p21(Cip1) and p27(Kip1) regulate cell cycle reentry after hypoxic stress but are not necessary for hypoxia-induced arrest

We investigated the role of the cyclin-dependent kinase inhibitors p2I(Cip1 ) and p27(Kip1) in cell cycle regulation during hypoxia and reoxygenation. While moderate hypoxia (1 or 0.1% oxygen) does not significantly impair bro modeoxyuridine incorporation, at very low oxygen tensions (0.01% oxygen) DN A replication is rapidly shut down in immortalized mouse embryo fibroblasts , This S-phase arrest is intact in fibroblasts lacking the cyclin kinase in hibitors p21(Cip1) and p27(Kip1), indicating that these molecules are not e ssential elements of the arrest pathway. Hypoxia-induced arrest is accompan ied by dephosphorylation of pRb and inhibition of cyclin-dependent kinase 2 , which results in part from inhibitory phosphorylation, Interestingly, cel ls lacking the retinoblastoma tumor suppressor protein also display arrest under hypoxia, suggesting that pRb is not an essential mediator of this res ponse, Upon reoxygenation, DNA synthesis resumes by 3.5 h and reaches aerob ic levels by 6 h, Cells lacking p21, however, resume DNA synthesis more rap idly upon reoxygenation than wild-type cells, suggesting that this inhibito r may play a role in preventing premature reentry into the cell cycle upon cessation of the hypoxic stress. While p27 null cells did not exhibit rapid reentry into the cell cycle, cells lacking both p21 and p27 entered S phas e even more aggressively than those lacking p21 alone, revealing a possible secondary role for p27 in this response. Cdk2 activity is also restored mo re rapidly in the double-knockout tells when returned to normoxia, These st udies reveal that restoration of DNA synthesis after hypoxic stress, but no t the S phase arrest itself, is regulated by p21 and p27.