Flow cytometry analysis confirmed the immunocytochemical counts. R115,777 plus Tam significantly increased the M30 positive cell population. The M30 positive cell population was much smaller following treatment with either R115,777 or Tam alone. These results were obtained in three independent experiments with little intra assay varia tions. however, the difficulty of cell counting has prevented our carrying out the extensive series of experiments required for isobologram constructions. Overall, these data show that R115,777, which induces neg ligible apoptosis by itself, when combined with Tam results in significant apoptosis induction in MCF 7 cells.

To differentiate between the involvement of the ER and AEBS pathways in this Tam effect, we compared the apoptosis inducing activities of ER or AEBS selective ligands Effects of R115,777 and different anti estrogens on caspase cleavage To define any possible involvement of the ER or AEBS path ways in apoptosis induction, a set of experiments similar to those described in Fig. 3c were performed using R115,777 in association with three different anti estrogens, Tam, ICI182,780 or PBPE. Flow cytometric analyses were per formed following M30 staining of MCF 7 cells treated for 5 days with these three different combinations of agents. As shown in Fig. 4, 10 ?M PBPE was able to increase the number of M30 positive cells to the same extent as 5 ?M Tam. In contrast, 5 nM ICI182,780 treatment resulted in only modest cytokeratin 18 cleavage.

The com bination of R115,777 with ICI182,780 resulted in slightly increased M30 staining compared with R115,777 treatment alone, suggesting that ER is only minimally involved in the synergistic induction of apoptosis resulting from exposure to the combination of R115,777 and Tam. A similar experiment revealed that the combination of R115,777 with Tam was as efficient in inducing M30 staining as was R115,777 plus PBPE, suggesting that the major part of the apoptosis induc ing effect of Tam either alone or in association with R115,777 is under the control of the AEBS pathway. These data, therefore, would be consistent with the proposal that the first level of cross talk between the two agents would be that one increases the intracellular concentration of the other, while the second level would be that one binds to the target of the other.

To examine these proposals we Entinostat first used bindings assays and verified that R115,777, like FTI 277, does not interact either with ERs or with AEBS. R115,777 does not affect the cellular uptake of tamoxifen We next examined whether R115,777 was able to increase the cellular uptake of Tam. Exposure to increasing concentra tions of R115,777 did not modify the cellular level of tritiated Tam in MCF 7 cells. Thus, the effects of combining R115,777 with Tam were not attrib utable to any increase of cellular Tam concentrations by the FTI.