Daly-Burns, B.; (2004) Development of an in-vitro model of human prostate. Masters thesis , University of London.

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Abstract

The mechanisms controlling the growth and development of Benign Prostate Hyperplasia (BPH), the most common non-malignant disorder found in men over the age of 70, are poorly understood. There is a shortage of relevant "in vitro" models suitable for studying this disease. The aim of this project was to develop a representative "in vitro" model system for the study of BPH. Epithelial and fibroblast cell lines (Pre2.8 and S2.13 respectively) were derived from the same biopsy of BPH and immortalised using the temperature sensitive SV40 large-T antigen construct. At 33 C the cells grow progressively under the influence of the SV40 large T-antigen, but at 39 C the conformation of the protein changes and the protein is no longer functional, so the cells stop dividing and are able to differentiate. When monolayer cultures were switched from 33 C to 37 C or 39 C, changes in morphology, cell size distribution and a reduction in cell proliferation were observed. Both cell lines expressed the 5a-reductase type I enzyme, but did not express androgen receptor (AR) and did not secrete PSA and PAP, hence they are likely to be undifferentiated cells. Pre2.8 epithelial cells have a basal cell phenotype at 33 C and undergo limited differentiation at 37 C and 39 C. DNA profiling was used to confirm the origin of the cells. The cell lines were shown to be Mycoplasma-free. Both cell lines showed amplification of DNA on chromosome 20q, a region known to be associated with cell immortalisation. In order to develop a representative model of BPH, Pre2.8 and S2.13 cells were mixed in 3-dimensional matrigel cultures and optimised in order to obtain the most suitable culture conditions to produce prostate-specific characteristics. The three-dimensional culture system consisted of epithelial cells surrounded by a layer of stromal cells and in some cases with the formation of acinus like structures. The 3-D model showed some prostate-specific characteristics, for instance the epithelial cells expressed the androgen receptor. The epithelial cells differentiated towards a luminal phenotype, expressing K8 and 18. In conclusion, a matched pair of epithelial and stromal BPH cell lines has been established which show some typical characteristics of prostate cells and have been grown m a three-dimensional culture The model may be useful for studying the cell interactions that control the growth of benign prostate Hyperplasia.

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