Integrated Cancer Prevention Center, Tel Aviv Sourasky Medical Center, Tel Aviv 64239, Israel.
J Biol Chem. 2011 Nov 25;286(47):40548-55. doi: 10.1074/jbc.M111.286534. Epub 2011 Oct 5.
CD24 is a cell surface, heavily glycosylated glycosylphosphatidylinositol-anchored mucin-like protein that is overexpressed in various human malignancies. To accurately analyze CD24 function and dissect its biological role in a defined genetic background, it is critical to tightly regulate its expression and be able to turn it on/off in a restricted environment and at a specific time. The tetracycline-induced expression system is most promising as it exhibits such regulation, lack of pleiotropic effects, and high and rapid induction levels. To evaluate the oncogenic and immunotherapeutic potential of CD24 by applying the Tet-On system, the human CD24 gene was cloned downstream to two tetracycline operator sequences, resulting in pCDNA4/TO-CD24, which was then transfected into tetracycline (Tet) repressor-expressing cells (293T-REx), allowing tight on/off regulation, thereby resulting in a very low background or leaky CD24 expression. Selected clones were chosen for further studies and characterized in vitro and in vivo, and several treatment modalities were examined. In addition, the role of CD24 in promoting cell proliferation and tumor growth was studied. The tetracycline-dependent system was successfully implemented. Tetracycline treatment induced CD24 expression in a dose- and time-dependent fashion, which was abrogated following treatment with anti-CD24 monoclonal antibodies (mAbs). CD24-induced expression led to an increased proliferation rate that was inhibited by mAb treatment. In vivo, significantly larger tumors were developed in tetracycline-fed mice. The CD24 Tet-On system is a good model to unravel the role and underlying CD24 pathogenesis in vivo. This valuable tool allows the successful study of novel treatment options, whose effectiveness depends on the CD24 expression level. This set of experiments supports CD24 oncogenic properties.
CD24 是一种细胞表面高度糖基化的糖基磷脂酰肌醇锚定粘蛋白样蛋白，在各种人类恶性肿瘤中过度表达。为了准确分析 CD24 的功能并在明确的遗传背景下剖析其生物学作用，必须严格调控其表达，并能够在受限环境和特定时间内开启/关闭它。四环素诱导表达系统最有前途，因为它具有这种调控、缺乏多效性效应以及高水平和快速诱导。为了通过应用 Tet-On 系统评估 CD24 的致癌和免疫治疗潜力，将人 CD24 基因克隆到两个四环素操纵子序列的下游，得到 pCDNA4/TO-CD24，然后将其转染到四环素（Tet）抑制剂表达细胞（293T-REx）中，从而实现严格的开/关调控，从而导致非常低的背景或漏 CD24 表达。选择选定的克隆进行进一步的研究，并在体外和体内进行了特征描述，并检查了几种治疗方式。此外，还研究了 CD24 在促进细胞增殖和肿瘤生长中的作用。四环素依赖性系统成功实施。四环素处理以剂量和时间依赖的方式诱导 CD24 表达，而用抗 CD24 单克隆抗体（mAb）处理后则消除了这种表达。CD24 诱导的表达导致增殖率增加，而 mAb 处理则抑制了增殖率。在体内，在给予四环素的小鼠中，肿瘤明显增大。CD24 Tet-On 系统是一种很好的模型，可以在体内揭示 CD24 发病机制的作用和潜在机制。该有用工具允许成功研究新的治疗选择，其有效性取决于 CD24 的表达水平。这组实验支持 CD24 的致癌特性。
