Antigens of tumours induced by naturally occurring murine sarcoma virus (MSV-FBJ). II. Detection of cell-surface antigens by indirect membrane immunofluorescence

Cell surface antigens expressed by cells transformed in vivo by FBJ virus, a wild type murine sarcoma virus (MSV) complex derived from a spontaneously arising sarcoma in a CF1 mouse, have been studied by indirect membrane immunofluorescence (MIF). Using mouse antisera raised by immunization of syngeneic CBA mice with transplanted FBJ sarcomata an antigen common to all FBJ tumours was detected which was also present on Gross (G) antigen positive tissues, viz. leukaemic and preleukaemic AKR lymphoid cells, but absent from the tissues of mice of G negative strains. Failure to demonstrate antigenic cross-reactivity in reciprocal MIF tests using FBJ immune sera and antisera to MSV-H (Harvey), an MSV isolate of Friend-Moloney-Rauscher (FMR) sub-group specificity, established the virus type-specificity of antigens expressed by sarcoma cells transformed by the respective MSV.     

Studies on mouse sarcoma virus. IV. The isoelectric point of the group-specific antigen

At least three classes of species-specific, group-specific (gsl) antigen-bearing molecules were separated by isoelectric focusing of tween-ether disrupted Moloney strain mouse sarcoma virus (M-MSV) derived from a permanent producer rat cell line (78 A1). The bulk of gs1 antigen, as measured by complement-fixation, was associated with a ³H-uridine-labelled component characterized by an isoelectric point (ip H) of 5.9 and a sedimentation coefficient of 2.7 S_{w, 20} Statistical evaluation showed the ipH value to be remarkably stable. Neither ipH nor content of ³H-uridine-label was altered by ribonuclease (RNase), in contrast to an ipH 5.0 component found occasionally. The other gs1 antigen-bearing molecules were only minor components and included an RNase-resistant, ipH 5.5 component, frequently observed as a shoulder on the major peak, and an inconstant, poorly labelled component with a more variable ipH value of about 6.6. The significance of these findings is discussed in view of previous reports on the subject and of recent information regarding the structure of murine tumor viruses.     

Antigens of tumours induced by naturally occurring murine sarcoma virus (MSV-FBJ). I. Detection of group and type specific antigens by complement fixation

Antigens associated with cells transformed in vivo by FBJ virus, a wild type murine sarcoma virus (MSV) complex originating from a spontaneously arising osteosarcoma in a CF1 mouse, have been partially characterized by complement fixation (CF). Using rat antisera against antigens specified by Gross leukaemia virus (GLV) the group specific (gs) antigen of C-type RNA murine tumour viruses was demonstrated in FBJ tumours as well as in GLV rat leukaemias, AKR lymphomata and sarcomata induced by MSV-H (Harvey), an MSV isolate of Friend-Moloney-Rauscher (FMR) subgroup specificity. Using mouse antisera against antigens present in FBJ cells the Gross (G) or wild type specificity of FBJ tumours was demonstrated by cross reactivity with antigens expressed on normal AKR lymphoid tissues and leukaemias. These antigens were absent from MSV-H induced sarcomata and in reciprocal tests mouse antisera to MSV-H failed to react with antigens present in FBJ tumour cells. No distinction between cellular and virion antigens expressed by FBJ cells was possible by CF although evidence for a cellular antigen with G specificity was obtained in tests using aged C57B1 antiserum containing a naturally occurring G antibody lacking significant virus neutralizing capacity. However, the likelihood that mouse FBJ antisera contain antibodies to type specific viral envelope antigens (VEA) as well as cellular antigen is discussed.     

Non-producer human cells induced by murine sarcoma virus.

Non-produces (NP) human cells were isolated from transformed foci induced by the Kirsten mouse sarcoma virus. These morphologically altered NP cells produced neither infectious virus nor complement-fixing antigens of the murine sarcoma-leukemia virus complex. However, the sarcoma virus genome could be rescued from these NF cells by co-cultivation with cells carrying "helper" Kirsten mouse leukemia virus or Woolly Monkey leukemia virus. The possible usefulness of these cells in efforts designed to detect covert or repressed RNA tumor viruses in various animal and human tissues is discussed.     

Effect of protease inhibitors on focus formation by murine sarcoma virus.

The effect of six protease inhibitors, isolated from various species of actinomycetes, on focus formation by murine sarcoma virus was examined. Pepstatin was the only inhibitor. The treatment of cells with pepstatin at various times possibly retards the early stage of infection with murine sarcoma virus.     

Genetic control of oncogenesis by murine sarcoma virus Moloney pseudotype. II. A dominant epistatic susceptibility gene.

We have shown in the preceding paper that AKR mice are highly resistant to M-MSV tumor development, and that resistance is transmitted as a dominant character. In the present studies the tumor-response pattern of F1 hybrids between resistant AKR and susceptible strains (C57Bl/6, BALB/c and B10BR) following injection with Moloney mouse sarcoma virus (M-MSV) resembles that of the non-AKR parent. Segregation is observed in first backcross (Bc1) and F2 mice, and the segregation ratios up to Bc3 mice fit a one-gene model. The data of triple cross hybrids suggests that this dominant susceptibility gene inhibits the phenotypic expression of M-MSV tumor resistance in some susceptible Fv1bb strains as well as in their hybrids with AKR. Neither Fv-1 nor H-2 exerts any significant influence on this complex system.     

Stimulatory effect of immunization on tumor induction by Moloney murine sarcoma virus.

Adult mice were immunized with varying doses of inactivated Moloney murine leukemia virus (M-MuLV). Eight weeks after immunization, mice were challenged with a dose of Moloney murine sarcoma virus (M-MuSV) that could induce tumors in approximately 50% of normal animals. Mice immunized with high doses of M-MuLV (10(10) particles) had significantly decreased tumor incidences, whereas mice immunized with low doses of M-MuLV (10(2) particles) had significnatly increased tumor incidences compared to those in nonimmunized controls. The stimulatory effect could be abrogated by the irradiation of mice with 450 rads 24 hours prior to M-MuSV challenge, whereas the inhibitory effect was resistant to this irradiation procedure. The results suggested that immunization with virus can either stimulate or inhibit virus-induced tumorigenesis, depending on the dose of virus used for immunization.     

Murine sarcoma virus: titration patterns and interference by a murine leukemia virus

A murine sarcoma virus (MSV-K) was assayed by conversion of 3T3 mouse cells into foci of morphologically altered cells. Murine erythroblastosis virus (MEV), a member of the murine leukemia virus group, induced resistance in 3T3 cultures specific to super-infection with focus-forming MSV-K. Interference by MEV was first detected at 14 days and reached maximal levels 21 days post infection. MSV-K preparations from transformed 3T3 cells contained competent virions, as judged by “one-hit” titration patterns. Since excess “helper” MEV in MSV-K stocks could effectively convert titration patterns to “one-hit” kinetics, the ratio of MEV to MSV-K was determined by interference experiments. The ratio of MEV to MSV was approximately 10:1. Thus it appears that a single particle is capable of producing a focus of altered cells in the absence of simultaneous infection with a separate particle of MEV.     

Transformation of rat liver epithelial cells by Kirsten murine sarcoma virus.

We studied the transformation of epithelial, diploid cell lines (RL-33 and RL-34) derived from W rat liver by the Kirsten murine sarcoma virus. On days 4-5 after virus infection, the epithelial cells began to pile up focally, forming small projections and releasing round cells from the foci. The epithelial cells grew in chains or as islets and grew in suspension above the cells attached to the bottom of the flasks when the cultures reached the confluent stage. The virus titration pattern was "one-hit." Three classes of transformed cells were isolated with respect to virus release and antigen expression: 1) virus producer, 2) non-producer, and 3) sarcoma-positive, leukemia-negative cells. When transplanted sc into newborn rats, the transformed cells produced sarcomas. The transformed cells formed within 1-3 days larger aggregates than those of their normal counterpart cells when suspended in liquid growth medium above an agar base. Aggregate properties (size, viability, and proliferation) of transformed cells correlated with growth in soft agar and tumorigenicity. RNA-dependent DNA polymerase and type C virus particles were readily induced in the normal rat liver epithelial cells after exposure to 5-iodo-2'-deoxyuridine.     

Chemical transformation of human revertant cells induced by murine sarcoma virus.

A human revertant cell line, derived from non-producer human osteosarcoma cells (NP/KHOS) induced by Kirsten murine sarcoma virus, was treated in vitro with various levels of polycyclic aromatic hydrocarbons (3-methylcholanthrene, 7,12-dimethylbenz(alpha)anthracene, and benzo(alpha)pyrene [BP] or dimethyl sulfoxide (control). Cells treated only with 3-methylcholanthrene and 7,12-dimethylbenz(alpha)anthracene underwent morphological alteration in vitro, and produced tumors when injected into NIH nude mice. These human revertant cells may be a useful in vitro tool in screening for the potential chemical carcinogens in human cell systems.     

Enhancement by asbestos of oncogenesis by Moloney murine sarcoma virus in CBA mice.

Five micrograms of finely ground crocidolite asbestos (UICC standard sample) were injected intraperitoneally into 3-week-old CBA mice, together with 10(5) FFU of Moloney murine sarcoma virus. Altogether 44 out of 61 mice (72.1%) so treated developed palpable intraperitoneal tumours, and half of these died of such tumours within 100 days. The same amount of quartz and carbon similarly administered gave lower tumour incidences, namely, 19.4% (3.2% fatal) and 11.9% (1.5% fatal) respectively. Only 1 out of 59 mice inoculated with the virus alone developed a palpable intraperitoneal tumour, and this regressed spontaneously within 10 days of its first appearance. No tumours were encountered in mice treated with either asbestos, quartz or carbon alone. All the neoplasms had the appearance, under the light microscope, of anaplastic sarcomas. Most of them were confined to the serosal surface of the abdominal cavity, although invasion of underlying tissues was observed in some of the animals that died. Electron microscopical examination of tumours revealed the presence of C-type particles budding off from cellular surface. Some neoplastic cells showed characteristic features of mesothelial lining cells. The role of milky spots (taches laiteuses) in oncogenesis by asbestos and virus, especially in the induction of mesothelioma, is discussed.     

Carcinogenesis by RNA sarcoma viruses. XIV. Infection of stationary cultures with murine sarcoma virus (Harvey)

No focus-forming virus was produced after exposure of stationary mouse embryo cell cultures (achieved by removal of serum) to the marine sarcoma virus (Harvey) complex. Addition of serum to these infected stationary cultures resulted in MSV (Harvey) production following cell division. Cytosine arabinoside, an inhibitor of DNA synthesis, was found to inhibit subsequent virus production when added to stationary cultures immediately after exposure to virus. These results are compatible with a requirement for both cell division and non-S-phase DNA synthesis for the production of this virus complex, and show a marked parallelism to results previously obtained with the avian sarcoma complex.     

Studies on group-specific antigens in tumours induced with avian tumour viruses in rats

Group-specific complement-fixing antigen (GS antigen) was studied by the complement-fixation test in various avian and mammalian tumours, induced with the following strains of avian tumour virus (ATV): Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV), Prague strain of RSV (PR-RSV), Fujinami sarcoma virus (FSV) and Bratislava 77 virus (B77V). All primary tumours induced in chicks by SR-RSV, FSV and B77V, as well as all duck tumours induced with B77V, contained high titres of GS antigen when studied with high-titre rabbit antiserum against purified GS antigen of avian myeloblastosis virus (AMV). In three out of six B77V-induced rat tumours (two virus-producing and one non-virogenic) high titres of GS antigen were detected when examined with rabbit antiserum and with some rat sera obtained from tumour-bearing animals. In three remaining B77V-induced virogenic rat tumours (C21, C22 and C23), GS antigen was not detected with rabbit antiserum; however, sera obtained from these three tumour-bearing rats contained high titres of GS antibodies when tested with different GS antigens. Positive results were obtained also with PR-RSV-induced virogenic rat tumour XC. However, all attempts to detect GS antigen in one SR-RSV induced virogenic rat tumour (MRI) gave negative results. Possible heterogeneity of ATV-induced GS antigen in mammalian tumours is discussed.     

The mechanism of interferon effect on cell transformation by murine sarcoma virus

Mouse interferon (IFN) was found to inhibit murine sarcoma virus (MSV)-induced neoplastic transformation of normal rat kidney (NRK) cells. This effect was observed upon examining the formation of foci of morphologically altered cells and colonies of anchorage-independent cells. IFN had no cytotoxic effect on MSV-transformed NRK cells, nor on their focus or colony-forming ability. It was therefore apparent that its inhibitory effect was directed against the viral role in cell transformation. In attempts to define the mechanism of this effect, we found that IFN delayed the initiation of the cytoplasmic viral DNA synthesis. However, the amount of this DNA eventually formed in IFN-treated cells was the same as in the control cells. Furthermore, the transport of this DNA to the nucleus was slower in IFN-treated cells, although all of it was finally transferred. However, while most of the viral DNA integrated into the genome of the control cells, very little integration occurred in IFN-treated cells. The unintegrated viral DNA of these cells was slowly degraded. Therefore, if the cells recovered from the antiviral effect of IFN when intact viral DNA molecules still existed in their nucleus, they could resume viral DNA integration and cell transformation. IFN was found to block viral DNA supercoiling. Since supercoiled viral DNA is considered to be a precursor to integrated provirus, it seems that the inhibition of both integration and cell transformation is due to this impaired coiling.     

In utero tumor induction by murine sarcoma virus (Moloney) in the rat. II. Histological and ultrastructural characteristics

On histological examination the twelve MSV-induced tumors in the uterus observed in our study proved to be of fetal origin. Ten of the 12 tumors had the histological and ultrastructural characteristics of a yolk-sac tumor. The two other tumors examined had a different morphological structure. One was classified as a teratoma with embryonal carcinoma, the other as a carcinosarcoma. In the primary neoplasms virus particles were found. Their ultrastructure was similar to that of MSV. The histology and the electron microscopy results are discussed in relation to the morphogenesis and the occurrence of these tumors in rodents as well as in man.     

Comparative studies on EBV antigens by immunofluorescence and immunoperoxidase techniques.

Three groups of EBV antigens, VCA, MA and EA, were compared by the techniques of electron microscopic immunoperoxidase (IP) and immunofluorescence (IF). P3HR-1 and EBV superinfected Raji cells served as targets for labelled sera from patients with BL, NPC and IM or from healthy donors. 125I peroxidase-labelled antibodies were also prepared to determine, autoradiographically, the penetration of the complex into the cell system, and to monitor the incubation and washing procedures. The development of a gentle sedimentation technique proved critical in handling the fragile target cells. VCA and MA were readily identified and localized by both procedures without significant modification of the basic techniques. Indentification of early antigens by IP required modification of the fixation method to include a brief treatment in acetone. The diffuse early antigen (EAD) was found to be associated with cellular ribosomes.