3H-thymidine is a defective tool with which to measure rates of DNA synthesis

Metabolic incorporation of 3H-thymidine into cellular DNA is a widely used protocol to monitor rates of DNA synthesis and cell proliferation. However, this radiochemical has also been reported to induce cell-cycle arrest and apoptosis in addition to DNA damage. Using stable isotope-labeled thymidine, we demonstrate that 3H-thymidine induces dose-dependent inhibition of the rate of DNA synthesis. This inhibition occurred within the first round of replication after addition of the radiolabeled tracer and demonstrates the cytotoxic effects of conventional doses of 3H-thymidine (typically greater than or equal to 1 microCi/ml). These results thus show that stable isotope methods are superior to radioisotopes for determining rates of DNA synthesis and cell replication. Because 3H-thymidine perturbs the very process it was employed to study, experiments using 3H-thymidine to monitor DNA synthesis and cell proliferation should be interpreted with caution.