Platelet-derived Growth Factor Receptor Family Research Articles

Biochemical Characterization of TAK-593, a Novel VEGFR/PDGFR Inhibitor with a Two-Step Slow Binding Mechanism

Inhibition of tumor angiogenesis leads to a lack of oxygen and nutrients in the tumor and therefore has become a standards of care for many solid tumor therapies. Dual inhibition of vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) protein kinase activities is a popular strategy for targeting tumor angiogenesis. We discovered that TAK-593, a novel imidazo[1,2-b]pyridazine derivative, potently inhibits tyrosine kinases from the VEGFR and PDGFR families. TAK-593 was highly selective for these families, with an IC(50) >1 μM when tested against more than 200 protein and lipid kinases. TAK-593 displayed competitive inhibition versus ATP. In addition, TAK-593 inhibited VEGFR2 and PDGFRβ in a time-dependent manner, classifying it as a type II kinase inhibitor. Analysis of enzyme-inhibitor preincubation experiments revealed that the binding of TAK-593 to VEGFR2 and PDGFRβ occurs via a two-step slow binding mechanism. Dissociation of TAK-593 from VEGFR2 was extremely slow (t(1/2) >17 h), and the affinity of TAK-593 at equilibrium (K(i)*) was less than 25 pM. Ligand displacement analysis with a fluorescent tracer confirmed the slow dissociation of TAK-593. The dissociation rate constants were in good agreement between the activity and ligand displacement data, and both analyses supported slow dissociation of TAK-593. The long residence time of TAK-593 may achieve an extended pharmacodynamic effect on VEGFR2 and PDGFRβ kinases in vivo that differs substantially from its observed pharmacokinetic profile.

Synthesis and structure–activity relationship of 6-arylureido-3-pyrrol-2-ylmethylideneindolin-2-one derivatives as potent receptor tyrosine kinase inhibitors

A series of new ureidoindolin-2-one derivatives were synthesized and evaluated as inhibitors of receptor tyrosine kinases. Investigation of structure–activity relationships at positions 5, 6, and 7 of the oxindole skeleton led to the identification of 6-ureido-substituted 3-pyrrolemethylidene-2-oxindole derivatives that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor (PDGFR) families of receptor tyrosine kinases. Several derivatives showed potency against the PDGFR inhibiting both its enzymatic and cellular functions in the single-digit nanomolar range. Among them, compound 35 was a potent inhibitor against tyrosine kinases, including VEGFR and PDGFR families, as well as Aurora kinases. Inhibitor 36 (non-substituted on the pyrrole or phenyl ring) had a moderate pharmacokinetic profile and completely inhibited tumor growth initiated with the myeloid leukemia cell line, MV4-11, in a subcutaneous xenograft model in BALB/c nude mice.

Response to Sunitinib Malate in Advanced Alveolar Soft Part Sarcoma

Alveolar soft part sarcoma (ASPS) is a rare, chemoresistant soft tissue sarcoma. ASPS harbors the t(17-X) (p11.2;q25) translocation, resulting in the ASPACR1-TFE3 fusion protein, causing MET autophosphorylation and activation of downstream signaling. The tumor vascular pattern prompted us to use sunitinib malate (SM), a tyrosine kinase inhibitor with antiangiogenic properties. Since July 2007, five patients with progressive metastatic ASPS have been treated with continuous SM 37.5 mg/d on a named basis. Four patients are evaluable for response. In four cases, cryopreserved material was available. Upstream and downstream targets of receptor tyrosine kinase (RTK) pathways, as well as mechanisms of activation, were investigated by biochemical profiles, including human phospho-receptor RTK antibody arrays and immunoprecipitation/Western blotting, molecular analyses, immunohistochemistry, and fluorescence in situ hybridization analyses. After 3 months, two patients had RECIST (response evaluation criteria in solid tumor) partial response, as well as positron emission tomography response and subjective improvement. One had a RECIST stable disease. One progressed and stopped treatment. One patient is still responding after 12 months. The upstream analysis showed activation of all the platelet-derived growth factor receptor (PDGFR) family members, as well as epidermal growth factor receptor, MET families, and RET. Vascular endothelial growth factor receptors (VEGFR1 and VEGFR2) were activated only in one case. The downstream target analysis showed strong activation of phosphatidylinositol 3-kinase/AKT, extracellular signal-regulated kinase 1/2, and mTOR and its targets (S6K and S6). The absence of any upstream mTOR effector deregulation and the presence of RTK cognate ligands support an autocrine-paracrine activation loop mechanism. SM may have antitumor activity in ASPS, possibly through a mechanism involving PDGFR and RET. The role of MET, epidermal growth factor receptor, and mTOR, as well as PDGFR inhibition, needs to be further explored.

3-Amino-benzo[d]isoxazoles as Novel Multitargeted Inhibitors of Receptor Tyrosine Kinases

A series of benzoisoxazoles and benzoisothiazoles have been synthesized and evaluated as inhibitors of receptor tyrosine kinases (RTKs). Structure-activity relationship studies led to the identification of 3-amino benzo[ d]isoxazoles, incorporating a N, N'-diphenyl urea moiety at the 4-position that potently inhibited both the vascular endothelial growth factor receptor (VEGFR) and platelet-derived growth factor receptor families of RTKs. Within this series, orally bioavailable compounds possessing promising pharmacokinetic profiles were identified, and a number of compounds demonstrated in vivo efficacy in models of VEGF-stimulated vascular permeability and tumor growth. In particular, compound 50 exhibited an ED 50 of 2.0 mg/kg in the VEGF-stimulated uterine edema model and 81% inhibition in the human fibrosarcoma (HT1080) tumor growth model when given orally at a dose of 10 mg/kg/day.

Silent mutations in KIT and PDGFRA and coexpression of receptors with SCF and PDGFA in Merkel cell carcinoma: implications for tyrosine kinase-based tumorigenesis

Merkel cell carcinoma is a rare and aggressive form of skin cancer of neuroendocrine origin. Its treatment involves wide excision and radiotherapy but no effective therapy exists for advanced disease. Upregulation of the platelet-derived growth factor receptor family of tyrosine kinases, PDGFRA and KIT, has a crucial role in cancer development. Several studies have shown expression of the tyrosine kinase receptor KIT (CD117) in Merkel cell carcinoma. In this study, we examined the expression and mutational status of KIT and PDGFRA in 14 primary and 18 metastatic Merkel cell carcinoma. The expression of KIT and PDGFRA and their respective ligands, stem cell factor (SCF) and PDGFA, was assessed by immunohistochemistry. In addition, we analyzed KIT exons 9, 11, 13 and 17, and PDGFRA exons 10, 12 and 18 for the presence of activating mutations. We found that only 53% of cases of Merkel cell carcinoma expressed KIT, which was mostly seen as diffuse weak staining, and SCF expression was observed only in 31% of cases. In contrast, 87 and 81% of cases expressed PDGFRA and PDGFA, respectively. We observed coexpression of SCF and KIT in only 5 of 32 cases (16%) whereas 25 of 31 cases (81%) showed coexpression of PDGFRA and its ligand PDGFA. While we documented silent mutations in exon 17 of KIT and exons 10, 12 and 18 of PDGFRA, we were not able to identify any known activating mutations. Our results indicate that there is no correlation between positive immunostaining and occurrence of activating mutations in KIT and PDGFRA. Moreover, the presence of KIT/SCF and PDGFRA/PDGFA coexpression in a proportion of cases may indicate an autocrine/paracrine stimulation loop. We think therefore that imatinib mesylate is less likely to be an effective therapy for Merkel cell carcinoma, unless activating mutations exist in other exons of these receptor kinases.

Discovery ofN-(4-(3-Amino-1H-indazol-4-yl)phenyl)-N‘-(2-fluoro-5-methylphenyl)urea (ABT-869), a 3-Aminoindazole-Based Orally Active Multitargeted Receptor Tyrosine Kinase Inhibitor

In our continued efforts to search for potent and novel receptor tyrosine kinase (RTK) inhibitors as potential anticancer agents, we discovered, through a structure-based design, that 3-aminoindazole could serve as an efficient hinge-binding template for kinase inhibitors. By incorporating an N,N'-diaryl urea moiety at the C4-position of 3-aminodazole, a series of RTK inhibitors were generated, which potently inhibited the tyrosine kinase activity of the vascular endothelial growth factor receptor and the platelet-derived growth factor receptor families. A number of compounds with potent oral activity were identified by utilizing an estradiol-induced mouse uterine edema model and an HT1080 human fibrosarcoma xenograft tumor model. In particular, compound 17p (ABT-869) was found to possess favorable pharmacokinetic profiles across different species and display significant tumor growth inhibition in multiple preclinical animal models.

Crystal Structure of the Tyrosine Kinase Domain of Colony-stimulating Factor-1 Receptor (cFMS) in Complex with Two Inhibitors

The cFMS proto-oncogene encodes for the colony-stimulating factor-1 receptor, a receptor-tyrosine kinase responsible for the differentiation and maturation of certain macrophages. Upon binding its ligand colony-stimulating factor-1 cFMS autophosphorylates, dimerizes, and induces phosphorylation of downstream targets. We report the novel crystal structure of unphosphorylated cFMS in complex with two members of different classes of drug-like protein kinase inhibitors. cFMS exhibits a typical bi-lobal kinase fold, and its activation loop and DFG motif are found to be in the canonical inactive conformation. Both ATP competitive inhibitors are bound in the active site and demonstrate a binding mode similar to that of STI-571 bound to cABL. The DFG motif is prevented from switching into the catalytically competent conformation through interactions with the inhibitors. Activation of cFMS is also inhibited by the juxtamembrane domain, which interacts with residues of the active site and prevents formation of the activated kinase. Together the structures of cFMS provide further insight into the autoinhibition of receptor-tyrosine kinases via their respective juxtamembrane domains; additionally the binding mode of two novel classes of kinase inhibitors will guide the design of novel molecules targeting macrophage-related diseases.

The 2.7 Å Crystal Structure of the Autoinhibited Human c-Fms Kinase Domain

c-Fms, a member of the Platelet-derived Growth Factor (PDGF) receptor family of receptor tyrosine kinases (RTKs), is the receptor for macrophage colony stimulating factor (CSF-1) that regulates proliferation, differentiation and survival of cells of the mononuclear phagocyte lineage. Abnormal expression of c-fms proto-oncogene is associated with a significant number of human pathologies, including a variety of cancers and rheumatoid arthritis. Accordingly, c-Fms represents an attractive therapeutic target. To further understand the regulation of c-Fms, we determined the 2.7 Å resolution crystal structure of the cytosolic domain of c-Fms that comprised the kinase domain and the juxtamembrane domain. The structure reveals the crucial inhibitory role of the juxtamembrane domain (JM) that binds to a hydrophobic site immediately adjacent to the ATP binding pocket. This interaction prevents the activation loop from adopting an active conformation thereby locking the c-Fms kinase into an autoinhibited state. As observed for other members of the PDGF receptor family, namely c-Kit and Flt3, three JM-derived tyrosine residues primarily drive the mechanism for autoinhibition in c-Fms, therefore defining a common autoinhibitory mechanism within this family. Moreover the structure provides an understanding of c-Fms inhibition by Gleevec as well as providing a platform for the development of more selective inhibitors that target the inactive conformation of c-Fms kinase.

The ability of sorafenib to inhibit oncogenic PDGFR and FLT3 mutants and overcome resistance to other small molecule inhibitors

Activated tyrosine kinases are implicated in the pathogenesis of chronic and acute leukemia, and represent attractive targets for therapy. Sorafenib (BAY43-9006, Nexavar) is a small molecule B-RAF inhibitor that is used for the treatment of renal cell carcinoma, and has been shown to have activity against receptor tyrosine kinases from the platelet-derived growth factor receptor (PDGFR) and vascular endothelial growth factor receptor (VEGFR) families. We investigated the efficacy of sorafenib at inhibiting mutants of the receptor tyrosine kinases PDGFRbeta, KIT, and FLT3, which are implicated in the pathogenesis of myeloid malignancies. We tested the effect of sorafenib on the proliferation of hematopoietic cells transformed by ETV6-PDGFRbeta, FLT3 with an internal tandem duplication or D835Y point mutation, and the KIT(D816V) mutant. The direct effect of sorafenib on the activity of these kinases and their downstream signaling was tested using phospho-specific antibodies. We show that sorafenib is a potent inhibitor of ETV6-PDGFRbeta and FLT3 mutants, including some of the mutants that confer resistance to PKC412 and other FLT3 inhibitors. Sorafenib induced a cell cycle block and apoptosis in the acute myeloid leukemia cell lines MV4-11 and MOLM-13, both expressing FLT3 with an internal tandem duplication, whereas no effect was observed on four other acute myeloid leukemia cell lines. The imatinib-resistant KIT(D816V) mutant, associated with systemic mastocytosis, was found to be resistant to sorafenib. These results warrant further clinical studies of sorafenib for the treatment of myeloid malignancies expressing activated forms of PDGFRbeta and FLT3.

Loss of T-Cell Protein Tyrosine Phosphatase Induces Recycling of the Platelet-derived Growth Factor (PDGF) β-Receptor but Not the PDGF α-Receptor

We have previously shown that the T-cell protein tyrosine phosphatase (TC-PTP) dephosphorylates the platelet-derived growth factor (PDGF) beta-receptor. Here, we show that the increased PDGF beta-receptor phosphorylation in TC-PTP knockout (ko) mouse embryonic fibroblasts (MEFs) occurs primarily on the cell surface. The increased phosphorylation is accompanied by a TC-PTP-dependent, monensin-sensitive delay in clearance of cell surface PDGF beta-receptors and delayed receptor degradation, suggesting PDGF beta-receptor recycling. Recycled receptors could also be directly detected on the cell surface of TC-PTP ko MEFs. The effect of TC-PTP depletion was specific for the PDGF beta-receptor, because PDGF alpha-receptor homodimers were cleared from the cell surface at the same rate in TC-PTP ko MEFs as in wild-type MEFs. Interestingly, PDGF alphabeta-receptor heterodimers were recycling. Analysis by confocal microscopy revealed that, in TC-PTP ko MEFs, activated PDGF beta-receptors colocalized with Rab4a, a marker for rapid recycling. In accordance with this, transient expression of a dominant-negative Rab4a construct increased the rate of clearance of cell surface receptors on TC-PTP ko MEFs. Thus, loss of TC-PTP specifically redirects the PDGF beta-receptor toward rapid recycling, which is the first evidence of differential trafficking of PDGF receptor family members.

Clinical Activity of Sorafenib and Sunitinib in Renal Cell Carcinoma Refractory to Previous Vascular Endothelial Growth Factor–Targeted Therapy: Two Case Reports

Sunitinib and sorafenib are multitargeted receptor tyrosine kinase inhibitors of the vascular endothelial growth factor and platelet-derived growth factor receptor families with antiangiogenic and antitumor activity in metastatic renal cell carcinoma. The utility of these agents in patients refractory to previous treatment with the other agent is unknown. We report 2 cases highlighting that efficacy of these agents is possible after failure of the other agent. Further prospective study is needed.

EGF and PDGF Receptor Tyrosine Kinases as Therapeutic Targets for Chronic Lung Diseases

Cell-surface receptor tyrosine kinases play pivotal roles in development, tissue repair, and normal cellular homeostasis. Aberrant expression or signaling patterns of these kinases has also been linked to the progression of a diversity of diseases, including cancer, atherosclerosis, asthma, and fibrosis. Two major families of receptor tyrosine kinases, the epidermal growth factor receptor (EGFR) and platelet-derived growth factor receptor (PDGFR) families, have received a great deal of attention as potential therapeutic targets for pulmonary diseases, as these receptors have been shown to play key roles in chronic tissue remodeling in asthma, bronchitis, and pulmonary fibrosis. The EGFR system on epithelial cells and underlying mesenchymal cells (fibroblasts, myofibroblasts, and smooth muscle cells) drives numerous phenotypic changes during the progression of these pulmonary diseases, including epithelial cell mucous cell metaplasia and mesenchymal cell hyperplasia, differentiation, and extracellular matrix production. The PDGFR system, located primarily on mesenchymal cells, transduces signals for cell survival, growth and chemotaxis. The variety of EGFR and PDGFR ligands produced by the airway epithelium or adjacent mesenchymal cells allows for intimate epithelial-mesenchymal cell communication. A full understanding of the complex mechanisms involving these receptors and ligands should lead to therapeutic strategies for the treatment of a wide range of fibroproliferative lung diseases.

Preclinical activity of ABT-869, a multitargeted receptor tyrosine kinase inhibitor

ABT-869 is a structurally novel, receptor tyrosine kinase (RTK) inhibitor that is a potent inhibitor of members of the vascular endothelial growth factor (VEGF) and platelet-derived growth factor (PDGF) receptor families (e.g., KDR IC50 = 4 nmol/L) but has much less activity (IC50s > 1 micromol/L) against unrelated RTKs, soluble tyrosine kinases, or serine/threonine kinases. The inhibition profile of ABT-869 is evident in cellular assays of RTK phosphorylation (IC50 = 2, 4, and 7 nmol/L for PDGFR-beta, KDR, and CSF-1R, respectively) and VEGF-stimulated proliferation (IC50 = 0.2 nmol/L for human endothelial cells). ABT-869 is not a general antiproliferative agent because, in most cancer cells, >1,000-fold higher concentrations of ABT-869 are required for inhibition of proliferation. However, ABT-869 exhibits potent antiproliferative and apoptotic effects on cancer cells whose proliferation is dependent on mutant kinases, such as FLT3. In vivo ABT-869 is effective orally in the mechanism-based murine models of VEGF-induced uterine edema (ED50 = 0.5 mg/kg) and corneal angiogenesis (>50% inhibition, 15 mg/kg). In tumor growth studies, ABT-869 exhibits efficacy in human fibrosarcoma and breast, colon, and small cell lung carcinoma xenograft models (ED50 = 1.5-5 mg/kg, twice daily) and is also effective (>50% inhibition) in orthotopic breast and glioma models. Reduction in tumor size and tumor regression was observed in epidermoid carcinoma and leukemia xenograft models, respectively. In combination, ABT-869 produced at least additive effects when given with cytotoxic therapies. Based on pharmacokinetic analysis from tumor growth studies, efficacy correlated more strongly with time over a threshold value (cellular KDR IC50 corrected for plasma protein binding = 0.08 microg/mL, >or=7 hours) than with plasma area under the curve or Cmax. These results support clinical assessment of ABT-869 as a therapeutic agent for cancer.

Origin and Molecular Evolution of Receptor Tyrosine Kinases with Immunoglobulin-Like Domains

Receptor tyrosine kinases (RTKs) are involved in the control of fundamental cellular processes in metazoans. In vertebrates, RTK could be grouped in distinct classes based on the nature of their cognate ligand and modular composition of their extracellular domain. RTK with immunoglobulin-like domains (IG-like RTK) encompass several RTK classes and have been found in early metazoans, including sponges. Evolution of IG-like RTK is characterized by extended molecular and functional diversification, which prompted us to study their evolutionary history. For that purpose, a nonredundant data set including annotated protein sequences of IG-like RTK (n = 85) was built, representing 19 species ranging from sponges to humans. Phylogenetic trees were generated from alignment of conserved regions using maximum likelihood approach. Molecular phylogeny strongly suggests that IG-like RTK diversification occurred according to a complex scenario. In particular, we propose that specific cis duplications of a common ancestor to both platelet-derived growth factor receptor (class III) and vascular endothelial growth factor receptor (class V) families preceded two trans duplications. In contrast, other IG-like RTK genes, like Musk and PTK7, apparently did not evolve by duplications, whereas fibroblast growth factor receptors (class IV) evolved through two rounds of trans duplications. The proposed model of IG-like RTK evolution is supported by high bootstrap values and by the clustering of genes encoding class III and class V RTKs at specific chromosomal locations in mouse and human genomes.

Platelet-derived growth factor receptor family mutations in gastrointestinal stromal tumours

Objective. Activating mutations of either KIT or platelet-derived growth factor receptor alpha (PDGFRA) genes are present in the majority of gastrointestinal stromal tumours (GISTs). The type of gene mutation is associated with the aggressiveness of the disease, response to imatinib therapy, and the tumour site in the gastrointestinal tract. However, a subgroup of GISTs does not harbour these mutations. Material and methods. Thirty-three GISTs were studied for mutations in exons encoding the juxtamembrane and the activation loop domains of KIT, PDGFRA, PDGFRB, CSF1R, and FLT3 genes using denaturing high-performance liquid chromatography and gene sequencing. Results. Twenty-two (67%) GISTs had mutation in KIT and 3 (9%) in PDGFRA. The three PDGFRA mutations were all detected in exon 18 of the gene. Three of the 5 GISTs that had weak to moderate KIT expression had a PDGFRA mutation as compared to none of the 26 cases with strong KIT immunopositivity (p=0.022). No mutations were found in PDGFRB, CSF1R or FLT3 in the 8 cases that did not harbour KIT or PDGFRA mutations. Conclusions.KIT and PDGFRA are the most commonly mutated type III receptor tyrosine kinase genes in GIST. GISTs with PDGFRA mutations often have reduced expression of the KIT protein in immunohistochemistry, suggesting that immunohistochemistry may be potentially useful in identification of such GISTs.

The crystal structure of placental growth factor in complex with domain 2 of vascular endothelial growth factor receptor-1.

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor (VEGF) family and plays an important role in pathological angiogenic events. PlGF exerts its biological activities through binding to VEGFR1, a receptor tyrosine kinase that consists of seven immunoglobulin-like domains in its extracellular portion. Here we report the crystal structure of PlGF bound to the second immunoglobulin-like domain of VEGFR1 at 2.5 A resolution and compare the complex to the closely related structure of VEGF bound to the same receptor domain. The two growth factors, PlGF and VEGF, share a sequence identity of approximately 50%. Despite this moderate sequence conservation, they bind to the same binding interface of VEGFR1 in a very similar fashion, suggesting that both growth factors could induce very similar if not identical signaling events.

JC virus-induced changes in cellular gene expression in primary human astrocytes.

Cell-type-specific transcription of the JC virus (JCV) promoter in glial cells initiates a series of events leading to viral replication in the brain and the development of the fatal demyelinating disease progressive multifocal leukoencephalopathy (PML) in patients with neurologic complications due to infection with human immunodeficiency virus type 1. Here we employed an in vitro infection of primary cultures of human astrocytes to compare the transcriptional profile of cellular genes after JCV infection by using an oligonucleotide-based microarray of 12600 genes. Transcription of nearly 355 genes was enhanced and expression of 130 genes was decreased to various degrees. Many transcripts that were increased upon JCV infection were found to encode proteins with properties that suggest their involvement in cell proliferation, including cyclin A and cyclin B1; signaling pathways, such as transforming growth factor beta receptor 1, platelet-derived growth factor receptor and fibroblast growth factor family receptor; and other regulatory events, such as inflammatory responses, including cyclo-oxygenase-2 (Cox-2). Microarray-based data for several cell cycle-regulatory genes were further examined by using Western blot analysis of in vitro infected astrocytes harvested early and late during the infection. Results demonstrate that protein levels of all upregulated genes were found to increase at some point during the infection time course. In parallel, immunohistochemical assessment of cell cycle proteins, including cyclins A, B1, E, and Cdk2, showed positive staining of astrocytes within PML lesions of brain tissue from patients with neuro-AIDS. Microarray analysis was found to be a useful predictor of gene expression in infected cells; however, it may not directly correlate with protein levels during infection with JCV.

Platelet Derived Growth Factor (PDGF) Responsive Epidermis Formed from Human Keratinocytes Transduced with the PDGFβ Receptor Gene

Platelet-derived growth factor is a major proliferative and migratory stimulus for connective tissue cells during the initiation of skin repair processes. In response to injury, locally produced platelet-derived growth factor is secreted by a diversity of cutaneous cell types whereas target activity is confined to cells of mesenchymal origin, e.g. dermal fibroblasts and smooth muscle cells. Although epidermal cells contribute to cutaneous platelet-derived growth factor activity by their ample capacity to secrete platelet-derived growth factor ligand, normal epidermal keratinocytes are not known to express any member of the platelet-derived growth factor receptor family. In order to study if epidermis may be genetically transformed to a platelet-derived growth factor sensitive compartment we aimed to introduce the gene encoding human platelet-derived growth factor receptor beta (PDGF beta R) into epidermal keratinocytes using a retrovirus-derived vector. Successful gene transfer to primary cells was confirmed by immunofluorescence staining, southern blotting, and ligand-induced receptor autophosphorylation. By culturing a mixture of PDGF beta R-transduced and unmodified keratinocytes at the air-liquid interface on devitalized dermis, we were able to establish a multilayered epithelium showing histologic similarities to that evolved from native keratinocytes or keratinocytes transduced with the reporter gene encoding enhanced green fluorescent protein. Receptor-modified epidermal tissue cultured for 6 days and examined by immunofluorescence microscopy was shown to contain PDGF beta R-expressing keratinocytes distributed in all layers of living epidermis. By continued tissue culture in serum-containing medium, the epidermis became increasingly cornified although receptor-positive cells were still observed within the viable basal compartment. Stimulation of PDGF beta R-transduced epidermis with recombinant platelet-derived growth factor BB had a mitogenic effect as reflected by an increased frequency of Ki-67 positive keratinocytes. The study demonstrates that transgene expression of human PDGF beta R can be achieved in epidermal keratinocytes by retroviral transduction, and that ligand activation of such gene-modified skin equivalent enhances cell proliferation. In perspective, viral PDGF beta R gene transfer to keratinocytes may be a useful approach in studies of receptor tyrosine kinase mediated skin repair and epithelialization.

C-kit immunoreactivity in endometrial adenocarcinomas and its clinicopathologic significance.

The proto-oncogene c-kit is a transmembrane-tyrosine-kinase receptor that is structurally related to the platelet-derived growth-factor receptor (PDGFR) and is involved in cell differentiation. C-kit has been found to be expressed in certain solid tumors and may play a role in their tumorigenesis. Recently, a tyrosine-kinase inhibitor specific for the PDGFR family, bcr-abl, and c-kit (STI571) has been reported to have therapeutic effects in tumors expressing the aberrant forms or high quantities of target proteins. Expression of c-kit has not been well evaluated in endometrial adenocarcinomas. In this study, c-kit immunoreactivity was evaluated on paraffin sections of 72 endometrial adenocarcinomas (57 endometrioid, 10 serous, and 5 clear cell) with a polyclonal antibody. Immunoreactivity of c-kit was analyzed semiquantitatively and correlated with various clinicopathologic factors. Cytoplasmic c-kit immunoreactivity was detected in 42 (58%) tumors. Thirty-four (60%) endometrioid, 8 (80%) serous, and 0 of the 5 clear-cell adenocarcinomas were c-kit positive. There was a significant correlation between c-kit positivity and the depth of myometrial invasion. Patients with c-kit-positive endometrial adenocarcinomas more frequently had metastases and shorter disease-free survival. Expression of c-kit may be a potentially adverse prognostic feature in endometrial adenocarcinoma. Patients with c-kit-positive advanced endometrial adenocarcinoma might benefit from tyrosine-kinase-inhibitor therapy.

Receptor tyrosine kinase mutations in myeloid neoplasms.

Receptor tyrosine kinase mutations in myeloid neoplasms.