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Membrane-associated inhibitor of DNA synthesis in senescent human diploid fibroblasts: characterization and comparison to quiescent cell inhibitor.

Cell membranes prepared from senescent human diploid fibroblasts (HDF) inhibited entry into S phase by 35% when added to the medium of replicating young HDF. This membrane-associated inhibitory activity was (i) sensitive to trypsin, heat, and periodate, which suggests that the inhibitor is a glycoprotein, and (ii) not able to inhibit DNA synthesis in simian virus 40-transformed HDF, which indicates that not all types of cells are sensitive to this inhibitor. Quiescent young HDF also have a surface membrane-associated inhibitor of DNA synthesis. A comparison of the senescent HDF and quiescent HDF inhibitor activities indicates that they may have the same chemical and physical nature and the same specific activity, but their regulation is different. The inhibitory activity of quiescent young HDF is abolished within 20 hr after refeeding with fresh serum-containing medium, whereas that of senescent HDF remains unchanged. Quiescent old HDF (two or three population doublings remaining) exhibit an intermediate response to serum with approximately two-thirds of the inhibitory activity abolished. The fraction of cells in S phase at 20-24 hr post-stimulation (37% in young HDF, 24% in old HDF, and 0% in senescent HDF) is inversely proportional to inhibitor levels. This suggests that inability to neutralize the inhibitory activity in response to serum stimulation could be involved in the inability of senescent HDF to enter S phase. Disappearance of the inhibitory activity from quiescent young HDF occurs late in G1 phase. Thus, the inhibitor may play a role in determining the length of the G0 to S phase transition in these cells.