Rabbit Papilloma Research Articles

Restriction mapping and physical characterization of the cottontail rabbit papillomavirus genome in transplantable VX2 and VX7 domestic rabbit carcinomas

The transplantable VX2 and VX7 carcinomas were analyzed for the presence, the nature, and the physical state of cottontail rabbit papillomavirus (CRPV) DNA sequences. Two CRPV DNA preparations, obtained from pooled cottontail rabbit papillomas, were found highly homogeneous as to their sensitivities to seven restriction endonucleases and were, therefore, considered as wild-type CRPV DNA. A cleavage map of the wild-type genome was constructed by locating 14 restriction sites. The carcinoma cells contained multiple copies of apparently whole viral genomes (42 and 444 genome equivalents per diploid VX2 and VX7 cell DNA contents, respectively), as determined by reassociation kinetic experiments and blot hybridization analyses of restricted VX2 and VX7 cell DNAs. Viral DNA molecules bearing a deletion of about 10% of the genome length, as well as minor sequence rearrangements, were also detected in VX7 carcinoma cells (about 25 copies per diploid cell DNA content). In both VX2 and VX7 cell DNAs, either unrestricted or treated with a no-cut endonuclease for the CRPV genome ( PvuI), viral DNA sequences were present exclusively as slow-migrating species with similar electrophoretic mobilities. The conversion of these species into one-genome-unit-length linear DNA molecules, by a one-cut endonuclease for both wild-type and deleted DNA molecules ( SmaI), shows that the viral sequences are not integrated in the cell genome. After cleavage of VX7 cell DNA by a no-cut endonuclease for deleted DNA molecules ( EcoRI), the deleted molecules migrated with a mobility corresponding to form I and II DNA molecules. This suggests that the viral genomes may be present in carcinoma cells as free catenated DNA molecules.

Variable-sized free episomes of Shope papilloma virus DNA are present in all non-virus-producing neoplasms and integrated episomes are detected in some.

The state of rabbit (Shope) papilloma virus DNA in virus-induced nonproducing tumors on domestic rabbits was investigated. Virus-specific sequences were resolved into many distinct bands by one-dimensional agarose gel electrophoresis. Two-dimensional gel electrophoresis, CsCl/propidium iodide density equilibrium centrifugation, partial digestion with a restriction endonuclease, and S1 nuclease digestion permitted us to identify the bands as free viral episomes representing circular molecules of increasing size. In some tumors (both papillomas and carcinomas), up to 25% of the virus-specific DNA was linear and comigrated with cellular DNA. Integration of at least some of these sequences was suggested by the detection of viral-cellular junction bands in one tumor after digestion of DNA with EcoRI and Sal I, enzymes that cut Shope DNA once. Finally, the physical states of viral DNA in papillomas and carcinomas were found to be similar, although free episomes were generally larger in carcinomas.

Transcription of the viral genome in papillomas and carcinomas induced by the shope virus

Single-phase molecular hybridization methods were employed for the detection of viral RNA nucleotide sequences in tumors induced by the Shope agent. DNA probes were prepared either from supercoiled viral DNA isolated from cottontail rabbit papillomas or from DNA cloned in the pBR 322 plasmid. In virus producing papillomas on cottontail rabbits, RNA sequences complementary to between 9 and 32% of the viral genome were found. Benign and malignant non-virus-producing tumors on domestic rabbits contained sequences complementary to between 6 and 12% of the viral genome. In all cases, the transcripts were present at low abundance, and no correlations were made between either abundance or complexity of transcripts and degree of anaplasia.

The Role of C-Type and Other Oncogenic Virus Particles in Cancer and Leukemia

During the first half of this century, cancer and allied diseases were generally thought to be caused by a variety of internal, presumably genetic and inherited, factors. The viral theory of cancer and leukemia was in disrepute. During those early years only a few tumors, such as chicken leukemia, chicken sarcoma, mouse mammary carcinoma, frogkidney carcinoma and some of the warts and papillomas in rabbits, dogs, cattle and human beings, were found to be transmissible by filtrates and were therefore believed to be caused by filterable viruses.1 , 2 Gradually, experimental tools became available, among them pedigreed strains of mice, propagation of . . .

Structural polypeptides of rabbit, bovine, and human papillomaviruses

The number and apparent molecular weight of the structural polypeptides of Shope rabbit papilloma virus (RPV), bovine papilloma virus (BPV), and human papilloma virus (HPV) were estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Up to 10 polypeptides were detected in highly purified BPV and HPV full particles; a close homology was found between the polypeptide composition of both viruses. Purified RPV virions gave a similar polypeptide pattern. The main components of the three papillomaviruses are the major polypeptide (VP1) with a mol wt of approximately 54,000 and the three smaller polypeptides (VP8, 9, 10) with mol wt of about 16,500, 15,500 and 12,500, respectively. VP8, VP9, and VP10 are never detected in empty capsids. When BPV virions were disrupted with alkaline buffer, the six lower-molecular-weight polypeptides (VP5 to 10) remained associated with viral DNA. This suggests that they are internal components of the virions and that the four higher-molecular-weight polypeptides (VP1 to 4) may represent external components. The polypeptide compositions of BPV and polyoma virus, another papovavirus, have been compared. The number of BPV and polyoma virus components (10 and 6, respectively) and the molecular weight of their major polypeptide (54,000 and 44,500, respectively) are different; however, the three main DNA-associated polypeptides of BPV (VP8, 9, 10) and the three histone-like components of polyoma virus (VP4, 5, 6) were shown to have identical apparent molecular weights. The possibility that some of the minor components of papillomaviruses may be proteolytic degradation products or cell protein contaiminants is discussed.

Unsuccessful trial of gene replacement in arginase deficiency

The Shope virus following cutaneous inoculation produces skin papillomas in rabbits and induces a virus-coded arginase in the squameous epithelium of these papillomas. The only discernible effect of the virus inoculated intravenously in rabbits and other animals was a decrease of the serum arginine concentration. Intravenous injection in 3 hyperargininemic patients, however, did not influence the underlying metabolic disease.

Evidence for and localization of vegetative viral DNA replication by autoradiographic detection of RNA-DNA hybrids in sections of tumors induced by Shope papilloma virus.

The occurrence and localization of vegetative viral DNA replication was studied in sections of tumors induced by the rabbit Shope papilloma virus, in cottontail and domestic rabbit papillomas, in primary domestic rabbit carcinoma, and in transplantable VX2 carcinoma, by in situ hybridization of radioactive RNA complementary to viral DNA. Vegetative viral DNA replication and viral protein synthesis were compared by means of cytological hybridization and immunofluorescence techniques on adjacent frozen sections. Vegetative viral DNA replication is completely repressed in the proliferating cellular layers of these tumors, which suggests a provirus state of the viral genome, as in other cells transformed by oncogenic DNA viruses. Vegetative viral DNA replication is induced, after initiation of the keratinization, in cells of cottonail rabbit papillomas, where it is usually followed by viral protein synthesis; this illustrates the influence of the physiological state of the host cell on the control of viral functions. Vegetative viral DNA replication is deteced only in a few cells of domestic rabbit papillomas, at the end of the keratinization process; this observation provides indirect evidence that the DNA synthesis specifically induced in these tumors after the onset of keratinization reflects mostly the induction of cellular DNA synthesis.

Growth of Human Skin on the Hamster

In the present study a method was developed which permits human skin to be placed in a readily accessible orthotopic position on the hamster. The hamster cheekpouch, an immunologically privileged tissue, diminishes the probability of rejection of foreign grafts. The hamster's own cheekpouch is excised and grafted to the lateral thoracic wall. After complete healing, grafts of human skin are placed in a bed cut into but not through the subcuticular tissue of the exteriorized cheekpouch. About 33% of the human skin grafts will survive for as long as 6–8 weeks without immunosuppressive treatment. With systemic cortisone administration, 50% of the grafts survive at least 14 weeks. Rabbit skin infected with Shope papilloma virus and grafted in the same manner almost always develops into typical Shope rabbit papillomas. Histotypical morphology of both normal human skin, rabbit skin, and rabbit papilloma is retained. This may prove adaptable to the study of the response of human skin to such agents as carcinogenic chemicals, irradiation, oncogenic viruses, and cytolytic viruses.

Immunologic Mechanisms in the Induction and Regression of Shope Papilloma Virus-Induced Epidermal Papillomas of Rats

Shope papilloma virus can readily induce epidermal papillomas in rabbits but rats show a high level of resistance to tumor induction. Tumors have not been induced in the skin of adult rats but when fetal rat skin is infected and grafted to syngeneic adult recipients (Lewis strain), papillomas develop in about 7–10% of the grafts. These tumors invariably regress within one week. The regression is always accompanied by an intensive lymphocytic infiltration. If this papilloma regression is mediated by an immune response, then it should be inhibited by immunosuppressive agents. For this purpose, adult rats were treated by a variety of methods. In the first series, varying doses of methylprednisolone were used. In the second series, anti-rat lymphocyte sera prepared in sheep or horses, were used either alone or in combination with adult thymectomy. All of the immunosuppressive methods resulted in prolongation of survival of the pappillomas. The best results were obtained with a combination of adult thymectomy and horse anti-rat lymphocyte serum. The original low incidence of papillomas (7–10%) was not increased by immunosuppression. It is concluded that an immunological mechanism is probably operative in the papilloma regression but that other mechanisms of resistance are responsible for the low initial incidence of papillomas.

Some aspects of the immune defense against cancer. I. In vitro studies on animal tumors.

Some aspects of the immune defense against cancer. I. In vitro studies on animal tumors.

Characterization of Surface Antigen on Cells Infected by Fibroma Virus

Abstract Cells infected with fibroma virus developed a surface antigen which could be detected by immunofluorescence. Sera from rabbits with regressed fibroma-induced tumors reacted with the surface of viable fibroma-infected cells, but not with cells infected with vaccinia virus or herpesvirus. Serum prepared against vaccinia virus, herpesvirus, SV40 virus and rabbit papilloma virus failed to react with fibroma-infected cells. The surface antigen appeared to be a late antigen and was dependent upon viral DNA synthesis. The antibody reaction was not to intact virions attached to the cell surface. The antigen persisted in association with the cell membrane long after infectious virus had decreased to low levels. A temporal relationship existed between the surface antigen measured by the fluorescent antibody technique and the antigen measured by the macrophage migration-inhibition test. Antibody reactive against the surface antigen developed in rabbits with growing fibromas and the antibody titer increased as the tumors regressed. The possible role of cell-mediated and humoral immune responses against this membrane-associated antigen in tumor regression is discussed.

Induction of papillomas in rabbits with nucleic acid extracts from Vx7 carcinomas.

Nucleic acid extracts from transplantable carcinomas Vx7 and Vx2, long maintained in domestic rabbits, were assayed for their ability to produce papillomas in animals of this kind. The Vx7 had been serially transferred 111 times when this was attempted and Vx2 was in its 203rd generation. The nucleic acid extracts from the Vx7 carcinomas consistently yielded papillomas whereas those from Vx2 completely failed to do so. The tumorigenicity of the Vx7 extracts was slight and regression of the induced papillomas often took place, as happens not infrequently to the growths caused by nucleic acid extracts obtained directly from papillomas. Malignant conversion, a common event in tumors induced by Shope papilloma virus, was also observed to occur among papillomas which were induced by the nucleic acid extracts from Vx7 carcinomas, and which kept persisting over a year on the skin of experimental animals.

Cellular immunity and its serum-mediated inhibition in Shope-virus-induced rabbit papillomas.

AbstractA colony‐inhibition technique was used to demonstrate lymph‐node‐cell (LNC) mediated immune reactions against tumor‐specific transplantation antigens of Shope‐virus‐induced papilloma and carcinoma cells. Lymph‐node cells from rabbits in which Shope papillomas had regressed spontaneously, as well as LNC from rabbits with persistent Shope papillomas, reduced the plating efficiency of Shope‐virus‐induced papilloma and carcinoma cells but did not affect normal skin fibroblasts from the same rabbits as the tumor cells.Serum from rabbits with persistent Shope papillomas or Shope carcinomas abrogated the inhibitory effect of regressor LNC on Shope tumor cells, while serum from regressor rabbits had no such effect. It is postulated that sera from “persistor” and carcinoma rabbits contain antibodies which mediate an efferent form of immunological enhancement.

Effect of High Level Supplemental Vitamin A on Avian Leukosis Agents in Chicks

INTRODUCTIONHIGH level supplemental vitamin A has been reported to both increase and decrease the resistance of animals to carcinogenic agents. Chu and Malmgren (1965) found vitamin A to have an inhibitory effect on tumor induction by carcinogenic polycyclic hydrocarbons. McMichael (1965) reported that high levels of vitamin A inhibited the growth of Shope rabbit papillomas; and Davies (1967) observed a similar effect with vitamin A on 7,12-dimethylbenz (α) anthracene (DMBA)-induced papillomas in rhino mouse skin. Polliack and Levij (1967), however, reported an increased incidence of DMBA-induced carcinoma in the hamster cheek pouch in response to vitamin A. March and Biely (1967) observed an increased incidence of avian leukosis in laying hens fed excess vitamin A. Glavind and Arffmann (1968) cited the possible importance of zation for carcinogenic properties of lipids conjugated double bonds and trans-isomeri-using the newt test.The objective of our studies was to ascertain whether the…

Serum-mediated protection of neoplastic cells from inhibition by lymphocytes immune to their tumor-specific antigens.

The combined effect of immune lymphocytes (lymphnode cells or blood lymphocytes) and serum from tumor-bearing donors was assessed in four tumor systems with the use of the colony inhibition assay: (a) Moloney virus-induced sarcomas in mice, (b) Shope papillomas in rabbits, (c) spontaneous mammary carcinomas in mice, and (d) two adenocarcinomas of the colon and two adenocarcinomas of the lung in humans. The neoplasms studied had previously been shown to possess tumor-specific antigens, against which cellular immunity could be detected in vitro. In all four systems, it was found that sera from hosts with progressively growing neoplasms could abrogate the inhibitory effect of lymphocytes which were immune to the specific antigens of the corresponding tumor type. Studies with Moloney sarcomas, in particular, showed that the serum effect had at least some degree of specificity.

A Micro-diffusion Technique for Visualizing Nucleic Acid Molecules

Contour lengths, molecular weights, and topological parameters of single molecules of nucleic acids can be studied accurately by electron microscopy. However, specialized equipment and highly purified samples of DNA or RNA in an amount of 10-100 μgm have been required. We have developed a simple micro-diffusion technique which uses less than 0.01 (μgm of nucleic acid. Droplets containing the nucleic acid are placed on a clean teflon sheet. Cytochrome C powder is added by needle to form a monofilm. After 20-30 minutes diffusion time, there is usually enough material attached for the film to be picked up on a suitable specimen grid, shadow-cast or stained, and examined in the electron microscope. Each droplet provides material for a single observation, but experiments may be carried out contiguously on the sheet.The method has been developed for viral nucleic acids, but can also be applied to solutions of virus particles where the genomes can be released by osmotic shock or by other physical procedures. Less than 1010 particles per ml are required for this modification. Examples of nucleic acids liberated from papovaviruses, SV40, rabbit papilloma, human papilloma, and from adeno-associated satellite virus will be presented. Viral nucleic acids (DNAs from SV40 and adenovirus, RNA from bacteriophage R17) prepared by conventional techniques will also be shown.

Nodamura virus, an ether- and chloroform-resistant arbovirus from Japan: physical and biological properties, with ecologic observations.

SummaryNodamura virus, an ether- and chloroformresistant arbovirus, uniformly passed filters with pore sizes of 100 but not 50 mµ, and 55 to 75 mµ virus-like particles were seen in infected mouse brain and heart. It produced limb paralysis, inflammatory and degenerative lesions in centralnervous-system tissues, skeletal muscle and brown fat, and death of suckling mice 5 to 15 days after intracranial, subcutaneous, or intraperitoneal inoculation. Suspensions of mouse limb muscle contained higher concentrations of infectious virus than brain suspensions. Antibody formation without disease occurred after one sc inoculation of a pig and after three or four ip or intramuscular inoculations of adult mice and rabbits; however, neutralizing antibody did not form in baby chicks after one sc inoculation. No overt illness occurred after inoculation of baby chicks (ic or im), weanling mice and rabbits (ic), weanling guinea pigs and Syrian hamsters (ic, ip, and im), or embryonated chicken eggs (chorioallantoic membrane, amniotic sac, or yolk sac). No cytopathologic effects were seen within 9 to 25 days after inoculation of primary cell cultures of Syrian hamster, porcine, calf, rabbit, suckling Swiss albino mouse and cynomolgus-monkey kidney, whole chicken embryo and human amnion, or of continuous cell cultures from human cervical carcinoma (HeLa), human liver and conjunctiva (Chang), C2H mouse subcutaneous tissue (L), rabbit papilloma, and rabbit skin.Infection of swine was common on farms near Tokyo during 1956–57, but only one of 54 herons and egrets, none of 29 rural residents, and none of 13 urban residents near Tokyo showed N antibody in plasma collected during 1956–57.

Structure of viruses of the papilloma-polyoma type IV. Analysis of tilting experiments in the electron microscope

Earlier papers in this series on the electron microscopy of negatively-stained human and rabbit papilloma viruses have shown both particles to be composed of 72 morphological units arranged on the T = 7 icosahedral surface lattice. This surface lattice is skew and can exist in either right- or left-handed forms: the hand of the human virus is dextro ( T = 7 d) and that of the rabbit virus is laevo ( T = 7 l). The absolute hand of the structure was determined in each case from images of particles which were stained dominantly on one side, but these “one-side” images form only a small proportion of the total. This paper presents the results of experiments in which “two-side” electron microscope images of the same virus particles obtained before and after tilting the supporting grid are compared with simulated images, i.e. superposition patterns from an appropriate model tilted through the same angle. The electron microscope images used were taken from special fields of view—close-packed arrays of particles in a sheet of negative stain—in which the particle structure is well preserved. The superposition patterns were computed and photographed from a display screen, the parameters of the model being varied until the detail in certain distinctive patterns agreed best with that in the corresponding electron microscope images (Plate I). Once the best model was obtained, a gallery of superposition patterns was calculated as a function of the spherical co-ordinates of the axis of view (e.g. Plate IV). When two superposition patterns are selected from the gallery which individually give the best fit for the pair of electron microscope images obtained from the same virus particle before and after tilting the grid, the axis and the angle of rotation to get from one pattern to the other, is, within experimental error, exactly the same as the axis and angle of tilt used in the experiment (Plate V). Moreover, the observed sense of rotation is consistent only with a dextro structure for the human virus and the laevo structure for the rabbit virus. There is very good correlation in detail between the electron microscope images and the simulated images computed for the model. This correspondence extends right to the periphery of the computed superposition patterns (Plate VI) and shows the faint outermost detail in the images to be meaningful, attesting to the faithfulness with which the negative stain preserves and outlines the structure. In the Appendix, the symmetry relationships between the superposition patterns obtained along different axes of view are discussed with the aid of a stereogram.

Inhibition by Methylprednisolone of Regression of the Shope Rabbit Papilloma<xref ref-type="fn" rid="FN1">2</xref>

Inhibition by Methylprednisolone of Regression of the Shope Rabbit Papilloma<xref ref-type="fn" rid="FN1">2</xref>

Immunofluorescent studies of virus-induced rabbit papilloma (Shope) in vitro.

SummaryShope virus-induced papillomas of domestic rabbits were cultivated in vitro and were subjected to immunofluorescent investigation. Two morphologically different cells, round or polygonal cells and triangular or columnar cells, were observed to grow out of explanted papilloma tissues. Among them, the majority of round or polygonal cells exhibited intense fluorescence reaction in nucleus. A certain number of triangular or columnar cells also reacted with specific fluorescence in nucleus but the intensity of the staining was weaker and less conspicuous. The implication of these findings was discussed in connection with previous immunofluorescent studies on the tumorous tissues of the Shope system.