Demonstration of T antigens on the surface of cells transformed with primate polyoma viruses

Primate polyoma virus-transformed hamster, mouse, and rat cell lines were examined by indirect immunofluorescence staining for cell surface-associated T antigens, by using a rabbit antiserum prepared against sodium dodecyl sulfate-denatured large T antigen of simian virus 40 (anti-SV40-SDS-T serum). Positive surface staining was shown not only on SV40-transformed cells, but also on BK and JC virus-transformed cells. In contrast, normal cells and cells transformed with mouse polyoma-, human adeno-, and murine sarcoma viruses were negative. The data on SV40-transformed cells confirmed the reports of others demonstrating the cell surface location of SV40 large T antigen, and the data on BK and JC virus-transformed cells proved that these cells have cell-surface T antigens that cross-react with anti-SV40-SDS-T serum.     

Characterization of phosphatidylthreonine in polyoma virus transformed fibroblasts.

A threonine phospholipid in polyoma virus-transformed hamster embryo fibroblasts has been characterized as phosphatidylthreonine. The identification has been made by chemical and enzymatic hydrolysis. Acid hydrolysis of the phospholipid produces free threonine. Mild alcoholysis produces a water-soluble derivative having the properties of glycerophosphorylthreonine. Hydrolysis with phospholipase C produces phosphorylthreonine which on prolonged acid hydrolysis yields threonine. Phosphatidylthreonine in the cell is more accessible to reaction with fluorodinitrobenzene than is phosphatidylserine. Phosphatidylthreonine also has been found as a major aminophospholipid in two other polyoma-transformed hamster cell lines and in the BHK-21/C13 line including the PVT-3 and TS-3 lines. the latter derived from BHK cells. Only a trace amount of phosphatidylthreonine occurs in normal liver, kidney and spleen of the adult mouse, in normal liver and kidney of the adult hamster, in whole mouse and hamster embryos, and in mouse 3T3 cells and SV40-transformed 3T3 cells.     

Cholesterol content and metabolism in normal and polyoma virus-transformed hamster embryo fibroblasts

The total cholesterol content of normal hamster embryo fibroblasts and polyoma virus-transformed hamster embryo fibroblasts were found to be similar. However, the free cholesterol: cholesterol ester ratio was 41.5 in normal cells as contrasted to 1.8 in their transformed counterparts. This difference is due in part to an increase in cholesterol esterification and a decrease in the hydrolysis of cholesterol esters in the transformed cell.     

Binding of concanavalin A by normal, herpes virus-transformed, and trypsin-treated hamster embryo fibroblasts

Using fluorescein isothiocyanate-labeled concanavalin A (ConA), it was shown that normal hamster embryo fibroblast cells appear to bind less ConA than do herpes virus-transformed cells. However, the binding capacity of normal HEF cells could be increased following trypsin treatment.     

Effects of acidified fetal bovine serum on the fibrinolytic activity and growth of cells in culture.

The fibrinolytic activity of cells in culture varied with the type of serum employed in the growth medium. Degradation of iodinated fibrin occurred slowly when Rous sarcoma virus-transformed chick embryo fibroblasts were grown in medium containing fetal bovine serum (FBS), and rapidly when chicken serum was employed. This difference reflected the low plasminogen and high inhibitor content of FBS. The inhibitors were found to be serum macromolecules that were precipitated with ammonium sulfate or polyethylene glycol, and were inactivated by boiling or upon exposure to acidic conditions. No inhibitor activity was detected in fetuin, one of the major proteins present in FBS. Acidified FBS was similar to chicken serum in that both supported high rates of cell-mediated fibrinolytic activity. Although virally transformed hamster, mouse and chicken cells grew well in acid-treated FBS, their normal counterparts did not. Apparently, acifification resulted in the formation of materials that were toxic to normal cells. These agents rapidly blocked cellular DNA synthesis.     

In vitro methylation of total and foldback DNAs in normal and virus-transformed cells

The levels of the in vitro methylation of total and palindromic DNAs in nuclei isolated from normal and virus-transformed cells are compared. The methylation rate of total DNA in normal rat kidney cells is much higher than that detected in normal mouse fibroblasts. However, for both cell species, while the maximal rate of DNA methylation is observed in the mid-logarithmic phase of the cell culture growth, palindromes are always found to be more heavily methylated than total DNA. The 5-methylcytosine content of DNA, especially of palindromes, is higher in virus-transformed cells than in untransformed cells.     

Rabbits as a source of antisera against SV40 virus-induced cell surface antigens.

Rabbits immunized with SV40 virus-transformed rabbit cells yielded antisera highly reactive in a mixed-hemadsorption assay against SV40-transformed hamster, mouse, and rabbit cells. Such antisera may prove of value in studies requiring large amounts of antibody angainst SV40-induced cell surface antigens.     

Control and virus-transformed baby hamster kidney cells resistant to ethidium bromide. I. Characterization and the respiratory enzymes

Cell lines resistant to ethidium bromide have been developed from cultured mammalian BHK21/C13 cells and these same cells transformed by Rous sarcoma virus (C13/B4). Cells resistant to 2 micrograms ethidium bromide per milliliter have been cloned. One clone of the control and one of the virus-transformed cell lines has been employed for characterization. The resistant cells, in the presence of 2 micrograms ethidium bromide/ml, grow at approximately the same rate as the untreated parental cells. The control cells possess a "normal" karyotype (44 chromosomes), while the corresponding ethidium bromide mutant has a reduced chromosome number of 41 and a number of translocations. The mitochondria displayed morphological alterations compared to the parental lines during the transition phase prior to the isolation of the ethidium bromide-resistant cells. The mitochondria of the ethidium bromide-resistant mutants appear somewhat enlarged with a normal morphology. The effect of ethidium bromide on selected respiratory enzymes in normal and virus-transformed ethidium bromide-resistant baby hamster kidney cells was determined. Ethidium bromide-resistant cells exhibited a depressed level of cytochrome aa3. This depression could not be reversed by growth in ethidium bromide-free media. Ethidium bromide-resistant cells possessed the same cytochrome b, c, and c1 levels per cell as their corresponding parental lines. Purified mitochondria isolated from virus-transformed ethidium bromide-resistant cells exhibited a depression in cytochrome oxidase-specific activity, while the ethidium bromide-resistant control cells did not. All cell lines studied showed a depression in NADH-ferricyanide and NADH-cytochrome c reductase-specific activities relative to their parental BHK21/C13 cells. No increase was observed in virus-transformed ethidium bromide-resistant cells. Ethidium bromide-resistant control cells exhibited a two-fold increase in oligomycin-insensitive adenosine triphosphatase activity relative to their parental cells. All of the cell lines studied possessed equivalent oligomycin-sensitive adenosine triphosphatase-specific activity except for the virus-transformed, dye-resistant mutant, whose activity was increased.     

Characterization of mouse integrin alpha3 subunit gene.

Integrin alpha3beta1 (VLA-3) is an adhesion receptor for extracellular matrix proteins including various isoforms of laminin. We have isolated mouse genomic clones encoding the integrin alpha3 subunit and deduced the exon/intron organization. The mouse integrin alpha3 subunit gene is encoded by 26 exons spanning 40 kb. The exon/intron structure of the integrin alpha3 subunit gene resembles that of the integrin alpha6 subunit gene, but differs somewhat from those of other members of the integrin family. We have demonstrated that the cytoplasmic domain splicing variants of the alpha3 subunits (alpha3A and alpha3B) are generated by alternative exon usage. We also cloned the 5'-flanking region and performed a preliminary analysis of its promoter activity in various tumor cell lines with different degrees of integrin alpha3 expression. Following transfection, activity in the luciferase assay was found to be roughly correlated with the expression level of integrin alpha3 as measured by flow cytometry. Furthermore, the luciferase assay was performed with normal and SV-40- or polyoma virus-transformed fibroblasts. In mouse, human, and hamster fibroblasts, higher levels of luciferase expression were observed in transformed cells than in normal cells. This result is consistent with our previous finding that integrin alpha3 expression at both the protein and mRNA levels is enhanced upon oncogenic transformation of fibroblasts by tumor viruses.     

Genealogies of clones of diploid fibroblasts. Cinemicrophotographic observations of cell division patterns in relation to population age

Addition of dibutyryl cyclic AMP (db-cAMP) plus theophylline to normal or virus-transformed 3T3 mouse fibroblasts, in concentrations sufficient to alter the growth characteristics, produced no qualitative changes in the pattern of incorporation of glucosamine (Glc.NH₂) into glycoproteins at their extreme periphery. Nor did these drugs qualitatively alter the patterns of incorporation of 1-¹⁴C-galactose (Gal) into the principal glycosphingolipids (GSL) of these cells. In particular, virus-transformed 3T3 cells, which do not synthesize certain complex gangliosides made by normal cells, were not caused to do so. It may, however, be significant that drug treatment did result in increased incorporation of precursor galactose into complex heteroglycans of both normal and virus-transformed cells. The implications of these observations are discussed.     

Viral and cellular fos proteins: a comparative analysis

The FBJ murine osteosarcoma virus (FBJ-MuSV) induces osteosarcomas in mice and transforms fibroblasts in vitro. It contains an oncogene termed v-fos derived from a normal cellular gene by recombination with an associated helper virus. The product of the v-fos gene is a 55,000 dalton protein, p55v-fos. This protein was found in the nuclei of cells containing amplified levels of the v-fos gene, and also in the nuclei of virus-transformed cells. The c-fos protein was localized in the nuclei of normal mouse amnion cells and in the nuclei of cells transformed by a recombinant plasmid that expresses the c-fos gene product. However, p55c-fos undergoes more extensive post-translational modification in the nucleus than p55v-fos. Immunofluorescence data indicate that the level of p55c-fos in normal mouse amnion cells is similar to that found in fibroblasts transformed by the v-fos or c-fos proteins.     

The uptake of actinomycin D by normal and virus transformed BHK21 hamster cells

The uptake of actinomycin-D (AMD) in the hamster cell line BHK21 clone 13, and its polyoma virus-transformed derivative, were compared. In the transformed cell the internal AMD concentration at equilibrium was lower and was reached more quickly. The AMD-binding capacities of nuclei from normal and transformed cells were similar, suggesting that some control of AMD uptake occurs at the plasma membrane. This may be a control on the efflux of AMD since this process has a higher rate constant in transformed cells.     

Unmasking of nerve growth factor membrane-specific binding sites in synchronized murine C 1300 neuroblastoma cells

The resistance to actinomycin D (AD) of a clone of SV40 virus-transformed hamster cells (TSV5 C12 A^R) was investigated. This line was found to be much less permeable to the antibiotic. Penetration of proflavine and puromycin is also greatly reduced without any prior selection for these drugs. The AD that enters the cells is capable of inhibiting RNA synthesis. Thus the resistance of these cells to AD seems to be due only to the decreased permeability of the drugs possibly resulting from an alteration of the cell membrane.     

Lymphotropic papovavirus transformation of hamster embryo cells

Hamster embryo cells were transformed by African green monkey lymphotropic papovavirus (LPV). The transformed cells contained intranuclear T-antigens demonstrable by fluorescent antibody staining with hamster anti-LPV serum. Analysis of uncloned and cloned lines of transformed cells for LPV sequences revealed that the viral DNA was present as free nonintegrated and integrated genomes; there were approximately 10 copies of free DNA and about one to two copies of integrated genomes per cell. The cells were highly tumorigenic when inoculated into hamsters and produced progressively growing tumors in 100% of newborn or 10-day-old hamsters that were inoculated with LPV-transformed cells. The serum from tumor-bearing hamsters reacted with LPV-transformed cells and also showed a weak reaction with simian virus 40-, BK virus-, and JC virus-transformed cells, thereby showing an antigenic relationship with the T-antigens of other primate polyomaviruses. The large T-antigen of LPV was found to be an 84,000-molecular-weight protein which was immunoprecipitated by hamster anti-LPV (antiviral) as well as by tumor serum.     

Characterization of the hamster genomic fragment cloned by TAR cloning technology with interspecific sequence information

CHO (Chinese Hamster ovary) cells are widely used for biotechnology and biomedical purposes, and now the EST library database of CHO cells is built. Based on this, the construction of the hamster genome library is under exertion. Though the transformation-associated recombination (TAR) cloning method is accounted as an innovative cloning technology without the construction of the genome library in human and mouse, there has been no trial to isolate the genomic fragment from hamster genome by TAR cloning. In this study, approximately 31 kb of hamster genomic fragment was isolated from the normal human/hamster mono-chromosomal somatic cell line (UV5HL9-5B) using universal hooks of rodent repeats sequence of B1 and B2 by TAR cloning. This fragment was analyzed by bioinformatics tools related to the genome alignment for the similarity analysis among rodent and primate, and was classified into rodents by phylogenetic analysis. One putative gene was found in this region which has homology with the human c14orf4 gene. A zinc finger protein domain was found in the translated hamster ORF. Therefore, we suggest that TAR cloning technique can be applied in CHO cells using mouse genomic information, and it can lead to the establishment of the hamster genome database.     

Several types of adherent cells elaborate a dialyzable lymphopoietic cofactor (Abelson Growth Promoter)

We have cloned a stromal cell from mouse bone marrow selected on the basis of its ability to promote the growth of an Abelson virus-transformed pre-B cell line. These stromal cells have smooth muscle features, and the ultrafiltrate of stromal cell-conditioned medium has proliferative effects on all feeder layer-responsive mouse pre-B cell lines tested, as well as on normal pre-B cells. One of these low MW substances is a protease-resistant factor with an MW of approximately 450 Da which we designate Abelson Growth Promoter (AGP). AGP was initially characterized as an activity which promotes the growth of Abelson virus-transformed mouse pre-B cells, but it also promotes the growth of a ras-transformed pre-B cell line as a single agent and in synergistic fashion with recombinant interleukin (IL) 7. AGP as a single agent has no effect on normal pre-B cells, which have a brisk response to IL-7. In contrast, transformed pre-B cells display a blunted response to IL-7. We propose that AGP plays a role in normal lymphopoiesis by expanding clones of pre-B cells which have been activated by other stromal cell-derived signals, such as IL-7, and directly promotes the growth of nascently transformed pre-B cells.     

Migration of transformed fibroblast-like cells on substrates with patterned relief-work (quantitative study)]

The quantitative estimation of migration ability of transformed fibroblast-like cells of various epecies (mouse , rat, hamster, man) cultured on the substratum with grooves of various depth (5--40 mcm) was performed. Of the majority of 11 cell lines examined, a reduced migration capacity up to its total disappeanance was registered as compared with the homological normal embryonic cells. A more pronounced reduction of migration ability was shown in mouse and rat transformed cells and less in human ones. Migration of transformed hamster cells was just equal to the weak migrtion of normal hamster embryonis cells. These data shown a weak response of the analysed transformed cells to the relief of underlying substratum.     

Vitiligo-related pigment cell differentiation antigens are expressed on malignant melanoma cells following phenotypic reversion induced by contact inhibitory factor

Most vitiligo sera contain antibodies to surface antigens on pigmented human melanocytes but not to human or mouse amelanotic melanoma cells. A density-dependent line of hamster amelanotic melanocytic cells (FF) produces a diffusible factor (CIF) which restores contact inhibition of growth as well as several other normal phenotypic characteristics to hamster, murine, and human melanoma cells. The ability of CIF to induce the expression of a phenotypic characteristic of pigmented human melanocytic cells, i.e., the vitiligo-related surface antigens, on hamster and mouse amelanotic melanoma cells was investigated. Vitiligo and normal sera were reacted with CIF-treated and untreated hamster and mouse amelanotic melanoma cells for both indirect-immunofluorescence assays and ELISA. Immunofluorescence testing showed that about 80% of hamster and mouse melanoma cells had pigment-cell antigens (in the absence of pigmentation) in a granular surface pattern after, but not prior to, CIF-induced morphologic reversion and confluent growth. Less than 5% of the control hamster and mouse melanoma cells expressed such antigens at confluence. These results were confirmed by ELISA. Metabolic-labeling studies with 35S-methionine showed that the vitiligo antigens were synthesized by the CIF-treated melanoma cells. The slowing of melanoma cell proliferation in isoleucine-deficient medium failed to elicit the expression of vitiligo antigens. Since antigen appearance following phenotypic reversion occurred without pigment induction, it is concluded that vitiligo-related surface antigens and pigmentation are distinct aspects of a differentiated function which may be non-coordinately expressed. The expression of pigment-cell differentiation antigens on amelanotic melanoma cells is an additional feature of the pleiotypic trans-species response to CIF.     

Size of virus-specific RNA in B-34, a hamster tumor cell producing nucleic acids of type C viruses from three species.

B-34 is the designation of a hamster tumor-derived cell line induced by the Harvey sarcoma virus. This cell line produces virions which contain structural proteins common to edogenous hamster viruses and nucleic acid sequences of hamster, mouse, and rat origin. The sedimentation characteristics of the intracellular virus-specific RNA was determined in sucrose gradients after treatment with dimethylsulfoxide by molecular hybridization using complementary DNA of strict virus specificity. Hamster virus-specific RNA sedimented at 35S (major peak) as is characteristic of productive infection by type C leukemia viruses of other species. Rat virus-specific RNA sedimented at 30S which is characteristic of the sarcoma virus-related genome found in nonproducer cells transformed by Kirsten sarcoma virus. Both Harvey and Kirsten sarcoma viruses contain a related but not necessarily identical 30S rat-specific component which is also found in normal cultured rat cells. Mouse cells producing Harvey sarcoma virus also contain a rat-specific 30S RNA. Mouse virus-derived sequences also sedimented at 30S in B-34 cells and in a similar size range in Harvey virus-infected mouse cells. The possibility that the mouse and rat-derived sequences are present on a single 30S RNA species which would then be related to sarcomagenic potential is one attractive hypothesis suggested by these data.     

Immunochemical purification of probe-labeled plasma membrane proteins: An approach to the molecular anatomy of the cell surface

The probe 2,4,6-trinitrobenzene sodium sulfonate may be used under appropriate conditions for selective labelling of plasma membrane proteins exposed at the outer cell surface. Labeled proteins, solubilized by detergents, can be purified by reverse immunoadsorption using antiprobe antibodies covalently linked to Sepharose 4B. This method has been applied to an investigation of the outer cell surface structure of chicken embryo and hamster fibroblasts. Coelectrophoresis in sodium dodecyl sulfate-polyacrylamide gels of probe-labeled membrane proteins purified from baby hamster kidney fibroblasts have shown that 7 major protein groups of different molecular weight are exposed on both control and Rous sarcoma or polyoma virus-transformed cells. Moreover, the transformed cells display a nonvirion component of 80–100 k daltons that is not labeled by the probe in normal cells. In fibroblasts transformed by a temperature sensitive Rous sarcoma virus mutant, that transforms at 37°C but not at 41°C, the expression of this component is related to the expression of the transformed phenotype.