MISREGULATED EXPRESSION OF THE CYCLIN-DEPENDENT KINASE-2 PROTEIN IN HUMAN FIBROBLASTS IS ACCOMPANIED BY THE INABILITY TO MAINTAIN A G(2) ARREST FOLLOWING DNA-DAMAGE

The misregulation of cell cycle checkpoints has been implicated in the onset of neoplasia. To thoroughly understand the differences in check point regulation between normal and transformed cells, we have; compar ed the cell cycle responses of normal and TAg-transformed fibroblasts to DNA damage by gamma-irradiation. Normal cell lines arrest in both G (1) and G(2) for in excess of 48 h after DNA damage; Surprisingly, bot h cyclindependent kinase 2 (CDK2) and;cyclin A proteins were specifica lly down-regulated within 24 h of DNA damage. In contrast, TAg transfo rmed cells did not down-regulate either cyclin A or CDK2 after DNA dam age and showed a significantly shortened G(2) arrest. To investigate t he role CDK2 down-regulation plays in cell cycle arrests, we generated normal cell lines that constitutively overexpress CDK2. These cells f ail to down-regulate both CDK2 protein and CDK2 protein kinase activit y after DNA damage and also show a G(2) checkpoint defect; although th e cells are able to normally initiate both a G(1) and a G(2) arrest, t hey prematurely escape the G(2)-M arrest after DNA damage. The escape from G(2) correlates with an illicit activation of cyclin B-associated protein kinase activity in these cells. These results suggest that th e misregulation of CDK2 contributes to the failure of checkpoint contr ol; by allowing cells to enter mitosis prematurely.