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Inhibition of Platelet-derived Growth Factor-induced Cell Growth Signaling by a Short Interfering RNA for EWS-Fli1 via Down-regulation of Phospholipase D2 in Ewing Sarcoma Cells

Satoshi Nozawa, Takatoshi Ohno, Yoshiko Banno, Taikoh Dohjima, Kazuhiko Wakahara, De-Gang Fan, Katsuji Shimizu
2005 Journal of Biological Chemistry  
EWS-Fli1, a fusion gene resulting from a chromosomal translocation t(11;22, q24;q12) and found in Ewing sarcoma and primitive neuroectodermal tumors, encodes a transcriptional activator and promotes cellular transformation. However, the precise biological functions of its products remain unknown. To investigate the role of EWS-Fli1 in cell growth signaling, we transfected Ewing sarcoma TC-135 cells with short interfering RNAs for EWS-Fli1. EWS-Fli1 knockdown reduced cell growth and
more » ... th and platelet-derived growth factor (PDGF)-BB-induced activation of the growth signaling enzymes. Interestingly, phospholipase D2 (but not the PDGF-BB receptor) showed marked down-regulation in the EWS-Fli1knocked down TC-135 cells compared with the control cells. In Ewing sarcoma TC-135 cells, the PDGF-BB-induced phosphorylation of growth signaling involving extracellular signal-regulated kinase, Akt, p70S6K, and the expression of cyclin D3 were markedly inhibited by transfection with short interfering RNA phospholipase (PL)-D2. The PDGF-BB-induced activation of growth signaling was also suppressed by 1-butanol, which prevents the production of phosphatidic acid by phospholipase D (but not by t-butyl alcohol), thereby implicating PLD2 in PDGF-BB-mediated signaling in TC-135 cells. These results suggest that EWS-Fli1 may play a role in the regulation of tumor proliferation-signaling enzymes via PLD2 expression in Ewing sarcoma cells. EXPERIMENTAL PROCEDURES Cell Culture-The Ewing sarcoma cell line TC-135 was kindly supplied by Dr. T. J. Triche (University of Southern California, Los Angeles, CA). The cells were maintained in RPMI 1640 medium (Invitrogen) containing 10% fetal bovine serum, 100 units/ml penicillin G, 100 g/ml streptomycin, and 0.25 g/ml amphotericin B at 37°C. Cell Growth Assay-Cell growth was determined by a WST-8 assay kit (Kishida Kagaku, Osaka, Japan). Briefly, cells (5 ϫ 10 3 cells/well) in 96-well plates were incubated overnight. Thereafter, the medium was replaced with new medium containing U0120, LY294002, rapamycin, or transfection reagent. After 48 -96 h of incubation, the WST-8 reagents were added to the culture. After 1 h of incubation, the absorbance at 450
doi:10.1074/jbc.m411626200 pmid:15919668 fatcat:bjagdv2u2jb67im4yianmfgf6y