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-beta 1 overrides the attachment-dependent transition in NRK and AKR-2B fibroblasts (lines in which TGF-beta 1 induces anchorage-independent growth), but not in NIH-3T3 or Balb/c 3T3 fibroblasts (lines in which TGF-beta 1 fails to induce anchorage-independent growth). These results show that (a) adhesion and TGF-beta 1 can have similar effects in stimulating cell cycle progression from G1 to S and (b) the differential effects of TGF-beta 1 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-beta 1 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-beta 1 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.
Han
E K
EK
Department of Biochemistry and Molecular Biophysics, College of Physicians and Surgeons, Columbia University, New York, NY 10032.
Guadagno
T M
TM
Dalton
S L
SL
Assoian
R K
RK
D016475:3T3 Cells; D000818:Animals; D002448:Cell Adhesion; D002452:Cell Count; D002999:Clone Cells; D003260:Contact Inhibition; D004815:Epidermal Growth Factor / Q000494:pharmacology; D016193:G1 Phase / Q000187:drug effects; D006918:Hydroxyurea; D051379:Mice; D008934:Mitogens / Q000494:pharmacology; D008938:Mitosis / Q000187:drug effects; D016296:Mutagenesis; D010641:Phenotype; D051381:Rats; D016196:S Phase / Q000187:drug effects; D016212:Transforming Growth Factor beta / Q000494:pharmacology*
D008934:Mitogens; D016212:Transforming Growth Factor beta; D004815:Epidermal Growth Factor; D006918:Hydroxyurea