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Workshop on estrogen and antiestrogen action; basic and clinical aspects
1626
WORKSHOP Kl
ON ESTROGEN
AND ANTIESTROGEN
ACTION;
BASIC AND CLINICAL
ASPECTS
An Endogenous Ligand For Type II Estrogen Binding Sites. B.M. Markaverich, R.R. Roberts, M.A. Alejandro and J.H. Clark, Dept. CT1 Biology, Baylor College of Medicine, Houston TX 77030. Dilution of uterine nuclear fractions from estrogen treated rats to quantitation of estrogen binding sites by [3H]-estradiol prior exchange results in an increase (3-4 fold) in the measurable quantities These increases of the type II site, while type I sites are unaffected. in type II sites following nuclear dilution occur independently of protein concentrat on and result from the dilution of a specific endogenous The inhibitor inhibitor of [j3HI-estradiol binding to these sites. activity is present in cytosol preparations from rat uterus, spleen, Preliminary characterization of diaphragm, skeletal muscle and serum. the inhibitor activity by Sephadex G-25 chromatography shows two distinct peaks which are similar in molecular weight (-300). These components (a and B) can be separated on LH-20 chromatography since the B-peak componPartial purifient is preferentially retained on this lipophilic resin. cation of the LH-20 B inhibitor component by high performance liquid chromatography and gas-liquid chromatography-mass spectrometric analysis suggest the putative inhibitor activity is steroidal in nature and conAnalysis of cytosol preparations on sists of two very similar molecules. tissues (uterus, liver, LH-20 chromatography shows that non-neoplastic components lactating mammary gland) contain both cx and B inhibitor whereas estrogen-induced rat mammary tumors contain very low to nonmeasurable quantities of the B-peak inhibitor activity.
K3 STUDIES WITH MONOCLONAL ANTIBODY RAISED AGAINST PARTIALLY PURIFIED OESTRADIOL RECEPTOR FROM HUMAN MYOMETRILJM. R.J.B.King*, A.I.Coffer and K.Lewis, Imperial Cancer Research Fund, London WCZA 3PX, United Kingdom. The 4s ER from human myomett-ium has been partially purified by oestradiol affinity chromatography and used to generate monoclonal antibodies. Screening for antibody was by immunoprecipitation of lz51E2 labelled myometrial receptor; an Ig GI (D5) and an IgM (C3) were obtained. D5 will precipitate (after addition of second antibody) receptor-bound 1251E2 in a dose-dependent manner, a reaction that is increased by KCNS, low pH and high salt. D5 is specific for human cytosol ER and will not react with human receptors for other classes of steroid, SHBG or nuclear ER; western blotting indicates that D5 recognizes a 30K protein that does not contain carbohydrate. IRMA and ELISA plate assays have been developed and applied to clinical material. Both breast and endometrial tumour cytosols show a highly significant correlation between D5 reactivity and ER content whilst ER-negative tumours do not react with D5. Cultured human breast tumour cells also show this quantitative relationship but with a different slope for the dose-response curve (more antigen) to that of solid breast tumours. Quantitative studies indicate that the antigen is present in all ER positive tissues in excess of ER content. Histochemistry shows a cytoplasmic location, regardless of fixation method, which parallels the ER content measured by the conventional 3HE2 method. D5 recognizes an antigen that is not the E2 bindin unit of the receptor but the ability to 9HE2 and the qualitative and quantitative immunoprecipitate receptor-bound correlation with ER emphasize the close relationship of the two antigens.
WORKSHOP Kl
ON ESTROGEN
AND ANTIESTROGEN
ACTION;
BASIC AND CLINICAL
ASPECTS
An Endogenous Ligand For Type II Estrogen Binding Sites. B.M. Markaverich, R.R. Roberts, M.A. Alejandro and J.H. Clark, Dept. CT1 Biology, Baylor College of Medicine, Houston TX 77030. Dilution of uterine nuclear fractions from estrogen treated rats to quantitation of estrogen binding sites by [3H]-estradiol prior exchange results in an increase (3-4 fold) in the measurable quantities These increases of the type II site, while type I sites are unaffected. in type II sites following nuclear dilution occur independently of protein concentrat on and result from the dilution of a specific endogenous The inhibitor inhibitor of [j3HI-estradiol binding to these sites. activity is present in cytosol preparations from rat uterus, spleen, Preliminary characterization of diaphragm, skeletal muscle and serum. the inhibitor activity by Sephadex G-25 chromatography shows two distinct peaks which are similar in molecular weight (-300). These components (a and B) can be separated on LH-20 chromatography since the B-peak componPartial purifient is preferentially retained on this lipophilic resin. cation of the LH-20 B inhibitor component by high performance liquid chromatography and gas-liquid chromatography-mass spectrometric analysis suggest the putative inhibitor activity is steroidal in nature and conAnalysis of cytosol preparations on sists of two very similar molecules. tissues (uterus, liver, LH-20 chromatography shows that non-neoplastic components lactating mammary gland) contain both cx and B inhibitor whereas estrogen-induced rat mammary tumors contain very low to nonmeasurable quantities of the B-peak inhibitor activity.
K3 STUDIES WITH MONOCLONAL ANTIBODY RAISED AGAINST PARTIALLY PURIFIED OESTRADIOL RECEPTOR FROM HUMAN MYOMETRILJM. R.J.B.King*, A.I.Coffer and K.Lewis, Imperial Cancer Research Fund, London WCZA 3PX, United Kingdom. The 4s ER from human myomett-ium has been partially purified by oestradiol affinity chromatography and used to generate monoclonal antibodies. Screening for antibody was by immunoprecipitation of lz51E2 labelled myometrial receptor; an Ig GI (D5) and an IgM (C3) were obtained. D5 will precipitate (after addition of second antibody) receptor-bound 1251E2 in a dose-dependent manner, a reaction that is increased by KCNS, low pH and high salt. D5 is specific for human cytosol ER and will not react with human receptors for other classes of steroid, SHBG or nuclear ER; western blotting indicates that D5 recognizes a 30K protein that does not contain carbohydrate. IRMA and ELISA plate assays have been developed and applied to clinical material. Both breast and endometrial tumour cytosols show a highly significant correlation between D5 reactivity and ER content whilst ER-negative tumours do not react with D5. Cultured human breast tumour cells also show this quantitative relationship but with a different slope for the dose-response curve (more antigen) to that of solid breast tumours. Quantitative studies indicate that the antigen is present in all ER positive tissues in excess of ER content. Histochemistry shows a cytoplasmic location, regardless of fixation method, which parallels the ER content measured by the conventional 3HE2 method. D5 recognizes an antigen that is not the E2 bindin unit of the receptor but the ability to 9HE2 and the qualitative and quantitative immunoprecipitate receptor-bound correlation with ER emphasize the close relationship of the two antigens.