Cadmium-induced DNA damage triggers G(2)/M arrest via chk1/2 and cdc2 in p53-deficient kidney proximal tubule cells.
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.
Bork
Ulrich
U
Department of Physiology & Pathophysiology, University of Witten/Herdecke, Witten, Germany.
Lee
Wing-Kee
WK
Kuchler
Anna
A
Dittmar
Thomas
T
Thévenod
Frank
F
D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002104:Cadmium / Q000494:pharmacology*; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000378:metabolism; D016923:Cell Death; D002455:Cell Division / Q000187:drug effects; D002478:Cells, Cultured; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D019926:Cyclin B / Q000378:metabolism*; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000378:metabolism; D016195:G2 Phase / Q000187:drug effects; D007687:Kidney Tubules, Proximal / Q000166:cytology* / Q000187:drug effects / Q000502:physiology; D010766:Phosphorylation / Q000187:drug effects; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000187:drug effects / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D015398:Signal Transduction; D019311:Staurosporine / Q000031:analogs & derivatives / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000172:deficiency*; D025521:Tumor Suppressor Proteins / Q000378:metabolism
D018797:Cell Cycle Proteins; D019926:Cyclin B; D004268:DNA-Binding Proteins; D047428:Protein Kinase Inhibitors; D017382:Reactive Oxygen Species; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D002104:Cadmium; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; C576887:ataxia telangiectasia and Rad3-related kinase, rat; D064447:Checkpoint Kinase 2; D064007:Ataxia Telangiectasia Mutated Proteins; D000071877:Checkpoint Kinase 1; C000605871:Chek1 protein, rat; C578632:Chek2 protein, rat; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine