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Fibroblast Growth Factor 2 Reactivates G1 Checkpoint in SK-N-MC Cells via Regulation of p21, Inhibitor of Differentiation Genes (Id1-3), and Epithelium-Mesenchyme Transition-Like Events

Centre for Hormone Research (S.H., S.H.X.W., M.R., C.L.R., G.A.W., V.C.R.), Embryology (D.F.N.), Murdoch Children’s Research Institute, Royal Children’s Hospital, 3052 Parkville, Victoria, Australia; Department of Paediatrics (S.H., S.H.X.W., M.R., C.L.R., D.F.N., G.A.W., V.C.R.), University of Melbourne, 3052 Parkville, Victoria, Australia; and MIND Centre (M.R.), Department of Medical Biochemistry, Aarhus University, 8000 Aarhus C, Denmark

Address all correspondence and requests for reprints to: Vincenzo Russo, Ph.D., Centre for Hormone Research, Murdoch Childrens Research Institute, Royal Children’s Hospital, Flemington Road, Parkville 3052, Victoria, Australia. E-mail: vince.russo{at}mcri.edu.au.

We have recently demonstrated that fibroblast growth factor (FGF)-2 promotes neuroblastoma cell differentiation and overrides their mitogenic response to IGF-I. However, the mechanisms involved are unknown. SK-N-MC cells were cultured with FGF-2 (50 ng/ml) and/or IGF-I (100 ng/ml) up to 48 h. Fluorescence-activated cell sorting analysis indicated that FGF-2 promotes G1/G0 cell cycle phase arrest. Gene expression by RT2-PCR and cellular localization showed up-regulation of p21. We then investigated whether FGF-2-induced differentiation of SK-N-MC cells (by GAP43 and NeuroD-6 expression) involves epithelium-mesenchyme transition interconversion. Real-time PCR (RT2-PCR) showed modulation of genes involved in maintenance of the epithelial phenotype and cell-matrix interactions (E-cadherin, Snail-1, MMPs). Zymography confirmed FGF-2 up-regulated MMP2 and induced MMP9, known to contribute to neuronal differentiation and neurite extension. Id1-3 expression was determined by RT2-PCR. FGF-2 induced Id2, while down-regulating Id1 and Id3. FGF-2 induced nuclear accumulation of ID2 protein, while ID1 and ID3 remained cytoplasmic. RNA interference demonstrated that Id3 regulates differentiation and cell cycle (increased Neuro-D6 and p21 mRNA), while d Id2 modulates epithelium-mesenchyme transition-like events (increased E-cadherin mRNA). In conclusion, we have shown for the first time that FGF-2 induces differentiation of neuroblastoma cells via activation of a complex gene expression program enabling modulation of cell cycle, transcription factors, and suppression of the cancer phenotype. The use of RNA interference indicated that Id-3 is a key regulator of these events, thus pointing to a novel therapeutic target for this devastating childhood cancer.