Analysis of the expression of cell cycle regulators in Ewing cell lines: EWS-FLI-1 modulates p57(KIP2) and c-Myc expression

Ewing tumour is characterized by specific chromosome translocations which f use EU'S to a subset of genes encoding ETS transcription factors, most freq uently FLI-1. We report the analysis of the expression of various cell cycl e regulators both in Ewing tumour derived cell lines and in different cellu lar 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 consist ently highly expressed whereas p57(KIP2), p15(INK4B) and p14(ARF) demonstra ted undetectable or low expression levels. The amount of p16(INK4A), p21(CI P1), 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 ev ident modification. High c-Myc and very low p57(KIP2) expression levels wer e also observed in neuroblastoma NGP cells constitutively expressing EWS-FL I-1 as compared to parental cells. Analysis of the p57(KIP2) promoter indic ated 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.