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.

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.

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

1982 ◽  
Vol 2 (3) ◽  
pp. 320-330 ◽  
Author(s):  
A S Menko ◽  
J Croop ◽  
Y Toyama ◽  
H Holtzer ◽  
D Boettiger
Keyword(s):  
Rous Sarcoma Virus ◽  
Chicken Embryo ◽  
Cytochalasin B ◽  
Dendritic Morphology ◽  
Dermal Fibroblasts ◽  
Transformed Cells ◽  
Temperature Sensitive ◽  
High Concentration ◽  
Rous Sarcoma ◽  
Sarcoma Virus

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.

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

1982 ◽  
Vol 2 (3) ◽  
pp. 320-330
Author(s):  
A S Menko ◽  
J Croop ◽  
Y Toyama ◽  
H Holtzer ◽  
D Boettiger
Keyword(s):  
Rous Sarcoma Virus ◽  
Chicken Embryo ◽  
Cytochalasin B ◽  
Dendritic Morphology ◽  
Dermal Fibroblasts ◽  
Transformed Cells ◽  
Temperature Sensitive ◽  
High Concentration ◽  
Rous Sarcoma ◽  
Sarcoma Virus

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.

scholarly journals Dissociation of inositol trisphosphate from diacylglycerol production in Rous sarcoma virus-transformed fibroblasts.

1989 ◽  
Vol 108 (2) ◽  
pp. 683-691 ◽  
Author(s):  
T J Martins ◽  
Y Sugimoto ◽  
R L Erikson
Keyword(s):  
Rous Sarcoma Virus ◽  
Inositol Trisphosphate ◽  
Metabolic Changes ◽  
Transformed Cells ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Early Effect ◽  
Rous Sarcoma ◽  
Sarcoma Virus ◽  
Inositol Metabolism

The metabolism of phosphatidylinositol (PI) and related intermediates was studied in uninfected and Rous sarcoma virus-(RSV) infected chicken embryo fibroblasts (CEFs). Cells infected with wild-type RSV exhibited twofold increases in steady-state concentrations of inositol trisphosphate (IP3) and inositol bisphosphate (IP2) as compared to uninfected CEFs. In addition, increased concentrations of IP3 and IP2 were observed in CEFs infected with the RSV temperature-sensitive transformation mutant NY72-4 when maintained at the permissive temperature (35 degrees C) for greater than 24 h. Slight increases were observed in the amounts of inositol lipids in RSV-transformed cells. Phosphoinositol metabolic changes were related to transformation and not to viral infection since CEFs infected with NY72-4, maintained at the nonpermissive temperature (41.5 degrees C), revealed amounts of phosphoinositols similar to that of uninfected cells. CEFs infected with a transformation-defective virus exhibited PI metabolic changes intermediate between those of transformed and nontransformed cells. NY72-4 CEF exhibited no increase in phosphoinositol concentrations before 8 h incubation at 35 degrees C, indicating that the transformation-specific changes in inositol metabolism were a delayed event. Furthermore, inositol turnover was not activated during this time. In contrast to the case of inositol metabolism, significant increases in diacylglycerol (DAG) concentrations were observed within 15-30 min after shift of NY72-4 CEFs to 35 degrees C. These findings suggest that (a) the major changes in inositol metabolism are specific for RSV-transformed cells; (b) transformation-specific changes in phosphoinositol content in RSV-infected CEFs are not an early effect of the expression of pp60v-src; and (c) increases in the DAG content of transformed cells occur before changes in inositol metabolism, indicating that DAG may be derived from other lipid sources.

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.

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.

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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