Inhibition of G(1) cyclin-dependent kinase activity in cell density-dependent growth arrest in human fibroblasts

The growth of normal fibroblasts in culture ceases as the cells reach satur ation density, Although cells in dense cultures express functionally active growth factor receptors, they are essentially refractory to the mitogenic activity of growth factors. Northern blot analysis revealed that immediate early genes, c-myc, c-fos and c-jun are induced by mitogen in dense culture s, However, these cells fail to express the late G(1) genes as E2F-1, cdc25 A, and cyclin A in response to mitogen stimulation, Furthermore, because pR b-phosphorylation is a key event in G(1) progression, here we show that in dense cultures, pRb remains in its active (hypophosphorylated) form after s timulation by mitogens. We also show that the kinase activity of cyclin-dep endent kinases that are indispensable for the phosphorylation of pRb in lat e G(1) phase was decreased on increasing cell density, The reduced kinase a ctivity may be caused by the observed increase in cyclin-dependent kinase i nhibitors and the reduction of cdc25A expression in dense cells.