IDENTIFICATION AND CLONING OF METASTASIS-ASSOCIATED GENE

转移性疾病是癌症死亡的主要原因。 患者，但肿瘤细胞扩散的分子机制 都没有被清楚地理解。本申请的目的是 识别和鉴定其直接表达的特定基因 与人表皮样癌细胞转移行为的关系 行，HEp3，这些细胞是致瘤和转移(T+M+)当 在鸡胚绒毛尿囊膜(CAM)上连续生长。然而， 在体外培养时，细胞迅速失去转移潜能(T+M-)。 一种基于种子和Aruffo方法的表达克隆策略 (1987)目前正被用来从Hep-3cDNA中分离克隆 用三种不同的抗HEp3单抗(MoAbs)构建文库。 这些抗体识别T+M+上优先表达的抗原 细胞，并在CAM实验中抑制HEp3转移，分离的cDNAs 将被测序，并与已知序列进行比较。为了进一步 证明这些抗原在介导HEp3中的直接作用 转移，T+M+细胞将被反义构建 下调靶抗原的表达。反义的 转基因将在鸡胚胎和裸鼠身上进行测试。 转移试验。抗转移单抗和单抗的抗转移作用 反义转染对HEp3侵袭、黏附基质蛋白的影响 细胞外基质的降解和迁移也会 下定决心。将对这些moAbs识别的抗原进行表征 生物化学来确定它们的亚细胞位置，碳水化合物 含量，以及与其他细胞蛋白质的关联性。这些实验 被设计用来识别转移相关的抗原和可能 确定它们在转移中的作用。这些抗原在大肠杆菌中的表达 其他肿瘤细胞株和正常人体组织中也会 下定决心。 本申请利用了几个 抗HEp3单抗，以及分子克隆和基因转移的力量 克隆、鉴定和鉴定正向调控肿瘤的基因 细胞转移。给定这些工具和适当的接收方细胞， 复杂多步转移的分子机制 级联是可以研究的。

Metastatic disease is responsible for the majority of deaths in cancer patients, yet molecular mechanisms underlying tumor cell dissemination are not clearly understood. The purpose of the present application is to identify and characterize specific genes whose expression are directly related to the metastatic behavior of the human epidermoid carcinoma cell line, HEp3, These cells are tumorigenic and metastatic (T+M+) when grown continuously on the chick chorioallantoic membrane (CAM). However, when grown in vitro the cells rapidly lose metastatic potential (T+M-). An expression cloning strategy, based on the methods of Seed and Aruffo (1987) is currently being used to isolate clones from a HEp-3 cDNA library using three different anti-HEp3 monoclonal antibodies (moAbs). These antibodies recognize antigens preferentially expressed on T+M+ cells, and inhibit HEp3 metastasis in the CAM assay, the isolated cDNAs will be sequenced and compared to known sequences. To further demonstrate the direct role of these antigens in mediating HEp3 metastasis, T+M+ cells will be transfected with anti-sense constructs to down regulated expression of target antigens. Anti-sense transfectants will be tested in the chick embryo and nude mouse metastasis assays. The effects of the anti-metastatic moAbs and antisense transfection on HEp3 invasion, adhesion to matrix proteins, degradation of the extracellular matrix, and migration will also be determined. The antigens recognized by these moAbs will be characterized biochemically to determine their subcellular location, carbohydrate content, and association with other cellular proteins. These experiments are designed to identify the metastatis associated antigens and possibly determine their role in metastasis. Expression of these antigens in other tumor cell lines and in normal human tissues will also be determined. The present application takes advantage of the specificity of several anti-HEp3 moAbs, and the power of molecular cloning and gene transfer to clone, identify and characterize genes which positively regulate tumor cell metastasis. Given these tools and the appropriate recipient cell, the molecular mechanisms involved in the complex, multistep metastatic cascade can be studied.