Regulation of the Balb/c-3T3 cell cycle-effects of growth factors

The platelet-derived growth factor (PDGF), which is found in serum but not in plasma, has been purified to homogeneity; it stimulates replication at a concentration of 10^?10M. Brief treatment with PDGF causes densityinhibited Balb/c-3T3 cells to become competent to synthesize DNA; pituitary fibroblast growth factor (FGF) or precipitates of calcium phosphate also induce competence. Continuous treatment with plasma allows competent, but not incompetent, cells to synthesize DNA. A critical component of plasma is somatomedin, a group of hormones with insulin-like activity; multiplication-stimulating activity (MSA) or insulin replace plasma somatomedin in promoting DNA synthesis. We have studied the molecular correlates of competence and the role of SV40 gene A products in regulating DNA synthesis. Treatment of quiescent cells with pure PDGF or FGF causes the preferential synthesis of five cytoplasmic proteins (approximate molecular weight 29,000, 35,000, 45,000, 60,000, and 72,000 detected by SDS-PAGE under reducing conditions). Two of these competence-associated proteins (29,000 and 35,000 daltons) are found within 40 min of PDGF addition; they are not induced by plasma, insulin, or epidermal growth factor (EGF), PDGF, FGF, or calcium phosphate induce an ultrastructure change within the centriole of 3T3 cells; this ultrastructural modification of the centriole is detectable by immunofluorescence within 2 h of PDGF treatment. Plasma, EGF, or MSA do not modify the centriole. SV40 induces replicative DNA synthesis in growth-arrested 3T3 cells but does not cause this alteration in centriole structure. Gene A variants of SV40, including a mutant with temperature-sensitive (ts) T-antigen (ts A209), a deletion in t-antigen (dl 884), and several ts A209 strains containing t-antigen deletions were used to induce DNA synthesis in Balb/c-3T3 cells. Like wild type SV40, all strains induced DNA synthesis equally well under permissive or nonpermissive conditions. Addition of PDGF or plasma had little effect on SV40-induced DNA synthesis. Thus, the viral function that induces replicative DNA synthesis in Balb/c-3T3 cells is not t and is not temperature sensitive. This SV40 gene function overrides the cellular requirement for hormonal growth factors. It does not induce transient centriole deciliation, a hormonally regulated event.     

Alteration of the chemotactic response of NIH/3T3 cells to PDGF by growth factors, transformation, and tumor promoters

The platelet-derived growth factor (PDGF) is a potent chemoattractant for cells that respond to PDGF as a mitogen. The chemotactic response of these cells to PDGF is inversely related to their rate of proliferation, with quiescent cells exhibiting a 25-fold greater chemotactic response than exponentially growing cells. Factors that stimulate the growth of quiescent cells (EGF, FGF, PDGF, and serum) decrease the cells' migratory response to PDGF but not to fibronectin, suggesting that the decreased migration is not due to a general paralysis of cell motility. Transformed lines of NIH/3T3 cells lose their ability to respond to PDGF as a chemoattractant but can still migrate in response to fibronectin. Similarly, after treatment of 3T3 cells with the tumor-promoter phorbol myristate acetate, which induces a transformation-like phenotype, the cells no longer respond to PDGF as a chemoattractant but retain their migratory response to fibronectin. Thus it appears that the growth state of the cells can alter their migratory response to PDGF. These data suggest that growth factors, transformation, and tumor promoters specifically alter the cells' ability to respond to the PDGF-mediated chemotactic signal. It appears that both transformation and tumor promoters accomplish this by altering PDGF-binding to the cell surface.     

Analysis of the reduced growth factor dependency of simian virus 40-transformed 3T3 cells.

We have measured in a defined serum-free medium the platelet-derived growth factor (PDGF) and insulin requirements of normal Swiss 3T3 cells, simian virus 40-transformed 3T3 cells, and partial revertants of simian virus 40-transformed 3T3 cells. Swiss 3T3 cells displayed strong requirements for both PDGF and insulin. Both of these requirements were significantly diminished in simian virus 40-transformed 3T3 cells. Analysis of the PDGF and insulin requirements of the revertants indicated that the loss of either of these two growth factor requirements was not necessarily linked to the other; rather, the growth factor requirements were specifically associated with other parameters of transformation. The reacquisition of a PDGF requirement cosegregated with reversion to density-dependent growth inhibition, whereas reacquisition of a normal insulin requirement cosegregated with reversion to a normal growth dependence on calf serum. Anchorage dependence was dissociable from both growth factor requirements. The relationship between the PDGF requirement and density-dependent growth inhibition was further analyzed in normal 3T3 cells by measuring the PDGF requirement at different cell densities. At high cell densities, the requirement for PDGF became significantly greater. We suggest that at least in part the ability of transformed cells to grow to high saturation densities results from their loss of a requirement for PDGF.     

Differential binding, biological and biochemical actions of recombinant PDGF AA, AB, and BB molecules on connective tissue cells.

We have compared the biological and biochemical properties of recombinant PDGF AA, AB, and BB using three types of fibroblastic cells: NIH/3T3, human skin fibroblast, and fetal bovine aortic smooth muscle. PDGF binding, receptor autophosphorylation, phosphatidyl inositol hydrolysis, as well as chemotactic and mitogenic responses of the cells were analyzed. PDGF-AB and PDGF-BB showed similar receptor binding, receptor autophosphorylation, and potent biological activity for all three of the cell types tested. In contrast, PDGF-AA was biologically active only for the NIH/3T3 cells in which binding sites for PDGF-AA were abundant, but was inactive for bovine aortic smooth muscle cells and human skin fibroblasts in which binding sites for PDGF-AA were absent. PDGF-AA could not induce any biochemical changes in the human skin fibroblasts or smooth muscle cells. Western blot studies with anti-Type alpha and beta PDGF receptor antibodies indicate that the NIH/3T3 cells contained both PDGF alpha and beta receptors, whereas the human skin fibroblasts and bovine smooth muscle cells contained only detectable levels of beta receptors. These results indicate that cells possessing high levels of PDGF beta receptors only are capable of responding equally well to either PDGF AB or BB.     

Platelet-derived growth factor does not induce c-fos in NIH 3T3 cells expressing the EJ-ras oncogene.

Platelet-derived growth factor (PDGF), the calcium ionophore A23187, and the tumor promoter phorbol myristate acetate stimulated c-fos mRNA levels in control NIH 3T3 cells. However, NIH 3T3 cells transformed by EJ-ras DNA transfection, which have diminished PDGF-stimulated phospholipase C activity, showed a 95% reduction in PDGF-stimulated c-fos mRNA levels. The responses to A23187 and phorbol myristate acetate were also attenuated, but not as severely as the PDGF-mediated induction. The reduction in PDGF-stimulated c-fos induction did not appear to be a general result of cellular transformation, since src-transformed NIH 3T3 cells displayed a strong PDGF-stimulated c-fos induction. Despite the reduction in PDGF-stimulated c-fos induction, EJ-ras-transformed cells still responded mitogenically to PDGF. These data suggest that the magnitude of c-fos induction cannot be directly correlated with PDGF-stimulated mitogenesis in EJ-ras-transformed NIH 3T3 cells.     

Dissociation of the chemotactic and mitogenic activities of platelet- derived growth factor by human neutrophil elastase

Because platelet-derived growth factor (PDGF) may be released at sites where neutrophil proteinases may also be released, we examined the effects of neutrophil elastase and cathepsin G upon the chemotactic and mitogenic activities of PDGF. Elastase abolished the chemotactic activity of PDGF for fibroblasts but had no effect on its chemotactic activity for monocytes, or on its mitogenic activity for 3T3 cells or its capacity to bind to 3T3 cells. Cathepsin G had no effect upon the chemotactic or mitogenic activities of PDGF. In contrast, trypsin eliminated the chemotactic activity of PDGF for monocytes and fibroblasts and the mitogenic activity of PDGF. After reduction and alkylation, PDGF retained full chemotactic activity for fibroblasts and monocytes but exhibited no mitogenic activity and only limited binding to 3T3 cells. These results indicate separate domains on PDGF for fibroblast chemotactic and mitogenic activity and for monocyte and fibroblast chemotactic activity and raise the possibility that the biological activities of PDGF may be modified selectively in vivo. The findings further suggest that the majority of PDGF receptors on fibroblasts mediate mitogenic activity and that only a minority of the PDGF receptors on fibroblasts are responsible for chemotactic activity.     

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.     

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.     

Cyclic AMP-dependent protein kinases from Balb 3T3 cells and other 3T3 derived lines

Cyclic AMP-dependent protein kinase and 3H-cAMP-binding activities were determined in normal Balb 3T3 cells and compared with the same preparations from SV40, chemical, and spontaneous transformants of 3T3 cells. The cytosolic protein kinase activities and protein kinase activity ratios were similar in all cell lines, although when the normal 3T3 cytosol was prepared by homogenization it contained less 3H-cAMP binding activity than the transformed 3T3 cytosols. The Triton X-100 treated particulate fractions from the normal and transformed 3T3 cells contained similar protein kinase and binding activities. The isozymic profile of cAMP-dependent protein kinases was examined by DEAE-chromatography. The 3T3 cells contained only type II isozyme in either cytosolic or membrane fractions. All transformants of the 3T3 cells contained both type I and type II isozymes. Other cell cultures, including chicken embryo fibroblasts, rat kidney cells, and human or calf endothelial cells contained type I and type II isozymes. Binding of the photoaffinity analogue of cAMP, 8-N3 cAMP, to the regulatory subunits of protein kinases in sonicates obtained from Balb 3T3 and SV 3T3 cells followed by separation on SDS polyacrylamide electrophoresis showed that the amount of RII subunit was approximately equal in the two cell lines. RI in Balb 3T3 cells was detectable but in a much lower quantity than in SV 3T3 cells. The cyclic AMP dependent-protein kinases from Balb 3T3 cells appears to be different from SV 3T3 cells by three criteria: 3H-cAMP binding in homogenates, DEAE chromatographic separation of isozymes, and 8-N3 cAMP binding.     

Similarities between fibroblast-derived growth factor and platelet-derived growth factor

Platelet-derived growth factor (PDGF), one of the most potent mitogens in serum for non-transformed cells, shares many biological and physical properties with fibroblast-derived growth factor (FDGF), a polypeptide produced by BHK cells transformed by SV40. Thus FDGF and PDGF have biological activity which is recoverable from sodium dodecyl sulphate (SDS) polyacrylamide gel electrophoresis, at positions indicating similar molecular weights. Further, the biological activity of both factors is heat-stable but sensitive to mercaptoethanol. FDGF and PDGF have similar abilities to induce DNA synthesis synergistically in the presence of either insulin, epidermal growth factor (EGF), vasopressin or colchicine. In contrast to other growth factors, (i) either FDGF or PDGF can induce DNA synthesis in the absence of other mitogens in 3T3 cells maintained in serum-free medium and (ii) a transient exposure of cultures to FDGF or PDGF causes a persistent stimulation of DNA synthesis. Either FDGF or PDGF enhances colony formation of non-transformed cells cultured in suspension in the presence of EGF and serum. FDGF is not PDGF adsorbed by SV40-BHK cells from serum, since SV40-BHK cells plated and grown in the absence of serum still produce FDGF. In view of the similarities between PDGF and FDGF, we suggest that they may belong to the same family of growth factors.     

Common elements in growth factor stimulation and oncogenic transformation: 85 kd phosphoprotein and phosphatidylinositol kinase activity

The phosphorylation of proteins on tyrosine in vivo and in vitro was examined in 3T3 cells stimulated by platelet-derived growth factor (PDGF) and transformed by polyoma middle T antigen (MTAg) by using an antibody directed against phosphotyrosine (P-tyr). Two common events were observed upon PDGF stimulation or MTAg transformation of cells: the appearance in the immunoprecipitates of an 85 kd phosphoprotein, and increased phosphatidylinositol (PI) kinase activity. In PDGF-stimulated cells, the 85 kd phosphoprotein and PI kinase activity appeared rapidly, within 1 min of growth factor addition. The PI kinase activity and 85 kd phosphorylation were also increased in anti-P-tyr immunoprecipitates from cells transformed by v-fms and v-sis, but not by SV40 T antigen. The presence of the tyrosine-phosphorylated 85 kd protein correlated with PI kinase activity during several purification steps. These results suggest that the 85 kd phosphoprotein, a putative PI kinase, is a substrate for both the PDGF receptor and MTAg/pp60c-src tyrosine kinase activities.     

Biological function of PDGF-induced PI-3 kinase activity: its role in alpha PDGF receptor-mediated mitogenic signaling

The tyrosine phosphorylation sites in the human alpha PDGF receptor (alpha PDGFR) required for association with PI-3 kinase have been identified as tyrosines 731 and 742. Mutation of either tyrosine substantially reduced PDGF-induced PI-3 kinase activity but did not impair the receptor-mediated mitogenic response. We sought to determine whether PDGF-induced PI-3 kinase activity could be further ablated so as to exclude a low threshold requirement for PDGFR signal transduction. Thus, we mutated both tyrosine 731 and 742 and expressed the double mutant (Y731F/Y742F) in 32D hematopoietic cells. In such transfectants, PDGF induced no detectable receptor-associated or anti-P- Tyr recoverable PI-3 kinase activity. Under the same conditions, neither mobility shift of raf-1 nor tyrosine phosphorylation of either PLC gamma or MAP kinase was impaired. 32D transfectants expressing the double mutant showed wild-type alpha PDGFR levels of mitogenic and chemotactic responses to PDGF. To examine the effect of the double mutation in cells that normally respond to PDGF, we generated chimeras in which the cytoplasmic domains of wild-type alpha PDGFR, Y731F, and Y731F/Y742F were linked to the extracellular domain of colony- stimulating factor-1 (CSF-1) receptor (fms). After introduction of the chimeric receptors into mouse NIH/3T3 fibroblasts, the ability of CSF-1 to stimulate growth of these transfectants was examined. Our data show that all these chimeric receptors exhibited similar abilities to mediate CSF-1-stimulated cell growth. These findings lead us to conclude that PDGF-induced PI-3 kinase activity is not required for PDGF-stimulated mitogenic pathway in both NIH/3T3 fibroblasts and 32D hematopoietic cells.     

Modulation of guanine nucleotides bound to Ras in NIH3T3 cells by oncogenes, growth factors, and the GTPase activating protein (GAP)

The mitogenic activity of membrane-associated tyrosine kinases such as Src and the PDGF receptor appear to depend on Ras function. Ras biochemical activity involves regulation of a GTP/GDP cycle and the GTPase activating protein (GAP). Recently, PDGF and v-Src have been shown to stimulate tyrosine phosphorylation of GAP, linking these pathways at the biochemical level. To test whether PDGF and v-Src affect the Ras GTP/GDP cycle, we have measured the guanine nucleotides complexed to Ras in NIH3T3 cells and compared the ratio of GTP to total GTP + GDP detected (percent GTP). In normal quiescent NIH3T3 cells, PDGF stimulated the basal amount of GTP complexed to Ras (7%) by 2.1-fold to 15%. The effect was dependent on PDGF concentration and was observed maximally within 10 min following PDGF challenge. Ras was complexed to 22% GTP in NIH3T3 cells transformed by v-src or v-abl. Overexpression of GAP by 110-fold in NIH3T3 cells reduced the basal level of GTP complexed to Ras to 2.4%; upon challenge with PDGF, Ras was complexed to 6.6% GTP. These results indicate that PDGF receptor activation and tyrosine kinase-encoding oncogene products can stimulate Ras into the GTP complex and that GAP in intact mammalian cells can decrease the amount of GTP complexed to Ras.     

High chemotactic response to platelet-derived growth factor of a teratocarcinoma differentiated mesodermal cell line

Summary Evidence is presented that a differentiated mesodermal line (MES-1) from P19 EC cells express a high chemotactic response to platelet-derived growth factor (PDGF) as assayed in a blind-well modified Boyden chamber. Compared to the NIH 3T3 fibroblasts the chemotactic response of MES-1 is increased by 10-fold at 0.3 ng/ml of PDGF, 4-fold at 1.25 ng/ml of PDGF, 2-fold at 2.5 ng/ml of PDGF. In contrast, PDGF induces the same increase in [³H]thymidine incorporation in both cell lines, made quiescent under reduced serum concentration. This high chemotactic response to PDGF seems specific for these mesodermal cells. Among the different teratocarcinoma cells tested, including stem cells (F9, PC 13, PCC4) and endodermal derivatives (PYS, F9 with retinoic acid, PSA 5E), only the visceral endodermlike cells (PSA5E) are slightly attracted by PDGF. This chemotactic response to PDGF is not related to the presence or characteristics of the type B PDGF receptors, which are less numerous in MES-1 cells (10⁵ receptors/cell, KDa 1,2 mM) compared to NIH 3T3 cells (64×10⁴ receptors per cell, KDa 1,8 nM). The MES-1 cell line might be of interest for studying the chemotactic effect of PDGF. These results also suggest a role for this soluble factor in cell migration during early embryogenesis. This investigation was supported by a grant of La Fondation pour la Recherche Médicale.     

Platelet-derived growth factor stimulation of GTPase-activating protein tyrosine phosphorylation in control and c-H-ras-expressing NIH 3T3 cells correlates with p21ras activation.

Platelet-derived growth factor (PDGF) stimulation of NIH 3T3 cells leads to the rapid tyrosine phosphorylation of the GTPase-activating protein (GAP) and an associated 64- to 62-kDa tyrosine-phosphorylated protein (p64/62). To assess the functions of these proteins, we evaluated their phosphorylation state in normal NIH 3T3 cells as well as in cells transformed by oncogenically activated v-H-ras or overexpression of c-H-ras genes. No significant GAP tyrosine phosphorylation was observed in unstimulated cultures, while PDGF-BB induced rapid tyrosine phosphorylation of GAP in all cell lines analyzed. In NIH 3T3 cells, we found that PDGF stimulation led to the recovery of between 37 and 52% of GAP molecules by immunoprecipitation with monoclonal antiphosphotyrosine antibodies. Furthermore, PDGF exposure led to a rapid and sustained increase in the levels of p21ras bound to GTP, with kinetics similar to those observed for GAP tyrosine phosphorylation. The PDGF-induced increases in GTP-bound p21ras in NIH 3T3 cells were comparable to the steady-state level observed in serum-starved c-H-ras-overexpressing transformants, conditions in which these cells maintained high rates of DNA synthesis. These results imply that the level of p21ras activation following PDGF stimulation of NIH 3T3 cells is sufficient to support mitogenic stimulation. Addition of PDGF to c-H-ras-overexpressing cells also resulted in a rapid and sustained increase in GTP-bound p21ras. In these cells GAP, but not p64/62, showed increased tyrosine phosphorylation, with kinetics similar to those observed for increased GTP-bound p21ras. All of these findings support a role for GAP tyrosine phosphorylation in p21ras activation and mitogenic signaling.     

Centriole deciliation associated with the early response of 3T3 cells to growth factors but not to SV40.

BALB/c-3T3 cells which are growth-arrested by high cell density or low serum have ciliated, unduplicated centrioles. Stimulation of these quiescent cells by serum is associated with a rapid (within 1–2 hr) deciliation of the centriole, followed by reciliation within 6–10 hr. This transient deciliation of the centriole is induced by the platelet-derived growth factor (PDGF) component of serum. The cells treated with PDGF became competent to replicate their DNA; most PDGF treated cells, however, did not progress from Go toward S phase unless they were incubated with the platelet-poor plasma component of serum. Addition of CaCl₂ or Fibroblast Growth Factor to the media mimicked PDGF by producing both centriole deciliation and competence to replicate DNA. In fact, over a range of concentrations of each of these factors, only doses which produced centriole deciliation were capable of producing competence for DNA synthesis. Plasma alone or factors such as Multiplication Stimulating Activity produced neither centriole deciliation nor competence; these agents were, however, required for the optimum progression of competent cells into DNA synthesis. In contrast, infection with SV40 induced host cell DNA synthesis without an initial transient deciliation of the centriole. Thus while growth factors may have to induce centriole deciliation for 3T3 cells to synthesize DNA, abortive transformation by SV40 overrides this requirement.     

Transforming growth factor alpha production and epidermal growth factor receptor expression in normal and oncogene transformed human mammary epithelial cells

We have characterized the expression of transforming growth factor alpha (TGF alpha) and its receptor, the epidermal growth factor receptor (EGF-R), in normal and malignantly transformed human mammary epithelial cells. Human mammary epithelial cells were derived from a reduction mammoplasty (184), immortalized by benzo-a-pyrene (184A 1N4), and further transformed by the oncogenes simian virus 40 T (SV40 T), v-Ha-ras, and v-mos alone or in combination using retroviral vectors. 184 and 184A 1N4 cells require EGF for anchorage-dependent clonal growth. In mass culture, they secrete TGF alpha at high concentrations and exhibit an attenuated requirement for exogenous EGF/TGF alpha. SV40 T transformed cells have 4-fold increased EGF-R, have acquired the ability to clone in soft agar with EGF/TGF alpha supplementation, but are not tumorigenic. Cells transformed by v-mos or v-Ha-ras are weakly tumorigenic and capable of both anchorage dependent and independent growth in the absence of EGF/TGF alpha. Cells transformed by both SV40 T and v-Ha-ras are highly tumorigenic, are refractory to EGF/TGF alpha, and clone with high efficiency in soft agar. The expression of v-Ha-ras is associated with a loss of the high (but not low) affinity binding component of the EGF-R. Malignant transformation and loss of TGF alpha/EGF responsiveness did not correlate with an increase in TGF alpha production. Thus, TGF alpha production does not appear to be a tumor specific marker for human mammary epithelial cells. Differential growth responses to EGF/TGF alpha, rather than enhanced production of TGF alpha, may determine the transition from normal to malignant human breast epithelium.     

Chemotaxis of 3T3 and SV3T3 cells to fibronectin is mediated through the cell-attachment site in fibronectin and a fibronectin cell surface receptor

Fibronectin (FN) is a multidomain extracellular matrix protein that induces attachment and chemotactic migration of fibroblastic cells. In this study we analyzed the molecular determinants involved in the FN-induced chemotactic migration of normal and SV40-transformed 3T3 cells. Two different monoclonal antibodies to the cell-binding site of FN blocked chemotaxis to a 140-kD FN fragment (Ca 140) containing the cell-binding domain. A monoclonal antibody to a determinant distant from the cell-binding site did not affect chemotaxis. A synthetic tetrapeptide, RGDS, which represents the major cell-attachment sequence, was able to compete with FN and the Ca 140 fragment in chemotaxis assays, but this peptide itself had no significant chemotactic activity. A larger peptide encompassing this sequence, GRGDSP, was chemotactic, while the peptide GRGESP, where a glutamic acid residue was substituted for aspartic acid, was inactive. Chemotactic migration could be prevented in a dose-dependent manner by a rabbit polyclonal antiserum to a 140-kD cell surface FN receptor. This antibody was more effective on normal than on transformed 3T3 cells. Neither the anti-FN receptor antiserum nor a monoclonal antibody to the cell-binding site of FN blocked migration induced by another potent chemoattractant, platelet-derived growth factor. These data indicate that FN-induced chemotaxis of 3T3 and SV3T3 cells is mediated via the RGDS cell-attachment site of FN and the 140-kD cell surface FN receptor. The interaction is specific and can be altered by transformation.     

Protamine inhibits platelet derived growth factor receptor activity but not epidermal growth factor activity

Protamine sulfate blocked 125I-PDGF binding to its specific physiological receptor on Swiss mouse 3T3 cells. Reduced 125I-PDGF binding in the presence of protamine sulfate correlated directly with a protamine sulfate dose-dependent decrease in the PDGF-dependent incorporation of [3H]-thymidine into 3T3 cells and a decreased PDGF-stimulated tyrosine-specific protein kinase activity in isolated membrane preparations of 3T3 cells. Protamine sulfate blocked 125I-PDGF binding to simian sarcoma virus transformed cells (SSV-NIH 3T3 and SSV-NP1 cells) and to nontransformed cells in a manner qualitatively identical to unlabelled PDGF. In contrast, protamine sulfate enhanced the specific binding of 125I-EGF by increasing the apparent number of EGF receptors on the cell surface. The increase in 125I-EGF receptor binding was not prevented by cycloheximide nor by actinomycin D. Protamine sulfate did not affect 125I-EGF binding to membranes from 3T3 cells or the EGF-stimulated 3T3 cell membrane tyrosine specific protein kinase activity, suggesting that protamine sulfate may have exposed a population of cryptic EGF receptors otherwise not accessible. Protamine sulfate was fractionated into four active fractions by Sephadex G-50 gel filtration columns; the half maximum inhibition concentration of 125I-PDGF binding to 3T3 cells of protamines I and II (MW approximately 11,000 daltons and 7,000 daltons, respectively) is approximately 0.4 microM. Protamine II (MW approximately 4,800 daltons) was equally active (half maximum inhibition concentration approximately 0.4 microM); protamine IV (MW approximately 3,300 daltons) was substantially less active (half maximum inhibition concentration approximately 2.8 microM). These investigations have extended previous observations that protamine sulfate is a potent inhibitor of PDGF binding and establish that protamine sulfate blocks PDGF binding at the physiological receptor, preventing PDGF initiated biological activities. Protamine sulfate can be used as a reagent to separate the influence of PDGF and EGF on cells with high specificity and has been used to demonstrate that the receptors on simian sarcoma virus transformed 3T3 cells qualitatively respond identically to protamine sulfate as to unlabelled PDGF and are likely identical to those on nontransformed 3T3 cells.     

Increased glutathione synthesis associated with platelet-derived growth factor stimulation of NIH3T3 fibroblasts

Previous data show a relation between GSH content and proliferation of normal and tumour cells. We recently demonstrated a specific involvement of GSH in the autophosphorylation activity of the platelet-derived growth factor (PDGF) receptor in NIH3T3 fibroblasts. In this study we demonstrate that the stimulation by PDGF of serum-starved NIH3T3 cells increases cellular GSH content, while no change in oxidized GSH content was measured. Experiments performed with actinomycin, cycloheximide and buthionine sulfoximide, a specific inhibitor of the rate-limiting enzyme of the de novo synthesis of GSH gamma-glutamylcysteine synthetase (gamma-GCS), confirm PDGF induction of GSH synthesis. These results provide the first demonstration that PDGF mediated transduction signals seem strictly related to mechanisms involved in the increase of gamma-GCS activity associated with increased gamma-GCS heavy subunit mRNA levels. In fact, serum and epidermal growth factor (EGF) stimulation of quiescent NIH3T3 and NIH3T3, which overexpress EGF receptor, does not affect GSH content or its synthesis. These data may be related to a possible GSH role in the redox regulation of cell proliferation mediated by PDGF.