FUNCTION OF EGF LIKE HOMEOTIC GENES IN CANCER AND THE IMMUNE SYSTEM

通过使用我们去年开发的抗体，我们已经证明：1） notch-1蛋白的稳态水平，而不是notch-1 mRNA的稳态水平， Friend小鼠终末分化过程中下调 红白血病细胞，表明可能的翻译后机制 控制缺口量-1; 2）观察到这种现象 与用于诱导终末分化的试剂无关 （六亚甲基双乙酰胺[HMBA]或丁酸钠）; 3） notch-1蛋白在哺乳动物中非常早地瞬时上调， HMBA诱导Friend细胞分化（1小时）通过一种途径， 似乎需要蛋白激酶C的钙离子非依赖性同种型。的 这种早期上调的机制和可能的作用目前 在研究中此外，4）notch-1的相似模式 当SY 5 Y人神经母细胞瘤细胞被转染时， 当Jurkat T细胞被激活时， 体外我们目前正在测试的假设是， notch-1的上调通过以下方式控制细胞分化的时间 使细胞保持在细胞周期中并变得有能力 接收第二信号，所述第二信号诱导生长停滞和终止 分化支持我们假设的初步证据是 通过用几种不同的硫代磷酸酯 反义notch-1寡核苷酸。这种治疗 显著降低（与有义寡核苷酸相比）总的 细胞数量和分化细胞的百分比。如果这 假设得到证实，操纵notch-1水平或其信号 转导途径可用作预期治疗的辅助剂 导致人类恶性肿瘤的终末分化和细胞死亡。 我们目前正在开发几种细胞的稳定转染克隆， 用反义notch-1构建体和用 致癌notch-1结构，以研究notch 活化、细胞周期和细胞分化。我们还产生了 针对推定的配体结合区的单克隆抗体 人类一级切口这些抗体将用于研究：1） notch-1在造血系统恶性肿瘤中的表达和2）信号 在体外分化模型中从野生型notch-1转导。 已发表的报告表明，许多人类实体瘤 与未转化的组织相比，Notch-1显著过表达。因此，在本发明中， 我们的最终目标是测试这些单克隆抗体中的一种或多种是否 抗体具有潜在的诊断和/或治疗应用， 人类恶性肿瘤最后，我们开发了两株notch-1 反义转基因小鼠我们目前正在扩大这些殖民地 到今年秋天我们就能生产出纯合子。这些小鼠 可能为我们提供了第一个部分notch-1缺陷的模型， 产后生活此外，这样的模型可以帮助我们预测 在体内操纵notch-1表达的可能作用， 治疗目的。

By using the antibodies we developed last year, we have shown that 1) steady state levels of notch-1 protein, but not of notch-1 mRNA, are downregulated during terminal differentiation of Friend murine erythroleukemia cells, indicating possible post-translational mechanisms controlling the amount of notch-1; 2) this phenomenon is observed independently of the agent used to induce terminal differentiation (hexamethylene-bis-acetamide [HMBA] or sodium butyrate); 3) levels of notch-1 protein are transiently upregulated very early during HMBA-induced Friend cell differentiation (1 h) through a pathway that seems to require Ca2+- independent isoforms of protein kinase C. The mechanism and possible role of this early upregulation are currently under investigation. In addition, 4) similar patterns of notch-1 expression are observed when SY5Y human neuroblastoma cells are differentiated with retinoic acid and when Jurkat T cells are activated in vitro. The hypothesis we are currently testing is that the early upregulation of notch-1 controls the timing of cell differentiation, by allowing the cell to remain in the cell cycle and become competent to receive a second signal which induces growth arrest and terminal differentiation. Preliminary evidence in support of our hypothesis was obtained by treating Friend cells with several different phosphorothioate antisense notch-1 oligonucleotides in addition to HMBA. Such treatment significantly reduces (compared to sense oligonucleotides) both the total number of cells and the percentage of differentiated cells. If this hypothesis is confirmed, manipulating notch-1 levels or its signal transduction pathway may be used as an adjuvant to treatments intended to cause terminal differentiation and cell death in human malignancies. We are currently developing stably transfected clones of several cellular differentiation models with antisense notch-1 constructs and with oncogenic notch-1 constructs to study the relationship between notch activation, cell cycle and cell differentiation. We have also generated monoclonal antibodies directed to the putative ligand-binding region of human notch-1. These antibodies will be used to study: 1) levels of notch-1 expression in hematopoietic malignancies and 2) signal transduction from wild-type notch-1 in in vitro differentiation models. Published reports suggest that a number of human solid tumors considerably overexpress notch-1 compared to untransformed tissues. Thus, our eventual objective is to test whether one or more of these monoclonal antibodies has potential diagnostic and/or therapeutic applications in human malignancies. Finally, we have developed 2 strains of notch-1 antisense transgenic mice. We are currently expanding colonies of these strains and we will have generated homozygotes by this fall. These mice may provide us with the first model of partial notch-1 deficiency during postnatal life. In addition, such a model may help us predict the possible effects of manipulating notch-1 expression in vivo for therapeutic purposes.