Unoccupied binding sites for oestrogen in nuclei of a breast tumour cell line (MCF-7)

Abstract

A method to measure occupied and unoccupied estrogen receptors directly in the crude nuclear fraction of [human breast tumor] MCF-7 cells was developed. The receptors had high affinity for estradiol (Kd .apprx. 0.7 nM) and binding specificity characteristics of estrogen receptors. A substantial amount of the unoccupied receptors were found in the crude nuclear fraction. Several experiments excluded the possibility that the unoccupied nuclear receptor might be a cytoplasmic contaminant. Multiple extractions with Tris buffer released .apprx. 75% of the total receptor content, leaving the rest unextractable in the crude nuclear fraction. Nuclei purified by centrifugation through 1.8 M-sucrose and treatment with 0.7% Triton X-100 or by centrifugation through 50% glycerol with 0.1% Triton X-100 contained similar amounts of unoccupied receptors to that found in the crude nuclear fraction. In cells cultured during 5 days after preconfluency a 3-fold increase in the amount of unoccupied cytoplasmic receptors occurred whereas the amount of unoccupied nuclear receptors did not change significantly. In cells exposed to increasing concentrations of estradiol the unoccupied cytoplasmic receptor was continuously depleted but no considerable change in the unoccupied nuclear receptor was found.