A CELL-CYCLE AND MUTATIONAL ANALYSIS OF ANCHORAGE-INDEPENDENT GROWTH - CELL-ADHESION AND TGF-BETA-1 CONTROL G1/S TRANSIT SPECIFICALLY

We have examined cell cycle control of anchorage-independent growth in nontransformed fibroblasts. In previous studies using G0-synchronized NRK and NIH-3T3 cells, we showed that anchorage-independent growth is regulated by an attachment-dependent transition at G1/S that resembles the START control point in the cell cycle of Saccharomyces cerevisiae. In the studies reported here, we have synchronized NRK and NIH-3T3 fibroblasts immediately after this attachment-dependent transition to determine if other portions of the fibroblast cell cycle are similarly regulated by adhesion. Our results show that S-, G2-, and M-phase progression proceed in the absence of attachment. Thus, we conclude that the adhesion requirement for proliferation of these cells can be explained in terms of the single START-like transition. In related studies, we show that TGF-beta1 overrides the attachment-dependent transition in NRK and AKR-2B fibroblasts (lines in which TGF-beta1 induces anchorage-independent growth), but not in NIH-3T3 or Balb/c 3T3 fibroblasts (lines in which TGF-beta1 fails to induce anchorage-independent growth). These results show that (a) adhesion and TGF-beta1 can have similar effects in stimulating cell cycle progression from GI to S and (b) the differential effects of TGF-beta1 on anchorage-independent growth of various fibroblast lines are directly reflected in the differential effects of the growth factor at G1/S. Finally, we have randomly mutagenized NRK fibroblasts to generate mutant lines that have lost their attachment/TGF-beta1 requirement for G1/S transit while retaining their normal mitogen requirements for proliferation. These clones, which readily proliferate in mitogen-supplemented soft agar, appear non-transformed in monolayer: they are well spread, nonrefractile, and contact inhibited. The existence of this new fibroblast phenotype demonstrates (a) that the growth factor and adhesion/TGF-beta1 requirements for cell cycle progression are genetically separable, (b) that the two major control points in the fibroblast cell cycle (G0/G1 and G1/S) are regulated by distinct extracellular signals, and (c) that the genes regulating anchorage-independent growth need not be involved in regulating contact inhibition, focus formation, or growth factor dependence.