Derived Growth Factor Receptor Beta Research Articles

Abstract 2506: Preclinical evaluation of sorafenib in combination with ionizing radiation in metastatic breast cancer model

Abstract Sorafenib tosylate (Bay 54-9085) is an oral, small molecule multikinase inhibitor of several targets including RAF/MEK/ERK signaling, vascular endothelial growth factor receptor-2 (VEGFR-2), VEGFR-3, and platelet derived growth factor receptor-beta (PDGFR-b). Sorafenib has shown clinical efficacy in solid tumors such as renal cell and hepatocellular carcinomas. Retrospective analysis of breast cancer patients has shown an unfavorable prognosis in patients with high expression level of VEGF, indicating that VEGF could be associated with efficacy of chemotherapy and radiotherapy. It has also been shown that radiation resistance is partially due to tumor cell production of angiogenic cytokines, particularly VEGF that protects endothelial cells through survival pathways. The aim of this study is to investigate if radiation response is enhanced through inhibition of p-VEGFR2/PDGFR-b by Sorafenib and if the combination of Sorafenib and radiation will increase the treatment response in a metastatic breast cancer model. Mouse metastatic mammary cancer cells, 4T1 were used in this study. Clonogenic assay was performed to assess the radiomodulating effect of Sorafenib. In addition, cell cycle analysis and annexin-V binding assay were performed 24 and 48 hrs post treatment respectively. To confirm our in vitro data, a tumor growth delay assay was completed in a syngeneic mouse model. Western blot analysis showed strong dose dependent inhibition of ERK1/2 phosphorylation by sorafenib. Our clonogenic assay showed a supra-additive effect of Sorafenib (7.5 μM) and radiation (4 Gy) with a dose enhancement factor (DEF) of 1.81. Sorafenib in combination with radiation demonstrated a significant G2/M cell cycle arrest (p<0.0001). Moreover, annexin-V staining assay showed a significant increase in the level of apoptosis in cells treated with both sorafenib and radiation (p<0.0004). Our in vivo results strongly suggest that radiation prolongs the anti tumor activity of sorafenib. In addition, tumor growth was delayed when radiation was combined with sorafenib concurrently (tumor growth delay was increased from 10.5 days in irradiated group to 18 days in combined treatment group). Our results demonstrate that sorafenib increases both the level of apoptosis and the sensitivity of 4T1 cancer cells to radiation. The higher potency of sorafenib combined with radiation can be partially due to the strong cell cycle arrest at G2/M phase. Moreover, our in vivo results show that sorafenib in combination with radiation exhibit a superior tumor growth control. Overall, our findings suggest that greater treatment response may be achieved when sorafenib is combined with radiation. More studies are being conducted to investigate the optimal schedule for this combination. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2506. doi:10.1158/1538-7445.AM2011-2506

Abstract 1367: Substituted Pyrimido[4,5-b]indoles with single agent combination chemotherapeutic potential

Abstract Combinations of cytotoxic agents with antiangiogenic agents have shown significant promise in cancer treatment, and several such clinical trials are currently underway. We previously identified two compounds with a 5-(thioaryl)-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold that each inhibit vascular endothelial growth factor receptor-2 (VEGFR-2) and platelet derived growth factor receptor-beta (PDGFR-β) for antiangiogenic effects and remarkably also inhibit human thymidylate synthase (hTS) for cytotoxic effects. In a COLO-205 xenograft mouse model one of these analogs demonstrated potent tumor growth inhibition, inhibition of metastasis, and antiangiogenic effects in vivo. Thus, these compounds afford combination chemotherapeutic potential in single agents. In an effort to optimize the structural requirements for antiangiogenic effects and hTS inhibition, six additional compounds were synthesized. Electron withdrawing (3,4-diClPh, 4-ClPh), electron donating (2,5-diOMePh, 4-OMePh) and bulky (1-naphthyl, 2-naphthyl) thioaryl groups in the 5-(thioaryl)-9H-pyrimido[4,5-b]indole-2,4-diamine scaffold were selected to establish optimum activity. The synthesis of these compounds involved the nucleophilic displacement of the common intermediate 5-bromo-9H-pyrimido[4,5-b]indole-2,4-diamine with appropriate benzenethiols. A novel four step synthetic scheme to the common intermediate was developed which is more efficient relative to the previously reported six step synthesis. Biological evaluation of these compounds in various RTKs indicated that in the VEGFR-2 assay the 2,5-diOMePh substituted compound was equipotent to the standard SU5416 used in this assay. In the hTS assay, the 2-naphthyl, 4-OMePh, and 4-ClPh substituted compounds were nanomolar inhibitors and were 3-fold better than or equipotent to the standard TS inhibitor raltitrexed. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1367. doi:10.1158/1538-7445.AM2011-1367

Dermatofibrosarcoma protuberans‐derived fibrosarcoma: Clinical history, biological profile and sensitivity to imatinib

Dermatofibrosarcoma Protuberans (DFSP) carries a translocation resulting in the COL1A1/PDGFB fusion-gene, responsible for platelet derived growth factor beta receptor (PDGFRB) activation. Fibrosarcomatous (FS) transformation in DFSP rarely occur. The fusion-gene and PDGFRB expression/activation pattern and imatinib role in DFSP-derived FS is less defined. We reviewed all consecutive patients operated for localized DFSP at our institution from 1994 to 2009, selecting cases with FS component. We also reviewed patients treated with imatinib for advanced FS-DFSP over the same period. When cryopreserved material was available, biochemical/molecular analyses were performed. Of 275 DFSPs, 13 (4.7%) showed a FS component. Fifteen percent of these patients developed metastases, one to the brain. Four patients with DFSP-derived FS received imatinib, with a Response Evaluation Criteria in Solid Tumor Partial Response. Response was followed by early secondary progression in two. One died for brain metastases. Three patients underwent surgery after imatinib. The fusion-gene was detected in all cases in both the classical and FS component, before and after imatinib. PDGFRB expression/activation was confirmed in all cases. mTOR was switched-off, despite the phosphorylation of its effectors. However, a strong phosphorylation of S6 and 4EBP1 was restricted to the FS component. In conclusion, DFSP-derived FS maintains the fusion-gene, being sensitive to imatinib. However, responses are short-lasting. Secondary resistance to imatinib is not related to PDGFRB.

Imatinib has deleterious effects on differentiating spermatogonia while sparing spermatogonial stem cell self renewal

Imatinib mesylate is among a growing number of effective cancer drugs that provide molecularly targeted therapy; however, imatinib causes reproductive defects in rodents. The availability of an in vitro system for screening the effect of drugs on spermatogenesis would be beneficial. The imatinib targets, KIT and platelet derived growth factor receptor beta (PDGFRB), were shown here to be expressed in “germline stem” (GS) cell cultures that contain spermatogonia, including spermatogonial stem cells (SSCs). GS cell cultures were utilized to determine whether imatinib affects SSC self renewal or differentiation. GS cells grown in imatinib retained self renewal based on multiple assays, including transplantation. However, growth in imatinib led to decreased numbers of differentiated spermatogonia and reduced culture growth consistent with the known requirement for KIT in survival and proliferation of spermatogonia. These results build upon the in vivo studies and support the possibility of utilizing GS cell cultures for preclinical drug tests.

Myeloid Neoplasm with PDGFRB Translocation, t(1;5)(q21;q33): A Congenital Presentation of An Imatinib Responsive Congenital JMML with Eosinophilia

AbstractAbstract 4092Eosinophilia is seen in several myeloproliferative disorders (MPD), including the newly WHO recognized category of MPDs associated with translocations involving alpha and beta chains of PDGFR (platelet derived growth factor receptor). A subset of these MPDs, which consistently involve the PDGFRB (platelet derived growth factor receptor beta) gene rearrangement at 5q31–33, demonstrate a t(1;5) (q23;q33). Only a few cases of MPD with this translocation have been described, with the youngest patient being 5 months old at presentation. Imatinib mesylate was recently shown to be efficacious in MPDs with PDGFR rearrangements.Here we describe the first case of a congenital presentation of a myeloproliferative neoplasm with t(1;5) (q21;q33) resulting in PDGFRB rearrangement. The patient, born at 34 weeks gestation, was noted at birth to have hepatomegaly and multiple nodular purpuric cutaneous lesions over his entire body. An extensive work up for congenital infections was negative. A biopsy of the skin lesion shortly after birth demonstrated myeloid proliferation with prominent eosinophilic infiltrate. He continued to demonstrate hepatosplenomegaly and nodular skin lesions at 3 months of age, at which time his white blood cell count (WBC) was 91K/uL with 20% eosinophils, hemoglobin 7g/dl, platelets 35K/uL. Chromosome analysis of peripheral blood revealed a t(1;5) (q21;q33) in 64% of the cells and FISH analysis showed evidence of 5q33 rearrangement involving the PDGFRB locus.The patient was initially treated with a 10 day course of hydroxyurea which resulted in a decrease in the total WBC count, but showed no improvement of the rash or hepatosplenomegaly. After the diagnosis was confirmed, he was started on Imatinib at a dose of 100 mg QD (370 mg/m2). His WBC decreased from 69K/uL to 21K/uL within 4 days and normalized within a month of initiating Imatinib. The dose of Imatinib was decreased to 50 mg QD (185mg/m2) after the first week of therapy. His counts remained normal and a repeat chromosomal analysis of peripheral blood after 10 months of therapy showed no evidence of the prior t(1;5) (q21;q33) abnormality. His dose, therefore, remains at 50 mg QD, which, taking into account his interval growth corresponds to 113 mg/m2. The child is currently 16 months old with normal peripheral blood counts. He is clinically well, growing and developing normally and is tolerating Imatinib without any significant side effects.Summary: Here we describe the first case of a congenital presentation of a myeloid neoplasm with a t(1;5) (q21;q33) translocation and eosinophilia. The child presented at birth with an extensive “blueberry muffin” pattern of cutaneous lesions, hepatomegaly, leukocytosis with eosinophilia, thrombocytopenia and anemia and demonstrated a complete and rapid response to Imatinib monotherapy. There are currently no guidelines on dosing Imatinib in newborns. Three remaining clinical challenges regarding this patient include choosing the best method for monitoring the leukemic clone, choosing the proper dose of Imatinib for this growing child and determining the potential role of bone marrow transplant in an otherwise well infant. Disclosures:No relevant conflicts of interest to declare.

Pericyte-specific expression of PDGF beta receptor in mouse models with normal and deficient PDGF beta receptor signaling

BackgroundPericytes are integral members of the neurovascular unit. Using mouse models lacking endothelial-secreted platelet derived growth factor-B (PDGF-B) or platelet derived growth factor receptor beta (PDGFRβ) on pericytes, it has been demonstrated that PDGF-B/PDGFRβ interactions mediate pericyte recruitment to the vessel wall in the embryonic brain regulating the development of the cerebral microcirculation and the blood-brain barrier (BBB). Relatively little is known, however, about the roles of PDGF-B/PDGFRβ interactions and pericytes in the adult brain in part due to a lack of adequate and/or properly characterized experimental models. To address whether genetic disruption of PDGFRβ signaling would result in a pericyte-specific insult in adult mice, we studied the pattern and cellular distribution of PDGFRβ expression in the brain in adult control mice and F7 mice that express two hypomorphic Pdgfrβ alleles containing seven point mutations in the cytoplasmic domain of PDGFRβ that impair downstream PDGFRβ receptor signaling.ResultsUsing dual fluorescent in situ hybridization, immunofluorescent staining for different cell types in the neurovascular unit, and a fluorescent in situ proximity ligation assay to visualize molecular PDGF-B/PDGFRβ interactions on brain tissue sections, we show for the first time that PDGFRβ is exclusively expressed in pericytes, and not in neurons, astrocytes or endothelial cells, in the adult brain of control 129S1/SvlmJ mice. PDGFRβ co-localized only with well-established pericyte markers such as Chondroitin Sulfate Proteoglycan NG2 and the xLacZ4 transgenic reporter. We next confirm pericyte-specific PDGFRβ expression in the brains of F7 mutants and show that these mice are viable in spite of substantial 40-60% reductions in regional pericyte coverage of brain capillaries.ConclusionsOur data show that PDGFRβ is exclusively expressed in pericytes in the adult 129S1/Sv1mJ and F7 mouse brain. Moreover, our findings suggest that genetic disruption of PDGFRβ signaling results in a pericyte-specific insult in adult F7 mutants and will not exert a primary effect on neurons because PDGFRβ is not expressed in neurons of the adult 129S1/SvlmJ and F7 mouse brain. Therefore, mouse models with normal and deficient PDGFRβ signaling on a 129S1/SvlmJ background may effectively be used to deduce the specific roles of pericytes in maintaining the cerebral microcirculation and BBB integrity in the adult and aging brain as well as during neurodegenerative and brain vascular disorders.

Polymorphisms in TGFB1 and PDGFRB are associated with Moyamoya disease in European patients

The etiology of Moyamoya disease (MMD) is still widely unknown. Several publications on Moyamoya describe differences of cytokine and growth factor concentrations in different specimen. We analyzed the DNA of patients with MMD for single nucleotide polymorphisms (SNPs) in and upstream of the genes for previously described associated cytokines and growth factors. Thirteen SNPs were genotyped in or upstream to four genes-basic fibroblast growth factor (BFGF), cellular retinoic acid-binding protein 1 (CRABP1), platelet derived growth factor receptor beta (PDGFRB), and transforming growth factor beta 1 (TGFB1)-comparing 40 DNA samples of MMD patients to 68 healthy controls from central Europe. Genotyping was performed by sequencing the SNP-containing genetic regions with custom made primers. We found association of two SNPs: rs382861 [A/C] (p = 0.0373, OR = 1.81, 95% CI = 1.03-3.17) in the promoter region of PDGFRB and rs1800471[C/G] (p = 0.0345, OR = 7.65, 95% CI = 0.97-59.95), located in the first exon of TGFB1. Our results indicate possible genetic risk factors for the genesis of MMD. TGFB1 and PDGFRB are involved in vascular growth and transformation processes which may play a role in the development of MMD. Further analyses in larger European cohorts and replication in patients of different ethnicity, as well as functional studies, may lead to possible early detection of patients at risk for developing MMD and subsequently to future preventive therapies.

Fate Tracing Reveals the Pericyte and Not Epithelial Origin of Myofibroblasts in Kidney Fibrosis

Understanding the origin of myofibroblasts in kidney is of great interest because these cells are responsible for scar formation in fibrotic kidney disease. Recent studies suggest epithelial cells are an important source of myofibroblasts through a process described as the epithelial-to-mesenchymal transition; however, confirmatory studies in vivo are lacking. To quantitatively assess the contribution of renal epithelial cells to myofibroblasts, we used Cre/Lox techniques to genetically label and fate map renal epithelia in models of kidney fibrosis. Genetically labeled primary proximal epithelial cells cultured in vitro from these mice readily induce markers of myofibroblasts after transforming growth factor beta(1) treatment. However, using either red fluorescent protein or beta-galactosidase as fate markers, we found no evidence that epithelial cells migrate outside of the tubular basement membrane and differentiate into interstitial myofibroblasts in vivo. Thus, although renal epithelial cells can acquire mesenchymal markers in vitro, they do not directly contribute to interstitial myofibroblast cells in vivo. Lineage analysis shows that during nephrogenesis, FoxD1-positive((+)) mesenchymal cells give rise to adult CD73(+), platelet derived growth factor receptor beta(+), smooth muscle actin-negative interstitial pericytes, and these FoxD1-derivative interstitial cells expand and differentiate into smooth muscle actin(+) myofibroblasts during fibrosis, accounting for a large majority of myofibroblasts. These data indicate that therapeutic strategies directly targeting pericyte differentiation in vivo may productively impact fibrotic kidney disease.

Abstract A35: Tyrosine kinase inhibitors block PDGFR-β signaling and suppress growth in malignant endothelial cells

Abstract Tyrosine kinase inhibitors (TKIs), including imatinib and dasatinib, have shown remarkable efficacy in controlling chronic myelogenous leukemia and gastrointestinal stromal tumors. Their role in treating other solid tumors is less clear. We are investigating the potential to control malignant endothelial cell (EC) growth with TKIs using a spontaneous canine hemangiosarcoma (HSA) model. Canine HSA is a highly malignant EC cancer with cellular features reminiscent of primitive endothelium. Controlling HSA cell growth has implications for improved outcomes of human and canine patients affected with EC cancer as well as for design of antiangiogenic strategies. We evaluated the effects of imatinib and dasatinib on constitutive tyrosine phosphorylation of platelet derived growth factor receptor-beta (PDGFR-β) that we previously determined to be abnormal in HSA cell cultures. Further, we correlated effects of these TKIs on HSA proliferation in vitro to determine if abnormal activation of PDGFR-β contributes to the HSA malignant phenotype. Dasatinib was approximately ten-fold more potent than imatinib in suppressing HSA cell growth. Dasatinib (100 nM) rapidly abrogated signal from full length and a possible truncated form (120 kDa) of phospho-PDGFR-β. Cells with constitutive activation of PDGFR-β also showed tyrosine phosphorylation of extracellular related kinase-1 (ERK-1), ERK-2, and Src suggesting these could be downstream partners of PDGFR-β in aberrant HSA signaling. Dasatinib markedly suppressed phosphorylation of Src in HSA cells. Taken together; these data suggest that tyrosine kinase inhibitors may affect endothelial cell growth in malignancy by blocking PDGFR-β or its downstream signaling partners. Citation Information: Cancer Res 2009;69(23 Suppl):A35.

VEGFR2+PDGFRβ+ circulating precursor cells participate in capillary restoration after hyperoxia acute lung injury (HALI)

The in vivo morphology and phenotype of circulating cells that spontaneously contribute to new vessel formation in adults remain unclear. Here, we use high-resolution imaging and flow cytometry to characterize the morphology and phenotype of a distinct population of circulating mononuclear cells contributing to spontaneous new vessel formation after hyperoxia acute lung injury (HALI). We identify a subpopulation of myeloid (CD11b/Mac1+) haematopoietic cells co-expressing vascular endothelial growth factor receptor 2 (VEGFR2) and platelet derived growth factor receptor beta (PDGFRβ). Moreover, we show that these CD11b+VEGFR2+PDGFRβ+ circulating precursor cells (CPCs) contribute structurally to the luminal surface of capillaries re-forming 2 weeks post-HALI. This indicates that these myeloid CPCs may function, at least transiently, as putative vascular precursors, and has important implications for capillary growth and repair in injury and in pathologies of the lung and other organs.

Review article: pharmacological therapy for hepatocellular carcinoma with sorafenib and other oral agents

Hepatocellular carcinoma (HCC) is the fifth most common malignancy worldwide. Unresectable disease patients have median survival of few months. There is a substantial need for novel treatments for patients with advanced HCC. To provide an update review of mechanism of hepatocarcinigenesis and systemic therapies for HCC and the relevant role of Sorafenib in patients with advanced disease. A Medline search was performed to identify pertinent original research and review articles. Selected references in these articles were also evaluated. Systemic chemotherapy for HCC has been quite ineffective. Preclinical studies demonstrated that Raf/MAPK-ERK kinase (MEK)/Extracellular signal regulated kinase (ERK) pathway has a role in HCC. HCC tumours are highly vascularized and vascular endothelial growth factor (VEGF) augments HCC development and metastasis. Sorafenib blocks tumour cell proliferation by targeting Raf/MEK/ERK signalling and exerts an antiangiogenic effect by targeting VEGF receptors-2/3 and platelet derived growth factor receptor beta tyrosine kinases. Currently available therapies are not effective for patients with advanced HCC. Sorafenib has demonstrated for the first time to prolong survival in patients with advanced HCC, and it is the new reference standard for systemic treatment in these patients.

Differential Cellular Gene Expression in Ganglioglioma

Gangliogliomas (GGs) are neuronal-glial tumors highly associated with epilepsy. We hypothesized that the expression of select gene families including neurotransmitter receptor subunits and growth factors would be distinct in neurons and astrocytes within GG compared with adjacent cortex and that these changes would yield insights into seizure onset and lesion formation. Candidate gene expression was defined in single immunohistochemically labeled neurons and astrocytes microdissected from GG specimens compared with neurons and astrocytes microdissected from morphologically intact cortex adjacent to the GG or normal control cortex. Differential expression of 16 genes including glutamate transporter (EAAC1) and receptor (NMDA2C, mGluR5), growth factor (hepatocyte growth factor), and receptor (platelet derived growth factor receptor beta, fibroblast growth factor receptor 3) mRNAs was detected in GG neurons compared with control neurons. In astrocytes, altered expression of p75NGF, mGluR3, TGFbeta3 and Glt-1 mRNAs was detected. Nestin mRNA, a gene that exhibits enhanced expression in balloon cell cortical dysplasia, was increased in GG neurons. Because of the morphological similarities between GG and cortical dysplasia, we show that there is activation of the mTOR cascade in GG as evidenced by enhanced expression of phospho-p70S6kinase and phosphoribosomal S6 proteins. We find differential candidate gene expression in neurons and astrocytes in GG compared with adjacent cortex and show that there is activation of the mTOR pathway. These changes highlight pathways that may be pivotal for epileptogenesis and lesion growth.

Identification of fibroblast heterogeneity in the tumor microenvironment

Tumors are unorganized organs that contain many different cell types. In the recent years, many studies have reported that primary tumors contain fibroblasts/myofibroblasts (carcinoma-associated fibroblasts), mesenchymal cells such as pericytes/mural cells and other vascular smooth muscle cells. Several different markers are used routinely to identify carcinoma-associated fibroblasts (CAFs) such as alpha-smooth muscle actin (α-SMA), vimentin, S100A4 protein/fibroblast specific protein-1 (FSP1) and type I collagen. Likewise markers such as platelet derived growth factor receptor-beta (PDGFRβ) and NG2 chondroitin sulfate proteoglycan (NG2) are used to identify mesenchymal cells such as pericytes and other vasculature associated smooth muscle cells. It is still unknown whether these markers overlap with each other or identify a unique population of cells within the tumor microenvironment. Therefore in the present study we utilized two different mouse models of cancer, the Rip1Tag2 mice that develop progressive pancreatic cancer and an orthotopic 4T1 breast cancer model, to study the overlap between six different mesenchymal markers commonly used in mouse cancer research. Our study demonstrates that among all the markers, S100A4/FSP1 identifies a unique population of fibroblasts with minimal overlap with markers for αSMA, PDGFRβ and NG2. Vimentin and type I collagen are not specific markers for fibroblasts in these tumors. αSMA, PDGFRβ and NG2 significantly overlap with each other in identifying a mixed population of fibroblasts (activated or resting), myofibroblasts, pericytes and vascular smooth muscle cells. Collectively, this study demonstrates that tumor microenvironment associated fibroblasts are a heterogeneous population and thus, the use of αSMA or vimentin as the only markers will not identify all the CAFs.

Molecular profiles of the mouse postnatal development of the esophageal epithelium showing delayed growth start

Studies on molecular mechanisms of self-renewal in normal stem cells are required for understanding the cancer stem cell. Self-renewal in many kinds of normal stem cells might be accelerated in the growth of a young organism and in the repair of damaged tissue. This study examined whether the esophagus in growing neonates provides an experimental system for studies on epithelial stem cell renewal. The esophageal epithelium consists of 3 layers, from the luminal side to the bottom: the differentiated, epibasal and basal cell layers. The basal cell layer is known to contain the stem cells for the esophageal epithelium. This basic architecture is observed both in mice and humans. We investigated the basal cells in the mouse neonate by immunostaining with a basal cell marker, nerve growth factor receptor (Ngfr), and compared the basal cell content in the esophageal epithelium between mice and humans. A mouse esophageal epithelial cell primary culture system was developed for studies on the basal cell growth and keratinocyte differentiation, and microarray analysis was conducted for obtaining expression profiles of the basal cells. It was revealed that the growth of the esophageal epithelium begins from postnatal day 3, and that the timing is consistent with membrane localization of Ngfr in the basal cell. An increase in the basal cell number by Ngf treatment is observed in in vitro mouse esophageal epithelium cultures. Furthermore, mRNA overexpression of Pdgfrb encoding platelet derived growth factor receptor beta and Egfr encoding epidermal growth factor receptor is associated with the timing of the growth of the esophageal epithelium in the neonatal mice. This study provides a new experimental model for studies on the growth of the basal cells, which are considered to include the stem cells, and on the enlargement of the body size in young organisms.

Imatinib mesylate responsiveness in aggressive systemic mastocytosis: Novel association with a platelet derived growth factor receptor beta mutation

Imatinib mesylate is a high-affinity competitive inhibitor of receptor tyrosine kinases associated with ABL, cKit, platelet derived growth factor receptors (PDGFR) alpha and beta, and certain core binding proteins [1]. It has been used successfully for neoplasms associated with activating mutations of these tyrosine kinases such as: chronic granulocytic leukemia (CGL), Ph+ acute lymphoblastic leukemia (ALL), gastrointestinal stromal tumor, chronic myeloproliferative disorders associated with eosinophilia

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.

A novel DNA vaccine encoding PDGFRβ suppresses growth and dissemination of murine colon, lung and breast carcinoma

Over the past several years it has become apparent that the tumor stroma represents a significant target for anti-cancer therapies. Therefore we evaluated the strategy of targeting the tumor stroma with a novel DNA vaccine encoding murine platelet derived growth factor receptor-beta (mPDGFRβ). Immunization with this vaccine induced cytotoxic lysis of mPDGFRβ-expressing target cells and protected mice from the growth and dissemination of murine colon, breast and lung carcinoma. Furthermore, this novel vaccine suppresses angiogenesis in vivo and reduces the numbers of tumor-associated, mPDGFRβ-expressing pericytes as suggested by a decrease in intra-tumoral expression of mPDGFRβ and NG2.

Vascular endothelialgrowth factor (vEGF) inhibition causes an increase in pericytes and platelet derived growth factor receptor beta(PDGFR-beta) in the vasculogenesis of pulmonary metastasis

Vascular endothelialgrowth factor (vEGF) inhibition causes an increase in pericytes and platelet derived growth factor receptor beta(PDGFR-beta) in the vasculogenesis of pulmonary metastasis

Glivec/STI571 Treatment Stimulates Megakaryopoiesis and Normalizes PDGF Receptor beta Kinase Expression in Thrombocytopenic Patients with Myeloid Metaplasia with Myelofibrosis.

Myeloid Metaplasia with Myelofibrosis (MMM) is a Philadelphia negative chronic myeloproliferative disorder associated with myeloid metaplasia in the spleen and liver, bone marrow fibrosis and neoangiogenesis. The myeloproliferation is characterized by an increased number of circulating CD34+ hematopoietic progenitors (up to 200 folds) with a prominent amplification of dystrophic megakaryocytes (Mk). Alteration of tyrosine kinase signals is suspected to play a key role in the myeloproliferation and in the increased sensitivity of hematopoietic progenitors to growth factors. Glivec is a small-molecule tyrosine kinase inhibitor of the ABL fusion gene, platelet derived growth factor receptor beta (PDGFRB) and SCF receptor (c-kit). In this study, we investigated the effect of Glivec/STI571 on the megakaryopoiesis and the kinase expression of 11 MMM patients enrolled in a European EUMNET clinical trial. We showed that 50 percents of the treated patients exhibited a tendency to a decrease in the CD34+ cell number from 261/μL to 56/μL with a normalization of the CD34+ cell count (1–10/μL) in 20 percents of these patients (2/10). We also observed a reduction of a CD34+CD41+ Mk subpopulation co-expressing the CD9 tetraspanin that is suggested to be a marker of MMM. Normalization of the CD9 gene expression level was further confirmed by quantitative RT-PCR in MMM mononuclear cells. In contrast, 55 percents of MMM patients (5/9 patients) showed an increase in their platelet count from 191 x 109/L to 282 x 109/L during treatment with Glivec. Interestingly, an increase in the platelet count was also observed in three of the five thrombopenic patients who exhibited an increase greater than 50 x 109/L (p= 0.02) being associated with an augmentation in the number of clonogenic megakaryocytic progenitors (CFU-Mk). The effect of Glivec treatment on megakaryopoiesis was further illustrated by a morphometric analysis on bone marrow histological sections showing a normalization of the size and shape of the dystrophic MMM Mk. We also analyzed by quantitative RT-PCR the effect of Glivec treatment on receptor kinase gene expression in circulating mononuclear cells from MMM patients and demonstrated a normalization of the receptor beta of the PDGF, a cytokine involved in Mk differentiation. Actually, the PDGFB expression that was down-regulated in MMM PBMC before Glivec as compared to normal PBMC (−1.14 +/− 0.76 and − 0.14 +/− 0.42, respectively; p= 0.02) was significantly increased during therapy, reaching a normal level in 60% of tested patients. In conclusion, our results show that Glivec treatment reduced the CD34+ cell number and stimulated the megakaryopoiesis of MMM thrombopenic patients, probably by restoring Mk progenitor differentiation.

Protein Gene Product 9.5 is Expressed by Fibroblasts in Human Cutaneous Wounds

In a study initially designed to evaluate reinnervation of human cutaneous wounds using an antibody to the neuroneal marker protein gene product (PGP) 9.5, we observed marked immunostaining of cells with morphologic features of fibroblasts in the wounds. PGP 9.5 has recently been shown to be an important enzyme in the highly conserved ubiquitin system of proteolysis. Because the ubiquitin system is known to play an important role in regulating the cell cycle, the presence of PGP 9.5 in cells at a wound site was of considerable interest. Our objectives were to clarify the time frame for the appearance of PGP 9.5 and ubiquitin in wounds, to verify that PGP 9.5 is produced by wound fibroblasts, and to evaluate a potential role for these proteins in the tissue repair process. Standard incisional human wounds were stained with antibodies specific for PGP 9.5 and ubiquitin. At 7 d, stellate cells with morphologic features of fibroblasts stained for PGP 9.5, whereas earlier wounds were generally negative. In 14 and 21 d incised wounds and in chronic granulation tissue from nonhealing ulcers there was strong cellular staining for PGP 9.5 and for ubiquitin. These stellate cells also showed expression of mRNA for PGP 9.5 by reverse transcriptase-polymerase chain reaction in situ hybridization. PGP 9.5 was detected in cultured fibroblasts both by reverse transcriptase-polymerase chain reaction and by northern blot analysis. Confocal microscopy showed colocalization of antibodies to PGP 9.5 and prolyl-4-hydroxylase (a fibroblast marker) as well as colocalization of PGP 9.5 and the platelet derived growth factor beta receptor. We conclude that ubiquitin and PGP 9.5 were expressed by fibroblasts during the granulation tissue and remodeling phases wound healing. The mRNA for PGP 9.5 was demonstrated in stellate cells in chronic wounds and in fibroblasts in culture. The appearance of these degradative proteins in later wounds suggests a downregulation function in the wound healing response.