Tumor-bearing Hamsters Research Articles

Antiserum to polyacrylamide gel-purified simian virus 40 T antigen

Antisera used previously to assay SV40 T antigen have been produced in tumor-bearing hamsters. This report describes the production and characterization of antiserum produced by injection of rabbits with denatured large-T antigen (Ag) purified by immunoprecipitation and electrophoresis. The resulting antiserum reacts with native large-T Ag as determined by indirect immunofluorescence, complement fixation, and indirect immunoprecipitation assays and binds to species such as “small-T Ag” and previously described proteolytic fragments of large-T Ag.

Adenovirus type 12 tumor antigen I. Separation from DNA polymerase α and immunoprecipitation of tumor-antigen polypeptides

Adenovirus type 12 (Ad12) tumor (T) antigen, extracted from Ad12-transformed hamster cells and partially purified (>500-fold) by chromatography on double-stranded DNA-cellulose, contains DNA polymerase α activity which is partially inhibited by antibodies reacting with T antigen. The DNA polymerase and T antigen can be completely separated by chromatography on single-stranded DNA-cellulose to which T antigen does not bind. Antibodies reacting with T antigen have no inhibitory effect on the isolated DNA polymerase. The fraction eluting in the void volume of a single-stranded DNA-cellulose column containing >1200-fold purified T antigen stimulates markedly the activity of DNA polymerase α with “native” DNA template; with the “activated” DNA template the stimulation is less prominent. This stimulation is not significantly affected by T antigen-reactive antibodies. An analogous fraction of control BHK21 cells has similar stimulatory effects on the isolated DNA polymerase α, indicating that the stimulatory activity is probably not attributable to T antigen. The T-antigen polypeptides were immunoprecipitated from >1200-fold-purified preparations by antibodies from tumor-bearing hamsters and were identified as a major peptide of molecular weight 50,000 and a minor species of molecular weight 11,000.

Simian virus 40 (SV40)-specific proteins associated with the nuclear matrix isolated from adenovirus type 2-SV40 hybrid virus-infected HeLa cells carry SV40 U-antigen determinants.

The distribution of simian virus 40 (SV40)-specific proteins in nuclear subfractions of pulse-chase-labeled HeLa cells infected with nondefective adenovirus type 2 (Ad2)-SV40 hybrid viruses was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND5 specifically associate with the nuclear matrix and are virtually absent from the high-salt nuclear extract. In Ad2+ND4-infected HeLa cells, the SV40-specific proteins with molecular weights of 64,000 (64K) and lower also specifically associate with the nuclear matrix. The SV40-specific 72K, 74K, and 95K proteins were found both in the nuclear matrix and in the high-salt nuclear extract. Analyses of the nuclear matrices isolated from hybrid virus-infected cells by immunofluorescence microscopy showed that SV40 U-antigen-positive sera from SV40 tumor-bearing hamsters react with SV40-specific proteins integrated into nuclear matrices of HeLa cells infected by Ad2+ND1, Ad2+ND2, and Ad2+ND4, but not with nuclear matrices of HeLa cells infected by Ad2+ND5. This suggests that SV40-specific proteins of Ad2+ND1, Ad2+ND2, and Ad2+ND4 integrated into the nuclear matrix carry SV40 U-antigen determinants. The apparent discrepancy in the subcellular localization of SV40-specific proteins in hybrid virus-infected cells when analyzed by biochemical cell fractionation procedures and when analyzed by immunofluorescence staining is discussed.

Effect of Somatostatin in the Syrian Hamster Bearing a Transplantable Islet-Cell Tumor

The influence of somatostatin (SRIF) on blood glucose, plasma insulin and plasma glucagon was studied in hamsters bearing a transplantable islet-cell tumor secreting insulin and glucagon as well as in normal controls. Fed anesthetized animals were infused intraperitoneally either at a dose of 10 microgram in 15 min or of 150 microgram in 30 min, and intravenously at a dose of 250 microgram in 30 min. Blood was withdrawn from the jugular vein before and after infusion. Before the infusions, tumor bearing animals (TB) had lower blood glucose, markedly elevated plasma glucagon and slightly lower plasma insulin by comparison with normal hamsters (N). Both doses of somatostatin infused by the intraperitoneal route produced a slight but significant hypoglycemia in TB hamsters but not in normals. Ten microgram SRIF did not affect insulin and plasma glucagon levels whereas 150 microgram SRIF significantly depressed plasma insulin in both types of hamsters (N and TB). This latter dose of SRIF decreased plasma glucagon in normal but not in tumor-bearing hamsters. Intravenous infusion of 250 microgram SRIF did not reduce the hyperglucagonemia of TB hamsters either. These results indicate that somatostatin does not reduce the hyperglucagonemia due to the transplantable islet-cell tumor but nevertheless decreases blood glucose and plasma insulin.

Interspecies-, species- and type-specific T antigenic determinants of human papovaviruses (JC and BK) and of Simian virus 40.

Immunofluorescence tests, absorption studies and quantitative analysis by a very sensitive 51Cr microcomplement fixation (CF) technique were used to define the degree of relatedness between the tumor (T) antigens induced by human papovaviruses, strain JC and BK, with simian virus 40(SV40) and mouse polyoma virus (PyV). Antisera against JCV, BKV, SV40 and PyV T were raised in tumor-bearing hamsters. The data obtained indicate that T antigens of JCV, BKV and SV40 possess various subspecificities which can be distinguished and looked upon as interspecies-, species- and type-specific antigenic determinants. It was found that JCV T and BKV T synthesized in transformed hamster cells share about the same amount (20%) of interspecies cross-reacting antigen with SV40 T from H-50 cell extracts (transformed hamster cells). Although hamster cells transformed by PyV showed definite PyV T reactivity, no cross-reactivity, at least with the sera used, was found with human papovavirus and SV40 T antigens. Furthermore, degree of heterogeneity was observed within the T antigen complex derived from different SV40-transformed cells.

Transformation by simian virus 40 induces virus-specific, related antigens in the surface membrane and nuclear envelope

Nucleus- and mitochondrion-free membranes from hamster lymphocytes transformed by simian virus 40 (SV40), GD248 cells, cause guinea pigs to produce immune sera that reveal the presence in GD248 plasma membranes and mitochondria of two types of glycoprotein that are not detected in membranes of normal lymphocytes [Schmidt-Ullrich, R., Thompson, W. S. & Wallach, D. F. H. (1977) Proc. Natl. Acad. Sci. USA 74, 643-647]. Indirect immune fluorescence of living, SV40-transformed T19 hamster reticulum cells, Balb/c 3T3 mouse fibroblasts, and W18 VA2 human fibroblasts, using the antisera against GD248 membrane, at 4 degrees produced a distinct cell surface fluorescence; however, above 20 degrees , staining at the nuclear perimeter, the SV40 U-antigen reaction, becomes equally prominent. In SV40-transformed cells that had been fixed in cold acetone, as well as in purified GD248 nuclei, thermostable U-antigen staining is dramatic, but there is no reaction for nuclear T-antigen. Rabbit antisera against T19 cells gave immunofluorescence reactions equivalent to those obtained with the antisera against GD248 cells. Normal guinea pig or rabbit sera and cells that had not been transformed by SV40 gave no reaction. Our sera from tumor-bearing hamsters gave only nuclear T-antigen fluorescence. The results indicate the presence of related, SV40-specific antigens in the surface membranes, nuclear envelope, and possibly other intracellular organelles of SV40-transformed cells.

Cancer-associated serum galactosyltransferase activity. Demonstration in an animal model system.

Two different lines of solid tumors were produced in outbred hamsters by subcutaneous injection of polyoma transformed BHK cells. Growth of the tumors correlated with the appearance in serum of an electrophoretically distinct peak of galactosyltransferase: NeuAc-, Gal-free fetuin acceptor activity on polyacrylamide gels. This slow moving peak of enzyme activity (GT-HH) was detected before solid tumors could be grossly observed and the amount of activity in this peak was also found to be linearly related with growth of the tumor. GT-IIH was not detectable in control animals and separated from a faster migrating major area of serum galactosyltransferase activity (GT-IH) found in sera of both control and tumor-bearing hamsters. These two activities were shown to maintain their respective mobilities on re-electrophoresis. Solubilized enzyme derived from excised tumors demonstrated an electrophoretic mobility on polyacrylamide gels identical to that for GT-IIH present in serum from tumor-bearing animals. In contrast, enzyme activity solubilized from livers of both control or tumor-bearing hamsters showed a mobility similar to that of the faster moving serum galactosyltransferase enzyme activity, i.e. GT-IH. In addition, medium derived from nonconfluent BHKpy cells in tissue culture contained galactosyltransferase activity which co-electrophoresed with the slower migrating characteristics of galactosyltransferase activities derived from serum (control and tumor-bearing), solid tumors, liver and BHKpy cells in tissue culture were compared. All kinetic properties were similar with the exception that the Km UDP-galactose of GT-IIH (1.0 X 10(-5) M) was half that of GT-IH (2.0 X 10(-5) M).

An immune complex assay for SV40 T antigen

An assay is described for the detection and quantitation of T antigen, a protein present in cells transformed or infected by simian virus 40. In principle it is a direct immunoprecipitation of the antigen with serum from tumor bearing hamsters, followed by collection of the immune complexes formed on glass fiber filters. The complexed immunoglobulin is then exposed to 125I-labeled protein A, a protein with affinity for the Fc region of many immunoglobulins. The amount of protein A bound is then related to the amount of immunoglobulin and thus of antigen on the filter. With the appropriate sera this assay can probably be used for many other large antigens of biological interest.

Membrane-associated antigen from the SV40-induced hamster fibrosarcoma, para-7. I. Role in immune complex formation and effector cell blockade.

PARA-7 membrane-associated antigen was prepared by treatment of the tumor cells with pH 9.4 glycine buffer, or by concentration of spent cell culture medium. When admixed with sensitized effector cells, both preparations could specifically block cellular cytotoxicity for PARA-7 target cells. Pretreatment of target cells with antigen did not result in blocking. Incubation of antigen extract with simian virus 40 (SV40) anti-serum caused neutralization of antibody-dependent cellular cytotoxicity (ADCC) with concomitant formation of a factor which blocked at the target cell level but not at the effector cell level. Serum from tumor-bearing hamsters exhibited blocking characteristics comparable to those of the antigen-SV40 teristics comparable to those of the antigen-SV40 antiserum preparation. Washing experiments indicated that PARA-7 antigen was more efficient than PARA-7 antigen-antibody complexes in blocking cell-mediated immunity in vitro. Material extracted from untransformed hamster embryo fibroblasts either by itself or when admixed with SV40 antiserum exhibited no significant blocking activity. These observations support the concept that loss of serum ADCC during progressive tumor growth is due to immune complex formation.

Carcinoembryonic antigen and glucose phosphate isomerase in a human colonic cancer model (GW-39)

Levels of carcinoembryonic antigen (CEA) and glucose phosphate isomerase (GPI) have been compared in the circulating blood of hamsters bearing intra-muscular grafts of GW-39 human colonic tumour. CEA in the sera of GW-39 tumour-bearing hamsters ranged from 2-6 to 8-4 ng/ml (mean = 4-5 +/- 1-7 ng/ml). GPI in the sera of normal hamsters ranged from 332 to 749 iu/1 (mean = 602 +/- 110 iu/1) while those with 14-week-old intra-muscular grafts of a hamster amelanotic melanoma, (A.Mel.3), or GW-39 human colonic carcinoma had a range of 664 to 1267 iu/1 (mean = 1024 +/- 220 iu/1) and 1430 to 4719 iu/1 (mean = 2065 +/- 601 iu/1) respectively. Thus, the ratio of enzyme activity in GW-39, A.Mel.3, and normal hamsters was 3-4:1-7:1, indicating a significant elevation (P less than 0-01) in animals bearing a human colon carcinoma or a hamster melanoma, with particularly high values obtained in hamsters with GW-39. Sequential determinations of CEA and GPI in a group of hamsters transplanted intra-muscularly with GW-39 tumours revealed that both markers increased proportionately with duration of tumour growth, suggesting that both serum CEA and GPI may be used as measures of tumour growth. The concentration of GPI in GW-39 human colonic carcinoma xenografts was also significantly higher than that measured in normal human colon, primary human colonic cancer, or normal hamster tissues. These results support the view that GPI, in addition to CEA, is a quantitatively increased marker in this tumour model, and is liberated into the circulation in proportion to the increase in tumour mass.

Induction of tumors by a guinea pig herpesvirus-transformed hamster cell line.

Syrian hamster embryo cells that had been transformed in vitro by guinea pig herpes-like virus (GPHLV) were found to be oncogenic when inoculated into hamster sc or ip. Of 71 animals inoculated, 30 showed tumors at the site of inoculation. Tumors appeared 4-23 weeks after inoculation of the transformed cells at passage 37 or higher. Inbred and randombred hamsters of all ages were susceptible. Upon microscopic examination the tumors were characterized as fibrosarcomas. The cultured hamster tumor cells were easily transplanted into hamsters, but produced no evidence of tumors when inoculated into guinea pigs. Infectious GPHLV was not isolated from the tumor cells, but GPHLV-specific surface antigens were detected in tumor cells by immunofluorescence of GPHLV antiserum produced in rabbits. Sera from tumor-bearing hamsters did not contain GPHLV-neutralizing antibodies, but sera from 4 of 23 hamsters bearing primary tumors and 12 of 41 bearing transplanted tumors produced nuclear fluorescence in cells infected with GPHLV, thus establishing the relationship between the guinea pig herpesvirus and the hamster tumors.

Effect of aprotinin on immunological resistance in tumour-bearing animals.

Previous studies suggested that aprotinin might enhance the host's immunological resistance to tumours. This possibility has now been further investigated by studying the behaviour of tumours in both hamsters and mice. A second tumour graft in tumour-bearing hamsters appeared more rapidly than the first. Prior administration of aprotinin abolished this effect. Pretreatment of non-cancerous mice with cortisone nullified the effectiveness of aprotinin in inhibiting the growth of a subsequent tumour graft. These results are interpreted as additional evidence that aprotinin enhances the immunological system against tumour cells.

Lacto-N-neotetraosylceramide (“paragloboside”) as a possible tumor) associated surface antigen of hamster NILpy tumor

A specific surface-labeled component of polyoma-transformed NIL cells (NILpy) was identified as “paragloboside” (βGall→4βG1cNAcl→3βGall→4 βG1c→ceramide) (C.G. Gahmberg and S. Hakomori, J. Biol. Chem. 250 , 2438, 1975) . The presence of sialylparagloboside in NIL cells, but not in NILpy, suggests that paragloboside accumulates in NILpy as a precursor, consequent to blocked synthesis of sialylparagloboside. Evidence is now furnished to support the possibility that this glycolipid is a tumorassociated surface antigen of NILpy tumor. Sera of NILpy tumor-bearing hamsters contain a specific antibody reactive with paragloboside. The amount of this antibody in sera was parallel to the size of tumors.

Oncogenic transformation of primary hamster cells by herpes simplex virus type 2 (HSV‐2) and an HSV‐2 temperature‐sensitive mutant

Six oncogenically transformed cell lines were obtained following infection of hamster embryo fibroblasts with UV-irradiated herpes simplex virus type 2 (HSV-2) or with an HSV-2 temperature-sensitive mutant. All lines produced undifferentiated fibrosarcomas in newborn hamsters, four of the six lines produced metastatic tumors in the lung, and sera from tumor-bearing hamsters contained neutralizing antibody to HSV-2. HSV-specific cell-surface antigen (s) was detected by immunogluorescence tests in all six of the lines established from primary tumors. Antibody to an early, HSV-specific, non-structural polypeptide (VP134) reacted by immunofluorescence with antigen(s) in fixed preparation of three of the six tumor-cell lines.

Sarcoma in a Hamster Inoculated With BK Virus, a Human Papovavirus<xref ref-type="fn" rid="fn2">2</xref>

An undifferentiated sarcoma occurred in 1 of 52 hamsters inoculated when newborn with BK virus (BKV), A SIMIAN VIRUS 40 (SV40)-related human papovavirus. It was transplantable and grew in tissue culture. Sera of tumor-bearing hamsters were without antibodies reactive to BKV virion antigens in hemagglutination-inhibition and neutralizationtexts, but contained antibodies reactive in immunofluorescence (IF) tests to SV40 T antigen in SV40-TRANSFORMED CELLS AND TO ANTIGENS IN CELLS ACUTELY INFECTED WITH BKV or SV40. The IF reaction between tumor cells and sera of tumor-bearing hamsters was minimal in texts of tumor cells of an early passage but satisfactory in tests of cells from a later passage. Fusion of tumor cells with permissive (monkey and human) cells, induced by inactivated Sendai virus, did not lead to recovery of infectious BKV. Immunization of hamster with BKV or SV40 failed to protect against challenge with tumor cells. There was considerable cross-reactivity between the T antigens of BKV and SV40 viruses

Tumor Antigens in Hamsters with Sarcomas Associated with Herpesvirus Type 2

Tumor-associated antigens (TTA) were demonstrated in preparations of hamster sarcomas associated with Herpes simplex virus type 2, as well as in the sera of tumor-bearing hamsters. An immunoadsorption-in-gel method was employed to demonstrate and purify the TAA and anti-TAA. These results suggest the potential use of this technique for the demonstration of TAA or of anti-TAA in humans or animals with cancer.

Malignant Transformation of Hamster Cells following Infection with a Bovine Herpesvirus (Infectious Bovine Rhinotracheitis Virus)

Hamster embryo cells, following infection with IBR virus, showed malignant transformation. Hamsters of all ages, inbred or random bred, inoculated with two of the transformed cell lines developed solid tumors. Preliminary characterization of the tumors induced by one of the cell lines has indicated undifferentiated sarcomas. Viral specific antigen was detected in about 5% of the transformed cells and 10% of primary tumor cells in culture. Viral specific antibody was detected in the serum of tumor-bearing hamsters by the indirect immunofluorescent method, but no neutralizing antibodies were found. Infectious virus has not been recovered from either the transformed or tumor cells by cocultivation with bovine embryonic kidney cells.

Demonstration of oncogenic potential of mammalian cells transformed by dna‐containing viruses following photodynamic inactivation

The oncogenic properties of hamster embryo cells transformed by herpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) and SV40 virus following photodynamic inactivation using neutral red were determined by subcutaneous inoculation into newborn Syrian hamsters. Cells transformed by all three viruses produced palpable tumors after different latent periods. Histopathological examination showed that HSV-2 tumors were fibrosarcomas and metastases were often seen in the lungs. HSV-2 primary tumors were reinoculated subcutaneously into weanling hamsters; they developed palpable tumors within 2 weeks. HSV-specific antigens were detected in the cytoplasm and/or on the surface of both the HSV-1 and HSV-2 tumor-cell cultures by the indirect immunofluorescence technique. The same method revealed SV40 tumor antigen in the nuclei of the SV40 tumor cells. Sera from HSV or SV40 tumor-bearing hamsters gave positive reactions when tested against HSV-infected hamster cells or SV40-infected monkey cells, respectively. These results demonstrate that herpes simplex virus and SV40, whose infectivity was lost following photodynamic inactivation, retained the virus genetic information necessary for transformation of normal cells to an oncogenic phenotype.

Presence of Herpesvirus-Associated Antigens on the Surfaces of Transformed, Tumor, and Metastatic Cells and Enhanced Antigenicity of Transformed Cells Using 5-Iodo-2’-deoxyuridine

Using a direct immunofluorescence technique, herpes simplex virus type 1 (HSV-1)-transformed cells and cell lines subsequently derived from tumors and metastases were examined for the presence of HSV-1-associated surface antigens. Fluorescein-labeled immunoglobulins derived from antisera were used and included anti-HSV-1 (from hamsters), sera from tumor-bearing hamsters, and antibodies prepared in rabbits against cell material shed early into the medium after infection of rabbit kidney cells with HSV-1. All immunoglobulins showed the greatest reactivity with cell lines derived from tumors and metastatic lesions. There appeared to be little or no reactivity on the surfaces of the transformed cells. Transformed cells treated with 5-iodo-2'-deoxyuridine for 48 h, however, showed an enhanced reactivity which approached that seen with the tumor-derived cell lines.

Spleen Cell-Mediated Cytotoxicity of Hamster Cells Transformed by Herpes Simplex Virus: Evidence for Virus-Specific Membrane Antigen

The nature of the host's immune response to isografts of hamster embryo fibroblasts (HEF) transformed by herpes simplex virus type 1 (HSV-1) was investigated by the microcytotoxicity assay. It was found that spleen cells from tumor-bearing hamsters killed homologous tumor cells but not HEF transformed by cytomegalovirus or PARA-(defective SV40)-adenovirus 7 (PARA-7). Cytotoxicity was lost as the tumor increased in size. Spleen cells from animals bearing isografts of HSV type 2 (HSV-2) transformed cells also killed HSV-1 target cells whereas spleen cells from PARA-7 tumor bearers did not. Further studies showed that animals immunized with HSV-1 or HSV-1-infected rabbit kidney cells produced spleen cells specifically cytotoxic for HSV-transformed cells. On the other hand, sera from virus-immunized hosts or tumor bearers had no effect on the cells in the presence of guinea pig complement. However, in blocking experiments such sera could significantly reduce spleen cell cytotoxicity. These experiments established that cells transformed by HSV could elicit a cellular immune response in the syngeneic host, and provided evidence that the immunity was directed against virus-specific antigens on the cell surface.