The long-term objective of the proposed research is to define the mechanism by which methyl p-hydrophenylactate (MeHPLA) controls mammalian cell proliferation. Our laboratory identified MeHPLA as an endogenous ligand for nuclear type II [3H] estradiol binding sites and this bioflavonoid or tyrosine metabolite appears to inhibit cell proliferation 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 goal of this project is to define the function 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, anti-estrogen) modulation of cellular proliferation. To accomplish these goals we have solubilized type II sites from rat uterine nuclear matrix and have purified the [3H] estradiol binding activity to near homogeneity to near homogeneity by dye ligand affinity chromatography (Affigel Blue) and HPLC. SDS-PAGE analysis of this highly purified material revealed a single 37 kDa protein which co-puries with the type II site [3H] estradiol binding activity. Affinity labeling studies indicate [3H] estradiol to the 37 kDa protein is blocked by the bioflavonoid luteolin and this protein is induced in the uterus by estrogen. Based upon our initial sequencing studies which have identified 10 amino acids of internal sequence, it appears that the 37 kDA protein may be unique. A major goal of this revised application is to obtain more exclusive amino acid sequence analysis of the 37 kDa protein to be used for the generation of oligonucleotide and anti- peptide antibody probes to the type II site (Specific Aim 1). 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 sequences and characterizes the gene(s) (Specific Aim 2). The cDNA and antibody probes will also be utilized to study estrogen, MeHPLA, and anti-estrogen (tamoxifen, ICI-164, 384 ICI- 182,780) modulation of type II gene expression and subsequent effects on cell proliferation in the rat uterus and in MCF-7 (ER+) and MDA-468 ( ER-) human breast cancer cells in vitro (Specific Aim 3). The availability of these molecular probes will facilitate future structure/function analyses which will define ligand binding and/or other functional domains of this nuclear regulatory protein(s).
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