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IDENTIFICATION AND CLONING OF METASTASIS-ASSOCIATED GENE

转移相关基因的鉴定和克隆

基本信息

批准号：
2008309
负责人：
JACQUELINE E TESTA
金额：
$ 17.19万
依托单位：
STATE UNIVERSITY NEW YORK STONY BROOK
依托单位国家：
美国
项目类别：
财政年份：
1995
资助国家：
美国
起止时间：
1995-01-01 至 1999-12-31
项目状态：
已结题

项目摘要

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.

转移性疾病是导致癌症死亡的主要原因患者，但肿瘤细胞播散的分子机制并不清楚。本申请的目的是为了鉴定和表征其表达直接影响基因表达的特定基因，与人表皮样癌细胞的转移行为有关细胞系HEp 3，这些细胞是致瘤性和转移性的（T+M+），当在鸡胚绒毛尿囊膜（CAM）上连续生长。然而，在这方面，当在体外生长时，细胞迅速失去转移潜能（T+M-）。基于Seed和Aruffo方法的表达克隆策略（1987）目前用于从HEp-3 cDNA中分离克隆使用三种不同的抗HEp 3单克隆抗体（moAb）构建文库。这些抗体识别优先在T+M+上表达的抗原细胞，并在CAM测定中抑制HEp 3转移，分离的cDNA 将被测序并与已知序列进行比较。进一步证明了这些抗原在介导HEp 3 转移，T+M+细胞将用反义构建体转染下调靶抗原的表达。反义将在鸡胚和裸鼠中测试转染子转移测定。抗转移性单克隆抗体和抗转移性单克隆抗体的作用反义转染对HEp 3侵袭、与基质蛋白的粘附、细胞外基质的降解和迁移也将被测定将对这些单克隆抗体识别的抗原进行表征以确定它们的亚细胞位置，碳水化合物内容，并与其他细胞蛋白质的关联。这些实验旨在识别转移相关抗原，决定它们在转移中的作用。这些抗原的表达在其它肿瘤细胞系和正常人组织中也将测定本申请利用了几个实施例的特异性。抗HEp 3单克隆抗体，以及分子克隆和基因转移的能力，克隆、鉴定和表征肿瘤正调控基因细胞转移有了这些工具和合适的受体细胞，参与复杂的多步骤转移的分子机制可以研究级联。