Dose effects of transfected c-Ha-ras oncogene in transformed cell clones

We have examined the expression of the transformed phenotype in a series of clonal lines of NIH/3T3 cells transfected with the human c-Ha-ras^{Val 12} oncogene and the neomycin phosphotransferase gene. Cells from individual transformed foci were cloned and subjected to detailed analyses of the ras sequences. Three clones were found that expressed approximately one, 2–4, or 4–8 copies of the human c-ras oncogene, respectively. A fourth clone had multiple copies of the transfected sequences, and expressed abundant c-Ha-ras RNA. Analysis of the tranformed phenotype of various clones indicated that cells expressing low levels of mutant c-Ha-ras had lost some of their extracellular fibronectin network, and were barely altered in their cytoskeleton. In contrast, cells expressing abundant c-Ha-ras had lost both their actin and fibronectin networks and showed an increase in plasminogen activator activity. Cells with amplified c-Ha-ras^{Val 12} grew better in low serum, formed large colonies in soft agar and showed enhanced activity of ornithine decarboxylase, the rate-controlling enzyme in polyamine biosynthesis. These results show that the dosage level of the mutant oncogene makes a significant contribution to the transformed phenotype of c-Ha-ras oncogene-transformed cells.     

Effect of cellular determination on oncogenic transformation by chemicals and oncogenes.

Three developmentally determined myogenic cell lines derived from C3H 10T1/2 C18 (10T1/2) mouse embryo cells treated with 5-azacytidine were compared with the parental 10T1/2 line for their susceptibility to oncogenic transformation by 3-methylcholanthrene or the activated human c-Ha-ras oncogene. Neither the 10T1/2 cells nor the myogenic derivatives grew in soft agar or formed tumors in nude mice. In contrast to 10T1/2 cells, the three myogenic derivatives were not susceptible to transformation by 3-methylcholanthrene, so that cellular determination altered the response of 10T1/2 cells to chemical carcinogen. On the other hand, all cell types were transformed to a tumorigenic phenotype following transfection with the activated c-Ha-ras gene. The transfected myogenic cells expressed both the c-Ha-ras gene and the muscle determination gene MyoD1. In contrast to other reports, the presence of as many as six copies of the c-Ha-ras gene per genome did not prevent the formation of striated muscle cells which expressed immunologically detectable muscle-specific myosin. The expression of the c-Ha-ras gene does not therefore necessarily preclude the expression of the determination gene for myogenesis or prevent end-stage myogenic differentiation.     

The ras oncogene and tumour metastasis: observations on murine cells transfected with activated human c-Ha-ras

Transfection of cells with cloned genes or total genomic DNA offers a means for studying aspects of neoplastic behaviour. We have used this method to examine whether incorporation of the cloned 6.6-kilobase (kb) fragment of DNA containing the mutant c-Ha-ras human oncogene can confer metastatic capability on murine NIH 3T3 cells. Cells co-transfected with the mutated ras gene and the neomycin resistance marker pSV2neo were selected by culture in neomycin. On subcutaneous inoculation into MF 1 nude mice, these cells proved to be tumourigenic with short latent periods (approximately 14 days)--nude mice were used to circumvent immunological rejection of the mouse cells expressing the product of the human oncogene. Transfectants were capable of lung colonisation after intravenous injection, but there was no evidence of spontaneous metastasis at autopsy, or on histological examination of the lungs and other organs, 90 days after inoculation. Incorporation of the transfected oncogene was confirmed by Southern blotting and its expression by dot-blot hybridisation and immunoprecipitation. The results in this experimental system indicate that transfection of a mutated human ras oncogene into non-neoplastic 3T3 cells can confer part of the metastatic phenotype, namely lung colonisation, but is not by itself sufficient to induce spontaneous metastatic behaviour.     

v-myb transformation of Xeroderma pigmentosum human fibroblasts: overexpression of the c-Ha-ras oncogene in the transformed cells

Human Xeroderma pigmentosum "normal" fibroblasts AS16 (XP4 VI) were transformed after transfection with a recombinant v-myb clone. In this clone (pKXA 3457) derived from avian myeloblastosis virus (AMV), the expression of the oncogene sequences is driven by the AMV U-5 LTR promoter. The transformed cells (ASKXA), which have integrated a rearranged v-myb oncogene, grow in agar, are not tumorigenic in nude mice, and express a 45-kDa v-myb protein. The HMW DNA of these cells transform chicken embryo fibroblasts. The c-Ha-ras oncogene is overexpressed in the ASKXA cells but not in the parental "normal" AS16 cells and a revertant clone (ASKXA Cl 1.1 G). Our results lead to the conclusion that the XP fibroblasts are phenotypically transformed by the presence of the transfected v-myb oncogene, which is able to induce an overexpression of the c-Ha-ras gene.     

Transformation of NIH 3T3 cells with basic fibroblast growth factor or the hst/K-fgf oncogene causes downregulation of the fibroblast growth factor receptor: reversal of morphological transformation and restoration of receptor number by suramin

When NIH 3T3 cells were transfected with the cDNA for basic fibroblast growth factor (bFGF), most cells displayed a transformed phenotype. Acquisition of a transformed phenotype was correlated with the expression of high levels of bFGF (Quarto et al., 1989). Cells that had been transformed as a result of transfection with bFGF cDNA had a decreased capacity to bind 125I-bFGF to high affinity receptors. NIH 3T3 cells transfected with bFGF cDNA that expressed lower levels of bFGF were not transformed and had a normal number of bFGF receptors. NIH 3T3 cells transfected with the hst/Kfgf oncogene, which encodes a secreted molecule with 45% homology to bFGF, also displayed a transformed phenotype and decreased numbers of bFGF receptors. However, NIH 3T3 cells transfected with the H-ras oncogene were transformed but had a normal number of bFGF receptors. Thus, transformation by bFGF-like molecules resulted in downregulation of bFGF receptors. Receptor number was not affected by cell density for both parental NIH 3T3 cells and transformed cells. In the cells transfected with bFGF cDNA that were not transformed, the receptors could be downregulated in response to exogenous bFGF. Conditioned medium from transformed transfected cells contained sufficient quantities of bFGF to downregulate bFGF receptors on parental NIH 3T3 cells. Thus, the downregulation of bFGF receptors seemed related to the presence of bFGF in an extracytoplasmic compartment. Treatment of the transformed transfected NIH 3T3 cells with suramin, which blocks the interaction of bFGF with its receptor, reversed the morphological transformation and restored receptors almost to normal numbers. These results demonstrate that in these cells bFGF transforms cells by interacting with its receptor and that bFGF and hst/K-fgf may use the same receptor.     

Characteristics of an infinite life span diploid human fibroblast cell strain and a near-diploid strain arising from a clone of cells expressing a transfected v-myc oncogene

Diploid human fibroblasts were transfected with a plasmid carrying a v-myc oncogene linked to the neo gene or with a vector control carrying a neo gene. Drug-resistant clones were isolated and subcultured as needed. All populations went into crisis and eventually senesced. But while they were senescing, viable-appearing clones were noted among the progeny of a transfected population that expressed the v-myc oncogene. After several months, these cells began replicating more rapidly. Karyotype analysis indicated that they were clonally derived since all of them had 45 chromosomes, including 2 marker chromosomes. This cell strain was designated MSU-1.1. Similar analysis showed that cells from an earlier passage were diploid. These cells were designated MSU-1.0. Both strains have undergone more than 200 population doublings since their siblings senesced, without any change in chromosome complement. Both strains express the v-myc protein and have the same integration site for the transfected v-myc and neo genes. The MSU-1.0 cells cannot grow without exogenously added growth factors. The MSU-1.1 cells grow moderately well under the same conditions and grow to a higher saturation density than MSU-1.0 cells. Since the chance of human cells acquiring an infinite life span in culture is very rare, the data suggest that MSU-1.1 cells are derived from MSU-1.0 cells. The expression of v-myc is probably required for acquisition of an infinite life span, since this phenotype did not develop in populations not expressing this oncogene. However, expression of v-myc is clearly not sufficient, since all of the progeny of the clone that gave rise to the MSU-1.0 cells expressed this oncogene, but the vast majority of them senesced.     

Morphology, cell-cell interactions, and migratory activity of IAR-2 epithelial cells transformed with the RAS oncogene: contribution of cell adhesion protein E-cadherin

The destruction of stable cell-cell adhesion and the acquisition of the ability to migrate are consistent stages of neoplastic evolution of tumor cells of epithelial origin. We studied the morphologic and mi gration characteristics of epithelial cells of Iar1162 and IAR1170 clones derived from a mixed culture of on cogene N-RasV12-transformed cell line IAR-2. It was found that the mutant oncogene RAS can cause two types of morphological changes in IAR-2 epithelial cells. Cells of one type (IAR1162 clones) underwent epithelial-mesenchymal transition: they stopped to express E-cadherin, acquired fibroblast-like morphology, and did not form tight junctions. Cells of the other type (IAR1170 clones) retained a morphology close to the morphology of nontransformed progenitor cells, formed E-cadherin-based adherens junctions and tight junctions, and formed a monolayer in confluent culture. However, in both IAR1162 and IAR1170 cells, the mutant oncogene RAS caused the destruction of marginal actin bundle and the reorganization of cell-cell adherens junctions. RAS-transformed IAR1162 and IAR1170 epithelial cells acquired the ability to migrate on a flat substrate as well as through narrow pores in membranes of migration chambers. A videomicroscopic study of transformed epithelial cell cultures demonstrated the instability of cell-cell contacts and the independent nature of cell migration. IAR 1170 epithelial cells, which had E-cadherin-based adherens junctions, were also able to move as a group (collective migration). 1162D3 cells, which lost the ability to express endogenous E-cadherin as a result of Ras-transformation, were transfected with a plasmid carrying the CDH1. As a result of transfection, clones of cells with different levels of expression of exogenous E-cadherin were obtained. The high level of expression of exogenous E-cadherin in transformed epithelial cells led to a decrease in the rate of migration on a two-dimensional substrate of the cells that were in contact with neighboring cells but almost had no effect on the migration of single cells, at the same time increasing the number of cells that migrated through the pores in migration chambers. Thus, the destruction of marginal actin bundle and the change in the spatial organization of cell-cell adherens junctions, irrespective of the presence or absence of E-cadherin, was accompanied by destruction of stable cell-cell adhesion and the appearance of locomotor activity in Ras-transformed epithelial cells. The retaining of E-cadherin in cell-cell adhesion junctions affects the locomotor activity of transformed epithelial cells and plays an important role in their collective migration.     

Transfection of murine P19S18 embryonal carcinoma cells with the oncogene neu induces an epithelioid phenotype.

An embryonal carcinoma cell line, P19S18, was transfected with the rat oncogene neu to investigate the function of its protein product, p185*, in a multipotential cellular environment. Levels of message for p185* were determined by in situ hybridization analysis and two highly expressing clones, PnnA and PnnB, were isolated. As demonstrated by indirect immunofluorescence and immunoprecipitation, these neu-transfected cells synthesized a full length rat p185*. The transfectants do not resemble typical embryonal carcinoma cells either before or after differentiation is induced by retinoic acid treatment. They are much larger, flatter, "epithelioid" cells. These cells have lost the expression of stage specific embryonic antigen-1 (SSEA-1), but do synthesize and assemble the basement membrane components laminin and fibronectin. These results suggest that expression of the neu oncogene in a multipotential cell line may induce the synthesis of proteins indicative of an epithelioid phenotype due to the presence of p185*.     

Oncogene-induced transformation of a rat embryo fibroblast cell line is enhanced by tumor promoters.

Rat embryo fibroblast cell line 6 was transfected with plasmid pT24, which contains the activated human bladder c-Ha-ras oncogene, and the cells were grown continuously in the absence or presence of the tumor promoters 12-O-tetradecanoyl phorbol-13-acetate (TPA) or teleocidin. The presence of TPA or teleocidin led to a 6- to 14-fold increase in the number of morphologically transformed foci. No transformed foci were seen when rat 6 cells were transfected with the normal c-Ha-ras oncogene in the absence or presence of TPA, or in cells simply treated with TPA or teleocidin. Enhancement of pT24-induced foci was seen even when the addition of TPA was delayed until day 16. In transfection studies with the drug resistance genes gpt and neo, TPA and teleocidin did not increase the number of Gpt+ or Neo+ colonies. When rat 6 cells were cotransfected with pT24 and neo genes and grown in the absence or presence of TPA, the presence of TPA did not increase the yield of Neo+ colonies but caused a fivefold increase in the number of Neo+ colonies that displayed a transformed morphology. Southern blot analyses of DNAs obtained from these clones indicated that TPA treatment did not influence the extent of integration of either the pT24 or neo gene. DNA samples from all of the morphologically transformed cells displayed a characteristic 2-kilobase SacI fragment homologous to pT24 DNA and expressed relatively high levels of the corresponding mRNA. Our findings indicate that in this system tumor promoters do not simply enhanced the process of DNA transfection per se. Thus, this model system may be useful for analyzing synergistic interactions between tumor promoters and activated oncogenes during multistage carcinogenesis. It may also serve as a simple screening test for detecting new tumor promoters.     

The effect of Ha-ras oncogene on cells immortalized by the gene for polyomavirus large T-antigen

Activated human Ha-ras oncogene cloned on the plasmid pEJras6,6 was transfected into REF (LT) cells immortalized by the gene for large T-antigen of the polyoma virus. The cells were shown to become completely transformed (in the terms of morphology and tumorogeneity) only after three cycles of transfection with the plasmid pEJras6,6. The integrated sequences of the plasmid pEJras6,6 and the ras oncogene product p21Ha-ras were detected in cells only after their selection in the nude mice (in the cell culture REF (LT) ras X 3tu obtained from the tumor and directly in the tumor cells). Thus, after sequential transfections with a c-Ha-ras oncogene we developed cell cultures on the different stages of transformation process.     

Different activities of the adenovirus types 5 and 12 E1A regions in transformation with the EJ Ha-ras oncogene.

We have compared the capacities of the E1A regions of nononcogenic adenovirus type 5 (Ad5) and highly oncogenic Ad12 to cooperate with the EJ bladder carcinoma Ha-ras-1 oncogene in the transformation of primary baby rat kidney cells. Both E1A regions, when cotransfected with the Ha-ras oncogene, transformed the primary cells with a low frequency. Ad5 E1A plus Ha-ras-transformed cells differed in phenotype from cells transformed by Ad12 E1A plus Ha-ras. The cells expressing Ad5 E1A appeared highly transformed and practically failed to adhere to plastic. This phenotype may be due to the virtually complete absence of fibronectin gene expression in these cells. In contrast, the cells expressing Ad12 E1A were flatter and adhered to plastic, whereas fibronectin gene expression was reduced but not absent. The oncogenic potential of the two types of E1A plus ras-transformed cells was tested by their injection into both athymic nude mice and weanling syngeneic rats. The Ad5 E1A plus ras-transformed cells were found to be highly oncogenic in both animal species, whereas the Ad12 E1A plus ras-transformed cells were only weakly oncogenic in both syngeneic rats and nude mice. The difference in oncogenic potential of the Ad5 E1A plus ras- and the Ad12 E1A plus ras-transformed cells is discussed in terms of the different capacities of the Ad5 and Ad12 E1A-encoded proteins to modulate cellular gene expression.     

The p53-mediated G1 checkpoint is retained in tumorigenic rat embryo fibroblast clones transformed by the human papillomavirus type 16 E7 gene and EJ-ras.

Rat embryo fibroblast clones transformed with the human papillomavirus type 16 E7 gene and the H-ras oncogene (ER clones) fall into two groups on the basis of endogenous p53 genotype, wild type or mutant. We have compared these clones with the aim of indentifying physiological differences that could be attributed to p53 protein function. We show that all ER clones, regardless of p53 gene status, are tumorigenic and metastatic in severe combined immunodeficiency mice. We demonstrate that only the wild-type p53 protein expressed in ER clones is functional on the basis of its site-specific double-stranded DNA-binding activity and its ability to confer a G1 delay on cells following treatment with ionizing radiation. These data indicate that disruption of the p53 growth-regulatory pathway is not a prerequisite for the malignant conversion of rat embryo fibroblasts expressing the E7 gene and mutant ras. Differences in phenotype that were correlated with loss of p53 protein function included the following: serum-independent growth of ER clones in culture, decreased tumor doubling time in vivo, and increased radioresistance. In addition, we demonstrate the p53-dependent G1 checkpoint alone does not determine radiosensitivity.     

Expression of anti-sense mRNA in H-ras transfected NIH/3T3 cells does not suppress the transformed phenotype

We have attempted to reverse the transformed phenotype of cells expressing the H-ras oncogene. A plasmid in which the first exon of the H-ras oncogene was coupled to the SV40 early promoter in an anti-sense orientation was constructed. This construct was introduced into a clone of H-ras-transformed NIH/3T3 cells. Simultaneous expression of both the SV40 anti-sense construct and H-ras was observed. Anti-sense RNA was present in a 10-20-fold excess over sense H-ras RNA. Only a small fraction of the cytoplasmic RNA was present in a sense: anti-sense duplexed form. The expression of anti-sense H-ras RNA was not accompanied by a phenotypic reversion of transformed cells. The only phenotypic reversion we observed was accompanied by a loss of transfected H-ras sequences. The loss of transfected H-ras sequences occurs with a high frequency in cells supertransfected with the SV40 anti-sense construct.     

Biological properties of "partial" transformation mutants of Rous sarcoma virus and characterization of their pp60src kinase

We have isolated mutants of Rous sarcoma virus from an unmutagenized stock of the Schmidt-Ruppin strain of Rous sarcoma virus. These mutants induce only a "partial" transformation, and the transformation properties induced show unusual properties or combinations. Cells infected with mutant CU2 have a unique "blebby" morphology, have lost surface fibronectin, form very small colonies in soft agar, and are nearly normal with respect to adhesiveness and hexose transport. Cells infected with mutant tsCU11 have a nearly normal morphology, but grow well in soft agar. Cells infected with mutant CU12 have a fusiform morphology, intermediate levels of hexose transport and fibronectin, and form very large colonies in soft agar. Because the appearance of the different parameters of transformation is dissociated in these mutant-infected cells, these data are interpreted as supporting a model in which the transforming protein pp60src interacts with more than one primary target in generating the transformed phenotype. All of the mutants display levels of pp60src kinase activity less than that of the wild type. In the case of mutant CU12, the lower kinase activity is in part a consequence of a lower steady-state amount of pp60src inside the cell.     

The mosquito dihydrofolate reductase gene functions as a dominant selectable marker in transfected cells

An Aedes albopictus dihydrofolate reductase gene was used to construct two chimeric DNA vectors that functioned as dominant selectable markers in transfected, wild type mosquito cells. Stably transformed clones were recovered after 10–15 days in the presence of selective medium containing 1 μM methotrexate. The transformed clones contained an estimated 100–500 copies of transfected DNA per nucleus. Combined data from Southern blots and in situ hybridization to metaphase chromosomes indicated that transfected DNA was likely integrated into chromosomes both as repeated structures and as randomly integrated single copy molecules, with minimal rearrangement of coding sequences. Transfected DNA was stably maintained under selective conditions, but in some cases was lost when cells were maintained for prolonged periods in the absence of methotrexate. These observations provide a general framework for further development of stable gene transfer systems for mosquito cells in culture.     

Ribozymes designed to inhibit transformation of NIH3T3 cells by the activated c-Ha-ras gene.

We have designed hammerhead ribozymes that cleave c-Ha-ras mRNA mutated at codon 12 (GGU----GUU). Plasmids containing the ribozyme-encoding genes were expressed under the control of the long terminal repeats of Rous sarcoma virus in NIH3T3 cells transfected with the activated c-Ha-ras gene. These ribozymes were found to inhibit formation of foci (by about 50%) by cleaving the oncogene mRNA, rather than by hybridizing to it. Furthermore, when the activated c-Ha-ras gene was cotransfected with the ribozyme-encoding gene, three morphologically flat colonies were found and isolated. We also found that expression of c-Ha-ras was suppressed in cells containing ribozymes.     

多胺生物合成的抑制对转化细胞c－Ha－ras癌基因表达的调控

抗多胺代谢剂──二氟甲基鸟氨酸（ＤＦＭＯ）作用于经含点突变的Ｈａ－ｒａｓ基因片段转染的转化细胞（ＨＲ－１细胞）引起细胞生长的抑制,其抑制率随ＤＦＭＯ浓度的增加而增大,此时细胞多停滞于Ｇ_１期;多胺合成的关键酶鸟氨酸脱羧酶（ＯＤＣ）活性显著下降;Ｈａ－ｒａｓ癌基因ｍＲＮＡ及ｒａｓＰ~（２１）蛋白的表达受到抑制;而外源性腐胺与ＤＦＭＯ的同时加入可防止上述一系列改变的发生,说明ＤＦＭＯ使ＨＲ－１细胞某些表型向亲本细胞逆转的作用是与细胞多胺生物合成的抑制直接相关。     

Complementary directed modifications of nucleic acids and oncogene-directed mutagenesis in vivo.

High reactivity of the polyalkylating ss oligomers that were sense or antisense 30-200-mers containing sequences complementary to E1 oncogenes of simian adenovirus SA7 and one alkylating residue -CH2CH2N(C2H5OH) (CH2)3N(Ph-p-CH2OH)CH2CH2Cl per each 25 bases of oligomers was demonstrated in vitro by alkylation of ss DNA of recombinant M 13 mp8E1 and mp9E1 phages with inserted E1 sequences of adenovirus oncogene and then by followed complete and selective elimination of E1 sequences from recombinant ss DNA. Treatment of rodent cell cultures transformed by oncogenic SA7 with polyalkylating oligomers which are complementary to the long region of the minus or plus chains of E1 DNA of SA7 revealed a rather high extent of mutant cell clones formation. The cells formed were normalized; they had lost some properties of the transformed cells. Dividing cell clones inherited the new phenotypic properties: morphology, slower and more limited proliferation, and higher dependence on bovine serum growth factors. Some of the mutant cell DNAs demonstrated different mutations in the E1A sequences of the integrated proviral oncogene. There were exchanges G to C (leu to val) in the 525 and C to A (asp to tyr) in the 555 positions of E1A oncogene. Besides a deletions in the 1057-1477 E1A region or/and a mutation in the 1457-1477 of E1A were observed. Thus the inherited cell normalization observed is performed due to oncogene-directed mutagenesis in vivo.