The expression profiles of chemokines, innate immune and apoptotic genes in tumors caused by Rous Sarcoma Virus (RSV-A) in chickens.

Innate immune genes play an important role in the immune responses to Rous sarcoma virus (RSV)-induced tumor formation and metastasis. Here, we determined in vivo expression of chemokines, innate immune and apoptotic genes in Synthetic Broiler Dam Line (SDL) chickens following RSV-A infection. The mRNA expression of genes was determined at the primary site of infection and in different organs of progressor, regressor and non-responder chicks, using RT-qPCR. Our results indicated a significant upregulation of: (1) chemokines, such as MIP1ß and RANTES, (2) the innate immune gene TLR4, and (3) p53, a tumor-suppressor gene, at the site of primary infection in progressor chickens. In contrast, inducible nitric oxide synthase (iNOS) gene expression was significantly downregulated in progressor chicks compared to uninfected, control chicks. All of the innate immune genes were significantly upregulated in the lungs and liver of the progressor and regressor chicks compared to control chicks. In the spleen of progressor chicks, RANTES, iNOS and p53 gene expression were significantly increased, whereas MIP1ß and TLR4 gene expression was significantly downregulated, compared to control chicks. The lungs and livers of non-responder chicks expressed a low level of iNOS and MIP1ß, whereas RANTES, TLR4, and p53 gene expression were significantly upregulated compared to uninfected control chicks. In addition, there was a significant downregulation of RANTES, MIP1ß, and TLR4 gene expression in non-responder chicks. These results suggest the different response to infection of chicks with RSV-A is due to differential changes in the expression of innate immune genes in different organs.

Apoptin, a tumor-selective killer.

Apoptin, a small protein from chicken anemia virus, has attracted great attention, because it specifically kills tumor cells while leaving normal cells unharmed. The subcellular localization of apoptin appears to be crucial for this tumor-selective activity. In normal cells, apoptin resides in the cytoplasm, whereas in cancerous cells it translocates into the nucleus. The nuclear translocation of apoptin is largely controlled by its phosphorylation. In tumor cells, apoptin causes the nuclear accumulation of survival kinases including Akt and is phosphorylated by CDK2. Thereby, apoptin redirects survival signals into cell death responses. Apoptin also binds as a multimeric complex to DNA and interacts with several nuclear targets, such as the anaphase-promoting complex, resulting in a G2/M phase arrest. The proapoptotic signal of apoptin is then transduced from the nucleus to cytoplasm by Nur77, which triggers a p53-independent mitochondrial death pathway. In this review, we summarize recent discoveries of apoptin's mechanism of action that might provide intriguing insights for the development of novel tumor-selective anticancer drugs.

An electron microscopic study of the chorioallantoic membrane following infection with Rous sarcoma virus.

Infection of the chick chorioallantoic membrane (CAM) with Rous sarcoma virus (RSV) has been thought by earlier workers (12, 20) to result in the transformation of the ectoderm and then the mesoderm of that organ. In the present study, CAM were infected with 10(4) PFU (pock-forming units) of RSV (Bryan high titre strain) and collected for electron microscopy at 2, 4, and 6 days postinfection. Observations of the fine structural changes in the CAM after RSV infection support a singular role of the mesenchyme in the initiation of the tumors. The ectodermal hyperplasia often associated with RSV tumors of the CAM appears to be a secondary response to the alteration of the underlying mesenchyme. These findings are discussed in detail, and an alternate course of RSV transformation of the CAM by way of the vascular bed is suggested.

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.

Rous sarcomas in chickens: enhanced growth coexisting with concomitant immunity.

Chickens bearing Rous sarcoma virus-induced tumors in one wing did not develop new tumors when subsequently inoculated with Rous sarcoma virus in the other wing. However, the second inoculation of Rous sarcoma virus caused accelerated growth of the established tumors. This phenomenon was found to be bursa-dependent. Paradoxically, established tumors in bursectomized chickens grew at a diminished rate if the chickens were reinoculated with Rous sarcoma virus.

Mediation of wound-related Rous sarcoma virus tumorigenesis by TGF-beta.

In Rous sarcoma virus (RSV)-infected chickens, wounding leads to tumor formation with nearly 100% frequency in tissues that would otherwise remain tumor-free. Identifying molecular mediators of this phenomenon should yield important clues to the mechanisms involved in RSV tumorigenesis. Immunohistochemical staining showed that TGF-beta is present locally shortly after wounding, but not unwounded controls. In addition, subcutaneous administration of recombinant transforming growth factor-beta 1 (TGF-beta 1) could substitute completely for wounding in tumor induction. A treatment protocol of four doses of 800 nanograms of TGF-beta resulted in v-src-expressing tumors with 100% frequency; four doses of only 10 nanograms still led to tumor formation in 80% of the animals. This effect was specific, as other growth factors with suggested roles in wound healing did not elicit the same response. Epidermal growth factor (EGF) or TGF-alpha had no effect, and platelet-derived growth factor (PDGF) or insulin-like growth factor-1 (IGF-1) yielded only occasional tumors after longer latency. TGF-beta release during the wound-healing response may thus be a critical event that creates a conducive environment for RSV tumorigenesis and may act as a cofactor for transformation in this system.

MHC-B haplotypes impact susceptibility and resistance to RSV-A infection.

We investigated the impact of haplotype of major histocompatibility complex (MHC)-B on the outcome of infection of Synthetic Dam Line (SDL) broiler strain with Rous Sarcoma Virus (RSV). Genomic analysis of MHC-B haplotypes, revealed a total of 12 known standard haplotypes that constituted to twenty-five different genotypes and one new haplotype of 217 bp size, designated BX. The inoculation of RSV-A in SDL chicks resulted in the development of tumors of progressive or regressive phenotypes with varying tumor profile index (TPI). Haplotypes B2, B21 and B22had low TPI scores (1 or 2) with less mortality and were resistant to RSV-A tumor. The haplotypes B13, B13.1., B15, B15.1. and B15.2. had significantly higher TPI scores (5 or 6), indicating a susceptibility to RSV-A. The genotype, Bx /Bx, had a mean TPI score of 3.67 ± 1.33, which was closer to the resistant haplotype. Sequence analysis of the new haplotype (BX) revealed 99.5% similarity with B2 haplotype. Metastases was observed in 44% of chicks and comprised of mixed fibrosarcoma and myxosarcoma.

Transformation of chicken embryo fibroblasts and tumor induction by the middle T antigen of polyomavirus carried in an avian retroviral vector.

The middle T antigen of polyomavirus transformed primary chicken embryo fibroblasts when expressed from a replication-competent avian retrovirus. This in vitro-constructed retrovirus, SRMT1, is a variant of Rous sarcoma virus that encodes the middle T antigen in place of v-src. Inoculation of SRMT1 into 1-week-old chickens rapidly induced hemangiomas and hemangiosarcomas. As shown with mammalian cells infected with polyomavirus, polyomavirus middle T antigen appears to be associated with p60c-src in chicken cells infected with SRMT1. When lysates of SRMT1-infected cells immunoprecipitated with either a monoclonal antibody against p60src or anti-T serum were assayed in an in vitro kinase reaction, the middle T antigen was heavily phosphorylated. To see whether an excess of p60c-src could alter the extent of phosphorylation of the middle T protein or the process of cell transformation by middle T, cells were doubly infected with SRMT1 and NY501, a virus which overexpresses p60c-src. Doubly infected chicken embryo fibroblasts transformed with the same kinetics and were morphologically indistinguishable from chicken embryo fibroblasts infected with SRMT1 alone. Phosphorylation of the middle T antigen was elevated two- to fivefold relative to cells infected only with SRMT1.

Arising podosomal structures are associated with neoplastic cell morphological phenotype induced by the microenvironment.

Increased numbers of rosettes of podosomes were observed in overgrown rat Rous sarcoma RsK4 cells. A possible role of these structures in nutrient uptake in tumour cell survival was investigated by exposure to acute starvation. A single cell suspension of RsK4 cells in Hanks balanced salt solution was allowed to interact with either clean uncoated or serum-coated for bait coverglasses. Confocal microscopy revealed contrasting 3D cell morphologies that were associated with conspicuous patterns of podosomal structures, which on the coated coverglasses resembled the sealing zones of osteoclasts, while on the uncoated coverglasses they resembled the marginal podosomes of migrating monocyte-derived cells. Thus, the arising podosomal structures, the involvement of which in an uptake of nutrients appeared feasible morphologically, were associated with the emerging 3D cell shapes guided by the microenvironment. Such phenotypic plasticity of neoplastic RsK4 cells in response to microenvironmental challenge suggested that uniqueness in cellular attributes within the neoplastic cell population could be crucial for the malignant potential.

Pathogenicity for rats of a strain of chicken sarcoma virus.

The RBI rat tumor, induced with cell suspensions from chicken sarcoma B77, is pathogenic for chicks as well as for rats. Cell suspensions from RBI tumors induced sarcomas in 100 percent of inoculated chicks. Cell suspensions of this chicken sarcoma induced early RBA sarcomas in 50 percent of 171 rats. Most of these rats died within 4 weeks after inoculation. The early tumors regressed in 15 of the 85 tumor-bearing rats, and the animals died of cystic hemorrhagic disease. The sarcoma was induced in 9 animals within 50 to 70 days after inoculation, and cystic hemorrhagic disease developed in 117. None of the 171 rats remained free from either tumor or cyst and only 12 survived for 3 months or longer. The tumors induced in rats were transplantable into rats and after transplantation of early-appearing tumors, tumors and cysts developed. Virus strongly infective in chicks was demonstrated by cell-free filtrates and virus preparations from RBI and RBA rat sarcomas induced by chicken sarcoma cells. Cell suspensions from the wall of small cysts induced tumors in chicks and cystic hemorrhagic disease and tumors in rats.

Differential susceptibility of avian sarcoma cells derived from different periods of tumor growth to natural killer cell activity.

Tumors which are induced in chickens by avian sarcoma virus usually grow actively for 2 to 3 weeks and then regress. We have cultured tumor cells from each of the active and regression periods of neoplastic growth and compared them in terms of sensitivity to natural killer (NK) activity and to specific cell-mediated cytotoxicity. The results indicate that those cells which derive from regressing sarcomas are sensitive to NK activity but resistant to specific cell-mediated cytolysis. In contrast, tumor cells derived from the period of active neoplastic growth can be lysed effectively in either type of assay, although they do not appear to be as susceptible as do "regressor" cells to the action of NK effectors. The addition of autologous virus to these various reaction mixtures is inhibitory to specific cell-mediated cytotoxicity but not to NK-mediated lysis. These data suggest that NK activity may play an important role in the elimination of tumor cells of the "regressor" phenotype, which otherwise appear to be resistant to immune cytolysis.

Effect of phorbol ester on growth of tumors induced by Rous sarcoma virus and on pp60src kinase activity in these tumors.

The inoculation of Rous sarcoma virus (RSV) into chickens results in the induction of tumors which usually grow progressively for several weeks and then regress. We have found that the direct injection of phorbol myristate acetate into growing RSV-induced sarcomas resulted in accelerated tumor regression in each of 11 cases studied. We further found that treatment of cultured Rous sarcoma cells with phorbol myristate acetate for 72 h resulted in a 50% diminution in numbers of viable cells, in comparison with controls, and a 60% reduction in DNA synthesis. We also investigated alterations in the pp60src kinase activity of cultured RSV-induced tumor cells after treatment with phorbol myristate acetate and found that pp60src kinase activity was reduced to 65-95% of control levels. This inhibition of pp60src kinase was both time- and concentration-dependent. Western blot analysis, using a tumor-bearing rabbit serum, indicated that the pp60src protein was reduced by 35-65% in treated cells, while Pr76, precursor of the structural RSV-proteins, was diminished by 10-35%. In contrast, nonviral proteins such as actin were not significantly affected.

Evaluation of the cocarcinogenic effect of wounding in Rous sarcoma virus tumorigenesis.

Chickens given injections of Rous sarcoma virus form sarcomas at the site of inoculation (primary tumor) and at the site of experimentally introduced wounds (wound tumor). This latter finding provides a model system to study systematically the mechanisms underlying the cocarcinogenic effects of wounding. Our experiments show the following. (a) Chickens inoculated with a Rous sarcoma virus-derived, replication-defective virus construct fail to elaborate wound tumors in spite of aggressively growing primary tumors. We thus rule out metastasis as a mechanism and conclude that infectious virus is required for wound tumor formation; (b) using bromodeoxyuridine incorporation and immunofluorescence on frozen sections we demonstrate proliferation in the unwounded wing in cell types which are normally targets for Rous sarcoma virus infection and transformation and conclude that proliferation per se is not sufficient to induce wound tumors; (c) using immunohistochemistry for the viral protein p19gag we show that wounding induces virus expression in fibroblasts of newly forming granulation tissue 2 days after injury. We also demonstrate expression of viral mRNA in wound tumors by in situ hybridization with a v-src probe. We discuss the possibility of activation of integrated, silent virus or the preferential infection of a special target cell population as a result of wounding as well as the potential role of wound factors in transformation.

Inflammation is responsible for the development of wound-induced tumors in chickens infected with Rous sarcoma virus.

When newly hatched chicks are given injections of Rous sarcoma virus, a tumor develops at the site of injection. In spite of the presence of the virus in the blood, no other tumors are found distant from the site of inoculation during the life span of the animal (4-6 weeks). However, if a wound is made away from the primary tumor, a tumor develops at the site of wounding. Work in our laboratory showed previously that these wound tumors do not develop as a result of metastasis, therefore, factors released upon wounding must contribute to the development of the wound tumors. In particular, we showed that transforming growth factor (TGF) beta, a growth factor implicated in wound healing, can replace wounding in tumor development. However, we also showed that epidermal growth factor and TGF-alpha, growth factors that also have roles in wound healing, do not induce tumors. To identify the critical event(s) and to determine the mechanism involved in wound tumor development, we have continued these studies. Here we show that: (a) wound tumor development correlates with the presence of circulating virus and inflammation; (b) the virus is present in serum and in heterophils of the peripheral blood; (c) cell division at the site of wounding precedes the expression of viral proteins; (d) in addition to TGF-beta, acidic and basic fibroblast growth factors can also replace wounding in tumor development; (e) these three factors (TGF-beta, acidic fibroblast growth factor, basic fibroblast growth factor) which promote tumors also induce inflammation, whereas epidermal growth factor and TGF-alpha do not; and (f) during the inflammatory response, blood vessel leakage occurs as tested by the release of fibrinogen into the tissues. To test the possibility that inflammation is the key element in the development of these wound tumors, we used beta-methylprednisolone, an antiinflammatory drug that inhibits inflammation (including blood vessel leakage), to determine if wound tumor development could be prevented. We found that when inflammation was inhibited, tumors were also inhibited; when inflammation could not be stopped, tumors developed as before. These results indicate that the effect of wounding on the development of wound tumors in Rous sarcoma virus-infected chicks is accomplished through the cytokines released by the inflammatory cells at the site of wounding. These inflammatory mediators play a critical role in providing the conducive environment for oncogene integration and activation, and subsequent development of tumors.(ABSTRACT TRUNCATED AT 400 WORDS)

Phosphorylation of integrin in differentiating ts-Rous sarcoma virus-infected myogenic cells.

The differentiation of primary myogenic cultures requires the attachment of the cells to an extracellular matrix substrate using an integrin family receptor. These integrin receptors can be phosphorylated on both their alpha and beta chains, and it has been postulated that phosphorylation regulates the receptor function. Quail myogenic clones transformed with ts-LA24A differentiated into mature myotubes following a temperature shift to nonpermissive temperature which inactivates the viral src kinas. Phosphorylation of integrin beta-1 chain and of at least one alpha chain was detected on both serine and tyrosine. An additional alpha chain(s) with a mobility similar to alpha 5 was not phosphorylated at either temperature. Following the induction of differentiation by a temperature shift, there was a marked decrease in integrin phosphorylation of both alpha and beta integrin chains. This decrease was more prominent for serine than for tyrosine, suggesting that src could not be the only kinase involved. The drop in integrin phosphorylation correlated with the initiation of differentiation, suggesting that integrin phosphorylation could be at least part of the mechanism by which myogenic differentiation is blocked by v-src.

Ascorbate stabilizes the differentiated state and reduces the ability of Rous sarcoma virus to replicate and to uniformly transform cell cultures.

In primary avian tendon cells, Rous sarcoma virus can coexist or completely take over the cell. Infection, at high multiplicity or under conditions that promote high virus production (no ascorbate and high serum concentrations), results in almost complete oncogenic transformation of the culture. This is indicated in part by a radical change in morphology, growth at high cell density, and a dramatic drop in the production of procollagen from approximately 50% to approximately 3% of total protein synthesis. In contrast, infection at low multiplicity, infection with a replication defective virus, or the presence of ascorbate restrict the ability of the virus to transform the culture. Thus, there appears to be a balance between the normal and transformed states of the cell that can be shifted depending on the cellular environment and the level of infection. Ascorbate stabilizes the normal state by reducing virus production and promoting the synthesis of differentiated proteins.

Experimental gliomas: an autoradiographic study of the endothelial component.

Autochthonous gliomas were induced in rats by intracerebral inoculation of avian of avian sarcoma virus and studied by 3H-thymidine autoradiography. Parenchymal glial tumor cells had a 3H-labeling index (LI) of 3.0 to 13.6%. Endothelial cells in tumor blood vessels had an LI of 2.6 to 34.3%, independent of and in most instances higher than the LI of the glial tumor. Endothelial cells of normal blood vessels had an average LI of 0.3%. This study documents the high proliferative rate of the endothelial cells in anaplastic experimental gliomas, and emphasizes the necessity for seeking direct, incontrovertible evidence to determine whether or not the rapidly proliferating endothelial cells are malignant.

Experimental allergic encephalomyelitis in Lewis rats bearing avian sarcoma virus-induced brain tumors.

Lewis rats bearing avian sarcoma virus (ASV)-induced brain tumors were injected with guinea pig spinal cord emulsion and complete Freund's Adjuvant to determine if they remained susceptible to induction of experimental allergic encephalomyelitis (EAE). The incidence of EAE among rats with small, and moderate sized gliomas was similar to non-tumor-bearing controls (P less than 0.5; P less than 0.9) while 18 of 24 (75%) animals with large gliomas developed EAE as compared to 31 of 33 (93%) controls (P less than 0.05). The histologic features and geographical distribution of "ordinary' EAE were seen in controls and were maintained in tumor-bearing rats. The presence of an intracranial tumor did not significantly alter the ability of Lewis rats to develop EAE.

Antiproliferative effects of 9-beta-d-arabinofuranosyladenine in a mammalian cell line devoid of adenosine deaminase activity.

The effect of ara-A on cellular growth, DNA synthesis, and RNA synthesis, and RNA synthesis was measured in an established cell line (B-mix K-44/6) devoid of adenosine deaminase activity. Cells adapted to growth in a medium supplemented with horse serum provided an environment totally lacking adenosine deaminase activity whereas cultivation of cells in a medium supplemented with calf serum provided a system capable of deaminating ara-A to ara-H (half-life = 14 hours). Under deaminase-free conditions early log phase cells underwent 1.5 population doublings during 28 hours compared with 0.25 doublings in the presence of 37 micronM ara-A. When cells were grown in medium supplemented with calf serum the additionof 37 to 225 micronM ara-A resulted in a cessation of mitosis for periods of 5 to 30 hours respectively. Following this quiescent period growth resumed at the original rate. With 600 micronM ara-A mitosis was reversibly inhibited up to 35 hours after drug addition. The effects of ara-A on RNA and DNA synthesis were monitored by continuously or pulse labeling B-mix K-44/6 cells with [3H]-uridine or [3H]thymidine. Ara-A did not influence RNA synthesis as judged by labeled uridine incorporation. Under deaminase-free conditions, 5.4 micronM ara-A inhibited labeled thymidine incorporation by 50%. In the presence of the enzyme, approximately twice the ara-A concentration was required for the same inhibition; furthermore the initial inhibition was followed by a partial recovery in the rate of thymidine incorporation. Examination of thymidine incorporation. Examination of thymidine nucleotide pools during ara-A treatment revealed to changes in the labeling of dTMP, dTDP, and dTTP. Thus inhibition of [3H]thymidine incorporation by ara-A accurately reflected inhibition of DNA synthesis. We conclude that, in spite of an initial inhibition of DNA synthesis and mitosis by ara-A, B-mix K-44/6 cells recover from the inhibitory effects if the drug is removed either by a change in the culture medium or by metabolism to ara-H.