Chicken macrophages synthesize and secrete avidin in culture

It was previously shown that avidin, a glycoprotein secreted in vivo by chicken oviduct, is produced by cultured transformed or damaged chicken embryo fibroblasts [27]. This report demonstrates synthesis and secretion of large amounts of avidin by macrophages isolated from chicken yolk sac. Avidin was secreted to the culture medium as shown by immunoprecipitation of metabolically labeled proteins. In the culture medium of macrophages the avidin concentration (up to 47.5 +/- 0.5 microgram/mg cellular protein) exceeded, in agreement with previous findings, that of fibroblasts (up to 7.3 +/- 0.7 microgram/mg) infected with transforming retroviruses (Rous sarcoma virus, its mutants temperature sensitive for transformation and OK 10 virus). No difference between the macrophage avidin and the egg white avidin was detected by both the heat-induced [14C] biotin exchange assay and immunoblotting (subunit Mr = 15600). By immunofluorescence 10 to 20% of the cells were positive for avidin, independent of the time in culture (1-30 days). The staining pattern varied between dense or granular perinuclear and strong reticulo-granular fluorescence throughout the cytoplasm. Double staining for avidin and the Golgi region by wheat germ agglutinin showed that avidin is concentrated, and might be processed, in the Golgi complex. The production of avidin by macrophages supports a role for avidin in host defence mechanisms.     

Localization of avidin in virus-transformed and damaged chicken embryo fibroblasts by immunofluorescence and the avidin-biotin-peroxidase complex (ABC) technique

Avidin, a high-affinity biotin-binding protein of chicken oviduct, was recently found to be synthesized and secreted by damaged or virus-transformed chicken embryo fibroblasts and by chicken macrophages. We have now localized avidin in fibroblasts that were transformed by Rous sarcoma virus. The cells released to the culture medium up to 12 micrograms avidin per 10(6) cells, as judged by the [14C] biotin-binding method. In immunofluorescence microscopy, avidin was localized to the cytoplasm of transformed and of untransformed damaged cells. Treatment with the ionophore monensin was used to determine whether avidin is processed through the Golgi region, which was localized using rhodamine-labeled wheat germ agglutinin. Under these conditions avidin was largely confined to the Golgi region. At the electron microscopic level avidin could be localized to the endoplasmic reticulum of transformed cells, using anti-avidin antibodies and the avidin-biotin-peroxidase complex (ABC) technique. Biotinyl peroxidase did not stain the endogenous avidin in cell layers processed for light or electron microscopy indicating that its biotin-binding sites were either saturated or denaturated. The possibility that endogenous avidin in tissues or cell cultures may bind biotinylated reagents should be controlled for in techniques involving the avidin-biotin interaction.     

Replication of Reticuloendotheliosis Viruses in Cell Culture: Acute Infection

Replication of reticuloendotheliosis viruses (REV) in cultures of chicken and duck fibroblasts leads to some cell death soon after infection. This cell killing was used to develop a plaque assay for Trager duck spleen necrosis virus (TDSNV) on duck embryo fibroblasts. A normal replicative cell cycle was required for normal virus production and the development of cytopathic effects in chicken cells exposed to TDSNV. The latent period was about two days. Stationary chicken embryo fibroblasts could be infected by REV; DNA synthesis was required, but protein synthesis was not.     

Role of the mitogenic property and kinase activity of p60src in tumor formation by Rous sarcoma virus

Expression of the src gene of Rous sarcoma virus in chicken embryo neuroretinal cells results in morphological transformation and sustained proliferation of this normally resting cell population. PA101 and PA104 are two mutants of Rous sarcoma virus which induce neuroretinal cell proliferation in the absence of morphological transformation. Their mitogenic property is temperature sensitive, and they both encode p60src proteins with low kinase activity. To study the role of the mitogenic function and protein kinase activity of p60src in tumorigenesis, we investigated the oncogenicity of PA101 and PA104. Both mutants were less tumorigenic than wild-type virus when injected into chicks. Tumorigenicity was further assayed by inoculating infected chicken embryo fibroblasts and neuroretinal cells onto the chorioallantoid membrane of embryonated duck eggs. This system provides a nonpermissive and immunodeficient environment for xenogenic cell grafting and allows the study of cell tumorigenicity within a temperature range of 37 to 39.5 degrees C. Chicken embryo fibroblasts and neuroretinal cells infected with PA101 were as tumorigenic as wild type-infected cells at 37 degrees C, but tumor development was significantly reduced at 39.5 degrees C. In contrast, both cell types infected with PA104 displayed sharply reduced tumorigenicity. Cell cultures derived from PA101 tumors induced on the chorioallantoid membrane were similar to the corresponding cells maintained in vitro in terms of morphology, production of plasminogen activator, relative amounts of phosphotyrosine in total cellular proteins, and phosphorylation of 34,000-molecular-weight protein. These results indicate that the expression of the mitogenic function of src does not account per se for cell tumorigenicity and that tumor formation is compatible with low levels of p60src protein kinase activity.     

Association of pp60src and src protein kinase activity with the plasma membrane of nonpermissive and permissive avian sarcoma virus-infected cells.

The intracellular localization of pp60src and src protein kinase activity in avian sarcoma virus (ASV)-infected chicken embryo fibroblasts and transformed and morphologically reverted field vole cells was examined by subcellular fractionation procedures. Fractionation by differential centrifugation of Dounce-homogenized cellular extracts prepared from vole cells showed that 83 to 91% of pp60src sedimented with particulate subcellular components from both transformed and revertant vole cells. A slightly lesser amount (60 to 70%) of pp60src was found associated with the particulate fraction from ASV-infected chicken embryo fibroblasts. The distribution of src protein kinase activity in the cytosol and particulate cell fractions was identical to that of pp60src, indicating no detectable differences in the activity of cytosol- and particulate-associated pp60src. When subcellular components of the cell were fractionated by discontinuous sucrose gradient centrifugation, similar amounts of both pp60src and src protein kinase activity cosedimented with the plasma membrane fractions from both transformed and revertant vole cells, as well as from ASV-infected chicken embryo fibroblasts. src protein kinase activity associated with plasma membrane fractions prepared from vole cells and ASV-infected chicken embryo fibroblasts was resistant to extraction with high salt concentrations, but partial elution was achieved with nonionic detergent. Thus, in both transformed and morphologically reverted vole cells, pp60src is intimately associated with the plasma membrane. Since transforming virus can be rescued from revertant vole cells by fusion to chicken embryo fibroblasts, revertant vole cell pp60src is capable of inducing morphological transformation. Thus, although the data presented herein suggest that transformation requires the association of pp60src with the plasma membrane, the binding of pp60src to the plasma membrane per se is insufficient to induce morphological transformation and requires the additional interaction with a specific target membrane protein which appears to be defective in revertant vole cells.     

Transfection of Chicken Embryo Cells with DNA Extracted from Avian Virus-Producing Neoplastic Cells

DNA isolated from avian virus-producing leukemic myeloblasts induced the production of viruses, but not morphological transformation, in cultivated chicken fibroblasts. The recovered virus had the same biological characteristics as the original avian myeloblastosis virus (AMV) and produced myeloblastosis and nephroblastomas when injected into chickens. Neutralization experiments with chicken anti-AMV-BAI strain A sera showed an antigenic community between the DNA-transfected virus and the original virus. Virus induced in fibroblasts after treatment with DNA from a viral nephroblastic nephroblastoma line only gave nephroblastoma when injected into chicken. Treatment of chicken embryo cells with DNA extracted from normal chicken embryos did not induce viral production.     

Efficient transformation by Prague A Rous sarcoma virus plasmid DNA requires the presence of cis-acting regions within the gag gene.

A region in addition to and outside the long terminal repeats (LTRs) in the gag gene of the Prague A strain of Rous sarcoma virus was found to be essential in cis for efficient cell transformation by cloned viral DNA. Transformation in chicken embryo fibroblasts, which requires infectious virus production and reinfection, was facilitated in cis by sequences between nucleotides 630 and 1659. Efficient transformation of NIH 3T3 cells in which secondary spread of virus is not necessary (as it is in chicken embryo fibroblasts) required sequences between nucleotides 630 and 1149. A src cDNA clone which also lacks this region demonstrated low transformation efficiency, indicating that the role of the cis element cannot be attributed to interference with RNA splicing. The gag gene segment required in cis for transformation, between nucleotides 630 and 1149, could substitute for the simian virus 40 enhancer in either orientation, and cells transfected with Rous sarcoma virus LTR-driven plasmids containing the gag cis element had a two- to threefold increase in steady-state viral RNA levels compared with plasmids lacking this region. Thus, additional cis-acting regulatory elements located outside the viral LTRs may modulate viral gene expression and contribute to the efficiency of cell transformation.     

Dimethyl-10,12-benz(a)acridine; evidence for differential effects on the synthesis of RNA of mammalian or avian fibroblasts and some RNA viruses.

We have studied the differential effect of dimethyl-10,12-benz(a)acridine (DBMAcr) on the synthesis of RNA of chicken or mouse fibroblasts in culture and that of some RNA-containing viruses such as Rous sarcoma virus and Mengovirus. DMBAcr at low concentrations blocks the cell multiplication of both normal and Rous sarcoma virus-transformed chicken fibroblasts in culture; it affects transformed cells more than normal ones. The cell growth inhibiting effect of DMBAcr is reversible after short periods of incubation. DMBAcr depresses the synthesis of cellular DNA and RNA in parallel. Concurrently the synthesis of protein proceedes at a relatively high rate in DMBAcr-treated cultures. Its inhibitory effect on cellular RNA synthesis is mostly due to a block in the formation of 28 S and 18 S ribosomal RNA species; in contrast, the synthesis of 45 S ribosomal RNA precursor is proceeding at almost control rate. Also, the synthesis of heterogeneous nuclear RNA is not blocked by DMBAcr. The production of Rous sarcoma virus in transformed fibroblasts is not affected by DMBAcr. Since this is correlated with persisting high rates of protein and heterogenous nuclear RNA synthesis, the effects of DMBAcr suggest that the synthesis of Rous sarcoma virus-RNA shares the specificity of messenger and heterogeneous nuclear RNA. DMBAcr inhibits the synthesis of viral RNA of Mengovirus under conditions where the synthesis of total cellular RNA is not appreciably depressed, suggesting its differential effect on the DNA-directed and the RNA-directed RNA synthesis.     

An avian retrovirus expressing chicken pp59c-myc possesses weak transforming activity distinct from v-myc that may be modulated by adjacent normal cell neighbors.

We demonstrate that EF168, an avian retrovirus that expresses the chicken pp59c-myc proto-oncogene, transforms quail embryo fibroblasts in vitro. An EF168-transformed quail clone, EF168-28, containing a single provirus, synthesizes several hundred copies of c-myc RNA and expresses elevated levels of the pp59c-myc gene product. The EF168 provirus in EF168-28 was isolated as a molecular clone, and the nucleotide sequence of its c-myc allele was confirmed as identical to that of exons 2 and 3 of the chicken c-myc proto-oncogene. Extended infection of quail embryo fibroblast cultures with EF168 induced a number of in vitro transformation-associated parameters similar to those elicited by the oncogenic v-myc-encoding retrovirus MC29, including alteration of cellular morphology, anchorage-independent growth, and induction of immortalized cell lines. Despite the fact that EF168 and MC29 shared these biological activities, further analysis revealed that EF168 initiated transformation in quail embryo fibroblasts, bone marrow, or adherent peripheral blood cultures 100- to 1,000-fold less efficiently than did MC29. Further, in contrast to MC29-induced foci, EF168 foci were smaller, morphologically diffuse, and less prominent. Analysis of newly infected cells demonstrated efficient expression of EF168 viral RNA in the absence of transformation. These differences suggest that while the pp59v-myc gene product can exert dominant transforming activity on quail embryo fibroblasts, its ability to initiate transformation is distinct from that of the pp110gag-v-myc gene product encoded by MC29 and may be suppressed by adjacent nontransformed cell neighbors.     

Transformation by Rous sarcoma virus: a cellular substrate for transformation-specific protein phosphorylation contains phosphotyrosine

Transformation of chicken embryo fibroblasts by Rous sarcoma virus (RSV) is caused by a single viral gene, src, which encodes a phosphoprotein, pp60src, with the enzymatic activity of a protein kinase. The relative abundance of a 36,000 molecular weight (36K) phosphorylated polypeptide which can be detected by two-dimensional electrophoresis of 32P-labeled phosphoproteins is greatly increased in RSV-transformed fibroblasts. We have reported previously that phosphorylation of the 36K polypeptide is an early event in the process of transformation and that protein synthesis is not required for its appearance. Here we identify a nonphosphorylated 36K polypeptide, present in both uninfected and transformed cells, which is homologous to the 36K phosphoprotein as judged by limited proteolysis and by tryptic peptide mapping. We conclude that the 36K phosphoprotein is generated by phosphorylation of this 36K polypeptide. It has recently been shown that pp60src phosphorylates tyrosine residues in vitro: phosphotyrosine and also phosphoserine are present in the 36K phosphoprotein isolated from RSV-transformed cells. On the basis of these results we propose that the 36K polypeptide present in chicken fibroblasts is a substrate for the protein kinase activity of pp60src. Phosphorylation of this polypeptide may be important in cellular transformation by Rous sarcoma virus.     

Molecular basis of retrovirus adaptation: nucleotide sequence of Rous sarcoma virus adapted to duck cells

The host range of retroviruses is rather complex and specific. It is controlled by the products of viral structural genes that interact with the determinants both on the surface and within the cell. The possibility to infect and transform duck embryo fibroblasts is shown for the Prague strain of chicken Rous sarcoma virus (subgroup C), though virus production in these cells is restricted. However, after the 6th passage the "adapted" virus gave the titre practically the same as it was for chicken embryo fibroblasts. Provirus of RSV adapted to the duck embryo fibroblasts and integrated into host DNA was isolated from the library of nucleotide sequences of duck embryo fibroblasts transformed by this virus. The nucleotide sequence of such provirus was determined. The alterations in gp85 coding region of the env gene which proved to be the result of recombination with endogeneous RAV-0 sequences were shown. The formation of viral particles with rather high titre was induced by the proviral transfection on both chicken and duck embryo fibroblasts. The contribution of the revealed alterations in the genome of transformation active virus and possible participation of its td mutant in the adaptation to the new host are discussed.     

Malignant transformation in Vitro of chinese hamster embryonic fibroblasts with the Schmidt-Ruppin strain of Rous sarcoma virus and karyological analysis of this process

Using a method of cocultivation of embryonic Chinese hamster cells (CHEF) with Rous sarcoma cells and infection of CHEF by RSV-SR, it was possible to obtain malignant transformation of hamster cells. The morphologically altered cells became apparent within 15–36 days. In the cells transformed by cocultivation, the genome of RSV was determined by the method of contact of the transformed cell and the chicken cell in vivo; the malignant character of the transformed cells was demonstrated by transfer to a homologous newborn host. Repeated attempts to detect virus production in transformed Chinese hamster cells failed. Prior to malignant transformation and in early transformed cultures the diploid stem-line was maintained. A slight decrease in the proportion of diploid cells in transformed cultures was revealed in some experiments and is discussed. Prolonged cultivation of these cells, as also of control fibroblasts, shifts the stem-line to the hyperdiploid or hypotetraploid region. The mechanism of malignant transformation by RSV is discussed with regard to the action of the viral genome and alteration of the genetic make-up of the cell by the virus.     

Calmodulin and Ca2+ in normal and transformed cells

Numerous lines of evidence implicate calcium and calmodulin (CaM) as regulators of cell growth and functional differentiation. In light of this evidence, several studies of the possible involvement of the CaM system in cellular transformation by RNA and DNA tumor viruses have been carried out. This paper summarizes the evidence linking calcium and CaM to the regulation of cell growth and critically examines the evidence that increases in CaM levels occur in transformed versus normal cells. A nontraumatic method for synchronizing both normal and transformed chick fibroblasts is presented. This method is utilized in a comparison of CaM level throughout the cell cycle of Rous sarcoma virus transformed and normal chick embryo fibroblasts. These studies best support the hypothesis that the observed differences in CaM levels between transformed and normal cultures under optimal growth conditions may largely reflect differences in the proportion of cells in a dividing versus a nondividing state.     

Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms.

Transformation of both rat and chicken fibroblasts by the src oncogene leads to a four- to fivefold increase in the rate of glucose transport and in the level of the glucose transporter protein. We have previously shown that, with chicken embryo fibroblasts, transformation leads to a reduction in the rate of degradation of the transporter, with little or no increase in the rate of its biosynthesis. We now show that, with the rat-1 cell line, the opposite result was obtained. src-induced transformation led to an increase in transporter biosynthesis, with little effect on turnover. A src-induced increase in transporter mRNA entirely accounted for the increase in biosynthesis of the protein. By contrast, in chicken embryo fibroblasts, the level of transporter mRNA was low and was not induced to rise by src transformation. Thus, src induced an increase in the level of the glucose transport protein by fundamentally different mechanisms in chicken embryo fibroblasts and rat-1 cells. To test whether this difference was due to rat-1 cells being an immortalized cell line, we measured transporter mRNA levels in primary fibroblast cultures from rat embryos and in parallel cultures transformed by src. Transporter mRNA was inducible by src in these cells. Thus, the difference in mRNA inducibility between chicken and rat cells is not due to immortalization.     

Conditions leading to the establishment of the N (a gene dependent) and A (a gene independent) transformed states after polyoma virus infection of rat fibroblasts.

Infection of normal rat fibroblasts (FR 3T3) with the early tsa mutant of polyoma virus may lead to either the A or the N phenotype, tsa-A transformants, originally derived by agar selection, are not temperature dependent for maintenance of the transformed phenotype, whereas tsa-N transormants revert at high temperature to normal growth control. A transformants did not result from an independent cellular mutation selected in agar medium, but rather from a transformation process distinct from that leading to the N state. It occurred in both liquid and agar media when the infected cells were maintained under growth-restricting conditions, such as absence of anchorage and contact inhibition at confluency. N transformation occurred in cells maintained in active growth after virus infection (sparse cultures on a solid substratum). Physiological conditions during a critical period after virus infection thus appear to be a crucial parameter of the transformation process.     

Transformation of mammalian fibroblasts and macrophages in vitro by a murine retrovirus encoding an avian v-myc oncogene

A murine retrovirus which expresses the avain v-myc OK10 oncogene was constructed. The virus, denoted MMCV, readily transforms fibroblasts of established lines, such as mouse NIH/3T3 and rat 208F cells, to anchorage-independent growth in agarose. The virus also transforms primary mouse cells: (i) virus-infected macrophages are induced to form large colonies in semi-solid media, and can easily be expanded into mass cultures; (ii) MMCV-infected fibroblastic cells from mouse limb buds undergo morphological transformation and grow in semi-solid medium. MMCV thus transforms both mouse fibroblastic cells and macrophages in vitro, in a fashion similar to the v-myc-containing avian viruses in chicken cells. The possibility of introducing a transforming myc gene into mammalian cells by virus infection provides a novel approach for studying the mechanism of myc transformation in cells from many lineages.     

Marker rescue of endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase.

Endogenous cellular genetic information related to the avian leukosis virus gene encoding RNA-directed DNA polymerase was studied, using a marker rescue assay to detect biological activity of subgenomic fragments of virus-related DNAs of uninfected avian cells. Recipient cultures of chicken embryo fibroblasts were treated with sonicated DNA fragments and were infected with a temperature-sensitive mutant of Rous sarcoma virus that encoded a thermolabile DNA polymerase. Wild-type progeny viruses were isolated by marker rescue with fragments of DNA of uninfected chicken, pheasant, quail, and turkey cells. The DNAs of these uninfected avian cells, therefore, appeared to contain endogenous genetic information related to the avian leukosis virus DNA polymerase gene.     

Assay of noninfectious fragments of DNA of avian leukosis virus-infected cells by marker rescue.

A marker rescue assay of noninfectious fragments of avian leukosis virus DNAs is describe. DNA fragments were prepared either by sonication of EcoRI-digestion of DNAs of chicken cells infected with wild-type Rous sarcoma virus, with a nontransforming avian leukosis virus, and with a mutant of Rous sarcoma virus temperature sensitive for transformation. Recipient cultures of chicken embryo fibroblasts were treated with noninfectious DNA fragments and infected with temperature-sensitive mutants of Rous sarcoma virus defective in DNA polymerase or in an internal virion structural protein. Wild-type progeny viruses which replicated at the nonpermissive temperature were isolated. Some of the wild-type progeny acquired both the wild-type DNA polymerase and the subgroup specificity of the Rous sarcona virus strain used for preparation of sonicated or EcoRI-digested DNA fragments. Therefore the genetic markers for DNA polymerase and envelope were linked and appeared to be located on the same EcoRi fragment of the DNA of Rous sarcoma virus-infected cells.