The Ewing tumor (ET) family of childhood malignancies express translocation-associated EWS-ETS chimeric oncoproteins that function as aberrant transcription factors. These molecules are thought to facilitate passage through the cell cycle, contributing to increased cell proliferation. We have been comparing signaling pathways activated in ET cell lines grown in conventional monolayers with those in anchorage-independent spheroid cultures. As expected, we found that monolayer ET cells show serum-dependent activation of ERK 1/2 and p38 MAP kinases. Serum also activates PI3 kinase-dependent PKB/AKT, involved in cell survival, and induces high levels of cyclin D1/D2 protein expression. However, when the same cells are grown in spheroid cultures, there is dramatic serum-independent activation of ERK 1/2, p38, and PKB/AKT, but paradoxically cyclin D1 and D2 expression is virtually undetectable. Fluorescence-activated cell sorting analysis indicates that at least 30% of spheroid cells are cycling. This pattern of serum-independent MAP kinase activation and cyclin D1/D2 suppression can be recapitulated in monolayer cells by treatment with PI3 kinase inhibitors. To further understand changes in immediate and early gene expression that may underlie these findings, we subjected monolayer and spheroid ET cells (with and without PI3 kinase inhibitors) to gene expression profiling using Affymetrix U95A gene chips (containing over 12,000 annotated human gene sequences). The results of these experiments are currently being analyzed to identify distinct expression profiles characteristic of monolayer versus spheroid ET cells. Morphological and ultrastructural analysis of the latter indicate many similarities with primary ETs, suggesting that spheroid cultures may be a better model to study the biology of these lesions.