Anchorage-dependent expression of cyclin A in primary cells requires a negative DNA regulatory element and a functional Rb

Abstract

Many cells, when cultured in suspension, fail to express cyclin A, a regulatory component of cell cycle kinases cdc2 and cdk2 and as a consequence, do not enter S phase. However, many cell type-specific differences are disclosed between not only normal and transformed cells, but also between cell lines whose proliferation is strictly anchorage-dependent. These apparent discrepancies are seen in established cell lines most probably because of adaptative events that have occurred during cell culture. We have therefore used primary cells to understand how cyclin A transcription is controlled by cell anchorage properties. To this aim, we have used embryonic fibroblasts from either wild type, Rb(−/−) or p107(−/−)/p130(−/−) mice and tested the effect of an ectopic expression of Rb mutants. In the experiments reported here, we show that anchorage-dependent expression of cyclin A (i) is reflected by the in vivo occupancy of a negative DNA regulatory element previously shown to be instrumental in the down regulation of cyclin A transcription in quiescent cells (Cell Cycle Responsive Element: CCRE) (ii) requires a functional Rb but neither p107 nor p130 (iii) mutation of the CCRE abolishes both adhesion-dependent regulation and response to Rb.