Permissive Temperature For Transformation Research Articles

Expression of the Soluble Lectin L-14 Gene Is Induced by TSH in Thyroid Cells and Suppressed by Retinoic Acid in Transformed Neural Cells

Expression of the soluble lectin L-14 is low in normal and very high in transformed thyroid cells. We show that L-14 gene expression is transiently induced upon thyrotropin stimulation of normal quiescent FRTL-5 rat thyroid cells. Permanent activation of L-14 gene expression is obtained in the same cells infected with a wild-type and a temperature sensitive mutant of Kirsten murine sarcoma virus, both at the permissive and non permissive temperature for transformation. We also find that L-14 mRNA is undetectable in rat brain but is abundant in rat oligodendrocytes precursors transformed by polyoma middle T oncogene. Retinoic acid treatment of these transformed cells leads to acquisition of a differentiated phenotype accompanied by a 30-fold decrease of L-14 mRNA levels. Removal of retinoic acid restores both the transformed undifferentiated phenotype and high L-14 expression. Taken together these results indicate that growth stimulation and induction of cell differentiation are accompanied by strong modulation of L-14 gene expression.

Specific increase in thymidine transport at a permissive temperature in the rat kidney cells infected with src ts-rous sarcoma virus

Thymidine transport was found to be increased two-fold at a permissive temperature in the cells of a normal rat kidney (NRK) line which was infected with a temperature-sensitive Rous sarcoma virus. This increase in thymidine transport was independent of cell density and coincided with the changes in cellular morphology that result from this temperature shift. A double reciprocal plot of the data demonstrated two saturable components (Km values of 60 microM and 250 microM at 39 degrees, non-permissive temperature) of the uptake of thymidine, and the drop of temperature (at 33 degrees, the permissive temperature for transformation) decreased Km to one half, but did not change Vmax. These results indicate that a qualitative alteration of thymidine transport took place in the presence of the active src gene product.

Detection of an 85,000-dalton phosphoprotein in ts110 murine sarcoma virus-infected cells with antiserum against a v-mos peptide.

We have used an antiserum directed against a synthetic v-mos peptide (anti-C3 serum) to screen ts110 murine sarcoma virus (MuSV)-infected cells for the presence of v-mos-encoded proteins. Anti-C3 serum specifically recognized an 85,000-dalton protein doublet (P85) from [35S]methionine-labeled ts110 MuSV-infected producer cells grown at 32 degrees C, the permissive temperature for transformation. The P85 doublet was also recognized by an antiserum directed against the viral gag protein p15. P85 was present but at 2- to 10-fold-lower levels in ts110 MuSV-infected producer cells grown at 39 degrees C, the restrictive temperature for transformation. The P85gag-mos fusion product was the only v-mos protein reproducibly detected in this ts110 MuSV-transformed cell line. Immunoprecipitation of 32P-labeled cells with anti-C3 serum revealed that the upper band of the P85 doublet is phosphorylated, containing mostly phosphoserine and some phosphothreonine. Cells acutely infected with ts110 MuSV contained slightly higher levels of P85 than did the ts110 MuSV-infected producer cell line. Anti-C3 serum specifically recognized a 33,000-dalton protein (p33) in the acutely infected cells labeled with [35S]methionine. p33 was present in trace amounts and may represent a previously unidentified ts110 MuSV-encoded v-mos protein.

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.

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.

Properties of two temperature-sensitive rous sarcoma virus transformed cerebellar cell lines

Cells from the cerebellum of 3-day-old BD-IX rats were obtained as permanent lines by transforming them with temperature-sensitive Rous sarcoma virus. The presence or absence of veratridine-stimulated Na +-uptake (voltage-dependent channels) was used to operationally classify them as neuronal or glial. When incubated at 34 °C, the permissive temperature for transformation, the cerebellar cells exhibit a transformed phenotype determined by anchorage independence, rounded morphology, high growth rate and absence of density-dependent inhibition of growth. In contrast, when the transformed cerebellar cell lines are kept at a temperature (38 °C) non-permissive for transformation, they exhibit a normal cellular phenotype with respect to the above properties. Moreover, changes toward neuronal morphology, increase in veratridine-stimulated Na +-uptake, decreased growth rate and the expression of the astrocyte specific protein, glial fibrillary acidic protein, suggest that a degree of differentiation is expressed at the non-permissive temperature.

Characterization of a temperature-sensitive membrane alteration in chick embryo fibroblasts infected with a temperature-sensitive mutant of Rous sarcoma virus

The intramembrane particles of freeze-fractured chick embryo fibroblasts transformed with a temperature-sensitive mutant of Rous sarcoma virus (TS68) are distributed differently at the permissive and non-permissive temperatures if, and only if, the cells are treated with glycerol before fixation. Few aggregates of intramembrane particles are present in glycerol-treated cells grown at the permissive temperature for transformation (36°C), while numerous large aggregates of particles are present at the non-permissive temperature (41°C). Changes in the distribution of particles after cells are shifted from 36 to 41°C are observed after 20 min, while a temperature shift from 41 to 36°C causes changes in glycerol-induced redistributions after 1 h. The changes observed in temperature shifts from 36 to 41°C and from 41 to 36°C do not require protein synthesis or RNA synthesis.

Effects of the src gene product on microfilament and microtubule organization in avian and mammalian cells infected with the same temperature-sensitive mutant of rous sarcoma virus

We have studied cytoskeletal organization in both mammalian (rat kidney cells) and avian cells (chick embryo fibroblasts) that have been transformed with temperature-sensitive src gene mutants of Rous sarcoma virus. The functioning of the src gene in rat cells affected the organization of actin-containing microfilament bundles, but not microtubules at the permissive temperature for transformation. These cells formed colonies in soft agar at permissive temperature, but not at nonpermissive temperature. In contrast, the src gene product affected both microtubule and microfilament organization in chick embryo fibroblasts at permissive temperature.

Transformation of chicken embryo retinal melanoblasts by a temperature-sensitive mutant of rous sarcoma virus

Retinal melanoblasts were transformed by a temperature-sensitive mutant of Rous sarcoma virus (ts-RSV). At the permissive temperature for transformation, the cells cease melanin synthesis, degrade their melanosomes and release much of their accumulated melanin into the medium. At the nonpermissive temperature, the cells assume an epithelioid morphology, actively synthesize melanin and become difficult to distinguish from normal uninfected control cultures. Both the transformed phenotype and the differentiated cell phenotype are temperature-dependent. Infected retinal melanoblasts which are incubated at the nonpermissive temperature and which accumulate a large amount of melanin are unable to transform in response to a temperature shift; instead, the cells degenerate and die. Retinal melanoblasts can be infected by subgroups A, B, C and D of RSV; however, their level of susceptibility to infection is about 1/40 compared to fibroblasts. Cultures infected by ts-RSV produce virus at both temperatures, suggesting that cell phenotype does not regulate virus synthesis.

Interactions of concanavalin A with chick embryo fibroblasts transformed by rous sarcoma virus Study with an RSV mutant thermosensitive for transformation

The interactions between concanvalin A and chick embryo fibroblasts, normal and infected with Rous sarcoma virus (RSV-BH) or its thermosensitive mutant RSV-BH-Ta, have been studied. Normal chick embryo cells and RSV-BH transformed cells showed at 4 and 25 °C a similar number of concanavalin A receptors per cell. Analysis of the binding data by the Scatchard relation showed that apparent changes in binding as a function of temperature are due to the thermodynamic properties of the process and and not to endocytosis. The lectin receptors on the cell surface of normal and RSV-BH infected cells showed homogeneity in their binding properties. Chick cells infected with RSV-BH-Ta showed a lectin binding behavior that was dependent on the temperature at which the cells were grown. At the permissive temperature for transformation (37 °C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 °C), the cells showed at least two sets of concanavalin A receptors. The new set of receptors on the cell surface had a lower lectin affinity than those observed in the same cells at 37 °C. Chick cells infected with RSV-BH showed an enhanced agglutinability by concanavalin A, as compared with normal cells. Cells infected with RSV-BH-Ta showed a reversal of the correlation between increased concanavalin A agglutinability and the transformed state. At the permissive temperature for transformation, the cells were not agglutinable, whereas at the nonpermissive temperature they presented agglutinability indexes as high as those observed with RSV-BH infected cells. This enhanced agglutinability observed with cells maintained at the nonpermissive temperature for transformation may be related to the new set of low affinity receptors present at 41 °C.

Regulation of sugar transport in chick embryo fibroblasts and in fibroblasts transformed by a temperature‐sensitive mutant of the rous sarcoma virus

The mode of induction of sugar transport by serum-stimulation of growth and hexose-starvation in chick embryo fibroblasts (CEF) has been studied using metabolic inhibitors. We have concluded from these studies that the sugar transport increases induced by serum-stimulation are regulated by post-transcriptional mechanisms while sugar transport increases induced by hexose-starvation are regulated by a transcriptional mechanism. CEF infected with a temperature-sensitive mutant of the Rous sarcoma virus. Ts68 and incubated at the nonpermissive temperature for transformation, 41 degrees, retain the capacity to regulate sugar transport in a manner similar to uninfected CEF. However, Ts68-infected CEF maintained at the permissive temperature for transformation, 37 degrees, have lost the ability to regulate sugar transport at the post-transcriptional and post-translational levels.