Production of growth factors by type 5 adenovirus transformed rat embryo cells

Transformation of Sprague-Dawley rat embryo (RE) cells and a cloned Fischer rat embryo cell line (CREF) with wild-type (Ad5) or a temperature-sensitive DNA-minus mutant (H5ts125) of type 5 adenovirus results in a reduction in binding of epidermal growth factor (EGF) to cell surface receptors. A reduction in EGF binding is also seen in a Syrian hamster embryo cell line transformed by a hexon mutant of Ad5. In contrast, a human embryonic kidney cell line (293) transformed by sheared Ad5 DNA or transfected clones of KB cells expressing the E1 transforming region of Ad5 do not show a decrease in receptor binding. When cocultivated, the adenovirus transformed rat cells were able to induce the growth in agar of normal CREF cells. Medium from Ad5 transformed RE cells stimulated the growth in agar of CREF cells and also inhibited [125I]-EGF binding in CREF cells. When fractionated by gel filtration, two peaks of [125I]-EGF inhibiting activities were obtained with apparent molecular weights of 35,000 and 16,000. These results provide the first evidence that cells transformed by an adenovirus can produce a growth factor(s) that inhibits EGF-receptor binding and induces anchorage-independent growth of normal cells.     

The co-carcinogenic activity of 4-nitropyridine-1-oxide (4-NPO) and prevention of transformation by type-specific anti-viral antibodies.

Fischer rat embryo cells chronically infected with Rauscher murine leukemia virus, and known to be sensitive to transformation by potent chemical carcinogens, were transformed by the weak carcinogen 4-nitropyridine-1-oxide. Transformed cells grew in semi-solid agar and produced tumors in newborn Fischer rats. Transformation was inhibited by antisera specific for the ecotropic Rauscher murine leukemia virus, but not by antisera of equal toxicity specific for xenotropic Swiss mouse AT-124 virus.     

The co-carcinogenic activity of 4-nitropyridine-1-oxide (4-npo) and prevention of transformation by type-specific anti-viral antibodies

Summary Fischer rat embryo cells chronically infected with Rauscher murine leukemia virus, and known to be sensitive to transformation by potent chemical carcinogens, were transformed by the weak carcinogen 4-nitropyridine-1-oxide. Transformed cells grew in semi-solid agar and produced tumors in newborn Fischer rats. Transformation was inhibited by antisera specific for the ecotropic Rauscher murine leukemia virus, but not by antisera of equal toxicity specific for xenotropic Swiss mouse AT-124 virus. This work was supported by contract NO1-CP-43240 within the Virus Cancer Program of the National Cancer Institute.     

Spontaneous Release of Endogenous Ecotropic Type C Virus from Rat Embryo Cultures

Type C viruses were isolated from embryo cultures of two different rat strains, Sprague-Dawley and Fischer. Both viruses (termed rat leukemia virus, RaLV) were released spontaneously from rat embryo cells, have a density of 1.14 to 1.15 g/cm(3) based on equilibrium sedimentation in sucrose gradients, contain 60-70S RNA, RNA-directed DNA polymerase, and rat type C virus-specific 30,000 molecular-weight-protein determinants. Molecular hybridization studies using the Sprague-Dawley RaLV 60-70S RNA show that the virus-specific nucleotide sequences are present in the DNA of rat embryos. Both Sprague-Dawley and Fischer RaLV can rescue the murine sarcoma virus genome from Kirsten murine sarcoma virus-transformed nonproducer cells and are neutralized by antisera to the RPL strain of RaLV. In contrast to previous RaLV's, these viruses propagate in their own cells of origin as well as in cells of heterologous rat strains.     

Transformation by the oncogene v-fms: The effects of castanospermine on transformation-related parameters

The effects of castanospermine on various parameters associated with transformation were examined in cells expressing the viral oncogene v-fms. Fischer rat embryo (FRE) cells transformed by the oncogene v-fms and grown in the presence of castanospermine reverted to a more normal cell morphology and accumulated fms protein within the endoplasmic reticulum. Treated cells attained contact inhibition of cell growth at a much lower cell density compared to the untreated controls. No effect of castanospermine on cell growth was observed for FRE cells transformed by a different oncogene v-fgr. Castanospermine-treated SM-FRE (v-fms transformed) cells reexpressed extracellular matrix fibronectin and exhibited an extensive actin-containing cytoskeleton similar to that of normal nontransformed FRE cells. Castanospermine treatment of SM-FRE cells resulted in a sixfold decrease in [3H]deoxyglucose uptake compared to that of the nonreverted SM-FRE cells. Again, no effect was observed in FRE cells transformed by the oncogene v-fgr (GR-FRE). These results further characterize the reversion caused by castanospermine and indicate that cell surface expression coordinately controls anchorage independent growth, cell morphology, contact inhibition of growth, and hexose uptake.     

Interactions of tumor-associated fetal antigens with rat histocompatibility antigens

The physical interactions of fetal antigens (tumor-associated fetal antigens; TAFA-I, TAFA-II, and TAFA-III) with rat histocompatibility antigens were studied. TAFA-I and TAFA-III are present on syngeneic (NBR) and allogeneic (Fisher F344, Wistar Furth, and White Buffalo) rat embryo fibroblasts and on tumor cells. TAFA-II was found only on NBR (syngeneic) rat embryo fibroblasts and on NBR tumor cells. Antibody-blocking experiments were used to examine the fetal and histocompatibility antigen topography on cell membranes of tumor cells transformed by chemical and viral carcinogens. Precoating the tumor cells with alloantisera inhibited the subsequent adsorption of anti-NBR embryo, anti-TAFA-I, and anti-TAFA-III sera, but not anti-TAFA-II serum. Immunofluorescent cocapping experiments indicated that TAFA-I and TAFA-III, as well as other fetal antigens found on cells from 14-day gestation NBR embryos cocap with histocompatibility antigens when tested on syngeneic embryo fibroblasts and on sarcoma cells. TAFA-I cocapped with White Buffalo (Buf) strain rat histocompatibility antigens on herpes simplex Type II virus-transformed cells. The specificity of the TAFA-histocompatibility interactions was confirmed by demonstrating that the different anti-TAFA sera did not have contaminating antiviral antigen specificity; and also that these interactions did not occur on normal adult fibroblasts or spleen cells.     

Mutations in the E1a gene of type 5 adenovirus result in oncogenic transformation of Fischer rat embryo cells

Transformation of a specific clone of Fischer rat embryo (CREF) cells with wild-type 5 adenovirus (Ad5) or the E1a plus E1b transforming gene regions of Ad5 results in epithelioid transformants that grow efficiently in agar but that do not induce tumors when inoculated into nude mice or syngeneic Fischer rats. In contrast, CREF cells transformed by a host-range Ad5 mutant, H5hrl, which contains a single base-pair deletion of nucleotide 1055 in E1a resulting in a 28-kd protein (calculated) in place of the wild-type 51-kd acidic protein, display a cold-sensitive transformation phenotype and an incomplete fibroblastic morphology but surprisingly do induce tumors in nude mice and syngeneic rats. Tumors develop in both types of animals following injection of CREF cells transformed by other cold-sensitive Ad5 E1a mutants (H5dl101 and H5in106), which contain alterations in their 13S mRNA and consequently truncated 289AA proteins. CREF cells transformed with only the E1a gene (0-4.5 m.u.) from H5hrl or H5dl101 also produce tumors in these animals. To directly determine the role of the 13S E1a encoded 289AA protein and the 12S E1a encoded 243AA protein in initiating an oncogenic phenotype in adenovirus-transformed CREF cells, we generated transformed cell lines following infection with the Ad2 mutant pm975, which synthesizes the 289AA E1a protein but not the 243AA protein, and the Ad5 mutant H5dl520 and the Ad2 mutant H2dl1500, which do not produce the 289AA E1a protein but synthesize the normal 243AA E1a protein. All three types of mutant adenovirus-transformed CREF cells induced tumors in nude mice and syngeneic rats. Tumor formation by these mutant adenovirus-transformed CREF cells was not associated with changes in the arrangement of integrated adenovirus DNA or in the expression of adenovirus early genes. These results indicate, therefore, that oncogenic transformation of CREF cells can occur in the presence of a wild-type 13S E1a protein or a wild-type 12S E1a protein when either protein is present alone, but does not occur when both wild-type E1a proteins are present.     

Influence of the deprivation of a single amino acid on cellular proliferation and survival in rat 3Y1 fibroblasts and their derivatives transformed by a wide variety of agents

We compared proliferation and survival of various syngeneic transformed cell lines under conditions of depletion of 15 amino acids in Dulbecco-Eagle's medium. We used a normal fibroblast line 3Y1 and 22 transformed sublines of 3Y1 which had been induced by one of seven transforming agents--simian virus 40, mouse polyomavirus, adenovirus type 12, E1A gene of adenovirus type 12, cDNA of Harvey murine sarcoma virus, Rous sarcoma virus, or N-methyl-N'-nitro-N-nitrosoguanidine. Unlike other untransformed cells examined (mouse BALB/c-3T3 line, mouse NIH-3T3 line, and primary Fischer rat embryo fibroblasts), 3Y1 ceased to proliferate and accumulated in a viable state with a G1-phase DNA content under 14 singular deprivations of amino acid. None of the transformed 3Y1 lines completely arrested in the G1 phase of the cell cycle and each showed different levels of survival, depending on each transforming agent. As for transformed 3Y1 cells induced by a given virus or a given transforming gene, any one of the three sublines shared the same trend with respect to proliferation and survival. Transformed derivatives induced by N-methyl-N'-nitro-N-nitrosoguanidine showed almost the same trend in proliferation, but the patterns of survival were not uniform. Our observations suggest that the unique responses of 3Y1 to amino acid depletion are differently modified by different transforming agents.     

Secreted proteins of normal andmyc-ras oncogene transformed rat embryo fibroblasts

Quiescent cultures of rat embryo fibroblasts synthesize and secrete several proteins in response to mitogenic stimulation. Two of these proteins have been characterized in this study and the effect of oncogenic transformation on these proteins was monitored. A serum induced 48,000 protein was shown to be related to plasminogen activator inhibitor while another serum-induced protein ofM _r 45,000 was found to be an inhibitor of DNA synthesis. Transformation of rat embryo fibroblasts with oncogenesmyc andras resulted in drastic reduction in the level of these proteins. The reduced levels of protease inhibitor may be responsible for the loss of anchorage dependence of the transformed cells. The DNA synthesis inhibitor protein may act as a negative growth regulator and reduced levels of this protein inmyc-ras transformed cells may accelerate the proliferation of these cells.     

Transformation of rat fibroblasts by cloned defective polyoma DNA

Defective polyoma DNA molecules isolated from mouse cells infected with high-multiplicity-passaged virus were cloned in pBR322, and the recombinant plasmids were tested for their capacity to transform Fischer rat 3T3 cells in culture. Recombinants carrying an intact proximal portion of the early region, i.e., the region coding for both small and middle T antigens, were able to induce the transformed phenotype. A recombinant plasmid containing a defective polyoma genome with a deletion of about 300 base pairs in the region coding for the C-terminal segment of middle T antigen failed to transform.     

Selective killing of transformed rat cells by minute virus of mice does not require infectious virus production.

Fischer rat fibroblasts, naturally resistant to killing by the fibrotropic strain of minute virus of mice [(parvovirus MVM(p)], became sensitive to MVM when transformed by polyomavirus. This sensitization did not involve an increase in the percentage of cells which synthesized viral capsid antigens or in the percentage of cells which produced infectious virus. The addition of anti-MVM antiserum to the growth medium of MVM-infected cells had only a small effect on their survival rates, indicating that the majority of the killing effect of MVM occurs in a single cycle of infection. The data indicate that cell killing by MVM is independent of infectious virus production and thus support the notion that the preferential cytolytic effect is affected by viral cytotoxic gene products which accumulate to intolerable levels in transformed cells but not in normal ones. Finally, using cells transformed with polyomavirus and genomic and subgenomic clones of polyomavirus, we showed that the extent of sensitization to killing by MVM depended on the transforming agent used.     

Studies on the transformation of rat embryo cells of low passage by carcinogenic fluorenylhydroxamic acids and their acetate esters

Rat embryo cells of low passage subjected to a single treatment with certain carcinogenic fluorenylhydroxamic acids and their respective acetate esters showed signs of transformation in vitro, such as changes in phenotype, growth in soft agar and agglutination with concanavalin A. In addition, certain changes in karyotype and loss of diploidy were observed. There was no evidence, either by electron microscopy or by assay of RNA-dependent DNA polymerase, for the presence of virus. None of these cell lines produced tumors after inoculation into the isologous host. The results of these study lead us to suggest that malignant transformation is a multistep process and that certain criteria of transformation of rat embryo cells are associated with the initial stage(s) in which the cells are transformed without being tumorigenic. The ultimate test for malignant transformation of rat embryo cells remains the production of tumors in a susceptible host after inoculation of treated cells.     

Transformation by the v-fms oncogene product: role of glycosylational processing and cell surface expression.

The effect of glycosylational-processing inhibitors on the synthesis, cell surface expression, endocytosis, and transforming function of the v-fms oncogene protein (gp140fms) was examined in McDonough feline sarcoma virus-transformed Fischer rat embryo (SM-FRE) cells. Swainsonine (SW), a mannosidase II inhibitor, blocked complete processing, but an abnormal v-fms protein containing hybrid carbohydrate structures was expressed on the cell surface. SW-treated SM-FRE cells retained the transformed phenotype. In contrast, two glucosidase I inhibitors (castanospermine [CA] and N-methyl-1-deoxynojirimycin [MdN]) blocked carbohydrate remodeling at an early stage within the endoplasmic reticulum and prevented cell surface expression of v-fms proteins. CA-treated SM-FRE cells reverted to the normal phenotype. Neither SW, CA, nor MdN affected either endocytosis or the tyrosine kinase activity associated with the v-fms gene product in vitro. These results demonstrate the necessity of carbohydrate processing for cell surface expression of the v-fms gene product and illustrate the unique ability to modulate the transformed state of SM-FRE cells with the glycosylational-processing inhibitors CA and MdN.     

Rimantadine but not amantadine protects Fischer rat embryo cells from transformation induced by 3-methylcholanthrene or benzo(a)pyrene.

The antiviral drugs amantadine hydrochloride and rimantadine hydrochloride were tested as to their oncogenic potential using a serial line of Fischer rat embryo cells that previously had been shown to be an accurate indicator of chemicals known to be oncogenic in animal studies. Neither compound was found to have transforming activity. At slightly toxic levels, rimanbadine hydrochloride, but not amantadine hydrochloride, protected the same cell line from the transformation induced by the polycyclic hydrocarbons 3-methylcholanthrene and benzo(a)pyrene.     

Transformation of rat cells by fusion-infection with Rous sarcoma virus

The transformation of a rat cell line, 3Y1, by nonmammalian tropic strains of avian sarcoma virus was tested using cell-virus fusion mediated by Sendai virus or polyethylene glycol. Furthermore, the establishment of several transformed 3Y1 cell clones induced by the Schmidt-Ruppin strain of Rous sarcoma virus (RSV), its derivative mutants, and the Bryan high-titer strain of RSV is reported. The presence and expression of the viral genomes in these cells were examined, and all transformed cell clones tested were found to contain rescuable RSV genomes when they had been fused with normal chicken embryo fibroblast cells or those preinfected with Rous-associated virus type 1. However, the gag gene product, pr76, was barely detectable in wild-type RSV-transformed cells, whereas it was produced in considerable amounts in cells transformed by env-deleted mutants, the Bryan high-titer strain of RSV and NY8 derived from the Schmidt-Ruppin strain of RSV.     

Differential expression of tropomyosin forms in the microfilaments isolated from normal and transformed rat cultured cells

Using a newly developed method for microfilament isolation (Matsumura, F., Yamashiro-Matsumura, S. and Lin, J. J.-C. (1983) J. Biol. Chem. 258, 6636-6644), we have analyzed protein composition of microfilaments in "normal" and transformed rat tissue culture cells. They include REF-52 (an established rat embryo cell line) cells, REF-52 transformed by DNA viruses (SV40 or adenovirus type 5), normal rat kidney cells, and normal rat kidney cells transformed by RNA viruses (Kirsten or Rous sarcoma virus). Microfilaments from normal rat culture cells contain three major tropomyosins (apparent Mr = 40,000, 36,500, and 32,400) and two relatively minor tropomyosins (apparent Mr = 35,000 and 32,000). In transformed cells the levels of one or two of the major tropomyosins (Mr = 40,000 and 36,500) are decreased and the levels of one or both of the minor tropomyosins (Mr = 35,000 and 32,000) are increased. These changes in tropomyosin patterns were also observed in temperature shift experiments with rat-1 cells transformed with a Rous sarcoma virus mutant, temperature-sensitive for transformation. Cell-free translation of whole cell mRNA generated similar tropomyosin patterns on two-dimensional gels, suggesting that changes in the pattern of tropomyosin expression were largely effected at the level of RNA rather than by post-translational modification. Such changes in the tropomyosin composition of microfilaments were consistently found to accompany the various morphological alterations associated with transformation. We suggest that alterations in the pattern of tropomyosin expression are involved in, or cause, rearrangement of stress fibers and that this may be responsible (in part) for morphological transformation.     

Studies on the transformation of rat embryo cells of low passage by carcinogenic fluorenylhydroxamic acids and their acetate esters

Summary Rat embryo cells of low passage subjected to a single treatment with certain carcinogenic fluorenylhydroxamic acids and their respective acetate esters showed signs of transformation in vitro, such as changes in phenotype, growth in soft agar and agglutination with concanavalin. A. In addition, certain changes in karyotype and loss of diploidy were observed. There was no evidence, either by electron microscopy or by assay of RNA-dependent DNA polymerase, for the presence of virus. None of these cell lines produced tumors after inoculation into the isologous host. The results of this study lead us to suggest that malignant transformation is a multistep process and that certain criteria of transformation of rat embryo cells are associated with the initial stage(s) in which the cells are transformed without being tumorigenic. The ultimate test for malignant transformation of rat embryo cells remains the production of tumors in a susceptible host after inoculation of treated cells. Supported by Grant CA-02571 from the National Cancer Institute (H. R. Gutmann) and by Grant CA-08832 from the National Cancer Institute (G. J. Vosika).     

Rimantadine but not amantadine protects fischer rat embryo cells from transformation induced by 3-methylcholanthrene or benzo(a)pyrene

Summary The antiviral drugs amantadine hydrochloride and rimantadine hydrochloride were tested as to their oncogenic potential using a serial line of Fischer rat embryo cells that previously had been shown to be an accurate indicator of chemicals known to be oncogenic in animal studies. Neither compound was found to have transforming activity. At slightly toxic levels, rimantadine hydrochloride, but not amantadine hydrochloride, protected the same cell line from the transformation induced by the polycyclic hydrocarbons 3-methylcholanthrene and benzo(a)pyrene. This work was supported by Contract N01-CP-43240 within The Virus Cancer Program of the National Cancer Institute.     

Arrangement of Integrated Avian Sarcoma Virus DNA Sequences Within the Cellular Genomes of Transformed and Revertant Mammalian Cells

We have examined the arrangement of integrated avian sarcoma virus (ASV) DNA sequences in several different avian sarcoma virus transformed mammalian cell lines, in independently isolated clones of avian sarcoma virus transformed rat liver cells, and in morphologically normal revertants of avian sarcoma virus transformed rat embryo cells. By using restriction endonuclease digestion, agarose gel electrophoresis, Southern blotting, and hybridization with labeled avian sarcoma virus complementary DNA probes, we have compared the restriction enzyme cleavage maps of integrated viral DNA and adjacent cellular DNA sequences in four different mouse and rat cell lines transformed with either Bratislava 77 or Schmidt-Ruppin strains of avian sarcoma virus. The results of these experiments indicated that the integrated viral DNA resided at a different site within the host cell genome in each transformed cell line. A similar analysis of several independently derived clones of Schmidt-Ruppin transformed rat liver cells also revealed that each clone contained a unique cellular site for the integration of proviral DNA. Examination of several morphologically normal revertants and spontaneous retransformants of Schmidt-Ruppin transformed rat embryo cells revealed that the internal arrangement and cellular integration site of viral DNA sequences was identical with that of the transformed parent cell line. The loss of the transformed phenotype in these revertant cell lines, therefore, does not appear to be the result of rearrangement or deletions either within the viral genome or in adjacent cellular DNA sequences. The data presented support a model for ASV proviral DNA integration in which recombination can occur at multiple sites within the mammalian cell genome. The integration and maintenance of at least one complete copy of the viral genome appear to be required for continuous expression of the transformed phenotype in mammalian cells.     

Detection of herpes simplex virus type 2 glycoproteins expressed in virus-transformed rat cells.

Rat embryo fibroblasts transformed by herpes simplex virus type 2 (HSV-2) were assayed for the expression of certain virus-specific glycoproteins on the surface membranes. Monospecific antisera to HSV-2-specific glycoproteins, designated gAgB, gC, and gX, were used in membrane immunofluorescence studies with HSV-2-transformed cell lines tREF-G-1, tREF-G-2, and a tumor-derived rat fibrosarcoma cells line produced in syngeneic rats inoculated with tREF-G-1 cells. Analysis of the three HSV-2-transformed cell lines showed that antisera to the gAgB and gX glycoproteins were reactive with these cells. In contrast, no significant reactivity was observed when anti-gC serum was reacted with the HSV-2-transformed cell lines. All three antiglycoprotein sera reacted positively with rat cells productively infected with HSV-2. Additionally, the HSV-2-transformed and tumor-derived cell lines showed positive internal immunofluorescence after reaction with antiserum to an early, nonstructural viral protein designated VP143 (molecular weight, 143,000). Infectivity of HSV-2 in standard plaque assays was neutralized by hyperimmune rat antisera to tREF-G-2 or rat fibrosarcoma cells and to HSV-2 virions and by sera from rats bearing the fibrosarcoma. Adsorption of rat-anti-HSV-2 serum with tREF-G-2 or rat fibrosarcoma cells reduced neutralizing activity to 10 and 12%, respectively, compared with 90% neutralization by antiserum adsorbed with nontransformed rat embryo fibroblast cells and 100% neutralization with unadsorbed antiserum. In summary, HSV-2-transformed rat cells retained and expressed genetic information necessary for the production of HSV-2 glycoproteins and a nonstructural protein after high passage in tissue culture or in the syngeneic host.