BACKGROUND: Virus-induced cellular genetic modifications result in the development of many human cancers. METHODS: In our experiments, we used the RVP3 cell line, which produce primary mouse virus-induced sarcoma in 100% of cases. Inbreed 4-week-old female C57BL/6 mice were injected subcutaneously in the interscapular region with RVP3 cells. Three groups of mice were used. For treatment, one and/or two intravenous injections of a complex of small non-coding RNAs (sncRNAs) a-miR-155, piR-30074, and miR-125b with a 2-diethylaminoethyl-dextran methyl methacrylate copolymer (DDMC) delivery system were used. The first group consisted of untreated animals (control). The second group was treated with one injection of complex DDMC/sncRNAs (1st group). The third group was treated with two injections of complex DDMC/sncRNAs (2nd group). The tumors were removed aseptically, freed of necrotic material, and used with spleen and lungs for subsequent RT-PCR and immunofluorescence experiments, or stained with Leishman-Romanowski dye. RESULTS: As a result, the mice fully recovered from virus-induced sarcoma after two treatments with a complex including the DDMC vector and a-miR-155, piR-30074, and miR-125b. In vitro studies showed genetic and morphological transformations of murine cancer cells after the injections. CONCLUSIONS: Treatment of virus-induced sarcoma of mice with a-miR-155, piR-30074, and miR-125b as active component of anti-cancer complex and DDMC vector as delivery system due to epigenetic-regulated transformation of cancer cells into cells with non-cancerous physiology and morphology and full recovery of disease.

• Keywords
• DDMC vector, Epigenetic therapy, Mice, Sarcoma, Small non-coding RNAs, Src tyrosine kinase,
• Publication type
• Journal Article MeSH

This article summarizes the essential steps in understanding the chicken Rous sarcoma virus (RSV) genome association with a nonpermissive rodent host cell genome. This insight was made possible by in-depth study of RSV-transformed rat XC cells, which were called virogenic because they indefinitely carry virus genetic information in the absence of any infectious virus production. However, the virus was rescued by association of XC cells with chicken fibroblasts, allowing cell fusion between both partners. This and additional studies led to the interpretation that the RSV genome gets integrated into the host cell genome as a provirus. Study of additional rodent virogenic cell lines provided evidence that the transcript of oncogene v-src can be transmitted to other retroviruses and produce cell transformation by itself. As discussed in the text, two main questions related to nonpermissiveness to retrovirus infection remain to be solved. The first is changes in the retrovirus envelope gene allowing virus entry into a nonpermissive cell. The second is the nature of the permissive cell functions required by the nonpermissive cell to ensure infectious virus production. Both lines of investigation are being pursued.

• Keywords
• cell transformation, nonpermissiveness to virus infection, virus integration, virus rescue,
• MeSH
• Cell Line MeSH
• Cell Fusion * MeSH
• Genome, Viral genetics   MeSH
• Gene Products, env genetics   MeSH
• Rats MeSH
• Chickens virology   MeSH
• Oncogene Protein pp60(v-src) genetics   MeSH
• Proviruses genetics   growth & development   MeSH
• Cell Transformation, Viral MeSH
• Rous sarcoma virus genetics   growth & development   MeSH
• Animals MeSH
• Check Tag
• Rats MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Gene Products, env MeSH
• Oncogene Protein pp60(v-src) MeSH

The group of closely related avian sarcoma and leukosis viruses (ASLVs) evolved from a common ancestor into multiple subgroups, A to J, with differential host range among galliform species and chicken lines. These subgroups differ in variable parts of their envelope glycoproteins, the major determinants of virus interaction with specific receptor molecules. Three genetic loci, tva, tvb, and tvc, code for single membrane-spanning receptors from diverse protein families that confer susceptibility to the ASLV subgroups. The host range expansion of the ancestral virus might have been driven by gradual evolution of resistance in host cells, and the resistance alleles in all three receptor loci have been identified. Here, we characterized two alleles of the tva receptor gene with similar intronic deletions comprising the deduced branch-point signal within the first intron and leading to inefficient splicing of tva mRNA. As a result, we observed decreased susceptibility to subgroup A ASLV in vitro and in vivo. These alleles were independently found in a close-bred line of domestic chicken and Indian red jungle fowl (Gallus gallus murghi), suggesting that their prevalence might be much wider in outbred chicken breeds. We identified defective splicing to be a mechanism of resistance to ASLV and conclude that such a type of mutation could play an important role in virus-host coevolution.

• MeSH
• Alpharetrovirus genetics   physiology   MeSH
• Genetic Predisposition to Disease * MeSH
• Introns MeSH
• Chickens genetics   metabolism   virology   MeSH
• Molecular Sequence Data MeSH
• Poultry Diseases genetics   metabolism   virology   MeSH
• Avian Proteins genetics   metabolism   MeSH
• Sarcoma, Avian genetics   metabolism   virology   MeSH
• Amino Acid Sequence MeSH
• Base Sequence MeSH
• Sequence Deletion * MeSH
• RNA Splicing * MeSH
• Receptors, Virus genetics   metabolism   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Avian Proteins MeSH
• Tva receptor MeSH Browser
• Receptors, Virus MeSH

Metastatic spreading of cancer cells is a highly complex process directed primarily by the interplay between tumor microenvironment, cell surface receptors, and actin cytoskeleton dynamics. To advance our understanding of metastatic cancer dissemination, we have developed a model system that is based on two v-src transformed chicken sarcoma cell lines-the highly metastatic parental PR9692 and a non-metastasizing but fully tumorigenic clonal derivative PR9692-E9. Oligonucleotide microarray analysis of both cell lines revealed that the gene encoding the transcription factor EGR1 was downregulated in the non-metastatic PR9692-E9 cells. Further investigation demonstrated that the introduction of exogenous EGR1 into PR9692-E9 cells restored their metastatic potential to a level indistinguishable from parental PR9692 cells. Microarray analysis of EGR1 reconstituted cells revealed the activation of genes that are crucial for actin cytoskeleton contractility (MYL9), filopodia formation (MYO10), the production of specific extracellular matrix components (HAS2, COL6A1-3) and other essential pro-metastatic abilities.

• MeSH
• Cell Adhesion MeSH
• Cell Line MeSH
• Cytoskeleton metabolism   MeSH
• Phenotype MeSH
• Kinetics MeSH
• Chickens MeSH
• Neoplasm Metastasis genetics   MeSH
• Cell Transformation, Neoplastic genetics   pathology   MeSH
• Oncogene Protein pp60(v-src) genetics   metabolism   MeSH
• Cell Movement MeSH
• Cell Proliferation MeSH
• Early Growth Response Protein 1 genetics   metabolism   MeSH
• Gene Expression Regulation, Neoplastic MeSH
• Sarcoma genetics   pathology   MeSH
• Gene Expression Profiling MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Oncogene Protein pp60(v-src) MeSH
• Early Growth Response Protein 1 MeSH

Retroviruses and retrovirus-derived vectors integrate nonrandomly into the genomes of host cells with specific preferences for transcribed genes, gene-rich regions, and CpG islands. However, the genomic features that influence the transcriptional activities of integrated retroviruses or retroviral vectors are poorly understood. We report here the cloning and characterization of avian sarcoma virus integration sites from chicken tumors. Growing progressively, dependent on high and stable expression of the transduced v-src oncogene, these tumors represent clonal expansions of cells bearing transcriptionally active replication-defective proviruses. Therefore, integration sites in our study distinguished genomic loci favorable for the expression of integrated retroviruses and gene transfer vectors. Analysis of integration sites from avian sarcoma virus-induced tumors showed strikingly nonrandom distribution, with proviruses found prevalently within or close to transcription units, particularly in genes broadly expressed in multiple tissues but not in tissue-specifically expressed genes. We infer that proviruses integrated in these genomic areas efficiently avoid transcriptional silencing and remain active for a long time during the growth of tumors. Defining the differences between unselected retroviral integration sites and sites selected for long-terminal-repeat-driven gene expression is relevant for retrovirus-mediated gene transfer and has ramifications for gene therapy.

• MeSH
• Chromosomes virology   MeSH
• Gene Expression MeSH
• Genetic Therapy methods   MeSH
• Genetic Vectors MeSH
• Virus Integration * MeSH
• Chickens MeSH
• Proviruses genetics   physiology   MeSH
• Sarcoma, Avian virology   MeSH
• Avian Sarcoma Viruses genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

We have examined the chicken TP53 tumor suppressor gene in v-src-transformed chicken tumor cells by reverse transcriptase-polymerase chain reaction and deoxyribonucleic acid (DNA) sequencing. Initially, we have detected frequent deletions of variable length in both DNA-binding and oligomerization domains of the TP53 in late as well as early in vitro passages of the chicken tumor cell line PR9692. This tumor cell line shows an immortal phenotype and acquires a metastatic potential that is unique in our experimental model of v-src-induced tumors in congenic chickens. Deletions in TP53 were also detected in an early passage of parallel in vivo subculture of the original v-src-induced tumor. In this case, tumor cells underwent replicative senescence later in tissue culture. Our results suggest that extensive deletions are efficient mechanisms of TP53 inactivation, occurring as early events during the immortalization of v-src-transformed chicken cells. Tumor cells with altered TP53 might, however, still be susceptible to growth control mechanisms, leading to withdrawal from the mitotic cycle in the early stage of the tumor lifeline.

• MeSH
• Genes, p53 * MeSH
• Genes, src * MeSH
• Chickens genetics   MeSH
• Neoplasm Metastasis MeSH
• Molecular Sequence Data MeSH
• Cell Transformation, Neoplastic * MeSH
• Cell Line, Tumor MeSH
• Base Sequence MeSH
• Sequence Alignment MeSH
• Cell Line, Transformed MeSH
• Gene Silencing MeSH
• Animals MeSH
• Check Tag
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH

CpG islands are important in the protection of adjacent housekeeping genes from de novo DNA methylation and for keeping them in a transcriptionally active state. However, little is known about their capacity to protect heterologous genes and assure position-independent transcription of adjacent transgenes or retroviral vectors. To tackle this question, we have used the mouse aprt CpG island to flank a Rous sarcoma virus (RSV)-derived reporter vector and followed the transcriptional activity of integrated vectors. RSV is an avian retrovirus which does not replicate in mammalian cells because of several blocks at all levels of the replication cycle. Here we show that our RSV-derived reporter proviruses linked to the mouse aprt gene CpG island remain undermethylated and keep their transcriptional activity after stable transfection into both avian and nonpermissive mammalian cells. This effect is most likely caused by the protection from de novo methylation provided by the CpG island and not by enhancement of the promoter strength. Our results are consistent with previous finding of CpG islands in proximity to active but not inactive proviruses and support further investigation of the protection of the gene transfer vectors from DNA methylation.

• MeSH
• Adenine Phosphoribosyltransferase genetics   MeSH
• Cell Line virology   MeSH
• CpG Islands * MeSH
• Defective Viruses genetics   MeSH
• DNA, Viral chemistry   genetics   MeSH
• DNA (Cytosine-5-)-Methyltransferases metabolism   MeSH
• Sarcoma, Experimental genetics   virology   MeSH
• Fibroblasts virology   MeSH
• Transcription, Genetic * MeSH
• Genetic Vectors genetics   physiology   MeSH
• Virus Integration MeSH
• Terminal Repeat Sequences MeSH
• Cricetinae MeSH
• Mesocricetus MeSH
• Chick Embryo MeSH
• DNA Methylation MeSH
• Mice MeSH
• Proviruses genetics   MeSH
• Gene Expression Regulation, Viral * MeSH
• Virus Replication MeSH
• Genes, Reporter MeSH
• Gene Silencing * MeSH
• Avian Sarcoma Viruses genetics   physiology   MeSH
• Animals MeSH
• Check Tag
• Cricetinae MeSH
• Chick Embryo MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Comparative Study MeSH
• Names of Substances
• Adenine Phosphoribosyltransferase MeSH
• DNA, Viral MeSH
• DNA (Cytosine-5-)-Methyltransferases MeSH

The growth pattern (progression/regression) of v-src DNA- and Rous sarcoma virus (RSV)-induced tumors was analogous on a panel of inbred chicken lines. The decisive role of the major histocompatibility complex [Mhc(B)] alleles in resistance to the progression of these tumors was formally proved in segregating backcross populations. The immune mechanism of tumor regression was demonstrated by both in vivo and in vitro assays. A protective effect of v-src-specific immunity against RSV challenge was shown in Rous sarcoma regressor, line CB (B12/B12). Immune cells from regressors of v-src DNA-induced tumors can protect syngeneic hosts from the development of tumor after challenge with both v-src DNA and RSV. Suppression of RSV-induced tumor cell growth in vitro was also achieved by the use of cocultivation with spleen cells from chickens in which v-src DNA-induced tumors had regressed. This in vitro sarcoma-specific response was Mhc(B)-restricted. Chickens of the congenic Rous sarcoma progressor line CC (B4/B4) are sometimes able to regress v-src DNA-induced tumors, but immune cells can only slow the growth of v-src DNA-induced tumors in syngeneic hosts. This suggests that the primary reason for the susceptibility of CC chickens is a weak v-src-specific immune response. Furthermore, some of the v-src DNA-induced tumors were transplantable across the Mhc(B) barrier. The growth of tumor allografts was able to be facilitated when immunological tolerance to the B-F/L region antigens (class I and class II) had been established. This demonstrated that a high tumorigenicity of the transplantable tumor was not due to the lack of Mhc(B) antigens on tumor cells.

• MeSH
• DNA, Viral genetics   MeSH
• Neoplasms, Experimental immunology   MeSH
• Genes, src * MeSH
• Major Histocompatibility Complex * MeSH
• Inbreeding MeSH
• Chickens immunology   MeSH
• Oncogene Protein pp60(v-src) immunology   MeSH
• Repetitive Sequences, Nucleic Acid MeSH
• Cell Transformation, Viral MeSH
• Avian Sarcoma Viruses genetics   MeSH
• Animals MeSH
• Check Tag
• Male MeSH
• Female MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• DNA, Viral MeSH
• Oncogene Protein pp60(v-src) MeSH