Continuous production of avian sarcoma virus B77 by rat tumour cells in tissue culture

The rat tumour RBI, previously induced by injection of fowl sarcoma B77 tissue into newborn rats, was adapted to long-term growth in tissue culture. Two cell lines, 16B177 and 17B177, were derived from the tumour. Both cell lines were capable of continuous production and release of infectious B77 virus over a period of more than two years' in vitro cultivation. The virus present in cell-free tissue culture media induced characteristic sarcomas upon inoculation into young chicks. Preliminary chromosomal analysis of the uncloned population of 17BI77 cells confirmed their rat origin and revealed that the hypotriploid stem-line predominates, being represented by 59 chromosomes.     

Cell-mediated and humoral immune responses to tumor-associated and viral antigens in relation to the pathogenic effect of Rous sarcoma virus in rats

After inoculation of the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) into a series of newborn rats (strain R/F) sarcomas developed in 47% of the animals, half of which also had hemorrhagic-cystic lesions. In 18% such hemorrhagic cysts occurred without demonstrable tumors, while no pathogenic effect of the virus was observed in the rest of the animals. The colony inhibition technique was used to detect lymph-node cell (LNC)-mediated and humoral immunity to the tumor-associated transplantation antigen (s). LNC-mediated immunity could be demonstrated in a high proportion of tumor-positive as well as tumor-negative rats (83 and 94%, respectively), while the rats with hemorrhagic-cystic lesions showed such immunity in a lower frequency (56%). Humoral cytotoxic activity could be demonstrated in a higher frequency in rats with cystic lesions than in rats without cysts, whether they had tumors or not (about 80 and 55%, respectively). Antiviral immune response was demonstrable in only a few animals. The results indicate that, as in several other systems, the development of tumors is not related to a deficient LNC-mediated immune response to the tumor-associated transplantation antigen (s). Nor could any difference be demonstrated in the frequency of humoral immune response to the antigen (s) between tumor-positive and tumor-negative animals. The results furthermore indicate that the hemorrhagic-cystic lesions, characteristic for the RSV effect, develop in animals which have a less efficient LNC-mediated immune response and that these lesions are of importance for the sustaining of humoral cytotoxic immune response.     

Transplantable primate tumors induced by Rous sarcoma virus. I. Induction of tumors transplantable into young marmosets

The tumors induced in white-lipped marmosets (Saguinus fuscicollis, S. nigricollis) by Rous sarcoma virus (RSV) of chicken origin (RSV-SR) were not transplantable to allogeneic hosts. In contrast, RSV rescued from these tumors (RSV-M) induced sarcomas that were transplantable to young but not to adult marmosets. The tumors induced by RSV-M and the transplants rapidly enlarged, metastasized to various organs, and killed the recipients 29-59 days post inoculation. Cell lines were readily established from all transplantable sarcomas. No virus expression was detected in transplantable tumor cell lines by electron microscopy or by biochemical and biological assays. However, RSV of the same subgroup as RSV-SR was rescued from both short-term and long-term tumor cell cultures by cocultivation with chicken embryo fibroblasts (CEF). The rescued viruses transformed marmoset cells 100-fold more efficiently than CEF cells, although CEF cells remained permissive for virus replication. Cytogenetic studies revealed extensive chromosome abnormalities in tumor transplants but not in RSV-M-induced sarcomas. All cell lines were hyperploid and contained structurally abnormal, large metacentric and telocentric chromosomes. Immunologic studies failed to detect group-specific (gs) antigen of the avian sarcoma-leukemia complex in either RSV-M-induced, transformed cells or tumor transplants. By complement-dependent cytotoxicity assays, with the use of marmoset anti-gs serum, RSV-associated antigen could be detected on the surfaces of tumor cells. No differences in the expression of this antigen existed between transplantable and nontransplantable marmoset sarcomas. All transplantable cell lines contained abnormal amounts of lipids and glycogen in comparison to RSV-SR-induced tumors and normal marmoset cell lines. The glycogen was associated with unique cytoplasmic membrane complexes and was surrounded by either single- or double-membraned vesicles.     

Cellular immunity in rats with primary brain tumors: inhibition of macrophage migration by soluble extracts of avian sarcoma virus-induced tumors.

Rats bearing primary tumors of the brain induced by avian sarcoma virus (ASV) were studied with the migration-inhibition factor (MIF) assay for the presence of cell-mediated immunity to tumor-associated antigens. Astrocytomas and sarcomas of the brain were induced in 34 neonatal F344 rats by the intracerebral inoculation of Bratislava-77 ASV. At weekly intervals from 4 to 9 weeks after the inoculation with virus, peritoneal exudate cells (PEC) from rats bearing brain tumors were tested an an MIF assay against soluble and KCl-treated extracts of a syngeneic, ASV-induced sarcoma. Incubation of the PEC with a soluble extract of syngeneic liver or with a KCl extract of a syngeneic, chemically induced tumor served as controls. Of 14 rats tested against the soluble tumor extract, 6 (43%) had statistically significant inhibition of migration (P less than or equal to 0.05). Of 23 animals tested against the KCl extract, 16 (70%) had significant inhibition. Immunity to the KCl extract was significant in most rats at each period. Ten rats were tested against a KCl extract of a hamster ASV-induced tumor; 7 gad significant inhibition of migration. None of 3 tested against a soluble extract of a syngeneic, chemically induced tumor had significant inhibition. Rats bearing ASV-induced brain tumors displayed cell-mediated immunity to tumor-associated antigen or antigens of ASV-induced tumors, which could be solubilized.     

Rous tumors in monkeys

Macaca rhesus, Macaca irus, Macaca nemestrina, Papio hamadryas and Cercopithecus aethiops monkeys are susceptible to Rous sarcoma virus (RSV) (Carr and Schmidt-Ruppin strains). After intramuscular or subcutaneous injection of RSV-infected chicken cell suspensions or cell-free extracts of the chicken Rous sarcoma virus, they develop tumors at the sites of inoculation. These tumors appear after different periods of time, ranging from 15 up to 35 days after inoculation of the virus-containing material. After an initial period of continuous growth (sometimes as long as 7 months), the tumors regress in most cases. Histologically, the tumors are polymorphous fibrosarcomas which do not produce metastases. The oncogenicity in monkeys of the Carr or Schmidt-Ruppin strains was of no substantial difference. Only newborn or very juvenile monkeys are susceptible to Rous virus. The virus may only in some cases be isolated from the simian tumors by inoculation of tumor cell suspensions into chickens. Monkeys inoculated with Rous sarcoma virus develop virus-neutralizing antibodies, in most cases very late.     

Transformation of rat and guinea-pig cells in vitro by SV40, and the transplantability of the transformed cells

Cell cultures of rat and guinea-pig kidneys inoculated with simian virus 40 (SV40) were found to undergo morphological changes characteristic for SV40 transformation. Cell lines of rapidly-growing transformed rat cells were obtained and found to be free from infectious SV40. They contained a specific antigen which was demonstrated in complement fixation tests with serum from hamsters bearing SV40 tumors. When the transformed rat cells were injected subcutaneously into the autologous hosts, tumors histologically classified as sarcomas developed in animals which had been pretreated by X-ray irradiation. Tumor cells from one of the rats were passaged in vivo and gave rise to sarcomas of high malignancy also in non-treated animals. A tumor line was thus established in rats. It contained the specific complement-fixing “tumor” antigen but no infectious SV40. Transformed guinea-pig cells autotransplanted into the irradiated host caused a small tumor which regressed within a few weeks. The results indicate that autotransplantation of in vitro- transformed cells into irradiated animals is a more promising way of obtaining SV40 tumors in different animals than the inoculation of newborns with virus.     

Sarcomas in ovarian mucinous tumors: a report of two cases.

Two sarcomas that appeared as distinct solitary nodules in the walls of ovarian mucinous cystic tumors are described. One of the tumors was a fibrosarcoma associated with a mucinous cystadenoma; the other was an undifferentiated sarcoma in a mucinous cystadenocarcinoma. Both patients died within 1 1/2 years of their operations with distant metastasis; one of them had had metastatic sarcoma in the para-aortic lymph nodes at the time of exploration. These two cases are the only well-documented examples of this unusual combination of tumor types.     

The effect of oestrogens, testosterone and progesterone on the induction of cervico-vaginal tumours in intact and castrate rats

This study investigates the carcinogenic effects of castration, stilbestrol, progesterone, testosterone, and combined estrogen and progesterone treatment on Lister strain rats. While castration seems to reduce the number of sarcomas in the cervico-vaginal tract when induced by 9,10-dimethyl-1,2-benzanthracene (DMBA), estrogens in doses sufficient to enlarge to normal size the castrate uterus inhibit the formation of sarcomas in intact rats, and fail to promote that in castrate animals. On the other hand, stilbestrol in doses insufficient to restore to normal the atrophy of the uterus, promotes and accelerates the formation of sarcomas and of epithelial tumors of the cervico-vaginal tract in castrate but not in intact rats. Progesterone in intact rats retards the induction of sarcomas but promotes papillomas of the cervix and vagina. Combined with estrogens, progesterone restores the rate of sarcoma formation to the level of intact rats. In both intact and in castrated rats testosterone seems to lengthen the induction period of sarcomas or to increase the formation of epithelial tumors; these effects are reduced when testosterone is combined with stilbestrol. Cholesterol increases the formation of sarcomas and epithelial tumors in castrate rats. Epithelial tumors as well as sarcomas arise at about the same time and rate in castrate animals given intermittently stilbestrol per os. The rate of tumor formation is greater than that in intact animals without additional treatments. The carcinogenic effects of estrogens, progesterone, cholesterol and testosterone are not correlated with the effect of the same substances on the normal tissues of the uterus, vaginal stroma, or other target organs.     

Inflammatory cells in solid murine neoplasms. II. Cell types found throughout the course of Moloney sarcoma regression or progression.

Regressing and progressing Moloney sarcomas, induced in BALB/c mice by the injection of cultured sarcoma cells (MSC)1, were sampled for histologic analysis and then disaggregated using mixtures of trypsin, collagenase and DNAse or collagenase and DNAse alone. The types of inflammatory cells (IC) found in resultant cell suspensions were determined 6, 11, 14 and 18 days post inoculation. Inflammatory infiltrates were composed almost exclusively of three cell types; neutrophils, T lymphocytes and macrophages. The extent to which each was found in tumors was related to the time post inoculation. Neutrophils were part of an early acute inflammatory response seen in both developing regressing and progressing sarcomas. The onset of regression was associated histologically with the appearance within tumors of a mononuclear inflammatory infiltrate. T lymphocytes and macrophages were the principal constituents. A higher percentage of T lymphocytes was recovered at all sampling times from regressing, compared to progressing, sarcomas. During development of the mononuclear inflammatory infiltrate there were relatively more large T cells in regressing, than in progressing tumors, and the percentage of macrophages was higher. Thereafter, the proportion of macrophages in the recovered cell population was approximately the same for both types of tumor. Such equality was more apparent than real, however, since IC were restricted to the peripheries of progressing sarcomas after the acute inflammatory phase, but continued to be found throughout regressing neoplasms. The effective ratio of macrophages and T lymphocytes to tumor cells therefore was much lower in progressing sarcomas than was suggested by percentage figures. The data presented support the concept that T lymphocytes are instrumental in causing the regression of Moloney sarcomas, possibly through interactions with macrophages.     

Studies on cellular immunity and its serum mediated inhibition in Moloney-virus-induced mouse sarcomas

A colony-inhibition technique was used to demonstrate lymph-node cell (LNC) mediated immune reactions against tumor-specific transplantation antigens of primary, Moloney sarcoma virus induced mouse sarcomas. Lymph-node cells from mice in which Moloney sarcomas had regressed spontaneously (regressors), as well as LNC from mice carrying progressively growing tumors (progressors), induced by virus inoculation at an age of 14 days or older, reduced the plating efficiency of Moloney sarcoma target cells. Serum from mice with progressively growing Moloney sarcomas, but not from mice with spontaneous mammary carcinomas or methylcholanthrene-induced sarcomas, abrogated the inhibitory effect of regressor LNC on Moloney sarcoma cells. Additional evidence for the specificity of the serum effect was obtained in experiments showing that the protective effect of progressor sera could be specifically removed by absorption with Moloney sarcoma cells. Regressor sera gave no protection against target cell inhibition by regressor LNC. It is suggested that progressor sera contain antibodies which mediate an efferent form of immunological enhancement.     

The horizontal development of cell-mediated tumor-specific immunity in Rous sarcoma virus tumorigenesis in rats

The horizontal development of lymphoid cell-mediated specific antitumor immunity was studied in R/F rats, inoculated with the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) during the neonatal period. Lymph-node cells (LNC) were harvested from individual animals on consecutive occasions and used in colony inhibition tests for the detection of LNC-mediated immunity to the Rous-specific tumor-associated transplantation antigen (s). None of the rats tested, which had been SR-RSV inoculated within 6 days after birth, had any demonstrable LNC-mediated immunity when tested within 4 weeks after virus inoculation. At the same time 68% of the rats SR-RSV-inoculated at a higher age disclosed a clear-cut immunity. In the former group sarcomas appeared after short latency periods (average 5 weeks), while the latter group developed sarcomas with long latency periods (average 11 weeks) or remained tumor-free. Also in tests performed later after SR-RSV-inoculation rats remaining tumor-free or developing very late tumors tended to develop an LNC-mediated immunity more rapidly than rats with tumors appearing earlier. The majority of both tumor-positive and tumor-negative animals developed at some time an LNC-mediated immunity, which often persisted for several months in negative animals but sometimes disappeared in rats with a large tumor burden. The results indicate that the kinetics of the LNC-mediated antitumor immune response can be of importance for the development of primary Rous sarcomas.     

Morphologic responses to a murine erythroblastosis virus

A recently described mouse erythroblastosis virus (MEV) was passed to newborn W/Fu rats. The rats developed generalized malignant lymphomas after 6-7 months. Two separate serial cell-free passage series were initiated. Rat lymphomas induced erythroblastosis in less than 20% of the rats that died within 4 weeks after inoculation. The surviving rats remained apparently healthy for months until they died from lymphomas. Inoculation of newborn C3Hf/Gs mice with the same Filtrates caused only lymphomas in 35-50% of the mice after 3-8 months. The lymphomas were morphologically indistinguishable from thymic lymphomas induced in mice and rats by Gross' leukemia virus. A different morphologic response was observed by serial passages of spleen filtrates or plasma from rats with erythroblastosis. All rats died within 4 weeks with erythroblastosis. In addition, multiple sarcomas and polyostotic osteolytic lesions were found in most rats of such erythroblastosis passages. The same rat erythroblastosis filtrates induced erythroblastosis and sarcomas but no osteolytic lesions in C3Hf/Gs mice. Erythroblastosis in rats was similar to the disease previously described in mice, except for a marked normoblastosis and cerebral hemorrhages which did not occur in erythroblastotic mice. The sarcomas arose in multiple sites and were histologically undifferentiated. The osteolytic lesions were likewise multicentric in origin, affecting particularly the vertebrae and long bones. Their exact nature could not be determined.     

V-onc product expression in early phase post perinatal retrovirus infection predictive of malignancy of induced lesions

Sixteen sarcomas developed in rats within 40 days post perinatal infection with feline sarcoma virus of Snyder-Theilen strain were examined to elucidate the association of the viral oncogene product (P85) expression with malignancy of developed sarcomas. Of these, 6 grew up to index-finger-tip-size, consisted of spindle shaped sarcoma cells and had no metastasis (group A), and 10 grew over thumb-tip-size, consisted of polygonal shaped sarcoma cells and had frequent metastasis (group B). One group A tumor and 8 group B tumors expressed P85. The v-onc product expression was thus found to be predictive of malignancy of induced tumors.     

A laboratory model for the study of the immunobiology of osteosarcoma.

Inoculation of Moloney sarcoma virus into the marrow cavity of the tibia of newborn Wistar-Lewis rats resulted in the appearance of an initially localized osteosarcoma in 97.7% of these animals. At least 77.9% of the rats developed lung metastases and died, usually within 6 weeks of inoculation. The remaining 22.1% showed regression of disease after initial growth of the tumor. Tumor cells were maintained in tissue culture and used as target cells for a visual and isotopic (3H-thymidine or 125IUdR) microcytotoxicity assay. Cell-mediated immunity could be measured by these methods throughout the course of the illness in animals with progressive disease as well as in those whose tumors eventually regressed. The presence of serum factors capable of modifying the level of CMI was documented. This Moloney-sarcoma-virus-induced rat osteosarcoma and human osteosarcoma thus appear to have several basic pathologic and immunologic similarities. The model may be useful for studying the effects of a variety of treatment protocols upon the clinical course and immune response to osteosarcoma.     

Influence of age at inoculation on avian oncornavirus-induced brain tumor incidence, tumor morphology, and postinoculation survival in F344 rats.

Intracranial neoplasms were induced by intracerebral inoculation of a standardized, cell-free inoculum of the Bratislava-77 strain of avian sarcoma virus in F344 rats at 1, 9,97 to 99, and 528 days of age. Deaths from diseases that occur spontaneously in aged F344 rats complicated assessment of tumor incidence in rats inoculated at 528 days; 20 of 30 rats inoculated at this age developed brain tumors. All rats inoculated at age 1 day (47 rats), at age 9 days (37 rats), and at 97 to 99 days of age (41 rats) developed brain tumors. The incidence of animals developing tumors was 100% in these three groups, but the incidence of multiple tumors declined with increasing age at inoculation. The mean and variance of postinoculation survival increased from 83.8 +/- 21.5 days for rats inoculated at 1 day of age to 284.6 +/- 151.5 days for rats inoculated at 97 to 99 days of age. Poorly differentiated astrocytomas and astrocytomas of mixed morphology were common among rats inoculated as neonates. Solitary, pilocytic astrocytomas were the most common tumors among rats inoculated as adults.     

The properties of sarcomas induced in Wistar rats byRous sarcoma virus (Schmidt-Ruppin)

The properties of single cell clones derived from sarcomas induced in young Wistar rats by RSV (S-R) are compared. Most sarcomas do not produce infectious RSV but are virogenic (i.e. capable of inducing Rous tumours) for chicks. One sarcoma was not virogenic. Clones were tested for virus release either alone in vitro or after passage on the chorioallantoic membrane of the embryonated egg. The avian sarcoma-leukosis group-specific (complement-fixing) antigen could not be detected in either virogenic or non-virogenic cells. A few primary Rous rat sarcomas showed the presence of virus-induced transplantation antigen but this could not be detected by graft-rejection in serially-passed tumours, or in any of the clones. The virogenic clones were more malignant for rats than the non-virogenic ones, but this property was not associated with the presence or absence of virus-specific antigens.     

Carcinogenic activity of petasitenine, a new pyrrolizidine alkaloid isolated from Petasites japonicus Maxim.

The carcinogenic activity of petasitenine, a new pyrrolizidine alkaloid isolated from young flower stalk of Petasites japonicus, was studied in ACI rats. All rats that had received a 0.05% solution of petasitenine in drinking water died or were killed in moribund condition 72 days after the start of experiment. They showed necrosis, hemorrhage, and remarkable proliferation of the bile ducts in the liver. In another group that had received a 0.01% solution, 8 of 10 animals surviving beyond 160 days developed tumors in the liver, i.e., hemangioendothelial sarcomas in 5 rats and liver cell adenomas in 5 rats, 2 of which simultaneously developed hemangioendothelial sarcomas. No tumors were observed in the livers of the control animals.     

Rous sarcomas induced by heterotransplantation of virogenic rat tumour cells in mice

The virogenic sarcoma RSL was induced in inbred Lewis rats by the Schmidt-Ruppin variant of Rous virus (SR-RSV). Heterotransplantation of RSL cells to newborn C57BL/10Sn mice gave rise to mouse sarcomas in approximately 10% of the recipients. The properties of one of these mouse sarcomas, designated as RSM, were compared with those of the original rat tumour line RSL. It was found that (1) RSL cells did not contain detectable amounts of infectious chicken, rat or mouse sarcoma virus. However, in contact with permissive chicken fibroblasts the RSL cells produced malignant conversion of avian cells accompanied by the formation of infectious RSV. RSL cells carried a tumour-associated transplantation antigen (TATA) of Rous sarcomas, a group-specific (gs) antigen of avian leukoviruses and a membrane antigen (MA), characteristic of Rous sarcomas, detectable by indirect immunofluorescence and cross-reacting with MA of mouse Rous sarcomas. The MA characteristic of tumours induced by mouse leukoviruses was not detected in the membrane of RSL cells. (2) RSM cells did not contain detectable amounts of oncogenic avian, mouse or rat viruses in an infectious form. Neither contact of these cells with permissive chicken c/o fibroblasts nor heterotransplantation of RSM tumours to chickens led to malignant conversion of avian cells. Gs antigen of avian leukoviruses was not detectable in RSM cells. However, TATA and MA characteristic of Rous sarcomas were present. Neither MA nor TATA induced by mouse leukoviruses was detected in RSM cells. The results obtained indicate that the non-infectious RSV genome present in rat virogenic cells is able to induce tumours in a further mammalian species.     

Properties of a murine sarcoma virus isolated from a tumor arising in an NZW-NZB F 1 hybrid mouse. I. Isolation and pathology of tumors induced in rodents

A potent sarcoma virus was isolated after in vivo passage of a tumor arising spontaneously in a New Zealand White × New Zealand Black (B/ W) F₁ hybrid mouse. Inoculation of the virus into mice, rats, hamsters, and Mastomys resulted in the rapid development of tumors at the sites of inoculation. The tumors induced in mice were sarcomas consisting of spindle cells and many inflammatory cells. Most tumors had a prominent angiomatous component. Tumor regressions occurred in some mice, while other tumors grew progressively, metastasized, and resulted in death. B/ W mice developed splenomegaly 4-6 weeks after virus inoculation. The normal architecture of their spleens was completely destroyed and replaced by angiomatous tissue, fibrosis, and inflammatory changes. Virus-induced tumors in hamsters consisted of undifferentiated polygonal cells. Virus-induced tumors of rats and Mastomys consisted of thin-walled blood vessels heavily infiltrated with polymorphonuclear leukocytes. Primary virus-induced tumors in hamsters, rats, and Mastomys did not regress, but some transplanted hamster tumors regressed. Electron microscopy of virus-induced and transplanted tumors revealed numerous type-C virus particles morphologically similar to the murine leukemia-sarcoma viruses. Tumor homogenates contained the group-specific (gs) antigen of the murine leukemia-sarcoma viruses.     

Comparison of the morphology of renal cysts and cystic renal tumors

Renal tumors appear uncommonly with cystic changes. They may develop due to necrosis though well-formed real cysts are also known. Such lesions may present problems in distinguishing them from benign renal cysts. Conditions leading to cyst formation are not known, however cell proliferation, altered extracellular matrix production and oncoprotein expression have been reported in cystic renal disorders. In the present study, we analysed the morphological features of 23 cystic renal tumors in comparison with 16 benign cysts using immunohistochemical and lectin binding methods. By our knowledge there has not been any piblication on such studies. The cystic renal tumors were represented predominantly in males and the size of the cysts was slightly larger than that of benign cysts. Tumorous cysts shared similar morphological appearance to solitary and multilocular cysts. They all showed strong epithelial membrane antigen reactivity on the luminal surface of the cells indicating distal tubular origin. Cell proliferation and p53 expression proved to be low excluding their role in the formation of the cysts. The amount of extracellular matrix and basement membrane was increased with an elevated type IV collagen and reduced fibronectin content. Polycystic kidney disease is different from tumorous cysts as cell proliferation, p53 oncoprotein expression and the composiition of extracellular matrix proved to be the opposite. As renal cell tumors arise from proximal tubules, neoplastic or metaplastic differentiation toward distal tubular direction seems to be the key even in cyst formation. Altered cell-matrix or cell-cell contact can modulate this transformation providing a basis for further results.