Selective Activation of the Glucocorticoid Receptor by Steroid Antagonists in Human Breast Cancer and Osteosarcoma Cells

Abstract

Steroid hormones regulate the transcription of numerous genes via high affinity receptors that act in concert with chromatin remodeling complexes, coactivators and corepressors. We have compared the activities of a variety of glucocorticoid receptor (GR) antagonists in breast cancer and osteosarcoma cell lines engineered to stably maintain the mouse mammary tumor virus promoter. In both cell types, GR activation by dexamethasone occurs via the disruption of mouse mammary tumor virus chromatin structure and the recruitment of receptor coactivator proteins. However, when challenged with a variety of antagonists the GR displays differential ability to activate transcription within the two cell types. For the breast cancer cells, the antagonists fail to activate the promoter and do not promote the association of the GR with either remodeling or coactivator proteins. In contrast, in osteosarcoma cells, the antiglucocorticoids, RU486 and RU43044, exhibit partial agonist activity. The capacity of these antagonists to stimulate transcription in the osteosarcoma cells is reflected in the ability of the RU486-bound receptor to remodel chromatin and associate with chromatin-remodeling proteins. Similarly, the observation that the RU486-bound receptor does not fully activate transcription is consistent with its inability to recruit receptor coactivator proteins.

Glucocorticoid receptor-mediated chromatin remodeling in vivo

“…The most prominent of these di erences being the lack of BAF250, a component known to directly interact with the GR, in the breast cancer cells. In contrast, RU486 was unable to induce GR binding with SRC1, p300/CBP, or pCIP in either of the cell lines as would be predicted from previous interaction studies (Collingwood et al, 1999;Fryer et al, 2000;Nie et al, 2000). The ability of the RU486-bound GR to bind BRG1 but not the coactivators parallels the weak agonist activity of RU486 compared to the glucocorticoid, dexamethasone.…”

Global Changes in and Characterization of Specific Sites of Phosphorylation in Mouse and Human Histone H1 Isoforms upon CDK Inhibitor Treatment Using Mass Spectrometry

“…In the cell line used for this work (UL3 cells derived from the osteosarcoma cell line U2OS 21 ), we found that only three isoforms of H1 were expressed, H1.0, H1.2, and H1.4. Using mass spectrometry, we show that both the H1.2 and H1.4 isoforms in UL3 cells are phosphorylated, and that phosphorylation is reduced in the presence of dexamethasone.…”

“…These structurally informative fragment ions allow the assignment of the site of phosphorylation to Ser-172 and Ser-187 in these two peptides, respectively. Figure 6C shows the MS/MS spectrum of the (M + H) + ion of m/z 524.28 which corresponds in mass to monophosphorylated tryptic peptide 2 (T2), TPVK (aa [18][19][20][21]. Although the S/N ratio in this spectrum is low, structurally diagnostic ions, especially y 3 and b 3 -98, are observed.…”

“…15,21 Cells were grown at 37 °C in Dulbecco's Modified Eagle's medium (DMEM) with 10% fetal bovine serum (Hyclone), penicillin-streptomycin (Invitrogen), and 6% CO 2 . Drug treatments were initiated when cells were growing exponentially.…”

Nuclear receptors and chromatin remodeling machinery

“…By contrast, the interaction between GR and BAF250 seems to be dependent on the presence of ligand . Although, BAF250 has been shown to be involved in GR-mediated transcriptional activity on unorganized chromatin, this subunit may be dispensable for GR-mediated chromatin remodeling at the MMTV promoter given that the remodeling process is equally as efficient in the human breast cancer cell line, T47D, which expresses BAF180 instead of BAF250 (Inoue et al, 2002;Fryer et al, 2000). Interestingly, data from the SW-13 cell line, which expresses BAF250 but not BAF180, suggest the BAF180 subunit is not required for GR-mediated remodeling at chromatin MMTV because the remodeling process is supported upon reintroduction of BRG1 (Trotter and Archer, 2004).…”