Kirsten murine sarcoma virus transformed cell lines and a spontaneously transformed rat cell-line produce transforming factors

We have examined culture fluids from a variety of Kirsten murine sarcoma virus (KiMSV) transformed rat and mouse cells for the presence of factors which induce normal Rat-1 cells to assume the transformed phenotype. All KiMSV transformants produced transforming factor (TF). Revertants of KiMSV transformed rat or mouse cells failed to relase TF as did normal rat or mouse cells. Cells transformed by a temperature sensitive mutant of KiMSV produced TF at the permissive temperature but not at the nonpermissive temperature. Further, cells from a spontaneous transformant of Rat-1 cells also produced TF. TF is a small polypeptide which competes for the epidermal growth factor receptor. Its effect upon normal cells is reversible and requires de novo RNA and protein synthesis. Cells treated with TF lose the actin fibers observed in normal fibroblasts, assume a transformed cell morphology, become anchorage independent for growth, grow in low concentrations of serum, grow to a high cell density, and have an increased rate of hexose uptake.     

Characterization of intra- and extracellular transforming growth factors from normal and avian sarcoma virus-transformed rat cells

Intra- and extracellular transforming growth factors (TGFs) have been characterized in an avian sarcoma virus-transformed rat cell line, 77N1, and the nontransformed parental cell line, NRK. Serum-free conditioned medium from 77N1 and cell extracts from NRK and 77N1 were subjected to ion exchange column chromatography. Intracellular TGFs in cell extracts of NRK and 77N1 cells showed a major peak of DNA synthesis-stimulating and colony-forming activities which eluted in the 0.10 to 0.15 M salt concentration region. Extracellular TGF in conditioned medium of 77N1 cells showed a major peak of activity which eluted at 0.05 to 0.06 M salt concentration. Furthermore partially purified extracellular TGF had a molecular weight of about 40,000 daltons, whereas that for intracellular TGF was about 12,000 daltons. These intra- and extracellular TGFs, as well as TGF gamma 2 which was purified from 77N1 cell extract, were heat- and acid-labile polypeptides sensitive to treatment with dithiothreitol. Radioimmunoprecipitation analyses with antiserum against TGF gamma 2 demonstrated that intra- and extracellular TGFs in 77N1 and NRK cells were immunologically identical or very closely related to each other and suggested the possibility that extracellular TGF from 77N1 cells was released into serum-free conditioned medium after formation of complex with other cellular components.     

Epidermal growth factor-like transforming growth factor. I. Isolation, chemical characterization, and potentiation by other transforming factors from feline sarcoma virus-transformed rat cells

An acid-stable transforming growth factor (TGF) that interacts with epidermal growth factor (EGF) receptors and is structurally related to EGF was isolated from serum-free culture fluids of Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells. Purification of this EGF-like TGF (eTGF) was achieved by molecular filtration chromatography and successive reverse-phase high pressure liquid chromatography steps on octadecyl support eluted with acetonitrile and 1-propanol gradients, respectively. Rat eTGF consists of a 7.4-kD single polypeptide chain that co-migrates with biological activity in dodecyl sulfate-polyacrylamide electrophoresis gels. Like preparations of a related TGF from human melanoma cells (Marquardt, H., and Todaro, G.J. (1982) J. Biol. Chem. 257, 5220-5225), but unlike EGF from rat, human, or mouse, rat eTGF has phenylalanine and lacks methionine. However, the sequence of the first 30 amino acid residues in rat eTGF is H2N-Val-Val-Ser-His-Phe-Asn-Lys-Cys-Pro-Asp-Ser-His-Thr-Gln-Tyr-Cys-Phe-His-Gly - Thr-(x)-Arg-Phe-Leu-Val-Gln-Glu-Glu-(Lys)-(Lys)-, which is significantly (20% and 28%) homologous to the NH2-terminal region of mouse EGF and human EGF, respectively. In addition to eTGF, molecular filtration chromatography of acid-soluble extracts from medium conditioned by FeSV-Fre cells resolved a 14-kD transforming factor(s) apparently devoid of intrinsic mitogenic activity but able to elicit a strong anchorage-independent growth response in the presence of eTGF or EGF. These results show that: 1) a 7.4-kDa TGF structurally and functionally related to EGF has been isolated from FeSV-Fre cells and 2) the full anchorage-independent growth-promoting activity of medium conditioned by FeSV-Fre cells is due to the coordinate action of at least two types of factors, the 7.4-kDa eTGF and a second 14-kDa transforming factor(s).     

Type beta transforming growth factor from feline sarcoma virus-transformed rat cells. Isolation and biological properties

We have isolated a strongly mitogenic, type beta transforming growth factor (beta TGF) released by Snyder-Theilen feline sarcoma virus-transformed rat embryo (FeSV-Fre) cells that induces phenotypic transformation of normal NRK cells when they are concomitantly stimulated by analogues of epidermal growth factor (EGF). Molecule filtration chromatography separates beta TGF from an EGF-like TGF (eTGF) which is also present in acid extracts from medium conditioned by FeSV-Fre cells (J. Massagué, (1983) J. Biol. Chem. 258, 13606-13613). Final purification of beta TGF is achieved by reverse phase high pressure liquid chromatography (HPLC) on octadecyl support, molecular filtration HPLC, and nonreducing dodecyl sulfate-polyacrylamide gel electrophoresis steps, yielding a 300,000-fold purified polypeptide with a final recovery of 21%. The purified rat beta TGF consists of two Mr = 11,000-12,000 polypeptide chains disulfide-linked as a Mr = 23,000 dimer. Induction of anchorage-independent proliferation of NRK cells by rat beta TGF depends on the simultaneous presence of eTGF or EGF. In the presence of a saturating (300 pM) concentration of either rat eTGF or mouse EGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with 4-6 pM rat beta TGF. In the presence of a saturating (20 pM) concentration of rat beta TGF, half-maximal anchorage-independent proliferation of NRK cells is obtained with either rat eTGF or mouse EGF at a 50-70 pM concentration. Rat beta TGF is also able to induce DNA synthesis and cell proliferation on growth-arrested NRK, human lung, and Swiss mouse 3T3 fibroblast monolayers, this effect being half-maximal at 2-3 pM beta TGF for NRK cells. These results identify eTGF and beta TGF as the two synergistically acting factors responsible for the transforming action of culture fluids from FeSV-Fre cells.     

Bifunctional activity of transforming growth factor type beta on the growth of NRK-49F cells, normal and transformed by Kirsten murine sarcoma virus

Transformation of rat NRK-49F cells (49F) by Kirsten murine sarcoma virus (Ki-MSV) renders these cells (Ki-49F cells) capable of autonomous anchorage independent (AI) growth. As compared to nontransformed 49F cells, the transformation by Ki-MSV does not modify the cell response to transforming growth factor-beta (TGF-beta) in monolayer conditions, but alters it in A I growth conditions. The growth of nontransformed or Ki-MSV-transformed adherent 49F cells is slowed down by porcine TGF-beta, and this effect is reversed by epidermal growth factor (EGF). This decrease in the cell growth rate, induced by TGF-beta, does not affect the cloning efficiency of untransformed and transformed adherent 49F cells. Contrarily, porcine TGF-beta decreases the A I cloning efficiency of Ki-49F cells in agar-gelled medium; this effect is only partly reversed by EGF, which does not synergise with TGF-beta to enhance the A I growth as in the case of untransformed 49F cells. Media conditioned by 49F cells, Ki-49F cells, and chicken embryo fibroblasts contain a latent TGF-beta whose capacity to promote the A I growth of 49F cells and to inhibit that of Ki-49F cells is unmasked by acidification. The same situation exists concerning TGF-beta from human platelets. Neutral extracts are inefficient in both tests of promotion and inhibition of A I growth and contain an acid-activable component with an apparent molecular weight of 600 kd. In acid extracts, a 5-9 kd apparent molecular weight component is responsible for the A I growth enhancement of 49F cells and the A I growth inhibition of Ki-49F cells. Further purification by reverse phase chromatography shows that both activities strictly coelute at the same point (32%) of an acetonitrile gradient. These results indicate that TGF-beta is present in physiological conditions as a latent form which requires activation for inhibiting the A I growth of transformed cells as well as for enhancing that of 49F cells.     

Transforming growth factors released from Kirsten sarcoma virus transformed cells do not compete for epidermal growth factor membrane receptors

Kirsten murine sarcoma virus (KiMSV)-transformed rat kidney cells (KNRK) release small polypeptides (Mr 12,500-15,300) into the culture medium that are capable of stimulating normal rat kidney cells (NRK) to form colonies in soft agar. The transforming growth factors (TGFs) did not compete with epidermal growth factor (EGF) for its receptor and did not induce specific phosphorylation of EGF receptor on NRK cell membranes. These properties differ from the TGFs isolated by other investigators. Our data further establish the heterogeneity of the materials produced by transformed cells that induce transformation-specific changes in normal cells.     

Separation of the transforming growth factor from cells of murine sarcoma induced by polymeric film

Use was made of the cell line PS-103 induced by subcutaneous implantation of plastic film to CBA mice. The conditioned medium from PS-103 was shown to stimulate anchorage independence of PS-103 itself and several pseudonormal cells: NRK and NIH/3T3. The peak of the colony-stimulating activity after gel filtration was displayed by fractions with a molecular weight of 15 000, which were acid- and heat-resistant. All those characteristics correlated with the traits of the transforming growth factors (TGF). The significance of the TGF for carcinogenesis induced by plastic films is discussed.     

Deoxyglucose transport of uninfected, murine sarcoma virus-transformed, and murine leukemia virus-infected mouse cells

The initial rates of deoxy-D-glucose transport by cultures of growing and density-inhibited mouse embryo cells and lines of mouse cells transformed spontaneously or after infection by murine leukemia virus or murine sarcoma virus were investigated as a function of the deoxyglucose concentration. The apparent K_m for deoxyglucose transport was about the same for all types of cells (1–2 mM). The V_max of secondary cultures of mouse embryo cells decreased from 6 nmoles/10⁶ cells/minute for sparse cultures to less than 1 nmole/10⁶ cells/minute for density-inhibited cultures. The V_max was about the same whether estimated in monolayer culture or in suspensions of cells dispersed by treatment with trypsin. The V_max for deoxyglucose transport by the established cells, whether transformed spontaneously or by virus infection, was 4 to 25 times higher than that for density-inhibited mouse embryo cells and was independent of the cell density of the cultures. Deoxyglucose transport was competitively inhibited by Cytochalasin B, Persantin, glucose and 3-O-methyl-D-glucose and the apparent K_i values of inhibition were similar for the mouse embryo cells and the various cell lines. Similarly, the sensitivity of the glucose transport systems to inactivation by p-chloromercuribenzoate was about the same for all types of cells. The results suggest that the glucose transport system of the normal mouse embryo cells and the cells of the various established lines is qualitatively the same, but that the number of functional transport sites differs for the various cell lines and decreases markedly in mouse embryo cells with an increase in cell density of the cultures.     

The purification of an acid- and heat-labile transforming growth factor from an avian sarcoma virus-transformed rat cell line

A new class of transforming growth factor (TGF), with chemical characteristics differing from previously reported TGFs, was isolated and purified from an avian sarcoma virus-transformed rat cell line, 77N1 . Purification steps were simple and consisted of ion-exchange chromatography on diethylaminoethyl (DEAE)-Sephacel, ammonium sulfate precipitation, Chromatofocusing, and DEAE-Sephadex A-25 chromatography. The purified TGF is a heat- and acid-labile protein with a molecular mass of 12,000 daltons and isoelectric point of 5.2-5.4. Because of the acid lability of this TGF, purification was carried out at neutral pH. The TGF induced DNA synthesis in growth-arrested BALB 3T3 cells and promoted anchorage-independent growth of nontransformed BALB 3T3 cells in soft agar; the latter activity is specific for the peptide growth factors, called TGFs, but it did not compete with epidermal growth factor (EGF) for binding to the EGF membrane receptors. The TGF activity was not potentiated by EGF. The purified preparation of the TGF stimulated BALB 3T3 cells to grow progressively in soft agar at a dose of 20 ng/ml.     

Nonfunctional epidermal growth factor receptor in cells transformed by Kirsten sarcoma virus

The cell membrane receptor for epidermal growth factor (EGF) appears to be a glycoprotein of Mr 170,000 and mediates the mitogenic and metabolic responses of cells with EGF receptors (EGF-R). Normal rat kidney (NRK) have about 3 X 10(5) EGF-R per cell. Upon transformation of NRK cells by Kirsten sarcoma virus, the transformed derivative (KNRK) loses the ability to bind 125I-EGF. Membranes from NRK and KNRK cells were included in EGF-dependent phosphorylation reactions to search for evidence of the EGF-R. A phosphorylated protein of Mr 170,000 was detected in both NRK and KNRK membranes. The Mr 170,000 protein was identified to be EGF-R by immunoprecipitation with monoclonal antibody to the receptor. Furthermore, two-dimensional peptide mapping using trypsin and chymotrypsin digestions of the iodinated receptors from both NRK and KNRK cells showed essentially identical patterns. These data indicate that the EGF-R is present in KNRK cells with apparently the same protein structure as the NRK counterpart.     

Morphological transformation of cells induced by Kirsten sarcoma virus transforming factor is independent of serine protease

Reports from several laboratories have suggested that the virus transformed state may be maintained either by ectopically produced growth factors of alternatively by ectopically produced serine proteases including plasminogen activator. Here we show that the maintenance of transformation induced by Kirsten sarcoma virus induced growth factor(s) is independent of serine proteases in that (1) the factors are not themselves serine proteases, and (2) the growth factors do not induce the expression of detectable serine proteases or plasminogen activator.     

Immortalization of murine connective tissue-type mast cells at multiple stages of their differentiation by coculture of splenocytes with fibroblasts that produce Kirsten sarcoma virus

Mature connective tissue mast cells (CTMC) have not been previously available as a cell line from any species. Here we describe 15 novel mast cell lines (KiSV-MC) that were derived by coculturing murine splenocytes with fibroblasts that produce a Ki-ras-containing murine sarcoma virus. Some of the KiSV-MC lines are similar to CTMC in that they synthesize predominantly heparin proteoglycans, and contain up to 35 micrograms of histamine and 2.2 units of carboxypeptidase A/10(6) cells in secretory granules which stain red with Safranin. Other cell lines display phenotypic characteristics intermediate to CTMC and mucosal-like mast cells in being predominantly Safranin-, having lower amounts of histamine and carboxypeptidase A, and in synthesizing chondroitin sulfate E proteoglycans in preference to heparin proteoglycans. When the individual KiSV-MC lines were compared, a linear relationship was found between the number of Safranin+ granules, the cellular contents of histamine and carboxypeptidase A, and the biosynthesis of heparin relative to chondroitin sulfate E proteoglycans. Upon sensitization with monoclonal IgE and exposure to hapten-specific antigen, the cells exocytose the contents of their secretory granules. Thus, these immortalized cells provide the first source of CTMC-like lines for chemical and functional analysis and illustrate that murine mast cells can express a continuum of phenotypes.     

Reduced binding of epidermal growth factor by avian sarcoma virus-transformed rat cells

Rat cells transformed by Rous sarcoma virus and Fujinami sarcoma virus bound 5-10% of the amount of epidermal growth factor (EGF) bound by normal cells. Scatchard plot analysis indicated that the reduction in binding by transformed cells was due to a decreased number of receptors rather than to altered binding affinity. In experiments with temperature sensitive mutants of Rous sarcoma virus and Fujinami sarcoma virus significant loss of EGF binding occurred within one hour of shift from non-permissive to permissive temperature. Conditioned media from various normal and transformed cell lines were examined for the ability to inhibit EGF binding to normal cells or to cause "down regulation" of EGF receptors. No activity of either type was found. EGF-dependent phosphorylation in isolated membrane preparations was also examined. Membranes from normal cells displayed EGF-dependent phosphorylation of a Mr 180,000 protein presumed to be the EGF receptor. This activity was absent in membranes from transformed cells. The data suggest a close correlation between activation of avian sarcoma virus transforming gene products and modulation of the EGF growth regulatory system.     

Effects of sodium butyrate on the membrane glycoconjugates of murine sarcoma virus-transformed rat cells

The temporal relationship between butyrate-induced cellular flattening of murine sarcoma virus-transformed rat cells (MSV-NRK) and alterations in certain surface-associated biochemical markers of transformation, e.g., surface glycopeptides, glycolipids, fibronectin, hexose uptake, and cell-substrate adhesion was examined. The induction of elevated levels of the ganglioside GM3 and of a GDla-like ganglioside were observed to precede or to parallel cellular flattening. Likewise, enhanced incorporation of radioisotopically labeled fucose into a novel fucose-containing component, i.e., glucopyranosyl (1 leads to 3) fucopyranosyl-threonine, was also observed to occur at an early stage of cellular flattening. In contrast, a shift in the molecular weight distribution of trypsin-sensitive, surface fucopeptides was observed to occur at a late stage of cellular flattening. Moreover, surface fibronectin was not detectable in the butyrate-flattened MSV-NRK cells despite the fact that the cells manifested significantly enhanced cell- substrate adhesion. Thus, butyrate appears to be a useful tool for understanding the sequential changes associated with expression of the transformed phenotype of MSV-NRK cells.     

Cytochalasin B-induced multinucleation of murine sarcoma virus-transformed cells. Inhibition by sodium butyrate.

Human neutrophils aggregate after exposure to the bivalent cation ionophore A23187, the synthetic chemotactic tripeptide formyl-methionyl-leucyl-phenylalanine, and the chemotactic fragment of human C5. Cells preincubated with cytochalasin B (CB) (0.5 μg/ml) showed an enhanced aggregation response to each of these three agents. By itself, CB did not aggregate the cells. The enhancing effect of CB on the aggregation response developed immediately after exposure to the drug and was readily reversible. Since A23187 and chemotactic factors stimulate the transmembrane flow of Ca²⁺, Na⁺, and K⁺ in the neutrophil, and since CB enhances this effect, CB may influence neutrophil aggregation through its influence on transmembrane cationic movements. It was also found that the enhancing effect of CB on neutrophil aggregation was abrogated by high (i.e., greater than 1.7 μg/ml) concentrations of the drug or by adding it to the neutrophils after the cells were exposed to a chemotactic factor.     

Increased collagen synthesis in Kirsten sarcoma virus-transformed BALB 3T3 cells grown in the presence of dibutyryl cyclic AMP

Growth of Kirsten sarcoma virus-transformed BALB 3T3 (Ki-3T3) cells in the presence of dibutyryl cyclic AMP (dbcAMP) resulted in alteration of morphology, inhibition of growth, and increased collagen synthesis as measured by incorporation of ¹⁴C-proline into collagenase-digestible protein. There was an increase in incorporation of ¹⁴C-proline into collagen when expressed not only as dpm per μg DNA or protein, but also as the relative rate of collagen synthesis compared to total cellular protein synthesis, which suggests that an alteration in amino acid transport cannot totally account for the increased incorporation into collagen. The three properties studied were all affected over a concentration range of 0.10 to 1.0 mM dbcAMP, but each had a slightly different dose-response curve. At 0.5 mM dbcGMP or sodium butyrate, there was no affect on growth, morphology, or the relative rate of collagen synthesis indicating specificity for the dibutyryl analog of cAMP. Growth of the parent line, BALB 3T3, was inhibited by 0.5 mM dbcAMP, but the relative rate of collagen synthesis did not increase. These results suggest that although growth, morphology, and collagen synthesis are altered in transformed cells so that they more closely resemble those of the parent line, each property may be regulated independently.     

Cells transformed by Rous sarcoma virus release transforming growth factors

Chicken embryo fibroblasts and hamster BHK cells transformed by Rous sarcoma virus (RSV) release in their culture media growth factors which enhance markedly anchorage-independent colony formation in gelified medium, at the restrictive temperature (41 degrees 5 C), of chicken embryo fibroblasts (CEF) infected by RSV mutants with a ts mutation of the src gene. This action is not observed with uninfected CEF, and, therefore, appears to require some expression of the viral src gene in the target cells. The enhancing factors are proteins related to the family of the transforming growth factors (TGFs) by their molecular weight (about 20 kd), their heat and acid resistance, and their sensitivity to dithiothreitol. They do not compete with 125I EGF for binding on the EGF receptors of the membrane of A431 cells. As chicken embryo fibroblasts are devoid of EGF receptors, their activity is not potentiated by EGF.