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1 Clinical Pharmacology, The Royal College of Surgeons, 123 St Stephens Green, Dublin 2, Ireland
* To whom correspondence should be addressed. E-mail: klawler{at}rcsi.ie.
To metastasise, tumour cells must adopt different morphological responses to resist shear forces encountered in circulating blood and invade through basement membranes. The Rho and Ras GTPases play a critical role in regulating this dynamic behaviour. Membrane blebbing has only recently been characterised as a rounded mode of cellular invasion promoted through Rho kinase (ROCK), however; the role of shear forces in modulating membrane blebbing activity is unknown. To further characterise membrane blebbing in esophageal metastatic cells (OC-1 cell line), we investigated the role of shear in cytoskeletal remodelling and signalling though ROCK and Ras. Our results show that actin and tubulin co-localise to the cortical ring of the OC-1 cell under static conditions. However, under shear, actin acquires a punctuate distribution, whilst tubulin localises to the leading edge of the OC-1 cell. We show that dynamic bleb formation is induced by shear alone independent of integrin mediated adhesion (**p < 0.001). Y-27632, a specific inhibitor of ROCK, causes a significant reduction in shear-induced bleb formation and inhibits integrin
v
3/Ras co-localisation at the leading edge of the cell. Direct measurement of Ras activation shows that the level of GTP-bound Ras is elevated in sheared OC-1 cells and that the shear-induced increase in Ras activity is inhibited by Y-27632. Finally, we show that shear stress significantly increases OC-1 cell invasion (**p < 0.007), an effect negated by the presence of Y-27632. Together our findings suggest a novel physiological role for ROCK and Ras in metastatic cell behaviour.
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