Harrisingh, Marie; (2002) Ras/Raf signalling in primary cells. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

Oncogenic activation of the Ras gene has been implicated in many human tumours. However, despite the ability of Ras to transform immortal cell lines, activated Ras is growth inhibitory in primary cells. We have previously shown that activation of Ras/Raf signalling in primary Schwann cells results in a proliferative arrest due to the induction of the cyclin dependent kinase inhibitor (CDKI) p21Cip1. In this thesis I examine the mechanisms involved in p21Cip1 induction and the roles of other CDKIs in the Ras/Raf induced cell-cycle arrest. I show that Raf activation is also associated with the induction of the CDKI pl5INK4b, however, in contrast to other primary cell types, p16INK4a levels decrease and the induction of p19ARF is not associated with p53 stabilisation. In certain cell types, the Ras induced proliferative arrest is associated with differentiation. I therefore investigated whether the Ras/Raf induced proliferative arrest in Schwann cells was associated with an induction of differentiation. Surprisingly in vitro I found that Raf/MAPK signalling blocks Schwann cell differentiation, as measured by the downregulation of differentiation markers. In addition, Raf is able to induce Schwann cell de-differentiation. In vivo, differentiated Schwann cells are found in the peripheral nervous system in association with axons and are capable of de-differentiating and proliferating in response to nerve damage throughout life. To investigate the effect of Ras/Raf activation on Schwann cell-neuron interactions I have set up a Schwann cell-dorsal root ganglion (DRG) co-culture system and I have generated transgenic mice expressing an inducible Raf protein in myelinating Schwann cells. Using the Schwann cell-DRG system I show that activation of Raf does not prevent the recognition or association of Schwann cells with axons. However, using time-lapse microscopy I have found that Raf activation results in subtle changes in the dynamics of Schwann cell-axon interactions.

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