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 resemble s the START control point in the cell cycle of Saccharomyces cerevisia e. 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 pro gression proceed in the absence of attachment. Thus, we conclude that the adhesion requirement for proliferation of these cells can be expla ined 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 anchor age-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) th e differential effects of TGF-beta1 on anchorage-independent growth of various fibroblast lines are directly reflected in the differential e ffects of the growth factor at G1/S. Finally, we have randomly mutagen ized NRK fibroblasts to generate mutant lines that have lost their att achment/TGF-beta1 requirement for G1/S transit while retaining their n ormal mitogen requirements for proliferation. These clones, which read ily proliferate in mitogen-supplemented soft agar, appear non-transfor med in monolayer: they are well spread, nonrefractile, and contact inh ibited. 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 co ntrol points in the fibroblast cell cycle (G0/G1 and G1/S) are regulat ed by distinct extracellular signals, and (c) that the genes regulatin g anchorage-independent growth need not be involved in regulating cont act inhibition, focus formation, or growth factor dependence.