Purification and characterization of a DNA synthesis inhibitor protein from mouse embryo fibroblasts.

Α DNA synthesis inhibitor protein was purified from the conditioned medium of cycloheximide treated mouse embryo fibroblasts. This protein has a molecular weight of 45,000 as determined by gel filtration and Polyacrylamide gel electrophoresis. The levels of the [35S] methionine la belled 45 kDa protein in the medium and matrix were monitored across two cell cycles in synchronized cultures. The 45 kDa protein was present in higher levels in the medium of non-S-phase cells depicting a peak between the two S- phases. The DNA synthesis inhibitor protein was immunologically related to a chicken DNA-binding protein which showed similar cell cycle specific variations at the intracellular level. The purified 45 kDa protein inhibited DNA synthesis in murine and human cells. In mouse embryo fibroblasts, the DNA synthesis was inhibited to an extent of 86% by 0·25 μg/ml of the inhibitor, while higher amounts of the inhibitor were required to arrest DNA synthesis in human skin fibroblasts: in these cells, 4 μg/ml of the inhibitor inhibited DNA synthesis to an extent of 50%. The high levels of the 45 kDa protein in the medium of non-S phase cells and its DNA synthesis inhibitory potential suggest that this protein may be involved in the regulation of DNA synthesis during the cell cycle.

Translocation of plasminogen activator inhibitor-1 during serum stimulated growth of mouse embryo fibroblasts.

Serum-stimulated mouse embryo fibroblasts specifically secrete two proteins of molecular weights 48,000 and 26,000. The 48 kDa protein showed affinity to concanavalin A and was precipitated by antibody to plasminogen activator inhibitor. Immunoflowcytometry using anti plasminogen activator inhibitor-1 serum indicate the presence of the 48 kDa protein in quiescent cells; this protein was virtually absent in serum-stimulated cells. The presence of the plasminogen activator inhibitor-1 related protein in quiescent cells and its absence in serum-stimulated cells in combination with the observation on the absence of this protein, in the medium of quiescent cells and its presence in the medium of stimulated cells indicate that the 48 kDa protein was transferred from the cells into the medium upon serum-stimulation. The serum-mediated transfer of plasminogen activator inhibitor-1 from the cells into the medium was inhibited by actinomycin-D suggesting that the transfer process required actinomycin-D sensitive events. Treatment of pre-labelled quiescent cells with medium containing 20% fetal calf serum resulted in the gradual transfer of the labelled 48 kDa protein to the extra cellular matrix. These studies indicate that exposure of quiescent cells to fetal calf serum results in the transfer of plasminogen activator inhibitor-1 from the cells to the growth medium via extracellular matrix. The translocation of the protease inhibitor from the cells to the matrix and medium may enable the cellular and possibly the membrane proteases to act on growth factors or their receptors thereby initiating the mitogenic response.