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Specific complement-fixing tumor antigens in human cells morphologically transformed by SV40 virus

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Article
Methods not involving the use of viable tumors were developed for production of high-titer antibodies. The methods involved use of tumor homogenates. Homogenates from all tumor lines tested contained the characteristic tumor antigen. Attempts to isolate virus failed, and virus antigens could not be demonstrated. Immunization with tumor homogenates, in Freund's complete adjuvant, resulted in high antibody levels. None of the animals so immunized developed tumors.
Article
Methods not involving the use of viable tumors were developed for production of high-titer antibodies. The methods involved use of tumor homogenates. Homogenates from all tumor lines tested contained the characteristic tumor antigen. Attempts to isolate virus failed, and virus antigens could not be demonstrated. Immunization with tumor homogenates, in Freund's complete adjuvant, resulted in high antibody levels. None of the animals so immunized developed tumors.
Article
In the immunofluorescent study it has been revealed that rabbit sera immunized with transformed cells induced by SV-40 DNA, produce circulating antibody capable of re:lcting with intranuclear antigens synthesized by SV-40 complyte virus transforming process, In addition, the result confirmed that SV-40 DNA replicates DNA-containing viruses in the host cell and that also the genome coding for the synthesis of SV-40 tumor antigen is resposible for viral DNA.
Article
Two types of morphological transformation of bovine kidney cells were obtained after inoculation with SV40. Primary cultures inoculated were transformed into cultures with epithelioid cells growing mainly in monolayer. When the kidney cells were subcultivated and infected at the 6th passage, another type of transformation, characterized by epithelioid and fibroblastic cells growing in a disorganized multilayer, was seen. Cell lines were obtained from both types of transformed cultures. The epithelioid cell cultures were found to be free of infectious SV40 whereas the cultures composed of epithelioid as well as fibroblastic cells yielded virus even at high passage levels. Both types of transformed cultures contained the complement-fixing “tumor antigen”. A comparison between the cultures as regards their susceptibility to various viruses showed that the epithelioid cell cultures were more susceptible to parainfluenza virus type 3 than the cultures with both epithelioid and fibroblastic cells. There were no differences in susceptibility to foot-and- mouth disease virus, bovine enterovirus, infectious bovine rhinotracheitis virus, pseudorabies virus, Newcastle disease virus or bovine viral diarrhoea virus.
Article
We have examined a non-lytic system (3T3) in which cells infected with the oncogenic virus SV40 do not support significant independent viral DNA replication. The infected 3T3 cells have two, rather different, possible fates. They may be only transiently infected, losing their SV40 T antigen and all other signs of viral presence in three or four generations, or they may become transformed, with the acquisition of SV40 T antigen synthesis as a heritable characteristic and the apparent integration of the SV40 genome in the cells. It appears that it is the occurrence of this process of integration that converts transient non-lytic infection to viral transformation. Utilizing interferon to study the expression of SV40 genetic material in transiently infected and transformed cells, we have provided indirect evidence to support the hypothesis that integration is the physical linkage of SV40 and host cell DNA in such a manner that transcription of m-RNA occurs without interruption across the point of junction. Studies with interferon and the adenovirus-SV40 hybrid virus E46+ support this interpretation. In cells infected with E46+ (in which the covalent linkage of SV40 and adenovirus DNA has been demonstrated) SV40 T antigen synthesis lost the interferon sensitivity characteristic of SV40 virus and acquired a relative interferon resistance indistinguishable from that of the adenovirus to whose DNA the SV40 DNA is linked. In cells simultaneously infected with non-hybrid adenoviruses and SV40, both SV40 and adenovirus T antigens were synthesized, but the SV40 T antigen synthesis exhibited its characteristic sensitivity to interferon. It is predicted that host-viral hybrid m-RNA molecules are synthesized in the SV40 transformed 3T3 cells and that adenovirus-SV40 hybrid m-RNA molecules are synthesized in cells infected with the adenovirusSV40 hybrid virus E46+.
Article
The properties of hamster cells containing SV40 tumor (T) antigen in the cytoplasm, rather than the nucleus, were determined. Eight cell lines were established from eight tumors induced by hamster embryo fibroblasts transformed in vitro by PARA (2cT)-adenovirus 7. Six cell lines contained only cytoplasmic SV40 T-positive cells while two were a mixture of nuclear T-positive and cytoplasmic T-positive cells. All the cell lines contained SV40 S antigen, all caused the production of SV40 T antibody in vivo, and four elicited the production of adenovirus T antibody. The cell lines seem to have acquired an infinite life span in vitro. The localization of T antigen apparently can be a stable phenomenon because five of the cell lines have retained T antigen exclusively in the cytoplasm for over 40 passages in tissue culture. The cytoplasmic T antigen could be detected by complement fixation in addition to immunofluorescence. Cytoplasmic T-positive cells were readily transplantable in vivo and contained SV40 TSTA demonstrable by both immunogenicity and immunosensitivity procedures. Two of the three cytoplasmic variants of PARA appear to be weakly oncogenic in newborn hamsters.
Article
A clonal line of SV40 virus-induced hamster tumor cells was exposed to polyoma virus in an attempt to obtain “doubly transformed” cells containing the complement-fixing (CF) tumor antigens (T antigens) specific for both viruses. Polyoma virus was found to persist indefinitely in the SV40 tumor cells in a “carrier state.” Cells freed of virus by antibody treatment were shown to possess the T antigens induced by both viruses. Thirty-six clonal isolates from the SV40-polyoma (SV-Py) doubly transformed cells were tested, and all contained both types of antigens. Antigens made from tumors produced by SV-Py cells were also positive for SV40 and polyoma T antigens, and tumor-bearing animals developed the corresponding antibodies. In transplantation-resistance tests, approximately 100 times greater cell concentration was required to produce tumors in hamsters previously immunized with polyoma, SV40, or a mixture of both viruses; these results indicate that the doubly transformed cells contained the transplantation as well as CF tumor antigens induced by both viruses.
Article
This chapter presents the experiments that are concerned with the in vitro studies on the mechanism by which carcinogens induce a change in the controls that determine contact inhibition. Carcinogenesis can be induced by viruses, nonviral chemicals, and physical agents. It is concerned with the change in this control induced by small DNA tumor viruses with particular reference to polyoma virus, carcinogenic hydrocarbons, and X-irradiation. The chapter presents three events that are required in the development of a visible tumor in vivo: (1) induction of the change in the control mechanism for cell replication; (2) fixation of this change in the cell so that it can be transmitted as a hereditary property of the tumor cell; (3) growth of a single tumor cell into a visible tumor. The experiments presented in the chapter concerned primarily with the first two events.
Article
Ten temperature-sensitive mutants of simian virus 40 have been isolated and characterized in permissive cells. The mutants could be divided into three functional groups and two complementation groups. Seven mutants produced T antigen, infectious viral deoxyribonucleic acid (DNA), and structural viral antigen but predominantly the empty shell type of viral particles. Two mutants produced T antigen and infectious viral DNA, but, although viral structural protein(s) could be detected immunologically, no V antigen or viral particles were found. These two functional groups of mutants did not complement each other. A single mutant was defective in the synthesis of viral DNA, viral structural antigens, and viral particles. T antigen could be detected in infected cells by fluorescent antibody but was reduced by complement fixation assay. This mutant stimulated cell DNA synthesis at the restrictive temperature and complemented the other two functional groups of mutants.
Article
Thus, there appears to be a correlation between the presence of HeLa(G) antigen and the heterotransplantability of these KB sublines to the cheek pouch; which is further emphasized by the absence of HeLa(G) antigen in the non-heterotransplantable sublines. The possible relationship of the HeLa(G) antigen to the cytochemical characteristics exhibited by populations of these KB sublines will be considered elsewhere.
Article
A passage line of a spontaneous hamster fibrosarcoma is contaminated by the virus. of lymphocytic choriomeningitis. Tumors from animals receiving implants when newborn contain high titers of infectious lymphocytic-choriomeningitis virus and complement-fixing antigen, and hamsters receiving implants when weanlings develop high titers of complement-fixing antibody against lymphocytic-choriomeningitis virus. In contrast with the specific reactions of tumorous hamsters to the initiating virus in virus-induced tumors, the development of complement-fixing antibody to lymphocytic-choriomeningitis virus does not depend on the development of tumors. Infant hamsters bearing the tumor have a generalized subclinical infection and seem able to spread virus to other hamsters and to humans.
Article
The application of tissue culture technology has revealed several new groups of viruses, comprising scores of different serotypes, as important causes of upper and lower respiratory tract disease in man. Other agents as yet unrecognized undoubtedly exist. Present epidemiologic data, although still incomplete, point up the unique importance of certain of these viruses in respiratory diseases. The particular type and severity of respiratory syndrome produced by a virus is determined by the immune status of the host, by the presence of complicating disease and by characteristics intrinsic in the infecting agent itself. Respiratory virus control might be particularly beneficial in certain groups, particularly persons with allergic sensitivity and chronic pulmonary disease.For control of viral respiratory disease, active immunization would provide significant protection but highly polyvalent vaccines might be necessary. Adjuvants could be helpful in this regard. Certain vaccines formerly in use were produced from strains of viruses which have recently been shown to be oncogenic in animals. In addition, hybridization of viruses can occur, resulting in the incorporation of the oncogenic potential of one agent into the genetic constitution of another. The significance of these biologic phenomena to vaccination programs has yet to be defined.Passive immunization would provide short-lived protection and would find application only in uniquely susceptible populations or at times of augmented risk. Active immunization of pregnant women, however, could provide increased breadth and duration to the transplacental passive immunity in the newborn.Interferon-inducing agents could potentially provide broad spectrum antiviral protection, but the extent and duration of their effectiveness are unknowns. Certain chemical agents have been shown to have prophylactic and therapeutic effects against a limited number of clinically severe viral diseases. Finally, limited data suggest that climatic control in places of public gathering might be worth evaluating as a means of controlling the spread of viral respiratory infections.
Article
Much of the research on virus-induced malignancy has been concerned with the question of the persistence of viral genome in virus-free tumors or transformed cells. An answer to this question is an essential preliminary to understanding the mechanism of viral oncogenesis. The major efforts, thus far, have been concerned with the presence of new antigens in the malignant cells. The first of these to be described, the transplantation resistance antigens, were found in polyoma virus and simian virus 40 (SV40) tumors or transformed cells, and are responsible for the rejection of tumor transplants in previously immunized animals (1-5). The second group of antigens found associated with virus-induced malignant cells are the tumor antigens, or neoantigens (hereafter referred to as T antigens). These antigens were first found in extracts of adenovirusinduced tumors (6) and have since been found in tumors induced by, and cells transformed by SV40 and polyoma viruses (7, 8). The T antigens are generally detected by complement fixation (CF) or fluorescent antibody (FA) procedures. This report will review the findings on one of the latter antigens, the SV40 T antigen. Discovery
Chapter
The principal members of the papova group are polyoma virus (Stewart et al., 1957), simian virus 40 (SV40), which is a vacuolating virus of monkeys (Sweet and Hilleman, 1960), and the papilloma viruses (Melnick, 1962). The name for this group of viruses is derived from the first two letters of the names of each of the viruses that were first included in the group, papilloma, polyoma, vacuolating virus (Melnick, 1962). The viruses are 40–57 nm in diameter and, as determined by negative staining, the outer shell has symmetry of the T = 7 icosahedral surface lattice and is composed of 72 morphological subunits (Finch and Klug, 1965; Anderer et al., 1967). The viruses contain no lipids and therefore are resistant to ether. Polyoma and SV40 do not share common antigens, nor is there evidence for the existence of any homology between their DNAs. The papovaviruses are capable of initiating a lytic cycle of replication or a latent infection. For the papilloma viruses it is difficult to obtain a suitable cell line in which the lytic cycle can be studied and for this reason studies that we will discuss concerning viral replication will deal exclusively with SV40 and polyoma.
Article
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. On a constaté que des cultures de cellules rénales de rat et de cobaye auxquelles avait été inoculé le virus simien SV40 subissaient les changements morphologiques caractéristiques de la transformation par ce virus. On a obtenu des lignées de cellules de rat transformées, ayant un taux de croissance élevé, qui étaient exemptes de SV40 infectieux mais contenaient un antigène spécifique; celui-ci a été mis en évidence par des épreuves de fixation du complément utilisant du sérum de hamsters porteurs de tumeurs à SV40. Après injection souscutanée des cellules de rat transformées aux hǒtes autologues, des tumeurs à histologie sarcomateuse se sont développées chez les animaux qui avaient été préalablement irradiés aux rayons X. Les cellules tumorales de l'un des rats ont été entretenues par passages in vivo et ont suscité des sarcomes d'une grande malignité měme chez les animaux non irradiés. On a ainsi constitué une lignée tumorale sur le rat. Elle contenait l'antigène «tumoral» spécifique fixant le complément mais pas de SV40 infectieux. Les cellules de cobaye transformées, après autotransplantation sur l'hǒte irradié, ont provoqué une petite tumeur qui a régressé en quelques semaines. Ces résultats montrent que l'autotransplantation à des animaux irradiés de cellules transformées in vitro est un moyen plus intéressant d'obtenir des tumeurs à SV40 sur différents animaux que l'inoculation du virus aux nouveaux-nés.
Chapter
Das Gebiet der Tumorimmunologie ist in den letzten Jahren rasch gewachsen, da immunologische Methoden auf vielen Gebieten der Krebsforschung angewandt werden. Insbesondere sind sie dazu benutzt worden, Veränderungen in den Tumorzellen selbst nachzuweisen, wie z.B. 1. die Entwicklung neuer antigener Determinanten in Krebszellen und in mit onkogenen Viren infizierten Zellen, 2. den Verlust oder die Verminderung normaler Zellkomponenten und 3. die Synthese löslicher Produkte durch Krebszellen, wie z.B. Myelomproteine. Immunologische Methoden sind aber auch bei der Untersuchung des Krebsträgers angewandt worden, nämlich beim Nachweis spezifischer Immunreaktionen gegenüber wachsenden Tumoren und bei der Untersuchung der Wirkung von Tumoren auf die immunologische Potenz des Tumorträgers.
Chapter
The principal members of the papova group are polyoma virus (Stewart et al., 1957), simian virus 40 (SV40), which is a vacuolating virus of monkeys (Sweet and Hilleman, 1960), and the papilloma viruses (Melnick, 1962). The name for this group of viruses is derived from the first two letters of the names of each of the viruses that were first included in the group, papilloma, polyoma, vacuolating virus (Melnick, 1962). The viruses are 40–57 nm in diameter and, as determined by negative staining, the outer shell has symmetry of the T = 7 icosahedral surface lattice and is composed of 72 morphological subunits (Finch and Klug, 1965; Anderer et al., 1967). The viruses contain no lipids and therefore are resistant to ether. Polyoma and SV40 do not share common antigens, and presently there is no evidence for the existence of homology betweeen their DNAs. The papovaviruses are capable of initiating a lytic cycle of replication or a latent infection. For the papilloma viruses it is difficult to obtain a suitable cell line in which the lytic cycle can be studied; therefore, studies that we will discuss concerning viral replication will deal exclusively with SV40 and polyoma.
Chapter
Although it has been known for many years that certain viruses have something to do with tumor induction, in most instances there has been no specific evidence that the individual tumor cell had been directly transformed by a given virus. This sort of evidence was available only in the case of those RNA viruses, such as the avian leukoses, where the infected transformed cell continues to produce infectious virus. In the DNA virus-induced tumors, which are frequently free of infectious virus, there was no easy, direct way of establishing the etiological relationship. However, with the discovery in recent years that virus-induced tumors contain specific new antigens, a ready means of identification became available. There is good reason to believe that these antigens will be present in all virus-induced tumors, since one type or another has been found in all classes of tumors thus far adequately examined.
Chapter
SV40 virus-induced tumors and cell cultures transformed by this virus possess new antigens. One antigen studied extensively has been the induced complement-fixation antigen, designated as SV40 “T” or neoantigen (Black et al., 1963; Rapp et al., 1964; Sabin et al., 1964; Habel et al., 1965; Gilden et al., 1965). The antigen can be detected by immunofluorescence techniques as described by Pope and Rowe (1964) employing sera from hamsters bearing noninfectious SV40-induced tumor transplants. In addition to the antigen measured by the complement-fixation (CF) reaction, evidence exists which points to the presence of cellular antigen(s) which are highly effective in promoting development of tumor resistance. This report is concerned with this latter type of antigen.
Article
Sera from hamsters bearing tumors induced by papovavirus SV40 reacted with tumor antigen prepared either from cells transformed by, or from cells infected with, the homologous virus. Serum titurs against both antogens were comparable. A high degree if correlation was obtained with the use of either complement-fixation or immunofluorescence to detect the antigen. The immunoflurescent intranuclear antigen present in cells transformed by SV40, an the early virus-induced intranuclear antigen found during the cytolytic cycle following infection of monkey cells by SV40, therefore appear to be similar, and they are either identival with or similar to the antigens detected by complement-fixation techniques. Synthesis of antibody against this antigen was detected only in hamsters bearing tumors induced by SV40 or by cells transformed by the virus. Hamsters immunized with SV40 usually reject cells transformed by the homologous virus; the few vaccinated animals which fail to reject such cells also fail to produce antibodies against tumor antigen although the tumor cells in such animals continue to synthesize the antigen.
Article
Studies were made on the effect of various doses of SV40 on the transformation rate of bovine cells, and the transformed cells were studied with regard to the presence of infectious SV40, the presence of the specific complement-fixing (CF) tumor antigen, and the transplantability into the autologous host.It was shown that the time required for transformation varied between 42 and 407 days, being shorter with larger doses of virus. All the transformed cell lines contained SV40 in small amounts. The CF tumor antigen was found in the transformed cell lines except for early passages. Autotransplantation of subcutaneous tissue transformedin vitro gave rise in one calf to a small nodule, histologically defined as a spindle-cell sarcoma, and in two other calves a late inflammatory reaction was observed.
Article
Bissett, Marjorie L. (University of Michigan, Ann Arbor), and Francis E. Payne. Development of antigens in human cells infected with simian virus 40. J. Bacteriol. 91 743–749. 1966.—An explanation for the apparent infrequency with which human cells transform in response to exposure to simian virus 40 (SV40) was sought by following the development of virus-induced antigens in human euploid cells, strain CR. For about 8 weeks after exposure to a high multiplicity of SV40, only a small proportion of the cells produced tumor (T) or viral (V) antigen detected by immunofluorescence. Double-tracer staining techniques revealed that the development of T and V antigen in about 1% of the CR cells resembled that in green monkey kidney cells, strain BS-C-1, in which SV40 replicates and destroys all the cells. T antigen was detected before V antigen; both antigens were detected in the nucleus, but only V antigen appeared later in the cytoplasm. All intact cells that contained V antigen also contained T antigen. Infected CR cell cultures, before and after transformation or when in “crisis,” contained only 0.1 to 1.0% of cells with both V and T antigen. Some CR cells contained only T antigen, and by 8 days after exposure to virus these cells were present as loose foci associated with an occasional cell containing V antigen. The proportion of CR cells with only T antigen increased from about 1% during the first 4 weeks to 8% at 7 weeks, and to nearly 100% at 11 weeks, when essentially all of the cells were epithelioid. Foci of epithelioid cells were first recognized in the 9th week. It was concluded that those CR cells that contained T antigen at any given time represented (i) a few cells that subsequently produced V antigen and lysed, and (ii) a progressively increasing population that produced only T antigen. If the latter population, in whole or in part, gave rise to the epithelioid transformed cells, then its initial size could account, at least in part, for the apparent infrequency with which human cells transform in response to SV40.
Article
Human diploid fibroblasts, WI-38, were infected with various agents and the levels of lysosomal enzymes determined by immunochemical quantitation. Esterase levels were raised by mumps virus and Toxoplasma gondii infection. The concentration of beta-D-glucuronidase was reduced by these same agents. Beta-D-N-acetyl glucosaminidase was greatly increased in cultures infected with T. gondii and decreased by mycoplasma infection. Two cultures transformed by SV40 showed reduced levels of esterase compared with WI-38, as did one of two transformed amnion cultures. A second amnion culture and a culture of transformed Detroit 551 fibroblasts were unchanged. The levels of acid phosphatase were sharply reduced in three of the four SV40 transformed cultures tested.
Article
Hamster tumors transplanted subcutaneously from primary intracranial tumors which developed after inoculation of the Bryan strain of Rous sarcoma virus, contained virusspecific tumor antigens indistinguishable from those induced by the Schmidt-Ruppin strain.
Article
Sweet und Hilleman (1960a und b) teilten 1960 mit, daß sich in vielen Nierengewebekulturen von Macacus rhesus und cynomolgus ein bis dahin unbekanntes Virus nachweisen läßt, wenn man diese Gewebekulturüberstände auf Nierengewebekulturen von Cercopithecus aethiops (Cerc. aeth.) überträgt. Während sich der Erreger in den Kulturen von Rhesusnierenzellen ohne einen bei üblicher Betrachtung sichtbaren cytopathischen Effekt vermehrte, führte die Multiplikation des Virus in Nierengewebekulturen von Cerc. aeth. zu einem charakteristischen cytopathischen Effekt, der zur Bezeichnung des Virus als „vacuolating agent“ Veranlassung gab. Kurz darauf berichteten Eddy et al. (1961), daß die Inoculation neugeborener Hamster mit Zellextrakten aus Nierengewebekulturen von Macacus rhesus zur Ausbildung maligner Tumoren vom Typ der Fibrosarkome führte. Durch weitere Untersuchungen von Eddy et al. (1962 a) sowie von Gieardi et al. (1962) wurde die Identität des „vacuolating agent“ oder Simian-Virus 40 (SV-40) (Hull) mit diesem onkogenen Faktor in Nierengewebekulturen von Rhesusaffen nachgewiesen. Melnick (1962) machte darauf aufmerksam, daß das Virus SV-40 zahlreiche Gemeinsamkeiten mit anderen tumorbildenden DNS-haltigen Viren (Papillomvirus des Kaninchens, Warzenvirus des Menschen, Polyomavirus und K-Virus der Maus) aufweist und hat vorgeschlagen, diese Viren auf Grund ihrer ausgeprägten Gemeinsamkeiten als „Papovaviren“ zusammenzufassen.
Article
With an immunofluorescent technique involving the use of serum of hamsters with SV40 tumors, nuclear fluorescence was detected in each of five cell lines, derived from four mammalian species, transformed by SV40 virus. Essentially all nuclei, including those of multinuclear cells, were fluorescent-stainable. Serum of hamsters bearing SV40 tumors was also found to give nuclear fluorescence in susceptible cells (AGMK or BSC-1) acutely infected with SV40 virus. These findings provide further evidence that cellular incorporation of the SV40 viral genome, with partial expression of the genome by synthesis of at least one virus-specific antigen, is an integral property of all SV40 transformed cells.
  • F Rapp
  • T Kitahara
  • J S Butel
  • J L Melnick
  • Proceedings These
  • Potentials
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  • Myelinated
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Rapp, F., T. Kitahara, J. S. Butel, and J. L. Melnick, these PROCEEDINGS, 52, 1138 (1964). CONTINUOUS CONDUCTION OF ACTION POTENTIALS BY SINGLE MYELINATED FIBERS OF DESHEATHED NERVE: TYPES OF NORMAL NERVE FIBERS* BY R. LORENTE DE NO AND V. HONRLTBIA THE ROCKEFELLER INSTITUTE Communicated October 8, 1964