title: [Influence of phosphoprotein associated with glycosphingolipid microdomains 1 on biologic behavior of human prostatic cancer cell line in-vitro].
abstract: OBJECTIVE
  To investigate the mechanism of influence of phosphoprotein associated with glycosphingolipid microdomains 1 (PAG1) on the biological behavior of human prostatic cancer cell line PC-3M-1E8.
  
  
  METHODS
  The expression of GST-Raf1-RBD recombinant protein, a specific binding domain of active Ras (GTP-Ras), was induced by IPTG in JM109 bacterium. SDS-PAGE and coomassie brilliant blue staining were performed using cleaved product of the bacterium to determine the expression of the fusion protein. GST-pull down essay was designed to detect the level of active Ras in PGA1 and vector transfected, respectively and in the native PC-3M-1E8 cells. Western blotting was used to detect the expression levels of downstream proteins of Ras signal pathway which may be related to the function of PAG1. Phalloidine labeled by tetramethylrhodamine-5-(and-6) isothiocyanate (TRITC) was used for the staining of intracellular F-actin, Laser passing confocal microscopy was adopted for observing change of the cell morphology and the arrangement of F-actin.
  
  
  RESULTS
  After IPTG induction, the GST-Raf1-RBD recombinant protein, with a molecular weight of 33 000, was noticed to be highly expressed in JM109 bacterium. GST-pull down assay revealed that the expression level of active Ras markedly decreased after PAG1-transfection while the total Ras remained unchanged. The expression of p-ERK and cyclin D1 in the PAG1-transfected cells decreased in accordance with the level of active Ras. However, the expression of p21(WAF1/CIP1) and p-Akt didn't show any variation. Additionally, the structure of F-actin was turbulent and the pseudopodia of cells diminished conspicuously after PAG1-transfection. There was a high expression of PAG1 in normal human prostate tissue, however, the positive rate of PAG1 immuno-staining decreased in cases of prostatic adenocarcinoma, correspondent with increasing of the grading index of cell differentiation established in the Gleason grading system for the diagnosis of prostate adenocarcinoma.
  
  
  CONCLUSIONS
  An over-expression of PAG1 in PC-3M-1E8 cells effectively suppresses the activation of Ras and ERK, as well as the cyclin D1 expression, leading to an inhibition of the proliferation ability of tumor cells. The turbulence of F-actin and reduction of pseudopodia of cells result in an impairment of cell mobility, invasiveness and metastatic capability. In human prostate and prostatic adenocarcinoma tissues, the expression of PAG1 is related to the cellular differentiation and malignancy.
authors.affiliation: Department of Pathology, Health Science Center, Peking University, Beijing, China.
authors.forename: Wen-juan
authors.initials: WJ
authors.lastname: Yu
authors.forename: Yue-wei
authors.initials: YW
authors.lastname: Wang
authors.forename: Jiang-feng
authors.initials: JF
authors.lastname: You
authors.forename: Jie-liang
authors.initials: JL
authors.lastname: Wang
authors.forename: Xiang-lin
authors.initials: XL
authors.lastname: Cui
authors.forename: Fei
authors.initials: F
authors.lastname: Pei
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
mesh_terms: D000199:Actins / Q000378:metabolism; D048868:Adaptor Proteins, Signal Transducing / Q000378:metabolism*; D000230:Adenocarcinoma / Q000378:metabolism / Q000473:pathology; D002453:Cell Cycle; D045744:Cell Line, Tumor; D002465:Cell Movement; D049109:Cell Proliferation; D019938:Cyclin D1 / Q000378:metabolism; D050759:Cyclin-Dependent Kinase Inhibitor p21 / Q000378:metabolism; D048049:Extracellular Signal-Regulated MAP Kinases / Q000378:metabolism; D006801:Humans; D008297:Male; D008565:Membrane Proteins / Q000378:metabolism*; D009361:Neoplasm Invasiveness; D011467:Prostate / Q000378:metabolism; D011471:Prostatic Neoplasms / Q000378:metabolism* / Q000473:pathology*; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D014162:Transfection; D018631:ras Proteins / Q000378:metabolism
chemical_list: D000199:Actins; D048868:Adaptor Proteins, Signal Transducing; C493956:CDKN1A protein, human; D050759:Cyclin-Dependent Kinase Inhibitor p21; D008565:Membrane Proteins; C498710:PAG1 protein, human; D019938:Cyclin D1; D051057:Proto-Oncogene Proteins c-akt; D048049:Extracellular Signal-Regulated MAP Kinases; D018631:ras Proteins