GENES WHICH POSITIVELY REGULATE TUMOR CELL METASTASIS

转移性疾病是造成大多数死亡的原因， 癌症患者，但肿瘤细胞的分子机制 传播不清楚。 编写本 建议是直接检测其表达的基因 与HEp-3细胞的转移行为有关， 癌细胞系 这些细胞在连续生长时具有转移性 鸡胚绒毛尿囊膜（CAM）。 然而，当生长在 在体外，细胞迅速失去转移潜能。实验来 确定正调节转移的HEp-3基因将需要 两种途径：1）转染。 一个来自高水平的cDNA文库， 转移性HEp-3细胞将以真核表达构建 载体pRc/RSV（Invitrogen）。 该质粒含有原核和 真核生物选择标记 非转移性HEp-3细胞将被 用该文库转染，并将选择稳定的转染子， G418的存在，在培养物中扩增，并在CAM中测试 转移测定。 该测定的选择压力将确保 只有表达适当基因的细胞才会转移。 转移性 细胞将通过酶促破坏小鸡器官来分离， 将所得细胞悬浮液接种于含有G418的培养基中。 到 回收cDNA，从回收的转移瘤细胞中分离的总DNA 将用Aat II切割转染子，Aat II是一种识别 pRc/RSV载体中的独特限制性位点。 DNA将被重新- 在低DNA浓度下环化并用于转化感受态E. coli细胞。 回收的cDNA将在转染中重新测试 实验如上。 那些诱导转移行为的将是 其特征在于核苷酸序列分析。2)鉴别筛选。 cDNA文库将从从高度分离的多聚A+ RNA制备。 转移性HEp-3细胞系。 图书馆将进行筛选， 从高度分离的poly A+ RNA合成的放射性标记的cDNA 转移性和非转移性HEp-3细胞系。 那些牌匾 优先被转移性细胞识别的RNA将被分离， 重新筛选如上所述。 第二次筛选后分离的克隆 将用于探测来自HEp-3细胞的RNA的北方印迹， 不同的转移潜能 与细胞杂交的cDNA 将根据转移潜力选择RNA用于 核苷酸序列分析

Metastatic disease is responsible for the majority of deaths in cancer patients, yet the molecular mechanisms underlying tumor cell dissemination are not clearly understood. The purpose of the present proposal is to detect the gene(s) whose expression is (are) directly related to the metastatic behavior of HEp-3 cells, a human epidermoid carcinoma cell line. These cells are metastatic when grown continuously on the chick chorioallantoic membrane (CAM). However, when grown in vitro the cells rapidly lose metastatic potential. Experiments to identify the HEp-3 gene(s) which positively regulate metastasis will take two avenues of approach: 1) Transfection. A cDNA library from highly metastatic HEp-3 cells will be constructed in the eukaryotic expression vector pRc/RSV (Invitrogen). This plasmid contains both prokaryotic and eukaryotic selection markers. Non-metastatic HEp-3 cells will be transfected with the library and stable transfectants will be selected in the presence of G418, expanded in culture, and tested in the CAM metastasis assay. The selection pressure of ,this assay will insure that only cells expressing the appropriate gene will metastasize. Metastatic cells will be isolated by enzymatically disrupting the chick organs and plating the resulting cell suspension in medium containing G418. To recover the cDNA, total DNA isolated from the recovered metastatic transfectants will be cut with Aat II, an enzyme which recognizes a unique restriction site in the pRc/RSV vector. DNA will be re- circularized at low DNA concentration and used to transform competent E. coli cells. 'The recovered cDNAs will be retested in transfection experiments as above. Those which induce metastatic behavior will be characterized by nucleotide sequence analysis. 2) Differential screening. A cDNA library will be made from poly A+ RNA isolated from a highly metastatic HEp-3 cell line. The library will be screened with radiolabeled cDNAs synthesized from poly A+ RNA isolated from highly metastatic and non-metastatic HEp-3 cell lines. Those plaques which are preferentially recognized by metastatic cell RNA will be isolated and rescreened as above. Those clones isolated after the second screening will be used to probe Northern blots of RNA from HEp-3 cells exhibiting different metastatic potentials. The cDNAs which hybridize to cellular RNA in accordance with metastatic potential will be selected for nucleotide sequence analysis.