The long term objective of the proposed research is to define the mechanism by which methyl p-hydroxyphenylactate (MeHPLA) controls mammalian cell proliferation. Our laboratory identified MeHPLA as an endogenous ligand for nuclear type II [3H]estradiol binding sites and the data suggest that this bioflavonoid or tyrosine metabolite inhibits cell growth through this binding interaction. Type II sites appear closely coupled to DNA replication and cellular proliferation, however, the precise function of this nuclear matrix protein in normal and abnormal cells is unknown. The overall goal of this project is to define the functions of MeHPLA and type II sites in normal and malignant cells and to study type II gene structure and expression as it relates to hormonal (MeHPLA, estrogen, antiestrogen) modulation of cellular proliferation. In order to accomplish these goals, our laboratory has developed techniques for the solubilization of type II sites from rat uterine nuclei and this protein has been purified to near homogeneity by DNA cellulose chromatography and ligand affinity chromatography on 2,6- bis((3-methoxy-4-hydroxyphenyl)-methylene)-cyclohexanone-Sepharose (GT- 18-Sepharose). SDS-PAGE analysis of GT-18-Sepharose purified material suggests the putative type II site is a 50 kDa protein which enriches in these preparations with the type II binding activity. Furthermore, like type II site binding activity, the 50 kDa protein is induced in the uterus by estrogen treatment. We are currently immunizing mice with highly purified type antigen (50 kDa protein) to generate antibodies to this nuclear MeHPLA binding site. A major goal of the proposed studies is to purify the type II site to homogeneity and obtain partial amino acid sequences for this protein which will be used for the generation of oligonucleotide and antipeptide antibody probes to the type II site (Specific Aim A). These oligonucleotide probes and antibodies will be used to screen a cDNA library prepared from estrogen-treated rat uterine tissue to identify type II site cDNA sequence and characterize this gene(s) (Specific Aim B). The cDNA and antibody probes will also be utilized to study estrogen, MeHPLA, and antiestrogen (tamoxifen, ICI- 164,384, ICI-182,780), modulation of type II gene expression and subsequent effects on cell proliferation in the rat uterus as well as in MCF-7 (ER+) and MDA-468 (ER-) human breast cancer cells in vitro (Specific Aim C). The availability of these molecular probes will also facilitate future structure/function analyses which will define ligand binding and/or other functional domains of this nuclear regulatory protein.
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