The goal of this proposal is to understand the role of naturally occurring polyamines in the function of estrogens in breast cancer. A combination of tissue culture, physico-chemical and recombinant DNA techniques will be used to achieve this goal. Two hormone-responsive cell lines (MCF-7 and T-47D) and two hormone-unresponsive cell lines (BT-20 and MDA-MB-231), all of human breast cancer origin, will be used for this study. The estrogenic stimulation of a polyamine biosynthetic enzyme, ornithine decarboxylase (ODC) will be studied by measuring the levels of ODC and polyamines as a function of estrogen administration. The antiproliferative effect of the ODC inhibitor, Alpha-difluromethylornithine (DFMO) will be examined in combination with antiestrogens on hormone responsive cell lines and with cisplatin, adriamycin, and their analogs on hormone-unresponsive cells. The role of polyamines on estrogen induced cell growth will be studied by correlating the synthesis of ODC with that of DNA, RNA and specific estrogen-induced proteins, such as 24 and 52K proteins. In order to understand the molecular mechanism of the action of polyamines on estrogen function, the stabilization and activation of estrogen receptors (ER) will be studied by density gradient analysis, DNA-cellulose competition assay and ligand dissociation rate in the presence of polyamines, and their derivatives. ER binding to the right-handed B-DNA and the left-handed Z-DNA will be examined to gain insight into the effect of polyamine-induced conformational transitions of DNA on ER-DNA interactions. These studies will be complemented by a mapping of the ER binding sites on DNA in the presence of polyamines. At the molecular level, the regulation of ODC gene by estrogen in human breast cancer will be studied using human ODC cDNA. Nuclear transcription assays, dot-blot analysis, Northern transfer and S1 nuclease analysis will be carried out to understand the transcriptional regulation of ODC in hormone-responsive and hormone-unresponsive cell lines. The results generated from these studies will advance our knowledge of the hormonal regulation of gene expression in breast cancer. These studies will also provide significant information for the development of novel therapeutic approaches involving polyamine inhibitors in combination with antiestrogens/antitumor drugs.
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