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Biomedical subjects

D Boettiger

Publications and source records attributed to D Boettiger.

At least 55 records · Page 3Linked to original sources
PubMed1984

Role of cell division in differentiation of myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus.

The relationship between a potential requirement for cell DNA synthesis and the expression of differentiated muscle cell functions was investigated using primary chicken embryo myoblasts infected with a temperature-sensitive mutant of Rous sarcoma virus (RSV). Under optimized conditions, transformed myoblasts growing at the permissive temperature could differentiate into multinucleated myotubes, express muscle-specific myosin, desmin and acetylcholine receptors in the absence of DNA synthesis and cell division following a shift to the non-permissive temperature. Furthermore, the experiments demonstrate that individual RSV-infected myoblasts have two options: either to divide and express the transformed phenotype or to withdraw from the cell cycle and differentiate into myotubes. The choice between these options appears to depend on the protein-kinase activity of pp60src, the src gene product.

Animals↗
PubMed1984

Effect of src infection on long-term marrow cultures: increased self-renewal of hemopoietic progenitor cells without leukemia.

Long-term marrow cultures prepared from mice have been infected with a molecular recombinant of Rous sarcoma virus and murine amphitropic leukemia virus. This resulted in introduction of the src gene into the cultured cells and expression of its protein kinase function. The infected cultures displayed an altered balance in the accumulation of cells in different compartments of granulocyte differentiation. There was a dramatic increase in the stem cell (CFU-S) compartment and the committed progenitor cell (GM-CFC) compartment and a decrease in mature granulocytes. The altered balance appears to be caused by intrinsic alterations in the CFU-S and GM-CFC themselves, which increase their "self-renewal" capacity at the expense of cell differentiation. Remarkably, unlike its effects in other systems, src did not produce a neoplastic transformation of the hemopoietic cells.

Animals↗
PubMed1984

Target cells for avian myeloblastosis virus in embryonic yolk sac and relationship of cell differentiation to cell transformation.

The yolk sac of the 12-day chicken embryo retains the blast stage progenitors to cells of the myeloid lineages with a very low level of contamination by more mature myeloid cells which have begun to express the characteristic myeloid cell markers. Both in vivo and in vitro experiments have supported the hypothesis that target cells for the BAI-A strain of avian myeloblastosis virus are contained within the myeloid lineages. An assay system for avian myeloblastosis virus was developed which utilizes this yolk sac cell system and which appears to be more sensitive than previous published assays. In addition, the kinetics of a liquid culture transformation system is presented in which at least 4% of the yolk sac cell population was transformed in a relatively synchronous fashion at 2 days after infection. The morphological transformation preceded an increased rate of cell proliferation. Cell separation procedures provided a 10- to 20-fold enrichment of target cells and demonstrated that the target cell population copurifies with macrophage colony-forming cells which are the committed progenitors to the macrophage lineage. In combination with earlier work, this work demonstrated that cells committed to the macrophage lineage at all stages of differentiation may serve as target cells for infection by avian myeloblastosis virus.

Animals↗
PubMed1984

Expression of the Rous sarcoma virus src gene in avian macrophages fails to elicit transformed cell phenotype.

Infection of avian macrophages with Rous sarcoma virus does not induce any changes in the morphology, growth behavior, or expression of macrophage-specific proteins. The absence of cellular transformation does not result from a block in the synthesis of viral proteins, since infectious viruses are released from a majority of cells in the culture. In this report, we examine the synthesis, processing, and functional activity of pp60src in Rous sarcoma virus-infected macrophages to determine whether the absence of transformation is due to an alteration in the functional expression of pp60src. Although the absolute level of pp60src was reduced compared with fibroblasts, the protein exhibited the same phosphorylation pattern and subcellular distribution and was able to phosphorylate immunoglobulin in the immune complex-protein kinase assay. These results imply that the failure of Rous sarcoma virus to transform macrophage may be due to a restriction in the cellular response to a functional src protein, perhaps due to the absence of cellular products which are essential for mediating pp60src-induced transformation.

Animals↗
PubMed1983

Transformation by Rous sarcoma virus induces similar patterns of glycosaminoglycan synthesis in chick embryo skin fibroblasts and vertebral chondroblasts.

Chick embryo skin fibroblasts and vertebral chondroblasts were infected with a temperature-sensitive mutant of Rous sarcoma virus, LA24A, and were grown at permissive (36 degrees C) and nonpermissive (41 degrees C) temperatures. During exponential growth, infected and parallel uninfected cultures were labeled with D-[3H]glucosamine, and newly synthesized glycosaminoglycans were identified by anion exchange chromatography and by selective enzymatic and chemical degradations. Control fibroblasts synthesized low levels of hyaluronic acid (HA), and dermatan sulfate (DS), moderate levels of heparan sulfate (HS), and high levels of chondroitin sulfate (CS). In contrast, control chondroblasts synthesized very low levels of HA and DS, no HS, and very high levels of CS. Following transformation and growth at 36 degrees C, both cell types showed a dramatic increase in HA synthesis and a significant decrease in CS synthesis. In addition, transformed chondroblasts initiated the synthesis of HS and increased their synthesis of DS to levels that matched those of transformed fibroblasts. The CS chains synthesized by control chondroblasts were partially undersulfated, while those synthesized by both normal and transformed fibroblasts were fully sulfated. Upon transformation, chondroblasts grown at 36 degrees C initiated the synthesis of fully sulfated CS chains. Most of the above biosynthetic alterations were completely reversed when infected cells were grown at 41 degrees C, indicating that they were dependent on the transforming gene product of LA24A. Clearly, the profound differences that distinguish normal fibroblasts from normal chondroblasts are lost upon transformation, and these two types of terminally differentiated cells converge toward a common, though not identical, biosynthetic program for glycosaminoglycans.

Animals↗
PubMed1983

Immunological characterization of the major chick cartilage proteoglycan and its intracellular localization in cultured chondroblasts: a comparison with Type II procollagen.

Polyclonal antibodies were raised in a rabbit against the major proteoglycan of chick sternal cartilage. A total of six antisera was obtained, three after the first booster injection (A1, A2, and A3) and three after the second booster injection (A4, A5, and A6). The A1 antiserum, which was characterized in most detail, immunoprecipitated native as well as chondroitinase ABC-digested or chondroitinase ABC/keratanase-digested cartilage proteoglycan synthesized by cultured chick chondroblasts, but failed to immunoprecipitate the major proteoglycan synthesized by chick skin fibroblasts. This antiserum was also able to immunoprecipitate the cartilage proteoglycan core protein newly synthesized by cultured chondroblasts, but no other major cell protein. However, the late bleed antisera obtained from the same rabbit after a second booster injection reacted with a new chondroblast-specific polypeptide(s) of approximately 60,000 mol wt in addition to the cartilage proteoglycan. By immunofluorescence procedures, the A1 antiserum stained the extracellular proteoglycan matrix of cultured chondroblasts but not that of skin fibroblasts. Following enzymatic removal of the extracellular matrix and cell membrane permeabilization, this antiserum stained primarily a large, juxtanuclear structure. Additional radioautographic evidence suggests that this structure represents the Golgi complex. Similar immunofluorescent staining with antibodies to the cartilage-characteristic Type II collagen revealed that type II procollagen was localized in numerous cytoplasmic, vacuole-like structures which were scattered throughout most of the chondroblast cytoplasm but were notably scanty in the Golgi complex area. In conclusion, our data suggest the transit of the major cartilage proteoglycan through the Golgi complex of cultured chondroblasts and possible differences in the intracellular distribution of newly synthesized cartilage proteoglycan and Type II procollagen.

Animals↗
PubMed1983

Altered cell spreading in cytochalasin B: a possible role for intermediate filaments.

Trypsinized chicken embryo dermal fibroblasts plated in the presence of cytochalasin B (CB) quickly attached to the substrate and within 24 h obtained an arborized morphology. This morphology is the result of the pushing out of pseudopodial processes along the substrate from the round central cell body. There were no microfilament bundles in the processes of these cells plated in the presence of CB; however, the processes were packed with highly oriented, parallel-aligned intermediate filaments. Only a few scattered microtubules were seen in these processes. These results demonstrated that in CB, cells are capable of a form of movement, i.e., the extension of pseudopodial processes, without the presence of the microfilament structures usually associated with extensions of the cytoplasm and pseudopodial movements. We also found that arborization did not depend on fibronectin since cells plated in CB did not have fibronectin fibers associated with the processes. Chicken fibroblasts transformed with tsLA24A, a Rous sarcoma virus which is temperature sensitive for pp60src, formed arborized cells with properties similar to those of uninfected fibroblasts when plated in the presence of CB at the nonpermissive temperature (41 degrees C). At the permissive temperature for transformation (36 degrees C), the cells attached to the substrate but remained round. These round cells were not only deficient in microfilament bundles but also lacked the highly organized intermediate filaments found in the processes of the arborized cells at 41 degrees C. Although both microfilament bundles and the fibronectin matrix were decreased after transformation with Rous sarcoma virus, neither was involved in the formation of processes in normal cells plated in CB. Therefore, the inability of the transformed cells to form or maintain processes in CB must be the result of another structural alteration in the transformed cells, such as that of the intermediate filaments.

Actins↗
PubMed1983

Infection of chick limb bud presumptive chondroblasts by a temperature-sensitive mutant of Rous sarcoma virus and the reversible inhibition of their terminal differentiation in culture.

Stage 21 to 22 chicken embryo limb bud cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and were grown in culture. Although control, uninfected cells yielded definitive chondroblasts (by day 4) which initiated the synthesis of the cartilage-characteristic proteoglycan, the transformed cells grown at the permissive temperature failed to do so. These effects were fully reversible after a shift to the nonpermissive temperature. In addition, infected cells at the nonpermissive temperature expressed traits of terminal chondrogenic maturation 2 to 3 days earlier than parallel, uninfected cells. Thus, Rous sarcoma virus-induced transformation reversibly blocks terminal limb bud cell chondrogenesis in culture, at the nonpermissive temperature, viral infection may also induce intracellular or extracellular conditions which favor or accelerate the process of chondrogenic cell maturation.

Animals↗
PubMed1983

Selective effect of Rous sarcoma virus src gene expression on contractile protein synthesis in chick embryo myotubes.

Myogenic precursor cells were infected with a temperature-sensitive mutant of Rous sarcoma virus and maintained at the permissive temperature for transformation. Following subculture, a population of cells was produced which failed to differentiate at the permissive temperature but produced a high proportion of myotubes in sister cultures shifted to the nonpermissive temperature. The myotube-containing cultures were further enriched for this cell type by the addition of 1-beta-D-arabinofuranosylcytosine to kill replicating mononucleated cells. This myotube population was suitable for testing the effect of viral-transforming gene expression in a postmitotic, terminally differentiated cell which expresses relatively low levels of the cellular homologue of the viral-transforming gene and is resistant to infection by the virus. The consequence of shifting these Rous sarcoma virus-infected myotube cultures to the permissive temperature was assessed in terms of protein synthesis. The total rate of incorporation of exogenous radioactive leucine, supplied at a concentration which saturated the intracellular pool, was similar between cultures held at the two temperatures, suggesting that the absolute rate of total protein synthesis was not affected by expression of viral-transforming gene. In contrast, the rate of synthesis of eight proteins the expression of which is specific for myotubes was suppressed reversibly. The rate of synthesis of five other proteins which are not selectively concentrated in myotubes was either unaffected or stimulated. Thus. the expression of viral-transforming gene in myotubes leads to differential effects on developmentally regulated proteins without inducing several properties of the transformed state classically observed for fibroblastic cells.

Animals↗
PubMed1982

Regulation of the synthesis of extracellular matrix components in chondroblasts transformed by a temperature-sensitive mutant of Rous sarcoma virus.

Regulation of cartilage extracellular matrix synthesis has been examined in chondroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus. Cells grown at the nonpermissive temperature synthesized large amounts of several chondroblast-specific polypeptides (type IV proteoglycan core protein, type II procollagen, a proteoglycan link protein(s) and a 60 kd protein) and very low levels of fibronectin. At the permissive temperature, synthesis of chondroblast-specific proteins was coordinately reduced, while fibronectin synthesis was greatly increased. These changes reflected comparable alterations in levels of translatable mRNAs encoding these proteins. This analysis also revealed the unexpected presence in the transformed cells of type I collagen mRNAs, which are not used in intact cells, indicating that a posttranscriptional control (or controls) may be acting in transformed chondroblasts.

Animals↗
PubMed1982

The response of chicken embryo dermal fibroblasts to cytochalasin B is altered by Rous sarcoma virus-induced cell transformation.

The drug cytochalasin B (CB), which disrupts the cellular microfilament network, allows the identification of as yet unclassified structural differences between normal and Rous sarcoma virus-transformed chicken embryo fibroblasts. When exposed to CB, normal chick fibroblasts attain an arborized or dendritic morphology. This results as the cytoplasm collapses upon the remaining structural and adhesive components of the cell. Rous sarcoma virus-transformed cells did not form or maintain these dendritic-like processes in the presence of CB and, as a result, rounded up but still remained attached to the substrate. With a temperature-sensitive mutant of Rous sarcoma virus, LA24A, it was possible to show that these effects are completely reversible and dependent on the expression of pp60src. The cytoskeleton in these CB-treated cells was examined by both immunofluorescence and electron microscopy. After exposure to CB, the microfilaments were found to be disrupted similarly throughout both the transformed and the nontransformed cells. In the nontransformed cells arborized by exposure to CB, the extended processes were found to contain intermediate filaments in an unusually high concentration and degree of organization. The distribution of these filaments in the central body of the arborized cells was random. This lower concentration and random distribution was similar to that seen throughout the transformed cells rounded up by exposure to CB. The failure of these transformed cells to arborize in CB indicates that the structural component(s) which is necessary for the formation or maintenance or both of the arborized state is altered by the expression of pp60src.

Animals↗
PubMed1981

Differential effects of transforming avian RNA tumor viruses on avian macrophages.

Functionally differentiated chicken macrophages were derived by in vitro differentiation of embryonic yolk sac cells and were characterized by several macrophage-specific cell markers. Uniform, infected, virus-producing cultures were obtained after exposure of these macrophages to avian myoblastosis virus (AMV), avian myelocytomatosis virus (MC29), myeloblastosis-associated virus (MAV-2), and Prague strain of Rous sarcoma virus (PR-B RSV). Both AMV and MC29 induced morphological transformation typical of the in vivo leukemias induced by these virus strains. Analysis of the expression of macrophage-specific markers in these two transformed cell types demonstrated that different markers of the mature macrophage were suppressed by each virus, even though the parental cell immediately preceding the transformation event was a mature macrophage in both cases. Cells infected with PR-B RSV and MAV-2 showed no observable difference from uninfected macrophages in terms of morphological characteristics, growth rate, or expression of the differentiated functions of macrophages. Ths system provides demonstrations of a cell type that produces infectious, transforming RSV but fails to respond by functional alterations induced by the transforming gene, src.

Acid Phosphatase↗