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Title: Studies of epidermal growth factors in prostate cancer using the cell lines DU 145 & LNCaP
Author: MacDonald, Anne
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1990
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Epidermal growth factor (EGF) receptor expression and modulation of growth rate by EGF were investigated in the androgen insensitive Du 145 and androgen sensitive LNCaP prostatic carcinoma cell lines. Competition and saturation analysis of binding data revealed one high affinity binding site for both DU 145 (lnmol/l±0.6) and LNCaP (2.8nmol/l±2.2), with DU 145 cells expressing high levels of receptor binding sites/cell (2.5xlOs±lxl06) and LNCaP cells expressing 10 fold lower receptor binding sites/cell (2xl04±lxl04). Addition of exogenous EGF or TGFa to scrum free cultures of DU 145 cells only minimally affected [3H]-Thymidine incorporation and cell proliferation, whereas EGF was a potent mitogen for LNCaP cells. Growth of LNCaP cells was also stimulated by the synthetic androgen Mibolcronc, but no additive effect on growth was observed when EGF and Mibolcrone were added together. Moreover, pre-incubation of LNCaP cells with Mibolerone did not affect either the number or affinity of the EGF receptor. Further characterization of the EGF receptor demonstrated time and temperature dependence of EGF binding to its receptor. The dissociation of EGF from the receptor was linear over the time period studied and binding sites on both cell lines were down regulated by pre-treatment with EGF. Specificity studies with ligands other than mEGF confirmed specificity, with only TGFa and hEGF competing with [125I]-mEGF for binding to the receptor. The presence of the EGF receptor was also verified by Western blotting. Cell lyzates from DU 145 and LNCaP cells were separated by sodium dodecylsulphate polyacrylamidc gel clectrophoresis (SDS-PAGE) and subsequently analysed for the presence of the EGF receptor using monoclonal antibodies specific for the native external domain and the internal domain of the EGF receptor. A band of molecular weight 170 kDa (which is the molecular weight of the EGF receptor) was visualized with DU 145 cell lyzates, but not from LNCaP cell lyzates. This was probably due to the low numbers of receptor binding sites expressed by this cell line. The truncated, v-erb B gene product was not detected from either cell line. The secretion of growth factors by some transformed cells is thought to enable these cells to proliferate in low serum concentrations as well as reducing the dependency upon exogenous growth factors. Since the growth of the DU 145 cell line was only minimally affected by exogenous EGF, serum free media conditioned by this cell line was analysed for growth factor production. EGF-like immunological activity as well as competitive activity in EGF-radio-receptor assays (RRA) was detected in the conditioned medium (CM) of these cells. Factors in the CM also inhibited and stimulated DNA synthesis of these cells and this effect was dose-responsive. Fractionation of the concentrated CM by gel filtration and reverse-phase high performance liquid chromatography (rHPLC) revealed several peaks of EGF-like competitive activity. One peak of rTGF-I immunological activity, which was separated by rHPLC, also demonstrated EGF-RRA competitive activity. None of the peaks demonstrating EGF-RRA competitive activity were related immunologically to hEGF.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available