NOTCH-1 IN REGULATION OF APOPTOSIS IN TUMOR CELLS

Notch基因编码跨膜受体，在发育过程中控制许多细胞类型的细胞分化。人类和小鼠有4个同源notch基因。 引起notch-1、notch-2和notch-4组成性激活的突变在体内和体外都是致癌的。 临床上，notch-1的组成性激活与T细胞急性淋巴细胞白血病（T-ALL）相关，并且在各种人类恶性肿瘤和肿瘤前病变（子宫颈、结肠和肺）中已经证明notch-1的表达显著增加，细胞内分布改变。 我们和其他人最近发现notch-1在各种实验模型的转化细胞中具有体外抗凋亡活性。 此外，我们发现在诱导分化的混合极性药物存在下下调小鼠红白血病（MEL）细胞中notch-1的表达导致大量凋亡。 notch-1是否影响未过度表达的非转化细胞的凋亡仍然未知。我们的假设是notch-1在调节notch-1表达的肿瘤细胞的凋亡易感性中起重要作用。 这意味着干扰notch-1表达或信号传导可用于增强化疗或放疗在表达notch-1的人类恶性肿瘤中的疗效。 我们的长期目标是使用基因疗法、重组notch-1拮抗剂或notch-1单克隆抗体开发针对notch-1的治疗方案，用于表达notch-1的人类恶性肿瘤的临床测试。 我们已经在这些类别中的每一个中生成了实验代理。我们的目标如下：1.为了确定notch-1是否参与正常细胞的凋亡调节，我们开发了新型转基因小鼠和notch-1拮抗剂来研究其在胸腺细胞凋亡中的作用。 2.通过研究已知与参与调节凋亡的notch-1相关的信号通路，阐明notch-1保护MEL细胞免于凋亡的机制。 3.目的：对notch-1反义逆转录病毒载体、重组notch-1拮抗剂和notch-1阻断单克隆抗体在小鼠肿瘤模型中的应用进行临床前研究。 实验药物将单独研究和与其他药物联合研究。 研究将在具有人宫颈癌细胞的免疫缺陷动物和具有由人乳头瘤病毒16（HPV 16）转化的同基因鼠肿瘤细胞的免疫活性动物中进行。这些实验将阐明notch-1作为癌症治疗靶点的可能用途以及notch-1靶向药物的机制。 此外，他们将提供这些药物的安全性信息，以及notch-1靶向药物在notch-1表达恶性肿瘤中的临床试验的初步概念证明。

Notch genes encode transmembrane receptors that control cell differentiation in numerous cell types during development. Humans and mice have 4 homologous notch genes. Mutations causing constitutive activation of notch-1,-2 and 4 are oncogenic in vivo and in vitro. Clinically, constitutive activation of notch-1 is associated with T-cell acute lymphoblastic leukemia (T-ALL), and strikingly increased expression with altered intracellular distribution of notch-1 have been demonstrated in various human malignancies and pre-neoplastic lesions (uterine cervix, colon and lung). We and others have recently discovered that notch-1 has anti-apoptotic activity in vitro in transformed cells in various experimental models. Additionally, we found that downregulating the expression of notch-1 in murine erythroleukemia (MEL) cells in the presence of differentiation- inducing hybrid polar drugs leads to massive apoptosis. Whether notch-1 affects apoptosis in non-transformed cells which do not overexress it remains unknown. Our hypothesis is that notch-1 plays an important role in regulating apoptosis susceptibility in notch-1 expressing tumor cells. This implies that interfering with notch-1 expression or signaling may be used to enhance the efficacy of chemotherapy or radiotherapy in human malignancies expressing notch-1. Our long term objective is the development of therapeutic regimens targeting notch-1 using gene therapy, recombinant notch-1 antagonists or notch-1 monoclonal antibodies for clinical testing in human malignancies expressing notch-1. We have generated experimental agents in each of these catagories. Our Aims our as follows: 1. To determine if notch-1 participates in apoptosis regulation in normal cells, by studying its role in thymocyte apoptosis using notch-1 antagonists and novel transgenic mice we have developed. 2. To elucidate the mechanism(s) through which notch-1 protects MEL cells from apoptosis, by investigating signaling pathways known to be linked to notch-1 which participate in regulating apoptosis. 3. To conduct pre-clinical studies of notch-1 inducible antisense retrovirus vectors, a recombinant notch-1 antagonists and a notch-1 blocking monoclonal antibody in mouse tumor models. The experimental agents will be studied alone and in combination with antineoplastic drugs. Studies will be conducted in both immunodeficient animals with human cervical cancer cells and immunocompetent animals with syngeneic murine tumor cells transformed by human papillomavirus 16 (HPV16). These experiments will elucidate the possible uses of notch-1 as a target for cancer treatment and the mechanisms of notch-1 targeting agents. Additionally, they will provide information on the safety profile of these agents and initial proof of concept for clinical trials of notch-1 targeting agents in notch-1 expressing malignancies.