The goal of this proposal is to dissect the role of the Rb family of proteins in cellular senescence, identify the targets of Rb during cellular senescence and identify genes that that contribute to the stability of senescence. Because components of the senescence machinery have been shown to play important roles in ageing and cancer, this approach could lead to the identification of new molecules with potential role in ageing and/or cancer. These questions will be addressed using the well characterize IMR90 human diploid fibroblast. These cells can be induced to undergo senescence by exogenous expression of oncogenic ras, etoposide, enforce expression of p16,or extensive passage. Senescent IMR90 cells display the characteristic associated with senescence such as irreversible cell cycle arrest, large flat morphology, senescence-associated beta-galactosidase (SA-beta-gal) activity and heterochromatic foci. These characteristics will be used together with RNAi technology to assess the role of the different Rb family members in senescence and to identify factors that contribute to the stability of senescence. RNAi technology will also be combined with expression microarray to identify the targets of Rb during senescence.