Investigation of the role of G1/S cell cycle mediators in cellular senescence.

Cellular senescence is a state of irreversible cell cycle arrest in which normal cells at the end of their lifespan fail to enter into DNA synthesis upon serum or growth factor stimulation. We examined whether proteins required for G1/S cell cycle progression were irreversibly down-regulated in senescent human fibroblasts. Both the 44- and 42-kDa forms of the MAP-kinase protein were expressed at similar levels in young and senescent cells. In contrast to young cells where both forms were phosphorylated on tyrosine in response to serum, the p42MAP-kinase was not tyrosine phosphorylated upon serum stimulation, whereas p44MAP-kinase was phosphorylated on tyrosine in serum-starved or serum-stimulated senescent cells. Examination of p53 protein in growing, quiescent, and senescent cells revealed no significant differences in levels between the different growth states. In contrast, cdk2 and cyclin A mRNAs were completely down-regulated in stimulated senescent fibroblasts, while the G1 cyclins, C, D1, and E mRNAs, were still expressed in stimulated senescent cells although at reduced levels compared to young cells. The expression of early G1 markers, but not late G1 markers, indicates that senescent cells may be blocked at a point in late G1. We investigated whether transfection of cyclin A, alone or in combination with cdc2, was sufficient for extension of lifespan or escape from senescence. Clones expressing the transfected human cyclin A or cdc2 genes senesced at a population doubling similar to controls, thereby showing that cyclin A or cdc2 expression alone was insufficient for escape from senescence.