NOTCH-1 IN REGULATION OF APOPTOSIS IN TUMOR CELLS

Notch基因编码跨膜受体，在发育过程中控制多种细胞类型的细胞分化。人类和小鼠有4个同源的缺口基因。导致Noch-1、-2和4结构性激活的突变在体内和体外都是致癌的。临床上，Noch-1的结构性激活与T细胞急性淋巴细胞白血病(T-ALL)有关，在多种人类恶性肿瘤和癌前病变(子宫颈、结肠和肺)中，Noch-1的表达显著增加，细胞内分布发生改变。我们和其他人最近在各种实验模型中发现Noch-1在体外转化的细胞中具有抗凋亡活性。此外，我们还发现，在诱导分化的杂交极性药物存在的情况下，下调小鼠红白血病(MEL)细胞中Noch-1的表达会导致大量的细胞凋亡。目前尚不清楚Noch-1是否影响不过度表达Noch-1的未转化细胞中的细胞凋亡。我们的假设是，Noch-1在调节表达Noch-1的肿瘤细胞的凋亡易感性中起重要作用。这意味着干扰Noch-1的表达或信号可用于提高表达Noch-1的人类恶性肿瘤的化疗或放射治疗的疗效。我们的长期目标是开发针对Noch-1的治疗方案，使用基因疗法、重组Noch-1拮抗剂或Noch-1单抗，用于临床测试表达Noch-1的人类恶性肿瘤。我们在这些类别中的每一个类别中都产生了实验试剂。我们的研究目的如下：1.利用Notch-1拮抗剂和我们建立的新型转基因小鼠，研究Noch-1在胸腺细胞凋亡中的作用，以确定Noch-1是否参与正常细胞的凋亡调控。2.通过研究已知的与Noch-1相关的参与调控细胞凋亡的信号通路，阐明Noch-1保护MEL细胞免于凋亡的机制(S)。3.在小鼠肿瘤模型中进行Noch-1可诱导的反义逆转录病毒载体、重组Noch-1拮抗剂和Noch-1封闭型单抗的临床前研究。实验药物将单独研究，并与抗肿瘤药物联合研究。研究将在免疫缺陷的人宫颈癌细胞动物和免疫活性动物的人乳头瘤病毒16(HPV16)转化的同基因小鼠肿瘤细胞中进行。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靶向剂的作用机制。此外，他们还将提供有关这些药物的安全性概况的信息，并为NOTCH-1靶向药物在表达NOTCH-1的恶性肿瘤的临床试验中提供初步的概念验证。

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.