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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.

Production of various forms of nonintegrated viral DNA was measured in cultured mouse cells carrying different Fv-1 alleles early after infection with N-tropic or B-tropic retroviruses. Quantitative analyses were performed by agarose gel electrophoresis, transfer to diazobenzyloxymethyl-paper, and molecular hybridization. In permissive infection of Fv-n cells (NIH Swiss and DBA mouse strains) with N-tropic virus and of Fv-1 b cells (BALB/c and C57BL/6 strains) with B-tropic virus, form III (double-stranded linear) DNA first appeared at 3-4 hr and reached a maximum at 8-10 hr; two form I (closed circle) DNAs appeared at 7-8 hr and reached a maximum at or beyond 12 hr. In the two Fv-1 b cells infected with N4ropic virus and in DBA (Fv-1 n) cells infected with B-tropic virus, formation of the two form I DNAs was quantitatively restricted but formation of form III DNA was unaltered. In Fv-1" NIH Swiss mouse embryo cells infected with B-tropic virus, the level of form III DNA was markedly depressed and hence the two form I DNAs were not detectable. In C57BL/6 cells as well as in DBA/2 cells 12 hr after infection, the quantity of form III DNA varied directly with the amount of restricted virus, whereas the quantity of form I DNA varied according to the square of the amount of restricted virus. The significance of these results for understanding the molecular basis of retrovirus replication and its restriction by the Fv-1 gene is discussed. The importance of the mouse Fv-1 gene locus in controlling infection by N-and B-tropic retroviruses has been well recognized in both animal studies and cell culture studies (1-5). This locus, mapped on chromosome 4 of the mouse (6), is presumably responsible for the production of specific inhibitor molecules in the cytoplasm of the cell (7-9). Inactivation of the restricted virus particles appears to be mediated by a virus protein, possibly the p30 core protein (10), which serves as a "target" for the Fv-1 gene product (11,12). However, the precise molecular mechanism of Fv-1 gene restriction is still unknown. Results of various previous investigations (13)(14)(15)(16)(17)(18)(19)(20) indicate that Fv-1 restriction occurs intracellularly at an early step of the virus replication cycle. Of particular significance are the observations (i) that, in Fv-1 restricted infection, the quantity of the cellgenome integrated viral DNA is markedly decreased whereas the formation of nonintegrated viral DNA appears to be unaffected (17-18), and (ii) that transfection by infectious integrated viral DNA is not restricted by the Fv-1 locus of the cell (19,20). These data imply that Fv-1 restriction may occur at or prior to the step of viral DNA integration.In the present study, we quantitatively measured the production of double-stranded linear and circular forms of viral DNA in Fv-1 permissive and restrictive cells early after infection with N-and B-tropic murine retroviruses. The results demonstrate that mouse cells may be assigned to either of two classes with regard to their ability to re...

Seven morphologically nontransformed (flat) revertants with reduced tumorigenicity in vivo have been isolated from populations of Kirsten sarcoma virus-transformed NIH 3T3 cells transfected with a cDNA expression library ofnormal human fibroblasts. Each revertant harbors 1-10 recombinant plis per cell and retains a rescuable transforming virus as well as high level expression of v-Ki-ras-specific RNA and the viral oncogene product, p21v-i-. Transformed phenotypes are suppressed in cell hybrids generated by fusing each revertant to v-Ki-rastransformed NIH 3T3 cells. From two of the revertant lines, plasmids capable ofgiving rise to flat secondary transfectants have been recovered. Thus, in some, if not all, of the revertants, transfected cDNAs seem to be responsible for the suppression of specific transformed phenotypes.Flat revertants from rodent fibroblast cell lines transformed by various agents have been isolated and characterized by several groups of investigators as an approach to define and, ultimately, resolve the mechanism(s) of malignant cell transformation (reviewed in refs. 1 and 2). This approach has been most successfully applied in defining and characterizing those genes that are responsible for the transforming activities of various retroviruses (e.g., see refs. 3 and 4). There are also additional reports describing flat revertants, which appear to have resulted from mutations in specific cellular genes rather than viral-encoded transforming genes (1,(5)(6)(7)(8). Such revertants may be useful in revealing cellular macromolecules that might either directly or indirectly participate in the process of cellular transformation. Previously, we described two nontumorigenic flat revertants isolated from populations of the DT line of Kirsten murine sarcoma virus (Ki-MSV)-transformed cells (7). These revertants seem to harbor dominant mutations in cellular genes, which in some way suppress certain malignant characteristics usually associated with Ki-MSV-induced transformation (7, 9-12). The putative gene or genes responsible for suppression of the malignant characteristics have not yet been identified.In this report, we show that flat revertants with reduced tumorigenicity can be isolated from Ki-MSV-transformed DT cells after transfection of normal human fibroblast cDNA expression library. The properties of some, if not all, of the revertants are consistent with the idea that expression of the introduced cDNA clones contributes to a reversal of the ras-induced transformed phenotype. MATERIALS AND METHODSCell Lines and Viruses. The derivations and methods for maintenance of NIH 3T3, DT (Ki/HGPRT-NIH 3T3), and Ki/TK-NIH have been described (7). DTneoR was derived from DT cells by transfection of pL2 DNA (13) followed by *G418 selection. Rescue for replication-defective sarcoma viruses, focus formation, and soft agar assays were performed as described (7, 9). Growth medium consisted of Dulbecco's modified Eagle's medium supplemented with 10% fetal calf serum (FCS) (KC Biological, Lenexa, KS) and 4 mM...

Oncogenes encoding serine/threonine or tyrosine kinases were introduced into the established rodent fibroblast cell line NIH 3T3 and tested for tumorigenic and metastatic behavior in T cell-deficient nude mice. Transforming oncogenes of the ras family were capable of converting fibroblast cell lines to fully metastatic tumors. Cell lines transformed by the kinase oncogenes mos, raf, src, fes, and fms formed experimental metastases and (in some cases) these genes were more efficient at metastatic conversion than a mutant ras gene. In contrast, cells transformed by either of two nuclear oncogenes, myc or p53, were tumorigenic when injected subcutaneously but were virtually nonmetastatic after intravenous injection. These data demonstrate that, in addition to ras, a structurally divergent group of kinase oncogenes can induce the metastatic phenotype.