The overall goal of this proposal is to elucidate the mechanism of action of polyamines in estrogen-mediated growth of breast cancer cells. Polyamines are organic cations present in all cells. Estrogens play a key role in the origin and progression of breast cancer and an antiestrogen, tamoxifen, is the most effective form of therapy for a subset of human breast cancer. Mechanism of action of estrogen involves the interaction of the hormone to a specific intracellular protein, estrogen receptor (ER) followed by the recognition of ER to specific DNA sequences in the genome. Recognition of ER by these sequences, estrogen response elements (EREs), stimulates the transcription of responsive genes, DNA synthesis, and cell division. However, factors that modulate this interaction and the conformational alterations that are involved in transcriptional control of estrogenic function are not known. Previous studies from our laboratory and others have shown that polyamines play an essential role in estrogenic function. We hypothesize that polyamines are involved in the formation of high affinity complexes with ERE and thus modulate breast cancer cell growth. To test this hypothesis, we will quantity the effects of polyamines on ER-ERE interaction in terms of equilibrium binding constants and conformational alterations of ERE, using oligonucleotides and plasmids containing EREs. Polyamine structural specificity will be examined using homologs and derivatives of polyamines. The ability of polyamines and ER to provoke conformational alterations in ERE will be studied by circular dichroism spectroscopy, enzyme-linked immunosorbent assay, gel electrophoresis, and by chemical probes that detect single-stranded and left-handed (Z-DNA) regions in ERE. Polyamine analogs that do not support ER-ERE interactions will be studied in tissue culture experiments with an estrogen-responsive breast cancer cell line MCF-7. Possible additive/synergistic growth inhibitory effects of polyamine analogs will be studied in combination with tamoxifen. The uptake and mechanism of action of polyamine analogs will be further explored in terms of their ability to alter polyamine biosynthetic enzymes and inhibit cell cycle progression. An understanding of the mechanism of action of polyamines in ER-ERE recognition is critical to the understanding of transcriptional control of gene expression and cell growth in breast cancer. This information is important to the development of novel strategies for the treatment of breast cancer.
