RESUMO
The role of c-Myc in estrogen regulation of vascular endothelial growth factor (VEGF) and of the vasculature function has been investigated in breast cancer cells and tumors. The studies were performed on MCF7 wild-type cells and MCF7-35im clone, stably transfected with an inducible c-Myc gene. In vitro and ex vivo methods for investigating molecular events were integrated with in vivo magnetic resonance imaging of the vascular function. The results showed that the c-Myc upregulation by estrogen is necessary for the transient induction of VEGF transcription; however, overexpression of c-Myc alone is not sufficient for this induction. Furthermore, both c-Myc and the activated estrogen receptor alpha (ERalpha) were shown to co-bind the VEGF promoter in close proximity, indicating a novel mechanism for estrogen regulation of VEGF. Studies of long-term estrogen treatment and overexpression of c-Myc alone demonstrated regulation of stable VEGF expression levels in vitro and in vivo, maintaining steady vascular permeability in tumors. However, withdrawal of estrogen from the tumors resulted in increased VEGF and elevated vascular permeability, presumably due to hypoxic conditions that were found to dominate VEGF overexpression in cultured cells. This work revealed a cooperative role for ERalpha and c-Myc in estrogen regulation of VEGF and the ability of c-Myc to partially mimic estrogen regulation of angiogenesis. It also illuminated the differences in estrogen regulation of VEGF during transient and long-term sustained treatments and under different microenvironmental conditions, providing a complementary picture of the in vitro and in vivo results.
Assuntos
Neoplasias da Mama/genética , Estradiol/farmacologia , Receptor alfa de Estrogênio/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genes myc/fisiologia , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Genes myc/efeitos dos fármacos , Humanos , Camundongos , Camundongos Nus , Neovascularização Patológica/genética , Regiões Promotoras Genéticas/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-myc/metabolismo , RNA Interferente Pequeno/farmacologia , Transplante Heterólogo , Células Tumorais Cultivadas , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
The discovery of metabolic and molecular markers that help improving the detection and diagnosis of breast cancer is an important goal to be achieved. A high composite-choline signal in magnetic resonance spectra of breast lesions has been demonstrated to improve the accuracy of breast cancer diagnosis. In the present study we revealed the principal molecular and biochemical steps associated with the induction of choline metabolism and phosphocholine accumulation in human breast cancer cell-lines in comparison with normal human mammary epithelial cells. We found upregulation of the expression levels of specific choline transporters: organic cation transporter-2 and choline high affinity transporter-1, as well as of the enzyme choline kinase alpha in the cancerous cells in comparison with that in the normal mammary epithelial cells. The expression levels of choline transporter like-1, organic cation transporter-1 and choline kinase beta were similar in normal and cancerous cells. We further showed that choline transport rates and choline kinase activity indeed increased by several fold in the cancer cells leading to the elevation of phosphocholine. The results strongly suggest that phosphocholine can serve as a biomarker of breast cancer reflecting upregulation of specific choline transporters and choline kinase genes.
Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias da Mama/metabolismo , Colina Quinase/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Transportador 1 de Cátions Orgânicos/metabolismo , Fosforilcolina/metabolismo , Biomarcadores Tumorais/genética , Mama/metabolismo , Colina Quinase/genética , Humanos , Proteínas de Membrana Transportadoras/genética , Transportador 1 de Cátions Orgânicos/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Simportadores , Células Tumorais CultivadasRESUMO
Vitronectin (Vn) stabilizes the inhibitory form of plasminogen activator inhibitor-1 (PAI-1), an important modulator of fibrinolysis. We have previously reported that Vn is specifically phosphorylated by PKA (at Ser378), a kinase we have shown to be released from platelets upon their physiological activation. Here we describe the molecular consequences of this phosphorylation and show (by circular dichroism, and by phosphorylation with casein kinase II) that it acts by modulating the conformation of Vn. The PKA phosphorylation of Vn is enhanced in the presence of either PAI-1, or heparin, or both. This enhanced phosphorylation occurs exclusively on Ser378 as shown with the Vn mutants Ser378Ala and Ser378Glu. The binding of PKA phosphorylated Vn to immobilized PAI-1 and to immobilized plasminogen is shown to be lower than that of Vn. The evidence compiled here suggests that this phosphorylation of Vn can modulate plasminogen activation and consequently control fibrinolysis.
Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Vitronectina/metabolismo , Caseína Quinase II , Fibrinólise , Heparina/farmacologia , Humanos , Fosforilação/efeitos dos fármacos , Plasminogênio/farmacologia , Inibidor 1 de Ativador de Plasminogênio/farmacologia , Ligação Proteica , Conformação Proteica , Proteínas Serina-Treonina Quinases/metabolismo , Serina/metabolismoRESUMO
The plasminogen activator inhibitor-1 (PAI-1) is stabilized in its inhibitory conformation by binding to Vitronectin (Vn). The anchorage of PAI-1 to the fibrin fibers was recently shown to be mediated by Vn, and as such to modulate fibrinolysis. Here we report the mapping of the fibrin binding sites in Vn using truncated recombinant Vns, and show that two segments of Vn are involved: one at its carboxyl terminus (within residues 348-459) and one at its amino terminus (within residues 1-44). This mapping sets the stage for (i) the design of specific inhibitors for the Vn-fibrin interaction; (ii) for studying the role of this interaction in the anchoring of endothelial cells and platelets onto the fibrin clot; and (iii) for getting a deeper insight into the mechanism of the Vn-fibrin interaction in fibrinolysis. (c)2002 Elsevier Science.