Pupa S M, Ménard S, Morelli D, Pozzi B, De Palo G, Colnaghi M I
Division of Experimental Oncology E, Istituto Nazionale Tumori, Milan, Italy.
Oncogene. 1993 Nov;8(11):2917-23.
A molecule that is immunologically related to the c-erbB-2 oncogene product (p185HER2/neu) was detected in the conditioned culture medium from neu-overexpressing tumor cell lines and in sera of advanced-stage breast carcinoma patients. Using a sensitive (in the range of 0.5 ng ml-1) double-determinant radioimmunoassay (DDIRMA) with two monoclonal antibodies (MAbs) directed against the neu extracellular domain (ECD), soluble oncoproteins were detected in supernatants from several neu-positive tumor cell lines, independent of the levels of membrane p185HER2 expression. The molecule detected did not react with a MAb directed against an intracytoplasmic epitope of the p185HER2. Western blot analysis of the concentrated supernatant revealed a protein of approximately 110 kDa molecular mass, which closely matches the predicted size of the glycosylated p185HER2 ECD. Immunoprecipitation of culture supernatant from cell surface-radioiodinated cells confirmed the 110 kDa molecular mass of the glycosylated shed protein, which migrated to 86 kDa after deglycosylation. Proteolytic cleavage of the p185HER2 molecule was demonstrated in release assays carried out with protease inhibitors. The combined use of leupeptin and EDTA completely inhibited release of the molecule. Analysis of sera from breast carcinoma patients and healthy donors by DDIRMA revealed the presence of soluble neu in 15% of pathologic sera but none of the normal sera. A good correlation was found between neu-overexpression in the primary tumor and the soluble marker in serum of patients with advanced disease; sera of early-stage patients were always negative, independent of neu-overexpression in the tumor. These results suggest the usefulness of soluble neu as an indicator of tumor aggressiveness but not as a diagnostic marker of breast cancer.
在过表达neu的肿瘤细胞系的条件培养基以及晚期乳腺癌患者的血清中，检测到一种与c-erbB-2癌基因产物（p185HER2/neu）具有免疫相关性的分子。使用一种灵敏的（检测范围为0.5 ng/ml）双决定簇放射免疫分析（DDIRMA），该分析采用两种针对neu细胞外结构域（ECD）的单克隆抗体（MAb），在几种neu阳性肿瘤细胞系的上清液中检测到了可溶性癌蛋白，其与膜p185HER2的表达水平无关。检测到的该分子不与针对p185HER2胞浆内表位的单克隆抗体发生反应。对浓缩上清液进行的蛋白质印迹分析显示，有一种分子量约为110 kDa的蛋白质，其大小与糖基化p185HER2 ECD的预测大小紧密匹配。对细胞表面放射性碘化细胞的培养上清液进行免疫沉淀，证实了糖基化脱落蛋白的分子量为110 kDa，去糖基化后迁移至86 kDa。在用蛋白酶抑制剂进行的释放试验中证实了p185HER2分子的蛋白水解切割。亮抑酶肽和乙二胺四乙酸（EDTA）联合使用完全抑制了该分子的释放。通过DDIRMA对乳腺癌患者和健康供体的血清进行分析，发现在15%的病理血清中存在可溶性neu，而正常血清中均未检测到。在晚期疾病患者中，原发性肿瘤中neu的过表达与血清中的可溶性标志物之间存在良好的相关性；早期患者的血清始终为阴性，与肿瘤中neu的过表达无关。这些结果表明，可溶性neu可作为肿瘤侵袭性的指标，但不能作为乳腺癌的诊断标志物。
