Related to this, our group and others have detected consistent ex pression http://www.selleckchem.com/products/dorsomorphin-2hcl.html of ER mRNA in MDA MB 231 cells. however, whether this expression may be related to membrane bound ER is actually unknown. More recently, Zhang and co workers indicated that 4OHT promoted the proliferation of ER negative breast cancer cells via the stimulation of the MAPK/ERK pathway, which is mediated by ER 36. Therefore, understand how 4OHT activates the MAPK cascade in ER negative breast cancer cells will require further studies. As a second approximation and to reduce the endogen ous levels of ER, we treated MDA MB 231 cells with fulvestrant. Fulvestrant is a potent anti oestrogen that pos sesses extremely high ER binding affinity and has two major effects on ER signalling.

First, it blocks ER sig nalling by inhibiting receptor dimerisation and nuclear localisation and, second, it blocks ER expression and ER mediated gene transcription. In addition, binding of fulvestrant to the ER has been proposed to induce proteasomal degradation of the aberrant receptor complex. As observed in Figure 2C, treatment with fulvestrant reduced the growth of cells treated with 4OHT. All to gether, these experiments support the notion that the effects of 4OHT on cell growth promotion in MDA MB 231 cells were dependent on an intact ER signalling. ER regulates E2F1 expression in ER negative breast cancer cells to mediate 4OHT resistance Recently, it has been proposed that ER regulates E2F1 expression to mediate tamoxifen resistance in ER positive cells.

The overexpression of E2F1 has been shown to be sufficient to promote breast cancer prolif eration by regulating the expression of cyclins and Cdks to mediate the G1 S transition. Although the E2F1 promoter lacks the classic oestrogen response elem ent 5 GGTCAnnnTGACC 3, ER is known to interact with several other transcription factors, including Sp1. In fact, it has been clearly demonstrated that tamoxifen increases ER/Sp1 interactions, modulating E2F1 expression in MCF7 tamoxifen resistant cells. Therefore, we performed a ChIP assay to deter mine whether 4OHT can promote the recruitment of ER to the E2F1 promoter. The results shown in Figure 3A indicated that compared to the control, 4OHT substantially increased the occupancy of ER on the E2F1 promoter. Consistent with the ChIP and proliferation data, semiquantitative RT PCR analysis indicated that the expression levels of E2F1 were markedly increased after exposure to 4OHT conditions. For these experiments, control cells and those subjected to 72 h of 4OHT treatment were GSK-3 harvested for gene expression analysis. Finally, to determine whether E2F1 con tributed to the increased proliferation after 4OHT exposure, we silenced E2F1 expression in MDA MB 231 cells using specific siRNAs.