Tsai C C, McGuire M H, Mellitt R J, Ritter R A, Xu J Z, Litwicki D J, Roodman S T
Department of Pathology, St. Louis University School of Medicine, Missouri 63104.
Cancer Res. 1990 Jan 1;50(1):152-8.
Three murine hybridomas (TMMR-1-3) were developed by repeated immunizations of mice with four different human osteosarcoma cell lines in an alternating sequence of inoculations. The monoclonal antibodies were screened for reactivities to cultured cell lines and tissue sections of osteosarcomas using flow cytometry and immunohistochemical techniques. TMMR-2 is a highly specific antibody (IgG1) that reacted with all 14 osteosarcoma tumors and eight human osteosarcoma cell lines tested, including the established human osteosarcoma cell lines HOS and Saos-2. Benign neoplastic cells from two osteoblastomas, osteoblasts from regions of reparative osteoid formation and neonatal new bone, are also reactive with TMMR-2. TMMR-1 has mesenchymal specificity while TMMR-3, although reactive with osseous differentiated cells, also reacted with mitotic cells of all cell types. Characterization of antigen structure by Western immunoblotting revealed that TMMR-2 reacted with a 100 degrees C heat labile mercaptoethanol-sensitive Mr 26,000 protein, and TMMR-3 recognized a mercaptoethanol-resistant Mr 97,000 protein whereas TMMR-1 reacted with a series of bands from 65,000 to 85,000 molecular weight, all of which were mercaptoethanol sensitive. TMMR-1 and TMMR-2 monoclonal antibodies showed complement-independent inhibition of [3H]thymidine incorporation into DNA, but did not exhibit cytotoxic activity. The results suggest that TMMR-2 is a specific antibody that recognizes an osteoblast/osteocyte surface antigen present in normal, reactive, and neoplastic disorders of bone. The inhibitory effects on DNA synthesis in cultured osteosarcoma cells by TMMR-2 indicate an important cell growth/proliferation role of this surface antigen. These monoclonal antibodies, in combination with other known antibodies, can be used to characterize mesenchymal cell surface antigenic structure and differentiation.
通过用四种不同的人骨肉瘤细胞系以交替接种序列反复免疫小鼠，获得了三种鼠杂交瘤（TMMR - 1 - 3）。使用流式细胞术和免疫组织化学技术筛选单克隆抗体与培养的细胞系和骨肉瘤组织切片的反应性。TMMR - 2是一种高度特异性抗体（IgG1），与所有测试的14个骨肉瘤肿瘤和8个人骨肉瘤细胞系反应，包括已建立的人骨肉瘤细胞系HOS和Saos - 2。来自两个成骨细胞瘤的良性肿瘤细胞、修复性类骨质形成区域的成骨细胞和新生儿新骨也与TMMR - 2反应。TMMR - 1具有间充质特异性，而TMMR - 3虽然与骨分化细胞反应，但也与所有细胞类型的有丝分裂细胞反应。通过蛋白质免疫印迹法表征抗原结构表明，TMMR - 2与一种100℃热不稳定、对巯基乙醇敏感的26,000分子量蛋白反应，TMMR - 3识别一种对巯基乙醇抗性的97,000分子量蛋白，而TMMR - 1与一系列分子量从65,000到85,000的条带反应，所有这些条带对巯基乙醇敏感。TMMR - 1和TMMR - 2单克隆抗体显示出对[3H]胸苷掺入DNA的补体非依赖性抑制，但不表现出细胞毒性活性。结果表明，TMMR - 2是一种特异性抗体，可识别存在于正常、反应性和肿瘤性骨疾病中的成骨细胞/骨细胞表面抗原。TMMR - 2对培养的骨肉瘤细胞中DNA合成的抑制作用表明该表面抗原在细胞生长/增殖中起重要作用。这些单克隆抗体与其他已知抗体结合，可用于表征间充质细胞表面抗原结构和分化。
