Primary keratinocytes have an adhesion dependent S phase checkpoint that is absent in immortalized cell lines

In order to understand the mechanism through which loss of anchorage inhibi ts growth, we have investigated the events that occur in murine keratinocyt es upon substratum detachment utilizing both primary cells and established immortalized cell lines, Our data has revealed that while both primary and immortalized cells undergo growth arrest in suspension, the nature of this arrest is markedly different, Primary cells exhibit a growth arrest that is characterized by rapid cessation of DNA synthesis resulting in a static S phase population. In contrast, an immortalized non-tumorgenic cell line, Ba lb MK, exhibits growth arrest as measured by thymidine incorporation, but d oes not prevent cells that have entered S phase from continuing into G(2)/M , and accumulating as a 4N population. In contrast to both primary and MK c ells, the tumorigenic SLC-1 cell line did not accumulate in a specific cell cycle interval and were able to undergo continuous growth in suspension, E xamination of cyclin A protein and its associated activity revealed that cy clin A protein levels decreased in primary but not MK cells; suggesting the continued presence of cyclin A may allow continued DNA synthesis observed in MK cells. Furthermore, we demonstrate the accumulation of suspension cul tured MK cells as a 4N population correlated with the loss of cyclin A/cdk2 kinase activity, which in turn occurred through the accumulation of p27(ki p1), whereas neither p27(kip1) accumulation nor loss of cyclin A activity w as observed in SLC-1 cells. Our results clearly reveal that the process of growth inhibition in suspension cultured cells may occur in several forms w ith distinct characteristics that are dependent on the status of cyclin/cdk complexes and CKI proteins. Tumor derived cells in suspension did not lose cyclin A dependent kinase activity and thus continued to grow and divide.