Transformation by the src oncogene alters glucose transport into rat and chicken cells by different mechanisms.

Transformation of both rat and chicken fibroblasts by the src oncogene leads to a four- to fivefold increase in the rate of glucose transport and in the level of the glucose transporter protein. We have previously shown that, with chicken embryo fibroblasts, transformation leads to a reduction in the rate of degradation of the transporter, with little or no increase in the rate of its biosynthesis. We now show that, with the rat-1 cell line, the opposite result was obtained. src-induced transformation led to an increase in transporter biosynthesis, with little effect on turnover. A src-induced increase in transporter mRNA entirely accounted for the increase in biosynthesis of the protein. By contrast, in chicken embryo fibroblasts, the level of transporter mRNA was low and was not induced to rise by src transformation. Thus, src induced an increase in the level of the glucose transport protein by fundamentally different mechanisms in chicken embryo fibroblasts and rat-1 cells. To test whether this difference was due to rat-1 cells being an immortalized cell line, we measured transporter mRNA levels in primary fibroblast cultures from rat embryos and in parallel cultures transformed by src. Transporter mRNA was inducible by src in these cells. Thus, the difference in mRNA inducibility between chicken and rat cells is not due to immortalization.     

Plasminogen activator is an apparent lymphocyte mitogen

Culture fluids of avian sarcoma virus (ASV)-transformed but not normal chicken embryo cells frequently elicited a mitogenic response in normal avian and murine lymphocytes. We examined the possibility that plasminogen activator (PA) might be responsible for the observed mitogenic effect. PA activity, present in culture medium, was correlated positively with lymphocyte mitogenic capacity. Treatment of cells with phorbol myristate acetate, which elevates PA levels, increased mitogenesis. Similar treatment with dexamethasone, which inhibits PA biosynthesis and/or secretion, reduced lymphocyte mitogenic activity. Addition to culture fluids of either benzamidine or diisopropylfluorophosphate, both specific PA inhibitors, blocked lymphocyte proliferative responsiveness to culture fluids. In contrast, neither epsilon-amino-caproic acid nor trasylol, which inhibits plasmin esterase activity but not PA, abrogated lymphocyte responsiveness. Furthermore, purified urokinase, an enzyme of similar substrate specificity to PA, had lymphocyte stimulatory activity. These results strongly suggest that PA can function as a lymphocyte mitogen.     

Vanadium compounds promote the induction of morphological transformation of hamster embryo cells with no effect on gap junctional cell communication

Vanadium compounds were found to promote the induction of morphological transformation of hamster embryo cells. Exposure of the cells to Na−O-vanadate, vanadin (V) oxide or vanadin (IV) oxide sulfate following pre-exposure to a low concentration of benzo[a]pyrene, potentiated the induction of transformed colonies similar to 12-O-tetradecanoylphorbol-13-acetate. Unlike this phorbol ester, vanadium compounds did not inhibit intercellular communication, or activate protein kinase C. Nor did vanadate influence the reoccurrence of communication after removal of a communication blocking phorbol ester. On the other hand, vanadate showed strong synergism with the phorbol ester on induction of transformed morphology in the phorbol ester sensitive cell line BPNi. This suggests that vanadium and tumor promoting phorbol esters mediate their effect on the induction of morphological transformation of hamster embryo cells through different mechanisms.     

The adhesion plaque protein, talin, is phosphorylated in vivo in chicken embryo fibroblasts exposed to a tumor-promoting phorbol ester.

Talin is a high molecular weight phosphoprotein that is localized at adhesion plaques. We have found that talin phosphorylation increases 3.0-fold upon exposure of chicken embryo fibroblasts to the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. Talin isolated from tumor promoter-treated cells is phosphorylated on serine and threonine residues. Vinculin, a 130 kDa talin-binding protein, also exhibits increased phosphorylation in vivo in response to tumor promoter, but to a lesser degree than does talin. Because tumor-promoting phorbol esters augment protein kinase C activity, we have compared the ability of purified protein kinase C to phosphorylate talin and vinculin in vitro. Both talin and vinculin were found to be substrates for protein kinase C; however, talin was phosphorylated to a greater extent than was vinculin. Cleavage of protein kinase C-phosphorylated talin by the calcium-dependent protease (Type II) revealed that while both the resulting 190-200 and 46 kDa proteolytic peptides were phosphorylated, the majority of label was contained within the 46-kDa fragment. Although incubation of chicken embryo fibroblasts with tumor-promoting phorbol ester induces a dramatic increase in talin phosphorylation, we detected no change in the organization of stress fibers and focal contacts in these cells. Exposure of the cells to tumor promoter did, however, result in a loss of actin and talin-rich cell surface elaborations that resemble focal contact precursor structures.     

Phorbol ester-induced morphological changes in transformed chick fibroblasts: evidence for direct catalytic involvement of plasminogen activator.

The tumor promoter phorbol myristate acetate (PMA) induces the production of the serine protease plasminogen activator (PA) in cultures of normal chick embryo fibroblasts (CEF) and synergistically enhances PA production in Rous sarcoma virus-transformed chick embryo fibroblasts (RSVCEF). Following PMA treatment of serum-free RSVCEF cultures, PA induction is accompanied by distinct morphological changes, including enhanced cell clustering and the formation of dense cellular aggregates. These alterations in the morphology of the PMA-treated transformed cells are inhibited by several protease inhibitors, including leupeptin, NPGB, SBTI, benzamidine and DFP, the specific inhibitor of serine enzymes. A number of protease inhibitors are ineffective in preventing the PMA-induced morphological changes; these include inhibitors of trypsin, chymotrypsin, elastase, thrombin and, most importantly, plasmin. The use of a fluorescent substrate to assay PA directly demonstrated that the pattern of inhibiton of PA activity correlates exactly with the inhibition of morphological changes. The of 3H-DFP to label and characterize serine zymes in the culture fluid from PMA-treated cells further indicated that PA is the serine protease responsible for the morphological changes. Thus PA itself can catalytically alter cellular behavior in culture independent of plasminogen, until not its only known natural substrate.     

Role of the mitogenic property and kinase activity of p60src in tumor formation by Rous sarcoma virus

Expression of the src gene of Rous sarcoma virus in chicken embryo neuroretinal cells results in morphological transformation and sustained proliferation of this normally resting cell population. PA101 and PA104 are two mutants of Rous sarcoma virus which induce neuroretinal cell proliferation in the absence of morphological transformation. Their mitogenic property is temperature sensitive, and they both encode p60src proteins with low kinase activity. To study the role of the mitogenic function and protein kinase activity of p60src in tumorigenesis, we investigated the oncogenicity of PA101 and PA104. Both mutants were less tumorigenic than wild-type virus when injected into chicks. Tumorigenicity was further assayed by inoculating infected chicken embryo fibroblasts and neuroretinal cells onto the chorioallantoid membrane of embryonated duck eggs. This system provides a nonpermissive and immunodeficient environment for xenogenic cell grafting and allows the study of cell tumorigenicity within a temperature range of 37 to 39.5 degrees C. Chicken embryo fibroblasts and neuroretinal cells infected with PA101 were as tumorigenic as wild type-infected cells at 37 degrees C, but tumor development was significantly reduced at 39.5 degrees C. In contrast, both cell types infected with PA104 displayed sharply reduced tumorigenicity. Cell cultures derived from PA101 tumors induced on the chorioallantoid membrane were similar to the corresponding cells maintained in vitro in terms of morphology, production of plasminogen activator, relative amounts of phosphotyrosine in total cellular proteins, and phosphorylation of 34,000-molecular-weight protein. These results indicate that the expression of the mitogenic function of src does not account per se for cell tumorigenicity and that tumor formation is compatible with low levels of p60src protein kinase activity.     

The non-phorbol ester tumor promoter okadaic acid does not promote morphological transformation or inhibit junctional communication in hamster embryo cells

Okadaic acid is a potent non-phorbol ester mouse skin tumor promoter. Unlike the phorbol ester tumor promoters, okadaic acid is unable to promote the induction of morphological transformation in Syrian hamster embryo cell colonies. On the contrary, okadaic acid seems to counteract the effect of phorbol esters on transformation. Also unlike phorbol ester tumor promoters, okadaic acid does not inhibit intercellular communication, neither in primary hamster embryo cells, nor in the phorbol ester sensitive cell line BPNi. Furthermore, okadaic acid has no effect on the reoccurrence of communication following removal of 12-O-tetradecanoylphorbol-13-acetate.     

Effects of phenylarsine oxide on expression of heme oxygenase-1 reporter constructs in transiently transfected cultures of chick embryo liver cells

Heme oxygenase catalyzes the first and rate-controlling step of heme catabolism. Induction of heme oxygenase-1 can be caused by numerous factors, including heme, other metalloporphyrins, transition metal ions, heat shock, ultraviolet light, phorbol esters, sodium arsenite, and phenylarsine oxide (PAO). Induction of this enzyme may protect cells from oxidative damage. Using heme oxygenase-1 promoter/reporter gene constructs, we have previously reported that the sodium arsenite-mediated induction of heme oxygenase-1 in chick embryo liver cells and chicken hepatoma (LMH) cells involves an AP-1 element. We have now investigated whether the PAO-mediated induction of heme oxygenase-1 also involves an AP-1 element. Primary cultures of chick embryo liver cells were transiently transfected with heme oxygenase-1 promoter/reporter gene constructs, treated with PAO, and reporter gene activities were measured. We found that the PAO-mediated increase in reporter gene activity was dose- and time-dependent. This activity was decreased by prior treatment with N-acetylcysteine. Studies with mutated constructs showed that both an AP-1 element and a metal responsive element are involved in the PAO-mediated induction of the heme oxygenase-1 reporter construct. Electrophoretic mobility shift assays showed that nuclear proteins from PAO-treated cells had increased binding to an AP-1 probe, and that this increase was abrogated by N-acetylcysteine. These findings support the hypothesis that the PAO-mediated induction of heme oxygenase-1 is caused by activation of AP-1 and MRE/cMyc elements and may involve nuclear proteins whose states of phosphorylation determine binding to regulatory elements, and thus the level of expression of heme oxygenase-1.     

Keloid fibroblasts are refractory to inhibition of DNA synthesis by phorbol esters. Altered response is accompanied by reduced sensitivity to prostaglandin E2 and altered down-regulation of phorbol ester binding sites.

To investigate abnormal growth regulation in keloid fibroblasts, responses to phorbol esters were examined. Treatment of quiescent cultures with phorbol 12-myristate 13-acetate (PMA) blocked a normally occurring (20-24 h) peak of serum-stimulated thymidine incorporation in normal and keloid cells. In keloid fibroblasts PMA induced a delayed peak of DNA synthesis. When indomethacin was added with PMA the delayed peak appeared in normal fibroblasts. The ED50 for inhibition of the 20-24-h peak was 1 nM, whereas the delayed peak required a 50-fold-higher PMA concentration. In both cell types PMA induced prostaglandin E2 (PGE2) synthesis, and exogenous PGE2 caused 50% inhibition of the 20-24-h peak. When PMA and indomethacin were added with PGE2 the delayed peak was inhibited 90% in normal fibroblasts, whereas inhibition of keloid cells was the same as with PGE2 alone. Normal and keloid fibroblasts had the same number of phorbol ester binding sites. However, in normal cells, phorbol 12,13-dibutyrate bound with greater affinity, and down-regulation of phorbol ester binding occurred to a greater extent. These findings suggest that altered expression of protein kinase C isozymes or another molecule that binds phorbol esters may play a role in abnormal growth regulation of keloid cells.     

Malignant transformation and tumor promoter treatment increase levels of a transcript for a secreted glycoprotein.

The major excreted protein of transformed mouse fibroblasts, a secreted, mannose 6-phosphate-containing glycoprotein, is induced in nontransformed cells by a variety of transforming agents, by phorbol esters, and by platelet-derived growth factor. We report here the molecular cloning of the cDNA encoding this protein and demonstrate that its induction is a consequence of enhanced mRNA levels for major excreted protein in both tetradecanoyl phorbol acetate-treated 3T3 cells and 3T3 cells transformed by a variety of retroviruses or retroviral oncogenes. These results indicate that tumor promoters and retroviral transformation might share a common pathway of action in cultured cells and that major excreted protein is a molecular marker for the growth response of cells to these agents.     

Postreplication repair of DNA in human fibroblasts after UV irradiation or treatment with metabolites of benzo(a)pyrene

We have examined the ability of normal fibroblasts and of excision-deficient xeroderma pigmentosum (XP) and XP variant fibroblasts to perform postreplication DNA repair after increasing doses of either ultraviolet (UV) irradiation or mutagenic benzo(a)pyrene derivatives. XP cells defective in the excision of both UV-induced pyrimidine dimers and guanine adducts induced by treatment with the 7,8-diol-9,10-epoxides of benzo(a)pyrene were partially defective in their ability to synthesize high molecular weight DNA after the induction of both classes of DNA lesions. This defect was more marked in XP variant cells, despite their ability to remove by excision repair both pyrimidine dimers and the diol epoxide-induced lesions to the same degree as observed in normal cells. The benzo(a)pyrene 9,10-oxide had no effect in any of the 3 cell lines. The response of the excision and postreplication DNA repair mechanisms operating in human fibroblasts treated with benzo(a)pyrene 7,8-diol-9,10-epoxides, therefore, appears to resemble closely that seen after the induction of pyrimidine dimers by UV irradiation.     

Transfection of Chicken Embryo Cells with DNA Extracted from Avian Virus-Producing Neoplastic Cells

DNA isolated from avian virus-producing leukemic myeloblasts induced the production of viruses, but not morphological transformation, in cultivated chicken fibroblasts. The recovered virus had the same biological characteristics as the original avian myeloblastosis virus (AMV) and produced myeloblastosis and nephroblastomas when injected into chickens. Neutralization experiments with chicken anti-AMV-BAI strain A sera showed an antigenic community between the DNA-transfected virus and the original virus. Virus induced in fibroblasts after treatment with DNA from a viral nephroblastic nephroblastoma line only gave nephroblastoma when injected into chicken. Treatment of chicken embryo cells with DNA extracted from normal chicken embryos did not induce viral production.     

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

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

Hexose and amino acid transport by chicken embryo fibroblasts infected with temperature-sensitive mutant of Rous sarcoma virus. Comparison of transport properties of whole cells and membrane vesicles

The effect of transformation on hexose and amino acid transport has been studied using whole cells and membrane vesicles of chicken embryo fibroblasts infected with the temperature-sensitive mutant of the Rous sarcoma virus, TS-68. In whole cells, TS-68-infected chicken embryo fibroblasts cultured at the permissive temperature (37 degrees C) had a 2-fold higher rate of 2-deoxy-D-glucose uptake than the same cells cultured at the non-permissive temperature (41 degrees C). However, both the non-transformed and transformed cells had comparable rates of alpha-aminoisobutyric acid transport. Membrane vesicles, isolated from TS-68-infected chicken embryo fibroblasts cultured at 41 degrees C or 37 degrees C, displayed carrier-mediated, intravesicular uptake of D-glucose and alpha-aminoisobutyric acid. Membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 37 degrees C had an approx. 50% greater initial rate of stereospecific hexose uptake than the membrane vesicles from fibroblasts cultured at 41 degrees C. The two types of membrane vesicle had similar uptake rates of alpha-aminoisobutyric acid. The results of hexose and amino acid uptake by the membrane vesicles correlated well with those observed with the whole cells. Km values for stereospecific D-glucose uptake by the membrane vesicles from TS-68-infected chicken embryo fibroblasts cultured at 41 and 37 degrees C were similar, but the V value was greater for the membrane vesicles from TS-68-infected cells cultured at 37 degrees C. Cytochalasin B competitively inhibited stereospecific hexose uptake in both types of membrane vesicle. These findings suggest that the membrane vesicles retained many of the features of hexose and amino acid transport observed in whole cells, and that the increased rate of hexose transport seen in the virally-transformed chicken embryo fibroblasts was due to an increase in the number or availability of hexose carriers.     

Induction of oncogenic transformation by low doses of X rays and dose-rate effect

Golden hamster embryo cells were used to study morphological transformation induced by low doses of X rays at different dose rates. X irradiation at lower dose rates was less effective in cell killing and induction of transformation than at higher dose rates. The general shapes of induction curves of transformants were almost the same at all dose rates. At shoulder doses (1 to 150 R), the transformation frequencies increased steeply with increasing dose at all dose rates. At doses higher than 150 R survivals declined exponentially, but the frequencies of transformants increased only slightly. However, irradiation at higher dose rates was more effective in induction of transformants than that at lower dose rates. We conclude that transformational damage introduced at a low dose rate, as well as sublethal damage, may be repaired during low-dose-rate irradiation, but transformational damage may be different from sublethal damage.     

Association of pp60src and src protein kinase activity with the plasma membrane of nonpermissive and permissive avian sarcoma virus-infected cells.

The intracellular localization of pp60src and src protein kinase activity in avian sarcoma virus (ASV)-infected chicken embryo fibroblasts and transformed and morphologically reverted field vole cells was examined by subcellular fractionation procedures. Fractionation by differential centrifugation of Dounce-homogenized cellular extracts prepared from vole cells showed that 83 to 91% of pp60src sedimented with particulate subcellular components from both transformed and revertant vole cells. A slightly lesser amount (60 to 70%) of pp60src was found associated with the particulate fraction from ASV-infected chicken embryo fibroblasts. The distribution of src protein kinase activity in the cytosol and particulate cell fractions was identical to that of pp60src, indicating no detectable differences in the activity of cytosol- and particulate-associated pp60src. When subcellular components of the cell were fractionated by discontinuous sucrose gradient centrifugation, similar amounts of both pp60src and src protein kinase activity cosedimented with the plasma membrane fractions from both transformed and revertant vole cells, as well as from ASV-infected chicken embryo fibroblasts. src protein kinase activity associated with plasma membrane fractions prepared from vole cells and ASV-infected chicken embryo fibroblasts was resistant to extraction with high salt concentrations, but partial elution was achieved with nonionic detergent. Thus, in both transformed and morphologically reverted vole cells, pp60src is intimately associated with the plasma membrane. Since transforming virus can be rescued from revertant vole cells by fusion to chicken embryo fibroblasts, revertant vole cell pp60src is capable of inducing morphological transformation. Thus, although the data presented herein suggest that transformation requires the association of pp60src with the plasma membrane, the binding of pp60src to the plasma membrane per se is insufficient to induce morphological transformation and requires the additional interaction with a specific target membrane protein which appears to be defective in revertant vole cells.     

Induction of angiogenesis in vitro by vanadate, an inhibitor of phosphotyrosine phosphatases

We have previously shown that capillary endothelial cells grown on the surface of three-dimensional collagen gels can be induced to invade the underlying fibrillar matrix and to form capillary-like tubular structures in response to tumor-promoting phorbol esters or the angiogenic agent fibroblast growth factor (FGF). Since both phorbol esters and FGF stimulate phosphorylation of tyrosine residues, we treated endothelial cells with vanadate, an inhibitor of phosphotyrosine-specific phosphatases, to determine whether this agent could induce the expression of an angiogenic phenotype in these cells. We show here that vanadate stimulates endothelial cells to invade collagen matrices and to organize into characteristic tubules resembling those induced by FGF or phorbol esters. We have further observed that vanadate concomitantly stimulates endothelial cells to produce plasminogen activators (PAs), proteolytic enzymes which are induced by phorbol esters and FGF, and which have been implicated in the neovascular response; this stimulation can be accounted for by an increase in the levels of urokinase-type PA and tissue type PA mRNA. These results suggest a role for tyrosine phosphorylation in the regulation of the angiogenic phenotype in capillary endothelial cells.