Lewis J G, Robertson J M, Elder P A
Department of Clinical Biochemistry, Christchurch Hospital, New Zealand.
J Steroid Biochem. 1989 Aug;33(2):171-8. doi: 10.1016/0022-4731(89)90291-4.
A mouse was immunised with an antigen(s) purified by oestradiol-Sepharose affinity chromatography of pooled oestrogen-receptor positive cytosols from human breast cancer tissue. One antibody secreting clone was identified which precipitated labelled antigen and which also stained MCF-7 cells. Culture supernatant and ascites fluid were used for immunofluorescence, SDS-PAGE-Western blotting, photoaffinity labelling and binding studies. The antibody staining of MCF-7 cells was inhibited by preincubation in oestrogen-receptor positive cytosol but was unaffected by oestrogen-receptor negative cytosol. MCF-7 cells stained whether cultured in the presence or absence of oestradiol. The oestrogen-receptor negative cell lines MDA-MB-231 and MDA-MB-330 did not stain. Binding studies with 16-alpha-iodooestradiol using breast cancer tissue cytosols followed by immunoprecipitation showed activity only with oestrogen-receptor positive cytosols with optimal binding activity at 4 degrees C, unaffected by molybdate, but reduced at 25 degrees C or in the presence of 0.4 M KCl. Binding studies with MCF-7, MDA-MB-231 and MDA-MB-330 cytosols and nuclear fractions only showed activity with the MCF-7 cytosol and MCF-7 particulate fractions. The antibody recognised a 48 K species in both MCF-7 cytosol and nuclear fractions but not in the cytosol and nuclear extracts of oestrogen-receptor negative cell lines. Photoaffinity labelling using 16 alpha-iodooestradiol suggests the 48 K antigen does not bind oestradiol directly. The relationship of this antigen to the classical oestrogen-receptor and receptor complex awaits further clarification.
用从人乳腺癌组织中提取的雌激素受体阳性胞质溶胶经雌二醇-琼脂糖亲和层析纯化的抗原免疫一只小鼠。鉴定出一个分泌抗体的克隆，它能沉淀标记抗原，也能对MCF-7细胞进行染色。培养上清液和腹水用于免疫荧光、SDS-聚丙烯酰胺凝胶电泳-蛋白质免疫印迹、光亲和标记和结合研究。MCF-7细胞的抗体染色可被雌激素受体阳性胞质溶胶预孵育所抑制，但不受雌激素受体阴性胞质溶胶的影响。无论在有或没有雌二醇的情况下培养，MCF-7细胞均被染色。雌激素受体阴性细胞系MDA-MB-231和MDA-MB-330未被染色。使用乳腺癌组织胞质溶胶进行16-α-碘雌二醇结合研究，随后进行免疫沉淀，结果显示仅与雌激素受体阳性胞质溶胶有活性，在4℃时结合活性最佳，不受钼酸盐影响，但在25℃或存在0.4M氯化钾时活性降低。对MCF-7、MDA-MB-231和MDA-MB-330胞质溶胶和核组分的结合研究仅显示与MCF-7胞质溶胶和MCF-7颗粒组分有活性。该抗体在MCF-7胞质溶胶和核组分中识别出一种48K的蛋白，但在雌激素受体阴性细胞系的胞质溶胶和核提取物中未识别到。使用16α-碘雌二醇的光亲和标记表明48K抗原不直接结合雌二醇。该抗原与经典雌激素受体及受体复合物的关系有待进一步阐明。
