USE OF I125-LABELED 5-IODO-2′-DEOXYURIDINE FOR THE MEASUREMENT OF DNA SYNTHESIS IN MAMMALIAN CELLS IN VITRO

Abstract

The use of isotopically labeled 5-iodo-2′-deoxyuridine (I¹²⁵UdR) for determination of the rate of deoxyribonucleic acid synthesis in mammalian cells in vitro has been investigated. The results obtained indicate that for this purpose I¹²⁵UdR is a suitable substitute for the more commonly used DNA precursor, tritium-labeled thymidine (H³TdR). I¹²⁵UdR appears to be incorporated specifically into the DNA of cells in culture, and has been demonstrated to compete with H³TdR, although the K_mfor H³TdR was smaller than that of I¹²⁵UdR by a factor of approximately 4. The amount of label incorporated into DNA of cells increased linearly with time. When the rate of DNA synthesis was reduced by exposure of the cells to various doses of X-rays, the ratio of I¹²⁵UdR incorporation to H³TdR incorporation into DNA of cells was found to be a constant, which supports the view that uptake of the analogue provides as reliable an indication of effects upon the rate of DNA synthesis as does that of H³TdR. The chief advantage of I¹²⁵UdR over H³TdR is that I¹²⁵is a gamma emitter, so that the difficulties encountered in detection of the low energy beta particles from H³may be avoided.