RAS INDUCES ANCHORAGE-INDEPENDENT GROWTH BY SUBVERTING MULTIPLE ADHESION-REGULATED CELL-CYCLE EVENTS

Anchorage-independent growth is a hallmark of transformed cells, but l ittle is known of the molecular mechanisms that underlie this phenomen on. We describe here studies of cell cycle control of anchorage-indepe ndent growth induced by the ras oncogene, with the use of a somatic ce ll mutant fibroblast line (ER-1-2) that is specifically defective in o ncogene-mediated, anchorage-independent growth. Control, nontransforme d PKC3-F4 cells and ER-1-2 cells cannot proliferate in semisolid mediu m. Three important cell cycle events are dependent on adhesion of thes e cells to a substratum: phosphorylation of the retinoblastoma protein , pRB; cyclin E-dependent kinase activity; and cyclin A expression. PK C3-F4 cells that express ras (PKC3-F4/ras cells) proliferate in nonadh erent cultures, and each of these three events occurs in the absence o f adhesion in PKC3-F4/ras cells. Thus, ras can override the adhesion r equirement of cellular functions that are necessary for cell cycle pro gression. ER-1-2 cells that express ras (ER-1-2/ras cells) possess hyp erphosphorylated forms of pRB and cyclin E-dependent kinase activity i n the absence of adhesion but remain adhesion dependent for expression of cyclin A. The adhesion dependence of pRB phosphorylation and cycli n E-dependent kinase activity is therefore dissociable from the adhesi on dependence of cyclin A expression. Furthermore, ectopic expression of cyclin A is sufficient to rescue anchorage-independent growth of ER -1-2/ras cells but does not induce anchorage-independent growth of PKC 3-F4 or ER-1-2 cells. However, like pRB phosphorylation and cyclin E-d ependent kinase activity, the kinase activity associated with ectopica lly expressed cyclin A is dependent on cell adhesion, and this depende nce is overcome by ras. Thus, the induction of anchorage-independent g rowth by ras may involve multiple signals that lead to both expression of cyclin A and activation of G(1) cyclin-dependent kinase activities in the absence of cell adhesion.