Density-dependent inhibition of mouse embryo fibroblast growth: involvement of IGFBP-3

Up until now the phenomenon of density dependent inhibition of growth has remained unexplained; one hypothesis suggests that autocrine growth inhibitory molecules are secreted in the medium of dense cultures and inhibit cell growth. From medium conditioned by mouse fibroblasts we purified a 45-kDa inhibitory factor which is an insulin-like growth factor binding protein (IGFBP-3). Based on different results we assumed that IGFBP-3 is a bifunctional molecule and has inhibitory function independent of its known function of binding IGF. In the present publication we attempted to verify whether IGFBP-3 is involved in DDI of growth. IGFBP-3 was secreted by mouse embryo fibroblasts (MEF). Its concentration in the medium increased with the cell density of the culture and was large at saturation density when DNA synthesis was minimum. Medium conditioned (CM) by dense culture was inhibitory compared to fresh medium and this inhibition disappeared when CM was preincubated with anti-IGFBP-3 IgG. Addition of FGFb to MEF dense cultures increased but transiently DNA synthesis which decreased as soon as 24 h after growth factor addition. By contrast accumulation of IGFBP-3 in the medium increased with time and was large at the time when DNA synthesis was minimum. Our results suggest that the rapid decrease of DNA synthesis in stimulated dense culture was the result of both depletion of the medium (particularly of FGFb) and the increase in concentration of inhibitory molecules like IGFBP-3. Addition of FGFb and preincubation of CM with anti-IGFBP-3 IgG were able to greatly reduce the inhibition.