Steroid receptors are gene regulatory proteins that provide an important model in eukaryotes to study gene transcription regulation. In recognition of the role of sex steroids in mammary cancer, study of progesterone regulated gene expression in T47D human breast cancer cells provides a relevant system with which to investigate regulatory mechanisms. Recently we, and others, have shown that in T47D cells, progestins regulate transcription directed by the mouse mammary tumor virus (MMTV) promoter/enhancer. We will exploit MMTV to investigate the fine structural details of the molecular interaction of human progesterone receptors (PR) with regulatory sequences. Toward this end we will (i) purify progesterone receptors using immunoaffinity methods and monoclonal antibodies that we have developed. PR will be purified in several forms: ligand-bound or unbound, transformed or untransformed, A and B receptors separately or together, complexed with heat shock proteins or free of this association. Heat shock proteins and another potential accessory protein, nuclear factor-one, will also be purified for these studies. These different receptor forms and potential accessory proteins will be employed (ii) to perform a detailed binding analysis with progesterone response elements from mouse mammary tumor virus. We will employ several types of assays including DNA-gel shift assays to quantitate binding affinities and association/dissociation rate constants of the various receptor forms with and without accessory proteins. (iii) A characterized library of mutant receptor recognition sequences will be examined for biological activity in T47D cells using a rapid, sensitive DNA-mediated gene transfer assay and a vector constructed expressly for this purpose. Specific binding parameters of the mutant receptor recognition sequences will be assayed in parallel. The expression and binding data with the mutant elements together will be used to assemble a map of the residues critical for receptor recognition and for hormone response. (iv) The induction by progestins of MMTV-directed gene expression will be utilized in a scheme to select for T47D cell variants resistant to progesterone action. Resistant cells will be characterized for the molecular basis of resistance by use of our anti-receptor monoclonal antibodies.
Showing the most recent 10 out of 46 publications
