In this report, confocal microscopy sellekchem experiments and western blot assays indicated that the MDA MB 231 breast cancer cell line did not express ER, which was in accordance with its ER negative sta tus. However, semiquantitative real time PCR designed to detect ER mRNA showed a consistent expression of this mRNA transcript, estimated to be approximately 3. 3 copies of ER mRNA for every 106 copies of B actin. Although the specificity of the amplification of this ER PCR product was verified by melting curve analysis, this product was barely detectable by agarose gel electrophoresis. Although ER mRNA was detected in MDA MB 231 cells, its expression levels, as expected, were much lower than in the ER positive cell line, MCF7.

Because the response of ER negative breast cancer cells to ER antagonists with respect to ER expression is not well known, we aimed to determine whether 4OHT can modulate ER expression in MDA MB 231 cells by analysing the ER mRNA levels in 4OHT treated cells. As observed in Figure 1C, 4OHT significantly induced the expression of ER by approximately 6 fold compared to untreated control cells. The ER PCR product was sequenced, and the published ER sequence was con firmed. Importantly, this increase in ER mRNA was accompanied by a significant increase in ER protein as determined by confocal microscopy and western blot experiments. There are at least two possible mechanisms by which ER gene expression may be lost in ER negative breast cancer cells. First, the activators necessary for ER tran scription may not be available or the transcriptional re pressors may predominate.

Alternatively, the ER gene may be selectively methylated and inaccessible to the existing transcriptional activators. Because 4OHT has not been identified as a demethylating agent, it seems more probable that this drug affected the pattern of ER transcription by modulating the range of its acti vators/repressors on its corresponding promoter. It is well established that the MAPK mediated hyperpho sphorylation of ER can contribute to resistance to tam oxifen in breast cancer and that serine 118 in ER is an important residue for the stimulation of ER activity by the selective ER modulator 4OHT. To demonstrate the involvement of the MAPK signalling pathway in the 4OHT induced up regulation of ER, we used U0126, a MAP ERK kinase 1/2 inhibitor.

As observed in Figure 1A C, U0126 inhibited ER mRNA Brefeldin_A and protein expression in cells co treated with 4OHT. ChIP assays also indicated that 4OHT increased the occupancy of ER on its own promoter and that U0126 completely abolished this occupation. It is well known that phosphorylation of ER at specific residues can stimulate ER activity in a ligand independent manner. By this mechanism of action ER is phosphory lated by active kinases, thereby activating ER to dimer ise, bind DNA, and regulate genes.