The effect of hydroxyurea on DNA synthesis of embryonic rat cells in culture

Abstract

3H-thymidine incorporation by secondary cultures of embryonic rat cells and by cultures of a permanent rat cell line is strongly inhibited a few minutes after addition of hydroxyurea (HU) to the culture medium (2 × 10-3 ᴍ and 5 × 10-4 ᴍ, resp.); normal embryonic rat cells are considerably less sensitive to the drug than are permanent cells. About 76 percent of the population of a secondary culture can be synchronized at the beginning of the S-period, if the cells at first are accumulated in G1 phase by incubation in serum-free medium and subsequently are stimulated by the addition of ten percent calf serum in the presence of 2 × 10-3 ᴍ HU. The size of DNA replicated in the presence of HU has been analysed by centrifugation in alkaline sucrose gradients. The results indicate that under these conditions DNA subunits are synthesized with sedimentation coefficients between 21S and 30S. These intermediates cannot be joined together, probably because a ligase is inhibited by HU. Secondary culture incubated in medium-containing 1 × 10-4 ᴍ HU incorporate twice as much 3H-thymidine as do control cells in HU-free medium. This is due to an increased number of cells in the S phase, as was shown by autoradiography. The kinetics of thymidine incorporation indicate that the duration of G1 phase is shortened by small doses of HU