Induction of type C virus-related functions in normal rat embryo fibroblasts by treatment with 5-iododeoxyuridine

Secondary cultures of rat embryo fibroblasts, derived from six different strains of normal laboratory and wild rats, were treated with 5-iododeoxyuridine in an attempt to establish the presence of endogenous type C viruses in rat cells. All the cell lines tested responded with a transient appearance of RNA-dependent DNA polymerase (RDP) activity which reached a peak 3 days after the beginning of treatment. However, no viral particles or rat gs-antigen could be detected at this stage. In the cells derived from two highly inbred strains, a spontaneous second burst of RDP activity was observed after 10–12 days, and on subculturing these cells a 20- to 40-fold increase of enzyme activity could be obtained. During this peak of activity both gs-antigen and viral particles capable of incorporating labelled uridine were detected. Electron microscopic examination revealed the presence of complete or immature virions in one cell strain. The virus induced was non-infective for monolayer cultures of 10 different animal species. Attempts to rescue infective virus by complementation with murine leukemia or sarcoma viruses failed. Prolonged cultivation of treated cultures did yield cells with trasformed morphology, but no oncogenicity could be demonstrated for these cells.     

Evidence for type-C retrovirus production by Burkitt's lymphoma-derived cell line

Burkitt's lymphoma cell line, P3HR-I, was found to secrete virions with properties of known type-C RNA tumor viruses. The viral particles had a buoyant density of 1.16 g/ml in sucrose gradients and contained a high-molecular-weight RNA and an RNA-instructed DNA polymerase. The viral polymerase was active in an endogenous reaction requiring the presence of the four deoxyriboside triphosphates and manganese ions, and was sensitive to RNase. The DNA product of the endogenous reaction specifically hybridized to P3HR-I viral 60 to 70S RNA. Electron microscopic examination of ultrathin sections of P3HR-I cells revealed immature, mature and budding virions typical of type-C retroviridae. Nucleic acid hybridization assays showed no sequence homoblastosis virus, murine oncornaviruses, simian sarcoma virus or RD114 virus.     

Surface localization of virus production on a glucocorticoid-stimulated oncornavirus-producing mouse mammary tumor cell line by scanning electron microscopy.

A chronically infected continuous mouse mammary tumor cell line containing virus particles of type B morphology, free of contaminating type C virions, has been grown in tissue culture. These cells were treated with dexamethasone, a synthetic glucocorticoid, a potent stimulator of mouse mammary tumor virus expression. Surfaces of untreated and dexamethasone-treated cells were investigated by scanning electron microscopy. Untreated cells demonstrated a moderate expression of mouse mammary tumor virus (80 particles/cell) distributed diffusely over the cell surface. However, virions on dexamethasone-treated cells were localized in clusters of 100 to greater than 2000 virus particles, often with more than one cluster per cell. Dexamethasone-treated cells typically showed a 10-fold increase in cell-associated virus over untreated cells. Concentrated extracellular fluids from untreated and dexamethasone-treated cultures were quantitated for free virus. Dexamethasone-treated culture fluids demonstrated a similar 10-fold increase of extracellular particles, in contrast to untreated cultures. This increase in virus particles on the cell surfaces as well as in the extracellular fluids supports the theory that dexamethasone has a stimulatory effect on viral replication, not just on the release of budding particles. The ultrastructure of budding mouse mammary tumor virus during dexamethasone stimulation, determined by scanning and transmission electron microscopy, and the significance of such an in vitro system for viral immunodiagnosis are discussed.     

Properties of a cell line from human adenocarcinoma of the rectum.

A new, highly differentiated line of cells derived from adenocarcinoma of the rectum (HT55) is described. This line is noteworthy for the following features: 1. The role played in its development by the use of UV-inactivated Sendai virus to attach tumour cell clumps to plastic bottles. 2. Evidence that it produces RNA-containing material of density 1-5--1-16 g/ml. 3. Induction of bone formation in the stroma when grown in athymic mice. 4. Stimulation of primary CBA mouse embryo fibroblasts to form a transient nodule when mixed with them and injected into adult CBA mice. The karyotype and growth-cycle characteristics of the line are described.     

Studies on the oncogenicity of bovine adenovirus type 3

The oncogenicity of bovine adenovirus type 3 in Syrian hamsters and CBA mice has been investigated. Two different pathological lesions were observed in the hamsters, but no unequivocal tumour production was demonstrated in the mice. Each type of lesion in the hamsters had a different latent period and was related to the original dose of virus. Low virus doses produced solid characteristic adenovirus-type tumours with a latent period of at least one month, whereas higher doses of virus characteristically produced haemorrhagic cystic lesions incorporating fibrous tumour plaques with a latent period of less than a month. The possibility that these two lesions resulted from the presence of two different agents in the inocula cannot be ruled out, although there is some evidence to suggest that this was not the case. Hamsters inoculated with virus developed complement-fixing, immunodiffusing and neutralizing antiviral antibodies regardless of tumour production, but an antibody response to ?T”? antigen was not consistently demonstrated. A classical transplantation immunity of the Habel-Sjögren type was produced. The cystic lesions, although appearing as low-grade neoplasms, might actually have been partly cytotoxic (or lytic) in nature. Thus the persistence of some virions or virion antigens into the period of immunological maturity may account for the antiviral antibody response in inoculated hamsters.     

Effect of ethidium bromide on transplanted virus-induced tumor cells.

Ethidium bromide (2,3-diamino-5-ethyl-6-phenylphenanthridinium bromide) significantly inhibited the RNA-dependent DNA polymerase of types A and C particles isolated from transplanted adenovirus 12-induced tumors of CBA mice. It was also cytotoxic for an established in vitro line of adenovirus 12-induced tumor cells of CBA mice and caused cell death, inhibition of [3H]thymidine uptake, and a significant reduction of cells in metaphase. Ethidium bromide significantly inhibited the in vivo growth of transplanted adenovirus 12-induced tumor cells of CBA mice, simian virus 40-induced tumor cells of hamsters, and murine leukemia virus-induced lymphoma cells of BALB/c mice. The compound may have exerted the antitumor activity by selectively affecting oncornavirus in the tumor cells.     

Enhanced oncornavirus expression in Marek's disease tumors from specific-pathogen-free chickens.

An oncornavirus was recovered from cell cultures of kidney tumors from specific-pathogen-free chickens inoculated with Marek's disease herpesvirus (MDHV). The MDHV inoculum was free of infectious avian leukosis virus (ALV). Direct examination of a variety of tissues from MDHV-inoculated chickens demonstrated increased levels of ALV-specific RNA compared to tissues from diluent-inoculated (control) chickens. DNA from cultured kidney tumor cells annealed to an ALV complementary DNA probe at the same rate and exhibited the same extent of homology as DNA from cultured control kidney cells. This finding indicated the absence of exogenous ALV proviral sequences. As with vertically transmitted endogenous ALV of subgroup E, the oncornavirus recovered from kidney tumor cell cultures failed to replicate in chicken embryo fibroblast (CEF) cultures of the C/E phenotype, but did replicate in turkey embryo fibroblasts (TEF), which are permissive for replication of endogenous ALV of subgroup E. These oncornavirus particles served as a helper virus to form Rous sarcoma virus pseudotypes, which produced foci in TEF cultures but not in CEF cultures of the C/E phenotype. Whether enhanced expression of endogenous oncornavirus contributes to MDHV-induced tumorigenesis is not known.     

In vitro anti-human immunodeficiency virus type 1 activity of biliverdin, a bile pigment.

Biliverdin (BV) is a bile pigment having anti-allergic properties. We examined the effect of BV on human immunodeficiency virus type 1 (HIV-1) in vitro. BV completely inhibited the cytopathic effect of HIV-1 in MT-4 cells at concentrations of 22.2 micrograms/ml or more. This inhibitory effect was also observed when BV was present during the adsorption period of HIV-1. However, BV was cytotoxic to MT-4 cells at concentrations above 800 micrograms/ml. At a concentration of 66.7 micrograms/ml, BV completely inhibited syncytia formation by HIV-infected and uninfected MOLT-4 cells. Moreover, after exposure of HIV-1 particles to BV for 2 h, the infectivity of the virus was reduced in a dose-dependent manner. It is speculated that the anti-HIV activity of BV is due to direct inactivation of virions and inhibition of virus binding to target cells.     

Virus-specific markers and virus-like particles in cell lines of tumors produced by CELO virus in Syrian golden hamsters.

Cell lines were established from 2 primary hepatocellular carcinomas (HC's) and 3 sarcomas produced in Syrian golden hamsters inoculated as newborns with chicken embryo lethal orphan (CELO) virus. Cell lines from 2 sarcomas (COT, CMT) and 1 HC (CEHEP) produced CELO virus-specific T-antigen. The antigen was not detected in cells of the third sarcoma line (RCT) until they had undergone more than 34 passages in vitro. Although 5-10% of cells in the second HC line (CILT/2) contained T-antigen during early passages, it was not demonstrable after the fifth subculture. Nevertheless, cells of both HC lines possessed CELO virus tumor-specific transplantation antigen. All 5 cell lines also contained hamster type R particles, and both HC lines had type C and intracytoplasmic type A particles. The percentage of carcinoma cells producing type R particles increased during cultivation in vitro, whereas the number of cells with type A particles decreased. Treatment with dibutyryl cyclic AMP and theophylline enhanced the number of cells producing type C particles in 1 HC line and type R particles in 2 sarcoma lines.     

Intracytoplasmic type A particles from mammary tumours and leukaemias of strain ICRC mice.

The ovarian-hormone-induced leukaemias of strain ICRC mice, with an abundance of intracytoplasmic type A particles in primary as well as transplanted lesions, were used to study morphological, biophysical, immunological and structural characteristics of type A particles. Mammary tumours of strains ICRC and C3H(Jax) were also used as sources for type A particles. The purified virions banded at the density of 1.20 g/ml in 12--60% linear sucrose-density gradient when subjected to spinning at 113,000 g for 4 h. The SDS-polyacrylamide-gel electrophoresis of type A particles from mammary tumours and leukaemias reproducibly resolved at least 8 polypeptides, 2 of these 54,000 and 24,000 dalton proteins, showing variable expression. Type A particles and B particles, despite the fact that each had a distinct polypeptide pattern, showed common antigens with different electrophoretic mobilities. Proteins of 24,000, 18,000 and 12,000 daltons from B particles were found to be antigenically related to those from type A particles. The bioassay studies carried out with purified A particles showed that 2/7 males of strain ICRC and 1/6 females of strain DBA-MTI developed leukaemias, as against none in the controls, when inoculated between the ages of 1-7 days. Spleen tumour and cervical tumour were seen in one female each of strain DBA-MTI.     

Activation of an endogenous mouse type C virus by UV-irradiated murine cytomegalovirus

Infection of Kirsten sarcoma virus-transformed non-producer BALB-3T3 mouse cells with UV-irradiated mouse cytomegalovirus (MCMV) resulted in activation of a xenotropic type C virus detected by infectious center formation in permissive rat or mink cells. The levels of type C virus activated by MCMV were related to the UV dose applied. Under optimal conditions the frequencies of activation varied from 3.0 to 8.0 x 10(-4). The capacity of MCMV to activate type C virus was abolished by heat inactivation and by neutralization with specific antiserum against MCMV. Virus induction decreased under conditions of cell exposure to hydroxyurea or actinomycin D, inhibitors of DNA and RNA synthesis, respectively, with actinomycin D having a greater inhibitory effect. This suggests that both DNA and RNA synthesis are required for UV-MCMV induction of murine xenotropic virus.     

Surface antigens on transplantable tumor cell lines producing mouse type C viruses.

A variety of transplantable mouse tumor lines were shown to contain murine type C viruses and virus-associated antigens. The type of virus isolated and antigens detected could not invariably be correlated with the original method of tumor induction, but testing of the majority of tumor lines for infectious virus at various levels of in vivo or in vitro passage yielded isolates that were consistent in tissue culture host range for each tumor. In contrast, during the course of in vivo transplantation, some of the lines underwent considerable change in the pattern of virus-associated cell-surface antigens. When the transplanted tumor lines were placed into culture, all showed some alteration in the detectable surface antigens. Upon retransplantation and passage of the cultured cells in mice, the surface antigens gradually returned to the original in vivo patterns and occasionally acquired additional type C virus-associated antigens not detected in the original tumor line. To test for association of antigens with infectious virus, appropriate tissue culture cell lines were infected with the viruses isolated from the tumors. In these infected indicator cells, some new virus-associated cell-surface and virion evelope antigens were detected, but the complete array of antigens found in the original tumor lines was not acquired. These findings indicated the presence of several different type C viruses in long transplanted cell lines and demonstrated that environmental and host cell factors may have major influences on expression of virus-associated antigens.     

In vitro Anti-human Immunodeficiency Virus Type 1 Activity of Biliverdin, a Bile Pigment

Biliverdin (BY) is a bile pigment having anti-allergic properties. We examined the effect of BV on human immunodeficiency virus type 1 (HIV-1) in vitro. BV completely inhibited the cytopathic effect of HIV-1 in MT-4 cells at concentrations of 22.2 μ g/ml or more. This inhibitory effect was also observed when BV was present during the adsorption period of HIV-1. However, BV was cytotoxic to MT-4 cells at concentrations above 800 μ g/ml. At a concentration of 66.7 μ g/ml, BV completely inhibited syncytia formation by HIV-infected and uninfected MOLT-4 cells. Moreover, after exposure of HIV-1 particles to BV for 2 h, the infectivity of the virus was reduced in a dose-dependent manner. It is speculated that the anti-HIV activity of BV is due to direct inactivation of virions and inhibition of virus binding to target cells.     

Separation of cells containing R-type virus-like particles from a simian virus 40-induced hamster tumor cell line.

Cells derived from a simian virus 40-induced hamster fibrosarcoma were separated into two distinct cell bands of differing buoyant densities. The lighter cell band or fraction (F1) had a buoyant density range of 1.025-1.032 g/ml and comprised 3.8% of the total cells applied to the gradient, whereas the heavier cell fraction (F2) had a buoyant density range of 1.054-1.074 g/ml and comprised 95.3% of the total cells applied. Both cell fractions were tumorigenic and did not differ greatly in cell type, viability, mitotic index, or their ability to incorporate [3H]thymidine. However, ultrastructurally, the F1 cells contained R-type virus-like particles within dilated intracisternal spaces and exhibited cytoplasmic vacuoles. In the F2 cells, few detectable R-type particles and cytoplasmic vacuoles were revealed by electron microscopy. The F2 cells demonstrated a twofold greater ability to incorporate [14C]protein hydrolysate into proteins than did the F1 cells.     

Uptake, metabolism, and persistence of 3-methylcholanthrene in rat embryo cells infected with murine leukemia virus.

The uptake and persistence of 3-methylcholanthrene have been followed in both unifected rat embryo tissue culture cells and in cells infected with type C RNA virus. No significant differences in these parameters were observed as a function of viral infection or cell passage level. Moreover, neither binding of 3-methylcholanthrene to nucleic acids or proteins nor carcinogen metabolism were altered by the viral carrier state. Although transformation of rat cells by chemical carcinogens alone has been reported by us and other authors, the low-passage rat embryo cells used in this study will not transform unless cells are carrying exogenous type C RNA virus. We thus suggest that the virus must play a more direct role in the transformation process rather than affecting the ability of the cell to absorb, retain, or metabolize the chemical.     

常春藤皂苷元影响内质网相关途径阻滞骨肉瘤细胞周期的实验研究

目的:研究常春藤皂苷元对骨肉瘤细胞周期的影响及机制。方法:用0、5、10、20、40 μg/ml的常春藤皂苷元处理骨肉瘤细胞，CCK-8法检测细胞增殖情况，细胞克隆实验测定细胞克隆形成能力，计算其半数抑制浓度。用半数抑制浓度的常春藤皂苷元处理骨肉瘤细胞，流式细胞术检测细胞周期和细胞凋亡，荧光探针法测定细胞内Ca2+浓度。用Western blot法检测细胞中结合免疫球蛋白（BIP）、内质网氧化还原酶1-Lα（Erol-Lα）、蛋白质二硫键异构酶（PDI）、剪切型Caspase-12（Cleaved Caspase-12）、细胞周期蛋白D1（Cyclin D1）蛋白水平。结果:5、10、20、40 μg/ml的常春藤皂苷元均可以抑制骨肉瘤细胞的增殖和克隆形成能力，与0 μg/ml常春藤皂苷元作用组比较，差异有统计学意义（P＜0.05）。半数抑制浓度的常春藤皂苷元处理后的骨肉瘤细胞G1期比例升高，细胞凋亡率也升高，细胞内Ca2+浓度升高，细胞中BIP、Erol-Lα、PDI、Cleaved Caspase-12蛋白水平升高，Cyclin D1蛋白水平降低，与0 μg/ml常春藤皂苷元作用组比较，差异有统计学意义（P＜0.05）。结论:常春藤皂苷元通过影响内质网相关途径将骨肉瘤细胞周期阻滞在G1期，诱导细胞凋亡发生。     

Characterization by molecular hybridization of two viral populations derived from a radiation leukemia virus.

Two leukemogenic viral populations were derived from a radiation leukemia virus of the C57BL mouse. One (FB), in which only B-tropic virus could be detected, was obtained in vivo by serial passage of cell-free extract in newborn rats. The second (3C), a complex containing at least B-tropic and xenotropic viruses, was produced in vitro by a permanent cell line (13-3C) established from the spleen of a virus-infected C57BL mouse. In molecular hybridization experiments, the 70S RNA of Gross leukemia virus hybridized 96 and 78% of FB and 3C radioactive complementary DNA's, respectively, with a relatively high thermal stability of the duplexes formed. In contrast, the 70S RNA of Rauscher leukemia virus hybridized 23 and 20% of the FB and 3C DNA probes, respectively, with a low thermal stability. The rat-grown FB virions exhibited 50% genome homology with the viruses produced in vitro on the 13-3C cells. Finally, hybridizing the FB and 3C probes with normal or leukemic mouse spleen DNA's resulted in 89 to 100% homology. The rat-grown virions did not appear to contain detectable rat cellular DNA sequences, while about 20 complete copies of their nucleotide sequences were detected in covalent linkage with FB-infected rat spleen DNA. These findings strongly support the endogenous murine origin of the investigated virions.