The long term objective of these studies is to elucidate the mechanisms by which hormones control growth and the expression of differentiated function in the normal mammary gland, and how these regulatory mechanisms have deviated in hormone-dependent breast cancer. Specific emphasis has been placed upon studying prolactin regulation of milk protein gene expression in normal and transformed mammary cell as a unique model system for understanding peptide hormone action on gene expression. These studies will also focus on the multihormonal control of milk protein gene expression by both peptide and steroid hormones and the interplay of these hormones at the transcriptional and post-transcriptional levels. Finally, the mammary gland system provides an excellent model in which to study those factors influencing specific mRNA turnover in mammalian cells. These studies may help elucidate the basic mechanisms by which hormones regulate mammary gland growth and differentiation and thereby enable the development of strategies for the prevention and improved treatment of breast cancer. The hormonal regulation of four well-defined milk protein genes (three members of the casein multi-gene family, and a novel whey acidic protein gene) will be studied in mammary gland explant cultures, in primary cultures of hormone-dependent DMBA-induced tumor cells and following transfection of these genes into several mammalian cell lines. We will complete our studies of the structure, including the role of DNA methylation, the organization and the boundaries of the hormonally-responsive casein and whey acidic protein gene domains. Genomic subclones will be employed with a nuclear """"""""run on"""""""" transcription assay and S1 mapping to determine the transcription units of these genes and determine possible hormonal effects on RNA processing. Pulse-chase studies in explant cultures will be employed to measure the effects of hormones on the individual half-lives of these mRNAs. The role of polyadenylation and the hormonal induction of 2'-5' oligo(A) and subsequent nuclease activation in influencing mRNA stability will be studied. The use of several different gene constructions, vector systems and recipient cell lines should improve the probability of success of gene transfection experiments designed to define DNA sequences involved in hormone regulation. Finally, we will test the validity of a controversial """"""""second messenger"""""""" model of prolactic action.
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