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.     

Regulation of amino acid transport in chicken embryo fibroblasts by purified multiplication-stimulating activity (MSA)

Enhanced amino acid transport is observed when quiescent cultures of chicken embryo fibroblasts are stimulated to proliferate by the addition of purified multiplication-stimulating activity (MSA). This increase in amino acid transport is an early event occuring prior to the onset of DNA synthesis in stimulated cells. Results indicate that the changes in transport activity, as measured by α-aminoisobutyric acid (AIB) uptake, are due to stimulation of only the Na⁺-dependent A transport system. There is little or no change in the activities of transport systems ASC, L, or Ly⁺ upon exposure to MSA. A kinetic analysis shows this increased activity is due to a change in Vmax while K_m remains unaltered. Continuous exposure to the stimulus is required to maintain the increased level of transport activity and the presence of inhibitors of RNA and protein synthesis significantly inhibits the response. Results also indicate that a similar specific increase in the A transport system is initiated when RSV tsNY68 infected cells are shifted to the permissive temperature. It appears that the A system of mediation is emerging as a strategic regulatory site for cell function.     

Role of the simian virus 40 gene A product in regulation of DNA synthesis in transformed cells.

Cells transformed by tsA mutants of simian virus 40 (SV40) are temperature sensitive for the maintenance of the transformed phenotype. The kinetics of induction of DNA synthesis were determined for hamster cell transformants shifted to the permissive temperature after a 48-h serum arrest at the nonpermissive temperature. DNAsynthesis was initiated in the tsA transformants by 8 h after shiftdown was maximal by 12 h. The presence or absence of fetal bovine serum at the time of temperature shift had no effect on the kinetics of initiation of DNA synthesis. Analysis of TTP in tsA transformants revealed similar levels of incorporation of [3H]thymidine into TTP at both permissive and nonpermissive temperatures. Autoradiography revealed that by 12 h after a shift to the permissive temperature, approximately 50% of the cells exhibited labeled nuclei after a 60-min pulse with [3H]thymidine, indicating that a majority of the cells were actively synthesizing DNA. By 8 to 12 h after a shiftup of confluent tsA transformants to the nonpermissive temperature, the number of labeled nuclei was reduced to approximately 16%, regardless of serum concentration. These data indicate that the SV40 gene A product, either directly or indirectly, regulates cellular DNA synthesis in transformed cells.     

Increased membrane transport of 2-deoxyglucose and 3-O-methylglucose is an early event in the transformation of chick embryo fibroblasts by Rous sarcoma virus.

Transformation of chicken embryo fibroblasts with Rous sarcoma virus results in cells with an enhanced rate of hexose uptake. We have examined transport of the glucose analogs 2-deoxyglucose and 3-O-methylglucose in cells infected with a temperature sensitive variant of the virus. In cells shifted from restrictive to permissive conditions for transformation, increased transport of the non-phosphorylatable analog 3-O-methylglucose occurs at the same time as that of 2-deoxyglucose, a phosphorylatable analog. This enhanced rate of transport can be observed within three hours of the temperature shift. There is a corresponding decrease in the transport rate of both analogs following shift to the restrictive temperature. These results suggest that increased transport is likely to be the primary event in causing transformation-specific changes in sugar metabolism. We have also examined uptake into the internal pools of both the phosphorylated and non-phosphorylated forms of 2-deoxyglucose in normal cells and in cells transformed by the wild-type virus. These data indicate a corresponding increase in the rate of accumulation of the free sugar in transformed cells and point to transport as the rate limiting step in the accumulation of 2-deoxyglucose in both normal and transformed chicken embryo cells.     

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

The interactions between concanavalin 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 degrees 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 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 degrees C), the binding process was similar to that observed for normal and RSV-BH infected cells. At the nonpermissive temperature (41 degrees 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 degrees 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 degrees C.     

Progress of aging in human diploid cells transformed with a tsA mutant of simian virus 40

Normal human diploid cells, TIG-1, ceased to proliferate at about the 62 population doubling level (PDL). Transformed clones isolated from TIG-1 cells infected with wtSV40 and those with tsA900 SV40 cultured at 34 °C were subcultured up to about 80 PDL. When the culture temperature of tsA SV40-transformed cells was shifted from 34 to 39.5 °C at 51 PDL, the growth curve of these transformed cells changed to that of normal young cells. When shifted to 39.5 °C after 62 PDL, cells immediately reached the end of their proliferative lifespan even under such favourable conditions for growth as low cell density in fresh medium. Growth of wtSV40-transformed cells did not change markedly at either temperature. These findings suggest that the clock of aging progresses in transformed cells as in normal cells, around 62 PDL being the senescent state in both cases, and that T-antigen of the tsA mutant of SV40 supports the extension of the lifespan of human cells only at the permissive temperature.     

Molecular weight dependence of hyaluronic acid produced during oncogenic transformation

Cultured chicken embryo fibroblasts synthesize two distinct molecular size classes of hyaluronic acid. The high molecular weight material (form I, 2.98 x 10(6) is the predominant species synthesized by transformed cells, whereas form II (1.42 x 10(5)) is the major product of non-transformed cells. A shift to synthesis of predominantly form I hyaluronic acid is an early transformation event in cells infected with LA24 Rous sarcoma virus and maintained at the permissive temperature for transformation (35 degrees C). Form I hyaluronic acid exhibits greater binding to preparations of cellular fibronectin and to both normal and transformed cells than does form II. Both forms bind more to transformed cells than to normal, uninfected cells. Hyaluronic acid (predominantly form I) isolated from transforming cells stimulates proliferation in growth-retarded, non-transformed cells.     

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 DNA replication by serum and the transforming function in cultured rat fibroblasts transformed by Rous sarcoma virus.

The onset and rate of semiconservative DNA replication were measured in stimulated cultured rat fibroblasts and their Rous sarcoma virus-transformed derivatives after a period of serum deprivation. Rat-1 (tsLA24/RSV) cells initiated DNA synthesis following a shift to the permissive temperature or addition of serum at the non-permissive temperature. Their rate of DNA replication was unaffected by the presence of serum at the permissive temperature, however, there was a serum requirement at the non-permissive temperature. The transition probability was less at the permissive temperature, independent of serum, than at the non-permissive temperature in the presence of serum. The amount of DNA induced to replicate by addition of serum at the non-permissive temperature or by a shift to the permissive temperature was similar. Using the untransformed Rat-1 cells and these cells transformed by wild-type RSV (Rat-1 (wt/RSV)), it was confirmed that the rate of entry into S phase (transition probability) was always lower in the transformed cell line at both 39° and 35°. In both cell lines the rate of DNA replication was independent of temperature, but the onset was delayed at the lower temperature. These results indicate that in the cell lines examined, (1) serum was able to commit the cells to replicate DNA (alter the transition probability) in both transformed and untransformed cells, but the transforming function was able to supplant a serum-dependent process during G₁ necessary for the initiation of DNA replication, and (2) the effects of the transforming function and serum factor(s) on the alteration of the transition probability are not additive, suggesting that the transforming function initiates a process which acts at the level of the commitment to DNA replication which may render the normal serum-related control mechanisms ineffective in the regulation of growth.     

Effects of withdrawal of a mitogenic stimulus on progression of fibroblasts into S phase: differences between serum and purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA) for chicken embryo fibroblasts was purified from serum-free medium conditioned by the growth of a rat liver cell line. A comparison between calf serum and purified MSA was made regarding the regulation of the fibroblast cell cycle. Addition of serum or MSA to stationary, quiescent cells stimulates them to enter the DNA synthetic phase after a characteristic lag period. Exposure to serum for shorter periods of time will irreverisbly commit cells to continue through the cell cycle and initiate DNA replication in the absence of serum. In contrast, the withdrawal of purified MSA from the medium results in an abrupt halt in the progression of cells towards S phase. The results of labeled thymidine incorporation and autoradiographic experiments clearly indicate that the point at which cells become irreversibly committed to enter the DNA synthetic period is at or near the G1-S boundary. The abrupt decay of the stimulation upon withdrawal of purified MSA provides a unique opportunity to investigate the biochemistry of this discrete phase of the cell cycle.     

Regulation of type VI collagen synthesis in transformed mesenchymal cells.

We have analysed the effects of oncogenic transformation on the expression of type VI collagen in mesenchymal cells. Synthesis of type VI collagen was almost completely inhibited in fibroblasts transformed by DNA or RNA tumour viruses or in cells derived from spontaneous mesenchymal tumours. Inhibition of type VI collagen synthesis appears, therefore, to be a common phenomenon of transformed mesenchymal cells. When introduced into normal cells by viral vectors, the 'nuclear' oncogene v-myc had an inhibitory effect similar to that of the 'cytoplasmic' oncogene v-src. Fibroblasts infected with a temperature-sensitive strain of Rous sarcoma virus (NY68) produced type VI collagen at the restrictive, but not at the permissive temperature. If such cells were shifted from the permissive to the restrictive temperature, synthesis of the individual subunits of type VI collagen was co-ordinately induced. These results demonstrate that the activity of a single oncogene product is sufficient to inhibit type VI collagen expression.     

Proliferation of Buffalo rat liver cells in serum-free medium does not depend upon multiplication-stimulating activity (MSA).

A line of Buffalo rat liver cells (BRL 3A) that multiplies in the absence of serum produces a family of polypeptides termed MSA that can partially satisfy the serum requirement for growth of chick embryo fibroblasts. Temin, Pierson and Dulak (1972) proposed that BRL cells multiply in serum-free medium because they produce MSA. This does not appear to be the case. We have studied three BRL cell lines: 3A2 and 3A have diverged from the same original isolate from normal liver; 61t is a spontaneous transformant of a different isolate. All three cell lines showed a 10 fold increase in cell number during 5 days in serum-free medium. However, 3A-conditioned medium stimulated 3H-thymidine incorporation into DNA in chick embryo fibroblasts and human skin fibroblasts; 3A2- and 61t-conditioned media did not. After ion-exchange chromatography or gel filtration of the conditioned media and measurement of MSA by 3H-thymidine incorporation or radioreceptor assay, MSA again was found in the 3A medium but not in the 3A2 or 61t media. The absence of MSA in the 3A2 and 61t media was not due to inactivation of MSA by these two cell lines. Addition of partially purified MSA to 3A2 cells did not increase their multiplication rate in serum-free medium. We conclude that the ability of the BRL cells to multiply in serum-free medium is independent of the level of MSA in the medium.     

Increased ouabain-sensitive 86Rubidium uptake after mitogenic stimulation of quiescent chicken embryo fibroblasts with purified multiplication-stimulating activity

Multiplication-stimulating activity (MSA), a protein which stimulates DNA synthesis and growth of chicken embryo fibroblasts, was purified from serum-free medium conditioned by the growth of a rat liver cell line. Purified MSA was shown to rapidly stimulate ouabain-sensitive Na+, K+-ATPase activity as measured by both enzyme assay and rate of 86Rubidium uptake. Labeled ouabain binding was also shown to increase after stimulation of quiescent cells by serum or purified MSA. Conditions which interfere with the ability of the cells to accumulate potassium, such as the presence of the specific inhibitor, ouabain; incubation in potassium-free medium; or the presence of the potassium ionophore, valinomycin, were all demonstrated to inhibit the stimulation of DNA synthesis by serum or purified MSA. These results suggest that an early event in the stimulation of DNA synthesis by purified MSA is an activation of membrane Na+, K+-ATPase with a resulting accumulation of potassium ions inside the cell.     

The viral Ki-ras gene must be expressed in the G2 phase if ts Kirsten sarcoma virus-infected NRK cells are to proliferate in serum-free medium.

NRK cells infected with a temperature-sensitive Kirsten sarcoma virus (ts371 KSV) are transformed at 36 degrees C, but are untransformed at 41 degrees C which inactivates the abnormally thermolabile oncogenic p21Ki product of the viral Ki-ras gene. At 41 degrees C, tsKSV-infected NRK cells were arrested in G0/G1 when incubated in serum-free medium, but could then be stimulated to transit G1, replicate DNA, and divide by adding serum at 41 degrees C or dropping the temperature to a p21-activating 36 degrees C without adding serum. When quiescent cells at 41 degrees C were stimulated to transit G1 in serum-free medium by activating p21 at 36 degrees C and then shifted back to the p21-inactivating 41 degrees C in the mid-S phase, they continued replicating DNA but could not transit G2. Reactivating p21 in the G2-arrested cells by once again lowering the temperature to 36 degrees C stimulated a rapid entry into mitosis. By contrast, while serum-stimulated quiescent G0 cells at 41 degrees C replicate DNA and divide, serum did not induce G2-arrested cells to enter mitosis, indicating that serum growth factors may trigger events in the G1 phase that ultimately determine G2 transit. These observations made with the viral ras product suggest that cellular ras proto-oncogene products have a role in G2 transit of normal cells.     

Failure of cultured chick embryo fibroblasts to incorporate collagen into their extracellular matrix when transformed by Rous sarcoma virus. An effect of transformation but not of virus production.

Whole chick embryo fibroblasts were infected with the Prague wild type Rous sarcoma virus and with a temperature sensitive mutant of this strain, RSVtsLA24. Normal fibroblasts and fibroblasts infected with the temperature-sensitive mutant and cultured at the nonpermissive temperature-sensitive mutant and cultured at the nonpermissive temperature, secreted procollagen into the medium and incorporated collagen into their extracellular matrix. On the other hand, transformed fibroblasts and fibroblasts infected with the temperature-sensitive mutant and cultured at the permissive temperature, were able to secrete procollagen into the medium, but there was no evidence that they were able to convert procollagen to collagen and incorporate collagen into an extracellular matrix. The inability of the infected cells to incorporate collagen into an extracellular matrix was found to be a result of transformation rather than of virus production in these cells.     

Temperature-dependent and transformation-associated changes in polyamine metabolism in normal and Rous sarcoma virus-infected chick embryo fibroblasts

Tertiary cultures of chick embryo fibroblasts infected and transformed by the wild-type Rous sarcoma virus, when actively growing at 35 degrees C, had higher putrescine levels than the respective uninfected cells. Transformed cells also had much higher specific activity of ornithine decarboxylase (EC 4.1.1.17) than the normal fibroblasts. At 41 degrees C the difference in putrescine levels between the normal and the transformed cells was less marked, and both cell types showed a relative accumulation of spermine. Cultures infected with the NY68 mutant virus, which is temperature-sensitive for transformation, showed at 41 degrees C normal cell morphology and intermediate polyamine patterns, while at 35 degrees C a transformed phenotype was found in both aspects. In shift-down experiments a change towards the permissive temperature pattern of polyamine metabolism was evident within 2-3 h. Difluoromethylornithine, a specific and irreversible inhibitor of ornithine decarboxylase efficiently reduced the enzyme activity as well as the levels of both putrescine and spermidine in all culture types and temperatures. Incubation of Rous sarcoma virus-transformed cells with 3 mM difluoromethylornithine for 36 h did not affect the maintenance of the transformed state. Likewise, when NY68-infected cultures were exposed to difluoromethylornithine at 41 degrees C for 12 h and then shifted down to 35 degrees C, the appearance of the transformed morphology took place concomitantly with that of the control cultures without respective changes in the polyamine levels. This suggests that the transformation-associated pattern of polyamines in chick embryo fibroblasts is not a prerequisite for morphological transformation of these cells.     

Modulation of adenylate cyclase by guanine nucleotides and Kirsten sarcoma virus mediated transformation

Certain tumour cells contain activated ras genes that code for 21 000 dalton proteins (p21). These proteins associate with the inner face of the plasma membrane and bind guanine nucleotides specifically. In order to determine whether p21s have functions similar to other GTP binding proteins, we investigated the regulation, by guanine nucleotides, of adenylate cyclase (AC) activity in membrane preparations isolated from fibroblasts (C127) transformed by a temperature sensitive mutant of Kirsten sarcoma virus (Ts 371). The degree of AC stimulation by GMP P(NH)P increased when these cells were shifted from the permissive temperature (33 degrees C) to the non-permissive temperature (39 degrees C). This effect was more pronounced at low Mg++ and low GMP P(NH)P concentrations. AC stimulation remained unchanged in rat fibroblasts infected with a temperature sensitive mutant of Rous Sarcoma virus. AC activity was depressed in C127 cells infected with wild type KiMSV. Our data illustrate the feasibility of correlating alterations in the AC system with ras gene expression and using such experimental approaches to elucidate the physiological functions of the p21 proteins.     

Nucleotide sequence of noncoding regions in Rous-associated virus-2: comparisons delineate conserved regions important in replication and oncogenesis.

Fujinami sarcoma virus (FSV) encodes a 140,000-dalton transforming protein, P140, which contains gag- and fps-specific sequences. The cellular localization of this protein was examined by fractionation of [35S]methionine-labeled, FSV-infected chicken embryo fibroblasts. In homogenates of cells infected by wild-type, temperature-resistant FSV prepared in either hypotonic or isotonic buffer, 60 to 80% of the P140 was particulate. Isopycnic separation on discontinuous sucrose gradients indicated that the majority of the particulate P140 was present in a light membrane fraction enriched for plasma membranes. Much of the particulate P140 could be solubilized by the addition of 0.6 M salt to a postnuclear supernatant, suggesting that P140 is not an integral membrane protein. Particulate P140 may be associated with membranes either directly as a peripheral membrane protein or indirectly via cytoskeletal elements. In cells infected by mutants of FSV temperature sensitive for cellular transformation, most of the P140 is particulate at the permissive temperature, whereas most is soluble at the nonpermissive temperature; this change in distribution is not a secondary consequence of the change in cellular phenotype, since it also occurs in nonconditionally transformed cells doubly infected with temperature-sensitive FSV and wild-type Rous sarcoma virus. The movement of P140 from the particulate to the soluble fraction occurs rapidly when cells infected by temperature-sensitive FSV are shifted from the permissive to the nonpermissive temperature. Furthermore, P140 moves from the soluble to the particulate fraction, although somewhat more slowly, when cells are shifted from the nonpermissive to the permissive temperature. These observations suggest that the association of P140 with plasma membranes or the cytoskeleton may play a role in transformation by FSV.