Transcription of mouse mammary tumor virus (MMTV) RNA from integrated proviral DNA is stimulated rapidly and specifically by glucocorticoids. Molecular and genetic approaches will be used to identify genes and gene products that are involved in glucocorticoid regulation of MMTV gene expression. A procedure employing ribonucleoside (Beta-S)triphosphates, Hg-Sepharose column chromatography, and nucleic acid filter hybridization has been established for measuring initiation of new MMTV RNA chains in preparations of cell nuclei. This cell free system will be used to determine whether glucocorticoids control MMTV RNA synthesis at the level of transcription initiation. The sites of initiation of MMTV RNA chains in vitro and in vivo will be compared by S1 nuclease mapping. Nuclei from MMTV-infected cells or, alternatively, nuclei from cells transfected with SV40/MMTV recombinant vectors, will be used to establish a system in which initiation of MMTV RNA chains is stimulated by the addition of glucocorticoid hormone and receptors. A combination of immunological and pharmacological procedures will be used to select novel glucocorticoid response variants from clones of MMTV-infected mouse lymphoma cells with two responses: stimulation of MMTV gene expression and glucocorticoid-induced cytolysis. Specific programs are proposed for obtaining and characterizing (biochemically and genetically) variants with lesions in MMTV structural genes, MMTV DNA sequences that regulate transcription and hormone inducibility, cellular genes necessary for posttranscriptional processing of MMTV proteins, and other cellular genes (including but not limited to the glucocorticoid receptor) that may be necessary for each hormone response. Programmed regulation of gene expression is essential for the normal development of an organism. Cancer is caused by a breakdown in the normal control of specific genes. The proposed project will increase understanding of the role of hormones in development, differentiation, and malignancy.
