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

Bork U, Lee WK, Kuchler A, Dittmar T, Thevenod F. Cadmium-induced DNA damage triggers G(2)/M arrest via chk1/2 and cdc2 in p53-deficient kidney proximal tubule cells. Am J Physiol Renal Physiol 298: F255-F265, 2010. First published November 18, 2009; doi: 10.1152/ajprenal.00273.2009.-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. G(2)/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 mu M) 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 mu M). Cd2+-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 mu M), which relieves G(2)/M transition block, abolished Cd2+-induced G(2) arrest 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 G(2)/M arrest. This may promote survival of premalignant PT cells and Cd2+ carcinogenesis.