Hormonal regulation of genes involved in purine and pyrimidine biosynthesis and metabolism in human breast cancer cells

ADA gene expression is induced by E2 in MCF-7 human breast cancer cells, whereas the antiestrogens 4-OH-T and ICI 182,780 exhibit estrogen receptor antagonist activities. In transient transfection studies with pADA211 which contains the −211 to +11 region of the ADA gene promoter linked to CAT reporter gene, E2 induced CAT activity. Ligand-induced transactivation was observed only in cells cotransfected with expression plasmids for wild-type ER or HE11 but not with HE15 or HE19. Deletion analysis of the ADA gene promoter showed that Sp1 binding site IV (−79 to −73) was primarily responsible for hormone-responsiveness.; IGF also induces ADA gene expression in MCF-7 cells. Deletion analysis demonstrates that IGF activates ERα/Sp1 interactions with multiple GC-rich sites in the promoter and this response is abrogated in cells transfected with ERα containing mutations at serine-118 or serine-163. IGF induces both MAPK and PI3-kinase phosphorylation cascades in MCF-7 cells; however, our results show that induction of ADA by IGF activation of ERα/Sp1 is dependent on the MAPK pathway.; E2 induces TS gene expression in MCF-7 cells and only the G-rich motif (−150 to −142) was required for E2 action. Gel mobility shift and in vitro DNA footprinting assays showed that both ERα and Sp1 proteins were required for hormone-induced transactivation that involved ERα/Sp1 binding to the G-rich site in which only Sp1 protein bound DNA. Both proteins also interacted in Drosophila cells in functional assays confirming the transcriptional activation of TS involved ERα/Sp1 interaction.; E2 induces DHFR gene expression in MCF-7 cells and this induction is blocked by cycloheximide. The E2-responsiveness required the −270 to +20 promoter region containing four G-rich sequences and an E2F binding site; however, only the E2F motif was required for E2 action. E2 increased binding of the E2F protein to the E2F motif in gel mobility shift assays. Western blot analysis showed that hormone induction of E2F is required for the subsequent induction of DHFR gene expression by E2. Studies using E2F expression plasmid and dominant negative E2F-1, DP-1 and Sp1 plasmids showed that DHFR activation by E2 was dependent on induction of E2F-1.