This study was aimed to investigate the abnormal expression of PDGFRA gene in eosinophilia by FISH. Translocations of PDGFRA gene in 13 patients with eosinophilia were detected by using 4q12 three-color probe and FISH technology. Fifteen people were used as control to establish the normal cut-off value of fluorescence signal of PDGFRA. The results indicated that 1 out of 13 patients with eosinophilia was corrected and was diagnosed as CML. The fusion gene of FIP1L1-PDGFRA (F/P) was found in 2 patients and the positive rate of F/P fusion gene detected by probe 4q12 was 17% in the 12 patients with eosinophilia. Other translocation forms involving PDGFRA gene were not found. It is concluded that a variety of translocation forms of PDGFRA gene can be detected in patients with eosinophilia by using 4q12 three-color probe and FISH technology, which can provide important information for assessing diagnosis and treatment.
Chromosomes, Human, Pair 4 ; Eosinophilia ; metabolism ; Humans ; In Situ Hybridization, Fluorescence ; Oncogene Proteins, Fusion ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; Translocation, Genetic

OBJECTIVEPlatelet-derived growth factor (PDGF) plays an important role during the pathophysiological changes in vascular remodeling. The study aimed to investigate the effect of truncated PDGF-alpha receptor on apoptosis and expression of c-sis mRNA of pulmonary artery smooth muscle cells (VSMCs).

METHODSTissue mass culture was done to get vascular smooth muscle cells of pulmonary artery in newborn pigs. Two methods were used to interfere VSMCs: adding adenoviral recombined body (Ad5CMV-PalphaRtr, ACP) with three different concentrations of truncated PDGF-alpha receptor into the cultures, or adding three concentrations of PDGF-BB after the treatment with mid-concentration of ACP. VSMC apoptosis, cellular cycle and expression of c-sis were observed using flow-cytometry, and the expression of c-sis mRNA was detected by reverse transcription polymerase chain reaction (RT-PCR).

RESULTSACP with mid- to- high concentrations could restrain the proliferation of VSMCs apparently with the increase of G(0)/G(1) cells. The apoptotic rate presented an ascending tendency. The differences among the groups were of statistically significant. Affected by mid- concentration of ACP, PDGF-BB did not exhibit a significantly accelerating effect on the changes of cellular cycle and VSMC apoptosis. The expression of c-sis mRNA was up-regulated under the effect of ACP. Affected by mid-concentration of ACP and PDGF-BB, c-sis mRNA expressed was down-regulated.

CONCLUSIONMid- to- high concentration of ACP is a powerful inhibitor of cellular proliferation for pulmonary artery VSMCs. It can significantly increase cells in number in G(0)/G(1) phase, apoptosis and c-sis mRNA expression.

Animals ; Animals, Newborn ; Apoptosis ; Gene Expression ; Genes, sis ; genetics ; Muscle, Smooth, Vascular ; cytology ; metabolism ; Pulmonary Artery ; cytology ; RNA, Messenger ; genetics ; metabolism ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; physiology ; Reverse Transcriptase Polymerase Chain Reaction ; Swine

OBJECTIVETo explore the effect of malignant transformation of the L839P, a new mutation site of the PDGFRA gene, on the pathogenesis of gastrointestinal stromal tumors.

METHODSAll recombinant plasmids were stably transfected into CHO cells by liposomes. Western blotting was used to detect the expression of PDGFRA protein. The cell growth curve was plotted by cell counting. Flow cytometry was used to detect the cell cycle and apoptosis of CHO cell, respectively. The stably transformed cells were inoculated subcutaneously into the back of nude mice and the mice were used to observe the tumorigenesis. Transient transfection of the mutant-type plasmids of PDGFRA gene and the wild-type plasmids of kit gene into the CHO cells was performed. Western blot was used to detect the expression of kit protein and its phosphorylated forms.

RESULTSPDGFRA protein expressed in the negative control, experimental group and positive control, except the empty vector. The growth curve showed that it was accelerated in the experimental group and positive control. The ratios of cells in proliferative phase were 28.4% (blank), 24.5% (negative control), 43.8% (experimental group) and 40.9% (positive control). Their apoptotic indexes were 1.8%, 1.9%, 1.5% and 1.6%, respectively. After three weeks, tumors were observed in the nude mice of experimental group and positive control, inoculated with the stably transformed cells. Moreover, the expression of phosphorylated protein of kit was enhanced after cotransfection of the mutant-type plasmids of PDGFRA and the wild-type plasmid of kit.

CONCLUSIONThe PDGFRA mutant L839P is a gain-of-function mutation and has obviously malignant transforming effect on normal cells, and may activate kit protein accelerating the tumorigenesis. Gastrointestinal stromal tumors;

Animals ; Apoptosis ; CHO Cells ; Cell Cycle ; Cell Proliferation ; Cell Transformation, Neoplastic ; Cricetinae ; Cricetulus ; Gastrointestinal Stromal Tumors ; etiology ; genetics ; pathology ; Mice ; Mice, Nude ; Mutation ; Plasmids ; Proto-Oncogene Proteins c-kit ; metabolism ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; metabolism ; Transfection

Metastasis represents by far the most feared complication of prostate carcinoma and is the main cause of death for patients. The skeleton is frequently targeted by disseminated cancer cells and represents the sole site of spread in more than 80% of prostate cancer cases. Compatibility between select malignant phenotypes and the microenvironment of colonized tissues is broadly recognized as the culprit for the organ-tropism of cancer cells. Here, we review our recent studies showing that the expression of platelet-derived growth factor receptor alpha (PDGFRα) supports the survival and growth of prostate cancer cells in the skeleton and that the soluble fraction of bone marrow activates PDGFRα in a ligand-independent fashion. Finally, we offer pre-clinical evidence that this receptor is a viable target for therapy.
Animals ; Antibodies, Monoclonal ; therapeutic use ; Bone Marrow ; enzymology ; pathology ; Bone Neoplasms ; prevention & control ; secondary ; Enzyme Activation ; Humans ; Male ; Prostatic Neoplasms ; drug therapy ; enzymology ; pathology ; Receptor, Platelet-Derived Growth Factor alpha ; antagonists & inhibitors ; genetics ; immunology ; metabolism ; Signal Transduction ; Transcriptional Activation

Antigens, CD34 ; metabolism ; Carcinoma, Signet Ring Cell ; genetics ; metabolism ; pathology ; surgery ; Codon ; Diagnosis, Differential ; Exons ; Female ; Follow-Up Studies ; Gastrectomy ; methods ; Gastrointestinal Neoplasms ; genetics ; metabolism ; pathology ; surgery ; Gastrointestinal Stromal Tumors ; genetics ; metabolism ; pathology ; surgery ; Humans ; Melanoma ; metabolism ; pathology ; Middle Aged ; Neurilemmoma ; metabolism ; pathology ; Point Mutation ; Proto-Oncogene Proteins c-kit ; metabolism ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; metabolism

OBJECTIVETo study the immunophenotype and c-kit or platelet derived growth factor receptor alpha (PDGFRA) gene mutations in CD117-negative gastrointestinal stromal tumors (GISTs).

METHODSTen cases of GISTs with typical histologic features but no CD117 expression were retrieved from the archival of Department of Pathology, Peking Union Medical College Hospital, China. The cases were further evaluated for the presence of c-kit exons 9, 11, 13 and 17 mutations and PDGFRA exons 12 and 18 mutations. DNA was extracted from the paraffin-embedded tumor tissue. The PCR products were sequenced directly for the mutations. An immunohistochemical study for CD117, CD34, smooth muscle actin, desmin, S-100 protein, WT-1 and DOG-1 was also performed.

RESULTSEight of the 10 cases had the mutation tests completed. C-kit mutation in exon 9 was detected in only one case. Amongst the 10 cases studied, CD34 was expressed in 9 cases. Smooth muscle actin was focally positive in 2 cases. None of them expressed desmin or S-100 protein. DOG-1 and WT-1 were diffusely positive in 5 and 4 cases, respectively. In addition, DOG1 was diffusely but weakly positive in 1 case and focally expressed in 2 cases. Three cases were focally positive for WT-1.

CONCLUSIONPathologic diagnosis of CD117-negative GISTs can be facilitated with the application of a panel of immunohistochemical markers, including DOG-1 and WT-1.

Actins ; metabolism ; Adult ; Aged ; Anoctamin-1 ; Antigens, CD34 ; metabolism ; Chloride Channels ; Exons ; Female ; Gastrointestinal Stromal Tumors ; genetics ; metabolism ; pathology ; Humans ; Immunophenotyping ; Male ; Membrane Proteins ; metabolism ; Middle Aged ; Mutation ; Neoplasm Proteins ; metabolism ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; metabolism ; WT1 Proteins ; metabolism ; Young Adult

Recent collaborative, large-scale genomic profiling of the most common and aggressive brain tumor glioblastoma multiforme(GBM) has significantly advanced our understanding of this disease. The gene encoding platelet-derived growth factor receptor alpha(PDGFRα) was identified as the third of the top 11 amplified genes in clinical GBM specimens. The important roles of PDGFRα signaling during normal brain development also implicate the possible pathologic consequences of PDGFRα over-activation in glioma. Although the initial clinical trials using PDGFR kinase inhibitors have been predominantly disappointing, diagnostic and treatment modalities involving genomic profiling and personalized medicine are expected to improve the therapy targeting PDGFRα signaling. In this review, we discuss the roles of PDGFRαsignaling during development of the normal central nervous system(CNS) and in pathologic conditions such as malignant glioma. We further compare various animal models of PDGF-induced gliomagenesis and their potential as a novel platform of pre-clinical drug testing. We then summarize our recent publication and how these findings will likely impact treatments for gliomas driven by PDGFRα overexpression. A better understanding of PDGFRα signaling in glioma and their microenvironment, through the use of human or mouse models, is necessary to design a more effective therapeutic strategy against gliomas harboring the aberrant PDGFRα signaling.
Animals ; Antineoplastic Agents ; therapeutic use ; Autocrine Communication ; Brain Neoplasms ; drug therapy ; genetics ; metabolism ; Central Nervous System ; cytology ; embryology ; metabolism ; Disease Models, Animal ; Glioblastoma ; drug therapy ; genetics ; metabolism ; Glioma ; drug therapy ; genetics ; metabolism ; Humans ; Neurons ; cytology ; metabolism ; Protein Kinase Inhibitors ; therapeutic use ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; metabolism

OBJECTIVETo investigate the status of c-kit and PDGFRA mutations of GIST in a the large sample of Chinese patients.

METHODOne hundred and sixty-five cases were evaluated for the presence of c-kit and PDGFRA mutations. Exon 9, 11, 13, 17 of c-kit and exon 12, 18 of PDGFRA were analyzed by PCR amplification and direct sequencing.

RESULTSImmunohistochemical demonstrations of KIT (CD117) were seen in 94% of the cases (155/165). Overall, c-kit mutations were identified in 76.1% (118/155) of CD117 positive cases: 67.1% (104/155) involving exon 11, 7.1% (11/155) involving exon 9, 1.3% (2/155) involving exon 13 and 0.6% (1/155) involving exon 17. The c-kit exon 11 mutations were mostly heterogeneous and clustered in the classic "hot spot" at the 5' end of the exon, including in-frame deletion and point mutation. The second "hot spots" were internal tandem duplications (ITD) at the 3' end of the exon, which were associated with female patient, older age, stomach location and low mitotic counts. The exon 9 mutations correlated with a distinct subset of GISTs involving the small bowel of young male patients. A new point mutation of L641P was identified in exon 13. PDGFRA mutations were present in 50% (5/10) of CD117-negative GISTs, all involving exon 18 with the majority of mutations being D842V. One novel in-frame deletion of IMHD mutation at codon 843 - 846 with S847T was identified. GISTs with PDGFRA mutations were often larger tumors arising from the omentum/mesentery of young male patients with high risk of aggressive behavior.

CONCLUSIONSThe vast majority of GISTs in this study harbored c-kit and PDGFRA mutations, there were non-random relations between the gene mutation patterns and the locations of GISTs. It appears that Chinese GIST patients have some unique mutation patterns. It is necessary to evaluate the gene mutations status of GISTs to guide target therapy.

Adolescent ; Adult ; Aged ; Aged, 80 and over ; Amino Acid Sequence ; Base Sequence ; Child ; DNA Mutational Analysis ; DNA, Neoplasm ; chemistry ; genetics ; Exons ; genetics ; Female ; Gastrointestinal Stromal Tumors ; genetics ; metabolism ; pathology ; Humans ; Immunohistochemistry ; Male ; Middle Aged ; Molecular Sequence Data ; Mutation ; Proto-Oncogene Proteins c-kit ; genetics ; metabolism ; Receptor, Platelet-Derived Growth Factor alpha ; genetics ; metabolism ; Sequence Homology, Amino Acid

To systematically clarify the effects of apolipoprotein E (aopE) and low-density lipoprotein receptor (LDLR) gene mutant on hyperlipidemia, vascular inflammation impairment and pathogenesis of atherosclerosis (AS), total RNA was isolated from fresh aortas of young apoE/LDLR double knockout (apoE(-/-)/LDLR(-/-)) and wild type (WT) mice using TRIzol reagent. Then RNA was reversely transcribed to first-strand cDNA by reverse transcriptase for reverse transcription polymerase chain reaction (RT-PCR) and real-time RT-PCR. Primer pairs were designed using primer design software according to the gene sequences available in GenBank. β-actin was used as an internal control. Then RT-PCR assay was used to analyze the expression patterns of interleukin-1β (IL-1β), tumor necrosis factor-&alpha; (TNF-&alpha;), nuclear factor-κB (NF-κB), granulocyte-macrophage colony-stimulating factor (GM-CSF), CD36, endothelin-1 (ET-1), toll-like receptor 2 (TLR2), monocyte chemoattractant protein-1 (MCP-1), vascular adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1) and platelet-derived growth factor-&alpha; (PDGF-&alpha;). SYBR Green quantitative real-time RT-PCR was used to validate gene expressions identified by RT-PCR. Blood samples were taken from the retro-orbital venous plexus, and serum levels of total cholesterol (TC), triglyceride (TG), low-density lipoprotein (LDL) and high-density lipoprotein (HDL) were measured by using biochemical techniques. Serum concentrations of circulating TNF-&alpha;, IL-1β and oxidized LDL (ox-LDL) were determined by ELISA. Frozen sections of aortic sinus were stained with Sudan IV to visualize intimal fatty lesions. The results showed that the relative expressions of IL-1β, GM-CSF, ET-1, TLR2, CD36, MCP-1, ICAM-1 and VCAM-1 in apoE(-/-)/LDLR(-/-) mice at the age of 1 month were higher than those in age-matched WT mice (P<0.05, P<0.01), respectively. The expressions of PDGF-&alpha; and TNF-&alpha; in apoE(-/-)/LDLR(-/-) mice at the age of 2 months were up-regulated compared to those in age-matched WT mice (P<0.05). All the expressions of target genes continued to be up-regulated (P<0.05, P<0.01) except that ET-1 expression at the age of 2 months, TLR2, VCAM-1 and ICAM-1 expressions at the age of 3 months were down-regulated to that in WT mice. NF-κB expression had no significant changes between two genotype mice at different ages. All the gene expressions kept unchanged in WT mice at different ages, except that IL-1b expressions were slightly up-regulated at the ages of 2 and 3 months. Serum levels of TC, TG, LDL, HDL, TNF-&alpha;, IL-1β and ox-LDL in apoE(-/-)/LDLR(-/-) mice at different ages were higher than those in age-matched WT mice (P<0.05, P<0.01), and were increasing with age. Primary atherosclerotic lesions were observed in 1-month old apoE(-/-)/LDLR(-/-) mice and were progressing with age. There were no lesions observed in all WT mice at different ages. The data suggest that hyperlipidemia due to apoE and LDLR gene mutant may stimulate the temporal expressions of AS-related genes and contribute to primary atherogenetic lesions and vascular inflammation impairment.
Animals ; Aorta ; metabolism ; Apolipoproteins E ; genetics ; Atherosclerosis ; genetics ; CD36 Antigens ; metabolism ; Chemokine CCL2 ; metabolism ; Endothelin-1 ; metabolism ; Gene Expression ; Granulocyte-Macrophage Colony-Stimulating Factor ; metabolism ; Hyperlipidemias ; metabolism ; Intercellular Adhesion Molecule-1 ; metabolism ; Interleukin-1beta ; blood ; metabolism ; Lipoproteins, LDL ; blood ; Mice ; Mice, Knockout ; NF-kappa B ; metabolism ; Platelet-Derived Growth Factor ; Receptors, LDL ; genetics ; Toll-Like Receptor 2 ; metabolism ; Tumor Necrosis Factor-alpha ; blood ; metabolism ; Vascular Cell Adhesion Molecule-1 ; metabolism

BACKGROUND: Eosinophilia may be associated with various primary and reactive conditions. The incidence and the causes of eosinophilia might have been changed according to the changes in the incidence of diseases such as cancer, chronic degenerative diseases, etc. We have conducted a retrospective study to investigate the incidence and causes of eosinophilia. METHODS: Eosinophilia and hypereosinophilia were defined when absolute eosinophil count was greater than 500/microL and 1,500/microL, respectively. Patient's clinical records were reviewed to find out the underlying clinical conditions responsible for causes of hypereosinophilia. Conventional chromosomal analysis, reverse transcriptase PCR and FISH for gene rearrangement were performed to check the presence of clonal eosinophilia. RESULTS: Out of 41,137 patients who had a hematology profile performed, 5,019 (12.2%) and 373 patients (0.9%) were found to have eosinophilia and hypereosinophilia, respectively. Among patients with hypereosinophilia, 227 patients (60.9%) had identifiable and/or possible causes. The major causes of hypereosinophilia were malignancy (35.2%), allergy and skin diseases (18.1%), infectious diseases (15.4%), hepatobiliary diseases (7.5%), bone marrow clonal diseases (6.6%) and parasite infections (6.6%). We also found a rare case of FIP1L1-PDGFRalpha positive chronic eosinophilic leukemia combined with light chain multiple myeloma. CONCLUSIONS: We found a difference in the distribution of causes of hypereosinophilia in comparison with previous Korean studies, and the most common cause of hypereosinophilia in the current study was malignancy. A rare case of clonal eosinophilia (chronic eosinophilic leukemia) associated with multiple myeloma was confirmed using molecular studies.
Adolescent ; Adult ; Age Factors ; Aged ; Aged, 80 and over ; Bone Marrow/pathology ; Child ; Child, Preschool ; Eosinophilia/epidemiology/*etiology/genetics ; Female ; Hospitals, University ; Humans ; Hypereosinophilic Syndrome/epidemiology/*etiology/genetics ; Infant ; Infant, Newborn ; Male ; Middle Aged ; Receptor, Platelet-Derived Growth Factor alpha/genetics/metabolism ; Retrospective Studies ; Sex Factors ; Young Adult ; mRNA Cleavage and Polyadenylation Factors/genetics/metabolism