Cadmium-induced DNA damage triggers G2/M arrest via chk1/2 and cdc2 in p53-deficient kidney proximal tubule cells

Abstract

Carcinogenesis is a multistep process that is frequently associated with p53 inactivation. The class 1 carcinogen cadmium (Cd2+) causes renal cancer and is known to inactivate p53. G2/mitosis (M) arrest contributes to stabilization of p53-deficient mutated cells, but its role and regulation in Cd2+-exposed p53-deficient renal cells are unknown. In p53-inactivated kidney proximal tubule (PT) cells, comet assay experiments showed that Cd2+(50–100 μM) 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 G2/M cell cycle arrest at 6 h, which were abolished by the antioxidant α-tocopherol (100 μM). Cd2+-induced G2/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 G2/M arrest mirrors DNA damage and toxicity. The Chk1/2 kinase inhibitor UCN-01 (0.3 μM), which relieves G2/M transition block, abolished Cd2+-induced G2arrest and increased apoptosis. This was accompanied by prevention of Cd2+-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 Cd2+-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 G2/M arrest. This may promote survival of premalignant PT cells and Cd2+carcinogenesis.