Repression of a G0-associated 65-kilodalton protein in actively proliferating and SV40-transformed mouse kidney cells

Abstract

The pattern of [35S]methionine-labeled proteins from primary cultures of mouse kidney epithelial cells arrested in G0 phase was analyzed by two-dimensional gel electrophoresis and compared with that observed from cultures of actively proliferating and SV40-transformed mouse kidney cells. A major polypeptide (p65) migrating with a molecular mass of 65 000 daltons and a pI of 5.8 was detected in quiescent cultures of cells which had exhausted their finite division potential. Under the experimental conditions used, these cells had lost sensitivity to growth factors and were irreversibly blocked in G0 phase of the cell cycle. In cultures of actively proliferating mouse kidney cells, the expression of p65 was not observed until just prior to arrest. Moreover, proliferating cultures of immortalized mouse kidney cells that had been reactivated from their quiescent state by infection with SV40 did not express p65. Subcellular localization studies suggest that p65 is associated with the crude nuclear fraction. In addition, p65 is glycosylated and binds the lectin concanavalin A. Pulse–chase experiments demonstrated that p65 was short lived with an estimated half life of 10 min. Thus, p65 appears to be a growth-arrest specific gene product whose expression is repressed during the proliferative state of mitotically active mouse kidney cells.Key words: G0 phase, senescence, proliferation, quiescence, SV40-transformed mouse cells.