Downregulation of PCNA potentiates p21-mediated growth inhibition in response to hyperoxia

Abstract

Prolonged exposure to hyperoxia inhibits cell proliferation in G1via increased expression of p21. While p21 inhibits proliferating cell nuclear antigen (PCNA)-dependent DNA synthesis, it can also directly lower PCNA abundance; however, it is unclear whether loss of PCNA contributes to growth arrest. Here, we investigate how PCNA loss affects ability of p21 to exert G1growth arrest of lung epithelial cells exposed to hyperoxia. In A549 cells that express p21 and growth arrest in G1during hyperoxia, small interfering RNA (siRNA) knockdown of p21 led to G1checkpoint bypass, increased cell death, and restoration of PCNA expression. Conditional overexpression of the PCNA binding domain of p21 in H1299 cells that do not normally express p21, or exposure to hyperoxia, caused a time-dependent loss of PCNA. Titrating PCNA levels using siRNA to approximate the low amount observed in cells expressing p21 resulted in S phase arrest. While lowering PCNA by itself caused S phase arrest, the combination of hyperoxia and siRNA against PCNA dramatically reduced PCNA abundance resulting in G1arrest. G1growth arrest was markedly enhanced upon the addition of p21 to these cells. Our findings suggest a model in which reducing expression of the abundant protein PCNA allows the less abundant protein p21 to be more effective at suppressing the processivity functions of remaining PCNA, thereby fully exerting the G1checkpoint. Given that high p21 expression is often associated with lower PCNA abundance, our findings are suggestive of a global growth inhibitory mechanism involving p21-mediated PCNA suppression.