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.
