The long term objective of the proposed research is to precisely define the function of type II binding sites in normal and malignant cells. Nuclear type II sites bind methyl p-hydroxyphenyllactate (MeHPLA) and this compound appears to regulate normal and malignant cell growth through this binding interaction. Although type II binding sites may be closely coupled to DNA replication and cellular proliferation, the precise function of this nuclear protein and MeHPLA in normal and malignant cell regulation is unknown. The general goal of this proposal is to study type II gene structure and expression and subsequent effects on cellular proliferation. In order to accomplish this goal, the following aims are proposed: determine the biological origin of MeHPLA in mammalian cells, synthesize an affinity ligand, and purify nuclear type II sites from the rat or rabbit uterus. Type II sites will be extracted from rat or rabbit uterine nuclei and purified by chromatographic techniques for separation based on molecular size, charge and ligand binding specificity (affinity chromatography). Monoclonal and polyclonal antibodies will be generated against purified type II antigen and these immunological probes will be employed for qualitative and quantitative analysis of type II sites and the estrogen receptor. Type II antibodies and oligonucleotide probes will be used to screen cDNA libraries for the type II gene(s). The gene will be characterized and the cDNA utilized as a probe to study estrogen, MeHPLA, tamoxifen and carcinogen (dimethylbenz[a]anthracene) modulation of type II gene expression in normal (rat uterus) and malignant cells in vitro (human breast cancer cells) and in vivo (rat and mouse mammary tumor).
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