Gharbi, Severine; (2003) Proteomic analysis of ErbB-2 overexpression in human mammary luminal epithelial cell lines. Doctoral thesis (Ph.D), UCL (University College London).

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Abstract

In the fight against cancer, constant effort is being made to find new markers of the genetic alterations associated with tumourigenesis in order to generate targeted therapies. An important molecular marker of breast cancers is ErbB-2. This receptor tyrosine kinase is found to be over expressed in 20 to 30% of human breast cancers and is now a target for breast cancer therapy. Understanding the role of ErbB-2 and other members of the ErbB receptor family has been the subject of intense effort and much is now known about the signalling pathways activated downstream of the ErbB family members. Despite these efforts, the mechanisms involved in ErbB-2-mediated tumourigenesis are still not fully understood. In order to further understand these mechanisms at the level of protein expression, a global proteomic analysis of ErbB-2 overexpression was carried out using a newly developed gel-based technique, two-dimensional difference gel electrophoresis (2D- DIGE). This approach, which uses differential covalent labelling of protein lysates with fluorphores, was developed and further optimised for sensitive, reproducible and quantitative comparison of protein expression in multiple samples. The differential protein expression occurring in response to ErbB-2 overexpression and growth factor treatment was then examined in a model human mammary luminal epithelial cell system. 2D-DIGE analysis and mass spectrometry were combined for the identification of molecular markers of ErbB-2 overexpression, some of which have been previously implicated in transformation or found to be up-regulated in other types of cancer. Protein identities as well as changes in expression were validated by immunoblotting. Subsequently, several of these proteins were further characterised and their connection with ErbB-2 overexpression investigated. Thus this study showed that 2D-DIGE analysis combined with mass spectrometry represents a powerful approach for the sensitive, reproducible and accurate identification of targets of ErbB-2-mediated cellular transformation. Together with post-genomic studies, this work has provided novel and important information on the regulation of the molecular processes involved in breast carcinogenesis.

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