Persistence of Ha-ras-induced metastatic potential of SP1 mouse mammary tumors despite loss of the Ha-ras shuttle vector.

Previous studies have shown that the SP1 mouse mammary adenocarcinoma cell line, which is tumorigenic but nonmetastatic, acquires metastatic potential when transfected with the activated human Ha-ras gene. In addition, the process of calcium phosphate-mediated DNA transfection, as well as treatment with the calcium ionophore A23187 or with phorbol 12-myristate 13-acetate, can also result in heritable changes in the malignant behavior of SP1 cells. It was of interest, therefore, to determine whether the metastatic consequences of Ha-ras oncogene expression in SP1 cells are a primary effect of the transfected gene or whether heritable secondary changes are induced by Ha-ras oncogene expression. In the latter case, continued expression of the Ha-ras oncogene would not be required to maintain the metastatic phenotype. To test this hypothesis we introduced the Ha-ras oncogene into SP1 cells on a shuttle vector in which maintenance of the vector was dependent on selection for resistance to the antibiotic G418. Subclones which had lost the transfected Ha-ras gene were subsequently isolated following growth in nonselective medium. The Ha-ras-transfected clones and the revertant subclones were found to be equally metastatic, indicating that transfection with the Ha-ras gene does induce stable secondary changes in the metastatic phenotype of SP1 cells.