Normal Chicken Cells Research Articles

Evaluation of a Recombinant Newcastle Disease Virus Expressing Human IL12 against Human Breast Cancer

The Newcastle disease virus (NDV) strain AF2240 is an avian avulavirus that has been demonstrated to possess oncolytic activity against cancer cells. However, to illicit a greater anti-cancer immune response, it is believed that the incorporation of immunostimulatory genes such as IL12 into a recombinant NDV backbone will enhance its oncolytic effect. In this study, a newly developed recombinant NDV that expresses IL12 (rAF-IL12) was tested for its safety, stability and cytotoxicity. The stability of rAF-IL12 was maintained when passaged in specific pathogen free (SPF) chicken eggs from passage 1 to passage 10; with an HA titer of 29. Based on the results obtained from the MTT cytotoxic assay, rAF-IL12 was determined to be safe as it only induced cytotoxic effects against normal chicken cell lines and human breast cancer cells while sparing normal cells. Significant tumor growth inhibition (52%) was observed in the rAF-IL12-treated mice. The in vivo safety profile of rAF-IL12 was confirmed through histological observation and viral load titer assay. The concentration and presence of the expressed IL12 was quantified and verified via ELISA assay. In summary, rAF-IL12 was proven to be safe, selectively replicating in chicken and cancer cells and was able to maintain its stability throughout several passages; thus enhancing its potential as an anti-breast cancer vaccine.

Detection and Characterization by Immunohistochemistry of the NGcGM3 Ganglioside in the Chicken Animal Model of Ovarian Cancer

In spite of ovarian cancer mortality rates have tended to level off and decrease during the last years in several countries, the disease is still the gynecological cancer with the highest death rate. The current therapies for ovarian cancer include surgery and chemotherapy based on platinum and a taxane. Nevertheless, these therapies are only efficient at the first stages of the disease and the treatments are lack of specificity, further contributing to the high mortality rates of ovarian cancer. The adult laying hen is a very interesting model for studying the development of the epithelial ovarian cancer since hen develops intense ovulation, spontaneous ovarian cancers and metabolic progression similar to women. It is known that normal chicken cells and human cells have no metabolic pathway for NeuGc biosynthesis due to a partial deletion in the gene that encodes CMP-Neu5Ac hydroxylase. Gangliosides are interesting type of molecules, different from proteins that have been identified as tumor antigens. Mammalian cells have these types of glycosphingolipids on their plasmatic membranes. Due to gangliosides are over-expressed in many tumors compared with the corresponding normal tissues, they are considered as very attractive compounds for immunotherapy, attributing to them a very important role in the growth and metastatic tumor processes. The aim of this study is to elucidate by immunohistochemistry the existence of NeuGc-GM3 in hen ovarian cancers. Slides with normal and cancer ovarian tissues from 3.5 years old chickens were incubated with the very specific anti- NeuGcGM3 ganglioside 14F7 Mab for immunohistochemical studies. These studies showed that the 14F7 Mab immunorecognition was mainly evidenced in epithelial ovarian tumors and not in normal tissues. The presence of NeuGc-containing gangliosides in chicken and human epithelial ovarian cancers makes the chicken an interesting and relevant animal model for further experimental and translational investigations on ganglioside based immunotherapy.

Identification of a cis-Element That Determines Autonomous DNA Replication in Eukaryotic Cells

A 36-bp human consensus sequence (CCTMDAWKSGBYTSMAAWTWBCMYTTRSCAAATTCC) is capable of supporting autonomous replication of a plasmid after transfection into eukaryotic cells. After transfection and in vitro DNA replication, replicated plasmid DNA containing a mixture of oligonucleotides of this consensus was found to reiterate the consensus. Initiation of DNA replication in vitro occurs within the consensus. One version, A3/4, in pYACneo, could be maintained under selection in HeLa cells, unrearranged and replicating continuously for >170 cell doublings. Stability of plasmid without selection was high (> or =0.9/cell/generation). Homologs of the consensus are found consistently at mammalian chromosomal sites of initiation and within CpG islands. Versions of the consensus function as origins of DNA replication in normal and malignant human cells, immortalized monkey and mouse cells, and normal cow, chicken, and fruit fly cells. Random mutagenesis studies suggest an internal 20-bp consensus sequence of the 36 bp may be sufficient to act as a core origin element. This cis-element consensus sequence is an opportunity for focused analyses of core origin elements and the regulation of initiation of DNA replication.

GTPase-activating protein interactions with the viral and cellular Src kinases.

GTPase-activating protein (GAP), which regulates the activities of Ras proteins, is implicated in mitogenic signal transduction by growth-factor receptors and oncoproteins with tyrosine kinase activity. Oncogenic viral Src (p60v-src) encoded in Rous sarcoma virus possesses elevated tyrosine kinase activity compared with its nononcogenic normal homolog, cellular Src (p60c-src). To examine molecular interactions between GAP and the two Src kinases, immunoprecipitates of Src or GAP prepared from cell lystates were resolved by gel electrophoresis and analyzed by an immunoblot procedure with antibodies to GAP or Src used as probes. Results suggest that p60c-src is associated with a complex containing GAP in immunoprecipitates from lysates of normal rat and chicken cells. However, GAP is not phosphorylated in p60c-src immunoprecipitates subjected to in vitro kinase reactions. By contrast, GAP undergoes tyrosyl phosphorylation in vitro when immunoprecipitates of p60v-src prepared from transformed cell lysates are incubated with ATP. Our findings suggest that p60v-src and p60c-src associate with complexes containing GAP and provide a biochemical link between both kinases and GAP/Ras signal transduction pathways. These results are consistent with the hypothesis that GAP has a role in mediating normal functions of p60c-src as well as oncogenic activities of p60v-src.

Differential localization of tropomyosin isoforms in cultured nonmuscle cells.

We have previously shown that chicken embryo fibroblast (CEF) cells and human bladder carcinoma (EJ) cells contain multiple isoforms of tropomyosin, identified as a, b, 1, 2, and 3 in CEF cells and 1, 2, 3, 4, and 5 in human EJ cells by one-dimensional SDS-PAGE (Lin, J. J.-C., D. M. Helfman, S. H. Hughes, and C.-S. Chou. 1985. J. Cell Biol. 100: 692-703; and Lin, J. J.-C., S. Yamashiro-Matsumura, and F. Matsumura. 1984. Cancer Cells 1:57-65). Both isoform 3 (TM-3) of CEF and isoforms 4,5 (TM-4,-5) of human EJ cells are the minor isoforms found respectively in normal chicken and human cells. They have a lower apparent molecular mass and show a weaker affinity to actin filaments when compared to the higher molecular mass isoforms. Using individual tropomyosin isoforms immobilized on nitrocellulose papers and sequential absorption of polyclonal antiserum on these papers, we have prepared antibodies specific to CEF TM-3 and to CEF TM-1,-2. In addition, two of our antitropomyosin mAbs, CG beta 6 and CG3, have now been demonstrated by Western blots, immunoprecipitation, and two-dimensional gel analysis to have specificities to human EJ TM-3 and TM-5, respectively. By using these isoform-specific reagents, we are able to compare the intracellular localizations of the lower and higher molecular mass isoforms in both CEF and human EJ cells. We have found that both lower and higher molecular mass isoforms of tropomyosin are localized along stress fibers of cells, as one would expect. However, the lower molecular mass isoforms are also distributed in regions near ruffling membranes. Further evidence for this different localization of different tropomyosin isoforms comes from double-label immunofluorescence microscopy on the same CEF cells with affinity-purified antibody against TM-3, and monoclonal CG beta 6 antibody against TM-a, -b, -1, and -2 of CEF tropomyosin. The presence of the lower molecular mass isoform of tropomyosin in ruffling membranes may indicate a novel way for the nonmuscle cell to control the stability and organization of microfilaments, and to regulate the cell motility.

Phosphorylation of the fibronectin receptor complex in cells transformed by oncogenes that encode tyrosine kinases.

The fibronectin (FN) receptor in avian cells has been characterized previously as a complex of three membrane glycoproteins of about Mr 160,000, Mr 140,000, and Mr 120,000 (simply termed protein band 1, band 2, and band 3, respectively). Monoclonal antibodies to the band 3 protein of the complex prevent FN and laminin binding both in vivo and in vitro and enable the detection of the receptor proteins in the plasma membrane and in adhesion plaques. Association of the FN receptor proteins with the adhesion-plaque protein talin also has been reported. We now find that the band 2 and band 3 proteins in the complex are phosphorylated in Rous sarcoma virus-transformed chicken cells but not in normal chicken cells. Phosphorylation occurs predominantly on tyrosine and is accompanied by a reorganization of the receptor complex in the membrane of the transformed cells. Whereas normal cells contain the FN receptor in focal contacts and cellular processes between cells, v-src-transformed cells exhibit a more diffuse distribution of this receptor. In addition to the viral v-src oncogene, cells transformed by other avian oncogenes that also encode tyrosine kinases (v-fps, v-erbB, and v-yes) also express the receptor complex proteins in the phosphorylated state regardless of whether the transforming protein is detectable in adhesion plaques. These results suggest that the altered FN and laminin receptor proteins may contribute to the transformed phenotype, but their significance and role in the transformed state remain to be established.

Protein product of proto-oncogene c-mil.

Using antipeptide antibodies with specificity for the carboxyl termini of v-raf and v-mil protein products, two proteins with apparent molecular weights of approximately 71,000/73,000 and 215,000 were detected in immunoprecipitates from normal uninfected chicken cells. The 71,000/73,000-molecular-weight protein was identified as the product of the c-mil proto-oncogene by the close structural relationship of its 42,000-molecular-weight carboxyl-terminal domain to the v-mil-encoded domain of the hybrid protein p100gag-mil specified by the avian retrovirus MH2. The amino-terminal domain of the cellular protein is encoded by 5' c-mil sequences that have not been transduced into the genome of MH2. The c-mil protein (p71/73c-mil) was found to be phosphorylated in vivo, and homologous proteins were detected at variable levels in a variety of vertebrate cells, including human cells.

Protein product of proto-oncogene c-mil.

Using antipeptide antibodies with specificity for the carboxyl termini of v-raf and v-mil protein products, two proteins with apparent molecular weights of approximately 71,000/73,000 and 215,000 were detected in immunoprecipitates from normal uninfected chicken cells. The 71,000/73,000-molecular-weight protein was identified as the product of the c-mil proto-oncogene by the close structural relationship of its 42,000-molecular-weight carboxyl-terminal domain to the v-mil-encoded domain of the hybrid protein p100gag-mil specified by the avian retrovirus MH2. The amino-terminal domain of the cellular protein is encoded by 5' c-mil sequences that have not been transduced into the genome of MH2. The c-mil protein (p71/73c-mil) was found to be phosphorylated in vivo, and homologous proteins were detected at variable levels in a variety of vertebrate cells, including human cells.

Pp60c‐src encoded by the proto‐oncogene c‐src is a product of sensory neurons

The advent of recombinant DNA technology has led to the identification in the DNA of normal animal cells of over 30 proto-oncogenes that are homologous to retroviral transforming genes. One of these encodes a protein kinase (pp60c-src) of unknown function, that is preferentially synthesized in brain and neural retina. Here the expression of pp60c-src in the peripheral nervous system was examined in sensory neurons from chick dorsal root ganglia with antisera raised against the transforming protein of Rous sarcoma virus (pp60v-src) expressed in Escherichia coli carrying the cloned v-src gene. This antiserum recognizes pp60c-src specifically in normal chicken cells. Western immunoblotting showed that dorsal root ganglia of stage 30 (day 6.5) chick embryos contained elevated levels of pp60c-src. Immunoperoxidase staining of neuron-enriched cultures prepared from chick dorsal root ganglia showed pp60c-src immunoreactivity in cells with neuronal morphology; flat, fibroblastic cells contained no detectable immunoreactivity. Indirect double immunofluorescence with pp60src antibodies and monoclonal antibodies against the 200-kD subunit of neurofilament protein confirmed that the cells expressing pp60c-src were neurons. Ninety-six percent of the neurofilament-positive cells were immunoreactive with pp60src antibodies, and conversely, all pp60c-src-positive cells were immunoreactive with neurofilament antibodies. pp60c-src immunofluorescence appeared to be distributed over the cell body, processes, and growth cones. These results clearly demonstrate that pp60c-src is a product of neurons and is expressed in sensory neurons in culture.

The ets sequence from the transforming gene of avian erythroblastosis virus, E26, has unique domains on human chromosomes 11 and 21: both loci are transcriptionally active.

Human DNA segments homologous to the ets region from the transforming gene of avian erythroblastosis virus, E26, were molecularly cloned and shown to be closely related to the viral equivalent by hybridization and partial sequence analysis. The transforming gene of E26 has a tripartite origin with the structure delta gag [1.2 kilobases (kb) from the viral gag gene]-myb(0.9 kb from the chicken myb gene)-ets (1.6 kb from the chicken ets gene). Human ets DNA is located on two distinct human chromosomes. The human ets-1 locus on chromosome 11 encodes a single mRNA of 6.8 kb; the second locus, ets-2 on chromosome 21, encodes three mRNAs of 4.7, 3.2, and 2.7 kb. The ets-related sequences of human DNA on chromosomes 11 and 21 are discontiguous, except for a small overlap region encoding 14 amino acids, where 12 are conserved between these two loci. By contrast, the chicken homolog has contiguous ets-1 and ets-2 sequences and is primarily expressed in normal chicken cells as a single 7.5-kb mRNA. We conclude that the ets sequence shared by the virus, the chicken, and humans is likely to contain at least two dissociable functional domains, ets-1 and ets-2. Thus, the tripartite transforming gene of E26 includes four distinct domains that may be functionally relevant for the transforming function of the virus (delta gag, myb, ets-1, and ets-2).

Transcription of three c-myc exons is enhanced in chicken bursal lymphoma cell lines.

The chicken c-myc gene, as defined by its homology to the v-myc gene of MC29 virus, is comprised of two exons. Using the techniques of runoff transcription, primer extension, and S1 nuclease protection, we demonstrate that there is a third c-myc exon of approximately equal to 345 base pairs (bp) located 0.7 kbp upstream of the 5' end of the v-myc homology. This first exon is transcribed and present in myc mRNA in normal chicken cells. We also examined RNA from five cell lines derived from avian leukosis virus-induced bursal lymphomas. In all these lines, the level of transcription of the 2.2- to 2.5-kbp myc mRNA is increased 30- to 60-fold over normal cells. The myc mRNA in four of these lines also contains increased levels of the first noncoding exon, and evidence is presented that the long terminal repeat (LTR) in the vicinity of c-myc is functioning as an enhancer of c-myc transcription rather than as a promoter in several of these cell lines. In two cell lines in which the viral LTR has integrated between the first and second exons in the proper orientation for downstream promotion of myc, the LTR does not exhibit promoter function. The pattern of c-myc transcription observed by others in a vast majority of avian leukosis virus-induced neoplasms is not observed in any of the five cell lines examined.

Characterization of two strains of avian sarcoma virus isolated from avian lymphatic leukosis virus-induced sarcomas

Two replication-defective avian sarcoma viruses, S1 and S2, which were independently isolated from tumors of chickens inoculated with avian lymphatic leukosis virus (LLV) were characterized. The genomes of S1 and S2 contain src-related sequences and are, respectively, about 3.9 and 4.5 kilobases long. pp60 src-related proteins with molecular weights of 62,000 (p62) were detected in cells infected with these viruses, and protein kinase activity was found to be associated with these proteins. No other viral proteins, such as gag, pol, and env gene products, were detected. These results suggested that the c-src sequence in normal chicken cells was incorporated into LLV genomes by recombination at the expense of most of the viral genes to generate highly defective new sarcoma viruses.

Isolation of a cDNA clone complementary to sequences for a 34-kilodalton protein which is a pp60v-src substrate.

We have isolated a partial cDNA clone containing sequences complementary to a mRNA encoding a 34- to 36-kilodalton normal chicken cell protein which is a substrate for pp60v-src kinase activity. Using this 34-kilodalton cDNA clone as a probe, we determined that the size of the 34-kilodalton mRNA was 1,100 nucleotides and the level of the 34-kilodalton RNA was the same in various tissues of mature chickens but was significantly higher in chicken embryo fibroblast cells.

Isolation of a cDNA clone complementary to sequences for a 34-kilodalton protein which is a pp60v-src substrate.

We have isolated a partial cDNA clone containing sequences complementary to a mRNA encoding a 34- to 36-kilodalton normal chicken cell protein which is a substrate for pp60v-src kinase activity. Using this 34-kilodalton cDNA clone as a probe, we determined that the size of the 34-kilodalton mRNA was 1,100 nucleotides and the level of the 34-kilodalton RNA was the same in various tissues of mature chickens but was significantly higher in chicken embryo fibroblast cells.

The differential expression of the cellular src-gene product pp60 src and its phosphokinase activity in normal chicken cells and tissues

Chick embryos of different ages and adult chickens were examined for the expression of pp60 c-src , the normal cellular homolog of the transforming protein of Rous sarcoma virus. In any brain extract, the pp60 c-src kinase activity was always high, whereas muscle extracts of embryos show an age-dependent decrease in kinase activity. Adult animals show either no or barely measurable activity in muscle tissue. In contrast, liver cell extracts of embryos show an age-dependent increase in pp60 c-src kinase activity, with adult chickens displaying the highest activity, very similar to that found in brain extracts. This demonstrates that increased expression of c-src is not necessarily correlated with cell proliferation, but suggests that, at an early stage of differentiation of mesenchymal cells, the relatively high expression of c-src could be responsible, at least in part, for the control of cell metabolism and proliferation.

A putative second cell-derived oncogene of the avian leukaemia retrovirus E26.

The acute avian leukaemia retroviruses AMV and E26 both induce myeloblastosis in vivo and transform myeloblasts in vitro. Both viruses contain the oncogene v-myb first described for AMV. Unlike AMV, E26 has the additional capacity to induce erythroblastosis in vivo and to transform erythroblasts. Previous analyses indicated that the genome of E26 also contained nucleotide sequences distinct from v-myb and unrelated to viral replicative genes. Using a molecularly cloned E26 provirus, we have now identified a novel nucleotide sequence designated v-ets (for E-twenty-six specific) of approximately 1.5 kilobase pairs (kbp) located next to v-myb. v-ets possesses all the structural characteristics of a putative new oncogene: it has a conserved cellular counterpart c-ets which is transcribed in some normal chicken cells as a major 7.5-kb polyadenylated RNA. Although our results now await elucidation of their biological significance, we propose that v-ets could be a new oncogene accounting for the additional transforming properties of E26, or potentiating the transforming properties of the v-myb oncogene.

Development and characterization of monoclonal antibodies to Marek's disease tumor-associated surface antigen.

Four monoclonal antibodies, A35, B94, EB29, and G152, against Marek's disease tumor-associated surface antigen have been developed and their specificities studied against a panel of Marek's disease and lymphoid leukosis primary tumors; Marek's disease, and lymphoid leukosis, and reticuloendotheliosis lymphoblastoid cell lines; and normal chicken cells. A35 and G152 are of the immunoglobulin M class, and B94 and EB29 are of the immunoglobulin G1 subclass.

Isolation and characterization of c-myc, a cellular homolog of the oncogene (v-myc) of avian myelocytomatosis virus strain 29.

The chicken genome contains nucleotide sequences homologous to transforming genes (oncogenes) of a number of avian retroviruses. We have isolated chicken DNA (c-myc) that is homologous to the oncogene (v-myc) of the avian myelocytomatosis virus MC29 and have compared the structures of the cellular and viral genes. Results from restriction endonuclease mapping of c-myc and from analysis of heteroduplexes between the DNAs of the cellular and viral genes show that c-myc is homologous to 1,500 nucleotides in v-myc DNA. This homologous region is interrupted in c-myc by an intron-like sequence of 1,100 nucleotides which is absent from v-myc. Nuclear RNA from normal chicken cells contains at least five species of transcripts from c-myc ranging from 2.5 to 6.5 kilobases in length. By contrast, cytoplasm contains only the 2.5-kilobase c-myc RNA. These features of the c-myc gene and its nuclear transcripts are characteristic of normal cellular genes and suggest that the myc gene is of cellular rather than viral origin. The exons in c-myc may define two functional domains in the gene and may therefore facilitate the dissection of the different oncogenic potentials of the MC29 virus.

The specific interaction of the Rous sarcoma virus transforming protein, pp60 src, with two cellular proteins

Sera from rabbits bearing tumors induced by Rous sarcoma virus (RSV) were previously found to contain antibody to the RSV transforming protein, pp60 src. Two additional transformation-specific phosphoproteins from RSV-transformed avian cells are immunoprecipitated with these sera. These proteins, having molecular weights of 90,000 (pp90) and 50,000 (pp50), are not precipitated from uninfected or transformation-defective virus-infected cells and are not related to any RSV structural proteins. Neither pp50 nor pp90 shares any partial or complete proteolytic cleavage peptides with pp60 src, suggesting that pp90 and pp50 do not represent either a precursor or a cleavage product of pp60 src. Sedimentation analysis of RSV-transformed cell lysates on glycerol gradients revealed that the RSV pp60 src protein is present as two forms, one of which represents the majority (95%) of pp60 src and sediments as a monomer, 60,000 molecular weight protein and the other of which sediments with pp90 and pp50 as an apparent 200,000 molecular weight complex. Lysates from cells transformed by viruses containing a temperature-sensitive defect in the src gene contain a greater percentage of pp60 src associated with pp90 and pp50 under both permissive (35°C) and nonpermissive (41°C) conditions compared to wild-type virus-infected cell lysates. Phosphoserine and phosphotyrosine were found associated with pp60 src molecules that sedimented as a monomer, whereas pp60 src molecules that are complexed with pp90 and pp50 contain phosphoserine and greatly reduced amounts of phosphotyrosine. Only the monomer form of pp60 src is capable of phosphorylating IgG in the immune complex phosphotransferase reaction. Normal uninfected chicken cells contain a protein that shares identical partial proteolytic cleavage peptides with the pp90 protein immunoprecipitated from RSV-transformed cells. This pp90 protein is one of the major cytoplasmic proteins in uninfected cells. Antibody directed against pp90 also immunoprecipitates pp60 src and pp50 from lysates of RSV-transformed chicken cells.

Characterization of the transformation-specific sequences of avian erythroblastosis virus in normal vertebrate cells

We have prepared a complementary DNA probe for the transformation-specific sequences of avian erythroblastosis virus (AEV) (cDNA AEV). This probe was used to characterize the cellular homologs of these sequences. We confirmed previous observations which had shown a wide-spread occurrence of nucleic acid sequences related to cDNA AEV in vertebrates (Roussel et al., Nature (London) 281, 452, 1979). In cells of invertebrates, sequences related to cDNA AEV could not be detected. Thermal denaturation of hybrids between cDNA AEV and chicken DNA revealed about 6% mismatching which is similar to that found with src and its homolog in chicken cells. In nonavian vertebrates cells cDNA AEV-related sequences showed an additional degree of mismatching of 3–4%. This evolutionary divergence of the AEV transformation-specific information is considerably less than that found for src and its cellular counterparts. The cellular sequences homologous to cDNA AEV were found to be located in chicken chromosomes 10–12 by in situ hybridization. Cellular sarc sequences are found in the same chromosome group. It was also shown that the cellular homolog of cDNA AEV in normal chicken cells is transcribed into RNA.