Mainou-Fowler Tryfonia, Overman Lynn Marie, Dignum Helen, Wood Katrina, Crosier Stephen, Angus Brian, Proctor Stephen John, Anderson John J
Department of Haematology, School of Clinical and Laboratory Sciences, William Leech Building, Medical School, University of Newcastle-upon-Tyne, Newcastle-upon-Tyne, NE2 4HH, UK.
Int J Oncol. 2008 Jan;32(1):59-68.
Anti-apoptotic factors including IAP-survivin and bcl-2 are involved in carcinogenesis and predict for disease outcome for patients with cancer. We used RT-PCR and specific primers to generate two recombinant IAP-survivin proteins; one encoding for the full-length protein and the second comprising the survivin sequence incorporating amino acids 98 to 142. Both proteins were used to immunize mice and as capture antigens to screen NS1/immune splenocyte hybridoma supernatants for anti-survivin antibody in ELISA assays. The antibody designated F2-9C3 was most effective and reacted with both recombinant proteins and with the native protein present in lysates of A549 (lung carcinoma) and Jurkat cells in Western blots, immunoprecipitation and formalin-fixed tissue sections. Immunohistochemical staining of normal and neoplastic tissues showed association of the F2-9C3 antibody with the mitotic spindles. Expression of survivin was not detected elsewhere in sections of normal tissue while all neoplastic tissues examined, including those from patients with diffuse large B-cell lymphoma (DLBCL), showed significant expression of survivin. The intensity and localization of staining in these tumours varied and was observed in cytoplasm and/or nuclei. High nuclear expression of survivin predicted the disease outcome in patients with DLBCL. This association was evident when relating intensity to patient survival (p=0.0321) and strengthened when a score was calculated based on both staining intensity and the proportion of the reactive tumour cells (p=0.0128; reduction in the mean survival times: 35% and 46%, respectively). Elevated expression of bcl-2 protein also identified the high-risk patients (p=0.0095; reduction in mean survival time: 37%). Over-expression of both factors was a more powerful indicator of poor prognosis than either marker alone (p=0.0054, 70% reduction in mean survival time). In conclusion, our novel F2-9C3 monoclonal antibody is effective in determination of expression of IAP-survivin in neoplastic tissue. Nuclear overexpression of IAP-survivin using this antibody predicts the disease outcome in patients with DLBCL and significantly improves the predictive power of bcl-2 in these patients.
包括IAP-生存素和bcl-2在内的抗凋亡因子参与肿瘤发生，并可预测癌症患者的疾病预后。我们使用逆转录聚合酶链反应（RT-PCR）和特异性引物生成了两种重组IAP-生存素蛋白；一种编码全长蛋白，另一种包含整合了98至142位氨基酸的生存素序列。这两种蛋白都用于免疫小鼠，并作为捕获抗原，在酶联免疫吸附测定（ELISA）中筛选NS1/免疫脾细胞杂交瘤上清液中的抗生存素抗体。命名为F2-9C3的抗体最为有效，在蛋白质印迹、免疫沉淀和福尔马林固定组织切片中，它能与两种重组蛋白以及A549（肺癌）和Jurkat细胞裂解物中的天然蛋白发生反应。正常组织和肿瘤组织的免疫组织化学染色显示F2-9C3抗体与有丝分裂纺锤体相关。在正常组织切片的其他部位未检测到生存素的表达，而所有检查的肿瘤组织，包括弥漫性大B细胞淋巴瘤（DLBCL）患者的肿瘤组织，均显示生存素的显著表达。这些肿瘤中染色的强度和定位各不相同，在细胞质和/或细胞核中均可观察到。生存素的高核表达可预测DLBCL患者的疾病预后。当将染色强度与患者生存率相关联时，这种关联很明显（p = 0.0321），当基于染色强度和反应性肿瘤细胞比例计算得分时，这种关联得到加强（p = 0.0128；平均生存时间分别缩短35%和46%）。bcl-2蛋白的表达升高也可识别高危患者（p = 0.0095；平均生存时间缩短37%）。两种因子的过表达比单独任何一种标志物更能有力地指示预后不良（p = 0.0054，平均生存时间缩短70%）。总之，我们新型的F2-9C3单克隆抗体可有效测定肿瘤组织中IAP-生存素的表达。使用该抗体检测到的IAP-生存素核过表达可预测DLBCL患者的疾病预后，并显著提高bcl-2对这些患者的预测能力。
