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Two flat revertants have been isolated from mutagen-treated populations of Kirsten murine sarcoma virus (KiMuSV)-transformed NIH/3T3 cells. These revertants, which appear to be cellular variants resistant to transformation by the KiMuSV oncogene v-Ki-ras, contain Ki-MuSV-specific DNA, elevated levels of the v-Ki-ras gene product p2l, and rescuable transforming virus. Cell hybridization studies indicated that the revertant phenotype is dominant in hybrids between revertant cells and cells transformed by Ki-MuSV or the closely related Harvey MuSV and BALB MuSV. Analysis of hybrid cells resulting from the fusion of these revertants to cell lines transformed by other retroviruses showed that the action of certain oncogenes structurally unrelated to v-Ki-ras also could be suppressed. Thus, there appear to be functional relationships and diversities among transforming genes (oncogenes) not readily apparent from their structural characteristics.Recent molecular studies have defined the structure of a number of transforming genes (oncogenes) and their products in considerable detail (reviewed in ref. 1). All of the known retroviral oncogenes are closely related to sequences present in normal vertebrate cells (cellular proto-oncogenes), from which they appear to have arisen (1, 2). The viral oncogenes associated with Kirsten and Harvey murine sarcoma viruses (Ki-and Ha-MuSVs), v-Ki-ras and v-Ha-ras, for example, are known to encode similar phosphorylated 21,000-dalton proteins (designated p2ls) with guanine nucleotide-binding activities (3-6). v-Ki-ras and v-Haras are structurally related to highly conserved cellular sequences (proto-oncogenes), some of which may be associated with the etiology of certain human tumors (refs. 7 and 8; reviewed in refs. 9 and 10).In spite of the relatively large body of information concerning the molecular structure of retrovirus oncogenes and the proteins that they specify, comparatively little is known about the cellular components with which these proteins interact and the mechanism(s) by which they transform cells. The nature of the cellular components involved in the expression of transformation can be defined theoretically by the isolation and molecular characterization of flat nontransformed variants (revertants) from populations of retrovirus-transformed cells. Although a number of such flat revertants have been isolated from cells transformed by retroviruses (refs. 11-21; reviewed in ref. 22), the great majority of these has been shown to lack expression of functioning viral oncogenes. A few revertant cell lines containing apparent alterations in host cell genomes have been isolated (13, 21); however, little is known about the specific cellular factors involved in the reversion process.This report describes two cellular revertants that are resistant to transformation by Ki-MuSV and certain other retroviruses. These revertants may be used to study the mechanism of transformation by the v-Ki-ras oncogene and to reveal functional relationships among different viral oncogene...

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To identify proteins whose production may be altered as a common event in the expression of structurally diverse oncogenes, we compared two-dimensional electropherograms of newly synthesized proteins from NIH/3T3 cell lines transformed by a variety of retroviral oncogenes, from cellular revertant lines, and from a line (433.3) which expresses the v-ras oncogene in response to corticosteroids. Most alterations in the synthesis of specific proteins detected by this approach appeared to be the result of selection during prolonged cultivation and were probably unrelated to the transformation process. However, we detected seven proteins whose synthesis was strongly suppressed in cell lines transformed by each of the six retroviral oncogenes we studied and whose production was fully or partially restored in two cellular revertant lines. Suppression of two of these proteins was also correlated with the initial appearance of morphological alteration during corticosteroid-induced oncogene expression in 433.3 cells. These proteins (p37/4.78 and p41/4.75) were identified as tropomyosins, a group of at least five cytoskeletal proteins. Transformation by the papovaviruses simian virus 40 and polyomavirus caused no suppression of synthesis of these tropomyosins. This indicates that suppression of tropomyosin synthesis is not a nonspecific response by cells to being forced to grow with the transformed phenotype but is specifically associated with oncogenesis by diverse retroviral oncogenes. The results are consistent with the hypothesis that the different biochemical processes initiated by expression of structurally diverse retroviral oncogenes may converge on a limited number of common targets, one of which is the mechanism which regulates the synthesis of tropomyosins.

Using an expression cloning assay, we have isolated a novel cDNA, referred to as rsp-1, which suppresses the v-Ras-transformed phenotype. When introduced into NIH 3T3 fibroblasts under the control of a metallothionein promoter, rsp-i confers resistance to v-Ras, but not to v-Mos or v-Src, and inhibits growth of the cells. The rsp-1 cDNA contains a 831-bp open reading frame encoding a 277-amino-acid leucine-rich protein. The rsp-l cDNA exhibits no significant homology to sequences in the DNA data bases. However, searches of the protein data bases revealed that it contains a series of leucine-based repeats which are homologous to the leucine repeats found in the regulatory region of the yeast adenylyl cyclase. rsp-1 specific RNA is detectable in a wide variety of cell lines and tissues, and the gene is conserved among eukaryotic species. These data suggest that rsp-l plays a role in Ras signal transduction.