RESUMEN
Sgf29, a component of the SPT-ADA-GCN5 acetyltransferase (SAGA) complex, binds H3K4me2/3 marks and leads to histone H3 acetylation. Previously, we found that downregulation of Sgf29 suppresses c-Myc-mediated malignant transformation. Nonetheless, the upstream regulator of the Sgf29 gene is not yet known. Here, we report that Sry (sex-determining region Y), an HMG (high-mobility group) domain containing transcription factor, directly upregulates Sgf29 gene expression. Sry expression was deregulated in two out of the four tested male rodent hepatocellular carcinoma (rHCC) cell lines. Luciferase reporter and chromatin immunoprecipitation assays indicated that Sry could bind HMG-boxes in the proximal promoter region of the Sgf29 gene. Knockdown of Sry robustly lowered anchorage-independent growth, invasiveness and tumorigenicity of rHCC cells, whereas ectopic expression of Sry conferred more malignant properties. Thus, these data show that Sry is involved in male-specific malignant conversion of rHCCs via Sgf29 upregulation.
Asunto(s)
Acetiltransferasas/metabolismo , Carcinogénesis , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/metabolismo , Proteína de la Región Y Determinante del Sexo/metabolismo , Acetiltransferasas/genética , Animales , Línea Celular , Línea Celular Tumoral , Regulación Neoplásica de la Expresión Génica , Técnicas de Silenciamiento del Gen , Dominios HMG-Box , Histona Acetiltransferasas/genética , Histona Acetiltransferasas/metabolismo , Masculino , Ratones , Ratones SCID , Invasividad Neoplásica , Regiones Promotoras Genéticas , Estructura Terciaria de Proteína , Ratas , Proteína de la Región Y Determinante del Sexo/genéticaRESUMEN
Assay of the platelet fibrinogen-binding receptor glycoprotein (GP) IIb/IIIa is widely performed using 125I-labeled fibrinogen (125I-fibrinogen). We successfully devised a receptor binding assay system with high selectivity and sensitivity using a stable chemiluminescent acridinium derivative-I-labeled fibrinogen (acridinium-fibrinogen). Human fibrinogen is saline was labeled with equimolar acridinium dissolved in dimethylformamide, and allowed to react with gel-filtered human platelets in the presence of ADP. Acridinium-fibrinogen binding to GPIIb/IIIa was assayed by measuring chemiluminescence emitted on addition of 0.1 N NaOH containing 0.06% H2O2 in a luminometer. Non-specific binding was measured in the presence of 10 mM EDTA. Acridinium-fibrinogen binding to human platelets was rapid and reversible, specific and saturable, and dependent on ADP concentrations. Scatchard plot analysis revealed one class of binding sites with Kd of 326 nM and Bmax of 7.8 pmol/10(8) platelets. These values were comparable to the data obtained by using 125I-fibrinogen. Unlabeled fibrinogen, RGDS, and HHLGGAKQAGDV (fibrinogen gamma-chain 400-411) displaced acridinium-fibrinogen from its binding site with Ki values of 322 nM, 9.2 microM and 31.3 microM, respectively. Thus, this binding assay system may be useful in measuring the binding between platelet GPIIb/IIIa and fibrinogen without using a radioisotope.