A cascade of events is triggered upon the addition of growth factor to quiescent mammalian cells, which ultimately restarts proliferation by inducing the transition from Go/G 1 to S-phase. We have studied cyclin D1, a putative G1 cyclin, in normal diploid human fibroblasts. Cyclin D1 accumulated and reached a maximum level before S-phase upon the addition of serum to quiescent cells. The protein was localized to the nucleus, and it disappeared from the nucleus as cells proceeded into S-phase. Microinjection of anti-cyclin D1 antibodies or antisense plasmid prevented cells from entering S-phase, and the kinetics of inhibition showed that cyclin D1 is required at a point in the cell cycle earlier than cyclin A. These results demonstrate that cyclin D1 is a critical target of proliferative signals in Gx.

The cellular action of growth factors, among them basic fibroblast growth factor (bFGF), is mediated by their interaction with a cell surface receptor, but the mechanism of transfer of mitogenic (or other) signals to the nucleus has not been identified. In this work, we show that bFGF is translocated to and accumulated in the nucleolus. Furthermore, the nucleolar localization of bFGF is correlated with a stimulation of transcription of ribosomal genes during Gq-.Gl transition induced by bFGF alone in adult bovine aortic endothelial cells (ABAE cells). Stimulation of ribosomal gene transcription is preceded by a significant increase of the major nonhistone nucleolar protein, nucleolin. In vitro, the growth factor has a direct effect on the enhancement of RNA polymerase I activity in isolated nuclei from quiescent sparse (GO) ABAE cells. The direct action of bFGF on the level of ribosomal gene transcription could correspond to an additional growthsignaling pathway, mediated by this growth factor.The family of fibroblast growth factors (FGFs) includes the factors described as endothelial cell growth factor, chondrosarcoma growth factor, and heparin-binding growth factors (1). Preliminary physical analysis of some of these mitogens has suggested their classification in two groups: acidic fibroblast growth factors (aFGFs) (2) and basic fibroblast growth factors (bFGFs) (3). In vitro, aFGFs and bFGFs are potent mitogens for a wide variety of mesoderm-and neuroectoderm-derived cells, including vascular and capillary endothelial cells (4) and, as in vivo (5), they induce the angiogenic response (6).The cellular action of FGFs is exerted through its interaction with specific cell surface receptors (7,8), but the intervening steps and the mechanism of transfer of mitogenic (or other) signals to the nucleus, leading to the "ple-otropic response" required to bring quiescent cells into full proliferation, are at present unknown.The proliferation state and ribosome biogenesis, which involve a series of coordinated nucleolar events, among them the transcription of ribosomal genes (rDNA), are closely related. The level of transcription of rDNA is modulated by cell growth conditions, growth-promoting hormones (9), and growth factors (10). A specific nucleolar protein, nucleolin, was shown in different eukaryotic cells to play a direct role in the control of the synthesis of the precursor to ribosomal RNA (pre-rRNA) and assembly of ribosomes (11,12). Barely detectable in resting cells, nucleolin represents up to 5% of nucleolar proteins in exponentially growing cells. In vitro, run-off experiments with rDNA as template have shown that endoproteolytic cleavage of phosphorylated nucleolin controls rDNA transcription (13).In this report, we have focused on the effects of bFGF on the reinitiation of ribosome biogenesis in cells undergoing the Gy-3G1 transition. We show by immunocytochemistry using a monospecific polyclonal anti-bFGF antibody that the reinitiation of pre-rRNA synthesis is preceded by the accumulation of nucleo...

Primary cultures of adult bovine aortic endothelial (ABAE) cells require bFGF to grow. G1‐arrested cells, obtained after 48 h without serum and bFGF, were found to enter S phase and grow synchronously for at least two generations on addition of bFGF. In growing cells bFGF was detected both in the cytoplasm (90%) and in the nucleus (10%) where it accumulates in the nucleolus. It was not detected in the nucleus of confluent cells. bFGF uptake was continuous in the cytoplasm throughout the cell cycle with a maximum in G2, while nuclear uptake occurred only in late G1. Cytoplasmic bFGF (18.4 kd) is cleaved into a 16.5 kd peptide in G1 (t1/2 = 30 min). In the nucleus the 18.4 kd form was the only one detected 2 h following bFGF addition and was then cleaved into the 16.5 kd in early S phase. These results are consistent with the possibility that in addition to the classical pathway of signal transduction, bFGF is directly translocated to the nucleus in late G1, and could play a role in replication and/or in transcription of rDNA.

Abstract. In mammalian cells inhibition of the cdc2function results in arrest in the G2-phase of the cell cycle. Several cdc2-related gene products have been identified recently and it has been hypothesized that they control earlier cell cycle events. Here we have studied the relationship between activation of one of these cdc2 homologs, the cdk2 protein kinase, and the progression through the cell cycle in cultured human fibroblasts. We found that cdk2 was activated and specifically localized to the nucleus during S phase and G2. Microinjection of afffinity-purified anti-cdk2 antibodies but not of affinity-purified anti-cdc2 antibodies, during G1, inhibited entry into S phase. The specificity of these effects was demonstrated by the fact that a plasmid-driven cdk2 overexpression counteracted the inhibition. These results demonstrate that the cdk2 protein kinase is involved in the activation of DNA synthesis.