Ewing tumour is characterized by speci®c chromosome translocations which fuse EWS 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 di erent 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-Myc and a considerable downregulation of p57 KIP2 . Other proteins did not show evident modi®cation. 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. Oncogene (2001) 20, 3258 ± 3265.

Neuroblastoma (NB) and the Ewing sarcoma (ES)/peripheral primitive neuroectodermal tumor (PNET) family are pediatric cancers derived from neural crest cells. Although NBs display features of the sympathetic nervous system, ES/PNETs express markers consistent with parasympathetic differentiation. To examine the control of these differentiation markers, we generated NB ؋ ES/PNET somatic cell hybrids. NB-specific markers were suppressed in the hybrids, whereas ES/PNET-specific markers were unaffected. These results suggested that the Ews/Fli-1 fusion gene, resulting from a translocation unique to ES/PNETs, might account for the loss of NB-specific markers. To test this hypothesis, we generated two different NB cell lines that stably expressed the Ews/Fli-1 gene. We observed that heterologous expression of the Ews/Fli-1 protein led to the suppression of NB-specific markers and de novo expression of ES/PNET markers. To determine the extent of changes in differentiation, we used the Affymetrix GeneChip Array system to observe global transcriptional changes of genes. This analysis revealed that the gene expression pattern of the Ews/Fli-1-expressing NB cells resembled that observed in pooled ES/PNET cell lines and differed significantly from the NB parental cells. Therefore, we propose that Ews/Fli-1 contributes to the etiology of ES/PNET by subverting the differentiation program of its neural crest precursor cell to a less differentiated and more proliferative state.