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  • The coupling of TEL/PDGFbetaR to distinct functional responses is modulated by the presence of cytokine: involvement of mitogen-activated protein kinases. 12714513

    The TEL/PDGFbetaR oncogenic fusion protein is the product of the t(5;12)(q33; p13) translocation recurrently found in patients with chronic myelomonocytic leukemia (CMML). To investigate the coupling of molecular signaling events activated by TEL/PDGFbetaR to functional responses, we expressed TEL/PDGFbetaR in interleukin 3 (IL-3)-dependent BaF/3 cells using the tetracycline-regulated expression system. Induction of TEL/PDGFbetaR expression led to increased cell survival following IL-3 withdrawal and constitutive activation of protein kinase B (PKB), signal transducer and activator of transcription 5 (STAT5), extracellular signal-regulated kinases 1/2 (ERK1/2), Jun N-terminal kinases 1/2 (JNK1/2), and p38 mitogen-activated protein kinase (MAPK) pathways. However, inducible expression of TEL/PDGFbetaR failed to generate factor-independent cells, whereas constitutive expression of TEL/PDGFbetaR did, albeit at low frequency, suggesting the duration of TEL/PDGFbetaR expression is important for transformation. Surprisingly, in cells induced to express TEL/PDGFbetaR, IL-3-dependent growth was dramatically reduced as a result of increased apoptosis of cells receiving combined IL-3 and TEL/PDGFbetaR signals. We demonstrate that TEL/PDGFbetaR expression augmented IL-3-induced activation of PKB, STAT5, ERK1/2, p38, and JNK1/2. Inhibition of neither phosphoinositide-3 kinases nor p38 MAPKs reduced the inhibition of IL-3-driven proliferation observed when TEL/PDGFbetaR was expressed. However, inhibition of MEKs or JNKs partially reversed the combined inhibitory effects of TEL/PDGFbetaR and IL-3 on proliferation and survival. These results suggest that the combination of TEL/PDGFbetaR and IL-3-induced signals activate apoptosis through ERK and JNK MAPK-dependent pathways. Given that in vivo hematopoietic cells are in contact with a variety of cytokines, our results have important implications for cellular responses in the pathogenesis of CMML.
    Document Type:
    Reference
    Product Catalog Number:
    Multiple
    Product Catalog Name:
    Multiple

  • Analysis of expression of growth factor receptors in replicatively and oxidatively senescent human fibroblasts. 16263123

    Replicatively and oxidatively senescent human fibroblasts demonstrate an impaired response to mitogens. To investigate whether this is due to downregulation of growth factor receptors we examined their expression in these two types of senescence. mRNA and protein levels of the insulin receptor and platelet-derived growth factor (PDGF) alpha-receptor decreased in replicatively senescent cells. The PDGF beta-receptor and insulin-like growth factor 1 receptor at the protein level also decreased but remained readily detectable. However, these major growth factor receptors remained unchanged in oxidatively premature senescent cells. This suggests that mechanisms underlying diminished responsiveness to mitogens might be different in replicative senescence and oxidatively premature senescence.
    Document Type:
    Reference
    Product Catalog Number:
    06-498

  • Platelet-derived growth factor and its receptor expression in human oligodendrogliomas. 9482185

    OBJECTIVE: Platelet-derived growth factor (PDGF) induces cellular proliferation and differentiation by activating intracellular signaling mechanisms via their cognate receptors. In previous studies, we demonstrated that human brain tumors such as meningiomas, astrocytomas, medulloblastomas, and ependymomas expressed the messenger ribonucleic acid for the PDGF subunits and their receptors. In the present study, we investigated the expression of the messenger ribonucleic acid PDGF A and B chains and the PDGF alpha and beta receptors in 17 cases of oligodendrogliomas. METHODS: Measurements of messenger ribonucleic acid levels were obtained using radioactive complementary deoxyribonucleic acid probes. Protein expression was analyzed with specific antibodies. RESULTS: Sixteen of 17 tumors expressed the PDGF A subunit and all the PDGF alpha receptors. Furthermore, all the tumors expressed PDGF B and PDGF beta receptor subunits. CONCLUSION: The results of this study suggest that oligodendrogliomas may have an autocrine loop stimulated by the interaction of PDGF and its receptor simultaneously produced by these tumors.
    Document Type:
    Reference
    Product Catalog Number:
    06-498

  • PDGF receptor protein tyrosine kinase expression in the balloon-injured rat carotid artery. 9261258

    Platelet-derived growth factor (PDGF) receptor gene expression has previously been demonstrated in balloon-injured rat carotid arteries to be regulated during repair of carotid injury. In the present study we showed that PDGF receptor protein expression and phosphorylation are changed over time after carotid artery injury. In control and 2-day-postinjury vessels, expression of PDGF alpha receptor protein was readily detectable, whereas PDGF beta receptor expression appeared very low. Between 2 and 7 days postinjury, a time interval previously shown to correspond with smooth muscle cell migration followed by the appearance of a neointima, PDGF alpha receptor expression had increased only slightly, to roughly 35% above control levels, and was maximal by day 7 postinjury, whereas PDGF beta receptor expression had doubled. From 7 to 14 days after carotid injury, intimal area was greatly increased and was associated with a further increase in PDGF beta receptor protein expression and receptor phosphorylation to a maximum between days 10 and 12. In contrast, PDGF alpha receptor expression had decreased slightly during this time interval. Moreover, phosphorylation of PDGF alpha receptors was barely detectable and did not change over the time course of injury. From 14 to 28 days after injury, intimal area was increased only slightly, whereas PDGF beta receptor protein and phosphorylation levels had diminished to roughly half of the 10-day injury values. In addition, the increase in PDGF beta receptor protein expression and tyrosine phosphorylation observed over the time of injury were also associated with a corresponding increase in the association of phosphatidylinositol 3' kinase (PI-3 kinase) with phosphorylated PDGF beta receptors. These findings show that balloon injury to rat carotid arteries results in temporally related changes in the expression of PDGF receptors and their state of tyrosine phosphorylation. Furthermore, tyrosine phosphorylation of PDGF beta receptors in the balloon-injured rat carotid artery in vivo resulted in the association of PI-3 kinase. These are important new findings, which add to our knowledge concerning the role and activity of PDGF receptors in the formation of a neointima.
    Document Type:
    Reference
    Product Catalog Number:
    06-498

  • Adventitial microvessel formation after coronary stenting and the effects of SU11218, a tyrosine kinase inhibitor. 16516095

    OBJECTIVES: The aim of this study was to delineate the temporal profile of adventitial microvessel (Ad-MV) formation after stenting, its relationship to arterial wall hypoxia, and the effects of a tyrosine kinase inhibitor (TKI), SU11218, on Ad-MV and in-stent intimal hyperplasia (IH). BACKGROUND: Adventitial microvessels have been reported after arterial injury; however, the underlying stimulus for this response and its relationship to IH is unknown. METHODS: Coronary stenting was performed in 40 pigs randomized to SU11218 (n = 20) or placebo (n = 20). Vessel wall hypoxia was assessed by pimonidazole adducts and hypoxia-inducible factor (HIF)-1 alpha expression. Adventitial microvessels were quantified by three-dimensional microscopic computed tomography (3D micro CT). Intimal hyperplasia was measured by intravascular ultrasound (IVUS), 3D micro CT, and morphometry. The effects of SU11218 were assessed in vitro on smooth muscle cell (SMC) and endothelial cell (EC) functions and in vivo on Ad-MV and IH. RESULTS: Hypoxia was evident in the vessel wall at 48 h and persisted for four weeks. Adventitial microvessels increased significantly at one week (24 +/- 7 microvessels/segment) and four weeks (23 +/- 7 microvessels/segment) compared with uninjured arteries (16 +/- 2 microvessels/segment; p 0.001) and correlated with IH (r = 0.77, p 0.001). The TKI SU11218 inhibited platelet-derived growth factor receptor-beta phosphorylation, EC and SMC DNA synthesis, and migration in a dose-dependent manner in vitro and significantly inhibited Ad-MV (16 +/- 5 vs. 23 +/- 7 microvessels/segment in placebo, p 0.001) and produced approximately 80% reduction in IH (0.52 +/- 0.51 mm2 vs. 2.47 +/- 1.66 mm2 in placebo, p 0.001) at four weeks in vivo. CONCLUSIONS: Arterial stenting causes arterial wall hypoxia followed by Ad-MV formation. The TKI SU11218 inhibits both Ad-MV formation and IH and represents a promising therapeutic agent to prevent in-stent restenosis.
    Document Type:
    Reference
    Product Catalog Number:
    06-498

  • MARCKS is a downstream effector in platelet-derived growth factor-induced cell motility in activated human hepatic stellate cells. 18329017

    Myristoylated alanine-rich protein kinase c substrate (MARCKS) has been suggested to be implicated in cell adhesion, secretion, motility and mitogenesis through regulation of the actin cytoskeletal structure. In the present study, a possible link between MARCKS and the platelet-derived growth factor (PDGF) signaling pathway was investigated in activated human hepatic stellate cells (hHSC), critical regulators of hepatic fibrogenesis. PDGF-BB stimulation resulted in a bi-directional movement of MARCKS that coincided with the phosphorylation of MARCKS and the activation of both PKCepsilon and PKCalpha. Biochemical inhibition of PKC kinase activity and small interfering RNA (siRNA) against PKCepsilon demonstrated that PKCepsilon is indispensable for PDGF-BB-induced MARCKS phosphorylation and cell migration. Immunoprecipitation studies revealed an association between MARCKS and the PDGFbeta-receptor, while the PDGFbeta-receptor and PKCalpha associated with focal adhesion kinase (FAK). Transient transfection with MARCKS DNA plasmid remarkably reduced PDGF-BB stimulated cell motility. In contrast, siRNA against MARCKS increased cell migration in RNAi treated cells in comparison to the scrambled control cells. In conclusion, the present study indicates that MARCKS play a major key role in PDGF-BB-induced chemotaxis in activated hHSC.
    Document Type:
    Reference
    Product Catalog Number:
    06-498

  • OSI-930: a novel selective inhibitor of Kit and kinase insert domain receptor tyrosine kinases with antitumor activity in mouse xenograft models. 16424037

    OSI-930 is a novel inhibitor of the receptor tyrosine kinases Kit and kinase insert domain receptor (KDR), which is currently being evaluated in clinical studies. OSI-930 selectively inhibits Kit and KDR with similar potency in intact cells and also inhibits these targets in vivo following oral dosing. We have investigated the relationships between the potency observed in cell-based assays in vitro, the plasma exposure levels achieved following oral dosing, the time course of target inhibition in vivo, and antitumor activity of OSI-930 in tumor xenograft models. In the mutant Kit-expressing HMC-1 xenograft model, prolonged inhibition of Kit was achieved at oral doses between 10 and 50 mg/kg and this dose range was associated with antitumor activity. Similarly, prolonged inhibition of wild-type Kit in the NCI-H526 xenograft model was observed at oral doses of 100 to 200 mg/kg, which was the dose level associated with significant antitumor activity in this model as well as in the majority of other xenograft models tested. The data suggest that antitumor activity of OSI-930 in mouse xenograft models is observed at dose levels that maintain a significant level of inhibition of the molecular targets of OSI-930 for a prolonged period. Furthermore, pharmacokinetic evaluation of the plasma exposure levels of OSI-930 at these effective dose levels provides an estimate of the target plasma concentrations that may be required to achieve prolonged inhibition of Kit and KDR in humans and which would therefore be expected to yield a therapeutic benefit in future clinical evaluations of OSI-930.
    Document Type:
    Reference
    Product Catalog Number:
    06-498

  • Calcium-dependent epidermal growth factor receptor transactivation mediates the angiotensin II-induced mitogen-activated protein kinase activation in vascular smooth musc ... 9535870

    We have recently reported that angiotensin II (Ang II)-induced mitogen-activated protein kinase (MAPK) activation is mainly mediated by Ca2+-dependent activation of a protein tyrosine kinase through Gq-coupled Ang II type 1 receptor in cultured rat vascular smooth muscle cells (VSMC). In the present study, we found Ang II rapidly induced the tyrosine phosphorylation of the epidermal growth factor (EGF) receptor and its association with Shc and Grb2. These reactions were inhibited by the EGF receptor kinase inhibitor, AG1478. The Ang II-induced phosphorylation of the EGF receptor was mimicked by a Ca2+ ionophore and completely inhibited by an intracellular Ca2+ chelator. Thus, AG1478 abolished the MAPK activation induced by Ang II, a Ca2+ ionophore as well as EGF but not by a phorbol ester or platelet-derived growth factor-BB in the VSMC. Moreover, Ang II induced association of EGF receptor with catalytically active c-Src. This reaction was not affected by AG1478. These data indicate that Ang II induces Ca2+-dependent transactivation of the EGF receptor which serves as a scaffold for pre-activated c-Src and for downstream adaptors, leading to MAPK activation in VSMC.
    Document Type:
    Reference
    Product Catalog Number:
    06-498

  • A dual inhibitor of platelet-derived growth factor beta-receptor and Src kinase activity potently interferes with motogenic and mitogenic responses to PDGF in vascular sm ... 10400906

    PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.
    Document Type:
    Reference
    Product Catalog Number:
    06-498

  • Inhibition of platelet-derived growth factor signaling attenuates pulmonary fibrosis. 15781583

    Pulmonary fibrosis is the consequence of a variety of diseases with no satisfying treatment option. Therapy-induced fibrosis also limits the efficacy of chemotherapy and radiotherapy in numerous cancers. Here, we studied the potential of platelet-derived growth factor (PDGF) receptor tyrosine kinase inhibitors (RTKIs) to attenuate radiation-induced pulmonary fibrosis. Thoraces of C57BL/6 mice were irradiated (20 Gy), and mice were treated with three distinct PDGF RTKIs (SU9518, SU11657, or Imatinib). Irradiation was found to induce severe lung fibrosis resulting in dramatically reduced mouse survival. Treatment with PDGF RTKIs markedly attenuated the development of pulmonary fibrosis in excellent correlation with clinical, histological, and computed tomography results. Importantly, RTKIs also prolonged the life span of irradiated mice. We found that radiation up-regulated expression of PDGF (A-D) isoforms leading to phosphorylation of PDGF receptor, which was strongly inhibited by RTKIs. Our findings suggest a pivotal role of PDGF signaling in the pathogenesis of pulmonary fibrosis and indicate that inhibition of fibrogenesis, rather than inflammation, is critical to antifibrotic treatment. This study points the way to a potential new approach for treating idiopathic or therapy-related forms of lung fibrosis.
    Document Type:
    Reference
    Product Catalog Number:
    06-498