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PubMed1977

Cyclic AMP-induced morphological transformation of cells infected by temperature-sensitive mouse sarcoma virus. Expression of transformation-associated markers.

Normal rat kidney (NRK) cells infected with a temperature-sensitive (ts) mutant of mouse sarcoma virus (NRK [MSV-1b]) express the transformed phenotype when grown under permissive conditions, but acquire the normal phenotype when grown under restrictive conditions. Addition of 3', 5' cyclic adenosine monophosphate (cAMP) to NRK (MSV-1b) cells grown at the restrictive temperature results in morphological transformation. To determine whether other markers associated with the transformed phenotype were coordinately expressed after cAMP exposure, concanavalin A (Con A) agglutinability, hexose transport rate, and incorporation of radioactively labeled fucose into fucolipid III and fucolipid IV (FL III and FL IV ) of the cells were examined. NRK cells transformed by wild-type MSV or NRK(MSV- 1b) grown under permissive conditions were agglutinated by low concentrations of Con A and exhibited relatively high maximal agglutination levels which were specifically inhibited by alpha-methyl-D-mannoside. In contrast, NRK (MSV-1b) cells grown under restrictive conditions were weakly agglutinated by Con A and exhibited reduced maximal agglutination levels, similar to uninfected NRK cells. Treatment of NRK (MSV-1b) cells at the restrictive temperature with cAMP resulted in morphological transformation and a change in the pattern of incorporation of labeled fucose inot FL III and FL IV to one comparable to that of NRK (MSV-1b) cells at the permissive temperature or to NRK cells transformed by wild-type MSV. In contrast, cAMP treatment resulted in no increase in Con A agglutinability or 2 deoxy-D- [(3)H]glucose transport relative to mock treated cultures. The results demonstrate that cAMP-induced morphological transformation and altered fucolipid composition of NRK (MSV-1b) cells are not correlated with alterations in hexose transport rate or Con A agglutinability.

Agglutination↗

PubMed1977

Continued presence of similar transformation-associated antigens related to murine oncornavirus proteins in -ransformed cells, morphological revertants, and cells restricted in the expression of transformation.

The possible alteration of transformation-related antigens in cells that revert to a normal phenotype but that continue to retain the viral genome, has been investigated in [3H]glucosamine-labeled extracts of rat cells exhibiting a reversible temperature-dependent restriction in the expression of transformation and in a comparison of a morphologically altered mouse cell transformed by the Kirsten sarcoma virus with a flat revertant mouse cell derived from the morphologically transformed cells. With the use of normal goat serum in the presence of dibutyryl cyclic 3':5'-adenosine monophosphate, some differences became obvious in the rat cells restricted in the expression of transformation. However, use of specific antiserum to murine leukemia virus revealed in every case the presence of major components that exhibited an electrophoretic mobility corresponding to about 100,000 daltons both in the parent and revertant mouse cells and in the rat cells exhibiting either untransformed or transformed growth properties. The glycoprotein components detected only by the immune serum may represent a cellular macromolecule antigenically related to an interspecies C-type viral species whose concentration is increased in transformed cells.

Antigens, Neoplasm↗

PubMed1979

Fibronectin expression is determined by the genotype of the transformed parental cells in heterokaryons between normal and transformed fibroblasts.

The expression of fibronectin, a cell surface-associated transformation-sensitive glycoprotein, was studied in hetero- and homokaryons of normal and SV40-transformed human fibroblasts. In immunofluorescence, fibroblast homokaryons had an intense surface-associated and intracelluar fibronectin fluorescence similar to that of normal fibroblasts. Transformed cells and their homokaryons had a minimal surface-associated and a weak intracellular fibronectin fluorescence. In heterokaryons formed between transformed and normal fibroblasts, the expression of fibronectin fell within 24 h to the level of the transformed cell homokaryons. The change was detectable already at 3 h after fusion and was gene-dose dependent. These results show that the transformed genotype determines fibronectin expression in the heterokaryons.

Cell Fusion↗

PubMed1976

Transformation-enhancing factor(s) produced by virus-transformed and established cells.

Chick embryo fibroblasts (CEF) and hamster BHK21 cells transformed by the Schmidt-Ruppin strain of Rous sarcoma virus (SR-RSV) release into the culture medium a factor or factors which enhance 2- to 7-fold the formation of transformed foci by chich embryo fibroblasts infected with the Bryan strain of RSV (B-RSV). The factor(s) also increase the number of foci failing to revert to normal phenotype at restrictive temperature (41 degrees C) in cultures infected with a temperature-sensitive mutant (FU-19) of SR-RSV which is defective for transformation. The factor(s) is produced also by BHK21 cells transformed by other tumor viruses and by BHK21 cells passaged for a long time, but not by normal CEF, CEF transformed by B-RSV, CEF infected by FU-19 at 41 degrees C, normal hamster embryo fibroblasts, established but density-inhibited mouse fibroblasts, or BHK21 cells of early passages. The relative enhancement of the number of B-RSV foci can be more than 100-fold when the medium contains fetal calf serum which suppresses focus formation in controls. The focus-enhacing factor(s) appears to act after infection and has been termed, operationally, transformation-enhancing factor(s) or TEF. The factor produced by RS2/3 cells which enhances the formation of B-RSV foci is non-dialyzable and thermolabile, and is presumably a protein. Its molecular weight is between 10(5) and 2 X 10(5) daltons.

Animals↗

PubMed1977

Transformation of human cystinotic fibroblasts by SV40: characteristics of transformed cells with limited and unlimited growth potential.

Human skin fibroblasts derived from patients with nephropathic cystinosis were transformed with SV40 virions, cloned and permitted to enter the degenerative stage of growth termed "crisis," characteristic of SV40 transformed human cells. Nephropathic cystinosis is an autosomal recessively inherited metabolic disorder resulting in the intracellular accumulation of the amino acid cystine. A transformed cystinotic cell line which was recovered from the crisis stage was indistinguishable from its transformed precrisis parental cell strain in growth rate in media containing either 1% or 10% serum, cloning efficiency on plastic, in semisolid media, or upon confluent monolayers of normal skin fibroblasts, expression of SV40 T antigen, or production of virus. However, the modal DNA content of the recovered postcrisis transformed cystinotic cell line was different from that of the cloned parental precrisis transformed cell strain, suggesting that the postcrisis line was derived from a small subpopulation of the precrisis strain. The DNA content of the established cystinotic cell line continued to be unstable during subsequent subculturing and gave rise to subclones with both more and less DNA per cell. This line now has an apparently infinite growth potential and still has the hallmark of the cystinotic parental line, the storage of abnormally large amounts of intracellular nonprotein cystine.

Antigens, Viral↗

PubMed1975

Inhibition of protease activity in cultures of rous sarcoma virus-transformed cells: effect on the transformed phenotype.

We have examined the role of proteolytic activity in the genesis and maintenance of the transformed phenotype by growing cultures of chick embryo fibroblasts transfromed by Rous sarcoma virus either in medium containing plasminogen-free serum or in medium to which protease inhibitors were added. Alterations in morphology, adhesiveness, and hexose transport were used as markers for the transformed state. Addition of the trypsin inhibitors NPGB or Soy Bean Trypsin Inhibitor at concentrations which inhibited transformation-associated fibrinolysis restored adhesiveness and morphology to near normal, but did not affect the rate of hexose transport. Growth of Rous-infected cells in plasminogen-free medium blocked the appearance of morphological and adhesive alterations, but allowed the rate of hexose transport to increase to the transformed level. Thus we were able to separate the appearance of transformation-specific changes in morphology and adhesiveness (which apparently require fibrinolytic activity) from the increased rate of hexose transport (which is independent of fibrinolytic activity). Another trypsin inhibitor, TLCK, although it did not inhibit fibrinolysis, was very effective at restoring adhesiveness and morphology as well as hexose transport to normal. This raises the possibility that there is another, perhaps earlier, protease involved in the genesis of the transformed phenotype.

Animals↗

PubMed1977

Restoration of normal morphology, adhesion and cytoskeleton in transformed cells by addition of a transformation-sensitive surface protein.

Transformed cells lack a large, external, transformation-sensitive (LETS) glycoprotein which is a major surface component of their normal counterparts. Addition of LETS glycoprotein isolated from normal cells to transfomed cells restores certain morphological features and adhesive properties characteristic of normal cells. LETS protein is detected on the cell surface both by iodination using lactoperoxidase and by immunofluorescent staining. The surface distribution pattern detected by immunofluorescence is strikingly similar to that of normal cells. After addition of LETS protein, transformed cells also exhibit well defined actin cables which are not seen in untreated, transformed cells. All these alterations can be blocked by treating LETS protein with specific antisera or by subjecting it to mild trypsinization prior to addition to transformed cells. The effects are rapidly reversible by mild trypsinization, which removes the added LETS protein. The high rate of uptake of 2-deoxyglucose, characteristic of transformed cells, is not affected by LETS protein. These results suggest that LETS protein may have a role in cell attachment and spreading, and affect the organization of cytoskeleton.

Actins↗

PubMed1979

The effect of 8-methoxypsoralen-plus ultraviolet light on cell-virus interaction: the transforming infection; effect of PUVA on the transformation of baby hamster kidney cells by polyoma virus.

Pre-treatment of baby hamster kidney (BHK) cells with 8-methoxypsoralen (8-MOP) plus ultraviolet (UV) light enhances the frequency of their transformation by polyoma (Py) virus. Of the doses tested, 0.5 microgram/ml 8-MOP plus 0.3 J/cm2 UV-light results in maximal (30-fold) stimulation of viral transformation. 8-MOP alone does not affect viral transformation and UV-light alone causes only a slight increase in the transformation frequency. Thus the drug and light act synergistically in promoting the effect. Treatment of BHK cells with drug plus light without Py infection does not lead to a transformed morphology. A drug-light combination (0.01 microgram/ml 8-MOP plus 1.2 J/cm2 UV) that inhibits cellular DNA synthesis to 75% of control at 28 hr after treatment results in a 6-fold stimulation of the transformation frequency.

Animals↗

PubMed1975

Transformation of Bacillus subtilis: transforming ability of deoxyribonucleic acid in lysates of L-forms or protoplasts.

The transformation of Bacillus subtilis by homologous deoxyribonucleic acid (DNA) made available by gently lysing a stable L-form or protoplast suspension was 3 to 10-fold more efficient than DNA isolated by conventional procedures. This increased transformation was not influenced by digestion with pronase, trypsin, or ribonuclease. Preincubation of isolated DNA with L-form lysates did not increase the transformation efficiency above that achieved with untreated, isolated DNA. In addition to displaying a higher efficiency of transformation, the DNA found in these gently prepared lysates was also able to co-transform heretofore unlinked markers at frequencies in excess of those found by congression. Comparison of the frequency of multiple marker transformations to single marker events as a function of DNA dilution conclusively proves that these markers originated from the same continuous strand of DNA.

Bacillus subtilis↗

PubMed1979

Detection of a transformation-related antigen in chemically induced sarcomas and other transformed cells of the mouse.

Antisera prepared against BALB/c Meth A sarcoma in syngeneic or compatible F1 mice recognize a protein with an apparent molecular weight of 53,000 in extracts of [35S]methionine-labeled transformed BALB/c cells. This component, designated p53, was not detected in normal adult mouse fibroblasts, lymphoid cells, or hematopoietic cells or in mouse embryo cells or 3T3 cells. An extensive variety of antisera, including alloantisera and heterologous antisera directed against structural antigens of murine leukemia viruses, was tested for reactivity with p53; other than Meth A antisera, only comparably prepared antisera against another BALB/c sarcoma, CMS4, had anti-p53 activity. All transformed mouse cells tested were found to express p53; these tests included chemically induced sarcomas, leukemias, spontaneously transformed fibroblasts, and cells transformed by simian virus 40 and murine sarcoma virus. The presence of p53 in tumors of no known viral etiology indicates coding by resident cellular genes; this does not exclude endogenous viruses as the source of coding sequences or the possibility that transforming viruses code directly for p53.

Animals↗

PubMed1976

SV40 transformation of mouse brain cells: critical role of gene A in maintenance of the transformed phenotype.

Brain cells derived from the NIH Swiss mouse strain have been established in tissue culture. Astrocytic neuroglial cells, identified by morphology and staining properties, predominate. The brain cell culture was successfully transformed with SV40 wild type virus and with a representative early (A239) and late (C219) mutant. When subjected to growth analysis the A239 transformant displayed selective loss of six characteristics of the transformed phenotype at the restrictive temperature (40.5 degrees C): doubling time, saturation density, ability to grow in low serum, efficiency of growth on plastic and on normal brain cell layers, and cloning in soft agar. Temperature shift experiment demonstrated the reversibility of the differences in saturation density. T-antigen was expressed at both temperatures. Alteration in uptake of 2-deoxyglucose was not a characteristic of the transformed phenotype. The cell lines may be utility in brain culture work and in studies on the mechanism of SV40 transformation.

Antigens↗

PubMed1976

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.

Agglutination Tests↗

PubMed1975

Fibrin overlay methods for the detection of single transformed cells and colonies of transformed cells.

Fibrin overlay methods are described which can detect the plasminogen activator produced by single transformed cells or small colonies of transformed cells. These methods were applied to malignant cells derived from humans, mice, hamsters, rats, and chicks. The lysis observed was plasminogen dependent. Transformation of chicken cells by Rous sarcoma virus was detected 4 days after infection. The number of lysis zones produced was proportional to the virus inoculum and was identical to the number of morphologically determined foci. These methods may also have application in model systems for scoring transformation by chemicals. Transformed mouse and chicken cells were detected at the single cell level and the number of lysis zones produced was dependent on the number of cells present, the time of incubation, and the concentration of plasminogen.

Animals↗

PubMed1979

Change of isozyme pattern during activation of a transforming gene product: its relation to other biochemical markers of cellular transformation and differentiation.

Isozyme patterns of leucine aminotransferase were studied in connection with glucose transport and DNA synthesis during the activation and deactivation of the transforming gene product in rat kidney cells transformed by one Rous sarcoma virus mutant (which has a temperature-sensitive lesion in its transforming gene. On temperature shift-down of confluent transformed cells grown at 40 degrees C in the presence of fresh serum, isozyme III of leucine aminotransferase appeared in 12--20 h, with increasing amounts from 24 to 48 h. Upon temperature shift-up, isozyme I became the predominant form in these cells within 4 days, the major change occurring within the first 24 h. The rate of protein turnover was similar to the rate of loss of isozymes I and III during temperature shift-down and shift-up, respectively. A stimulation of incorporation of [3H]thymidine into DNA was observed within 8--12 h after temperature shift-down of the transformed cells. For the maintenance of stimulated DNA synthesis for at least 16 h, continued exposure to the permissive temperature is not necessary. Stimulation of glucose transport occurred prior to the stimulation of [3H]thymidine incorporation. The isozymes of leucine aminotransferase also changed during the in vitro differentiation of Yaffee L6A cells in such a way that isozyme I represented the major part of this enzyme in the fused myotube, and isozyme III was more predominant in the less differentiated state (mononucleated cells).

Animals↗

PubMed1977

Glucose depletion accounts for the induction of two transformation-sensitive membrane proteinsin Rous sarcoma virus-transformed chick embryo fibroblasts.

Chick embryo fibroblasts transformed by Rous sarcoma virus have an increased content of two membrane proteins of molecular weights 78,000 and 95,000. The increased content of the 95,000-dalton protein and the principal increase in the content of the 78,000-dalton protein are not an early consequence of cell transformation but instead are secondary to the rapid depletion of glucose from the growth medium of transformed cells. When glucose is maintained at high levels in the growth medium of transformed cells, the synthesis of the 95,000-dalton protein is arrested and that of the 78,000-dalton protein is markedly suppressed. Upon removal of glucose from the growth medium of normal cells, these proteins increase to levels comparable to those of transformed cells. Because the amount of these two proteins is influenced by the presence or absence of glucose, we suggest they be referred to as "glucose-regulated proteins." GRP-78 and GRP-95. These proteins may have an important role in regulating the utilization of glucose in cultured cells.

Animals↗

PubMed1978

Stable transformation of mouse, rabbit and monkey cells and abortive transformation of human cells by BK virus, a human papovavirus.

Semi-permissive mouse, rabbit and monkey cells were stably transformed by BK virus (BKV). The specificity of transformation was demonstrated by the presence of BKV tumour (T) antigen in nuclei of transformed cells and by virus rescue with Sendai virus-mediated fusion or transfection. Two out of seven BKV-transformed cell lines were oncogenic. Permissive human cells were only abortively transformed by BKV, since morphologically modified cells persisted in culture for a few passages and eventually died.

Animals↗

PubMed1979

Transforming DNA sequences in rat cells transformed by DNA fragments of highly oncogenic human adenovirus type 12.

Rat cell lines tranformed by viral DNA fragments, EcoRI-C and HindIII-G, of adenovirus type 12 DNA were analyzed for the viral transforming DNA sequences present in cell DNAs. Cell lines transformed by the EcoRI-C fragment of adenovirus type 12 DNA (leftmost 16.5% of the viral genome) contain most of the HindIII-G sequences of the HindIII-G fragment, but at a different frequency depending on the portions of the fragment. The sequence of the AccI-H fragment of adenovirus type 12 DNA (the left part of the HindIII-G; leftmost 4.5% of the viral genome) was detected dominantly in cells transformed by the HindIII-G fragment Southern blot analysis showed that viral DNA sequences are present at multiple integration sites in high-molecular-weight cell DNA from cells transformed by the EcoRI-C or HindIII-G fragment of adenovirus type 12 DNA. These results suggest that most of the HindIII-G sequences in cells transformed by the HindIII-G fragment are present as fragmented forms.

Adenoviruses, Human↗

PubMed1976

Altered microfilament structure in cells transformed with a temperature-sensitive transformation mutant of murine sarcoma virus.

The structure and distribution of microfilaments were examined by electron microscopy in uninfected normal rat kidney (NRK) cells, murine sarcoma virus (MSV)-transformed NRK cells, and NRK cells infected with a cold-sensitive transformation mutant of MSV, i.e., NRK (MSV-1b) cells, grown at both permissive (39 degrees) and nonpermissive (33 degrees) temperature. The uninfected cells contained numerous microfilaments which were especially prominent at sites of intercellular adherens junctions. In contrast, the MSV-transformed cells contained few microfilaments and did not form adherens junctions. At 33 degrees, the NRK (MSV-1b) cells appeared normal but formed an altered form of adherens junction with disorganized microfilaments. At 39 degrees, these cells resembled NRK cells transformed by wild-type MSV but still formed a few of the altered type of adherens junctions. Disorganized adherens junction microfilaments were also found in cells newly infected with wild-type MSV. These results suggest that the perturbed assembly of microfilaments at adherens junctions may be an intermediate stage in the loss of adherens junctions during viral transformation.

Cell Line↗