In contrast to the well characterized primary responses of glucocorticoid hormones receptor interaction with viral and cellular target genes, secondary responses, requiring protein synthesis, are poorly understood. This proposal addresses at two levels the mechanism of glucocorticoid inhibition of cell growth, a secondary glucocorticoid induced response in the DDT1 MF-2 smooth muscle tumor cell line. First, it will be determined if expression of p29 protein, a secondary response to glucocorticoid administration, is directly related to cell cycle arrest in DDT1 MF-2 cells. Second, the mechanism(s) regulating p29 gene expression will be investigated. There are three interrelated specific aims to accomplish these objectives.
SPECIFIC AIM 1 is to clone and characterize p29 cDNA. Cloning is accomplished by differential hybridization and/or oligonucleotide screening of a lambda gt-10 library. The cDNA will be identified by immunoprecipitation of cell-free translation products synthesized using hybrid selected mRNA. cDNA's will be sequenced and further characterized.
SPECIFIC AIM 2 will be to determine p29's biological role in cell cycle arrest through introduction of sense and antisense vectors. Evolutionary conservation of the gene will be examined by Northern and Southern blotting.
SPECIFIC AIM 3 is to clone the p29 gene from an EMBL-3 DDT1 MF-2 genomic library. The gene will be characterized by restriction and heteroduplex mapping. The 5' end of the gene will be identified, sequenced and hormonal regulation of the enhancer-promoter will be studied. The long term goal to be realized from this grant is to purify and clone the glucocorticoid regulated protein(s) that regulate p29 gene expression. Elucidation of the pathways controlling cell growth will be a framework to allow for rational design of intervention protocols for the control of cancer and will also provide a much deeper understanding of the mechanisms of cell differentiation.
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