One of the hallmarks of oncogenic transformation is anchorage-independent growth (27). Here we demonstrate that responses to substrate rigidity play a major role in distinguishing the growth behavior of normal cells from that of transformed cells. We cultured normal or H-ras-transformed NIH 3T3 cells on flexible collagen-coated polyacrylamide substrates with similar chemical properties but different rigidity. Compared with cells cultured on stiff substrates, nontransformed cells on flexible substrates showed a decrease in the rate of DNA synthesis and an increase in the rate of apoptosis. These responses on flexible substrates are coupled to decreases in cell spreading area and traction forces. In contrast, transformed cells maintained their growth and apoptotic characteristics regardless of substrate flexibility. The responses in cell spreading area and traction forces to substrate flexibility were similarly diminished. Our results suggest that normal cells are capable of probing substrate rigidity and that proper mechanical feedback is required for regulating cell shape, cell growth, and survival. The loss of this response can explain the unregulated growth of transformed cells.

) was first identified as a v-src substrate in chicken embryo fibroblasts (1). It was subsequently found to be a ubiquitous non-receptor protein tyrosine kinase (2), colocalizing with integrins at focal adhesions in adherent cells (3-5). The C-terminal domain contains multiple binding sites for focal adhesion proteins that associate with integrin clusters, such as paxillin, p130cas (6), and talin (7,8). It is believed that integrin-dependent autophosphorylation of FAK recruits and activates Src family kinases, which in turn trigger downstream signaling events. A particularly important site of autophosphorylation, Tyr-397, was identified at the juncture between the N-terminal and the catalytic domain (9). Phosphorylation of this site promotes the binding of FAK with the SH2 domain of Src family kinase (9) and other proteins carrying this domain, such as phospholipase C (10).The biological function of FAK is still a subject of much speculation. There is evidence that FAK is essential for integrinstimulated cell migration (11-17), cell spreading (16), and proliferation (11,18,19). Overexpression of FAK increases the migration rate (20), whereas abolition of FAK expression impairs cell migration and leads to embryonic lethality (21). Tyr-397 autophosphorylation site is required for the maximal adhesion-induced FAK activation and for FAK-enhanced cell spreading and migration (16). So far, speculations have centered on the possible role of FAK in the detachment of cells from the substrate, as initially suggested by the apparent increase in the size of focal adhesions in FAK-null cells (21). However, because the turnover of focal adhesion is closely coupled to cell migration, it is possible that the increase in size or stability of the focal adhesion is associated with other defects in cell migration.In this study, we pursued the possibility that FAK is involved in focal adhesion-mediated responses of cells to physical signals. There is increasing evidence that mechanical signals regulate not only cell migration, but also cell growth, apoptosis, and gene expression (22)(23)(24)(25)(26). Experiments with flexible substrates demonstrated that both mechanical forces and substrate rigidity could profoundly affect cell shape and migration rate (27). For example, when 3T3 cells are stretched with mechanical forces, protrusions that extended toward forces expand into dominant lamellipodia whereas other protrusions retract (28). In addition, cells plated on soft substrates showed both increased motility (27) and decreased growth (26). The localization of FAK at the juncture between integrins and the cytoskeleton makes it an attractive candidate for converting external mechanical stimulations into intracellular chemical events.To address this possibility, we have used a tetracycline repression system to achieve inducible expression of wild-type FAK (WT-FAK) or Tyr-397 mutant FAK (F397-FAK) in fibroblasts derived from FAK-knockout mouse embryos (16). Cells under either gene inhibition or expression conditions were plated on flex...