Cyclin D2 is a member of the family of D-type cyclins that is implicated in cell cycle regulation, differentiation, and oncogenic transformation. To better understand the role of this cyclin in the control of cell proliferation, cyclin D2 expression was monitored under various growth conditions in primary human and established murine fibroblasts. In different states of cellular growth arrest initiated by contact inhibition, serum starvation, or cellular senescence, marked increases (5-to 20-fold) were seen in the expression levels of cyclin D2 mRNA and protein. Indirect immunofluorescence studies showed that cyclin D2 protein localized to the nucleus in G 0 , suggesting a nuclear function for cyclin D2 in quiescent cells. Cyclin D2 was also found to be associated with the cyclin-dependent kinases CDK2 and CDK4 but not CDK6 during growth arrest. Cyclin D2-CDK2 complexes increased in amounts but were inactive as histone H1 kinases in quiescent cells. Transient transfection and needle microinjection of cyclin D2 expression constructs demonstrated that overexpression of cyclin D2 protein efficiently inhibited cell cycle progression and DNA synthesis. These data suggest that in addition to a role in promoting cell cycle progression through phosphorylation of retinoblastoma family proteins in some cell systems, cyclin D2 may contribute to the induction and/or maintenance of a nonproliferative state, possibly through sequestration of the CDK2 catalytic subunit.In eukaryotes, cell proliferation is regulated by the cooperative activity of a set of cell cycle control genes (reviewed in references 56 and 57). These genes include those that encode cyclin-dependent kinases (CDKs) and the proteins that regulate their behavior, cyclins and CDK inhibitors. Analysis of cyclin and CDK inhibitor expression has indicated that phasespecific oscillations in the abundance of some of these proteins are responsible, in part, for the orderly and linear progression of cells through key cell cycle checkpoints. In mammalian cells, an important cell cycle checkpoint called the restriction point is located at a position just prior to the onset of DNA synthesis (51). The expression of a subset of cell cycle control genes occurs during the G 1 phase of the cycle, placing them in a temporal position to influence this key cell cycle decision (56). Among such candidate G 1 control genes are those encoding cyclins D and E and the CDK inhibitors belonging to the p21 WAF/CIP/SDI and p16 INK4 families. The D-type cyclins consist of three family members, cyclins D1, D2, and D3. The cyclin D1 gene was identified as a delayed-early gene that was inducible by colony-stimulating factor (37), by its ability to complement G 1 cyclin-deficient yeast strains (33,74), and as the PRAD-1/bcl-1 proto-oncogene that underwent gene rearrangements, gene amplification, and deregulated expression in a variety of tumor types (reviewed in reference 43). Microinjection experiments with anti-cyclin D1 antibodies have suggested that cyclin D1 may be required for progression ...

We have examined the role of cyclin D1 and cyclin-dependent kinase-4 (CDK4) in the cell cycle progression and proliferation of MCF-7 breast cancer cells. Forced expression of cyclin D1 using a tetracycline-regulated expression system, and suppression of endogenous cyclin D1 and CDK4 using small interfering RNA (siRNA) were used to validate this protein complex as a drug target in cancer drug discovery. Overexpression of cyclin D1 increased both phosphorylation of the retinoblastoma gene product (RB) and passage through the G1-S phase transition, resulting in increased proliferation of cells. When cyclin D1 expression was shut off, growth rates fell below those seen in control cell lines transfected with the vector, indicating an increased dependence on this protein for proliferation. Inhibition of endogenous cyclin D1 or CDK4 expression by RNA interference resulted in hypophosphorylation of RB and accumulation of cells in G1. These results support the prevailing view that pharmacological inhibition of cyclin D1/CDK4 complexes is a useful strategy to inhibit the growth of tumors. Furthermore, since MCF-7 cells appear to be dependent on this pathway for their continued proliferation, it is a suitable cell line to test novel cyclin D1/CDK4 inhibitors.

Human diploid fibroblasts (HDFs) undergo a limited number of population doublings in vitro and are widely used as a model of cellular aging. Despite growing evidence that cellular aging occurs as a result of altered gene expression, little is known about the activity of transcription factors that regulate gene expression in aging cells. Here we survey the relevant literature regarding altered gene expression and the role of transcription factors during cellular aging, focusing upon the serum response factor (SRF). SRF is hyperphosphorylated in senescent HDFs and fails to bind to the serum-response element in the c-fos promoter. Differential phosphorylation during replicative aging may contribute, at least in part, to the altered activity of SRF and possibly other transcription factors and to subsequent changes in the expression of serum-regulated genes in senescent HDFs.

Human diploid fibroblasts (HDFs) undergo a limited number of population doublings in culture before reaching the end of their proliferative life span, an event termed in vitro cellular senescence. Considerable evidence suggests that altered expression of key genes involved in the mitogenic response may be responsible for the inability of senescent cells to proliferate. Here we examined the expression and activity of the early growth response-1 (egr-1) gene, an "immediate-early" gene that is believed to link extracellular mitogenic signals to cell-cycle progression. We found that egr-1 was strongly downregulated in senescent HDFs at the level of mRNA, protein, and DNA binding activity. Decreased DNA binding activity of Egr-1 in vitro corresponded to decreased transcriptional activation in vivo. To further understand the mechanism of egr-1 downregulation, we examined the potential role of the serum response elements (SREs) present in the egr-1 promoter. Electrophoretic mobility shift studies using young and old cell nuclear extracts showed a marked decrease in serum response factor (SRF) binding activity to the SRE in old compared to young cells. Loss of SRF binding activity has been correlated with the loss of expression of another growth-related immediate-early gene (c-fos). These results suggest a common mechanism for the downregulation of c-fos, egr-1, and other SRE-dependent, mitogen-responsive genes during cellular senescence.