GENES WHICH POSITIVELY REGULATE TUMOR CELL METASTASIS

转移性疾病是导致大多数人死亡的原因 癌症患者，但肿瘤细胞背后的分子机制 对传播的理解不是很清楚。现在的目的是 建议直接检测表达的基因(S) 人表皮样癌Hep-3细胞转移行为的研究 癌细胞系。这些细胞在连续生长时是转移的。 在鸡绒毛膜尿囊膜上。然而，当生长在 在体外，这些细胞会迅速失去转移潜能。实验以实现 鉴定正向调控转移的HEp-3基因(S) 途径有两条：1)转染法。高密度脂蛋白基因文库的构建 转移性Hep-3细胞将在真核细胞中表达 载体PRC/RSV(Invitgen)。此质粒包含原核生物和 真核选择标记。非转移性Hep-3细胞将 用文库转染法筛选出稳定的转染体 G418的存在，在培养中扩大，并在CAM中进行测试 转移试验。这个测试的选择压力将确保 只有表达适当基因的细胞才会转移。转移性 细胞将通过酶作用破坏鸡的器官来分离 将得到的细胞悬液接种在含有G418的培养液中。至 从已恢复的转移瘤中提取cDNA、总DNA 转染体将被AAT II切割，AAT II是一种识别 PRC/RSV载体中的唯一限制位点。DNA将被重新- 在低DNA浓度下循环，用于转化感受态E. Coli细胞。“回收的DNA将在转染法中重新测试。 如上的实验。那些导致转移行为的人将是 以核苷酸序列分析为特征。2)鉴别筛查。 从一株高度分离的多聚体A RNA中构建了一个cDNA文库 转移性Hep-3细胞株。这座图书馆将用 从高度分离的猪瘟病毒多聚A RNA中合成放射性标记的cDNA 转移和非转移Hep-3细胞株。那些斑块是 被转移细胞优先识别的RNA将被分离并 如上所述重新筛选。二次筛选后分离的克隆 将用于探测Hep-3细胞的RNA的Northern blotts 不同的转移潜能。与细胞杂交的cDNA 将根据转移潜能选择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.