Analysis of the expression of cell cycle regulators in Ewing cell lines:: EWS-FLI-1 modulates p57KIP2 and c-Myc expression

Ewing tumour is characterized by specific chromosome translocations which fuse EU'S to a subset of genes encoding ETS transcription factors, most frequently FLI-1. We report the analysis of the expression of various cell cycle regulators both in Ewing tumour derived cell lines and in different cellular models with either inducible or constitutive EWS-FLI-1 cDNA expression. In Ewing cell lines, cyclin D1, CDK4, Rb, p27(KIP1) and c-Myc were consistently highly expressed whereas p57(KIP2), p15(INK4B) and p14(ARF) demonstrated undetectable or low expression levels. The amount of p16(INK4A), p21(CIP1), p18(INKAC) and CDK6 was variable from one cell line to the other, The inducible expression of EWS-FLI-1 led to a strong upregulation of c-MSc and a considerable downregulation of p57(KIP2). Other proteins did not show evident modification. High c-Myc and very low p57(KIP2) expression levels were also observed in neuroblastoma NGP cells constitutively expressing EWS-FLI-1 as compared to parental cells. Analysis of the p57(KIP2) promoter indicated that EWS-FLI-1 downregulates, possibly through an indirect mechanism, the transcription of this gene, Finally, we show that ectopic expression of p57(KIP2) in Ewing cells blocks proliferation through a complete G1 arrest. These results suggest that the modulation of p57(KIP2) expression by EWS-FLI-1 is a fundamental step in Ewing tumorigenesis.