Validation of cyclin D1/CDK4 as an anticancer drug target in MCF-7 breast cancer cells: Effect of regulated overexpression of cyclin D1 and siRNA-mediated inhibition of endogenous cyclin D1 and CDK4 expression.

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.

A novel transcriptional inhibitory element differentially regulates the cyclin D1 gene in senescent cells.

Senescent human diploid fibroblasts are unable to initiate DNA synthesis following mitogenic stimulation and adopt a unique gene expression profile distinct from young or quiescent cells. In this study, a novel transcriptional regulatory element was identified in the 5'-untranslated region of the cyclin D1 gene. We show that this element differentially suppresses cyclin D1 expression in young versus senescent fibroblasts. Electrophoretic mobility shift assays revealed abundant complexes forming with young cell nuclear extracts compared with senescent cell nuclear extracts. Binding was maintained in young quiescent cells, showing that loss of this activity was specific to senescent cells and not an effect of cell cycle arrest. Site-directed mutagenesis within this cyclin D1 inhibitory element (DIE) abolished binding activity and selectively increased cyclin D1 promoter activity in young but not in senescent cells. Sequences with homology to the DIE were found in the 5'-untranslated regions of other genes known to be up-regulated during cellular aging, suggesting that protein(s) that bind the DIE might be responsible for the coordinate increase in transcription of many genes during cellular aging. This study provides evidence that loss of transcriptional repressor activity contributes to the up-regulation of cyclin D1, and possibly additional age-regulated genes, during cellular senescence.