RAS SIGNALS TO THE CELL-CYCLE MACHINERY VIA MULTIPLE PATHWAYS TO INDUCE ANCHORAGE-INDEPENDENT GROWTH

Several specific cell cycle activities are dependent on cell-substratu m adhesion in nontransformed cells, and the ability of the Ras oncopro tein to induce anchorage-independent growth is linked to its ability t o abrogate this adhesion requirement. Ras signals,ia multiple downstre am effector proteins, a synergistic combination of which may be requir ed for the highly altered phenotype of fully transformed cells. We des cribe here studies on cell cycle regulation of anchorage-independent g rowth that utilize Ras effector loop mutants in NW 3T3 and Rat 6 cells . Stable expression of activated H-Ras (12V) induced soft agar colony formation by both cell types, but each of three effector loop mutants (12V, 35S, 12V, 37G, and 12V, 40C) was defective in producing this res ponse. Expression of all three possible pairwise combinations of these mutants synergized to induce anchorage-independent growth of NIH 3T3 cells, but only the 12V, 35S-12V,37G and 12V, 37G-12V, 40C combination s were complementary in Rat 6 cells. Each individual effector loop mut ant partially relieved adhesion dependence of pRB phosphorylation, cyc lin E-dependent kinase activity, and expression of cyclin A in NIH 3T3 , but not Rat 6, cells. The pairwise combinations of effector loop mut ants that were synergistic in producing anchorage-independent growth i n Rat 6 cells also led to synergistic abrogation of the adhesion requi rement for these cell cycle activities. The relationship between compl ementation in producing anchorage-independent growth and enhancement o f cell cycle activities was not as clear in NM 3T3 cells that expresse d pairs of mutants, implying the existence of either thresholds for th ese activities or additional requirements in the induction of anchorag e-independent growth. Ectopic expression of cyclin D1, E, or A synergi zed with individual effector loop mutants to induce soft agar colony f ormation in NM 3T3 cells, cyclin A being particularly effective. Taken together, these data indicate that Ras utilizes multiple pathways to signal to the cell cycle machinery and that these pathways synergize t o supplant the adhesion requirements of specific cell cycle events, le nding to anchorage-independent growth.