Mouse Platelet-derived Growth Factor Research Articles

Convection-enhanced delivery of etoposide is effective against murine proneural glioblastoma

Glioblastoma subtypes have been defined based on transcriptional profiling, yet personalized care based on molecular classification remains unexploited. Topoisomerase II (TOP2) contributes to the transcriptional signature of the proneural glioma subtype. Thus, we targeted TOP2 pharmacologically with etoposide in proneural glioma models. TOP2 gene expression was evaluated in mouse platelet derived growth factor (PDGF)(+)phosphatase and tensin homolog (PTEN)(-/-)p53(-/-) and PDGF(+)PTEN(-/-) proneural gliomas and cell lines, as well as human glioblastoma from The Cancer Genome Atlas. Correlation between TOP2 transcript levels and etoposide susceptibility was investigated in 139 human cancer cell lines from the Cancer Cell Line Encyclopedia public dataset and in mouse proneural glioma cell lines. Convection-enhanced delivery (CED) of etoposide was tested on cell-based PDGF(+)PTEN(-/-)p53(-/-) and retroviral-based PDGF(+)PTEN(-/-) mouse proneural glioma models. TOP2 expression was significantly higher in human proneural glioblastoma and in mouse proneural tumors at early as well as late stages of development compared with normal brain. TOP2B transcript correlated with susceptibility to etoposide in mouse proneural cell lines and in 139 human cancer cell lines from the Cancer Cell Line Encyclopedia. Intracranial etoposide CED treatment (680 μM) was well tolerated by mice and led to a significant survival benefit in the PDGF(+)PTEN(-/-)p53(-/-) glioma model. Moreover, etoposide CED treatment at 80 μM but not 4 μM led to a significant survival advantage in the PDGF(+)PTEN(-/-) glioma model. TOP2 is highly expressed in proneural gliomas, rendering its pharmacological targeting by intratumoral administration of etoposide by CED effective on murine proneural gliomas. We provide evidence supporting clinical testing of CED of etoposide with a molecular-based patient selection approach.

Single variable domain antibody as a versatile building block for the construction of IgG-like bispecific antibodies

Bispecific antibodies (BsAb) have been traditionally utilized to redirect cytotoxic effector cells and agents to kill tumor cells expressing the target antigens. Recently a new concept is emerging to develop BsAb that simultaneously block the functions of two tumor-associated targets, eg., growth factor receptors, for enhanced antitumor efficacies. Broad clinical applications of BsAb have been, and still are, significantly hampered by the difficulty in producing the materials in sufficient quantity and quality by traditional approaches. Here we describe a recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, using a single variable domain (sVD) antibody as a versatile building block. In this method, a sVD of a defined specificity is genetically fused to either the N-terminus of the light chain or the C-terminus of the heavy chain of a functional IgG antibody of a different specificity. A model BsAb was constructed using a sVD to mouse platelet derived growth factor receptor alpha and a conventional IgG antibody to mouse platelet derived growth factor receptor beta. The BsAb were expressed in mammalian cells and purified to homogeneity by a one-step Protein A affinity chromatography. Further, the BsAb retained the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. Importantly, the BsAb inhibited the activation of both its target receptors in tumor cells stimulated by both platelet derived growth factor AA and BB, whereas the parent monospecific antibody only inhibited the activation of a single receptor stimulated by its cognate ligand. This format of BsAb should be readily applicable to the production of other BsAb recognizing any pairs of antigens.

Single Variable Domain-IgG Fusion: A NOVEL RECOMBINANT APPROACH TO Fc DOMAIN-CONTAINING BISPECIFIC ANTIBODIES

Both laboratory and early clinical studies to date have demonstrated that bispecific antibodies (BsAb) may have potentially significant application in cancer therapy. The clinical development of BsAb as therapeutics has been hampered, however, by the difficulty in preparing the materials in sufficient quantity and quality by traditional methods. In recent years, a variety of recombinant methods has been developed for efficient production of BsAb, both as antibody fragments and as full-length IgG-like molecules. Here we describe a novel recombinant approach for the production of an Fc domain-containing, IgG-like tetravalent BsAb, with two antigen-binding sites to each of its target antigens, by genetically fusing a single variable domain antibody to the N terminus of the light chain of a functional IgG antibody of different specificity. A model BsAb was constructed using a single variable domain antibody to mouse platelet-derived growth factor receptor alpha and a conventional IgG antibody to mouse vascular endothelial growth factor receptor 2. The BsAb was expressed in mammalian cells and purified to homogeneity by one-step protein A affinity chromatography. Furthermore, the BsAb retains the antigen binding specificity and the receptor neutralizing activity of both of its parent antibodies. This design and expression of Fc domain-containing, IgG-like BsAb should be applicable to the construction of similar BsAb from antibodies recognizing any pair of antigens.

Biology of platelet-derived growth factors in development.

Platelet-derived growth factor (PDGF) was one of the first growth factors to be characterized, and the PDGF family of ligand and receptors has remained an archetype system for studies of the mechanisms of action of growth factors and receptor tyrosine kinases for more than two decades. The small size of the family has also facilitated genetic studies and, in particular, manipulations of the mouse PDGF and PDGF receptor genes have given important insights into the role of this family during mammalian development. These studies have shown that discrete populations of mesenchymal and neuroectodermal progenitor cells depend on PDGF signaling for their growth and distribution within developing organs. Other studies suggest that the same, or similar, cells may be targeted by exaggerated PDGF signaling in a number of pathological processes, including different types of cancer. The present review summarizes current views on the roles of PDGFs in developmental processes, and discusses the critical importance of the amount, spatial distribution, and bioavailability of the PDGF proteins for acquisition of the correct number and location of target cells.

Evolutionary divergence of platelet-derived growth factor alpha receptor signaling mechanisms.

Receptor tyrosine kinases (RTKs) direct diverse cellular and developmental responses by stimulating a relatively small number of overlapping signaling pathways. Specificity may be determined by RTK expression patterns or by differential activation of individual signaling pathways. To address this issue we generated knock-in mice in which the extracellular domain of the mouse platelet-derived growth factor alpha receptor (PDGFalphaR) is fused to the cytosolic domain of Drosophila Torso (alpha(Tor)) or the mouse fibroblast growth factor receptor 1 (alpha(FR)). alpha(Tor) homozygous embryos exhibit significant rescue of neural crest and angiogenesis defects normally found in PDGFalphaR-null embryos yet fail to rescue skeletal or extraembryonic defects. This phenotype was associated with the ability of alpha(Tor) to stimulate the mitogen-activated protein (MAP) kinase pathway to near wild-type levels but failure to completely activate other pathways, such as phosphatidylinositol (PI) 3-kinase. The alpha(FR) chimeric receptor fails to rescue any aspect of the PDGFalphaR-null phenotype. Instead, alpha(FR) expression leads to a gain-of-function phenotype highlighted by ectopic bone development. The alpha(FR) phenotype was associated with a failure to limit MAP kinase signaling and to engage significant PI3-kinase response. These results suggest that precise regulation of divergent downstream signaling pathways is critical for specification of RTK function.

Mechanism for the transcriptional repression by c-Myc on PDGF beta-receptor.

c-Myc plays a key role in the cell cycle dependent control of the PDGF beta-receptor mRNA. The mouse platelet-derived growth factor (PDGF) beta-receptor promoter contains a CCAAT motif, and NF-Y plays an essential role in its transcription. Coexpression of c-Myc represses PDGF beta-receptor luciferase reporter activity, and the CCAAT motif in the promoter is indispensable for this repression. Here we show that c-Myc binds NF-Y subunits, YB and YC, by immunoprecipitation from cotransfected COS-1 cells. The in vitro-translated c-Myc also binds the glutathione S-transferase (GST)-NF-YB fusion protein and GST-NF-YC, but not GST-NF-YA. The most C-terminal region of HAP domains of NF-YB and NF-YC, and the Myc homology boxes, but not the C-terminal bHLHZip domain, are indispensable for the coimmunoprecipitation, and also for the repression of PDGF beta-receptor. c-Myc binds NF-Y complex without affecting the efficiency of NF-Y binding to DNA. However, the expression of Myc represses the transcriptional activation of NF-YC when fused to the GAL4 DNA binding domain. Furthermore, this repression was seen only when Myc homology boxes are present, and NF-YC contains the c-Myc binding region.

Molecular characterization of a new member of the immunoglobulin superfamily that potentially functions in T-cell activation and development.

Differential hybridization cloning has been used to isolate a novel chicken thymic activation and developmental sequence (cTADS). The nucleotide sequence of the cTADS cDNA predicts an open reading frame of 439 amino acids. The inferred cTADS protein possesses a hydrophobic membrane-spanning domain and putative intracellular kinase activation domains. Its extracellular domain shares similarities with the immunoglobulin protein superfamily, featuring two conserved immunoglobulin folds that resemble C1 and C2 constant regions. The cTADS sequence shows similarity to a subfamily of proteins involved in cellular adhesion: chicken neural cell adhesion molecule and human opioid-binding adhesion molecule, and to proteins that have a biological role in intracellular signaling: mouse platelet-derived growth factor receptor and human fibroblast growth factor receptor. cTADS is differentially expressed in chicken thymic cells during embryonic development and during activation through the T-cell receptor. Sequence similarities and expression patterns suggest that cTADS could be involved in cell recognition and adhesion, and/or peptide ligand binding.

Expression of platelet-derived growth factor proteins and their receptor alpha and beta mRNAs during fracture healing in the normal mouse.

Platelet-derived growth factor (PDGF), abundant in bone tissue, has been reported to stimulate mesenchymal cell proliferation and migration. To elucidate the functional roles of PDGF during fracture healing, we investigated the expression of PDGF-A and -B chain proteins and receptor alpha and beta mRNAs in fractured mouse tibiae. Twelve-week-old male BALB/c mice were operated on to make a closed fracture on the proximal tibia. On days 2, 4, 7, 10, 14, 21, and 28 after the operation, the fractured tibiae were excised, fixed with 4% paraformaldehyde, decalcified with 20% EDTA, and embedded in paraffin to prepare 7-microm sections. Immunohistochemistry using polyclonal antibodies against human PDGF-A and -B chains was carried out by the avidin-biotin-peroxidase method. For in situ hybridization, we used digoxigenin-labeled single-stranded DNA probes specific for mouse PDGF receptors alpha and beta generated by unidirectional polymerase chain reaction. In the inflammatory phase on days 2-4 after the fracture, mesenchymal cells gathering at the fracture site expressed the PDGF-B chain and beta receptor mRNA. At the stage of cartilaginous callus formation on day 7, the immunoreactivity for PDGF-A and -B chains on proliferating and hypertrophic chondrocytes and the signals of alpha and beta receptor mRNAs on proliferating chondrocytes became manifest. At the stage of bony callus and bone remodeling on days 14-21, the predominant expression of the PDGF-B chain and beta receptor was observed on both osteoclasts and osteoblasts. On day 28, signals for PDGF ligand proteins and receptor mRNAs diminished. The coincidental localization of PDGF ligands and their receptors implies a paracrine and autocrine mechanism. Our data suggested that PDGF contributed in part to the promotion of the chondrogenic and osteogenic changes of mesenchymal cells from the early to the midphase of fracture healing; the functions mediated by the beta receptor, including cell migration, might be prerequisites to the recruitment of mesenchymal cells in the initial step and to the interaction between osteoclasts and osteoblasts in the bone remodeling phase.

Nuclear factor Y controls the basal transcription activity of the mouse platelet-derived-growth-factor beta-receptor gene.

To determine the regulatory mechanism of the expression of the mouse platelet-derived growth factor (PDGF) beta-receptor gene, a 1.9-kb 5' flanking genomic fragment was cloned and analyzed. Site-directed mutagenesis of a CCAAT motif, located 60 bp upstream of the transcriptional-start site, completely abolished the promoter activity [Ballagi, A. E., Ishisaki, A., Nelin, J.-O. & Funa, K. (1995) Biochem. Biophys. Res. Commun. 210, 165-1751. The sequence around the intact CCAAT motif was protected by in vitro DNase-I-footprinting analysis. Electrophoresis-mobility-shift assays with anti-[nuclear factor Y(NF-Y)]Ig revealed binding of the NF-Y complex to the CCAAT box. Furthermore, the double-stranded oligonucleotides corresponding to the sequence around the CCAAT motif were conjugated with DNA-affinity magnetic beads. The binding proteins were affinity purified and identified as the NF-Y transcription factor by western blotting. Our results indicate that NF-Y controls the basal transcription activity of the mouse PDGF beta-receptor gene.

PDGF-A Signaling Is a Critical Event in Lung Alveolar Myofibroblast Development and Alveogenesis

A mouse platelet-derived growth factor A chain (PDGF-A) null allele is shown to be homozygous lethal, with two distinct restriction points, one prenatally before E10 and one postnatally. Postnatally surviving PDGF-A-deficient mice develop lung emphysema secondary to the failure of alveolar septation. This is apparently caused by the loss of alveolar myofibroblasts and associated elastin fiber deposits. PDGF α receptor–positive cells in the lung having the location of putative alveolar myofibroblast progenitors were specifically absent in PDGF-A null mutants. We conclude that PDGF-A is crucial for alveolar myofibroblast ontogeny. We have previously shown that PDGF-B is required in the ontogeny of kidney mesangial cells. The PDGFs therefore appear to regulate the generation of specific populations of myofibroblasts during mammalian development. The two PDGF null phenotypes also reveal analogous morphogenetic functions for myofibroblast-type cells in lung and kidney organogenesis. 8 Present address: Gävle Hospital, S-801 87 Gävle, Sweden.

Cross-linking of the p55 Tumor Necrosis Factor Receptor Cytoplasmic Domain by a Dimeric Ligand Induces Nuclear Factor-κB and Mediates Cell Death

We have fused the cytoplasmic domain of the p55 tumor necrosis factor (TNF) receptor to the extracellular and transmembrane domain of the mouse platelet-derived growth factor (PDGF) receptor. Mouse mammary gland epithelial (NMuMG) cells were stably transfected with the PDGFR-TR55 chimeric receptor. These cells lack endogenous PDGF receptor expression and do not respond to PDGF. In the PDGFR-TR55 transfectants, PDGF elicited a cytotoxic response, which is indistinguishable from that induced by the wild type p55 TNF receptor. In addition, PDGF-induced activation of the PDGFR-TR55 chimeric receptor resulted in nuclear translocation of NF-κB. The data presented suggest that cross-linking of the p55 TNF receptor cytoplasmic domain by a dimeric ligand such as PDGF is sufficient to generate cellular responses that do not differ from those observed with the trimeric ligand TNF.

Activation of Na+/H+ exchange by platelet-derived growth factor involves phosphatidylinositol 3'-kinase and phospholipase C gamma.

The effect of site-specific mutations in the mouse platelet-derived growth factor (PDGF) beta-receptor on activation of the Na+/H+ exchanger was examined in normal murine mammary gland epithelial (NMuMG) and Chinese hamster ovary (CHO) cells. These cells, which do not normally express PDGF receptors, were stably transfected with PDGF beta-receptor cDNA. Intracellular pH and Ca2+ were monitored using fluorescent probes. In both NMuMG and CHO cells expressing wild-type PDGF beta-receptors, PDGF B/B activated the amiloride-sensitive Na+/H+ exchanger. In both cell types, cell alkalinization was reduced by approximately 50% with a receptor mutant Y708F,Y719F which cannot bind phosphatidylinositol (PI) 3'-kinase. An inhibitor of PI 3'-kinase, LY294002, also inhibited alkalinization by 43% in cells with wild-type, but not Y708F,Y719F receptors. PDGF-induced intracellular Ca2+ release was not affected by this mutation. Both alkalinization and Ca2+ release were reduced by nearly 100% with the mutant Y977F,Y989F, which cannot bind phospholipase C gamma (PLC gamma). Y739F, a mutant that fails to bind the GTPase-activating protein did not affect PDGF-induced alkalinization. In protein kinase C (PKC) down-regulated NMuMG cells (wild-type receptor), PDGF no longer activated the Na+/H+ exchanger. In contrast, in PKC down-regulated CHO cells (wild-type receptor), PDGF-induced alkalinization was attenuated by only 37%. This residual activity was unaffected by the Y708F,Y719F mutation, but was completely eliminated by removal of medium Ca2+. These findings indicate that phospholipase C gamma is essential for activation of Na+/H+ exchange. PI 3'-kinase participates in PKC-dependent activation of Na+/H+ exchange by PDGF. In CHO cells, there is a second, Ca(2+)-dependent mechanism for activation of the exchanger.

Evidence for Both Tyrosine Kinase and G-Protein-Coupled Pathways Leading to Starfish Egg Activation

To investigate possible pathways leading to egg activation at fertilization, the ability of exogenously introduced tyrosine kinase and G-protein-coupled recaptors to mimic events of fertilization was examined. Oocytes of the starfish Asterina miniata were injected with RNA for a chimeric receptor consisting of the extracellular domain of the β form of the mouse platelet-derived growth factor (PDGF) receptor and the transmembrane/intracellular domain of the human fibroblast growth factor (FGF) receptor, or with RNA for the rat serotonin 1c receptor. These oocytes were cultured for 1 to 3 days and then matured with 1-methyladenine. In response to PDGF or serotonin, the injected eggs underwent responses like those at fertilization: cortical granule exocytosis, a rise in intracellular free calcium, and DNA synthesis. Some of these artificially activated eggs cleaved, and some of the PDGF-activated eggs were observed to form larvae, A PDGF/FGF receptor with a point mutation which eliminated its ability to interact with phospholipase C-γ did not cause fertilization-like responses. Thus components of a signaling pathway involving phospholipase C-γ, characteristic of tyrosine kinase receptors, as well as components of a pathway involving a G-protein and phospholipase C-β, characteristic of G-protein-coupled receptors, appear to be present in starfish eggs. Either or both could function in egg activation at fertilization.

Signal transduction through a biomolecular receptor tyrosine protein kinase composed of a platelet-derived growth factor receptor-CD4 chimera and the nonreceptor tyrosine protein kinase Lck.

We have generated a novel "receptor tyrosine kinase" by fusing the extracellular and transmembrane domain of the mouse platelet-derived growth factor receptor (PDGFR) to the cytoplasmic domain of CD4 and coexpressing the construct with the murine cytoplasmic tyrosine protein kinase p56lck. NMuMG cells, which are mouse mammary gland epithelial cells that lack endogenous platelet-derived growth factor (PDGF) receptor expression, were stably transfected with both PDGFR-CD4 and p56lck. The PDGFR-CD4 chimeric protein was expressed at the cell surface and formed a complex with p56lck. Addition of PDGF to these cells led to increased tyrosine phosphorylation of a 56-kDa protein likely to be p56lck and several unidentified cellular proteins. The enzymatic activity of p56lck was increased after treatment with PDGF, indicating that dimerization (or oligomerization) mediated by ligand binding at the cell surface is capable of inducing the activation not only of receptor tyrosine kinases but nonreceptor tyrosine kinases as well. However, the PDGFR-CD4.p56lck complex was, in contrast to the wild type PDGF receptor, not able to induce a PDGF-dependent mitogenic response or DNA synthesis in NMuMG cells. Analysis of several known substrates of the PDGFR-signaling pathway indicates an early block in the transduction of the signal generated by p56lck.

Mouse platelet-derived growth factor receptor alpha gene is deleted in W19H and patch mutations on chromosome 5.

The mouse W19H mutation is an x-ray-induced deletion of more than 2 centimorgans on chromosome 5 encompassing the white spotting mutation W (encoded by the Kit protooncogene), patch (Ph), and recessive lethal (l) loci. The platelet-derived growth factor receptor alpha gene (PDGFRA) like Kit encodes a transmembrane receptor tyrosine kinase. By using mouse-Chinese hamster somatic cell hybrids and haplotype analysis in interspecific backcross mice, mouse Pdgfra was mapped to chromosome 5 in tight linkage with Kit. Hybridization of a PDGFRA probe to DNAs from W19H/ + heterozygous mice and patch heterozygous mice, and their wild-type littermates, demonstrated deletion of Pdgfra. Pulsed-field gel electrophoresis indicated that Kit and Pdgfra are linked on a 630-kilobase Mlu I DNA fragment. Thus the W19H deletion removes at least two receptor tyrosine kinases and the results suggest Pdgfra as a candidate for the Ph locus.

Thrombin-stimulated immunoprecipitation of phosphatidylinositol 3-kinase from human platelets.

Growth factors and transforming proteins that activate tyrosine phosphorylation have been shown to cause an increased labeling of 3-phosphate-containing phosphatidylinositols. Turnover correlates with the formation of a complex between phosphatidylinositol 3-kinase, the activated protein-tyrosine kinase, and other proteins thought to participate in transmembrane signaling. When human platelets are treated with thrombin, labeling of 3-phosphate-containing phosphatidylinositols is stimulated with a time course and concentration dependence consistent with a role for these lipids in platelet activation. We now report that when human platelets are stimulated with thrombin, a complex forms between phosphatidylinositol 3-kinase, a protein-serine/threonine kinase, and an uncharacterized platelet membrane protein. The complex is immunoprecipitated from detergent lysates of thrombinstimulated platelets by a rabbit antiserum prepared against a peptide from the cytoplasmic domain of the mouse platelet-derived growth factor (PDGF) receptor. The antigen is not the PDGF receptor, since complex formation is not stimulated by PDGF and thrombin-induced complexes are not precipitated by another rabbit antiserum against the same peptide or by monoclonal anti-human PDGF receptor antibodies. Formation of the complex is rapid (within 30 sec) and occurs at thrombin concentrations that stimulate platelet aggregation and secretion (50% of maximal complex formation at 0.03 unit of thrombin per ml). We propose that the complex initiates formation of 3-phosphate-containing phosphatidylinositols that may function in platelet activation.

Cloning and expression of a cDNA coding for the human platelet-derived growth factor receptor: evidence for more than one receptor class.

The complete nucleotide sequence of a cDNA encoding the human platelet-derived growth factor (PDGF) receptor is presented. The cDNA contains an open reading frame that codes for a protein of 1106 amino acids. Comparison to the mouse PDGF receptor reveals an overall amino acid sequence identity of 86%. This sequence identity rises to 98% in the cytoplasmic split tyrosine kinase domain. RNA blot hybridization analysis of poly(A)+ RNA from human dermal fibroblasts detects a major (approximately 5.7 kb) and a minor (approximately 4.8 kb) transcript using the cDNA as a probe. Baby hamster kidney cells, transfected with an expression vector containing the receptor cDNA, express an approximately equal to 190-kDa cell surface protein that is recognized by an anti-human PDGF receptor antibody. The recombinant PDGF receptor is functional in the transfected baby hamster kidney cells as demonstrated by ligand-induced phosphorylation of the receptor. Binding properties of the recombinant PDGF receptor were also assessed with pure preparations of BB and AB isoforms of PDGF (i.e., PDGF dimers composed of two B chains or an A and a B chain). Unlike human dermal fibroblasts, which bind both isoforms with high affinity, the transfected baby hamster kidney cells bind only the BB isoform of PDGF with high affinity. This observation is consistent with the existence of more than one PDGF receptor class.

Platelet-derived growth factor receptors expressed by cDNA transfection couple to a diverse group of cellular responses associated with cell proliferation.

We have expressed the mouse platelet-derived growth factor (PDGF) receptor cDNA under transcriptional control of simian virus 40 promoter in Chinese hamster ovary cells, a cell type that lacks PDGF receptors. Stable transfectants expressed receptor protein that was recognized by antireceptor antibodies. PDGF stimulated a diverse group of responses in the transfected cells including tyrosine phosphorylation of the receptor and a 32-kDa cellular substrate, receptor internalization, phosphoinositide turnover, cytoplasmic alkalinization, changes in the intracellular calcium concentration, thymidine uptake into DNA, and cell division. There were no responses to PDGF in the control Chinese hamster ovary cells. After prolonged exposure to PDGF, the transfectants became refractory to subsequent PDGF stimulation and the receptor protein was no longer detectable. These data validate the authenticity of the PDGF receptor cDNA, show that transfection of a single type of receptor cDNA sequence confers PDGF sensitivity to a diverse group of cellular responses, and demonstrate that effector molecules that mediate the PDGF responses are present in cells that lack PDGF receptors.