Biomedical subjects

P C Balduzzi

Publications and source records attributed to P C Balduzzi.

At least 19 recordsLinked to original sources

PubMed1991

Modulation of retinal differentiation by oncogenes: effect of the v-src gene on expression of choline acetyltransferase and glutamine synthetase.

Expression of the protooncogene c-src in chick neural retina is developmentally regulated and associated with neural differentiation. In the present study, chick neural retina (NR) cell cultures from 7 day embryos were exposed to the exogenous src oncogene, the c-src counterpart, to establish the effect of expression of v-src on specific retinal cellular differentiation. NR cells from 7 day chick embryos were placed in monolayer or rotation culture and infected with Rous sarcoma virus (RSV) containing a single transforming gene. Other cultures were infected with a transforming defective mutant of RSV which still possesses mitogenic activity for NR cells. While control cultures showed typical neuronal and Muller cell morphologies at the light and electron microscopic level, NR cells infected with RSV exhibited dramatic morphological alterations in monolayer culture and cell aggregates. However, the mutant src gene induced mitosis without accompanying transforming properties. When aggregate cultures were treated with hydrocortisone to induce glutamine synthetase (GS) expression in Muller cells, control cultures showed the typical immunofluorescence pattern of GS staining, while RSV infected cultures showed no GS fluorescence. Cultures infected with mutant RSV showed some staining for GS. In contrast, choline acetyltransferase activity was shown to increase in both monolayer and aggregate cultures of retinal cells following v-src expression. These data indicate that the presence of excess v-src in differentiating cultures of NR inhibits the expression of some neural specific enzymes and enhances the presence of other specific proteins. Moreover, continually growing cultures of oncogene-altered retinal cells may be useful as models to study gene expression in development of the nervous system.

Animals↗

PubMed1987

Infection of neuroretinal cells in vitro by avian sarcoma viruses UR1 and UR2: transformation, cell growth stimulation, and changes in transducin levels.

Infection in vitro of differentiating chick embryo neuroretinal cells with avian sarcoma viruses UR1 and UR2 results in mitogenic stimulation and morphologic conversion of both support neuronal cells. This was shown by the continuous propagation of transformed cells for over 4 months and growth of reaggregated colonies in liquid medium as well as in soft agar. Production of the transforming proteins p 150 gag-fps and p68 gag-ros of UR1 and UR2, respectively, was similar to that of transformed chick embryo fibroblasts, as judged from in vitro kinase activity assays. The two protein subunits, T beta and T gamma, but not T alpha of the GTP binding protein transducin, found in the retina of many animal species, were present in control neuroretinal cells. Infection with Rous sarcoma virus or UR2 resulted in an inhibition of T gamma synthesis and enhancement of T beta-like protein production.

Animals↗

PubMed1986

Transfection and recombination with molecularly cloned derivatives of avian sarcoma virus UR2.

A cloned version of avian sarcoma virus UR2, plasmid pKD6, which includes the full, nonpermuted proviral sequence between two LTR regions, has been prepared. The plasmid is biologically active in transfection experiments, even when intact. Two transformation-defective mutants with nonoverlapping deletions within the transforming gene ros were constructed from pKD6. These mutants recombine to produce transforming virus when mixed DNA from both is used to transfect chick embryo fibroblasts along with helper virus DNA. However, recombination was not readily detected when cells were coinfected with fluids harvested from cultures separately transfected with DNA from each mutant. This, and marker rescue experiments with a temperature-sensitive mutant of UR2 defective in transformation but able to replicate, suggest that deletion mutants of UR2 do not propagate efficiently.

Animals↗

PubMed1986

The human c-ros gene (ROS) is located at chromosome region 6q16----6q22.

The human homolog, c-ros, of the transforming gene, v-ros, of the avian sarcoma virus, UR2, has been isolated from a human genomic library. A single-copy fragment from the human c-ros genomic clone has been used to map the human c-ros homolog (ROS) to human chromosome region 6q16----6q22 by somatic cell hybrid analysis and chromosomal in situ hybridization. Thus, the c-ros gene joins the c-myb oncogene, which is distal to the c-ros gene on the long arm of human chromosome 6, as a candidate for involvement in chromosome 6q deletions and rearrangements seen in various malignancies.

Chromosome Mapping↗

PubMed1986

Specific inhibition of tyrosine kinase activity by an antibody to the v-ros oncogene product.

Antibodies present in two peritoneal exudates of rats bearing abdominal tumors induced by UR2-transformed rat cells were characterized. The ability to immunoprecipitate p68gag-ros and to inhibit the protein and phospholipid kinase activities of this protein was investigated. One of the exudates specifically inhibited tyrosyl phosphorylation by p68gag-ros but not the activity of other known tyrosyl kinases, such as p150gag-fps of UR1 avian sarcoma virus, p60src, and the insulin receptor. It precipitated p68gag-ros but not Pr76 or other gag-related proteins from UR2-infected cells. Phosphorylation of phosphatidylinositol was not affected by this exudate, suggesting that this activity is not intrinsic to p68gag-ros. Another exudate precipitated p68gag-ros but not gag-related proteins from UR2-infected cells or p140gag-fps from Fujinami sarcoma virus-infected cells. These results demonstrated that the antibodies in these exudates recognized epitopes present in the ros portion of the fused protein p68gag-ros, but only one of the two exudates inhibited the intrinsic tyrosyl kinase of p68gag-ros.

Animals↗

PubMed1984

Changes in the synthesis and phosphorylation of cellular proteins in chick fibroblasts transformed by two avian sarcoma viruses.

35S- and 32P-labeled proteins from control chick embryo fibroblasts and from fibroblasts transformed by UR2 sarcoma virus, or by a temperature-sensitive mutant (tsLA29) of Rous sarcoma virus, were separated by two-dimensional electrophoresis on giant gels to detect transformation-specific changes in protein synthesis and total phosphorylation. A nontransforming avian retrovirus, UR2-associated virus (UR2AV), was also studied. Virus-coded proteins appear in whole cell lysates of all infected cells. The structural proteins can be identified by comparison with proteins immunoprecipitated with antivirus serum. The transforming proteins pp60src and p68ros, present in cells transformed with Rous sarcoma virus and UR2, respectively, are phosphorylated in vivo. Eighteen increases and eight decreases in cellular phosphoproteins are associated with transformation, and revert toward normal levels when cells infected with tsLA29 are incubated at 42 degrees C. These changes are more extensive than previously reported, but none represent new phosphorylations, since all phosphoproteins seen in transformed cells also appear to be phosphorylated to a certain extent in control cells. Fifteen cellular proteins show increased relative rates of synthesis apparently related either to transformation or to growth at 42 degrees C. Four other proteins are increased exclusively in cells incubated at 42 degrees C, but not at 37 degrees C, whether transformed or not. Eleven additional increases in the synthesis of cellular proteins, many quite large, and one seemingly a de novo induction, appear to be specific for transformation. These changes occur in cells transformed by either UR2 or Rous sarcoma virus at 37 degrees C, do not occur with UR2AV infection, and tend to revert in cells infected with tsLA29 incubated at 42 degrees C. These 11 changes may represent increases in cellular gene expression that are related specifically to the maintenance of the transformed state.

Animals↗

PubMed1984

Cytoskeletal changes induced by two avian sarcoma viruses: UR2 and Rous sarcoma virus.

UR2-transformed cells were examined by immunofluorescence and compared to control cells and cells transformed by Rous Sarcoma Virus (RSV). Actin and tubulin which are normally depolymerized in RSV-transformed cells appeared to be unaffected by UR2 transformation. Cell surface fibronectin which is normally lost from RSV-infected cells, appears more abundantly on UR2-transformed cells than on normal cells. Vinculin was shown to be in adhesion plaques in UR2-transformed cells as well as in control fibroblasts but diffuse in the cytoplasm of RSV-transformed cells. Polyacrylamide gel electrophoresis of [35S]methionine-labeled fibronectin and vinculin immunoprecipitated from lysates of normal and transformed cells indicated that cell associated fibronectin synthesized during the labeling period is reduced by 60% in RSV-transformed cells but occurs in normal amounts in UR2-transformed cells. However, immunoprecipitation of radiolabeled fibronectin released in supernatant fluids of normal and transformed cells showed a decreased amount of fibronectin in fluids from UR2-transformed cells, but a considerable increase in the medium from RSV-infected cells as compared to uninfected cultures. These data suggest that more fibronectin binds to the surface of UR2-transformed cells then to normal cells, but is readily released from RSV-transformed cells. Vinculin was reduced by about 50% of normal levels in both RSV- and UR2-transformed cells. Immunofluorescence studies using antibody to virion structural proteins (gag) show that the nuclei of UR2-transformed cells are not fluorescent. This indicates a cytoplasmic location or membrane association for p68ros, the transforming protein of UR2, which contains gag determinants. Overall, these data suggest that changes in the major cytoskeletal proteins of fibroblasts are not essential for the neoplastic properties of cells but are rather a phenotypic expression of transformation, since UR2, which causes tumors in vivo, induces only minor cytoskeletal alterations of cells transformed in vitro.

Actins↗

PubMed1984

Transforming protein of avian sarcoma virus UR2 is associated with phosphatidylinositol kinase activity: possible role in tumorigenesis.

The transforming protein of avian sarcoma virus UR2, p68v-ros, has an associated tyrosine-specific protein kinase activity similar to that of p60v-src and several other oncogene products. However, this activity has not been linked unequivocally to transformation, and the physiological action of these proteins remains in doubt. We now have found that immunoprecipitated p68v-ros also is associated with phosphatidylinositol (PtdIns) kinase (ATP:PtdIns 4-phosphotransferase, EC 2.7.1.67) activity. PtdIns 4,5-bisphosphate [PtdIns(4,5)P2] specifically inhibits both this activity and the autophosphorylation of p68v-ros. Moreover, cells transformed by UR2 showed significant increases in 32P-labeling of PtdIns 4-phosphate (PtdIns4P) and PtdIns(4,5)P2 and in the formation of their catabolites, inositol 1,4-bisphosphate and inositol 1,4,5-trisphosphate, as compared to uninfected cells. These results suggest that a physiologically relevant function of oncogene kinases might be the phosphorylation of PtdIns and that increased turnover of PtdIns4P and PtdIns(4,5)P2 might play a role in transformation by increasing the formation of diacylglycerol, a catabolite of polyphosphoinositides that activates kinase C. This protein copurifies with the phorbol ester receptor, and its activation is likely to be intimately linked with mitogenesis. This hypothesis suggests a mechanism whereby certain oncogene proteins might cause the unrestricted growth typical of transformed cells and could explain why tumor promoters mimic many of the effects of transformation.

1-Phosphatidylinositol 4-Kinase↗

PubMed1982

Genetic structure and transforming sequence of avian sarcoma virus UR2.

We have recently shown that a newly isolated avian sarcoma virus, UR2, is defective in replication and contains no sequences homologous to the src gene of Rous sarcoma virus. In this study, we analyzed the genetic structure and transforming sequence of UR2 by oligonucleotide fingerprinting. The sizes of the genomic RNAs of UR2 and its associated helper virus, UR2AV, were determined to be 24S and 35S, respectively, by sucrose gradient sedimentation. The molecular weight of the 24S UR2 genomic RNA was estimated to be 1.1 x 10(6), corresponding to 3,300 nucleotides, by gel electrophoresis under the native and denatured conditions. RNase T1 oligonucleotide mapping indicated that UR2 RNA contains seven unique oligonucleotides in the middle of the genome and shares eight 5'- and six 3'-terminal oligonucleotides with UR2AV RNA. From these data, we estimated that UR2 RNA contains a unique sequence of about 12 kilobases in the middle of the genome, and contains 1.4 and 0.7 kilobases of sequences shared with UR2AV RNA at the 5' and 3' ends, respectively. Partial sequence analysis of the UR2-specific oligonucleotides by RNase A digestion revealed that there are no homologous counterparts to these oligonucleotides in the RNAs of other avian sarcoma and acute leukemia viruses studied to date. UR2-transformed non-virus-producing cells contain a single 24S viral RNA which is most likely the message coding for the transforming protein of UR2. On the basis of the uniqueness of the transforming sequence, we concluded that UR2 is a new member of the defective avian sarcoma viruses.

Alpharetrovirus↗

PubMed1982

Adsorption of Rous sarcoma virus to genetically susceptible and resistant chicken cells studied by laser flow cytometry.

Quantitative binding of Rous sarcoma virus (RSV) of different antigenic subgroups to chicken cells was examined by using a laser flow cytometer/cell sorter. RSV of subgroups A, C, and E, labeled with the fluorescent membrane probe rhodamine-18, bound 2 to 10 times more to genetically susceptible chicken embryo fibroblasts than to resistant cells, as measured by flow cytometry on a single-cell basis. This suggested that susceptible cells possess both specific and nonspecific receptors for virus adsorption, whereas resistant cells bind virus only by means of nonspecific sites. Polybrene at low concentration increased eightfold the binding of virus. Higher levels of Polybrene inhibited adsorption. Cell binding sites were saturable, and attachment of labeled virus could be partially blocked by preexposure of cells to unlabeled RSV. Virus surface glycoproteins played an important role in adsorption, since their removal with bromelain decreased binding of virus to susceptible cells. Maximal binding of RSV to both susceptible and resistant cells occurred within 10 min, although the level of binding was up to 10-fold higher for susceptible cells. Binding to all cell types showed a broad distribution. This implies that there are considerable differences in the number of virions bound per cell.

Adsorption↗

PubMed1982

Cellular sequences related to three new onc genes of avian sarcoma virus (fps, yes, and ros) and their expression in normal and transformed cells.

Two onc genes of avian sarcoma viruses unrelated to the src gene have recently been identified: fps of Fujinami sarcoma virus/PRCII/UR1 and yes of Y73/Esh sarcoma virus. In the first part of this study we demonstrated that UR2, the most recently isolated avian sarcoma virus, contains in its genome a unique sequence, ros, nonhomologous to src, fps, and yes sequences or to transforming genes of avian acute leukemia viruses. Using cDNAs specific to the inserts of avian sarcoma virus genomes, we examined the existence and the transcription of cellular nucleotide sequences related to the three new onc genes of avian sarcoma virus (fps, yes and ros) in various cells. The progenitor cellular sequences for these onc genes (c-onc) were present in uninfected chicken DNA in one or few copies per haploid genome. These c-onc sequences were detectable in cellular DNA of a wide variety of vertebrates, and the homology between viral and cellular onc was inversely related to the phylogenetic distance of animal species. The pattern of expression of these c-onc genes in different tissues of chickens was found to be unique to each gene. The expression of c-fps and c-ros genes was generally repressed in many tissues, but c-fps was expressed at higher levels in bone marrow (2.5 copies per cell) and lung (1.1 copies per cell), whereas c-ros was mainly transcribed in kidney (2.5 copies per cell). On the other hand, c-yes transcripts were easily detectable in all tissues analyzed and were found at high levels in kidney (26 copies per cell). These c-onc expressions were unaffected by infection with avian sarcoma viruses that contained other onc genes. In a few cultures of chicken and quail transformed cells derived from tumors induced by chemical carcinogens, we found that the levels of transcription of the four c-onc genes remained unaltered, compared with that in normal tissues.

Alpharetrovirus↗

PubMed1982

Avian sarcoma virus UR2 encodes a transforming protein which is associated with a unique protein kinase activity.

UR2 is a newly characterized avian sarcoma virus whose genome contains a unique sequence that is not related to the sequences of other avian sarcoma virus transforming genes thus far identified. This unique sequence, termed ros, is fused to part of the viral gag gene. The product of the fused gag-ros gene of UR2 is a protein of 68,000 daltons (P68) immunoprecipitable by antiserum against viral gag proteins. In vitro translation of viral RNA and in vivo pulse-chase experiments showed that P68 is not synthesized as a large precursor and that it is the only protein product encoded in the UR2 genome, suggesting that it is involved in cell transformation by UR2. In vivo, P68 was phosphorylated at both serine and tyrosine residues. Immunoprecipitates of P68 with anti-gag antisera had a cyclic nucleotide-independent protein kinase activity that phosphorylated P68, rabbit immunoglobulin G in the immune complex, and alpha-casein. The phosphorylation by P68 was specific to tyrosine of the substrate proteins. P68 was phosphorylated in vitro at only one tyrosine site, and the tryptic phosphopeptide of in vitro-labeled P68 was different from those of Fujinami sarcoma virus P140 and avian sarcoma virus Y73-P90. A comparison of the protein kinases encoded by UR2, Rous sarcoma virus, Fujinami sarcoma virus, and avian sarcoma virus Y73 revealed that UR2-P68 protein kinase is distinct from the protein kinases encoded by those viruses by several criteria. Our results suggest that several different protein kinases encoded by viral transforming genes have the same functional specificity and cause essentially the same cellular alterations.

Alpharetrovirus↗

PubMed1981

Genetic structure, transforming sequence, and gene product of avian sarcoma virus UR1.

We analyzed the genetic structure and gene products of the newly isolated avian sarcoma virus UR1, which recently has been shown to be replication defective and to contain no sequences homologous to the src gene of Rous sarcoma virus. The sizes of the genomic RNAs of UR1 and its associated helper virus, UR1AV, were determined to be 29S and 35S (5.9 and 8.5 kilobases), respectively, by gel electrophoresis and sucrose gradient sedimentation. RNase T1 oligonucleotide mapping of purified viral RNAs indicated that UR1 RNA contains eight unique oligonucleotides in the middle of the genome and shares four 5'-terminal and three 3'-terminal oligonucleotides with UR1AV RNA. The unique sequences of UR1 and Fujinami sarcoma virus were found to be closely related to each other by molecular hybridization of UR1 RNA with DNA complementary to the unique sequence of Fujinami sarcoma virus RNA, but minor differences were found by oligonucleotides fingerprinting. In the regions flanking the unique sequences, UR1 and Fujinami sarcoma viral RNAs contain distinct oligonucleotides, which are shared with oligonucleotides of the respective helper viral RNAs. Cell transformed with UR1 produce a single 29S RNA species which contains a UR1 unique sequence; this species is most likely the mRNA coding for the transforming protein. In UR1-transformed cells, a phosphoprotein fo 150,000 daltons (p150) was detected by immunoprecipitation with antiserum against gag proteins. p150 was associated with a protein kinase activity that was capable of phosphorylating p150 itself, immunoglobulin G of antiserum, and a soluble substrate, alpha-casein. This enzyme transferred phosphate exclusively to tyrosine residues of substrates in vitro, but p 150 labeled in vivo with 32P contained both phosphoserine and phosphotyrosine. The in vitro kinase reaction was not affected by the presence of cyclic AMP or cyclic GMP and strongly preferred Mn2+ over Mg2+. Thus, the properties of UR1 protein are almost identical to those of Fujinami sarcoma virus protein.

Alpharetrovirus↗

PubMed1981

Some biological properties of two new avian sarcoma viruses.

The new avian retroviruses UR1 and UR2 were isolated from spontaneous tumors of chickens by cocultivation of tumor material with susceptible chicken embryo fibroblasts. In vitro, UR1 induced formation of small foci of round and fusiform cells. On the other hand, cells infected by UR2 assumed an extremely elongated morphology. In vivo, both viruses induced fibrosarcomas and myxosarcomas with short latencies. Infectivity assays with and without mitomycin C showed that both viruses were defective for replication, but transformed nonproducing cell clones were obtained only with UR1. UR1-infected transformed nonproducing clones did not release particles detectable by reverse transcriptase assays, and fusion of transformed nonproducing cells with quail cells chronically infected with Rous sarcoma virus (a Bryan strain) failed to rescue infectious virus. This suggested that UR1 does not code for functional envelope glycoproteins. In this regard, UR1 appeared to be similar to Fujinami, PRCII, and Y73 viruses. The helper viruses of partially purified stocks of UR1 and UR2 appeared to belong to subgroup A, but these helper viruses were distinguishable from each other, as shown by host range experiments and neutralization tests. Hybridization studies with DNA complementary to the src gene of Rous sarcoma virus and RNAs extracted from both UR1 and UR2 showed no homology between the genomes of the new isolates and the transforming gene of Rous sarcoma virus.

Alpharetrovirus↗

PubMed1980

Studies on recombination in heterologous crosses of Rous sarcoma virus.

Ts mutants in src from the Prague and Schmidt-Ruppin strains of Rous sarcoma virus (RSV) were used to superinfect chicken and quail cells chronically infected with the Bryan strain of RSV. No wild-type recombinants and few double-defective virions were produced. These results indicate that genetic recombination does not occur with high frequency in these heterologous crosses even if all infectious virus is derived from doubly infected cells.

Animals↗

PubMed1976

Cooperative transformation studies with temperature-sensitive mutants of Rous sarcoma virus.

Stocks of Rous sarcoma virus Bryan strain were mutagenized using a bromodeoxyuridine treatment immediately after infection. Thirty temperature-sensitive (ts) mutants defective in transformation (td) were isolated by a replica plating technique. Twenty of these mutants were preliminarily characterized and found to be defective in late functions related to transformation. These mutants were used in experiments of cooperative transformation with four Prague strain td ts mutants of different co-transformation group. A small number of Bryan ts mutants were found to cooperate with some of the Prague mutants in transforming chicken embryo cells at the nonpermissive temperature. However, the amount of co-transformation observed was lower than that observed with cooperating Prague ts mutants and no clear-cut pattern of cotransformation was obtained in Prague and Bryan crosses. Indirect evidence indicates that cooperative transformation is the result of recombination events.

Animals↗

PubMed1975

Plaque assay of avian sarcoma viruses using casein.

The caseinolytic activity of several strains of Rous sarcoma virus (RSV), conditional and nonconditional mutants of RSV, and nontransforming avian leukosis viruses was investigated. Only those viruses capable of transforming chick fibroblasts in vitro induced lysis of casein incorporated into an agar overlay. Lysis produced distinct clear areas in the turbid casein-agar gel which allowed a quantitative "plaque" assay of cell transformation. Casein plaque formation could not be separated from morphological conversion in cultures infected by wild-type RSV strains. In plates infected by mutants temperature sensitive for transformation, the caseinolytic activity appeared to be affected by temperature to a lesser extent than morphological conversion.

Avian Leukosis Virus↗

PubMed1973

Mechanism of oncogenic transformation by Rous sarcoma virus. 3. Role of proviral DNA in morphologic conversion of chicken embryo fibroblasts.

Multiplication of Rous sarcoma virus and morphological conversion of chicken embryo fibroblasts are mediated by a DNA provirus. The role of the provirus in induction of morphological conversion has been shown by experiments of light inactivation of bromodeoxyuridine (BUdR)-sensitized proviral DNA. In the experiments reported here, inactivation of focus formation by BUdR and light could be obtained in cells in which the ability to produce virus has become resistant to X irradiation. This property is considered here to reflect the integrated state of the provirus. These experiments indicate that the role of proviral DNA extends beyond induction of morphological conversion and that an intact provirus is required for the maintenance of the transformed state. These experiments also indicate that no irreversible process leading to morphological conversion is initiated by a nonintegrated or by an integrated provirus.

Animals↗