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

Many cells, when cultured in suspension, fail to express cyclin A, a regula tory component of cell cycle kinases cdc2 and cdk2 and as a consequence, do not enter S phase, However, many cell type-specific differences are disclo sed between not only normal and transformed cells, but also between cell li nes whose proliferation is strictly anchorage-dependent, These apparent dis crepancies are seen in established cell lines most probably because of adap tative events that have occurred during cell culture, We have therefore use d primary cells to understand how cyclin A transcription is controlled by c ell anchorage properties, To this aim, we have used embryonic fibroblasts f rom either wild type, Rb(-/-) or p107(-/-)/p130(-/-) mice and tested the ef fect of an ectopic expression of Rb mutants. In the experiments reported he re, we show that anchorage-dependent expression of cyclin A (i) is reflecte d by the irt 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 fu nctional Rb but neither p107 nor p130 (iii) mutation of the CCRE abolishes both adhesion-dependent regulation and response to Rb.