Genetic control of susceptibility of mice to Rous sarcoma virus tumorigenesis

Studies with congenic resistant strains of mice indicate that susceptibility to Rous sarcoma virus tumorigenesis is influenced by a gene or genes associated with the major histocompatibility complex (H-2). These genes manifest dominant relative susceptibility. Preliminary studies indicate that the CBA/J strain harbors a gene or genes for relative susceptibility, which are recessive. These results are compared with other studies onH-2-associated genes affecting murine viral oncogenesis.     

Genetic linkage between immune response to GAT and the fate of RSV-induced tumors in chickens

Recent studies suggest that the gene locus controlling the fate of tumors induced by Rous sarcoma virus (RSV) is linked to theB histocompatibility complex. Birds carrying the dominant allele regress the tumor; homozygous recessives being unable to do so, develop large tumors and die. These are called progressors.The Bryan strain of RSV was inoculated into 220 6 week old Leghorns homozygous forB ¹ B ¹,B ² B ², orB ¹⁹ B ¹⁹ of which the percentages of progressors were 79, 22 and 56, respectively. The balance of each were regressors and survived.TheB ¹ B ¹ test birds were derived from special matings, i.e., high and low immune responders to the amino acid polymer, GAT. Of 67 tests progeny of theB ¹ B ¹ GAT-low mating, 63 or 94% proved to be progressors, and 6% were regressors. Of 84 test progeny of theB ¹ B ¹ GAT-high matings, 67% were progressors, and 33% were regressors. The difference between the high and low GAT responders is highly significant and indicates that the locus controlling the fate of RSV-induced tumors is closely linked to the locus controlling immune response to GAT. The latter maps within theIr region of theB histocompatibility complex.     

Tumor growth and antibodies after RSV-challenge in normal chickens and in chickens congenitally infected with avian leukosis virus.

Normal chickens and chickens congenitally infected with an avian leukosis virus (ALV) of antigenic subgroup A were challenged with strains of Rous sarcoma virus (RSV) of two different antigenic subgroups (B and C) and tumor induction and growth as well as humoral antibody to viral envelope antigen (VEA) and tumor-specific surface antigen (TSSA) were measured. There was no effect of congenital ALV infection on RSV tumor incidence or latent period but the growth rate and size of the tumors were much higher in congenitally infected birds as compared to controls. Whereas most tumors in the RSV-challenged normal birds regressed, tumors in ALV-infected birds grew progressively. There were no striking differences in the number of birds in either group in the incidence of anti-TSSA or anti-VEA antibodies nor did the presence of either type of antibody reflect the tumor status of the host.     

Development of anti-pp60src serum with antigen produced in Escherichia coli

We have purified p60^src from bacterial recombinants which direct the synthesis of the Rous sarcoma virus transforming gene (src) product. This protein was injected into rabbits, and they produced a highly cross-reactive serum which can recognize the src protein from many different strains of Rous sarcoma virus.     

Genetic analysis of a divergent selection for resistance to Rous sarcomas in chickens?. This article is dedicated to the memory of Pierrick Thoraval (1960–2000).

Selection for disease resistance related traits is a tool of choice for evidencing and exploring genetic variability and studying underlying resistance mechanisms. In this framework, chickens originating from a base population, homozygote for the B¹⁹ major histocompatibility complex (MHC) were divergently selected for either progression or regression of tumors induced at 4 weeks of age by a SR-D strain of Rous sarcoma virus (RSV). The first generation of selection was based on a progeny test and subsequent selections were performed on full-sibs. Data of 18 generations including a total of 2010 birds measured were analyzed for the tumor profile index (TPI), a synthetic criterion of resistance derived from recording the volume of the tumors and mortality. Response to selection and heritability of TPI were estimated using a restricted maximum likelihood method with an animal model. Significant progress was shown in both directions: the lines differing significantly for TPI and mortality becoming null in the "regressor" line. Heritability of TPI was estimated as 0.49 ± 0.05 and 0.53 ± 0.06 within the progressor and regressor lines respectively, and 0.46 ± 0.03 when estimated over lines. Preliminary results showed within the progressor line a possible association between one Rfp-Y type and the growth of tumors.     

Localization of N6-methyladenosine in the Rous sarcoma virus genome.

The 10,000-nucleotide RNA genome of the Prague strain, subgroup B (PR-B) of Rous sarcoma virus, was found to contain 11.6 ± 0.5 residues of m⁶Ap by quantitative analysis of ³²P-labeled virion RNA after complete RNAase digestion. Approximately ten of the m⁶Ap residues are located, without obvious clustering, in that region of the genome between 500 and 4000 nucleotides from the 3′ poly(A) end. The src gene, which is required for transformation, and part of the env gene, which codes for the major viral envelope glycoprotein, have previously been mapped in this region of the viral genome. A transformation-defective deletion mutant of PR-B Rous sarcoma virus, which lacks the src gene, has 7.0 ± 0.2 m⁶Ap residues per RNA subunit. This supports our mapping of a portion of the m⁶A residues in src and suggests that this methylation is specific to certain regions of the genome. The possible significance of this result for Rous sarcoma virus RNA processing and translation is discussed.     

Tests of association of immunoglobulin allotype genes and viral oncogenesis in chickens

Chickens from Regional Poultry Research Laboratory (RPRL) inbred line 6₃ are resistant to virally-induced Marek's disease (MD) and lymphoid leukosis (LL) and are relatively strong regressors of virally-induced Rous sarcomas. In contrast, RPRL line 100 chickens are highly susceptible to MD and LL and are weaker regressors of Rous sarcomas than line 6₃. RPRL lines 100 and 6₃ differ for alleles at the IgG-1 (G-1) allotype locus, but have identical IgM-1 (M-1) allotype alleles. To test the possible association of the G-1 locus with variations in resistance to virally-induced tumors, homozygous and heterozygous genotypes among F₃ crosses were infected. F₃ chickens with different G-1 types were comparable in their resistance to MD tumors following inoculation with the JM strain of the MD virus, and for their ability to regress Rous sarcoma tumors induced by the Rous sarcoma virus (RSV) RAV-1. However, following RAV-1 virus infection a smaller proportion of G-1 ^a /G-1 ^a F₃ or F₄ birds developed LL tumors than G-1 ^a /G-1 ^e and G-1 ^e /G-1 ^e birds. Genes determining immunoglobulin heavy chains were therefore associated with a recessive resistance to B-cell lymphomagenesis in chickens.Deceased     

Expression Analysis of Genes Involved in the RB/E2F Pathway in Astrocytic Tumors

Astrocytic gliomas, which are derived from glial cells, are considered the most common primary neoplasias of the central nervous system (CNS) and are histologically classified as low grade (I and II) or high grade (III and IV). Recent studies have shown that astrocytoma formation is the result of the deregulation of several pathways, including the RB/E2F pathway, which is commonly deregulated in various human cancers via genetic or epigenetic mechanisms. On the basis of the assumption that the study of the mechanisms controlling the INK4/ARF locus can help elucidate the molecular pathogenesis of astrocytic tumors, identify diagnostic and prognostic markers, and help select appropriate clinical treatments, the present study aimed to evaluate and compare methylation patterns using bisulfite sequencing PCR and evaluate the gene expression profile using real-time PCR in the genes CDKN2A, CDKN2B, CDC6, Bmi-1, CCND1, and RB1 in astrocytic tumors. Our results indicate that all the evaluated genes are not methylated independent of the tumor grade. However, the real-time PCR results indicate that these genes undergo progressive deregulation as a function of the tumor grade. In addition, the genes CDKN2A, CDKN2B, and RB1 were underexpressed, whereas CDC6, Bmi-1, and CCND1 were overexpressed; the increase in gene expression was significantly associated with decreased patient survival. Therefore, we propose that the evaluation of the expression levels of the genes involved in the RB/E2F pathway can be used in the monitoring of patients with astrocytomas in clinical practice and for the prognostic indication of disease progression.     

Nucleotide sequences of feline retroviral oncogenes (v-fes) provide evidence for a family of tyrosine-specific protein kinase genes

The nucleotide sequences encoding the transforming polyproteins of the Snyder-Theilen and Gardner-Arnstein strains of feline sarcoma virus (FeSV) have been determined. These sequences include a viral transforming gene (v-fes), derived from cellular proto-oncogene sequences (c-fes) of domestic cats by recombination with feline leukemia virus (FeLV). The v-fes sequences are predicted to encode a polypeptide domain strikingly similar to that specified by the transforming gene (v-fps) of the avian Fujinami sarcoma virus. In addition, the 3′ 0.8 kilobase pairs of v-fes encode amino acid sequences homologous to the carboxy-terminal portion of pp60^src, the transforming protein encoded by the avian Rous sarcoma virus src gene. Thus different feline and avian retroviral transforming genes, all of which encode functionally related proteins with associated tyrosine-specific kinase activities, must be derived from divergent members of the same protooncogene family.     

Changes in amino-terminal sequences of pp60src lead to decreased membrane association and decreased in vivo tumorigenicity

We have suggested previously that the amino-terminal 8 kilodaltons of pp60^src may serve as a structural hydrophobic domain through which pp60^src attaches to plasma membranes. Two isolates of recovered avian sarcoma viruses (rASVs), 1702 and 157, encode pp60^src proteins that have alterations in this amino-terminal region. The rASV 1702 src protein (56 kilodaltons) and the 157 src protein (62.5 kilodaltons) show altered membrane association, and fractionate largely as soluble, cytoplasmic proteins in aqueous buffers, in contrast with the membrane association of more than 80% of the src protein of standard avian sarcoma virus under the identical fractionation procedure. Plasma membranes purified from cells transformed by these rASVs contain less than 10% of the amount of pp60^src found in membranes purified from cells transformed by Rous sarcoma virus or control rASVs. The altered membrane association of these src proteins had little or no effect on the properties of chick embryo fibroblasts transformed in monolayer culture. In contrast, rASV 1702 showed reduced in vivo tumorigenicity compared with Rous sarcoma virus or with other rASVs that encode membrane-associated src proteins. Rous sarcoma virus-induced tumors are malignant, poorly differentiated sarcomas that are lethal to their hosts. rASV 1702 induces a benign, differentiated sarcoma that regresses and is not lethal to its hosts. These data support the role of amino-terminal sequences in the membrane association of pp60^src, and suggest that the amino terminus of pp60^src may have a critical role in the promotion of in vivo tumorigenicity.     

The role of NK cells in tumor growth assessed by induction of their suppressor cells in Japanese quails

In Japanese quails treated with chicken amniotic fluid (ChAmF) which had been previously shown to induce suppressor cells to natural killer (NK) cells, tumors appeared with shortened incubation periods after inoculation with Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) compared with untreated quails. The tumors in ChAmF-treated quails subsequently grew in a similar pattern to those in untreated quails, whereas by challenging with a lower dose of the virus, enhanced tumor growth was observed as well as earlier onset of tumors in ChAmF-treated quails than in untreated ones. This enhancing effect on tumor growth due to suppression of NK-cell activity was transferred to normal quails with spleen cells obtained from ChAmF-treated quails, since RSV-induced tumors appeared earlier in the recipients of ChAmF-treated spleen cells than in those of untreated spleen cells. These findings show that suppression of NK-cell activity by ChAmF administration rendered quails higher susceptibility to tumor induction by SR-RSV challenge. In other words, NK-cell activity was strongly suggested to contribute to the early protection against tumor growth in the system of Rous sarcoma in Japanese quails.     

Universal intracellular transducer ras and its role in the development of drosophila

Ras genes were first identified in the 1960s as transforming oncogenes that caused tumors in rats infected with Harvey and Kirsten sarcoma viruses (Ha-ras and Ki-ras oncogenes, accordingly). Subsequently, transforming ras genes were found in human cancer cells. Further investigations of neuroblastoma cells resulted in the finding of the third ras gene in the human, which was called N-ras. Ras gene products play an important role in the processes of cellular proliferation and differentiation and are controlled by receptor tyrosine kinases. Using drosophila as a model object allowed us to perform a successful genetic analysis while studying the functions of ras genes. Three polytene chromosome bands were detected in D. melanogaster with the help of the v-Ha ras sampling. According to Bridges’ map, all three bands (Dras1, Dras2, Dras3) were mapped to regions 85D, 64B, and 62B of chromosome 3. Among them, only Dras1 has a common origin with ras genes of mammals. Although there are numerous investigations of the role played by ras genes in the development of insects, this problem is still not fully understood. The importance of ras gene variations in the course of the evolutionary process has been insufficiently studied as well. Currently, Ras target proteins are actively identified, their signal pathways, as well as effects of influencing these pathways in the drosophila tissues, are studied in the cells of yeast and mammals. The main functions of Ras protein is in the signaling pathways controlling mutations during drosophila’s morphogenesis and the connections of ras gene with phenotypic symptoms of tumors.     

Phosphorylation of the Mr = 34,000 protein in normal and Rous sarcoma virus-transformed rat fibroblasts

Antiserum was raised against the M_r = 34,000 chick cell protein which may serve as a substrate for the Rous sarcoma virus transforming gene product. The antiserum specifically immunoprecipitated 2 proteins from [³⁵S]methionine labeled Rous sarcoma virus-transformed rat cell extracts (a M_r = 35,000 and a M_r = 38,000 protein). Partial protease treatment revealed these two proteins to be very closely related. The protein of apparent M_r = 38,000 was phosphorylated and the phosphate was present exclusively on tyrosine residues. The effect of epidermal growth factor on phosphorylation of the M_r = 35,000 protein was examined in several normal rat fibroblast cell lines. EGF treatment had no effect on phosphorylation of the M_r = 35,000 protein for any normal cell line and also failed to elevate overall levels of phosphotyrosine.     

The Hypoxia-Inducible Epigenetic Regulators Jmjd1a and G9a Provide a Mechanistic Link between Angiogenesis and Tumor Growth

Hypoxia promotes stem cell maintenance and tumor progression, but it remains unclear how it regulates long-term adaptation toward these processes. We reveal a striking downregulation of the hypoxia-inducible histone H3 lysine 9 (H3K9) demethylase JMJD1A as a hallmark of clinical human germ cell-derived tumors, such as seminomas, yolk sac tumors, and embryonal carcinomas. Jmjd1a was not essential for stem cell self-renewal but played a crucial role as a tumor suppressor in opposition to the hypoxia-regulated oncogenic H3K9 methyltransferase G9a. Importantly, loss of Jmjd1a resulted in increased tumor growth, whereas loss of G9a produced smaller tumors. Pharmacological inhibition of G9a also resulted in attenuation of tumor growth, offering a novel therapeutic strategy for germ cell-derived tumors. Finally, Jmjd1a and G9a drive mutually opposing expression of the antiangiogenic factor genes Robo4, Igfbp4, Notch4, and Tfpi accompanied by changes in H3K9 methylation status. Thus, we demonstrate a novel mechanistic link whereby hypoxia-regulated epigenetic changes are instrumental for the control of tumor growth through coordinated dysregulation of antiangiogenic gene expression.     

Tumor induction by the LTR,v-src,LTR DNA in four B (MHC) congenic lines of chickens

We report that the cloned DNA harboring the long terminal repeat (LTR), v-src, LTR proviral structure is tumorigenic in chickens of the Prague congenic lines. The growth rate of these tumors is by far the highest in the recombinant CC.R1 line, the B haplotype of which is composed of the B-F/L ⁴ and B-G ¹² subregions originating from different naturally occurring haplotypes. Some of the tumors induced by the LTR, v-src, LTR DNA are repeatedly transplantable in syngeneic chickens, maintain unaltered provirus, and express v-src mRNA. Differences in the response to challenge with Rous sarcoma virus (RSV) and LTR, v-src, LTR DNA on a given experimental model are compared and possible involvement of an interaction between B-F/L and B-G region gene is considered. Regression of the LTR, v-src, LTR DNA-induced tumors did not prevent the formation and growth of tumors induced subsequently by RSV.     

Alloantigen system L affects the outcome of rous sarcomas

This study was designed to examine the alloantigen system L effects on Rous sarcomas in three B complex genotypes. The parental stock was 50% Modified Wisconsin Line 3 x White Leghorn Line NIU 4 and 50% inbred Line 6.15-5. Pedigree matings of two B(2)B(5) L(1)L(2) sires to five B(2)B(5) L(1)L(2) dams per sire produced experimental chicks segregating for B and L genotypes. Chicks were inoculated with 20 pock-forming units (pfu) of Rous sarcoma virus (RSV) at 6 weeks of age. Tumors were scored six times over 10 weeks postinoculation after which the tumor scores were used to assign a tumor profile index (TPI) to each chicken. Tumor growth over time and TPI were evaluated by repeated-measures analysis of variance and analysis of variance, respectively. Six trials were conducted with a total of 151 chickens. The major histocompatibility (B) complex affected the responses as the B(2)B(2) and B(2)B(5) genotypes had significantly lower tumor growth over time and TPI than the B(5)B(5) genotype. Separate analyses revealed no significant L system effect in B(2)B(2) or B(2)B(5) backgrounds. However, L genotype significantly affected (P < 0.05) both tumor growth over time and TPI in B(5)B(5) chickens. B(5)B(5) L(1)L(2) birds had TPI significantly lower than B(5)B(5) L(1)L(1) chickens but not B(5)B(5) L(2)L(2). Mortality was lower in the B(5)B(5) L(1)L(2) birds than in B(5)B(5) L(2)L(2) chickens. The L system, or one closely linked, affects the growth and ultimate outcome of Rous sarcomas. The response may depend upon the genetic background as well as MHC type.     

Evidence that the phosphorylation of tyrosine is essential for cellular transformation by Rous sarcoma virus

All cells transformed by Rous sarcoma virus contain levels of phosphotyrosine in protein which are 6–10 fold greater than the very low levels present in uninfected cells. The increase is due largely to modification of cellular polypeptides. The abundance of phosphorylated tyrosines in protein in cells infected with tsLA29, a mutant of Rous sarcoma virus which is temperature-sensitive for cellular transformation, increases to 60% of maximum within 60 min of a shift to the permissive temperature and drops to a level close to that in uninfected cells within 60 min of a shift to the restrictive temperature. In light of the fact that pp60^src phosphorylates tyrosine in vitro, these results suggest strongly that the modification of one or more cellular polypeptides by way of pp60^src is critical for cellular transformation by Rous sarcoma virus. There is, however, no increase in the abundance of phosphotyrosine in protein in mouse cells transformed by Kirsten sarcoma virus, Moloney sarcoma virus, or SV40 virus, in chick embryo cells infected with avian myelocytomatosis virus MC29, and in rat and hamster cells transformed by polyoma virus. Thus increased phosphorylation of tyrosine is neither a universal mechanism of transformation nor an inevitable secondary cellular response to transformation.     

Human Sarcoma Growth Is Sensitive to Small-Molecule Mediated AXIN Stabilization

Sarcomas are mesenchymal tumors showing high molecular heterogeneity, reflected at the histological level by the existence of more than fifty different subtypes. Genetic and epigenetic evidences link aberrant activation of the Wnt signaling to growth and progression of human sarcomas. This phenomenon, mainly accomplished by autocrine loop activity, is sustained by gene amplification, over-expression of Wnt ligands and co-receptors or epigenetic silencing of endogenous Wnt antagonists. We previously showed that pharmacological inhibition of Wnt signaling mediated by Axin stabilization produced in vitro and in vivo antitumor activity in glioblastoma tumors. Here, we report that targeting different sarcoma cell lines with the Wnt inhibitor/Axin stabilizer SEN461 produces a less transformed phenotype, as supported by modulation of anchorage-independent growth in vitro. At the molecular level, SEN461 treatment enhanced the stability of the scaffold protein Axin1, a key negative regulator of the Wnt signaling with tumor suppressor function, resulting in downstream effects coherent with inhibition of canonical Wnt signaling. Genetic phenocopy of small molecule Axin stabilization, through Axin1 over-expression, coherently resulted in strong impairment of soft-agar growth. Importantly, sarcoma growth inhibition through pharmacological Axin stabilization was also observed in a xenograft model in vivo in female CD-1 nude mice. Our findings suggest the usefulness of Wnt inhibitors with Axin stabilization activity as a potentialyl clinical relevant strategy for certain types of sarcomas.     

Cholera toxin treatment stimulates tumorigenicity of Rous sarcoma virus-transformed cells.

Chinese hamster ovary cells transformed by Rous sarcoma virus form tumors poorly in nude mice. Tumorigenicity was markedly stimulated by pretreatment of the cells with cholera toxin, which raises cyclic AMP levels and activates cyclic AMP-dependent protein kinase. Increased tumorigenicity was manifested by a severalfold increase in the rate of tumor formation, as well as earlier appearance and more rapid growth of tumors. In contrast, spontaneously transformed Chinese hamster ovary cells showed decreased tumorigenicity after cholera toxin treatment. The activation of tumorigenic potential in Rous sarcoma virus-transformed Chinese hamster ovary cells by cholera toxin correlated with increased phosphorylation of the viral oncogene product pp60src and stimulation of its tyrosine kinase activity.