid carcinoma cell lines but not in normal thyroid cells. Apoptotic cell death induced by Ets-Z was dependent on the reduction of c-MYC protein levels, because it was prevented by overexpression of c-myc. Taken together, these data indicate that the induction of Ets-1 and Ets-2 transcription factors plays a pivotal role in thyroid cell neoplastic transformation.
Assuntos
Transformação Celular Neoplásica/metabolismo , Proteínas de Ligação a DNA , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Repressoras , Glândula Tireoide/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Transativadores/biossíntese , Fatores de Transcrição/biossíntese , Apoptose/fisiologia , Sequência Consenso , DNA/metabolismo , Genes myc/genética , Humanos , Proteína Proto-Oncogênica c-ets-1 , Proteína Proto-Oncogênica c-ets-2 , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-ets , Glândula Tireoide/patologia , Neoplasias da Glândula Tireoide/patologia , Transativadores/fisiologia , Fatores de Transcrição/fisiologia , Ativação Transcricional/fisiologia , Células Tumorais CultivadasRESUMO
Point mutations of the RET receptor tyrosine kinase are responsible for the inheritance of multiple endocrine neoplasia (MEN) type 2 syndromes and are also present in a fraction of sporadic medullary thyroid carcinomas. Somatic rearrangements of the RET gene generating the chimeric RET/papillary thyroid carcinoma (PTC) oncogenes are the predominant molecular lesions associated with papillary carcinoma, the most frequent thyroid malignancy in humans. Oncogenic mutations cause constitutive activation of the kinase function of RET, which, in turn, results in the autophosphorylation of RET tyrosine residues critical for signaling. In vitro kinase assays previously revealed six putative RET autophosphorylation sites. The aim of the present study was to assess the phosphorylation of two such residues, tyrosines 1015 and 1062 (Y1015 and Y1062), in the in vivo signaling of RET and RET-derived oncogenes. Using phosphorylated RET-specific antibodies, we demonstrate that both Y1015 and Y1062 are rapidly phosphorylated upon ligand triggering of RET. Moreover, regardless of the nature of the underlying activating mutation, the concomitant phosphorylation of Y1015 and Y1062 is a common feature of the various oncogenic RET products (MEN2A, MEN2B, and PTC). This study shows that Ab-pY1062 is a useful tool with which to detect activated RET in human tumor cells and surgical samples. Finally, the microinjection of Ab-pY1062 antibodies into living cells demonstrates that Ret/PTC1 signaling is required to maintain the mitogenesis of a human carcinoma cell line expressing the Ret/PTC1 oncoprotein.
Assuntos
Proteínas de Drosophila , Proteínas Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores Proteína Tirosina Quinases/metabolismo , Tirosina/metabolismo , Sequência de Aminoácidos , Animais , Anticorpos Bloqueadores/farmacologia , Células COS , DNA/biossíntese , Fibroblastos , Humanos , Imuno-Histoquímica , Microinjeções , Dados de Sequência Molecular , Proteínas Oncogênicas/genética , Oncogenes/genética , Fosforilação , Mutação Puntual/fisiologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-ret , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores Proteína Tirosina Quinases/genética , Neoplasias da Glândula Tireoide/metabolismo , Transfecção , Células Tumorais Cultivadas , Tirosina/genéticaRESUMO
Retinoic acid (RA) treatment of embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) induces growth arrest and terminal differentiation along the neuronal pathway. In the present study, we provide a functional link between RA and p27 function in the control of neuronal differentiation in NT2/D1 cells. We report that RA enhances p27 expression, which results in increased association with cyclin E/cyclin-dependent kinase 2 complexes and suppression of their activity; however, antisense clones, which have greatly reduced RA-dependent p27 inducibility (NT2-p27AS), continue to synthesize DNA and are unable to differentiate properly in response to RA as determined by lack of neurite outgrowth and by the failure to modify surface antigens. As to the mechanism involved in RA-dependent p27 upregulation, our data support the concept that RA reduces p27 protein degradation through the ubiquitin/proteasome-dependent pathway. Taken together, these findings demonstrate that in embryonal carcinoma cells, p27 expression is required for growth arrest and proper neuronal differentiation.
Assuntos
Antineoplásicos/farmacologia , Carcinoma Embrionário/tratamento farmacológico , Carcinoma Embrionário/patologia , Proteínas de Ciclo Celular , Neurônios/patologia , Tretinoína/farmacologia , Proteínas Supressoras de Tumor , Antineoplásicos/uso terapêutico , Carcinoma Embrionário/genética , Carcinoma Embrionário/metabolismo , Diferenciação Celular/efeitos dos fármacos , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Cisteína Endopeptidases/genética , Cisteína Endopeptidases/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Associadas aos Microtúbulos/metabolismo , Complexos Multienzimáticos/genética , Complexos Multienzimáticos/metabolismo , Neurônios/metabolismo , Complexo de Endopeptidases do Proteassoma , Tretinoína/uso terapêutico , Células Tumorais CultivadasRESUMO
Treatment of human prostate carcinoma-derived LNCaP cells with androgen or oestradiol triggers simultaneous association of androgen receptor and oestradiol receptor beta with Src, activates the Src/Raf-1/Erk-2 pathway and stimulates cell proliferation. Surprisingly, either androgen or oestradiol action on each of these steps is inhibited by both anti-androgens and anti-oestrogens. Similar findings for oestradiol receptor alpha were observed in MCF-7 or T47D cells stimulated by either oestradiol or androgens. Microinjection of LNCaP, MCF-7 and T47D cells with SrcK(-) abolishes steroid-stimulated S-phase entry. Data from transfected Cos cells confirm and extend the findings from these cells. Hormone-stimulated Src interaction with the androgen receptor and oestradiol receptor alpha or beta is detected using glutathione S:-transferase fusion constructs. Src SH2 interacts with phosphotyrosine 537 of oestradiol receptor alpha and the Src SH3 domain with a proline-rich stretch of the androgen receptor. The role of this phosphotyrosine is stressed by its requirement for association of oestradiol receptor alpha with Src and consequent activation of Src in intact Cos cells.
Assuntos
Neoplasias da Próstata/patologia , Receptores Androgênicos/metabolismo , Receptores de Estradiol/metabolismo , Esteroides/farmacologia , Quinases da Família src/metabolismo , Antagonistas de Androgênios/farmacologia , Androgênios/farmacologia , Animais , Células COS , Divisão Celular/efeitos dos fármacos , Estradiol/farmacologia , Antagonistas de Estrogênios/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Substâncias Macromoleculares , Masculino , Metribolona/farmacologia , Modelos Biológicos , Ligação Proteica/efeitos dos fármacos , Receptores Androgênicos/genética , Receptores de Estradiol/genética , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Transfecção , Células Tumorais Cultivadas , Domínios de Homologia de src/efeitos dos fármacos , Quinases da Família src/genéticaRESUMO
Hexamethylen-bisacetamide (HMBA) represents the prototype of a group of hybrid polar compounds, which induce differentiation in a variety of transformed cells including human embryonal carcinoma cells. Therefore, HMBA has been used in the differentiation therapy of cancer for patients with both hematological and solid malignancies. Upon HMBA treatment, the embryonal carcinoma cell line NTERA-2 clone D1 (NT2/D1) accumulates in G1 and undergoes terminal differentiation. Here we demonstrate that growth arrest and differentiation of NT2/D1 cells induced by HMBA involve increased expression of the cyclin-dependent kinase inhibitor p27, enhanced association of p27 with cyclin E/CDK2 complexes and suppression of kinase activity associated to cyclin E/CDK2 (but not to cyclin D3/CDK4). When HMBA differentiation was induced in the presence of p27 antisense oligonucleotides, NT2/D1 cells failed to arrest growth properly and, in parallel with the reduction of the anti-apoptotic Bcl-2 gene expression, cells underwent massive programmed cell death. Conversely, constitutive expression of p27 into NT2/D1 cells induced a marked reduction in the growth potential of these cells and partially reproduced HMBA-induced modification of surface antigen expression (down-regulation of SSEA-3 expression and up-regulation of VINIS-53 expression). Expression of p21 induced growth arrest but not differentiation. Likewise, inhibition of CDK2 by transfection of a dominant negative CDK2 in NT2/D1 cells or treatment with the kinase inhibitor olomucine induced growth arrest but not differentiation. Therefore, we propose that p27 represents a crucial molecule in HMBA signaling that cannot be replaced by p21. Furthermore, the results obtained with CDK2 inhibitors demonstrate that the block of CDK2 activity is sufficient for growth arrest but not for cell differentiation and suggest that, at least in these cells, growth arrest and differentiation are regulated by two overlapping but different pathways.
Assuntos
Apoptose/fisiologia , Quinases relacionadas a CDC2 e CDC28 , Carcinoma Embrionário/patologia , Proteínas de Ciclo Celular , Proteínas Associadas aos Microtúbulos/fisiologia , Proteínas de Neoplasias/fisiologia , Proteínas Supressoras de Tumor , Acetamidas/farmacologia , Antígenos de Neoplasias/biossíntese , Antígenos de Neoplasias/genética , Antígenos de Superfície/biossíntese , Antígenos de Superfície/genética , Antígenos Glicosídicos Associados a Tumores , Apoptose/efeitos dos fármacos , Carcinoma Embrionário/metabolismo , Diferenciação Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclina E/metabolismo , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/genética , Ciclinas/fisiologia , Regulação Neoplásica da Expressão Gênica , Glicoesfingolipídeos/biossíntese , Glicoesfingolipídeos/genética , Humanos , Cinetina , Substâncias Macromoleculares , Proteínas Associadas aos Microtúbulos/biossíntese , Proteínas Associadas aos Microtúbulos/genética , Proteínas de Neoplasias/biossíntese , Proteínas de Neoplasias/genética , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Purinas/farmacologia , Proteína do Retinoblastoma/metabolismo , Roscovitina , Antígenos Embrionários Estágio-Específicos , Células Tumorais CultivadasRESUMO
The majority of thyroid carcinomas maintain the expression of the cell growth suppressor p27, an inhibitor of cyclin-dependent kinase-2 (Cdk2). However, we find that 80% of p27-expressing tumors show an uncommon cytoplasmic localization of p27 protein, associated with high Cdk2 activity. To reproduce such a situation, a mutant p27 devoid of its COOH-terminal nuclear-localization signal was generated (p27-NLS). p27-NLS accumulates in the cytoplasm and fails to induce growth arrest in 2 different cell lines, indicating that cytoplasm-residing p27 is inactive as a growth inhibitor, presumably because it does not interact with nuclear Cdk2. Overexpression of cyclin D3 may account in part for p27 cytoplasmic localization. In thyroid tumors and cell lines, cyclin D3 expression was associated with cytoplasmic localization of p27. Moreover, expression of cyclin D3 in thyroid carcinoma cells induced cytoplasmic retention of cotransfected p27 and rescued p27-imposed growth arrest. Endogenous p27 also localized prevalently to the cytoplasm in normal thyrocytes engineered to stably overexpress cyclin D3 (PC-D3 cells). In these cells, cyclin D3 induced the formation of cytoplasmic p27-cyclin D3-Cdk complexes, which titrated p27 away from intranuclear complexes that contain cyclins A-E and Cdk2. Our results demonstrate a novel mechanism that may contribute to overcoming the p27 inhibitory threshold in transformed thyroid cells.
Assuntos
Quinases relacionadas a CDC2 e CDC28 , Proteínas de Ciclo Celular , Ciclinas/metabolismo , Proteínas Associadas aos Microtúbulos/metabolismo , Neoplasias da Glândula Tireoide/metabolismo , Proteínas Supressoras de Tumor , Núcleo Celular/metabolismo , Ciclina D3 , Quinase 2 Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p27 , Quinases Ciclina-Dependentes/antagonistas & inibidores , Quinases Ciclina-Dependentes/metabolismo , Ciclinas/genética , Citoplasma/metabolismo , Genes Supressores de Tumor , Humanos , Proteínas Associadas aos Microtúbulos/genética , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais , Especificidade por Substrato , Neoplasias da Glândula Tireoide/patologia , Células Tumorais CultivadasRESUMO
Anchorage-independent myelomonocytic cells acquire adherence within minutes of differentiation stimuli, such as the proteolytically inactive N-terminal fragment of urokinase binding to its cognate glycosylphosphatidylinositol (GPI)-anchored receptor. Here, we report that urokinase-treated differentiating U937 monocyte-like cells exhibit a rapid and transient inhibition of p56/59(hck) and p55(fgr) whereas no changes in the activity of other Src family kinases, such as p53/56(lyn) and p59(fyn) were observed. U937 transfectants expressing a kinase-defective (Lys267 to Met) p56/59(hck) variant exhibit enhanced adhesiveness and a marked F-actin redistribution in thin protruding structures. Conversely, urokinase as well as expression of wild-type or constitutively active (Tyr499 to Phe) p56/59(hck) stimulates the directional migration of uninduced U937 cells. Accordingly, expression of constitutively active or kinase inactive p56/59(hck) selectively prevents urokinase receptor-dependent induction of either adhesion or motility, indicating that a specific activation state of p56/59(hck) is required for each cell response. In conclusion, modulation of the intracellular p56/59(hck) tyrosine kinase activity switches cell motility towards adherence, providing a mutually exclusive mechanism to regulate these properties during monocyte/macrophage differentiation in vivo.
Assuntos
Movimento Celular , Macrófagos/enzimologia , Monócitos/enzimologia , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Receptores de Superfície Celular/metabolismo , Actinas/metabolismo , Animais , Calcitriol/farmacologia , Adesão Celular/efeitos dos fármacos , Diferenciação Celular , Movimento Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Ativação Enzimática , Humanos , Macrófagos/citologia , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Camundongos , Monócitos/citologia , Monócitos/efeitos dos fármacos , Monócitos/metabolismo , Mutação , Fenótipo , Fosforilação/efeitos dos fármacos , Inibidores de Proteínas Quinases , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-hck , Receptores de Ativador de Plasminogênio Tipo Uroquinase , Temperatura , Transfecção , Fator de Crescimento Transformador beta/farmacologia , Células Tumorais Cultivadas , Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Ativador de Plasminogênio Tipo Uroquinase/farmacologiaRESUMO
The recent findings that oestradiol and progestins activate the Src/Ras/Erks signalling pathway raise the question of the role of this stimulation. Microinjection experiments of human mammary cancer-derived cells (MCF-7 and T47D) with cDNA of catalytically inactive Src or anti-Ras antibody prove that Src and Ras are required for oestradiol and progestin-dependent progression of cells through the cell cycle. The antitumoral ansamycin antibiotic, geldanamycin, disrupts the steroid-induced Ras-Raf-1 association and prevents Raf-1 activation and steroid-induced DNA synthesis. Furthermore, the selective MEK 1 inhibitor, PD 98059, inhibits oestradiol and progestin stimulation of Erk-2 and the steroid-dependent S-phase entry. The MDA-MB231 cells, which do not express oestradiol receptor, fail to respond to oestradiol in terms of Erk-2 activation and S-phase entry. Fibroblasts are made equally oestradiol-responsive in terms of DNA synthesis by transient transfection with either the wild-type or the transcriptionally inactive mutant oestradiol receptor (HE241G). Co-transfection of catalytically inactive Src as well as treatment with PD98059 inhibit the oestradiol-dependent S-phase entry of fibroblasts expressing either the wild-type oestrogen receptor or its transcriptionally inactive mutant. The data presented support the view that non-transcriptional action of the two steroids plays a major role in cell cycle progression.
Assuntos
Neoplasias da Mama/metabolismo , Estradiol/farmacologia , Quinases de Proteína Quinase Ativadas por Mitógeno , Progestinas/farmacologia , Células 3T3 , Animais , Benzoquinonas , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular , DNA de Neoplasias/biossíntese , Feminino , Flavonoides/farmacologia , Genes ras , Genes src , Humanos , Lactamas Macrocíclicas , MAP Quinase Quinase 1 , Camundongos , Ligação Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-raf/metabolismo , Quinonas/farmacologia , Receptores de Estradiol/metabolismo , Fase S , Transdução de Sinais , Transcrição Gênica , Proteínas ras/metabolismoRESUMO
The proto-oncogene RET encodes a transmembrane growth neurotrophic receptor with tyrosine kinase (TK) activity. RET mutations are associated with several human neoplastic and nonneoplastic diseases, including thyroid papillary carcinoma, multiple endocrine neoplasia type 2 syndromes, and Hirschsprung's disease. Activation of receptor TKs results in the binding and activation of downstream signaling proteins, among which are nonreceptor TKs of the Src family. To test the involvement of c-Src in Ret-mediated signaling, we measured the levels of c-Src activity in NIH3T3 cells coexpressing Ret and the accessory GFR alpha-1 receptor or an epidermal growth factor receptor/Ret chimeric receptor when the cells were stimulated by glial cell line-derived neurotrophic factor or epidermal growth factor, respectively. Ret stimulation resulted in the activation of c-Src. We also measured the levels of Src kinase activity in cell lines expressing isoforms of the Ret receptor activated by different mutations. These cells showed higher Src kinase activity than the normal counterpart. Furthermore, we show that Ret is able to associate with the SH2 domain of Src in a phosphotyrosine-dependent fashion. Microinjection of a kinase inactive mutant of c-Src blocked Ret-mediated mitogenic effect. These experiments demonstrate that activated Ret is able to bind and stimulate c-Src kinase and that Src activation is essential for the mitogenic activity of Ret.
Assuntos
Ciclo Celular , Proteínas de Drosophila , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Proto-Oncogenes , Receptores Proteína Tirosina Quinases/genética , Receptores Proteína Tirosina Quinases/metabolismo , Quinases da Família src/metabolismo , Células 3T3 , Animais , Linhagem Celular , Ativação Enzimática , Receptores de Fator Neurotrófico Derivado de Linhagem de Célula Glial , Humanos , Cinética , Camundongos , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-ret , Proteínas Recombinantes de Fusão/metabolismo , Fase S , Transdução de Sinais , TransfecçãoRESUMO
Growth factors such as platelet-derived growth factor (PDGF) elicit the transcriptional activation of a large number of immediate early genes (many of which encode transcription factors), and ultimately DNA synthesis. Both AP1 and Myc are activated in fibroblasts in response to growth factor stimulation, and various experiments suggest their importance in proliferation. Src family kinases are required for PDGF (and other growth factors) to induce DNA synthesis. We have examined which transcription factors, when constitutively expressed, 'rescue' the block elicited by dominant negative Src. We report here that Myc, but not Fos and/or Jun, was able to rescue the block. In contrast, Fos and Jun, but not Myc, rescued the block induced by dominant negative Ras. Our data suggest that Src kinases control the transcriptional activation of Myc.
Assuntos
Fator de Crescimento Derivado de Plaquetas/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-fos/metabolismo , Proteínas Proto-Oncogênicas c-myc/metabolismo , Transdução de Sinais , Ativação Transcricional , Células 3T3 , Animais , Sequência de Bases , Proteína Tirosina Quinase CSK , DNA/biossíntese , Ativação Enzimática , Camundongos , Dados de Sequência Molecular , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Tirosina Quinases/genética , Proteínas Proto-Oncogênicas c-fos/genética , Proteínas Proto-Oncogênicas c-myc/genética , Fase S , Proteínas ras/metabolismo , Quinases da Família srcRESUMO
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.
Assuntos
Replicação do DNA/efeitos dos fármacos , Substâncias de Crescimento/farmacologia , Proteínas Tirosina Quinases/metabolismo , Células 3T3 , Sequência de Aminoácidos , Animais , Anticorpos/farmacologia , Bombesina/farmacologia , DNA/biossíntese , Fator de Crescimento Epidérmico/farmacologia , Genes src , Lisofosfolipídeos/farmacologia , Fator Estimulador de Colônias de Macrófagos/farmacologia , Camundongos , Dados de Sequência Molecular , Oligopeptídeos/síntese química , Oligopeptídeos/imunologia , Faloidina/farmacologia , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Tirosina Quinases/biossíntese , Coelhos/imunologia , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/metabolismoRESUMO
In NIH3T3 fibroblasts, the ubiquitous helix-loop-helix (HLH) protein E2A (E12/E47) and the myogenic HLH proteins MyoD, MRF4 and myogenin are growth-inhibitory, while two ubiquitous Id proteins lacking the basic region are not. The dimerization domain mediates inhibition. However, in addition to the HLH region, E2A contains two inhibitory regions over-lapping with the main transcriptional activation domains. The growth-suppressive activity of the intact E47 as well as MyoD was counteracted by the Id proteins. When E47 lacking the HLH domain was overexpressed, Id could no longer reverse growth inhibition. By increasing the amount of E47 with an inducible system or neutralizing the endogenous Id with microinjected anti-Id antibodies, withdrawal from the cell cycle occurred within hours before the G1-S transition point. The combined results suggest that the Id proteins are required for G1 progression. The antagonism between the E2A and Id proteins further suggests that both are involved in regulatory events prior to or near the restriction point in the G1 phase of the cell cycle.
Assuntos
Proteínas de Ligação a DNA/fisiologia , Fase G1/fisiologia , Sequências Hélice-Alça-Hélice , Proteínas Repressoras , Fatores de Transcrição , Células 3T3 , Animais , Ciclo Celular/efeitos dos fármacos , Ciclo Celular/fisiologia , Análise Mutacional de DNA , DNA Complementar , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/imunologia , Estrogênios/farmacologia , Fase G1/efeitos dos fármacos , Proteína 1 Inibidora de Diferenciação , Camundongos , Fase S/efeitos dos fármacos , Fase S/fisiologia , Fatores de Transcrição TCF , Proteína 1 Semelhante ao Fator 7 de TranscriçãoRESUMO
Id1, Id2, and Id3 (HLH462) dimerize with members of the basic helix-loop-helix protein family, but due to the absence of the basic region, the resulting heterodimers cannot bind DNA. Therefore Id-type proteins negatively regulate DNA binding of the basic helix-loop-helix proteins. Here we report that Id1, Id2, and Id3 are induced shortly after serum stimulation in arrested NIH 3T3. Antisense oligonucleotides against the Id mRNAs delay the reentry of arrested cells into the cell cycle elicited by stimulation with serum or growth factors. Antisense oligonucleotides against all three Id mRNAs are more effective than individual ones. Combined, these results indicate that Id proteins are involved in the control of growth induction.