A single amino acid substitution converts a transmembrane protein activator of the platelet-derived growth factor ß receptor into an inhibitor.

Receptors for PDGF play an important role in cell proliferation and migration and have been implicated in certain cancers. The 44-amino acid E5 protein of bovine papillomavirus binds to and activates the PDGFß receptor (PDGFßR), resulting in oncogenic transformation of cultured fibroblasts. Previously, we isolated an artificial 36-amino acid transmembrane protein, pTM36-4, which transforms cells because of its ability to activate the PDGFßR despite limited sequence similarity to E5. Here, we demonstrated complex formation between the PDGFßR and three pTM36-4 mutants: T21E, T21Q, and T21N. T21Q retained wild type transforming activity and activated the PDGFßR in a ligand-independent manner as a consequence of binding to the transmembrane domain of the PDGFßR, but T21E and T21N were severely defective. In fact, T21N substantially inhibited E5-induced PDGFßR activation and transformation in both mouse and human fibroblasts. T21N did not prevent E5 from binding to the receptor, and genetic evidence suggested that T21N and E5 bind to nonidentical sites in the transmembrane domain of the receptor. T21N also inhibited transformation and PDGFßR activation induced by v-Sis, a viral homologue of PDGF-BB, as well as PDGF-induced mitogenesis and signaling by preventing phosphorylation of the PDGFßR at particular tyrosine residues. These results demonstrated that T21N acts as a novel inhibitor of the PDGFßR and validated a new strategy for designing highly specific short transmembrane protein inhibitors of growth factor receptors and possibly other transmembrane proteins.

Autocrine PDGF stimulation in malignancies.

Platelet-derived growth factor (PDGF) isoforms are important mitogens for different types of mesenchymal cells, which have important functions during the embryonal development and in the adult during wound healing and tissue homeostasis. In tumors, PDGF isoforms are often over-expressed and contribute to the growth of both normal and malignant cells. This review focuses on tumors expressing PDGF isoforms together with their tyrosine kinase receptors, thus resulting in autocrine stimulation of growth and survival. Patients with such tumors could benefit from treatment with inhibitors of either PDGF or PDGF receptors.

Distinct clinical characteristics between patients with nonerosive reflux disease and those with reflux esophagitis.

BACKGROUND & AIMS: It has been postulated that nonerosive reflux disease (NERD) and erosive reflux disease (ERD) are 2 distinct entities of gastroesophageal reflux disease. The aim of this study was to compare the clinical characteristics between patients with NERD and those with ERD. METHODS: We prospectively recruited consecutive patients presenting with weekly attacks of heartburn or acid regurgitation. Exclusion criteria included gastric surgery, recent use of nonsteroidal anti-inflammatory drug or proton pump inhibitor, and peptic ulcer disease. Concomitant functional dyspepsia, irritable bowel syndrome, and psychological disorders were documented. Endoscopy, esophageal manometry, acid perfusion test, and 24-hour ambulatory pH monitoring were performed. Risk factors of NERD were determined by multivariate analysis. RESULTS: Two hundred fourteen patients (NERD, 113; ERD, 111) were studied. NERD patients were characterized by higher prevalence of Helicobacter pylori (36.3% vs 18%, P = .005), functional dyspepsia (64.6% vs 42.3%, P = .003), irritable bowel syndrome (44.2% vs 15.3%, P < .001), psychological disorders (9% vs 0.9%, P = .04), and positive acid perfusion test (40.7% vs 19.8%, P = .004). ERD patients had more hiatal hernias (35.1% vs 17.1%, P = .009), higher esophageal acid exposure (total time esophageal pH <4, 4.2% +/- 2.1% vs 5.9% +/- 2.3%; P = .01), and esophageal dysmotility (P < .05). With multivariate analysis, H pylori (odds ratio, 1.8; 95% confidence interval [CI], 1.1-3.2), irritable bowel syndrome (odds ratio, 2.8; 95% CI, 1.6-5.3), and positive acid perfusion test (odds ratio, 1.9; 95% CI, 1.4-2.8) were independent risk factors for NERD. CONCLUSIONS: Patients with NERD and ERD have distinct differences in clinical characteristics. NERD is characterized by higher prevalence of functional gastrointestinal disorders and esophageal acid hypersensitivity.

IL-27 subunits and its receptor (WSX-1) mRNAs are markedly up-regulated in inflammatory cells in the CNS during experimental autoimmune encephalomyelitis.

IL-27 (EBI3p28) is a recently discovered heterodimeric cytokine, which is functionally related to IL-23p40p19 and IL-12p40p35. IL-27 acts in synergy with IL-12 early during Th1 development from naive T cells. IL-27 functions through the WSX-1 and the gpl30 receptor subunits, which shares homology with the IL-12Rbeta2 subunit. We have previously reported that IL-23 is up-regulated in CD11b+ microglia/macrophages in the CNS during the early phase of experimental autoimmune encephalomyelitis (EAE), and thus may contribute to the early induction of EAE. In the present study, we examined the expression of IL-27 and its receptor in the CNS, spleen, and lymph nodes at different stages of EAE actively induced with myelin oligodendrocyte glycoprotein peptide(35-55). Our findings show that IL-27 EBI3 and p28 mRNA were up-regulated to a maximum level at the peak of disease in APC from the CNS and lymph nodes, but not in the spleen. Moreover, IL-27 receptor (WSX-1) expression was greatly up-regulated during the early stage of EAE in both the CNS and lymph nodes. Taken together, our data show that subunits of IL-27 and its receptor (WSX-1) mRNAs are markedly up-regulated in inflammatory cells in the CNS at the peak of disease. Thus, IL-27 produced by infiltrating cells in the CNS may regulate in a paracrine manner the Th1 response in EAE.

Regulatory mechanisms for the expression and activity of platelet-derived growth factor receptor.

PDGF is one of the most potent serum mitogens, and the signalling mechanism by way of its receptor tyrosine-kinase has been extensively studied since its first purification in 1979. The identification of homology between the simian sarcoma virus oncogene, v-sis, and the B-chain of PDGF, as well as the frequent over-expression of both the ligands and receptors in various tumours and stroma led to the proposal of the PDGF-mediated autocrine and paracrine hypothesis. Consistent with the important roles of PDGF in the growth and survival of cells, the expression and activity of PDGF receptors are tightly controlled by both positive and negative feedback mechanisms at different levels. The deregulation of the control system can result in serious pathological conditions such as chronic inflammation and tumours. Understanding the molecular mechanisms for the regulatory system and the signalling pathway of PDGF is essential in order to find effective therapies in the diseases where PDGF is involved.

Cloning, expression, characterization, and role in autocrine cell growth of cell surface retention sequence binding protein-1.

Cell surface retention sequence binding protein-1 (CRSBP-1) is a cell surface binding protein for the cell surface retention sequence (CRS) motif of the v-sis gene product (platelet-derived growth factor-BB). It has been shown to be responsible for cell surface retention of the v-sis gene product in v-sis-transformed cells (fibroblasts) and has been hypothesized to play a role in autocrine growth and transformation of these cells. Here we demonstrate that the CRSBP-1 cDNA cloned from bovine liver libraries encodes a 322-residue type I membrane protein containing a 23-residue signal peptide, a 215-residue cell surface domain, a 21-residue transmembrane domain, and a 63-residue cytoplasmic domain. CRSBP-1 expressed in transfected cells is an approximately 120-kDa disulfide-linked homodimeric glycoprotein and exhibits dual ligand (CRS-containing growth regulators (v-sis gene product and insulin-like growth factor binding protein-3, IGFBP-3) and hyaluronic acid) binding activity. CRSBP-1 overexpression (by stable transfection of cells with CRSBP-1 cDNA) enhances autocrine loop signaling, cell growth, and tumorigenicity (in mice) of v-sis-transformed cells. CRSBP-1 expression also enhances autocrine cell growth mediated by IGFBP-3 in human lung carcinoma cells (H1299 cells), which express very little, if any, endogenous CRSBP-1 and exhibits a mitogenic response to exogenous IGFBP-3, stably transfected with IGFBP-3 cDNA. However, CRSBP-1 overexpression does not affect growth of normal and transformed cells that do not produce these CRS-containing growth regulators. These results suggest that CRSBP-1 plays a role in autocrine regulation of cell growth mediated by growth regulators containing CRS.

Overexpression of the sis oncogene in a canine osteosarcoma cell line.

Specific oncogenes that contribute to the pathogenesis of canine osteosarcoma (OS) have not been identified. In the process of characterizing four OS cell lines, we have found one cell line, CO8, that overexpresses the sis oncogene, which encodes the platelet-derived growth factor (PDGF)-beta. The expression of an important downstream transcriptional target of the PDGF signaling pathway, c-myc, is also elevated fourfold. Conditioned medium from CO8 alone specifically induces tyrosine phosphorylation and therefore the activation of the PDGF-alpha and PDGF-beta receptors on murine 3T3 cells. All of the canine OS lines tested contain PDGF receptors and therefore are capable of responding to PDGE Given the importance of PDGF in promoting cell proliferation, migration, and cell survival, the activation of the sis oncogene and the resultant growth factor autocrine loop potentially contribute to the pathogenesis of a subset of canine osteosarcomas.

Epigallocathechin-3 gallate selectively inhibits the PDGF-BB-induced intracellular signaling transduction pathway in vascular smooth muscle cells and inhibits transformation of sis-transfected NIH 3T3 fibroblasts and human glioblastoma cells (A172).

Enhanced activity of receptor tyrosine kinases such as the PDGF beta-receptor and EGF receptor has been implicated as a contributing factor in the development of malignant and nonmalignant proliferative diseases such as cancer and atherosclerosis. Several epidemiological studies suggest that green tea may prevent the development of cancer and atherosclerosis. One of the major constituents of green tea is the polyphenol epigallocathechin-3 gallate (EGCG). In an attempt to offer a possible explanation for the anti-cancer and anti-atherosclerotic activity of EGCG, we examined the effect of EGCG on the PDGF-BB-, EGF-, angiotensin II-, and FCS-induced activation of the 44 kDa and 42 kDa mitogen-activated protein (MAP) kinase isoforms (p44(mapk)/p42(mapk)) in cultured vascular smooth muscle cells (VSMCs) from rat aorta. VSMCs were treated with EGCG (1-100 microM) for 24 h and stimulated with the above mentioned agonists for different time periods. Stimulation of the p44(mapk)/p42(mapk) was detected by the enhanced Western blotting method using phospho-specific MAP kinase antibodies that recognized the Tyr204-phosphorylated (active) isoforms. Treatment of VSMCs with 10 and 50 microM EGCG resulted in an 80% and a complete inhibition of the PDGF-BB-induced activation of MAP kinase isoforms, respectively. In striking contrast, EGCG (1-100 microM) did not influence MAP kinase activation by EGF, angiotensin II, and FCS. Similarly, the maximal effect of PDGF-BB on the c-fos and egr-1 mRNA expression as well as on intracellular free Ca2+ concentration was completely inhibited in EGCG-treated VSMCs, whereas the effect of EGF was not affected. Quantification of the immunoprecipitated tyrosine-phosphorylated PDGF-Rbeta, phosphatidylinositol 3'-kinase, and phospholipase C-gamma1 by the enhanced Western blotting method revealed that EGCG treatment effectively inhibits tyrosine phosphorylation of these kinases in VSMCs. Furthermore, we show that spheroid formation of human glioblastoma cells (A172) and colony formation of sis-transfected NIH 3T3 cells in semisolid agar are completely inhibited by 20-50 microM EGCG. Our findings demonstrate that EGCG is a selective inhibitor of the tyrosine phosphorylation of PDGF-Rbeta and its downstream signaling pathway. The present findings may partly explain the anti-cancer and anti-atherosclerotic activity of green tea.

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.

Suramin enters and accumulates in low pH intracellular compartments of v-sis-transformed NIH 3T3 cells.

Using acridine orange as a reporter compound, we demonstrate that suramin enters and accumulates in low pH intracellular compartments (endosomes, lysosomes, and trans-Golgi complex) of normal and v-sis-transformed NIH 3T3 cells. The concentration of suramin in these acidic compartments is estimated to be > 150 microM, higher than the concentration known to completely inhibit interaction of the platelet-derived growth factor (PDGF) receptor and v-sis gene product. These results support the hypothesis that suramin reverses the transformed phenotype of v-sis-transformed cells by entering the cell via endocytosis and blocking interaction of the v-sis gene product and PDGF receptor in intracellular organelles.

Identification of amino acids in the transmembrane and juxtamembrane domains of the platelet-derived growth factor receptor required for productive interaction with the bovine papillomavirus E5 protein.

The bovine papillomavirus E5 protein forms a stable complex with the cellular platelet-derived growth factor (PDGF) beta receptor, resulting in receptor activation and cell transformation. Amino acids in both the putative transmembrane domain and extracytoplasmic carboxyl-terminal domain of the E5 protein appear important for PDGF receptor binding and activation. Previous analysis indicated that the transmembrane domain of the receptor was also required for complex formation and receptor activation. Here we analyzed receptor chimeras and point mutants to identify specific amino acids in the PDGF beta receptor required for productive interaction with the E5 protein. These receptor mutants were analyzed in murine Ba/F3 cells, which do not express endogenous receptor. Our results confirmed the importance of the transmembrane domain of the receptor for complex formation, receptor tyrosine phosphorylation, and mitogenic signaling in response to the E5 protein and established that the threonine residue in this domain is required for these activities. In addition, a positive charge in the extracellular juxtamembrane domain of the receptor was required for E5 interaction and signaling, whereas replacement of the wild-type lysine with either a neutral or acidic amino acid inhibited E5-induced receptor activation and transformation. All of the receptor mutants defective for activation by the E5 protein responded to acute treatment with PDGF and to stable expression of v-Sis, a form of PDGF. The required juxtamembrane lysine and transmembrane threonine are predicted to align precisely on the same face of an alpha helix packed in a left-handed coiled-coil geometry. These results establish that the E5 protein and v-Sis recognize distinct binding sites on the PDGF beta receptor and further clarify the nature of the interaction between the viral transforming protein and its cellular target.

Counter-selection for over-expressed human CD44s in primary tumors versus lung metastases in a mouse fibrosarcoma model.

Human CD44 standard isoform cDNA (hCD44s) was transfected into sis-transformed Balb/c 3T3 cells and into ras-revertant IIIA4 cells (both tumorigenic but nonmetastatic). Transfectants were injected subcutaneously into athymic nude mice to elucidate the functional role of hCD44s over-expression in progression and metastasis. The transfectants (but not parental cells) were capable of lung micrometastasis and of binding exogenously-added hyaluronan. hCD44s protein expression was conserved in lung micrometastases suggesting that it may have been necessary for their formation. In contrast, no hCD44s protein was detected in large subcutaneous (s.c.) tumors but normal levels of murine CD44 were detected. A second round of tumor development, using these two tumor cell classes, demonstrated that hCD44s-nonexpressing s.c. tumor cells re-expressed it in lung micrometastases. Conversely, hCD44s-expressing lung micrometastatic cells, when injected into a second group of mice, downregulated hCD44s expression in order to grow sizable s.c. tumors. S.c. tumor cells still contained the hCD44s gene but its expression was inhibited by epigenetic mechanisms, one of which was shown to be methylation of the hCD44s gene. These studies demonstrate (a) opposing selective pressures on CD44s over-expression for s.c. tumor growth and for metastatic spread to the lung and (b) further credence for the significance of CD44 for metastatic spread of fibrosarcomas. Therefore, CD44s may be a critical component of the metastatic phenotype induced by specific oncogenes.

Platelet-derived growth factor (PDGF)-signaling mediates radiation-induced apoptosis in human prostate cancer cells with loss of p53 function.

Platelet-derived growth factor (PDGF) signals a diversity of cellular responses in vitro, including cell proliferation, survival, transformation, and chemotaxis. PDGF functions as a "competence factor" to induce a set of early response genes expressed in G1 including p21WAF1/CIP1, a functional mediator of the tumor suppressor gene p53 in G1/S checkpoint. For PDGF-stimulated cells to progress beyond G1 and transit the cell cycle completely, progression factors in serum such as insulin and IGF-1 are required. We have recently shown a novel role of PDGF in inducing apoptosis in growth-arrested murine fibroblasts. The PDGF-induced apoptosis is rescued by insulin, suggesting that G1/S checkpoint is a critical determinant for PDGF-induced apoptosis. Because recent studies suggest that radiation-induced signal transduction pathways interact with growth factor-mediated signaling pathways, we have investigated whether activation of the PDGF-signaling facilitates the radiation-induced apoptosis in the absence of functional p53. For this study we have used the 125-IL cell line, a mutant p53-containing, highly metastatic, and hormone-unresponsive human prostate carcinoma cell line. PDGF signaling is constitutively activated by transfection with a p28v-sis expression vector, which was previously shown to activate PDGF alpha- and beta- receptors. Although the basal level of p21WAF1/CIP1 expression and radiation-induced apoptosis were not detectable in control 125-IL cells as would be predicted in mutant p53-containing cells, activation of PDGF-signaling induced expression of p21WAF1/CIP1 and radiation-induced apoptosis. Our study suggests that the level of "competence" growth factors including PDGF may be one of the critical determinants for radiation-induced apoptosis, especially in cells with loss of p53 function at the site of radiotherapy in vivo.

Platelet-derived growth factor-B/v-sis confers a tumorigenic and metastatic phenotype to human T98G glioblastoma cells.

Autocrine stimulation by platelet-derived growth factor-B (PDGF-B)-like factors has been widely implicated as an important mechanism in the cause and/or maintenance of a variety of human tumors. However, normal human cells appear to be resistant to transformation by PDGF-B-like molecules, and a direct demonstration of the tumor-promoting or tumor-maintaining property of a PDGF-B autocrine system is lacking. T98G human glioblastoma cells are nontumorigenic in athymic mice. We show that these cells express predominantly PDGF-beta type receptors and continuously secrete small amount of PDGF-B/c-sis. Addition of suramin or specific anti-PDGF-B/v-sis antibody inhibits proliferation in culture. Conversely, multiple clonal lines that stably overexpress PDGF-B/v-sis (T98Gsis cells) exhibit a striking 200-250% increased proliferation rate and an enhanced colony-forming frequency in soft agar. Clonal lines with stable expression of PDGF-B/v-sis (T98Gsis cells) reliably (80%) develop tumors in 4-6 weeks, whereas the empty-vector control cells are nontumorigenic. Moreover, in some cases, T98Gsis cells disseminate to form bilateral and multifocal pulmonary metastases. The results show that T98G cells contain functional PDGF receptors that, upon sufficient stimulation, can cause greatly increased mitogenic response, which may account for the development of the malignant phenotype. Metastatic tumor formation in athymic mice by PDGF stimulation has not been reported previously. The mechanism may depend on preexisting changes such as the lost p53 function of these cells. T98Gsis cells provide a model of growth factor-dependent tumorigenesis and metastases, which may be helpful in elucidating these relationships.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation.

Identification, purification, and characterization of cell-surface retention sequence-binding proteins from human SK-Hep cells and bovine liver plasma membranes.

Cell-surface retention is a newly identified mechanism associated with the secretion of certain polypeptide growth factors and cytokines. This novel form of secretion appears to be mediated by cell-surface retention sequences (CRS) in the polypeptide molecules. To test the hypothesis that high-affinity CRS-binding proteins (CRS-BPs) are responsible for the cell-surface retention, we identified and characterized the high-affinity binding sites on various cell types for 125I-labeled CRS peptide (sis) and CRS peptide (VEGF), each of which contained the putative CRS motifs of platelet-derived growth factor B (c-sis) and vascular endothelial cell growth factor, respectively. Scatchard plot analysis revealed a single class of high-affinity binding sites with Kd = 0.5-0.7 nM and approximately 22,000-55,000 sites/cell. High-affinity binding activity could be demonstrated between pH 4.5 and 8.0, but was much greater below 6.0 (maximum pH 5.0-5.5). The ligand binding activity was inhibited by heparin, polylysine, and protamine, but not by cytochrome c. CRS-BPs responsible for the high-affinity binding were identified as 60-72-kDa proteins by ligand affinity labeling. CRS-BPs were purified from human SK-Hep cells and bovine liver plasma membranes by Triton X-100 extraction followed by affinity column chromatography on wheat germ lectin-Sepharose 4B and CRS peptide (sis)-Affi-Gel 10. Purified CRS-BPs exhibited ligand binding properties (pH profile and inhibitor sensitivity) similar to those of the high-affinity binding sites for CRS peptides on cultured cells. The major CRS-BPs (p60, p66, and p72) purified from bovine liver plasma membranes were found to have identical N-terminal amino acid sequence and were assumed to represent different forms of the same gene product, which we have designated CRS-BP1.

The v-sis oncoprotein loses transforming activity when targeted to the early Golgi complex.

The location of autocrine interactions between the v-sis protein and PDGF receptors remains uncertain and controversial. To examine whether receptor-ligand interactions can occur intracellularly, we have constructed fusion proteins that anchor v-sis to specific intracellular membranes. Fusion of a cis-Golgi retention signal from a coronavirus E1 glycoprotein to v-sis protein completely abolished its transforming ability when transfected into NIH3T3 cells. Fusion proteins incorporating mutations in this retention signal were not retained within the Golgi complex but instead were transported to the cell surface, resulting in efficient transformation. All chimeric proteins were shown to dimerize properly. Derivatives of some of these constructs were also constructed bearing the cytoplasmic tail from the glycoprotein of vesicular stomatitis virus (VSV-G). These constructs allowed examination of subcellular localization by double-label immunofluorescence, using antibodies that distinguish between the extracellular PDGF-related domain and the VSV-G cytoplasmic tail. Colocalization of sis-E1-G with Golgi markers confirmed its targeting to the early Golgi complex. The sis-E1 constructs, targeted to the early Golgi complex, exhibited no proteolytic processing whereas the mutant forms of sis-E1 exhibited normal proteolytic processing. Treatment with suramin, a polyanionic compound that disrupts ligand/receptor interactions at the cell surface, was able to revert the transformed phenotype induced by the mutant sis-E1 constructs described here. Our results demonstrate that autocrine interactions between the v-sis oncoprotein and PDGF receptors within the early Golgi complex do not result in functional signal transduction. Another v-sis fusion protein was constructed by attaching the transmembrane domain and COOH-terminus of TGN38, a protein that localizes to the trans-Golgi network (TGN). This construct was primarily retained intracellularly, although some of the fusion protein reached the surface. Deletion of the COOH-terminal region of the TGN38 retention signal abrogated the TGN-localization, as evidenced by very prominent cell surface localization, and resulted in increased transforming activity. The behavior of the sis-TGN38 derivatives is discussed within the context of the properties of TGN38 itself, which is known to recycle from the cell surface to the TGN.

Platelet-derived growth factor (PDGF) B and A homodimers transform murine fibroblasts depending on the genetic background of the cell.

The endogenously overexpressed c-sis (platelet-derived growth factor (PDGF) B-chain) proto-oncogene and its viral counterpart v-sis effectively transform NIH 3T3 cells by an autocrine mechanism, whereas the PDGF A-chain gene does not. However, PDGF B continuously cultured with confluent NIH 3T3 cells fails to induce phenotypic transformation as would be predicted by the autocrine model. We now have established conditions in which subconfluent, logarithmically growing NIH 3T3 cells are efficiently transformed by exogenous PDGF B but not PDGF A. Clonally selected cells from transformed foci remain transformed when continuously cultured with PDGF B but revert to the non-transformed phenotype without PDGF B or if PDGF A is substituted for PDGF B, suggesting that PDGF B complements a genetically stable NIH 3T3 cell phenotype to induce transformation and initiates a signal different from that of PDGF A required to induce transformation. Neither PDGF A nor PDGF B transform normal rat kidney (NRK) cells under identical conditions of culture. However, we now demonstrate that exogenous PDGF A and PDGF B independently transform NRK cells that endogenously overexpress the anti-apoptotic bcl-2 gene. These results suggest that both PDGF A and PDGF B effectively transform murine fibroblasts with a compatible genotype, that transformation by the PDGF isoforms requires complementation with a compatible genotype in a multistep transformational process, and that the autocrine mechanism is required but not sufficient for transformation of murine fibroblasts by PDGF.

Transformation-specific pattern of phosphorylation of c-Jun, Jun-B, Jun-D and Egr-1 in v-sis transformed cells.

The degree of phosphorylation of c-Jun, Jun-B, Jun-D and Egr-1 transcription factors was examined during normal growth and during a prolonged period of defined transformation of NIH-3T3 cells which conditionally express v-sis [Mercola, D. et al. (1992) Oncogene, 7, 1793-1803]. During the asynchronous growth of normal cells phosphorylation of all factors was low and constant at all stages of growth from low density (c. 25 x 10(3) cells/cm2) through log-phase of growth to saturation density (c. 100 x 10(3) cells/cm2). Upon induction of v-sis, a marked and coordinate increase in phosphorylation occurred for c-Jun, Jun-B and Egr-1 to approximately 320%, 230% and 420% respectively above basal levels which was stable for the 2.5 day transformation period. The phosphorylation of Jun-D increased to over 600% and, after about 20 h, steadily declined to near basal levels at 54 h post-induction. Moreover, at any time phosphorylation and v-sis expression were fully reversible upon removal of the inducer. It appears that increased phosphorylation of the Jun family members and Egr-1 is not necessary for normal growth of NIH-3T3 but is dependent upon the expression of v-sis. Thus, normal and transformed cells may be distinguished. For c-Jun, the v-sis enhanced phosphorylation occurs at serines 63 and 73 and is required for transformation by several oncogenes [Smeal, T. et al. (1992) Mol. Cell. Biol., 12, 3507-3513]. The results described here show that the phosphorylation of additional factors is a stable and specific correlate of transformation which have have regulatory significance during transformation.