Metabolic labeling of human cells with tritiated nucleosides results in activation of the ATM-dependent p53 signaling pathway and acceleration of DNA repair

Abstract

We investigated the effects of metabolic labeling with [³H]thymidine, [³H]uridine, and [¹⁴C]thymidine on human cells in terms of cell growth, p53 signaling, and nucleotide excision repair. Labeling with [³H] 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 [³H]thymidine significantly accelerated the repair of ultraviolet (UV) light-induced cyclobutane pyrimidine dimers, as monitored by a sensitive immunofluorescence assay. Unlike [³H] labeling, [¹⁴C] 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 [³H] and [¹⁴C] labeling is discussed. Our immunofluorescence approach detected pyrimidine dimers after exposure to UV fluences as low as 1 J/m² (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.