title: [Mechanism of the mitogen-activated protein kinase phosphatase-5 regulating the growth and invasion of a human prostate cancer cell line].
abstract: OBJECTIVE
  To investigate the effects of mitogen-activated protein kinase phosphatase-5 (MKP5) on the biological behaviors of cell and the activation of mitogen-activated protein kinase (MAPK) induced by P2Y purinergic receptor agonist ATP in the prostate cancer cell line 1E8.
  
  
  METHODS
  1E8 cells were cultured. The recombinant plasmid pcDL-SRalpha296-MKP5 containing the human wild type MKP5 cDNA and blank vector pcDL-SRalpha were co-transfected with pcDNA3 vector plasmid into the 1E8 cells respectively to get strong expression clones. RT-PCR was used to detect the expression of MKP5 mRNA. ATP, MEK inhibitor PD 98059, and p38 inhibitor SB 203580 were added into the culture. Then the total protein was extracted, SDS-PAGE and Western blotting were used to detect the activation of p38 and ERK1/2 induced by ATP with phosphospecific antibodies directed against the dually phosphorylated, active forms of p38 and ERK1/2. The in vitro growth curve was drawn, soft agar colony formation test was made, and Matrigel membrane-penetrating experiment was made to test the invasion ability of 1E8 cells.
  
  
  RESULTS
  The activity of p38 and that of ERK1/2 of the 1E8 cells untreated by ATP were almost undetected. ATP time-dependently stimulated the activities of ERK1/2 and p38 kinases, inhibited the in vitro growth and colony formation on soft agar, and promoted the in vitro invasion of 1E8 cells. MKP5-transfection effectively inhibited the p38 activity induced by ATP and blocked the effects by ATP stimulation on 1E8 cells. The activity of p38 kinase was significantly decreased both in the control and MKP5 transfected cells that were pretreated with SB203580 and then with ATP with an inhibition rates of 83.14% and 58.00% (P < 0.001 and P = 0.003). The growth of cells transfected with MKP5 was significantly inhibited (P < 0.001) and further inhibited by addition of ATP. The inhibition of growth of MKP5-transfected cells by ATP could be partially reversed by pretreatment of PD98059 (P < 0.05). The number of cells transfected with MKP5 that penetrated the membrane was significantly less than that of the control cells (P = 0.000 9). ATP increased the penetrating ability of different cells (all P < 0.01), and this action was partially inhibited by MKP5 transfection and addition of SB203580 (P < 0.001), but not by the addition of PD98059. ATP treatment decreased the colony formation on soft agar of different cells, especially of the MKP5 transfected cells (P < 0.001). Such action was partially reversed by SB203580 and PD98059.
  
  
  CONCLUSIONS
  The p38 and ERK1/2 pathways exert different effects on the in vitro growth, invasion and colony formation of 1E8 prostate cancer cells related to ATP. The p38 pathway may be more important in the invasive capability of 1E8 cells. MKP5 plays an important role in the regulation of p38 action.
authors.affiliation: Department of Pathology, Peking University Health Science Center, Beijing 100083, China.
authors.forename: Hui-ying
authors.initials: HY
authors.lastname: He
authors.forename: Wei-gang
authors.initials: WG
authors.lastname: Fang
authors.forename: Jie
authors.initials: J
authors.lastname: Zheng
authors.forename: Jiang-feng
authors.initials: JF
authors.lastname: You
authors.forename: Wan-jie
authors.initials: WJ
authors.lastname: Heng
authors.forename: Yan
authors.initials: Y
authors.lastname: Li
mesh_terms: D000255:Adenosine Triphosphate / Q000494:pharmacology; D002455:Cell Division; D045744:Cell Line, Tumor; D054637:Dual-Specificity Phosphatases; D006801:Humans; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D054639:Mitogen-Activated Protein Kinase Phosphatases; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D009361:Neoplasm Invasiveness; D011471:Prostatic Neoplasms / Q000473:pathology*; D017027:Protein Tyrosine Phosphatases / Q000235:genetics / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D014162:Transfection; D048051:p38 Mitogen-Activated Protein Kinases
chemical_list: D047908:Intracellular Signaling Peptides and Proteins; D012333:RNA, Messenger; D000255:Adenosine Triphosphate; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; C120407:DUSP10 protein, human; D054639:Mitogen-Activated Protein Kinase Phosphatases; D054637:Dual-Specificity Phosphatases; D017027:Protein Tyrosine Phosphatases