No abstract

Our results show that an insulin-like growth factor binding protein, IGFBP-3, purified from rat serum, is an inhibitor of chick embryo fibroblast (CEF) growth. It abolished DNA synthesis in CEF stimulated by IGF-I as well as by human serum. Rat IGFBP-3 and IDF45 (an inhibitory diffusible factor secreted by mouse cells) had the same activities, confirming that they have an intrinsic capacity to inhibit serum stimulation and may be considered as growth inhibitors. Our data show that inhibition by IGFBP-3 of serum stimulation was not simply the result of its inhibition of IGF present in the serum: 1) While anti-IGF-I IgG was able to completely inhibit stimulation induced by added IGF-I, it did not decrease stimulation induced by 1% human serum. Anti-IGF-II IgG inhibited the stimulation induced by added IGF-II, but only 25% decreased the stimulation induced by 0.7% serum. The percent inhibition was not significantly increased when the concentration of serum was decreased to 0.2%, which induced 140% stimulation of DNA synthesis; 2) stimulation by 0.2% serum was much more inhibited by IGFBP-3 than by IgG anti IGF-II; 3) after separation of IGF-I and IGF-II from serum by chromatography of acidified serum proteins on BioGel P150, the remaining serum proteins (with a molecular mass greater than 45 kDa) which were depleted in IGF-I and -II (verified by RIA determination) still stimulated DNA synthesis, and this stimulation was 80% inhibited by IGFBP-3.

We purified to homogeneity a growth inhibiting diffusible factor (IDF45) secreted by dense cultures of mouse 3T3 cells and which was able to inhibit 100% of DNA synthesis stimulated by serum in chick embryo fibroblasts (CEF) (Blat et al., 1989a). We then demonstrated that this factor was an IGF-binding protein (Blat et al., 1989b). Indeed, its N-terminal amino acid sequence was homologous to that of rat IGFBP-3. Our present results show that basic fibroblast growth factor (bFGF) induced, respectively, a fivefold and threefold increase in DNA synthesis in mouse embryo fibroblasts (MEF) and CEF. IDF-45 inhibited the stimulation induced by bFGF by about 65%, while stimulation induced by insulin, PDGF, or EGF was only weakly or not at all inhibited by IDF45. When bFGF stimulation was determined in the presence of a high concentration of insulin in conditions which minimize the effect of endogenous IGF-I or -II, this stimulation was decreased by about 50% in the presence of IDF45. This result suggests that addition of bFGF stimulates IGF secretion, thereby resulting in partial loss of inhibition, by IDF45, of bFGF stimulation.

In 3T3 Swiss mouse fibroblasts, incorporation of phosphate into cells and phosphorylation of small organic compounds were increased by shaking dense cultures. This response was not obtained with SV40 transformed Swiss 3T3 cells (SV-3T3). It appeared likely that these results could be accounted for by an inhibitor released from 3T3 cells but not from SV-3T3 cells. Our new method of co-incubation of sparse and dense cultures allowed us to demonstrate inhibition of growth and phosphate metabolism in sparse 3T3 cultures which were shaken in the presence of dense cultures. The inhibition was much less when the cultures were co-cultivated but not shaken. The inhibition of phosphate incorporation in acid-soluble and acid-insoluble fractions of sparse cultures was observed as early as 20 minutes of co-incubation in the presence of dense cultures, so this inhibition is not the result of depletion of growth factors in the medium. Our experiments suggest that an inhibitor(s) was released from dense cultures of 3T3 cells.

Different data have suggested that the density-dependent inhibition of growth in normal 3T3 cells is due to the release of inhibitory factor(s) into the medium. The present results provide more evidence supporting this hypothesis. From conditioned medium of dense cultures of 3T3 cells, we fractionated a thermolabile inhibitory diffusible factor (I.D.F.) with a molecular weight of about 40.000 This factor decreased the [11C]-inosine incorporation into purine nucleotides and into nucleic acids of stimulated 3T3 cells. DNA synthesis, determined by [11C]-thymidine incorporation into nucleic acids, and protein synthesis (determined by the protein content of the cultures maintained in the presence or absence of I.D.F.), were also decreased. The possibility that I.D.F. is involved in density-dependent inhibition of growth is discussed.