Rous Sarcoma Virus-induced Tumors Research Articles

Anti-neoplastic effect of avian reovirus σ C protein on Rous sarcoma virus-induced tumors in chicken

This study investigated the anti-neoplastic potential of avian reovirus σC (sigma C) protein on Rous sarcoma virus-induced fibrosarcoma in chicken. The recombinant vector expressing σC protein was injected intra-tumorally into specific pathogen free chicken with fibro-sarcoma at the dose 100µg per bird, while control birds were mock-treated with 100µg of empty vector per bird. Recombinant σC protein induced apoptosis in tumors of treated birds resulting in progressive tumor regression, while similar changes were absent in tumors of mock-treated controls. The σC protein-induced apoptosis in tumors was quantified by flow cytometry and the mean level of apoptosis up to 66% was observed in treated tumors, whereas any significant level of apoptosis was absent in mock-treated controls.

Isozyme patterns in Rous sarcoma virus-induced tumors in the rat.

Electrophoretic isozyme patterns of 9 enzyme systems were studied in normal tissues of an inbred rat strain, in 11 Rous sarcoma virus-induced tumors in this strain, in cultured cells from one of the primary tumors, and in 22 tumors from reimplanted cells of 5 clonal isolates from the cells in culture. All primary tumors showed largely similar deviations of the isozyme patterns in comparison with the tissue of origin (subcutaneous connective tissue). Changes of the patterns occurred in the cultured primary tumor, but the characteristic patterns of primary tumors returned in the tumors after implantation in vivo of the cultured cells. The experiments indicated that the change in isozyme patterns induced by the virus was the same each time a normal tissue was transformed into a Rous tumor. The changes accompanying the explantation of the tumor cells in vitro were of another character and were reversible on reimplantation of the cells in vivo.

Anti-neoplastic effect of chicken anemia virus VP3 protein (apoptin) in Rous sarcoma virus-induced tumours in chicken

The anti-neoplastic effect of chicken anemia virus VP3 protein (apoptin) was investigated in vitro in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblast (CEF) cells and in RSV-induced tumours of specific-pathogen-free (SPF) chicks in vivo. The apoptin gene was cloned in the pVAX expression vector and in vitro expression of the recombinant vector pVAX-CAV-VP3 was confirmed. Two groups of SPF chicks, each containing ten chicks, were used. Chicks in groups I and II were inoculated with RSV at 1 day old. Group I served as the control, receiving pVAX vector without insert, and group II received recombinant vector pVAX-CAV-VP3 containing the apoptin gene, on day 10. An in vitro study confirmed that apoptin induced apoptosis in RSV-transformed CEF cells, which was demonstrated by observation of the characteristic changes of apoptosis using the indirect immunofluorescence technique and acridine orange/ethidium bromide staining. In vivo study also indicated that apoptin induced apoptosis and caused tumour regression by an intratumoral-delivery method. Apoptotic changes, such as nuclear condensation, fragmentation of the chromatin and formation of apoptic bodies in the tumour cells, were demonstrated by histopathology and acridine orange/ethidium bromide staining. No apoptotic changes were seen in the tumours of the control group. The results of the present study showed that apoptin had an anti-neoplastic effect in vivo and in vitro in RSV-induced tumours. The anti-neoplastic effect is due to apoptin-induced apoptosis. Further improvements in the dose, delivery method and delivery frequency of the apoptin-expressing recombinant vector could help to develop apoptin as an anti-neoplastic drug.

Genetic analysis of the growth curve of Rous sarcoma virus-induced tumors in chickens

White Leghorn chicks homozygous for B19 MHC haplotype were selected for 18 generations on tumor regression after inoculation in the wing web with an SR-D strain of Rous sarcoma virus (RSV) at 4 wk of age. Each chick was assigned a tumor profile index (TPI) based on age at death and size of the tumor. During 18 generations, 2,010 birds were divergently selected on TPI for either progression or regression of the tumor (P and R lines). A Brody growth curve was fitted for each bird. Brody function parameters included the asymptotic tumor volume (A), the factor for increased growth in progression phase (K1), the factor for decreased growth in regression phase (K2), age at maximum volume (Tmax), and maximum volume of the tumor (Vmax). Tumor growth curves were found to be different according to line, sex, and restriction fragment pattern Y complex Rfp-Y MHC haplotype (Yw*15, Yw*16, and Yw*17). Within the P line, birds from the Yw*16 haplotype reached Vmax at an earlier age than Yw*15 and Yw*17, but with a lower Vmax value. Within the R line, tumor growth curves of birds from Yw*16 and Yw*17 haplotypes were similar. Rank correlations between the different parameters and TPI were low (between -0.26 and 0.36). Heritability estimated by the sire component was high for Vmax (0.73). Heritabilities of Tmax and K2 were moderate (0.20 to 0.23 for Tmax and 0.18 to 0.21 for K2) allowing these traits to be used as selection criteria. Heritabilities of A and K1 were lower than 0.12. Modeling the growth curve should contribute to better distinction between progressors and regressors.

The fingerprints of the host innate immunity on the cells of primary virus-induced tumors

As shown earlier, the cells transformed in vitro by various oncogenes, during subsequent in vivo growth were gradually replaced by descendant tumor cells, which co-expressed highly increased H 2O 2-catabolizing antioxidant activity (H 2O 2 CA), and the ability to release PGE 2 (PGE S) in contact with natural killer cells; v-src was the only oncogene, which in vitro induced cells transformation characterised with the expression of [H 2O 2 CA+PGE S] phenotype. Expression of [H 2O 2 CA+PGE S] phenotype was providing tumor cells with significantly increased resistance to cytotoxic activities of macrophages and NK cells. However, the possible involvement of [H 2O 2 CA+PGE S] phenotype in primary carcinogenesis remained obscure. Here, using three models of the primary virus-induced Syrian hamster tumors we demonstrated that Rous Sarcoma Virus-induced tumors arising after short latent period expressed [H 2O 2 CA + PGE S] phenotype at appearance. During the latent periods of SV40- and SA7(C8)-induced tumors the cells expressing [H 2O 2 CA+PGE S] phenotype gradually replaced the original [H 2O 2 CA+PGE S]-phenotype-negative cell populations. The effectiveness of such selection correlated with the duration of in vivo tumor development. Thus it was shown, that selection of tumor cells expressing [H 2O 2 CA+PGE S] phenotype is beginning and may be completed during the latent period of primary carcinogenesis. Taken together, data of this and preceding our studies on [H 2O 2 CA+PGE S] phenotype demonstrate that in vivo the host innate immunity antitumor reactions are apparently responsible for the selection of rare tumor cell variants capable to defend themselves against CTA of Mph and NK.

Nonmajor histocompatibility complex alloantigen effects on the fate of Rous sarcomas

Rous sarcoma virus-induced tumor outcome is controlled by the MHC (B). Additional data, using controlled segregation in families, has indicated non-MHC effects as well, but few studies have focused on blood groups other than the B complex. Segregating combinations of genes encoding erythrocyte (Ea) alloantigen systems A, C, D, E, H, I, P, and L in B2B5 and B5B5 MHC (B) backgrounds were examined for their effects on Rous sarcomas. Six-week-old chickens were inoculated in the wing-web with 30 pfu of Rous sarcoma virus (RSV). Tumors were scored six times over a 10-wk period. A tumor profile index (TPI) was assigned to each chicken based on the six tumor size scores. Response was evaluated using tumor size at each measurement period, TPI, and mortality. The genotypes of Ea systems A, C, D, E, H, I, and P had no significant effect on any parameter in either B complex population. The Ea-L system had an effect on Rous sarcomas in the B2B5 intermediate responders and B5B5 progressors. Tumor size, TPI, and mortality were all significantly lower in B2B5 L1L1 chickens than in B2B5 L1L2 chickens. Mortality was lower in the B5B5 L1L1 birds than in B5B5 L1L2 chickens. It appears that the Ea-L system, or one closely linked, is acting in a manner independent of the B complex in response to RSV challenge.

Protective Effects of Type I and Type II Interferons toward Rous Sarcoma Virus-Induced Tumors in Chickens

Growth of tumors induced by Rous sarcoma virus (RSV) is controlled by alleles at the major histocompatibility complex locus in chickens, indicating that immunological host defense mechanisms play a major role. We show here that the resistance phenotype of CB regressor chickens can be partially reverted by treating the animals with a monoclonal antibody that neutralizes the major serotype of chicken type I interferon, ChIFN-α. Injection of recombinant ChIFN-α into susceptible CC progressor chickens resulted in a dose-dependent inhibition of RSV-induced tumor development. This treatment was not effective, however, in CC chickens challenged with a DNA construct expressing the v-src oncogene, suggesting that the beneficial effect of type I interferon in this system resulted from its intrinsic antiviral activity and probably not from indirect immunmodulatory effects. By contrast, recombinant chicken interferon-γ strongly inhibited tumor growth when given to CC chickens that were challenged with the v-src oncogene, indicating that the two cytokines target different steps of tumor development.

The 9E3/CEF4 gene and its product the chicken chemotactic and angiogenic factor (cCAF): Potential roles in wound healing and tumor development

The 9E3/CEF4 gene was one of the first inducible chemokine genes to be discovered. Its product, the chicken chemotactic and angiogenic factor (cCAF), is highly homologous to IL-8. It is expressed at low levels in tissues of mesenchymal origin but is highly expressed shortly after wounding and persists throughout the period of granulation tissue formation. It also is highly expressed around Rous sarcoma virus-induced tumors. The most potent natural stimulator of this gene is thrombin and in vivo cCAF is chemotactic for monocytes and lymphocytes and is angiogenic. The chemotactic properties can be potentially important in the inflammatory response and in the deterrence of tumors, whereas the angiogenic properties could be important in wound repair and tumor growth. The very rapid stimulation of 9E3 by thrombin and fast synthesis and secretion of cCAF suggest that this is a new type of stress response protein whose rapid production is designed to protect tissues rather than individual cells.

Activation of complement in quails bearing Rous sarcoma virus-induced tumors.

The concentration of serum C3 in quails bearing tumors induced by Rous sarcoma virus (RSV) was elevated in parallel with tumor growth, whereas serum C3 levels in quails inoculated with avian leukosis virus, which lacks transforming activity, showed a pattern similar to that in mock-infected quails. C3 deposition was also observed in almost all tumor cells at the tumor developing stage. These findings obtained in vivo suggest that the cells transformed by RSV activated the C3 on their surfaces.

The Leukocyte Response of Japanese Quail to Rous Sarcoma Virus-Induced Tumors

In male Japanese quail, different circulating leukocyte responses were observed for progressors (birds developing a massive tumor that persisted throughout the experiment) and regressors (birds developing a tumor that gradually disappeared) after initial challenge with Rous sarcoma virus (RSV). Blood was sampled before and at weekly intervals postinoculation. Blood smears were prepared and stained with Diff Quik, and a light microscope (1000 x) was used in a direct count of 50 fields. Leukocytes were classified as heterophils, lymphocytes, monocytes, or eosinophils. The significant increase (P < 0.05) in total leukocytes at 14 days in regressors and progressors was consistent with the increase in tumor growth. The regressors' individual percentage of leukocytes did not deviate from control values, whereas the progressors' percentages of heterophils and monocytes were significantly higher (P < 0.05) and of lymphocytes significantly lower (P < 0.05) than those of controls by 14 days postinoculation. Indicative of this was the progressors' heterophil to lymphocyte ratio, which was significantly higher (P < 0.05) than that of controls 14 days post RSV challenge and remained elevated throughout the experiment. These findings suggest that the progressors' immune response is suppressed by proliferation of malignant cells. Therefore, the heterophil to lymphocyte ratio may be used in addition to tumor size to identify those birds that will regress RSV-induced tumors.

Influence of different B-complex recombinants on the outcome of Rous sarcomas in chickens.

Seven major histocompatibility (B) complex recombinants were evaluated for anti-Rous sarcoma response. In experiment 1, the BR5(F21-G19) recombinant haplotype both homozygous and in heterozygous combinations with B19 and B21 haplotypes were compared to B19/B19 and B21/B21 chickens to determine the relative influence of the BF versus BG chromosomal segments on regression of Rous sarcoma virus-induced tumours. In experiment 2, six recombinant haplotypes BR1(F24-G23), BR2(F2-G23), BR3(F2-G23), BR4(F2-G23), BR6(F21-G23) and BR8(F2-G2a,23) present in chickens heterozygous for normal haplotypes B19, B23 or B26 were compared for anti-sarcoma response. A total of 1328 chickens were blood typed for B alloantigens at 17 days of age, inoculated in the wingweb with Rous sarcoma virus at 6 weeks and monitored for anti-tumour immune response over a 10-week period. Genotypes which shared the same BF haplotype, but differed in their BG regions, had similar anti-tumour responses, implicating the BF but not the BG region in tumour regression. Chickens carrying BF2 or BF21 had a strong anti-tumour response, while BF24 conferred a weaker response, regardless of the accompanying normal haplotype.

Frequency of Chicken CD4+ and CD8+ Cells. Genetic Control and Effect of Rous Sarcoma Virus Infection

In chickens from congenic inbred lines CB and CC that differ only in the major histocompatibility complex (MHC), we observed significantly different percentages of CD4+ and CD8+ cells in peripheral blood lymphocytes (PBL) and spleen. Positive cells were detected by indirect immunofluorescence test as analysed by flow cytometry. In both PBL and spleen cell suspensions, the number of CD4+ cells was significantly higher in CB than in CC chickens, whereas in CC birds there was a higher percentage of CD8+ cells than in CB. These statistically significant differences were under the MHC control. We found no statistically significant influence of regressions or progression of Rous sarcoma virus-induced tumours on the percentage of peripheral T cells and on the interleukin-2 production in vitro.

Inheritance of fibrosarcomatous liver tumours in White Leghorn chicks inoculated vu the chorioallantoic membrane with subgroup A Rous sarcoma virus: A four‐allele genetic model

An investigation was made using White Leghorn fowl to study the genetic control of subgroup A Rous sarcoma virus-induced fibrosarcomatous liver tumours (LT) in chicks inoculated via the chorioallantoic membrane (CAM). A total of 723 CAM-inoculated embryos were hatched in three experiments. In Expt 1, CAM-susceptibility and LT-mortality were examined on a within sire family basis to ascertain the inter-relationship between the two parameters. In Expt 2, sires that had records of high or low LT(+) deaths in the families were selected to produce progeny within high and low incidence lines in the next generation to ascertain the amenability of the trait (LT death) to selection pressure. Survivors of LT-assay (Expt 1) were mated inter se in Expt 3 to study the inheritance of the two traits according to known or proposed genetic models. It was shown that LT mortality is a genetic trait because of its amenability to selection, with a high realised heritability (h(2)(R)= 1.16). In the three experiments, most CAM-susceptibles (S) died of LT(+), and most CAM-resistants(R) survived, but there were some conversely associated phenotypes i.e. S(LT-) and R(LT+). The conventional 2-allele model of the tva (tumour virus a) locus with pleiotropic effect, or a 2-locus model with linkage, were considered inadequate to explain the occurrence of conversely associated phenotypes on a within family basis. However, a 4-allele model of the tva locus showed a good fit to the results of this study.

Localization of 9E3/CEF-4 in avian tissues: expression is absent in Rous sarcoma virus-induced tumors but is stimulated by injury.

The avian gene 9E3/CEF-4, a member of the superfamily of genes that includes KC and gro, is expressed abundantly in exponentially growing cultures of chick embryo fibroblasts (CEFs) and at high levels in CEFs transformed with Rous sarcoma virus (RSV). The product of this gene is a secreted protein that has homologies and structural similarities to inflammatory mediators. The function of 9E3 is obscure and its expression in vivo has not yet been investigated. We studied by in situ hybridization and RNA blots the pattern of 9E3 mRNA distribution in the wings of normal, wounded, and RSV-infected newly hatched chicks. We found that the message for 9E3 is high in specific tissues in normal wings; whereas connective tissue, tendon, and bone express the gene, muscle fibers, endothelium, epidermis, and bone marrow do not. The distribution coincides with that of interstitial collagen. Wounding results in marked elevation of the mRNA within the granulation tissue formed during healing and in adjacent tissues, especially those showing neovascularization. Similar elevation of mRNA occurs immediately adjacent to RSV tumors but, surprisingly, the tumor tissue itself shows no detectable levels of this message. Cells explanted from the tumors and grown in culture also show no expression of 9E3, in marked contrast to the very high level found in similarly cultured RSV-transformed CEFs. These results show that there are intrinsic differences between transformed embryonic cells in tissue culture and RSV target cells in the hatched chick. However, the expression of the gene in the periphery of tumors leaves open the possibility that 9E3 may still be involved in RSV carcinogenesis. The abundant expression of 9E3 in normal tissues indicates that the product of this gene plays a normal physiological role in tissues growing by cell division, perhaps as a growth regulator. The elevated expression of 9E3 in areas of neovascularization, makes it possible that the product of this gene could act as an angiogenic factor. Finally, expression in conjunction with high collagen levels and in wounded tissues may point to a role in wound response and/or repair, possibly via alteration of extracellular matrix.

Isolation of a catalytically competent phosphorylated tyrosine kinase from Rous sarcoma virus-induced rat tumor by immunoadsorption to and hapten elution from phosphotyrosine binding antibodies

A procedure has been developed for the isolation of a catalytically competent phosphorylated tyrosine kinase (RSV Y-kinase) from avian sarcoma virus-induced rat tumors. The procedure involves reaction of partially purified RSV Y-kinase with ATP to effect tyrosyl phosphorylation of catalytically competent RSV Y-kinase. Tyrosyl phosphorylated RSV Y-kinase was isolated from the heterogenous reaction mixture by immunoadsorption on immobilized phosphotyrosyl binding antibodies and elution with the hapten p-nitrophenyl phosphate. Estimation of the phosphate content of the purified phosphorylated RSV Y-kinase indicated that 1–3 tyrosyl groups had been phosphorylated upon reaction with ATP. The specific activity toward histone 2B of the purified phosphorylated RSV Y-kinase was at least 30-fold greater than that estimated for the RSV Y-kinase prepared previously by immunoadsorption on immobilized antiserum from tumor bearing rabbits.

Suppression of Rous sarcoma virus-induced tumor formation by preinfection with viruses encoding src protein with novel N termini

Two recovered avian sarcoma viruses (rASVs), rASV157 and rASV1702, encode src products which contain novel, nonmyristoylated N-terminal amino acids. These viruses transform chicken embryo fibroblasts and cause tumors in chicks. However, the tumors rASVs induce are small and regress within 2 weeks. To determine whether this regression results from weak tumorigenicity or from the active immunity of the host, we injected 1-week-old chicks with rASV and several days later injected the chicks with challenge virus of a different subgroup. Of the rASV1702-preinfected chicks challenged 5 days later with Rous sarcoma virus (RSV), 40% showed no subsequent tumor formation and 60% formed tumors which regressed within 1 week. The potency of this protective effect depended on the dosage of preinfection virus used and increased as the interval between preinfection and challenge infection was lengthened (when the interval was 9 days, none of the challenged chicks formed tumors). rASV157-preinfected chicks challenged with RSV after 9 days showed only partial protection: 42% formed tumors which regressed, whereas 58% formed tumors which continued to grow. Challenging rASV-preinfected chicks with Fujinami sarcoma virus or a RSV vector encoding the v-fps oncogene or polyomavirus middle T resulted in no suppression of tumor formation. Preinfection with src mutants or a RSV vector encoding polyomavirus middle T antigen, both of which induce slow-growing tumors, failed to elicit the protective effect. Finally, a novel N-terminal domain encoded by rASV1702 src was shown to be involved in but not sufficient for full protection. These data indicate that determinants on or induced by rASV157 and rASV1702 can elicit a potent protection against the tumorigenic potential of RSV-encoded p60v-src.

Response of Major Histocompatibility (B) Complex Haplotypes B22, B26, and B30 to Rous Sarcomas

The response to Rous sarcoma virus-induced tumors was measured over a 2-yr period using Line UNH 105 New Hampshire chickens segregating for three major histocompatibility (B) complex haplotypes, B22, B26, and B30. Twelve sires and 49 dams were used to produce 489 pedigreed progeny consisting of six B genotypes B22/B22, B22/B26, B22/B30, B26/B26, B26/B30, and B30/B30. Six-wk-old chicks were inoculated in the wingweb with a pseudotype of Bryan high-titer Rous sarcoma virus designated as BH RSV (RAV-1). Resultant tumors were scored six times during a 10-wk period, which served as a basis for a tumor profile index (TPI) assigned to each chick.The B22/B26 chickens (x̄ TPI = 2.6) had the greatest antitumor response, which was significantly different from that of B26/B26 (x̄ TPI = 3.0) and B30/B30 chickens (x̄ TPI = 3.4), the lowest responders. Lower mean TPI of two heterozygotes, B22/B26 (2.6) and B26/B30 (2.9), compared with those of appropriate homozygotes, suggested complementation between B22 and B26 as well as B26 and B30. Chi-square analysis revealed an additional beneficial effect of the B22 haplotype in tumor-bearing chickens, as B22-containing genotypes had a significantly higher survival rate than genotypes not containing B22.

Analysis of B-G and Immune Response Genes in the Iowa State University S1 Chicken Line by Hybridization of Sperm Deoxyribonucleic Acid with a Major Histocompatibility Complex Class II Probe

Sperm DNA was isolated from chickens of the Iowa State University S1 line. Birds were from sublines selected for B-G antigen, humoral immune response to glutamic acid-alanine-tyrosine (IrGAT), and response to Rous sarcoma virus-induced (RSV) tumors. The DNA was digested with restriction enzymes and subjected to Southern blot analysis with a DNA probe specific for a class II gene of the chicken major histocompatibility complex (MHC).Restriction fragment length polymorphisms (RFLP) associated with B-G antigen type were found after digestion of DNA with three enzymes, PvuII, BglII, or Sau3A, out of a total of 15 tested. No RFLP were shown to be associated with IrGAT or RSV type. This study shows that RFLP analysis of DNA may be a useful addition to or alternative to serological evaluation of MHC haplotype in the chicken.

Transplantation resistance to a Rous sarcoma virus-induced tumor in mice immunized with v-src protein.

It is of great interest in tumor immunology to know whether oncogene products could be used not only as tumor markers for cancer diagnosis, but also as immunogens for cancer therapy. BALB/c mice immunized with syngeneic fibroblasts, Escherichia coli cells producing p60v-src, or the purified p60v-src protein extracted from the E. coli producer cells showed transplantation resistance to a Rous sarcoma virus-induced tumor but not a Kirsten sarcoma virus-induced tumor. In contrast, mice immunized with cells not producing p60v-src or their derived proteins or with chicken ovalbumin did not show any significant resistance. These findings suggest that p60v-src can act as a specific transplantation rejection antigen in mice.

Genetic Analysis of Immunocompetence Measures in a White Leghorn Chicken Line

Immunocompetence of the Iowa State University S1 White Leghorn chicken line was studied. This line was divided into eight sublines based upon erythrocyte antigen B (Ea-B) allele (B1 B1 or B19 B19), antibody response to glutamic acid60-alanine30-tyrosine10 (GAT) (high or low), and response to Rous sarcoma virus-induced tumors (progression or regression). Antibody responses to Pasteurella multocida (PM), Mycoplasma gallisepticum (MG), and infectious bursal disease virus vaccines were evaluated by enzyme-linked immunosorbent assay. Phagocytic activity and T cell-mediated response were measured by carbon clearance and phytohemagglutinin (PHA) injection assays, respectively.Significant haplotype (subline) differences and sire family differences were observed in all three measurements. Significant sex differences were observed in phagocytic activity and T cell-mediated responses. Haplotypes with high antibody responses to GAT had significantly higher antibody titers to PM and MG vaccines than haplotypes with low antibody responses. Significant positive correlations were observed between antibody levels to the two vaccines. A significant negative correlation was seen between phagocytic activity and T cell-mediated response of females. The data suggest that the total immunocompetence profile of an individual must be considered to select for optimum immune responsiveness.