Metabolic labeling of human cells with tritiated nucleosides results in activation of the ATM-dependent p53 signaling pathway and acceleration of DNA repair.
We investigated the effects of metabolic labeling with [(3)H]thymidine, [(3)H]uridine, and [(14)C]thymidine on human cells in terms of cell growth, p53 signaling, and nucleotide excision repair. Labeling with [(3)H] nucleosides resulted in growth inhibition by both p53-dependent and -independent mechanisms. Tritium labeling also led to nuclear accumulation of p53 and induction of the p53-regulated gene p21(WAF1) and its encoded protein (p21). ATM-deficient cells, however, did not increase their p53 and p21 protein levels in response to radiolabeling. Thus, labeling of human cells with tritiated nucleosides activates the radiation-responsive, ATM-dependent, DNA-damage surveillance network. Labeling of normal cells with [(3)H]thymidine significantly accelerated the repair of ultraviolet (UV) light-induced cyclobutane pyrimidine dimers, as monitored by a sensitive immunofluorescence assay. Unlike [(3)H] labeling, [(14)C] labeling did not produce any impact on proliferation, p53 signaling, or DNA repair. In the light of these findings, the validity of results obtained with nucleic acid synthesis and DNA repair assays that involve [(3)H] and [(14)C] labeling is discussed. Our immunofluorescence approach detected pyrimidine dimers after exposure to UV fluences as low as 1 J/m(2) (the lowest fluence examined). This approach may prove particularly useful for monitoring DNA damage and its repair following exposure to extremely low levels of genotoxic agents.
Mirzayans
Razmik
R
Department of Oncology, University of Alberta, Cross Cancer Institute, Edmonton, Alberta, Canada T6G 1Z2. davem@cancerboard.ab.ca
Pollock
Scott
S
Scott
April
A
Gao
Cindy Q
CQ
Murray
David
D
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins; D002455:Cell Division; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins / Q000032:analysis; D004249:DNA Damage; D004260:DNA Repair; D004268:DNA-Binding Proteins; D005455:Fluorescent Antibody Technique; D006801:Humans; D009705:Nucleosides / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000502:physiology*; D012333:RNA, Messenger / Q000032:analysis; D015398:Signal Transduction / Q000502:physiology*; D014316:Tritium; D016159:Tumor Suppressor Protein p53 / Q000032:analysis / Q000502:physiology*; D025521:Tumor Suppressor Proteins
C493956:CDKN1A protein, human; D018797:Cell Cycle Proteins; D050759:Cyclin-Dependent Kinase Inhibitor p21; D016213:Cyclins; D004268:DNA-Binding Proteins; D009705:Nucleosides; D012333:RNA, Messenger; D016159:Tumor Suppressor Protein p53; D025521:Tumor Suppressor Proteins; D014316:Tritium; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; D017346:Protein Serine-Threonine Kinases