Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.538225
Title: Characterisation of the cellular roles of the non-inhibitory serpin, maspin
Author: Ravenhill, Lorna Elizabeth
Awarding Body: University of East Anglia
Current Institution: University of East Anglia
Date of Award: 2010
Availability of Full Text:
Access through EThOS:
Access through Institution:
Abstract:
Maspin is a non-inhibitory member of the serine protease inhibitor (serpin) family. Maspin functions as a class II tumour suppressor, resulting from its ability to negatively influence cell migration, invasion, proliferation and angiogenesis, whilst positively regulating cell adhesion and apoptosis. The expression of maspin has been shown to correlate with an increased survival rate for patients diagnosed with several cancers, including those of the breast and the prostate. The overall aim of this work was to characterise the cellular roles of maspin focusing on its effects on cellular morphology, migration and adhesion and the underlying influence of the extracellular matrix. In addition, we aimed to provide an insight into the currently unclear molecular mechanism that maspin uses to exert its tumour suppressor effects. Insect Drosophila S2 cells were used to express and produce wild type maspin protein. The biological activity of recombinant maspin was confirmed through cell migration and cell adhesion assays. We also manipulated wild type maspin gene using siRNA in mammalian cell lines and used site directed mutagenesis to generate constructs point mutated at sites potentially involved in protein-protein interaction. These mutant constructs were transfected alongside wild type maspin into maspin-null breast and prostate cancer cell lines in an attempt to identify structural motifs critical to maspin's biological function. The addition of recombinant maspin protein or the transfection of wild type maspin gene promoted a mesenchymal-epithelial transition (MET), decreased cell motility and enhanced cell adhesion. Mutation to maspin's α-helix G attenuated its epithelialmesenchymal transition (EMT) and disabled its ability to reduce migration and increase adhesion. The influence of maspin on these cell behaviours was mimicked by wild type and mutant maspin peptides that spanned its α-helical G region; supporting the evidence that this helical structure is important. We also showed that maspin changed the affinity state of β1 integrins; an ability also dependent on its α-helix G. The importance of maspin to mediate its non-metastatic effects, via its α-helix G, were consistent on collagen I, fibronectin and fibrillar matrices. However, an intact α-helical G region was not essential for maspin's ability to decrease cell migration, nor its ability to increase MET and adhesion, when plated onto laminin. Thus, in the presence of laminin, maspin must use a different mechanism of action.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.538225  DOI: Not available
Share: