scholarly journals Inhibition of chicken embryo lens differentiation and lens junction formation in culture by pp60v-src.

1988 ◽  
Vol 8 (4) ◽  
pp. 1414-1420 ◽  
Author(s):  
A S Menko ◽  
D Boettiger
Keyword(s):  
Gap Junctions ◽  
Rous Sarcoma Virus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rous Sarcoma ◽  
Junction Protein ◽  
Junction Formation ◽  
Cell Layers ◽  
Sarcoma Virus ◽  
Gap Junction Protein

A culture system was developed which permitted the differentiation of chicken lens epithelial cells to lentoid bodies which contained several cell layers, accumulated high levels of delta-crystallin, and produced extensive gap junctions. This differentiation process was prevented when the cells were infected with a temperature-sensitive src mutant of Rous sarcoma virus and maintained at the permissive temperature. These transformed cells continued to proliferate and also synthesized the major lens gap junction protein, MP28, at near-normal rates. However, this MP28 was not assembled to produce gap junctions. Cultures shifted to the nonpermissive temperature formed lentoid bodies similar to those in uninfected lens cultures, including the establishment of gap junctions containing MP28.

Inhibition of chicken embryo lens differentiation and lens junction formation in culture by pp60v-src

1988 ◽  
Vol 8 (4) ◽  
pp. 1414-1420
Author(s):  
A S Menko ◽  
D Boettiger
Keyword(s):  
Gap Junctions ◽  
Rous Sarcoma Virus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rous Sarcoma ◽  
Junction Protein ◽  
Junction Formation ◽  
Cell Layers ◽  
Sarcoma Virus ◽  
Gap Junction Protein

A culture system was developed which permitted the differentiation of chicken lens epithelial cells to lentoid bodies which contained several cell layers, accumulated high levels of delta-crystallin, and produced extensive gap junctions. This differentiation process was prevented when the cells were infected with a temperature-sensitive src mutant of Rous sarcoma virus and maintained at the permissive temperature. These transformed cells continued to proliferate and also synthesized the major lens gap junction protein, MP28, at near-normal rates. However, this MP28 was not assembled to produce gap junctions. Cultures shifted to the nonpermissive temperature formed lentoid bodies similar to those in uninfected lens cultures, including the establishment of gap junctions containing MP28.

Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts

1990 ◽  
Vol 10 (4) ◽  
pp. 1754-1763
Author(s):  
D S Crow ◽  
E C Beyer ◽  
D L Paul ◽  
S S Kobe ◽  
A F Lau
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rous Sarcoma Virus ◽  
Gap Junctional Communication ◽  
Rous Sarcoma ◽  
Junctional Communication ◽  
Junction Protein ◽  
Sarcoma Virus ◽  
Gap Junction Protein ◽  
Gap Junctional

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.

scholarly journals Phosphorylation of connexin43 gap junction protein in uninfected and Rous sarcoma virus-transformed mammalian fibroblasts.

1990 ◽  
Vol 10 (4) ◽  
pp. 1754-1763 ◽  
Author(s):  
D S Crow ◽  
E C Beyer ◽  
D L Paul ◽  
S S Kobe ◽  
A F Lau
Keyword(s):  
Gap Junctions ◽  
Gap Junction ◽  
Rous Sarcoma Virus ◽  
Gap Junctional Communication ◽  
Rous Sarcoma ◽  
Junctional Communication ◽  
Junction Protein ◽  
Sarcoma Virus ◽  
Gap Junction Protein ◽  
Gap Junctional

Gap junctions are membrane channels that permit the interchange of ions and other low-molecular-weight molecules between adjacent cells. Rous sarcoma virus (RSV)-induced transformation is marked by an early and profound disruption of gap-junctional communication, suggesting that these membrane structures may serve as sites of pp60v-src action. We have begun an investigation of this possibility by identifying and characterizing putative proteins involved in junctional communication in fibroblasts, the major cell type currently used to study RSV-induced transformation. We found that uninfected mammalian fibroblasts do not appear to contain RNA or protein related to connexin32, the major rat liver gap junction protein. In contrast, vole and mouse fibroblasts contained a homologous 3.0-kilobase RNA similar in size to the heart tissue RNA encoding the gap junction protein, connexin43. Anti-connexin43 peptide antisera specifically reacted with three proteins of approximately 43, 45 and 47 kilodaltons (kDa) from communicating fibroblasts. Gap junctions of heart cells contained predominantly 45- and 47-kDa species similar to those found in fibroblasts. Uninfected fibroblast 45- and 47-kDa proteins were phosphorylated on serine residues. Phosphatase digestions of 45- and 47-kDa proteins and pulse-chase labeling studies indicated that these proteins represented phosphorylated forms of the 43-kDa protein. Phosphorylation of connexin protein appeared to occur shortly after synthesis, followed by an equally rapid dephosphorylation. In comparison with these results, connexin43 protein in RSV-transformed fibroblasts contained both phosphotyrosine and phosphoserine. Thus, the presence of phosphotyrosine in connexin43 correlates with the loss of gap-junctional communication observed in RSV-transformed fibroblasts.

Aggregation properties of chondrocytes infected with a temperature-sensitive mutant of Rous sarcoma virus

1980 ◽  
Vol 43 (1) ◽  
pp. 407-417
Author(s):  
A. Tanaka ◽  
A. Kaji
Keyword(s):  
Normal Level ◽  
Rous Sarcoma Virus ◽  
Cell Aggregation ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Level Of Aggregation

Aggregation capacity of chicken embryo chondrocytes decreases when transformed by Rous sarcoma viruses. Cell-to-cell aggregation capacity of chondrocytes infected with a T class temperature-sensitive mutant (tsNY68) (with the temperature-sensitive lesion at the src gene) of Rous sarcoma virus is dependent upon the temperature at which these cells are grown. When grown at the permissive temperature (36 degrees C), where the transforming gene is expressed, aggregation capacity was lower than normal while infected cells grown at the non-permissive temperature (41.5 degrees C) had similar capacity to aggregate to that of normal chondrocytes. However, after a prolonged period of culture (10 days), chondrocytes transformed by wild type SR-RSV regained the normal level of aggregation capacity. Cells transformed by tsNY68 and incubated at the permissive temperature for 10 days also regained the normal level of aggregation capacity. It appears therefore that RSV-transformed chondrocytes first become less adhesive but during long-term cultivation they regain their property to aggregate. The decrease of aggregation capacity due to T class mutants of RSV at 36 degrees C is dependent on constant maintenance of protein synthesis because addition of cycloheximide restored the aggregation capacity even at the permissive temperature.

scholarly journals Expression of the v-src or v-fps oncogene increases fructose 2,6-bisphosphate in chick-embryo fibroblasts. Novel mechanism for the stimulation of glycolysis by retroviruses

1986 ◽  
Vol 236 (2) ◽  
pp. 595-599 ◽  
Author(s):  
L Bosca ◽  
M Mojena ◽  
J Ghysdael ◽  
G G Rousseau ◽  
L Hue
Keyword(s):  
Chick Embryo ◽  
Rous Sarcoma Virus ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Kinase Activation ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Embryo Fibroblasts ◽  
Rate Limiting ◽  
Stimulation Of

The concentration of fructose 2,6-bisphosphate and the activity of 6-phosphofructo-2-kinase are increased after infection of chick-embryo fibroblasts with the Rous sarcoma virus, or with a temperature-sensitive mutant of this virus at the permissive, but not at the non-permissive, temperature. This is observed after transformation by retroviruses carrying either the v-src or v-fps, but not the v-mil and/or v-myc, oncogenes. Comparison of the effects of the Rous sarcoma virus with those of phorbol myristate acetate on fructose 2,6-bisphosphate suggests that both result from the stimulation of a step which is rate-limiting for 6-phosphofructo-2-kinase activation and which is also controlled by protein kinase C.

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

1983 ◽  
Vol 3 (1) ◽  
pp. 113-125
Author(s):  
A S Menko ◽  
Y Toyama ◽  
D Boettiger ◽  
H Holtzer
Keyword(s):  
Intermediate Filaments ◽  
Rous Sarcoma Virus ◽  
Cytochalasin B ◽  
Dermal Fibroblasts ◽  
Transformed Cells ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rous Sarcoma ◽  
Microfilament Bundles ◽  
Sarcoma Virus

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.

Phosphorylation of talin at tyrosine in Rous sarcoma virus-transformed cells

1987 ◽  
Vol 7 (1) ◽  
pp. 371-378
Author(s):  
J E DeClue ◽  
G S Martin
Keyword(s):  
Rous Sarcoma Virus ◽  
Peptide Mapping ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Sensitive Mutant ◽  
Tyrosine Residues ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Chicken Embryo Fibroblasts ◽  
In Cells

The cytoskeletal protein talin was found to undergo enhanced phosphorylation at tyrosine residues in chicken embryo fibroblasts following transformation by Rous sarcoma virus. An increase in the tyrosine phosphorylation of talin was also observed within 6 h in cells infected by the temperature-sensitive mutant tsNY68 after a shift from the nonpermissive to the permissive temperature. The overall extent of phosphorylation was 0.07 mol of phosphate per mol of talin and was not appreciably altered by transformation. In uninfected cells talin was shown to be phosphorylated at multiple sites by tryptic peptide mapping. Following transformation most of these sites remained phosphorylated, to the same or to a lesser extent, while novel, phosphotyrosine-containing phosphopeptides appeared. Talin was phosphorylated at tyrosine in cells infected by Rous sarcoma virus mutants which induce altered or partial transformation morphologies; thus the increased phosphorylation of talin at tyrosine occurred irrespective of the morphology induced. Transformation by Y73 also induced elevated levels of phosphotyrosine in talin, whereas transformation by the avian erythroblastosis and Fujinami sarcoma viruses did not.

scholarly journals 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.

1983 ◽  
Vol 3 (8) ◽  
pp. 1518-1526 ◽  
Author(s):  
D Boettiger ◽  
R Soltesz ◽  
H Holtzer ◽  
M Pacifici
Keyword(s):  
Rous Sarcoma Virus ◽  
Limb Bud ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Infected Cells

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.

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

1983 ◽  
Vol 3 (1) ◽  
pp. 113-125 ◽  
Author(s):  
A S Menko ◽  
Y Toyama ◽  
D Boettiger ◽  
H Holtzer
Keyword(s):  
Intermediate Filaments ◽  
Rous Sarcoma Virus ◽  
Cytochalasin B ◽  
Dermal Fibroblasts ◽  
Transformed Cells ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Rous Sarcoma ◽  
Microfilament Bundles ◽  
Sarcoma Virus

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.

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

1983 ◽  
Vol 3 (8) ◽  
pp. 1518-1526
Author(s):  
D Boettiger ◽  
R Soltesz ◽  
H Holtzer ◽  
M Pacifici
Keyword(s):  
Rous Sarcoma Virus ◽  
Limb Bud ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Nonpermissive Temperature ◽  
Sensitive Mutant ◽  
Temperature Sensitive Mutant ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Infected Cells

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.

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