Behavior of myc and ras oncogenes in transformation of rat embryo fibroblasts.

The requirements for transformation of rat embryo fibroblasts (REFs) by transfected ras and myc oncogenes were explored. Under conditions of dense monolayer culture, neither oncogene was able to transform REFs on its own. However, the introduction of a ras oncogene together with a selectable neomycin resistance marker into REFs allowed killing of the normal nontransfected cells and the outgrowth of colonies of ras transformants, 10% of which survived crisis and became tumorigenic. These cells expressed greater than 10-fold-higher levels of ras p21 than tumorigenic cells cotransfected with ras and myc oncogenes. The myc oncogene similarly was unable to induce tumorigenic conversion of REFs unless especially refractile colonies of oncogene-bearing cells, produced by use of a cotransfected selectable marker, were picked and subcultured. Tumorigenic conversion of REFs by single transfected oncogenes appears to require special culture conditions and high levels of gene expression.     

Multistage carcinogenesis induced by ras and myc oncogenes in a reconstituted organ

ras and myc oncogenes were able to induce distinct phenotypic alterations, resembling different types of premalignant lesions, when introduced into approximately 0.1% of the cells used to reconstitute the mouse prostate gland. While ras induced dysplasia in combination with angiogenesis, myc induced a hyperplasia of the otherwise normally developed organ. ras and myc together induced primarily carcinomas. However, tumor progression was also associated with additional genetic alterations involving gene amplification. Our data indicate that specific types of benign premalignant lesions may reflect the activation of different single oncogenes, and that the consecutive activation of multiple oncogenes could be a causal event in the step-like progression of tumorigenesis.     

Ras-induced hyperplasia occurs with mutation of p53, but activated ras and myc together can induce carcinoma without p53 mutation.

Using a reconstituted mouse prostate organ, the effects on endogenous p53 expression of the ras oncogene or of the ras + myc oncogenes were investigated. In this system the ras gene alone causes mild hyperplasia, but the combination of ras and myc leads to the formation of carcinomas. Surprisingly, while p53 mutations were found in cells derived from the reconstituted organs containing ras alone, no such mutations were found in the ras + myc-transformed cells. Their growth, unlike that of the cells containing ras alone, was not inhibited by transfection with plasmids encoding wild-type human p53. We suggest that expression of both activated ras and myc genes bypasses the need for p53 mutation by neutralizing the tumor suppressor activity of normal p53.     

Rat embryo cells immortalized with transfected oncogenes are transformed by gamma irradiation.

Cesium-137 gamma rays were used to transform rat embryo cells (REC) which were first transfected with activated c-myc or c-Ha-ras oncogenes to produce immortal cell lines (REC:myc and REC:ras). When exposed to 6 Gy of 137Cs gamma rays, some cells became morphologically transformed with focus formation frequencies of approximately 3 x 10(-4) for REC:myc and approximately 1 x 10(-4) for REC:ras, respectively. Cells isolated from foci of gamma-ray-transformed REC:myc (REC:myc:gamma) formed anchorage-independent colonies and were tumorigenic in nude mice, but foci from gamma-ray-transformed REC:ras (REC:ras:gamma) did not exhibit either of these criteria of transformation. Similar to the results with gamma irradiation, we observed a sequence-dependent phenomenon when myc and ras were transfected into REC, one at a time. REC immortalized by ras transfection were not converted to a tumorigenic phenotype by secondary transfection with myc, but REC transfected with myc were very susceptible to transformation by subsequent ras transfection. This suggests that myc-immortalized cells are more permissive to transformation via secondary treatments. In sequentially transfected REC, myc expression was high whether it was transfected first or second, whereas ras expression was highest when the ras gene was transfected secondarily into myc-containing REC. Molecular analysis of REC:ras:gamma transformants showed no alterations in structure of the transfected ras or of the endogenous ras, myc, p53, or fos genes. The expression of ras and p53 was increased in some isolates of REC:ras:gamma, but myc and fos expression were not affected. Similarly, REC:myc:gamma transformants did not demonstrate rearrangement or amplification of the transfected or the endogenous myc genes, or of the potentially cooperating Ha-, Ki-, or N-ras genes. Northern hybridization analysis revealed increased expression of N-ras in two isolates, REC:myc:gamma 33 and gamma 41, but no alterations in the expression of myc, raf, Ha-ras, or Ki-ras genes in any REC:myc transformant. DNA from several transformed REC:myc:gamma cell lines induced focus formation in recipient C3H 10T1/2 and NIH 3T3 cells. The NIH 3T3 foci tested positive when hybridized to a probe for rat repetitive DNA. A detailed analysis of the NIH 3T3 transformants generated from REC:myc:gamma 33 and gamma 41 DNA failed to detect Ha-ras, Ki-ras, raf, neu, trk, abl, fms, or src oncogenes of rat origin.(ABSTRACT TRUNCATED AT 400 WORDS)     

Enhanced growth characteristics of hybridoma cells infected with myc- and ras-containing retroviruses

The introduction of retrovirally encoded myc or ras genes into the mouse hybridoma cell line PQXB 1/2, in which antibody production is unstable, resulted in altered growth characteristics: infected cells showed increased growth rates, higher peak cell densities, and slower decline of viability following maximum density than did control cultures. In addition, some oncogene-infected cultures maintained initial levels of antibody production during 16 weeks in culture, while antibody levels fell by 90% in uninfected PQXB cells. Both myc and ras oncogenes were expressed at only low levels in control and infected cells. These results suggest that oncogene expression in hybridomas can lead to enhanced growth characteristics and can stabilize antibody production.     

Large E1B proteins of adenovirus types 5 and 12 have different effects on p53 and distinct roles in cell transformation.

The formation of complexes between oncoproteins of DNA tumor viruses and the cellular protein p53 is thought to result in inactivation of the growth suppressor function of p53. In cells transformed by nononcogenic human adenovirus type 5 (Ad5), the 55-kDa protein encoded by E1B forms a stable complex with p53 and sequesters it in the cytoplasm. However, the homologous 54-kDa protein of highly oncogenic Ad12 does not detectably associate with p53. Yet in Ad12-transformed cells, p53 is metabolically stable, is present at high levels in the nucleus, and contributes to the oncogenicity of the cells. Such properties have previously been described for mutant forms of p53. Here, we show that stable p53 in Ad12-transformed cells is wild type rather than mutant and that stabilization of p53 is a direct consequence of the expression of the Ad12 E1B protein. We also compared the effects of the E1B proteins on transformation of rodent cells by different combinations of oncogenes. A synergistic interaction was observed for the gene encoding the 54-kDa E1B protein of Ad12 with myc plus ras oncogenes, resembling the effect of mutant p53 on myc plus ras. In contrast, the Ad5 55-kDa E1B protein strongly inhibited transformation by myc plus ras but stimulated transformation by E1A plus ras. The data are explained in terms of different interactions of the two E1B proteins with endogenous p53. The results suggest that in cultured rat cells, endogenous wild-type p53 plays an essential role in cell proliferation, even in the presence of myc plus ras. The dependence on p53 is lost, however, when the adenovirus E1A oncogene is present.     

Active ras and myc oncogenes can be compatible, but Sv40 large T antigen is specifically suppressed with normal differentiation of mouse embryonic stem cells

The pathobiological effects of oncogenes on normal differentiation of mouse embryonic stem cells from 4-day embryos were examined by introducing active ras, myc, and SV40 large T genes, all driven by mouse metallothionein I enhancer and promoter. Stem cell clones R5, M3, and T2 for ras, myc, and SV40 T genes, respectively, were particularly chosen for analyses because of their higher levels of transgene expression and their diploid chromosomal constitutions. These stem cells were then introduced into host 4-day embryos and the embryos were allowed to develop in the uterus of foster mothers. The stem cells colonized the tissues as extensively as the parent cells and gave rise to adult chimera with no apparent loss or abnormality of the embryos. The active ras and myc oncogenes introduced were expressed not only in the stem cells, but also in the developing embryos and in a variety of tissues of adult chimeras. However, although T antigen was originally expressed in the stem cells, it was not expressed in either developing embryos or tissues of adult chimeras. Induced by retinoic acid treatment in vitro or by subcutaneous grafting, this suppression of T-gene expression was also confirmed in differentiated progeny cells from several stem cell clones expressing T antigen. Permanent lines of fibroblast-like cells could be established at higher frequency from primary cultures of tissues of chimera, subcutaneous differentiated cells, and in vitro differentiated cells derived from T2 cells, and all these clones reexpressed T antigen. The results suggest that active myc and ras genes can be compatible with normal differentiation of the stem cells, but the expression of T antigen is specifically suppressed with recognition of its coding domain.     

Transfer of ‘immortalizing’ oncogenes into rat fibroblasts induces both high rates of sister chromatid exchange and appearance of abnormal karyotypes

Cell lines established after transfer into FR3T3 rat fibroblast cells of 'immortalizing' oncogenes (plt gene (large T protein) of polyoma virus, v-myc gene of MC29 virus, rearranged forms of c-myc) exhibited increased rates of sister chromatid exchange (SCE). This was observed neither in cells which expressed one of the oncogenes responsible for the terminal stages of tumorigenic transformation (polyoma virus pmt (middle T protein), mutated ras genes), nor in cell lines carrying oncogenes of both types. Abnormal chromosome numbers were observed in cell lines expressing plt or myc, but not after transformation by pmt or ras oncogenes.     

Differential responsiveness of myc- and ras-transfected cells to growth factors: selective stimulation of myc-transfected cells by epidermal growth factor.

To identify functional relationships between oncogenes and growth factors, we compared the effects of transfected myc and ras oncogenes on the responsiveness of Fischer rat 3T3 cells to three growth factors: epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF-beta). Control cells did not grow in soft agar under any conditions. ras-Transfected cells grew in soft agar under all conditions tested and were insensitive to the stimulatory effects of exogenous growth factors. These cells secreted elevated levels of both EGF-like factors and TGF-beta, suggesting that the lack of responsiveness of these cells to exogenous growth factors arose from autocrine stimulation. myc-Transfected cells displayed conditional anchorage-independent growth: they formed numerous colonies in soft agar in the presence of EGF but relatively few colonies in the presence of PDGF or TGF-beta. Secretion of EGF-like factors and TGF-beta by these cells was not elevated above that of control cells. These results suggest a model for the mechanism of cooperation between myc and ras oncogenes in which ras-like genes induce growth factor production, while myc-like genes increase the responsiveness of cells to these factors.     

Differential induction of cytolytic susceptibility by E1A, myc, and ras oncogenes in immortalized cells.

The E1A oncogene of adenovirus serotypes 2 and 5 induces susceptibility to the cytolytic effects of natural killer lymphocytes and activated macrophages when expressed in infected and transformed mammalian cells (cytolysis-susceptible phenotype). E1A and the oncogenes v-myc, long-terminal-repeat-promoted c-myc, and activated c-ras share the ability to immortalize transfected low-passage rodent cells. The cytolytic phenotypes of well-characterized rodent cell lines immortalized by these three oncogenes were defined. In contrast to target cells expressing the intact E1A gene, myc- and ras-expressing, immortalized primary transfectants were resistant to lysis by both types of killer cell populations. The same patterns of susceptibility (E1A) and resistance (myc and ras) to cytolysis were observed in oncogene-transfected continuous rat (REF52) and mouse (NIH 3T3) cell lines, indicating that differences in the cytolytic phenotypes associated with expression of these oncogenes are not due to cell selection during immortalization. The results suggest that the E1A oncogene may possess a functional domain that is different from those of other oncogenes, such as myc and ras, and that the activity linked to this postulated domain is dissociable from the process of immortalization.     

bcr/abl and src but not myc and ras replace v-abl in lymphoid transformation.

Lymphoid cells transformed by temperature-sensitive Abelson virus die at the nonpermissive temperature. This property was exploited to show that bcr/abl and v-src but not myc and ras can replace the transforming signal of v-abl, a result suggesting that the former but not the latter oncogenes transform lymphoid cells via a similar pathway.     

Synergistic effects of the myc and ras oncogenes on ganglioside synthesis by BALB/c 3T3 fibroblasts

The effects of oncogene activation on glycosphingolipid (GSL) synthesis by a mouse fibroblast clonal cell line were studied. A transfectant that expressed the activated ras gene showed a definite change in the composition of acidic GSLs, probably an increase in polysialoganglioside, while one that expressed the myc gene showed only a slight change. Neither transfectant grew in soft agar. However, another transfectant, which expressed both the myc and ras genes, and grew in soft agar, showed a more dramatic increase in the acidic GSL component. Thus, activations of the myc and ras oncogenes have a synergistic effect on GSL synthesis during transformation.     

Basal levels of max are sufficient for the cotransformation of C3H10T1/2 cells by ras and myc.

The ras and myc oncoproteins cooperate to transform the established murine fibroblast cell line C3H10T1/2. To determine the impact of overexpression of the myc oncoprotein on the phenotype of C3H10T1/2 cells, two C3H10T1/2-myc clonal cell lines, SVc-myc 11A and myc neo 13A, were isolated and characterized. Although both C3H10T1/2-myc cell lines are morphologically indistinguishable from wild-type C3H10T1/2 cells and possess growth properties similar to those of C3H10T1/2 cells, each displays a predisposition to transformation following transfection with the activated form of the human H-ras gene. In C3H10T1/2 cells overexpressing the v-myc or H-ras oncogenes, the levels of mRNA encoding max, the recently identified oligomerization partner of myc, remain unchanged, suggesting that the endogenous level of max in C3H10T1/2 cells is sufficient for a high frequency of transformation by ras and myc. Based on these studies, the C3H10T1/2-myc clonal cell lines we describe are suitable model systems for examining the molecular role of the myc protein in transformation and for characterizing additional factors that synergize with myc in multistep transformation.     

The HL-60 transforming sequence: a ras oncogene coexisting with altered myc genes in hematopoietic tumors

The oncogene of the HL-60 human promyelocytic leukemia cell line has been passed serially through NIH/3T3 mouse fibroblasts. Oncogene-specific probes prepared from the resulting tertiary transfectants by molecular cloning have been used to show that loss of the transfected oncogene from NIH/3T3 cells correlates with reversion to nontransformed morphology. Analysis of cells transfected by the oncogenes of other tumors and tumor cell lines indicates that the transforming gene of the HL-60 leukemia cell line is closely related to oncogenes of a Burkitt's lymphoma, an acute myelogenous leukemia, an adenocarcinoma of the colon, a neuroblastoma, and two sarcomas. This oncogene is distantly related to the viral oncogenes of Kirsten and Harvey sarcoma viruses. It has been termed N-ras. The active N-ras oncogene coexists with altered versions of the myc oncogene in the HL-60 and AW Ramos human tumors. This suggests a multistep mechanism involving both ras and myc genes in the creation of these tumors.     

Activation of ras and myc proto-oncogenes in human breast carcinoma and neuroblastoma

DNA from human breast carcinoma (SK-BR-3) and neuroblastoma (LA-N-1) cell lines are capable of inducing foci of transformed NIH 3T3 cells after DNA-mediated gene transfer. The blot hybridization analysis of DNA from primary and secondary NIH 3T3 transformants identified additional sequences homologous to the c-Ha-ras 1 oncogene, and revealed amplification of nucleotide sequences homologous to the v-myc oncogene. Restriction fragments of the amplified myc-related sequences correspond to c-myc (SK-BR-3) and N-myc (LA-N-1) loci of the human genome. The results show that active Ha-ras oncogenes can coexist with altered myc oncogenes in breast carcinomas and neuroblastomas. This suggests that a multi-step mechanism involves both ras and myc genes and their cooperation in the development of these tumors.     

Influence of myc overexpression on the phenotypic properties of Chinese hamster lung cells resistant to antitumor agents.

In the Chinese hamster lung fibroblast cell line DC-3F, the development of resistance to different drugs, through several mechanisms like MDR expression or alteration of the DNA topoisomerase II activity, has been shown to be associated with a decreased tumorigenicity. Multiple studies have shown that the myc oncogene, in cooperation with ras, plays a major role in the oncogenic transformation of fibroblasts. As an approach to a better understanding of the relationship between the different phenotypic traits, we analyzed the expression of myc and ras oncogenes in the drug-sensitive DC-3F cells and in variants resistant to 9-hydroxyellipticine (9-OH-E) (DNA topoisomerase II alteration) or to actinomycin D (AD) (multidrug (MDR) expression). Southern and Northern blot analyses revealed about a 10-fold amplification and a 20-fold overexpression of the c-myc gene in the DC-3F cells as compared to the normal lung fibroblasts. Both amplification and overexpression are markedly decreased in the two resistant variants, ras gene copy number and expression were found to be identical in all cell types. In order to analyze the contribution of the decreased myc expression on the different phenotypic traits, the DC-3F/9-OH-E cells were transfected with the plasmid pSV-c-myc, and six clones expressing high amounts of the transfected myc were isolated and characterized. Morphological and caryological alterations, as well as an increased cloning efficiency in soft agar, indicated that the myc gene product was made in these cells. However, the tumorigenicity of the sensitive parental cells was not restored, thus showing that the decreased myc expression alone does not account for the loss of tumorigenicity in the resistant cells. 9-OH-E resistance was not modified in the transfected cells, while the cross-resistance of these cells to MDR-sensitive drugs, such as vincristine, actinomycin D, and taxol, was reversed roughly in proportion of the expression of the transfected myc.     

Transcription of cellular oncogenes in human tumors

The analysis of 11 various oncogenes expression in different human tumors showed that each tumor is characterised by a specific functioning program of these genes. In 40-50% of tumors the oncogenes ras, fos and myc are expressed. All other oncogenes are either considered to be "silent" or are expressed only in few cases. The increased expression of sis and myb oncogenes is observed in metastases.