Induction and progression of the transformed phenotype in cloned rat embryo fibroblast cells: Studies employing type 5 adenovirus and wild‐type and mutant Ha‐ras oncogenes

Abstract

Transformation of cloned rat embryo fibroblast (CREF) cells with the wild‐type 5 adenovirus (wtAd5) transforming genes E1A and E1B (which extend from 0 to 11.2 map units) results in morphologically transformed cells that exhibit an increased saturation density in monolayer culture and display an anchorage‐independent phenotype. WtAd5‐transformed CREF (wtAd5 CREF) cells do not, however, induce tumors when injected subcutaneously into athymic nude mice or syngeneic Fischer rats. We have analyzed the effect of the ras oncogene and sitespecific mutants in the ras oncogene that result in p21 proteins with altered biochemical properties on the oncogenic and metastatic properties of singly (ras) and doubly (ras + wtAd5) transformed CREF cells. Transformants expressing the wild‐type ras p21 protein and ras mutants producing p21 proteins that retained GTP‐binding properties grew in agar, induced tumors in nude mice and syngeneic rats, and metastasized to the lungs of rats when injected into their tail veins. In contrast, cells transformed with the ras mutant 116K (which contains a mutation at residue 116 that produces a Lys instead of an Asn and does not bind GTP or induce CREF cells to grow in agar) did not become morphologically transformed and were not oncogenic when injected subcutaneously into either nude mice or Fischer rats; further, such cells were not metastatic when injected into the tail veins of Fischer rats. When the wild‐type ras or the ras mutants, including 116K, were expressed in nontumorigenic E1A‐plus‐E1B‐expressing wtAd5 CREF cells, transformed cells induced tumors in both types of animals. The CREF cells doubly transformed with 116K + wtAd5, unlike transformants containing the wild‐type ras and the other ras mutants that still retained GTP binding, were still unable to induce lung metastases. In addition, 116K + wtAd5—transformed CREF cells also did not display any alterations in morphology distinguishable from wtAd5 CREF cells and were not able to grow in agar with increased efficiency. These results indicate that the loss of GTP‐binding ability by this mutant p21 ras protein eliminated the ability of these proteins to induce an oncogenic phenotype in an immortal but normal CREF cell line. However, the mutant ras could cooperate with wtAd5 transforming genes in transformed CREF cells to make these cells progress to an oncogenic (but not metastatic) phenotype.