Immunologic analysis of human breast cancer progesterone receptors. 1. Immunoaffinity purification of transformed receptors and production of monoclonal antibodies.
Immunologic analysis of human breast cancer progesterone receptors. 1. Immunoaffinity purification of transformed receptors and production of monoclonal antibodies.
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人乳腺癌孕酮受体的免疫学分析。
DOI：
10.1021/bi00393a045
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发表时间：
1987
期刊：
影响因子：
2.9
通讯作者：
Edwards,DP
中科院分区：
文献类型：
--
作者：
Estes,PA;Suba,EJ;Lawler-Heavner,J;Elashry-Stowers,D;Wei,LL;Toft,DO;Sullivan,WP;Horwitz,KB;Edwards,DP
病理学和医学系，科罗拉多大学健康科学中心，丹佛，科罗拉多80262，以及生物化学和分子生物学系，马约诊所，罗切斯特，明尼苏达州55905接收日期：1986年11月24日;修订后的Mandarin，1987年4月10日摘要：一种单克隆抗体（MAb），命名为PR-6，产生抗鸡输卵管孕酮受体[Sullivan，W. P.，Beito，T. G.，正确的，J，Krco，CJ，& Toft，DO（1986）Endocrinology（巴尔的摩）119，1549-1557]与Mr 120000人B受体交叉反应。用PR-6制备的免疫基质从人乳腺癌T47 D细胞中纯化孕激素受体（PR）。一步免疫亲和色谱法导致B受体富集（通过PR-6免疫印迹和[3* H]普罗美孕酮光亲和标记鉴定）至比活性为1915 pmol/mg蛋白（或23%纯度），产率为27%。通过凝胶电泳和光密度扫描判断的B蛋白的纯度和产率约高1.7倍，这是由于在洗脱步骤中激素结合活性的部分损失。通过二乙氨基乙基层析的第二个纯化步骤进一步富集至3720 pmol/mg蛋白质（或44%纯度），基本上产生两种蛋白质，120-千道尔顿（kDa）B受体和76-kDa非类固醇结合蛋白，每种蛋白质的量大致相等。在这些条件下纯化的B受体被转化并具有生物活性。它们保持为未降解的120 kDa的双联体，并保留激素和DNA结合活性。分离的B受体不含90-kDa非类固醇结合蛋白，观察到该蛋白与其它系统中的8 S未转化受体相关，也不含先前描述的与鸡PR共纯化的非激素结合105-108-kDa B抗原。命名为B-30（IgGO）、B-64（IgG [）和B-II（IgM），对B受体具有特异性。第四种MA B，A/B-52（IgG [），与A和B受体反应。IgG单克隆抗体对人PR是单特异性的，因为它们识别和吸收天然受体-激素复合物，取代4S受体在含蔗糖梯度的盐上的沉降，并且通过粗T47 D胞质溶胶的免疫印迹测定，仅与受体多肽反应。尽管小鼠仅注射B受体，但识别A和B受体的A/B-52的产生提供了这两种蛋白质共享结构同源性区域的证据。这些新的单克隆抗体为进一步研究人类受体的结构和功能以及乳腺肿瘤中PR的临床免疫检测提供了有价值的试剂。孕激素受体（PR）1在分子水平上的作用机制还不清楚。该领域的进一步进展需要纯化受体以及开发特异性抗体作为检测受体的直接探针。含有PR的激素反应性乳腺癌细胞为此类研究提供了极好的模型系统。T47 D人乳腺癌细胞系（Keydar等人，1979）是特别好的模型，因为这些细胞含有与正常靶组织相似的不常见的高PR含量（即，250000分子/细胞），并且通过几个可测量的参数含有功能性受体系统（Horwitz等，1982; Mockus和Horwitz，1983; Vignon等人，1983; Chalbos和Rochefort，1984 a，B; Horwitz和Fredenberg，
Departments of Pathology and Medicine, University of Colorado Health Sciences Center, Denver, Colorado 80262, and Department of Biochemistry and Molecular Biology, Mayo Clinic, Rochester, Minnesota 55905 Received November 24, 1986; Revised Manuscript Received April 10, 1987 abstract: A monoclonal antibody (MAb), designated PR-6, produced against chick oviduct progesterone receptors [Sullivan, W. P., Beito, T. G., Proper, J., Krco, CJ, & Toft, DO (1986) Endocrinology (Baltimore) 119, 1549-1557] cross-reacts with the Mr 120000 human B receptors. An immunomatrix prepared with PR-6 was used to purify progesterone receptors (PR) from T47D humanbreast cancer cells. Single-step immunoaffinity chromatographyresults in enrichment of B receptors (identified by immunoblot with PR-6 and by photoaffinity labeling with [3* H] promegestone) to a specific activity of 1915 pmol/mg of protein (or 23% purity) and with 27% yield. Purity and yields as judged by gel electrophoresis and densitometric scanning of the B protein were approximately 1.7-fold higher due to partial loss in hormone binding activity at the elution step. A second purification step by diethylaminoethyl chromatography gives further enrichmentto 3720 pmol/mg of protein (or 44% purity) to yield essentially two proteins, 120-kilodalton (kDa) B receptors and a 76-kDa non-steroid binding protein, each in approximately equivalent amounts. B receptors purified under these conditions are transformed and biologically active. They were maintained as undegraded 120-kDa doublets and retained both hormone and DNA binding activities. Isolated B receptors were free of the 90-kDa non-steroid binding protein observed to be associated with 8S untransformed receptors in other systems and were free also of the non-hormone binding 105-108-kDa B antigen described previously to copurify with chick PR. These purified B receptors were used as immunogen for production of four monoclonal antibodies against human PR. Three of the MAbs, designated as B-30 (IgGO, B-64 (IgG [), and B-ll (IgM), are specific for B receptors. The fourth MAb, A/B-52 (IgG [), reacts with both A and B receptors. The IgG MAbs are monospecific for human PR since they recognize and absorb native receptor-hormone complexes, displace the sedimentation of 4S receptors on salt containing sucrose gradients, and, by immunoblot assay of crude T47D cytosol, react onlywith receptor polypeptides. Although mice were injected with B receptors only, production of A/B-52 which recognized both A and B receptors provides evidence that these two proteins share regions of structural homology. These new MAbs are valuable reagents for further studies of human receptor structure and function and for clinical immunodetection of PR in breast tumors. e mechanism of action of progesterone receptors (PR) 1 at the molecular level is not well understood. Further progress in this area requires purification of receptors as well as development of specific antibodies as direct probes for detection of receptors. Hormone-responsive breast cancer cells con-taining PR provide an excellent model system for such studies. The T47D human breast cancer cell line (Keydar et al., 1979) is a particularly good model since these cells contain an un-usually high PR content (ie, 250000 molecules/cell) com-pared to normal target tissues, and by several measurable parameters contain a functional receptorsystem (Horwitz et al., 1982; Mockus & Horwitz, 1983; Vignon et al., 1983; Chalbos & Rochefort, 1984a, b; Horwitz & Freidenberg,