George Th. Diamandopoulos

Leukemia, Lymphoma, and Osteosarcoma Induced in the Syrian Golden Hamster by Simian Virus 40

Leukemia, lymphoma, and osteogenic and anaplastic sarcomas develop in Syrian golden hamsters inoculated intravenously at 3 weeks of age with simian virus 40, which is a popova virus. Previously, only RNA and herpes DNA viruses have been recognized as capable of inducing leukemia and lymphoma in mammals. The significance of these findings is emphasized in relation to the nature of viral agents that may be involved in analogous diseases of man.

Development of S and T antigens and oncogenicity in hamster embryonic cell lines exposed to SV40

Abstract Cultures of 7 nononcogenic lines of Syrian hamster cells originating from the same pool of embryonic tissues were exposed to SV40. Four months later tests for “Surface” (S) and “Tumor” (T) antigens and for oncogenicity were performed on cells deriving from these cultures and on cells from homologous unexposed cultures which were maintained under the same conditions. Cells from 4 of the virus-exposed lines exhibited S antigen, and 2 of these also exhibited T antigen. Neither antigen was detected in cells from unexposed cultures. Among cells from exposed cultures only those exhibiting one or both of the antigens were found to induce tumors in hamsters. Among those from unexposed cultures, 2 had clearly become oncogenic. The latter were homologous with the virus-exposed cultures which exhibited S antigen only. Results of tests for S and T antigens on the cells of tumors resulting from implantation of exposed and unexposed cells revealed an exact qualitative correlation between the distribution of these antigens in the tumor cells and in the homologous cells prior to implantation. The proportion, however, of T-positive cells which was low in the cultures before implantation was found to approach 100% in the homologous tumor cells. In general the results provide evidence that the synthesis of S antigen is specifically determined by the SV40 genome and may take place independently of the synthesis of T antigen. Because of concurrent “spontaneous” oncogenic transformation in homologous cultures not exposed to SV40, no association between the capacity to synthesize S antigen and the development of oncogenic potential was established.

An Improved Immunoperoxidase Technique for Identifying SV40 V and T Antigens by Light Microscopy

Discussion and Summary Development of the peroxidase reaction for a period of 10 min has consistently resulted in strong nuclear staining of SV40 V and T positive cells. These cells are easily distinguished from antigen negative cells except for some finely granular cytoplasmic staining present in the latter cells. Attempts to eliminate this nonspecific staining by adsorbing either the serum or the conjugate with liver powder or with brain homogenate were unsuccessful. On the contrary, such adsorption weakened the activity of the serum. Incubation in absolute methanol or in 0.5% H2O2 (18) before exposure to serum did not eliminate the nonspecific staining but resulted in some nuclear damage and in a general decrease in staining intensity. Lowering the concentration of H2O2 (0.1%, 0.01%, 0.001%/) for this pretreatment did not improve the overall staining reaction and also resulted in occasional nuclear damage. Finally, partial purification of the conjugate by removing unbound peroxidase did not improve the staining effect as compared to that of the crude preparation due both to the efficiency of the peroxidase conjugation and to the lack of affinity of free peroxidase for the cells employed. Throughout the immunoperoxidase test individual staining jars and separate forceps should be used for each cover glass in order to avoid transferring antigen-positive cells or antiserum to control preparations. In addition, three separate sets of staining jars should be used for each cover glass: one for the predevelopment part of the test, another for the development, and a third for the final rinses and dehydration, to avoid formation and deposition on the cover glasses of an intractable dark brown precipitate. (Flecks of dark precipitate may be noted on the nucleus and cytoplasm of some cells in Fig. 3.) On the basis of our own experience and that of others (19, 20), it can be stated that, in addition to its slightly greater sensitivity, the indirect immunoperoxidase method offers the following major advantages when it is compared to the immunofluorescence technique. First, it allows examination of the preparations under brightfield light microscopy, thus eliminating the requirement for darkfield equipment and an ultraviolet light source. Second, since the procedure is based on a gentle although highly efficient conjugation reaction, neither the antibody nor the enzyme activity appears to be reduced to any appreciable degree while, at the same time, the conjugate remains stable. Third, the color intensity of the staining reaction is not dependent upon narrow pH changes or upon the ratio of peroxidase to antibody in the conjugation reaction, since it depends upon the accumulation of a reaction product. Admittedly, the contrast of a dark deposit against a light background, characteristic of the immunoperoxidase method, is not so marked as that of a few photons of fluorescent light against a black background characteristic of the immunofluorescence technique. Finally and most importantly, since the preparations are permanently mounted, no alterations of the label can occur. As a result, the preparations can be examined repeatedly over prolonged periods for the purpose of differential cell counts, for demonstration, and for photomicrography. In light of these advantages, it appears that the immunoperoxidase procedure is a simple, sensitive technique that can be a useful adjunct to the immunofluorescence procedure in identifying viral and virus-related cellular antigens.

The Tumor Imprint Technique For Demonstrating SV40 T Antigen By Immunofluorescence

Summary A quick, reliable and inexpensive test is described for identifying the T or “tumor” antigen, in cells rendered neoplastic by the oncogenic DNA virus SV40. The approach is based on the feasibility of adapting the tumor imprint technique to the indirect immunofluorescence test. This method is of particular advantage when one wishes to examine leukemic or lymphosarcomatous cells, which lack the capacity to grow as monolayers in culture.

Increased resistance to herpes simplex virus of hamster and human cells transformed by SV40.

Summary Lines of cells derived originally from various hamster and human tissues and transformed in vitro by SV40 consistently exhibited increased resistance to infection with herpes simplex virus, as compared with control cells not exposed to SV40. Increased resistance did not appear to depend upon differences in cellular capacity to adsorb HSV or to allow penetration into the cell; nor could it be related to enhanced production of interferon. It is suggested that the SV40 genome in the nucleus of transformed cells interferes in a manner as yet undefined with the synthesis of HSV which occurs at the same site.

Plasma cell and lymphocytic reaction in the Syrian hamster to transplanted homologous tumor cells transformed in vitro "spontaneously" or by SV 40.

Both primary and metastatic tumors, induced in Syrian hamsters by various homologous oncogenic cells, transformedin vitro “spontaneously” or by SV40, were studied histologically. The majority of the neoplasms were associated with a plasma cell and lymphocytic reaction by the host. Although this cellular response was minimal and was found only rarely in the “spontaneously” oncogenic cells, it was moderate to severe and was present in a very large proportion of cases in the SV40-transformed and SV40-exposed cell lines. This difference may be an expression of the additional antigens found in the latter tumor cells that are SV40-dependent. In tumors induced by SV40-transformed cells, the mononuclear cell reaction was quite often associated with foci of degenerating tumor cells. These degenerative changes are interpreted as a sign of host defense response. Since the cellular infiltrate is composed of plasma cells and lymphocytes, it suggests that this reaction might be immunologic in nature.