POSITIVE SELECTION OF GROWTH-INHIBITORY GENES

We have isolated a limited set of cDNAs that limit cell proliferation using a unique assay based on the dilution of a lipophilic fluorescent dye as transfected cells divide. The identification of growth-inhibit ory factors has been limited by the lack of a strong assay for growth inhibitors. A growth-inhibited cell does not grow and so is at a selec tive disadvantage in vitro when compared with any growing cell. Severa l assays have been used to screen for growth-inhibitory genes; however , these approaches are either very difficult to implement, leaky, or n ot comprehensive. We have developed an assay that selects for cDNAs ca pable of inhibiting proliferation in which cells are nonspecifically l abeled with a lipophilic fluorescent dye, PKH-2, and subsequently tran sfected with a cDNA library made from growth-inhibited cells. With eac h cell division, the amount of dye per cell is reduced by one-half. Ov er time, growth-inhibited cells will retain more dye per cell relative to actively growing cells. The population is then analyzed by fluores cence-activated cell sorting, and the brightest cells in the populatio n are isolated. This assay has allowed us to select pools of cDNAs enr iched for growth-inhibitory activities and may provide a general metho d for identifying growth-inhibitory genes active in varying biological contexts. We report here the successful application of the dye retent ion assay to the selection of cDNAs that inhibit epithelial cell proli feration.