RESUMO
We describe a novel surgical technique in 31 women with histopathologically confirmed placenta accreta spectrum (PAS) disorders managed by a multidisciplinary team using a prophylactic infrarenal abdominal aortic cross-clamping technique during caesarean hysterectomy. We conclude that this new surgical procedure is a relatively safe technique to potentially control operative blood loss. Our work may stimulate others to develop protocols assessing this innovative technique to improve the surgical outcome of PAS disorders.
Assuntos
Perda Sanguínea Cirúrgica/prevenção & controle , Cesárea/métodos , Hemostasia Cirúrgica/métodos , Histerectomia/métodos , Placenta Acreta , Hemorragia Pós-Parto , Adulto , Aorta Abdominal , Cesárea/efeitos adversos , Constrição , Duração da Terapia , Feminino , Humanos , Histerectomia/efeitos adversos , Avaliação de Resultados em Cuidados de Saúde , Equipe de Assistência ao Paciente , Placenta Acreta/diagnóstico , Placenta Acreta/cirurgia , Hemorragia Pós-Parto/etiologia , Hemorragia Pós-Parto/prevenção & controle , Gravidez , Taiwan , Ultrassonografia Doppler em Cores/métodosAssuntos
Placenta Acreta , Aorta Abdominal/cirurgia , Cesárea , Constrição , Feminino , Humanos , Histerectomia , Placenta Acreta/cirurgia , GravidezAssuntos
Placenta Acreta , Placenta Prévia , Cesárea , Constrição , Feminino , Humanos , Placenta Acreta/diagnóstico por imagem , Placenta Acreta/cirurgia , GravidezRESUMO
It has been confirmed that trimethylation of lysine 27 on histone H3 (H3K27me3) plays an important role in epigenetic process of tumorigenesis. However, the status of H3K27me3 in ovarian cancer and its impact on patients' clinicopathologic characteristics and prognosis are unclear. In the present study, the immunohistochemistry (IHC) was utilized to detect protein expression of H3K27me3 in 12 normal ovaries, 26 ovarian cystadenomas, 31 borderline ovarian tumors and 168 ovarian carcinomas by tissue microarray. The association between H3K27me3 expression with clinicopathologic features and patient prognosis were also evaluated using various statistical models. The expression of H3K27me3 was decreased in 2 of 12 (16.7%) cases of the normal ovaries, 8 of 26 (30.8%) cases of cystadenomas, 12 of 31 (38.7%) cases of borderline ovarian tumors, and 93 of 168 (55.4%) cases of primary ovarian carcinomas, respectively (P<0.05). Further correlation analysis suggested that decreased expression of H3K27me3 in ovarian carcinomas was significantly correlated with more advanced pM and FIGO stages (P<0.05). In addition, a significant association between decreased expression of H3K27me3 and shortened patient survival (mean 66 months versus 101 months, p=0.019) was demonstrated by univariate survival analysis of the ovarian carcinoma cohorts. Importantly, H3K27me3 expression provided a significant independent prognostic factor in multivariate analysis (p=0.028). These findings confirmed that decreased expression of H3K27me3 in primary ovarian cancer might be correlated with the acquisition of an invasive and/or aggressive phenotype of tumor, and might serve as an independent biomarker for poor prognosis in patients with ovarian carcinoma.
RESUMO
BACKGROUND: Distant metastasis is the major cause of cancer-related death, and epithelial-to-mesenchymal transition (EMT) has a critical role in this process. Accumulating evidence indicates that EMT can be regulated by microRNAs (miRNAs). miR-29c has been implicated as a tumor suppressor in several human cancers. However, the role of miR-29c in the progression of colorectal cancer (CRC) metastasis remains largely unknown. PATIENTS AND METHODS: The expression of miR-29c was examined by qRT-PCR in a cohort of primary CRC (PC) and distant liver metastasis (LM) tissues. A series of in vivo and in vitro assays were carried out in order to elucidate the functions of miR-29c and the molecular mechanisms underlying the pathogenesis of metastatic CRC. RESULTS: miR-29c was markedly downregulated in PCs with distant metastasis and determined to be an independent predictor of shortened patient survival. But LM tissues showed higher levels of miR-29c than that in PC tissues. In CRC cells, miR-29c dramatically suppressed cell migration and invasion abilities in vitro and cancer metastasis in vivo. In addition, miR-29c inhibited EMT and negatively regulated Wnt/ß-catenin signaling pathway. Guanine nucleotide binding protein alpha13 (GNA13) and protein tyrosine phosphatase type IVA (PTP4A) were identified as direct targets of miR-29c, which acted through ERK/GSK3ß/ß-catenin and AKT/GSK3ß/ß-catenin pathways, respectively, to regulate EMT. Furthermore, significant associations between miR-29c, its target genes (GNA13 and PTP4A) and EMT markers were validated in both PC and LM tissues. CONCLUSION: Our findings highlight the important role of miR-29c in regulating CRC EMT via GSK-3ß/ß-catenin signaling by targeting GNA13 and PTP4A and provide new insights into the metastatic basis of CRC.
Assuntos
Proteínas de Ciclo Celular/biossíntese , Neoplasias Colorretais/genética , Proteínas de Membrana/biossíntese , MicroRNAs/genética , Proteínas Tirosina Fosfatases/biossíntese , beta Catenina/genética , Proteínas de Ciclo Celular/genética , Linhagem Celular Tumoral , Movimento Celular/genética , Neoplasias Colorretais/patologia , Transição Epitelial-Mesenquimal/genética , Feminino , Regulação Neoplásica da Expressão Gênica , Quinase 3 da Glicogênio Sintase/genética , Glicogênio Sintase Quinase 3 beta , Humanos , Proteínas de Membrana/genética , Metástase Neoplásica , Proteínas Tirosina Fosfatases/genética , Via de Sinalização WntRESUMO
In THP-1 monocytoid cells infected with HIV, viral expression can be regulated in several ways: (a) latency (no viral expression); (b) restricted expression (chronic low-level viral expression with little or no detectable virus released); and (c) continuous production. In cells with restricted HIV expression, nuclear factor(s) were found that blocked tat-associated DNA binding complex formation, suggesting that initiation of transcription was negatively regulated. Also, viral particles were seen budding into and accumulating within intracytoplasmic vacuoles with little virus released, suggesting multiple levels of regulation. These cells with restricted expression had no detectable viral antigens on the cell surface and were not lysed by IL-2-activated large granular lymphocytes. However, they could cause viral-mediated T cell cytolysis in cell-cell assays, suggesting viral transmission through cell contact. In addition, cells with latent HIV were identified and could still produce infectious virus after 5-azacytidine exposure 10 mo later. LPS and other treatments could increase viral production in cells with restricted but not latent expression, suggesting they occur by distinct mechanisms. These infected cells provide a reservoir for viral transmission to uninfected T cells that itself is not detected by immune surveillance mechanisms.
Assuntos
HIV-1/fisiologia , Monócitos/microbiologia , Replicação Viral , Antígenos Virais/análise , Linhagem Celular , Sobrevivência Celular , DNA Viral/análise , HIV-1/genética , HIV-1/ultraestrutura , Humanos , Microscopia Eletrônica , Monócitos/citologia , Fenótipo , Reação em Cadeia da Polimerase , RNA Viral/análise , Sequências Repetitivas de Ácido NucleicoRESUMO
The cDNA coding for human monocyte-derived neutrophil-specific chemotactic factor (MDNCF) was cloned from LPS-stimulated human monocyte mRNA. The cDNA sequence codes for a polypeptide consisting of 99 amino acids, including a putative signal sequence. Comparison of the deduced amino acid sequence with the NH2-terminal amino acid sequence of natural MDNCF shows that the mature functional protein comprises 72 amino acids, beginning with serine at residue 28. The deduced amino acid sequence shows striking similarity to several platelet-derived factors, a v-src-induced protein, a growth-regulated gene product (gro), and an IFN-gamma inducible protein. The availability of the MDNCF cDNA enabled us to use it as a probe to identify inducers of MDNCF mRNA expression in human PBMC. MDNCF mRNA was increased greater than 10-fold within 1 h after stimulation with LPS, IL-1, or TNF, but not by IFN-gamma, IFN-alpha, or IL-2. Furthermore, we also determined that LPS, IL-1, and TNF stimulated the mononuclear cells to produce biologically active MDNCF. This observation may account for the in vivo capacity of IL-1 and TNF to induce netrophil infiltrates.
Assuntos
Fatores Quimiotáticos/genética , Quimiotaxia de Leucócito , Interleucina-1/farmacologia , Monócitos/fisiologia , Fator de Necrose Tumoral alfa/farmacologia , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Inflamação/fisiopatologia , Lipopolissacarídeos/farmacologia , Dados de Sequência Molecular , Neutrófilos/fisiologia , Biossíntese de Proteínas , RNA Mensageiro/genéticaRESUMO
BACKGROUND: The amplified in breast cancer 1 (AIB1) gene has been considered to play an oncogenic role in human cancers, but its clinical/prognostic significance in non-small-cell lung cancer (NSCLC) is still unclear. PATIENTS AND METHODS: The methods of immunohistochemistry and FISH were utilized to examine protein expression and amplification of AIB1 in 230 informative surgically resected NSCLCs and in 30 samples of normal lung tissues. RESULTS: Overexpression and amplification of AIB1 were found in 48.3% and 8.2% of NSCLCs, respectively. AIB1 overexpression was associated with AIB1 gene amplification and cell proliferation but not related to estrogen receptor (ER)-alpha, ER-beta, progesterone receptor or androgen receptor status. A positive correlation between AIB1 overexpression and an ascending pathologic node stage in lung adenocarcinoma (ADC) was observed (P = 0.043). Univariate survival analysis demonstrated a significant association of AIB1 overexpression with shortened patient survival, especially for those with stage III disease (P < 0.001). Importantly, AIB1 expression was evaluated as the most significant predictor for survival in multivariate analysis (hazards ratio = 2.069, P < 0.001). CONCLUSION: Overexpression of AIB1 might provide a selective advantage for lymph node metastasis of lung ADC and serve as a useful biomarker for poor prognosis for NSCLC patients.
Assuntos
Carcinoma Pulmonar de Células não Pequenas/patologia , Neoplasias Pulmonares/patologia , Coativador 3 de Receptor Nuclear/genética , Carcinoma Pulmonar de Células não Pequenas/genética , Carcinoma Pulmonar de Células não Pequenas/cirurgia , Feminino , Humanos , Hibridização in Situ Fluorescente , Pulmão/metabolismo , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/cirurgia , Masculino , Pessoa de Meia-Idade , Prognóstico , Receptores Androgênicos/metabolismo , Receptores de Estrogênio/metabolismo , Receptores de Progesterona/metabolismo , Taxa de SobrevidaRESUMO
BACKGROUND: Our previous study suggested that melanoma nuclear protein 18 (Mel-18) acted as a tumor suppressor in human breast cancer. This study was designed to investigate the clinical and prognostic significance of Mel-18 in breast cancer patients. PATIENTS AND METHODS: Mel-18 was detected by immunohistochemistry in paraffin-embedded tissues from 287 breast cancer patients, of which 287 were from primary cancer sites, 63 from matched adjacent noncancerous sites, and 35 from metastatic lymph nodes. Differences in Mel-18 expression and clinical characteristics were compared by χ² test. Prognostic outcomes correlated with Mel-18 were examined using Kaplan-Meier analysis and Cox proportional hazards model. RESULTS: The decreased Mel-18 expression is incremental depending upon the magnitude of cancer progression (P < 0.001). Mel-18 was conversely correlated with the pathological classifications (P < 0.001 for T, N, and M classifications, respectively), clinical staging (P < 0.001), and progesterone receptor (P = 0.030). Furthermore, patients with higher level of Mel-18 showed prolonged overall survivals (P < 0.001). The diminished Mel-18 expression may be a risk factor for the patients' survival (P < 0.001). CONCLUSIONS: Lower Mel-18 expression is correlated with advanced clinicopathologic classifications and a poor overall survival in breast cancer patients. These findings suggest that Mel-18 may serve as a useful marker in prognostic evaluation for patients.
Assuntos
Neoplasias da Mama/diagnóstico , Neoplasias da Mama/mortalidade , Carcinoma/diagnóstico , Carcinoma/mortalidade , Proteínas Repressoras/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Carcinoma/metabolismo , Carcinoma/patologia , Estudos de Casos e Controles , Feminino , Humanos , Pessoa de Meia-Idade , Estadiamento de Neoplasias , Complexo Repressor Polycomb 1 , Prognóstico , Proteínas Repressoras/análise , Análise de SobrevidaAssuntos
Linfócitos T CD4-Positivos/virologia , Linfócitos T CD8-Positivos/virologia , Infecções por HIV/virologia , Hepatite C/virologia , Coinfecção , Biologia Computacional , Regulação da Expressão Gênica , HIV/isolamento & purificação , Infecções por HIV/complicações , Hepacivirus/isolamento & purificação , Hepatite C/complicações , Humanos , Análise em Microsséries , Reação em Cadeia da Polimerase em Tempo Real , TranscriptomaRESUMO
Posttranslational modification by covalent attachment of polyisoprene intermediates to a carboxyterminal CAAX-box motif is required for the biologic function of proteins such as p21ras, the supergene family of ras-related proteins, nuclear lamins, and subunits of heterotrimeric G-proteins. Cells grown in the presence of lovastatin, which inhibits HMG-CoA reductase and prevents synthesis of intermediates required for protein prenylation, develop a round, refractile morphology. Our data indicate that this is due to the selective loss of actin cables without gross changes in the microtubular lattice or intermediate filament structure. Microinjection of a competitive peptide inhibitor of protein prenyltransferases into the cytoplasm of cells induces an identical change in morphology with loss of actin cables. Mevalonate (MVA) reverses the lovastatin-induced morphologic change by inducing a rapid repolymerization of actin cables with coincident reversion to the flat morphology. Furthermore, microinjection of farnesyl-pyrophosphate or geranylgeranyl-pyrophosphate into lovastatin-treated cells also results in rapid morphologic reversion. The morphologic reversion induced by MVA requires the presence of serum, and is independent of extracellular calcium. The addition of cycloheximide to the growth medium prevents lovastatin-induced loss of actin cables, and causes morphologic reversion of lovastatin-treated cells by a mechanism that is independent of MVA. A1F4- induces morphologic reversion in a manner indistinguishable from MVA. These data indicate that prenylated protein(s) play a critical role in regulating the state of intracellular actin, and that GGPP can rescue the lovastatin-induced morphologic phenotype in the absence of upstream intermediates of cholesterol biosynthesis. We have begun to dissect the signaling events that mediate this pathway.
Assuntos
Citoesqueleto de Actina/ultraestrutura , Actinas/metabolismo , Lovastatina/farmacologia , Proteínas/metabolismo , Citoesqueleto de Actina/efeitos dos fármacos , Sangue , Cálcio/farmacologia , Linhagem Celular , Dimetilaliltranstransferase/metabolismo , Humanos , Ácido Mevalônico/farmacologia , Fosfatos de Poli-Isoprenil/farmacologia , Polímeros , Processamento de Proteína Pós-Traducional , SesquiterpenosRESUMO
The biological activity of Ras proteins is thought to be controlled by the guanine nucleotide exchange factor and the guanosine triphosphatase activating protein (GAP). Treatment of rat pheochromocytoma PC-12 cells with nerve growth factor (NGF) increased the amount of active Ras guanosine triphosphate complex and stimulated the activities of both the guanine nucleotide exchange factor and GAP. In PC-12 cells that overexpressed the tyrosine kinase encoded by the trk proto-oncogene (a component of the high-affinity NGF receptor), the NGF-induced activation of the regulatory proteins was potentiated. These results suggest that the NGF receptor system enhances the activities of both the guanine nucleotide exchange factor and GAP and that the activation of Ras might be controlled by the balance in activity between these two regulatory proteins.
Assuntos
Fatores de Crescimento Neural/farmacologia , Proteínas/metabolismo , Animais , Cromatografia por Troca Iônica , Proteínas Ativadoras de GTPase , Fatores de Troca do Nucleotídeo Guanina , Guanosina 5'-O-(3-Tiotrifosfato)/farmacologia , Guanosina Difosfato/metabolismo , Guanosina Trifosfato/metabolismo , Cinética , Peso Molecular , Células PC12 , Fosfatos/metabolismo , Proteínas/isolamento & purificação , Frações Subcelulares/metabolismo , Proteínas Ativadoras de ras GTPase , Fatores ras de Troca de Nucleotídeo GuaninaRESUMO
Several immunologically distinct isozymes of inositol phospholipid-specific phospholipase C (PLC) have been purified from bovine brain. Murine NIH 3T3 fibroblasts were found to express PLC-gamma, but the expression of PLC-beta was barely detectable by radioimmunoassay or protein immunoblot. A mixture of monoclonal antibodies was identified that neutralizes the biological activity of both endogenous and injected purified PLC-gamma. When co-injected with oncogenic Ras protein or PLC-gamma, this mixture of antibodies inhibited the induction of DNA synthesis that characteristically results from the injection of these proteins into quiescent 3T3 cells. However, when oncogenic Ras protein or PLC-gamma was co-injected with a neutralizing monoclonal antibody to Ras, only the DNA synthesis induced by the Ras protein was inhibited--that induced by PLC was unaffected. These results suggest that the Ras protein is an upstream effector of PLC activity in phosphoinositide-specific signal transduction and that PLC-gamma activity is necessary for Ras-mediated induction of DNA synthesis.
Assuntos
DNA/biossíntese , Isoenzimas/metabolismo , Proteína Oncogênica p21(ras)/farmacologia , Fosfolipases Tipo C/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Linhagem Celular , Fibroblastos , Substâncias de Crescimento/farmacologia , Hibridomas , Immunoblotting , Interfase , Isoenzimas/imunologia , Microinjeções , Proteína Oncogênica p21(ras)/imunologia , Radioimunoensaio , Transdução de Sinais , Fosfolipases Tipo C/imunologia , Fosfolipases Tipo C/farmacologiaRESUMO
IL-6 is an autocrine growth factor for U266 myeloma cells and their growth is inhibited by IFN-alpha or IL-6 mAb. We asked, therefore, whether IFN-alpha-induced growth inhibition involved IL-6. IFN-alpha and mAb against IL-6, the IL-6R alpha-(gp80) or beta-chain (gp130) potently inhibited U266 cells. Remarkably, this effect occurred despite IFN-alpha-augmented secretion of endogenous IL-6. However, examining the IL-6R revealed that IFN-alpha drastically curtailed expression of the IL-6R alpha- and beta-chain. This effect occurred on two different levels (protein and mRNA) and by two different mechanisms (directly and indirectly through IL-6). First, IFN-alpha, but not IL-6, greatly decreased gp80 and, to a lesser extent, gp130 mRNA levels which resulted in a loss of IL-6 binding sites. Second, IFN-alpha-induced IL-6 predominantly down-regulated membrane-bound gp130. IFN-alpha-mediated decrease of gp80 levels was not detected on IL-6-independent myeloma (RPMI 8226) or myeloid cells (U937). We conclude that IFN-alpha inhibited IL-6-dependent myeloma cell growth by depriving U266 cells of an essential component of their autocrine growth loop, a functional IL-6R.
Assuntos
Regulação para Baixo , Interferon-alfa/farmacologia , Interleucina-6/farmacologia , Mieloma Múltiplo/metabolismo , Receptores de Interleucina/biossíntese , Marcadores de Afinidade , Divisão Celular/efeitos dos fármacos , Reagentes de Ligações Cruzadas , Citocinas/farmacologia , Relação Dose-Resposta a Droga , Humanos , Receptores de Interleucina/genética , Receptores de Interleucina-6 , Células Tumorais CultivadasRESUMO
Glioblastoma multiforme is the most aggressive form of human brain tumor, which has no effective cure. Previously, we have demonstrated that overexpression of the C-terminal fragment of the human telomerase reverse transcriptase (hTERTC27) inhibits the growth and tumorigenicity of human cervical cancer HeLa cells. In this study, the therapeutic effect and molecular mechanisms of hTERTC27-mediated cancer gene therapy were further explored in vivo in established human glioblastoma xenografts in nude mice. We showed that intratumoral injection of adeno-associated virus carrying hTERTC27 (rAAV-hTERTC27) is highly effective in reducing the growth of the subcutaneously transplanted glioblastoma tumors. Histological analyses showed that rAAV-hTERTC27 treatment leads to profound necrosis, apoptosis, infiltration of polymorphonuclear neutrophils and reduced microvessel density in the tumor samples. To study the molecular mechanism of rAAV-hTERTC27-mediated antitumor effects, we analyzed the global gene expression profiles of the rAAV-hTERTC27-treated tumor tissues and cell line as compared with that of the control rAAV-green fluorescent protein-treated samples by DNA microarray. Our results suggest that hTERTC27 exerts its effect through complex mechanisms, which involve genes regulating apoptosis, cell adhesion, cell cycle, immune responses, metabolism, signal transduction, transport, transcription and telomere maintenance.
Assuntos
Dependovirus/genética , Terapia Genética , Glioblastoma/terapia , Animais , Técnicas de Cultura de Células , Modelos Animais de Doenças , Técnicas de Transferência de Genes , Vetores Genéticos , Glioblastoma/genética , Glioblastoma/imunologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Análise de Sequência com Séries de Oligonucleotídeos , Fragmentos de Peptídeos/biossíntese , Fragmentos de Peptídeos/genética , Fragmentos de Peptídeos/uso terapêutico , Telomerase/genética , Telomerase/uso terapêuticoRESUMO
Using Xenopus oocytes as a model system, we investigated the possible involvement of ras proteins in the pathway leading to phosphorylation of ribosomal protein S6. Our results indicate that microinjection of oncogenic T24 H-ras protein (which contains valine at position 12) markedly stimulated S6 phosphorylation on serine residues in oocytes, whereas normal ras protein (which contains glycine at position 12) was without effect. The S6 phosphorylation activity in the cell extract from T24 ras protein-injected oocytes was increased significantly. In addition, injection of protein kinase C potentiated the induction of maturation and S6 phosphorylation by the oncogenic ras protein. A similar potentiation was detected when T24 ras protein-injected oocytes were incubated with active phorbol ester. These findings suggest that ras proteins activate the pathway linked to S6 phosphorylation and that protein kinase C has a synergistic effect on the ras-mediated pathway.
Assuntos
Proteína Oncogênica p21(ras)/administração & dosagem , Proteína Quinase C/administração & dosagem , Proteínas Ribossômicas/metabolismo , Animais , Sinergismo Farmacológico , Eletroforese em Gel Bidimensional , Ativação Enzimática , Microinjeções , Oócitos , Fosforilação , Proteínas Quinases/metabolismo , Proteína S6 Ribossômica , Proteínas Quinases S6 Ribossômicas , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Tempo , Xenopus laevisRESUMO
Microinjection of transforming p21 ras protein induces maturation of Xenopus laevis oocytes, and the induction is blocked by coinjection of monoclonal antibody (Y13-259) against p21 ras proteins. Similar to other inducing agents, the effect of p21 ras protein is mediated via the appearance of maturation or meiosis-promoting factor activity. In addition, the neutralizing antibody markedly reduces oocyte maturation after insulin induction, whereas it fails to inhibit progesterone induction. Our results suggest that insulin induces maturation of oocytes via a different pathway than that of steroidal agents. The induction by insulin is ras dependent, and the action of ras may be directed at the steps before meiosis-promoting factor autocatalytic activation. These results suggest a role of p21 ras protein in the events associated with amphibian oocyte maturation.
Assuntos
Insulina/farmacologia , Oócitos/efeitos dos fármacos , Proteínas Proto-Oncogênicas/fisiologia , Animais , Anticorpos Monoclonais/administração & dosagem , Feminino , Meiose/efeitos dos fármacos , Microinjeções , Oócitos/crescimento & desenvolvimento , Progesterona/farmacologia , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas Proto-Oncogênicas/farmacologia , Xenopus laevisRESUMO
Full-length and N-terminal deletion mutants of human c-raf-1 cDNA were cloned into Escherichia coli expression plasmids. Bacterially expressed c-raf proteins were purified by anion-exchange, gel filtration, and affinity chromatography. Microinjection of mutant c-raf proteins into G0-arrested NIH 3T3 cells induced DNA synthesis and morphological transformation, whereas microinjection of full-length c-raf had no effect. The amino terminus of the raf protein has an important negative regulatory influence; alteration of this region resulted in increased kinase activity and oncogenicity.
Assuntos
Transformação Celular Neoplásica , Replicação do DNA , Proteínas Proto-Oncogênicas/metabolismo , Animais , Células Cultivadas , Clonagem Molecular , Escherichia coli/genética , Humanos , Camundongos , Microinjeções , Mutação , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas/administração & dosagem , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-raf , Proteínas Recombinantes/administração & dosagem , Proteínas Recombinantes/metabolismo , Mapeamento por RestriçãoRESUMO
Microinjection of p21ras induced c-fos protein accumulation in three types of 3T3 cells. The induction was rapid and efficient and persisted for many hours. In addition, anti-ras antibody dramatically reduced c-fos accumulation after serum stimulation of injected cells. However, cells which expressed p21ras continuously did not maintain a high level of c-fos expression.
Assuntos
Transformação Celular Neoplásica , Proteínas Oncogênicas Virais/genética , Oncogenes , Proteínas Proto-Oncogênicas/genética , Animais , Células Cultivadas , Cinética , Camundongos , Proteína Oncogênica p21(ras) , Proteínas Oncogênicas Virais/administração & dosagem , Proteínas Proto-Oncogênicas/administração & dosagem , Proteínas Proto-Oncogênicas c-fos , Proteínas Proto-Oncogênicas p21(ras)RESUMO
Microinjection of monoclonal antibody Y13-259, which reacts with all known mammalian and yeast ras-encoded proteins, has previously been shown to prevent NIH 3T3 cells from entering the S phase (L. S. Mulcahy, M. R. Smith, and D. W. Stacey, Nature [London] 313:241-243, 1985). We have now found several transformation-competent mutant v-rasH genes whose protein products in transformed NIH 3T3 cells are not immunoprecipitated by this monoclonal antibody. These mutant proteins are, however, precipitated by a different anti-ras antibody. Each of these mutants lacks Met-72 of v-rasH. In contrast to the result for cells transformed by wild-type v-rasH, Y13-259 microinjection of NIH 3T3 cells transformed by these mutant ras genes did not prevent the cells from entering the S phase. These results imply that a transformation-competent ras gene can supply a normal essential function for NIH 3T3 cells. When the proteins encoded by the mutant ras genes were overproduced in Escherichia coli, several mutant proteins that lacked Met-72 failed to bind Y13-259 in a Western blot. However, a ras protein from a mutant lacking amino antibody, but a ras protein from a mutant lacking amino acids 72 to 84 did not. These results suggest that Y13-259 may bind to a higher ordered structure that has been restored in the mutant lacking amino acids 72 to 82.