The goals of this proposal are to clone putative glucocorticoid (G) regulated tumor suppressor or negative growth regulatory gene(s) that mediate cell growth arrest and examine how they are regulated by glucocorticoids (Gs).
Aim 1 is to clone cDNAs transcriptionally regulated by glucocorticoid (G) in DDT1 MF2 cells using subtractive hybridization procedures. Candidate genes will be directionally inserted into expression vectors under the control of a glucocorticoid (G) dependent promoter and stably transfected into DDT1 MF2 cells followed by direct selection for growth in the presence of glucocorticoid (G).
Aim 2 is to characterize candidate gene(s) cloned from Aim 1. The cloned cDNAs will be subcloned into vectors with glucocorticoid (G) independent promoters for analysis by transfection into DDT1 MF2 and DDT1 MF2 GR cells. Concurrently, candidate gene(s) and their products will be characterized by sequencing, southern and northern blotting, and immunoblotting.
Aim 3 is to study the regulatory mechanisms of the gene(s) that mediate cell growth arrest. Hormonal regulatory mechanisms will be studied by characterizing the features of cis-regulatory elements that respond to glucocorticoids (Gs) or glucocorticoid (G) regulated trans-acting proteins.
