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G Tarone

Publications and source records attributed to G Tarone.

At least 91 records · Page 5Linked to original sources
PubMed1985

Rous sarcoma virus-transformed fibroblasts adhere primarily at discrete protrusions of the ventral membrane called podosomes.

Rous sarcoma virus-transformed BHK cells (RSV/B4-BHK) adhere to a fibronectin-coated substratum primarily at specific dot-shaped sites. Such sites contain actin and vinculin and represent close contacts with the substratum as revealed by interference reflection microscopy. Only a few adhesion plaques and actin filament bundles can be detected in these cells as compared to untransformed parental fibroblasts. In thin sections examined with transmission electron microscopy (TEM) these adhesion sites correspond to short protrusions of the ventral cell surface that contact the substratum at their apical portion. These structures, which may represent cellular feet, are therefore called podosomes. By screening a number of different transformed fibroblasts plated on a fibronectin-coated substratum we find that podosomes are common to mammalian and avian cell lines transformed either by Rous sarcoma virus (RSV) or by Fujinami avian sarcoma virus (FSV), whose oncogenes encode specific tyrosine kinases. Using antibodies reacting with phosphotyrosine in immunofluorescence experiments, we show that phosphotyrosine-containing molecules are concentrated in podosomes. Podosomes are not detected in fibroblasts transformed by other retroviruses (Snyder-Theilen sarcoma virus, Abelson leukemia virus and Kirsten sarcoma virus) or by DNA tumor viruses (polyoma, SV40), indicating that podosome-mediated adhesion in transformed fibroblasts is related to the peculiar properties of some oncoproteins and possibly to their tropism for adhesion systems. Podosomes and adhesion plaques, although similar in cytoskeletal protein composition, have different mechanisms and kinetics of formation. Assembly of podosomes, in fact (i) does not require fetal calf serum (FCS) in the adhesion medium, that is necessary for the organization of adhesion plaques; (ii) does not require protein synthesis; and (iii) is insensitive to the ionophore monensin, that prevents adhesion plaque formation. Moreover, during attachment to fibronectin-coated dishes, podosomes appear in the initial phase (60 min) of attachment, while adhesion plaques require a minimum of 180 min. In conclusion podosomes of RSV- and FSV-transformed fibroblasts represent a phenotypic variant of adhesion structures.

Actins↗
PubMed1984

Detection of phosphotyrosine-containing proteins in the detergent-insoluble fraction of RSV-transformed fibroblasts by azobenzene phosphonate antibodies.

The Rous sarcoma virus (RSV) oncogene product pp60src is known to trigger the acquisition of the transformed phenotype by phosphorylating host cell target molecule(s) at tyrosine residues. To identify phosphotyrosine-containing proteins, rabbit antibodies were raised against the synthetic hapten p-azobenzene-phosphonate (ABP) that specifically cross-reacts with phosphorylated tyrosine. By immuno-decoration of proteins extracted from RSV-transformed mouse fibroblasts and transferred to nitrocellulose sheets, phosphoproteins of 130, 70 and 60 kd were identified. These molecules were found to be associated with the cellular fraction insoluble in non-ionic detergent. Moreover, ABP antibodies precipitated detergent-insoluble proteins of 130, 70 and 60 kd, plus two additional components of 85 and 65 kd, that had been phosphorylated in vitro by [gamma-32P]ATP under conditions allowing the kinase reaction catalyzed by pp60src. Phosphoproteins of closely related mol. wts. were immunoprecipitated from RSV-transformed avian fibroblasts. The radioactivity co-migrated with authentic phosphotyrosine in two-dimensional chromatography. The 60-kd protein comigrated with pp60src, while the identity between the 130-kd protein and vinculin was disproved by the lack of cross-reaction with appropriate antisera. In transformed mouse and duck fibroblasts ABP antibodies, employed in indirect immunofluorescence microscopy, stained diffusely the cytoplasm and intensely decorated restricted areas of the ventral cell plasma membrane. These data show that antibodies reacting with phosphotyrosine may be usefully employed in the identification and in the intracellular localization of molecules that are potential targets of the pp60src protein kinase.

Animals↗
PubMed1984

A cell surface integral membrane glycoprotein of 85,000 mol wt (gp85) associated with triton X-100-insoluble cell skeleton.

The Triton X-100-insoluble skeleton of baby hamster kidney BHK cells consists of the nucleus, intermediate-size filaments, and actin fibers. By transmission electron microscopy, membrane fragments were found to be associated with these insoluble structures. When radioiodinated or [3H]glucosamine-labeled cells were extracted with 0.5% Triton, most plasma membrane glycoproteins were solubilized except for a glycoprotein with a molecular weight of 85,000 (gp85) that remained associated with the insoluble skeletons. Immunoprecipitation with a specific antiserum indicated that the gp85 is not a proteolytic degradation product of fibronectin, an extracellular matrix glycoprotein insoluble in detergent. A monoclonal antibody of BHK cells specific for gp85 was produced. Immunofluorescence analysis with this monoclonal antibody indicated that gp85 is not associated with the extracellular matrix, but is confined to the cell membrane. Both in fixed and unfixed intact cells, fluorescence was concentrated in dots preferentially aligned in streaks on the cell surface. Gp85 was found to behave as an integral membrane protein interacting with the hydrophobic core of the lipid bilayer since it was extracted from membrane preparations by ionic detergents such as SDS, but not by 0.1 N NaOH (pH 12) in the absence of detergents, a condition known to release peripheral molecules. Association of gp85 with the cell skeleton was unaffected by increasing the Triton concentration up to 5%, but it was affected when actin filaments were dissociated or when a protein-denaturing agent (6 M urea) was used in the presence of Triton, suggesting that protein-protein interactions are involved in the association of gp85 with the cell skeleton. We conclude that gp85 is an integral plasma membrane glycoprotein that might have a role in cell surface-cytoskeleton interaction.

Animals↗
PubMed1984

Monoclonal antibodies to the collagen binding domain of human plasma fibronectin.

Five independent hybrids producing monoclonal antibodies to human plasma fibronectin have been obtained by fusing P3/X63-Ag8 myeloma cells with immune mouse splenocytes. The specificity of these monoclonal antibodies (MABs) for fibronectin was demonstrated by three independent tests: binding to the purified soluble molecule, immunofluorescence staining of insoluble extracellular matrices produced by endothelial cells in vitro, immunostaining of fibronectin tryptic peptides after separation on SDS-PAGE and transfer to nitrocellulose sheets. Two antibodies (MAB 29 and 52) recognized selectively human fibronectin while the others (MAB 5, 30 and 59) reacted also with plasma fibronectin from calf, hamster and chicken. Four distinct epitopes were recognized by the MABs studied. MAB 5, 30, 52 and 59 reacted with distinct antigenic sites, while MAB 29 and 52 bind to the same site. Antigenic fragments were identified by immunostaining of fibronectin tryptic peptides. MAB 5 reacted with a collagen binding fragment with a molecular weight of 120 K. In addition, each of the MAB 29, 30, 52 and 59 reacted with peptides with a molecular weight of 40 K that bind to gelatin. Since these antibodies do not inhibit fibronectin-collagen interaction, it is concluded that their corresponding epitopes are clustered in a region close, but not coincident, to the collagen binding site of fibronectin.

Animals↗
PubMed1984

Mouse immune interferon enhances fibronectin production of elicited macrophages.

By using mouse immune interferon (IFN-gamma) either produced by recombinant DNA technology or partially purified from a T cell lymphoma, L12-R4, we conclusively demonstrated that mouse IFN-gamma enhances fibronectin synthesis and secretion of proteose-peptone-elicited macrophages, but not of normal macrophages. The enhancing activity of both IFN-gamma preparations was acid-labile and was neutralized by a rabbit antiserum made against native IFN-gamma. In addition, IFN-gamma induced the disappearance, as revealed by immunofluorescence studies, of the characteristic fibronectin streaks from the cell surface of elicited macrophages. Taking into account the opsonizing activity of fibronectin, these findings offer some explanations for the IFN-gamma-mediated increase of macrophage phagocytosis and tumor cell killing.

Adjuvants, Immunologic↗
PubMed1983

Immunofluorescence study of fibronectin distribution in the developing chick embryo striated muscles.

This paper reports immunofluorescence data concerning fibronectin distribution in chick embryo striated muscle buds, i.e. myotomes and heart bud, in stages following their differentiation. At earliest developmental stages (12-15 HH) no fibronectin could be observed between the still poorly differentiated myoblasts of myotomes and of the splanchnopleure. Fibronectin, however, was observed among differentiated myoblasts of the myocardial ventral portion. In myotomes at later stages (16-18 HH), a few thin strands of fibronectin were observed between the myotomal and dermatomal laminae; this pattern became more marked between stages 17 and 26 HH. It is concluded that during myogenesis, fibronectin is absent at the earliest differentiation stages. A faint fibronectin-containing matrix is detectable at later stages and becomes more evident only in mature muscle tissue. This pattern is consistent with a role of fibronectin in the process of tissue organization.

Animals↗
PubMed1982

Cell surface molecules and fibronectin-mediated cell adhesion: effect of proteolytic digestion of membrane proteins.

Proteases have been used as a tool to investigate the role of surface molecules in fibronectin-mediated cell adhesion. Proteolytic digestion of membrane-proteins by pronase (1 mg/ml for 20 min at 37 degrees C) completely inhibited adhesion of baby hamster kidney (BHK) fibroblasts on fibronectin-coated plastic dishes. Various degrees of inhibition were also obtained after treatment with proteinase K, chymotrypsin, papain, subtilopeptidase A, and thermolysin. Protein synthesis was required to restore the adhesive properties of pronase-treated cells, showing the protein nature of the molecules involved in adhesion to fibronectin. A peculiar feature of these proteins was their resistance to cleavage by trypsin. After prolonged trypsin treatment (1 mg/ml for 20 min at 37 degrees C), cells adhered and spread on fibronectin-coated dishes, even when protein synthesis was inhibited by 4 microM cycloheximide. Under these conditions only three glycoproteins (gp) of molecular weight 130,000, 120,000, and 80,000 were left on the cell surface. These were precipitated by a rabbit antiserum against BHK cells that also inhibited adhesion of trypsin-treated cells. gp120 and gp80 were left at the cell surface after mild pronase digestion (0.2 mg/ml for 20 min at 37 degrees C), under conditions not affecting adhesion. These data suggest that these glycoproteins may be involved in fibronectin-mediated cell adhesion in some yet unknown way.

Animals↗
PubMed1981

Mouse fibroblasts transformed by Rous sarcoma virus express a virus-specific non-virion transplantation antigen.

Rous sarcoma virus (RSV)-transformed fibroblasts from different animals species a serologically detectable virus-induced non-virion cell surface antigen (VCSA), whose expression is controlled by the transforming viral src gene and by a cellular gene. Now, by in vivo immunization, we have found that RSV-transformed fibroblasts from different mouse strains share a virus-specific transplantation antigen. In fact, only animals immunized with irradiated syngeneic or allogeneic fibroblasts transformed by RSV, but not animals immunized with cells transformed by different oncogenic agents, rejected a lethal dose of syngeneic RSV-induced tumor cells. Immunoprecipitation tests with monospecific antisera showed that the expression of this antigen did not correlate with the presence of intracellular viral proteins other than the src gene product pp60src. However, hyperimmunization of mice with hamster or quail fibroblasts transformed by RSV, that express a high level of pp60src, did not induce transplantation resistance. It is concluded that the expression of both the serologically-defined VCSA and the transplantation antigen is the result of the interaction of pp60src with host cell gene product(s) rather than the simple exposure of pp60src at the outer cell surface.

Animals↗
PubMed1981

Transformation-sensitive protein with molecular weight of 45,000 secreted by mouse fibroblasts.

The [35S]methionine-labeled proteins released in the medium conditioned by normal and transformed mouse fibroblasts have been analyzed by sodium dodecyl sulfate-polyacrylamide electrophoresis. Three major proteins, fibronectin, procollagens, and a protein with a molecular weight of 45,000 (45K protein) have been identified. The 45K protein, which has not yet been described, accounts for about 30% of the proteins released by control 3T3 fibroblasts or mouse embryo cultures. Quantitation of the radioactivity incorporated by the 45K protein indicated a 10- to 15-fold decrease in 3T3 fibroblasts transformed by Kirsten, Abelson, or Rous sarcoma viruses. The amounts of fibronectin and procollagens released in the medium by transformed cells were also reduced by factors of 3- and 5-fold, respectively. Pulse chase experiments have shown that the decreased level of the 45K protein in the medium of transformed cells cannot be explained by a reduced rate of secretion or by extracellular proteolytic degradation. It is not known, however, whether decreased synthesis is responsible for this alteration. The "tumor promoter" phorbol myristate acetate, which is known to induce many of the alterations associated with neoplastic cells, also induced 3T3 fibroblasts to release the 45K protein in amounts comparable to that of transformed cells. Thus, this protein represents a new molecular marker of oncoviral transformation.

Animals↗
PubMed1980

Identification and partial characterization of five major membrane glycoproteins of BHK fibroblasts.

Five major membrane glycoproteins of the BHK-B4 hamster fibroblast plasma membrane have been identified by binding specific rabbit antibodies to the cell surface and by recovering the detergent solubilized immunocomplexes with Protein A-Sepharose immunoadsorption. These glycoproteins, designated as gp45, gp65, gp95, gp130 and gp140, are exposed at the cell surface since: (i) they were accessible to antibodies in intact viable cells; (ii) they were radioiodinated by the lactoperoxidase-glucose oxidase procedure; and (iii) they were cleaved by proteolytic enzymes in conditions affecting only the cell surface. Among these glycoproteins the gp130 is the predominant component and its exposed portion is characterized by lack of sensitivity to trypsin cleavage. Glycoproteins of different molecular weight, but immunologically related to the major hamster membrane glycoproteins, have been detected at the surface of both rat and mouse fibroblasts.

Animals↗
PubMed1980

Interaction between cellular and viral genes in the expression of the RSV-induced transformation-specific cell-surface antigen VCSA.

Transformation of BHK hamster fibroblasts by an env- strain of Rous sarcoma virus (RSV) leads to the appearance at the cell surface of a virus-induced nonvirion antigen (VCSA), specific for transformation, whose expression is controlled by the transforming src gene. Previous work has shown that a rabbit anti-VCSA serum lyses specifically, in the presence of complement, 51Cr-labelled RSV-transformed cells from different animal species. Now, by competition experiments with a panel of different unlabelled cells we show that the VCSA expressed on RSV-transformed hamster fibroblasts is a complex of at least three distinct antigenic specificities: (1) one expressed on all RSV-transformed fibroblasts, regardless their species and the subgroup or strain of the transforming virus; (2) one cross-reacting with a cell-surface antigen (CSA) expressed at various degrees on untransformed avian fibroblasts, but not on mammalian fibroblasts; (3) one species-specific, present only on RSV-transformed hamster fibroblasts. It is concluded that VCSA is a complex of several antigenic determinants, and that some of these differ in different cells transformed by RSV. This observation indicates that VCSA expression at the cell surface is likely to be the result of the interaction between the viral src gene product pp60src with host cell gene(s) or gene product(s), rather than the simple expression of this molecule at the cell surface.

Animals↗
PubMed1980

Target antigens for antibodies and complement at the cell surface of RSV-transformed fibroblasts.

Using cells transformed by Rous sarcoma virus (RSV), an RNA tumour virus whose genetic and structural composition is fully known, the virus-induced surface antigens acting as targets for antibodies and complement were studied. Among the virus structural proteins, only the envelope antigen gp85, but not the core group-specific proteins or reverse transcriptase, were able to mediate immune lysis in the 51Cr-release assay. The group-specific antigenic determinants of gp85 were predominantly involved. The virus-induced cell surface antigen (VCSA), specific for transformation, was the only other molecule effective. Since different cells express either of these antigens, further support is given to the non-identity of virus structural antigens and VCSA.

Animals↗
PubMed1979

Proteolytic degradation of human erythrocyte band 3 by membrane-associated protease activity.

Antisera directed against the cytoplasmic portion of human erythrocyte Band 3 were used to follow the degradation of the band 3 molecule. Small amounts of Band 3 were degraded when well-washed red cell membrane ghosts were incubated in the cold; this process was greatly accelerated by incubating ghosts were incubated in the cold; this process was greatly accelerated by incubating ghosts at 37 degrees C. Band 3 labeled with pyridoxal-phosphate was digested at comparable rates. Band 3 digestion also took place when alkali-extracted ghost membranes were incubated at 37 degrees for prolonged periods. These results suggest that human erythrocytes contain tightly bound, membrane-associated proteolytic activity.

Antigen-Antibody Reactions↗
PubMed1979

Cell surface changes during muscle differentiation in vitro: a study with the probe 2,4,6-trinitrobenzene sulphonate.

Cell surface changes during muscle differentiation in vitro, were investigated using the non permeant probe 2,4,6-trinitrobenzene sulphonate (TNBS) in order to label the aminogroups of proteins exposed on the outer surface of the plasma membrane. Surface proteins of chick myotubes and 'mature' unfused myoblasts (myoblasts grown for 7 days in a calcium-depleted medium) were found to bind an equal amount of probe, which is twice the amount bound by surface proteins in 'immature' myoblasts (1--2 days of culture) and fibroblasts. This indicates that a 'remodelling' of the plasma membrane outer surface takes place in the course of muscle cell differentiation even in the absence of cell fusion. Moreover, the total amount of TNBS bound to the surface was 4--5 times greater in myotubes than in unfused myoblasts. This appears to result from the surface expansion which occurs in myotubes during the development of the T tubule system.

Animals↗
PubMed1979

Relationship between Rous sarcoma virus-induced expression of membrane antigen and phenotypic transformation.

Rous sarcoma virus-transformed hamster BHK fibroblasts express a virus-induced cell surface antigen undetectable in cells either transformed by unrelated viruses or infected by transformation-defective strains of Rous sarcoma virus. To clarify whether this antigen plays any role in the process of malignant transformation or is expressed at the cell surface only as a consequence of the acquisition of the transformed phenotype, we have investigated its expression at the cell surface of Rous sarcoma virus-transformed BHK cells treated with dibutyryl cyclic adenosine 3':5'-monophosphate and at the surface of parental BHK cells transiently transformed by the tumor promoter phorbol myristate acetate. In the dibutyryl cyclic adenosine 3':5'-monophosphate-treated cells, in which most of the parameters of the transformed phenotype are reverted to normality, but the product of the transforming gene is still present, virus-induced cell surface antigen is expressed. In the mirror experiment, this antigen is not expressed by phenotypically transformed but genetically normal phorbol myristate acetate-treated cells. It is concluded that the tumor membrane antigen studied is intimately associated with the expression of the function(s) controlled by the transforming gene.

Animals↗
PubMed1978

Immunochemical purification of probe-labeled plasma membrane proteins: an approach to the molecular anatomy of the cell surface.

The probe 2,4,6-trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labeling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate-polyacrylamide gels of probe-labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus-transformed cells. Moreover, the transformed cells display a nonvirion component of 80--100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37 degrees C but not at 41 degrees C, the expression of this component is related to the expression of the transformed phenotype.

Animals↗