Reoxygenation following hypoxia activates DNA-damage checkpoint signaling pathways that suppress cell-cycle progression in cultured human lymphocytes

Abstract

Cellular responses to DNA damage after hypoxia and reoxygenation (H/R) were examined in human lymphocytes. Cultured lymphocytes exposed to H/R showed a lower cytokinesis block proliferation index and a higher frequency of micronuclei in comparison to control cells. Western blots showed that H/R exposure induced p53 expression; however, p21 and Bax expression did not increase, indicating that H/R did not affect p53 transactivational activity. Phosphorylation of p53 (Ser15), Chk1 (Ser345), and Chk2 (Thr68) was also observed, suggesting that H/R activates p53 through checkpoint signals. In addition, H/R exposure caused the phosphorylation and negative regulation of Cdc2 and Cdc25C, proteins that are involved in cell-cycle arrest at the G2/M checkpoint. The S-phase checkpoint, regulated by the ATM-p95/NBS1-SMC1 pathway, was also triggered in H/R-exposed lymphocytes. These results demonstrate that H/R exposure triggers checkpoint signaling and induces cell-cycle arrest in cultured human lymphocytes.