Wang Q F, Khoury R H, Smith P C, McConnell D S, Padmanahban V, Midgley A R, Schneyer A L, Crowley W F, Sluss P M
National Cooperative Pogram for Infertility Research, Massachusetts General Hospital, Boston, 02114, USA.
J Clin Endocrinol Metab. 1996 Apr;81(4):1434-41. doi: 10.1210/jcem.81.4.8636347.
The follistatin/activin/inhibin system increasingly appears to have important growth and differentiating effects in a variety of cell types, including cancer. We have developed a two-site immunoradiometric assay for measurement of human follistatin using two monoclonal antibodies against recombinant human follistatin. This cloned protein donor assay is sensitive (0.5 ng/mL), specific for free human follistatin, and precise (<5% within assay coefficient of variation). Using this assay, native human follistatin could be measured in human pituitary extracts, follicular fluid, and granulosa-luteal cell-conditioned medium. To identify and characterize human follistatin secreted by ovarian cancer cells, we screened five human ovarian carcinoma cell lines currently available from the American Type Culture Collection (Rockville, MD). One of these, a cell line derived from a teratocarcinoma (designated PA-1, American Type Culture Collection, CRL1572), secreted large (3 microg/10(6) cells per 24 h) quantities of immunoreactive follistatin constituitively. Increasing volumes of conditioned medium from these cultured cells generated response curves parallel to those of recombinant human follistatin 288 reference protein, human follicular fluid, or culture medium from human granulosa-luteal cells. Secretion of follistatin by PA-1 cells was time and cell-number dependent with 297.9 +/- 15.2, 654 +/- 29.8, and 940 +/- 49.1 ng follistatin secreted over 24 h by 1 x 10(5), 2 x 10(5), and 3 x 10(5) cells, respectively. Western and ligand blot analysis revealed that the immunoreactive follistatin secreted by PA-1 cells and isolated by sulfate-cellufine chromatography was identical to the molecular weight variants (32,000 and 35,000 Mr) of recombinant human follistatin 288. PA-1 cell-conditioned medium suppressed basal secretion of FSH by cultured rat anterior pituitary cells in a dose-dependent fashion. This follistatin bioactivity was completely removed by adsorption with either solid-phase monoclonal antifollistatin or a dextran-sulfate chromatography gel. Because activin suppressed the proliferation of PA-1 cells, secretion of bioactive follistatin may represent an autocrine mechanism opposing activin to maintain the rapid growth rate of PA-1 cells. These observations demonstrate that the ovarian teratocarcinoma cell line, PA-1, secretes considerable amounts of human follistatin that is biologically active, capable of binding human activin, and antigenically similar to recombinant human follistatin 288. The monoclonal antibodies and two-site assay reported herein should be useful in assessing the regulation of follistatin secretion and as a diagnostic tool, especially if follistatin measurements prove to be a marker for some ovarian cancers.
卵泡抑素/激活素/抑制素系统似乎在包括癌症在内的多种细胞类型中日益显示出重要的生长和分化作用。我们利用两种抗重组人卵泡抑素的单克隆抗体，开发了一种用于检测人卵泡抑素的双位点免疫放射分析方法。这种克隆蛋白供体分析方法灵敏（0.5 ng/mL），对游离人卵泡抑素具有特异性，且精密度高（批内变异系数<5%）。利用该分析方法，可在人垂体提取物、卵泡液和颗粒黄体细胞条件培养基中检测到天然人卵泡抑素。为了鉴定和表征卵巢癌细胞分泌的人卵泡抑素，我们筛选了目前可从美国模式培养物集存库（马里兰州罗克维尔）获得的5种人卵巢癌细胞系。其中之一，一种源自畸胎癌的细胞系（命名为PA-1，美国模式培养物集存库，CRL1572），能组成性地分泌大量（每24小时3 μg/10⁶细胞）的免疫反应性卵泡抑素。来自这些培养细胞的条件培养基体积增加时，产生的反应曲线与重组人卵泡抑素288参考蛋白、人卵泡液或人颗粒黄体细胞培养基的反应曲线平行。PA-1细胞分泌卵泡抑素具有时间和细胞数量依赖性，1×10⁵、2×10⁵和3×10⁵个细胞在24小时内分别分泌卵泡抑素297.9±15.2、654±29.8和940±49.1 ng。蛋白质免疫印迹和配体印迹分析显示，PA-1细胞分泌并经硫酸纤维素柱层析分离的免疫反应性卵泡抑素与重组人卵泡抑素288的分子量变体（32000和35000 Mr）相同。PA-1细胞条件培养基以剂量依赖性方式抑制培养的大鼠垂体前叶细胞基础FSH分泌。用固相单克隆抗卵泡抑素或葡聚糖硫酸酯层析凝胶吸附可完全消除这种卵泡抑素生物活性。由于激活素抑制PA-1细胞增殖，生物活性卵泡抑素的分泌可能代表一种自分泌机制，对抗激活素以维持PA-1细胞的快速生长速率。这些观察结果表明，卵巢畸胎癌细胞系PA-1分泌大量具有生物活性、能够结合人激活素且抗原性与重组人卵泡抑素288相似的人卵泡抑素。本文报道的单克隆抗体和双位点分析方法在评估卵泡抑素分泌的调节以及作为诊断工具方面应具有实用性，特别是如果卵泡抑素检测被证明是某些卵巢癌的标志物时。
