Specific inhibition of estrogen receptor alpha function by antisense oligodeoxyribonucleotides

We have tested the effect of a range of antisense oligodeoxyribonucleotides (ODN) directed against the human estrogen receptor alpha (ER alpha) on ER alpha protein expression and function. Antisense ER alpha ODN transfected i nto the ER alpha -positive human breast carcinoma cell line MCF7-K2 showed variable responses dependent on the oligo used. The most active antisense O DN (oligo 7) decreased the levels of ER alpha protein by 61% as measured by Western blot analysis. Exogenous 17 beta -estradiol (17 beta -E-2), but no t 17 alpha -E-2, augmented this effect, with a threshold effect at 10(-8) M 17 beta -E-2. The inhibitory effect of antisense ER alpha oligo 7 was conf irmed by measurement of functional ER alpha protein. H-3-17 beta -E-2 bindi ng to MCF7 cell extracts was inhibited to approximately 40% of control valu es in the presence of oligo 7. Antisense-transfected MCF7-K2 cell cultures produced a further 30% binding reduction in the presence of exogenous 17 be ta -E-2. An inhibitory effect on 17 beta -E-2-dependent cell function was c onfirmed by the demonstration that ER alpha oligo 7-transfected MCF7-K2 cel ls failed to exhibit 17 beta -E-2-stimulated cell proliferation. Exogenous 17 beta -E-2 enhanced the inhibitory effect of the antisense ODN by increas ing ODN transfection efficiency but without ER alpha catabolism via the pro teosomal pathway, suggesting an effect of 17 beta -E-2 on the plasma membra ne and the existence of different ER alpha degradation pathways in the MCF7 -K2 cell subclone. As 17 beta -E-2 had no effect on ER alpha protein degrad ation, we conclude that the observed reduction of ER alpha protein levels i s due solely to the presence of the antisense ER alpha ODN. Antisense ER al pha ODN molecules, therefore, may form the basis of effective therapies aga inst ER alpha -dependent malignancies.