Steroid hormone and growth factor action in normal and cancer cell

The endometrium is an epithelio-mesenchymal structure. The growth and differentiation of normal endometrial cells is regulated by the interactions of both polypeptide and steroid hormones. An understanding of how hormones affect proliferation and metabolic activities, necessarily involves knowledge of the regulatory processes of these cellular functions. The regulation of the frequency of replication in animal cells is a complex phenomenon for which much of the molecular basis is currently unknown. Cell cultures, which are exempt from tissue and systemic influences are ideal for investigating whether individual growth factors are acting directly on the endometrial cell. Primary cultures of both normal and malignant epithelial cells were investigated. These cells have been grown both as pure epithelial cultures and as mixed populations of epithelial and stromal cells. The purpose of this study was first, to evaluate some of the critical data now available on various aspects of steroid regulated growth, and second to develop in vitro new models of steroid promoted growth that can accommodate current information. Different cell types, from both rat uterus and human endometrium, were separated by enzymatic and mechanical techniques. Epithelial cells were tentatively identified by comparison of their morphologic features in culture with the well documented features of endometrial cells in vivo. Moreover, epithelial nature was also confirmed using immunocytochemical criteria. Growth dynamics of these cells in culture were analysed by the classical techniques of 3H-thymidine incorporation, cell proliferation index and morphological criteria. Serum performs many functions in cell culture. In addition to providing classical hormones and growth factors, it compensates for the deficiences of defined media, supplying additional nutrients and trace elements. To determine the minimum requirements for foetal calf serum in cultures of normal and malignant cells derived from endometrium, this study reports a detailed examination of growth requirements of endometrial cells during the early culture. The results presented in Chapter 4 strongly suggest that it is possible to eliminate serum from culture medium and that the main function of serum in cell culture is to furnish hormones and other growth factors. The most important factors are EGF, transferrin, insulin and glucocorticoids. The role of serum in attachment and spreading of these cells was also assessed. Charcoal stripping of serum at 4C enhanced the attachment, whereas, the same treatment of serum at 56°C depressed attachment and spreading of normal epithelial cells. Increased growth was observed in endometrial cancer cells in 56°C-stripped serum. Coating plastic substrates with different preparations increased the speed of attachment of the cells. Culture conditions that were employed resulted in immediate selection of epithelial cells that were able to grow in response to specific growth factors. The presence of significant but unknown concentrations of biologically available hormones in serum obviously hampers attribution of a response to a particular growth factor(s). In a detailed study of serum involvement in steroid responsiveness, two diverse observations were made. Firstly, serum seemed to be essential for the inhibitory effects of certain hormones. Secondly stimulation of growth by particular growth factors was most readily achieved in serum-free conditions. Oestradiol stimulated the rate of DNA synthesis in presence of charcoal-stripped serum or serum-free medium. Tamoxifen and progesterone showed wide ranging effects, depending on their concentrations and the serum levels. Medroxyprogesterone Acetate (MPA 10.;-7 M) required 10% serum to show inhibitory effects. Glucocorticoidsstimulated the process regardless of serum levels.