Abstract
Carcinogenesis is a multistep process that is frequently associated with p53 inactivation. The class 1 carcinogen cadmium (Cd(2+)) causes renal cancer and is known to inactivate p53. G(2)/mitosis (M) arrest contributes to stabilization of p53-deficient mutated cells, but its role and regulation in Cd(2+)-exposed p53-deficient renal cells are unknown. In p53-inactivated kidney proximal tubule (PT) cells, comet assay experiments showed that Cd(2+) (50-100 microM) induced DNA damage within 1-6 h. This was associated with peak formation of reactive oxygen species (ROS) at 1-3 h, measured with dihydrorhodamine 123, and G(2)/M cell cycle arrest at 6 h, which were abolished by the antioxidant alpha-tocopherol (100 microM). Cd(2+)-induced G(2)/M arrest was enhanced approximately twofold on release from cell synchronization (double thymidine block or nocodazole) and resulted in approximately twofold increase of apoptosis, indicating that G(2)/M arrest mirrors DNA damage and toxicity. The Chk1/2 kinase inhibitor UCN-01 (0.3 microM), which relieves G(2)/M transition block, abolished Cd(2+)-induced G(2) arrest and increased apoptosis. This was accompanied by prevention of Cd(2+)-induced cyclin-dependent kinase cdc2 phosphorylation at tyrosine 15, as shown by immunofluorescence microscopy and immunoblotting. The data indicate that in p53-inactivated PT cells Cd(2+)-induced ROS formation and DNA damage trigger signaling of checkpoint activating kinases ataxia telangiectasia-mutated kinase (ATM) and ataxia telangiectasia and Rad3-related kinase (ATR) to cause G(2)/M arrest. This may promote survival of premalignant PT cells and Cd(2+) carcinogenesis.