Transforming growth factor-beta and retinoic acid modulate phenotypic transformation of normal rat kidney cells induced by epidermal growth factor and platelet-derived growth factor

In this study we have investigated the ability of epidermal growth factor (EGF), platelet-derived growth factor (PDGF), and transforming growth factor-beta (TGF beta) together with retinoic acid (RA) at saturating concentrations to induce phenotypic transformation of normal rat kidney (NRK) cells in a growth factor-defined medium. This medium contains serum in which all growth factor activity has been chemically inactivated, thereby eliminating the effects of growth factors from serum in the assay. It is shown that neither TGF eta nor a ligand binding to the EGF receptor is essential for phenotypic transformation of NRK cells, since anchorage-independent growth is also induced by EGF in combination with RA and by PDGF in combination with RA and TGF beta. Our data indicate strong similarities between TGF beta and RA in their ability to act as modulators for phenotypic transformation. In addition, both agents enhance the number of EGF receptors in NRK cells, without affecting the number of PDGF receptors. On the other hand, TGF beta has mitogenic effects on a number of non-transformed cell lines, such as Swiss 3T3 fibroblasts, particularly when assayed in the absence of insulin, whereas RA is mitogenic for these cells only in the presence of insulin. These data demonstrate that phenotypic transformation of NRK cells requires specific combinations of polypeptide growth factors and modulating agents, but that this process can be induced under many more conditions than previously described. Moreover, our data point toward both parallels and differences in the activities of TGF beta and RA.     

Monoclonal antibody C3.1 is a platelet derived growth factor (PDGF) antagonist

A monoclonal antibody C3.1 raised against NR6 cells and partially purified PDGF receptor blocked PDGF stimulated [3H] thymidine incorporation in Swiss mouse 3T3 cells and immunoprecipitated a 180 kDa phosphoprotein from NR6 cells. The phosphoprotein bound to a C3.1 sepharose 4B affinity matrix and could be specifically eluted with PDGF but not by EGF or basic FGF. These preliminary results suggests that the ability of C3.1 to inhibit PDGF stimulated mitogenesis may be due to its direct or allosteric interaction at the PDGF receptor binding site.     

Growth factors modify the epidermal growth factor receptor through multiple pathways

Previous results have shown that tumor promoters modify the properties of the epidermal growth factor (EGF) receptor through the activation of protein kinase C. Diacylglycerol-generating factors such as platelet-derived growth factor (PDGF) and p28sis should activate protein kinase C and alter EGF receptor properties in a similar manner. To test directly the involvement of protein kinase C in the action of media from v-sis-transformed cells on the EGF receptor, Swiss 3T3 cells were first extensively treated with various concentrations of the tumor-promoter phorbol dibutyrate (PDBu) This treatment reduced levels of active protein kinase C in the cells, making them less responsive to subsequent rechallenge with the tumor promoter. The results demonstrate that there are at least two components to the action of media from v-sis transformed cells on EGF binding: a labile factor that confers protein kinase C independence and a stable factor that appears to be dependent on protein kinase C. The action of the first factor cannot be mimicked by transforming growth factor-beta or EGF in either the presence or absence of PDGF. The action of the second factor is similar to that of PDGF. These findings indicate that heterologous regulation of the EGF receptor can occur through both protein kinase C-dependent and -independent pathways.     

Aortic endothelial cells synthesize basic fibroblast growth factor which remains cell associated and platelet-derived growth factor-like protein which is secreted

Cultured bovine aortic endothelial cells synthesize growth factors which markedly differ in the regulation of their storage and secretion. Endothelial cell lysates, but not conditioned medium, contain a growth factor activity that appears to be basic fibroblast growth factor (FGF) by the following criteria: (1) it elutes from heparin-Sepharose at 1.4-1.6 M NaCl; (2) it is mitogenic for bovine aortic and capillary endothelial cells; (3) it is heat sensitive but stable to dithiothreitol; (4) it has a molecular weight of about 18,000 daltons; and (5) it cross-reacts with antiserum directed against basic FGF. In contrast, endothelial cell conditioned medium, but not lysates, contains a growth factor activity that (1) elutes from heparin-Sepharose at 0.4-0.5 M NaCl; (2) is mitogenic for fibroblasts and vascular smooth muscle cells but not for capillary endothelial cells; (3) is heat stable and dithiothreitol sensitive; and (4) competes with platelet-derived growth factor (PDGF) for binding to fibroblasts. From these criteria, it appears that endothelial cells secrete into the medium growth factors some of which are PDGF-like, but secrete little if any basic FGF. It is suggested that endothelial cell-associated basic FGF acts in an autocrine fashion to stimulate endothelial cell proliferation in response to endothelial cell perturbation or injury. On the other hand, the endothelial cell-secreted growth factors which are smooth muscle cell but not endothelial cell mitogens might exert a paracrine function on neighboring cells of the vessel wall.     

Basic and acidic fibroblast growth factors modulate the epidermal growth factor receptor by a protein kinase C-independent pathway

Human acidic and basic fibroblast growth factors (aFGF and bFGF) inhibit epidermal growth factor (EGF) receptor binding in mouse Swiss 3T3 cells. Scatchard analysis indicates that aFGF and bFGF cause a decrease in the high affinity EGF receptor population, similar to that observed for activators of protein kinase C such as phorbol esters, platelet-derived growth factor (PDGF) and bombesin. However, unlike phorbol esters, aFGF and bFGF inhibit EGF binding in protein kinase C-deficient cells. The time course and dose response of inhibition of EGF binding by both aFGF and bFGF are very similar, with an ID50 of approximately 0.10 ng/ml. In contrast to bombesin but like PDGF, neither aFGF nor bFGF act on the EGF receptor through a pertussis toxin-sensitive G protein. These results indicate that both acidic and basic FGF depress high affinity EGF binding in Swiss 3T3 cells with similar potency through a protein kinase C/Gi-independent pathway.     

Cell surface retention sequence binding protein-1 interacts with the v-sis gene product and platelet-derived growth factor beta-type receptor in simian sarcoma virus-transformed cells

The cell surface retention sequence (CRS) binding protein-1 (CRSBP-1) is a newly identified membrane glycoprotein which is hypothesized to be responsible for cell surface retention of the oncogene v-sis and c-sis gene products and other secretory proteins containing CRSs. In simian sarcoma virus-transformed NIH 3T3 cells (SSV-NIH 3T3 cells), a fraction of CRSBP-1 was demonstrated at the cell surface and underwent internalization/recycling as revealed by cell surface 125I labeling and its resistance/sensitivity to trypsin digestion. However, the majority of CRSBP-1 was localized in intracellular compartments as evidenced by the resistance of most of the 35S-metabolically labeled CRSBP-1 to trypsin digestion, and by indirect immunofluorescent staining. CRSBP-1 appeared to form complexes with proteolytically processed forms (generated at and/or after the trans-Golgi network) of the v-sis gene product and with a approximately 140-kDa proteolytically cleaved form of the platelet-derived growth factor (PDGF) beta-type receptor, as demonstrated by metabolic labeling and co-immunoprecipitation. CRSBP-1, like the v-sis gene product and PDGF beta-type receptor, underwent rapid turnover which was blocked in the presence of 100 microM suramin. In normal and other transformed NIH 3T3 cells, CRSBP-1 was relatively stable and did not undergo rapid turnover and internalization/recycling at the cell surface. These results suggest that in SSV-NIH 3T3 cells, CRSBP-1 interacts with and forms ternary and binary complexes with the newly synthesized v-sis gene product and PDGF beta-type receptor at the trans-Golgi network and that the stable binary (CRSBP-1.v-sis gene product) complex is transported to the cell surface where it presents the v-sis gene product to unoccupied PDGF beta-type receptors during internalization/recycling.     

The v-sis oncogene product but not platelet-derived growth factor (PDGF) A homodimers activate PDGF alpha and beta receptors intracellularly and initiate cellular transformation.

The v-sis oncogene product p28v-sis and the platelet-derived growth factor (PDGF) B chain share 92% homology with each other and over 50% homology with the PDGF A chain. Exogenously added homodimers of PDGF A and PDGF B and of p28v-sis are potent mitogens but only PDGF B and p28v-sis induce transformation when endogenously expressed with a strong promoter. Because exogenous PDGF AA and PDGF BB both initiate a full mitogenic response, understanding the mechanisms underlying the difference in their transforming potential may clarify how growth factor genes act as oncogenes. In this work, we compared cells expressing high levels of PDGF A and v-sis. We observed that transformation by v-sis correlated directly with the rapid degradation (t1/2 approximately 20 min) of the alpha and beta PDGF receptors, with a failure of either the alpha or beta receptor to be fully processed and with the association of high levels of phosphatidylinositol (PI) 3-kinase with immunoprecipitates of the PDGF receptors. In contrast, in cells expressing essentially equal levels of PDGF A, transformation was not detected, alpha and beta PDGF receptor processing was normal, and association of PI 3-kinase with receptors in immunoprecipitates was not found above control values. The ability of v-sis to autoactivate PDGF receptors within processing compartments and to initiate activation of the PI 3-kinase signaling pathway coupled with the failure of PDGF A to activate its receptor intracellularly and to induce transformation when endogenously expressed at high levels suggests that the internal autoactivation of PDGF receptors may be essential for transformation by v-sis.     

Loss of epidermal growth factor receptors and release of transforming growth factors do not correlate with sarcoma virus-transformation in clonally-related NIH/3T3-derived cell lines.

Transformation of NIH/3T3 cells by Kirsten murine sarcoma virus (MSV) caused a dramatic reduction in the number of cell-surface receptors for epidermal growth factor (EGF). However, the number of EGF receptors remained at a very low level in a non-tumourigenic revertant cell line isolated from the virus-transformed cells, indicating that an increase in EGF receptors is not a requirement for the phenotypic reversion of Kirsten MSV-transformed 3T3 cells. Serum-free conditioned medium from normal and virus-transformed cell lines contained similar amounts of cell growth-promoting activity as assayed by the ability to stimulate DNA synthesis in quiescent Swiss 3T3 cell cultures. However, the concentrated conditioned medium from these cell lines showed no evidence of beta-transforming growth factor (TGF) activity as assayed by promotion of anchorage-independent growth of untransformed normal rat kidney (NRK) fibroblasts in agarose. The cellular release of alpha-TGF activity was assayed by measuring the ability of concentrated conditioned medium to inhibit the binding of 125I-EGF to Swiss 3T3 cells. Conditioned medium protein from the virus-transformed cell line inhibited 125I-EGF binding but only to the same extent as conditioned medium protein prepared from the untransformed cell line. The alpha-TGF secretion by these cell lines was estimated to be 30-45-fold lower than the level of alpha-TGF released by a well-characterized alpha-TGF-producing cell line (3B11). These results suggest that the induction of TGF release is not a necessary event in the transformation of NIH/3T3 cells by Kirsten MSV.     

Rapid turnover of the platelet-derived growth factor receptor in sis-transformed cells and reversal by suramin. Implications for the mechanism of autocrine transformation

In cells transformed by either v-sis or c-sis, the majority of the newly synthesized platelet-derived growth factor (PDGF) receptors fail to reach the cell surface and are rapidly degraded. This rapid turnover (t1/2 less than 30 min) appears to result from interaction of the sis gene product with the PDGF receptor in the endoplasmic reticulum and/or Golgi apparatus during their intracellular routing from the endoplasmic reticulum to the plasma membrane or extracellular compartment. Several lines of evidence support this hypothesis. 1) Both the 160-kDa precursor and the intracellular 180-kDa mature form of the PDGF receptor possessed ligand binding activity for PDGF; 2) both the 160-kDa precursor and the 180-kDa mature form of the receptor in sis-transformed cells were found to be activated (phosphorylated); 3) protamine, a competitive inhibitor for PDGF or v-sis gene product binding to the cell-surface receptor, did not affect the rapid turnover of the PDGF receptor in sis-transformed cells; 4) suramin, an inhibitor for PDGF or v-sis gene product binding to the PDGF receptor, not only reversed the rapid turnover of the PDGF receptor in sis-transformed cells, but also increased the secretion of sis gene products; and 5) rapid turnover of the PDGF receptor was only observed in sis-transformed cells but not in cells transformed by other oncogenes. We suggest that the persistence of a mitogenic signal from cellular organelles, arising from the intracellular interaction of sis gene products with newly synthesized PDGF receptors, is the mechanism for autocrine transformation by sis.     

Transmodulation of epidermal growth factor binding by platelet-derived growth factor and 12-O-tetradecanoylphorbol-13-acetate is not sodium-dependent in Balb/c/3T3 cells

The addition of platelet-derived growth factor (PDGF) to many types of cells causes a rapid decrease in high affinity binding of 125I-epidermal growth factor (EGF), a process which has been termed transmodulation. Treatment with the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) also results in the transmodulation of the EGF receptor in many cell types. PDGF can transmodulate EGF binding through a mechanism that is not dependent on protein kinase C activity. A recent report (Wattenberg, E. V., McNeil, P. L., Fujiki, H., and Rosner, M. R. (1989) J. Biol. Chem. 264, 213-219) described the requirement for a sodium ion influx in the down-modulation of the EGF receptor stimulated by a non-TPA-type tumor promoter, palytoxin, in Swiss 3T3 cells. We tested for a similar sodium requirement in Balb/c/3T3 and Swiss 3T3 cells stimulated by PDGF or TPA in Balb cells treated with TPA for prolonged periods to down-regulate protein kinase C activity. Our results clearly show that the PDGF- and TPA-stimulated transmodulation of the EGF receptor does not require external sodium nor is the process affected by amiloride. In each of these experiments, the loss of 125I-EGF binding occurred to a similar extent and at a similar rate in the presence or absence of sodium. Intracellular pH also did not appear to have a role in the response. The sodium ionophore, monensin, was previously shown to bring about the down-modulation of 125I-EGF binding in Swiss cells. However, our results indicate that monensin-induced transmodulation of the EGF receptor occurs with or without external sodium, suggesting that the loss of binding is not the result of a sodium ion influx. These findings demonstrate that an increase in intracellular sodium does not cause nor is it required for PDGF- or TPA-stimulated EGF receptor transmodulation.     

A v-sis oncogene protein produced in bacteria competes for platelet-derived growth factor binding to its receptor

The oncogene of simian sarcoma virus, v-sis, encodes a protein which is homologous to human platelet-derived growth factor (PDGF). This v-sis-encoded protein was expressed in bacteria using an inducible promotor of lambda phage. Soluble extracts from these bacteria contained a substance which competed with 125I-PDGF for PDGF receptor sites in fibroblast membranes. The receptor competition activity was correlated with the presence of the v-sis-encoded protein as assessed by genetic and immunochemical criteria. These results directly demonstrate that the v-sis oncogene product is functionally related to PDGF.     

Growth factor requirements of oncogene-transformed NIH 3T3 and BALB/c 3T3 cells cultured in defined media.

We report conditions for the efficient growth of NIH 3T3 and BALB/c 3T3 cells cultured in a defined medium supplemented with either platelet-derived growth factor (PDGF) or pituitary-derived fibroblast growth factor (FGF). The oncogenes v-mos, v-src, v-sis, and c-H-ras Val 12 can induce morphological transformation of these cells and can release them from the mitogen requirement for growth, while the oncogene v-fos cannot abrogate the PDGF-FGF requirement. The radically different behavior of normal and transformed NIH 3T3 cells in PDGF-FGF-free defined medium can form the basis of a sensitive new fibroblast transformation assay.     

Autocrine stimulation by the v-sis gene product requires a ligand- receptor interaction at the cell surface

Autocrine expression of a growth factor and its receptor in the same cell raises the possibility of an intracellular receptor-ligand interaction within the cell, in addition to a receptor-ligand interaction at the cell surface. We have constructed a NIH3T3 cell line which contains the v-sis gene under the inducible control of the Drosophila melanogaster hsp70 promoter. Expression of both v-sis RNA and protein is rapidly induced by a short period of heat-shock. We have analyzed the cellular site of interaction between the v-sis protein and the platelet-derived growth factor receptor in these cells. Autophosphorylation of the PDGF receptor and induction of the c-fos gene were found to occur at 45 and 50 min, respectively, after heat-induced synthesis of the v-sis protein. Monensin treatment of the heat-induced cells prevented autophosphorylation of the mature PDGF receptor and also prevented subsequent induction of c-fos. Autophosphorylation of the PDGF receptor and c-fos induction were also prevented by the addition of suramin to the medium. These results demonstrate that autocrine stimulation, as monitored by c-fos induction and by PDGF receptor autophosphorylation, requires an interaction between the v-sis protein and the PDGF receptor that occurs at the cell surface, rather than an intracellular location.     

Transformation of diploid human fibroblasts by DNA transfection with the v-sis oncogene

The simian sarcoma virus (SSV) oncogene (v-sis) has a high degree of homology to the cellular gene coding for the B peptide of human platelet-derived growth factor (PDGF), a potent fibroblast mitogen. The cellular homolog of v-sis is activated in some mesenchymal human tumors and cell lines derived from them. To determine the phenotype produced by v-sis in diploid human fibroblasts, we constructed plasmids containing the SSV provirus and drug-resistance markers and transfected them into early-passage human cells. Fibroblasts that had integrated the plasmid were selected for drug resistance and shown to contain and express the v-sis oncogene by DNA and RNA hybridization. The v-sis-expressing cells grew to higher saturation densities than control cells transfected with the vector plasmid alone and formed large, well defined foci. This allowed selection of transfectants directly for focus formation. The v-sis transformed cells continued to grow well in the absence of serum, whereas age-matched, vector-transfected control cells ceased replicating under these conditions so that the final difference in density between the two populations was tenfold. Incorporation of thymidine in serum-free medium by the v-sis-transformed cells was independent of exogenous PDGF. In contrast, PDGF increased thymidine incorporation in such medium by the control cells to the level found in the v-sis-transformed cells with or without added PDGF. These results suggest that expression of the v-sis oncogene in diploid human fibroblasts causes sufficient endogenous synthesis of the B chain of PDGF to allow transformants to grow to abnormally high cell densities. When individual v-sis-transformed cells were grown on a background of normal cells, this higher cell density at confluence could be visualized as a focus.     

Altered growth factor sensitivity in EL2 rat fibroblasts: influence of this biological characteristic on cell growth

Extensive evidence indicate that platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) play a key role in the stimulation of the 3T3 fibroblast replication: in this connection, PDGF and EGF act as a competence and a progression factor, respectively. We have previously demonstrated that EGF alone leads density-arrested EL2 rat fibroblasts to synthesize DNA and proliferate in serum-free cultures. Here, we have analyzed the role of EGF in the control of EL2 cell proliferation. Our data show a dose-related effect of EGF on DNA synthesis and cell growth, with maximal stimulation for both parameters at 20 ng/ml. On the other hand, autocrine production of PDGF or PDGF-like substances by EL2 cells is seemingly excluded by experiments with anti-PDGF serum or medium conditioned by EL2 fibroblasts. EGF binding studies show that EL2 cells possess high affinity EGF receptors, at a density level 3 to 4-fold higher than other fibroblastic lines. In addition, EL2 cells show a normal down-regulation of EGF receptors, following exposure to EGF, but PDGF, fibroblast growth factor (FGF), transforming growth factor beta (TGF beta) and bombesin have not decreased the affinity of EGF receptor for its ligand. Moreover, in EL2 cells, the EGF is able to induce the synthesis of putative intracellular regulatory proteins that govern the PDGF-induced competence in 3T3 cells. Our data indicate that EGF in EL2 cells may act as both a competence and a progression factor, via induction of the mechanisms, regulated in other cell lines by cooperation between different growth factors.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cytolytic T cells recognize the two amino-terminal domains of H-2 K antigens in tandem in influenza A infected cells

Simian sarcoma virus-transformed NIH 3T3 (SSV-NIH 3T3) and SSV-NRK cells secrete a potent growth-promoting activity identical with the platelet-derived growth factor (PDGF) in mitogenic assays. The secreted activity is blocked by anti-PDGF antisera and competes with 125I-PDGF for receptor binding, suggesting that the secreted protein is the transforming protein of SSV, p28v-sis, or its processed product. Secreted p28v-sis appears to stimulate autocrine cell growth of SSV-transformed cells because anti-PDGF antisera block 3H-thymidine incorporation into growing SSV-NIH 3T3 and SSV-NRK cells. SSV-transformed cells have reduced numbers of high-affinity 125I-PDGF receptors; PDGF/p28v-sis receptor was purified from SSV-NIH 3T3 cells and retained active protein tyrosine kinase activity stimulated by PDGF. The rate of tumor growth in athymic nude mice injected with SSV-transformed cells was compared with levels of secreted growth factor activity. The rate of tumor growth in nude mice correlated directly with levels of p28v-sis secreted by SSV-transformed cells.     

Interactions between the receptors for platelet-derived growth factor and epidermal growth factor

Preincubation of Swiss 3T3 cells or human fibroblasts with purified platelet-derived growth factor (PDGF) at 4 degrees C or 37 degrees C rapidly inhibits subsequent binding of 125I-epidermal growth factor (125I-EGF). The effect does not result from competition by PDGF for binding to the EGF receptor since (a) very low concentrations of PDGF are effective, (b) cells with EGF receptors but no PDGF receptors are not affected, and (c) the inhibition persists even if the bound PDGF is eluted before incubating the cells with 125I-EGF. PDGF does not affect 125I-insulin binding nor does EGF affect 125I-PDGF binding under these conditions. Endothelial cell-derived growth factor also competes for binding to PDGF receptors and inhibits 125I-EGF binding. The inhibition demonstrated by PDGF seems to result from an increase in the Kd for 125I-EGF binding with no change in the number of EGF receptors.     

Cultured endothelial cells produce multiple growth factors for connective tissue cells

Cultured bovine aortic endothelial cells (BAEC) secrete into their medium a growth-promoting factor that stimulates many connective tissue cells in culture. We now report that this growth-promoting activity is due to at least two different proteins which are biochemically separable and immunologically distinct. Cation exchange chromatography (Carboxymethyl-Sephadex) of concentrated BAEC-conditioned medium yields two major peaks of growth-promoting activity which adsorb at pH 8 and elute with a salt gradient. One of these peaks contains as well a protein that inhibits the binding of radioiodinated platelet-derived growth factor (PDGF) to its receptor on target cells. The PDGF-like mitogen is purified approx. 25-fold by this chromatographic step. A second peak of mitogenic activity exhibits no binding to the PDGF receptor. Both the PDGF-like mitogenic activity and the PDGF-distinct mitogenic activity are highly cationic, stable to boiling, sensitive to beta-mercaptoethanol, and between 30 and 50 kD in molecular weight. Complementary studies with human umbilical vein endothelial cells in culture were performed. These human cells also produce both growth-promoting activity and a protein that binds to the PDGF receptor. The latter activity is greatly inhibited by a specific antiserum to human PDGF, whereas the growth-promoting activity of the conditioned medium is minimally affected. The degree of inhibition of the two activities is, however, quantitatively consistent: 3.5 ng of PDGF-like activity in the radioreceptor assay is inhibited, while 5 ng of PDGF-like activity in the DNA synthesis assay is inhibited. The data from the two species are consistent with the proposal that cultured endothelial cells produce at least two distinct mitogens, one of which is biochemically and immunologically related to PDGF.     

The phenotypic characteristics of simian sarcoma virus-transformed human fibroblasts suggest that the v-sis gene product acts solely as a PDGF receptor agonist in cell transformation.

Previous studies have indicated that the oncogene v-sis of simian sarcoma virus (SSV) encodes a growth factor that is structurally and functionally similar to platelet-derived growth factor (PDGF). In the present investigation we have analysed the phenotypic characteristics of human foreskin fibroblasts transformed by SSV. It was found that the PDGF receptors were extensively down-regulated. This finding is consistent with a high, local, extracellular concentration of a PDGF-like factor, synthesized by the transformed cell. The receptors were up-regulated by suramin, a drug that is known to dissociate PDGF and the v-sis product from the PDGF receptors. A cell-associated v-sis product of mol. wt 24,000 was identified by immunoprecipitation with PDGF antibodies; release of this component was induced by a high concentration of exogenous PDGF, indicating that a fraction of the product is associated with the PDGF receptors. SSV was not found to be an immortalizing virus; when serially passaged, SSV-transformed cells had essentially the same life-span as their non-transformed counterparts. Moreover, SSV did not induce growth in soft agar beyond the level afforded by exogenously added PDGF. Thus, the present study favors the notion that SSV transformation is mediated by a growth factor that mimics PDGF but has no further cellular effects.