IDENTIFICATION AND CLONING OF METASTASIS-ASSOCIATED GENE

转移性疾病是导致大多数癌症死亡的原因

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.