Vol. 11, No. 6
MOLECULAR AND CELLULAR BIOLOGY, June 1991, p. 3148-3154 0270-7306/91/063148-07$02.00/0 Copyright © 1991, American Society for Microbiology
BALB/c-3T3 Fibroblasts Resistant to Growth Inhibition by Beta Interferon Exhibit Aberrant Platelet-Derived Growth Factor, Epidermal Growth Factor, and Fibroblast Growth Factor Signal Transduction LAURA J. MUNDSCHAU AND DOUGLAS V. FALLER* Division of Pediatric Hematology and Oncology, Dana Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115 Received 13 December 1990/Accepted 11 March 1991
Several lines of evidence now exist to suggest an interaction between the platelet-derived growth factor (PDGF) growth-stimulatory signal transduction pathway and the beta interferon (IFN-j8) growth-inhibitory signal transduction pathway. The most direct examples are inhibition of PDGF-mediated gene induction and mitogenesis by IFN-4 and the effects of activators and inhibitors of the IFN-inducible double-stranded RNAdependent eIF2 kinase on expression of PDGF-inducible genes. To further investigate the nature of this PDGF/ IFN-, interaction, we selected BALB/c-3T3 cells for resistance to growth inhibition by IFN-, and analyzed the phenotypes of resulting clonal lines (called IRB cells) with respect to PDGF signal transduction. Although selected only for IFN resistance, the IRB cells were found to be defective for induction of growth-related genes c-fos, c-myc, and JE in response to PDGF. This block to signal transduction was not due to loss or inactivation of PDGF receptors, as immunoprecipitation of PDGF receptors with antiphosphotyrosine antibodies showed them to be present at equal levels in the BALB/c-3T3 and IRB cells and to be autophosphorylated normally in response to PDGF. Furthermore, treatment with other peptide growth factors (PDGF-AA, fibroblast growth factor, and epidermal growth factor) also failed to induce c-fos, c-myc, or JE expression in IRB cells. All of these growth factors, however, were able to induce another early growth-related gene, Egr-1. The block to signaling was not due to a defect in inositol phosphate metabolism, as PDGF treatment induced normal calcium mobilization and phosphotidylinositol-3-kinase activation in these cells. Activation of protein kinase C by phorbol esters did induce c-fos, c-myc, and JE in IRB cells, indicating that signaling pathways distal to this enzyme remained intact. We have previously shown that IFN-inducible enzyme activities, including doublestranded RNA-dependent eIF2 kinase and 2',5'-oligoadenylate synthetase, are normal in IRB cells. The finding that the induction of multiple growth-related genes by several independent growth factors is inhibited in these IFN-resistant cells suggests that there is a second messenger common to both growth factor and IFN signaling pathways and that this messenger is defective in these cells.
dent eIF2 kinase (15) have profound effects on plateletderived growth factor (PDGF)-inducible genes. Poly(I-C), a double-stranded RNA and activator of this kinase, rapidly induces steady-state mRNA levels of c-fos, c-myc, KC, and JE genes in fibroblasts (7, 25; our unpublished results). Conversely, 2-aminopurine, a guanidine analog and inhibitor of this kinase, prevents induction of transcription of c-fos and c-myc by serum or PDGF (22, 24; our unpublished observations). Given these results, which strongly suggest an interaction of IFN-inducible activities and PDGF-signaling pathways, we isolated cells which are defective in their response to the antiproliferative activity of IFN and analyzed their ability to respond to PDGF and other mitogens. Spontaneous mutants exhibiting IFN resistance were produced by long-term exposure of BALB/c-3T3 fibroblasts to very high doses of IFN-P (2,000 U/ml). We have previously described the phenotype of two of the resultant IFN-resistant BALB/c-3T3 (IRB) clones (called IRB 2 and IRB 5) with respect to IFN responsiveness (lla). Briefly, the IRB cells are resistant to the antiproliferative effect of IFN-,B but were found to retain the ability to establish an antiviral state and to increase class I major histocompatibility complex antigen cell surface expression in response to IFN. Two of the enzyme systems thought to be primarily responsible for producing the antiviral state,
Treatment of cultured cells with anti-interferon (IFN) antibodies has provided evidence that many cell types utilize the antiproliferative activity of IFN to inhibit their own proliferation via an autocrine IFN feedback mechanism (5, 6, 9, 18). The significance of this growth control mechanism is suggested by numerous reports that neutralization of the autocrine IFN or the spontaneous development of resistance to its antiproliferative effects can result in neoplasia or failure to differentiate, both of which may lead to malignancy (3, 9). Since the mechanism by which IFN inhibits cell growth is unknown, it remains to be established whether autocrine IFN has its effect by directly interacting with, and inhibiting, growth-stimulatory signaling pathways or instead by activating a separate growth-inhibitory mechanism, independent of the growth-stimulatory pathways. Most of the evidence accumulated so far has suggested a mechanism of direct interaction of IFN-stimulated machinery with growth factor-induced signaling pathways. For example, alpha IFN (IFN-ao) treatment of proliferating Daudi cells decreases c-myc expression (3) and decreases expression and activation of p34CDC2Hs a putative human cell cycle control protein (21). Furthermore, activators and inhibitors of the IFN-inducible double-stranded RNA-depen*
Corresponding author. 3148
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double-stranded RNA-dependent eIF2 kinase and 2',5'-oligoadenylate synthetase, were found to be inducible and activatable in the IRB cells to levels equal to or greater than those of the IFN-sensitive parental BALB/c-3T3 cells. In the work presented here, we have investigated the effect of the IFN-resistant phenotype of the IRB cells on their ability to respond to PDGF and other growth factors. We demonstrate that the ability of the IRB cells to induce c-fos, c-myc, and JE transcript levels in response to PDGF, fibroblast growth factor (FGF), and epidermal growth factor (EGF) is profoundly inhibited. These results suggest that interaction between the PDGF growth-stimulatory signal and the IFN growth-inhibitory signal occurs because these two pathways have at least one component of their signal transduction machinery in common and that this component is defective in the IRB cells. MATERIALS AND METHODS
Cell culture and selection of IFN-,-resistant lines. Clone A31 of BALB/c-3T3 cells was obtained from the American Tissue Culture Collection, Rockville, Md. IFN-resistant clones were selected by maintaining these cells in Dulbecco modified Eagle medium supplemented with 10% heat-inactivated donor bovine calf serum (BCS) (Hazelton Research Products, Inc., Lenexa, Kans.), 2 mM L-glutamine, 100 U of penicillin per ml, 100 ,ug of streptomycin per ml, and 2,000 U of IFN-, per ml (murine IFN-,; specific activity, 5.3 x 107 IU/mg; Lee Biomolecular Research Products, San Diego, Calif.) until proliferating colonies were apparent (6 to 8 weeks). These colonies were picked and cloned by limiting dilution in the presence of IFN. Two clones, called IRB 2 and IRB 5, were chosen at random for further study. These clones were normally carried in the presence of 800 U of IFN-, per ml; however, their IFN-resistant phenotype remained stable for more than 25 passages whether or not IRB cells were carried in the presence of exogenous IFN. PDGFAA, PDGF-BB, and recombinant EGF were obtained from Amgen (Thousand Oaks, Calif.). Recombinant basic FGF was a generous gift of California Biotechnologies (San Diego, Calif.). Preparation of conditioned media. Confluent 150-mm plates of BALB/c-3T3 or IRB cells were incubated with 0.5% serum-containing medium for 24 to 48 h. After centrifugation to remove particulate matter, this depleted medium was used immediately to treat other cells. RNA blot analysis of total cellular RNA. RNA was isolated by extraction with guanidine thiocyanate and separated on formaldehyde-agarose gels, and RNA blot analysis was carried out as previously described (12). [32P]DNA probes. 32P-labeled DNA probes were made by the random oligonucleotide primer method (4). The actin gene probe was a 0.7-kb PstI fragment of the murine ,-actin gene. The ATPase gene probe was a pBR322-derived Tetr plasmid containing a 3.7-kb fragment of a rat sodiumpotassium ATPase a1 subunit cDNA (gift from Russell M. Medford, Emory University). Egr-J transcripts were detected with a 1.6-kb BglII fragment of murine Egr-J (kindly provided by V. Sukhatme). The myc probe was a 0.45-kb PstI fragment of the second exon of murine c-myc. The fos probe was a 1.1-kb EcoRI-Sall fragment of murine c-fos, and the JE probe was a 0.750-kb PstI fragment of murine JE. [3lHjthymidine incorporation. Cells (104 per well) were plated into 96-well tissue culture plates with media containing 0, 0.5, 1, or 10% BCS and 5 ,uCi of [3H]thymidine per ml
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([3H]thymidine stock; specific activity, 18.7 Ci/mmol; New England Nuclear, Boston, Mass.) and incubated at 37°C. After 48 h, labeling medium was removed, wells were washed twice with cold phosphate-buffered saline, and 100 ,ul of 0. 1% Nonidet P-40 was added per well to lyse the cells. Nucleic acids were harvested onto glass fiber filters, and incorporated thymidine was quantitated by scintillation counting. Detection of PDGF-stinulated PDGF receptor autophosphorylation. Confluent 60-mm wells of BALB/c-3T3, IRB 2, and IRB 5 cells were metabolically labeled for 4 h by incubation in methionine-free medium containing 5% dialyzed serum and 100 ,uCi of [35S]methionine (1,000 Ci/mmol; New England Nuclear) per ml. One well of each cell type was also treated with 1,000 U of IFN-,B per ml for 8.5 h, beginning 4.5 h prior to [35S]methionine addition. Labeling medium was aspirated, and the cells were washed once with 37°C phosphate-buffered saline and once with 37°C isotonic solution (120 mM KCl, 30 mM NaCl, 2 mM MnCl2, 10 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES; pH 7.4]). Cells were then overlaid with 1.5 ml of isotonic solution with or without PDGF-BB at 10 ng/ml (Amgen) and incubated at room temperature on a rotary shaker for 5 min. Cell lysis and antiphosphotyrosine immunoprecipitation were carried out as previously described (17). The immunoprecipitated proteins were separated on a 5 to 15% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel, which was treated with an autoradiographic enhancer (Entensify; New England Nuclear), dried, and exposed to film. Alternatively, PDGF receptor phosphorylation in response to PDGF was assayed by using digitonin permeabilization and labeling with [_y-32P]ATP as previously described (17). Phosphatidylinositol-3-kinase assay. Confluent monolayers of cells were made quiescent by 24 h of incubation in medium containing 0.5% BCS. PDGF (20 ng/ml; BB homodimer; Amgen) was added, and incubation continued at 37°C for 1 h. Preparation of cytoplasmic extracts and assay of phosphatidylinositol-3-kinase were carried out as previously described (23). Measurement of calcium mobilization. Confluent monolayers of BALB/c-3T3 and IRB cells were serum deprived (0.5% BCS) overnight. The conditioned media were then aspirated from the plates and reserved. Cells were removed from the tissue culture plates with EDTA, suspended in their respective conditioned media, and incubated for 30 min at 37°C. Cells were then pelleted and resuspended at 106 cells per ml in buffer A (140 mM NaCl, 4 mM KCI, 1.8 mM CaCl2, 0.8 mM Mg2SO4, 1 mM KH2PO4, 10 mM glucose, 2 mM glutamine, 0.1% bovine serum albumin and 10 mM HEPES [pH 7.4]) for 15 to 30 min with 1 ,uM acetoxymethylester of indo-1 (Molecular Probes, Eugene, Oreg.). After a wash in buffer A, cells were resuspended at 5 x 106/ml. Indo-lloaded cells were kept at room temperature until used. For assay of cellular Ca2 , cells were diluted to 5 x 105/ml in buffer A and allowed to equilibrate at 37°C until a stable baseline was obtained. Calcium mobilization in response to PDGF was measured as a function of changes in fluorescence intensity, using an SLM-Aminco (Urbana, Ill.) SPF500 spectrofluorometer with excitation set at 355 nm and emission set at 405 and 480 nm. Sample temperature was maintained at 37°C, and the sample was stirred continuously. RESULTS IRB cells proliferate under low-serum conditions. Incorporation of [3H]thymidine as a measure of mitogenic activity
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Serum Concentration FIG. 1. Serum requirements of IRB cells. BALB/c-3T3, IRB 2, and IRB 5 cells were plated at 104 cells per well in 96-well tissue culture plates with medium containing 0, 0.5, 1, or 10% calf serum and 5 ,uCi of [3H]thymidine per ml (specific activity, 18.7 Ci/mmol). was compared between BALB/c-3T3 and IRB cells under different serum conditions (Fig. 1). All three cell lines showed active DNA synthesis in 10% serum conditions. However, the parental 3T3 cells showed a 25-fold reduction in thymidine incorporation under conditions of 0, 0.5, or 1% serum, while the IRB cells were inhibited only minimally (0 to 0.5-fold) under the same conditions. This finding suggested that the IRB cells were largely growth factor independent. Induction of c-myc and JE mRNA levels by serum but not by PDGF in IRB cells. Confluent cells were rendered quiescent by 24 h of incubation in 0.5% serum-containing medium and then stimulated with serum or PDGF. Whole cell RNA was analyzed by transfer to nitrocellulose and hybridization for the presence of c-myc and JE mRNA. In the parental cells, both c-myc and JE transcript expression was increased more than 10-fold by either PDGF (Fig. 2, lanes 1 and 2) or serum (not shown). In contrast, PDGF treatment of the IRB clones caused little or no induction of c-myc or JE transcripts (Fig. 2, lanes 3 to 5 and 8 to 10). These genes remained inducible by other factors. For example, treatment of the IRB cells with serum induced c-myc. In five repetitions of this experiment,no induction of these genes by PDGF was observed in the IRB cells. It is noteworthy that basal, unstimulated c-myc and JE mRNA levels were substantially higher in the IRB cells than in the parental BALB/c-3T3 cells. This may be the result of incomplete growth arrest in the IRB cells by starvation in 0.5% serum. (Other potential explanations are discussed below.) In contrast, c-fos basal expression was very low in both the IRB cells and the parental BALB/c-3T3 cells. Yet, in agreement with the findings for c-myc and JE, treatment with PDGF did not result in c-fos expression in IRB cells (Fig. 3), whereas serum treatment producedfos gene induction. IFN-y treatment does not reconstitute c-myc induction by PDGF in IRB cells. Cells resistant to class I IFN (IFN-P and -ar) are sometimes still responsive to class II IFN (IFN--y), and there has been a report that IFN--y is able to act synergistically with IFN-13 to produce an antiviral state in a cell line resistant to both interferons individually (11). Furthermore, interaction between the signaling pathways of the two classes of IFN has been demonstrated in HeLa cells (1, 10) and Daudi cells (8). We speculated, therefore, that if
1 2 3 4 5 6 7 8 9 10 11 12 FIG. 2. Evidence that c-myc or JE expression is not induced in response to PDGF in IRB cells. Total cellular RNA was extracted from BALB/c-3T3 (lanes 1 and 2), IRB 2 (lanes 3 to 7), or IRB 5 (lanes 8 to 12) confluent monolayers after incubation in 0.5% serumcontaining medium for 24 h (BALB/c-3T3 cells) or 48 h (IRB cells) and one of the following treatments: no additions (lanes 1, 3, and 8), PDGF-BB (10 ng/ml) for 60 min (lanes 2, 5, and 10), 10% donor calf serum for 60 min (lanes 4 and 9), IFN--y (1,000 U/ml; specific activity, 1.3 x 106 IU/mg; Genentech, San Francisco, Calif.) for 8 h followed by PDGF (10 ng/ml) for an additional 60 min (lanes 6 and 11), or IFN--y (1,000 U/ml) for 9 h (lanes 7 and 12). Each lane contained 20 p.g of total cellular RNA separated on a 1% agaroseformaldehyde gel. The RNA was transferred to nitrocellulose and hybridized with a 32P-labeled probe specific for c-myc exon 2. The same filter was stripped and reprobed for JE transcript. Ethidium bromide staining of the nitrocellulose filter confirmed equal loading and transfer of RNA (not shown). The autoradiograms are shown.
activation of an IFN signaling pathway is required for PDGF signal transduction and if IFN--y and IFN-P share some portion of their signaling pathways, perhaps PDGF responsiveness in the IFN-p-resistant IRB cells could be reconstituted by addition of exogenous IFN--y. BALB/c-3T3 and IRB cells were therefore treated with PDGF or serum after 1 ATPase
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fos _ egr-1 FIG. 3. Induction of c-fos and Egr-l in IRB cells by PDGF-BB, PDGF-AA, FGF, and EGF. Total cellular RNA was extracted from BALB/c-3T3 (lanes 1 to 6) or IRB 5 (lanes 7 to 12) confluent monolayers after incubation for 24 h in 0.5% serum-containing medium, followed by 30 min of treatment with no additions (lanes 1 and 7), 10% donor calf serum (lanes 2 and 8), 10 ng of PDGF-BB per ml (lanes 3 and 9), 20 ng of PDGF-AA per ml (lanes 4 and 10), 12 ng of EGF per ml (lanes 5 and 11), or 20 ng of FGF per ml (lanes 6 and 12). Each lane contained 20 ,ug of total cellular RNA separated on a 1% agarose-formaldehyde gel. The RNA was transferred to nitrocellulose and simultaneously hybridized with 32P-labeled probes specific for the c-fos and ATPase genes. The same filter was stripped and reprobed for Egr-I transcript. The ATPase gene probe serves as a control for equal loading of RNA and even transfer. The autoradiograms are shown.
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GROWTH FACTOR RESPONSIVENESS AND IFN-P RESISTANCE
pretreatment with recombinant murine IFN-,y, and the level of c-myc and JE transcript was analyzed. We found no evidence that IFN--y could synergize with PDGF for the induction of c-myc in IRB cells (Fig. 2). We cannot rule out an induction of JE by PDGF in the presence of IFN-y, as any such change was obscured by the induction of JE expression by IFN-y alone. Induction of the JE gene by IFN-y has been previously reported in macrophages (16). PDGF-AA, FGF, and EGF, like PDGF-BB, all fail to induce c-fos expression in IRB cells. We next investigated whether the mitogenic signaling defect of the IRB cells affected other growth factors in addition to PDGF-BB. Induction of c-fos was used as a marker for activation of the signal transduction pathway because of its low background levels in unstimulated cells. Although serum, PDGF-BB, PDGF-AA, FGF, and EGF all induced c-fos expression at least 20-fold in the parental BALB/c-3T3 cells, only serum treatment induced any c-fos expression in the IRB cells (Fig. 3). Overexposure of the autoradiogram in Fig. 3 also failed to show any c-fos induction in IRB cells by any factor other than serum. Lack of responsiveness to PDGF is not due to absence of PDGF receptors or defects in the activation of PDGF receptors in IRB cells. There are four general mechanisms which could potentially account for the phenotype observed, i.e., a block in the induction of multiple growth-related genes by many different growth factors: (i) loss of growth factor receptors on the cell surface; (ii) transformation by ras; (iii) expression of an autocrine inhibitor of early growth-related gene expression; and (iv) a defect in a second messenger system. The presence of functional PDGF receptors on the surface of the IRB cells was determined by assaying for PDGF receptor autophosphorylation in IRB cells. The experiment was performed by metabolic labeling of the cells with [35S] methionine, stimulation with PDGF, and immunoprecipitation with antiphosphotyrosine antibody. The data from [35S] methionine labeling are presented in Fig. 4. Both IRB clones demonstrate receptor autophosphorylation in response to PDGF comparable to that seen in their parental cells and low or undetectable basal levels of receptor kinase activation. As a control, pretreatment of BALB/c-3T3 cells for 8 h with 800 U of IFN-, per ml did not prevent or reduce PDGF activation of its receptor (Fig. 4, lanes 3, 6, and 9). An alternative assay for PDGF receptor autophosphorylation, using antiphosphotyrosine immunoprecipitation of cells labeled by digitonin permeabilization in the presence of [y_32p] ATP, gave identical results (data not shown). The labeled 180-kDa band was identified as authentic PDGF receptor by demonstrating immunoprecipitation with anti-PDGF receptor antibody (generous gift of Tom Daniel) in other experiments (not shown). Inspection of the proteins immunoprecipitated by the antiphosphotyrosine antibody in the PDGFstimulated samples also revealed the presence of newly phosphorylated proteins other than the PDGF receptor in both BALB/c-3T3 and IRB cells. Although the identities of these proteins have not been formally proven, their molecular weights and their appearance after PDGF stimulation make it likely that they represent the PDGF receptorassociated second messenger proteins phospholipase C (145 kDa), phosphatidylinositol-3-kinase (85 kDa), Raf-1 (74 kDa), Src (60 kDa), and pp42 (42 kDa). Further evidence that the defect in IRB responsiveness to growth factors is not at the level of the receptor was provided by RNA blot analysis of growth factor induction of another early response gene, Egr-J (Fig. 3). Egr-J has serum induction kinetics similar to those of c-myc (19). In contrast to the other early genes examined, induction of Egr-l mRNA
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FIG. 4. Autophosphorylation of the PDGF receptor in response to PDGF in IRB cells. Confluent 60-mm wells of BALB/c-3T3 (lanes 1 to 3), IRB 2 (lanes 4 to 6), and IRB 5 (lanes 7 to 9) cells were metabolically labeled for 4 h by incubation in methionine-free medium containing 5% dialyzed serum and 100 ,Ci of [35S]methionine (New England Nuclear) per ml. One well of each cell type was also treated with 1,000 U of IFN-P per ml for 8.5 h, beginning 4.5 h prior to [35S]methionine addition (lanes 3, 6, and 9). Labeling medium was removed, and the cells were overlaid with 1.5 ml of isotonic solution with 10 ng of PDGF-BB per ml (lanes 2, 3, 5, 6, 8, and 9) or without treatment (lanes 1, 4, and 7). After 5 min of incubation, the cells were lysed with 1% Triton X-100. Immunoprecipitation was carried out by adsorption to antiphosphotyrosineSepharose beads at 4°C for 4 h. The proteins eluted from the beads were electrophoresed on a 5 to 15% gradient SDS-polyacrylamide gel which was treated with an autoradiographic enhancer (Entensify; New England Nuclear), dried, and exposed to film. The autoradiogram is shown. The PDGF receptor protein has been identified in other studies using a specific antibody (16a, 17), and its position on the autoradiogram is indicated. Sizes on the left are given in kilodaltons.
levels by serum, PDGF-BB, PDGF-AA, FGF, and EGF in IRB cells was found to be identical to that seen in the parental cells, indicating that normal levels of functional receptors for PDGF-BB, PDGF-AA, EGF, and FGF were present on IRB cells. We have previously reported a phenotype in Kirsten or Harvey ras-transformed BALB/c-3T3 fibroblasts similar to the IRB phenotype: (i) decreased serum requirements, (ii) inhibition of PDGF-mediated induction of c-fos, c-myc, and JE (26), and (iii) higher basal (unstimulated) levels of myc and JE expression (unpublished observations). We therefore probed RNA blots from the IRB cells for expression of ras transcripts. No ras mRNA expression above that of the parental 3T3 cells was observed (data not shown). Furthermore, although different in morphologic appearance from their parental cells, IRB cells do not resemble ras-transformed 3T3 cells or any transformed fibroblast cell type that we have observed. They remain adherent, contact inhibited, and polygonal in shape without the characteristic morphology of ras-transformed cells (lla). We and others have reported that the ras-transformed phenotype, including the block in growth factor induction of c-fos, c-myc, and JE, can be at least partially reversed by treatment of the cells with dibutyryl cyclic AMP (14, 16a). Treatment of IRB cells with dibutyryl cyclic AMP had no effect on cell morphology or PDGF induction of c-fos (data not shown). Finally, we have reported that autophosphorylation of tyrosine residues on
3152
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the PDGF receptor in response to PDGF is inhibited in v-ras-containing cells (17), yet PDGF-induced autophosphorylation of the receptor occurs normally in IRB cells. We therefore conclude that the block in growth factor induction of c-fos, c-myc, and JE in IRB cells is not caused by the presence of an activated or overexpressed ras gene. PDGF induction of growth-related genes in IRB cells is not blocked by an autocrine inhibitor. To determine whether an autocrine inhibitor of growth-related gene induction was being produced by the IRB cells, experiments using IRB cell-conditioned media were performed. Confluent plates of BALB/c-3T3 cells were incubated for 24 h with 0.5% serumcontaining medium conditioned by incubation with confluent monolayers of BALB/c-3T3, IRB 2, or IRB 5 cells for 24 h to mimic the conditions present during the assays for growthrelated gene transcripts at the time of PDGF stimulation (described above; Fig. 2 and 3). The IRB-conditioned media had no inhibitory effect on PDGF induction of c-myc or JE in BALB/c-3T3 cells (Fig. 5). On the contrary, IRB-conditioned media alone stimulated expression of these genes, while BALB/c-3T3-conditioned media did not have this effect. Protein kinase C is intact in IRB cells. PDGF receptoractivated second messenger pathways were compared between the parental and IFN-resistant cells. Activation of protein kinase C has been postulated as an essential component of the PDGF signaling pathway which results in the induction of growth-related genes. In BALB/c-3T3 cells, 10% calf serum and direct activation of protein kinase C by phorbol ester (phorbol myristate acetate [PMA] at 100 ng/ml) gave comparable stimulation of c-fos mRNA levels (Fig. 6A, lanes 1 to 3). In IRB 5 cells both serum and phorbol ester also induced c-fos expression, but phorbol ester was less effective than serum and the response to both stimulators was weaker in IRB cells than in the parental cells (Fig. 6A, lanes 4 to 6). Lower phorbol ester responsiveness in the IRB cells than in the parental cells is seen for c-myc and JE induction as well (Fig. 6B), suggesting that protein kinase C may be somewhat down-regulated or inhibited in IRB cells. Phosphoinositide turnover in response to PDGF appears normal in IRB cells. In BALB/c-3T3 cells, PDGF stimulation
6
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myc 1 2 3 4 5 6 FIG. 5. Evidence that IRB cells do not secrete an autocrine inhibitor capable of blocking growth-related gene induction. Whole cell RNA was harvested from confluent dishes of BALB/c-3T3 cells treated for 24 h with conditioned medium from BALB/c-3T3 (lanes 1 and 2), IRB 2 (lanes 3 and 4), or IRB 5 (lanes 5 and 6) cells and then stimulated with PDGF-BB for 1 h (lanes 2, 4, and 6) or left unstimulated (lanes 1, 3, and 5). The RNA (20 p.g) was size fractionated on a 1% formaldehyde-agarose gel, transferred to a nitrocellulose membrane, and hybridized with a c-myc (exon 2)specific probe. The membrane was stripped and reprobed for JE transcripts. The autoradiograms are shown.
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FIG. 6. Evidence that direct stimulation of protein kinase C with PMA induces c-fos, c-myc, and JE in IRB cells. Confluent monolayers of cells were made quiescent by 24 h of incubation in 0.5% serum-containing medium and received one of three treatments for 1 h prior to harvesting of RNA: no additions (lanes 1 and 4 in panel A; lanes 1, 3, and 5 in panel B), 10% serum (lanes 2 and 5 in panel A); or PMA (100 ng/ml) (lanes 3 and 6 in panel A; lanes 2, 4, and 6 in panel B). Lanes in panel A: 1 to 3, parental BALB/c-3T3; lanes 4 to 6, IRB 5. Lanes in panel B: 1 and 2, BALB/c-3T3; 3 and 4, IRB 2; 5 and 6, IRB 5. Total cellular RNA (20 ,ug) was size fractionated on a 1% agarose-formaldehyde gel, transferred to nitrocellulose, and hybridized with 32P-labeled DNA probes specific for the c-fos and ATPase genes (A) or the c-myc and JE genes (B). Autoradiograms are shown.
results in phosphatidylinositol hydrolysis by phospholipase C, activation of phosphatidylinositol-3-kinase activity, and mobilization of calcium in response to the inositol phosphate products of these two enzyme activities (2). A defect in one of these components of inositol phosphate metabolism could potentially result in loss of a second messenger essential to gene induction by PDGF. Consequently, biochemical events resulting from activation of inositol hydrolysis in response to PDGF were analyzed in the IRB cells. Stimulation of phosphatidylinositol-3-kinase activity by PDGF was assayed by measuring phosphorylation of two different exogenous phosphatidylinositol substrates by cellular cytoplasmic extracts from PDGF-stimulated and unstimulated cells in the presence of [y-32PIATP. The results show that IRB 2 and IRB 5 demonstrated normal induction of phosphatidylinositol-3-kinase activity in comparison with the parental cells (Fig. 7). Calcium mobilization in response to PDGF was assessed by indo-1 loading and spectrofluorometry during PDGF stimulation. By this method, mobilization of calcium in IRB 2 and IRB 5 cells was found to be essentially normal in comparison with the BALB/c-3T3 parental line (Fig. 8). DISCUSSION It is well established that IFN allows cells to move out of the cell cycle (into Go) and then prevents them from reentering the growth cycle despite the addition of growth factors
GROWTH FACTOR RESPONSIVENESS AND IFN-P RESISTANCE
VOL . 1 l, 1991
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MOLECULAR AND CELLULAR BIOLOGY, June 1991, p. 3148-3154 0270-7306/91/063148-07$02.00/0 Copyright © 1991, American Society for Microbiology
BALB/c-3T3 Fibroblasts Resistant to Growth Inhibition by Beta Interferon Exhibit Aberrant Platelet-Derived Growth Factor, Epidermal Growth Factor, and Fibroblast Growth Factor Signal Transduction LAURA J. MUNDSCHAU AND DOUGLAS V. FALLER* Division of Pediatric Hematology and Oncology, Dana Farber Cancer Institute, Harvard Medical School, 44 Binney Street, Boston, Massachusetts 02115 Received 13 December 1990/Accepted 11 March 1991
Several lines of evidence now exist to suggest an interaction between the platelet-derived growth factor (PDGF) growth-stimulatory signal transduction pathway and the beta interferon (IFN-j8) growth-inhibitory signal transduction pathway. The most direct examples are inhibition of PDGF-mediated gene induction and mitogenesis by IFN-4 and the effects of activators and inhibitors of the IFN-inducible double-stranded RNAdependent eIF2 kinase on expression of PDGF-inducible genes. To further investigate the nature of this PDGF/ IFN-, interaction, we selected BALB/c-3T3 cells for resistance to growth inhibition by IFN-, and analyzed the phenotypes of resulting clonal lines (called IRB cells) with respect to PDGF signal transduction. Although selected only for IFN resistance, the IRB cells were found to be defective for induction of growth-related genes c-fos, c-myc, and JE in response to PDGF. This block to signal transduction was not due to loss or inactivation of PDGF receptors, as immunoprecipitation of PDGF receptors with antiphosphotyrosine antibodies showed them to be present at equal levels in the BALB/c-3T3 and IRB cells and to be autophosphorylated normally in response to PDGF. Furthermore, treatment with other peptide growth factors (PDGF-AA, fibroblast growth factor, and epidermal growth factor) also failed to induce c-fos, c-myc, or JE expression in IRB cells. All of these growth factors, however, were able to induce another early growth-related gene, Egr-1. The block to signaling was not due to a defect in inositol phosphate metabolism, as PDGF treatment induced normal calcium mobilization and phosphotidylinositol-3-kinase activation in these cells. Activation of protein kinase C by phorbol esters did induce c-fos, c-myc, and JE in IRB cells, indicating that signaling pathways distal to this enzyme remained intact. We have previously shown that IFN-inducible enzyme activities, including doublestranded RNA-dependent eIF2 kinase and 2',5'-oligoadenylate synthetase, are normal in IRB cells. The finding that the induction of multiple growth-related genes by several independent growth factors is inhibited in these IFN-resistant cells suggests that there is a second messenger common to both growth factor and IFN signaling pathways and that this messenger is defective in these cells.
dent eIF2 kinase (15) have profound effects on plateletderived growth factor (PDGF)-inducible genes. Poly(I-C), a double-stranded RNA and activator of this kinase, rapidly induces steady-state mRNA levels of c-fos, c-myc, KC, and JE genes in fibroblasts (7, 25; our unpublished results). Conversely, 2-aminopurine, a guanidine analog and inhibitor of this kinase, prevents induction of transcription of c-fos and c-myc by serum or PDGF (22, 24; our unpublished observations). Given these results, which strongly suggest an interaction of IFN-inducible activities and PDGF-signaling pathways, we isolated cells which are defective in their response to the antiproliferative activity of IFN and analyzed their ability to respond to PDGF and other mitogens. Spontaneous mutants exhibiting IFN resistance were produced by long-term exposure of BALB/c-3T3 fibroblasts to very high doses of IFN-P (2,000 U/ml). We have previously described the phenotype of two of the resultant IFN-resistant BALB/c-3T3 (IRB) clones (called IRB 2 and IRB 5) with respect to IFN responsiveness (lla). Briefly, the IRB cells are resistant to the antiproliferative effect of IFN-,B but were found to retain the ability to establish an antiviral state and to increase class I major histocompatibility complex antigen cell surface expression in response to IFN. Two of the enzyme systems thought to be primarily responsible for producing the antiviral state,
Treatment of cultured cells with anti-interferon (IFN) antibodies has provided evidence that many cell types utilize the antiproliferative activity of IFN to inhibit their own proliferation via an autocrine IFN feedback mechanism (5, 6, 9, 18). The significance of this growth control mechanism is suggested by numerous reports that neutralization of the autocrine IFN or the spontaneous development of resistance to its antiproliferative effects can result in neoplasia or failure to differentiate, both of which may lead to malignancy (3, 9). Since the mechanism by which IFN inhibits cell growth is unknown, it remains to be established whether autocrine IFN has its effect by directly interacting with, and inhibiting, growth-stimulatory signaling pathways or instead by activating a separate growth-inhibitory mechanism, independent of the growth-stimulatory pathways. Most of the evidence accumulated so far has suggested a mechanism of direct interaction of IFN-stimulated machinery with growth factor-induced signaling pathways. For example, alpha IFN (IFN-ao) treatment of proliferating Daudi cells decreases c-myc expression (3) and decreases expression and activation of p34CDC2Hs a putative human cell cycle control protein (21). Furthermore, activators and inhibitors of the IFN-inducible double-stranded RNA-depen*
Corresponding author. 3148
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double-stranded RNA-dependent eIF2 kinase and 2',5'-oligoadenylate synthetase, were found to be inducible and activatable in the IRB cells to levels equal to or greater than those of the IFN-sensitive parental BALB/c-3T3 cells. In the work presented here, we have investigated the effect of the IFN-resistant phenotype of the IRB cells on their ability to respond to PDGF and other growth factors. We demonstrate that the ability of the IRB cells to induce c-fos, c-myc, and JE transcript levels in response to PDGF, fibroblast growth factor (FGF), and epidermal growth factor (EGF) is profoundly inhibited. These results suggest that interaction between the PDGF growth-stimulatory signal and the IFN growth-inhibitory signal occurs because these two pathways have at least one component of their signal transduction machinery in common and that this component is defective in the IRB cells. MATERIALS AND METHODS
Cell culture and selection of IFN-,-resistant lines. Clone A31 of BALB/c-3T3 cells was obtained from the American Tissue Culture Collection, Rockville, Md. IFN-resistant clones were selected by maintaining these cells in Dulbecco modified Eagle medium supplemented with 10% heat-inactivated donor bovine calf serum (BCS) (Hazelton Research Products, Inc., Lenexa, Kans.), 2 mM L-glutamine, 100 U of penicillin per ml, 100 ,ug of streptomycin per ml, and 2,000 U of IFN-, per ml (murine IFN-,; specific activity, 5.3 x 107 IU/mg; Lee Biomolecular Research Products, San Diego, Calif.) until proliferating colonies were apparent (6 to 8 weeks). These colonies were picked and cloned by limiting dilution in the presence of IFN. Two clones, called IRB 2 and IRB 5, were chosen at random for further study. These clones were normally carried in the presence of 800 U of IFN-, per ml; however, their IFN-resistant phenotype remained stable for more than 25 passages whether or not IRB cells were carried in the presence of exogenous IFN. PDGFAA, PDGF-BB, and recombinant EGF were obtained from Amgen (Thousand Oaks, Calif.). Recombinant basic FGF was a generous gift of California Biotechnologies (San Diego, Calif.). Preparation of conditioned media. Confluent 150-mm plates of BALB/c-3T3 or IRB cells were incubated with 0.5% serum-containing medium for 24 to 48 h. After centrifugation to remove particulate matter, this depleted medium was used immediately to treat other cells. RNA blot analysis of total cellular RNA. RNA was isolated by extraction with guanidine thiocyanate and separated on formaldehyde-agarose gels, and RNA blot analysis was carried out as previously described (12). [32P]DNA probes. 32P-labeled DNA probes were made by the random oligonucleotide primer method (4). The actin gene probe was a 0.7-kb PstI fragment of the murine ,-actin gene. The ATPase gene probe was a pBR322-derived Tetr plasmid containing a 3.7-kb fragment of a rat sodiumpotassium ATPase a1 subunit cDNA (gift from Russell M. Medford, Emory University). Egr-J transcripts were detected with a 1.6-kb BglII fragment of murine Egr-J (kindly provided by V. Sukhatme). The myc probe was a 0.45-kb PstI fragment of the second exon of murine c-myc. The fos probe was a 1.1-kb EcoRI-Sall fragment of murine c-fos, and the JE probe was a 0.750-kb PstI fragment of murine JE. [3lHjthymidine incorporation. Cells (104 per well) were plated into 96-well tissue culture plates with media containing 0, 0.5, 1, or 10% BCS and 5 ,uCi of [3H]thymidine per ml
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([3H]thymidine stock; specific activity, 18.7 Ci/mmol; New England Nuclear, Boston, Mass.) and incubated at 37°C. After 48 h, labeling medium was removed, wells were washed twice with cold phosphate-buffered saline, and 100 ,ul of 0. 1% Nonidet P-40 was added per well to lyse the cells. Nucleic acids were harvested onto glass fiber filters, and incorporated thymidine was quantitated by scintillation counting. Detection of PDGF-stinulated PDGF receptor autophosphorylation. Confluent 60-mm wells of BALB/c-3T3, IRB 2, and IRB 5 cells were metabolically labeled for 4 h by incubation in methionine-free medium containing 5% dialyzed serum and 100 ,uCi of [35S]methionine (1,000 Ci/mmol; New England Nuclear) per ml. One well of each cell type was also treated with 1,000 U of IFN-,B per ml for 8.5 h, beginning 4.5 h prior to [35S]methionine addition. Labeling medium was aspirated, and the cells were washed once with 37°C phosphate-buffered saline and once with 37°C isotonic solution (120 mM KCl, 30 mM NaCl, 2 mM MnCl2, 10 mM N-2hydroxyethylpiperazine-N'-2-ethanesulfonic acid [HEPES; pH 7.4]). Cells were then overlaid with 1.5 ml of isotonic solution with or without PDGF-BB at 10 ng/ml (Amgen) and incubated at room temperature on a rotary shaker for 5 min. Cell lysis and antiphosphotyrosine immunoprecipitation were carried out as previously described (17). The immunoprecipitated proteins were separated on a 5 to 15% gradient sodium dodecyl sulfate (SDS)-polyacrylamide gel, which was treated with an autoradiographic enhancer (Entensify; New England Nuclear), dried, and exposed to film. Alternatively, PDGF receptor phosphorylation in response to PDGF was assayed by using digitonin permeabilization and labeling with [_y-32P]ATP as previously described (17). Phosphatidylinositol-3-kinase assay. Confluent monolayers of cells were made quiescent by 24 h of incubation in medium containing 0.5% BCS. PDGF (20 ng/ml; BB homodimer; Amgen) was added, and incubation continued at 37°C for 1 h. Preparation of cytoplasmic extracts and assay of phosphatidylinositol-3-kinase were carried out as previously described (23). Measurement of calcium mobilization. Confluent monolayers of BALB/c-3T3 and IRB cells were serum deprived (0.5% BCS) overnight. The conditioned media were then aspirated from the plates and reserved. Cells were removed from the tissue culture plates with EDTA, suspended in their respective conditioned media, and incubated for 30 min at 37°C. Cells were then pelleted and resuspended at 106 cells per ml in buffer A (140 mM NaCl, 4 mM KCI, 1.8 mM CaCl2, 0.8 mM Mg2SO4, 1 mM KH2PO4, 10 mM glucose, 2 mM glutamine, 0.1% bovine serum albumin and 10 mM HEPES [pH 7.4]) for 15 to 30 min with 1 ,uM acetoxymethylester of indo-1 (Molecular Probes, Eugene, Oreg.). After a wash in buffer A, cells were resuspended at 5 x 106/ml. Indo-lloaded cells were kept at room temperature until used. For assay of cellular Ca2 , cells were diluted to 5 x 105/ml in buffer A and allowed to equilibrate at 37°C until a stable baseline was obtained. Calcium mobilization in response to PDGF was measured as a function of changes in fluorescence intensity, using an SLM-Aminco (Urbana, Ill.) SPF500 spectrofluorometer with excitation set at 355 nm and emission set at 405 and 480 nm. Sample temperature was maintained at 37°C, and the sample was stirred continuously. RESULTS IRB cells proliferate under low-serum conditions. Incorporation of [3H]thymidine as a measure of mitogenic activity
3150
MUNDSCHAU AND FALLER
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Serum Concentration FIG. 1. Serum requirements of IRB cells. BALB/c-3T3, IRB 2, and IRB 5 cells were plated at 104 cells per well in 96-well tissue culture plates with medium containing 0, 0.5, 1, or 10% calf serum and 5 ,uCi of [3H]thymidine per ml (specific activity, 18.7 Ci/mmol). was compared between BALB/c-3T3 and IRB cells under different serum conditions (Fig. 1). All three cell lines showed active DNA synthesis in 10% serum conditions. However, the parental 3T3 cells showed a 25-fold reduction in thymidine incorporation under conditions of 0, 0.5, or 1% serum, while the IRB cells were inhibited only minimally (0 to 0.5-fold) under the same conditions. This finding suggested that the IRB cells were largely growth factor independent. Induction of c-myc and JE mRNA levels by serum but not by PDGF in IRB cells. Confluent cells were rendered quiescent by 24 h of incubation in 0.5% serum-containing medium and then stimulated with serum or PDGF. Whole cell RNA was analyzed by transfer to nitrocellulose and hybridization for the presence of c-myc and JE mRNA. In the parental cells, both c-myc and JE transcript expression was increased more than 10-fold by either PDGF (Fig. 2, lanes 1 and 2) or serum (not shown). In contrast, PDGF treatment of the IRB clones caused little or no induction of c-myc or JE transcripts (Fig. 2, lanes 3 to 5 and 8 to 10). These genes remained inducible by other factors. For example, treatment of the IRB cells with serum induced c-myc. In five repetitions of this experiment,no induction of these genes by PDGF was observed in the IRB cells. It is noteworthy that basal, unstimulated c-myc and JE mRNA levels were substantially higher in the IRB cells than in the parental BALB/c-3T3 cells. This may be the result of incomplete growth arrest in the IRB cells by starvation in 0.5% serum. (Other potential explanations are discussed below.) In contrast, c-fos basal expression was very low in both the IRB cells and the parental BALB/c-3T3 cells. Yet, in agreement with the findings for c-myc and JE, treatment with PDGF did not result in c-fos expression in IRB cells (Fig. 3), whereas serum treatment producedfos gene induction. IFN-y treatment does not reconstitute c-myc induction by PDGF in IRB cells. Cells resistant to class I IFN (IFN-P and -ar) are sometimes still responsive to class II IFN (IFN--y), and there has been a report that IFN--y is able to act synergistically with IFN-13 to produce an antiviral state in a cell line resistant to both interferons individually (11). Furthermore, interaction between the signaling pathways of the two classes of IFN has been demonstrated in HeLa cells (1, 10) and Daudi cells (8). We speculated, therefore, that if
1 2 3 4 5 6 7 8 9 10 11 12 FIG. 2. Evidence that c-myc or JE expression is not induced in response to PDGF in IRB cells. Total cellular RNA was extracted from BALB/c-3T3 (lanes 1 and 2), IRB 2 (lanes 3 to 7), or IRB 5 (lanes 8 to 12) confluent monolayers after incubation in 0.5% serumcontaining medium for 24 h (BALB/c-3T3 cells) or 48 h (IRB cells) and one of the following treatments: no additions (lanes 1, 3, and 8), PDGF-BB (10 ng/ml) for 60 min (lanes 2, 5, and 10), 10% donor calf serum for 60 min (lanes 4 and 9), IFN--y (1,000 U/ml; specific activity, 1.3 x 106 IU/mg; Genentech, San Francisco, Calif.) for 8 h followed by PDGF (10 ng/ml) for an additional 60 min (lanes 6 and 11), or IFN--y (1,000 U/ml) for 9 h (lanes 7 and 12). Each lane contained 20 p.g of total cellular RNA separated on a 1% agaroseformaldehyde gel. The RNA was transferred to nitrocellulose and hybridized with a 32P-labeled probe specific for c-myc exon 2. The same filter was stripped and reprobed for JE transcript. Ethidium bromide staining of the nitrocellulose filter confirmed equal loading and transfer of RNA (not shown). The autoradiograms are shown.
activation of an IFN signaling pathway is required for PDGF signal transduction and if IFN--y and IFN-P share some portion of their signaling pathways, perhaps PDGF responsiveness in the IFN-p-resistant IRB cells could be reconstituted by addition of exogenous IFN--y. BALB/c-3T3 and IRB cells were therefore treated with PDGF or serum after 1 ATPase
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fos _ egr-1 FIG. 3. Induction of c-fos and Egr-l in IRB cells by PDGF-BB, PDGF-AA, FGF, and EGF. Total cellular RNA was extracted from BALB/c-3T3 (lanes 1 to 6) or IRB 5 (lanes 7 to 12) confluent monolayers after incubation for 24 h in 0.5% serum-containing medium, followed by 30 min of treatment with no additions (lanes 1 and 7), 10% donor calf serum (lanes 2 and 8), 10 ng of PDGF-BB per ml (lanes 3 and 9), 20 ng of PDGF-AA per ml (lanes 4 and 10), 12 ng of EGF per ml (lanes 5 and 11), or 20 ng of FGF per ml (lanes 6 and 12). Each lane contained 20 ,ug of total cellular RNA separated on a 1% agarose-formaldehyde gel. The RNA was transferred to nitrocellulose and simultaneously hybridized with 32P-labeled probes specific for the c-fos and ATPase genes. The same filter was stripped and reprobed for Egr-I transcript. The ATPase gene probe serves as a control for equal loading of RNA and even transfer. The autoradiograms are shown.
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GROWTH FACTOR RESPONSIVENESS AND IFN-P RESISTANCE
pretreatment with recombinant murine IFN-,y, and the level of c-myc and JE transcript was analyzed. We found no evidence that IFN--y could synergize with PDGF for the induction of c-myc in IRB cells (Fig. 2). We cannot rule out an induction of JE by PDGF in the presence of IFN-y, as any such change was obscured by the induction of JE expression by IFN-y alone. Induction of the JE gene by IFN-y has been previously reported in macrophages (16). PDGF-AA, FGF, and EGF, like PDGF-BB, all fail to induce c-fos expression in IRB cells. We next investigated whether the mitogenic signaling defect of the IRB cells affected other growth factors in addition to PDGF-BB. Induction of c-fos was used as a marker for activation of the signal transduction pathway because of its low background levels in unstimulated cells. Although serum, PDGF-BB, PDGF-AA, FGF, and EGF all induced c-fos expression at least 20-fold in the parental BALB/c-3T3 cells, only serum treatment induced any c-fos expression in the IRB cells (Fig. 3). Overexposure of the autoradiogram in Fig. 3 also failed to show any c-fos induction in IRB cells by any factor other than serum. Lack of responsiveness to PDGF is not due to absence of PDGF receptors or defects in the activation of PDGF receptors in IRB cells. There are four general mechanisms which could potentially account for the phenotype observed, i.e., a block in the induction of multiple growth-related genes by many different growth factors: (i) loss of growth factor receptors on the cell surface; (ii) transformation by ras; (iii) expression of an autocrine inhibitor of early growth-related gene expression; and (iv) a defect in a second messenger system. The presence of functional PDGF receptors on the surface of the IRB cells was determined by assaying for PDGF receptor autophosphorylation in IRB cells. The experiment was performed by metabolic labeling of the cells with [35S] methionine, stimulation with PDGF, and immunoprecipitation with antiphosphotyrosine antibody. The data from [35S] methionine labeling are presented in Fig. 4. Both IRB clones demonstrate receptor autophosphorylation in response to PDGF comparable to that seen in their parental cells and low or undetectable basal levels of receptor kinase activation. As a control, pretreatment of BALB/c-3T3 cells for 8 h with 800 U of IFN-, per ml did not prevent or reduce PDGF activation of its receptor (Fig. 4, lanes 3, 6, and 9). An alternative assay for PDGF receptor autophosphorylation, using antiphosphotyrosine immunoprecipitation of cells labeled by digitonin permeabilization in the presence of [y_32p] ATP, gave identical results (data not shown). The labeled 180-kDa band was identified as authentic PDGF receptor by demonstrating immunoprecipitation with anti-PDGF receptor antibody (generous gift of Tom Daniel) in other experiments (not shown). Inspection of the proteins immunoprecipitated by the antiphosphotyrosine antibody in the PDGFstimulated samples also revealed the presence of newly phosphorylated proteins other than the PDGF receptor in both BALB/c-3T3 and IRB cells. Although the identities of these proteins have not been formally proven, their molecular weights and their appearance after PDGF stimulation make it likely that they represent the PDGF receptorassociated second messenger proteins phospholipase C (145 kDa), phosphatidylinositol-3-kinase (85 kDa), Raf-1 (74 kDa), Src (60 kDa), and pp42 (42 kDa). Further evidence that the defect in IRB responsiveness to growth factors is not at the level of the receptor was provided by RNA blot analysis of growth factor induction of another early response gene, Egr-J (Fig. 3). Egr-J has serum induction kinetics similar to those of c-myc (19). In contrast to the other early genes examined, induction of Egr-l mRNA
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FIG. 4. Autophosphorylation of the PDGF receptor in response to PDGF in IRB cells. Confluent 60-mm wells of BALB/c-3T3 (lanes 1 to 3), IRB 2 (lanes 4 to 6), and IRB 5 (lanes 7 to 9) cells were metabolically labeled for 4 h by incubation in methionine-free medium containing 5% dialyzed serum and 100 ,Ci of [35S]methionine (New England Nuclear) per ml. One well of each cell type was also treated with 1,000 U of IFN-P per ml for 8.5 h, beginning 4.5 h prior to [35S]methionine addition (lanes 3, 6, and 9). Labeling medium was removed, and the cells were overlaid with 1.5 ml of isotonic solution with 10 ng of PDGF-BB per ml (lanes 2, 3, 5, 6, 8, and 9) or without treatment (lanes 1, 4, and 7). After 5 min of incubation, the cells were lysed with 1% Triton X-100. Immunoprecipitation was carried out by adsorption to antiphosphotyrosineSepharose beads at 4°C for 4 h. The proteins eluted from the beads were electrophoresed on a 5 to 15% gradient SDS-polyacrylamide gel which was treated with an autoradiographic enhancer (Entensify; New England Nuclear), dried, and exposed to film. The autoradiogram is shown. The PDGF receptor protein has been identified in other studies using a specific antibody (16a, 17), and its position on the autoradiogram is indicated. Sizes on the left are given in kilodaltons.
levels by serum, PDGF-BB, PDGF-AA, FGF, and EGF in IRB cells was found to be identical to that seen in the parental cells, indicating that normal levels of functional receptors for PDGF-BB, PDGF-AA, EGF, and FGF were present on IRB cells. We have previously reported a phenotype in Kirsten or Harvey ras-transformed BALB/c-3T3 fibroblasts similar to the IRB phenotype: (i) decreased serum requirements, (ii) inhibition of PDGF-mediated induction of c-fos, c-myc, and JE (26), and (iii) higher basal (unstimulated) levels of myc and JE expression (unpublished observations). We therefore probed RNA blots from the IRB cells for expression of ras transcripts. No ras mRNA expression above that of the parental 3T3 cells was observed (data not shown). Furthermore, although different in morphologic appearance from their parental cells, IRB cells do not resemble ras-transformed 3T3 cells or any transformed fibroblast cell type that we have observed. They remain adherent, contact inhibited, and polygonal in shape without the characteristic morphology of ras-transformed cells (lla). We and others have reported that the ras-transformed phenotype, including the block in growth factor induction of c-fos, c-myc, and JE, can be at least partially reversed by treatment of the cells with dibutyryl cyclic AMP (14, 16a). Treatment of IRB cells with dibutyryl cyclic AMP had no effect on cell morphology or PDGF induction of c-fos (data not shown). Finally, we have reported that autophosphorylation of tyrosine residues on
3152
MOL. CELL. BIOL.
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the PDGF receptor in response to PDGF is inhibited in v-ras-containing cells (17), yet PDGF-induced autophosphorylation of the receptor occurs normally in IRB cells. We therefore conclude that the block in growth factor induction of c-fos, c-myc, and JE in IRB cells is not caused by the presence of an activated or overexpressed ras gene. PDGF induction of growth-related genes in IRB cells is not blocked by an autocrine inhibitor. To determine whether an autocrine inhibitor of growth-related gene induction was being produced by the IRB cells, experiments using IRB cell-conditioned media were performed. Confluent plates of BALB/c-3T3 cells were incubated for 24 h with 0.5% serumcontaining medium conditioned by incubation with confluent monolayers of BALB/c-3T3, IRB 2, or IRB 5 cells for 24 h to mimic the conditions present during the assays for growthrelated gene transcripts at the time of PDGF stimulation (described above; Fig. 2 and 3). The IRB-conditioned media had no inhibitory effect on PDGF induction of c-myc or JE in BALB/c-3T3 cells (Fig. 5). On the contrary, IRB-conditioned media alone stimulated expression of these genes, while BALB/c-3T3-conditioned media did not have this effect. Protein kinase C is intact in IRB cells. PDGF receptoractivated second messenger pathways were compared between the parental and IFN-resistant cells. Activation of protein kinase C has been postulated as an essential component of the PDGF signaling pathway which results in the induction of growth-related genes. In BALB/c-3T3 cells, 10% calf serum and direct activation of protein kinase C by phorbol ester (phorbol myristate acetate [PMA] at 100 ng/ml) gave comparable stimulation of c-fos mRNA levels (Fig. 6A, lanes 1 to 3). In IRB 5 cells both serum and phorbol ester also induced c-fos expression, but phorbol ester was less effective than serum and the response to both stimulators was weaker in IRB cells than in the parental cells (Fig. 6A, lanes 4 to 6). Lower phorbol ester responsiveness in the IRB cells than in the parental cells is seen for c-myc and JE induction as well (Fig. 6B), suggesting that protein kinase C may be somewhat down-regulated or inhibited in IRB cells. Phosphoinositide turnover in response to PDGF appears normal in IRB cells. In BALB/c-3T3 cells, PDGF stimulation
6
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myc 1 2 3 4 5 6 FIG. 5. Evidence that IRB cells do not secrete an autocrine inhibitor capable of blocking growth-related gene induction. Whole cell RNA was harvested from confluent dishes of BALB/c-3T3 cells treated for 24 h with conditioned medium from BALB/c-3T3 (lanes 1 and 2), IRB 2 (lanes 3 and 4), or IRB 5 (lanes 5 and 6) cells and then stimulated with PDGF-BB for 1 h (lanes 2, 4, and 6) or left unstimulated (lanes 1, 3, and 5). The RNA (20 p.g) was size fractionated on a 1% formaldehyde-agarose gel, transferred to a nitrocellulose membrane, and hybridized with a c-myc (exon 2)specific probe. The membrane was stripped and reprobed for JE transcripts. The autoradiograms are shown.
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FIG. 6. Evidence that direct stimulation of protein kinase C with PMA induces c-fos, c-myc, and JE in IRB cells. Confluent monolayers of cells were made quiescent by 24 h of incubation in 0.5% serum-containing medium and received one of three treatments for 1 h prior to harvesting of RNA: no additions (lanes 1 and 4 in panel A; lanes 1, 3, and 5 in panel B), 10% serum (lanes 2 and 5 in panel A); or PMA (100 ng/ml) (lanes 3 and 6 in panel A; lanes 2, 4, and 6 in panel B). Lanes in panel A: 1 to 3, parental BALB/c-3T3; lanes 4 to 6, IRB 5. Lanes in panel B: 1 and 2, BALB/c-3T3; 3 and 4, IRB 2; 5 and 6, IRB 5. Total cellular RNA (20 ,ug) was size fractionated on a 1% agarose-formaldehyde gel, transferred to nitrocellulose, and hybridized with 32P-labeled DNA probes specific for the c-fos and ATPase genes (A) or the c-myc and JE genes (B). Autoradiograms are shown.
results in phosphatidylinositol hydrolysis by phospholipase C, activation of phosphatidylinositol-3-kinase activity, and mobilization of calcium in response to the inositol phosphate products of these two enzyme activities (2). A defect in one of these components of inositol phosphate metabolism could potentially result in loss of a second messenger essential to gene induction by PDGF. Consequently, biochemical events resulting from activation of inositol hydrolysis in response to PDGF were analyzed in the IRB cells. Stimulation of phosphatidylinositol-3-kinase activity by PDGF was assayed by measuring phosphorylation of two different exogenous phosphatidylinositol substrates by cellular cytoplasmic extracts from PDGF-stimulated and unstimulated cells in the presence of [y-32PIATP. The results show that IRB 2 and IRB 5 demonstrated normal induction of phosphatidylinositol-3-kinase activity in comparison with the parental cells (Fig. 7). Calcium mobilization in response to PDGF was assessed by indo-1 loading and spectrofluorometry during PDGF stimulation. By this method, mobilization of calcium in IRB 2 and IRB 5 cells was found to be essentially normal in comparison with the BALB/c-3T3 parental line (Fig. 8). DISCUSSION It is well established that IFN allows cells to move out of the cell cycle (into Go) and then prevents them from reentering the growth cycle despite the addition of growth factors
GROWTH FACTOR RESPONSIVENESS AND IFN-P RESISTANCE
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