A cell cycle and mutational analysis of anchorage-independent growth: cell adhesion and TGF-beta 1 control G1/S transit specifically

Abstract

Abstract. We have examined cell cycle control of anchorage-independent growth in nontransformed fibroblasts. In previous studies using Go-synchronized NRK and NIH-3T3 cells, we showed that anchorageindependent growth is regulated by an attachmentdependent 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-/~I overrides the attachment-dependent transition in NRK and AKR-2B fibroblasts (lines in which TGF-/$1 induces anchorageindependent growth), but not in NIH-3T3 or Balb/c 3T3 fibroblasts (lines in which TGF-/31 fails to induce anchorage-independent growth). These results show that (a) adhesion and TGF-/31 can have similar effects in stimulating cell cycle progression from G1 to S and (b) the differential effects of TGF-fll on anchorageindependent 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-~l 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-~/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.

Developmentally regulated expression of calponin isoforms and the effect of h2-calponin on cell proliferation

“…Its presence in both smooth muscle and nonmuscle cells indicates that it may have a cytoskeletal function. Considering calponin's inhibitory activity on actin-myosin interactions, h2-calponin may also play an inhibitory role in regulating the functions of the actin cytoskeleton, such as coordinating changes in cell shape and intracellular molecular trafficking, both of which are critical events in cytokinesis (15). Therefore, h2-calponin may act as a balancing mechanism to maintain the physiological levels of actin filament activity in both smooth muscle and nonmuscle cells.…”

“…The contraction of the contractile ring is most likely generated by the interaction between actin and myosin (2,6,22,27). The actin cytoskeleton has been demonstrated to participate in anchorage-dependent cell division (15), and actin-myosin interactions have been shown to power cell proliferation by driving cytoplasmic streaming. In vitro experiments have shown that calponin inhibits the relative movements of actin and myosin (40).…”

“…Actin-myosin interaction also powers cell proliferation by driving cytoplasmic streaming, which may contribute to the division of the cytosolic components of the cell during cytokinesis. Accordingly, through the inhibition of actin-myosin interaction, calponin may play a role in regulating the functions of the actin cytoskeleton, such as coordinating changes in cell shape and intracellular molecular trafficking, both of which are critical events in cytokinesis (15). Indeed, forced expression of chicken gizzard calponin in cultured smooth muscle cells and fibroblasts showed an inhibition of cell proliferation (19).…”

“…Cleavage furrow ingression requires a contractile cortical ring of actin and myosin (28,38,39); thus the activity of the actin cytoskeleton has an effect on cell division (15). Actin-myosin interaction also powers cell proliferation by driving cytoplasmic streaming, which may contribute to the division of the cytosolic components of the cell during cytokinesis.…”

Anchorage-Dependent Transcription of the Cyclin A Gene

“…Established human and rodent fibroblasts undergo cell cycle arrest in the G 1 phase of the cell cycle when cultured in suspension (19,36,41). Beyond a defined time point in G 1 , cells are no longer dependent on adhesion to complete the cell cycle (6,21). Cell adhesion and spreading involves interaction of integrins with the extracellular matrix and the actin-based cytoskeleton.…”