Changes in the nuclear distribution of cyclin (PCNA) but not its synthesis depend on DNA replication

Synthesis of cyclin in serum-stimulated quiescent 3T3 cells increases shortly before DNA synthesis after 10 h of stimulation, reaching a maximum after 16 h. Inhibition of DNA synthesis by hydroxyurea does not affect the increase of cyclin following stimulation, as determined by quantitative two-dimensional gel electrophoresis. The levels of cyclin decrease dramatically at the end of the S-phase. Cells kept in the presence of hydroxyurea (G1/S boundary) do not show this decrease in cyclin, indicating that its amounts are regulated by events occurring during the S-phase. Immunofluorescence studies of serum-stimulated quiescent cells in the presence of hydroxyurea, using proliferating cell nuclear antigen (PCNA) autoantibodies, confirm the results obtained by protein analysis. They also reveal that there are dramatic changes in the nuclear distribution of cyclin and that these depend on DNA synthesis or events occurring during the S-phase. Cyclin (PCNA) is no longer detectable at the end of the S-phase. However, pulse-chase experiments indicate that this protein is very stable, suggesting that it possibly interacts with other macromolecules rendering it inaccessible to the antibody. These results strengthen the notion that cyclin is an important component of the events leading to DNA replication and cell division.