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

Abstract

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.

Subdivision of S‐phase by analysis of nuclear 5‐bromodeoxyuridine staining patterns

“…For instance, the nuclear distribution of cyclin, a proliferating cell nuclear antigen (PCNA), which is detectable only during the S-phase and is probably involved in the stimulation of DNA polymerase-d (3), indeed shows a striking similarity with the BrdUrd patterns as described above (5). (20) respectively) are compared, showing in both cases three distinct peaks in early, mid, and late S a n d a shoulder or small peak very late in the S-phase.…”

Comparison of Proliferating Cell Nuclear Antigen to Tritiated Thymidine As a Marker of Proliferating Hepatocytes in Rats

“…DNA pol 6, in conjunction with PCNA, is also responsible for leading strand DNA synthesis in replicating cells, whereas PCNA involved in DNA repair mechanisms is independent of DNA pol 6 (13). Studies of cells in culture have identified PCNA in G1, S, G2, and M phases of the cell cycle with peak expression of the protein during S phase (14)(15)(16). The advent of commercially available antibodies of PCNA has allowed the immunohistochemical detection and evaluation of this endogenous cell-cycle-related protein as a marker of replicating cells, which is of particular importance with the recent interest in the role of cell proliferation in chemical carcinogenesis (17,18).…”

Progressin: A novel proliferation‐sensitive and cell cycle‐regulated human protein whose rate of synthesis increases at or near the G1/S transition border of the cell cycle

“…In contrast to histones, the synthesis of progressin, cyclin (PCNA) and dividin is independent of DNA replication ( [26]; this article). In vivo replicative DNA synthesis on the other hand has not been observed in the absence of synthesis of these proteins.…”

“…Indirect immunofluorescence analysis of methanol-fixed cells confirmed that the thymidine-treated cells were blocked at the Gl/S transition border of the cell cycle ( fig.3D). Untreated cells on the other hand progressed into S-phase as judged by the late patterns of cyclin (PCNA) antigen distribution ( fig.4C) [17, 24,26].…”