RESUMO
Genetic ras mutations are infrequent in breast cancer but Ras may be pathologically activated in breast cancer by overexpression of growth factor receptors which signal through Ras. Using a highly sensitive, coupled enzymatic assay, we measured Ras activation in 20 breast cancers, two fibroadenomas, and seven normal breast samples. Ras was highly activated compared to benign tissue in 11 of the 20 cancers; 7 of these 11 cancers expressed both the epidermal growth factor (EGF) and ErbB-2/neu/HER-2 receptors with the remaining four cancers with high Ras activation expressing one of these two receptors. In the other nine cancers, Ras activation was similar to that observed in benign breast tissue with none of these cancers expressing the EGF receptor while one expressed the ErbB-2 receptor. None of the cancers tested had an activating K-ras mutation nor did any of the cancers express a truncated EGF receptor or the c-FMS receptor. The activity of mitogen-activated protein (MAP) kinase was high in the cancers, and reflected the degree of Ras activation. In cultured mammary tumor cell lines, we showed that Ras activation was ligand dependent in cells overexpressing the ErbB-2 receptor. Thus, Ras was abnormally activated in breast cancers overexpressing the EGF and/or ErbB-2 receptors indicating there are sufficient ligands in vivo to activate these receptors, and this work provides a basis for new target-based treatments of this disease.
Assuntos
Neoplasias da Mama/genética , Fibroadenoma/genética , Regulação Neoplásica da Expressão Gênica , Genes ras/genética , Proteínas ras/biossíntese , Feminino , Humanos , Imuno-Histoquímica , Ligantes , Neoplasias Mamárias Animais , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Mutação Puntual , Células Tumorais CultivadasRESUMO
We have shown that nitric oxide (NO) regulates c-fos gene expression via cGMP-dependent protein kinase (G-kinase), but NO's precise mechanism of action is unclear. We now demonstrate that: (1) NO targets two transcriptional elements in the fos promoter, i.e., the fos AP-1 binding site and the cAMP-response element (CRE); (2) NO activation of these two enhancer elements requires the CRE binding protein CREB because a dominant negative CREB fully inhibits NO transactivation of reporter genes whereas dominant negative Fos or CCAAT enhancer binding proteins have no effect; (3) CREB is phosphorylated by G-kinase in vitro and its phosphorylation increases in vivo when G-kinase is activated either directly by cGMP or indirectly by NO via soluble guanylate cyclase; (4) NO activation of fos promoter elements requires nuclear translocation of G-kinase but not activation of mitogen-activated protein kinases.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , Genes fos , Sistema de Sinalização das MAP Quinases , Óxido Nítrico/fisiologia , Transporte Ativo do Núcleo Celular , Animais , Linhagem Celular , Núcleo Celular/metabolismo , Cricetinae , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/genética , Proteínas Quinases Dependentes de GMP Cíclico/genética , Elementos Facilitadores Genéticos , Genes Reporter , Guanilato Ciclase/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mutação , Fosforilação , Regiões Promotoras Genéticas , Ativação Transcricional , Transfecção , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
Nitric oxide (NO) regulates the expression of multiple genes but in most cases its precise mechanism of action is unclear. We used baby hamster kidney (BHK) cells, which have very low soluble guanylate cyclase and cGMP-dependent protein kinase (G-kinase) activity, and CS-54 arterial smooth muscle cells, which express these two enzymes, to study NO regulation of the human fos promoter. The NO-releasing agent Deta-NONOate (ethanamine-2,2'-(hydroxynitrosohydrazone)bis-) had no effect on a chloramphenicol acetyltransferase (CAT) reporter gene under control of the fos promoter in BHK cells transfected with an empty vector or in cells transfected with a G-kinase Ibeta expression vector. In BHK cells transfected with expression vectors for guanylate cyclase, Deta-NONOate markedly increased the intracellular cGMP concentration and caused a small (2-fold) increase in CAT activity; the increased CAT activity appeared to be from cGMP activation of cAMP-dependent protein kinase. In BHK cells co-transfected with guanylate cyclase and G-kinase expression vectors, CAT activity was increased 5-fold in the absence of Deta-NONOate and 7-fold in the presence of Deta-NONOate. Stimulation of CAT activity in the absence of Deta-NONOate appeared to be largely from endogenous NO since we found that: (i) BHK cells produced high amounts of NO; (ii) CAT activity was partially inhibited by a NO synthase inhibitor; and (iii) the inhibition by the NO synthase inhibitor was reversed by exogenous NO. In CS-54 cells, we found that NO increased fos promoter activity and that the increase was prevented by a guanylate cyclase inhibitor. In summary, we found that NO activates the fos promoter by a guanylate cyclase- and G-kinase-dependent mechanism.
Assuntos
Proteínas Quinases Dependentes de GMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Guanilato Ciclase/metabolismo , Óxido Nítrico/fisiologia , Transcrição Gênica , Animais , Células Cultivadas , Cricetinae , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Proteína Quinase Dependente de GMP Cíclico Tipo I , Ativação Enzimática , Humanos , Compostos Nitrosos/metabolismo , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas c-fos/genética , Solubilidade , Ativação Transcricional , TransfecçãoRESUMO
Activation of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (A-kinase) promotes hemoglobin synthesis in several erythropoietin-dependent cell lines, whereas A-kinase-deficient murine erythroleukemia (MEL) cells show impaired hemoglobin production; A-kinase may regulate the erythroid transcription factor NF-E2 by directly phosphorylating its p45 subunit or by changing p45 interactions with other proteins. We have mapped the major A-kinase phosphorylation site of p45 to Ser(169); Ala substitution for Ser(169) resulted in a protein that was no longer phosphorylated by A-kinase in vitro or in vivo. The mutant protein formed NF-E2 complexes that bound to DNA with the same affinity as wild-type p45 and functioned normally to restore beta-globin gene expression in a p45-deficient MEL cell line. Transactivation properties of the (Ser (169)--> Ala) mutant p45 were also indistinguishable from wild-type p45 when Gal4-p45 fusion constructs were tested with a Gal4-dependent reporter gene. Transactivation of the reporter by both mutant and wild-type p45 was significantly enhanced when A-kinase was activated by membrane-permeable cAMP analogs or when cells were cotransfected with the catalytic subunit of A-kinase. Stimulation of p45 transactivation by A-kinase required only the N-terminal transactivation domain of p45, suggesting that A-kinase regulates the interaction of p45 with downstream effectors.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Proteínas de Ligação a DNA/metabolismo , Fatores de Transcrição/metabolismo , Animais , AMP Cíclico/fisiologia , Fatores de Ligação de DNA Eritroide Específicos , Eritropoese , Eritropoetina/fisiologia , Histona Acetiltransferases , Camundongos , Mutagênese Sítio-Dirigida , Fator de Transcrição NF-E2 , Subunidade p45 do Fator de Transcrição NF-E2 , Coativador 3 de Receptor Nuclear , Fosfosserina/metabolismo , Relação Estrutura-Atividade , Transativadores/fisiologia , Ativação TranscricionalRESUMO
Diethylcarbamazine (DEC, 1-diethylcarbamyl-4-methylpiperazine) is an antiparasitic piperazine derivative used in the treatment of lymphatic filariasis. DEC-N-oxide is a major metabolite in humans which has antifilarial activity. Gas chromatographic analysis of DEC in plasma can be complicated by the presence of the metabolite, since the thermally unstable DEC-N-oxide is converted to a material which coelutes with DEC under the conditions of the analysis. We now report a method to separate DEC-N-oxide from DEC in plasma using solid-phase extraction with subsequent gas chromatographic analysis using a nitrogen specific detector. 1-Diethylcarbamyl-4-ethylpiperazine (E-DEC) was the internal standard. The standard curve of DEC is linear in the range of 10 to 200 ng/ml. The limit of detection is 4 ng/ml. Reproducibility at 10, 100 and 200 ng/ml concentration points of the standard curve gives coefficients of variation of 6.1%, 7.8% and 1.6%, respectively. Recovery following solid-phase extraction is 99.3% for DEC and 94.8% for the internal standard. This sensitive and specific analytical method is suitable for pharmacokinetic studies of DEC.
Assuntos
Cromatografia Gasosa/métodos , Dietilcarbamazina/sangue , Filaricidas/sangue , Varredura Diferencial de Calorimetria , Dietilcarbamazina/química , Dietilcarbamazina/isolamento & purificação , Estabilidade de Medicamentos , Temperatura Alta , Humanos , Concentração de Íons de Hidrogênio , Oxirredução , Sensibilidade e Especificidade , TermodinâmicaRESUMO
To develop hemoglobin (Hb) derivatives with an increased circulatory half-life, Hb was chemically modified with long chain fatty acid analogs. One compound, sodium 1-hexadecyl 6-(2-iodoacetamido)hexyl phosphate, specifically modified the Cys-93 beta residues of human hemoglobin (HbA) as determined by sulfhydryl titration analysis. The resulting modified Hb derivative, FAHbA, was isolated and was shown to have a two-fold longer circulatory half-life than native HbA in a rat low-dose acute transfusion model.