Using this tool, we compared ligand dependent upregulated genes in cells stably expressing either WT or KR recep tors. Upon progestin treatment, SUMO deficient PR, but not WT, significantly upregulated gene sets assigned to multiple proliferative and pro survival biological func tions. We showed that breast can selleck bio cer cells stably expressing SUMO deficient PR Inhibitors,Modulators,Libraries exhibit increased growth in soft agar relative to cells stably expressing either WT or phosphorylation deficient Inhibitors,Modulators,Libraries S294A PR. We performed MTT proliferation assays using our inducible models. The advantage Inhibitors,Modulators,Libraries of this isogenic system is the elimination of clonal variation in cell growth death rates and phenoty pic drift that can occur in stable cell line models.

Cells were plated at equal density on day zero and treated with or without the AP21967 compound to induce PR expression, prior to exposure to either vehicle or progestin. Progestin treated cells expressing iWT or iKR PRs grew faster than their un induced or untreated Inhibitors,Modulators,Libraries counterparts. However, by day six of continu ous exposure to both AP21967 and R5020, significantly more cells were present in cultures expressing iKR rela tive to those expressing iWT receptors, while all control groups remained very similar. Western blotting demon strated that inducible PR expression was sustained when AP21967 was added to the cell culture media and that comparable levels of iWT and iKR PR protein were expressed. MTT assays measure viable cells over time and PRs have been implicated in breast cancer cell pro survival. Thus, we also measured cleavage of PARP as an indirect indicator of apoptosis.

Inhibitors,Modulators,Libraries PARP is tar geted for cleavage at Asp214 by activated caspase 3 and is a sensitive measure of committed apoptotic signaling. PR expression was induced by AP21967 treatment and cells were pre treated with R5020 for six hours to activate the respective iWT or SUMO deficient iKR gene expression programs. Following R5020 pre treat ment, doxorubicin was added to the cell culture med ium to induce apoptosis for one day, after which the cell lysate was harvested and the relative levels of cleaved PARP were measured by western blotting. Notably, doxorubicin treated cells expressing SUMO deficient iKR PR had reduced levels of PARP cleavage relative to cells expressing iWT PR, especially in cells nearly pre treated with R5020. Doxorubicin treatment reduced both WT and KR PR protein expression. However, in multiple repeat experiments normalized to protein expression changes, cells expressing iKR PR consistently exhibited reduced PARP cleavage relative to cells expressing iWT PR. These findings were validated in T47D cells stably expressing PRs.