Figure 1: Experimental design. After serum starvation for 24 h, cells were trypsinized and collected in DME containing 2% BSA, washed, and plated in DME containing 0.2% BSA on various different substrates (point a). Growth factors were added 16 h later (point b), and the ability of each substrate to support progression through the G1 phase of the cell cycle was compared.

Mentions: For synchronization in G0, subconfluent NIH-3T3 cultures were incubated for 24 h in DME without serum (see Fig. 1 for a schematic representation of the experimental procedure). Cells were then detached with 0.05% trypsin-EDTA and collected in DME containing 2% BSA and 0.5 mg/ml soybean trypsin inhibitor (Sigma-Aldrich), washed in DME, and finally collected in DME containing 0.2% BSA and counted. For suspension cultures, cells were replated on wells that had been coated for 1 h at room temperature with 1% agar in PBS and subsequently been incubated overnight in DME. For adherent cultures, cells were replated on wells that had been precoated overnight at 4°C with 10 μg/ml fibronectin, 20 μg/ml laminin, 20 μg/ml collagen or 100 μg/ml poly-l-lysine and subsequently been blocked with 2% heat-denatured BSA for 2 h at 37°C. After 16 h incubation on the various substrates, growth factors were added to a final concentration of 15 ng/ml bFGF (a gift from Gera Neufeld, Technion-Israel Institute of Technology, Haifa, Israel), 10 ng/ml EGF (Sigma-Aldrich), 6.25 μg/ml insulin, 6.25 μg/ml transferrin, 6.25 ng/ml selenous acid, 5.35 μg/ml linoleic acid (ITS1; Collaborative Biomedical Products), and 1 μg/ml heparin (Sigma-Aldrich).

Danen EH, Sonneveld P, Sonnenberg A, Yamada KM - J. Cell Biol. (2000)

Bottom Line: Among several extracellular matrix components tested, only fibronectin supported growth factor-induced, E2F-dependent S phase entry.Although all substrates supported the mitogen-activated protein kinase (MAPK) response to growth factors, RhoA activity was specifically enhanced on fibronectin.This ability of fibronectin to stimulate both Ras/MAPK- and RhoA-dependent signaling can explain its potent cooperation with growth factors in the stimulation of cell cycle progression.

Affiliation: Division of Cell Biology, The Netherlands Cancer Institute, 1066 CX Amsterdam, The Netherlands. edanen@nki.nl

Abstract: In cellular transformation, activated forms of the small GTPases Ras and RhoA can cooperate to drive cells through the G1 phase of the cell cycle. Here, we show that a similar but substrate-regulated mechanism is involved in the anchorage-dependent proliferation of untransformed NIH-3T3 cells. Among several extracellular matrix components tested, only fibronectin supported growth factor-induced, E2F-dependent S phase entry. Although all substrates supported the mitogen-activated protein kinase (MAPK) response to growth factors, RhoA activity was specifically enhanced on fibronectin. Moreover, induction of cyclin D1 and suppression of p21(Cip/Waf) occurred specifically, in a Rho-dependent fashion, in cells attached to fibronectin. This ability of fibronectin to stimulate both Ras/MAPK- and RhoA-dependent signaling can explain its potent cooperation with growth factors in the stimulation of cell cycle progression.