RESUMO
Distant metastasis represents the major lethal cause of breast cancer. To understand the molecular mechanisms of breast cancer metastasis and identify markers with metastatic potential, we established a highly metastatic variant of parental MDA-MB-231 cells (MDA-MB-231HM). Using two-dimensional electrophoresis (2-DE), we performed a proteomic comparison of the two kinds of cells. As much as 51 protein spots were differentially expressed between the selected variant and its parental counterpart in at least 3 experiments. Ten unique proteins were identified using matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF) mass spectrometry (MS), liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS), and database searching software. Among them, nine proteins were up-regulated in MDA-MB-231HM cells, including Macrophage-capping protein (CapG), Galectin-1, Chloride intracellular channel protein 1, Endoplasmic reticulum protein ERp29 precursor, Stathmin-1 (STMN1), Isoform 1 of uridine-cytidine kinase 2(UCK2), Rho GDP-dissociation inhibitor 2 (ARHGDIB), isocitrate dehydrogenase [NADP] cytoplasmic (IDH1), and N-myc downstream regulated gene 1 (NDRG1) protein. Only transgelin-2 was down-regulated. Differential expression was confirmed for three proteins including CapG, STMN1, and transgelin-2 by Western blotting analysis. Transgelin-2 was chosen for further verification by immunohistochemistry. The results suggested that 2-DE would be an efficient way to screen the proteins responsible for specific biological function. Furthermore, the findings imply that different proteins may be involved in the metastatic process in breast carcinomas.
Assuntos
Neoplasias da Mama/química , Eletroforese em Gel Bidimensional/métodos , Proteínas de Neoplasias/análise , Proteômica/métodos , Animais , Western Blotting , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Feminino , Humanos , Imuno-Histoquímica , Camundongos , Metástase Neoplásica , Espectrometria de Massas por Ionização por Electrospray , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por MatrizRESUMO
BACKGROUND: PreS/S gene was derived from hepatitis B virus (HBV) integration fragment of human hepatocellular carcinoma genome, containing the promoter of preS/S and the C terminal truncated preS/S open reading frame. PreS/S protein may have important roles in processing hepatoma in some HBV-infected patients. The aim of the study was to study the activity of HBV preS/S protein on proliferating cell nuclear antigen (PCNA) promoter and to localize compartment of the preS/S protein in the liver cell line L02. METHODS: The authors studied the effect of the 3 -truncated preS/S on human PCNA promoter by co-transfecting the expression plasmids of luciferase reporter gene, used the immunohistochemical method to localize the preS/S protein. RESULTS: The expression product of the plasmid, pKSH7C-HpaI which contained the 3 -truncated preS/S and the flanking cellular sequences, stimulated the expression of PCNA promoter dose dependently,and its effect was 0.5 folds higher than control. Immunohistochemistry showed that the preS/S protein located in the cytosolic region of the liver cell line L02. CONCLUSIONS: The HBV preS/S protein could stimulate the PCNA promoter of the liver cell, its effect was not direct, which suggests that the effect of preS/S protein on PCNA promoter was probably through the cell signal transduction pathway.
Assuntos
Antígenos de Superfície da Hepatite B/genética , Vírus da Hepatite B/genética , Antígeno Nuclear de Célula em Proliferação/genética , Precursores de Proteínas/genética , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/virologia , Humanos , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/virologia , Regiões Promotoras Genéticas , Células Tumorais Cultivadas , Integração ViralRESUMO
Proliferating cell nuclear antigen (PCNA) is an auxiliary factor of DNA polymerase delta and epsilon and functions in DNA replication and repair. Using PCR methods, 17 sites within the human PCNA promoter from 60 to 538 were subjected to a 8 bp substitution mutagenesis. Wild type promoter and each mutated promoters were inserted into luciferase expression vector pGL2-Basic. These nonmutated and mutated PCNA promoters were assayed by transient transfection of the plasmids into HeLa, HepG2, L-02 and MCF-7 cell line, respectively. It was found that several sites were common cis acting elements and several sites were cell-specific cis acting elements. Data were further presented using in vitro transcription assay with HeLa and HepG2 nuclear extract.