RESUMO
S-acylation/deacylation cycles and vesicular transport are critical for an adequate subcellular distribution of S-acylated Ras proteins. H-Ras is dually acylated on cysteines 181 and 184, but it is unknown how these residues individually contribute to H-Ras trafficking. In this study, we characterized the acylation and deacylation rates and membrane trafficking of monoacylated H-Ras mutants to analyze their contributions to H-Ras plasma membrane and endomembrane distribution. We demonstrated that dually acylated H-Ras interacts with acyl-protein thioesterases (APTs) 1 and 2 at the plasma membrane. Moreover, single-acylation mutants of H-Ras differed not only in their subcellular distribution, where both proteins localized to different extents at both the Golgi complex and plasma membrane, but also in their deacylation rates, which we showed to be due to different sensitivities to APT1 and APT2. Fluorescence photobleaching and photoactivation experiments also revealed that 1) although S-acylated, single-acylation mutants are incorporated with different efficiencies into Golgi complex to plasma membrane vesicular carriers, and 2) the different deacylation rates of single-acylated H-Ras influence differentially its overall exchange between different compartments by nonvesicular transport. Taken together, our results show that individual S-acylation sites provide singular information about H-Ras subcellular distribution that is required for GTPase signaling.
Assuntos
Membrana Celular/metabolismo , Genes ras/fisiologia , Transporte Proteico/fisiologia , Acilação , Animais , Células CHO , Linhagem Celular , Membrana Celular/fisiologia , Cricetulus , Cisteína/metabolismo , Complexo de Golgi/metabolismo , Mutação , Proteínas/metabolismo , Transdução de Sinais , Tioléster Hidrolases/metabolismoRESUMO
Class I PI3K is composed of heterodimeric lipid kinases regulating essential cellular functions including proliferation, apoptosis and metabolism. Class I PI3K isoforms are commonly amplified in different cancer types and the PI3Kalpha catalytic subunit, PIK3CA, has been found mutated in a variable proportion of tumours of different origin. Furthermore, PI3K has been shown to mediate oncogenic signalling induced by several oncogenes such as HER2 or Ras. These facts suggest that PI3K might be a good target for anticancer drug discovery. Today, the rise of PI3K inhibitors and their first in vivo results have cleared much of the path for the development of PI3K inhibitors for anticancer therapy. Here we will review the PI3K pathway and the pharmacological results of PI3K inhibition.
Assuntos
Inibidores Enzimáticos/uso terapêutico , Neoplasias/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Animais , Antineoplásicos/uso terapêutico , Sistemas de Liberação de Medicamentos , Genes ras/fisiologia , Humanos , Redes e Vias Metabólicas/efeitos dos fármacos , Redes e Vias Metabólicas/fisiologia , Modelos Biológicos , Neoplasias/metabolismoRESUMO
Aminolevulinic acid (ALA)-based photodynamic therapy (PDT) has been successfully employed in the treatment of certain tumours. Porphyrins endogenously generated from ALA induce tumour regression after illumination with light of an appropriate wavelength. The aim of this work was to compare porphyrin production from ALA and sensitivity to photodynamic treatment in a tumour/normal cell line pair. We employed the HB4a cell line from normal mammary luminal epithelium and its counterpart transfected with the oncogen H-Ras (VAL/12 Ras). After 3 h of exposure to ALA, HB4a-Ras cells produce a maximum of 150 ng porphyrins per 10(5) cells whereas HB4a produce 95 ng porphyrins per 10(5) cells. In addition, HB4a-Ras cells show a plateau of porphyrin synthesis at 1 mM whereas HB4a porphyrins peak at the same concentration, and then decrease quickly. This higher porphyrin synthesis in the tumorigenic cell line does not lead to a higher response to the photodynamic treatment upon illumination. Lethal doses 50, LD(50), determined by MTT assay were 0.015 J cm(-2) and 0.039 J cm(-2) for HB4a and HB4a-Ras respectively after 3 h exposure to 1 mM ALA. The conclusion of this work is that a tumour cell line obtained by transfection of the Ras oncogene, although producing higher porphyrin synthesis from ALA, is more resistant to ALA-PDT than the parental non-tumour line, however the mechanism is not related to photosensitiser accumulation, but very likely to cell survival responses.
Assuntos
Ácido Aminolevulínico/farmacologia , Antineoplásicos/farmacologia , Genes ras/fisiologia , Fotoquimioterapia , Mama/citologia , Linhagem Celular , Relação Dose-Resposta a Droga , Feminino , Genes ras/genética , Humanos , Porfirinas/metabolismo , Fatores de TempoRESUMO
The RECK gene is widely expressed in normal human tissues but is downregulated in tumor cell lines and oncogenically transformed fibroblasts. RECK encodes a membrane-anchored glycoprotein that suppresses tumor invasion and angiogenesis by regulating matrix-metalloproteinases (MMP-2, MMP-9 and MT1-MMP). Understanding of the transcriptional regulation of tumor/metastasis suppressor genes constitutes a potent approach to the molecular basis of malignant transformation. In order to uncover the mechanisms of control of RECK gene expression, the RECK promoter has been cloned and characterized. One of the elements responsible for the Ras-mediated downregulation of mouse RECK gene is the Sp1 site, to which Sp1 and Sp3 factors bind. Other regulatory events, such as DNA methylation of the RECK promoter and histone acetylation/deacetylation have been studied to understand the underlying mechanisms of RECK expression. Understanding of the mechanisms which control RECK gene transcription may lead to the development of new strategies for cancer prevention and treatment.
Assuntos
Glicoproteínas de Membrana/genética , Metástase Neoplásica/genética , Neovascularização Patológica/genética , Transcrição Gênica , Acetilação , Animais , Metilação de DNA , Regulação para Baixo , Proteínas Ligadas por GPI , Regulação da Expressão Gênica , Genes Supressores de Tumor , Genes ras/fisiologia , Histona Desacetilases/metabolismo , Humanos , Metaloproteinases da Matriz , Glicoproteínas de Membrana/metabolismo , Invasividade Neoplásica/genética , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/fisiologiaRESUMO
O câncer colorretal ainda é uma das neoplasias de maior importância no mundo ocidental. O grande desenvolvimento da genética e biologia molecular nos últimos anos permitiu um melhor conhecimento dos mecanismos biomoleculares no câncer, e em especial, no câncer colorretal. Oncogenes (K-ras), genes supressores de tumor (p53, DCC e APC) e genes reparadores de DNA (hMSH2, MLH1, PMS1 e 2) estäo envolvidos na progressäo da seqüência adenoma-carcinoma no cólon e no reto. Algumas características anatômicas, histopatológicas, epidemiológicas e o comportamento biológico dos tumores parecem estar relacionados com alteraçöes genéticas específicas nestes genes. O conhecimento dos mecanismos genéticos que promovem a carcinogênese dos tumores colorretais abre novas perspectivas para o diagnóstico, tratamento, prognóstico e seguimento dos pacientes acometidos por esta neoplasia
Assuntos
Adenocarcinoma/genética , Neoplasias Colorretais/genética , Biologia Molecular , Adenocarcinoma/metabolismo , Adenocarcinoma/ultraestrutura , Células/ultraestrutura , Neoplasias Colorretais/metabolismo , Neoplasias Colorretais/ultraestrutura , Metilação de DNA , Genes APC/fisiologia , Genes Neoplásicos/genética , Genes p53/genética , Genes ras/fisiologia , Polipose Adenomatosa do Colo/diagnósticoRESUMO
All-trans-retinoic acid (RA) is a master regulator of cell differentiation and in this process it greatly influences cell adhesion and the elaboration of the extracellular matrix. Therefore, we were interested in the effect of RA on the biosynthesis of fibronectin (FN). RA reduced the level of intracellular FN in a time- and concentration-dependent fashion in NIH-3T3 cells, but not in NIH-3T3 cells transformed by an activated Ha-ras oncogene. Since the steady-state level of FN transcripts did not change after treatment of the cells with RA for various times or concentrations, RA probably acts at the translational level. In NIH-3T3 cells, RA had distinct effects on different receptors, from decreasing retinoic acid receptor (RAR)alpha to increasing RAR beta expression to no effect on RAR gamma. Transformation of NIH-3T3 cells with an activated Ha-ras oncogene downmodulated RAR expression and also abolished responsiveness to RA. A variety of approaches permitted the following conclusions: 1) RA-dependent FN downmodulation is mediated by RARs, 2) retinoid X receptors (RXRs) mediate the observed reduction of RAR alpha by RA, and 3) the blockade of RA responsiveness by Ha-ras-transfected cells cannot be overcome by overexpression of RAR alpha. These studies have identified fibronectin and RAR alpha as RA targets in fibroblast cells and have shown that oncogenic transformation renders the cells resistant to RA action.
Assuntos
Fibronectinas/fisiologia , Genes ras/fisiologia , Proteínas Proto-Oncogênicas p21(ras)/fisiologia , Tretinoína/fisiologia , Regulação da Expressão Gênica , Humanos , Membranas IntracelularesAssuntos
Humanos , Carcinoma Pulmonar de Células não Pequenas/classificação , Carcinoma Pulmonar de Células não Pequenas/complicações , Carcinoma Pulmonar de Células não Pequenas/diagnóstico , Carcinoma Pulmonar de Células não Pequenas/tratamento farmacológico , Carcinoma Pulmonar de Células não Pequenas/epidemiologia , Carcinoma Pulmonar de Células não Pequenas/etiologia , Carcinoma Pulmonar de Células não Pequenas/fisiopatologia , Carcinoma Pulmonar de Células não Pequenas/terapia , Genes ras/imunologia , Genes ras/fisiologiaRESUMO
EJ-A is a Balb-3T3 transfectant cell line that bears a small number of EJ-ras oncogene copies/cell, has low EJ-ras expression, and resembles the parental cell line in displaying a non-transformed phenotype. The glucocorticoid hormone dexamethasone reversibly induces transformation traits in EJ-A cells, namely: 1) morphological transformation; 2) increased growth rate and saturation density; 3) reduced G1 length; and 4) independence of the FGF requirement to initiate DNA synthesis. Western blot analysis revealed that dexamethasone does not increase the p21ras protein intracellular level. beta-IFN, added to the culture medium, does not suppress the dexamethasone-induced growth stimulation and morphological transformation. Therefore, glucocorticoid hormones can complement low EJ-ras expression to transform Balb-3T3 cells, by a mechanism that is likely to be independent of p21ras increase and beta-IFN decrease.