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Analysis of Insulin-Like Growth Factor-II in Human Breast Cancer Cells.

Lee, Adrian. (1993) Analysis of Insulin-Like Growth Factor-II in Human Breast Cancer Cells. Doctoral thesis, University of Surrey (United Kingdom)..

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Abstract

This thesis describes the detection and partial characterisation of large precursor forms of insulin-like growth factor II (IGF-II), secreted by MCF-7 human breast cancer cells transfected with IGF-II cDNA (MI7) or control vector (MN2). Due to technical difficulties with radioimmunoassay, and the fact that this method provides no information on the size or nature of secreted IGF-II, an immunoblotting technique has been developed and optimised, using a commercially available monoclonal antibody. The technique is specific, sensitive, and semi-quantitative, by the use of enhanced chemiluminescence. MI7 cells secreted IGF-II in large precursor forms (22 and 15kDa) and the mature 7kDa growth factor, whilst control cells (MN2) secreted no detectable IGF-II. Conditioned media from MI7 cells caused proliferation of MCF-7 cells, and the 15kDa form was separated from the 22 and 7kDa IGF-II by cation-exchange chromatography. Secretion of large precursors may be due to saturation of the processing machinery normally involved in cleavage of prepro-IGF-II to the mature growth factor, and these data are discussed in relation to processing of IGF-II, and the biological activity of large precursors. In contrast, four non-transfected breast cancer cell lines secreted mainly mature 7kDa IGF-II, with trace amounts of a large 15kDa form. Oestradiol induced secretion of mature IGF-II in oestrogen-responsive cells (T47D and MCF-7 McG), whilst having no effect on higher constitutive levels in oestrogen-unresponsive MDA-231 and HBL-100 cells. MCF-7 (9) cells rendered oestrogen-unresponsive by transfection with a mixed cDNA library also secreted large precursors of IGF-II, whilst oestrogen-responsive wild-type cells secreted no IGF-II. Conditioned media from two ovarian cancer cell lines contained large precursors of IGF-II, PEO14 oestrogen receptor negative cells secreting higher levels of IGF-II (22, 15 and 7kDa), than PE04 oestrogen receptor positive cells which only secreted 15 and 7kDa IGF-II. The levels of IGF-H were higher when conditioned media was prepared in 5% serum than with serum-free media. These data are discussed in relation to the role of IGF-II in oestrogen-mediated proliferation and progression to hormone-independence.

Item Type: Thesis (Doctoral)
Divisions : Theses
Authors : Lee, Adrian.
Date : 1993
Additional Information : Thesis (Ph.D.)--University of Surrey (United Kingdom), 1993.
Depositing User : EPrints Services
Date Deposited : 06 May 2020 12:15
Last Modified : 06 May 2020 12:17
URI: http://epubs.surrey.ac.uk/id/eprint/855776

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