Induction of the neoplastic phenotype of EBV-established B lymphocytes by the human Ha-ras oncogene

We report on the use of human B lymphocytes immortalized by the Epstein-Barr virus (EBV) as targets for transformation by the c-Ha-ras oncogene of bladder carcinoma cells T24. Several stably transformed cell lines were obtained and their in vivo and in vitro growth properties as well as levels of expression of the ras gene were studied. The transformed phenotype in these cells was correlated to ras oncoprotein expression level; only the cell lines which overproduce p21 ras, by at least six-fold, were tumorigenic in nude mice. In this regard, our ras transformed cells behave as lymphoblastoid cells transformed by the c-myc oncogene, suggesting that c-myc and c-Ha-ras might act on the same regulatory level.     

Transformation of human mammary epithelial cells by oncogenic retroviruses

We have introduced viral oncogenes into human mammary epithelial cells through the use of murine retroviruses. A continuous cell line (184A1N4) derived from benzo(a)pyrene treatment of normal breast epithelial cells was used as a recipient for the ras, mos, and T-antigen oncogenes. Each of these oncogenes enabled the 184A1N4 cells to grow in a selective medium, thus demonstrating the potential utility of these cells for oncogene detection and isolation. 184A1N4 cells transformed by T-antigen were nontumorigenic in athymic mice, but v-ras transformants were weakly tumorigenic. Transformants bearing both the T-antigen and ras oncogenes were strongly tumorigenic, however. The karyotype of these double transformants shows a high degree of stability. These results demonstrate the stepwise acquisition of the fully malignant phenotype by normal human epithelial cells in vitro.     

Inhibition of interferon-gamma antiviral and antiproliferative activities by ras oncogene expression

In this study, we evaluated the effect of ras oncogene activation on cell response to interferons (IFNs). For this purpose, we treated NIH 3T3 murine fibroblasts transformed by transfection with K-, Ha-, or N-ras oncogenes, either mutated or amplified, for 24 hours with IFN-gamma or IFN-alpha. We evaluated cell response by measuring virus replication, [3H]thymidine incorporation, 2',5'-oligoadenylate synthetase activation, and class I antigen induction. Transformed cells were much less responsive to IFN-gamma antiviral and antiproliferative activities than normal NIH 3T3 cells. Similarly, the induction of 2',5'-oligoadenylate synthetase following IFN-gamma treatment was completely depressed in transformed cells. Only class I antigens, measured at the cell surface and mRNA levels, appeared partially inducible by IFN-gamma in ras-transformed cells. When the same cell lines were treated with IFN-alpha, we observed full response. Because both normal and ras-transformed NIH 3T3 cells were able to bind [125I]IFN-gamma with comparable Kd values (8.3 X 10(-11) M vs. 3 X 10(-11) M, respectively), these findings suggest that ras oncogenes may differentially impair IFN-gamma activities by affecting activation of IFN-inducible genes downstream from the receptor binding event.     

Enhanced ras oncogene mediated cell transformation and tumorigenesis by adenovirus 2 mutants lacking the C-terminal region of E1a protein

Mutants of adenovirus 2 E1a defective in coding for the C-terminal 61 or 67 amino acids of a 243 amino acid (243R) protein are defective in immortalization of primary baby rat kidney (BRK) cells. However, they cooperate with T24 ras in oncogenic transformation more efficiently than wt. BRK cells transformed by the E1a C-terminal mutants and T24 ras induce rapidly growing tumors in syngeneic rats and athymic mice whereas cells transformed by the wt 243R and ras oncogene are not tumorigenic in syngeneic rats and can only induce slowly growing tumors in athymic mice. Cells transformed by the E1a mutants and ras oncogene also induce rapid metastatic tumors whereas cells transformed by the wt 243R and T24 ras can not do so. The increased tumorigenic ability exhibited by the 243R mutants does not appear to be due to differential levels of expression of p21 ras. Our results suggest that the C-terminal region of the 243R protein may have a novel function in suppression of cell transformation, tumorigenesis and tumor progression.     

Attenuated expression of the serum responsive T1 gene in ras transformed fibroblasts due to the inhibition of c-fos gene activity

The T1 gene encodes a protein, which shares homology with the IL-1 receptors. In fibroblasts, T1 is induced by growth factors and in response to the onset of oncogene expression. The c-fos gene is transiently activated in these situations and was shown to be the major mediator of T1 gene induction. In contrast, the sustained expression of a ras oncogene in NIH3T3 cells resulted in the downregulation of basal T1 gene activity and the attenuation of T1 gene induction in response to mitogenic signals. Likewise, the immediate early genes encoding c-Fos, FosB, and Fra-2 are repressed in these cells. T1 gene repression could be overcome by the forced expression of c-fos in ras transformed fibroblasts. Thus, the lack of c-fos gene expression is the likely cause for ras mediated T1 gene repression. Fra-1, in contrast to the other three members of the Fos family, is permanently synthesized in high amounts in ras transformed NIH3T3 fibroblasts. We show that AP-1, which is abundant in these cells throughout the whole cell cycle, consists predominantly of Fra-1/c-Jun and Fra1/JunD heterodimers. We provide evidence that Fra1/c-Jun heterodimers are responsible for the repression of c-fos gene induction following serum stimulation. The introduction of a dominant negative version of c-Jun into ras transformed fibroblasts was able to rescue c-fos gene induction in response to serum stimulation, further demonstrating that AP-1 is indeed involved in c-fos gene repression. We conclude that oncogenic ras mediates the activation of the fra-1 gene which results in elevated AP-1 activity throughout the cell cycle. Fra-1 containing AP-1 complexes repress the c-fos and possibly other immediate early genes thereby preventing the induction of certain delayed early genes such as the T1 gene in response to mitogenic stimulation.     

ras transformation of simian virus 40-immortalized rat hepatocytes: an in vitro model of hepatocarcinogenesis.

Primary rat hepatocytes were transfected with simian virus 40 DNA and cultured in a chemically defined medium. Proliferating colonies developed after 2-3 weeks. Three cell lines were established by cloning albumin-secreting colonies, as identified by an immunooverlay assay. Two of the cell lines, ALB-6 and ALB-8, expressed all five liver-specific mRNAs studied, albumin, alpha-1-antitrypsin, fibrinogen, alpha-1-acid glycoprotein, and histidase. ALB-6 cells were nontumorigenic in nude mice while ALB-8 cells were weakly tumorigenic with only one of four injected nude mice developing a slowly growing tumor. Further transfection of ALB-6 and ALB-8 cells with an activated c-Ha-ras or N-ras oncogene resulted in strongly tumorigenic cells. The tumors induced by ras-transformed ALB-6 cells were moderately differentiated hepatocellular carcinomas. The tumors derived from ras-transformed ALB-8 cells were poorly differentiated, while the slowly growing tumors induced by untransfected or control DNA-transfected ALB-8 cells were well-differentiated trabecular hepatocellular carcinomas, suggesting histological dedifferentiation of cells following ras transformation. However, the synthetic capabilities of the cells were not lost in that the ras-transfected cultures and the tumors induced by ras-transformed cells retained the ability to synthesize the five liver-specific mRNAs. Thus we have developed an in vitro model of carcinogenesis in which, by sequential exposure to SV40 DNA and a ras oncogene, primary rat hepatocytes are transformed.     

Suppression of tumorigenicity in human cell hybrids derived from cell lines expressing different activated ras oncogenes

Four different human tissue-derived cell lines, each previously shown to express either a Ha-, Ki-, or N-ras-activated oncogene, were fused in four different paired combinations. The three combinations that involved the tumor line HT1080 (activated N-ras oncogene) were found to be tumorigenic in nude mice, but to different degrees. However, the fusion of the tumor lines EJ and SW480 (activated Ha-ras and Ki-ras, respectively) resulted in hybrid cells suppressed for tumorigenicity. The EJ x SW480 hybrids were found to harbor and express both of the activated ras oncogenes. The results suggest that tumorigenic suppression can occur in the presence of two transforming oncogenes of the ras family and that tumorigenicity associated with ras oncogene activation involves additional mechanisms that may differ among tumor cells.     

Effects of ouabain on NIH/3T3 cells transformed with retroviral oncogenes and on human tumor cell lines

Both murine and human cell lines transformed by the v-Ki-ras gene have been shown to be much more sensitive to the toxic effects of the cardiac glycoside ouabain than their respective controls. This differential toxicity has previously been used in the isolation of flat revertant clones from populations of Kirsten murine sarcoma virus transformed NIH/3T3 cells. Here, we have undertaken a further characterization of this phenomenon in murine and human tumor cells. Two different techniques, a 51Cr-release assay and a quantitative Crystal violet elution assay, have been employed to compare the sensitivities to ouabain of normal and v-Ki-ras-transformed NIH/3T3 cells. In each assay, ras-transformed NIH/3T3 cell lines displayed an increased sensitivity to ouabain as compared to the parental NIH/3T3 cell line, both in dose-response and in time-course experiments. In a separate study, ouabain was also able to inhibit the growth in semi-solid medium of 2 v-Ki-ras-transformed NIH/3T3 cell lines (DT and K-NIH) in a dose-dependent fashion. The same concentrations of ouabain were effective in both the 51Cr-release and Crystal violet assays. To address the question of whether increased sensitivity to ouabain is a specific result of transformation with the ras oncogene or is a common event which accompanies transformation by other oncogenes, we have screened a variety of transformed NIH/3T3 derivatives. All of these lines displayed an increased sensitivity to ouabain when compared to the parental NIH/3T3 cell line.     

Characterization of activated proto-oncogenes in chemically transformed syrian hamster embryo cells

The Syrian hamster embryo (SHE) cell transformation model has been used by many investigators to study the multistep process of neoplastic transformation induced by chemical carcinogens. In this study we have attempted to determine if activated proto-oncogenes are present in the transformed cells induced by a variety of chemical carcinogens. Twelve carcinogen-induced hamster cell lines, established by treatment of normal SHE cells with benzo[a]pyrene, diethylstilbestrol, or asbestos, were examined. One spontaneously transformed cell line (BHK-A) was also studied. Some of the cell lines were also tested for oncogene activation at the preneoplastic stage, before they acquired tumorigenic potential. DNAs from normal, preneoplastic, and neoplastic cells were tested by transfection into mouse NIH 3T3 cells, and morphologically transformed foci were scored on the contact-inhibited monolayer of 3T3 cells. The frequency of focus formation for normal SHE cell DNA was <0.0008 foci/μg DNA, while approximately 40% (5 of 12) of the DNAs from carcinogen-induced, tumorigenic hamster cell lines induced foci at a frequency of ? 0.012 foci/μg DNA. The other seven carcinogen-induced cell lines and the BHK-A cells were negative (<0.002 foci/μg DNA). When the DNAs from transformed foci induced by the five positive cell lines were retransfected into NIH 3T3 cells, the frequency of secondary foci of 3T3 cells was as much as 50-fold higher (1.34 foci/μg DNA) than with the primary transfectants. DNAs from transformed foci or tumors derived from transformed foci were screened by Southern blot analyses with known oncogenes and with a hamster repetitive DNA probe for the presence of transfected hamster oncogenes. Newly acquired hamster Ha-ras sequences were detected in transformed 3T3 cells induced by four of the five hamster tumor DNAs. Immunoprecipitation of lysates of several secondary transformants with a ras monoclonal antibody (Y13–259) showed altered gel mobility of the p21^ras protein consistent with a mutation at codon 12. These activated ras genes were detected by the NIH 3T3 assay in the tumorigenic hamster cells but not in the preneoplastic, immortal cell from which they were derived. The activated Ha-ras proto-oncogene was detected in cell lines induced by each of the three different carcinogens studied. Cells from transformed foci inauced by DNA from one of the hamster tumor cell lines (BP6T) contained hamster sequences but did not show newly acquired Haras, Ki-ras, or N-ras genes on Southern analysis or altered p21^ras protein. The transforming gene in this cell line appears to be a non-^ras oncogene. These observations indicate that ～40% of the chemically transformed Syrian hamster tumor cell lines have activated Ha-ras oncogenes. The activation of Ha-ras proto-oncogene is a late, postimmortalization step in the neoplastic progression of SHE cells. Only one cell line with a non-^ras oncogene was detected in the NIH 3T3 focus assay, and ～60% of the cell lines were inactive in this assay, indicating the need to develop alternative assay systems for oncogene activation. Some of the preneoplastic Syrian hamster cell lines may be useful for this purpose.     

Modulation of differentiation in PA-1 human teratocarcinoma cells after N-ras oncogene-induced tumorigenicity.

We have been studying the effect of oncogenes on differentiation using the human ovarian teratoma-derived cell line PA-1. From this study we have characterized variants representing four stages relevant to multistage carcinogenesis, two non-tumorigenic and two tumorigenic. The two non-tumorigenic cell variants differ in that one is resistant to transformation by ras oncogenes whereas the other can be transformed to tumorigenicity. When these non-tumorigenic PA-1 variants are treated with retinoic acid (RA), a morphogen, they stop dividing, begin to express homeobox genes, and change in morphology. Transfection of an activated N-ras oncogene into ras-resistant non-tumorigenic PA-1 cells does not alter the RA responsiveness of the cells, indicating that expression of the activated oncogene is not sufficient for blocking RA-induced differentiation. Spontaneous activation of an N-ras oncogene leading to tumorigenic transformants and gene transfer-induced N-ras transformants are resistant to these effects of RA. However, another spontaneous transformant of PA-1 cells that does not contain an activated N-ras is responsive to RA. We prepared somatic cell hybrids of the RA-non-responsive, N-ras-transformed and tumorigenic PA-1 cell and the RA-responsive, ras-resistant non-tumorigenic PA-1 cell; the hybrid cell lines continue to express the oncogene but are non-tumorigenic. These non-tumorigenic hybrids are responsive to RA with regard to morphological changes, growth arrest and induction of homeobox gene expression. Tumorigenic revertants of these hybrids arise as a result of the loss of some chromosomes; these hybrid cells express the oncogene but have lost RA responsiveness. These results indicate that tumorigenic transformation in general is not sufficient to induce RA resistance, and resistance to differentiation may be oncogene-specific. In addition, the expression of an activated N-ras oncogene alone is insufficient to induce resistance to RA and ras-induced tumorigenicity is necessary. Therefore, some feature of cellular metabolism that is altered by and discordantly segregates with tumorigenic transformation controls responsiveness to RA. This controlling element is presumably a tumor suppressor.     

Oncogene-mediated multistep transformation of C3H10T1/2 cells

We have examined the response of the mouse embryonic cell line C3H10T1/2 to transfection with the activated human c-H-ras oncogene and the gag-myc oncogene from avian myelocytomatosis virus 29. C3H10T1/2 cells are not morphologically transformed following transfection with the gag-myc oncogene. A low level of focus formation is observed following transfection of the c-H-ras oncogene. When C3H10T1/2 cells are cotransfected with the ras and myc oncogenes, focus formation is increased by an average of 13 fold. In addition, C3H10T1/2 ras/myc foci have a distinct, transformed morphology which correlates with an increased potential for anchorage-independent growth. Although morphological transformation in this system is largely a function of ras oncogene expression, our studies demonstrate that it is potentiated by the presence of a functional gag-myc protein. Oncogene-mediated multistep transformation, which was first described in primary embryo cultures, is not a general property of established cell lines. The C3H10T1/2 cell line is an exception and provides a model system in which partially transformed phenotypes, in a progression toward malignant transformation, can be isolated and studied.     

Specific viral oncogenes cause differential effects on cell-to-cell communication, relevant to the suppression of the transformed phenotype by normal cells

We have studied growth regulation in mixed cultures of normal and oncogene-transformed 3T3 cells. The NIH 3T3 cells transformed by myc, src, and ras showed comparable cloning efficiency in semisolid medium. However, when they were plated on plastic with an excess of normal mouse embryo fibroblasts, BALB/c 3T3 ClA31-1-1, ras- and src-transformed cells were able to form distinct foci on the layer of density-arrested normal cells, whereas myc-transformed cells lacked this ability. In order to determine whether suppression or expression of the transformed phenotype could be correlated with the ability of the different cell populations to communicate, gap-junctional intercellular communication (IC) was measured by the Lucifer yellow dye transfer assay in coculture of normal and transformed cells. The dye was observed to spread from BALB/c 3T3 to myc-NIH 3T3 cells, indicating the presence of IC between these two cell types. In contrast no passage of Lucifer yellow was observed between src-NIH 3T3 or ras-NIH 3T3 and BALB/c 3T3. Addition of a phorbol ester tumor promoter, phorbol-12,13-didecanoate, efficiently rescued proliferation and focus formation by myc-transformed cells. The tumor promoter was able to inhibit IC in BALB/c 3T3 cells, although this response greatly varied among the different oncogene transformed clones. Tumorigenicity in nude mice strongly correlated with growth behavior in vitro: myc-transformed cells were either nontumorigenic or slowly tumorigenic, and src- and ras-transformed cells were highly tumorigenic. These data suggest an important role of IC in modulating abnormal growth behavior in vitro and in vivo.     

Human oncogenes detected by a defined medium culture assay

Oncogenes in DNAs from human tumor cell lines have been detected by a new transformation assay. Cellular DNAs are transfected into NIH3T3 murine fibroblasts, and transformed cells are selected by maintaining cell cultures in a defined medium lacking platelet-derived or fibroblast growth factors. DNAs from eight of 17 human tumor cell lines have yielded transformants by this method. Activated cellular ras genes account for three of the transforming activities. The SAOS2 osteosarcoma cell line contains an activated oncogene distinct from 18 known oncogenes. Another cellular oncogene was detected as the consequence of a fortuitous transfection-mediated DNA rearrangement.     

Activation of the c-Ha-ras-1 proto-oncogene by methylation in vitro with alpha-acetoxy-N-nitrosodimethylamine

alpha-Acetoxy-N-nitrosodimethylamine, an activated derivative of the carcinogen N-nitrosodimethylamine, methylated in vitro a plasmid containing the human c-Ha-ras-1 proto-oncogene, resulting in the generation of a transforming oncogene, assayed by transfection into NIH 3T3 cells. The resulting transformed cells were tumorigenic and metastatic in immune-deprived mice. Further transfection using tumor DNA led to the formation of three secondary NIH 3T3 transformants. DNA from these secondary transformants contained human ras gene sequences. Two of the three secondary transformants contained G----A mutations at guanine 35 in codon 12, and the third secondary transformant retained the wild-type sequence at codons 12, and 61. For the latter, the activating mutation was not determined. These results demonstrate that a simple methylating agent can activate a normal human ras proto-oncogene to a transforming oncogene.     

Induction of expression of osteopontin (OPN; secreted phosphoprotein) in metastatic, ras-transformed NIH 3T3 cells.

We have previously shown that transfection of NIH 3T3 cells with the T24 H-ras oncogene converts the cells to a tumorigenic and metastatic phenotype, in proportion to levels of ras expression. We hypothesize that ras-induced increases in malignancy occur via altered expression of various genes. We have identified OPN (osteopontin; also known as Secreted Phosphoprotein, 2ar, Eta-1, and transformation-associated phosphoprotein) as a ras-induced gene in these cells. We report here that expression of OPN RNA and secretion of OPN protein are increased in a series of ras-transformed NIH 3T3 cells, in proportion to levels of expression of ras. Detection of secreted OPN protein was facilitated by a barium citrate precipitation procedure. Although the function of this protein in tumor cells is not known, OPN contains a conserved GRGDS (glycine-arginine-glycine-aspartic acid-serine) amino acid sequence, which may function as a cell attachment site for this protein. We speculate that increased expression of OPN contributes to the increased malignancy of ras oncogene-transformed NIH 3T3 cells, perhaps by alterations in either adhesive properties or integrin-mediated signal transduction pathways.     

Melittin resistance: a counterselection for ras transformation.

The prevalence of activated ras oncogenes in human primary tumors suggests a central role for this oncogene in human cancer. Despite its ubiquitous distribution, the biochemical role of the oncogene remains unclear, and hence attempts to control its activity have been frustrated. This study demonstrates the ability of melittin, a 26 amino acid, amphipathic peptide from bee venom, to specifically select against cells in culture that express high levels of the ras oncogene. Acquisition of resistance to increasing concentrations of melittin is accompanied by corresponding decreases in the levels of expression of the ras oncoprotein and the number of copies of the ras gene. This results in a concomitant reversion of transformed cells to a normal morphology in a strict dose-dependent manner. Melittin is a known activator of cellular phospholipase A2 (PLA2), and these results suggest an interrelationship between ras and PLA2. In addition these studies indicate that melittin preferentially hyperactivates PLA2 in ras oncogene-transformed cells, resulting in their selective destruction.