SV40-transformed Mouse Fibroblasts Research Articles

Both tumor necrosis factor and nitric oxide participate in lysis of simian virus 40-transformed cells by activated macrophages.

SV40 transformation of rodent fibroblasts generally produces cells that are highly sensitive to killing by activated macrophages. The cell line SV-COL-E8 (E8) is typical of SV40-transformed mouse fibroblasts in that it is readily lysed when exposed to activated macrophages. This killing is not due solely to TNF, because soluble TNF alone is incapable of lysing these cells. TNF is, however, necessary for lysis since antibodies to TNF will prevent macrophage-mediated lysis. Similarly, E8 is not sensitive to nitric oxide (NO); however, NO is also necessary for lysis since inhibition of NO generation (by coincubation with the arginine analogue NG-monomethyl-1-arginine) with Fe(II)) blocks lysis of E8 by activated macrophages. Cytolysis by macrophages is contact dependent, suggesting that the cell-associated TNF precursor may be involved in mediating cytolysis. However, transfected cell lines bearing cell-associated TNF precursor do not mediate killing of E8. Thus, killing of E8 either involves both TNF and NO in addition to a third, as yet unidentified, lytic mechanism, or killing requires the contact-dependent delivery of TNF and NO from the macrophage to its target.

Mutational analysis of regulation of MHC and anti-viral genes.

CTL-mediated selection for loss of expression of Mta by H-2-heterozygous SV40-transformed mouse fibroblasts (line 24SV) produced an unusual phenotypic class of maternally transmitted Ag negative mutants defective in both MHC expression and in anti-viral activity. Severely reduced surface expression of class I MHC Ag from multiple loci of both haplotypes correlated with low levels of MHC H chain and beta 2-microglobulin mRNA. Inasmuch as IFN can up-regulate class I expression and some fibroblasts elaborate autocrine IFN-beta, we examined whether IFN could restore wild-type expression of class I MHC Ag. However, IFN could not restore wild-type expression. Moreover, the fold-increases in class I Ag and mRNA expression were significantly reduced in mutant cells compared to wild-type cells. These results suggested that the mutants might have generalized defects in IFN response. Inasmuch as the induction of an anti-viral state is a hallmark of IFN responses, we exposed cells to IFN-alpha, -beta, or -gamma and challenged with virus. 24SV cells, exposed to any of the three IFNs, were completely protected from destruction by vesicular stomatitis, mengovirus or respiratory syncytial viruses. In contrast, MHC and anti-viral defective mutants could not be protected from virus-induced lysis by any IFN. Somatic cell hybridization analyses indicated that both basal MHC and IFN-inducible phenotypes were recessive to wild-type, and that a trans-acting regulatory factor required for basal MHC expression is defectively expressed in the mutants. Such a factor may integrate the organismal response to virus infection, encompassing both immune and nonimmune anti-viral responses.

Natural cytotoxic cells from rat liver and spleen kill human glioma cells.

Natural cytotoxic cells from rat spleen and rat liver, (isolated using the collagenase method) were found to be cytotoxic against different lines of human gliomas: T406, T508, T705, HeRo, HeRoCl 1, HeRoCl 8, and HeRo-SV 7/114. After 18 h the lytic units ranged from 75 to 251 in the liver and from 5 to 24 in the spleen. Analyzing the ratio of lysis at 4 h/18 h, it may be concluded that this natural killing is predominantly macrophage (Kupffer cell)-dependent. Lysis by Kupffer cells cannot be increased by transforming glioma cells with SV40. Experiments with SV40-transformed mouse fibroblasts (3T3) and virus-transformed human cell lines (SV80) suggested a "SV40" receptor on Kupffer cells. Thus Kupffer cells have receptors for glioma cells and SV40-dependent membrane structures.

Heterogeneity in distribution and content of p53 in SV40-transformed mouse fibroblasts

The high level and intranuclear location of the cellular phosphoprotein p53 are usually regarded as invariant features of SV40-transformed fibroblast lines. During the development of improved methods for immunocytochemical detection of p53 using SVA31 E7 mouse fibroblasts, we have observed unexpected and marked variations in its distribution. Cells grown on plastic coverslips were fixed in acetone and the content and distribution of p53 and SV40 large T-antigen analysed by an indirect immunoperoxidase procedure using monoclonal antibodies PAb122 and PAb 248 for p53, and PAb416 for large T. First, we observed in all cultures an apparent reversal of intracellular compartmentalization, with strong cytoplasmic and absent nuclear/chromatin positivity in mitotic cells and young daughter cells. More importantly, for a short period, between 18–24 h after trypsinization and cell passage, we observed a marked overall reduction in detectable nuclear p53 content in all cells with both PAb122 and PAb248 antibodies. The first observation also held for SV40 large T-antigen, the second only for p53. These variations have important practical implications for the immunocytochemical analysis of cellular content and intracellular compartmentalization of p53. The biological implications of our findings are also discussed.

Highly adherent mutants of SV40-transformed mouse fibroblasts: genetic and biochemical characterization.

Mutants of SV40-transformed mouse fibroblasts have been isolated that have greatly increased cell-substratum adherence. The adherent phenotype (COL-) is recessive, and all mutants analyzed belong to one complementation group. No consistent qualitative differences between wild-type and mutant cells were found with respect to the protein content of the substrate-attached material (SAM), a cell surface fraction left after removal of cells from the substrate with a gentle Ca2+-chelating agent. However, the mutants yielded 2.5-10-fold more SAM than the parental cell line, and the SAM deposited by mutants was able to mediate attachment of transformed cells to a much greater degree than was the SAM from the parental cell line. The mutation, which appears to control the generation of footpads, was shown to cosegregate with resistance to the drug 6-thioguanine, which suggests X-linkage.

Transformation of mouse mammary epithelial cells by papovavirus SV40

Primary and established murine mammary epithelial cells and wild-type SV40 were employed to study the phenomenon of epithelial cell transformation. Thirteen independent transformed cell lines were derived. All contained SV40 intranuclear T antigen. Eight transformed mammary cell lines were examined ultrastructurally and all were found to exhibit pronounced epithelial cell characteristics, including desmosomes and tight junctions. Growth studies revealed that while normal mammary cells were unable to grow in low serum (2% FBS), established Cl S1 mammary cells and SV40-transformed mammary epithelial cells replicated well. Cell densities achieved by the transformants were only slightly elevated in high serum (13% FBS) over normal cell values. All the transformants formed colonies on plastic and exhibited anchorage-independent growth in methylcellulose. Five of the transformed lines were tumorigenic in syngeneic animals, in marked contrast to the lack of transplantability usually observed with SV40-transformed mouse fibroblasts. Anchorage-independent growth was not a predictor of tumorigenic potential in this system. The transformants exhibited a spectrum of responsiveness to exogenous growth factors. This study establishes that the SV40-murine mammary cell system is a valid model for analyses of the process and consequences of epithelial cell transformation, in general, and mammary cell transformation in particular.

Cell surface and cytoskeletal interactions in a temperature-sensitive strain of SV40-transformed mouse fibroblasts.

In order to assess the role of cytoskeletal structure in modulating cell surface topography during cell transformation, cytoskeletal organization of 3T3 mouse cells transformed with a tsA mutant of simian virus 40 (SV40) was studied in detail by correlative light and electron microscopy. Detergent-extracted, critical-point dried whole cells observed in the electron microscope were seen to contain well-organized microfilament bundles (stress fibers) traversing the longitudinal axis of cells grown at the restrictive temperature (39 degrees C). When grown at the permissive temperature (32 degrees C), cells prepared in this manner were not observed to contain such structures. However, when semithin sections (0.5 micron) were viewed by transmission electron microscopy at 120 kV, short microfilament bundles were seen in 32 degrees C-grown cells. There was an alteration in the morphology of these structures at sites of attachment to the substratum (focal contacts), and they were shorter in length than microfilament bundles of 39 degrees C-grown cells. A difference was also observed between the two phenotypes in the layer of microfilaments associated with the dorsal cell surface. Since it is this layer that directly determines cell surface architecture, it is proposed that changes in microfilament bundle-generated surface tension are responsible for alterations of this layer, leading to an altered cell surface morphology. Tension may be modified by disturbances in focal contacts (or adjacent regions) or altered actin-associated protein(s).

Regulation of polyamine content in cultured fibroblasts.

The content of putrescine and of the polyamines (spermidine and spermine) and the activities of their biosynthetic enzymes were measured in 3T3 mouse fibroblasts and SV40-transformed mouse fibroblasts over the entire period from subculturing in fresh medium until confluence. The transformed cells had a substantially higher content of putrescine and spermidine than the 3T3 cells and higher activities of all of the biosynthetic enzymes. However, the ratio of spermine synthase to spermidine synthase was higher in the 3T3 cells, which correlated with their higher spermine-to-spermidine ratio. All of the biosynthetic enzymes increased in activity during cell growth. Ornithine decarboxylase increased 20-fold with a maximum at 24-36 h after culturing whereas S-adenosylmethionine decarboxylase increased 3-fold at the same time. Spermidine synthase increased 10- to 16-fold during the growth period whereas spermine synthase increased 2- to 3-fold. The relative enzyme activities and the changes in total polyamine content suggested that 1) the activity of S-adenosylmethionine decarboxylase limited the production of the polyamines and 2) the relative amounts of spermidine and spermine synthase determined the predominant polyamine that the available decarboxylated S-adenosylmethionine is used to synthesize. When 3T3 cells become quiescent at confluence, there was a substantial fall in the intracellular spermidine level because of a greatly increased excretion of spermidine into the medium. Spermine content also fell because there was an increased conversion of spermine into spermidine, which was then excreted. The specific excretion of spermidine did not occur with the transformed SV-3T3 cells.

Stimulation of DNA polymerase alpha by a nuclear DNA/protein complex.

A nuclear DNA complex containing DNA polymerase and SV40 T-antigen was isolated from nuclei of SV40-transformed mouse fibroblasts. DNA polymerase could be separated from the complex. The remaining DNA/T-antigen-containing complex stimulated DNA polymerase alpha activity about 10-fold. The complex contained 4 major proteins with molecular weights of 46, 54, 76, and 94 kilo-dalton (KD). The stimulation activity was retained by protein A-Sepharose loaded with specific IgG from SV40-tumor bearer serum, or from antisera against the 94 KD and 76 KD components and was partially inhibited in the presence of these antisera. The stimulation activity was completely abolished by treatment of the complex with trypsin or DNase I.

Fluorescence photobleaching recovery measurements of surface lateral mobilities on normal and SV40-transformed mouse fibroblasts.

Lateral mobilities of fluorescent cell surface probes have been measured on normal (3T3) and transformed (SV3T3) cultured mouse fibroblasts. There is little discernible difference in the mobilities of a lipid analogue (diI), a fluorescent ganglioside derivative (GM1), and tetramethylrhodamine-labeled succinylated concanavalin A. The two cell lines showed expected differences in their abilities to grow in agar, to grow without serum, and to be agglutinated by lectins, indicating that changes of these properties in transformed cells are probably not mediated through increased overall membrane fluidity but are associated with distinct alterations in the mobilities of cell surface receptors. Both fluorescent dextran derivatives and antimouse cell surface antibodies were distinctly less mobile on SV3T3 cells, and the mobile fraction of Con A receptors was lower on SV3T3 cells.

Procaine-Mediated Modification of Membranes and of the Response to X Irradiation and Hyperthermia in Mammalian Cells

Procaine, a membrane-specific agent, was found to protect the response of oxygenated mammalian cells toward x irradiation in vitro. In addition, the drug also potentiates hyperthermic killing of cells. In an effort to gain further insights into the mechanisms underlying the action of procaine as a modifier of cell injury in x-irradiated and heated cells, the effects of procaine on cellular morphology, membrane fluidity, and cellular electrokinetic properties were studied. It was found that procaine can reversibly influence the cellular morphology. Using scanning electron microscopy, we were able to demonstrate the presence of numerous smooth blebs on the cell surface of either normal or SV40-transformed 3T3 mouse fibroblasts treated with procaine. When 1,6-diphenyl-1,3,5-hexatriene was used as a membrane probe, the overall fluidity of cell membranes from 3T3 cells grown as monolayers or L5178Y lymphoma cells grown in suspensions was not significantly changed by procaine; however, when perylene was used as a probe, the overall fluidity of cell membranes of procaine-treated cells was found to increase significantly. Lastly, procaine was found to alter the electrophoretic mobility of cells. These results indicate that procaine may exert its modification of radiation- and heat-induced effects via a still elusive membrane-mediated mechanism(s). It is anticipatedmore » that membrane-active agents with different specificities may be employed as tools to probe the role of membranes in the radiation or heat response of cells.« less

Nucleotide Sequence of Three Isoaccepting Lysine tRNAs from Rabbit Liver and SV40‐Transformed Mouse Fibroblasts

The lysine isoacceptor tRNAs differ in two aspects from the majority of the other mammalian tRNA species: they do not contain ribosylthymine (T) in loop IV, and a 'new' lysine tRNA, which is practically absent in non-dividing tissue, appears at elevated levels in proliferating cells. We have therefore purified the three major isoaccepting lysine tRNAs from rabbit liver and the 'new' lysine tRNA isolated from SV40-transformed mouse fibroblasts, and determined their nucleotide sequences. Our basic findings are as follows. a) The three major lysine tRNAs (species 1, 2 and 3) from rabbit liver contain 2'-O-methylribosylthymine (Tm) in place of T. tRNA1Lys and tRNA2Lys differ only by a single base pair in the middle of the anticodon stem; the anticodon sequence C-U-U is followed by N-threonyl-adenosine (t6A). TRNA3Lys has the anticodon S-U-U and contains two highly modified thionucleosides, S (shown to be 2-thio-5-carboxymethyl-uridine methyl ester) and a further modified derivative of t6 A (2-methyl-thio-N6-threonyl-adenosine) on the 3' side of the anticodon. tRNA3Lys differs in 14 and 16 positions, respectively, from the other two isoacceptors. b) Protein synthesis in vitro, using synthetic polynucleotides of defined sequence, showed that tRNA2Lys with anticodon C-U-U recognized A-A-G only, whereas tRNA3Lys, which contains thio-nucleotides in and next to the anticodon, decodes both lysine codons A-A-G and A-A-A, but with a preference for A-A-A. In a globin-mRNA-translating cell-free system from ascites cells, both lysine tRNAs donated lysine into globin. The rate and extent of lysine incorporation, however, was higher with tRNA2Lys than with tRNA3Lys, in agreement with the fact that alpha-globin and beta-globin mRNAs contain more A-A-G than A-A-A- codons for lysine. c) A comparison of the nucleotide sequences of lysine tRNA species 1, 2 and 3 from rabbit liver, with that of the 'new' tRNA4Lys from transformed and rapidly dividing cells showed that this tRNA is not the product of a new gene or group of genes, but is an undermodified tRNA derived exclusively from tRNA2Lys. Of the two dihydrouridines present in tRNA2Lys, one is found as U in tRNA4Lys; the purine next to the anticodon is as yet unidentified but is known not be t6 A. In addition we have found U, T and psi besides Tm as the first nucleoside in loop IV.

Spontaneous glycosylation of glycosaminoglycan substrates by adherent fibroblasts

SV40-transformed mouse fibroblasts migrate upon, and spontaneously glycosylate, plastic substrates derivatized with chondroitin-6-sulfate and hyaluronic acid. Autoradiography of cultures prelabeled with 3H-glucose and 3H-galactose demonstrates the presence of silver grains near adherent cells. Silver grains are particularly dense near cells cultured on chondroitin sulfate. No significant grains are observed on control dishes or dishes derivatized with polygalacturonic acid. Radioactive material left by cells is not removed by boiling the dishes in 8 M urea and 10% sodium dodecylsulfate, suggesting that it is covalently linked to the derivatized plastic. Acid hydrolysis shows that the radioactive material consists of glucuronic acid and N-acetylglucosamine when the prelabeled cells are cultured on hyaluronic acid. When cells are cultured on chondroitin sulfate, the radioactive product consists only of glucuronic acid and N-acetylgalactosamine sulfate. Sonicates of prelabeled cells or the supernatants from cultures of intact prelabeled cells add no SDS-urea-resistant radioactivity to dishes derivatized with these glycosaminoglycans.

A GTP-specific protein kinase in plasma membranes of mouse fibroblasts

Endogenous protein phosphorylation in plasma membranes isolated from SV 40-transformed mouse fibroblasts was studied in the presence of [γ- 32P]GTP. The phosphoprotein pattern showed one main component with 100 k which could not be phosphorylated with ATP. Optimal phosphorylation of pp 100 was dependent on protein concentration. Strict substrate specificity of the corresponding endogenous kinase could be demonstrated by comparative electrophoresis with a plasma membrane sample phosphorylated with [γ- 32P]ATP and by isotope dilution experiments. GTP-specific phosphorylation of pp 100 was not dependent on cyclic nucleotides but influenced by GDP, GMP and AMP.

Short-time phosphorylation of plasma membrane proteins by endogenous kinases

Endogenous protein phosphorylation in plasma membranes isolated from SV 40-transformed mouse fibroblasts was studied in the presence and absence of cyclic nucleotides. Using low concentrations of membrane protein the kinetics of ATP-dependent 32P-incorporation showed a rapid phosphorylation reaction up to 2 sec of incubation which was stimulated by cAMP and inhibited by cGMP. This short-time phosphorylation reaction was followed by a rapid dephosphorylation and a slower rephosphorylation. This phenomenon was dependent on protein concentration.

Relation of cell type and cell density in tissue culture to the isoaccepting spectra of the nucleoside Q containing tRNAs: tRNATYr, tRNAHis, tRNAAsnand tRNAAsp

An examination, using reversed-phase chromatography and cyanogen bromide treatment, of tRNATyr, tRNAHis, tRNAAsn, and tRNAAsp from SV40-transformed mouse fibroblasts grown to different cell densities, untransformed cells grown to confluence, and mouse liver indicates that: (1) The tissue cultured mouse fibroblasts examined here are hypomodified with respect to nucleoside Q, while liver tRNA is almost completely modified with respect to Q. (2) Cell density and/or proliferative state do not present as major variables in controlling the expression of Q in the present system. (3) SV40 virus transformation is not a major variable controlling the expression of Q in the present system. The present results support previous use of cyanogen bromide effected shifts in chromatographic elution as an assay for nucleoside Q.

An effect of ATP on the permeability of transport-competent plasma membrane vesicles from mouse fibroblasts

ATP and Mg ++ produced a striking inhibition of Na +-stimulated amino acid and phosphate transport activity assayed in vesicles isolated from the plasma membrane of SV40-transformed and nontransformed mouse fibroblasts. The α-β and β-γ-methylene analogs of ATP were ineffective. Purified membrane vesicles contain an endogenous protein kinase activity which catalyzes autophosphorylation of membrane proteins in the presence of ATP. This provides an experimental approach to investigate the role of membrane protein phosphorylation in mediating permeability changes in response to changes in cellular proliferative or transformed state.

Biochemical studies of a protein which restores contact inhibition of growth to malignant melanocytes

A factor prepared from conditioned medium of a contact-inhibited hamster melanocytic cell line (FF), and which restores the capacity for contact inhibition of growth to malignant melanocytes, is a glycoprotein of molecular weight about 160000 whose amino acid composition shows an acid-base ratio of 2.04. Although the melanocyte contact inhibitory factor (MCIF) is a constituent of both contact-inhibited (FF) and non-contact-inhibited (RPMI 1846) melanocytes, being present in a fraction which includes endoplasmic reticulum and plasma membrane, it is spontaneously released only into culture media of the contact-inhibited (FF) cells. Addition of MCIF to cultures of highly malignant hamster melanocytes (RPMI 1846) is followed by a rapid fall of intracellular cGMP and rise in cAMP. A protein with electrophoretic mobility identical with that of MCIF is present in conditioned culture media of other contact-inhibited cell lines including a human epidermal line and human and mouse fibroblasts, but is absent from media of hamster melanoma, and much reduced in cultures of SV40-transformed mouse fibroblasts. MCIF may be a widely distributed growth regulatory macromolecule concerned with facilitating cell-cell interactions required for the generation and/or reception of normal signals leading to cessation of growth.

Biochemical studies of a protein which restores contact inhibition of growth to malignant melanocytes

A factor prepared from conditioned medium of a contact-inhibited hamster melanocytic cell line (FF), and which restores the capacity for contact inhibition of growth to malignant melanocytes, is a glycoprotein of molecular weight about 160000 whose amino acid composition shows an acid-base ratio of 2.04. Although the melanocyte contact inhibitory factor (MCIF) is a constituent of both contact-inhibited (FF) and non-contact-inhibited (RPMI 1846) melanocytes, being present in a fraction which includes endoplasmic reticulum and plasma membrane, it is spontaneously released only into culture media of the contact-inhibited (FF) cells. Addition of MCIF to cultures of highly malignant hamster melanocytes (RPMI 1846) is followed by a rapid fall of intracellular cGMP and rise in cAMP. A protein with electrophoretic mobility identical with that of MCIF is present in conditioned culture media of other contact-inhibited cell lines including a human epidermal line and human and mouse fibroblasts, but is absent from media of hamster melanoma, and much reduced in cultures of SV40-transformed mouse fibroblasts. MCIF may be a widely distributed growth regulatory macromolecule concerned with facilitating cell-cell interactions required for the generation and/or reception of normal signals leading to cessation of growth.

Differences in the Lipid Distribution in Subcellular Fractions of Mouse Fibroblasts Derived from Logarithmic and Stationary Growth

The lipid class compositions of subcellular fractions of SV40-transformed mouse fibroblasts derived from the logarithmic and stationary growth phase were compared. Cell populations of the stationary growth phase showed a relative decrease of the protein content and an increase of triglycerides and alkoxydiglycerides which could be located in the non-sedimenting fraction and in the nuclei and mitochondria containing fraction, respectively. Futheron, distinct shifts in the subcellular distribution of those lipid classes could be observed which exhibited no relative overall increase or decrease when the cells of both growth phases were compared. In the crude plasma membrane fraction the ratio "lipid class/protein" remained about constant with the exception of the phospholipids and alkoxydiglycerides.