Ellis, Rowena Louise; (2000) The role of rho family proteins in the transformation of human breast and colon epithelial cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Transformation by Ras and other oncogenes is generally accompanied by dramatic alterations in cell morphology and re-organisation of the actin cytoskeleton, suggesting a role for Rho GTPases. There is now accumulating evidence for the requirement of Rho GTPases in Ras-induced transformation of rodent fibroblasts, but at present little is known about their involvement in human cancers. Therefore, the role played by Rho GTPases in the transformation of human breast and colon epithelial cells was investigated. Mutant active and dominant negative forms of Rho proteins were injected into cells to study the short-term effects of each protein on cell behaviour. Each protein induced morphological changes as predicted from previous research, although the extent of the response was dependent on the initial morphology of individual cell lines. In summary, RhoA induced stress fibre formation, Racl injection resulted in membrane ruffling and Cdc42 gave rise to filopodia. The effects of Rho proteins on cell motility were studied in a motile Ras-transformed breast epithelial cell line. Active forms of RhoA, Racl and Cdc42 each reduced cell motility, while inhibition of either RhoA or Racl had no effect. However, injection of dominant negative Cdc42 significantly reduced the migration speed of the Ras-transformed cells. A tetracycline-regulated expression system was used to study the long-term effects of mutant active or dominant negative Rho proteins in DLD-1 cells derived from a human colon carcinoma. Expression of active forms of RhoA, Racl and Cdc42 inhibited colony formation in soft agar and disrupted growth on Matrigel. Their expression also altered the cellular organisation of DLD-1 cells. RhoA disrupted nuclear division through deregulated centrosome duplication, resulting in cells that exhibited multi-septated nuclei and continued to synthesise DNA without undergoing division. Racl altered membrane trafficking as illustrated by cells showing surface blebs and increased levels of pinocytosis. Finally, Cdc42 altered cell morphology, although in a manner more associated with a Racl phenotype, as cells spread and showed large lamellipodia and membrane ruffles. These observations differ from those obtained previously in other cell types and indicate that the effects of Rho family proteins are cell-type dependent. This work emphasises the importance of analysing protein function in a range of cell types as well as the effects of both short- and long-term protein expression, in order to understand the precise roles of proteins in specific situations.

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