The overall goal of this application is to investigate the role of polyamines in the estrogenic regulation of breast cancer cell proliferation. Previous studies demonstrated that polyamines alter the binding of estrogen receptor (ER) to estrogen response elements (ERE). Recent studies have described the polyamine induced DNA conformational changes producing B* DNA in the ERE sequences which in turn facilitate the formation of ER/ERE complexes. The results suggest that polyamines play an important role in estrogen action on cell cycle kinetics and the regulation cell cycle proteins, such as cyclin D1. The main hypothesis to be tested is that polyamines have a regulatory role in breast cancer cell growth through their ability to modulate ER/ERE binding and alter the function cell cycle machinery. With the goals of testing this hypothesis and developing therapeutically active agents, the role of polyamines in ER/ERE interactions will be examined using: (i) circular dichroism spectroscopy to determine the ERE sequence and polyamine structural requirements which must be satisfied for the induction of B* conformation, (ii) electrophoretic mobility shift assays and competitive ER/ERE binding assays to quantify the effects of polyamine and polyamine analogue on ER/ERE-complex formation, and (iii) DNA-footprint analysis to localize the polyamine-induced conformational changes at the ER binding site. Efficacy in modulation ER/ERE binding will be used as a first test to identify the therapeutic potential of a new class of cyclopolyamines. Selected analogues will be investigated for their effects on: (i) proliferation and cell cycle kinetics, (ii) cellular polyamine levels and regulation of polyamine metabolic enzymes, and (iii) alteration in polyamine transport in the estrogen responsive breast cancer line, MCF-7. The mechanism of polyamine analogues in altering cell cycle kinetics will be examined using the regulation of cyclin D1 mRNA and protein as targets. Interactions between polyamine analogues and cyclin D1 will be further investigated in MCF-7 cells over-expressing cyclin D1. Novel aspects of estrogenic signal transduction through ER-associated proteins will be examined by characterization of polyamine analogue and estrogen- responsiveness of these proteins and determination of their role in cell growth. Results of these studies will provide new insights into the mechanism of action of polyamines in estrogen function in breast cancer and contribute to the identification and design of novel therapeutic agents.
