By transfecting the adenovirus 5 E1A gene into neu-transformed NIH3T3 cells, we previously showed that E1A can dramatically repress neu-encoded p185 expression and, concomitantly, suppress the features of transformation and metastasis of neu+E1A transfectants. From these results we concluded that suppression of transformation and metastasis by E1A in neu-transformed cells may be through repression of neu gene expression. However, E1A has recently been shown to also repress the transformation features of other human cancer cells that do not overexpress neu. This observation raised a possibility that repression of neu gene expression in our neu+E1A cells might not be the only mechanism for transformation and metastasis suppression. To study whether other molecular mechanisms might be involved in suppression of transformation and metastasis by E1A in our neu+E1A cells, we reexpressed p185 oncoprotein in the neu+E1A cells by transfecting them with a plasmid containing activated rat neu complementary DNA and we examined whether E1A can suppress transformation and metastasis when the neu-encoded p185 protein is reexpressed. All the features of transformed cells including cell morphology, DNA synthesis rate, colony formation in soft agar, and tumorigenicity in nu/nu mice were restored in the cell lines that reexpressed neu. In addition, the levels of neu reexpression corresponded to the degree of malignant transformation. However, the in vivo metastatic tumor formation by these p185 reexpressing cells was still significantly inhibited by E1A. When metastasis-associated properties were examined in the cell lines that reexpressed p185, we found that cell motility was recovered by reexpression of pl85 to the degree corresponding to the p185 reexpression level, but secretion of membrane-degrading gelatinases and invasion through the basement membrane preparation Matrigel by these cells were still inhibited by E1A. The data demonstrated that reexpression of p185 in neu+E1A cells can counteract the tumor-suppressing function of E1A but not completely recover the neu-induced metastatic phenotype. We conclude from these results that (a) repression of new oncogene expression was indeed the molecular mechanism by which E1A suppressed tumor formation in neu-transformed 3T3 cells, and (b) suppression of metastasis by E1A in neu-transformed 3T3 cells was via multiple molecular mechanisms in addition to repressing neu. Our model system clearly demonstrated that tumorigenicity and metastasis are related but separable phenomena.
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This research was in part supported by Department of Health and Human Services Cancer Center Core Support Grant CA-16672 from the National Cancer Institute.