Department of Molecular Biology and Genetics, Bilkent University, Ankara, Turkey.
Exp Mol Pathol. 2010 Oct;89(2):182-9. doi: 10.1016/j.yexmp.2010.05.010. Epub 2010 May 31.
Smad-interacting protein 1 (SIP1, also known as ZEB2) represses the transcription of E-cadherin and mediates epithelial-mesenchymal transition in development and tumor metastasis. Due to the lack of human SIP1-specific antibodies, its expression in human tumor tissues has not been studied in detail by immunohistochemistry. Hence, we generated two anti-SIP1 monoclonal antibodies, clones 1C6 and 6E5, with IgG1 and IgG2a isotypes, respectively. The specificity of these antibodies was shown by Western blotting studies using siRNA mediated downregulation of SIP1 and ZEB1 in a human osteosarcoma cell line. In the same context, we also compared them with 5 commercially available SIP1 antibodies. Antibody specificity was further verified in an inducible cell line system by immunofluorescence. By using both antibodies, we evaluated the tissue expression of SIP1 in paraffin-embedded tissue microarrays consisting of 22 normal and 101 tumoral tissues of kidney, colon, stomach, lung, esophagus, uterus, rectum, breast and liver. Interestingly, SIP1 predominantly displayed a cytoplasmic expression, while the nuclear localization of SIP1 was observed in only 6 cases. Strong expression of SIP1 was found in distal tubules of kidney, glandular epithelial cells of stomach and hepatocytes, implicating a co-expression of SIP1 and E-cadherin. Squamous epithelium of the esophagus and surface epithelium of colon and rectum were stained with moderate to weak intensity. Normal uterus, breast and lung tissues remained completely negative. By comparison with their normal tissues, we observed SIP1 overexpression in cancers of the kidney, breast, lung and uterus. However, SIP1 expression was found to be downregulated in tumors from colon, rectum, esophagus, liver and stomach tissues. Finally we did nuclear/cytoplasmic fractionation in 3 carcinoma cell lines and detected SIP1 in both fractions, nucleus being the dominant one. To our best knowledge, this is the first comprehensive immunohistochemical study of the expression of SIP1 in a series of human cancers. Our finding that SIP1 is not exclusively localized to nucleus suggests that the subcellular localization of SIP1 is regulated in normal and tumor tissues. These novel monoclonal antibodies may help elucidate the role of SIP1 in tumor development.
Smad 相互作用蛋白 1（SIP1，也称为 ZEB2）抑制 E-钙黏蛋白的转录，并在发育和肿瘤转移中介导上皮-间充质转化。由于缺乏人 SIP1 特异性抗体，其在人肿瘤组织中的表达尚未通过免疫组织化学进行详细研究。因此，我们生成了两种抗 SIP1 单克隆抗体，克隆 1C6 和 6E5，分别具有 IgG1 和 IgG2a 同种型。这些抗体的特异性通过 siRNA 介导的人骨肉瘤细胞系中 SIP1 和 ZEB1 的下调的 Western 印迹研究显示。在相同的背景下，我们还将它们与 5 种市售的 SIP1 抗体进行了比较。通过免疫荧光在可诱导的细胞系系统中进一步验证了抗体的特异性。使用这两种抗体，我们评估了 SIP1 在包含 22 个正常和 101 个肾、结肠、胃、肺、食管、子宫、直肠、乳腺和肝肿瘤组织的石蜡包埋组织微阵列中的组织表达。有趣的是，SIP1 主要显示细胞质表达，而仅在 6 例中观察到 SIP1 的核定位。在肾脏的远曲小管、胃的腺上皮细胞和肝细胞中发现 SIP1 的强表达，表明 SIP1 和 E-钙黏蛋白的共表达。食管的鳞状上皮和结肠和直肠的表面上皮染色呈中度至弱强度。正常子宫、乳腺和肺组织完全为阴性。与正常组织相比，我们观察到 SIP1 在肾、乳腺、肺和子宫癌中的过表达。然而，在来自结肠、直肠、食管、肝和胃组织的肿瘤中发现 SIP1 表达下调。最后，我们在 3 种癌细胞系中进行核/细胞质分馏，并在两个馏分中检测到 SIP1，核为主。据我们所知，这是 SIP1 在一系列人类癌症中的首次全面免疫组织化学研究。我们发现 SIP1 并非专门定位于核，表明 SIP1 的亚细胞定位在正常和肿瘤组织中受到调节。这些新型单克隆抗体可能有助于阐明 SIP1 在肿瘤发生中的作用。
