c-Kit modifies the inflammatory status of smooth muscle cells

PubMed Central

Song, Lei; Martinez, Laisel; Zigmond, Zachary M.; Hernandez, Diana R.; Lassance-Soares, Roberta M.; Selman, Guillermo

2017-01-01

Background c-Kit is a receptor tyrosine kinase present in multiple cell types, including vascular smooth muscle cells (SMC). However, little is known about how c-Kit influences SMC biology and vascular pathogenesis. Methods High-throughput microarray assays and in silico pathway analysis were used to identify differentially expressed genes between primary c-Kit deficient (KitW/W–v) and control (Kit+/+) SMC. Quantitative real-time RT-PCR and functional assays further confirmed the differences in gene expression and pro-inflammatory pathway regulation between both SMC populations. Results The microarray analysis revealed elevated NF-κB gene expression secondary to the loss of c-Kit that affects both the canonical and alternative NF-κB pathways. Upon stimulation with an oxidized phospholipid as pro-inflammatory agent, c-Kit deficient SMC displayed enhanced NF-κB transcriptional activity, higher phosphorylated/total p65 ratio, and increased protein expression of NF-κB regulated pro-inflammatory mediators with respect to cells from control mice. The pro-inflammatory phenotype of mutant cells was ameliorated after restoring c-Kit activity using lentiviral transduction. Functional assays further demonstrated that c-Kit suppresses NF-κB activity in SMC in a TGFβ-activated kinase 1 (TAK1) and Nemo-like kinase (NLK) dependent manner. Discussion Our study suggests a novel mechanism by which c-Kit suppresses NF-κB regulated pathways in SMC to prevent their pro-inflammatory transformation. PMID:28626608

Masitinib (AB1010), a Potent and Selective Tyrosine Kinase Inhibitor Targeting KIT

PubMed Central

Dubreuil, Patrice; Letard, Sébastien; Ciufolini, Marco; Gros, Laurent; Humbert, Martine; Castéran, Nathalie; Borge, Laurence; Hajem, Bérengère; Lermet, Anne; Sippl, Wolfgang; Voisset, Edwige; Arock, Michel; Auclair, Christian; Leventhal, Phillip S.; Mansfield, Colin D.; Moussy, Alain; Hermine, Olivier

2009-01-01

Background The stem cell factor receptor, KIT, is a target for the treatment of cancer, mastocytosis, and inflammatory diseases. Here, we characterise the in vitro and in vivo profiles of masitinib (AB1010), a novel phenylaminothiazole-type tyrosine kinase inhibitor that targets KIT. Methodology/Principal Findings In vitro, masitinib had greater activity and selectivity against KIT than imatinib, inhibiting recombinant human wild-type KIT with an half inhibitory concentration (IC50) of 200±40 nM and blocking stem cell factor-induced proliferation and KIT tyrosine phosphorylation with an IC50 of 150±80 nM in Ba/F3 cells expressing human or mouse wild-type KIT. Masitinib also potently inhibited recombinant PDGFR and the intracellular kinase Lyn, and to a lesser extent, fibroblast growth factor receptor 3. In contrast, masitinib demonstrated weak inhibition of ABL and c-Fms and was inactive against a variety of other tyrosine and serine/threonine kinases. This highly selective nature of masitinib suggests that it will exhibit a better safety profile than other tyrosine kinase inhibitors; indeed, masitinib-induced cardiotoxicity or genotoxicity has not been observed in animal studies. Molecular modelling and kinetic analysis suggest a different mode of binding than imatinib, and masitinib more strongly inhibited degranulation, cytokine production, and bone marrow mast cell migration than imatinib. Furthermore, masitinib potently inhibited human and murine KIT with activating mutations in the juxtamembrane domain. In vivo, masitinib blocked tumour growth in mice with subcutaneous grafts of Ba/F3 cells expressing a juxtamembrane KIT mutant. Conclusions Masitinib is a potent and selective tyrosine kinase inhibitor targeting KIT that is active, orally bioavailable in vivo, and has low toxicity. PMID:19789626

FES kinase participates in KIT-ligand induced chemotaxis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Voisset, Edwige, E-mail: Edwige.Voisset@inserm.fr; Institut Paoli-Calmettes, Marseille; Universite de la Mediterranee, Aix-Marseille II

2010-02-26

FES is a cytoplasmic tyrosine kinase activated by several membrane receptors, originally identified as a viral oncogene product. We have recently identified FES as a crucial effector of oncogenic KIT mutant receptor. However, FES implication in wild-type KIT receptor function was not addressed. We report here that FES interacts with KIT and is phosphorylated following activation by its ligand SCF. Unlike in the context of oncogenic KIT mutant, FES is not involved in wild-type KIT proliferation signal, or in cell adhesion. Instead, FES is required for SCF-induced chemotaxis. In conclusion, FES kinase is a mediator of wild-type KIT signalling implicatedmore » in cell migration.« less

qPCR in gastrointestinal stromal tumors: Evaluation of reference genes and expression analysis of KIT and the alternative receptor tyrosine kinases FLT3, CSF1-R, PDGFRB, MET and AXL

PubMed Central

2010-01-01

Background Gastrointestinal stromal tumors (GIST) represent the most common mesenchymal tumors of the gastrointestinal tract. About 85% carry an activating mutation in the KIT or PDGFRA gene. Approximately 10% of GIST are so-called wild type GIST (wt-GIST) without mutations in the hot spots. In the present study we evaluated appropriate reference genes for the expression analysis of formalin-fixed, paraffin-embedded and fresh frozen samples from gastrointestinal stromal tumors. We evaluated the gene expression of KIT as well as of the alternative receptor tyrosine kinase genes FLT3, CSF1-R, PDGFRB, AXL and MET by qPCR. wt-GIST were compared to samples with mutations in KIT exon 9 and 11 and PDGFRA exon 18 in order to evaluate whether overexpression of these alternative RTK might contribute to the pathogenesis of wt-GIST. Results Gene expression variability of the pooled cDNA samples is much lower than the single reverse transcription cDNA synthesis. By combining the lowest variability values of fixed and fresh tissue, the genes POLR2A, PPIA, RPLPO and TFRC were chosen for further analysis of the GIST samples. Overexpression of KIT compared to the corresponding normal tissue was detected in each GIST subgroup except in GIST with PDGFRA exon 18 mutation. Comparing our sample groups, no significant differences in the gene expression levels of FLT3, CSF1R and AXL were determined. An exception was the sample group with KIT exon 9 mutation. A significantly reduced expression of CSF1R, FLT3 and PDGFRB compared to the normal tissue was detected. GIST with mutations in KIT exon 9 and 11 and in PDGFRA exon 18 showed a significant PDGFRB downregulation. Conclusions As the variability of expression levels for the reference genes is very high comparing fresh frozen and formalin-fixed tissue there is a strong need for validation in each tissue type. None of the alternative receptor tyrosine kinases analyzed is associated with the pathogenesis of wild-type or mutated GIST. It

Temporal quantitation of mutant Kit tyrosine kinase signaling attenuated by a novel thiophene kinase inhibitor OSI-930.

PubMed

Petti, Filippo; Thelemann, April; Kahler, Jen; McCormack, Siobhan; Castaldo, Linda; Hunt, Tony; Nuwaysir, Lydia; Zeiske, Lynn; Haack, Herbert; Sullivan, Laura; Garton, Andrew; Haley, John D

2005-08-01

OSI-930, a potent thiophene inhibitor of the Kit, KDR, and platelet-derived growth factor receptor tyrosine kinases, was used to selectively inhibit tyrosine phosphorylation downstream of juxtamembrane mutant Kit in the mast cell leukemia line HMC-1. Inhibition of Kit kinase activity resulted in a rapid dephosphorylation of Kit and inhibition of the downstream signaling pathways. Attenuation of Ras-Raf-Erk (phospho-Erk, phospho-p38), phosphatidyl inositol-3' kinase (phospho-p85, phospho-Akt, phospho-S6), and signal transducers and activators of transcription signaling pathways (phospho-STAT3/5/6) were measured by affinity liquid chromatography tandem mass spectrometry, by immunoblot, and by tissue microarrays of fixed cell pellets. To more globally define additional components of Kit signaling temporally altered by kinase inhibition, a novel multiplex quantitative isobaric peptide labeling approach was used. This approach allowed clustering of proteins by temporal expression patterns. Kit kinase, which dephosphorylates rapidly upon kinase inhibition, was shown to regulate both Shp-1 and BDP-1 tyrosine phosphatases and the phosphatase-interacting protein PSTPIP2. Interactions with SH2 domain adapters [growth factor receptor binding protein 2 (Grb2), Cbl, Slp-76] and SH3 domain adapters (HS1, cortactin, CD2BP3) were attenuated by inhibition of Kit kinase activity. Functional crosstalk between Kit and the non-receptor tyrosine kinases Fes/Fps, Fer, Btk, and Syk was observed. Inhibition of Kit modulated phosphorylation-dependent interactions with pathways controlling focal adhesion (paxillin, leupaxin, p130CAS, FAK1, the Src family kinase Lyn, Wasp, Fhl-3, G25K, Ack-1, Nap1, SH3P12/ponsin) and septin-actin complexes (NEDD5, cdc11, actin). The combined use of isobaric protein quantitation and expression clustering, immunoblot, and tissue microarray strategies allowed temporal measurement signaling pathways modulated by mutant Kit inhibition in a model of mast cell

Synthesis and biological evaluation of di-aryl urea derivatives as c-Kit inhibitors.

PubMed

Ravez, Séverine; Arsenlis, Stéphane; Barczyk, Amélie; Dupont, Anthony; Frédérick, Raphaël; Hesse, Stéphanie; Kirsch, Gilbert; Depreux, Patrick; Goossens, Laurence

2015-11-15

Inhibition of receptor tyrosine kinases (RTKs) continued to be a successful approach for the treatment of many types of human cancers and many potent small molecules kinase inhibitors have been discovered the last decade. In the present study, we describe the synthesis of thienopyrimidine derivatives and their pharmacological evaluation against nine kinases (EGFR, PDGFR-ß, c-Kit, c-Met, Src, Raf, VEGFR-1, -2 and -3). Most of the synthesized compounds showed from moderate to potent activities against c-Kit with IC50 values in the nanomolar range. Among them, 4-anilino(urea)thienopyrimidine analogs showed selectivity and potent c-Kit inhibition with IC50 values less than 6 nM. Docking simulation was performed for the most promising compound 9 into the c-Kit active site to determine the potential binding mode. This study reveal that the 4-anilino(urea)thienopyrimidine is an interesting scaffold to design novel potent and selective c-Kit inhibitors which may make promising candidates for cancers where c-Kit receptors are overexpressed. Copyright © 2015 Elsevier Ltd. All rights reserved.

Essential requirement for PP2A inhibition by the oncogenic receptor c-KIT suggests PP2A reactivation as a strategy to treat c-KIT+ cancers

PubMed Central

Roberts, Kathryn G.; Smith, Amanda M.; McDougall, Fiona; Carpenter, Helen; Horan, Martin; Neviani, Paolo; Powell, Jason A.; Thomas, Daniel; Guthridge, Mark A.; Perrotti, Danilo; Sim, Alistair T.R.; Ashman, Leonie K.; Verrills, Nicole M.

2010-01-01

Oncogenic mutations of the receptor tyrosine kinase c-KIT play an important role in the pathogenesis of gastrointestinal stromal tumors (GIST), systemic mastocytosis, and some acute myeloid leukemias (AML). Whilst juxtamembrane mutations commonly detected in GIST are sensitive to tyrosine kinase inhibitors, the kinase domain mutations frequently encountered in systemic mastocytosis and AML confer resistance and are largely unresponsive to targeted inhibition by the existing agent imatinib. In this study we show that myeloid cells expressing activated c-KIT mutants that are imatinib-sensitive (V560G) or –resistant (D816V) can inhibit the tumor suppressor activity of protein phosphatase 2A (PP2A). This effect was associated with reduced expression of PP2A structural (A) and regulatory subunits (B55α; B56α; B56γ and B56δ). Overexpression of PP2A-Aα in D816V c-KIT cells induced apoptosis and inhibited proliferation. In addition, pharmacological activation of PP2A by FTY720 reduced proliferation, inhibited clonogenic potential and induced apoptosis of mutant c-KIT+ cells, whilst having no effect on WT c-KIT cells or empty vector controls. FTY720 treatment caused dephosphorylation of the D816V c-KIT receptor and its downstream signaling targets pAkt, pSTAT5 and pERK1/2. Additionally, in vivo administration of FTY720 delayed the growth of V560G and D816V c-KIT tumors, inhibited splenic and bone marrow infiltration, and prolonged survival. Our findings show that PP2A inhibition is essential for c-KIT-mediated tumorigenesis, and that reactivating PP2A may offer an attractive strategy to treat drug-resistant c-KIT+ cancers. PMID:20551067

Signal transduction by normal isoforms and W mutant variants of the Kit receptor tyrosine kinase.

PubMed

Reith, A D; Ellis, C; Lyman, S D; Anderson, D M; Williams, D E; Bernstein, A; Pawson, T

1991-09-01

Germline mutations at the Dominant White Spotting (W) and Steel (Sl) loci have provided conclusive genetic evidence that c-kit mediated signal transduction pathways are essential for normal mouse development. We have analysed the interactions of normal and mutant W/c-kit gene products with cytoplasmic signalling proteins, using transient c-kit expression assays in COS cells. In addition to the previously identified c-kit gene product (Kit+), a second normal Kit isoform (KitA+) containing an in-frame insertion, Gly-Asn-Asn-Lys, within the extracellular domain, was detected in murine mast cell cultures and mid-gestation placenta. Both Kit+ and KitA+ isoforms showed increased autophosphorylation and enhanced association with phosphatidylinositol (PI) 3' kinase and PLC gamma 1, when stimulated with recombinant soluble Steel factor. No association or increase in phosphorylation of GAP and two GAP-associated proteins, p62 and p190, was observed. The two isoforms had distinct activities in the absence of exogenous soluble Steel factor; Kit+, but not KitA+, showed constitutive tyrosine phosphorylation that was accompanied by a low constitutive level of association with PI-3' kinase and PLC gamma 1. Introduction of the point substitutions associated with W37 (Glu582----Lys) or W41 (Val831----Met) mutant alleles into c-kit expression constructs abolished (W37) or reduced (W41) the Steel factor-induced association of the Kit receptor with signalling proteins in a manner proportional to the overall severity of the corresponding W mutant phenotype. These data suggest a diversity of normal Kit signalling pathways and indicate that W mutant phenotypes result from primary defects in the Kit receptor that affect its interaction with cytoplasmic signalling proteins.

A potencial theranostic agent for EGF-R expression tumors: (177)Lu-DOTA-nimotuzumab.

PubMed

Calzada, Victoria; Zhang, Xiuli; Fernandez, Marcelo; Diaz-Miqueli, Arlhee; Iznaga-Escobar, Normando; Deutscher, Susan L; Balter, Henia; Quinn, Thomas P; Cabral, Pablo

2012-10-01

In this work Nimotuzumab (monoclonal antibody, recognizes the EGF-R) was radiolabeled with (177)Lu as a potential cancer therapy radiopharmaceutical. In-vitro cell binding studies and in-vivo biodistribution and imaging studies were performed to determine the radiochemical stability, targeting specificity and pharmacokinetics of the (177)Lu-labeled antibody. Nimotuzumab was derivatized with DOTA-NHS at room temperature for 2 hours. DOTA-Nimotuzumab was radiolabeled with (177)LuCl3 (15 MBq/mg) at 37°C for 1 h. The radiochemical purity was assessed by ITLC, silica gel and by RP-HPLC. Binding specificity studies were performed with EGF-R positive A431 human epithelial carcinoma and EGF-R negative MDA-MB-435 breast carcinoma cells. Biodistribution studies were performed in healthy female CD-1 mice at 1 h, 4 h, 24 h, and A431 xenografted nude mice at 10 min, 1 h, 4 h, 24 h, 48 h, and 96 h. SPECT-CT imaging studies were performed in A431 xenografted mice at 24 h post injection. DOTA-Nimotuzumab was efficiently labeled with (177) LuCl(3) at 37°C. The in vitro stability of labeled product was optimal over 24 h in buffered saline and mouse serum. Specific recognition of EGF-R by (177)Lu-DOTA-Nimotuzumab was observed in A431 cell binding studies. Biodistribution studies demonstrated increasing tumor uptake of (177)Lu-DOTA-Nimotuzumab over time, with tumor to muscle ratios of 6.26, 10.68, and 18.82 at 4 h, 24 h, and 96 h post injection. Imaging of A431 xenografted mice showed high uptake in the tumor. (177)Lu-DOTA-Nimotuzumab has the potential to be a promising therapy agent, which may be useful in the treatment of patients with EGF-R positive cancer.

CysLT1 receptor-induced human airway smooth muscle cells proliferation requires ROS generation, EGF receptor transactivation and ERK1/2 phosphorylation

PubMed Central

Ravasi, Saula; Citro, Simona; Viviani, Barbara; Capra, Valérie; Rovati, G Enrico

2006-01-01

Background Cysteine-containing leukotrienes (cysteinyl-LTs) are pivotal inflammatory mediators that play important roles in the pathophysiology of asthma, allergic rhinitis, and other inflammatory conditions. In particular, cysteinyl-LTs exert a variety of effects with relevance to the aetiology of asthma such as smooth muscle contraction, eosinophil recruitment, increased microvascular permeability, enhanced mucus secretion and decreased mucus transport and, finally, airway smooth muscle cells (ASMC) proliferation. We used human ASMC (HASMC) to identify the signal transduction pathway(s) of the leukotriene D4 (LTD4)-induced DNA synthesis. Methods Proliferation of primary HASMC was measured by [3H]thymidine incorporation. Phosphorylation of EGF receptor (EGF-R) and ERK1/2 was assessed with a polyclonal anti-EGF-R or anti-phosphoERKl/2 monoclonal antibody. A Ras pull-down assay kit was used to evaluate Ras activation. The production of reactive oxygen species (ROS) was estimated by measuring dichlorodihydrofluorescein (DCF) oxidation. Results We demonstrate that in HASMC LTD4-stimulated thymidine incorporation and potentiation of EGF-induced mitogenic signaling mostly depends upon EGF-R transactivation through the stimulation of CysLT1-R. Accordingly, we found that LTD4 stimulation was able to trigger the increase of Ras-GTP and, in turn, to activate ERK1/2. We show here that EGF-R transactivation was sensitive to pertussis toxin (PTX) and phosphoinositide 3-kinase (PI3K) inhibitors and that it occurred independently from Src activity, despite the observation of a strong impairment of LTD4-induced DNA synthesis following Src inhibition. More interestingly, CysLT1-R stimulation increased the production of ROS and N-acetylcysteine (NAC) abolished LTD4-induced EGF-R phosphorylation and thymidine incorporation. Conclusion Collectively, our data demonstrate that in HASMC LTD4 stimulation of a Gi/o coupled CysLT1-R triggers the transactivation of the EGF-R through the

SHP-1 Binds and Negatively Modulates the c-Kit Receptor by Interaction with Tyrosine 569 in the c-Kit Juxtamembrane Domain

PubMed Central

Kozlowski, Maya; Larose, Louise; Lee, Fai; Le, Duc Mingh; Rottapel, Robert; Siminovitch, Katherine A.

1998-01-01

The SH2 domain-containing SHP-1 tyrosine phosphatase has been shown to negatively regulate a broad spectrum of growth factor- and cytokine-driven mitogenic signaling pathways. Included among these is the cascade of intracellular events evoked by stem cell factor binding to c-Kit, a tyrosine kinase receptor which associates with and is dephosphorylated by SHP-1. Using a series of glutathione S-transferase (GST) fusion proteins containing either tyrosine-phosphorylated segments of the c-Kit cytosolic region or the SH2 domains of SHP-1, we have shown that SHP-1 interacts with c-Kit by binding selectively to the phosphorylated c-Kit juxtamembrane region and that the association of c-Kit with the larger of the two SHP-1 isoforms may be mediated through either the N-terminal or C-terminal SHP-1 SH2 domain. The results of binding assays with mutagenized GST-Kit juxtamembrane fusion proteins and competitive inhibition assays with phosphopeptides encompassing each c-Kit juxtamembrane region identified the tyrosine residue at position 569 as the major site for binding of SHP-1 to c-Kit and suggested that tyrosine 567 contributes to, but is not required for, this interaction. By analysis of Ba/F3 cells retrovirally transduced to express c-Kit receptors, phenylalanine substitution of c-Kit tyrosine residue 569 was shown to be associated with disruption of c-Kit–SHP-1 binding and induction of hyperproliferative responses to stem cell factor. Although phenylalanine substitution of c-Kit tyrosine residue 567 in the Ba/F3–c-Kit cells did not alter SHP-1 binding to c-Kit, the capacity of a second c-Kit-binding tyrosine phosphatase, SHP-2, to associate with c-Kit was markedly reduced, and the cells again showed hyperproliferative responses to stem cell factor. These data therefore identify SHP-1 binding to tyrosine 569 on c-Kit as an interaction pivotal to SHP-1 inhibitory effects on c-Kit signaling, but they indicate as well that cytosolic protein tyrosine phosphatases other

Nociceptive tuning by stem cell factor/c-Kit signaling.

PubMed

Milenkovic, Nevena; Frahm, Christina; Gassmann, Max; Griffel, Carola; Erdmann, Bettina; Birchmeier, Carmen; Lewin, Gary R; Garratt, Alistair N

2007-12-06

The molecular mechanisms regulating the sensitivity of sensory circuits to environmental stimuli are poorly understood. We demonstrate here a central role for stem cell factor (SCF) and its receptor, c-Kit, in tuning the responsiveness of sensory neurons to natural stimuli. Mice lacking SCF/c-Kit signaling displayed profound thermal hypoalgesia, attributable to a marked elevation in the thermal threshold and reduction in spiking rate of heat-sensitive nociceptors. Acute activation of c-Kit by its ligand, SCF, resulted in a reduced thermal threshold and potentiation of heat-activated currents in isolated small-diameter neurons and thermal hyperalgesia in mice. SCF-induced thermal hyperalgesia required the TRP family cation channel TRPV1. Lack of c-Kit signaling during development resulted in hypersensitivity of discrete mechanoreceptive neuronal subtypes. Thus, c-Kit can now be grouped with a small family of receptor tyrosine kinases, including c-Ret and TrkA, that control the transduction properties of sensory neurons.

Disruption of c-Kit Signaling in Kit(W-sh/W-sh) Growing Mice Increases Bone Turnover.

PubMed

Lotinun, Sutada; Krishnamra, Nateetip

2016-08-16

c-Kit tyrosine kinase receptor has been identified as a regulator of bone homeostasis. The c-Kit loss-of-function mutations in WBB6F1/J-Kit(W/W-v) mice result in low bone mass. However, these mice are sterile and it is unclear whether the observed skeletal phenotype is secondary to a sex hormone deficiency. In contrast, C57BL/6J-Kit(W-sh)/(W-sh) (W(sh)/W(sh)) mice, which carry an inversion mutation affecting the transcriptional regulatory elements of the c-Kit gene, are fertile. Here, we showed that W(sh)/W(sh) mice exhibited osteopenia with elevated bone resorption and bone formation at 6- and 9-week-old. The c-Kit W(sh) mutation increased osteoclast differentiation, the number of committed osteoprogenitors, alkaline phosphatase activity and mineralization. c-Kit was expressed in both osteoclasts and osteoblasts, and c-Kit expression was decreased in W(sh)/W(sh)osteoclasts, but not osteoblasts, suggesting an indirect effect of c-Kit on bone formation. Furthermore, the osteoclast-derived coupling factor Wnt10b mRNA was increased in W(sh)/W(sh) osteoclasts. Conditioned medium from W(sh)/W(sh) osteoclasts had elevated Wnt10b protein levels and induced increased alkaline phosphatase activity and mineralization in osteoblast cultures. Antagonizing Wnt10b signaling with DKK1 or Wnt10b antibody inhibited these effects. Our data suggest that c-Kit negatively regulates bone turnover, and disrupted c-Kit signaling couples increased bone resorption with bone formation through osteoclast-derived Wnt 10 b.

Radotinib induces high cytotoxicity in c-KIT positive acute myeloid leukemia cells.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-06-05

Previously, we reported that radotinib, a BCR-ABL1 tyrosine kinase inhibitor, induced cytotoxicity in acute myeloid leukemia (AML) cells. However, the effects of radotinib in the subpopulation of c-KIT-positive AML cells were unclear. We observed that low-concentration radotinib had more potent cytotoxicity in c-KIT-positive cells than c-KIT-negative cells from AML patients. To address this issue, cell lines with high c-KIT expression, HEL92.1.7, and moderate c-KIT expression, H209, were selected. HEL92.1.7 cells were grouped into intermediate and high c-KIT expression populations. The cytotoxicity of radotinib against the HEL92.1.7 cell population with intermediate c-KIT expression was not different from that of the population with high c-KIT expression. When H209 cells were grouped into c-KIT expression-negative and c-KIT expression-positive populations, radotinib induced cytotoxicity in the c-KIT-positive population, but not the c-KIT-negative population. Thus, radotinib induces cytotoxicity in c-KIT-positive cells, regardless of the c-KIT expression intensity. Therefore, radotinib induces significant cytotoxicity in c-KIT-positive AML cells, suggesting that radotinib is a potential target agent for the treatment of c-KIT-positive malignancies including AML. Copyright © 2017 Elsevier B.V. All rights reserved.

Direct association of Csk homologous kinase (CHK) with the diphosphorylated site Tyr568/570 of the activated c-KIT in megakaryocytes.

PubMed

Price, D J; Rivnay, B; Fu, Y; Jiang, S; Avraham, S; Avraham, H

1997-02-28

The Csk homologous kinase (CHK), formerly MATK, has previously been shown to bind to activated c-KIT. In this report, we characterize the binding of SH2(CHK) to specific phosphotyrosine sites on the c-KIT protein sequence. Phosphopeptide inhibition of the in vitro interaction of SH2(CHK)-glutathione S-transferase fusion protein/c-KIT from SCF/KL-treated Mo7e megakaryocytic cells indicated that two sites on c-KIT were able to bind SH2(CHK). These sites were the Tyr568/570 diphosphorylated sequence and the monophosphorylated Tyr721 sequence. To confirm this, we precipitated native CHK from cellular extracts using phosphorylated peptides linked to Affi-Gel 15. In addition, purified SH2(CHK)-glutathione S-transferase fusion protein was precipitated with the same peptide beads. All of the peptide bead-binding studies were consistent with the direct binding of SH2(CHK) to phosphorylated Tyr568/570 and Tyr721 sites. Binding of FYN and SHC to the diphosphorylated Tyr568/570 site was observed, while binding of Csk to this site was not observed. The SH2(CHK) binding to the two sites is direct and not through phosphorylated intermediates such as FYN or SHC. Site-directed mutagenesis of the full-length c-KIT cDNA followed by transient transfection indicated that only the Tyr568/570, and not the Tyr721, is able to bind SH2(CHK). This indicates that CHK binds to the same site on c-KIT to which FYN binds, possibly bringing the two into proximity on associated c-KIT subunits and leading to the down-regulation of FYN by CHK.

Localisation of epidermal growth factor (EGF), its specific receptor (EGF-R) and aromatase at the materno-fetal interface during placentation in the pregnant mare.

PubMed

Allen, W R Twink; Gower, Susan; Wilsher, Sandra

2017-02-01

Implantation and placentation in the mare does not commence until as late as day 40 after ovulation. The reasons for this and the growth factors and/or hormones which drive placentation when it does finally occur are of considerable academic and practical interest. Placental interface tissues recovered from 11 accurately aged and perfused-fixed horse uteri between 20 and 68 days of gestation were stained immunocytochemically for Epidermal Growth Factor (EGF), its specific receptor (EGF-R) and for the steroid hormone enzyme, aromatase. EGF was present in endometrial gland and lumenal epithelia from day 20 but staining intensity increased noticeably for the protein between days 30 and 40, coincidentally with the commencing secretion of equine Chorionic Gonadotrophin (eCG) from the endometrial cups and immediately prior to attachment and commencing interdigitation between the allantochorion and endometrium. EGF-R, on the other hand, was expressed strongly on the cell surface membrane of both non-invasive and invasive trophoblast and it similarly increased in staining intensity between days 30 and 40. Aromatase, the enzyme necessary for conversion of C-19 androgens to C-18 oestrogens, was expressed strongly and constantly from as early as day 12 in the non-invasive trophoblast of the allantochorion, but not the invasive trophoblast of the chorionic girdle, the progenitor tissue of the endometrial cups. The findings support the hypothesis that, in equine pregnancy, the maternal growth factor EGF synergises with maternally and fetally secreted oestrogens to drive the rapid growth and extensive vascularisation of the non-invasive, epitheliochorial, microcotyledonary placenta which results in the birth of the precocious foal after only 11 months gestation. Copyright © 2016 Elsevier Ltd. All rights reserved.

Molecular Characterization of PDGFR-α/PDGF-A and c-KIT/SCF in Gliosarcomas

PubMed Central

Reis, Rui M.; Martins, Albino; Ribeiro, Susana A.; Basto, Diana; Longatto-Filho, Adhemar; Schmitt, Fernando C.; Lopes, José M.

2005-01-01

Gliosarcomas are rare and poorly characterized malignant brain tumors that exhibit a biphasic tissue pattern with areas of gliomatous and sarcomatous differentiation. These tumors are histological variants of glioblastoma, displaying a similar genetic profile and dismal prognosis. Up-regulation of PDGFR subfamily of tyrosine kinase members, PDGFR-α and c-Kit, and their intracellular effectors RAS/RAF/MAPK has a crucial role in the cancer development. In addition, signal transduction mediated by activating mutations of c-Kit and PDGFR can be effectively blocked by specific tyrosine kinase inhibitors, such as Imatinib mesylate. The aim of this study was to characterize the molecular alterations of PDGFR signaling in gliosarcomas. Six cases were analyzed by immunohistochemistry for the expression of PDGFR-α, c-Kit and their ligands PDGF-A and SCF, respectively. The cases were further evaluated for the presence of activating mutations of PDGFR-α (exons 12 and 18) and c-kit (exons 9, 11, 13, and 17), as well as B-RAF (exons 11 and 15). Expression of PDGF-A was found in all cases and co-expression of PDGFR-α was observed in three cases. Four cases showed expression of SCF, and c-Kit was observed only in one case that also expressed SCF. Generally, immunoreaction predominates in the glial component. The mutational analysis of PDGFR-α showed the presence of an IVS17-50insT intronic insertion in two cases, one of them also with a 2472C > T silent mutation; this silent mutation was also found in another case. Glioma cell line analysis of IVS17-50insT insertion showed no influence on PDGFR-α gene splicing. No mutations were detected in c-kit and B-RAF oncogenes. Our Results indicate that activating mutations of PDGFR-α, c-kit and B-RAF are absent in gliosarcomas. Nevertheless, the presence of a PDGFR-a/PDGFA and c-Kit/SCF autocrine/paracrine stimulation loop in a proportion of cases, supports the potential role of specific tyrosine kinase inhibitors in the treatment of

Synthesis and characterization of N-parinaroyl analogs of ganglioside GM3 and de-N-acetyl GM3. Interactions with the EGF receptor kinase

NASA Technical Reports Server (NTRS)

Song, W.; Welti, R.; Hafner-Strauss, S.; Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

1993-01-01

A specific plasma membrane glycosphingolipid, known as ganglioside GM3, can regulate the intrinsic tyrosyl kinase activity of the epidermal growth factor (EGF) receptor; this modulation is not associated with alterations in hormone binding to the receptor. GM3 inhibits EGF receptor tyrosyl kinase activity in detergent micelles, in plasma membrane vesicles, and in whole cells. In addition, immunoaffinity-purified EGF receptor preparations contain ganglioside GM3 (Hanai et al. (1988) J. Biol. Chem. 263, 10915-10921), implying that the glycosphingolipid is intimately associated with the receptor kinase in cell membranes. Both the nature of this association and the molecular mechanism of kinase inhibition remain to be elucidated. In this report, we describe the synthesis of a fluorescent analog of ganglioside GM3, in which the native fatty acid was replaced with trans-parinaric acid. This glycosphingolipid inhibited the receptor kinase activity in a manner similar to that of the native ganglioside. A modified fluorescent glycosphingolipid, N-trans-parinaroyl de-N-acetyl ganglioside GM3, was also prepared. This analog, like the nonfluorescent de-N-acetyl ganglioside GM3, had no effect on receptor kinase activity. Results from tryptophan fluorescence quenching and steady-state anisotropy measurements in membranes containing these fluorescent probes and the human EGF receptor were consistent with the notion that GM3, but not de-N-acetyl GM3, interacts specifically with the receptor in intact membranes.

Cell proliferation and inhibition of apoptosis are related to c-Kit activation in leukaemic lymphoblasts.

PubMed

Reyes-Sebastian, Josefina; Montiel-Cervantes, Laura Arcelia; Reyes-Maldonado, Elba; Dominguez-Lopez, Maria Lilia; Ortiz-Butron, Rocio; Castillo-Alvarez, Aida; Lezama, Ruth Angélica

2018-03-01

Receptor tyrosine kinase (RTK) activity may contribute to carcinogenesis. The c-Kit receptor, a member of the RTK family, is expressed in immature haematopoietic system cells. Acute lymphoblastic leukaemia (ALL) presents incompletely differentiated lymphoblasts, and consequently, c-Kit expression can be detected in these cells. The BCR-ABL kinase, which is usually present in both ALL and chronic myeloid leukaemia, can trigger signalling pathways with neoplastic effects. However, a certain number of ALL patients and chronic myeloid leukaemia patients do not express this kinase, raising the question of which other proteins that intervene in signalling pathways may be involved in the development of these diseases. To test whether c-Kit has proliferative effects and affects the inhibition of apoptosis of leukaemic lymphoblasts that do not express BCR-ABL. We cultured RS4:11 lymphoblasts and analysed the expression and activation of c-Kit by immunofluorescence, and flow cytometry, evaluation of cell proliferation, apoptosis, cyclin D1 and Bak expression were carried out by flow cytometry; activation of AKT and survivin expression were tested by immunoblot. The c-Kit receptor was found to induce proliferation and to increase the expression of cyclin D1 via the PI3K/AKT/NF-kB signalling pathway. Additionally, the c-Kit/PI3K/AKT pathway increased the inhibition of apoptosis and survivin expression. Similarly, c-Kit was observed to reduce the expression of the pro-apoptotic Bak protein. These results suggest that, in leukaemic lymphoblasts, c-Kit triggers a signalling pathway with proliferative and anti-apoptotic effects; information to this effect has not yet been reported in the literature.

Discovery of 4-Methyl-N-(4-((4-methylpiperazin-1-yl)methyl)-3-(trifluoromethyl)phenyl)-3-((1-nicotinoylpiperidin-4-yl)oxy)benzamide (CHMFL-ABL/KIT-155) as a Novel Highly Potent Type II ABL/KIT Dual Kinase Inhibitor with a Distinct Hinge Binding.

PubMed

Wang, Qiang; Liu, Feiyang; Wang, Beilei; Zou, Fengming; Qi, Ziping; Chen, Cheng; Yu, Kailin; Hu, Chen; Qi, Shuang; Wang, Wenchao; Hu, Zhenquan; Liu, Juan; Wang, Wei; Wang, Li; Liang, Qianmao; Zhang, Shanchun; Ren, Tao; Liu, Qingsong; Liu, Jing

2017-01-12

The discovery of a novel potent type II ABL/c-KIT dual kinase inhibitor compound 34 (CHMFL-ABL/KIT-155), which utilized a hydrogen bond formed by NH on the kinase backbone and carbonyl oxygen of 34 as a unique hinge binding, is described. 34 potently inhibited purified ABL (IC 50 : 46 nM) and c-KIT kinase (IC 50 : 75 nM) in the biochemical assays and displayed high selectivity (S Score (1) = 0.03) at the concentration of 1 μM among 468 kinases/mutants in KINOMEscan assay. It exhibited strong antiproliferative activities against BCR-ABL/c-KIT driven CML/GISTs cancer cell lines through blockage of the BCR-ABL/c-KIT mediated signaling pathways, arresting cell cycle progression and induction of apoptosis. 34 possessed a good oral PK property and effectively suppressed the tumor progression in the K562 (CML) and GIST-T1 (GISTs) cells mediated xenograft mouse model. The distinct hinge-binding mode of 34 provided a novel pharmacophore for expanding the chemical structure diversity for the type II kinase inhibitors discovery.

c-KIT positive schistosomal urinary bladder carcinoma are frequent but lack KIT gene mutations.

PubMed

Shams, Tahany M; Metawea, Mokhtar; Salim, Elsayed I

2013-01-01

Urinary bladder squamous cell carcinoma (SCC), one of the most common neoplasms in Egypt, is attributed to chronic urinary infection with Schistosoma haematobium (Schistosomiasis). The proto-oncogene c-KIT, encoding a tyrosine kinase receptor and implicated in the development of a number of human malignancies, has not been studied so far in schistosomal urinary bladder SCCs. We therefore determined immunohistochemical (IHC) expression of c-KIT in paraffin sections from 120 radical cystectomies of SCCs originally obtained from the Pathology Department of Suez Canal University (Ismailia, Egypt). Each slide was evaluated for staining intensity where the staining extent of >10% of cells was considered positive. c-KIT overexpression was detected in 78.3% (94/120) of the patients, the staining extents in the tumor cells were 11-50% and >50% in 40 (42.6%) and 54 (57.4%) respectively. The positive cases had 14.9%, 63.8%, 21.3% as weak, moderate and strong intensity respectively. Patients with positive bilharzial ova had significantly higher c-KIT expression than patients without (95.2% vs. 38.9%, P=0.000). Mutation analysis of exons 9-13 was negative in thirty KIT positive cases. The high rate of positivity in SBSCC was one of the striking findings; However, CD117 may be a potential target for site specific immunotherapy to improve the outcome of this tumor.

Protein-tyrosine-phosphatase-mediated epidermal growth factor (EGF) receptor transinactivation and EGF receptor-independent stimulation of mitogen-activated protein kinase by bradykinin in A431 cells.

PubMed Central

Graness, A; Hanke, S; Boehmer, F D; Presek, P; Liebmann, C

2000-01-01

Transactivation of the epidermal growth factor (EGF) receptor (EGFR) has been proposed to represent an essential link between G-protein-coupled receptors and the mitogen-activated protein kinase (MAPK) pathway in various cell types. In the present work we report, in contrast, that in A431 cells bradykinin transinactivates the EGFR and stimulates MAPK activity independently of EGFR tyrosine phosphorylation. Both effects of bradykinin are mediated by a pertussis-toxin-insensitive G-protein. Three lines of evidence suggest the activation of a protein tyrosine phosphatase (PTP) by bradykinin: (i) treatment of A431 cells with bradykinin decreases both basal and EGF-induced EGFR tyrosine phosphorylation, (ii) this effect of bradykinin can be blocked by two different PTP inhibitors, and (iii) bradykinin significantly increased the PTP activity in total A431 cell lysates when measured in vitro. The transmembrane receptor PTP sigma was identified as a putative mediator of bradykinin-induced downregulation of EGFR autophosphorylation. Activation of MAPK in response to bradykinin was insensitive towards AG 1478, a specific inhibitor of EGFR tyrosine kinase, but was blocked by wortmannin or bisindolylmaleimide, inhibitors of phosphatidylinositol 3-kinase (PI3-K) and protein kinase C (PKC) respectively. These results also suggest that the bradykinin-induced activation of MAPK is independent of EGFR and indicate a pathway involving PI3-K and PKC. In addition, bradykinin evokes a rapid and transient increase in Src kinase activity. Although Src does not participate in bradykinin-induced stimulation of PTP activity, inhibition of Src by 4-amino-5-(4-methylphenyl)-7-(t-butyl)pyrazolo(3,4-d)pyrimidine leads to an increase in MAPK activation by bradykinin. Our results suggest that in A431 cells the G(q/11)-protein-coupled bradykinin B(2) receptor may stimulate PTP activity and thereby transinactivate the EGFR, and may simultaneously activate MAPK by an alternative signalling pathway

Comparative experimental/theoretical studies on the EGFR dimerization under the effect of EGF/EGF analogues binding: Highlighting the importance of EGF/EGFR interactions at site III interface.

PubMed

Mehrabi, Masomeh; Mahdiuni, Hamid; Rasouli, Hassan; Mansouri, Kamran; Shahlaei, Mohsen; Khodarahmi, Reza

2018-04-14

Epidermal growth factor receptors (EGFRs) and their cytoplasmic tyrosine kinases play significant roles in cell proliferation and signaling. All the members of the EGFR/ErbB family are primary goals for cancer therapy, particularly for tumors of breast, cervix, ovaries, kidney, esophagus, prostate and non-small-cell lung carcinoma and head and neck tumors. However, the therapeutic ability of accessible anti-ErbB agents is limited. Therefore, recognizing EGF analogues or small organic molecules with high affinity for the extracellular domain of the EGFR is a critical target on cancer research. An effective EGF analogue should have a comparable binding affinity for EGFR in order to create an effective ligand competitive inhibition against circulating wild EGF while fails to transduce appropriate downstream signaling into the cancer cell. In our earlier study we have developed a mutant form of human EGF (mEGF, lacking the four critical amino acid residues; Gln 43 , Tyr 44 , Arg 45 and Asp 46 at the C-terminal of the protein) and its binding properties and mitogenic activity were assessed. The mEGF showed high affinity for EGFR binding domains but caused poor EGFR dimerization and phosphorylation and especially, mEGF induced EGFR internalization. However, underlying mechanism of action of EGF analogues is still unclear and thus considered to be worthwhile for further study. With regard to different effects of the EGF analogue on EGFR activating process, computational analysis of wild EGF/EGFR and mEGF/EGFR complexes (along with EGFt/EGFR complex) were done. Results of the protein dissection identified several interactions within "ligand/EGFR" that are common among EGF and EGFt/mEGF. These results disclose that while several interactions are conserved within EGF/EGFR interfaces, EGF/EGFR interactions on site III interface controls the affinity, EGFR dimerization and subsequent downstream signaling through a heterogeneous set of non-covalent interactions. These findings

The clinical significance of c-Kit mutations in metastatic oral mucosal melanoma in China.

PubMed

Ma, Xuhui; Wu, Yunteng; Zhang, Tian; Song, Hao; Jv, Houyu; Guo, Wei; Ren, Guoxin

2017-10-10

c-Kit mutations are frequently detected in mucosal melanomas, but their clinical significance in metastatic oral mucosal melanomas (OMM) remains unclear. The main purpose of this study was to investigate the clinical and pathological features of metastatic OMMs with c-Kit mutations and the efficiency of the tyrosine kinase inhibitor imatinib in treating metastatic OMMs. We found thatresidual primary lesion and neck lymph nodes could act as independent prognostic factors in metastatic OMM patients. c-Kit mutations were detected in 22 out of 139 (15.8%) metastatic OMM patients. Under chemotherapy, the overall survival (OS) of c-Kit mutant patients was significantly shorter than that of wild-type patients. The Ki67 expression was significantly higher in c-Kit mutant patients than in wild-type patients. In distant metastatic OMM patients with c-Kit mutations, the treatment with c-Kit inhibitor resulted in a better OS. In conclusion, residual primary lesion, cervical lymph nodes and c-Kit mutations act as adverse prognostic factors of metastatic OMMs. The Kit inhibitor imatinib could benefit metastatic OMM patients with c-Kit mutations.

Effect of epidermal growth factor (EGF) on the phosphorylation of mitogen-activated protein kinase (MAPK) in the bovine oviduct in vitro: Alteration by heat stress.

PubMed

Wijayagunawardane, Missaka P B; Hambruch, Nina; Haeger, Jan-Dirk; Pfarrer, Christiane

2015-01-01

Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival.

Effect of epidermal growth factor (EGF) on the phosphorylation of mitogen-activated protein kinase (MAPK) in the bovine oviduct in vitro: Alteration by heat stress

PubMed Central

WIJAYAGUNAWARDANE, Missaka P. B.; HAMBRUCH, Nina; HAEGER, Jan-Dirk; PFARRER, Christiane

2015-01-01

Epidermal growth factor (EGF) has been shown to be involved in control of the oviductal microenvironment. To elucidate the potential mechanisms responsible for the detrimental effect of heat stress and to identify the relation with the endocrine status, the effects of EGF on the level of phosphorylated mitogen-activated-protein kinase (MAPK) and proliferation of bovine oviductal epithelial cells (OECs) exposed to different cyclic ovarian steroidal environments (luteal phase (LP), follicular phase (FP) and postovulatory phase (PO)) and temperatures (mild heat stress (40 C) and severe heat stress (43 C)) were investigated. Western blot was performed to evaluate phosphorylated MAPK, while proliferation was analyzed by MTT assay. Stimulation of OECs with EGF alone or with EGF in the PO and FP environments significantly increased the amount of phosphorylated MAPK, with MAPK 44 phosphorylation being highest during exposure to PO conditions. These effects were not observed in the LP. Heat treatment completely blocked effects of EGF on phosphorylated MAPK. Additionally, severe heat stress led to a significantly lower basal level of phosphorylated MAPK. PD98059 (MAPK inhibitor) completely abolished EGF-stimulated MAPK phosphorylation and OECs proliferation. Overall the results indicate that EGF has the potential to increase the amount of phosphorylated MAPK in OECs and therefore could be involved in regulation of the bovine oviductal microenvironment. However, these regulatory mechanisms may be compromised in the presence of heat stress (high ambient temperature), leading to low fertility rates and impaired embryo survival. PMID:26050642

Type II cGMP‑dependent protein kinase inhibits the migration, invasion and proliferation of several types of human cancer cells.

PubMed

Wu, Min; Wu, Yan; Qian, Hai; Tao, Yan; Pang, Ji; Wang, Ying; Chen, Yongchang

2017-10-01

Previous studies have indicated that type II cyclic guanosine monophosphate (cGMP)‑dependent protein kinase (PKG II) could inhibit the proliferation and migration of gastric cancer cells. However, the effects of PKG II on the biological functions of other types of cancer cells remain to be elucidated. Therefore, the aim of the present study was to investigate the effects of PKG II on cancer cells derived from various types of human tissues, including A549 lung, HepG2 hepatic, OS‑RC‑2 renal, SW480 colon cancer cells and U251 glioma cells. Cancer cells were infected with adenoviral constructs coding PKG II (Ad‑PKG II) to up‑regulate PKG II expression, and treated with 8‑(4‑chlorophenylthio) (8‑pCPT)‑cGMP to activate the kinase. A Cell Counting kit 8 assay was used to detect cell proliferation. Cell migration was measured using a Transwell assay, whereas a terminal deoxynucleotidyl transferase 2'‑deoxyuridine, 5'‑triphosphate nick‑end labeling assay was used to detect cell apoptosis. A pull‑down assay was used to investigate the activation of Ras‑related C3 botulinum toxin substrate (Rac) 1 and western blotting was used to detect the expression of proteins of interest. The present results demonstrated that EGF (100 ng/ml, 24 h) promoted the proliferation and migration of cancer cells, and it suppressed their apoptosis. In addition, treatment with EGF enhanced the activation of Rac1, and up‑regulated the protein expression of proliferating cell nuclear antigen, matrix metalloproteinase (MMP)2, MMP7 and B‑cell lymphoma (Bcl)‑2, whereas it down‑regulated the expression of Bcl‑2‑associated X protein. Transfection of cancer cells with Ad‑PKG II, and PKG II activation with 8‑pCPT‑cGMP, was identified to counteract the effects triggered by EGF. The present results suggested that PKG II may exert inhibitory effects on the proliferation and migration of various types of cancer cells.

Phosphorylation of the Activation Loop Tyrosine 823 in c-Kit Is Crucial for Cell Survival and Proliferation*

PubMed Central

Agarwal, Shruti; Kazi, Julhash U.; Rönnstrand, Lars

2013-01-01

The receptor tyrosine kinase c-Kit, also known as the stem cell factor receptor, plays a key role in several developmental processes. Activating mutations in c-Kit lead to alteration of these cellular processes and have been implicated in many human cancers such as gastrointestinal stromal tumors, acute myeloid leukemia, testicular seminomas and mastocytosis. Regulation of the catalytic activity of several kinases is known to be governed by phosphorylation of tyrosine residues in the activation loop of the kinase domain. However, in the case of c-Kit phosphorylation of Tyr-823 has been demonstrated to be a late event that is not required for kinase activation. However, because phosphorylation of Tyr-823 is a ligand-activated event, we sought to investigate the functional consequences of Tyr-823 phosphorylation. By using a tyrosine-to-phenylalanine mutant of tyrosine 823, we investigated the impact of Tyr-823 on c-Kit signaling. We demonstrate here that Tyr-823 is crucial for cell survival and proliferation and that mutation of Tyr-823 to phenylalanine leads to decreased sustained phosphorylation and ubiquitination of c-Kit as compared with the wild-type receptor. Furthermore, the mutated receptor was, upon ligand-stimulation, quickly internalized and degraded. Phosphorylation of the E3 ubiquitin ligase Cbl was transient, followed by a substantial reduction in phosphorylation of downstream signaling molecules such as Akt, Erk, p38, Shc, and Gab2. Thus, we propose that activation loop tyrosine 823 is crucial for activation of both the MAPK and PI3K pathways and that its disruption leads to a destabilization of the c-Kit receptor and decreased survival of cells. PMID:23803604

MicroRNA-132 targets HB-EGF upon IgE-mediated activation in murine and human mast cells.

PubMed

Molnár, Viktor; Érsek, Barbara; Wiener, Zoltán; Tömböl, Zsófia; Szabó, Péter M; Igaz, Péter; Falus, András

2012-03-01

MicroRNAs provide an additional layer in the regulation of gene expression acting as repressors with several targets at the posttranscriptional level. This study describes microRNA expression patterns during differentiation and activation of mast cells. The expression levels of 567 different mouse miRNAs were compared by microarray between c-Kit+ committed progenitors, mucosal mast cells, resting and IgE-crosslinked BMMCs in vitro. The strongest upregulation of miR-132 upon IgE-mediated activation was validated in human cord blood-derived mast cells as well. HB-EGF growth factor also upregulated upon activation and was ranked high by more prediction algorithms. Co-transfection of miR-132 mimicking precursor and the 3'UTR of human Hbegf-containing luciferase vector proves that the predicted binding site is functional. In line with this, neutralization of miR-132 by anti-miR inhibitor leads to sustained production of HB-EGF protein in activated mast cells. Our data provide a novel example for negative regulation of a growth factor by an upregulated miRNA. © Springer Basel AG 2011

Cigarette smoke induces aberrant EGF receptor activation which mediates lung cancer development and resistance to tyrosine kinase inhibitors

PubMed Central

Filosto, Simone; Becker, Cathleen R.; Goldkorn, Tzipora

2015-01-01

The EGF Receptor (EGFR) and its downstream signaling are implicated in lung cancer development. Therefore, much effort was spent in developing specific tyrosine kinase inhibitors (TKIs) that bind to the EGFR ATP-pocket, blocking EGFR phosphorylation/signaling. Clinical use of TKIs is effective in a subset of lung cancers with mutations in the EGFR kinase domain, rendering the receptor highly susceptible to TKIs. However, these benefits are limited, and emergence of additional EGFR mutations usually results in TKI resistance and disease progression. Previously, we demonstrated one mechanism linking cigarette smoke (CS) to EGFR-driven lung cancer. Specifically, exposure of lung epithelial cells to CS-induced oxidative stress stimulates aberrant EGFR phosphorylation/activation with impaired receptor ubiquitination/degradation. The abnormal stabilization of the activated receptor leads to uncontrolled cell growth and tumorigenesis. Here we describe for the first time a novel post-translational mechanism of EGFR resistance to TKIs. Exposure of airway epithelial cells to CS causes aberrant phosphorylation/activation of EGFR, resulting in a conformation that is different from that induced by the ligand EGF. Unlike EGF-activated EGFR, CS-activated EGFR binds c-Src and caveolin-1 and does not undergo canonical dimerization. Importantly, the CS-activated EGFR is not inhibited by TKIs (AG1478; Erlotinib; Gefitinib); in fact, the CS exposure induces TKI-resistance even in the TKI-sensitive EGFR mutants. Our findings demonstrate that CS exposure stimulates not only aberrant EGFR phosphorylation impairing receptor degradation, but also induces a different EGFR conformation and signaling that are resistant to TKIs. Together, these findings offer new insights into CS-induced lung cancer development and TKI resistance. PMID:22302097

Oncogenic signaling by Kit tyrosine kinase occurs selectively on the Golgi apparatus in gastrointestinal stromal tumors

PubMed Central

Obata, Y; Horikawa, K; Takahashi, T; Akieda, Y; Tsujimoto, M; Fletcher, J A; Esumi, H; Nishida, T; Abe, R

2017-01-01

Gastrointestinal stromal tumors (GISTs) are caused by gain-of-function mutations in the Kit receptor tyrosine kinase. Most primary GIST patients respond to the Kit inhibitor imatinib, but this drug often becomes ineffective because of secondary mutations in the Kit kinase domain. The characteristic intracellular accumulation of imatinib-sensitive and -resistant Kit protein is well documented, but its relationship to oncogenic signaling remains unknown. Here, we show that in cancer tissue from primary GIST patients as well as in cell lines, mutant Kit accumulates on the Golgi apparatus, whereas normal Kit localizes to the plasma membrane (PM). In imatinib-resistant GIST with a secondary Kit mutation, Kit localizes predominantly on the Golgi apparatus. Both imatinib-sensitive and imatinib-resistant Kit (Kit(mut)) become fully auto-phosphorylated only on the Golgi and only if in a complex-glycosylated form. Kit(mut) accumulates on the Golgi during the early secretory pathway, but not after endocytosis. The aberrant kinase activity of Kit(mut) prevents its export from the Golgi to the PM. Furthermore, Kit(mut) on the Golgi signals and activates the phosphatidylinositol 3-kinase–Akt (PI3K–Akt) pathway, signal transducer and activator of transcription 5 (STAT5), and the Mek–Erk pathway. Blocking the biosynthetic transport of Kit(mut) to the Golgi from the endoplasmic reticulum inhibits oncogenic signaling. PM localization of Kit(mut) is not required for its signaling. Activation of Src-family tyrosine kinases on the Golgi is essential for oncogenic Kit signaling. These results suggest that the Golgi apparatus serves as a platform for oncogenic Kit signaling. Our study demonstrates that Kit(mut)’s pathogenicity is related to its mis-localization, and may offer a new strategy for treating imatinib-resistant GISTs. PMID:28192400

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT.

PubMed

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors' sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib.

Alkyl isothiocyanates suppress epidermal growth factor receptor kinase activity but augment tyrosine kinase activity.

PubMed

Nomura, Takahiro; Uehara, Yoshimasa; Kawajiri, Hiroo; Ryoyama, Kazuo; Yamori, Takao; Fuke, Yoko

2009-10-01

We have reported the in vitro and in vivo anticancer activities of 6-(methylsulfinyl)hexyl isothiocyanate (6-MITC) derived from a Japanese spice, wasabi. In order to obtain some clues about the mechanism of the anticancer activity, we have studied the effect of alkyl isothiocyanates (MITCs) on protein kinase activities. The anti-autophosphorylation activity of MITCs with respect to the epidermal growth factor (EGF)-stimulated receptor kinase of A431 epidermoid carcinoma cells was examined by incorporation of radioactive ATP into an acid-insoluble fraction. Their anti-phosphorylation activity with respect to the non-receptor protein kinase was analyzed by a standard SDS-PAGE method. All the tested MITCs interfered with the EGF-stimulated receptor kinase activity in a dose-dependent manner, although their effects were less than 1/10 of that of erbstatin in microg/ml. On the other hand, the MITCs did not interfere with non-receptor kinases (kinase A, kinase C, tyrosine kinase and calmodulin dependent kinase III), but enhanced non-receptor tyrosine kinase. A possible anticancer mechanism of MITCs may involve the suppression of EGF receptor kinase activity and augmentation of non-receptor PTK.

[INHIBITORS OF MAP-KINASE PATHWAY U0126 AND PD98059 DIFFERENTLY AFFECT ORGANIZATION OF TUBULIN CYTOSKELETON AFTER STIMULATION OF EGF RECEPTOR ENDOCYTOSIS].

PubMed

Zlobina, M V; Steblyanko, Yu Yu; Shklyaeva, M A; Kharchenko, V V; Salova, A V; Kornilova, E S

2015-01-01

To confirm the hypothesis about the involvement of EGF-stimulated MAP-kinase ERK1/2 in the regulation of microtubule (MT) system, the influence of two widely used ERK1/2 inhibitors, U0126 and PD98059, on the organization of tubulin cytoskeleton in interphase HeLa cells during EGF receptor endocytosis has been investigated. We have found that addition of U0126 or PD98059 to not-stimulated with EGF ells for 30 min has no effect on radially organized MT system. However, in the case of U0126 addition before EGF endocytosis stimulation, the number of MT per cell decreased within 15 min after such stimulation and was followed by complete MT depolymerization by 60-90 min. Stimulation of EGF endocytosis in the presence of PD98059 resulted only in insignificant depolymerization of MT and it could be detected mainly from their minus-ends. At the same time, MT regions close to plasma membrane became stabilized, which was proved by increase in tubulin acetylation level. This situation was characteristic for all period of the experiment. It has been also found that the inhibitors affect endocytosis dynamics of EGF-receptor complexes. Quantitative analysis demonstrated that the stimulation of endocytosis in the presence of U0126 generated a greater number of endosomes compared to control cells, and their number did not change significantly during the experiment. All these endosomes were localized peripherally. Effect of PD98059 resulted in the formation of lower number of endosomes that in control, but they demonstrated very slow clusterization despite the presence of some intact MT. Both inhibitors decreased EGFR colocolization with early endosomal marker EEA1, which indicated a delay in endosome fusions and maturation. The inhibitors were also shown to affect differently phospho-ERK 1 and 2 forms: U0126 completely inhibited phospho-ERK1 and 2, white, in the presence of PD98059, the two ERK forms demonstrated sharp transient activation in 15 min after stimulation, but only

Targeting c-KIT (CD117) by dasatinib and radotinib promotes acute myeloid leukemia cell death.

PubMed

Heo, Sook-Kyoung; Noh, Eui-Kyu; Kim, Jeong Yi; Jeong, Yoo Kyung; Jo, Jae-Cheol; Choi, Yunsuk; Koh, SuJin; Baek, Jin Ho; Min, Young Joo; Kim, Hawk

2017-11-10

Dasatinib and radotinib are oral BCR-ABL tyrosine kinase inhibitors that were developed as drugs for the treatment of chronic myeloid leukemia. We report here that the c-KIT (CD117) targeting with dasatinib and radotinib promotes acute myeloid leukemia (AML) cell death, and c-KIT endocytosis is essential for triggering c-KIT-positive AML cell death by dasatinib and radotinib during the early stages. In addition, dasatinib and radotinib reduce heat shock protein 90β (HSP90β) expression and release Apaf-1 in c-KIT-positive AML cells. Finally, this activates a caspase-dependent apoptotic pathway in c-KIT-positive AML cells. Moreover, the inhibition of c-KIT endocytosis by dynamin inhibitor (DY) reversed cell viability and c-KIT expression by dasatinib and radotinib. HSP90β expression was recovered by DY in c-KIT-positive AML cells as well. Furthermore, the effect of radotinib on c-KIT and HSP90β showed the same pattern in a xenograft animal model using HEL92.1.7 cells. Therefore, dasatinib and radotinib promote AML cell death by targeting c-KIT. Taken together, these results indicate that dasatinib and radotinib treatment have a potential role in anti-leukemic therapy on c-KIT-positive AML cells.

Ableson Kinases Negatively Regulate Invadopodia Function and Invasion in Head and Neck Squamous Cell Carcinoma by Inhibiting an HB-EGF Autocrine Loop

PubMed Central

Hayes, Karen E.; Walk, Elyse L.; Ammer, Amanda Gatesman; Kelley, Laura C.; Martin, Karen H.; Weed, Scott A.

2014-01-01

Head and neck squamous cell carcinoma (HNSCC) has a proclivity for locoregional invasion. HNSCC mediates invasion in part through invadopodia-based proteolysis of the extracellular matrix (ECM). Activation of Src, Erk1/2, Abl and Arg downstream of epidermal growth factor receptor (EGFR) modulates invadopodia activity through phosphorylation of the actin regulatory protein cortactin. In MDA-MB-231 breast cancer cells, Abl and Arg function downstream of Src to phosphorylate cortactin, promoting invadopodia ECM degradation activity and thus assigning a pro-invasive role for Ableson kinases. We report that Abl kinases have an opposite, negative regulatory role in HNSCC where they suppress invadopodia and tumor invasion. Impairment of Abl expression or Abl kinase activity with imatinib mesylate enhanced HNSCC matrix degradation and 3D collagen invasion, functions that were impaired in MDA-MB-231. HNSCC lines with elevated EGFR and Src activation did not contain increased Abl or Arg kinase activity, suggesting Src could bypass Abl/Arg to phosphorylate cortactin and promote invadopodia ECM degradation. Src transformed Abl−/−/Arg−/− fibroblasts produced ECM degrading invadopodia containing pY421 cortactin, indicating that Abl/Arg are dispensable for invadopodia function in this system. Imatinib treated HNSCC cells had increased EGFR, Erk1/2 and Src activation, enhancing cortactin pY421 and pS405/418 required for invadopodia function. Imatinib stimulated shedding of the EGFR ligand heparin-binding EGF-like growth factor (HB-EGF) from HNSCC cells, where soluble HB-EGF enhanced invadopodia ECM degradation in HNSCC but not in MDA-MB-231. HNSCC cells treated with inhibitors of the EGFR invadopodia pathway indicated that EGFR and Src are required for invadopodia function. Collectively our results indicate that Abl kinases negatively regulate HNSCC invasive processes through suppression of an HB-EGF autocrine loop responsible for activating a EGFR-Src-cortactin cascade

Insight on Mutation-Induced Resistance from Molecular Dynamics Simulations of the Native and Mutated CSF-1R and KIT

PubMed Central

Da Silva Figueiredo Celestino Gomes, Priscila; Chauvot De Beauchêne, Isaure; Panel, Nicolas; Lopez, Sophie; De Sepulveda, Paulo; Geraldo Pascutti, Pedro; Solary, Eric; Tchertanov, Luba

2016-01-01

The receptors tyrosine kinases (RTKs) for the colony stimulating factor-1, CSF-1R, and for the stem cell factor, SCFR or KIT, are important mediators of signal transduction. The abnormal function of these receptors, promoted by gain-of-function mutations, leads to their constitutive activation, associated with cancer or other proliferative diseases. A secondary effect of the mutations is the alteration of receptors’ sensitivity to tyrosine kinase inhibitors, compromising effectiveness of these molecules in clinical treatment. In particular, the mutation V560G in KIT increases its sensitivity to Imatinib, while the D816V in KIT, and D802V in CSF-1R, triggers resistance to the drug. We analyzed the Imatinib binding affinity to the native and mutated KIT (mutations V560G, S628N and D816V) and CSF-1R (mutation D802V) by using molecular dynamics simulations and energy calculations of Imatinib•target complexes. Further, we evaluated the sensitivity of the studied KIT receptors to Imatinib by measuring the inhibition of KIT phosphorylation. Our study showed that (i) the binding free energy of Imatinib to the targets is highly correlated with their experimentally measured sensitivity; (ii) the electrostatic interactions are a decisive factor affecting the binding energy; (iii) the most deleterious impact to the Imatinib sensitivity is promoted by D802V (CSF-1R) and D816V (KIT) mutations; (iv) the role of the juxtamembrane region, JMR, in the imatinib binding is accessory. These findings contribute to a better description of the mutation-induced effects alternating the targets sensitivity to Imatinib. PMID:27467080

Bone-induced c-kit expression in prostate cancer: a driver of intraosseous tumor growth

PubMed Central

Mainetti, Leandro E.; Zhe, Xiaoning; Diedrich, Jonathan; Saliganan, Allen D.; Cho, Won Jin; Cher, Michael L.; Heath, Elisabeth; Fridman, Rafael; Kim, Hyeong-Reh Choi; Bonfil, R. Daniel

2014-01-01

Loss of BRCA2 function stimulates prostate cancer (PCa) cell invasion and is associated with more aggressive and metastatic tumors in PCa patients. Concurrently, the receptor tyrosine kinase c-kit is highly expressed in skeletal metastases of PCa patients and induced in PCa cells placed into the bone microenvironment in experimental models. However, the precise requirement of c-kit for intraosseous growth of PCa and its relation to BRCA2 expression remain unexplored. Here, we show that c-kit expression promotes migration and invasion of PCa cells. Alongside, we found that c-kit expression in PCa cells parallels BRCA2 downregulation. Gene rescue experiments with human BRCA2 transgene in c-kit-transfected PCa cells resulted in reduction of c-kit protein expression and migration and invasion, suggesting a functional significance of BRCA2 downregulation by c-kit. The inverse association between c-kit and BRCA2 gene expressions in PCa cells was confirmed using laser capture microdissection in experimental intraosseous tumors and bone metastases of PCa patients. Inhibition of bone-induced c-kit expression in PCa cells transduced with lentiviral short hairpin RNA reduced intraosseous tumor incidence and growth. Overall, our results provide evidence of a novel pathway that links bone-induced c-kit expression in PCa cells to BRCA2 downregulation and supports bone metastasis. PMID:24798488

Angiotensin II stimulates calcium-dependent activation of c-Jun N-terminal kinase.

PubMed Central

Zohn, I E; Yu, H; Li, X; Cox, A D; Earp, H S

1995-01-01

In GN4 rat liver epithelial cells, angiotensin II (Ang II) and other agonists which activate phospholipase C stimulate tyrosine kinase activity in a calcium-dependent, protein kinase C (PKC)-independent manner. Since Ang II also produces a proliferative response in these cells, we investigated downstream signaling elements traditionally linked to growth control by tyrosine kinases. First, Ang II, like epidermal growth factor (EGF), stimulated AP-1 binding activity in a PKC-independent manner. Because increases in AP-1 can reflect induction of c-Jun and c-Fos, we examined the activity of the mitogen-activated protein (MAP) kinase family members Erk-1 and -2 and the c-Jun N-terminal kinase (JNK), which are known to influence c-Jun and c-Fos transcription. Ang II stimulated MAP kinase (MAPK) activity but only approximately 50% as effectively as EGF; again, these effects were independent of PKC. Ang II also produced a 50- to 200-fold activation of JNK in a PKC-independent manner. Unlike its smaller effect on MAPK, Ang II was approximately four- to sixfold more potent in activating JNK than EGF was. Although others had reported a lack of calcium ionophore-stimulated JNK activity in lymphocytes and several other cell lines, we examined the role of calcium in GN4 cells. The following results suggest that JNK activation in rat liver epithelial cells is at least partially Ca(2+) dependent: (i) norepinephrine and vasopressin hormones that increase inositol 1,4,5-triphosphate stimulated JNK; (ii) both thapsigargin, a compound that produces an intracellular Ca(2+) signal, and Ca(2+) ionophores stimulated a dramatic increase in JNK activity (up to 200-fold); (iii) extracellular Ca(2+) chelation with ethylene glycol tetraacetic acid (EGTA) inhibited JNK activation by ionophore and intracellular chelation with 1,2-bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid tetraacetoxymethyl-ester (BAPTA-AM) partially inhibited JNK activation by Ang II or thapsigargin; and (iv) JNK

Perforating folliculitis in a patient treated with nilotinib: a further evidence of C-kit involvement.

PubMed

Llamas-Velasco, Mar; Steegmann, Juan Luis; Carrascosa, Raquel; Fraga, Javier; García Diez, Amaro; Requena, Luis

2014-07-01

We present a case of perforating folliculitis in a patient treated with nilotinib, a kinase inhibitor. A 48-year-old man presented with a severely pruritic follicular rash for several months that started after reaching a complete molecular response of his myeloid chronic while treated with nilotinib. Clinical examination showed predominantly follicular pinpoint papules on trunk and proximal extremities and a biopsy showed a slightly dilated hair follicle with a focal disruption of the infundibular follicular epithelium. Other diseases related with perforating folliculitis were ruled out. The temporary relationship between the treatment and the appearance of the lesions suggests some pathogenic role of nilotinib. Relationship with nilotinib is also supported by previous similar cases related with sorafenib therapy. Both drugs inhibit c-kit and PDGF-R. PDGF-R has been previously involved in murine and human in vitro models of hair follicle cycle. So, our case supports in vivo the previous evidence of the importance of PDGF-R, a kinase, in the normal hair follicle development.

Oncogenic activation of v-kit involves deletion of a putative tyrosine-substrate interaction site.

PubMed

Herbst, R; Munemitsu, S; Ullrich, A

1995-01-19

The transforming gene of the Hardy-Zuckerman-4 strain of feline sarcoma virus, v-kit, arose by transduction of the cellular c-kit gene, which encodes the receptor tyrosine kinase (RTK) p145c-kit. To gain insight into the molecular basis of the v-kit transforming potential, we characterized the feline c-kit by cDNA cloning. Comparison of the feline v-kit and c-kit sequences revealed, in addition to deletions of the extracellular and transmembrane domains, three additional mutations in the v-kit oncogene product: deletion of tyrosine-569 and valine-570, the exchange of aspartate at position 761 to glycine, and replacement of the C-terminal 50 amino acids by five unrelated residues. Examinations of individual v-kit mutations in the context of chimeric receptors yielded inhibitory effects for some mutants on both autophosphorylation and substrate phosphorylation functions. In contrast, deletion of tyrosine-569 and valine-570 significantly enhanced transforming and mitogenic activities of p145c-kit, while the other mutations had no significant effects. Conservation in subclass III RTKs and the identification of the corresponding residue in beta PDGF-R, Y579, as a binding site for src family tyrosine kinases suggests an important role for Y568 in kit signal regulation and the definition of its oncogenic potential. Repositioning of Y571 by an inframe two codon deletion may be the crucial alteration resulting in enhancement of v-kit oncogenic activity.

HEPARIN-BINDING EGF CLEAVAGE MEDIATES ZINC-INDUCED EGF RECEPTOR PHOSPHORYLATION

EPA Science Inventory

We have previously shown that exposure to zinc ions can activate epidermal growth factor (EGF) receptor (EGFR) signaling in murine fibroblasts and A431 cells through a mechanism involving Src kinase. While studying the effects of zinc ions in normal human bronchial epithelial cel...

The first EGF domain of coagulation factor IX attenuates cell adhesion and induces apoptosis.

PubMed

Ishikawa, Tomomi; Kitano, Hisataka; Mamiya, Atsushi; Kokubun, Shinichiro; Hidai, Chiaki

2016-07-01

Coagulation factor IX (FIX) is an essential plasma protein for blood coagulation. The first epidermal growth factor (EGF) motif of FIX (EGF-F9) has been reported to attenuate cell adhesion to the extracellular matrix (ECM). The purpose of the present study was to determine the effects of this motif on cell adhesion and apoptosis. Treatment with a recombinant EGF-F9 attenuated cell adhesion to the ECM within 10 min. De-adhesion assays with native FIX recombinant FIX deletion mutant proteins suggested that the de-adhesion activity of EGF-F9 requires the same process of FIX activation as that which occurs for coagulation activity. The recombinant EGF-F9 increased lactate dehydrogenase (LDH) activity release into the medium and increased the number of cells stained with annexin V and activated caspase-3, by 8.8- and 2.7-fold respectively, indicating that EGF-F9 induced apoptosis. Activated caspase-3 increased very rapidly after only 5 min of administration of recombinant EGF-F9. Treatment with EGF-F9 increased the level of phosphorylated p38 mitogen-activated protein kinase (MAPK), but not that of phosphorylated MAPK 44/42 or c-Jun N-terminal kinase (JNK). Inhibitors of caspase-3 suppressed the release of LDH. Caspase-3 inhibitors also suppressed the attenuation of cell adhesion and phosphorylation of p38 MAPK by EGF-F9. Our data indicated that EGF-F9 activated signals for apoptosis and induced de-adhesion in a caspase-3 dependent manner. © 2016 The Author(s).

Differential role of EGF and BFGF in human GBM-TIC proliferation: relationship to EGFR-tyrosine kinase inhibitor sensibility.

PubMed

Bajetto, A; Porcile, C; Pattarozzi, A; Scotti, L; Aceto, A; Daga, A; Barbieri, F; Florio, T

2013-01-01

Glioblastoma multiforme (GBM) is among the most devastating human tumors being rapidly fatal despite aggressive surgery, radiation and chemotherapies. It is characterized by extensive dissemination of tumor cells within the brain that hinders complete surgical resection. GBM tumor initiating-cells (TICs) are a rare subpopulation of cells responsible for tumor development, growth, invasiveness and recurrence after chemotherapy. TICs from human GBM can be selected in vitro using the same conditions permissive for the growth of normal neural cells, of which share some features including marker expression, self-renewal capacity, long-term proliferation, and ability to differentiate into neuronal and glial cells. EGFR overexpression and its constitutive activation is one of the most important signaling alteration identified in GBM, and its pharmacological targeting represents an attractive therapeutic goal. We previously demonstrated that human GBM TICs have different sensitivity to the EGFR kinase inhibitors erlotinib and gefitinib, depending on the differential modulation of downstream signaling cascades. In this work we investigated the mechanisms of resistance to erlotinib in two human GBM TIC cultures, analyzing EGF and bFGF individual contribution to proliferation, clonogenicity, and migration. We demonstrated the presence of a small cell subpopulation whose proliferation is supported by EGF and a larger one mainly dependent on bFGF. Thus, insensitivity to EGFR kinase inhibitors as far as TIC proliferation results from a predominant FGFR activation that hides the inhibitory effects induced on EGFR signaling. Conversely, EGF and bFGF induced cell migration with similar efficacy. In addition, unlike neural stem/progenitors cells, the removal of chondroitin sulphate proteoglycans from cell surface was unable to discern EGF- and bFGF-dependent subpopulations in GBM TICs.

Saccharomyces boulardii Inhibits EGF Receptor Signaling and Intestinal Tumor Growth in Apcmin Mice

PubMed Central

Chen, Xinhua; Fruehauf, Johannes; Goldsmith, Jeffrey D.; Xu, Hua; Katchar, Kianoosh K; Koon, Hon-Wai; Zhao, Dezheng; Kokkotou, Efi G.; Pothoulakis, Charalabos; Kelly, Ciarán P.

2009-01-01

Saccharomyces boulardii (Sb) is a probiotic yeast with anti-inflammatory and antimicrobial activities and has been used for decades in the prevention and treatment of a variety of human gastrointestinal disorders. We reported previously that Sb modulates host inflammatory responses through down regulation of Erk1/2 MAP kinase activities both in vitro and in vivo. The aim of this study was to identify upstream mediators responsible for Erk1/2 inactivation and to examine the effects of Sb on tumor development in ApcMin mice. We found that the EGF receptor was deactivated upon exposure to Sb leading to inactivation of both the EGFR-Erk and EGFR-Akt pathways. In human colonic cancer cells, Sb prevented EGF induced proliferation, reduced cell colony formation and promoted apoptosis. HER-2, HER-3 and IGF-1R were also found to be inactivated by Sb. Oral intake of Sb reduced intestinal tumor growth and dysplasia in C57BL/6J Min/+ (ApcMin) mice. Thus, in addition to its anti-inflammatory effects, S. boulardii inhibits EGFR and other receptor tyrosine kinase signaling and thereby may also serve a novel therapeutic or prophylactic role in intestinal neoplasia. PMID:19482027

Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition

PubMed Central

Shi, Xiarong; Sousa, Leiliane P.; Mandel-Bausch, Elizabeth M.; Tome, Francisco; Reshetnyak, Andrey V.; Hadari, Yaron; Schlessinger, Joseph; Lax, Irit

2016-01-01

Large genomic sequencing analysis as part of precision medicine efforts revealed numerous activating mutations in receptor tyrosine kinases, including KIT. Unfortunately, a single approach is not effective for inhibiting cancer cells or treating cancers driven by all known oncogenic KIT mutants. Here, we show that each of the six major KIT oncogenic mutants exhibits different enzymatic, cellular, and dynamic properties and responds distinctly to different KIT inhibitors. One class of KIT mutants responded well to anti-KIT antibody treatment alone or in combination with a low dose of tyrosine kinase inhibitors (TKIs). A second class of KIT mutants, including a mutant resistant to imatinib treatment, responded well to a combination of TKI with anti-KIT antibodies or to anti-KIT toxin conjugates, respectively. We conclude that the preferred choice of precision medicine treatments for cancers driven by activated KIT and other RTKs may rely on clear understanding of the dynamic properties of oncogenic mutants. PMID:27482095

Comparison of lesional skin c-KIT mutations with clinical phenotype in patients with mastocytosis.

PubMed

Chan, I J; Tharp, M D

2018-06-01

Activating c-KIT mutations cause abnormal mast cell growth and appear to play a role in mastocytosis. However, the correlation of c-KIT mutations with disease phenotypes is poorly characterized. To evaluate the correlation of c-KIT mutations with clinical presentations and laboratory findings. Total cellular RNA was isolated from the skin lesions of 43 adults and 7 children with mastocytosis, and PCR amplicons of cDNA were sequenced for c-KIT mutations. The most common activating mutation, KIT-D816V, was identified in 72% of adults and 57% of children. Additional activating mutations, namely, V560G and the internal tandem duplications (ITDs) 502-503dupAY, were detected in 12% of adults and 8% of children. V560G occurred more commonly in our patients than previously reported, and it appeared to be associated with more advanced disease. Otherwise, the presence or absence of activating mutations did not correlate with skin lesion morphology, disease extent or total serum tryptase levels. Four adults had expression only of wild-type KIT, while two others had expression of a truncated KIT lacking tyrosine kinase activity; yet these patients were clinically indistinguishable from those patients with activating c-KIT mutations. Activating c-KIT mutations exist in a significant portion of patients with mastocytosis, but not all patients showed expression of these mutations. Except for V560G, the presence or absence of activating c-KIT mutations did not predict the extent of disease. These observations suggest that although activating c-KIT mutations are associated with mast cell growth, other genes probably play a role in the cause of mastocytosis. © 2018 British Association of Dermatologists.

Spatio-temporal imaging of EGF-induced activation of protein kinase A by FRET in living cells

NASA Astrophysics Data System (ADS)

Wang, Jin Jun; Chen, Xiao-Chuan; Xing, Da

2004-07-01

Intracellular molecular interaction is important for the study of cell physiology, yet current relevant methods require fixation or microinjection and lack temporal or spatial resolution. We introduced a new method -- fluorescence resonance energy transfer (FRET) to detect molecular interaction in living cells. On the basis of FRET principle, A-kinase activity reporter (AKAR) protein was designed to consist of the fusions of cyan fluorescent protein (CFP), a phosphoamino acid binding domain, a consensus substrate for protein kinase-A (PKA), and yellow fluorescent protein (YFP). In this study, the designed pAKAR plasmid was used to transfect a human lung cancer cell line (ASTC-a-1). When the AKAR-transfected cells were treated by forskolin (Fsk), we were able to observe the efficient transfer of energy from excited CFP to YFP within the AKAR molecule by fluorescence microcopy, whereas no FRET was detected in the transfected cells without the treatment of Fsk. When the cells were treated by Epidermal growth factor (EGF), the change of FRET was observed at different subcellular locations, reflecting PKA activation inside the cells upon EGF stimulation. The successful design of a fluorescence reporter of PKA activation and its application demonstrated the superiority of this technology in the research of intracellular protein-protein interaction.

Intratumoral CD3+ T-Lymphocytes Immunoexpression and Its Association with c-Kit, Angiogenesis, and Overall Survival in Malignant Canine Mammary Tumors

PubMed Central

Carvalho, Maria Isabel; Pires, Isabel; Dias, Marlene; Prada, Justina; Gregório, Hugo; Lobo, Luis; Queiroga, Felisbina

2015-01-01

In this study 80 malignant CMT were submitted to immunohistochemical detection of CD3, c-kit, VEGF, and CD31, together with clinicopathological parameters of tumor aggressiveness. CD3+ T-cells and c-kit overexpression revealed a positive correlation with VEGF (r = 0.503, P < 0.0001; r = 0.284, P = 0.023 for CD3 and c-kit, resp.) and CD31 (r = 0.654, P < 0.0001; r = 0.365, P = 0.003 for CD3 and c-kit, resp.). A significant association (P = 0.039) and a positive correlation (r = 0.263, P = 0.039) between CD3 and c-kit were also observed. High CD3/VEGF, c-kit/VEGF, and CD3/c-kit tumors were associated with elevated grade of malignancy (P < 0.0001 for all groups), presence of intravascular emboli (P < 0.0001 for CD3/VEGF and CD3/c-kit; P = 0.002 for c-kit/VEGF), and presence of lymph node metastasis (P < 0.0001 for all groups). Tumors with high CD3/VEGF (P = 0.006), c-kit/VEGF (P < 0.0001), and CD3/c-kit (P = 0.002) were associated with poor prognosis. Interestingly high c-kit/VEGF tumors retained their significance by multivariate analysis arising as independent prognostic factor. PMID:26346272

The epidermal growth factor receptor (EGF-R) is present on the basolateral, but not the apical, surface of enterocytes in the human gastrointestinal tract.

PubMed Central

Playford, R J; Hanby, A M; Gschmeissner, S; Peiffer, L P; Wright, N A; McGarrity, T

1996-01-01

BACKGROUND: While it is clear that luminal epidermal growth factor (EGF) stimulates repair of the damaged bowel, its significance in maintaining normal gut growth remains uncertain. If EGF is important in maintaining normal gut growth, the EGF receptor (EGF-R) should be present on the apical (luminal) surface in addition to the basolateral surface. AIMS/SUBJECTS/METHODS: This study examined the distribution of the EGF-R in the epithelium throughout the human gastro-intestinal tract using immunohistochemistry, electron microscopy, and western blotting of brush border preparations. RESULTS: Immunostaining of the oesophagus showed circumferential EGF-R positivity in the cells of the basal portions of the stratified squamous epithelium but surface cells were EGF-R negative. In the normal stomach, small intestine, and colon, immunostaining localised the receptor to the basolateral surface with the apical membranes being consistently negative. EGF-R positivity within the small intestine appeared to be almost entirely restricted to the proliferative (crypt) region. Western blotting demonstrated a 170 kDa protein in whole tissue homogenates but not in the brush border vesicle preparations. CONCLUSIONS: As the EGF-R is located only on the basolateral surfaces in the normal adult gastrointestinal tract, the major role of luminal EGF is probably to stimulate repair rather than to maintain normal gut growth. Images Figure 1 Figure 2 Figure 3 PMID:8977341

Activated c-Kit receptor in the heart promotes cardiac repair and regeneration after injury

PubMed Central

Di Siena, S; Gimmelli, R; Nori, S L; Barbagallo, F; Campolo, F; Dolci, S; Rossi, P; Venneri, M A; Giannetta, E; Gianfrilli, D; Feigenbaum, L; Lenzi, A; Naro, F; Cianflone, E; Mancuso, T; Torella, D; Isidori, A M; Pellegrini, M

2016-01-01

The role of endogenous c-Kit receptor activation on cardiac cell homeostasis and repair remains largely unexplored. Transgenic mice carrying an activating point mutation (TgD814Y) in the kinase domain of the c-Kit gene were generated. c-KitTgD814Y receptor was expressed in the heart during embryonic development and postnatal life, in a similar timing and expression pattern to that of the endogenous gene, but not in the hematopoietic compartment allowing the study of a cardiac-specific phenotype. c-KitTgD814Y mutation produced a constitutive active c-Kit receptor in cardiac tissue and cells from transgenic mice as demonstrated by the increased phosphorylation of ERK1/2 and AKT, which are the main downstream molecular effectors of c-Kit receptor signaling. In adult transgenic hearts, cardiac morphology, size and total c-Kit+ cardiac cell number was not different compared with wt mice. However, when c-KitTgD814Y mice were subjected to transmural necrotic heart damage by cryoinjury (CI), all transgenic survived, compared with half of wt mice. In the sub-acute phase after CI, transgenic and wt mice showed similar heart damage. However, 9 days after CI, transgenic mice exhibited an increased number of c-Kit+CD31+ endothelial progenitor cells surrounding the necrotic area. At later follow-up, a consistent reduction of fibrotic area, increased capillary density and increased cardiomyocyte replenishment rate (as established by BrdU incorporation) were observed in transgenic compared with wt mice. Consistently, CD45−c-Kit+ cardiac stem cells isolated from transgenic c-KitTgD814Y mice showed an enhanced endothelial and cardiomyocyte differentiation potential compared with cells isolated from the wt. Constitutive activation of c-Kit receptor in mice is associated with an increased cardiac myogenic and vasculogenic reparative potential after injury, with a significant improvement of survival. PMID:27468693

SMOC Binds to Pro-EGF, but Does Not Induce Erk Phosphorylation via the EGFR.

PubMed

Thomas, J Terrig; Chhuy-Hy, Lina; Andrykovich, Kristin R; Moos, Malcolm

2016-01-01

In an attempt to identify the cell-associated protein(s) through which SMOC (Secreted Modular Calcium binding protein) induces mitogen-activated protein kinase (MAPK) signaling, the epidermal growth factor receptor (EGFR) became a candidate. However, although in 32D/EGFR cells, the EGFR was phosphorylated in the presence of a commercially available human SMOC-1 (hSMOC-1), only minimal phosphorylation was observed in the presence of Xenopus SMOC-1 (XSMOC-1) or human SMOC-2. Analysis of the commercial hSMOC-1 product demonstrated the presence of pro-EGF as an impurity. When the pro-EGF was removed, only minimal EGFR activation was observed, indicating that SMOC does not signal primarily through EGFR and its receptor remains unidentified. Investigation of SMOC/pro-EGF binding affinity revealed a strong interaction that does not require the C-terminal extracellular calcium-binding (EC) domain of SMOC or the EGF domain of pro-EGF. SMOC does not appear to potentiate or inhibit MAPK signaling in response to pro-EGF, but the interaction could provide a mechanism for retaining soluble pro-EGF at the cell surface.

Cardiac c-Kit Biology Revealed by Inducible Transgenesis.

PubMed

Gude, Natalie A; Firouzi, Fareheh; Broughton, Kathleen M; Ilves, Kelli; Nguyen, Kristine P; Payne, Christina R; Sacchi, Veronica; Monsanto, Megan M; Casillas, Alexandria R; Khalafalla, Farid G; Wang, Bingyan J; Ebeid, David E; Alvarez, Roberto; Dembitsky, Walter P; Bailey, Barbara A; van Berlo, Jop; Sussman, Mark A

2018-06-22

Biological significance of c-Kit as a cardiac stem cell marker and role(s) of c-Kit+ cells in myocardial development or response to pathological injury remain unresolved because of varied and discrepant findings. Alternative experimental models are required to contextualize and reconcile discordant published observations of cardiac c-Kit myocardial biology and provide meaningful insights regarding clinical relevance of c-Kit signaling for translational cell therapy. The main objectives of this study are as follows: demonstrating c-Kit myocardial biology through combined studies of both human and murine cardiac cells; advancing understanding of c-Kit myocardial biology through creation and characterization of a novel, inducible transgenic c-Kit reporter mouse model that overcomes limitations inherent to knock-in reporter models; and providing perspective to reconcile disparate viewpoints on c-Kit biology in the myocardium. In vitro studies confirm a critical role for c-Kit signaling in both cardiomyocytes and cardiac stem cells. Activation of c-Kit receptor promotes cell survival and proliferation in stem cells and cardiomyocytes of either human or murine origin. For creation of the mouse model, the cloned mouse c-Kit promoter drives Histone2B-EGFP (enhanced green fluorescent protein; H2BEGFP) expression in a doxycycline-inducible transgenic reporter line. The combination of c-Kit transgenesis coupled to H2BEGFP readout provides sensitive, specific, inducible, and persistent tracking of c-Kit promoter activation. Tagging efficiency for EGFP+/c-Kit+ cells is similar between our transgenic versus a c-Kit knock-in mouse line, but frequency of c-Kit+ cells in cardiac tissue from the knock-in model is 55% lower than that from our transgenic line. The c-Kit transgenic reporter model reveals intimate association of c-Kit expression with adult myocardial biology. Both cardiac stem cells and a subpopulation of cardiomyocytes express c-Kit in uninjured adult heart

A change in structural integrity of c-Kit mutant D816V causes constitutive signaling.

PubMed

Raghav, Pawan Kumar; Singh, Ajay Kumar; Gangenahalli, Gurudutta

2018-03-01

Several signaling pathways, ligands, and genes that regulate proliferative and self-renewal properties of the Hematopoietic Stem Cells (HSCs) have been studied meticulously. One of the signaling pathways that play a crucial role in the process of hematopoiesis is the Stem Cell Factor (SCF) mediated c-Kit pathway. The c-Kit is a Receptor Tyrosine Kinase (RTK), which is expressed in the cells including HSCs. It undergoes dimerization upon binding with its cognate ligand SCF. As a result, phosphorylation of the Juxtamembrane (JM) domain of c-Kit takes place at Tyr568 and Tyr570 residues. These phosphorylated residues become the docking sites for protein tyrosine phosphatases (PTPs) namely SHP-1 and SHP-2, which in turn cause dephosphorylation and negative regulation of the downstream signaling responsible for the cell proliferation. Interestingly, it has been reported that the mutation of c-Kit at D816V makes it independent of SCF stimulation and SHP-1/SHP-2 inhibition, thereby, causing its constitutive activation. The present study was commenced to elucidate the structural behavior of this mutation in the JM and A-loop region of c-Kit using Molecular Dynamics (MD) simulations of the wild-type and mutant c-Kit in unphosphorylated and phosphorylated states. The energy difference computed between the wild type and mutant (D816V) c-Kit, and protein-protein docking and complex analysis revealed the impact of this single residue mutation on the integrity dynamics of c-Kit that makes it independent of SHP-1/SHP-2 negative regulation. Copyright © 2018 Elsevier B.V. All rights reserved.

Clinicopathological characteristics of KIT and protein kinase C-δ expression in adenoid cystic carcinoma: comparison with chromophobe renal cell carcinoma and gastrointestinal stromal tumour.

PubMed

Park, Cheol Keun; Kim, Won Kyu; Kim, Hoguen

2017-10-01

KIT overexpression is frequently observed in adenoid cystic carcinomas (AdCCs), chromophobe renal cell carcinomas (ChRCCs), and gastrointestinal stromal tumours (GISTs). Persistent KIT activation has been reported to be mediated by protein kinase C (PKC)-δ in a subset of colon cancers with wild-type KIT overexpression, and by PKC-θ in GISTs with mutant KIT overexpression. To elucidate the clinical implications of PKC-δ and PKC-θ expression in KIT-expressing tumours, we investigated the expression of KIT, PKC-δ and PKC-θ in AdCCs and ChRCCs in comparison with GISTs. KIT expression, PKC-δ expression and PKC-θ expression were analysed in whole sections from 41 AdCCs, 40 ChRCCs and 56 GISTs by immunohistochemistry. Membranous expression of KIT was found in 34 AdCCs and all ChRCCs, whereas cytoplasmic expression of KIT was found in 46 GISTs. In AdCCs, PKC-δ expression was associated with histological grade (P = 0.049), lymphovascular invasion (P = 0.004), perineural invasion (P = 0.002), and KIT positivity (P = 0.002). PKC-δ positivity was associated with shorter relapse-free survival (RFS) (P = 0.017) and a tendency for there to be shorter overall survival (OS) (P = 0.090) in patients with AdCCs. No clinicopathological associations were observed between PKC-δ and KIT expression in ChRCCs. In GISTs, PKC-θ expression was associated with higher mitotic count (P = 0.011) and high grade according to the modified National Institutes of Health criteria (P < 0.001). PKC-θ positivity was associated with shorter RFS (P = 0.016) and a tendency for there to be shorter OS (P = 0.051) in patients with GISTs. PKC-δ expression is associated with KIT expression and the prognosis of patients with AdCCs, suggesting that PKC-δ may be a potential therapeutic target for AdCCs. © 2017 The Authors. Histopathology published by John Wiley & Sons Ltd.

Insufficiency of pro-heparin-binding epidermal growth factor-like growth factor shedding enhances hypoxic cell death in H9c2 cardiomyoblasts via the activation of caspase-3 and c-Jun N-terminal kinase.

PubMed

Uetani, Teruyoshi; Nakayama, Hironao; Okayama, Hideki; Okura, Takafumi; Higaki, Jitsuo; Inoue, Hirofumi; Higashiyama, Shigeki

2009-05-01

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a cardiogenic and cardiohypertrophic growth factor. ProHB-EGF, a product of the Hb-egf gene and the precursor of HB-EGF, is anchored to the plasma membrane. Its ectodomain region is shed by a disintegrin and metalloproteases (ADAMs) when activated by various stimulations. It has been reported that an uncleavable mutant of Hb-egf, uc-Hb-egf, produces uc-proHB-EGF, which is not cleaved by ADAMs and causes dilation of the heart in knock-in mice. This suggests that the shedding of proHB-EGF is essential for the development and survival of cardiomyocytes: however, the molecular mechanism involved has remained unclear. In this study, we investigated the relationship between uc-proHB-EGF expression and cardiomyocyte survival. Human uc-proHB-EGF was adenovirally introduced into the rat cardiomyoblast cell line H9c2, and the cells were cultured under normoxic and hypoxic conditions. Uc-proHB-EGF-expressing H9c2 cells underwent apoptosis under normoxic conditions, which distinctly increased under hypoxic conditions. Furthermore, we observed an increased Caspase-3 activity, reactive oxygen species accumulation, and an increased c-Jun N-terminal kinase (JNK) activity in the uc-proHB-EGF-expressing H9c2 cells. Treatment of the uc-proHB-EGF transfectants with inhibitors of Caspase-3, reactive oxygen species, and JNK, namely, Z-VAD-fmk, N-acetylcysteine, and SP600125, respectively, significantly reduced hypoxic cell death. These data indicate that insufficiency of proHB-EGF shedding under hypoxic stress leads to cardiomyocyte apoptosis via Caspase-3- and JNK-dependent pathways.

Activated protein C cofactor function of protein S: a critical role for Asp95 in the EGF1-like domain

PubMed Central

Andersson, Helena M.; Arantes, Márcia J.; Crawley, James T. B.; Luken, Brenda M.; Tran, Sinh; Dahlbäck, Björn; Rezende, Suely M.

2010-01-01

Protein S has an established role in the protein C anticoagulant pathway, where it enhances the factor Va (FVa) and factor VIIIa (FVIIIa) inactivating property of activated protein C (APC). Despite its physiological role and clinical importance, the molecular basis of its action is not fully understood. To clarify the mechanism of the protein S interaction with APC, we have constructed and expressed a library of composite or point variants of human protein S, with residue substitutions introduced into the Gla, thrombin-sensitive region (TSR), epidermal growth factor 1 (EGF1), and EGF2 domains. Cofactor activity for APC was evaluated by calibrated automated thrombography (CAT) using protein S–deficient plasma. Of 27 variants tested initially, only one, protein S D95A (within the EGF1 domain), was largely devoid of functional APC cofactor activity. Protein S D95A was, however, γ-carboxylated and bound phospholipids with an apparent dissociation constant (Kdapp) similar to that of wild-type (WT) protein S. In a purified assay using FVa R506Q/R679Q, purified protein S D95A was shown to have greatly reduced ability to enhance APC-induced cleavage of FVa Arg306. It is concluded that residue Asp95 within EGF1 is critical for APC cofactor function of protein S and could define a principal functional interaction site for APC. PMID:20308596

The docking protein Gab1 is the primary mediator of EGF-stimulated activation of the PI-3K/Akt cell survival pathway

PubMed Central

Mattoon, Dawn R; Lamothe, Betty; Lax, Irit; Schlessinger, Joseph

2004-01-01

Background Gab1 is a docking protein that recruits phosphatidylinositol-3 kinase (PI-3 kinase) and other effector proteins in response to the activation of many receptor tyrosine kinases (RTKs). As the autophosphorylation sites on EGF-receptor (EGFR) do not include canonical PI-3 kinase binding sites, it is thought that EGF stimulation of PI-3 kinase and its downstream effector Akt is mediated by an indirect mechanism. Results We used fibroblasts isolated from Gab1-/- mouse embryos to explore the mechanism of EGF stimulation of the PI-3 kinase/Akt anti-apoptotic cell signaling pathway. We demonstrate that Gab1 is essential for EGF stimulation of PI-3 kinase and Akt in these cells and that these responses are mediated by complex formation between p85, the regulatory subunit of PI-3 kinase, and three canonical tyrosine phosphorylation sites on Gab1. Furthermore, complex formation between Gab1 and the protein tyrosine phosphatase Shp2 negatively regulates Gab1 mediated PI-3 kinase and Akt activation following EGF-receptor stimulation. We also demonstrate that tyrosine phosphorylation of ErbB3 may lead to recruitment and activation of PI-3 kinase and Akt in Gab1-/- MEFs. Conclusions The primary mechanism of EGF-induced stimulation of the PI-3 kinase/Akt anti-apoptotic pathway occurs via the docking protein Gab1. However, in cells expressing ErbB3, EGF and neuroregulin can stimulate PI-3 kinase and Akt activation in a Gab1-dependent or Gab1-independent manner. PMID:15550174

Non-Ligand-Induced Dimerization is Sufficient to Initiate the Signalling and Endocytosis of EGF Receptor.

PubMed

Kourouniotis, George; Wang, Yi; Pennock, Steven; Chen, Xinmei; Wang, Zhixiang

2016-07-25

The binding of epidermal growth factor (EGF) to EGF receptor (EGFR) stimulates cell mitogenesis and survival through various signalling cascades. EGF also stimulates rapid EGFR endocytosis and its eventual degradation in lysosomes. The immediate events induced by ligand binding include receptor dimerization, activation of intrinsic tyrosine kinase and autophosphorylation. However, in spite of intensified efforts, the results regarding the roles of these events in EGFR signalling and internalization is still very controversial. In this study, we constructed a chimeric EGFR by replacing its extracellular domain with leucine zipper (LZ) and tagged a green fluorescent protein (GFP) at its C-terminus. We showed that the chimeric LZ-EGFR-GFP was constitutively dimerized. The LZ-EGFR-GFP dimer autophosphorylated each of its five well-defined C-terminal tyrosine residues as the ligand-induced EGFR dimer does. Phosphorylated LZ-EGFR-GFP was localized to both the plasma membrane and endosomes, suggesting it is capable of endocytosis. We also showed that LZ-EGFR-GFP activated major signalling proteins including Src homology collagen-like (Shc), extracellular signal-regulated kinase (ERK) and Akt. Moreover, LZ-EGFR-GFP was able to stimulate cell proliferation. These results indicate that non-ligand induced dimerization is sufficient to activate EGFR and initiate cell signalling and EGFR endocytosis. We conclude that receptor dimerization is a critical event in EGF-induced cell signalling and EGFR endocytosis.

ROLE OF RAS IN METAL-INDUCED EGF RECEPTOR AND NFKB SIGNALING IN HUMAN AIRWAY EPITHELIAL CELLS

EPA Science Inventory

We have shown previously that EGF receptor signaling is triggered by some metals associated with ambient air particles. Western blot using phospho-specific antibodies showed that As, Zn and V activated EGF receptor tyrosine kinase and the downstream kinases, MEK1/2 and ERK1/2. Us...

Isolation, identification, and culture of goat spermatogonial stem cells using c-kit and PGP9.5 markers.

PubMed

Heidari, Banafsheh; Rahmati-Ahmadabadi, Maryam; Akhondi, Mohammad Mehdi; Zarnani, Amir Hassan; Jeddi-Tehrani, Mahmood; Shirazi, Abolfazl; Naderi, Mohammad Mehdi; Behzadi, Bahareh

2012-10-01

Presently the techniques for making transgenic animals are cumbersome, required costly instruments and trained man-power. The ability of spermatogonial stem cells (SSCs) to integrate foreign genes has provided the opportunity for developing alternate methods for generation of transgenic animals. One of the big challenges in this field is development of the methods to identify and purify donor SSCs by antibody mediated cell sorting. The present study was aimed to identify goat subpopulations of SSCs using polyclonal antibodies against PGP9.5 and c-kit molecular markers as well as the growth characteristics of SSCs during short term culture. One month old goats' testicular samples were subjected for immunohistochemical and immunocytochemical evaluations. The enzymatically isolated SSCs were cultured in DMEM plus FCS supplemented with (treatment) or without (control) growth factors (GDNF, LIF, FGF, and EGF) for 2 weeks. At the end of culture the morphological characteristics of SSCs colonies and immunocytochemical staining were evaluated. The number and size of colonies in treatment groups were significantly (P < 0.01) higher than corresponding values in controls. The presence of PGP 9.5 and c-kit antigens was confirmed in immunocytochemical evaluation. In immunocytochemical evaluation, the proportion of c-kit and PGP9.5 positive cells were significantly (P < 0.001) higher in control and treatment groups, respectively. The presence of PGP9.5 and c-kit antigens was confirmed in goat SSCs. Moreover, culture medium supplementation with growth factors could effectively retain the undifferentiation status of SSCs, reflected as a higher population of PGP9.5 positive cells, after short term culture.

Cell type-specific deficiency of c-kit gene expression in mutant mice of mi/mi genotype.

PubMed Central

Isozaki, K.; Tsujimura, T.; Nomura, S.; Morii, E.; Koshimizu, U.; Nishimune, Y.; Kitamura, Y.

1994-01-01

The mi locus of mice encodes a novel member of the basic-helix-loop-helix-leucine zipper protein family of transcription factors (hereafter called mi factor). In addition to microphthalmus, osteopetrosis, and lack of melanocytes, mice of mi/mi genotype are deficient in mast cells. Since the c-kit receptor tyrosine kinase plays an important role in the development of mast cells, and since the c-kit expression by cultured mast cells from mi/mi mice is deficient in both mRNA and protein levels, the mast cell deficiency of mi/mi mice has been attributed at least in part to the deficient expression of c-kit. However, it remained to be examined whether the c-kit expression was also deficient in tissues of mi/mi mice. In the present study, we examined the c-kit expression by mi/mi skin mast cells using in situ hybridization and immunohistochemistry. Moreover, we examined the c-kit expression by various cells other than mast cells in tissues of mi/mi mice. We found that the c-kit expression was deficient in mast cells but not in erythroid precursors, testicular germ cells, and neurons of mi/mi mice. This suggested that the regulation of the c-kit transcription by the mi factor was dependent on cell types. Mice of mi/mi genotype appeared to be a useful model to analyze the function of transcription factors in the whole-animal level. Images Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 PMID:7524330

Icotinib, a selective EGF receptor tyrosine kinase inhibitor, for the treatment of non-small-cell lung cancer.

PubMed

Tan, Fenlai; Shi, Yuankai; Wang, Yinxiang; Ding, Lieming; Yuan, Xiaobin; Sun, Yan

2015-01-01

Advanced non-small-cell lung cancer (NSCLC) is the main cause for cancer-related mortality. Treatments for advanced NSCLC are largely palliative and a benefit plateau appears to have reached with the platinum-based chemotherapy regimens. EGF receptor (EGFR) tyrosine kinase inhibitors gefitinib, erlotinib and afatinib came up with prolonged progression-free survival and improved quality of life, especially in EGFR-mutated patients. Icotinib is an oral selective EGFR tyrosine kinase, which was approved by China Food and Drug administration in June 2011 for treating advanced NSCLC. Its approval was based on the registered Phase III trial (ICOGEN), which showed icotinib is noninferior to gefitinib. This review will discuss the role of icotinib in NSCLC, and its potential application and ongoing investigations.

Interkinase domain of kit contains the binding site for phosphatidylinositol 3' kinase.

PubMed Central

Lev, S; Givol, D; Yarden, Y

1992-01-01

Our previous analysis of the signal transduction pathway used by the c-kit-encoded receptor for the stem cell factor (SCF) indicated efficient coupling to the type I phosphatidylinositol 3' kinase (PI3K). In an attempt to localize the receptor's site of interaction with PI3K, we separately deleted either the noncatalytic 68-amino-acid-long interkinase domain or the carboxyl-terminal portion distal to the catalytic sequences. Loss of ligand-induced association of PI3K with the former deletion mutant and retention of the PI3K association by the carboxyl-terminally deleted receptor implied interactions of PI3K with the kinase insert. This was further supported by partial inhibition of the association by an anti-peptide antibody directed against the kinase insert and lack of effect of an antibody directed to the carboxyl tail of the SCF receptor. A bacterially expressed kinase insert domain was used as a fusion protein to directly test its presumed function as a PI3K association site. This protein bound PI3K from cell lysate as demonstrated by PI3K activity and by an associated phosphoprotein of 85 kDa. The association was dependent on phosphorylation of the tyrosine residues on the expressed kinase insert. On the basis of these observations, we conclude that the kinase insert domain of the SCF receptor selectively interacts with the p85 regulatory subunit of PI3K and that this association requires phosphorylation of tyrosine residues in the kinase insert region, with apparently no involvement of the bulk cytoplasmic structure or tyrosine kinase function of the receptor. Images PMID:1370584

Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells With D816V-KIT

PubMed Central

Bandara, Geethani; Muñoz-Cano, Rosa; Tobío, Araceli; Yin, Yuzhi; Komarow, Hirsh D.; Desai, Avanti; Metcalfe, Dean D.; Olivera, Ana

2018-01-01

Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 depletion, and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK isoforms reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DDR in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation. PMID:29643855

Targeting Sphingosine Kinase Isoforms Effectively Reduces Growth and Survival of Neoplastic Mast Cells With D816V-KIT.

PubMed

Bandara, Geethani; Muñoz-Cano, Rosa; Tobío, Araceli; Yin, Yuzhi; Komarow, Hirsh D; Desai, Avanti; Metcalfe, Dean D; Olivera, Ana

2018-01-01

Mastocytosis is a disorder resulting from an abnormal mast cell (MC) accumulation in tissues that is often associated with the D816V mutation in KIT, the tyrosine kinase receptor for stem cell factor. Therapies available to treat aggressive presentations of mastocytosis are limited, thus exploration of novel pharmacological targets that reduce MC burden is desirable. Since increased generation of the lipid mediator sphingosine-1-phosphate (S1P) by sphingosine kinase (SPHK) has been linked to oncogenesis, we studied the involvement of the two SPHK isoforms (SPHK1 and SPHK2) in the regulation of neoplastic human MC growth. While SPHK2 inhibition prevented entry into the cell cycle in normal and neoplastic human MCs with minimal effect on cell survival, SPHK1 inhibition caused cell cycle arrest in G2/M and apoptosis, particularly in D816V-KIT MCs. This was mediated via activation of the DNA damage response (DDR) cascade, including phosphorylation of the checkpoint kinase 2 (CHK2), CHK2-mediated M-phase inducer phosphatase 3 depletion, and p53 activation. Combination treatment of SPHK inhibitors with KIT inhibitors showed greater growth inhibition of D816V-KIT MCs than either inhibitor alone. Furthermore, inhibition of SPHK isoforms reduced the number of malignant bone marrow MCs from patients with mastocytosis and the growth of D816V-KIT MCs in a xenograft mouse model. Our results reveal a role for SPHK isoforms in the regulation of growth and survival in normal and neoplastic MCs and suggest a regulatory function for SPHK1 in the DDR in MCs with KIT mutations. The findings also suggest that targeting the SPHK/S1P axis may provide an alternative to tyrosine kinase inhibitors, alone or in combination, for the treatment of aggressive mastocytosis and other hematological malignancies associated with the D816V-KIT mutation.

Differential Effects of CSF-1R D802V and KIT D816V Homologous Mutations on Receptor Tertiary Structure and Allosteric Communication

PubMed Central

Da Silva Figueiredo Celestino Gomes, Priscila; Panel, Nicolas; Laine, Elodie; Pascutti, Pedro Geraldo; Solary, Eric; Tchertanov, Luba

2014-01-01

The colony stimulating factor-1 receptor (CSF-1R) and the stem cell factor receptor KIT, type III receptor tyrosine kinases (RTKs), are important mediators of signal transduction. The normal functions of these receptors can be compromised by gain-of-function mutations associated with different physiopatological impacts. Whereas KIT D816V/H mutation is a well-characterized oncogenic event and principal cause of systemic mastocytosis, the homologous CSF-1R D802V has not been identified in human cancers. The KIT D816V oncogenic mutation triggers resistance to the RTK inhibitor Imatinib used as first line treatment against chronic myeloid leukemia and gastrointestinal tumors. CSF-1R is also sensitive to Imatinib and this sensitivity is altered by mutation D802V. Previous in silico characterization of the D816V mutation in KIT evidenced that the mutation caused a structure reorganization of the juxtamembrane region (JMR) and facilitated its departure from the kinase domain (KD). In this study, we showed that the equivalent CSF-1R D802V mutation does not promote such structural effects on the JMR despite of a reduction on some key H-bonds interactions controlling the JMR binding to the KD. In addition, this mutation disrupts the allosteric communication between two essential regulatory fragments of the receptors, the JMR and the A-loop. Nevertheless, the mutation-induced shift towards an active conformation observed in KIT D816V is not observed in CSF-1R D802V. The distinct impact of equivalent mutation in two homologous RTKs could be associated with the sequence difference between both receptors in the native form, particularly in the JMR region. A local mutation-induced perturbation on the A-loop structure observed in both receptors indicates the stabilization of an inactive non-inhibited form, which Imatinib cannot bind. PMID:24828813

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells

PubMed Central

Singh, Satyendra; Klarmann, Kimberly D.; Coppola, Vincenzo; Keller, Jonathan R.; Tessarollo, Lino

2016-01-01

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies. PMID:27835883

RanBPM (RanBP9) regulates mouse c-Kit receptor level and is essential for normal development of bone marrow progenitor cells.

PubMed

Puverel, Sandrine; Kiris, Erkan; Singh, Satyendra; Klarmann, Kimberly D; Coppola, Vincenzo; Keller, Jonathan R; Tessarollo, Lino

2016-12-20

c-Kit is a tyrosine kinase receptor important for gametogenesis, hematopoiesis, melanogenesis and mast cell biology. Dysregulation of c-Kit function is oncogenic and its expression in the stem cell niche of a number of tissues has underlined its relevance for regenerative medicine and hematopoietic stem cell biology. Yet, very little is known about the mechanisms that control c-Kit protein levels. Here we show that the RanBPM/RanBP9 scaffold protein binds to c-Kit and is necessary for normal c-Kit protein expression in the mouse testis and subset lineages of the hematopoietic system. RanBPM deletion causes a reduction in c-Kit protein but not its mRNA suggesting a posttranslational mechanism. This regulation is specific to the c-Kit receptor since RanBPM reduction does not affect other membrane proteins examined. Importantly, in both mouse hematopoietic system and testis, RanBPM deficiency causes defects consistent with c-Kit loss of expression suggesting that RanBPM is an important regulator of c-Kit function. The finding that this regulatory mechanism is also present in human cells expressing endogenous RanBPM and c-Kit suggests a potential new strategy to target oncogenic c-Kit in malignancies.

MNK1/2 inhibition limits oncogenicity and metastasis of KIT-mutant melanoma.

PubMed

Zhan, Yao; Guo, Jun; Yang, William; Goncalves, Christophe; Rzymski, Tomasz; Dreas, Agnieszka; Żyłkiewicz, Eliza; Mikulski, Maciej; Brzózka, Krzysztof; Golas, Aniela; Kong, Yan; Ma, Meng; Huang, Fan; Huor, Bonnie; Guo, Qianyu; da Silva, Sabrina Daniela; Torres, Jose; Cai, Yutian; Topisirovic, Ivan; Su, Jie; Bijian, Krikor; Alaoui-Jamali, Moulay A; Huang, Sidong; Journe, Fabrice; Ghanem, Ghanem E; Miller, Wilson H; Del Rincón, Sonia V

2017-11-01

Melanoma can be stratified into unique subtypes based on distinct pathologies. The acral/mucosal melanoma subtype is characterized by aberrant and constitutive activation of the proto-oncogene receptor tyrosine kinase C-KIT, which drives tumorigenesis. Treatment of these melanoma patients with C-KIT inhibitors has proven challenging, prompting us to investigate the downstream effectors of the C-KIT receptor. We determined that C-KIT stimulates MAP kinase-interacting serine/threonine kinases 1 and 2 (MNK1/2), which phosphorylate eukaryotic translation initiation factor 4E (eIF4E) and render it oncogenic. Depletion of MNK1/2 in melanoma cells with oncogenic C-KIT inhibited cell migration and mRNA translation of the transcriptional repressor SNAI1 and the cell cycle gene CCNE1. This suggested that blocking MNK1/2 activity may inhibit tumor progression, at least in part, by blocking translation initiation of mRNAs encoding cell migration proteins. Moreover, we developed an MNK1/2 inhibitor (SEL201), and found that SEL201-treated KIT-mutant melanoma cells had lower oncogenicity and reduced metastatic ability. Clinically, tumors from melanoma patients harboring KIT mutations displayed a marked increase in MNK1 and phospho-eIF4E. Thus, our studies indicate that blocking MNK1/2 exerts potent antimelanoma effects and support blocking MNK1/2 as a potential strategy to treat patients positive for KIT mutations.

Structural Basis for Activation of the Receptor Tyrosine Kinase KIT by Stem Cell Factor

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yuzawa,S.; Opatowsky, Y.; Zhang, Z.

2007-01-01

Stem Cell Factor (SCF) initiates its multiple cellular responses by binding to the ectodomain of KIT, resulting in tyrosine kinase activation. We describe the crystal structure of the entire ectodomain of KIT before and after SCF stimulation. The structures show that KIT dimerization is driven by SCF binding whose sole role is to bring two KIT molecules together. Receptor dimerization is followed by conformational changes that enable lateral interactions between membrane proximal Ig-like domains D4 and D5 of two KIT molecules. Experiments with cultured cells show that KIT activation is compromised by point mutations in amino acids critical for D4-D4more » interaction. Moreover, a variety of oncogenic mutations are mapped to the D5-D5 interface. Since key hallmarks of KIT structures, ligand-induced receptor dimerization, and the critical residues in the D4-D4 interface, are conserved in other receptors, the mechanism of KIT stimulation unveiled in this report may apply for other receptor activation.« less

Conformational stability of the epidermal growth factor (EGF) receptor as influenced by glycosylation, dimerization and EGF hormone binding.

PubMed

Taylor, Eric S; Pol-Fachin, Laercio; Lins, Roberto D; Lower, Steven K

2017-04-01

The epidermal growth factor receptor (EGFR) is an important transmembrane glycoprotein kinase involved the initiation or perpetuation of signal transduction cascades within cells. These processes occur after EGFR binds to a ligand [epidermal growth factor (EGF)], thus inducing its dimerization and tyrosine autophosphorylation. Previous publications have highlighted the importance of glycosylation and dimerization for promoting proper function of the receptor and conformation in membranes; however, the effects of these associations on the protein conformational stability have not yet been described. Molecular dynamics simulations were performed to characterize the conformational preferences of the monomeric and dimeric forms of the EGFR extracellular domain upon binding to EGF in the presence and absence of N-glycan moieties. Structural stability analyses revealed that EGF provides the most conformational stability to EGFR, followed by glycosylation and dimerization, respectively. The findings also support that EGF-EGFR binding takes place through a large-scale induced-fitting mechanism. Proteins 2017; 85:561-570. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Transcriptome and proteome analysis of tyrosine kinase inhibitor treated canine mast cell tumour cells identifies potentially kit signaling-dependent genes

PubMed Central

2012-01-01

Background Canine mast cell tumour proliferation depends to a large extent on the activity of KIT, a tyrosine kinase receptor. Inhibitors of the KIT tyrosine kinase have recently been introduced and successfully applied as a therapeutic agent for this tumour type. However, little is known on the downstream target genes of this signaling pathway and molecular changes after inhibition. Results Transcriptome analysis of the canine mast cell tumour cell line C2 treated for up to 72 hours with the tyrosine kinase inhibitor masitinib identified significant changes in the expression levels of approximately 3500 genes or 16% of the canine genome. Approximately 40% of these genes had increased mRNA expression levels including genes associated with the pro-proliferative pathways of B- and T-cell receptors, chemokine receptors, steroid hormone receptors and EPO-, RAS and MAP kinase signaling. Proteome analysis of C2 cells treated for 72 hours identified 24 proteins with changed expression levels, most of which being involved in gene transcription, e.g. EIA3, EIA4, TARDBP, protein folding, e.g. HSP90, UCHL3, PDIA3 and protection from oxidative stress, GSTT3, SELENBP1. Conclusions Transcriptome and proteome analysis of neoplastic canine mast cells treated with masitinib confirmed the strong important and complex role of KIT in these cells. Approximately 16% of the total canine genome and thus the majority of the active genes were significantly transcriptionally regulated. Most of these changes were associated with reduced proliferation and metabolism of treated cells. Interestingly, several pro-proliferative pathways were up-regulated, which may represent attempts of masitinib treated cells to activate alternative pro-proliferative pathways. These pathways may contain hypothetical targets for a combination therapy with masitinib to further improve its therapeutic effect. PMID:22747577

EGF receptor lysosomal degradation is delayed in the cells stimulated with EGF-Quantum dot bioconjugate but earlier key events of endocytic degradative pathway are similar to that of native EGF

PubMed Central

Leontieva, Ekaterina A.; Kornilova, Elena S.

2017-01-01

Quantum dots (QDs) complexed to ligands recognizing surface receptors undergoing internalization are an attractive tool for live cell imaging of ligand-receptor complexes behavior and for specific tracking of the cells of interest. However, conjugation of quasi-multivalent large QD-particle to monovalent small growth factors like EGF that bound their tyrosine-kinase receptors may affect key endocytic events tightly bound to signaling. Here, by means of confocal microscopy we have addressed the key endocytic events of lysosomal degradative pathway stimulated by native EGF or EGF-QD bioconjugate. We have demonstrated that the decrease in endosome number, increase in mean endosome integrated density and the pattern of EEA1 co-localization with EGF-EGFR complexes at early stages of endocytosis were similar for the both native and QD-conjugated ligands. In both cases enlarged hollow endosomes appeared after wortmannin treatment. This indicates that early endosomal fusions and their maturation proceed similar for both ligands. EGF-QD and native EGF similarly accumulated in juxtanuclear region, and live cell imaging of endosome motion revealed the behavior described elsewhere for microtubule-facilitated motility. Finally, EGF-QD and the receptor were found in lysosomes. However, degradation of receptor part of QD-EGF-EGFR-complex was delayed compared to native EGF, but not inhibited, while QDs fluorescence was detected in lysosomes even after 24 hours. Importantly, in HeLa and A549 cells the both ligands behaved similarly. We conclude that during endocytosis EGF-QD behaves as a neutral marker for degradative pathway up to lysosomal stage and can also be used as a long-term cell marker. PMID:28574831

The stem cell factor (SCF)/c-KIT system in carcinogenesis of reproductive tissues: What does the hormonal regulation tell us?

PubMed

Figueira, Marília I; Cardoso, Henrique J; Correia, Sara; Maia, Cláudio J; Socorro, Sílvia

2017-10-01

The tyrosine kinase receptor c-KIT and its ligand, the stem cell factor (SCF) are expressed in several tissues of male and female reproductive tract, playing an important role in the regulation of basic biological processes. The activation of c-KIT by SCF controls, cell survival and death, cell differentiation and migration. Also, the SCF/c-KIT system has been implicated in carcinogenesis of reproductive tissues due to its altered expression pattern or overactivation in consequence of gain-of-functions mutations. Over the years, it has also been shown that hormones, the primary regulators of reproductive function and causative agents in the case of hormone-dependent cancers, are also able to control the SCF/c-KIT tissue levels. Therefore, it is liable to suppose that disturbed SCF/c-KIT expression driven by (de)regulated hormone actions can be a relevant step towards carcinogenesis. The present review describes the SCF and c-KIT expression in cancers of reproductive tissues, discussing the implications of the hormonal regulation of the SCF/c-KIT system in cancer development. Understanding the relationship between hormonal imbalance and the SCF/c-KIT expression and activity would be relevant in the context of novel therapeutic approaches in reproductive cancers. Copyright © 2017 Elsevier B.V. All rights reserved.

Discovery of a highly selective KIT kinase primary V559D mutant inhibitor for gastrointestinal stromal tumors (GISTs).

PubMed

Yu, Kailin; Liu, Xuesong; Jiang, Zongru; Hu, Chen; Zou, Fengming; Chen, Cheng; Ge, Juan; Wu, Jiaxin; Liu, Xiaochuan; Wang, Aoli; Wang, Wenliang; Wang, Wenchao; Qi, Ziping; Wang, Beilei; Wang, Li; Yan, Hezhong; Wang, Jiaoxue; Ren, Tao; Tang, Jun; Liu, Qingsong; Liu, Jing

2017-12-19

KIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors (GISTs). Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031, which displayed about 10-20 fold selectivity over KIT wt in the biochemical assay (IC 50 : 28 nM over 168 nM; Kd: 266 nM versus 6640 nM) and in cell (EC 50 : 176 nM versus 2000 nM for pY703) examination. It also displayed 15∼400-fold selectivity over other primary mutants such as L576P and secondary mutants including T670I, V654A (ATP binding pocket) as well as N822K and D816V (activation loop). In addition, it exhibited a selectivity S score (1) of 0.01 among 468 kinases/mutants in the KINOMEScan ™ assay. CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo (Tumor Growth Inhibition: 68.5%). Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GISTs.

Discovery of a highly selective KIT kinase primary V559D mutant inhibitor for gastrointestinal stromal tumors (GISTs)

PubMed Central

Yu, Kailin; Liu, Xuesong; Jiang, Zongru; Hu, Chen; Zou, Fengming; Chen, Cheng; Ge, Juan; Wu, Jiaxin; Liu, Xiaochuan; Wang, Aoli; Wang, Wenliang; Wang, Wenchao; Qi, Ziping; Wang, Beilei; Wang, Li; Yan, Hezhong; Wang, Jiaoxue; Ren, Tao; Tang, Jun; Liu, Qingsong; Liu, Jing

2017-01-01

KIT kinase V559D mutation is the most prevalent primary gain-of-function mutation in Gastrointestinal Stromal Tumors (GISTs). Here we reported a highly selective KIT V559D inhibitor CHMFL-KIT-031, which displayed about 10-20 fold selectivity over KIT wt in the biochemical assay (IC50: 28 nM over 168 nM; Kd: 266 nM versus 6640 nM) and in cell (EC50: 176 nM versus 2000 nM for pY703) examination. It also displayed 15∼400-fold selectivity over other primary mutants such as L576P and secondary mutants including T670I, V654A (ATP binding pocket) as well as N822K and D816V (activation loop). In addition, it exhibited a selectivity S score (1) of 0.01 among 468 kinases/mutants in the KINOMEScan™ assay. CHMFL-KIT-031 showed potent inhibitory efficacy for KIT V559D mediated signaling pathways in cell and anti-tumor activity in vivo (Tumor Growth Inhibition: 68.5%). Its superior selectivity would make it a good pharmacological tool for further dissection of KIT V559D mediated pathology in the GISTs. PMID:29340041

Immunohistochemical expression of c-KIT protein in feline soft tissue fibrosarcomas.

PubMed

Smith, A J; Njaa, B L; Lamm, C G

2009-09-01

C-KIT is the cellular homolog of the feline sarcoma viral oncogene v-KIT, which encodes the tyrosine kinase receptor protein KIT. Mutations and varied expression of this gene have been demonstrated within multiple neoplasms in people and domestic animals. The purpose of this study was to determine if KIT protein is expressed in feline soft tissue fibrosarcomas (ST FSA) using immunohistochemistry (IHC). The computer database at the Oklahoma Animal Disease Diagnostic Laboratory was searched from January 1, 2006, to December 31, 2007, for any domestic cat with an ST FSA. Routinely stained slides from 46 feline ST FSAs were reviewed and graded based on the scale outlined by Kuntz et al. Immunohistochemistry for KIT protein was performed on one representative section from each cat. There were a total of 12/46 (26%) cats that were immunoreactive for KIT. Immunoreactivity was detected in greater than 80% of the neoplastic cells in 4/46 (9%) cats. Immunoreactivity was detected in less than 10% of the neoplastic cells in 8/46 (17%) cats. Immunoreactivity was characterized by evenly distributed cytoplasmic stippling within the neoplastic spindle-shaped cells and/or multinucleated giant cells. Based on these results, KIT immunoreactivity can be detected within feline ST FSAs using IHC. The results of this study also indicate that KIT immunoreactivity in feline ST FSA does not correlate with the histologic grade (P = .141, X(2) = 2.166), survivability (P = .241, X(2) = 1.373), or whether the neoplasm was a spontaneous or an injection site FSA (P = .074, X(2) = 3.184).

SCF/C-Kit/JNK/AP-1 Signaling Pathway Promotes Claudin-3 Expression in Colonic Epithelium and Colorectal Carcinoma.

PubMed

Wang, Yaxi; Sun, Tingyi; Sun, Haimei; Yang, Shu; Li, Dandan; Zhou, Deshan

2017-04-06

Claudin-3 is a major protein of tight junctions (TJs) in the intestinal epithelium and is critical for maintaining cell-cell adhesion, barrier function, and epithelium polarity. Recent studies have shown high claudin-3 levels in several solid tumors, but the regulation mechanism of claudin-3 expression remains poorly understood. In the present study, colorectal cancer (CRC) tissues, HT-29 and DLD-1 CRC cell lines, CRC murine model (C57BL/6 mice) and c-kit loss-of-function mutant mice were used. We demonstrated that elevated claudin-3 levels were positively correlated with highly expressed c-kit in CRC tissues based upon analysis of protein expression. In vitro, claudin-3 expression was clearly increased in CRC cells by overexpressed c-kit or stimulated by exogenous recombinant human stem cell factor (rhSCF), while significantly decreased by the treatment with c-kit or c-Jun N-terminal kinase (JNK) inhibitors. Chromatin immunoprecipitation (ChIP) and luciferase reporter assay showed that SCF/c-kit signaling significantly promoted activator protein-1 (AP-1) binding with CLDN-3 promoter and enhanced its transcription activity. Furthermore, decreased expression of claudin-3 was obtained in the colonic epithelium from the c-Kit loss-of-function mutant mice. In conclusion, SCF/c-kit-JNK/AP-1 signaling pathway significantly promoted claudin-3 expression in colonic epithelium and CRC, which could contribute to epithelial barrier function maintenance and to CRC development.

Discovery of Aryl Aminoquinazoline Pyridones as Potent, Selective, and Orally Efficacious Inhibitors of Receptor Tyrosine Kinase c-Kit

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hu, Essa; Tasker, Andrew; White, Ryan D.

2008-12-09

Inhibition of c-Kit has the potential to treat mast cell associated fibrotic diseases. We report the discovery of several aminoquinazoline pyridones that are potent inhibitors of c-Kit with greater than 200-fold selectivity against KDR, p38, Lck, and Src. In vivo efficacy of pyridone 16 by dose-dependent inhibition of histamine release was demonstrated in a rodent pharmacodynamic model of mast cell activation.

Developmental defects in zebrafish for classification of EGF pathway inhibitors

DOE Office of Scientific and Technical Information (OSTI.GOV)

Pruvot, Benoist; Curé, Yoann; Djiotsa, Joachim

2014-01-15

One of the major challenges when testing drug candidates targeted at a specific pathway in whole animals is the discrimination between specific effects and unwanted, off-target effects. Here we used the zebrafish to define several developmental defects caused by impairment of Egf signaling, a major pathway of interest in tumor biology. We inactivated Egf signaling by genetically blocking Egf expression or using specific inhibitors of the Egf receptor function. We show that the combined occurrence of defects in cartilage formation, disturbance of blood flow in the trunk and a decrease of myelin basic protein expression represent good indicators for impairmentmore » of Egf signaling. Finally, we present a classification of known tyrosine kinase inhibitors according to their specificity for the Egf pathway. In conclusion, we show that developmental indicators can help to discriminate between specific effects on the target pathway from off-target effects in molecularly targeted drug screening experiments in whole animal systems. - Highlights: • We analyze the functions of Egf signaling on zebrafish development. • Genetic blocking of Egf expression causes cartilage, myelin and circulatory defects. • Chemical inhibition of Egf receptor function causes similar defects. • Developmental defects can reveal the specificity of Egf pathway inhibitors.« less

The human tyrosine kinase Kit and its gatekeeper mutant T670I, show different kinetic properties: Implications for drug design.

PubMed

Kissova, Miroslava; Maga, Giovanni; Crespan, Emmanuele

2016-10-01

The tyrosine kinase Kit, a receptor for Stem Cell Factor, is involved, among others, in processes associated to cell survival, proliferation and migration. Upon physiological conditions, the activity of Kit is tightly regulated. However, primary mutations that lead to its constitutive activation are the causal oncogenic driver of gastrointestinal stromal tumours (GISTs). GISTs are known to be refractory to conventional therapies but the introduction of Imatinib, a selective inhibitor of tyrosine kinases Abl and Kit, significantly ameliorated the treatment options of GISTs patients. However, the acquisition of secondary mutations renders Kit resistant towards all available drugs. Mutation involving gatekeeper residues (such as V654a and T670I) influence both the structure and the catalytic activity of the enzyme. Therefore, detailed knowledge of the enzymatic properties of the mutant forms, in comparison with the wild type enzyme, is an important pre-requisite for the rational development of specific inhibitors. In this paper we report a thorough kinetic analysis of the reaction catalyzed by the Kit kinase and its gatekeeper mutated form T670I. Our results revealed the different mechanisms of action of these two enzymes and may open a new avenue for the future design of specific Kit inhibitors. Copyright © 2016 Elsevier Ltd. All rights reserved.

SH2/SH3 adaptor proteins can link tyrosine kinases to a Ste20-related protein kinase, HPK1.

PubMed

Anafi, M; Kiefer, F; Gish, G D; Mbamalu, G; Iscove, N N; Pawson, T

1997-10-31

Ste20-related protein kinases have been implicated as regulating a range of cellular responses, including stress-activated protein kinase pathways and the control of cytoskeletal architecture. An important issue involves the identities of the upstream signals and regulators that might control the biological functions of mammalian Ste20-related protein kinases. HPK1 is a protein-serine/threonine kinase that possesses a Ste20-like kinase domain, and in transfected cells activates a protein kinase pathway leading to the stress-activated protein kinase SAPK/JNK. Here we have investigated candidate upstream regulators that might interact with HPK1. HPK1 possesses an N-terminal catalytic domain and an extended C-terminal tail with four proline-rich motifs. The SH3 domains of Grb2 bound in vitro to specific proline-rich motifs in the HPK1 tail and functioned synergistically to direct the stable binding of Grb2 to HPK1 in transfected Cos1 cells. Epidermal growth factor (EGF) stimulation did not affect the binding of Grb2 to HPK1 but induced recruitment of the Grb2.HPK1 complex to the autophosphorylated EGF receptor and to the Shc docking protein. Several activated receptor and cytoplasmic tyrosine kinases, including the EGF receptor, stimulated the tyrosine phosphorylation of the HPK1 serine/threonine kinase. These results suggest that HPK1, a mammalian Ste20-related protein-serine/threonine kinase, can potentially associate with protein-tyrosine kinases through interactions mediated by SH2/SH3 adaptors such as Grb2. Such interaction may provide a possible mechanism for cross-talk between distinct biochemical pathways following the activation of tyrosine kinases.

The role of small molecule Kit protein-tyrosine kinase inhibitors in the treatment of neoplastic disorders.

PubMed

Roskoski, Robert

2018-04-25

The Kit proto-oncogene was found as a consequence of the discovery of the feline v-kit sarcoma oncogene. Stem cell factor (SCF) is the Kit ligand and it mediates Kit dimerization and activation. The Kit receptor contains an extracellular segment that is made up of five immunoglobulin-like domains (D1/2/3/4/5), a transmembrane segment, a juxtamembrane segment, a protein-tyrosine kinase domain that contains an insert of 77 amino acid residues, and a carboxyterminal tail. Activating somatic mutations in Kit have been documented in various neoplasms including gastrointestinal stromal tumors (GIST), mast cell overexpression (systemic mastocytosis), core-binding factor acute myeloid leukemias (AML), melanomas, and seminomas. In the case of gastrointestinal stromal tumors, most activating mutations occur in the juxtamembrane segment and these mutants are initially sensitive to imatinib. As with many targeted anticancer drugs, resistance to Kit antagonists occurs in about two years and is the result of secondary KIT mutations. An activation segment exon 17 D816V mutation is one of the more common resistance mutations in Kit and this mutant is resistant to imatinib and sorafenib. Type I protein kinase inhibitors interact with the active enzyme form with DFG-D of the proximal activation segment directed inward toward the active site (DFG-D in ). In contrast, type II inhibitors bind to their target with the DFG-D pointing away from the active site (DFG-D out ). Based upon the X-ray crystallographic structures, imatinib, sunitinib, and ponatinib are Type II Kit inhibitors. We used the Schrödinger induced fit docking protocol to model the interaction of midostaurin with Kit and the result indicates that it binds to the DFG-D in conformation of the receptor and is thus classified as type I inhibitor. This medication inhibits the notoriously resistant Kit D816V mutant and is approved for the treatment of systemic mastocytosis and is effective against tumors bearing the D816V

Huntingtin-interacting protein 1: a Merkel cell carcinoma marker that interacts with c-Kit.

PubMed

Ames, Heather M; Bichakjian, Christopher K; Liu, Grace Y; Oravecz-Wilson, Katherine I; Fullen, Douglas R; Verhaegen, Monique E; Johnson, Timothy M; Dlugosz, Andrzej A; Ross, Theodora S

2011-10-01

Merkel cell carcinoma (MCC) is a neoplasm thought to originate from the neuroendocrine Merkel cells of the skin. Although the prevalence of MCC has been increasing, treatments for this disease remain limited because of a paucity of information regarding MCC biology. We have found that the endocytic oncoprotein Huntingtin-interacting protein 1 (HIP1) is expressed at high levels in ∼90% of MCC tumors and serves as a more reliable histological cytoplasmic stain than the gold standard, cytokeratin 20. Furthermore, high anti-HIP1 antibody reactivity in the sera of a cohort of MCC patients predicts the presence of metastases. Another protein that is frequently expressed at high levels in MCC tumors is the stem cell factor (SCF) receptor tyrosine kinase, c-Kit. In working toward an understanding of how HIP1 might contribute to MCC tumorigenesis, we have discovered that HIP1 interacts with SCF-activated c-Kit. These data not only identify HIP1 as a molecular marker for management of MCC patients but also show that HIP1 interacts with and slows the degradation of c-Kit.

Kit receptor tyrosine kinase dysregulations in feline splenic mast cell tumours.

PubMed

Sabattini, S; Barzon, G; Giantin, M; Lopparelli, R M; Dacasto, M; Prata, D; Bettini, G

2017-09-01

This study investigated Kit receptor dysregulations (cytoplasmic immunohistochemical expression and/or c-KIT mutations) in cats affected with splenic mast cell tumours. Twenty-two cats were included. Median survival time was 780 days (range: 1-1219). An exclusive splenic involvement was significantly (P = 0.042) associated with longer survival (807 versus 120 days). Eighteen tumours (85.7%) showed Kit cytoplasmic expression (Kit pattern 2, 3). Mutation analysis was successful in 20 cases. Fourteen missense mutations were detected in 13 out of 20 tumours (65%). Eleven (78.6%) were located in exon 8, and three (21.6%) in exon 9. No mutations were detected in exons 11 and 17. Seven mutations corresponded to the same internal tandem duplication in exon 8 (c.1245_1256dup). Although the association between Kit cytoplasmic expression and mutations was significant, immunohistochemistry cannot be considered a surrogate marker for mutation analysis. No correlation was observed between c-Kit mutations and tumour differentiation, mitotic activity or survival. © 2016 John Wiley & Sons Ltd.

Gq protein mediates UVB-induced cyclooxygenase-2 expression by stimulating HB-EGF secretion from HaCaT human keratinocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Seo, MiRan; Juhnn, Yong-Sung, E-mail: juhnn@snu.ac.kr

Ultraviolet (UV) radiation induces cyclooxygenase-2 expression to produce cellular responses including aging and carcinogenesis in skin. We hypothesised that heterotrimeric G proteins mediate UV-induced COX-2 expression by stimulating secretion of soluble HB-EGF (sHB-EGF). In this study, we aimed to elucidate the role and underlying mechanism of the {alpha} subunit of Gq protein (G{alpha}q) in UVB-induced HB-EGF secretion and COX-2 induction. We found that expression of constitutively active G{alpha}q (G{alpha}qQL) augmented UVB-induced HB-EGF secretion, which was abolished by knockdown of G{alpha}q with shRNA in HaCaT human keratinocytes. G{alpha}q was found to mediate the UVB-induced HB-EGF secretion by sequential activation of phospholipasemore » C (PLC), protein kinase C{delta} (PKC{delta}), and matrix metaloprotease-2 (MMP-2). Moreover, G{alpha}qQL mediated UVB-induced COX-2 expression in an HB-EGF-, EGFR-, and p38-dependent manner. From these results, we concluded that G{alpha}q mediates UV-induced COX-2 expression through activation of EGFR by HB-EGF, of which ectodomain shedding was stimulated through sequential activation of PLC, PKC{delta} and MMP-2 in HaCaT cells.« less

Pathologic Stimulus Determines Lineage Commitment of Cardiac C-kit+ Cells.

PubMed

Chen, Zhongming; Zhu, Wuqiang; Bender, Ingrid; Gong, Wuming; Kwak, Il-Youp; Yellamilli, Amritha; Hodges, Thomas J; Nemoto, Natsumi; Zhang, Jianyi; Garry, Daniel J; van Berlo, Jop H

2017-12-12

Although cardiac c-kit + cells are being tested in clinical trials, the circumstances that determine lineage differentiation of c-kit + cells in vivo are unknown. Recent findings suggest that endogenous cardiac c-kit + cells rarely contribute cardiomyocytes to the adult heart. We assessed whether various pathological stimuli differentially affect the eventual cell fates of c-kit + cells. We used single-cell sequencing and genetic lineage tracing of c-kit + cells to determine whether various pathological stimuli would result in different fates of c-kit + cells. Single-cell sequencing of cardiac CD45 - c-kit + cells showed innate heterogeneity, indicative of the existence of vascular and mesenchymal c-kit + cells in normal hearts. Cardiac pressure overload resulted in a modest increase in c-kit-derived cardiomyocytes, with significant increases in the numbers of endothelial cells and fibroblasts. Doxorubicin-induced acute cardiotoxicity did not increase c-kit-derived endothelial cell fates but instead induced cardiomyocyte differentiation. Mechanistically, doxorubicin-induced DNA damage in c-kit + cells resulted in expression of p53. Inhibition of p53 blocked cardiomyocyte differentiation in response to doxorubicin, whereas stabilization of p53 was sufficient to increase c-kit-derived cardiomyocyte differentiation. These results demonstrate that different pathological stimuli induce different cell fates of c-kit + cells in vivo. Although the overall rate of cardiomyocyte formation from c-kit + cells is still below clinically relevant levels, we show that p53 is central to the ability of c-kit + cells to adopt cardiomyocyte fates, which could lead to the development of strategies to preferentially generate cardiomyocytes from c-kit + cells. © 2017 American Heart Association, Inc.

The Intracellular Juxtamembrane Domain of the Epidermal Growth Factor (EGF) Receptor Is Responsible for the Allosteric Regulation of EGF Binding*S⃞♦

PubMed Central

Macdonald-Obermann, Jennifer L.; Pike, Linda J.

2009-01-01

We have previously shown that the binding of epidermal growth factor (EGF) to its receptor can best be described by a model that involves negative cooperativity in an aggregating system (Macdonald, J. L., and Pike, L. J. (2008) Proc. Natl. Acad. Sci. U. S. A. 105, 112–117). However, despite the fact that biochemical analyses indicate that EGF induces dimerization of its receptor, the binding data provided no evidence for positive linkage between EGF binding and dimer assembly. By analyzing the binding of EGF to a number of receptor mutants, we now report that in naive, unphosphorylated EGF receptors, ligand binding is positively linked to receptor dimerization but the linkage is abolished upon autophosphorylation of the receptor. Both phosphorylated and unphosphorylated EGF receptors exhibit negative cooperativity, indicating that mechanistically, cooperativity is distinct from the phenomenon of linkage. Nonetheless, both the positive linkage and the negative cooperativity observed in EGF binding require the presence of the intracellular juxtamembrane domain. This indicates the existence of inside-out signaling in the EGF receptor system. The intracellular juxtamembrane domain has previously been shown to be required for the activation of the EGF receptor tyrosine kinase (Thiel, K. W., and Carpenter, G. (2007) Proc. Natl. Acad. Sci. U. S. A. 104, 19238–19243). Our experiments expand the role of this domain to include the allosteric control of ligand binding by the extracellular domain. PMID:19336395

Acupuncture Ameliorates Postoperative Ileus via IL-6-miR-19a-KIT Axis to Protect Interstitial Cells of Cajal.

PubMed

Deng, Jingjing; Yang, Shu; Yuan, Qing; Chen, Yuzhong; Li, Dandan; Sun, Haimei; Tan, Xinghua; Zhang, Fuchun; Zhou, Deshan

2017-01-01

Acupuncture is a therapy effective in treating postoperative ileus (POI); its underlying mechanisms remain unclear. MicroRNAs (miRNAs) participate in inflammation and injury to the interstitial cells of Cajal (ICCs), both of which are considered to be contributors to POI. C-kit, encoding KIT, a specific marker of ICCs, is predicted to be targeted by miR-19a, an inflammation-related miRNA. Therefore, we investigated a possible link between inflammation, miR-19a, and ICCs in POI, as well as the mechanism by which these factors are affected by acupuncture. The effects of acupuncture on POI were assessed in patients after colorectal resection and in colocolic anastomosis mice. Immunofluorescence staining demonstrated that KIT[Formula: see text]/ano1[Formula: see text] ICCs dramatically decreased around the colonic incision in mice, which was negatively correlated with the pronounced increase in macrophage. However, this decrease was not due to apoptosis. IL-6R was expressed in ICCs, and IL-6 level was significantly increased, as measured by ELISA, in accompaniment with high miR-19a expression. The increase in IL-6 and miR-19a levels was negatively correlated with the decrease in KIT[Formula: see text]/ano1[Formula: see text] ICCs. A luciferase reporter assay demonstrated that miR-19a directly targeted C-kit, indicating that miR-19a caused ICC damage. Interestingly, acupuncture inhibited macrophage activation, IL-6 release, and miR-19a upregulation, while promoting KIT and ano1 restoration in ICCs. High serum miR-19a level in patients after colorectal resection was also reduced by acupuncture. Conclusively, the IL-6 released by macrophages during gastrointestinal surgery upregulated miR-19a, which downregulated KIT in ICCs and finally resulted in POI. Acupuncture can interfere with the "IL-6-miR-19a-KIT" axis, suggesting that it may be a therapeutic mechanism that works against POI.

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

PubMed

Garton, Andrew J; Crew, Andrew P A; Franklin, Maryland; Cooke, Andrew R; Wynne, Graham M; Castaldo, Linda; Kahler, Jennifer; Winski, Shannon L; Franks, April; Brown, Eric N; Bittner, Mark A; Keily, John F; Briner, Paul; Hidden, Chris; Srebernak, Mary C; Pirrit, Carrie; O'Connor, Matthew; Chan, Anna; Vulevic, Bojana; Henninger, Dwight; Hart, Karen; Sennello, Regina; Li, An-Hu; Zhang, Tao; Richardson, Frank; Emerson, David L; Castelhano, Arlindo L; Arnold, Lee D; Gibson, Neil W

2006-01-15

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.

Auto-phosphorylation Represses Protein Kinase R Activity.

PubMed

Wang, Die; de Weerd, Nicole A; Willard, Belinda; Polekhina, Galina; Williams, Bryan R G; Sadler, Anthony J

2017-03-10

The central role of protein kinases in controlling disease processes has spurred efforts to develop pharmaceutical regulators of their activity. A rational strategy to achieve this end is to determine intrinsic auto-regulatory processes, then selectively target these different states of kinases to repress their activation. Here we investigate auto-regulation of the innate immune effector protein kinase R, which phosphorylates the eukaryotic initiation factor 2α to inhibit global protein translation. We demonstrate that protein kinase R activity is controlled by auto-inhibition via an intra-molecular interaction. Part of this mechanism of control had previously been reported, but was then controverted. We account for the discrepancy and extend our understanding of the auto-inhibitory mechanism by identifying that auto-inhibition is paradoxically instigated by incipient auto-phosphorylation. Phosphor-residues at the amino-terminus instigate an intra-molecular interaction that enlists both of the N-terminal RNA-binding motifs of the protein with separate surfaces of the C-terminal kinase domain, to co-operatively inhibit kinase activation. These findings identify an innovative mechanism to control kinase activity, providing insight for strategies to better regulate kinase activity.

CD117/c-kit in Cancer Stem Cell-Mediated Progression and Therapeutic Resistance

PubMed Central

Young, Tyler R.; Mobley, Mary E.

2018-01-01

Metastasis is the primary cause of cancer patient morbidity and mortality, but due to persisting gaps in our knowledge, it remains untreatable. Metastases often occur as patient tumors progress or recur after initial therapy. Tumor recurrence at the primary site may be driven by a cancer stem-like cell or tumor progenitor cell, while recurrence at a secondary site is driven by metastatic cancer stem cells or metastasis-initiating cells. Ongoing efforts are aimed at identifying and characterizing these stem-like cells driving recurrence and metastasis. One potential marker for the cancer stem-like cell subpopulation is CD117/c-kit, a tyrosine kinase receptor associated with cancer progression and normal stem cell maintenance. Further, activation of CD117 by its ligand stem cell factor (SCF; kit ligand) in the progenitor cell niche stimulates several signaling pathways driving proliferation, survival, and migration. This review examines evidence that the SCF/CD117 signaling axis may contribute to the control of cancer progression through the regulation of stemness and resistance to tyrosine kinase inhibitors. PMID:29518044

Protein Kinase B Activation and Lamellipodium Formation Are Independent Phosphoinositide 3-Kinase-Mediated Events Differentially Regulated by Endogenous Ras

PubMed Central

van Weering, David H. J.; de Rooij, Johan; Marte, Barbara; Downward, Julian; Bos, Johannes L.; Burgering, Boudewijn M. T.

1998-01-01

Regulation of phosphoinositide 3-kinase (PI 3-kinase) can occur by binding of the regulatory p85 subunit to tyrosine-phosphorylated proteins and by binding of the p110 catalytic subunit to activated Ras. However, the way in which these regulatory mechanisms act to regulate PI 3-kinase in vivo is unclear. Here we show that several growth factors (basic fibroblast growth factor [bFGF], platelet-derived growth factor [PDGF], and epidermal growth factor [EGF; to activate an EGF receptor-Ret chimeric receptor]) all activate PI 3-kinase in vivo in the neuroectoderm-derived cell line SKF5. However, these growth factors differ in their ability to activate PI 3-kinase-dependent signaling. PDGF and EGF(Ret) treatment induced PI 3-kinase-dependent lamellipodium formation and protein kinase B (PKB) activation. In contrast, bFGF did not induce lamellipodium formation but activated PKB, albeit to a small extent. PDGF and EGF(Ret) stimulation resulted in binding of p85 to tyrosine-phosphorylated proteins and strong Ras activation. bFGF, however, induced only strong activation of Ras. In addition, while RasAsn17 abolished bFGF activation of PKB, PDGF- and EGF(Ret)-induced PKB activation was only partially inhibited and lamellipodium formation was unaffected. Interestingly, in contrast to activation of only endogenous Ras (bFGF), ectopic expression of activated Ras did result in lamellipodium formation. From this we conclude that, in vivo, p85 and Ras synergize to activate PI 3-kinase and that strong activation of only endogenous Ras exerts a small effect on PI 3-kinase activity, sufficient for PKB activation but not lamellipodium formation. This differential sensitivity to PI 3-kinase activation could be explained by our finding that PKB activation and lamellipodium formation are independent PI 3-kinase-induced events. PMID:9528752

Aberrant expressions of c-KIT and DOG-1 in mucinous and nonmucinous colorectal carcinomas and relation to clinicopathologic features and prognosis.

PubMed

Foda, Abd Al-Rahman Mohammad; Mohamed, Mie Ali

2015-10-01

c-KIT and DOG-1 are 2 highly expressed proteins in gastrointestinal stromal tumors. Few studies had investigated c-KIT, but not DOG-1, expression in colorectal carcinoma (CRC). This study aims to investigate expressions of c-KIT and DOG-1 in colorectal mucinous carcinoma and nonmucinous carcinoma using manual tissue microarray technique. In this work, we studied tumor tissue specimens from 150 patients with colorectal mucinous (MA) and nonmucinous adenocarcinoma (NMA). High-density manual tissue microarrays were constructed using modified mechanical pencil tip technique, and immunohistochemistry for c-KIT and DOG-1 was done. We found that aberrant c-KIT expression was detected in 12 cases (8%); 6 cases (4%) showed strong expression. Aberrant DOG-1 expression was detected in 15 cases (10%); among them, only 4 cases (2.7%) showed strong expression. Nonmucinous adenocarcinoma showed a significantly high expression of c-KIT, but not DOG-1, than MA. Aberrant c-KIT and DOG-1 expressions were significantly unrelated but were associated with excessive microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. In conclusion, aberrant c-KIT and DOG-1 expressions in CRC are rare events, either in NMA or MA. Nonmucinous adenocarcinoma showed a significantly higher expression of c-KIT, but not DOG-1, than MA. The expressions of both in CRC are significantly unrelated but are associated with microscopic abscess formation. Neither c-KIT nor DOG-1 expression showed a significant impact on disease-free survival or overall survival. So, c-KIT and DOG-1 immunostaining is not a cost-effective method of identifying patients with CRC who may benefit from treatment with tyrosine kinase inhibitors. Copyright © 2015 Elsevier Inc. All rights reserved.

Huntingtin Interacting Protein 1: a Merkel Cell Carcinoma Marker That Interacts with c-Kit

PubMed Central

Ames, Heather M.; Bichakjian, Christopher K.; Liu, Grace Y.; Oravecz-Wilson, Katherine I.; Fullen, Douglas R.; Verhaegen, Monique; Johnson, Timothy M.; Dlugosz, Andrzej A.; Ross, Theodora S.

2011-01-01

Merkel Cell Carcinoma (MCC) is a neoplasm thought to originate from the neuroendocrine Merkel cells of the skin. While the prevalence of MCC has been increasing, treatments for this disease remain limited due to a paucity of information regarding MCC biology. We have found that the endocytic oncoprotein Huntingtin interacting protein 1 (HIP1) is expressed at high levels in close to 90% of MCC tumors and serves as a more reliable histological cytoplasmic stain than the gold standard, cytokeratin 20 (CK20). Furthermore, high anti-HIP1 antibody reactivity in the sera of a cohort of MCC patients predicts the presence of metastases. Another protein that is frequently expressed at high levels in MCC tumors is the stem cell factor (SCF) receptor tyrosine kinase, c-Kit. In working towards an understanding of how HIP1 might contribute to MCC tumorigenesis, we have discovered that HIP1 interacts with SCF activated c-Kit. These data not only identify HIP1 as a molecular marker for management of MCC patients but also show that HIP1 interacts with and slows the degradation of c-Kit. PMID:21697888

miR-148b-3p functions as a tumor suppressor in GISTs by directly targeting KIT.

PubMed

Wang, Yu; Li, Jun; Kuang, Dong; Wang, Xiaoyan; Zhu, Yuanli; Xu, Sanpeng; Chen, Yaobing; Cheng, Henghui; Zhao, Qiu; Duan, Yaqi; Wang, Guoping

2018-04-16

Gain-of-function mutations and overexpression of KIT are characteristic features of gastrointestinal stromal tumor (GIST). Dysregulation in miRNA expression may lead to KIT overexpression and tumorigenesis. miRNA microarray analysis and real-time PCR were used to determine the miRNA expression profiles in a cohort of 69 clinical samples including 50 CD117 IHC+ /KIT mutation GISTs and 19 CD117 IHC- /wild-type GISTs. GO enrichment and KEGG pathway analyses were performed to reveal the predicted targets of the dysregulated miRNAs. Of the dysregulated miRNAs whose expression was inversely correlated with that of KIT miRNAs were predicted by bioinformatics analysis and confirmed by luciferase reporter assay. Cell counting kit-8 (CCK-8) and flow cytometry were used to measure the cell proliferation, cycle arrest and apoptosis. Wound healing and transwell assays were used to evaluate migration and invasion. A xenograft BALB/c nude mouse model was applied to investigate the tumorigenesis in vivo. Western blot and qRT-PCR were used to investigate the protein and mRNA levels of KIT and its downstream effectors including ERK, AKT and STAT3. Of the six miRNAs whose expression was inversely correlated with that of KIT, we found that miR-148b-3p was significantly downregulated in the CD117 IHC+ /KIT mutation GIST cohort. This miRNA was subsequently found to inhibit proliferation, migration and invasion of GIST882 cells. Mechanistically, miR-148b-3p was shown to regulate KIT expression through directly binding to the 3'-UTR of the KIT mRNA. Restoration of miR-148b-3p expression in GIST882 cells led to reduced expression of KIT and the downstream effectors proteins ERK, AKT and STAT3. However, overexpression of KIT reversed the inhibitory effect of miR-148b-3p on cell proliferation, migration and invasion. Furthermore, we found that reduced miR-148b-3p expression correlated with poor overall survival (OS) and disease-free survival (DFS) in GIST patients. miR-148b-3p functions as

Stromal and epithelial cells react differentially to c-kit in fibroepithelial tumors of the breast.

PubMed

Logullo, Angela F; Nonogaki, Suely; Do Socorro Maciel, Maria; Mourão-Neto, Mário; Soares, Fernando Augusto

2008-01-01

The CD117 protein is a tyrosine-kinase receptor encoded by the c-kit gene that frequently bears activating mutations in gastrointestinal tumors. Conflicting findings regarding CD117 expression in other stromal tumors, including phyllodes tumors (PTs), have been reported in the literature. The purpose of this study was to evaluate c-kit expression in the stroma and epithelia of fibroepithelial breast tumors and its correlation with clinical pathological variables. Ninety-six fibroepithelial tumors of the breast, including 14 fibroadenomas (FAs), 12 juvenile FAs and 70 PTs, were classified according to stromal cellularity, atypia, epithelial hyperplasia, mitosis and borders into 45 benign (PTB), 17 borderline (PTBL) and 8 malignant (PTM) tumors. CD117 expression was identified in the stromal component in only two cases of PTBL. Overall, 38 cases (39.6%) showed positive CD117 in the epithelial component, including 20 FAs (10 regular, 10 juvenile) and 18 PTs (11 PTBs and 8 PTBLs). Other cases, including all PTMs, 6 FAs (4 regular, 2 juvenile), 34 PTBs and 10 PTBLs, showed no positivity in the epithelial component. Expression of c-kit did not correlate with diagnosis or malignancy (p>0.05). In conclusion, c-kit is expressed more often in the epithelial than in the stromal component in fibroepithelial tumors of the breast, and is associated with benign lesions.

Nuclear translocation of the cytoplasmic domain of HB-EGF induces gastric cancer invasion.

PubMed

Shimura, Takaya; Yoshida, Michihiro; Fukuda, Shinji; Ebi, Masahide; Hirata, Yoshikazu; Mizoshita, Tsutomu; Tanida, Satoshi; Kataoka, Hiromi; Kamiya, Takeshi; Higashiyama, Shigeki; Joh, Takashi

2012-05-30

Membrane-anchored heparin-binding epidermal growth factor-like growth factor (proHB-EGF) yields soluble HB-EGF, which is an epidermal growth factor receptor (EGFR) ligand, and a carboxy-terminal fragment of HB-EGF (HB-EGF-CTF) after ectodomain shedding. We previously reported that HB-EGF-CTF and unshed proHB-EGF which has the cytoplasmic domain of proHB-EGF (HB-EGF-C), translocate from the plasma membrane to the nucleus and regulate cell cycle after shedding stimuli. However, the significance of nuclear exported HB-EGF-C in human gastric cancer is unclear. We investigated the relationship between intracellular localization of HB-EGF-C and clinical outcome in 96 gastric cancer patients treated with gastrectomy. Moreover, we established stable gastric cancer cell lines overexpressing wild-type HB-EGF (wt-HB-EGF) and mutated HB-EGF (HB-EGF-mC), which prevented HB-EGF-C nuclear translocation after shedding. Cell motility between these 2 gastric cancer cell lines was investigated using a transwell invasion assay and a wound healing assay. Of the 96 gastric cancer cases, HB-EGF-C immunoreactivity was detected in both the nucleus and cytoplasm in 19 cases (19.8 %) and in the cytoplasm only in 25 cases (26.0 %). The nuclear immunoreactivity of HB-EGF-C was significantly increased in stage pT3/4 tumors compared with pT1/2 tumors (T1/2 vs. T3/4: 11.1 % vs. 36.4 %, P < 0.01). The growth of wt-HB-EGF- and HB-EGF-mC-expressing cells significantly increased compared with control cells, but the growth of HB-EGF-mC-expressing cells was significantly decreased compared with wt-HB-EGF-expressing cells. Gastric cancer cell invasion obviously increased in wt-HB-EGF-expressing cells, but invasion in HB-EGF-mC-expressing cells showed a slight increase compared with control cells. Moreover, wt-HB-EGF overexpression increased the effectiveness of wound healing, but had no significant effect in HB-EGF-mC-expressing cells. Both the function of HB-EGF as an EGFR ligand and a novel signal for

Expression profiling of c-kit and its impact after esiRNA silencing during gonadal development in catfish.

PubMed

Laldinsangi, C; Senthilkumaran, B

2018-04-03

C-kit receptor is a member of a family of growth factor receptors that have tyrosine kinase activity, and are involved in the transduction of growth regulatory signals across plasma membrane by activation of its ligand, kitl/scf. The present study analysed mRNA and protein expression profiles of c-kit in the gonads of catfish, Clarias gariepinus, using real time PCR, in situ hybridization and immunohistochemistry. Tissue distribution analysis revealed higher expression mainly in the catfish gonads. Ontogeny studies showed minimal expression during early developmental stages and highest during 50-75 days post hatch, and the dimorphic expression in gonads decreased gradually till adulthood, which might suggest an important role for this gene around later stages of sex differentiation and gonadal development. Expression of C-kit was analysed at various phases of gonadal cycle in both male and female, which showed minimal expression during the resting phase, and higher expression in male compared to females during the pre-spawning phase. In vitro and in vivo induction using human chorionic gonadotropin elevated the expression of c-kit indicating the regulatory influence of hypothalamo-hypophyseal axis. In vivo transient gene silencing using c-kit-esiRNA in adult catfish during gonadal recrudescence showed a decrease in c-kit expression, which affected the expression level of germ cell meiotic marker sycp3, as well as several factors and steroidogenic enzyme genes involved in germ cell development. Decrease in the levels of serum 11-KT and T were also observed after esiRNA silencing. The findings of this study suggest that c-kit has an important role in the process of germ cell proliferation, development and maturation during gonadal development and recrudescence in catfish. Copyright © 2018. Published by Elsevier Inc.

Oncoprotein protein kinase antibody kit

DOEpatents

Karin, Michael [San Diego, CA; Hibi, Masahiko [San Diego, CA; Lin, Anning [La Jolla, CA

2008-12-23

An isolated polypeptide (JNK) characterized by having a molecular weight of 46 kD as determined by reducing SDS-PAGE, having serine and threonine kinase activity, phosphorylating the c-Jun N-terminal activation domain and polynucleotide sequences and method of detection of JNK are provided herein. JNK phosphorylates c-Jun N-terminal activation domain which affects gene expression from AP-1 sites.

Loss of c-KIT expression in thyroid cancer cells.

PubMed

Franceschi, Sara; Lessi, Francesca; Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria

2017-01-01

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression.

Loss of c-KIT expression in thyroid cancer cells

PubMed Central

Panebianco, Federica; Tantillo, Elena; La Ferla, Marco; Menicagli, Michele; Aretini, Paolo; Apollo, Alessandro; Naccarato, Antonio Giuseppe; Marchetti, Ivo; Mazzanti, Chiara Maria

2017-01-01

Papillary thyroid carcinoma is the most frequent histologic type of thyroid tumor. Few studies investigated the role of c-KIT expression in thyroid tumors, suggesting a role for this receptor and its ligand in differentiation and growth control of thyroid epithelium and a receptor loss following malignant transformation. We investigated and correlated c-KIT expression levels and two known markers of thyrocytes differentiation, PAX8 and TTF-1, in malignant and benign cytological thyroid samples. Moreover, we performed functional studies on human papillary thyroid carcinoma cell line to associated c-KIT expression to thyrocytes differentiation and tumor proliferation. c-KIT and PAX8 expression resulted higher in benign samples compared to the malignant ones, and the expression levels of these two genes were significantly correlated to each other. We also observed that c-KIT overexpression led to an increase of PAX8 expression level together with a decrease of proliferation. Furthermore, c-KIT overexpressing cells showed a regression of typical morphological features of malignancy. Taken together these results suggest that c-KIT could be involved in the differentiation of thyroid cells and in tumor progression. PMID:28301608

Epidermal growth factor-induced phosphatidylinositol 3-kinase activation and DNA synthesis. Identification of Grb2-associated binder 2 as the major mediator in rat hepatocytes.

PubMed

Kong, M; Mounier, C; Wu, J; Posner, B I

2000-11-17

In previous work we showed that the phosphatidylinositol 3-kinase (PI3-kinase), not the mitogen-activated protein kinase, pathway is necessary and sufficient to account for insulin- and epidermal growth factor (EGF)-induced DNA synthesis in rat hepatocytes. Here, using a dominant-negative p85, we confirmed the key role of EGF-induced PI3-kinase activation and sought to identify the mechanism by which this is effected. Our results show that EGF activates PI3-kinase with a time course similar to that of the association of p85 with three principal phosphotyrosine proteins (i. e. PY180, PY105, and PY52). We demonstrated that each formed a distinct p85-associated complex. PY180 and PY52 each constituted about 10% of EGF-activated PI3-kinase, whereas PY105 was responsible for 80%. PY105 associated with Grb2 and SHP-2, and although it behaved like Gab1, none of the latter was detected in rat liver. We therefore cloned a cDNA from rat liver, which was found to be 95% homologous to the mouse Grb2-associated binder 2 (Gab2) cDNA sequence. Using a specific Gab2 antibody, we demonstrated its expression in and association with p85, SHP-2, and Grb2 upon EGF treatment of rat hepatocytes. Gab2 accounted for most if not all of the PY105 species, since immunoprecipitation of Gab2 with specific antibodies demonstrated parallel immunodepletion of Gab2 and PY105 from the residual supernatants. We also found that the PI3-kinase activity associated with Gab2 was totally abolished by dominant negative p85. Thus, Gab2 appears to be the principal EGF-induced PY protein recruiting and activating PI3-kinase and mitogenesis.

miR-96 promotes osteogenic differentiation by suppressing HBEGF-EGFR signaling in osteoblastic cells.

PubMed

Yang, Mingfu; Pan, Yong; Zhou, Yue

2014-12-20

MicroRNAs (miRNAs) are a class of small non-coding RNAs with important roles in various biological and pathological processes, including osteoblast differentiation. Here, we identified miR-96 as a positive regulator of osteogenic differentiation in a mouse osteoblastic cell line (MC3T3-E1) and in mouse bone marrow-derived mesenchymal stem cells. Moreover, we found that miR-96 down-regulates post-transcriptional expression of heparin-binding EGF-like growth factor (HB-EGF) by specifically binding to the 3'untranslated region of HB-EGF mRNA. Furthermore, in MC3T3-E1 cells, miR-96-induced HB-EGF down-regulation suppressed the phosphorylation of epidermal growth factor receptor (EGFR) and of extracellular signal-regulated kinase 1 (ERK1) and AKT, which both lie downstream of EGFR activation. Taken together, miR-96 promotes osteogenic differentiation by inhibiting HB-EGF and by blocking the HB-EGF-EGFR signaling pathway in osteoblastic cells. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

c-kit+ Cells Minimally Contribute Cardiomyocytes to the Heart

PubMed Central

van Berlo, Jop H.; Kanisicak, Onur; Maillet, Marjorie; Vagnozzi, Ronald J.; Karch, Jason; Lin, Suh-Chin J.; Middleton, Ryan C.; Marbán, Eduardo; Molkentin, Jeffery D.

2014-01-01

If and how the heart regenerates after an injury event is highly debated. c-kit-expressing cardiac progenitor cells have been reported as the primary source for generation of new myocardium after injury. Here we generated two genetic approaches in mice to examine if endogenous c-kit+ cells contribute differentiated cardiomyocytes to the heart during development, with aging or after injury in adulthood. A cDNA encoding either Cre recombinase or a tamoxifen inducible MerCreMer chimeric protein was targeted to the Kit locus in mice and then bred with reporter lines to permanently mark cell lineage. Endogenous c-kit+ cells did produce new cardiomyocytes within the heart, although at a percentage of ≈0.03% or less, and if a preponderance towards cellular fusion is considered, the percentage falls below ≈0.008%. In contrast, c-kit+ cells amply generated cardiac endothelial cells. Thus, endogenous c-kit+ cells can generate cardiomyocytes within the heart, although likely at a functionally insignificant level. PMID:24805242

KIT Mutations Are Common in Testicular Seminomas

PubMed Central

Kemmer, Kathleen; Corless, Christopher L.; Fletcher, Jonathan A.; McGreevey, Laura; Haley, Andrea; Griffith, Diana; Cummings, Oscar W.; Wait, Cecily; Town, Ajia; Heinrich, Michael C.

2004-01-01

Expression of KIT tyrosine kinase is critical for normal germ cell development and is observed in the majority of seminomas. Activating mutations in KIT are common in gastrointestinal stromal tumors and mastocytosis. In this study we examined the frequency and spectrum of KIT mutations in 54 testicular seminomas, 1 ovarian dysgerminoma and 37 non-seminomatous germ cell tumors (NSGCT). Fourteen seminomas (25.9%) contained exon 17 point mutations including D816V (6 cases), D816H (3 cases), Y823D (2 cases), and single examples of Y823C, N822K, and T801I. No KIT mutations were found in the ovarian dysgerminoma or the NSGCTs. In transient transfection assays, mutant isoforms D816V, D816H, Y823D, and N822K were constitutively phosphorylated in the absence of the natural ligand for KIT, stem cell factor (SCF). In contrast, activation of T801I and wild-type KIT required SCF. Mutants N822K and Y823D were inhibited by imatinib mesylate (Gleevec, previously STI571) whereas D816V and D816H were both resistant to imatinib mesylate. Biochemical evidence of KIT activation, as assessed by KIT phosphorylation and KIT association with phosphatidylinositol (PI) 3-kinase in tumor cell lysates, was largely confined to seminomas with a genomic KIT mutation. These findings suggest that activating KIT mutations may contribute to tumorigenesis in a subset of seminomas, but are not involved in NSGCT. PMID:14695343

Decoupling the Functional Pleiotropy of Stem Cell Factor by Tuning c-Kit Signaling

PubMed Central

Ho, Chia Chi M.; Chhabra, Akanksha; Starkl, Philipp; Schnorr, Peter-John; Wilmes, Stephan; Moraga, Ignacio; Kwon, Hye-Sook; Gaudenzio, Nicolas; Sibilano, Riccardo; Wehrman, Tom S.; Gakovic, Milica; Sockolosky, Jonathan T.; Tiffany, Matthew R.; Ring, Aaron M.; Piehler, Jacob; Weissman, Irving L.; Galli, Stephen J.; Shizuru, Judith A.; Garcia, K. Christopher

2017-01-01

SUMMARY Most secreted growth factors and cytokines are functionally pleiotropic because their receptors are expressed on diverse cell types. While important for normal mammalian physiology, pleiotropy limits the efficacy of cytokines and growth factors as therapeutics. Stem cell factor (SCF) is a growth factor that acts through the c-Kit receptor tyrosine kinase to elicit hematopoietic progenitor expansion, but can be toxic when administered in vivo because it concurrently activates mast cells. We engineered a mechanism-based SCF partial agonist that impaired c-Kit dimerization, truncating downstream signaling amplitude. This SCF variant elicited biased activation of hematopoietic progenitors over mast cells in vitro and in vivo. Mouse models of SCF-mediated anaphylaxis, radioprotection, and hematopoietic expansion revealed that this SCF partial agonist retained therapeutic efficacy while exhibiting virtually no anaphylactic off-target effects. The approach of biasing cell activation by tuning signaling thresholds and outputs has applications to many dimeric receptor-ligand systems. PMID:28283060

Calmodulin-mediated activation of Akt regulates survival of c-Myc-overexpressing mouse mammary carcinoma cells.

PubMed

Deb, Tushar B; Coticchia, Christine M; Dickson, Robert B

2004-09-10

c-Myc-overexpressing mammary epithelial cells are proapoptotic; their survival is strongly promoted by epidermal growth factor (EGF). We now demonstrate that EGF-induced Akt activation and survival in transgenic mouse mammary tumor virus-c-Myc mouse mammary carcinoma cells are both calcium/calmodulin-dependent. Akt activation is abolished by the phospholipase C-gamma inhibitor U-73122, by the intracellular calcium chelator BAPTA-AM, and by the specific calmodulin antagonist W-7. These results implicate calcium/calmodulin in the activation of Akt in these cells. In addition, Akt activation by serum and insulin is also inhibited by W-7. EGF-induced and calcium/calmodulin-mediated Akt activation occurs in both tumorigenic and non-tumorigenic mouse and human mammary epithelial cells, independent of their overexpression of c-Myc. These results imply that calcium/calmodulin may be a common regulator of Akt activation, irrespective of upstream receptor activator, mammalian species, and transformation status in mammary epithelial cells. However, only c-Myc-overexpressing mouse mammary carcinoma cells (but not normal mouse mammary epithelial cells) undergo apoptosis in the presence of the calmodulin antagonist W-7, indicating the vital selective role of calmodulin for survival of these cells. Calcium/calmodulin-regulated Akt activation is mediated directly by neither calmodulin kinases nor phosphatidylinositol 3-kinase (PI-3 kinase). Pharmacological inhibitors of calmodulin kinase kinase and calmodulin kinases II and III do not inhibit EGF-induced Akt activation, and calmodulin antagonist W-7 does not inhibit phosphotyrosine-associated PI-3 kinase activation. Akt is, however, co-immunoprecipitated with calmodulin in an EGF-dependent manner, which is inhibited by calmodulin antagonist W-7. We conclude that calmodulin may serve a vital regulatory function to direct the localization of Akt to the plasma membrane for its activation by PI-3 kinase.

Epidermal growth factor (EGF) receptor-ligand based molecular staging predicts prognosis in head and neck squamous cell carcinoma partly due to deregulated EGF- induced amphiregulin expression.

PubMed

Gao, Jian; Ulekleiv, Camilla H; Halstensen, Trond S

2016-09-26

Increased expression of epidermal growth factor receptor (EGFR) and its ligands is associated with poor prognosis and chemoresistance in many carcinoma types, but its role in head and neck squamous cell carcinoma (HNSCC) is unclear. Our aim was to clarify whether mRNA expression of EGFR-ligands was linked to prognosis and cisplatin resistance, and if so, which ligand was most important and how was the expression regulated. To examine the prognostic effect of EGFR-ligand expression, we analyzed tumorous mRNA expression in 399 HNSCC patients. The intracellular signaling pathways controlling epidermal growth factor (EGF)-induced amphiregulin (AREG) expression were examined in three oral squamous cell carcinoma (OSCC) cell lines. Effect of AREG on cisplatin resistance was examined by viability assays in four-, and by association in 11 OSCC cell lines. The patients were divided into five groups according to the median mRNA expression levels of four EGFR ligands, i.e. AREG, EGF, heparin-binding EGF-like growth factor (HBEGF) and beta-cellulin (BTC). The number of increased-expressed EGFR-ligands were progressively correlated to five-year survival, even in advanced TNM-stage IV patients, where five-year mortality increased from 26 % if tumor expressed none to one EGFR-ligand, to 45 % in three to four ligand expressing tumors. Thus, staging the tumor according to these EGFR-ligand mRNA expression pattern completely out performed TNM staging in predicting prognosis. Multivariate analysis identified AREG as the dominating predictor, and AREG was overexpressed in OSCC compared to tumors from other sites. Both EGF and HBEGF stimulation induced strong AREG increase in OSCC cell lines, which was partially mediated by the extracellular signal-regulated kinase 1/2 pathway, and negatively regulated by p38, c-Jun N-terminal kinase, and phosphoinositide-3 kinase. Although increased AREG mRNA expression predicted unfavorable prognosis in platinum treated HNSCC patients, AREG did

MNK1/2 inhibition limits oncogenicity and metastasis of KIT-mutant melanoma

PubMed Central

Zhan, Yao; Guo, Jun; Yang, William; Goncalves, Christophe; Rzymski, Tomasz; Dreas, Agnieszka; Żyłkiewicz, Eliza; Mikulski, Maciej; Brzózka, Krzysztof; Golas, Aniela; Kong, Yan; Ma, Meng; Huang, Fan; Huor, Bonnie; Guo, Qianyu; da Silva, Sabrina Daniela; Torres, Jose; Cai, Yutian; Topisirovic, Ivan; Su, Jie; Bijian, Krikor; Alaoui-Jamali, Moulay A.; Huang, Sidong; Journe, Fabrice; Ghanem, Ghanem E.; Miller, Wilson H.

2017-01-01

Melanoma can be stratified into unique subtypes based on distinct pathologies. The acral/mucosal melanoma subtype is characterized by aberrant and constitutive activation of the proto-oncogene receptor tyrosine kinase C-KIT, which drives tumorigenesis. Treatment of these melanoma patients with C-KIT inhibitors has proven challenging, prompting us to investigate the downstream effectors of the C-KIT receptor. We determined that C-KIT stimulates MAP kinase–interacting serine/threonine kinases 1 and 2 (MNK1/2), which phosphorylate eukaryotic translation initiation factor 4E (eIF4E) and render it oncogenic. Depletion of MNK1/2 in melanoma cells with oncogenic C-KIT inhibited cell migration and mRNA translation of the transcriptional repressor SNAI1 and the cell cycle gene CCNE1. This suggested that blocking MNK1/2 activity may inhibit tumor progression, at least in part, by blocking translation initiation of mRNAs encoding cell migration proteins. Moreover, we developed an MNK1/2 inhibitor (SEL201), and found that SEL201-treated KIT-mutant melanoma cells had lower oncogenicity and reduced metastatic ability. Clinically, tumors from melanoma patients harboring KIT mutations displayed a marked increase in MNK1 and phospho-eIF4E. Thus, our studies indicate that blocking MNK1/2 exerts potent antimelanoma effects and support blocking MNK1/2 as a potential strategy to treat patients positive for KIT mutations. PMID:29035277

EGF stimulates Mg{sup 2+} influx in mammary epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Trapani, Valentina; Arduini, Daniela; Luongo, Francesca

2014-11-28

Highlights: • EGF stimulation potentiates Mg{sup 2+} influx into epithelial cells. • EGF-induced Mg{sup 2+} influx does not depend on the concomitantly induced Ca{sup 2+} signal. • EGF-induced Ca{sup 2+} signal is dependent on the presence of extracellular Mg{sup 2+}. • New players in EGF-mediated signaling might be exploited as therapeutic targets. - Abstract: Magnesium is well established as a fundamental factor that regulates cell proliferation. However, the molecular mechanisms linking mitogenic signals, extracellular magnesium availability and intracellular effectors are still largely unknown. In the present study we sought to determine whether EGF regulates magnesium homeostasis in normal HC11 mammarymore » epithelial cells. To this end, we measured Mg{sup 2+} and Ca{sup 2+} fluxes by confocal imaging in live cells loaded with specific fluorescent ion indicators (Mag-Fluo-4 and Fluo-4, respectively). EGF stimulation induces a rapid and sustained increase in intracellular Mg{sup 2+}, concomitantly with a rise in intracellular calcium. The increase in intracellular Mg{sup 2+} derives from an influx from the extracellular compartment, and does not depend on Ca{sup 2+}. On the contrary, the increase in intracellular Ca{sup 2+} derives from intracellular stores, and is impaired in the absence of extracellular magnesium. Inhibition of the EGF receptor tyrosine kinase by Tyrphostin AG1478 markedly inhibits EGF-induced Mg{sup 2+} and Ca{sup 2+} signals. These findings demonstrate that not only does Mg{sup 2+} influx represent an important step in the physiological response of epithelial cells to EGF, but unexpectedly the EGF-induced Mg{sup 2+} influx is essential for the Ca{sup 2+} signal to occur.« less

Stage-specific expression of DDX4 and c-kit at different developmental stages of the porcine testis.

PubMed

Lee, Ran; Lee, Won-Young; Park, Hyun-Jung; Ha, Woo-Tae; Woo, Jae-Seok; Chung, Hak-Jae; Lee, Ji-Heon; Hong, Kwonho; Song, Hyuk

2018-03-01

Spermatogenesis begins with spermatogonial stem cells (SSCs), which are located in the basement membrane of the adult testes. Previous studies have described specific biomarkers for undifferentiated porcine spermatogonia or SSCs; however, these markers are not sufficient to understand spermatogenesis at different developmental stages. The objective of this study was characterize the expression of DEAD-Box polypeptide 4 (DDX4, also known as VASA) and tyrosine-protein kinase kit (c-kit), as potential markers of male germ cells in the porcine testis. In porcine testis tissue at prepubertal stages (5, 30, and 60 days), DDX4 and c-kit protein expression was detected in the most undifferentiated spermatogonia, which also express protein gene product 9.5 (PGP9.5). However, in porcine testis tissues from pubertal and postpubertal stages (90, 120, and 150 days), DDX4 and c-kit were not detected in PGP9.5-positive undifferentiated spermatogonia. The DDX4 expression pattern was similar to that of c-kit in the porcine testis. In adult porcine testes, DDX4-expressing cells were located on the lumenal side, compared to synaptonemal complex protein 3-positive primary spermatocytes, but DDX-4 was not co-expressed with acrosin, a known acrosome marker. In addition, DDX4 was detected in PGP9.5-expressing porcine SSCs in culture. Based on our results, we suggest that DDX4 and c-kit are putative markers of undifferentiated spermatogonia in the prepubertal porcine testis. While in the postpubertal porcine testis, they are markers of differentiated spermatocytes. These findings may facilitate future studies of porcine spermatogenesis. Copyright © 2018 Elsevier B.V. All rights reserved.

A potent anti-HB-EGF monoclonal antibody inhibits cancer cell proliferation and multiple angiogenic activities of HB-EGF.

PubMed

Sato, Shuji; Drake, Andrew W; Tsuji, Isamu; Fan, Jinhong

2012-01-01

Heparin-binding epidermal growth factor-like growth factor (HB-EGF) is a member of the epidermal growth factor family and has a variety of physiological and pathological functions. Modulation of HB-EGF activity might have a therapeutic potential in the oncology area. We explored the therapeutic possibilities by characterizing the in vitro biological activity of anti-HB-EGF monoclonal antibody Y-142. EGF receptor (EGFR) ligand and species specificities of Y-142 were tested. Neutralizing activities of Y-142 against HB-EGF were evaluated in EGFR and ERBB4 signaling. Biological activities of Y-142 were assessed in cancer cell proliferation and angiogenesis assays and compared with the anti-EGFR antibody cetuximab, the HB-EGF inhibitor CRM197, and the anti-vascular endothelial growth factor (VEGF) antibody bevacizumab. The binding epitope was determined with alanine scanning. Y-142 recognized HB-EGF as well as the EGFR ligand amphiregulin, and bound specifically to human HB-EGF, but not to rodent HB-EGF. In addition, Y-142 neutralized HB-EGF-induced phosphorylation of EGFR and ERBB4, and blocked their downstream ERK1/2 and AKT signaling. We also found that Y-142 inhibited HB-EGF-induced cancer cell proliferation, endothelial cell proliferation, tube formation, and VEGF production more effectively than cetuximab and CRM197 and that Y-142 was superior to bevacizumab in the inhibition of HB-EGF-induced tube formation. Six amino acids in the EGF-like domain were identified as the Y-142 binding epitope. Among the six amino acids, the combination of F115 and Y123 determined the amphiregulin cross-reactivity and that F115 accounted for the species selectivity. Furthermore, it was suggested that the potent neutralizing activity of Y-142 was derived from its recognition of R142 and Y123 and its high affinity to HB-EGF. Y-142 has a potent HB-EGF neutralizing activity that modulates multiple biological activities of HB-EGF including cancer cell proliferation and angiogenic

EGF receptor tyrosine kinase inhibitors diminish transforming growth factor-alpha-induced pulmonary fibrosis.

PubMed

Hardie, William D; Davidson, Cynthia; Ikegami, Machiko; Leikauf, George D; Le Cras, Timothy D; Prestridge, Adrienne; Whitsett, Jeffrey A; Korfhagen, Thomas R

2008-06-01

Transforming growth factor-alpha (TGF-alpha) is a ligand for the EGF receptor (EGFR). EGFR activation is associated with fibroproliferative processes in human lung disease and animal models of pulmonary fibrosis. We determined the effects of EGFR tyrosine kinase inhibitors gefitinib (Iressa) and erlotinib (Tarceva) on the development and progression of TGF-alpha-induced pulmonary fibrosis. Using a doxycycline-regulatable transgenic mouse model of lung-specific TGF-alpha expression, we determined effects of treatment with gefitinib and erlotinib on changes in lung histology, total lung collagen, pulmonary mechanics, pulmonary hypertension, and expression of genes associated with synthesis of ECM and vascular remodeling. Induction in the lung of TGF-alpha caused progressive pulmonary fibrosis over an 8-wk period. Daily administration of gefitinib or erlotinib prevented development of fibrosis, reduced accumulation of total lung collagen, prevented weight loss, and prevented changes in pulmonary mechanics. Treatment of mice with gefitinib 4 wk after the induction of TGF-alpha prevented further increases in and partially reversed total collagen levels and changes in pulmonary mechanics and pulmonary hypertension. Increases in expression of genes associated with synthesis of ECM as well as decreases of genes associated with vascular remodeling were also prevented or partially reversed. Administration of gefitinib or erlotinib did not cause interstitial fibrosis or increases in lavage cell counts. Administration of small molecule EGFR tyrosine kinase inhibitors prevented further increases in and partially reversed pulmonary fibrosis induced directly by EGFR activation without inducing inflammatory cell influx or additional lung injury.

Correlations of EGF G1380A, bFGF C754G and VEGF T460C polymorphisms with malignant melanoma susceptibility and prognosis: A case-control study.

PubMed

Wang, Xin-Hua; Long, Zi-Wen

2017-06-20

This case-control study aims to investigate the correlations of EGF G1380A, bFGF C754G and VEGF T460C polymorphisms with the susceptibility and prognosis of malignant melanoma. A total of 153 patients with multiple primary melanomas were collected as the case group and another 170 healthy individuals were selected as the control group. ELISA and PCR-RFLP were performed to test the serum level of VEGF and to analyze the genotype as well as allele frequencies of VEGF T460C, EGF G1380A, and bFGF C754G, respectively. The patients were assigned into complete remission (CR), partial remission (PR) and non-remission groups after treatment. HE and CD34 staining were conducted in tissue samples of CR and PR patients. Event-free survival (EFS) and overall survival (OS) were measured. AA genotype of EGF G1380A and GG genotype of bFGF C754G had higher frequency distribution in the case group than the control group. Patients with AA genotype of EGF G1380 and GG genotype of bFGF C754G had an elevated VEGF level in comparison to other genotypes. Patients with GA+GG genotypes of EGF G1380A and CG+CC genotypes of bFGF C754G had higher EFS and OS than those with AA genotype and those with GG genotype, respectively. According to the haplotype analysis, the case group had a notably higher frequency of TAG and CAG along with while lower frequency of TGG and CGC compared with the control group. Logistic regression analysis revealed that the polymorphisms of EGF G1380A and bFGF C754G as well as the haploid TAG increased the susceptibility of malignant melanoma. The results indicated that EGF G1380A and bFGF C754G gene polymorphisms were associated with the susceptibility and prognosis of malignant melanoma, and that the polymorphisms of EGF G1380A and bFGF C754G as well as the haploid TAG increased the susceptibility of malignant melanoma. Copyright © 2017 Elsevier B.V. All rights reserved.

Epidermal growth factor-stimulated protein phosphorylation in rat hepatocytes

DOE Office of Scientific and Technical Information (OSTI.GOV)

Connelly, P.A.; Sisk, R.B.; Johnson, R.M.

1987-05-01

Epidermal growth factor (EGF) causes a 6-fold increase in the phosphorylation state of a cytosolic protein (pp36, M/sub r/ = 36,000, pI = 5.5) in hepatocytes isolated from fasted, male, Wistar rats. Stimulation of /sup 32/P incorporation is observed as early as 1 min following treatment of hepatocytes with EGF and is still present at 30 min after exposure to the growth factor. The phosphate incorporated into pp36 in response to EGF is located predominantly in serine but not tyrosine residues. Phosphorylation of pp36 does not occur in response to insulin or to agents which specifically activate the cAMP-dependent proteinmore » kinase (S/sub p/ -cAMPS), protein kinase C (PMA) or Ca/sup 2 +//calmodulin-dependent protein kinases (A23187) in these cells. Prior treatment of hepatocytes with the cAMP analog, S/sub p/-cAMPS, or ADP-ribosylation of N/sub i/, the inhibitory GTP-binding protein of the adenylate cyclase complex, does not prevent EGF-stimulated phosphorylation of pp36. However, as seen in other cell types, pretreatment of hepatocytes with PMA abolishes all EGF-mediated responses including phosphorylation of pp36. These results suggest that EGP specifically activates an uncharacterized, serine protein kinase in hepatocytes that is distal to the intrinsic EGF receptor tyrosine protein kinase. The rapid activation of this kinase suggests that it may play an important role in the early response of the cell to EGF.« less

No GIST-type c-kit gain of function mutations in neuroblastic tumours

PubMed Central

Korja, M; Finne, J; Salmi, T T; Haapasalo, H; Tanner, M; Isola, J

2005-01-01

Aims: Neuroblastic tumours (NTs) have been shown to respond to imatinib treatment in vivo and in vitro, possibly via inactivating the c-kit receptor. The purpose of this study was to identify gastrointestinal stromal tumour (GIST)-type c-kit gene associated mutations in exons 9, 11, 13, and 17 in NTs to recognise a subset of tumours that would probably respond to imatinib treatment. Methods: Expression of the c-kit protein was detected immunohistochemically in a total of 37 archival paraffin wax embedded NTs using polyclonal rabbit antihuman c-kit antibody. After immunohistochemistry, c-kit gene associated chromosomal mutations in all cases of NT were detected with denaturing high performance liquid chromatography (HPLC). Results: Denaturing HLPC analysis did not reveal GIST-type mutations in four immunohistochemically detected c-kit positive or in 33 c-kit negative NTs. Conclusions: c-kit receptor expression and GIST-type c-kit gene mutations are rare events in NTs. Oncogenic activation of c-kit in NTs presumably differs from that of GISTs, which may influence their responsiveness to imatinib treatment. Whether c-kit has an essential role in the pathogenesis of NTs remains to be investigated. PMID:15976348

Association of paediatric mastocytosis with a polymorphism resulting in an amino acid substitution (M541L) in the transmembrane domain of c-KIT.

PubMed

Foster, R; Byrnes, E; Meldrum, C; Griffith, R; Ross, G; Upjohn, E; Braue, A; Scott, R; Varigos, G; Ferrao, P; Ashman, L K

2008-11-01

The receptor tyrosine kinase c-KIT plays a key role in normal mast cell development. Point mutations in c-KIT have been associated with sporadic or familial mastocytosis. Two unrelated pairs of apparently identical twins affected by cutaneous mastocytosis attending the Mastocytosis Clinic at the Royal Children's Hospital, Melbourne, provided an opportunity to assess the possible contribution of c-KIT germline mutations or polymorphisms in this disease. Tissue biopsy, blood and/or buccal swab specimens were collected from 10 children with mastocytosis. To detect germline mutations/polymorphisms in c-KIT, we studied all coding exons by denaturing high pressure liquid chromatography. Exons showing mismatches were examined by direct sequencing. The influence of the substitution identified was further examined by expressing the variant form of c-KIT in factor-dependent FDC-P1 cells. In both pairs of twins, a heterozygous ATG to CTG transition in codon 541 was observed, resulting in the substitution of a methionine residue in the transmembrane domain by leucine (M541L). In each case, one parent was also heterozygous for this allele. Expression of M541L KIT in FDC-P1 cells enabled them to grow in human KIT ligand (stem cell factor, SCF) but did not confer factor independence. Compared with cells expressing wild-type KIT at a similar level, M541L KIT-expressing cells displayed enhanced growth at low levels of SCF, and heightened sensitivity to the KIT inhibitor, imatinib mesylate. The data suggest that the single nucleotide polymorphism resulting in the substitution M541L may predispose to paediatric mastocytosis.

Photoaffinity labelling of the ATP-binding site of the epidermal growth factor-dependent protein kinase.

PubMed

Kudlow, J E; Leung, Y

1984-06-15

Epidermal growth factor (EGF), after binding to its receptor, activates a tyrosine-specific protein kinase which phosphorylates several substrates, including the EGF receptor itself. The effects of a photoaffinity analogue of ATP, 3'-O-(3-[N-(4-azido-2-nitrophenyl)amino]propionyl)adenosine 5'-triphosphate (arylazido-beta-alanyl-ATP) on the EGF-dependent protein kinase in A431 human tumour cell plasma membrane vesicles was investigated. This analogue was capable of inactivating the EGF-receptor kinase in a photodependent manner. Partial inactivation occurred at an analogue concentration of 1 microM and complete inactivation occurred at 10 microM when a 2 min light exposure was used. Arylazido-beta-alanine at 100 microM and ATP at 100 microM were incapable of inactivating the enzyme with 2 min of light exposure. The photodependent inactivation of the enzyme by the analogue could be partially blocked by 20 mM-ATP and more effectively blocked by either 20 mM-adenosine 5'-[beta gamma-imido]triphosphate or 20 mM-guanosine 5'-[beta gamma-imido]triphosphate, indicating nucleotide-binding site specificity. Arylazido-beta-alanyl-[alpha-32P]ATP was capable of labelling membrane proteins in a photodependent manner. Numerous proteins were labelled, the most prominent of which ran with an apparent Mr of 53000 on polyacrylamide-gel electrophoresis. A band of minor intensity was seen of Mr corresponding to the EGF receptor (170000). Immunoprecipitation of affinity-labelled and solubilized membranes with an anti-(EGF receptor) monoclonal antibody demonstrated that the Mr 170000 receptor protein was photoaffinity labelled by the analogue. The Mr 53000 peptide was not specifically bound by the anti-receptor antibody. The affinity labelling of the receptor was not enhanced by EGF, suggesting that EGF stimulation of the kinase activity does not result from changes in the affinity of the kinase for ATP. These studies demonstrate that arylazido-beta-alanyl-ATP interacts with the ATP

Cross-talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells

PubMed Central

Liu, Xing; Qin, Jiaqiang; Luo, Qing; Bi, Yang; Zhu, Gaohui; Jiang, Wei; Kim, Stephanie H; Li, Mi; Su, Yuxi; Nan, Guoxin; Cui, Jing; Zhang, Wenwen; Li, Ruidong; Chen, Xiang; Kong, Yuhan; Zhang, Jiye; Wang, Jinhua; Rogers, Mary Rose; Zhang, Hongyu; Shui, Wei; Zhao, Chen; Wang, Ning; Liang, Xi; Wu, Ningning; He, Yunfeng; Luu, Hue H; Haydon, Rex C; Shi, Lewis L; Li, Tingyu; He, Tong-Chuan; Li, Ming

2013-01-01

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine. PMID:23844832

Specific Inhibitors of Platelet-Derived Growth Factor or Epidermal Growth Factor Receptor Tyrosine Kinase Reduce Pulmonary Fibrosis in Rats

PubMed Central

Rice, Annette B.; Moomaw, Cindy R.; Morgan, Daniel L.; Bonner, James C.

1999-01-01

The proliferation of myofibroblasts is a central feature of pulmonary fibrosis. In this study we have used tyrosine kinase inhibitors of the tyrphostin class to specifically block autophosphorylation of the platelet-derived growth factor receptor (PDGF-R) or epidermal growth factor receptor (EGF-R). AG1296 specifically inhibited autophosphorylation of PDGF-R and blocked PDGF-stimulated [3H]thymidine uptake by rat lung myofibroblasts in vitro. AG1478 was demonstrated as a selective blocker of EGF-R autophosphorylation and inhibited EGF-stimulated DNA synthesis in vitro. In a rat model of pulmonary fibrosis caused by intratracheal instillation of vanadium pentoxide (V2O5), intraperitoneal delivery of 50 mg/kg AG1296 or AG1478 in dimethylsulfoxide 1 hour before V2O5 instillation and again 2 days after instillation reduced the number of epithelial and mesenchymal cells incorporating bromodeoxyuridine (Brdu) by ∼50% at 3 and 6 days after instillation. V2O5 instillation increased lung hydroxyproline fivefold 15 days after instillation, and AG1296 was more than 90% effective in preventing the increase in hydroxyproline, whereas AG1478 caused a 50% to 60% decrease in V2O5-stimulated hydroxyproline accumulation. These data provide evidence that PDGF and EGF receptor ligands are potent mitogens for collagen-producing mesenchymal cells during pulmonary fibrogenesis, and targeting tyrosine kinase receptors could offer a strategy for the treatment of fibrotic lung diseases. PMID:10393853

A variant c-KIT mutation, D816H, fundamental to the sequential development of an ovarian mixed germ cell tumor and systemic mastocytosis with chronic myelomonocytic leukemia.

PubMed

Mitchell, Sarah G; Bunting, Silvia T; Saxe, Debra; Olson, Thomas; Keller, Frank G

2017-04-01

An activating point mutation of the c-KIT tyrosine kinase receptor gene, D816H, has been described in germ cell tumors (GCTs). We report an adolescent diagnosed with an ovarian mixed GCT and systemic mastocytosis with chronic myelomonocytic leukemia (SM-CMML). The teratoma and dysgerminoma differed by copy number aberrations via single nucleotide polymorphism (SNP) microarray, but were inclusive of the same c-KIT D816H point mutation (c.2446G>C) also identified in blood and bone marrow mast cells. These findings indicate not only a clonal origin of the GCT and hematologic malignancy, but also suggest a rare KIT mutation may be playing a fundamental role in malignancy development. © 2016 Wiley Periodicals, Inc.

EGF receptor uses SOS1 to drive constitutive activation of NFκB in cancer cells

PubMed Central

De, Sarmishtha; Dermawan, Josephine Kam Tai; Stark, George R.

2014-01-01

Activation of nuclear factor κB (NFκB) is a central event in the responses of normal cells to inflammatory signals, and the abnormal constitutive activation of NFκB is important for the survival of most cancer cells. In nonmalignant human cells, EGF stimulates robust activation of NFκB. The kinase activity of the EGF receptor (EGFR) is required, because the potent and specific inhibitor erlotinib blocks the response. Down-regulating EGFR expression or inhibiting EGFR with erlotinib impairs constitutive NFκB activation in several different types of cancer cells and, conversely, increased activation of NFκB leads to erlotinib resistance in these cells. We conclude that EGF is an important mediator of NFκB activation in cancer cells. To explore the mechanism, we selected an erlotinib-resistant cell line in which the guanine nucleotide exchange factor Son of Sevenless 1 (SOS1), well known to be important for EGF-dependent signaling to MAP kinases, is overexpressed. Increased expression of SOS1 increases NFκB activation in several different types of cancer cells, and ablation of SOS1 inhibits EGF-induced NFκB activation in these cells, indicating that SOS1 is a functional component of the pathway connecting EGFR to NFκB activation. Importantly, the guanine nucleotide exchange activity of SOS1 is not required for NFκB activation. PMID:25071181

Cross-talk between EGF and BMP9 signalling pathways regulates the osteogenic differentiation of mesenchymal stem cells.

PubMed

Liu, Xing; Qin, Jiaqiang; Luo, Qing; Bi, Yang; Zhu, Gaohui; Jiang, Wei; Kim, Stephanie H; Li, Mi; Su, Yuxi; Nan, Guoxin; Cui, Jing; Zhang, Wenwen; Li, Ruidong; Chen, Xiang; Kong, Yuhan; Zhang, Jiye; Wang, Jinhua; Rogers, Mary Rose; Zhang, Hongyu; Shui, Wei; Zhao, Chen; Wang, Ning; Liang, Xi; Wu, Ningning; He, Yunfeng; Luu, Hue H; Haydon, Rex C; Shi, Lewis L; Li, Tingyu; He, Tong-Chuan; Li, Ming

2013-09-01

Mesenchymal stem cells (MSCs) are multipotent progenitors, which give rise to several lineages, including bone, cartilage and fat. Epidermal growth factor (EGF) stimulates cell growth, proliferation and differentiation. EGF acts by binding with high affinity to epidermal growth factor receptor (EGFR) on the cell surface and stimulating the intrinsic protein tyrosine kinase activity of its receptor, which initiates a signal transduction cascade causing a variety of biochemical changes within the cell and regulating cell proliferation and differentiation. We have identified BMP9 as one of the most osteogenic BMPs in MSCs. In this study, we investigate if EGF signalling cross-talks with BMP9 and regulates BMP9-induced osteogenic differentiation. We find that EGF potentiates BMP9-induced early and late osteogenic markers of MSCs in vitro, which can be effectively blunted by EGFR inhibitors Gefitinib and Erlotinib or receptor tyrosine kinase inhibitors AG-1478 and AG-494 in a dose- and time-dependent manner. Furthermore, EGF significantly augments BMP9-induced bone formation in the cultured mouse foetal limb explants. In vivo stem cell implantation experiment reveals that exogenous expression of EGF in MSCs can effectively potentiate BMP9-induced ectopic bone formation, yielding larger and more mature bone masses. Interestingly, we find that, while EGF can induce BMP9 expression in MSCs, EGFR expression is directly up-regulated by BMP9 through Smad1/5/8 signalling pathway. Thus, the cross-talk between EGF and BMP9 signalling pathways in MSCs may underline their important roles in regulating osteogenic differentiation. Harnessing the synergy between BMP9 and EGF should be beneficial for enhancing osteogenesis in regenerative medicine. © 2013 The Authors. Journal of Cellular and Molecular Medicine Published by Foundation for Cellular and Molecular Medicine/Blackwell Publishing Ltd.

Thrombin-induced p38 mitogen-activated protein kinase activation is mediated by epidermal growth factor receptor transactivation pathway

PubMed Central

Kanda, Yasunari; Mizuno, Katsushige; Kuroki, Yasutomi; Watanabe, Yasuhiro

2001-01-01

Thrombin is a potent mitogen for vascular smooth muscle cells (VSMC) and has been implicated its pathogenic role in vascular remodelling. However, the signalling pathways by which thrombin mediates its mitogenic response are not fully understood.We have previously reported that thrombin activates p38 mitogen-activated protein kinase (p38 MAPK) by a tyrosine kinase-dependent mechanism, and that p38 MAPK has a role in thrombin-induced mitogenic response in rat VSMC.In the present study, we examine the involvement of epidermal growth factor (EGF) receptor in thrombin-induced p38 MAPK activation. We found that thrombin induced EGF receptor tyrosine phosphorylation (transactivation) in A10 cells, a clonal VSMC cell line. A selective inhibitor of EGF receptor kinase (AG1478) inhibited the p38 MAPK activation in a dose-dependent manner, whereas it had no effect on the response to platelet-derived growth factor (PDGF). EGF receptor phosphorylation induced by thrombin was inhibited by BAPTA-AM and GF109203X, which suggest a requirement for intracellular Ca2+ increase and protein kinase C.We next examined the effect of AG1478 on thrombin-induced DNA synthesis. AG1478 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. In contrast, PDGF-induced DNA synthesis was not affected by AG1478.In conclusion, these data suggest that the EGF receptor transactivation and subsequent p38 MAPK activation is required for thrombin-induced proliferation of VSMC. PMID:11309236

Epac-protein kinase C alpha signaling in purinergic P2X3R-mediated hyperalgesia after inflammation.

PubMed

Gu, Yanping; Li, Guangwen; Chen, Yong; Huang, Li-Yen Mae

2016-07-01

Sensitization of purinergic P2X3 receptors (P2X3Rs) is a major mechanism contributing to injury-induced exaggerated pain responses. We showed in a previous study that cyclic adenosine monophosphate (cAMP)-dependent guanine nucleotide exchange factor 1 (Epac1) in rat sensory dorsal root ganglia (DRGs) is upregulated after inflammatory injury, and it plays a critical role in P2X3R sensitization by activating protein kinase C epsilon (PKCε) inside the cells. protein kinase C epsilon has been established as the major PKC isoform mediating injury-induced hyperalgesic responses. On the other hand, the role of PKCα in receptor sensitization was seldom considered. Here, we studied the participation of PKCα in Epac signaling in P2X3R-mediated hyperalgesia. The expression of both Epac1 and Epac2 and the level of cAMP in DRGs are greatly enhanced after complete Freund adjuvant (CFA)-induced inflammation. The expression of phosphorylated PKCα is also upregulated. Complete Freund adjuvant (CFA)-induced P2X3R-mediated hyperalgesia is not only blocked by Epac antagonists but also by the classical PKC isoform inhibitors, Go6976, and PKCα-siRNA. These CFA effects are mimicked by the application of the Epac agonist, 8-(4-chlorophenylthio)-2 -O-methyl-cAMP (CPT), in control rats, further confirming the involvement of Epacs. Because the application of Go6976 prior to CPT still reduces CPT-induced hyperalgesia, PKCα is downstream of Epacs to mediate the enhancement of P2X3R responses in DRGs. The pattern of translocation of PKCα inside DRG neurons in response to CPT or CFA stimulation is distinct from that of PKCε. Thus, in contrast to prevalent view, PKCα also plays an essential role in producing complex inflammation-induced receptor-mediated hyperalgesia.

Phosphorylation of the Yeast Choline Kinase by Protein Kinase C

PubMed Central

Choi, Mal-Gi; Kurnov, Vladlen; Kersting, Michael C.; Sreenivas, Avula; Carman, George M.

2005-01-01

The Saccharomyces cerevisiae CKI1-encoded choline kinase catalyzes the committed step in phosphatidylcholine synthesis via the Kennedy pathway. The enzyme is phosphorylated on multiple serine residues, and some of this phosphorylation is mediated by protein kinase A. In this work, we examined the hypothesis that choline kinase is also phosphorylated by protein kinase C. Using choline kinase as a substrate, protein kinase C activity was dose- and time-dependent, and dependent on the concentrations of choline kinase (Km = 27 μg/ml) and ATP (Km = 15 μM). This phosphorylation, which occurred on a serine residue, was accompanied by a 1.6-fold stimulation of choline kinase activity. The synthetic peptide SRSSS25QRRHS (Vmax/Km = 17.5 mM-1 μmol min-1 mg-1) that contains the protein kinase C motif for Ser25 was a substrate for protein kinase C. A Ser25 to Ala (S25A) mutation in choline kinase resulted in a 60% decrease in protein kinase C phosphorylation of the enzyme. Phosphopeptide mapping analysis of the S25A mutant enzyme confirmed that Ser25 was a protein kinase C target site. In vivo, the S25A mutation correlated with a decrease (55%) in phosphatidylcholine synthesis via the Kennedy pathway whereas an S25D phosphorylation site mimic correlated with an increase (44%) in phosphatidylcholine synthesis. Whereas the S25A (protein kinase C site) mutation did not affect the phosphorylation of choline kinase by protein kinase A, the S30A (protein kinase A site) mutation caused a 46% reduction in enzyme phosphorylation by protein kinase C. A choline kinase synthetic peptide (SQRRHS30LTRQ) containing Ser30 was a substrate (Vmax/Km = 3.0 mM−1 μmol min−1 mg−1) for protein kinase C. Comparison of phosphopeptide maps of the wild type and S30A mutant choline kinase enzymes phosphorylated by protein kinase C confirmed that Ser30 was also a target site for protein kinase C. PMID:15919656

Effect of the anti-receptor ligand-blocking 225 monoclonal antibody on EGF receptor endocytosis and sorting

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jaramillo, Maria L.; Leon, Zully; Grothe, Suzanne

The anti-receptor antibody, 225 mAb, is known to block binding of ligand to the epidermal growth factor receptor (EGFR). However, the effect of this neutralizing antibody on EGFR endocytosis, trafficking and degradation remains unclear. Here, we demonstrate that endocytosis of {sup 125}I-225 mAb occurs, albeit with a slower rate than that of EGF. Using pulse chase assays, we show that internalized {sup 125}I-225 mAb is recycled to the surface much more efficiently than internalized {sup 125}I-EGF. Also, we found that internalization of {sup 125}I-225 mAb, in contrast to that of EGF, is independent of receptor tyrosine kinase activity, as evidencedmore » by its insensitivity to AG1478, a specific EGFR tyrosine kinase inhibitor. Analysis of the levels of cell surface and total EGFR showed that treatment with 225 mAb results in a 30-40% decrease in surface EGFR and a relatively slow downregulation of total EGFR. Taken together, these data indicate that 225 mAb induces internalization and downregulation of EGFR via a mechanism distinct from that underlying EGF-induced EGFR internalization and downregulation.« less

The SH2 and SH3 domains of mammalian Grb2 couple the EGF receptor to the Ras activator mSos1.

PubMed

Rozakis-Adcock, M; Fernley, R; Wade, J; Pawson, T; Bowtell, D

1993-05-06

Many tyrosine kinases, including the receptors for hormones such as epidermal growth factor (EGF), nerve growth factor and insulin, transmit intracellular signals through Ras proteins. Ligand binding to such receptors stimulates Ras guanine-nucleotide-exchange activity and increases the level of GTP-bound Ras, suggesting that these tyrosine kinases may activate a guanine-nucleotide releasing protein (GNRP). In Caenorhabditis elegans and Drosophila, genetic studies have shown that Ras activation by tyrosine kinases requires the protein Sem-5/drk, which contains a single Src-homology (SH) 2 domain and two flanking SH3 domains. Sem-5 is homologous to the mammalian protein Grb2, which binds the autophosphorylated EGF receptor and other phosphotyrosine-containing proteins such as Shc through its SH2 domain. Here we show that in rodent fibroblasts, the SH3 domains of Grb2 are bound to the proline-rich carboxy-terminal tail of mSos1, a protein homologous to Drosophila Sos. Sos is required for Ras signalling and contains a central domain related to known Ras-GNRPs. EGF stimulation induces binding of the Grb2-mSos1 complex to the autophosphorylated EGF receptor, and mSos1 phosphorylation. Grb2 therefore appears to link tyrosine kinases to a Ras-GNRP in mammalian cells.

The EGF receptor family as targets for cancer therapy.

PubMed

Mendelsohn, J; Baselga, J

2000-12-27

Human carcinomas frequently express high levels of receptors in the EGF receptor family, and overexpression of at least two of these receptors, the EGF receptor (EGFr) and closely related ErbB2, has been associated with a more aggressive clinical behavior. Further, transfection or activation of high levels of these two receptors in nonmalignant cell lines can lead to a transformed phenotype. For these reasons therapies directed at preventing the function of these receptors have the potential to be useful anti-cancer treatments. In the last two decades monoclonal antibodies (MAbs) which block activation of the EGFr and ErbB2 have been developed. These MAbs have shown promising preclinical activity and 'chimeric' and 'humanized' MAbs have been produced in order to obviate the problem of host immune reactions. Clinical activity with these antibodies has been documented: trastuzumab, a humanized anti-ErbB2 MAb, is active and was recently approved in combination with paclitaxel for the therapy of patients with metastatic ErbB2-overexpressing breast cancer; IMC-C225, a chimeric anti-EGFr MAb, has shown impressive activity when combined with radiation therapy and reverses resistance to chemotherapy. In addition to antibodies, compounds that directly inhibit receptor tyrosine kinases have shown preclinical activity and early clinical activity has been reported. A series of phase III studies with these antibodies and direct tyrosine kinase inhibitors are ongoing or planned, and will further address the role of these active anti-receptor agents in the treatment of patients with cancer.

Elevated expression of the proto-oncogene c-kit in patients with mastocytosis.

PubMed

Nagata, H; Worobec, A S; Semere, T; Metcalfe, D D

1998-02-01

The stem cell factor (SCF)c-kit receptor interaction plays a critical role in the development and survival of mast cells. Several studies have also associated c-kit receptor mutations with the human diseases, mastocytosis and piebaldism. Overexpression of c-kit has been reported to be associated with myeloproliferative disorders and myelodysplastic syndromes. Using peripheral blood mononuclear cells (PBMCs) from 11 patients with indolent mastocytosis (category I), mastocytosis with an associated hematologic disorder (category II), or aggressive mastocytosis (category III); a patient with CMML unassociated with mastocytosis, and PBMCs from 13 normal subjects, we examined the level of expression of c-kit mRNA along with other c-kit isoforms to determine if overexpression of the c-kit receptor was associated with mastocytosis. Using quantitative competitive PCR, c-kit mRNA levels on average were found to be statistically elevated in the five patients with mastocytosis with an associated hematologic disorder and in the patient with aggressive mastocytosis as compared with controls, but not elevated in patients with indolent mastocytosis. The relative mRNA expression for the two c-kit isoforms was not significantly different in the mastocytosis patients compared with controls. This demonstration of the overexpression of c-kit mRNA in mastocytosis, and particularly those patients with clinical evidence of myelodysplastic syndrome, adds evidence to support the conclusion that mastocytosis, at least in some patients, is a feature of myelodysplasia; and suggests that determination of c-kit mRNA expression in PBMCs may provide an additional approach to assessing prognosis.

c-Kit mediates chemoresistance and tumor-initiating capacity of ovarian cancer cells through activation of Wnt/β-catenin-ATP-binding cassette G2 signaling.

PubMed

Chau, W K; Ip, C K; Mak, A S C; Lai, H-C; Wong, A S T

2013-05-30

Cisplatin and paclitaxel are standard chemotherapy for metastatic ovarian cancer, but with limited efficacy. Cancer stem/progenitor cells (or tumor-initiating cells, TICs) are hypothesized to be chemoresistant, and the existence of TICs in ovarian cancer has been previously demonstrated. However, the key signals and molecular events regulating the formation and expansion of ovarian tumor-initiating cells (OTICs) remain elusive. Here, we show that c-Kit is not just a marker of OTICs, but also a critical mediator of the phenotype that can be a viable target for the treatment of ovarian cancer. In contrast to non-OICs, c-Kit was overexpressed in OTICs. Moreover, the use of small interfering RNA to inhibit c-Kit expression markedly attenuated the number and size of OTIC subpopulations, inhibited the expression of stem cell markers and decreased the tumorigenic capabilities of OTICs. Imatinib (Gleevec), a clinical drug that blocks c-Kit kinase activity, also demonstrated its inhibition potency on OTICs. In addition, cisplatin/paclitaxel, which killed non-OTICs, with c-Kit knockdown or imatinib revealed that this was critically required for intervening ovarian cancer progression and recurrence in vitro and in xenograft tumors in vivo. Similar results were obtained with OTICs derived from ovarian carcinoma patients. Studies into the mechanisms suggest an important role for the activation of Wnt/β-catenin and ATP-binding cassette G2 downstream of c-Kit. The tumor-promoting microenvironment, such as hypoxia, could promote OTICs via upregulation of c-Kit expression. These results unravel an integral role for c-Kit in ovarian neoplastic processes and shed light on its mechanisms of action.

The usefulness of c-Kit in the immunohistochemical assessment of melanocytic lesions

PubMed Central

Pilloni, L.; Bianco, P.; Difelice, E.; Cabras, S.; Castellanos, M.E.; Atzori, L.; Ferreli, C.; Mulas, P.; Nemolato, S.; Faa, G.

2011-01-01

C-Kit (CD117), the receptor for the stem cell factor, a growth factor for melanocyte migration and proliferation, has shown differential immunostaining in various benign and malignant melanocytic lesions. The purpose of this study is to compare c-Kit immunostaining in benign nevi and in primary and metastatic malignant melanomas, to determine whether c-Kit can aid in the differential diagnosis of these lesions. c-Kit immunostaining was performed in 60 cases of pigmented lesions, including 39 benign nevi (5 blue nevi, 5 intra-dermal nevi, 3 junctional nevi, 15 cases of primary compound nevus, 11 cases of Spitz nevus), 18 cases of primary malignant melanoma and 3 cases of metastatic melanoma. The vast majority of nevi and melanomas examined in this study were positive for c-Kit, with minimal differences between benign and malignant lesions. C-Kit cytoplasmatic immunoreactivity in the intraepidermal proliferating nevus cells, was detected in benign pigmented lesions as well as in malignant melanoma, increasing with the age of patients (P=0.007) in both groups. The patient’s age at presentation appeared to be the variable able to cluster benign and malignant pigmented lesions. The percentage of c-Kit positive intraepidermal nevus cells was better associated with age despite other variables (P=0.014). The intensity and percentage of c-Kit positivity in the proliferating nevus cells in the dermis was significantly increased in malignant melanocytic lesions (P=0.015 and P=0.008) compared to benign lesions (compound melanocytic nevi, Spitz nevi, intradermal nevi, blue nevi). Immunostaning for c-Kit in metastatic melanomas was negative. Interestingly in two cases of melanoma occurring on a pre-existent nevus, the melanoma tumor cells showed strong cytoplasmatic and membranous positivity for c-kit, in contrast with the absence of any immunoreactivity in pre-existent intradermal nevus cells. C-Kit does not appear to be a strong immunohistochemical marker for distinguishing

Modulation of focal adhesion constituents and their down-stream events by EGF: On the cross-talk of integrins and growth factor receptors.

PubMed

Eberwein, Philipp; Laird, Dougal; Schulz, Simon; Reinhard, Thomas; Steinberg, Thorsten; Tomakidi, Pascal

2015-10-01

Within the concept of integrin growth factor receptor (GFR) cross-talk, little is known about the effects of GFRs on focal adhesions (FAs). Therefore, we tested the hypothesis whether EGF can modulate constituents of FAs and subsequent down-stream events. To this end, EGF-treated keratinocytes were subjected to combined fluorescence imaging and western blotting, to quantify expression and/or activation of molecules, involved in integrin GFR cross-talk, and receptor proximal and distal signaling events. Generally, EGF response revealed an amplified redistribution or activation of molecules under study, which will be explained in detail from the plasma membrane to the cell interior. In addition to significant activation of EGF receptor (EGFR) at tyrosine Tyr845, a remarkable redistribution was detectable for the focal adhesion constituents, integrin ß1 and ß3, and zyxin. Increased activation also applied to focal adhesion kinase (FAK) by phosphorylation at Tyr397, Tyr576, and Src at Tyr418, while total FAK remained unchanged. Risen activity was seen as well for the analyzed distal down-stream events, p190RhoGAP and MAP kinases p42/44. Intriguingly, Src-specific inhibitor Herbimycin A abrogated the entire EGF response except FAK Tyr397 phosphorylation, independent of EGF presence. Mechanistically, our results show that EGF modulates adhesion in a dual fashion, by firstly redistributing focal adhesion constituents to adhesion sites, but also by amplifying levels of activated RhoA antagonist p190RhoGAP, important for cell motility. Further, the findings suggest that the observed EGF response underlies an EGFR integrin cross-talk under recruitment of receptor proximal FAK and Src, and MAP kinase and p190RhoGAP as receptor distal events. Copyright © 2015 Elsevier B.V. All rights reserved.

Prostaglandin E2-stimulated prostanoid EP4 receptors induce prolonged de novo prostaglandin E2 synthesis through biphasic phosphorylation of extracellular signal-regulated kinases mediated by activation of protein kinase A in HCA-7 human colon cancer cells.

PubMed

Fujino, Hiromichi; Seira, Naofumi; Kurata, Naoki; Araki, Yumi; Nakamura, Hiroyuki; Regan, John W; Murayama, Toshihiko

2015-12-05

Approximately two decades have passed since E-type prostanoid 4 (EP4) receptors were cloned, and the signaling pathways mediated by these receptors have since been implicated in cancer development through the alliance of Gαi-protein/phosphatidylinositol 3-kinase (PI3K)/extracellular signal-regulated kinases (ERKs) activation. Although prostanoid EP4 receptors were initially identified as Gαs-coupled receptors, the specific/distinctive role(s) of prostanoid EP4 receptor-induced cAMP/protein kinase A (PKA) pathways in cancer development have not yet been elucidated in detail. We previously reported using HCA-7 human colon cancer cells that prostaglandin E2 (PGE2)-stimulated prostanoid EP4 receptors induced cyclooxygenase-2 (COX-2) as an initiating event in development of colon cancer. Moreover, this induction of COX-2 was mediated by transactivation of epidermal growth factor (EGF) receptors. However, direct activation of EGF receptors by EGF also induced similar amounts of COX-2 in this cell line. Thus, the emergence of unique role(s) for prostanoid EP4 receptors is expected by clarifying the different signaling mechanisms between PGE2-stimulated prostanoid EP4 receptors and EGF-stimulated EGF receptors to induce COX-2 and produce PGE2. We here demonstrated that prostanoid EP4 receptor activation by PGE2 in HCA-7 cells led to PKA-dependent re-activation of ERKs, which resulted in prolonged de novo synthesis of PGE2. Although EGF-stimulated EGF receptors in cells also induced COX-2 and the de novo synthesis of PGE2, the activation of this pathway was transient and not mediated by PKA. Therefore, the novel mechanism underlying prolonged de novo synthesis of PGE2 has provided an insight into the importance of prostanoid EP4 receptor-mediated Gαs-protein/cAMP/PKA pathway in development of colon cancer. Copyright © 2015 Elsevier B.V. All rights reserved.

Single-cell analysis of the fate of c-kit-positive bone marrow cells

NASA Astrophysics Data System (ADS)

Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D.; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa

2017-10-01

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

Single-cell analysis of the fate of c-kit-positive bone marrow cells.

PubMed

Czarna, Anna; Sanada, Fumihiro; Matsuda, Alex; Kim, Junghyun; Signore, Sergio; Pereira, João D; Sorrentino, Andrea; Kannappan, Ramaswamy; Cannatà, Antonio; Hosoda, Toru; Rota, Marcello; Crea, Filippo; Anversa, Piero; Leri, Annarosa

2017-01-01

The plasticity of c-kit-positive bone marrow cells (c-kit-BMCs) in tissues different from their organ of origin remains unclear. We tested the hypothesis that c-kit-BMCs are functionally heterogeneous and only a subgroup of these cells possesses cardiomyogenic potential. Population-based assays fall short of identifying the properties of individual stem cells, imposing on us the introduction of single cell-based approaches to track the fate of c-kit-BMCs in the injured heart; they included viral gene-tagging, multicolor clonal-marking and transcriptional profiling. Based on these strategies, we report that single mouse c-kit-BMCs expand clonally within the infarcted myocardium and differentiate into specialized cardiac cells. Newly-formed cardiomyocytes, endothelial cells, fibroblasts and c-kit-BMCs showed in their genome common sites of viral integration, providing strong evidence in favor of the plasticity of a subset of BMCs expressing the c-kit receptor. Similarly, individual c-kit-BMCs, which were infected with multicolor reporters and injected in infarcted hearts, formed cardiomyocytes and vascular cells organized in clusters of similarly colored cells. The uniform distribution of fluorescent proteins in groups of specialized cells documented the polyclonal nature of myocardial regeneration. The transcriptional profile of myogenic c-kit-BMCs and whole c-kit-BMCs was defined by RNA sequencing. Genes relevant for engraftment, survival, migration, and differentiation were enriched in myogenic c-kit-BMCs, a cell subtype which could not be assigned to a specific hematopoietic lineage. Collectively, our findings demonstrate that the bone marrow comprises a category of cardiomyogenic, vasculogenic and/or fibrogenic c-kit-positive cells and a category of c-kit-positive cells that retains an undifferentiated state within the damaged heart.

c-kit expression profile and regulatory factors during spermatogonial stem cell differentiation

PubMed Central

2013-01-01

Background It has been proven that c-kit is crucial for proliferation, migration, survival and maturation of spermatogenic cells. A periodic expression of c-kit is observed from primordial germ cells (PGCs) to spermatogenetic stem cells (SSCs), However, the expression profile of c-kit during the entire spermatogenesis process is still unclear. This study aims to reveal and compare c-kit expression profiles in the SSCs before and after the anticipated differentiation, as well as to examine its relationship with retinoic acid (RA) stimulation. Results We have found that there are more than 4 transcripts of c-kit expressed in the cell lines and in the testes. The transcripts can be divided into short and long categories. The long transcripts include the full-length canonical c-kit transcript and the 3′ end short transcript. Short transcripts include the 3.4 kb short transcript and several truncated transcripts (1.9-3.2 kb). In addition, the 3.4 kb transcript (starting from intron 9 and covering exons 10 ~ 21) is discovered to be specifically expressed in the spermatogonia. The extracellular domain of Kit is obtained in the spermatogonia stage, but the intracellular domain (50 kDa) is constantly expressed in both SSCs and spermatogonia. The c-kit expression profiles in the testis and the spermatogonial stem cell lines vary after RA stimulation. The wave-like changes of the quantitative expression pattern of c-kit (increase initially and decrease afterwards) during the induction process are similar to that of the in vivo male germ cell development process. Conclusions There are dynamic transcription and translation changes of c-kit before and after SSCs’ anticipated differentiation and most importantly, RA is a significant upstream regulatory factor for c-kit expression. PMID:24161026

C-MORE Science Kits as a Classroom Learning Tool

ERIC Educational Resources Information Center

Foley, J. M.; Bruno, B. C.; Tolman, R. T.; Kagami, R. S.; Hsia, M. H.; Mayer, B.; Inazu, J. K.

2013-01-01

To support teachers in enhancing ocean literacy, the Center for Microbial Oceanography: Research and Education (C-MORE) has developed a series of portable, hands-on science kits on selected topics in oceanography. This paper provides an overview of kit content, describes how the kits were developed, and evaluates their efficacy as a curriculum…

Factors for C-Kit Expression in Cardiac Outgrowth Cells and Human Heart Tissue.

PubMed

Matsushita, Satoshi; Minematsu, Kazuo; Yamamoto, Taira; Inaba, Hirotaka; Kuwaki, Kenji; Shimada, Akie; Yokoyama, Yasutaka; Amano, Atsushi

2017-12-12

We determined the factors associated with the expression of c-kit in the heart and the proliferation of c-kit-positive (c-kit pos ) cardiac stem cells among the outgrowth cells cultured from human cardiac explants.Samples of the right atrium (RA), left atrium (LA), and left ventricle obtained from patients during open-heart surgery were processed for cell culture of outgrowth cells and tissue analysis. The total number of growing cells and the population of c-kit pos cells were measured and compared with c-kit expression in native tissues and characteristics of the patients according to the region of the heart.We analyzed 452 samples from 334 patients. Atrial fibrillation (AF) in the patients reduced the number of outgrowth cells from the RA and LA, and aging was a co-factor for the LA. The c-kit pos population from the RA was associated with serum brain natriuretic peptide (BNP). C-kit expression in native tissue was also associated with BNP expression. However, we observed no relationship in expression between outgrowth cells and native tissue. In addition, the RA tissue provided the highest number of c-kit pos cells, and the left ventricle provided the lowest.C-kit was weakly expressed in response to damage. In addition, no correlation between outgrowth cells and native tissue was found for c-kit expression.

Specificity and mechanism of protein kinase C activation by sn-1,2-diacylglycerols.

PubMed Central

Ganong, B R; Loomis, C R; Hannun, Y A; Bell, R M

1986-01-01

The specificity of protein kinase C activation by sn-1,2-diacylglycerols and analogues was investigated by using a Triton X-100 mixed micellar assay [Hannun, Y. A., Loomis, C. R. & Bell, R. M. (1985) J. Biol. Chem. 260, 10039-10043]. Analogues containing acyl or alkyl chains eight carbons in length were synthesized because sn-1,2-dioctanoylglycerol is an effective cell-permeant activator of protein kinase C. These analogues were tested as activators and antagonists of rat brain protein kinase C to determine the exact structural features important for activity. The analogues established that activation of protein kinase C by diacylglycerols is highly specific. Several analogues established that both carbonyl moieties of the oxygen esters are required for maximal activity and that the 3-hydroxyl moiety is also required. None of the analogues were antagonists. These data, combined with previous investigations, permitted formulation of a model of protein kinase C activation. A three-point attachment of sn-1,2-diacylglycerol to the surface-bound protein kinase C-phosphatidylserine-Ca2+ complex is envisioned to cause activation. Direct ligation of diacylglycerol to Ca2+ is proposed to be an essential step in the mechanism of activation of protein kinase C. Images PMID:3456578

Immunohistochemical study of C-kit expression in subtypes of renal cell carcinoma.

PubMed

Norouzinia, Farahnaz; Abbasi, Fariba; Dindarian, Sina; Mohammadi, Sedra; Meisami, Farid; Bagheri, Mahdi; Mohammadi, Hozan

2018-01-01

Renal cell carcinomas (RCCs) include about 2% of adult neoplasms and 90-95% of all renal tumors. Mostly, it is possible to distinguish RCC subtypes using hematoxylin-eosin staining. However, overlapping morphologic features cause some difficulties in making a precise diagnosis. In order to render an accurate diagnosis, additional methods such as immunohistochemical staining for c-kit have been recommended. In this study, we aimed to investigate c-kit gene expression in various subtypes of RCC. We reviewed 65 diagnosed RCC cases. Formalin- fixed, paraffin- embedded specimens were available for the cases. The expression of c-kit was evaluated using immunohistochemistry. The correlation between c-kit expression and clinicopathological parameters including patients' age and gender in addition to grade, stage, and size of the tumor were investigated. Six cases of 39 clear cell types (15.4%), 8 of 13 papillary types (61.5%), 11 of 12 chromophobe types (91.7%), and no sarcomatoid type were positive for c-kit expression. Based on chi-square test results, there was a significant relationship between RCC subtypes and c-kit expression (p=0.001). There was no significant correlation between age, sex, grade, stage, and size of the tumor and c-kit expression. The expression of c-kit in RCC may have diagnostic significance in subtypes of RCC especially papillary and chromophobe subtypes of RCC.

Crenolanib is a type I tyrosine kinase inhibitor that inhibits mutant KIT D816 isoforms prevalent in systemic mastocytosis and core binding factor leukemia.

PubMed

Kampa-Schittenhelm, Kerstin Maria; Frey, Julia; Haeusser, Lara A; Illing, Barbara; Pavlovsky, Ashly A; Blumenstock, Gunnar; Schittenhelm, Marcus Matthias

2017-10-10

Activating D816 mutations of the class III receptor tyrosine kinase KIT are associated with the majority of patients with systemic mastocytosis (SM), but also core binding factor (CBF) AML, making KIT mutations attractive therapeutic targets for the treatment of these cancers. Crenolanib is a potent and selective inhibitor of wild-type as well as mutant isoforms of the class III receptor tyrosine kinases FLT3 and PDGFRα/β. Notably, crenolanib inhibits constitutively active mutant-FLT3 isoforms resulting from amino acid substitutions of aspartic acid at codon 835, which is homologous to codon 816 in the KIT gene - suggesting sensitivity against mutant-KIT D816 isoforms as well. Here we demonstrate that crenolanib targets KIT D816 in SM and CBF AML models: crenolanib inhibits cellular proliferation and initiates apoptosis of mastocytosis cell lines expressing these mutations. Target-specificity was confirmed using an isogenic cell model. In addition, we demonstrate that KIT D816 mutations are targetable with clinically achievable doses of crenolanib. Further, a rationale to combine cladribine (2-CDA), the therapeutic standard in SM, with crenolanib is provided. In conclusion, we demonstrate that crenolanib is an inhibitor of mutant-KIT D816 isoforms at clinically achievable concentrations, and thus may be a potential treatment for SM and CBF AML as a monotherapy or in combination approaches.

Cellular progesterone receptor phosphorylation in response to ligands activating protein kinases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rao, K.V.; Peralta, W.D.; Greene, G.L.

1987-08-14

Progesterone receptors were immunoprecipitated with monoclonal antibodies KD68 from lysates of human breast carcinoma T47D cells labelled to steady state specific activity with /sup 32/Pi. The 120 kDa /sup 32/P-labelled progesterone receptor band was resolved by polyacrylamide gel electrophoresis and identified by autoradiography. Phosphoamino acid analysis revealed serine phosphorylation, but no threonine or tyrosine phosphorylation. Treatment of the /sup 32/Pi-labelled cells with EGF, TPA or dibutyryl cAMP had no significant quantitative effect on progesterone receptor phosphorylation, though the EGF receptor and the cAMP-dependent protein kinases have been reported to catalyze phosphorylation of purified avian progesterone receptor preparations in cell freemore » systems. Progesterone receptor phosphorylation on serine residues was increased by 2-fold in cells treated with 10 nM progesterone; EGF had no effect on progesterone-mediated progesterone receptor phosphorylation.« less

Ascofuranone suppresses EGF-induced HIF-1α protein synthesis by inhibition of the Akt/mTOR/p70S6K pathway in MDA-MB-231 breast cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Jeong, Yun-Jeong; Cho, Hyun-Ji; Magae, Junji

2013-12-15

Hypoxia-inducible factor (HIF)-1 plays an important role in tumor progression, angiogenesis and metastasis. In this study, we investigated the potential molecular mechanisms underlying the anti-angiogenic effect of ascofuranone, an isoprenoid antibiotic from Ascochyta viciae, in epidermal growth factor (EGF)-1 responsive human breast cancer cells. Ascofuranone significantly and selectively suppressed EGF-induced HIF-1α protein accumulation, whereas it did not affect the expression of HIF-1β. Furthermore, ascofuranone inhibited the transcriptional activation of vascular endothelial growth factor (VEGF) by reducing protein HIF-1α. Mechanistically, we found that the inhibitory effects of ascofuranone on HIF-1α protein expression are associated with the inhibition of synthesis HIF-1α throughmore » an EGF-dependent mechanism. In addition, ascofuranone suppressed EGF-induced phosphorylation of Akt/mTOR/p70S6 kinase, but the phosphorylation of ERK/JNK/p38 kinase was not affected by ascofuranone. These results suggest that ascofuranone suppresses EGF-induced HIF-1α protein translation through the inhibition of Akt/mTOR/p70S6 kinase signaling pathways and plays a novel role in the anti-angiogenic action. - Highlights: • Inhibitory effect of ascofuranone on HIF-1α expression is EGF-specific regulation. • Ascofuranone decreases HIF-1α protein synthesis through Akt/mTOR pathways. • Ascofuranone suppresses EGF-induced VEGF production and tumor angiogenesis.« less

EGF stimulates the activation of EGF receptors and the selective activation of major signaling pathways during mitosis.

PubMed

Wee, Ping; Shi, Huaiping; Jiang, Jennifer; Wang, Yuluan; Wang, Zhixiang

2015-03-01

Mitosis and epidermal growth factor (EGF) receptor (EGFR) are both targets for cancer therapy. The role of EGFR signaling in mitosis has been rarely studied and poorly understood. The limited studies indicate that the activation of EGFR and downstream signaling pathways is mostly inhibited during mitosis. However, we recently showed that EGFR is phosphorylated in response to EGF stimulation in mitosis. Here we studied EGF-induced EGFR activation and the activation of major signaling pathways downstream of EGFR during mitosis. We showed that EGFR was strongly activated by EGF during mitosis as all the five major tyrosine residues including Y992, Y1045, Y1068, Y1086, and Y1173 were phosphorylated to a level similar to that in the interphase. We further showed that the activated EGFR is able to selectively activate some downstream signaling pathways while avoiding others. Activated EGFR is able to activate PI3K and AKT2, but not AKT1, which may be responsible for the observed effects of EGF against nocodazole-induced cell death. Activated EGFR is also able to activate c-Src, c-Cbl and PLC-γ1 during mitosis. However, activated EGFR is unable to activate ERK1/2 and their downstream substrates RSK and Elk-1. While it activated Ras, EGFR failed to fully activate Raf-1 in mitosis due to the lack of phosphorylation at Y341 and the lack of dephosphorylation at pS259. We conclude that contrary to the dogma, EGFR is activated by EGF during mitosis. Moreover, EGFR-mediated cell signaling is regulated differently from the interphase to specifically serve the needs of the cell in mitosis. Copyright © 2015 Elsevier Inc. All rights reserved.

Smurf1 plays a role in EGF inhibition of BMP2-induced osteogenic differentiation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lee, Hye-Lim; Park, Hyun-Jung; Kwon, Arang

2014-05-01

It has been demonstrated that epidermal growth factor (EGF) plays a role in supporting the proliferation of bone marrow stromal cells in bone but inhibits their osteogenic differentiation. However, the mechanism underlying EGF inhibition of osteoblast differentiation remains unclear. Smurf1 is an E3 ubiquitin ligase that targets Smad1/5 and Runx2, which are critical transcription factors for bone morphogenetic protein 2 (BMP2)-induced osteoblast differentiation. In this study, we investigated the effect of EGF on the expression of Smurf1, and the role of Smurf1 in EGF inhibition of osteogenic differentiation using C2C12 cells, a murine myoblast cell line. EGF increased Smurf1 expression,more » which was blocked by inhibiting the activity of either JNK or ERK. Chromatin immunoprecipitation and Smurf1 promoter assays demonstrated that c-Jun and Runx2 play roles in the EGF induction of Smurf1 transcription. EGF suppressed BMP2-induced expression of osteogenic marker genes, which were rescued by Smurf1 knockdown. EGF downregulated the protein levels of Runx2 and Smad1 in a proteasome-dependent manner. EGF decreased the transcriptional activity of Runx2 and Smurf1, which was partially rescued by Smurf1 silencing. Taken together, these results suggest that EGF increases Smurf1 expression via the activation of JNK and ERK and the subsequent binding of c-Jun and Runx2 to the Smurf1 promoter and that Smurf1 mediates the inhibitory effect of EGF on BMP2-induced osteoblast differentiation. - Highlights: • EGF increases the expression level of Smurf1 in mesenchymal precursor cells. • EGF reduces the protein levels and transcriptional activity of Runx2 and Smad1. • EGF suppresses BMP2-induced osteogenic differentiation, which is rescued by Smurf1 knockdown.« less

Changes in c-Kit expression levels during the course of radiation therapy for nasopharyngeal carcinoma.

PubMed

Jiang, Feng; Hu, Wei; Zhang, Bicheng; Xu, Jing; Shui, Yongjie; Zhou, Xiaofeng; Ren, Xiaoqiu; Chen, Xiaozhong; Shen, Li; Wei, Qichun

2016-10-01

In the era of intensity-modulated radiotherapy, distant metastasis is currently the main cause of treatment failure for nasopharyngeal carcinoma (NPC). Additional therapeutic strategies are required to control the metastasis and improve survival. One strategy is targeted therapy, for example against c-Kit. In the current study, the frequency of c-Kit expression was determined immunohistochemically in 106 NPC patients. c-Kit expression changes during the course of radiation therapy were detected in 41 cases via weekly biopsy. Twelve cases (11.3%) had c-Kit expression scores of 3 + and 16 (15.1%) had scores of 2 + . Thus, c-Kit overexpression (2 + or 3 + ) was observed in 28 (26.4%) patients. There were 35 (33.0%) and 43 (40.6%) patients with c-Kit expression scores of 1 + and 0, respectively. Furthermore, a trend of decreased c-Kit expression was observed after commencing radiotherapy according to the 41 NPC patients who were biopsied weekly. Therefore, c-Kit overexpression was identified to be common in NPC, and evaluating c-Kit as a therapeutic target for metastatic NPC via c-Kit overexpression subsequent to first line treatment may be of interest. To the best of our knowledge, the present study is the first to demonstrate a trend of decreased c-Kit expression during the course of radiotherapy.

Egr-1 is a critical regulator of EGF-receptor-mediated expansion of subventricular zone neural stem cells and progenitors during recovery from hypoxia–hypoglycemia

PubMed Central

Alagappan, Dhivyaa; Balan, Murugabaskar; Jiang, Yuhui; Cohen, Rachel B.; Kotenko, Sergei V.; Levison, Steven W.

2013-01-01

We recently established that the EGF-R (epidermal growth factor receptor) (EGF-R) is an essential regulator of the reactive expansion of SVZ (subventricular zone) NPs (neural precursors) that occurs during recovery from hypoxic-ischemic brain injury. The purpose of the current studies was to identify the conditions and the transcription factor (s) responsible for inducing the EGF-R. Here, we show that the increase in EGF-R expression and the more rapid division of the NPs can be recapitulated in in vitro by exposing SVZ NPs to hypoxia and hypoglycemia simultaneously, but not separately. The EGF-R promoter has binding sites for multiple transcription factors that includes the zinc finger transcription factor, Egr-1. We show that Egr-1 expression increases in NPs, but not astrocytes, following hypoxia and hypoglycemia where it accumulates in the nucleus. To determine whether Egr-1 is necessary for EGF-R expression, we used SiRNAs (small interfering RNA) specific for Egr-1 to decrease Egr-1 expression. Knocking-down Egr-1 decreased basal levels of EGF-R and it abolished the stress-induced increase in EGF-R expression. By contrast, HIF-1 accumulation did not contribute to EGF-R expression and FGF-2 only modestly induced EGF-R. These studies establish a new role for Egr-1 in regulating the expression of the mitogenic EGF-R. They also provide new information into mechanisms that promote NP expansion and provide insights into strategies for amplifying the numbers of stem cells for CNS (central nervous system) regeneration. PMID:23763269

Role of G protein-coupled receptors (GPCR), matrix metalloproteinases 2 and 9 (MMP2 and MMP9), heparin-binding epidermal growth factor-like growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) in trenbolone acetate-stimulated bovine satellite cell proliferation.

PubMed

Thornton, K J; Kamange-Sollo, E; White, M E; Dayton, W R

2015-09-01

Implanting cattle with steroids significantly enhances feed efficiency, rate of gain, and muscle growth. However, the mechanisms responsible for these improvements in muscle growth have not been fully elucidated. Trenbolone acetate (TBA), a testosterone analog, has been shown to increase proliferation rate in bovine satellite cell (BSC) cultures. The classical genomic actions of testosterone have been well characterized; however, our results indicate that TBA may also initiate a quicker, nongenomic response that involves activation of G protein-coupled receptors (GPCR) resulting in activation of matrix metalloproteinases 2 and 9 (MMP2 and MMP9) that release membrane-bound heparin-binding epidermal growth factor-like growth factor (hbEGF), which then binds to and activates the epidermal growth factor receptor (EGFR) and/or erbB2. Furthermore, the EGFR has been shown to regulate expression of the IGF-1 receptor (IGF-1R), which is well known for its role in modulating muscle growth. To determine whether this nongenomic pathway is potentially involved in TBA-stimulated BSC proliferation, we analyzed the effects of treating BSC with guanosine 5'-O-2-thiodiphosphate (GDPβS), an inhibitor of all GPCR; a MMP2 and MMP9 inhibitor (MMPI); CRM19, a specific inhibitor of hbEGF; AG1478, a specific EGFR tyrosine kinase inhibitor; AG879, a specific erbB2 kinase inhibitor; and AG1024, an IGF-1R tyrosine kinase inhibitor on TBA-stimulated proliferation rate (H-thymidine incorporation). Assays were replicated at least 9 times for each inhibitor experiment using BSC cultures obtained from at least 3 different animals. Bovine satellite cell cultures were obtained from yearling steers that had no previous exposure to androgenic or estrogenic compounds. As expected, BSC cultures treated with 10 n TBA showed ( < 0.05) increased proliferation rate when compared with control cultures. Additionally, treatment with 5 ng hbEGF/mL stimulated proliferation in BSC cultures ( < 0.05). Treatment

Investigation of c-KIT and Ki67 expression in normal, preneoplastic and neoplastic canine prostate.

PubMed

Fonseca-Alves, Carlos Eduardo; Kobayashi, Priscilla Emiko; Palmieri, Chiara; Laufer-Amorim, Renée

2017-12-06

c-KIT expression has been related to bone metastasis in human prostate cancer, but whether c-KIT expression can be similarly classified in canine prostatic tissue is unknown. This study assessed c-KIT and Ki67 expression in canine prostate cancer (PC). c-KIT gene and protein expression and Ki67 expression were evaluated in forty-four canine prostatic tissues by immunohistochemistry, RT-qPCR and western blot. Additionally, we have investigated c-KIT protein expression by immunoblotting in two primary canine prostate cancer cell lines. Eleven normal prostates, 12 proliferative inflammatory atrophy (PIA) prostates, 18 PC, 3 metastatic lesions and two prostate cancer cell cultures (PC1 and PC2) were analysed. The prostatic tissue exhibited varying degrees of membranous, cytoplasmic or membranous/cytoplasmic c-KIT staining. Four normal prostates, 4 PIA and 5 prostatic carcinomas showed positive c-KIT expression. No c-KIT immunoexpression was observed in metastases. Canine prostate cancer and PIA samples contained a higher number of Ki67-positive cells compared to normal samples. The median relative quantification (RQ) for c-KIT expression in normal, PIA and prostate cancer and metastatic samples were 0.6 (0.1-2.5), 0.7 (0.09-2.1), 0.7 (0.09-5.1) and 0.1 (0.07-0.6), respectively. A positive correlation between the number of Ki67-positive cells and c-KIT transcript levels was observed in prostate cancer samples. In the cell line, PC1 was negative for c-KIT protein expression, while PC2 was weakly positive. The present study identified a strong correlation between c-KIT expression and proliferative index, suggesting that c-KIT may influence cell proliferation. Therefore, c-KIT heterogeneous protein expression among the samples (five positive and thirteen negative prostate cancer samples) indicates a personalized approach for canine prostate cancer.

GM-CSF Inhibits c-Kit and SCF Expression by Bone Marrow-Derived Dendritic Cells

PubMed Central

Barroeta Seijas, Amairelys Belen; Simonetti, Sonia; Vitale, Sara; Runci, Daniele; Quinci, Angela Caterina; Soriani, Alessandra; Criscuoli, Mattia; Filippi, Irene; Naldini, Antonella; Sacchetti, Federico Maria; Tarantino, Umberto; Oliva, Francesco; Piccirilli, Eleonora; Santoni, Angela; Di Rosa, Francesca

2017-01-01

Stem cell factor (SCF), the ligand of c-kit, is a key cytokine for hematopoiesis. Hematopoietic precursors express c-kit, whereas differentiated cells of hematopoietic lineage are negative for this receptor, with the exception of NK cells, mast cells, and a few others. While it has long been recognized that dendritic cells (DCs) can express c-kit, several questions remain concerning the SCF/c-kit axis in DCs. This is particularly relevant for DCs found in those organs wherein SCF is highly expressed, including the bone marrow (BM). We characterized c-kit expression by conventional DCs (cDCs) from BM and demonstrated a higher proportion of c-kit+ cells among type 1 cDC subsets (cDC1s) than type 2 cDC subsets (cDC2s) in both humans and mice, whereas similar levels of c-kit expression were observed in cDC1s and cDC2s from mouse spleen. To further study c-kit regulation, DCs were generated with granulocyte-macrophage colony-stimulating factor (GM-CSF) from mouse BM, a widely used protocol. CD11c+ cells were purified from pooled non-adherent and slightly adherent cells collected after 7 days of culture, thus obtaining highly purified BM-derived DCs (BMdDCs). BMdDCs contained a small fraction of c-kit+ cells, and by replating them for 2 days with GM-CSF, we obtained a homogeneous population of c-kit+ CD40hi MHCIIhi cells. Not only did BMdDCs express c-kit but they also produced SCF, and both were striking upregulated if GM-CSF was omitted after replating. Furthermore, a small but significant reduction in BMdDC survival was observed upon SCF silencing. Incubation of BMdDCs with SCF did not modulate antigen presentation ability of these cells, nor it did regulate their membrane expression of the chemokine receptor CXCR4. We conclude that the SCF/c-kit-mediated prosurvival circuit may have been overlooked because of the prominent use of GM-CSF in DC cultures in vitro, including those human DC cultures destined for the clinics. We speculate that DCs more prominently rely

Identification of tyrosine phosphorylation sites in human Gab-1 protein by EGF receptor kinase in vitro.

PubMed

Lehr, S; Kotzka, J; Herkner, A; Klein, E; Siethoff, C; Knebel, B; Noelle, V; Brüning, J C; Klein, H W; Meyer, H E; Krone, W; Müller-Wieland, D

1999-01-05

Grb2-associated binder-1 (Gab-1) has been identified recently in a cDNA library of glioblastoma tumors and appears to play a central role in cellular growth response, transformation, and apoptosis. Structural and functional features indicate that Gab-1 is a multisubstrate docking protein downstream in the signaling pathways of different receptor tyrosine kinases, including the epidermal growth factor receptor (EGFR). Therefore, the aim of the study was to characterize the phosphorylation of recombinant human Gab-1 (hGab-1) protein by EGFR in vitro. Using the pGEX system to express the entire protein and different domains of hGab-1 as glutathione S-transferase proteins, kinetic data for phosphorylation of these proteins by wheat germ agglutinine-purified EGFR and the recombinant EGFR (rEGFR) receptor kinase domain were determined. Our data revealed similar affinities of hGab-1-C for both receptor preparations (KM = 2.7 microM for rEGFR vs 3.2 microM for WGA EGFR) as well as for the different recombinant hGab-1 domains. To identify the specific EGFR phosphorylation sites, hGab-1-C was sequenced by Edman degradation and mass spectrometry. The entire protein was phosphorylated by rEGFR at eight tyrosine residues (Y285, Y373, Y406, Y447, Y472, Y619, Y657, and Y689). Fifty percent of the identified radioactivity was incorporated in tyrosine Y657 as the predominant peak in HPLC analysis, a site exhibiting features of a potential Syp (PTP1D) binding site. Accordingly, GST-pull down assays with A431 and HepG2 cell lysates showed that phosphorylated intact hGab-1 was able to bind Syp. This binding appears to be specific, because it was abolished by changing the Y657 of hGab-1 to F657. These results demonstrate that hGab-1 is a high-affinity substrate for the EGFR and the major tyrosine phosphorylation site Y657 in the C terminus is a specific binding site for the tyrosine phosphatase Syp.

KIT mutations in Russian patients with mucosal melanoma.

PubMed

Abysheva, Svetlana N; Iyevleva, Aglaya G; Efimova, Nina V; Mokhina, Yulia B; Sabirova, Feruza A; Ivantsov, Alexandr O; Artemieva, Anna S; Togo, Alexandr V; Moiseyenko, Vladimir M; Matsko, Dmitry E; Imyanitov, Evgeny N

2011-12-01

A single institution series of 48 mucosal melanomas (MMs) has been analyzed for the presence of KIT mutations using high-resolution melting and sequencing of abnormally melted DNA fragments. The analysis of exons 9, 11, 13, and 17 has revealed eight of 48 (17%) nonsynonymous alterations, including zero of seven head and neck, six of 24 anorectal, one of 15 genitourinary, one of one gastric, and zero of one mediastinal MMs. Seven of these mutations were potentially associated with the tumor sensitivity to KIT tyrosine kinase inhibitors. One tumor harbored somatically acquired silent nucleotide substitution c.1383A>G (T461T). This study adds to the evidence that a substantial portion of MMs carry a therapeutically relevant mutation in the KIT oncogene.

Loss of c-Kit function impairs arteriogenesis in a mouse model of hindlimb ischemia.

PubMed

Hernandez, Diana R; Artiles, Adriana; Duque, Juan C; Martinez, Laisel; Pinto, Mariana T; Webster, Keith A; Velazquez, Omaida C; Vazquez-Padron, Roberto I; Lassance-Soares, Roberta M

2018-04-01

Arteriogenesis is a process whereby collateral vessels remodel usually in response to increased blood flow and/or wall stress. Remodeling of collaterals can function as a natural bypass to alleviate ischemia during arterial occlusion. Here we used a genetic approach to investigate possible roles of tyrosine receptor c-Kit in arteriogenesis. Mutant mice with loss of c-Kit function (Kit W/W-v ), and controls were subjected to hindlimb ischemia. Blood flow recovery was evaluated pre-, post-, and weekly after ischemia. Foot ischemic damage and function were assessed between days 1 to 14 post-ischemia while collaterals remodeling were measured 28 days post-ischemia. Both groups of mice also were subjected to wild type bone marrow cells transplantation 3 weeks before hindlimb ischemia to evaluate possible contributions of defective bone marrow c-Kit expression on vascular recovery. Kit W/W-v mice displayed impaired blood flow recovery, greater ischemic damage and foot dysfunction after ischemia compared to controls. Kit W/W-v mice also demonstrated impaired collateral remodeling consistent with flow recovery findings. Because arteriogenesis is a biological process that involves bone marrow-derived cells, we investigated which source of c-Kit signaling (bone marrow or vascular) plays a major role in arteriogenesis. Kit W/W-v mice transplanted with bone marrow wild type cells exhibited similar phenotype of impaired blood flow recovery, greater tissue ischemic damage and foot dysfunction as nontransplanted Kit W/W-v mice. This study provides evidence that c-Kit signaling is required during arteriogenesis. Also, it strongly suggests a vascular role for c-Kit signaling because rescue of systemic c-Kit activity by bone marrow transplantation did not augment the functional recovery of Kit W/W-v mouse hindlimbs. Copyright © 2017 Elsevier Inc. All rights reserved.

Protein-protein interactions between SWCNT/chitosan/EGF and EGF receptor: a model of drug delivery system.

PubMed

Rungnim, Chompoonut; Rungrotmongkol, Thanyada; Kungwan, Nawee; Hannongbua, Supot

2016-09-01

Epidermal growth factor (EGF) was used as the targeting ligand to enhance the specificity of a cancer drug delivery system (DDS) via its specific interaction with the EGF receptor (EGFR) that is overexpressed on the surface of some cancer cells. To investigate the intermolecular interaction and binding affinity between the EGF-conjugated DDS and the EGFR, 50 ns molecular dynamics simulations were performed on the complex of tethered EGFR and EGF linked to single-wall carbon nanotube (SWCNT) through a biopolymer chitosan wrapping the tube outer surface (EGFR·EGF-CS-SWCNT-Drug complex), and compared to the EGFR·EGF complex and free EGFR. The binding pattern of the EGF-CS-SWCNT-Drug complex to the EGFR was broadly comparable to that for EGF, but the binding affinity of the EGF-CS-SWCNT-Drug complex was predicted to be somewhat better than that for EGF alone. Additionally, the chitosan chain could prevent undesired interactions of SWCNT at the binding pocket region. Therefore, EGF connected to SWCNT via a chitosan linker is a seemingly good formulation for developing a smart DDS served as part of an alternative cancer therapy.

[Molecular mechanisms of primary and secondary resistance, molecular-genetic features and characteristics of KIT/PDGFRA non-mutated GISTs].

PubMed

Kalfusová, Alena; Kodet, Roman

2017-01-01

Gastrointestinal stromal tumors (GISTs) are the most common mesenchymal tumors of the gastrointestinal tract. Most of them arise due to activating mutations in KIT (75 - 85 %) or PDGFRA (less than 10 %) genes. Identification of the activating mutations in KIT and PDGFRA genes, which code for receptor tyrosine kinases (RTKs), has improved the outcome of targeted therapy of metastatic, unresectable or recurrent GISTs. Primary and/or secondary resistance represents a significant problem in the targeted therapy by Imatinib mesylate (IM) in patients with GIST. An important mechanism of the secondary resistance is the evolvement of secondary mutations. Except for primary and secondary resistance, there is another problem of disease progression - a failure of tumor cells eradication even in the long term therapy of tyrosine kinase inhibitors. GISTs without mutations in KIT/PDGFRA genes constitute 10 - 15% GISTs in adults, and a majority (85 %) of pediatric GISTs. KIT/PDGFRA wild-type GISTs represent a heterogeneous group of tumors with several molecular-genetics and/or morphologic differences. KIT/PDGFRA wild-type GISTs are different in their molecular features, for example in mutations in the BRAF, KRAS, NF1 genes or defects of succinate dehydrogenase (SDH) subunits. KIT/PDGFRA wild-type GISTs are generally less sensitive to targeted therapy by tyrosine kinase inhibitors in comparison with KIT/PDGFRA mutated GISTs. Inhibitors of BRAF, PI3K (mTOR) or inhibitors of IGF1R and VEGFR receptors provide alternative therapeutic strategies.

The heparin-binding domain of HB-EGF mediates localization to sites of cell-cell contact and prevents HB-EGF proteolytic release

DOE Office of Scientific and Technical Information (OSTI.GOV)

Prince, Robin N.; Schreiter, Eric R.; Zou, Peng

2010-07-01

Heparin-binding EGF-like growth factor (HB-EGF) is a ligand for EGF receptor (EGFR) and possesses the ability to signal in juxtacrine, autocrine and/or paracrine mode, with these alternatives being governed by the degree of proteolytic release of the ligand. Although the spatial range of diffusion of released HB-EGF is restricted by binding heparan-sulfate proteoglycans (HSPGs) in the extracellular matrix and/or cellular glycocalyx, ascertaining mechanisms governing non-released HB-EGF localization is also important for understanding its effects. We have employed a new method for independently tracking the localization of the extracellular EGFlike domain of HB-EGF and the cytoplasmic C-terminus. A striking observation wasmore » the absence of the HB-EGF transmembrane proform from the leading edge of COS-7 cells in a wound-closure assay; instead, this protein localized in regions of cell-cell contact. A battery of detailed experiments found that this localization derives from a trans interaction between extracellular HSPGs and the HBEGF heparin-binding domain, and that disruption of this interaction leads to increased release of soluble ligand and a switch in cell phenotype from juxtacrine-induced growth inhibition to autocrine-induced proliferation. Our results indicate that extracellular HSPGs serve to sequester the transmembrane pro-form of HB-EGF at the point of cell-cell contact, and that this plays a role in governing the balance between juxtacrine versus autocrine and paracrine signaling.« less

Differential activation of the Ras/extracellular-signal-regulated protein kinase pathway is responsible for the biological consequences induced by the Axl receptor tyrosine kinase.

PubMed

Fridell, Y W; Jin, Y; Quilliam, L A; Burchert, A; McCloskey, P; Spizz, G; Varnum, B; Der, C; Liu, E T

1996-01-01

To understand the mechanism of Axl signaling, we have initiated studies to delineate downstream components in interleukin-3-dependent 32D cells by using a chimeric receptor containing the recombinant epidermal growth factor (EGF) receptor extracellular and transmembrane domains and the Axl kinase domain (EAK [for EGF receptor-Axl kinase]). We have previously shown that upon exogenous EGF stimulation, 32D-EAK cells are capable of proliferation in the absence of interleukin-3. With this system, we determined that EAK-induced cell survival and mitogenesis are dependent upon the Ras/extracellular-signal-regulated protein kinase (ERK) cascade. Although the phosphatidylinositol-3 kinase pathway is activated upon EAK signaling, it appears to be dispensable for the biological actions of the Axl kinase. Furthermore, we demonstrated that different threshold levels of Ras/ERK activation are needed to induce a block to apoptosis or proliferation in 32D cells. Recently, we have identified an Axl ligand, GAS6. Surprisingly, GAS6-stimulated 32D-Axl cells exhibited no blockage to apoptosis or mitogenic response which is correlated with the absence of Ras/ERK activation. Taken together, these data suggest that different extracellular domains dramatically alter the intracellular response of the Axl kinase. Furthermore, our data suggest that the GAS6-Axl interaction does not induce mitogenesis and that its exact role remains to be determined.

Domain-specific phosphomimetic mutation allows dissection of different protein kinase C (PKC) isotype-triggered activities of the RNA binding protein HuR.

PubMed

Schulz, Sebastian; Doller, Anke; Pendini, Nicole R; Wilce, Jacqueline A; Pfeilschifter, Josef; Eberhardt, Wolfgang

2013-12-01

The ubiquitous mRNA binding protein human antigen R (HuR) participates in the post-transcriptional regulation of many AU-rich element (ARE)-bearing mRNAs. Previously, by using in vitro kinase assay, we have identified serines (Ser) 158, 221 and 318 as targets of protein kinase C (PKC)-triggered phosphorylation. In this study, we tested whether GFP- or GST-tagged HuR constructs bearing a phosphomimetic Ser (S)-to-Asp (D) substitution at the different PKC target sites, would affect different HuR functions including HuR nucleo-cytoplasmic redistribution and binding to different types of ARE-containing mRNAs. The phosphomimetic GFP-tagged HuR protein bearing a phosphomimetic substitution in the hinge region of HuR (HuR-S221D) showed an increased cytoplasmic abundance when compared to wild-type HuR. Conversely, data from in vitro kinase assay and electrophoretic mobility shift assay (EMSA), implicates that phosphorylation at Ser 221 is not relevant for mRNA binding of HuR. Quantification of in vitro binding affinities of GST-tagged wild-type HuR and corresponding HuR proteins bearing a phosphomimetic substitution in either RRM2 (HuR-S158D) or in RRM3 (HuR-S318D) by microscale thermophoresis (MST) indicates a specific binding of wild-type HuR to type I, II or type III-ARE-oligonucleotides in the high nanomolar range. Interestingly, phosphomimetic mutation at position 158 or 318 had a negative influence on HuR binding to type I- and type II-ARE-mRNAs whereas it significantly enhanced HuR affinity to a type III-ARE substrate. Our data suggest that differential phosphorylation of HuR by PKCs at different HuR domains coordinates subcellular HuR distribution and leads to a preferential binding to U-rich bearing target mRNA. © 2013.

Expression of c-Kit isoforms in multiple myeloma: differences in signaling and drug sensitivity.

PubMed

Montero, Juan Carlos; López-Pérez, Ricardo; San Miguel, Jesús F; Pandiella, Atanasio

2008-06-01

c-Kit is expressed in the plasma cells from 30% of patients with multiple myeloma. Two different isoforms of c-Kit, characterized by the presence or absence of the tetrapeptide sequence GNNK in the extracellular domain, have been described. However, their expression and function in myeloma cells are unknown. We explored the function and expression of these c-Kit isoforms in myeloma cells. Expression of c-Kit isoforms was investigated by reverse transcriptase polymerase chain reaction in fresh plasma cells from patients and cell lines. The function of these c-Kit isoforms was analyzed upon expression in myeloma cells. Signaling was investigated by western blotting using antibodies specific for activated forms of several signaling proteins. The impact of c-Kit on the action of drugs commonly used in the treatment of multiple myeloma was investigated by MTT proliferation assays. Fresh plasma cells from patients as well as myeloma cell lines expressed the two isoforms of c-Kit. Retroviral infection of myeloma cells with vectors that code for c-Kit-GNNK+ or c-Kit-GNNK- forms demonstrated differences in the kinetics of phosphorylation between these isoforms. Stem cell factor-induced activation of the GNNK- form was faster and more pronounced than that of the GNNK+ form, whose activation, however, lasted for longer. The c-Kit receptors weakly activated the Erk1/2 and Erk5 pathways. Both receptors, however, efficiently coupled to the PI3K/Akt pathway, and stimulated p70S6K activation. The latter was sensitive to the mTOR inhibitor, rapamycin. Studies of drug sensitivity indicated that cells expressing the GNNK- form were more resistant to the anti-myeloma action of bortezomib and melphalan. Our data indicate that c-Kit expression in multiple myeloma cells is functional, and coupled to survival pathways that may modulate cell death in response to therapeutic compounds used in the treatment of this disease.

Expression of EGF and EGFR strongly correlates with metastasis of pancreatic ductal carcinoma.

PubMed

Pryczynicz, Anna; Guzińska-Ustymowicz, Katarzyna; Kemona, Andrzej; Czyzewska, Jolanta

2008-01-01

The epidermal growth factor family members: EGF, EGFR and the c-erbB-2(HER-2/neu) gene product have been found to play a role in carcinomas of the stomach, liver, breast, ovary and lungs. Recent reports have indicated that they are also involved in the growth of pancreatic ductal carcinoma, its invasiveness and metastasis. Thirty-six patients with pancreatic ductal carcinoma were analysed with respect to sex, age, histological type, malignancy grade (G), pTN status (pTN), local lymph node involvement and distant metastasis. The tumor levels of EGF, EGFR and c-erbB-2 expression were determined immunohistochemically. Expression of c-erbB-2 was observed in 24/36 cases, EGF in 13/36 cases and EGFR in 18/36 cases. Overexpression of EGF and EGFR was associated with metastasis to lymph nodes and other organs. A correlation was also found between EGF expression and the presence of EGFR in the tumour. The expression of c-erbB-2 protein was not found to correlate with any parameters. EGF and EGFR play a key role in neoplastic spread through lymph node involvement and metastasis to other organs.

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/Wv mutant mouse colon

PubMed Central

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice. PMID:26727725

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/Wv mutant mouse colon.

PubMed

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/Wv mice carrying W and Wv mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/Wv mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/Wv mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/Wv mutant colon.The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers,but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/Wv mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/Wv mutant mice.

Transforming and differentiation-inducing potential of constitutively activated c-kit mutant genes in the IC-2 murine interleukin-3-dependent mast cell line.

PubMed Central

Hashimoto, K.; Tsujimura, T.; Moriyama, Y.; Yamatodani, A.; Kimura, M.; Tohya, K.; Morimoto, M.; Kitayama, H.; Kanakura, Y.; Kitamura, Y.

1996-01-01

Two mutations of c-kit receptor tyrosine kinase (KIT), valine-559 to glycine (G559) and aspartic acid-814 to valine (V814), resulted in its constitutive activation. To examine the transforming and differentiation-inducing potential of the mutant KIT, we used the murine interleukin-3-dependent IC-2 mast cell line as a transfectant. The IC-2 cells contained few basophilic granules and did not express KIT on the surface. The KITG559 or KITV814 gene was introduced into IC-2 cells using a retroviral vector. KITG559 and KITV814 expressed in IC-2 cells were constitutively phosphorylated on tyrosine and demonstrated kinase activity in the absence of stem cell factor, which is a ligand for KIT. IC-2 cells expressing either KITG559 or KITV814 (IC-2G559 or IC-2V814 cells) showed factor-independent growth in suspension culture and produced tumors in nude athymic mice. In addition, IC-2G559 and IC-2V814 cells showed a more mature phenotype compared with the phenotype of the original IC-2 cells, especially after transplantation into nude mice. The number of basophilic granules and the content of histamine increased remarkably. KITG559 and KITV814 also influenced the transcriptional phenotype of mouse mast cell proteases (MMCP) in IC-2 cells. The expression of MMCP-2, MMCP-4, and MMCP-6 was much greater in IC-2G559 and IC-2V814 cells than in the original IC-2 cells. The results indicated that constitutively activated KIT had not only oncogenic activity but also differentiation-inducing activity in mast cells. Images Figure 1 Figure 4 Figure 5 Figure 6 PMID:8546206

Mutational status of EGFR and KIT in thymoma and thymic carcinoma.

PubMed

Yoh, Kiyotaka; Nishiwaki, Yutaka; Ishii, Genichiro; Goto, Koichi; Kubota, Kaoru; Ohmatsu, Hironobu; Niho, Seiji; Nagai, Kanji; Saijo, Nagahiro

2008-12-01

This study was conducted to evaluate the prevalence of EGFR and KIT mutations in thymomas and thymic carcinomas as a means of exploring the potential for molecularly targeted therapy with tyrosine kinase inhibitors. Genomic DNA was isolated from 41 paraffin-embedded tumor samples obtained from 24 thymomas and 17 thymic carcinomas. EGFR exons 18, 19, and 21, and KIT exons 9, 11, 13, and 17, were analyzed for mutations by PCR and direct sequencing. Protein expression of EGFR and KIT was evaluated immunohistochemically. EGFR mutations were detected in 2 of 20 thymomas, but not in any of the thymic carcinomas. All of the EGFR mutations detected were missense mutations (L858R and G863D) in exon 21. EGFR protein was expressed in 71% of the thymomas and 53% of the thymic carcinomas. The mutational analysis of KIT revealed only a missense mutation (L576P) in exon 11 of one thymic carcinoma. KIT protein was expressed in 88% of the thymic carcinomas and 0% of the thymomas. The results of this study indicate that EGFR and KIT mutations in thymomas and thymic carcinomas are rare, but that many of the tumors express EGFR or KIT protein.

c-kit Positive Cardiac Outgrowth Cells Demonstrate Better Ability for Cardiac Recovery Against Ischemic Myopathy.

PubMed

Li, Chuan; Matsushita, Satoshi; Li, Zhengqing; Guan, Jianjun; Amano, Atsushi

2017-10-01

Resident cardiac stem cells are expected to be a therapeutic option for patients who suffer from severe heart failure. However, uncertainty remains over whether sorting cells for c-kit, a stem cell marker, improves therapeutic outcomes. Cardiac outgrowth cells cultured from explants of rat heart atrium were sorted according to their positivity (+) or negativity (-) for c-kit. These cells were exposed to hypoxia for 3 d, and subsequently harvested for mRNA expression measurement. The cell medium was also collected to assess cytokine secretion. To test for a functional benefit in animals, myocardial infarction (MI) was induced in rats, and c-kit+ or c-kit- cells were injected. The left ventricular ejection fraction (LVEF) was measured for up to 4 weeks, after which the heart was harvested for biological and histological analyses. Expression of the angiogenesis-related genes, VEGF and ANGPTL2, was significantly higher in c-kit+ cells after 3 d of hypoxic culture, although we found no such difference prior to hypoxia. Secretion of VEGF and ANGPTL2 was greater in the c-kit+ group than in the c-kit- group, while hypoxia tended to increase cytokine expression in both groups. In addition, IGF-1 was significantly increased in the c-kit+ group, consistent with the relatively low expression of cleaved-caspase 3 revealed by western blot assay, and the relatively low count of apoptotic cells revealed by histochemical analysis. Administration of c-kit+cells into the MI heart improved the LVEF and increased neovascularization. These results indicate that c-kit+cells may be useful in cardiac stem cell therapy.

NHERF-1 regulation of EGF and neurotensin signalling in HT-29 epithelial cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kruger, Wade A.; Monteith, Gregory R.; Poronnik, Philip, E-mail: philip.poronnik@sydney.edu.au

2013-03-22

Highlights: ► NHERF-1 expression was abundant throughout HT-29 cells consistent with a cancerous phenotype. ► Knockdown of NHERF-1 lead to a significant reduction in cell proliferation. ► EGF and neurotensin-mediated proliferation was inhibited by knockdown of NHERF-1. ► Neurotensin-mediated Ca{sup 2+} response was abolished by knockdown of NHERF-1. -- Abstract: Neurotensin receptors (NT-R) and the epidermal growth factor receptors (EGF-R) are commonly overexpressed in many epithelial origin tumours. In addition to their role as mitogenic mediators through specific cell signalling, recent studies indicate that the activity/expression of scaffold proteins responsible for the assembly and coordination of the signalling complexes maymore » also have central roles in epithelial transformation. In particular, the “epithelial” PSD-95/Dlg/Zo-1 (PDZ) scaffold/adapter protein, Na{sup +}/H{sup +} exchanger regulatory factor isoform one (NHERF-1), has been identified as a potential regulator of cellular transformation. NHERF-1 is a known regulator of EGF-R function and plays numerous roles in G-protein-coupled receptor signalling. Because of the synergistic signalling between these two potent mitogens, we investigated a potential role for NHERF-1 in the molecular mechanism linking the aberrant proliferative phenotype initiated by some G-Protein-coupled receptor activators in the colon adenocarcinoma HT-29 cell line. Knockdown (80%) of endogenous NHERF-1 leads to significant reduction in proliferation rate; an effect that could not be recovered by exogenous application of either NT or EGF. Inhibition of the EGF-R with AG1487 also inhibited proliferation and this effect could not be recovered with NT. Knockdown of NHERF-1 significantly altered the expression of the EGF-R, and almost completely abolished the NT-mediated increases in intracellular free Ca{sup 2+}. Knockdown of NHERF-1 also attenuated UTP-mediated purinergic Ca{sup 2+} signalling. Taken together

Existence of c-Kit negative cells with ultrastructural features of interstitial cells of Cajal in the subserosal layer of the W/W(v) mutant mouse colon.

PubMed

Tamada, Hiromi; Kiyama, Hiroshi

2015-01-01

Interstitial cells of Cajal (ICC) are mesenchymal cells that are distributed along the gastrointestinal tract and function as pacemaker cells or intermediary cells between nerves and smooth muscle cells. ICC express a receptor tyrosine kinase c-Kit, which is an established marker for ICC. The c-kit gene is allelic with the murine white-spotting locus (W), and some ICC subsets were reported to be missing in heterozygous mutant W/W(v) mice carrying W and W(v) mutated alleles. In this study, the characterization of interstitial cells in the subserosal layer of W/W(v) mice was analyzed by immunohistochemistry and electron microscopy. In the proximal and distal colon of W/W(v) mutant mice, no c-Kit-positive cells were detected in the subserosal layer by immunohistochemistry. By electron microscopy, the interstitial cells, which were characterized by the existence of caveolae, abundant mitochondria and gap junctions, were observed in the W/W(v) mutant colon. The morphological characteristics were comparable to those of the multipolar c-Kit positive ICC seen in the subserosa of proximal and distal colon of wild-type mice. Fibroblasts were also located in the same layers, but the morphology of the fibroblasts was distinguishable from that of ICC in wild type mice or of ICC-like cells in W/W(v) mutant mice. Collectively, it is concluded that c-Kit-negative interstitial cells showing a typical ICC ultrastructure exist in the proximal and distal colon of W/W(v) mutant mice.

[Expression of c-kit in North African nasopharyngeal carcinomas: correlation with age and LMP1].

PubMed

Charfi, S; Khabir, A; Ayadi, L; Mseddi, M; Makni, H; Gorbel, A; Daoud, J; Frikha, M; Jlidi, R; Busson, P; Boudawara, T S

2007-09-01

To determine the level and prognostic significance of c-kit expression in the two age groups of North African nasopharyngeal carcinomas. A retrospective study of 99 NPC specimens from Tunisian patients was investigated by immunohistochemistry. Immunohistochemical data were correlated with Epstein-Barr virus LMP1 expression and pathological, clinical and survival parameters. c-kit was detected in 79% of the cases for patients under 30 years of age (juvenile form) but in only 56% of specimens in patients over 30 years (P=0.039) and was significantly over-expressed for patients with lymph node involvement (P=0.015). LMP1 score was 5.78 (+/-1.84) for c-kit negative tumors compared to 8,23 (+/-2.39) for c-kit positive tumors (P=0.002). Multivariate analysis including age, lymph nodes involvement and LMP1 expression as co-variables, showed that only age (P=0.027) and LMP1 expression (P=0.005) were significantly correlated to the c-kit expression. c-kit is highly expressed in the juvenile form of North African nasopharyngeal carcinomas. There is a significant association between LMP1 and c-kit expression. The contrasted levels of C-kit expression in the two age groups strengthen the hypothesis that these clinical forms result from distinct oncogenic mechanisms.

SDF1 gradient associates with the distribution of c-Kit+ cardiac cells in the heart.

PubMed

Renko, Outi; Tolonen, Anna-Maria; Rysä, Jaana; Magga, Johanna; Mustonen, Erja; Ruskoaho, Heikki; Serpi, Raisa

2018-01-18

Identification of the adult cardiac stem cells (CSCs) has offered new therapeutic possibilities for treating ischemic myocardium. CSCs positive for the cell surface antigen c-Kit are known as the primary source for cardiac regeneration. Accumulating evidence shows that chemokines play important roles in stem cell homing. Here we investigated molecular targets to be utilized in modulating the mobility of endogenous CSCs. In a four week follow-up after experimental acute myocardial infarction (AMI) with ligation of the left anterior descending (LAD) coronary artery of Sprague-Dawley rats c-Kit+ CSCs redistributed in the heart. The number of c-Kit+ CSCs in the atrial c-Kit niche was diminished, whereas increased amount was observed in the left ventricle and apex. This was associated with increased expression of stromal cell-derived factor 1 alpha (SDF1α), and a significant positive correlation was found between c-Kit+ CSCs and SDF1α expression in the heart. Moreover, the migratory capacity of isolated c-Kit+ CSCs was induced by SDF1 treatment in vitro. We conclude that upregulation of SDF1α after AMI associates with increased expression of endogenous c-Kit+ CSCs in the injury area, and show induced migration of c-Kit+ cells by SDF1.

Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression through suppression of p300 stabilization and NFκB/c-Jun activation in breast cancer MDA-MB-231 cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Ying-Jung; Lee, Yuan-Chin; Huang, Chia-Hui

Triple-negative breast cancers (TNBCs) are highly invasive and have a higher rate of distant metastasis. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in EGF/EGFR-mediated malignant progression and metastasis of TNBCs. Various studies have revealed that treatment with gallic acid down-regulates MMP-9 expression in cancer cells, and that conjugation of phytochemical compounds with gold nanoparticles (AuNPs) increases the anti-tumor activity of the phytochemical compounds. Thus, the effect of gallic acid-capped AuNPs (GA-AuNPs) on MMP-9 expression in EGF-treated TNBC MDA-MB-231 cells was analyzed in the present study. The so-called green synthesis of AuNPs by means of gallic acid was performed at pHmore » 10, and the resulting GA-AuNPs had spherical shape with an average diameter of approximately 50 nm. GA-AuNPs notably suppressed migration and invasion of EGF-treated cells, and inhibited EGF-induced MMP-9 up-regulation. GA-AuNPs abrogated EGF-induced Akt/p65 and ERK/c-Jun phosphorylation, leading to down-regulation of MMP-9 mRNA and protein expression in EGF-treated cells. Meanwhile, EGF-induced p300 stabilization was found to be involved in MMP-9 expression, whereas GA-AuNPs inhibited the EGF-promoted stability of the p300 protein. Although GA-AuNPs and gallic acid suppressed EGF-induced MMP-9 up-regulation via the same signaling pathway, the effective concentration of gallic acid was approximately 100-fold higher than that of GA-AuNPs for inhibition of MMP-9 expression in EGF-treated cells to a similar extent. Collectively, our data indicate that, in comparison with gallic acid, GA-AuNPs have a superior ability to inhibit EGF/EGFR-mediated MMP-9 expression in TNBC MDA-MB-231 cells. Our findings also point to a way to improve the anti-tumor activity of gallic acid. - Highlights: • Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression. • EGF-induced MMP-9 expression via p300 stabilization and NFκB/c-Jun activation. • Gallic

Nck-Interacting Ste20 Kinase Couples Eph Receptors to c-Jun N-Terminal Kinase and Integrin Activation

PubMed Central

Becker, Elena; Huynh-Do, Uyen; Holland, Sacha; Pawson, Tony; Daniel, Tom O.; Skolnik, Edward Y.

2000-01-01

The mammalian Ste20 kinase Nck-interacting kinase (NIK) specifically activates the c-Jun amino-terminal kinase (JNK) mitogen-activated protein kinase module. NIK also binds the SH3 domains of the SH2/SH3 adapter protein Nck. To determine whether Nck functions as an adapter to couple NIK to a receptor tyrosine kinase signaling pathway, we determined whether NIK is activated by Eph receptors (EphR). EphRs constitute the largest family of receptor tyrosine kinases (RTK), and members of this family play important roles in patterning of the nervous and vascular systems. In this report, we show that NIK kinase activity is specifically increased in cells stimulated by two EphRs, EphB1 and EphB2. EphB1 kinase activity and phosphorylation of a juxtamembrane tyrosine (Y594), conserved in all Eph receptors, are both critical for NIK activation by EphB1. Although pY594 in the EphB1R has previously been shown to bind the SH2 domain of Nck, we found that stimulation of EphB1 and EphB2 led predominantly to a complex between NIK/Nck, p62dok, RasGAP, and an unidentified 145-kDa tyrosine-phosphorylated protein. Tyrosine-phosphorylated p62dok most probably binds directly to the SH2 domain of Nck and RasGAP and indirectly to NIK bound to the SH3 domain of Nck. We found that NIK activation is also critical for coupling EphB1R to biological responses that include the activation of integrins and JNK by EphB1. Taken together, these findings support a model in which the recruitment of the Ste20 kinase NIK to phosphotyrosine-containing proteins by Nck is an important proximal step in the signaling cascade downstream of EphRs. PMID:10669731

Identification of a polymorphism in the transmembrane domain of the protooncogene c-kit in healthy subjects.

PubMed

Nagata, H; Worobec, A S; Metcalfe, D D

1996-01-01

c-Kit is the receptor for stem cell factor (SCF) and is found on hematopoietic stem cells, mast cells, melanocytes, and germ cells. Aggregation of c-Kit by SCF regulates cell proliferation, differentiation, and survival. In the process of examining c-Kit, a polymorphism in the transmembrane domain of the protooncogene c-Kit was identified. This polymorphism consisted of an A-to-C transversion at nucleotide (nt) 1642, and was deduced to substitute leucine for methionine at codon 541. The frequency of the allele with 'C' at nt 1642 was 0.09 in 64 unrelated subjects. Analysis of a two-generation family with the polymorphism suggested that this polymorphism did not result in disease. This is the first report of a polymorphism in the transmembrane domain of c-Kit, and may be of value in understanding and following the function of c-Kit in normal subjects and in those with other abnormalities of c-Kit.

[4-t-butylphenyl]-N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03) inhibits SCF/c-kit signaling in 501mel human melanoma cells and abolishes melanin production in mice and brownish guinea pigs.

PubMed

Na, Yong Joo; Baek, Heung Su; Ahn, Soo Mi; Shin, Hyun Jung; Chang, Ih-Seop; Hwang, Jae Sung

2007-09-01

It is well known that c-kit is related to pigmentation as well as to the oncology target protein. The objective of this study was to discover a skin-whitening agent that regulates c-kit activity. We have developed a high-throughput screening system using recombinant human c-kit protein. Approximately 10,000 synthetic compounds were screened for their effect on c-kit activity. Phenyl-imidazole sulfonamide derivatives showed inhibitory activity on c-kit phosphorylation in vitro. The effects of one derivative, [4-t-butylphenyl]-N-(4-imidazol-1-yl phenyl)sulfonamide (ISCK03), on stem-cell factor (SCF)/c-kit cellular signaling in 501mel human melanoma cells were examined further. Pretreatment of 501mel cells with ISCK03 inhibited SCF-induced c-kit phosphorylation dose dependently. ISCK03 also inhibited p44/42 ERK mitogen-activated protein kinase (MAPK) phosphorylation, which is known to be involved in SCF/c-kit downstream signaling. However ISCK03 did not inhibit hepatocyte growth factor (HGF)-induced phosphorylation of p44/42 ERK proteins. To determine the in vivo potency of ISCK03, it was orally administered to depilated C57BL/6 mice. Interestingly, oral administration of ISCK03 induced the dose-dependent depigmentation of newly regrown hair, and this was reversed with cessation of ISCK03 treatment. Finally, to investigate whether the inhibitory effect of ISCK03 on SCF/c-kit signaling abolished UV-induced pigmentation, ISCK03 was applied to UV-induced pigmented spots on brownish guinea pig skin. The topical application of ISCK03 promoted the depigmentation of UV-induced hyperpigmented spots. Fontana-Masson staining analysis showed epidermal melanin was diminished in spots treated with ISCK03. These results indicate that phenyl-imidazole sulfonamide derivatives are potent c-kit inhibitors and might be used as skin-whitening agents.

Simultaneous detection of human CYP2C19 polymorphisms and antibiotic resistance of Helicobacter pylori using a personalised diagnosis kit.

PubMed

Zhang, Jun; Zhong, Jing; Ding, Jian; Shi, Jiemin; Tang, Tao; Liu, Qiqi; Huang, Huilian; Dai, Licheng; Yang, Ningmin

2018-06-01

A personalised diagnosis kit for Helicobacter pylori that employs visual gene chip technology for the simultaneous detection of CYP2C19 polymorphisms and clarithromycin/levofloxacin antibiotic resistance was evaluated. Gastric antrum mucosa biopsy specimens of 394 patients were tested using the kit. DNA sequencing and antibiotic susceptibility testing of the H. pylori were also performed. In total, 267 (67.8%) of the 394 specimens were positive for H. pylori using the kit and DNA sequencing, and 136 (34.5%) were positive by culturing. For human CYP2C19 and the bacterial 23S rRNA and gyrA genes, the concordance rates were 92.4% (364/394), 96.6% (258/267) and 97.0% (259/267) between the kit and DNA sequencing results, respectively. For clarithromycin and levofloxacin resistance, the concordance rates were 90.4% (123/136) and 81.6% (111/136) between the kit and antibiotic susceptibility testing results. The personalised diagnosis kit for H. pylori provides useful information for the choice of proton pump inhibitor and antibiotic in combination therapy. Copyright © 2018 International Society for Chemotherapy of Infection and Cancer. Published by Elsevier Ltd. All rights reserved.

Analysis of the surface expression of c-kit and occurrence of the c-kit Asp816Val activating mutation in T cells, B cells, and myelomonocytic cells in patients with mastocytosis.

PubMed

Akin, C; Kirshenbaum, A S; Semere, T; Worobec, A S; Scott, L M; Metcalfe, D D

2000-02-01

The Asp816Val c-kit activating mutation is detectable in the peripheral blood cells of some patients with mastocytosis and in lesional skin biopsies obtained from adult patients with urticaria pigmentosa. These observations led to the conclusion that this mutation is present in mast cells and mast cell precursors that express c-kit. However, the distribution of the Asp816Val mutation among hematopoietic lineages is unknown. To determine the distribution of the Asp816Val mutation among hematopoietic lineages and to explore its relationship to clinical disease, we examined cells bearing differentiation markers for myelomonocytic cells as well as T and B lymphocytes, in both peripheral blood and bone marrow obtained from patients with mastocytosis. The presence of Asp816Val c-kit mutation in cells magnetically sorted from peripheral blood or bone marrow according to surface differentiation markers was studied by reverse transcriptase polymerase chain reaction (RT-PCR) restriction fragment length polymorphism (RFLP) analysis. The surface expression of c-kit was determined by flow cytometry. The mutation was detectable by RT-PCR in at least one cell lineage in the bone marrow in 7 of 7 patients examined and in the peripheral blood of 11 of 11 adult patients with urticaria pigmentosa and indolent disease. The mutation was identified most frequently in B cells and myeloid cells. Flow cytometric analysis demonstrated that the differentiated cells expressing mutated c-kit were negative for surface KIT. These results are consistent with the conclusion that the c-kit Asp816Val mutation occurs in an early progenitor cell and is carried by myelomonocytic cells, T cells, and B cells in addition to mast cells. However, unlike mast cells, these myelomonocytic cells, T cells, and B cells do not concomitantly express surface c-kit and thus may be less susceptible to the effects of this mutation.

Selective inhibition of the platelet-derived growth factor signal transduction pathway by a protein-tyrosine kinase inhibitor of the 2-phenylaminopyrimidine class.

PubMed Central

Buchdunger, E; Zimmermann, J; Mett, H; Meyer, T; Müller, M; Regenass, U; Lydon, N B

1995-01-01

The platelet-derived growth factor (PDGF) receptor is a member of the transmembrane growth factor receptor protein family with intrinsic protein-tyrosine kinase activity. We describe a potent protein-tyrosine kinase inhibitor (CGP 53716) that shows selectivity for the PDGF receptor in vitro and in the cell. The compound shows selectivity for inhibition of PDGF-mediated events such as PDGF receptor autophosphorylation, cellular tyrosine phosphorylation, and c-fos mRNA induction in response to PDGF stimulation of intact cells. In contrast, ligand-induced autophosphorylation of the epidermal growth factor (EGF) receptor, insulin receptor, and the insulin-like growth factor I receptor, as well as c-fos mRNA expression induced by EGF, fibroblast growth factor, and phorbol ester, was insensitive to inhibition by CGP 53716. In antiproliferative assays, the compound was approximately 30-fold more potent in inhibiting PDGF-mediated growth of v-sis-transformed BALB/c 3T3 cells relative to inhibition of EGF-dependent BALB/Mk cells, interleukin-3-dependent FDC-P1 cells, and the T24 bladder carcinoma line. When tested in vivo using highly tumorigenic v-sis- and human c-sis-transformed BALB/c 3T3 cells, CGP 53716 showed antitumor activity at well-tolerated doses. In contrast, CGP 53716 did not show antitumor activity against xenografts of the A431 tumor, which overexpresses the EGF receptor. These findings suggest that CGP 53716 may have therapeutic potential for the treatment of diseases involving abnormal cellular proliferation induced by PDGF receptor activation. Images Fig. 1 Fig. 2 Fig. 3 PMID:7708684

Growth control of genetically modified cells using an antibody/c-Kit chimera.

PubMed

Kaneko, Etsuji; Kawahara, Masahiro; Ueda, Hiroshi; Nagamune, Teruyuki

2012-05-01

Gene therapy has been regarded as an innovative potential treatment against serious congenital diseases. However, applications of gene therapy remain limited, partly because its clinical success depends on therapeutic gene-transduced cells acquiring a proliferative advantage. To address this problem, we have developed the antigen-mediated genetically modified cell amplification (AMEGA) system, which uses chimeric receptors to enable the selective proliferation of gene-transduced cells. In this report, we describe mimicry of c-Kit signaling and its application to the AMEGA system. We created an antibody/c-Kit chimera in which the extracellular domain of c-Kit is replaced with an anti-fluorescein single-chain Fv antibody fragment and the extracellular D2 domain of the erythropoietin receptor. A genetically modified mouse pro-B cell line carrying this chimera showed selective expansion in the presence of fluorescein-conjugated BSA (BSA-FL) as a growth inducer. By further engineering the transmembrane domain of the chimera to reduce interchain interaction we attained stricter ligand-dependency. Since c-Kit is an important molecule in the expansion of hematopoietic stem cells (HSCs), this antibody/c-Kit chimera could be a promising tool for gene therapy targeting HSCs. Copyright © 2011 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.

Trivanillic polyphenols with anticancer cytostatic effects through the targeting of multiple kinases and intracellular Ca2+ release

PubMed Central

Lamoral-Theys, Delphine; Wauthoz, Nathalie; Heffeter, Petra; Mathieu, Véronique; Jungwirth, Utte; Lefranc, Florence; Nève, Jean; Dubois, Jacques; Dufrasne, François; Amighi, Karim; Berger, Walter; Gailly, Philippe; Kiss, Robert

2012-01-01

Abstract Cancer cells exhibit de-regulation of multiple cellular signalling pathways and treatments of various types of cancers with polyphenols are promising. We recently reported the synthesis of a series of 33 novel divanillic and trivanillic polyphenols that displayed anticancer activity, at least in vitro, through inhibiting various kinases. This study revealed that minor chemical modifications of a trivanillate scaffold could convert cytotoxic compounds into cytostatic ones. Compound 13c, a tri-chloro derivative of trivanillic ester, displayed marked inhibitory activities against FGF-, VEGF-, EGF- and Src-related kinases, all of which are implicated not only in angiogenesis but also in the biological aggressiveness of various cancer types. The pan-anti-kinase activity of 13c occurs at less than one-tenth of its mean IC50in vitro growth inhibitory concentrations towards a panel of 12 cancer cell lines. Of the 26 kinases for which 13c inhibited their activity by >75%, eight (Yes, Fyn, FGF-R1, EGFR, Btk, Mink, Ret and Itk) are implicated in control of the actin cytoskeleton organization to varying degrees. Compound 13c accordingly impaired the typical organization of the actin cytoskeleton in human U373 glioblastoma cells. The pan-anti-kinase activity and actin cytoskeleton organization impairment provoked by 13c concomitantly occurs with calcium homeostasis impairment but without provoking MDR phenotype activation. All of these anticancer properties enabled 13c to confer therapeutic benefits in vivo in a mouse melanoma pseudometastatic lung model. These data argue in favour of further chemically modifying trivanillates to produce novel and potent anticancer drugs. PMID:21810170

Lysosome trafficking is necessary for EGF-driven invasion and is regulated by p38 MAPK and Na+/H+ exchangers.

PubMed

Dykes, Samantha S; Steffan, Joshua J; Cardelli, James A

2017-10-04

Tumor invasion through a basement membrane is one of the earliest steps in metastasis, and growth factors, such as Epidermal Growth Factor (EGF) and Hepatocyte Growth Factor (HGF), stimulate this process in a majority of solid tumors. Basement membrane breakdown is one of the hallmarks of invasion; therefore, tumor cells secrete a variety of proteases to aid in this process, including lysosomal proteases. Previous studies demonstrated that peripheral lysosome distribution coincides with the release of lysosomal cathepsins. Immunofluorescence microscopy, western blot, and 2D and 3D cell culture techniques were performed to evaluate the effects of EGF on lysosome trafficking and cell motility and invasion. EGF-mediated lysosome trafficking, protease secretion, and invasion is regulated by the activity of p38 mitogen activated protein kinase (MAPK) and sodium hydrogen exchangers (NHEs). Interestingly, EGF stimulates anterograde lysosome trafficking through a different mechanism than previously reported for HGF, suggesting that there are redundant signaling pathways that control lysosome positioning and trafficking in tumor cells. These data suggest that EGF stimulation induces peripheral (anterograde) lysosome trafficking, which is critical for EGF-mediated invasion and protease release, through the activation of p38 MAPK and NHEs. Taken together, this report demonstrates that anterograde lysosome trafficking is necessary for EGF-mediated tumor invasion and begins to characterize the molecular mechanisms required for EGF-stimulated lysosome trafficking.

FUNCTIONAL DEREGULATION OF KIT: LINK TO MAST CELL PROLIFERATIVE DISEASES AND OTHER NEOPLASMS

PubMed Central

Cruse, Glenn; Metcalfe, Dean D.; Olivera, Ana

2014-01-01

SYNOPSIS Signaling through the receptor tyrosine kinase KIT mediates differentiation, proliferation and survival of hematopoietic precursor cells and mast cells. Constitutive KIT signaling due to somatic point mutations in c-Kit is an important occurrence in the development of mast cell proliferation disorders and other hematological malignancies. In this review, we discuss the common gain-of-function mutations found in these malignancies, particularly in mast cell proliferation disorders, and summarize the current understanding of the molecular mechanisms by which transforming point mutations in KIT may affect KIT structure and function and lead to altered downstream signaling and cellular transformation. Drugs targeting KIT have shown mixed success in the treatment of these diseases. A brief overview of the most common KIT inhibitors currently used, the reasons for the varied clinical results of such inhibitors and a discussion of potential new strategies are provided. PMID:24745671

Oncogenic Kit signals on endolysosomes and endoplasmic reticulum are essential for neoplastic mast cell proliferation

PubMed Central

Obata, Yuuki; Toyoshima, Shota; Wakamatsu, Ei; Suzuki, Shunichi; Ogawa, Shuhei; Esumi, Hiroyasu; Abe, Ryo

2014-01-01

Kit is a receptor-type tyrosine kinase found on the plasma membrane. It can transform mast cells through activating mutations. Here, we show that a mutant Kit from neoplastic mast cells from mice, Kit(D814Y), is permanently active and allows cells to proliferate autonomously. It does so by activating two signalling pathways from different intracellular compartments. Mutant Kit from the cell surface accumulates on endolysosomes through clathrin-mediated endocytosis, which requires Kit’s kinase activity. Kit(D814Y) is constitutively associated with phosphatidylinositol 3-kinase, but the complex activates Akt only on the cytoplasmic surface of endolysosomes. It resists destruction because it is under-ubiquitinated. Kit(D814Y) also appears in the endoplasmic reticulum soon after biosynthesis, and there, can activate STAT5 aberrantly. These mechanisms of oncogenic signalling are also seen in rat and human mast cell leukemia cells. Thus, oncogenic Kit signalling occurs from different intracellular compartments, and the mutation acts by altering Kit trafficking as well as activation. PMID:25493654

Characterization of a mast cell line that lacks the extracellular domain of membrane c-kit.

PubMed

Mekori, Y A; Oh, C K; Dastych, J; Goff, J P; Adachi, S; Bianchine, P J; Worobec, A; Semere, T; Pierce, J H; Metcalfe, D D

1997-04-01

Expression of the c-kit proto-oncogene receptor on mast cells is essential for their normal proliferation and maturation as well as for several biological responses such as chemotaxis and attachment. In the present study we report that the interleukin-3 (IL-3)-dependent mast cell line CFTL-15 lacks the extracellular domain of the c-kit receptor. This observation was made after noting that the c-kit ligand stem cell factor (SCF) could not prevent IL-3 deprivation-induced mast cell apoptosis and that CFTL-15 cells did not proliferate in response to SCF. Flow cytometric analysis employing monoclonal anti-c-kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequently showed a diminished expression of c-kit on CFTL-15 cells. There was no identifiable message for the extracellular domain of c-kit in these cells, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). These previously unrecognized properties of the CFTL-15 mast cell line allowed the examination of other biological consequences of the lack of c-kit on mast cells. Analysing the ability of these cells to adhere to surface-bound fibronectin, it was found that addition of SCF did not increase their adhesion to this substrate, in opposition to what is reported with other mast cells. Similarly, CFTL-15 mast cells did not adhere to fibroblasts, which is known to require c-kit expression. Also, there was no protein tyrosine phosphorylation in these cells in response to SCF. CFTL-15 cells underwent apoptosis on removal of IL-3 coincident with a decrease in endogenous Bcl-2 mRNA. Overexpression of Bcl-2 cDNA prolonged survival of Bcl-2-transfected CFTL-15 cells upon withdrawal of IL-3. Thus, the CFTL-15 cell line that lacks surface c-kit is not able to proliferate in response to SCF, undergoes apoptosis in the presence of SCF, and does not adhere to fibroblasts. These results confirm earlier studies on the functional consequences of c-kit and provide a novel

Inhibition of AMP Kinase by the Protein Phosphatase 2A Heterotrimer, PP2APpp2r2d*

PubMed Central

Joseph, Biny K.; Liu, Hsing-Yin; Francisco, Jamie; Pandya, Devanshi; Donigan, Melissa; Gallo-Ebert, Christina; Giordano, Caroline; Bata, Adam; Nickels, Joseph T.

2015-01-01

AMP kinase is a heterotrimeric serine/threonine protein kinase that regulates a number of metabolic processes, including lipid biosynthesis and metabolism. AMP kinase activity is regulated by phosphorylation, and the kinases involved have been uncovered. The particular phosphatases counteracting these kinases remain elusive. Here we discovered that the protein phosphatase 2A heterotrimer, PP2APpp2r2d, regulates the phosphorylation state of AMP kinase by dephosphorylating Thr-172, a residue that activates kinase activity when phosphorylated. Co-immunoprecipitation and co-localization studies indicated that PP2APpp2r2d directly interacted with AMP kinase. PP2APpp2r2d dephosphorylated Thr-172 in rat aortic and human vascular smooth muscle cells. A positive correlation existed between decreased phosphorylation, decreased acetyl-CoA carboxylase Acc1 phosphorylation, and sterol response element-binding protein 1c-dependent gene expression. PP2APpp2r2d protein expression was up-regulated in the aortas of mice fed a high fat diet, and the increased expression correlated with increased blood lipid levels. Finally, we found that the aortas of mice fed a high fat diet had decreased AMP kinase Thr-172 phosphorylation, and contained an Ampk-PP2APpp2r2d complex. Thus, PP2APpp2r2d may antagonize the aortic AMP kinase activity necessary for maintaining normal aortic lipid metabolism. Inhibiting PP2APpp2r2d or activating AMP kinase represents a potential pharmacological treatment for many lipid-related diseases. PMID:25694423

Serine Phosphorylation of the Insulin-like Growth Factor I (IGF-1) Receptor C-terminal Tail Restrains Kinase Activity and Cell Growth*

PubMed Central

Kelly, Geraldine M.; Buckley, Deirdre A.; Kiely, Patrick A.; Adams, David R.; O'Connor, Rosemary

2012-01-01

Insulin-like growth factor I receptor (IGF-1R) signaling is essential for cell, organ, and animal growth. The C-terminal tail of the IGF-1R exhibits regulatory function, but the mechanism is unknown. Here, we show that mutation of Ser-1248 (S1248A) enhances IGF-1R in vitro kinase activity, autophosphorylation, Akt/mammalian target of rapamycin activity, and cell growth. Ser-1248 phosphorylation is mediated by GSK-3β in a mechanism that involves a priming phosphorylation on Ser-1252. GSK-3β knock-out cells exhibit reduced IGF-1R cell surface expression, enhanced IGF-1R kinase activity, and signaling. Examination of crystallographic structures of the IGF-1R kinase domain revealed that the 1248SFYYS1252 motif adopts a conformation tightly packed against the kinase C-lobe when Ser-1248 is in the unphosphorylated state that favors kinase activity. S1248A mutation is predicted to lock the motif in this position. In contrast, phosphorylation of Ser-1248 will drive profound structural transition of the sequence, critically affecting connection of the C terminus as well as exposing potential protein docking sites. Decreased kinase activity of a phosphomimetic S1248E mutant and enhanced kinase activity in mutants of its predicted target residue Lys-1081 support this auto-inhibitory model. Thus, the SFYYS motif controls the organization of the IGF-1R C terminus relative to the kinase domain. Its phosphorylation by GSK-3β restrains kinase activity and regulates receptor trafficking and signaling. PMID:22685298

Differential regulation of betacellulin and heparin-binding EGF-like growth factor in cultured zebrafish ovarian follicle cells by EGF family ligands.

PubMed

Tse, Anna Chung-Kwan; Ge, Wei

2009-05-01

Recently the roles of epidermal growth factor (EGF) family ligands in vertebrate ovaries have received increasing attention, including betacellulin (BTC), amphiregulin (AR), heparin-binding EGF-like growth factor (HB-EGF), transforming growth factor alpha (TGFalpha), epiregulin, and EGF itself. In the zebrafish (Danio rerio), four members of EGF family have been identified by either molecular cloning or genome sequencing, which are EGF, TGFalpha, BTC, and HB-EGF. Although they are mostly expressed in the oocytes in the ovary, the present study demonstrated the expression of all the four EGF family ligands (egf, btc, tgfa, and hbegf) in cultured zebrafish follicle cells albeit at very low levels. Treatment of the cultured follicle cells with EGF, BTC, and HB-EGF demonstrated differential effects of these ligands on the expression of themselves. While the expression of egf was rather non-responsive to EGF, BTC, and HB-EGF, the expression of btc was consistently down-regulated by all the three molecules. In contrast, hbegf increased its expression in response to these molecules. These results suggest that there is an EGF signaling network in the zebrafish ovarian follicle, and the functionality of this network is self-regulated by its own members.

Epidermal growth factor induction of front–rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2

PubMed Central

Ho, Ernest; Dagnino, Lina

2012-01-01

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front–rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front–rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front–rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front–rear polarity and forward movement. PMID:22160594

Epidermal growth factor induction of front-rear polarity and migration in keratinocytes is mediated by integrin-linked kinase and ELMO2.

PubMed

Ho, Ernest; Dagnino, Lina

2012-02-01

Epidermal growth factor (EGF) is a potent chemotactic and mitogenic factor for epidermal keratinocytes, and these properties are central for normal epidermal regeneration after injury. The involvement of mitogen-activated protein kinases as mediators of the proliferative effects of EGF is well established. However, the molecular mechanisms that mediate motogenic responses to this growth factor are not clearly understood. An obligatory step for forward cell migration is the development of front-rear polarity and formation of lamellipodia at the leading edge. We show that stimulation of epidermal keratinocytes with EGF, but not with other growth factors, induces development of front-rear polarity and directional migration through a pathway that requires integrin-linked kinase (ILK), Engulfment and Cell Motility-2 (ELMO2), integrin β1, and Rac1. Furthermore, EGF induction of front-rear polarity and chemotaxis require the tyrosine kinase activity of the EGF receptor and are mediated by complexes containing active RhoG, ELMO2, and ILK. Our findings reveal a novel link between EGF receptor stimulation, ILK-containing complexes, and activation of small Rho GTPases necessary for acquisition of front-rear polarity and forward movement.

MUC5AC, a Gel-Forming Mucin Accumulating in Gallstone Disease, Is Overproduced via an Epidermal Growth Factor Receptor Pathway in the Human Gallbladder

PubMed Central

Finzi, Laetitia; Barbu, Véronique; Burgel, Pierre-Regis; Mergey, Martine; Kirkwood, Kimberly S.; Wick, Elizabeth C.; Scoazec, Jean-Yves; Peschaud, Frédérique; Paye, François; Nadel, Jay A.; Housset, Chantal

2006-01-01

Despite evidence that mucin overproduction is critical in the pathogenesis of gallstones, the mechanisms triggering mucin production in gallstone disease are unknown. Here, we tested the potential implication of an inflammation-dependent epidermal growth factor receptor (EGF-R) pathway in the regulation of gallbladder mucin synthesis. In gallbladder tissue sections from subjects with cholesterol gallstones, mucus accumulation was associated with neutrophil infiltration and with increased expressions of EGF-R and of tumor necrosis factor-α (TNF-α). In primary cultures of human gallbladder epithelial cells, TNF-α induced EGF-R overexpression. In the presence of TNF-α, EGF-R ligands (either EGF or transforming growth factor-α) caused significant increases in MUC5AC mRNA and protein production, whereas expression of the other gallbladder mucins MUC1, MUC3, and MUC5B was unchanged. In addition, on gallbladder tissue sections from subjects with gallstones, increased MUC5AC immunoreactivity was detected in the epithelium and within mucus gel in the lumen. Studies in primary cultures demonstrated that MUC5AC up-regulation induced by the combination of TNF-α with EGF-R ligands was completely blunted by inhibitors of EGF-R tyrosine kinase and mitogen-activated protein/extracellular signal-related kinase kinase. In conclusion, an inflammation-dependent EGF-R cascade causes overproduction of the gel-forming mucin MUC5AC, which accumulates in cholesterol gallstone disease. The ability to interrupt this cascade is of potential interest in the prevention of cholesterol gallstones. PMID:17148666

Carboxyl group footprinting mass spectrometry and molecular dynamics identify key interactions in the HER2-HER3 receptor tyrosine kinase interface.

PubMed

Collier, Timothy S; Diraviyam, Karthikeyan; Monsey, John; Shen, Wei; Sept, David; Bose, Ron

2013-08-30

The HER2 receptor tyrosine kinase is a driver oncogene in many human cancers, including breast and gastric cancer. Under physiologic levels of expression, HER2 heterodimerizes with other members of the EGF receptor/HER/ErbB family, and the HER2-HER3 dimer forms one of the most potent oncogenic receptor pairs. Previous structural biology studies have individually crystallized the kinase domains of HER2 and HER3, but the HER2-HER3 kinase domain heterodimer structure has yet to be solved. Using a reconstituted membrane system to form HER2-HER3 kinase domain heterodimers and carboxyl group footprinting mass spectrometry, we observed that HER2 and HER3 kinase domains preferentially form asymmetric heterodimers with HER3 and HER2 monomers occupying the donor and acceptor kinase positions, respectively. Conformational changes in the HER2 activation loop, as measured by changes in carboxyl group labeling, required both dimerization and nucleotide binding but did not require activation loop phosphorylation at Tyr-877. Molecular dynamics simulations on HER2-HER3 kinase dimers identify specific inter- and intramolecular interactions and were in good agreement with MS measurements. Specifically, several intermolecular ionic interactions between HER2 Lys-716-HER3 Glu-909, HER2 Glu-717-HER3 Lys-907, and HER2 Asp-871-HER3 Arg-948 were identified by molecular dynamics. We also evaluated the effect of the cancer-associated mutations HER2 D769H/D769Y, HER3 E909G, and HER3 R948K (also numbered HER3 E928G and R967K) on kinase activity in the context of this new structural model. This study provides valuable insights into the EGF receptor/HER/ErbB kinase structure and interactions, which can guide the design of future therapies.

Antiproliferation effect of imatinib mesylate on MCF7, T-47D tumorigenic and MCF 10A nontumorigenic breast cell lines via PDGFR-β, PDGF-BB, c-Kit and SCF genes

PubMed Central

Kadivar, Ali; Kamalidehghan, Behnam; Akbari Javar, Hamid; Karimi, Benyamin; Sedghi, Reihaneh; Noordin, Mohamed Ibrahim

2017-01-01

Recent cancer molecular therapies are targeting main functional molecules to control applicable process of cancer cells. Attractive targets are established by receptor tyrosine kinases, such as platelet-derived growth factor receptors (PDGFRs) and c-Kit as mostly irregular signaling, which is due to either over expression or mutation that is associated with tumorigenesis and cell proliferation. Imatinib mesylate is a selective inhibitor of receptor tyrosine kinase, including PDGFR-β and c-Kit. In this research, we studied how imatinib mesylate would exert effect on MCF7 and T-47D breast cancer and MCF 10A epithelial cell lines, the gene and protein expression of PDGFR-β, c-Kit and their relevant ligands platelet-derived growth factor (PDGF)-BB and stem cell factor (SCF). The MTS assay was conducted in therapeutic relevant concentration of 2–10 µM for 96, 120 and 144 h treatment. In addition, apoptosis induction and cytostatic activity of imatinib mesylate were investigated with the terminal deoxynucleotidyl transferase dUTP nick end labeling TUNEL and cell cycle assays, respectively, in a time-dependent manner. Comparative real-time PCR and Western blot analysis were conducted to evaluate the expression and regulation of imatinib target genes and proteins. Our finding revealed that imatinib mesylate antiproliferation effect, apoptosis induction and cytostatic activity were significantly higher in breast cancer cell lines compared to MCF 10A. This effect might be due to the expression of PDGFR-β, PDGF-BB, c-Kit and SCF, which was expressed by all examined cell lines, except the T-47D cell line which was not expressed c-Kit. However, examined gene and proteins expressed more in cancer cell lines. Therefore, imatinib mesylate was more effective on them. It is concluded that imatinib has at least two potential targets in both examined breast cancer cell lines and can be a promising drug for targeted therapy to treat breast cancer. PMID:28260860

Identification of a secondary mutation in the KIT kinase domain correlated with imatinib-resistance in a canine mast cell tumor.

PubMed

Nakano, Yuko; Kobayashi, Masato; Bonkobara, Makoto; Takanosu, Masamine

2017-06-01

Imatinib-resistance is a major therapeutic problem in human chronic myeloid leukemia, human gastrointestinal stromal tumors, and canine mast cell tumors. In the present study, we identified the secondary mutation c.2006C>T in c-KIT exon 14 in a mast cell tumor obtained from a dog carrying c.1663-1671del in exon 11 and showing resistance to imatinib. The mutation in exon 14 resulted in substitution of threonine with isoleucine at position 669, which was located at the center of the ATP binding site as a gatekeeper and played an important role in binding to imatinib. Transfectants were constructed to survey the functions of these mutations in exons 11 and 14. The transfectant with mutant KIT encoded by c-KIT carrying c.1663-1671del showed constitutive ligand-independent phosphorylation that was suppressed by imatinib, indicating a gain-of-function mutation. Furthermore, the transfectant with mutant KIT encoded by c-KIT carrying both c.1663-1671del and c.2006C>T caused ligand-independent phosphorylation, which was not suppressed by imatinib. From these results, we concluded that the mutation c.2006C>T in c-KIT exon 14 was an imatinib-resistance mutation in a canine mast cell tumor. These findings revealed, for the first time, a mechanism of imatinib resistance in a clinical case of canine mast cell tumor. Copyright © 2017 Elsevier B.V. All rights reserved.

Stem cell factor (SCF) protects osteoblasts from oxidative stress through activating c-Kit-Akt signaling

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Lei; Wu, Zhong; Yin, Gang

2014-12-12

Highlights: • SCF receptor c-Kit is functionally expressed in primary and transformed osteoblasts. • SCF protects primary and transformed osteoblasts from H{sub 2}O{sub 2}. • SCF activation of c-Kit in osteoblasts, required for its cyto-protective effects. • c-Kit mediates SCF-induced Akt activation in cultured osteoblasts. • Akt activation is required for SCF-regulated cyto-protective effects in osteoblasts. - Abstract: Osteoblasts regulate bone formation and remodeling, and are main target cells of oxidative stress in the progression of osteonecrosis. The stem cell factor (SCF)-c-Kit pathway plays important roles in the proliferation, differentiation and survival in a range of cell types, but littlemore » is known about its functions in osteoblasts. In this study, we found that c-Kit is functionally expressed in both osteoblastic-like MC3T3-E1 cells and primary murine osteoblasts. Its ligand SCF exerted significant cyto-protective effects against hydrogen peroxide (H{sub 2}O{sub 2}). SCF activated its receptor c-Kit in osteoblasts, which was required for its cyto-protective effects against H{sub 2}O{sub 2}. Pharmacological inhibition (by Imatinib and Dasatinib) or shRNA-mediated knockdown of c-Kit thus inhibited SCF-mediated osteoblast protection. Further investigations showed that protection by SCF against H{sub 2}O{sub 2} was mediated via activation of c-Kit-dependent Akt pathway. Inhibition of Akt activation, through pharmacological or genetic means, suppressed SCF-mediated anti-H{sub 2}O{sub 2} activity in osteoblasts. In summary, we have identified a new SCF-c-Kit-Akt physiologic pathway that protects osteoblasts from H{sub 2}O{sub 2}-induced damages, and might minimize the risk of osteonecrosis caused by oxidative stress.« less

Myeloid Conditioning with c-kit-Targeted CAR-T Cells Enables Donor Stem Cell Engraftment.

PubMed

Arai, Yasuyuki; Choi, Uimook; Corsino, Cristina I; Koontz, Sherry M; Tajima, Masaki; Sweeney, Colin L; Black, Mary A; Feldman, Steven A; Dinauer, Mary C; Malech, Harry L

2018-05-02

We report a novel approach to bone marrow (BM) conditioning using c-kit-targeted chimeric antigen receptor T (c-kit CAR-T) cells in mice. Previous reports using anti-c-kit or anti-CD45 antibody linked to a toxin such as saporin have been promising. We developed a distinctly different approach using c-kit CAR-T cells. Initial studies demonstrated in vitro killing of hematopoietic stem cells by c-kit CAR-T cells but poor expansion in vivo and poor migration of CAR-T cells into BM. Pre-treatment of recipient mice with low-dose cyclophosphamide (125 mg/kg) together with CXCR4 transduction in the CAR-T cells enhanced trafficking to and expansion in BM (<1%-13.1%). This resulted in significant depletion of the BM c-kit + population (9.0%-0.1%). Because congenic Thy1.1 CAR-T cells were used in the Thy1.2-recipient mice, anti-Thy1.1 antibody could be used to deplete CAR-T cells in vivo before donor BM transplant. This achieved 20%-40% multilineage engraftment. We applied this conditioning to achieve an average of 28% correction of chronic granulomatous disease mice by wild-type BM transplant. Our findings provide a proof of concept that c-kit CAR-T cells can achieve effective BM conditioning without chemo-/radiotherapy. Our work also demonstrates that co-expression of a trafficking receptor can enhance targeting of CAR-T cells to a designated tissue. Published by Elsevier Inc.

C-kit mutations in gastrointestinal stromal tumours.

PubMed

Morey, Adrienne L; Wanigesekera, G David; Hawkins, Nicholas J; Ward, Robyn L

2002-08-01

Gastrointestinal stromal tumours (GISTs), once assumed to be of smooth muscle origin, generally express CD117 and CD34, similar to the interstitial cells of Cajal. Assessment of malignant potential in GISTs is problematic, especially on small biopsies. Some recent data indicate that mutations in the juxtamembrane domain (exon 11) of the c-kit (CD117) proto-oncogene may be associated with a worse prognosis. In this study, the frequency of c-kit exon 11 mutations has been determined in a series of 18 gut stromal tumours. Immunophenotype was assessed by immunoperoxidase stains for smooth muscle actin, desmin, S100, CD34 and CD117, and each tumour classified as being of low, uncertain (intermediate) or high malignant potential based on standard histological criteria. DNA from each tumour was extracted from fresh (n = 5) or formalin-fixed, paraffin-embedded (n= 13) tissues using the direct lysis method. Exon 11 was amplified by PCR and sequencing of both sense and antisense strands was performed on two occasions using an ABI 377 sequencer. Mutations in exon 11 were detected in three of 14 confirmed GISTs, two being point mutations at codon 560 and one a 3-bp deletion resulting in the in-frame deletion of glutamine at codon 561. All three tumours were of high or intermediate malignant potential histologically. Three other 'high risk' primary GISTs and a metastatic GIST deposit were negative for exon 11 mutations. Data on this relatively small cohort of Australian patients indicate that c-kit exon 11 mutation analysis does not correlate well with histological assessment of malignant potential, and cannot be regarded as a reliable objective marker for poor prognosis in GISTs.

EGF receptor ligands: recent advances.

PubMed

Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J

2016-01-01

Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR.

EGF receptor ligands: recent advances

PubMed Central

Singh, Bhuminder; Carpenter, Graham; Coffey, Robert J.

2016-01-01

Seven ligands bind to and activate the mammalian epidermal growth factor (EGF) receptor (EGFR/ERBB1/HER1): EGF, transforming growth factor-alpha (TGFA), heparin-binding EGF-like growth factor (HBEGF), betacellulin (BTC), amphiregulin (AREG), epiregulin (EREG), and epigen (EPGN). Of these, EGF, TGFA, HBEGF, and BTC are thought to be high-affinity ligands, whereas AREG, EREG, and EPGN constitute low-affinity ligands. This focused review is meant to highlight recent studies related to actions of the individual EGFR ligands, the interesting biology that has been uncovered, and relevant advances related to ligand interactions with the EGFR. PMID:27635238

A decrease in ubiquitination and resulting prolonged life-span of KIT underlies the KIT overexpression-mediated imatinib resistance of KIT mutation-driven canine mast cell tumor cells.

PubMed

Kobayashi, Masato; Kuroki, Shiori; Kurita, Sena; Miyamoto, Ryo; Tani, Hiroyuki; Tamura, Kyoichi; Bonkobara, Makoto

2017-10-01

Overexpression of KIT is one of the mechanisms that contributes to imatinib resistance in KIT mutation-driven tumors. Here, the mechanism underlying this overexpression of KIT was investigated using an imatinib-sensitive canine mast cell tumor (MCT) line CoMS, which has an activating mutation in KIT exon 11. A KIT-overexpressing imatinib-resistant subline, rCoMS1, was generated from CoMS cells by their continuous exposure to increasing concentrations of imatinib. Neither a secondary mutation nor upregulated transcription of KIT was detected in rCoMS1 cells. A decrease in KIT ubiquitination, a prolonged KIT life-span, and KIT overexpression were found in rCoMS1 cells. These events were suppressed by withdrawal of imatinib and were re-induced by re‑treatment with imatinib. These findings suggest that imatinib elicited overexpression of KIT via suppression of its ubiquitination. These results also indicated that imatinib-induced overexpression of KIT in rCoMS1 cells was not a permanently acquired feature but was a reversible response of the cells. Moreover, the pan deubiquitinating enzyme inhibitor PR619 prevented imatinib induction of KIT overexpression, suggesting that the imatinib-induced decrease in KIT ubiquitination could be mediated by upregulation and/or activation of deubiquitinating enzyme(s). It may be possible that a similar mechanism of KIT overexpression underlies the acquisition of imatinib resistance in some human tumors that are driven by KIT mutation.

Synthesis and evaluation of 7-substituted-5,6-dihydrobenzo[c]acridine derivatives as new c-KIT promoter G-quadruplex binding ligands.

PubMed

Guo, Qian-Liang; Su, Hua-Fei; Wang, Ning; Liao, Sheng-Rong; Lu, Yu-Ting; Ou, Tian-Miao; Tan, Jia-Heng; Li, Ding; Huang, Zhi-Shu

2017-04-21

It has been shown that treatment of cancer cells with c-KIT G-quadruplex binding ligands can reduce their c-KIT expression levels thus inhibiting cell proliferation and inducing cell apoptosis. Herein, a series of new 7-substituted-5,6-dihydrobenzo[c]acridine derivatives were designed and synthesized. Subsequent biophysical evaluation demonstrated that the derivatives could effectively bind to and stabilize c-KIT G-quadruplex with good selectivity against duplex DNA. It was found that 12-N-methylated derivatives with a positive charge introduced at 12-position of 5,6-dihydrobenzo[c]acridine ring had similar binding affinity but lower stabilizing ability to c-KIT G-quadruplex DNA, compared with those of nonmethylated derivatives. Further molecular modeling studies showed possible binding modes of G-quadruplex with the ligands. RT-PCR assay and Western blot showed that compound 2b suppressed transcription and translation of c-KIT gene in K562 cells, which was consistent with the property of an effective G-quadruplex binding ligand targeting c-KIT oncogene promoter. Further biological evaluation showed that compound 2b could induce apoptosis through activation of the caspase-3 cascade pathway. Copyright © 2017 Elsevier Masson SAS. All rights reserved.

Protein kinase D1 drives pancreatic acinar cell reprogramming and progression to intraepithelial neoplasia

NASA Astrophysics Data System (ADS)

Liou, Geou-Yarh; Döppler, Heike; Braun, Ursula B.; Panayiotou, Richard; Scotti Buzhardt, Michele; Radisky, Derek C.; Crawford, Howard C.; Fields, Alan P.; Murray, Nicole R.; Wang, Q. Jane; Leitges, Michael; Storz, Peter

2015-02-01

The transdifferentiation of pancreatic acinar cells to a ductal phenotype (acinar-to-ductal metaplasia, ADM) occurs after injury or inflammation of the pancreas and is a reversible process. However, in the presence of activating Kras mutations or persistent epidermal growth factor receptor (EGF-R) signalling, cells that underwent ADM can progress to pancreatic intraepithelial neoplasia (PanIN) and eventually pancreatic cancer. In transgenic animal models, ADM and PanINs are initiated by high-affinity ligands for EGF-R or activating Kras mutations, but the underlying signalling mechanisms are not well understood. Here, using a conditional knockout approach, we show that protein kinase D1 (PKD1) is sufficient to drive the reprogramming process to a ductal phenotype and progression to PanINs. Moreover, using 3D explant culture of primary pancreatic acinar cells, we show that PKD1 acts downstream of TGFα and Kras, to mediate formation of ductal structures through activation of the Notch pathway.

Heparin-binding EGF-like growth factor is present in human amniotic fluid and breast milk.

PubMed

Michalsky, M P; Lara-Marquez, M; Chun, L; Besner, G E

2002-01-01

Heparin-binding EGF-like growth factor (HB-EGF) is a member of the epidermal growth factor (EGF) family that has been implicated in the healing of various organ injuries. Endogenous HB-EGF production is upregulated in response to injury to the kidney, liver, brain, skin, and intestine. Exogenous administration of HB-EGF protects against intestinal epithelial cell apoptosis and necrosis and intestinal ischemia/reperfusion (I/R) injury. This study examines the presence of endogenous HB-EGF in human amniotic fluid and breast milk, fluids that are in intimate contact with the developing and neonatal gastrointestinal tract. Breast milk samples were collected from lactating women and amniotic fluid was gathered from full-term uteri (cesarian sections) or preterm uteri (amniocentesis). Crude and partially purified breast milk and amniotic fluid samples were analyzed for HB-EGF levels using an HB-EGF-specific enzyme-linked immunosorbent assay (ELISA). Analysis results showed detectable HB-EGF levels in human amniotic fluid and breast milk, ranging from 0.2 to 230 pg/mL. Breast milk and amniotic fluid subjected to heparin affinity or HB-EGF-affinity column chromatography showed bioactivity eluting at positions consistent with those known for native HB-EGF. This study represents the first report of detectable HB-EGF in human amniotic fluid and breast milk. The presence of HB-EGF in these fluids may serve a role in the development of the gastrointestinal tract in utero, and in protection against gut mucosal injury after birth. Copyright 2002 by W.B. Saunders Company.

Characterization of a mast cell line that lacks the extracellular domain of membrane c-kit.

PubMed Central

Mekori, Y A; Oh, C K; Dastych, J; Goff, J P; Adachi, S; Bianchine, P J; Worobec, A; Semere, T; Pierce, J H; Metcalfe, D D

1997-01-01

Expression of the c-kit proto-oncogene receptor on mast cells is essential for their normal proliferation and maturation as well as for several biological responses such as chemotaxis and attachment. In the present study we report that the interleukin-3 (IL-3)-dependent mast cell line CFTL-15 lacks the extracellular domain of the c-kit receptor. This observation was made after noting that the c-kit ligand stem cell factor (SCF) could not prevent IL-3 deprivation-induced mast cell apoptosis and that CFTL-15 cells did not proliferate in response to SCF. Flow cytometric analysis employing monoclonal anti-c-kit antibodies, and immunogold labelling with analysis by electron microscopy, subsequently showed a diminished expression of c-kit on CFTL-15 cells. There was no identifiable message for the extracellular domain of c-kit in these cells, as determined by reverse transcriptase-polymerase chain reaction (RT-PCR). These previously unrecognized properties of the CFTL-15 mast cell line allowed the examination of other biological consequences of the lack of c-kit on mast cells. Analysing the ability of these cells to adhere to surface-bound fibronectin, it was found that addition of SCF did not increase their adhesion to this substrate, in opposition to what is reported with other mast cells. Similarly, CFTL-15 mast cells did not adhere to fibroblasts, which is known to require c-kit expression. Also, there was no protein tyrosine phosphorylation in these cells in response to SCF. CFTL-15 cells underwent apoptosis on removal of IL-3 coincident with a decrease in endogenous Bcl-2 mRNA. Overexpression of Bcl-2 cDNA prolonged survival of Bcl-2-transfected CFTL-15 cells upon withdrawal of IL-3. Thus, the CFTL-15 cell line that lacks surface c-kit is not able to proliferate in response to SCF, undergoes apoptosis in the presence of SCF, and does not adhere to fibroblasts. These results confirm earlier studies on the functional consequences of c-kit and provide a novel

Ultrastructural study of relationships between c-kit immunoreactive interstitial cells and other cellular elements in the human colon.

PubMed

Mazzia, C; Porcher, C; Julé, Y; Christen, M O; Henry, M

2000-05-01

C-kit immunocytochemistry was performed on ultrathin sections of human distal colon. Our attention was focused on relationships between c-kit immunoreactive interstitial cells (c-kit ICs) and muscular cells and nervous elements located in the external muscular layers of the colonic wall. C-kit ICs established membrane apposition with both nerve fibers and smooth muscle cells of, respectively, the longitudinal and circular muscle layers, the myenteric area, and the extremus submucosus plexus. C-kit ICs also surrounded the external submucosus plexus and established membrane appositions with nerve elements located inside the myenteric ganglia. These membrane appositions were observed either at the level of the c-kit IC bodies or at that of their cytoplasmic processes. In some cases, membrane appositions were observed concomitantly between the c-kit ICs, nerve fibers, and smooth muscle cells. In all the regions studied, the c-kit ICs were also found to be located in the close vicinity of blood vessels and to have established close contacts with non-immunoreactive fibroblast-like cells. The results of the present study shed essential light on the relationships of c-kit ICs with the neighboring muscle cells and nerve elements, and confirm that the intercalated c-kit ICs well fit with the so-called "interstitial cells of Cajal".

Molecular structure of EmbR, a response element of Ser/Thr kinase signaling in Mycobacterium tuberculosis

PubMed Central

Alderwick, Luke J.; Molle, Virginie; Kremer, Laurent; Cozzone, Alain J.; Dafforn, Timothy R.; Besra, Gurdyal S.; Fütterer, Klaus

2006-01-01

Ser/Thr phosphorylation has emerged as a critical regulatory mechanism in a number of bacteria, including Mycobacterium tuberculosis. This problematic pathogen encodes 11 eukaryotic-like Ser/Thr kinases, yet few substrates or signaling targets have been characterized. Here, we report the structure of EmbR (2.0 Å), a putative transcriptional regulator of key arabinosyltransferases (EmbC, -A, and -B), and an endogenous substrate of the Ser/Thr-kinase PknH. EmbR presents a unique domain architecture: the N-terminal winged-helix DNA-binding domain forms an extensive interface with the all-helical central bacterial transcriptional activation domain and is positioned adjacent to the regulatory C-terminal forkhead-associated (FHA) domain, which mediates binding to a Thr-phosphorylated site in PknH. The structure in complex with a phospho-peptide (1.9 Å) reveals a conserved mode of phospho-threonine recognition by the FHA domain and evidence for specific recognition of the cognate kinase. The present structures suggest hypotheses as to how EmbR might propagate the phospho-relay signal from its cognate kinase, while serving as a template for the structurally uncharacterized Streptomyces antibiotic regulatory protein family of transcription factors. PMID:16477027

Interaction Between a Novel p21 Activated Kinase (PAK6) and Androgen Receptor in Prostate Cancer

DTIC Science & Technology

2005-02-01

Beest , M., van Es, J., 51. Easwaran, V., Pishvaian, M., Salimuddin, and Byers, S. (1999) Curr. Biol. 9, Loureiro, J., Ypma, A., Hursh, D., Jones, T...antibody was generated and used in the study. 40 Immunofluorescence. Cells were cultured in 8-well Lab Tek chambered cover slides (Nalge Nunc...C19220, Transduction Labs , Lex- To further evaluate the roles of IGF-11 and EGF in ington, KY). Proteins were detected using the ECL kit (Am- AR

Adult cardiac stem cells are multipotent and robustly myogenic: c-kit expression is necessary but not sufficient for their identification.

PubMed

Vicinanza, Carla; Aquila, Iolanda; Scalise, Mariangela; Cristiano, Francesca; Marino, Fabiola; Cianflone, Eleonora; Mancuso, Teresa; Marotta, Pina; Sacco, Walter; Lewis, Fiona C; Couch, Liam; Shone, Victoria; Gritti, Giulia; Torella, Annalaura; Smith, Andrew J; Terracciano, Cesare Mn; Britti, Domenico; Veltri, Pierangelo; Indolfi, Ciro; Nadal-Ginard, Bernardo; Ellison-Hughes, Georgina M; Torella, Daniele

2017-12-01

Multipotent adult resident cardiac stem cells (CSCs) were first identified by the expression of c-kit, the stem cell factor receptor. However, in the adult myocardium c-kit alone cannot distinguish CSCs from other c-kit-expressing (c-kit pos ) cells. The adult heart indeed contains a heterogeneous mixture of c-kit pos cells, mainly composed of mast and endothelial/progenitor cells. This heterogeneity of cardiac c-kit pos cells has generated confusion and controversy about the existence and role of CSCs in the adult heart. Here, to unravel CSC identity within the heterogeneous c-kit-expressing cardiac cell population, c-kit pos cardiac cells were separated through CD45-positive or -negative sorting followed by c-kit pos sorting. The blood/endothelial lineage-committed (Lineage pos ) CD45 pos c-kit pos cardiac cells were compared to CD45 neg (Lineage neg /Lin neg ) c-kit pos cardiac cells for stemness and myogenic properties in vitro and in vivo. The majority (~90%) of the resident c-kit pos cardiac cells are blood/endothelial lineage-committed CD45 pos CD31 pos c-kit pos cells. In contrast, the Lin neg CD45 neg c-kit pos cardiac cell cohort, which represents ⩽10% of the total c-kit pos cells, contain all the cardiac cells with the properties of adult multipotent CSCs. These characteristics are absent from the c-kit neg and the blood/endothelial lineage-committed c-kit pos cardiac cells. Single Lin neg c-kit pos cell-derived clones, which represent only 1-2% of total c-kit pos myocardial cells, when stimulated with TGF-β/Wnt molecules, acquire full transcriptome and protein expression, sarcomere organisation, spontaneous contraction and electrophysiological properties of differentiated cardiomyocytes (CMs). Genetically tagged cloned progeny of one Lin neg c-kit pos cell when injected into the infarcted myocardium, results in significant regeneration of new CMs, arterioles and capillaries, derived from the injected cells. The CSC's myogenic regenerative capacity is

DIRECT MODULATION OF THE PROTEIN KINASE A CATALYTIC SUBUNIT α BY GROWTH FACTOR RECEPTOR TYROSINE KINASES

PubMed Central

Caldwell, George B.; Howe, Alan K.; Nickl, Christian K.; Dostmann, Wolfgang R.; Ballif, Bryan A.; Deming, Paula B.

2011-01-01

The cyclic-AMP-dependent protein kinase A (PKA) regulates processes such as cell proliferation and migration following activation of growth factor receptor tyrosine kinases (RTKs), yet the signaling mechanisms that link PKA with growth factor receptors remain largely undefined. Here we report that RTKs can directly modulate the function of the catalytic subunit of PKA (PKA-C) through post-translational modification. In vitro kinase assays revealed that both the epidermal growth factor and platelet derived growth factor receptors (EGFR and PDGFR, respectively) tyrosine phosphorylate PKA-C. Mass spectrometry identified tyrosine 330 (Y330) as a receptor-mediated phosphorylation site and mutation of Y330 to phenylalanine (Y330F) all but abolished the RTK-mediated phosphorylation of PKA-C in vitro. Y330 resides within a conserved region at the C-terminal tail of PKA-C that allosterically regulates enzymatic activity. Therefore, the effect of phosphorylation at Y330 on the activity of PKA-C was investigated. The Km for a peptide substrate was markedly decreased when PKA-C subunits were tyrosine phosphorylated by the receptors as compared to un-phosphorylated controls. Importantly, tyrosine-phosphorylated PKA-C subunits were detected in cells stimulated with EGF, PDGF and FGF2 and in fibroblasts undergoing PDGF-mediated chemotaxis. These results demonstrate a direct, functional interaction between RTKs and PKA-C and identify tyrosine phosphorylation as a novel mechansim for regulating PKA activity. PMID:21866565

Comparative gene expression profiling of ADAMs, MMPs, TIMPs, EMMPRIN, EGF-R and VEGFA in low grade meningioma.

PubMed

Rooprai, Harcharan K; Martin, Andrew J; King, Andrew; Appadu, Usha D; Jones, Huw; Selway, Richard P; Gullan, Richard W; Pilkington, Geoffrey J

2016-12-01

MMPs (matrix metalloproteinases), ADAMs (a disintegrin and metalloproteinase) and TIMPs (tissue inhibitors of metalloproteinases) are implicated in invasion and angiogenesis: both are tissue remodeling processes involving regulated proteolysis of the extracellular matrix, growth factors and their receptors. The expression of these three groups and their correlations with clinical behaviour has been reported in gliomas but a similar comprehensive study in meningiomas is lacking. In this study, we aimed to evaluate the patterns of expression of 23 MMPs, 4 TIMPs, 8 ADAMs, selective growth factors and their receptors in 17 benign meningiomas using a quantitative real-time polymerase chain reaction (qPCR). Results indicated very high gene expression of 13 proteases, inhibitors and growth factors studied: MMP2 and MMP14, TIMP-1, -2 and -3, ADAM9, 10, 12, 15 and 17, EGF-R, EMMPRIN and VEGF-A, in almost every meningioma. Expression pattern analysis showed several positive correlations between MMPs, ADAMs, TIMPs and growth factors. Furthermore, our findings suggest that expression of MMP14, ADAM9, 10, 12, 15 and 17, TIMP-2, EGF-R and EMMPRIN reflects histological subtype of meningioma such that fibroblastic subtype had the highest mRNA expression, transitional subtype was intermediate and meningothelial type had the lowest expression. In conclusion, this is the first comprehensive study characterizing gene expression of 8 ADAMs in meningiomas. These neoplasms, although by histological definition benign, have invasive potential. Taken together, the selected elevated gene expression pattern may serve to identify targets for therapeutic intervention or indicators of biological progression and recurrence.

An EGFR wild type-EGFRvIII-HB-EGF feed forward loop regulates the activation of EGFRvIII

PubMed Central

Li, Li; Chakraborty, Sharmistha; Yang, Chin-Rang; Hatanpaa, Kimmo J.; Cipher, Daisha J.; Puliyappadamba, Vineshkumar Thidil; Rehman, Alizeh; Jiwani, Ameena J.; Mickey, Bruce; Madden, Christopher; Raisanen, Jack; Burma, Sandeep; Saha, Debabrata; Wang, Zhixiang; Pingle, Sandeep C.; Kesari, Santosh; Boothman, David A.; Habib, Amyn A.

2014-01-01

EGFRvIII is a key oncogene in glioblastoma (GBM). EGFRvIII results from an in frame deletion in the extracellular domain of EGFR, does not bind ligand, and is thought to be constitutively active. While EGFRvIII dimerization is known to activate EGFRvIII, the factors that drive EGFRvIII dimerization and activation are not well understood. Here we present a new model of EGFRvIII activation and propose that oncogenic activation of EGFRvIII in glioma cells is driven by co-expressed activated EGFR wild type (EGFRwt). Increasing EGFRwt leads to a striking increase in EGFRvIII tyrosine phosphorylation and activation while silencing EGFRwt inhibits EGFRvIII activation. Both the dimerization arm and the kinase activity of EGFRwt are required for EGFRvIII activation. EGFRwt activates EGFRvIII by facilitating EGFRvIII dimerization. We have previously identified HB-EGF, a ligand for EGFRwt, as a gene induced specifically by EGFRvIII. In this study we show that HB-EGF, is induced by EGFRvIII only when EGFRwt is present. Remarkably, altering HB-EGF recapitulates the effect of EGFRwt on EGFRvIII activation. Thus, increasing HB-EGF leads to a striking increase in EGFRvIII tyrosine phosphorylation while silencing HB-EGF attenuates EGFRvIII phosphorylation, suggesting that an EGFRvIII-HB-EGF-EGFRwt feed forward loop regulates EGFRvIII activation. Silencing EGFRwt or HB-EGF leads to a striking inhibition of EGFRvIII induced tumorigenicity, while increasing EGFRwt or HB-EGF levels resulted in accelerated EGFRvIII mediated oncogenicity in an orthotopic mouse model. Furthermore, we demonstrate the existence of this loop in human GBM. Thus, our data demonstrate that oncogenic activation of EGFRvIII in GBM is likely maintained by a continuous EGFRwt-EGFRvIII-HBEGF loop, potentially an attractive target for therapeutic intervention. PMID:24077285

Active G protein-coupled receptors (GPCR), matrix metalloproteinases 2/9 (MMP2/9), heparin-binding epidermal growth factor (hbEGF), epidermal growth factor receptor (EGFR), erbB2, and insulin-like growth factor 1 receptor (IGF-1R) are necessary for trenbolone acetate-induced alterations in protein turnover rate of fused bovine satellite cell cultures.

PubMed

Thornton, K J; Kamanga-Sollo, E; White, M E; Dayton, W R

2016-06-01

TBA exhibit increased ( < 0.05) pAKT protein levels. These data indicate the TBA-mediated increases in protein synthesis likely involve GPCR, MMP2/9, hbEGF, EGFR, erbB2, and IGF-1R. However, the mechanism through which TBA mediates changes in protein degradation is different and appears to involve only the EGFR and erbB2. Furthermore, it appears the protein kinase B pathway is involved in TBA's effects on fused BSC cultures.

CD45{sup low}c-Kit{sup high} cells have hematopoietic properties in the mouse aorta-gonad-mesonephros region

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nobuhisa, Ikuo, E-mail: nobuhisa.scr@mri.tmd.ac.jp; Department of Cell Fate Modulation, Institute of Molecular Embryology and Genetics/Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, 860-0811; Yamasaki, Shoutarou

Long-term reconstituting hematopoietic stem cells first arise from the aorta of the aorta-gonad-mesonephros (AGM) region in a mouse embryo. We have previously reported that in cultures of the dispersed AGM region, CD45{sup low}c-Kit{sup +} cells possess the ability to reconstitute multilineage hematopoietic cells, but investigations are needed to show that this is not a cultured artifact and to clarify when and how this population is present. Based on the expression profile of CD45 and c-Kit in freshly dissociated AGM cells from embryonic day 9.5 (E9.5) to E12.5 and aorta cells in the AGM from E13.5 to E15.5, we defined sixmore » cell populations (CD45{sup -}c-Kit{sup -}, CD45{sup -}c-Kit{sup low}, CD45{sup -}c-Kit{sup high}, CD45{sup low}c-Kit{sup high}, CD45{sup high}c-Kit{sup high}, and CD45{sup high}c-Kit{sup very} {sup low}). Among these six populations, CD45{sup low}c-Kit{sup high} cells were most able to form hematopoietic cell colonies, but their ability decreased after E11.5 and was undetectable at E13.5 and later. The CD45{sup low}c-Kit{sup high} cells showed multipotency in vitro. We demonstrated further enrichment of hematopoietic activity in the Hoechst dye-effluxing side population among the CD45{sup low}c-Kit{sup high} cells. Here, we determined that CD45{sup low}c-Kit{sup high} cells arise from the lateral plate mesoderm using embryonic stem cell-derived differentiation system. In conclusion, CD45{sup low}c-Kit{sup high} cells are the major hematopoietic cells of mouse AGM.« less

Negative Cooperativity in the EGF Receptor

PubMed Central

Pike, Linda J.

2012-01-01

Scatchard analyses of the binding of EGF to its receptor yield concave up Scatchard plots, indicative of some type of heterogenity in ligand binding affinity. This was typically interpreted as being due to the presence of two independent binding site–one of high affinity representing ≤10% of the receptor population and one of low affinity making up the bulk of the receptors. However, the concept of two independent binding sites is difficult to reconcile with the X-ray structures of the dimerized EGF receptor that show symmetric binding of the two ligands. A new approach to the analysis of 125I-EGF binding data combined with the structure of the singly-occupied Drosophila EGF receptor have now shown that this heterogeneity is due to the presence of negative cooperativity in the EGF receptor. Concerns that negative cooperativity precludes ligand-induced dimerization of the EGF receptor confuse the concepts of linkage cooperativity. Linkage refers to the effect of ligand on the assembly of dimers while cooperativity refers to the effect of ligand binding to one subunit on ligand binding to the other subunit within a preassembled dimer. Binding of EGF to its receptor is positively linked with dimer assembly but shows negative cooperativity within the dimer. PMID:22260659

Prognostic and predictive values of oncogenic BRAF, NRAS, c-KIT and MITF in cutaneous and mucous melanoma.

PubMed

Pracht, M; Mogha, A; Lespagnol, A; Fautrel, A; Mouchet, N; Le Gall, F; Paumier, V; Lefeuvre-Plesse, C; Rioux-Leclerc, N; Mosser, J; Oger, E; Adamski, H; Galibert, M-D; Lesimple, T

2015-08-01

Mutations of BRAF, NRAS and c-KIT oncogenes are preferentially described in certain histological subtypes of melanoma and linked to specific histopathological features. BRAF-, MEK- and KIT-inhibitors led to improvement in overall survival of patients harbouring mutated metastatic melanoma. To assess the prevalence and types of BRAF, NRAS, c-KIT and MITF mutations in cutaneous and mucous melanoma and to correlate mutation status with clinicopathological features and outcome. Clinicopathological features and mutation status of 108 samples and of 98 consecutive patients were, respectively, assessed in one retrospective and one prospective study. Clinicopathological features were correlated with mutation status and the predictive value of these mutations was studied. This work identified significant correlations between BRAF mutations and melanoma occurring on non-chronic sun-damaged skin and superficial spreading melanoma (P < 0.05) on one hand, and between NRAS mutations and nodular melanoma (P < 0.05) on the other hand. Younger age (P < 0.05), microscopic (P < 0.05) and macroscopic (P < 0.05) lymphatic involvement at diagnosis of primary melanoma were significantly linked to BRAF mutations. A mutated status was a positive predictive factor of a response to BRAF inhibitors (OR = 3.44). Mutated melanoma showed a significantly (P = 0.038) higher objective response rate to cytotoxic chemotherapy (26.3%) than wild-type tumours (6.7%). Clinical and pathological characteristics of the primary melanoma differed between wild-type and BRAF- or NRAS-mutated tumours. Patients with BRAF-mutated tumours were younger at diagnosis of primary melanoma. Patients carrying mutations showed better responses better to specific kinase inhibitors and interestingly also to systemic cytotoxic chemotherapy. © 2015 European Academy of Dermatology and Venereology.

Prognostic significance of c-KIT in vulvar cancer: bringing this molecular marker from bench to bedside

PubMed Central

2012-01-01

Background Vulvar carcinomas are rare tumors, and there is limited data regarding molecular alterations. To our knowledge there are no published studies on c-KIT and squamous cell carcinomas of the vulva (VSCC). Although there are a significant number of other tumor types which express c-KIT, there remains controversy as to its relationship to patient outcome. Thus, we wished to investigate such controversial findings to determine the prognostic importance of c-KIT by evaluating its protein and mRNA expression in VSCCs, correlating these findings with clinicopathological features and Human Papillomavirus (HPV) infection. Methods c-KIT expression was scored by immunohistochemistry (IHC) as positive or negative in 139 formalin-fixed paraffin-embedded (FFPE) cases of vulvar carcinomas arrayed in a tissue microarray (TMA) using the DAKO® A4502 rabbit polyclonal c-KIT antibody (diluted 1:100). c-KIT mRNA was evaluated by qRT-PCR in 34 frozen samples from AC Camargo Hospital Biobank (17 tumoral and 17 non-tumoral samples) using TaqMan probes–Applied Biosystems [Hs00174029_m1]. HPV genotyping was assessed in 103 samples using Linear Array® HPV Genotyping Test kit (Roche Molecular Diagnostics, Basel, Switzerland). All results obtained were correlated with clinical and pathological data of the patients. Results c-KIT protein was positive by immunohistochemistry in 70.5% of the cases and this was associated with a higher global survival (p = 0.007), a higher recurrence-free survival (p < 0.0001), an absence of associated lesions (p = 0.001), lymph node metastasis (p = 0.0053), and HPV infection (p = 0.034). Furthermore, c-KIT mRNA quantitation revealed higher levels of transcripts in normal samples compared to tumor samples (p = 0,0009). Conclusions Our findings indicate that those vulvar tumors staining positively for c-KIT present better prognosis. Thus, positivity of c-KIT as evaluated by IHC may be a good predictor for use of more conservative

Resveratrol Improves Cell Cycle Arrest in Chronic Prostatitis Rats, by C-kit/SCF Suppression.

PubMed

He, Yi; Zeng, Huizhi; Yu, Yang; Zhang, Jiashu; Zeng, Xiaona; Gong, Fengtao; Liu, Qi; Yang, Bo

2017-08-01

Chronic prostatitis (CP) with complex pathogenesis is difficult for treatment. c-kit has been associated with the control of cell proliferation of prostate cells. This study aims to evaluate the role of resveratrol, an activator of Sirt1, in regulating the expression of c-kit in CP and investigate the consequent effects on cell cycle. Rat model of CP was established through subcutaneous injections of diphtheria-pertussis-tetanus vaccine and subsequently treated with resveratrol. Hematoxylin and eosin staining was performed to identify the histopathological changes in prostates. Western blotting and immunohistochemical staining examined the expression level of c-kit, stem cell factor (SCF), Sirt1, and cell cycle-associated proteins. The model group exhibited severe diffuse chronic inflammation, characterized by leukocyte infiltration and papillary frond protrusion into the gland cavities, and a notable increase in prostatic epithelial height. Gland lumen diameter was also significantly smaller; the activity of c-kit/SCF in the CP rats was increased significantly compared to the control group. Meanwhile, the cell cycle proteins are dysregulated significantly in CP rats. Resveratrol treatment significantly improved these factors by Sirt1 activation. Dysregulation of cell cycle was involved in the pathological processes of CP, which was improved after resveratrol treatment by the downregulation of c-kit/SCF by activating Sirt1.

[Immunoenzymatic assays of c-erbB-2 oncoprotein and epidermal growth factor receptor in breast cancer: correlation with clinical and biological parameters].

PubMed

Métayé, T; Bareille Saint-Gaudens, A; Millet, C; Ingrand, P; Daban, A; Bégon, F

1996-01-01

Two new immunoenzymatic assays for c-erbB-2 oncoprotein and epidermal growth factor receptor (EGF-R) (Oncogene Science) in human breast cancer were validated. Correlations between these assays and some clinical and biological parameters were also studied. The repeatability and reproducibility of standard curves for the two methods gave a coefficient of variation (CV) of less than 4% and about 10% respectively. The accuracy of c-erbB-2 oncoprotein and EGF-R assays was examined by using dilution and recovery tests throughout the standard curves. The linear relations between theoretical and measured values, for these tests, had slopes close to 1 and an intercept near 0. The median value for EGF-R, measured on solubilized membranes of 290 primary tumors, was 0.12 fmol/micrograms protein, the mean value was 0.37 (range 0 to 35.7). For c-erbB-2 oncoprotein, the median value, measured using the same population, was 2.75 human neu unit/micrograms protein, the mean value was 7.85 (range 1 to 125). There was an inverse relationship between EGF-R values and those for the estrogen receptor (ER), progesterone receptor and pS2 protein as well as menopausal status. C-erbB-2 oncoprotein concentrations were positively correlated with ER, pS2 protein and cathepsin D. Furthermore, a significant positive correlation was observed between EGF-R levels and c-erbB-2 oncoprotein levels. In conclusion, immunoenzymatic assays of EGF-R and c-erbB-2 oncoprotein are easy to use, sensitive and reliable. The accurate standardisation of immunoenzymatic assays could contribute to the clinical use of EGF-R and c-erbB-2 oncoprotein as prognostic factors in breast cancer.

A Novel Positive Feedback Loop Mediated by the Docking Protein Gab1 and Phosphatidylinositol 3-Kinase in Epidermal Growth Factor Receptor Signaling

PubMed Central

Rodrigues, Gerard A.; Falasca, Marco; Zhang, Zhongtao; Ong, Siew Hwa; Schlessinger, Joseph

2000-01-01

The Gab1 protein is tyrosine phosphorylated in response to various growth factors and serves as a docking protein that recruits a number of downstream signaling proteins, including phosphatidylinositol 3-kinase (PI-3 kinase). To determine the role of Gab1 in signaling via the epidermal growth factor (EGF) receptor (EGFR) we tested the ability of Gab1 to associate with and modulate signaling by this receptor. We show that Gab1 associates with the EGFR in vivo and in vitro via pTyr sites 1068 and 1086 in the carboxy-terminal tail of the receptor and that overexpression of Gab1 potentiates EGF-induced activation of the mitogen-activated protein kinase and Jun kinase signaling pathways. A mutant of Gab1 unable to bind the p85 subunit of PI-3 kinase is defective in potentiating EGFR signaling, confirming a role for PI-3 kinase as a downstream effector of Gab1. Inhibition of PI-3 kinase by a dominant-interfering mutant of p85 or by Wortmannin treatment similarly impairs Gab1-induced enhancement of signaling via the EGFR. The PH domain of Gab1 was shown to bind specifically to phosphatidylinositol 3,4,5-triphosphate [PtdIns(3,4,5)P3], a product of PI-3 kinase, and is required for activation of Gab1-mediated enhancement of EGFR signaling. Moreover, the PH domain mediates Gab1 translocation to the plasma membrane in response to EGF and is required for efficient tyrosine phosphorylation of Gab1 upon EGF stimulation. In addition, overexpression of Gab1 PH domain blocks Gab1 potentiation of EGFR signaling. Finally, expression of the gene for the lipid phosphatase PTEN, which dephosphorylates PtdIns(3,4,5)P3, inhibits EGF signaling and translocation of Gab1 to the plasma membrane. These results reveal a novel positive feedback loop, modulated by PTEN, in which PI-3 kinase functions as both an upstream regulator and a downstream effector of Gab1 in signaling via the EGFR. PMID:10648629

Protein Kinase C alpha (PKCα) dependent signaling mediates endometrial cancer cell growth and tumorigenesis

PubMed Central

Haughian, James M.; Reno, Elaine M.; Thorne, Alicia M.; Bradford, Andrew P.

2009-01-01

Endometrial cancer is the most common invasive gynecologic malignancy, yet molecular mechanisms and signaling pathways underlying its etiology and pathophysiology remain poorly characterized. We sought to define a functional role for the protein kinase C (PKC) isoform, PKCα, in an established cell model of endometrial adenocarcinoma. Ishikawa cells depleted of PKCα protein grew slower, formed fewer colonies in anchorage-independent growth assays and exhibited impaired xenograft tumor formation in nude mice. Consistent with impaired growth, PKCα knockdown increased levels of the cyclin dependent kinase (CDK) inhibitors p21Cip1/WAF1 (p21) and p27Kip1 (p27). Despite the absence of functional phosphatase and tensin homologue (PTEN) protein in Ishikawa cells, PKCα knockdown reduced Akt phosphorylation at serine 473 and concomitantly inhibited phosphorylation of the Akt target, glycogen synthase kinase-3β (GSK-3β). PKCα knockdown also resulted in decreased basal ERK phosphorylation and attenuated ERK activation following EGF stimulation. p21 and p27 expression was not increased by treatment of Ishikawa cells with ERK and Akt inhibitors, suggesting PKCα regulates CDK expression independently of Akt and ERK. Immunohistochemical analysis of grade 1 endometrioid adenocarcinoma revealed aberrant PKCα expression, with foci of elevated PKCα staining, not observed in normal endometrium. These studies demonstrate a critical role for PKCα signaling in endometrial tumorigenesis by regulating expression of CDK inhibitors p21 and p27 and activation of Akt and ERK dependent proliferative pathways. Thus, targeting PKCα may provide novel therapeutic options in endometrial tumors. PMID:19672862

Membrane estrogen receptor alpha is an important modulator of bone marrow C-Kit+ cells mediated cardiac repair after myocardial infarction

PubMed Central

Su, Feng; Zhang, Wentian; Liu, Jianfang

2015-01-01

It has been validated that c-kit positive (c-kit+) cells in infarcted myocardium are from bone marrow (BM). Given the recent study that in the heart, estrogen receptor alpha (ERα) is involved in adaptive mechanisms by supporting cardiomyocytes survival via post-infarct cardiac c-kit+ cells, we tested a novel hypothesis that membrane ERα (mERа) supports survival of BM c-kit+ cells and enhance protective paracrine function for cardiac repair. Our data showed that myocardial infarction (MI) leads to an increase in c-kit+ first in bone marrow and then specifically within the infarcted myocardium. Also up-regulated mERа in post-infarct BM c-kit+ cells was found in day 3 post MI. In vitro co-culture system, mERа+ enhances the beneficial effects of BM c-kit+ cells by increasing their viability and reducing apoptosis. Post-infarct c-kit+ mERа+ cells population expresses predominant ERα and holds self-renewal as well as cardiac differentiation potentials after MI. In vivo, BM c-kit+ cells reduced infarct size, fibrosis and improved cardiac function. In conclusion, BM c-kit+ mERа+ exerted significantly cardiac protection after MI. A potential important implication of this study is that the manipulation of BM c-kit+ stem cells with ERа-dependent fashion may be helpful in recovering functional performance after cardiac tissue injury. PMID:26191121

Regulation of CD93 cell surface expression by protein kinase C isoenzymes.

PubMed

Ikewaki, Nobunao; Kulski, Jerzy K; Inoko, Hidetoshi

2006-01-01

Human CD93, also known as complement protein 1, q subcomponent, receptor (C1qRp), is selectively expressed by cells with a myeloid lineage, endothelial cells, platelets, and microglia and was originally reported to be involved in the complement protein 1, q subcomponent (C1q)-mediated enhancement of phagocytosis. The intracellular molecular events responsible for the regulation of its expression on the cell surface, however, have not been determined. In this study, the effect of protein kinases in the regulation of CD93 expression on the cell surface of a human monocyte-like cell line (U937), a human NK-like cell line (KHYG-1), and a human umbilical vein endothelial cell line (HUV-EC-C) was investigated using four types of protein kinase inhibitors, the classical protein kinase C (cPKC) inhibitor Go6976, the novel PKC (nPKC) inhibitor Rottlerin, the protein kinase A (PKA) inhibitor H-89 and the protein tyrosine kinase (PTK) inhibitor herbimycin A at their optimum concentrations for 24 hr. CD93 expression was analyzed using flow cytometry and glutaraldehyde-fixed cellular enzyme-linked immunoassay (EIA) techniques utilizing a CD93 monoclonal antibody (mAb), mNI-11, that was originally established in our laboratory as a CD93 detection probe. The nPKC inhibitor Rottlerin strongly down-regulated CD93 expression on the U937 cells in a dose-dependent manner, whereas the other inhibitors had little or no effect. CD93 expression was down-regulated by Go6976, but not by Rottlerin, in the KHYG-1 cells and by both Rottlerin and Go6976 in the HUV-EC-C cells. The PKC stimulator, phorbol myristate acetate (PMA), strongly up-regulated CD93 expression on the cell surface of all three cell-lines and induced interleukin-8 (IL-8) production by the U937 cells and interferon-gamma (IFN-gamma) production by the KHYG-1 cells. In addition, both Go6976 and Rottlerin inhibited the up-regulation of CD93 expression induced by PMA and IL-8 or IFN-gamma production in the respective cell

Both sides of the same coin: Rac1 splicing regulating by EGF signaling.

PubMed

Fu, Xiang-Dong

2017-04-01

EGF, a well-studied mitogen for cancer cells, is revealed to induce an E3 ubiquitin ligase adaptor SPSB1, which recruits the Elongin B/C-Collin complex to trigger ubiquitylation of the negative splicing regulator hnRNP A1. This event is synergized with EGF-activated SR proteins to alter alternative splicing of a key small GTPase Rac1 to enhance cell migration, highlighting converging EGF signals on both negative and positive splicing regulators to jointly promote a key cancer pathway.

Aldosterone interaction with epidermal growth factor receptor signaling in MDCK cells.

PubMed

Gekle, Michael; Freudinger, Ruth; Mildenberger, Sigrid; Silbernagl, Stefan

2002-04-01

Epidermal growth factor (EGF) regulates cell proliferation, differentiation, and ion transport by using extracellular signal-regulated kinase (ERK)1/2 as a downstream signal. Furthermore, the EGF-receptor (EGF-R) is involved in signaling by G protein-coupled receptors, growth hormone, and cytokines by means of transactivation. It has been suggested that steroids interact with peptide hormones, in part, by rapid, potentially nongenomic, mechanisms. Previously, we have shown that aldosterone modulates Na(+)/H(+) exchange in Madin-Darby canine kidney (MDCK) cells by means of ERK1/2 in a way similar to growth factors. Here, we tested the hypothesis that aldosterone uses the EGF-R as a heterologous signal transducer in MDCK cells. Nanomolar concentrations of aldosterone induce a rapid increase in ERK1/2 phosphorylation, cellular Ca(2+) concentration, and Na(+)/H(+) exchange activity similar to increases induced by EGF. Furthermore, aldosterone induced a rapid increase in EGF-R-Tyr phosphorylation, and inhibition of EGF-R kinase abolished aldosterone-induced signaling. Inhibition of ERK1/2 phosphorylation reduced the Ca(2+) response, whereas prevention of Ca(2+) influx did not abolish ERK1/2 phosphorylation. Our data show that aldosterone uses the EGF-R-ERK1/2 signaling cascade to elicit its rapid effects in MDCK cells.

Increased c-kit and stem cell factor expression in the pulmonary vasculature of nitrofen-induced congenital diaphragmatic hernia.

PubMed

Takahashi, Toshiaki; Friedmacher, Florian; Zimmer, Julia; Puri, Prem

2016-05-01

Persistent pulmonary hypertension(PPH) in congenital diaphragmatic hernia (CDH) is caused by increased vascular cell proliferation and endothelial cell (EC) dysfunction, thus leading to obstructive changes in the pulmonary vasculature. C-Kit and its ligand, stem cell factor(SCF), are expressed by ECs in the developing lung mesenchyme, suggesting an important role during lung vascular formation. Conversely, absence of c-Kit expression has been demonstrated in ECs of dysplastic alveolar capillaries. We hypothesized that c-Kit and SCF expression is increased in the pulmonary vasculature of nitrofen-induced CDH. Timed-pregnant rats received nitrofen or vehicle on gestational day 9(D9). Fetuses were sacrificed on D15, D18, and D21, and divided into control and CDH group. Pulmonary gene expression levels of c-Kit and SCF were analyzed by qRT-PCR. Immunofluorescence double staining for c-Kit and SCF was combined with CD34 to evaluate protein expression in ECs of the pulmonary vasculature. Relative mRNA levels of c-Kit and SCF were significantly increased in lungs of CDH fetuses on D15, D18, and D21 compared to controls. Confocal laser scanning microscopy confirmed markedly increased vascular c-Kit and SCF expression in mesenchymal ECs of CDH lungs on D15, D18, and D21 compared to controls. Increased expression of c-Kit and SCF in the pulmonary vasculature of nitrofen-induced CDH lungs suggest that increased c-Kit signaling during lung vascular formation may contribute to vascular remodeling and thus to PPH. Copyright © 2016 Elsevier Inc. All rights reserved.

Wounding-induced synthesis of hyaluronic acid in organotypic epidermal cultures requires the release of heparin-binding egf and activation of the EGFR.

PubMed

Monslow, James; Sato, Nobuyuki; Mack, Judith A; Maytin, Edward V

2009-08-01

Hyaluronic acid (HA), a glycosaminoglycan located between keratinocytes in the epidermis, accumulates dramatically following skin wounding. To study inductive mechanisms, a rat keratinocyte organotypic culture model that faithfully mimics HA metabolism was used. Organotypic cultures were needle-punctured 100 times, incubated for up to 24 hours, and HA analyzed by histochemical and biochemical methods. Within 15 minutes post-injury, HA levels had elevated two-fold, increasing to four-fold by 24 hours. HA elevations far from the site of injury suggested the possible involvement of a soluble HA-inductive factor. Media transfer experiments (from wounded cultures to unwounded cultures) confirmed the existence of a soluble factor. From earlier evidence, we hypothesized that an EGF-like growth factor might be responsible. This was confirmed as follows: (1) EGFR kinase inhibitor (AG1478) completely prevented wounding-induced HA accumulation. (2) Rapid tyrosine-phosphorylation of EGFR correlated well with the onset of increased HA synthesis. (3) A neutralizing antibody that recognizes heparin binding EGF-like growth factor (HB-EGF) blocked wounding-induced HA synthesis by > or =50%. (4) Western analyses showed that release of activated HB-EGF (but neither amphiregulin nor EGF) occured after wounding. In summary, rapid HA accumulation after epidermal wounding occurs through a mechanism requiring cleavage of HB-EGF and activation of EGFR signaling.

Improved expression of recombinant plant-made hEGF.

PubMed

Thomas, David Rhys; Walmsley, Amanda Maree

2014-11-01

The yield of recombinant hEGF was increased approximately tenfold through a range of optimisations. Further, the recombinant protein was found to have biological activity comparable to commercial hEGF. Human epidermal growth factor (hEGF) is a powerful mitogen that can enhance the healing of a wide range of injuries, including burns, cuts, diabetic ulcers and gastric ulcers. However, despite its clinical value, hEGF is only consistently used for the treatment of chronic diabetic ulcers due to its high cost. In this study, hEGF was transiently expressed in Nicotiana benthamiana plants and targeted to the apoplast, ER and vacuole. Several other approaches were also included in a stepwise fashion to identify the optimal conditions for the expression of recombinant hEGF. Expression was found to be highest in the vacuole, while targeting hEGF to the ER caused a decrease in total soluble protein (TSP). Using a codon optimised sequence was found to increase vacuolar targeted hEGF yield by ~34 %, while it was unable to increase the yield of ER targeted hEGF. The use of the P19 silencing inhibitor was able to further increase expression by over threefold, and using 5-week-old plants significantly increased expression compared to 4- or 6-week-old-plants. The combined effect of these optimisations increased expression tenfold over the initial apoplast targeted construct to an average yield of 6.24 % of TSP. The plant-made hEGF was then shown to be equivalent to commercial E. coli derived hEGF in its ability to promote the proliferation of mouse keratinocytes. This study supports the potential for plants to be used for the commercial production of hEGF, and identifies a potential limitation for the further improvement of recombinant protein yields.

[Effects of naloxone on the expression of stem cell factor and C-kit receptor in combined oxygen-glucose deprivation of primary cultured human embryonic neuron in vitro].

PubMed

Zhu, Bo; Li, Lan-ying; Lü, Guo-yi; Xue, Yu-liang; Ye, Tie-hu

2010-04-01

To explore the effects of naloxone on the expression of c-kit receptor (c-kit R) and its ligand stem cell factor (SCF) in human embryo neuronal hypoxic injury. Serum-free cerebral cortical cultures prepared from embryonic human brains were deprived of both oxygen and glucose which would set up an environment more likely with that of in vivo ischemic injury. Neurons in 24-well culture plates were randomly divided into four groups: control group, hypoxia group, naloxone 0.5 microg/ml group and naloxone 10 microg/ml group. MTT assay and biological analysis were performed to study the cell death and the changes of extracellular concentrations of lactate dehydrogenase (LDH) after combined oxygen-glucose deprivation. Neurons in 25 ml culture flasks were also randomly allocated into four groups as previously described. Intracellular total RNA were extracted at different time points: pre-hypoxia, immediately after hypoxia, and 3, 6, 12, and 24 hours after reoxygenation. The changes of SCF/c-kit R mRNA expression in hypoxic neurons treated with different concentrations of naloxone pre and post oxygen-glucose deprivation were determined with RT-PCR. The cell vitality detected by MTT assay decreased significantly in hypoxia group and naloxone 0.5 microg/ml group when compared with control group (P<0.01), while no significant difference was found between naloxone 0.5 microg/ml group and hypoxia group or between naloxone 10 microg/ml group and control group. Extracellular concentration of LDH significantly increased in hypoxia group (P<0.05), while no difference was found between naloxone 0.5 microg/ml group and control group, between naloxone 0.5 microg/ml and hypoxia group, or between naloxone 10 microg/ml and control group (all P>0.05). Immediately after oxygen-glucose deprivation, the expression of SCF/c-kit R mRNA increased significantly (P<0.01). Among those the expression of SCF presented a distribution of double-peak value within 24 hours. After treated with different

Gata4-Dependent Differentiation of c-Kit+ Derived Endothelial Cells Underlies Artefactual Cardiomyocyte Regeneration in the Heart.

PubMed

Maliken, Bryan D; Kanisicak, Onur; Karch, Jason; Khalil, Hadi; Fu, Xing; Boyer, Justin G; Prasad, Vikram; Zheng, Yi; Molkentin, Jeffery D

2018-04-17

Background -While c-Kit + adult progenitor cells were initially reported to produce new cardiomyocytes in the heart, recent genetic evidence suggests that such events are exceedingly rare. However, to determine if these rare events represent true de novo cardiomyocyte formation we deleted the necessary cardiogenic transcription factors Gata4 and Gata6 from c-Kit-expressing cardiac progenitor cells (CPCs). Methods - Kit allele-dependent lineage tracing and fusion analysis was performed in mice following simultaneous Gata4 and Gata6 cell-type specific deletion to examine rates of putative de novo cardiomyocyte formation from c-Kit + cells. Bone marrow transplantation experiments were used to define the contribution of Kit allele-derived hematopoietic cells versus Kit lineage-dependent cells endogenous to the heart in contributing to apparent de novo lineage-traced cardiomyocytes. A Tie2 CreERT2 transgene was also used to examine the global impact of Gata4 deletion on the mature cardiac endothelial cell network, which was further evaluated with select angiogenesis assays. Results -Deletion of Gata4 in Kit lineage-derived endothelial cells or in total endothelial cells using the Tie2 CreERT2 transgene, but not from bone morrow cells, resulted in profound endothelial cell expansion, defective endothelial cell differentiation, leukocyte infiltration into the heart and a dramatic increase in Kit allele-dependent lineage-traced cardiomyocytes. However, this increase in labeled cardiomyocytes was an artefact of greater leukocyte-cardiomyocyte cellular fusion due to defective endothelial cell differentiation in the absence of Gata4 Conclusions -Past identification of presumed de novo cardiomyocyte formation in the heart from c-Kit + cells using Kit allele lineage tracing appears to be an artefact of labeled leukocyte fusion with cardiomyocytes. Deletion of Gata4 from c-Kit + endothelial progenitor cells or adult endothelial cells negatively impacted angiogenesis and

Protein Tyrosine Phosphatase PRL2 Mediates Notch and Kit Signals in Early T Cell Progenitors.

PubMed

Kobayashi, Michihiro; Nabinger, Sarah C; Bai, Yunpeng; Yoshimoto, Momoko; Gao, Rui; Chen, Sisi; Yao, Chonghua; Dong, Yuanshu; Zhang, Lujuan; Rodriguez, Sonia; Yashiro-Ohtani, Yumi; Pear, Warren S; Carlesso, Nadia; Yoder, Mervin C; Kapur, Reuben; Kaplan, Mark H; Daniel Lacorazza, Hugo; Zhang, Zhong-Yin; Liu, Yan

2017-04-01

The molecular pathways regulating lymphoid priming, fate, and development of multipotent bone marrow hematopoietic stem and progenitor cells (HSPCs) that continuously feed thymic progenitors remain largely unknown. While Notch signal is indispensable for T cell specification and differentiation, the downstream effectors are not well understood. PRL2, a protein tyrosine phosphatase that regulates hematopoietic stem cell proliferation and self-renewal, is highly expressed in murine thymocyte progenitors. Here we demonstrate that protein tyrosine phosphatase PRL2 and receptor tyrosine kinase c-Kit are critical downstream targets and effectors of the canonical Notch/RBPJ pathway in early T cell progenitors. While PRL2 deficiency resulted in moderate defects of thymopoiesis in the steady state, de novo generation of T cells from Prl2 null hematopoietic stem cells was significantly reduced following transplantation. Prl2 null HSPCs also showed impaired T cell differentiation in vitro. We found that Notch/RBPJ signaling upregulated PRL2 as well as c-Kit expression in T cell progenitors. Further, PRL2 sustains Notch-mediated c-Kit expression and enhances stem cell factor/c-Kit signaling in T cell progenitors, promoting effective DN1-DN2 transition. Thus, we have identified a critical role for PRL2 phosphatase in mediating Notch and c-Kit signals in early T cell progenitors. Stem Cells 2017;35:1053-1064. © 2016 AlphaMed Press.

Phosphorylation of TNF-alpha converting enzyme by gastrin-releasing peptide induces amphiregulin release and EGF receptor activation.

PubMed

Zhang, Qing; Thomas, Sufi M; Lui, Vivian Wai Yan; Xi, Sichuan; Siegfried, Jill M; Fan, Huizhou; Smithgall, Thomas E; Mills, Gordon B; Grandis, Jennifer Rubin

2006-05-02

G protein-coupled receptors induce EGF receptor (EGFR) signaling, leading to the proliferation and invasion of cancer cells. Elucidation of the mechanism of EGFR activation by G protein-coupled receptors may identify new signaling paradigms. A gastrin-releasing peptide (GRP)/GRP receptor-mediated autocrine pathway was previously described in squamous cell carcinoma of head and neck. In the present study, we demonstrate that TNF-alpha converting enzyme (TACE), a disintegrin and metalloproteinse-17, undergoes a Src-dependent phosphorylation that regulates release of the EGFR ligand amphiregulin upon GRP treatment. Further investigation reveals the phosphatidylinositol 3-kinase (PI3-K) as the intermediate of c-Src and TACE, contributing to their association and TACE phosphorylation. Phosphoinositide-dependent kinase 1 (PDK1), a downstream target of PI3-K, has been identified as the previously undescribed kinase to directly phosphorylate TACE upon GRP treatment. These findings suggest a signaling cascade of GRP-Src-PI3-K-PDK1-TACE-amphiregulin-EGFR with multiple points of interaction, translocation, and phosphorylation. Furthermore, knockdown of PDK1 augmented the antitumor effects of the EGFR inhibitor erlotinib, indicating PDK1 as a therapeutic target to improve the clinical response to EGFR inhibitors.

Transplantation of Epigenetically Modified Adult Cardiac c-Kit+ Cells Retards Remodeling and Improves Cardiac Function in Ischemic Heart Failure Model

PubMed Central

Zakharova, Liudmila; Nural-Guvener, Hikmet; Feehery, Lorraine; Popovic-Sljukic, Snjezana

2015-01-01

Cardiac c-Kit+ cells have a modest cardiogenic potential that could limit their efficacy in heart disease treatment. The present study was designed to augment the cardiogenic potential of cardiac c-Kit+ cells through class I histone deacetylase (HDAC) inhibition and evaluate their therapeutic potency in the chronic heart failure (CHF) animal model. Myocardial infarction (MI) was created by coronary artery occlusion in rats. c-Kit+ cells were treated with mocetinostat (MOCE), a specific class I HDAC inhibitor. At 3 weeks after MI, CHF animals were retrogradely infused with untreated (control) or MOCE-treated c-Kit+ cells (MOCE/c-Kit+ cells) and evaluated at 3 weeks after cell infusion. We found that class I HDAC inhibition in c-Kit+ cells elevated the level of acetylated histone H3 (AcH3) and increased AcH3 levels in the promoter regions of pluripotent and cardiac-specific genes. Epigenetic changes were accompanied by increased expression of cardiac-specific markers. Transplantation of CHF rats with either control or MOCE/c-Kit+ cells resulted in an improvement in cardiac function, retardation of CHF remodeling made evident by increased vascularization and scar size, and cardiomyocyte hypertrophy reduction. Compared with CHF infused with control cells, infusion of MOCE/c-Kit+ cells resulted in a further reduction in left ventricle end-diastolic pressure and total collagen and an increase in interleukin-6 expression. The low engraftment of infused cells suggests that paracrine effects might account for the beneficial effects of c-Kit+ cells in CHF. In conclusion, selective inhibition of class I HDACs induced expression of cardiac markers in c-Kit+ cells and partially augmented the efficacy of these cells for CHF repair. Significance The study has shown that selective class 1 histone deacetylase inhibition is sufficient to redirect c-Kit+ cells toward a cardiac fate. Epigenetically modified c-Kit+ cells improved contractile function and retarded remodeling of the

Sphingosine-1-phosphate mediates epidermal growth factor-induced muscle satellite cell activation

DOE Office of Scientific and Technical Information (OSTI.GOV)

Nagata, Yosuke, E-mail: cynagata@mail.ecc.u-tokyo.ac.jp; Ohashi, Kazuya; Wada, Eiji

2014-08-01

Skeletal muscle can regenerate repeatedly due to the presence of resident stem cells, called satellite cells. Because satellite cells are usually quiescent, they must be activated before participating in muscle regeneration in response to stimuli such as injury, overloading, and stretch. Although satellite cell activation is a crucial step in muscle regeneration, little is known of the molecular mechanisms controlling this process. Recent work showed that the bioactive lipid sphingosine-1-phosphate (S1P) plays crucial roles in the activation, proliferation, and differentiation of muscle satellite cells. We investigated the role of growth factors in S1P-mediated satellite cell activation. We found that epidermalmore » growth factor (EGF) in combination with insulin induced proliferation of quiescent undifferentiated mouse myoblast C2C12 cells, which are also known as reserve cells, in serum-free conditions. Sphingosine kinase activity increased when reserve cells were stimulated with EGF. Treatment of reserve cells with the D-erythro-N,N-dimethylsphingosine, Sphingosine Kinase Inhibitor, or siRNA duplexes specific for sphingosine kinase 1, suppressed EGF-induced C2C12 activation. We also present the evidence showing the S1P receptor S1P2 is involved in EGF-induced reserve cell activation. Moreover, we demonstrated a combination of insulin and EGF promoted activation of satellite cells on single myofibers in a manner dependent on SPHK and S1P2. Taken together, our observations show that EGF-induced satellite cell activation is mediated by S1P and its receptor. - Highlights: • EGF in combination with insulin induces proliferation of quiescent C2C12 cells. • Sphingosine kinase activity increases when reserve cells are stimulated with EGF. • EGF-induced activation of reserve cells is dependent on sphingosine kinase and ERK. • The S1P receptor S1P2 is involved in EGF-induced reserve cell activation. • EGF-induced reserve cell activation is mediated by S1P and

Detection of single nucleotide polymorphisms (SNP) in equine coat color genes using SNaPshotTM multiplex kit or pluronic F-108 tri-block copolymer and capillary electrophoresis.

PubMed

Martin, Lauren; Damaso, Natalie; Mills, DeEtta

2016-10-01

Molecular methods for the detection of mammalian coat color phenotypes have expanded greatly within the past decade. Many phenotypes are associated with a single nucleotide polymorphism mutation in the genetic sequence. Traditionally, these mutations are detected through sequencing, hybridization assays or mini-sequencing. However, these techniques can be expensive and tedious. Previously, CE-SSCP using the F-108 polymer was able to distinguish SNPs for the melanocortin-1 receptor (mc1r) coat color gene in horses (Equus caballus) that differed by one nucleotide substitution. The objective of this study was to expand the detection of coat color SNPs in horses. The genes for the solute carrier family member 2 (slc45a2/matp), type III receptor protein-tyrosine kinase (kit) and mc1r genes using CE-SSCP and F-108 polymer were compared to mini-sequencing with the SNaPshot TM kit. The F-108 polymer reproducibly resolved homozygous and heterozygous individuals for the mc1r and kit markers, but was unable to resolve heterozygous individuals for slc45a2 at 38ºC. The need for temperatures <15ºC, the SNP position being close to the 5'-end, and conformational structures/free energy with similar values resulted in the inability to resolve the secondary structures. Despite this limitation, the CE-SSCP method could be used to provide a rapid phenotypic description for equine forensic investigations. © 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Cigarette Smoke Activates the Proto-Oncogene c-Src to Promote Airway Inflammation and Lung Tissue Destruction

PubMed Central

Geraghty, Patrick; Hardigan, Andrew

2014-01-01

The diagnosis of chronic obstructive pulmonary disease (COPD) confers a 2-fold increased lung cancer risk even after adjusting for cigarette smoking, suggesting that common pathways are operative in both diseases. Although the role of the tyrosine kinase c-Src is established in lung cancer, less is known about its impact in other lung diseases, such as COPD. This study examined whether c-Src activation by cigarette smoke contributes to the pathogenesis of COPD. Cigarette smoke increased c-Src activity in human small airway epithelial (SAE) cells from healthy donors and in the lungs of exposed mice. Similarly, higher c-Src activation was measured in SAE cells from patients with COPD compared with healthy control subjects. In SAE cells, c-Src silencing or chemical inhibition prevented epidermal growth factor (EGF) receptor signaling in response to cigarette smoke but not EGF stimulation. Further studies showed that cigarette smoke acted through protein kinase C α to trigger c-Src to phosphorylate EGF receptor and thereby to induce mitogen-activated protein kinase responses in these cells. To further investigate the role of c-Src, A/J mice were orally administered the specific Src inhibitor AZD-0530 while they were exposed to cigarette smoke for 2 months. AZD-0530 treatment blocked c-Src activation, decreased macrophage influx, and prevented airspace enlargement in the lungs of cigarette smoke–exposed mice. Moreover, inhibiting Src deterred the cigarette smoke–mediated induction of matrix metalloproteinase-9 and -12 in alveolar macrophages and lung expression of cathepsin K, IL-17, TNF-α, MCP-1, and KC, all key factors in the pathogenesis of COPD. These results indicate that activation of the proto-oncogene c-Src by cigarette smoke promotes processes linked to the development of COPD. PMID:24111605

Vasopressin up-regulates the expression of growth-related immediate-early genes via two distinct EGF receptor transactivation pathways

PubMed Central

Fuentes, Lida Q.; Reyes, Carlos E.; Sarmiento, José M.; Villanueva, Carolina I.; Figueroa, Carlos D.; Navarro, Javier; González, Carlos B.

2008-01-01

Activation of V1a receptor triggers the expression of growth-related immediate-early genes (IEGs), including c-Fos and Egr-1. Here we found that pre-treatment of rat vascular smooth muscle A-10 cell line with the EGF receptor inhibitor AG1478 or the over-expression of an EGFR dominant negative mutant (HEBCD533) blocked the vasopressin-induced expression of IEGs, suggesting that activation of these early genes mediated by V1a receptor is via transactivation of the EGF receptor. Importantly, the inhibition of the metalloproteinases, which catalyzed the shedding of the EGF receptor agonist HB-EGF, selectively blocked the vasopressin-induced expression c-Fos. On the other hand, the inhibition of c-Src selectively blocked the vasopressin-induced expression of Egr-1. Interestingly, in contrast to the expression of c-Fos, the expression of Egr-1 was mediated via the Ras/MEK/MAPK-dependent signalling pathway. Vasopressin-triggered expression of both genes required the release of intracellular calcium, activation of PKC and β-arrestin 2. These findings demonstrated that vasopressin up-regulated the expression of c-Fos and Erg-1 via transactivation of two distinct EGF receptor-dependent signalling pathways. PMID:18571897

Opposing roles of KIT and ABL1 in the therapeutic response of gastrointestinal stromal tumor (GIST) cells to imatinib mesylate.

PubMed

Rausch, Jessica L; Boichuk, Sergei; Ali, Areej A; Patil, Sneha S; Liu, Lijun; Lee, Donna M; Brown, Matthew F; Makielski, Kathleen R; Liu, Ying; Taguchi, Takahiro; Kuan, Shih-Fan; Duensing, Anette

2017-01-17

Most gastrointestinal stromal tumors (GISTs) are caused by activating mutations of the KIT receptor tyrosine kinase. The small molecule inhibitor imatinib mesylate was initially developed to target the ABL1 kinase, which is constitutively activated through chromosomal translocation in BCR-ABL1-positive chronic myeloid leukemia. Because of cross-reactivity of imatinib against the KIT kinase, the drug is also successfully used for the treatment of GIST. Although inhibition of KIT clearly has a major role in the therapeutic response of GIST to imatinib, the contribution of concomitant inhibition of ABL in this context has never been explored. We show here that ABL1 is expressed in the majority of GISTs, including human GIST cell lines. Using siRNA-mediated knockdown, we demonstrate that depletion of KIT in conjunction with ABL1 - hence mimicking imatinib treatment - leads to reduced apoptosis induction and attenuated inhibition of cellular proliferation when compared to depletion of KIT alone. These results are explained by an increased activity of the AKT survival kinase, which is mediated by the cyclin-dependent kinase CDK2, likely through direct phosphorylation. Our results highlight that distinct inhibitory properties of targeted agents can impede antitumor effects and hence provide insights for rational drug development. Novel KIT-targeted agents to treat GIST should therefore comprise an increased specificity for KIT while at the same time displaying a reduced ability to inhibit ABL1.

EGF Functionalized Polymer-Coated Gold Nanoparticles Promote EGF Photostability and EGFR Internalization for Photothermal Therapy

PubMed Central

Silva, Catarina Oliveira; Petersen, Steffen B.; Reis, Catarina Pinto; Rijo, Patrícia; Molpeceres, Jesús; Fernandes, Ana Sofia; Gonçalves, Odete; Gomes, Andreia C.; Correia, Isabel; Vorum, Henrik; Neves-Petersen, Maria Teresa

2016-01-01

The application of functionalized nanocarriers on photothermal therapy for cancer ablation has wide interest. The success of this application depends on the therapeutic efficiency and biocompatibility of the system, but also on the stability and biorecognition of the conjugated protein. This study aims at investigating the hypothesis that EGF functionalized polymer-coated gold nanoparticles promote EGF photostability and EGFR internalization, making these conjugated particles suitable for photothermal therapy. The conjugated gold nanoparticles (100–200 nm) showed a plasmon absorption band located within the near-infrared range (650–900 nm), optimal for photothermal therapy applications. The effects of temperature, of polymer-coated gold nanoparticles and of UVB light (295nm) on the fluorescence properties of EGF have been investigated with steady-state and time-resolved fluorescence spectroscopy. The fluorescence properties of EGF, including the formation of Trp and Tyr photoproducts, is modulated by temperature and by the intensity of the excitation light. The presence of polymeric-coated gold nanoparticles reduced or even avoided the formation of Trp and Tyr photoproducts when EGF is exposed to UVB light, protecting this way the structure and function of EGF. Cytotoxicity studies of conjugated nanoparticles carried out in normal-like human keratinocytes showed small, concentration dependent decreases in cell viability (0–25%). Moreover, conjugated nanoparticles could activate and induce the internalization of overexpressed Epidermal Growth Factor Receptor in human lung carcinoma cells. In conclusion, the gold nanoparticles conjugated with Epidermal Growth Factor and coated with biopolymers developed in this work, show a potential application for near infrared photothermal therapy, which may efficiently destroy solid tumours, reducing the damage of the healthy tissue. PMID:27788212

BI-D1870 is a specific inhibitor of the p90 RSK (ribosomal S6 kinase) isoforms in vitro and in vivo

PubMed Central

Sapkota, Gopal P.; Cummings, Lorna; Newell, Felicity S.; Armstrong, Christopher; Bain, Jennifer; Frodin, Morten; Grauert, Matthias; Hoffmann, Matthias; Schnapp, Gisela; Steegmaier, Martin; Cohen, Philip; Alessi, Dario R.

2006-01-01

Hormones and growth factors induce the activation of a number of protein kinases that belong to the AGC subfamily, including isoforms of PKA, protein kinase B (also known as Akt), PKC, S6K p70 (ribosomal S6 kinase), RSK (p90 ribosomal S6 kinase) and MSK (mitogen- and stress-activated protein kinase), which then mediate many of the physiological processes that are regulated by these extracellular agonists. It can be difficult to assess the individual functions of each AGC kinase because their substrate specificities are similar. Here we describe the small molecule BI-D1870, which inhibits RSK1, RSK2, RSK3 and RSK4 in vitro with an IC50 of 10–30 nM, but does not signi-ficantly inhibit ten other AGC kinase members and over 40 other protein kinases tested at 100-fold higher concentrations. BI-D1870 is cell permeant and prevents the RSK-mediated phorbol ester- and EGF (epidermal growth factor)-induced phosphoryl-ation of glycogen synthase kinase-3β and LKB1 in human embry-onic kidney 293 cells and Rat-2 cells. In contrast, BI-D1870 does not affect the agonist-triggered phosphorylation of substrates for six other AGC kinases. Moreover, BI-D1870 does not suppress the phorbol ester- or EGF-induced phosphorylation of CREB (cAMP-response-element-binding protein), consistent with the genetic evidence indicating that MSK, and not RSK, isoforms mediate the mitogen-induced phosphorylation of this transcription factor. PMID:17040210

Control of Human Endometrial Stromal Cell Motility by PDGF-BB, HB-EGF and Trophoblast-Secreted Factors

PubMed Central

Schwenke, Maren; Knöfler, Martin; Velicky, Philipp; Weimar, Charlotte H. E.; Kruse, Michelle; Samalecos, Annemarie; Wolf, Anja; Macklon, Nick S.; Bamberger, Ana-Maria; Gellersen, Birgit

2013-01-01

Human implantation involves extensive tissue remodeling at the fetal-maternal interface. It is becoming increasingly evident that not only trophoblast, but also decidualizing endometrial stromal cells are inherently motile and invasive, and likely contribute to the highly dynamic processes at the implantation site. The present study was undertaken to further characterize the mechanisms involved in the regulation of endometrial stromal cell motility and to identify trophoblast-derived factors that modulate migration. Among local growth factors known to be present at the time of implantation, heparin-binding epidermal growth factor-like growth factor (HB-EGF) triggered chemotaxis (directed locomotion), whereas platelet-derived growth factor (PDGF)-BB elicited both chemotaxis and chemokinesis (non-directed locomotion) of endometrial stromal cells. Supernatants of the trophoblast cell line AC-1M88 and of first trimester villous explant cultures stimulated chemotaxis but not chemokinesis. Proteome profiling for cytokines and angiogenesis factors revealed neither PDGF-BB nor HB-EGF in conditioned media from trophoblast cells or villous explants, while placental growth factor, vascular endothelial growth factor and PDGF-AA were identified as prominent secretory products. Among these, only PDGF-AA triggered endometrial stromal cell chemotaxis. Neutralization of PDGF-AA in trophoblast conditioned media, however, did not diminish chemoattractant activity, suggesting the presence of additional trophoblast-derived chemotactic factors. Pathway inhibitor studies revealed ERK1/2, PI3 kinase/Akt and p38 signaling as relevant for chemotactic motility, whereas chemokinesis depended primarily on PI3 kinase/Akt activation. Both chemotaxis and chemokinesis were stimulated upon inhibition of Rho-associated, coiled-coil containing protein kinase. The chemotactic response to trophoblast secretions was not blunted by inhibition of isolated signaling cascades, indicating activation of

gC1q-R/p32, a C1q-binding protein, is a receptor for the InlB invasion protein of Listeria monocytogenes.

PubMed

Braun, L; Ghebrehiwet, B; Cossart, P

2000-04-03

InlB is a Listeria monocytogenes protein that promotes entry of the bacterium into mammalian cells by stimulating tyrosine phosphorylation of the adaptor proteins Gab1, Cbl and Shc, and activation of phosphatidyl- inositol (PI) 3-kinase. Using affinity chromatography and enzyme-linked immunosorbent assay, we demonstrate a direct interaction between InlB and the mammalian protein gC1q-R, the receptor of the globular part of the complement component C1q. Soluble C1q or anti-gC1q-R antibodies impair InlB-mediated entry. Transient transfection of GPC16 cells, which are non-permissive to InlB-mediated entry, with a plasmid-expressing human gC1q-R promotes entry of InlB-coated beads. Furthermore, several experiments indicate that membrane recruitment and activation of PI 3-kinase involve an InlB-gC1q-R interaction and that gC1q-R associates with Gab1 upon stimulation of Vero cells with InlB. Thus, gC1q-R constitutes a cellular receptor involved in InlB-mediated activation of PI 3-kinase and tyrosine phosphorylation of the adaptor protein Gab1. After E-cadherin, the receptor for internalin, gC1q-R is the second identified mammalian receptor promoting entry of L. monocytogenes into mammalian cells.

Cetuximab in combination with anti-human IgG antibodies efficiently down-regulates the EGF receptor by macropinocytosis

DOE Office of Scientific and Technical Information (OSTI.GOV)

Berger, Christian; Madshus, Inger Helene; Department of Pathology, Oslo University Hospital, Rikshospitalet, Post box 4950 Nydalen, 0424 Oslo

The monoclonal antibody C225 (Cetuximab) blocks binding of ligand to the epidermal growth factor receptor (EGFR). In addition, it is known that incubation with C225 induces endocytosis of the EGFR. This endocytosis has previously been shown to be increased when C225 is combined with an additional monoclonal anti-EGFR antibody. However, the effects of antibody combinations on EGFR activation, endocytosis, trafficking and degradation have been unclear. By binding a secondary antibody to the C225-EGFR complex, we here demonstrate that a combination of antibodies can efficiently internalize and degrade the EGFR. Although the combination of antibodies activated the EGFR kinase and inducedmore » ubiquitination of the EGFR, the kinase activity was not required for internalization of the EGFR. In contrast to EGF-induced EGFR down-regulation, the antibody combination efficiently degraded the EGFR without initiating downstream proliferative signaling. The antibody-induced internalization of EGFR was found not to depend on clathrin and/or dynamin, but depended on actin polymerization, suggesting induction of macropinocytosis. Macropinocytosis may cause internalization of large membrane areas, and this could explain the highly efficient internalization of the EGFR induced by combination of antibodies. -- Highlight: Black-Right-Pointing-Pointer Cetuximab induced endocytosis of EGFR increases upon combination with anti-human IgG. Black-Right-Pointing-Pointer Antibody combination causes internalization of EGFR by macropinocytosis. Black-Right-Pointing-Pointer Antibody-induced internalization of EGFR is independent of EGFR kinase activity. Black-Right-Pointing-Pointer Antibody combination may have a zipper effect and cross-link EGFRs on neighboring cells.« less

C-kit-positive gastric metastasis of lobular carcinoma of the breast masquerading as gastrointestinal stromal tumor.

PubMed

Vennapusa, Bharathi; Oman, Sarah A; Parasher, Gulshan; Cerilli, Lisa A

2010-10-01

A 61-year-old woman with no significant past history underwent gastric biopsies demonstrating a strongly c-kit-positive epithelioid malignancy, initially thought to represent gastrointestinal stromal tumor (GIST). Subsequent clinical and immunohistochemical evaluation proved the neoplasm to represent metastatic lobular carcinoma. This case illustrates that although c-kit is highly specific and sensitive for GIST, its expression may occur in a variety of other neoplasms, some of which morphologically resemble GIST and may present in the gastrointestinal tract as metastases. Therefore, a review of other c-kit-positive lesions is also highlighted.

Calcium Channels, Rho-Kinase, Protein Kinase-C, and Phospholipase-C Pathways Mediate Mercury Chloride-Induced Myometrial Contractions in Rats.

PubMed

Koli, Swati; Prakash, Atul; Choudhury, Soumen; Mandil, Rajesh; Garg, Satish K

2018-05-21

-induced myometrial contraction in rats. M receptor: Muscarinic receptor; PIP2: phospho-inositol bisphosphate; PLC: phospholipase-C; DAG: diacyl glycerol; IP3: inositol triphosphate; IP3R: inositol triphosphate receptor; PKC; protein kinase-C; MLCP: myosin light chain phosphatise; MYPT: myosin phosphatase; SR: sarco-endoplasmic reticulum.

Tas13D inhibits growth of SMMC-7721 cell via suppression VEGF and EGF expression.

PubMed

He, Huai-Zhen; Wang, Nan; Zhang, Jie; Zheng, Lei; Zhang, Yan-Min

2012-01-01

Taspine, isolated from Radix et Rhizoma Leonticis has demosntrated potential proctiective effects against cancer. Tas13D, a novel taspine derivative synthetized by structure-based drug design, have been shown to possess interesting biological and pharmacological activities. The current study was designed to evaluate its antiproliferative activity and underlying mechanisms. Antiproliferative activity of tas13D was evaluated by xenograft in athymic mice in vivo, and by 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) and cell migration assays with human liver cancer (SMMC-7721) cell lines in vitro. Docking between tas13D and VEGFR and EGFR was studied by with a Sybyl/Surflex module. VEGF and EGF and their receptor expression was determined by ELISA and real-time PCR methods, respectively. Our present study showed that tas13D inhibited SMMC-7721 xenograft tumor growth, bound tightly with the active site of kinase domains of EGFR and VEGFR, and reduced SMMC-7721 cell proliferation (IC=34.7 μmol/L) and migration compared to negative controls. VEGF and EGF mRNAs were significantly reduced by tas13D treatment in a dose-dependent manner, along with VEGF and EGF production. The obtained results suggest that tas13D inhibits tumor growth and cell proliferation by inhibiting cell migration, downregulating mRNA expression of VEGF and EGF, and decreasing angiogenic factor production. Tas13D deserves further consideration as a chemotherapeutic agent.

AML1/ETO trans-activates c-KIT expression through the long range interaction between promoter and intronic enhancer.

PubMed

Tian, Ying; Wang, Genjie; Hu, Qingzhu; Xiao, Xichun; Chen, Shuxia

2018-04-01

The AML1/ETO onco-fusion protein is crucial for the genesis of t(8;21) acute myeloid leukemia (AML) and is well documented as a transcriptional repressor through dominant-negative effect. However, little is known about the transactivation mechanism of AML1/ETO. Through large cohort of patient's expression level data analysis and a series of experimental validation, we report here that AML1/ETO transactivates c-KIT expression through directly binding to and mediating the long-range interaction between the promoter and intronic enhancer regions of c-KIT. Gene expression analyses verify that c-KIT expression is significantly high in t(8;21) AML. Further ChIP-seq analysis and motif scanning identify two regulatory regions located in the promoter and intronic enhancer region of c-KIT, respectively. Both regions are enriched by co-factors of AML1/ETO, such as AML1, CEBPe, c-Jun, and c-Fos. Further luciferase reporter assays show that AML1/ETO trans-activates c-KIT promoter activity through directly recognizing the AML1 motif and the co-existence of co-factors. The induction of c-KIT promoter activity is reinforced with the existence of intronic enhancer region. Furthermore, ChIP-3C-qPCR assays verify that AML1/ETO mediates the formation of DNA-looping between the c-KIT promoter and intronic enhancer region through the long-range interaction. Collectively, our data uncover a novel transcriptional activity mechanism of AML1/ETO and enrich our knowledge of the onco-fusion protein mediated transcription regulation. © 2017 Wiley Periodicals, Inc.

A cGMP kinase mutant with increased sensitivity to the protein kinase inhibitor peptide PKI(5-24).

PubMed

Ruth, P; Kamm, S; Nau, U; Pfeifer, A; Hofmann, F

1996-01-01

Synthetic peptides corresponding to the active domain of the heat-stable inhibitor protein PKI are very potent inhibitors of cAMP-dependent protein kinase, but are extremely weak inhibitors of cGMP-dependent protein kinase. In this study, we tried to confer PKI sensitivity to cGMP kinase by site-directed mutagenesis. The molecular requirements for high affinity inhibition by PKI were deduced from the crystal structure of the cAMP kinase/PKI complex. A prominent site of interaction are residues Tyr235 and Phe239 in the catalytic subunit, which from a sandwich-like structure with Phe10 of the PKI(5-24) peptide. To increase the sensitivity for PKI, the cGMP kinase codons at the corresponding sites, Ser555 and Ser559, were changed to Tyr and Phe. The mutant cGMP kinase was stimulated half maximally by cGMP at 3-fold higher concentrations (240 nM) than the wild type (77 nM). Wild type and mutant cGMP kinase did not differ significantly in their Km and Vmax for three different substrate peptides. The PKI(5-24) peptide inhibited phosphotransferase activity of the mutant cGMP kinase with higher potency than that of wild type, with Ki values of 42 +/- .3 microM and 160 +/- .7 microM, respectively. The increased affinity of the mutant cGMP kinase was specific for the PKI(5-24) peptide. Mutation of the essential Phe10 in the PKI(5-24) sequence to an Ala yielded a peptide that inhibited mutant and wild type cGMP kinase with similar potency, with Ki values of 160 +/- 11 and 169 +/- 27 microM, respectively. These results suggest that the mutations Ser555Tyr and Ser559Phe are required, but not sufficient, for high affinity inhibition of cGMP kinase by PKI.

Convection enhanced delivery of boronated EGF as a molecular targeting agent for neutron capture therapy of brain tumors

PubMed Central

Yang, Weilian; Wu, Gong; Huo, Tianyao; Tjarks, Werner; Ciesielski, Michael; Fenstermaker, Robert A.; Ross, Brain D.; Wikstrand, Carol J.; Riley, Kent J.; Binns, Peter J.

2010-01-01

In the present study, we have evaluated a boronated dendrimer-epidermal growth factor (BD-EGF) bioconjugate as a molecular targeting agent for boron neutron capture therapy (BNCT) of the human EGFR gene-transfected F98 rat glioma, designated F98EGFR. EGF was chemically linked to a heavily boronated polyamidoamine dendrimer (BD) by means of the heterobifunctional reagent, mMBS. Biodistribution studies were carried out at 6 h and 24 h following intratumoral (i.t.) injection or intracerebral (i.c.) convection enhanced delivery (CED) of 125I-labeled or unlabeled BD-EGF (40 μg 10B/10 μg EGF) to F98 glioma bearing rats. At 24 h. there was 43% more radioactivity in EGFR(+) tumors following CED compared to i.t. injection, and a doubling of the tumor boron concentration (22.3 μg/g vs. 11.7 μg/g). CED of BD-EGF resulted in a 7.2× increase in the volume of distribution within the infused cerebral hemisphere and a 1.9× increase in tumor uptake of BD-EGF compared with i.t. injection. Based on these favorable bio-distribution data, BNCT was carried out at the Massachusetts Institute of Technology nuclear reactor 14 days following i.c. tumor implantation and 24 h. after CED of BD-EGF. These animals had a MST of 54.1 ± 4.7 days compared to 43.0 ± 2.8 days following i.t. injection. Rats that received BD-EGF by CED in combination with i.v. boronophenylalanine (BPA), which has been used in both experimental and clinical studies, had a MST of 86.0 ± 28.1 days compared to 39.8 ± 1.6 days for i.v. BPA alone (P < 0.01), 30.9 ± 1.4 days for irradiated controls and 25.1 ± 1.0 days for untreated controls (overall P < 0.0001). These data have demonstrated that the efficacy of BNCT was significantly increased (P < 0.006), following i.c CED of BD-EGF compared to i.t injection, and that the survival data were equivalent to those previously reported by us using the boronated anti-human-EGF mAb, C225 (cetuximab). PMID:19588228

Expression of c-kit receptor in human cholangiocarcinoma and in vivo treatment with imatinib mesilate in chimeric mice

PubMed Central

Kamenz, Thomas; Caca, Karel; Blüthner, Thilo; Tannapfel, Andrea; Mössner, Joachim; Wiedmann, Marcus

2006-01-01

AIM: To investigate the c-kit expression in biliary tract cancer cell lines and histological sections from patients with extrahepatic cholangiocarcinoma (CC) and to evaluate the efficacy of in vitro and in vitro treatment with imatinib mesilate. METHODS: The protein expression of c-kit in the human biliary tract cancer cell lines Mz-ChA-2 and EGI-1 and histological sections from 19 patients with extrahepatic CC was assessed by immunoblotting, immunocytochemistry, and immunohistochemistry. The anti-proliferative effect of imatinib mesilate on biliary tract cancer cell lines Mz-ChA-2 and EGI-1 was studied in vitro by automated cell counting. In addition, immunodeficient NMRI mice (TaconicTM) were subcutaneously injected with 5 x 106 cells of cell lines MzChA-2 and EGI-1. After having reached a tumour volume of 200 mm3, daily treatment was started intraperitoneally with imatinib mesilate at a dose of 50 mg/kg or normal saline (NS). Tumor volume was calculated with a Vernier caliper. After 14 d, mice were sacrificed with tumors excised and tumor mass determined. RESULTS: Immunoblotting revealed presence of c-kit in Mz-ChA-2 and absence in EGI-1 cells. Immunocytochemistry with c-kit antibodies displayed a cytoplasmatic and membraneous localization of receptor protein in Mz-ChA-2 cells and absence of c-kit in EGI-1 cells. c-kit was expressed in 7 of 19 (37%) extrahepatic human CC tissue samples, 2 showed a moderate and 5 a rather weak immunostaining. Imatinib mesilate at a low concentration of 5 µmol/L caused a significant growth inhibition in the c-kit positive cell line Mz-ChA-2 (31%), but not in the c-kit negative cell line EGI-1 (0%) (P < 0.05). Imatinib mesilate at an intermediate concentration of 10 µmol/L inhibited cellular growth of both cell lines (51% vs 57%). Imatinib mesilate at a higher concentration of 20 µmol/L seemed to have a general toxic effect on both cell lines. The IC50 values were 9.7 µmol/L and 11 µmol/L, respectively. After 14 d of in vitro

Resveratrol Improved the Progression of Chronic Prostatitis via the Downregulation of c-kit/SCF by Activating Sirt1.

PubMed

He, Yi; Zeng, Huizhi; Yu, Yang; Zhang, Jiashu; Zeng, Xiaona; Gong, Fengtao; Duan, Xingping; Liu, Qi; Yang, Bo

2017-07-19

The regulation mechanism of inflammation inducing prostate carcinogenesis remains largely unknown. Therefore, we investigated the role of the c-kit/SCF pathway, which has been associated with the control of prostate carcinogenesis, in chronic prostatitis (CP) rats and evaluated the anti-prostatitis effect of resveratrol. We performed hemolysin and eosin staining to evaluate the histopathological changes in prostates. Multiple approaches evaluated the expression levels of c-kit, stem cell factor (SCF), Sirt1, and carcinogenesis-associated proteins. The CP group exhibited severe diffuse chronic inflammation. Meanwhile, the prostate cells appeared atypia; the activity of c-kit/SCF was upregulated, and carcinogenesis-associated proteins are dysregulated significantly in CP rats. Resveratrol treatment significantly improved these factors by Sirt1 activation. In summary, CP could further cause prostate carcinogenesis, which may be associated with activated c-kit/SCF signaling. Resveratrol treatment could improve the progression of CP via the downregulation of c-kit/SCF by activating Sirt1.

Generation of Functional Blood Vessels from a Single c-kit+ Adult Vascular Endothelial Stem Cell

PubMed Central

Fang, Shentong; Wei, Jing; Pentinmikko, Nalle; Leinonen, Hannele; Salven, Petri

2012-01-01

In adults, the growth of blood vessels, a process known as angiogenesis, is essential for organ growth and repair. In many disorders including cancer, angiogenesis becomes excessive. The cellular origin of new vascular endothelial cells (ECs) during blood vessel growth in angiogenic situations has remained unknown. Here, we provide evidence for adult vascular endothelial stem cells (VESCs) that reside in the blood vessel wall endothelium. VESCs constitute a small subpopulation within CD117+ (c-kit+) ECs capable of undergoing clonal expansion while other ECs have a very limited proliferative capacity. Isolated VESCs can produce tens of millions of endothelial daughter cells in vitro. A single transplanted c-kit-expressing VESC by the phenotype lin−CD31+CD105+Sca1+CD117+ can generate in vivo functional blood vessels that connect to host circulation. VESCs also have long-term self-renewal capacity, a defining functional property of adult stem cells. To provide functional verification on the role of c-kit in VESCs, we show that a genetic deficit in endothelial c-kit expression markedly decreases total colony-forming VESCs. In vivo, c-kit expression deficit resulted in impaired EC proliferation and angiogenesis and retardation of tumor growth. Isolated VESCs could be used in cell-based therapies for cardiovascular repair to restore tissue vascularization after ischemic events. VESCs also provide a novel cellular target to block pathological angiogenesis and cancer growth. PMID:23091420

C-MORE Science Kits: Putting Technology in the Hands of K-12 Teachers and Students

NASA Astrophysics Data System (ADS)

Achilles, K.; Weersing, K.; Daniels, C.; Puniwai, N.; Matsuzaki, J.; Bruno, B. C.

2008-12-01

The Center for Microbial Oceanography: Research and Education (C-MORE) is a NSF Science and Technology Center based at the University of Hawaii. The C-MORE education and outreach program offers a variety of resources and professional development opportunities for science educators, including online resources, participation in oceanography research cruises, teacher-training workshops, mini-grants to incorporate microbial oceanography-related content and activities into their classroom and, most recently, C- MORE science kits. C-MORE science kits provide hands-on classroom, field, and laboratory activities related to microbial oceanography for K-12 students. Each kit comes with complete materials and instructions, and is available free of charge to Hawaii's public school teachers. Several kits are available nationwide. C-MORE science kits cover a range of topics and technologies and are targeted at various grade levels. Here is a sampling of some available kits: 1) Marine Murder Mystery: The Case of the Missing Zooxanthellae. Students learn about the effect of climate change and other environmental threats on coral reef destruction through a murder-mystery experience. Participants also learn how to use DNA to identify a suspect. Grades levels: 3-8. 2) Statistical sampling. Students learn basic statistics through an exercise in random sampling, with applications to microbial oceanography. The laptops provided with this kit enable students to enter, analyze, and graph their data using EXCEL. Grades levels: 6-12. 3) Chlorophyll Lab. A research-quality fluorometer is used to measure the chlorophyll content in marine and freshwater systems. This enables students to compare biomass concentrations in samples collected from various locations. Grades levels: 9-12. 4) Conductivity-Temperature-Depth (CTD). Students predict how certain variables (e.g., temperature, pressure, chlorophyll, oxygen) vary with depth. A CTD, attached to a laptop computer, is deployed into deep water

Role of binding ligand in toxic hybrid proteins: a comparison of EGF-ricin, EGF-ricin A-chain, and ricin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Herschman, H.R.

1984-10-30

To analyze the influence of ricin B-chain on (i) the toxicity of hybrid-protein conjugates, (ii) the rate of cellular uptake of conjugates, and (iii) the rate at which ricin A-chain (RTA) is delivered to the cytoplasm, toxic hybrid proteins have been constructed consisting of epidermal growth factor (EGF) coupled in disulfide linkage either to ricin or to RTA. EGF-ricin is no more toxic on A431 cells than EGF-RTA. The two conjugates demonstrate similar kinetics of cellular uptake (defined as antibody irreversible toxicity). EGF-RTA and EGF-ricin, like ricin, required a 2-2 1/2 hour period at 37/sup 0/ before the onset ofmore » protein synthesis inhibition occurred. Results suggest that (i) RTA determines the processes which carry it, either in conjugate or toxin, from the plasma membrane binding site to the cytoplasm following endocytosis, and (ii) the ricin B chain is not required for these processes.« less

Estrogen-induced transcription factor EGR1 regulates c-Kit transcription in the mouse uterus to maintain uterine receptivity for embryo implantation.

PubMed

Park, Mira; Kim, Hye-Ryun; Kim, Yeon Sun; Yang, Seung Chel; Yoon, Jung Ah; Lyu, Sang Woo; Lim, Hyunjung Jade; Hong, Seok-Ho; Song, Haengseok

2018-07-15

Early growth response 1 (Egr1) is a key transcription factor that mediates the action of estrogen (E 2 ) to establish uterine receptivity for embryo implantation. However, few direct target genes of EGR1 have been identified in the uterus. Here, we demonstrated that E 2 induced EGR1-regulated transcription of c-Kit, which plays a crucial role in cell fate decisions. Spatiotemporal expression of c-Kit followed that of EGR1 in uteri of ovariectomized mice at various time points after E 2 treatment. E 2 activated ERK1/2 and p38 to induce EGR1, which then activated c-Kit expression in the uterus. EGR1 transfection produced rapid and transient induction of c-KIT in a time- and dose-dependent manner. Furthermore, luciferase assays to measure c-Kit promoter activity confirmed that a functional EGR1 binding site(s) (EBS) was located within -1 kb of the c-Kit promoter. Site-directed mutagenesis and chromatin immunoprecipitation-PCR for three putative EBS within -1 kb demonstrated that the EBS at -818/-805 was critical for EGR1-dependent c-Kit transcription. c-Kit expression was significantly increased in the uterus on day 4 and administration of Masitinib, a c-Kit inhibitor, effectively interfered with embryo implantation. Collectively, our results showed that estrogen induces transcription factor EGR1 to regulate c-Kit transcription for uterine receptivity for embryo implantation in the mouse uterus. Copyright © 2017 Elsevier B.V. All rights reserved.

Single molecule force spectroscopy for in-situ probing oridonin inhibited ROS-mediated EGF-EGFR interactions in living KYSE-150 cells.

PubMed

Pi, Jiang; Jin, Hua; Jiang, Jinhuan; Yang, Fen; Cai, Huaihong; Yang, Peihui; Cai, Jiye; Chen, Zheng W

2017-05-01

As the active anticancer component of Rabdosia Rubescens, oridonin has been proved to show strong anticancer activity in cancer cells, which is also found to be closely related to its specific inhibition effects on the EGFR tyrosine kinase activity. In this study, atomic force microscopy based single molecule force spectroscopy (AFM-SMFS) was used for real-time and in-situ detection of EGF-EGFR interactions in living esophageal cancer KYSE-150 cells to evaluate the anticancer activity of oridonin for the first time. Oridonin was found to induce apoptosis and also reduce EGFR expression in KYSE-150 cells. AFM-SMFS results demonstrated that oridonin could inhibit the binding between EGF and EGFR in KYSE-150 cells by decreasing the unbinding force and binding probability for EGF-EGFR complexes, which was further proved to be closely associated with the intracellular ROS level. More precise mechanism studies based on AFM-SMFS demonstrated that oridonin treatment could decrease the energy barrier width, increase the dissociation off rate constant and decrease the activation energy of EGF-EGFR complexes in ROS dependent way, suggesting oridonin as a strong anticancer agent targeting EGF-EGFR interactions in cancer cells through ROS dependent mechanism. Our results not only suggested oridonin as a strong anticancer agent targeting EGF-EGFR interactions in ROS dependent mechanism, but also highlighted AFM-SMFS as a powerful technique for pharmacodynamic studies by detecting ligand-receptor interactions, which was also expected to be developed into a promising tool for the screening and mechanism studies of drugs. Copyright © 2016 Elsevier Ltd. All rights reserved.

Endothelial NOS is required for SDF-1alpha/CXCR4-mediated peripheral endothelial adhesion of c-kit+ bone marrow stem cells.

PubMed

Kaminski, Alexander; Ma, Nan; Donndorf, Peter; Lindenblatt, Nicole; Feldmeier, Gregor; Ong, Lee-Lee; Furlani, Dario; Skrabal, Christian A; Liebold, Andreas; Vollmar, Brigitte; Steinhoff, Gustav

2008-01-01

In the era of intravascular approaches for regenerative cell therapy, the underlying mechanisms of stem cell migration to non-marrow tissue have not been clarified. We hypothesized that next to a local inflammatory response implying adhesion molecule expression, endothelial nitric oxide synthase (eNOS)-dependent signaling is required for stromal- cell-derived factor-1 alpha (SDF-1alpha)-induced adhesion of c-kit+ cells to the vascular endothelium. SDF-1alpha/tumor necrosis factor-alpha (TNF-alpha)-induced c-kit+-cell shape change and migration capacity was studied in vitro using immunohistochemistry and Boyden chamber assays. In vivo interaction of c-kit+ cells from bone marrow with the endothelium in response to SDF-1alpha/TNF-alpha stimulation was visualized in the cremaster muscle microcirculation of wild-type (WT) and eNOS (-/-) mice using intravital fluorescence microscopy. In addition, NOS activity was inhibited with N-nitro-L-arginine-methylester-hydrochloride in WT mice. To reveal c-kit+-specific adhesion behavior, endogenous leukocytes (EL) and c-kit+ cells from peripheral blood served as control. Moreover, intercellular adhesion molecule-1 (ICAM-1) and CXCR4 were blocked systemically to determine their role in inflammation-related c-kit+-cell adhesion. In vitro, SDF-1alpha enhanced c-kit+-cell migration. In vivo, SDF-1alpha alone triggered endothelial rolling-not firm adherence-of c-kit+ cells in WT mice. While TNF-alpha alone had little effect on adhesion of c-kit+ cells, it induced maximum endothelial EL adherence. However, after combined treatment with SDF-1alpha+TNF-alpha, endothelial adhesion of c-kit+ cells increased independent of their origin, while EL adhesion was not further incremented. Systemic treatment with anti-ICAM-1 and anti-CXCR4-monoclonal antibody completely abolished endothelial c-kit+-cell adhesion. In N-nitro-L-arginine-methylester-hydrochloride-treated WT mice as well as in eNOS (-/-) mice, firm endothelial adhesion of c-kit+ cells

Oncogenic Kit signalling on the Golgi is suppressed by blocking secretory trafficking with M-COPA in gastrointestinal stromal tumours.

PubMed

Obata, Yuuki; Horikawa, Keita; Shiina, Isamu; Takahashi, Tsuyoshi; Murata, Takatsugu; Tasaki, Yasutaka; Suzuki, Kyohei; Yonekura, Keita; Esumi, Hiroyasu; Nishida, Toshirou; Abe, Ryo

2018-02-28

Most gastrointestinal stromal tumours (GISTs) are caused by constitutively active mutations in Kit tyrosine kinase. The drug imatinib, a specific Kit inhibitor, improves the prognosis of metastatic GIST patients, but these patients become resistant to the drug by acquiring secondary mutations in the Kit kinase domain. We recently reported that a Kit mutant causes oncogenic signals only on the Golgi apparatus in GISTs. In this study, we show that in GIST, 2-methylcoprophilinamide (M-COPA, also known as "AMF-26"), an inhibitor of biosynthetic protein trafficking from the endoplasmic reticulum (ER) to the Golgi, suppresses Kit autophosphorylation at Y703/Y721/Y730/Y936, resulting in blockade of oncogenic signalling. Results of our M-COPA treatment assay show that Kit Y703/Y730/Y936 in the ER are dephosphorylated by protein tyrosine phosphatases (PTPs), thus the ER-retained Kit is unable to activate downstream molecules. ER-localized Kit Y721 is not phosphorylated, but not due to PTPs. Importantly, M-COPA can inhibit the activation of the Kit kinase domain mutant, resulting in suppression of imatinib-resistant GIST proliferation. Our study demonstrates that Kit autophosphorylation is spatio-temporally regulated and may offer a new strategy for treating imatinib-resistant GISTs. Copyright © 2017 The Author(s). Published by Elsevier B.V. All rights reserved.

Gallic acid-capped gold nanoparticles inhibit EGF-induced MMP-9 expression through suppression of p300 stabilization and NFκB/c-Jun activation in breast cancer MDA-MB-231 cells.

PubMed

Chen, Ying-Jung; Lee, Yuan-Chin; Huang, Chia-Hui; Chang, Long-Sen

2016-11-01

Triple-negative breast cancers (TNBCs) are highly invasive and have a higher rate of distant metastasis. Matrix metalloproteinase-9 (MMP-9) plays a crucial role in EGF/EGFR-mediated malignant progression and metastasis of TNBCs. Various studies have revealed that treatment with gallic acid down-regulates MMP-9 expression in cancer cells, and that conjugation of phytochemical compounds with gold nanoparticles (AuNPs) increases the anti-tumor activity of the phytochemical compounds. Thus, the effect of gallic acid-capped AuNPs (GA-AuNPs) on MMP-9 expression in EGF-treated TNBC MDA-MB-231 cells was analyzed in the present study. The so-called green synthesis of AuNPs by means of gallic acid was performed at pH10, and the resulting GA-AuNPs had spherical shape with an average diameter of approximately 50nm. GA-AuNPs notably suppressed migration and invasion of EGF-treated cells, and inhibited EGF-induced MMP-9 up-regulation. GA-AuNPs abrogated EGF-induced Akt/p65 and ERK/c-Jun phosphorylation, leading to down-regulation of MMP-9 mRNA and protein expression in EGF-treated cells. Meanwhile, EGF-induced p300 stabilization was found to be involved in MMP-9 expression, whereas GA-AuNPs inhibited the EGF-promoted stability of the p300 protein. Although GA-AuNPs and gallic acid suppressed EGF-induced MMP-9 up-regulation via the same signaling pathway, the effective concentration of gallic acid was approximately 100-fold higher than that of GA-AuNPs for inhibition of MMP-9 expression in EGF-treated cells to a similar extent. Collectively, our data indicate that, in comparison with gallic acid, GA-AuNPs have a superior ability to inhibit EGF/EGFR-mediated MMP-9 expression in TNBC MDA-MB-231 cells. Our findings also point to a way to improve the anti-tumor activity of gallic acid. Copyright © 2016 Elsevier Inc. All rights reserved.

c-Kit-Mediated Functional Positioning of Stem Cells to Their Niches Is Essential for Maintenance and Regeneration of Adult Hematopoiesis

PubMed Central

Kimura, Yuki; Ding, Bisen; Imai, Norikazu; Nolan, Daniel J.; Butler, Jason M.; Rafii, Shahin

2011-01-01

The mechanism by which hematopoietic stem and progenitor cells (HSPCs) through interaction with their niches maintain and reconstitute adult hematopoietic cells is unknown. To functionally and genetically track localization of HSPCs with their niches, we employed novel mutant loxPs, lox66 and lox71 and Cre-recombinase technology to conditionally delete c-Kit in adult mice, while simultaneously enabling GFP expression in the c-Kit-deficient cells. Conditional deletion of c-Kit resulted in hematopoietic failure and splenic atrophy both at steady state and after marrow ablation leading to the demise of the treated adult mice. Within the marrow, the c-Kit-expressing GFP+ cells were positioned to Kit ligand (KL)-expressing niche cells. This c-Kit-mediated cellular adhesion was essential for long-term maintenance and expansion of HSPCs. These results lay the foundation for delivering KL within specific niches to maintain and restore hematopoiesis. PMID:22046410

Expression and Mutational Analysis of c-kit in Ovarian Surface Epithelial Tumors

PubMed Central

Lee, Myung-Hoon; Park, Tae-In; Bae, Han-Ik

2006-01-01

Coexpression of Kit ligand and c-kit has been reported in some gynecologic tumors. To determine whether imatinib mesylate is useful in ovarian epithelial tumors, we performed immunohistochemical and mutational analysis. The cases consisted of 33 cases, which included 13 serous cystadenocarcinomas, 1 borderline serous tumor, 8 mucinous cystadenocarcinomas, 6 borderline mucinous tumors and 5 clear cell carcinomas. Five cases of serous cystadenoma and 5 cases of mucinous cystadenoma were also included. In the immunohistochemical study, 3 cases (3/6, 50%) of borderline mucinous cystic tumor and two cases (2/8, 25%) of mucinous cystadenocarcinoma show positive staining for KIT protein. Only one case (1/13, 7.7%) of serous cystadenocarcinoma had positive staining. On mutational analysis, no mutation was identified at exon 11. However, two cases of borderline mucinous tumors and one case of mucinous cystadenocarcinoma had mutations at exon 17. In these cases, the immunohistochemistry also shows focal positive staining at epithelial component. Although, KIT protein expression showed higher incidence in mucinous tumors than serous tumors, they lack KIT-activating mutations in exon 11. Thus, ovarian surface epithelial tumors are unlikely to respond to imatinib mesylate. PMID:16479070

c-KIT receptor expression is strictly associated with the biological behaviour of thyroid nodules

PubMed Central

2012-01-01

Background A large amount of information has been collected on the molecular tumorigenesis of thyroid cancer. A low expression of c-KIT gene has been reported during the transformation of normal thyroid epithelium to papillary carcinoma suggesting a possible role of the gene in the differentiation of thyroid tissue rather than in the proliferation. The initial presentation of thyroid carcinoma is through a nodule and the best way nowadays to evaluate it is by fine-needle aspiration (FNA). However many thyroid FNAs are not definitively benign or malignant, yielding an indeterminate or suspicious diagnosis which ranges from 10 to 25% of FNAs. BRAF mutational analysis is commonly used to assess the malignancy of thyroid nodules but unfortunately it still leaves indeterminate diagnoses. The development of molecular initial diagnostic tests for evaluating a thyroid nodule is needed in order to define optimal surgical approach for patients with uncertain diagnosis pre- and intra-operatively. Methods In this study we extracted RNA from 82 FNA smears, 46 malignant and 36 benign at the histology, in order to evaluate by quantitative Real Time PCR the expression levels of c-KIT gene. Results We have found a highly preferential decrease rather than increase in transcript of c-KIT in malignant thyroid lesions compared to the benign ones. To explore the diagnostic utility of c-KIT expression in thyroid nodules, its expression values were divided in four arbitrarily defined classes, with class I characterized by the complete silencing of the gene. Class I and IV represented the two most informative groups, with 100% of the samples found malignant or benign respectively. The molecular analysis was proven by ROC (receiver operating characteristic) analysis to be highly specific and sensitive improving the cytological diagnostic accuracy of 15%. Conclusion We propose the use of BRAF test (after uncertain cytological diagnosis) to assess the malignancy of thyroid nodules at first

C-Kit Promotes Growth and Migration of Human Cardiac Progenitor Cells via the PI3K-AKT and MEK-ERK Pathways

PubMed Central

Al-Maqtari, Tareq; Cao, Pengxiao; Keith, Matthew C. L.; Wysoczynski, Marcin; Zhao, John; Moore IV, Joseph B.; Bolli, Roberto

2015-01-01

A recent phase I clinical trial (SCIPIO) has shown that autologous c-kit+ cardiac progenitor cells (CPCs) improve cardiac function and quality of life when transplanted into patients with ischemic heart disease. Although c-kit is widely used as a marker of resident CPCs, its role in the regulation of the cellular characteristics of CPCs remains unknown. We hypothesized that c-kit plays a role in the survival, growth, and migration of CPCs. To test this hypothesis, human CPCs were grown under stress conditions in the presence or absence of SCF, and the effects of SCF-mediated activation of c-kit on CPC survival/growth and migration were measured. SCF treatment led to a significant increase in cell survival and a reduction in cell death under serum depletion conditions. In addition, SCF significantly promoted CPC migration in vitro. Furthermore, the pro-survival and pro-migratory effects of SCF were augmented by c-kit overexpression and abrogated by c-kit inhibition with imatinib. Mechanistically, c-kit activation in CPCs led to activation of the PI3K and the MAPK pathways. With the use of specific inhibitors, we confirmed that the SCF/c-kit-dependent survival and chemotaxis of CPCs are dependent on both pathways. Taken together, our findings suggest that c-kit promotes the survival/growth and migration of human CPCs cultured ex vivo via the activation of PI3K and MAPK pathways. These results imply that the efficiency of CPC homing to the injury site as well as their survival after transplantation may be improved by modulating the activity of c-kit. PMID:26474484

Is receptor oligomerization causally linked to activation of the EGF receptor kinase?

NASA Technical Reports Server (NTRS)

Rintoul, D. A.; Spooner, B. S. (Principal Investigator)

1992-01-01

Transduction of a signal from an extracellular peptide hormone to produce an intracellular response is often mediated by a cell surface receptor, which is usually a glycoprotein. The secondary intracellular signal(s) generated after hormone binding to the receptor have been intensively studied. The nature of the primary signal generated by ligand binding to the receptor is understood less well in most cases. The particular case of the epidermal growth factor (EGF) receptor is analyzed, and evidence for or against two dissimilar models of primary signal transduction is reviewed. Evidence for the most widely accepted current model is found to be unconvincing. Evidence for the other model is substantial but indirect; a direct test of this model remains to be done.

A Chrysin Derivative Suppresses Skin Cancer Growth by Inhibiting Cyclin-dependent Kinases*

PubMed Central

Liu, Haidan; Liu, Kangdong; Huang, Zunnan; Park, Chan-Mi; Thimmegowda, N. R.; Jang, Jae-Hyuk; Ryoo, In-Ja; He, Long; Kim, Sun-Ok; Oi, Naomi; Lee, Ki Won; Soung, Nak-Kyun; Bode, Ann M.; Yang, Yifeng; Zhou, Xinmin; Erikson, Raymond L.; Ahn, Jong-Seog; Hwang, Joonsung; Kim, Kyoon Eon; Dong, Zigang; Kim, Bo-Yeon

2013-01-01

Chrysin (5,7-dihydroxyflavone), a natural flavonoid widely distributed in plants, reportedly has chemopreventive properties against various cancers. However, the anticancer activity of chrysin observed in in vivo studies has been disappointing. Here, we report that a chrysin derivative, referred to as compound 69407, more strongly inhibited EGF-induced neoplastic transformation of JB6 P+ cells compared with chrysin. It attenuated cell cycle progression of EGF-stimulated cells at the G1 phase and inhibited the G1/S transition. It caused loss of retinoblastoma phosphorylation at both Ser-795 and Ser-807/811, the preferred sites phosphorylated by Cdk4/6 and Cdk2, respectively. It also suppressed anchorage-dependent and -independent growth of A431 human epidermoid carcinoma cells. Compound 69407 reduced tumor growth in the A431 mouse xenograft model and retinoblastoma phosphorylation at Ser-795 and Ser-807/811. Immunoprecipitation kinase assay results showed that compound 69407 attenuated endogenous Cdk4 and Cdk2 kinase activities in EGF-stimulated JB6 P+ cells. Pulldown and in vitro kinase assay results indicated that compound 69407 directly binds with Cdk2 and Cdk4 in an ATP-independent manner and inhibited their kinase activities. A binding model between compound 69407 and a crystal structure of Cdk2 predicted that compound 69407 was located inside the Cdk2 allosteric binding site. The binding was further verified by a point mutation binding assay. Overall results indicated that compound 69407 is an ATP-noncompetitive cyclin-dependent kinase inhibitor with anti-tumor effects, which acts by binding inside the Cdk2 allosteric pocket. This study provides new insights for creating a general pharmacophore model to design and develop novel ATP-noncompetitive agents with chemopreventive or chemotherapeutic potency. PMID:23888052

Maternal heparin-binding-EGF deficiency limits pregnancy success in mice

PubMed Central

Xie, Huirong; Wang, Haibin; Tranguch, Susanne; Iwamoto, Ryo; Mekada, Eisuke; DeMayo, Francesco J.; Lydon, John P.; Das, Sanjoy K.; Dey, Sudhansu K.

2007-01-01

An intimate discourse between the blastocyst and uterus is essential for successful implantation. However, the molecular basis of this interaction is not clearly understood. Exploiting genomic Hbegf mutant mice, we show here that maternal deficiency of heparin-binding EGF-like growth factor (HB-EGF) defers on-time implantation, leading to compromised pregnancy outcome. We also demonstrate that amphiregulin, but not epiregulin, partially compensates for the loss of HB-EGF during implantation. In search of the mechanism of this compensation, we found that reduced preimplantation estrogen secretion from ovarian HB-EGF deficiency is a cause of sustained expression of uterine amphiregulin before the initiation of implantation. To explore the significance specifically of uterine HB-EGF in implantation, we examined this event in mice with conditional deletion of uterine HB-EGF and found that this specific loss of HB-EGF in the uterus still defers on-time implantation without altering preimplantation ovarian estrogen secretion. The observation of normal induction of uterine amphiregulin surrounding the blastocyst at the time of attachment in these conditional mutant mice suggests a compensatory role of amphiregulin for uterine loss of HB-EGF, preventing complete failure of pregnancy. Our study provides genetic evidence that HB-EGF is critical for normal implantation. This finding has high clinical relevance, because HB-EGF signaling is known to be important for human implantation. PMID:17986609

Maternal heparin-binding-EGF deficiency limits pregnancy success in mice.

PubMed

Xie, Huirong; Wang, Haibin; Tranguch, Susanne; Iwamoto, Ryo; Mekada, Eisuke; Demayo, Francesco J; Lydon, John P; Das, Sanjoy K; Dey, Sudhansu K

2007-11-13

An intimate discourse between the blastocyst and uterus is essential for successful implantation. However, the molecular basis of this interaction is not clearly understood. Exploiting genomic Hbegf mutant mice, we show here that maternal deficiency of heparin-binding EGF-like growth factor (HB-EGF) defers on-time implantation, leading to compromised pregnancy outcome. We also demonstrate that amphiregulin, but not epiregulin, partially compensates for the loss of HB-EGF during implantation. In search of the mechanism of this compensation, we found that reduced preimplantation estrogen secretion from ovarian HB-EGF deficiency is a cause of sustained expression of uterine amphiregulin before the initiation of implantation. To explore the significance specifically of uterine HB-EGF in implantation, we examined this event in mice with conditional deletion of uterine HB-EGF and found that this specific loss of HB-EGF in the uterus still defers on-time implantation without altering preimplantation ovarian estrogen secretion. The observation of normal induction of uterine amphiregulin surrounding the blastocyst at the time of attachment in these conditional mutant mice suggests a compensatory role of amphiregulin for uterine loss of HB-EGF, preventing complete failure of pregnancy. Our study provides genetic evidence that HB-EGF is critical for normal implantation. This finding has high clinical relevance, because HB-EGF signaling is known to be important for human implantation.

KIT pathway alterations in mucosal melanomas of the vulva and other sites.

PubMed

Omholt, Katarina; Grafström, Eva; Kanter-Lewensohn, Lena; Hansson, Johan; Ragnarsson-Olding, Boel K

2011-06-15

A significant proportion of mucosal melanomas contain alterations in KIT. The aim of this study was to characterize the pattern of KIT, NRAS, and BRAF mutations in mucosal melanomas at specific sites and to assess activation of the KIT downstream RAF/MEK/extracellular signal-regulated kinase (ERK) and phosphoinositide 3-kinase (PI3K)/AKT pathways in mucosal melanoma specimens. Seventy-one primary mucosal melanomas from various sites were studied. Mutation analysis was done by DNA sequencing. Expression of KIT, phosphorylated (p)-ERK, and p-AKT was evaluated by immunohistochemistry. KIT mutations were detected in 35% (8 of 23) of vulvar, 9% (2 of 22) of anorectal, 7% (1 of 14) of nasal cavity, and 20% (1 of 5) of penile melanomas. No KIT mutations were found in 7 vaginal melanomas. The difference in KIT mutation frequency between vulvar and nonvulvar cases was statistically significant (P = 0.014). The overall frequencies of NRAS and BRAF mutations were 10% and 6%, respectively. Notably, vaginal melanomas showed a NRAS mutation rate of 43%. KIT gene amplification (≥4 copies), as assessed by quantitative real-time PCR, was observed in 19% of cases. KIT expression was associated with KIT mutation status (P < 0.001) and was more common in vulvar than nonvulvar tumors (P = 0.016). Expression of p-ERK and p-AKT was observed in 42% and 59% of tumors, respectively, and occurred irrespective of KIT/NRAS/BRAF mutation status. NRAS mutation was associated with worse overall survival in univariate analysis. Results show that KIT mutations are more common in vulvar melanomas than other types of mucosal melanomas and that both the RAF/MEK/ERK and PI3K/AKT pathways are activated in mucosal melanoma specimens. ©2011 AACR.

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells.

PubMed

Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo

2017-01-01

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to

M-COPA suppresses endolysosomal Kit-Akt oncogenic signalling through inhibiting the secretory pathway in neoplastic mast cells

PubMed Central

Hara, Yasushi; Obata, Yuuki; Horikawa, Keita; Tasaki, Yasutaka; Suzuki, Kyohei; Murata, Takatsugu; Shiina, Isamu; Abe, Ryo

2017-01-01

Gain-of-function mutations in Kit receptor tyrosine kinase result in the development of a variety of cancers, such as mast cell tumours, gastrointestinal stromal tumours (GISTs), acute myeloid leukemia, and melanomas. The drug imatinib, a selective inhibitor of Kit, is used for treatment of mutant Kit-positive cancers. However, mutations in the Kit kinase domain, which are frequently found in neoplastic mast cells, confer an imatinib resistance, and cancers expressing the mutants can proliferate in the presence of imatinib. Recently, we showed that in neoplastic mast cells that endogenously express an imatinib-resistant Kit mutant, Kit causes oncogenic activation of the phosphatidylinositol 3-kinase-Akt (PI3K-Akt) pathway and the signal transducer and activator of transcription 5 (STAT5) but only on endolysosomes and on the endoplasmic reticulum (ER), respectively. Here, we show a strategy for inhibition of the Kit-PI3K-Akt pathway in neoplastic mast cells by M-COPA (2-methylcoprophilinamide), an inhibitor of this secretory pathway. In M-COPA-treated cells, Kit localization in the ER is significantly increased, whereas endolysosomal Kit disappears, indicating that M-COPA blocks the biosynthetic transport of Kit from the ER. The drug greatly inhibits oncogenic Akt activation without affecting the association of Kit with PI3K, indicating that ER-localized Kit-PI3K complex is unable to activate Akt. Importantly, M-COPA but not imatinib suppresses neoplastic mast cell proliferation through inhibiting anti-apoptotic Akt activation. Results of our M-COPA treatment assay show that Kit can activate Erk not only on the ER but also on other compartments. Furthermore, Tyr568/570, Tyr703, Tyr721, and Tyr936 in Kit are phosphorylated on the ER, indicating that these five tyrosine residues are all phosphorylated before mutant Kit reaches the plasma membrane (PM). Our study provides evidence that Kit is tyrosine-phosphorylated soon after synthesis on the ER but is unable to

c-Kit mutation reduce intestinal epithelial cell proliferation and migration, but not influence intestinal permeability stimulated by lipopolysaccharide.

PubMed

Xue, Hong; Wang, Feng Yun; Kang, Qian; Tang, Xu Dong

2018-06-20

The proto-oncogene c-kit, as a marker of interstitial cells of Cajal (ICCs) in the gastrointestinal tract, plays an important role in the ICCs. Although limited evidences showed c-kit is present in the colonic epithelium but its roles remain unclear. In the present study, we aimed to investigate the expression, location and function of c-kit in the intestinal epithelium. Immunofluorescence, western blotting, and RT-PCR were performed to detect the expression and location of c-kit in the intestinal mucosa of WT mice. We investigated intestinal epithelial proliferation and migration in vivo by performing 5-Bromodeoxyuridine (BrdU) incorporation and Ki-67 staining in WT and Wads m/m mice. An Ussing chamber with fluorescein-isothiocyanate dextran 4000 was used to detect the transepithelial electric resistance (TER), short circuit current (ISC) and permeability across ex vivo colon segments under control and endotoxaemia conditions. We demonstrated that c-kit was located and expressed in the gut crypt compartment in WT mice, which was demonstrated in the c-kit mutant mice (Wads m/m ). In addition, both the number of proliferating cells and the percentage of the distance migrated were lower in the Wads m/m mice than those in the WT mice. Moreover, the intestinal permeability, TER and tight junction were unaltered in the Wads m/m mice under endotoxic conditions compared with those in both the control condition and the WT mice. Altogether, these observations imply that the expression of c-kit in the colonic epithelium is involved in the proliferation and permeability of the colonic epithelium. Copyright © 2018. Published by Elsevier GmbH.

Activated protein kinase C binds to intracellular receptors in rat hepatocytes.

PubMed

Robles-Flores, M; García-Sáinz, J A

1993-12-01

The aim of this study was to identify in rat hepatocytes cellular polypeptides that bind protein kinase C (PKC) and may influence its activity and its compartmentation. At least seven proteins, with apparent M(r) values between 12,000 and 36,000, that behave like Receptors for Activated C-Kinase (RACKs) were found in the Triton-X-100-insoluble fraction of these cells; i.e. PKC bound to these polypeptides when it was in its active form. RACKS seem to be PKC substrates. Studies using isotype-specific PKC antibodies suggested some selectivity of RACKs, i.e. RACKs in the M(r) approximately 28,000-36,000 region bound PKC-alpha and PKC-beta in the presence of phosphatidylserine, diolein and Ca2+, whereas those of M(r) approximately 12,000-14,000 bound all isoforms studied, and, in contrast with the other RACKs, they did this even in the absence of Ca2+. Peptide I (KGDYEKILVALCGGN), which has a sequence suggested to be involved in the PKC-RACKs interaction [Mochly-Rosen, Khaner, Lopez and Smith (1991) J. Biol. Chem. 266, 14866-14868], inhibited PKC activity. Preincubation of RACKs with antisera directed against peptide I prevented PKC binding to them. The data suggest that peptide I blocks PKC binding to RACKs by two mechanisms: inhibition of PKC activity and competition with a putative binding site.

Characterization of various types of mast cells derived from model mice of familial gastrointestinal stromal tumors with KIT-Asp818Tyr mutation

PubMed Central

Kajimoto, Noriko; Nakai, Norihiro; Ohkouchi, Mizuka; Hashikura, Yuka; Liu-Kimura, Ning-Ning; Isozaki, Koji; Hirota, Seiichi

2015-01-01

Sporadic mast cell neoplasms and gastrointestinal stromal tumors (GISTs) often have various types of somatic gain-of-function mutations of the c-kit gene which encodes a receptor tyrosine kinase, KIT. Several types of germline gain-of-function mutations of the c-kit gene have been detected in families with multiple GISTs. All three types of model mice for the familial GISTs with germline c-kit gene mutations at exon 11, 13 or 17 show development of GIST, while they are different from each other in skin mast cell number. Skin mast cell number in the model mice with exon 17 mutation was unchanged compared to the corresponding wild-type mice. In the present study, we characterized various types of mast cells derived from the model mice with exon 17 mutation (KIT-Asp818Tyr) corresponding to human familial GIST case with human KIT-Asp820Tyr to clarify the role of the c-kit gene mutation in mast cells. Bone marrow-derived cultured mast cells (BMMCs) derived from wild-type mice, heterozygotes and homozygotes were used for the experiments. Immortalized BMMCs, designated as IMC-G4 cells, derived from BMMCs of a homozygote during long-term culture were also used. Ultrastructure, histamine contents, proliferation profiles and phosphorylation of various signaling molecules in those cells were examined. In IMC-G4 cells, presence of additional mutation(s) of the c-kit gene and effect of KIT inhibitors on both KIT autophosphorylation and cell proliferation were also analyzed. We demonstrated that KIT-Asp818Tyr did not affect ultrastructure and proliferation profiles but did histamine contents in BMMCs. IMC-G4 cells had an additional novel c-kit gene mutation of KIT-Tyr421Cys which is considered to induce neoplastic transformation of mouse mast cells and the mutation appeared to be resistant to a KIT inhibitor of imatinib but sensitive to another KIT inhibitor of nilotinib. IMC-G4 cells might be a useful mast cell line to investigate mast cell biology. PMID:26722383

Filamin A Modulates Kinase Activation and Intracellular Trafficking of Epidermal Growth Factor Receptors in Human Melanoma Cells

PubMed Central

Fiori, Jennifer L.; Zhu, Tie-Nian; O'Connell, Michael P.; Hoek, Keith S.; Indig, Fred E.; Frank, Brittany P.; Morris, Christa; Kole, Sutapa; Hasskamp, Joanne; Elias, George; Weeraratna, Ashani T.; Bernier, Michel

2009-01-01

The actin-binding protein filamin A (FLNa) affects the intracellular trafficking of various classes of receptors and has a potential role in oncogenesis. However, it is unclear whether FLNa regulates the signaling capacity and/or down-regulation of the activated epidermal growth factor receptor (EGFR). Here it is shown that partial knockdown of FLNa gene expression blocked ligand-induced EGFR responses in metastatic human melanomas. To gain greater insights into the role of FLNa in EGFR activation and intracellular sorting, we used M2 melanoma cells that lack endogenous FLNa and a subclone in which human FLNa cDNA has been stably reintroduced (M2A7 cells). Both tyrosine phosphorylation and ubiquitination of EGFR were significantly lower in epidermal growth factor (EGF)-stimulated M2 cells when compared with M2A7 cells. Moreover, the lack of FLNa interfered with EGFR interaction with the ubiquitin ligase c-Cbl. M2 cells exhibited marked resistance to EGF-induced receptor degradation, which was very active in M2A7 cells. Despite comparable rates of EGF-mediated receptor endocytosis, internalized EGFR colocalized with the lysosomal marker lysosome-associated membrane protein-1 in M2A7 cells but not M2 cells, in which EGFR was found to be sequestered in large vesicles and subsequently accumulated in punctated perinuclear structures after EGF stimulation. These results suggest the requirement of FLNa for efficient EGFR kinase activation and the sorting of endocytosed receptors into the degradation pathway. PMID:19213840

Ethanol increases affinity of protein kinase C for phosphatidylserine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chin, J.H.

1986-03-01

Protein kinase C is a calcium-dependent enzyme that requires phospholipid for its activation. It is present in relatively high concentration in the brain and may be involved in neuronal function. The present experiments test whether the membrane disorder induced by ethanol affects the activity of kinase C by changing its interaction with membrane lipid. Fractions rich in kinase C were purified from rat brain cytosol by DEAE-cellulose chromatography and Sephadex G-200 gel filtration. Enzyme activity was assayed by measuring the phosphorylation of histone H1. As expected, phosphatidylserine activated the enzyme, and the stimulation was further increased by the addition ofmore » calcium and/or diacylglycerol. At low concentration of free calcium (0.5-1..mu..M), ethanol (800 mM0 enhanced kinase C activity if the presence of phospholipid. similar results were observed in the absence of calcium. Double reciprocal plots of the data showed that ethanol increased the affinity of the enzyme for phosphatidylserine without affecting the V/sub max. The stimulation of kinase C activity by ethanol was not observed at high calcium concentrations. These experiments suggest that ethanol may activated protein kinase C at physiological levels of calcium by facilitating its transfer into the hydrophobic membrane environment.« less

Statistical modeling of dental unit water bacterial test kit performance.

PubMed

Cohen, Mark E; Harte, Jennifer A; Stone, Mark E; O'Connor, Karen H; Coen, Michael L; Cullum, Malford E

2007-01-01

While it is important to monitor dental water quality, it is unclear whether in-office test kits provide bacterial counts comparable to the gold standard method (R2A). Studies were conducted on specimens with known bacterial concentrations, and from dental units, to evaluate test kit accuracy across a range of bacterial types and loads. Colony forming units (CFU) were counted for samples from each source, using R2A and two types of test kits, and conformity to Poisson distribution expectations was evaluated. Poisson regression was used to test for effects of source and device, and to estimate rate ratios for kits relative to R2A. For all devices, distributions were Poisson for low CFU/mL when only beige-pigmented bacteria were considered. For higher counts, R2A remained Poisson, but kits exhibited over-dispersion. Both kits undercounted relative to R2A, but the degree of undercounting was reasonably stable. Kits did not grow pink-pigmented bacteria from dental-unit water identified as Methylobacterium rhodesianum. Only one of the test kits provided results with adequate reliability at higher bacterial concentrations. Undercount bias could be estimated for this device and used to adjust test kit results. Insensitivity to methylobacteria spp. is problematic.

Internal associations and dynamic expression of c-kit and nanog genes in ventricular remodelling induced by adriamycin.

PubMed

Liu, Zhen; Li, Shuo; Liu, Lingling; Guo, Zhikun; Wang, Pengfei

2016-09-01

The present study aimed to investigate the dynamic expression of the c-kit and nanog genes in rats with left ventricular remodelling induced by adriamycin (ADR), and explore its internal association and mechanism of action. Sprague-Dawley male rats were randomly divided into a normal control group and a heart failure model group. Heart failure was induced by a single intraperitoneal injection of ADR (4 mg/kg) weekly for six weeks. The normal control group was given the same amount of saline. At the eighth week, rat cardiac function was examined to demonstrate the formation of heart failure. The rat hearts were harvested frozen and sectioned, and the expression levels of the nanog and c-kit genes in the myocardial tissue samples were detected using immunohistochemistry, immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). Hematoxylin and eosin staining demonstrated various pathological changes in the myocardial cells in the heart failure model group, whereas myocardial infarction was not observed in the normal control group. Immunohistochemistry and immunofluorescence demonstrated that nanog-positive cells were predominantly expressed in the vascular endothelium, with a few myocardial cells and stem cells in normal myocardium. The expression levels of c-kit and nanog in the myocardium of the rats with heart failure decreased significantly. c-kit-positive cells clustered together in the epicardium and its vicinity, and c-kit expression significantly decreased in the myocardium of rats with heart failure, as compared with normal rats. In both groups, some cells co-expressed both the c-kit and nanog genes. The RT-PCR results demonstrated that the expression levels of the two genes in the heart failure model group were significantly lower compared with those in the normal control group (P<0.05). In conclusion, the c-kit- and nanog-positive stem cells decreased in the myocardium of the rats with left ventricular remodelling induced by ADR

Overexpression of c-kit(CD117), relevant with microvessel density, is an independent survival prognostic factor for patients with HBV-related hepatocellular carcinoma.

PubMed

Yan, Weiwei; Zhu, Zhenyu; Pan, Fei; Huang, Ang; Dai, Guang-Hai

2018-01-01

To explore new biomarkers for indicating the recurrence and prognosis in hepatitis B virus (HBV)-related hepatocellular carcinoma (HCC) patients after tumor resection, we investigated the expression and prognostic value of c-kit(CD117) in HBV-related HCC. Immunohistochemistry was used to estimate the expression of c-kit(CD117) and CD34 in the liver cancer tissues. The correlations between the expression of these biomarkers and the clinicopathologic characteristics were analyzed. The positive rate of c-kit(CD117) expression in 206 HCC cases was 48.1%, and c-kit expression was significantly related with CD34-positive microvessel density. CD34-microvessel density numbers were much higher in c-kit(+) HCC tissues than in c-kit(-) HCC tissues (44.13±17.01 vs 26.87±13.16, P =0.003). The expression of c-kit was significantly higher in patients with Edmondson grade III-IV ( P <0.001) and TNM stage III ( P <0.001). Moreover, Kaplan-Meier survival analysis showed that c-kit ( P <0.001) expression was correlated with reduced disease-free survival (DFS). Multivariate analysis identified c-kit as an independent poor prognostic factor of DFS in HCC patients ( P <0.001). Increased c-kit expression could be considered as an independent unfavorable prognostic factor for predicting DFS in HBV-related HCC patients after surgery. These results could be used to identify patients at a higher risk of early tumor recurrence and poor prognosis.

SNP analyses of growth factor genes EGF, TGF{beta}-1, and HGF reveal haplotypic association of EGF with autism

DOE Office of Scientific and Technical Information (OSTI.GOV)

Toyoda, Takao; Thanseem, Ismail; Kawai, Masayoshi

Autism is a pervasive neurodevelopmental disorder diagnosed in early childhood. Growth factors have been found to play a key role in the cellular differentiation and proliferation of the central and peripheral nervous systems. Epidermal growth factor (EGF) is detected in several regions of the developing and adult brain, where, it enhances the differentiation, maturation, and survival of a variety of neurons. Transforming growth factor-{beta} (TGF{beta}) isoforms play an important role in neuronal survival, and the hepatocyte growth factor (HGF) has been shown to exhibit neurotrophic activity. We examined the association of EGF, TGF{beta}1, and HGF genes with autism, in amore » trio association study, using DNA samples from families recruited to the Autism Genetic Resource Exchange; 252 trios with a male offspring scored for autism were selected for the study. Transmission disequilibrium test revealed significant haplotypic association of EGF with autism. No significant SNP or haplotypic associations were observed for TGF{beta}1 or HGF. Given the role of EGF in brain and neuronal development, we suggest a possible role of EGF in the pathogenesis of autism.« less

Case report of a KIT-mutated melanoma patient with an excellent response to apatinib and temozolomide combination therapy.

PubMed

Luo, Cong; Shen, Jiayu; Ying, Jieer; Fang, Xianhua; Wang, Xiaohong; Fu, Zhixuan; Liu, Peng

2017-01-01

Malignant melanoma is one kind of malignant disease which has high rates of mortality, metastasis, and poor prognosis. The therapeutic landscape is rapidly changing with the development of novel agents in recent decades, such as anti-PD-1 agents, anti-CTLA-4 agents, and BRAF inhibitors. However, since most of these novel agents are very expensive, not all patients can afford them. Apatinib is a novel oral small-molecule tyrosine kinase inhibitor targeting the intracellular domain of vascular endothelial growth factor receptor 2 (VEGFR-2) and may also be effective on Ret, c-KIT, and c-src. Temozolomide (TMZ) is a second-generation alkylating agent and a cytotoxic drug for melanoma treatment. In this work, we reported a case of metastatic melanoma with an excellent response to apatinib/TMZ combination therapy with progression-free survival for more than one year. This patient showed high expression of CD117, VEGFR-3, and KIT mutation in exon 11, suggesting that apatinib may induce clinical response via inhibiting VEGFR and c-KIT. Apatinib/TMZ combination therapy could be a new option for the treatment of advanced melanoma with KIT mutation.

Investigating Associations Between Proliferation Indices, C-kit, and Lymph Node Stage in Canine Mast Cell Tumors.

PubMed

Krick, Erika Lauren; Kiupel, Matti; Durham, Amy C; Thaiwong, Tuddow; Brown, Dorothy C; Sorenmo, Karin U

Previous studies have evaluated cellular proliferation indices, KIT expression, and c-kit mutations to predict the clinical behavior of canine mast cell tumors (MCTs). The study purpose was to retrospectively compare mitotic index, argyrophilic nucleolar organizer regions (AgNORs)/nucleus, Ki-67 index, KIT labeling pattern, and internal tandem duplication mutations in c-KIT between stage I and stage II grade II MCTs. Medical records and tumor biopsy samples from dogs with Grade II MCTs with cytological or histopathological regional lymph node evaluation were included. Signalment, tumor location and stage, and presence of a recurrent versus de novo tumor were recorded. Mitotic index, AgNORs/nucleus, Ki-67, KIT staining pattern, and internal tandem duplication mutations in exon 11 of c-KIT were evaluated. Sixty-six tumors (51 stage I; 15 stage II) were included. Only AgNORs/nucleus and recurrent tumors were significantly associated with stage (odds ratio 2.8, 95% confidence interval [CI] 1.0-8.0, P = .049; odds ratio 8.8, 95% CI 1.1-69.5; P = .039). Receiver-operator characteristic analysis showed that the sensitivity and specificity of AgNORs/cell ≥ 1.87 were 93.3% and 27.4%, respectively, (area under the curve: 0.65) for predicting stage. Recurrent tumors and higher AgNORs/nucleus are associated with stage II grade II MCTs; however, an AgNOR cutoff value that reliably predicts lymph node metastasis was not determined.

ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES SRC-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)

EPA Science Inventory

ZINC-INDUCED EGF RECEPTOR SIGNALING REQUIRES Src-MEDIATED PHOSPHORYLATION OF THE EGF RECEPTOR ON TYROSINE 845 (Y845)
Weidong Wu1, Lee M. Graves2, Gordon N. Gill3 and James M. Samet4 1Center for Environmental Medicine and Lung Biology; 2Department of Pharmacology, University o...

Molecular Alterations of KIT Oncogene in Gliomas

PubMed Central

Gomes, Ana L.; Reis-Filho, Jorge S.; Lopes, José M.; Martinho, Olga; Lambros, Maryou B. K.; Martins, Albino; Schmitt, Fernando; Pardal, Fernando; Reis, Rui M.

2007-01-01

Gliomas are the most common and devastating primary brain tumours. Despite therapeutic advances, the majority of gliomas do not respond either to chemo or radiotherapy. KIT, a class III receptor tyrosine kinase (RTK), is frequently involved in tumourigenic processes. Currently, KIT constitutes an attractive therapeutic target. In the present study we assessed the frequency of KIT overexpression in gliomas and investigated the genetic mechanisms underlying KIT overexpression. KIT (CD117) immunohistochemistry was performed in a series of 179 gliomas of various grades. KIT activating gene mutations (exons 9, 11, 13 and 17) and gene amplification analysis, as defined by chromogenic in situ hybridization (CISH) and quantitative real-time PCR (qRT-PCR) were performed in CD117 positive cases. Tumour cell immunopositivity was detected in 15.6% (28/179) of cases, namely in 25% (1/4) of pilocytic astrocytomas, 25% (5/20) of diffuse astrocytomas, 20% (1/5) of anaplastic astrocytomas, 19.5% (15/77) of glioblastomas and one third (3/9) of anaplastic oligoastrocytomas. Only 5.7% (2/35) of anaplastic oligodendrogliomas showed CD117 immunoreactivity. No association was found between tumour CD117 overexpression and patient survival. In addition, we also observed CD117 overexpression in endothelial cells, which varied from 0–22.2% of cases, being more frequent in high-grade lesions. No KIT activating mutations were identified. Interestingly, CISH and/or qRT-PCR analysis revealed the presence of KIT gene amplification in 6 glioblastomas and 2 anaplastic oligoastrocytomas, corresponding to 33% (8/24) of CD117 positive cases. In conclusion, our results demonstrate that KIT gene amplification rather than gene mutation is a common genetic mechanism underlying KIT expression in subset of malignant gliomas. Further studies are warranted to determine whether glioma patients exhibiting KIT overexpression and KIT gene amplification may benefit from therapy with anti-KIT RTK inhibitors. PMID

Autocrine expression of the epidermal growth factor receptor ligand heparin-binding EGF-like growth factor in cervical cancer.

PubMed

Schrevel, Marlies; Osse, E Michelle; Prins, Frans A; Trimbos, J Baptist M Z; Fleuren, Gert Jan; Gorter, Arko; Jordanova, Ekaterina S

2017-06-01

In cervical cancer, the epidermal growth factor receptor (EGFR) is overexpressed in 70-90% of the cases and has been associated with poor prognosis. EGFR-based therapy is currently being explored in cervical cancer. We investigated which EGFR ligand is primarily expressed in cervical cancer and which cell type functions as the major source of this ligand. We hypothesized that macrophages are the main source of EGFR ligands and that a paracrine loop between tumor cells and macrophages is responsible for ligand expression. mRNA expression analysis was performed on 32 cervical cancer cases to determine the expression of the EGFR ligands amphiregulin, β-cellulin, epidermal growth factor (EGF), epiregulin, heparin-binding EGF-like growth factor (HB‑EGF) and transforming growth factor α (TGFα). Subsequently, protein expression was determined immunohistochemically on 36 additional cases. To assess whether macrophages are the major source of EGFR ligands, immunohistochemical double staining was performed on four representative tissue slides. Expression of the chemokines granulocyte-macrophage colony-stimulating factor (GM-CSF) and C-C motif ligand 2 (CCL2) was determined by mRNA in situ hybridization. Of the known EGFR ligands, HB‑EGF had the highest mRNA expression and HB‑EGF and EGFR protein expression were highly correlated. Tumor specimens with high EGFR expression showed higher numbers of macrophages, and higher expression of GM-CSF and CCL2, but only a small subset (9%) of macrophages was found to be HB‑EGF-positive. Strikingly, 78% of cervical cancer specimens were found to express HB‑EGF. Standardized assessment of staining intensity, using spectral imaging analysis, showed that HB‑EGF expression was higher in the tumor compartment than in the stromal compartment. These results suggest that HB‑EGF is an important EGFR ligand in cervical cancer and that cervical cancer cells are the predominant source of HB‑EGF. Therefore, we propose an autocrine

Functional TRPV2 and TRPV4 channels in human cardiac c-kit(+) progenitor cells.

PubMed

Che, Hui; Xiao, Guo-Sheng; Sun, Hai-Ying; Wang, Yan; Li, Gui-Rong

2016-06-01

The cellular physiology and biology of human cardiac c-kit(+) progenitor cells has not been extensively characterized and remains an area of active research. This study investigates the functional expression of transient receptor potential vanilloid (TRPV) and possible roles for this ion channel in regulating proliferation and migration of human cardiac c-kit(+) progenitor cells. We found that genes coding for TRPV2 and TRPV4 channels and their proteins are significantly expressed in human c-kit(+) cardiac stem cells. Probenecid, an activator of TRPV2, induced an increase in intracellular Ca(2+) (Ca(2+) i ), an effect that may be attenuated or abolished by the TRPV2 blocker ruthenium red. The TRPV4 channel activator 4α-phorbol 12-13-dicaprinate induced Ca(2+) i oscillations, which can be inhibited by the TRPV4 blocker RN-1734. The alteration of Ca(2+) i by probenecid or 4α-phorbol 12-13-dicprinate was dramatically inhibited in cells infected with TRPV2 short hairpin RNA (shRNA) or TRPV4 shRNA. Silencing TRPV2, but not TRPV4, significantly reduced cell proliferation by arresting cells at the G0/G1 boundary of the cell cycle. Cell migration was reduced by silencing TRPV2 or TRPV4. Western blot revealed that silencing TRPV2 decreased expression of cyclin D1, cyclin E, pERK1/2 and pAkt, whereas silencing TRPV4 only reduced pAkt expression. Our results demonstrate for the first time that functional TRPV2 and TRPV4 channels are abundantly expressed in human cardiac c-kit(+) progenitor cells. TRPV2 channels, but not TRPV4 channels, participate in regulating cell cycle progression; moreover, both TRPV2 and TRPV4 are involved in migration of human cardiac c-kit(+) progenitor cells. © 2016 The Authors. Journal of Cellular and Molecular Medicine published by John Wiley & Sons Ltd and Foundation for Cellular and Molecular Medicine.

Differential effects of c-fms and c-kit ligands on the lineage development of the lymphohematopoietic cell line EML C1.

PubMed

Tsai, S

1996-01-01

The lymphohematopoietic progenitors represent < 0.01% of nucleated marrow cells. We have shown that murine lymphohematopoietic progenitors can be immortalized by a recombinant retroviral vector harboring a dominant-negative retinoic acid (RA) receptor. The immortalized progenitors proliferate as a stem-cell factor-dependent clonal line designated EML C1. The EML C1 cell line spontaneously generates prepro-B-lymphocytes and erythroid and myeloid progenitors. Upon stimulation with interleukin 7 and marrow stromal cells, the prepro-B-lymphocytes express recombination-activating gene 1 (RAG-1) and undergo D-J rearrangements of the immunoglobulin heavy-chain genes. With erythropoietin, the erythroid progenitors proliferate and differentiate into red cells. Generation of the common progenitors for neutrophils and macrophages [colony-forming units-granulocyte-macrophage (CFU-GM)] is suppressed in EML C1 cells but is inducible by high concentrations of RA. An additional block in neutrophil differentiation occurs at the promyelocyte stage, but this can also be overcome by high concentrations of RA. Although c-fms is homologous to c-kit, which encodes the receptor for stem-cell factor (SCF), EML C1 cells neither express c-fms nor respond to macrophage colony-stimulating factor (M-CSF), the ligand for c-fms. Transduction and expression of c-fms cDNA in EML C1 cells confers responsiveness to M-CSF. This finding indicates that c-kit and c-fms share substantially overlapping signal-transduction pathways. However, c-fms-transduced EML C1 cells (EML C1/c-fms cells) exhibit different development patterns when stimulated by SCF alone or by M-CSF alone. When stimulated by SCF alone, EML C1/c-fms cells show mostly erythroid and B-lymphoid development. When stimulated by M-CSF alone, development switches to mostly myeloid (neutrophil and macrophage) development. This observation suggests that c-kit and c-fms must have unique signal-transduction pathways in addition to the common ones.

[Construction and expression of the targeting super-antigen EGF-SEA fusion gene].

PubMed

Xie, Yang; Peng, Shaoping; Liao, Zhiying; Liu, Jiafeng; Liu, Xuemei; Chen, Weifeng

2014-05-01

To construct expression vector for the SEA-EGF fusion gene. Clone the SEA gene and the EGF gene segment with PCR and RT-PCR independently, and connect this two genes by the bridge PCR. Insert the fusion gene EGF-SEA into the expression vector PET-44. Induced the secretion of the fusion protein SEA-EGF by the antileptic. The gene fragment encoding EGF and SEA mature peptide was successfully cloned. The fusion gene EGF-SEA was successfully constructed and was inserted into expression vector. The new recombinant expression vector for fusion gene EGF-SEA is specific for head and neck cancer, laid the foundation for the further study of fusion protein SEA-EGF targeting immune therapy in head and neck tumors.

Improvement of the Xpert Carba-R Kit for the Detection of Carbapenemase-Producing Enterobacteriaceae

PubMed Central

Fusaro, Mathieu

2016-01-01

The Xpert Carba-R kit, version 2 (v2), which has been improved for the efficient detection of blaOXA-181 and blaOXA-232 genes, was tested on a collection of 150 well-characterized enterobacterial isolates that had a reduced susceptibility to carbapenems. The performance of the Xpert Carba-R v2 was high, as it was able to detect the five major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48). Thus, it is now well adapted to the carbapenemase-producing Enterobacteriaceae epidemiology of many countries worldwide. PMID:27021332

C-kit+ cells isolated from developing kidneys are a novel population of stem cells with regenerative potential

PubMed Central

Rangel, Erika B; Gomes, Samirah A; Dulce, Raul A; Premer, Courtney; Rodrigues, Claudia O; Kanashiro-Takeuchi, Rosemeire M; Oskouei, Behzad; Carvalho, Decio A; Ruiz, Phillip; Reiser, Jochen; Hare, Joshua M

2013-01-01

The presence of tissue specific precursor cells is an emerging concept in organ formation and tissue homeostasis. Several progenitors are described in the kidneys. However, their identity as a true stem cell remains elusive. Here, we identify a neonatal kidney-derived c-kit+ cell population that fulfills all of the criteria as a stem cell. These cells were found in the thick ascending limb of Henle's loop and exhibited clonogenicity, self-renewal, and multipotentiality with differentiation capacity into mesoderm and ectoderm progeny. Additionally, c-kit+ cells formed spheres in nonadherent conditions when plated at clonal density and expressed markers of stem cells, progenitors, and differentiated cells. Ex-vivo expanded c-kit+ cells integrated into several compartments of the kidney, including tubules, vessels, and glomeruli, and contributed to functional and morphological improvement of the kidney following acute ischemia-reperfusion injury in rats. Together these findings document a novel neonatal rat kidney c-kit+ stem cell population that can be isolated, expanded, cloned, differentiated, and employed for kidney repair following acute kidney injury. These cells have important biological and therapeutic implications. PMID:23733311

BRAF, NRAS and C-KIT Advanced Melanoma: Clinico-pathological Features, Targeted-Therapy Strategies and Survival.

PubMed

Ponti, Giovanni; Manfredini, Marco; Greco, Stefano; Pellacani, Giovanni; Depenni, Roberta; Tomasi, Aldo; Maccaferri, Monia; Cascinu, Stefano

2017-12-01

The mutational status of stage III and IV melanomas should be recognized in order to allow for targeted therapies. The aim of our study was the characterization of BRAF, NRAS and C-KIT melanoma patients, in order to define their optimal management. Between 1991 and 2015, 63 mutated melanoma patients were treated and monitored during their diagnostic and therapeutic management at a single institution. BRAF-mutated melanoma patients were the most common, representing 70% of the study population, while NRAS- and C-KIT-mutated melanoma represented 19% and 11% respectively. BRAF-mutated melanomas were mostly located at sites of intermittent sun exposure, and were associated with higher Breslow thickness and an increased number of mitosis. NRAS mutated melanoma were mainly observed in chronic sun-damaged areas and had a negative prognostic value, with shorter time to progression and a high incidence of central nervous system involvement. C-KIT mutated melanoma were located at acral and mucosal sites. Overall survival observed in the three groups of patients revealed wide differences. BRAF, NRAS and C-KIT melanomas constitute distinct clinico-pathological entities. BRAF-mutated melanoma benefit from both anti-BRAF and anti-MEK targeted therapies while triple-negative melanomas could benefit from novel anti-CTLA-4 and anti-PD-L1 immunotherapeutic approaches. Copyright© 2017, International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.

Neoplasia Driven by Mutant c-KIT Is Mediated by Intracellular, Not Plasma Membrane, Receptor Signaling▿

PubMed Central

Xiang, Zhifu; Kreisel, Frederike; Cain, Jennifer; Colson, AnnaLynn; Tomasson, Michael H.

2007-01-01

Activating mutations in c-KIT are associated with gastrointestinal stromal tumors, mastocytosis, and acute myeloid leukemia. In attempting to establish a murine model of human KITD816V (hKITD816V)-mediated leukemia, we uncovered an unexpected relationship between cellular transformation and intracellular trafficking. We found that transport of hKITD816V protein was blocked at the endoplasmic reticulum in a species-specific fashion. We exploited these species-specific trafficking differences and a set of localization domain-tagged KIT mutants to explore the relationship between subcellular localization of mutant KIT and cellular transformation. The protein products of fully transforming KIT mutants localized to the Golgi apparatus and to a lesser extent the plasma membrane. Domain-tagged KITD816V targeted to the Golgi apparatus remained constitutively active and transforming. Chemical inhibition of intracellular transport demonstrated that Golgi localization is sufficient, but plasma membrane localization is dispensable, for downstream signaling mediated by KIT mutation. When expressed in murine bone marrow, endoplasmic reticulum-localized hKITD816V failed to induce disease in mice, while expression of either Golgi-localized HyKITD816V or cytosol-localized, ectodomain-deleted KITD816V uniformly caused fatal myeloproliferative diseases. Taken together, these data demonstrate that intracellular, non-plasma membrane receptor signaling is sufficient to drive neoplasia caused by mutant c-KIT and provide the first animal model of myelomonocytic neoplasia initiated by human KITD816V. PMID:17060458

The role of the C8 proton of ATP in the catalysis of shikimate kinase and adenylate kinase

PubMed Central

2012-01-01

Background It has been demonstrated that the adenyl moiety of ATP plays a direct role in the regulation of ATP binding and/or phosphoryl transfer within a range of kinase and synthetase enzymes. The role of the C8-H of ATP in the binding and/or phosphoryl transfer on the enzyme activity of a number of kinase and synthetase enzymes has been elucidated. The intrinsic catalysis rate mediated by each kinase enzyme is complex, yielding apparent KM values ranging from less than 0.4 μM to more than 1 mM for ATP in the various kinases. Using a combination of ATP deuterated at the C8 position (C8D-ATP) as a molecular probe with site directed mutagenesis (SDM) of conserved amino acid residues in shikimate kinase and adenylate kinase active sites, we have elucidated a mechanism by which the ATP C8-H is induced to be labile in the broader kinase family. We have demonstrated the direct role of the C8-H in the rate of ATP consumption, and the direct role played by conserved Thr residues interacting with the C8-H. The mechanism by which the vast range in KM might be achieved is also suggested by these findings. Results We have demonstrated the mechanism by which the enzyme activities of Group 2 kinases, shikimate kinase (SK) and adenylate kinase 1 (AK1), are controlled by the C8-H of ATP. Mutations of the conserved threonine residues associated with the labile C8-H cause the enzymes to lose their saturation kinetics over the concentration range tested. The relationship between the role C8-H of ATP in the reaction mechanism and the ATP concentration as they influence the saturation kinetics of the enzyme activity is also shown. The SDM clearly identified the amino acid residues involved in both the catalysis and regulation of phosphoryl transfer in SK and AK1 as mediated by C8H-ATP. Conclusions The data outlined serves to demonstrate the “push” mechanism associated with the control of the saturation kinetics of Group 2 kinases mediated by ATP C8-H. It is therefore conceivable

Allelic Heterogeneity at the Equine KIT Locus in Dominant White (W) Horses

PubMed Central

Haase, Bianca; Brooks, Samantha A; Schlumbaum, Angela; Azor, Pedro J; Bailey, Ernest; Alaeddine, Ferial; Mevissen, Meike; Burger, Dominik; Poncet, Pierre-André; Rieder, Stefan; Leeb, Tosso

2007-01-01

White coat color has been a highly valued trait in horses for at least 2,000 years. Dominant white (W) is one of several known depigmentation phenotypes in horses. It shows considerable phenotypic variation, ranging from ∼50% depigmented areas up to a completely white coat. In the horse, the four depigmentation phenotypes roan, sabino, tobiano, and dominant white were independently mapped to a chromosomal region on ECA 3 harboring the KIT gene. KIT plays an important role in melanoblast survival during embryonic development. We determined the sequence and genomic organization of the ∼82 kb equine KIT gene. A mutation analysis of all 21 KIT exons in white Franches-Montagnes Horses revealed a nonsense mutation in exon 15 (c.2151C>G, p.Y717X). We analyzed the KIT exons in horses characterized as dominant white from other populations and found three additional candidate causative mutations. Three almost completely white Arabians carried a different nonsense mutation in exon 4 (c.706A>T, p.K236X). Six Camarillo White Horses had a missense mutation in exon 12 (c.1805C>T, p.A602V), and five white Thoroughbreds had yet another missense mutation in exon 13 (c.1960G>A, p.G654R). Our results indicate that the dominant white color in Franches-Montagnes Horses is caused by a nonsense mutation in the KIT gene and that multiple independent mutations within this gene appear to be responsible for dominant white in several other modern horse populations. PMID:17997609

'W' mutant forms of the Fms receptor tyrosine kinase act in a dominant manner to suppress CSF-1 dependent cellular transformation.

PubMed

Reith, A D; Ellis, C; Maroc, N; Pawson, T; Bernstein, A; Dubreuil, P

1993-01-01

Point mutations in highly conserved amino acid residues in the catalytic domain of the Kit receptor tyrosine kinase (RTK) are responsible for the coat color, fertility and hematopoietic defects of mice bearing mutant alleles at the dominant white-spotting (W) locus. The dominant nature of structural Kit mutations suggests that expression of other kinase-defective RTKs might also specifically interfere with signal transduction by normal receptors. To test this possibility, we have investigated the functional consequences of introducing analogous mutations into the RTK encoded by the c-fms proto-oncogene. Both Fms37 (glu582-->lys) and Fms42 (asp776-->asn) mutant proteins, corresponding to the strongly dominant-negative W37 and W42 mutant c-kit alleles, had undetectable in vitro kinase activity and were unable to transform Rat-2 fibroblasts in the presence of exogenous CSF-1. Moreover, expression of Fms37 or Fms42 proteins in Rat-2 cells specifically inhibited anchorage-independent growth mediated by the normal Fms receptor in the presence of exogenous CSF-1 and conferred a dominant loss of Fms-associated PI3-kinase activity on CSF-1 stimulation. Mutant RTKs, bearing point substitutions identical to those present in mild or severe W mutants, may provide a generally applicable strategy for inducing dominant loss of function defects in RTK-mediated signalling pathways.

TGF{beta} induces proHB-EGF shedding and EGFR transactivation through ADAM activation in gastric cancer cells

DOE Office of Scientific and Technical Information (OSTI.GOV)

Ebi, Masahide; Kataoka, Hiromi, E-mail: hkataoka@med.nagoya-cu.ac.jp; Shimura, Takaya

2010-11-19

Research highlights: {yields} TGF{beta} induces EGFR transactivation through proHB-EGF shedding by activated ADAM members in gastric cancer cells. {yields} TGF{beta} induces nuclear translocation of HB-EGF-CTF cleaved by ADAM members. {yields} TGF{beta} enhances cell growth by EGFR transactivation and HB-EGF-CTF nuclear translocation and ADAM inhibitors block these effects. {yields} Silencing of ADAM17 also blocks EGFR transactivation, HB-EGF-CTF nuclear translocation and cancer cell growth by TGF{beta}. {yields} ADAM17 may play a crucial role in this TGF{beta}-HB-EGF signal transduction. -- Abstract: Background and aims: Transforming growth factor-beta (TGF{beta}) is known to potently inhibit cell growth. Loss of responsiveness to TGF{beta} inhibition on cellmore » growth is a hallmark of many types of cancer, yet its mechanism is not fully understood. Membrane-anchored heparin-binding EGF-like growth factor (proHB-EGF) ectodomain is cleaved by a disintegrin and metalloproteinase (ADAM) members and is implicated in epidermal growth factor receptor (EGFR) transactivation. Recently, nuclear translocation of the C-terminal fragment (CTF) of pro-HB-EGF was found to induce cell growth. We investigated the association between TGF{beta} and HB-EGF signal transduction via ADAM activation. Materials and methods: The CCK-8 assay in two gastric cancer cell lines was used to determine the effect for cell growth by TGF{beta}. The effect of two ADAM inhibitors was also evaluated. Induction of EGFR phosphorylation by TGF{beta} was analyzed and the effect of the ADAM inhibitors was also examined. Nuclear translocation of HB-EGF-CTF by shedding through ADAM activated by TGF{beta} was also analyzed. EGFR transactivation, HB-EGF-CTF nuclear translocation, and cell growth were examined under the condition of ADAM17 knockdown. Result: TGF{beta}-induced EGFR phosphorylation of which ADAM inhibitors were able to inhibit. TGF{beta} induced shedding of proHB-EGF allowing HB-EGF-CTF to

SunWise[R] Meteorologist Tool Kit

ERIC Educational Resources Information Center

US Environmental Protection Agency, 2007

2007-01-01

The SunWise Program is designed to help meteorologists raise sun safety awareness by addressing the science of the sun, the risk of overexposure to its ultraviolet (UV) radiation, and what students and their families can do to protect themselves from overexposure. This Tool Kit has been designed for use all over the United States and its…

Interaction of AIP with protein kinase A (cAMP-dependent protein kinase).

PubMed

Schernthaner-Reiter, Marie Helene; Trivellin, Giampaolo; Stratakis, Constantine A

2018-05-02

Germline mutations in the aryl hydrocarbon receptor-interacting protein (AIP) gene cause mostly somatotropinomas and/or prolactinomas in a subset of familial isolated pituitary adenomas (FIPA). AIP has been shown to interact with phosphodiesterases (PDEs) and G proteins, suggesting a link to the cyclic AMP (cAMP)-dependent protein kinase (PKA) pathway. Upregulation of PKA is seen in sporadic somatotropinomas that carry GNAS1 mutations, and those in Carney complex that are due to PRKAR1A mutations. To elucidate the mechanism of AIP-dependent pituitary tumorigenesis, we studied potential functional and physical interactions of AIP with PKA's main subunits PRKAR1A (R1α) and PRKACA (Cα). We found that AIP physically interacts with both R1α and Cα; this interaction is enhanced when all three components are present, but maintained during Cα-R1α dissociation by PKA pathway activation, indicating that AIP binds Cα/R1α both in complex and separately. The interaction between AIP and R1α/Cα is reduced when the frequent AIP pathogenic mutation p.R304* is present. AIP protein levels are regulated both by translation and the ubiquitin/proteasome pathway and Cα stabilizes both AIP and R1α protein levels. AIP reduction by siRNA leads to an increase of PKA pathway activity, which is disproportionately enhanced during PDE4-inhibition. We show that AIP interacts with the PKA pathway on multiple levels, including a physical interaction with both the main regulatory (R1α) and catalytic (Cα) PKA subunits and a functional interaction with PDE4-dependent PKA activation. These findings provide novel insights on the mechanisms of AIP-dependent pituitary tumorigenesis.

Protein kinase inhibitor peptide (PKI): a family of endogenous neuropeptides that modulate neuronal cAMP-dependent protein kinase function.

PubMed

Dalton, George D; Dewey, William L

2006-02-01

Signal transduction cascades involving cAMP-dependent protein kinase are highly conserved among a wide variety of organisms. Given the universal nature of this enzyme it is not surprising that cAMP-dependent protein kinase plays a critical role in numerous cellular processes. This is particularly evident in the nervous system where cAMP-dependent protein kinase is involved in neurotransmitter release, gene transcription, and synaptic plasticity. Protein kinase inhibitor peptide (PKI) is an endogenous thermostable peptide that modulates cAMP-dependent protein kinase function. PKI contains two distinct functional domains within its amino acid sequence that allow it to: (1) potently and specifically inhibit the activity of the free catalytic subunit of cAMP-dependent protein kinase and (2) export the free catalytic subunit of cAMP-dependent protein kinase from the nucleus. Three distinct PKI isoforms (PKIalpha, PKIbeta, PKIgamma) have been identified and each isoform is expressed in the brain. PKI modulates neuronal synaptic activity, while PKI also is involved in morphogenesis and symmetrical left-right axis formation. In addition, PKI also plays a role in regulating gene expression induced by cAMP-dependent protein kinase. Future studies should identify novel physiological functions for endogenous PKI both in the nervous system and throughout the body. Most interesting will be the determination whether functional differences exist between individual PKI isoforms which is an intriguing possibility since these isoforms exhibit: (1) cell-type specific tissue expression patterns, (2) different potencies for the inhibition of cAMP-dependent protein kinase activity, and (3) expression patterns that are hormonally, developmentally and cell-cycle regulated. Finally, synthetic peptide analogs of endogenous PKI will continue to be invaluable tools that are used to elucidate the role of cAMP-dependent protein kinase in a variety of cellular processes throughout the nervous

"String theory" of c-kit(pos) cardiac cells: a new paradigm regarding the nature of these cells that may reconcile apparently discrepant results.

PubMed

Keith, Matthew C L; Bolli, Roberto

2015-03-27

Although numerous preclinical investigations have consistently demonstrated salubrious effects of c-kit(pos) cardiac cells administered after myocardial infarction, the mechanism of action remains highly controversial. We and others have found little or no evidence that these cells differentiate into mature functional cardiomyocytes, suggesting paracrine effects. In this review, we propose a new paradigm predicated on a comprehensive analysis of the literature, including studies of cardiac development; we have (facetiously) dubbed this conceptual construct "string theory" of c-kit(pos) cardiac cells because it reconciles multifarious and sometimes apparently discrepant results. There is strong evidence that, during development, the c-kit receptor is expressed in different pools of cardiac progenitors (some capable of robust cardiomyogenesis and others with little or no contribution to myocytes). Accordingly, c-kit positivity, in itself, does not define the embryonic origins, lineage capabilities, or differentiation capacities of specific cardiac progenitors. C-kit(pos) cells derived from the first heart field exhibit cardiomyogenic potential during development, but these cells are likely depleted shortly before or after birth. The residual c-kit(pos) cells found in the adult heart are probably of proepicardial origin, possess a mesenchymal phenotype (resembling bone marrow mesenchymal stem/stromal cells), and are capable of contributing significantly only to nonmyocytic lineages (fibroblasts, smooth muscle cells, and endothelial cells). If these 2 populations (first heart field and proepicardium) express different levels of c-kit, the cardiomyogenic potential of first heart field progenitors might be reconciled with recent results of c-kit(pos) cell lineage tracing studies. The concept that c-kit expression in the adult heart identifies epicardium-derived, noncardiomyogenic precursors with a mesenchymal phenotype helps to explain the beneficial effects of c-kit

Dual Targeting of Insulin Receptor and KIT in Imatinib-Resistant Gastrointestinal Stromal Tumors.

PubMed

Chen, Weicai; Kuang, Ye; Qiu, Hai-Bo; Cao, Zhifa; Tu, Yuqing; Sheng, Qing; Eilers, Grant; He, Quan; Li, Hai-Long; Zhu, Meijun; Wang, Yuexiang; Zhang, Rongqing; Wu, Yeqing; Meng, Fanguo; Fletcher, Jonathan A; Ou, Wen-Bin

2017-09-15

Oncogenic KIT or PDGFRA receptor tyrosine kinase (RTK) mutations are compelling therapeutic targets in gastrointestinal stromal tumors (GIST), and treatment with the KIT/PDGFRA inhibitor imatinib is the standard of care for patients with metastatic GIST. Most GISTs eventually acquire imatinib resistance due to secondary mutations in the KIT kinase domain, but it is unclear whether these genomic resistance mechanisms require other cellular adaptations to create a clinically meaningful imatinib-resistant state. Using phospho-RTK and immunoblot assays, we demonstrate activation of KIT and insulin receptor (IR) in imatinib-resistant GIST cell lines (GIST430 and GIST48) and biopsies with acquisition of KIT secondary mutations, but not in imatinib-sensitive GIST cells (GIST882 and GIST-T1). Treatment with linsitinib, a specific IR inhibitor, inhibited IR and downstream intermediates AKT, MAPK, and S6 in GIST430 and GIST48, but not in GIST882, exerting minimal effect on KIT phosphorylation in these cell lines. Additive effects showing increased apoptosis, antiproliferative effects, cell-cycle arrest, and decreased pAKT and pS6 expression, tumor growth, migration, and invasiveness were observed in imatinib-resistant GIST cells with IR activation after coordinated inhibition of IR and KIT by linsitinib (or IR shRNA) and imatinib, respectively, compared with either intervention alone. IGF2 overexpression was responsible for IR activation in imatinib-resistant GIST cells, whereas IR activation did not result from IR amplification, IR mutation, or KIT phosphorylation. Our findings suggest that combinatorial inhibition of IR and KIT warrants clinical evaluation as a novel therapeutic strategy in imatinib-resistant GISTs. Cancer Res; 77(18); 5107-17. ©2017 AACR . ©2017 American Association for Cancer Research.

Improvement of the Xpert Carba-R Kit for the Detection of Carbapenemase-Producing Enterobacteriaceae.

PubMed

Dortet, Laurent; Fusaro, Mathieu; Naas, Thierry

2016-06-01

The Xpert Carba-R kit, version 2 (v2), which has been improved for the efficient detection of blaOXA-181 and blaOXA-232 genes, was tested on a collection of 150 well-characterized enterobacterial isolates that had a reduced susceptibility to carbapenems. The performance of the Xpert Carba-R v2 was high, as it was able to detect the five major carbapenemases (NDM, VIM, IMP, KPC, and OXA-48). Thus, it is now well adapted to the carbapenemase-producing Enterobacteriaceae epidemiology of many countries worldwide. Copyright © 2016, American Society for Microbiology. All Rights Reserved.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor.

PubMed

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Veach, Darren; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R; DeMatteo, Ronald P; Besmer, Peter

2017-10-03

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic Kit V558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant Kit V558Δ/+ mice, double-mutant Kit V558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in Kit V558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing Kit V558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha-restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant Kit V558Δ;T669I/+ tumors.

Direct engagement of the PI3K pathway by mutant KIT dominates oncogenic signaling in gastrointestinal stromal tumor

PubMed Central

Bosbach, Benedikt; Rossi, Ferdinand; Yozgat, Yasemin; Loo, Jennifer; Zhang, Jennifer Q.; Berrozpe, Georgina; Warpinski, Katherine; Ehlers, Imke; Kwok, Andrew; Manova, Katia; Antonescu, Cristina R.; DeMatteo, Ronald P.; Besmer, Peter

2017-01-01

Gastrointestinal stromal tumors (GISTs) predominantly harbor activating mutations in the receptor tyrosine kinase KIT. To genetically dissect in vivo the requirement of different signal transduction pathways emanating from KIT for tumorigenesis, the oncogenic KitV558Δ mutation was combined with point mutations abrogating specific phosphorylation sites on KIT. Compared with single-mutant KitV558Δ/+ mice, double-mutant KitV558Δ;Y567F/Y567F knock-in mice lacking the SRC family kinase-binding site on KIT (pY567) exhibited attenuated MAPK signaling and tumor growth. Surprisingly, abrogation of the PI3K-binding site (pY719) in KitV558Δ;Y719F/Y719F mice prevented GIST development, although the interstitial cells of Cajal (ICC), the cells of origin of GIST, were normal. Pharmacologic inhibition of the PI3K pathway in tumor-bearing KitV558Δ/+ mice with the dual PI3K/mTOR inhibitor voxtalisib, the pan-PI3K inhibitor pilaralisib, and the PI3K-alpha–restricted inhibitor alpelisib each diminished tumor proliferation. The addition of the MEK inhibitor PD-325901 or binimetinib further decreased downstream KIT signaling. Moreover, combining PI3K and MEK inhibition was effective against imatinib-resistant KitV558Δ;T669I/+ tumors. PMID:28923937

Identification of a novel phosphorylation site in c-jun directly targeted in vitro by protein kinase D

DOE Office of Scientific and Technical Information (OSTI.GOV)

Waldron, Richard T.; Whitelegge, Julian P.; Faull, Kym F.

Protein kinase D (PKD) phosphorylates the c-jun amino-terminal in vitro at site(s) distinct from JNK [C. Hurd, R.T. Waldron, E. Rozengurt, Protein kinase D complexes with c-jun N-terminal kinase via activation loop phosphorylation and phosphorylates the c-jun N-terminus, Oncogene 21 (2002) 2154-2160], but the sites have not been identified. Here, metabolic {sup 32}P-labeling of c-jun protein in COS-7 cells indicated that PKD phosphorylates c-jun in vivo at a site(s) between aa 43-93, a region containing important functional elements. On this basis, the PKD-mediated phosphorylation site(s) was further characterized in vitro using GST-c-jun fusion proteins. PKD did not incorporate phosphate intomore » Ser63 and Ser73, the JNK sites in GST-c-jun(1-89). Rather, PKD and JNK could sequentially phosphorylate distinct site(s) simultaneously. By mass spectrometry of tryptic phosphopeptides, Ser58 interposed between the JNK-binding portion of the delta domain and the adjacent TAD1 was identified as a prominent site phosphorylated in vitro by PKD. These data were further supported by kinase reactions using truncations or point-mutations of GST-c-jun. Together, these data suggest that PKD-mediated phosphorylation modulates c-jun at the level of its N-terminal functional domains.« less

Blockade of epidermal growth factor- or heregulin-dependent ErbB2 activation with the anti-ErbB2 monoclonal antibody 2C4 has divergent downstream signaling and growth effects.

PubMed

Jackson, James G; St Clair, Patricia; Sliwkowski, Mark X; Brattain, Michael G

2004-04-01

Due to heterodimerization and a variety of stimulating ligands, the ErbB receptor system is both diverse and flexible, which proves particularly advantageous to the aberrant signaling of cancer cells. However, specific mechanisms of how a particular receptor contributes to generating the flexibility that leads to aberrant growth regulation have not been well described. We compared the utilization of ErbB2 in response to epidermal growth factor (EGF) and heregulin stimulation in colon carcinoma cells. Anti-ErbB2 monoclonal antibody 2C4 blocked heregulin-stimulated phosphorylation of ErbB2 and ErbB3; activation of mitogen-activated protein kinase (MAPK), phosphatidylinositol 3'-kinase (PI3K), and Akt; proliferation; and anchorage-independent growth. 2C4 blocked EGF-mediated phosphorylation of ErbB2 and inhibited PI3K/Akt and anchorage-independent growth but did not affect ErbB1 or MAPK. Immunoprecipitations showed that ErbB3 and Grb2-associated binder (Gab) 1 were phosphorylated and associated with PI3K activity after heregulin treatment and that Gab1 and Gab2, but not ErbB3, were phosphorylated and associated with PI3K activity after EGF treatment. These data show that monoclonal antibody 2C4 inhibited all aspects of heregulin signaling as well as anchorage-independent and monolayer growth. Furthermore, we identify ErbB2 as a critical component of EGF signaling to the Gab1/Gab2-PI3K-Akt pathway and anchorage-independent growth, but EGF stimulation of MAPK and monolayer growth can occur efficiently without the contribution of ErbB2.

The cannabinoid receptor inverse agonist AM251 regulates the expression of the EGF receptor and its ligands via destabilization of oestrogen-related receptor α protein

PubMed Central

Fiori, JL; Sanghvi, M; O'Connell, MP; Krzysik-Walker, SM; Moaddel, R; Bernier, M

2011-01-01

BACKGROUND AND PURPOSE AM251 is an inverse agonist of the cannabinoid 1 receptor (CB1R) that can exert ‘off-target’ effects in vitro and in CB1R knock-out mice. AM251 is also potent at modulating tumour cell growth, suggesting that growth factor-mediated oncogenic signalling could be regulated by AM251. Since dysregulation of the EGF receptor has been associated with carcinogenesis, we examined AM251 regulation of EGF receptor (EGFR) expression and function. EXPERIMENTAL APPROACH The various biological functions of AM251 were measured in CB1R-negative human cancer cells. Pharmacological and genetic approaches were used to validate the data. KEY RESULTS The mRNA levels for EGFR and its associated ligands, including HB-EGF, were induced several fold in PANC-1 and HCT116 cells in response to AM251. This event was associated with enhanced expression of EGFR on the cell surface with concomitant increase in EGF-induced cellular responses in AM251-treated cells. Exposure to XCT790, a synthetic inverse agonist of the orphan nuclear oestrogen-related receptor α (ERRα), also induced EGFR and HB-EGF expression to the same extent as AM251, whereas pretreatment with the ERRα-selective agonist, biochanin A, blunted AM251 actions. AM251 promoted the degradation of ERRα protein without loss of the corresponding mRNA. Knock-down of ERRα by siRNA-based approach led to constitutive induction of EGFR and HB-EGF levels, and eliminated the biological responses of AM251 and XCT790. Finally, AM251 displaced diethylstilbestrol prebound to the ligand-binding domain of ERRα. CONCLUSIONS AND IMPLICATIONS AM251 up-regulates EGFR expression and signalling via a novel non-CB1R-mediated pathway involving destabilization of ERRα protein in selected cancer cell lines. PMID:21449913

Metalloproteinase-dependent transforming growth factor-alpha release mediates neurotensin-stimulated MAP kinase activation in human colonic epithelial cells.

PubMed

Zhao, Dezheng; Zhan, Yanai; Koon, Hon Wai; Zeng, Huiyan; Keates, Sarah; Moyer, Mary P; Pothoulakis, Charalabos

2004-10-15

Expression of the neuropeptide neurotensin (NT) and its high affinity receptor (NTR1) is increased during the course of Clostridium difficile toxin A-induced acute colitis, and NTR1 antagonism attenuates the severity of toxin A-induced inflammation. We recently demonstrated in non-transformed human colonic epithelial NCM460 cells that NT treatment caused activation of a Ras-mediated MAP kinase pathway that significantly contributes to NT-induced interleukin-8 (IL-8) secretion. Here we used NCM460 cells, which normally express low levels of NTR1, and NCM460 cells stably transfected with NTR1 to identify the upstream signaling molecules involved in NT-NTR1-mediated MAP kinase activation. We found that inhibition of the epidermal growth factor receptor (EGFR) by either an EGFR neutralizing antibody or by its specific inhibitor AG1478 (0.2 microm) blocked NT-induced MAP kinase activation. Moreover, NT stimulated tyrosine phosphorylation of the EGFR, and pretreatment with a broad spectrum metalloproteinase inhibitor batimastat reduced NT-induced MAP kinase activation. Using neutralizing antibodies against the EGFR ligands EGF, heparin-binding-EGF, transforming growth factor-alpha (TGFalpha), or amphiregulin we have shown that only the anti-TGFalpha antibody significantly decreases NT-induced phosphorylation of EGFR and MAP kinases. Furthermore, inhibition of the EGF receptor by AG1478 significantly reduced NT-induced IL-8 promoter activity and IL-8 secretion. This is the first report demonstrating that NT binding to NTR1 transactivates the EGFR and that this response is linked to NT-mediated proinflammatory signaling. Our findings indicate that matrix metalloproteinase-mediated release of TGFalpha and subsequent EGFR transactivation triggers a NT-mediated MAP kinase pathway that leads to IL-8 gene expression in human colonic epithelial cells.

[99mTc] HYNIC-hEGF, a potential agent for imaging of EGF receptors in vivo: preparation and pre-clinical evaluation.

PubMed

Babaei, Mohammad Hossein; Almqvist, Ylva; Orlova, Anna; Shafii, Mohammad; Kairemo, Kalevi; Tolmachev, Vladimir

2005-06-01

Expression of epidermal growth factor receptors (EGFR) has prognostic and predictive value in many kinds of tumors. Imaging of expression of EGFR in vivo may give valuable diagnostic information. The epidermal growth factor (EGF), a natural ligand, is a possible candidate for the targeting of EGFR. The present study describes a method for preparation of (99m)Tc-EGF via the hydrazinopyridine-3-carboxylic acid (HYNIC) conjugation using tricine and ethylenediamine-N,N'-diacetic acid (EDDA) as co-ligands. Both conjugates bound EGFR expressing cells with nanomolar affinity, and demonstrated good intracellular retention. The complex with EDDA demonstrated much higher stability in blood serum and during cysteine challenge. Biodistribution of (99m)Tc-EDDA-HYNIC-EGF in normal mice demonstrated fast blood clearance of conjugate, and its ability to bind EGFR in vivo. (99m)Tc-EDDA-HYNIC-EGF is a promising candidate for visualization of EGFR expression in vivo.

Cabozantinib Is Active against Human Gastrointestinal Stromal Tumor Xenografts Carrying Different KIT Mutations.

PubMed

Gebreyohannes, Yemarshet K; Schöffski, Patrick; Van Looy, Thomas; Wellens, Jasmien; Vreys, Lise; Cornillie, Jasmien; Vanleeuw, Ulla; Aftab, Dana T; Debiec-Rychter, Maria; Sciot, Raf; Wozniak, Agnieszka

2016-12-01

In the majority of gastrointestinal stromal tumors (GIST), oncogenic signaling is driven by KIT mutations. Advanced GIST is treated with tyrosine kinase inhibitors (TKI) such as imatinib. Acquired resistance to TKI is mainly caused by secondary KIT mutations, but can also be attributed to a switch of KIT dependency to another receptor tyrosine kinase (RTK). We tested the efficacy of cabozantinib, a novel TKI targeting KIT, MET, AXL, and vascular endothelial growth factor receptors (VEGFR), in patient-derived xenograft (PDX) models of GIST, carrying different KIT mutations. NMRI nu/nu mice (n = 52) were bilaterally transplanted with human GIST: UZLX-GIST4 (KIT exon 11 mutation, imatinib sensitive), UZLX-GIST2 (KIT exon 9, imatinib dose-dependent resistance), or UZLX-GIST9 (KIT exon 11 and 17 mutations, imatinib resistant). Mice were grouped as control (untreated), imatinib (50 mg/kg/bid), and cabozantinib (30 mg/kg/qd) and treated orally for 15 days. Cabozantinib resulted in significant tumor regression in UZLX-GIST4 and -GIST2 and delayed tumor growth in -GIST9. In all three models, cabozantinib inhibited the proliferative activity, which was completely absent in UZLX-GIST4 and significantly reduced in -GIST2 and -GIST9. Increased apoptotic activity was observed only in UZLX-GIST4. Cabozantinib inhibited the KIT signaling pathway in UZLX-GIST4 and -GIST2. In addition, compared with both control and imatinib, cabozantinib significantly reduced microvessel density in all models. In conclusion, cabozantinib showed antitumor activity in GIST PDX models through inhibition of tumor growth, proliferation, and angiogenesis, in both imatinib-sensitive and imatinib-resistant models. Mol Cancer Ther; 15(12); 2845-52. ©2016 AACR. ©2016 American Association for Cancer Research.

Evaluation of Altered Stromal/Epithelial Tissue Arrangement of the c-Kit Messaging System in the Control of Breast Cancer

DTIC Science & Technology

2008-07-31

Lipofectamine-mediated transfection of pcDNA3/c-Kit. Chemosensitivity to the c-Kit modulating drugs, imatinib and alpha - fetoprotein -derived peptide (AFPep), was...Cancer Res. 10:3528-3534. 7. Bennett JA, Mesfin FB, Andersen TT, Gierthy JF, and Jacobson HI. (2002). A peptide derived from α- fetoprotein prevents

AMD3100 treatment attenuates pulmonary angiogenesis by reducing the c-kit (+) cells and its pro-angiogenic activity in CBDL rat lungs.

PubMed

Shen, Cheng-Cheng; Chen, Bing; Gu, Jian-Teng; Ning, Jiao-Lin; Zeng, Jing; Yi, Bin; Lu, Kai-Zhi

2018-03-01

Recent studies have shown that pulmonary angiogenesis is an important pathological process in the development of hepatopulmonary syndrome (HPS), and growing evidence has indicated that Stromal cell-derived factor 1/C-X-C chemokine receptor type 4 (SDF-1/CXCR4) axis is involved in pulmonary vascular disease by mediating the accumulation of c-kit+ cells. This study aimed to test the effect of AMD3100, an antagonist of CXCR4, in HPS pulmonary angiogenesis. Common bile duct ligation (CBDL) rats were used as experimental HPS model and were treated with AMD3100 (1.25mg/kg/day, i.p.) or 0.9% saline for 3weeks. The sham rats underwent common bile duct exposure without ligation. The c-kit+ cells accounts and its angiogenic-related functions, prosurvival signals, pulmonary angiogenesis and arterial oxygenation were analysed in these groups. Our results showed that pulmonary SDF-1/CXCR4, Akt, Erk and VEGF/VEGFR2 were significantly activated in CBDL rats, and the numbers of circulating and pulmonary c-kit+ cells were increased in CBDL rats compared with control rats. Additionally, the angiogenic-related functions of c-kit+ cells and pulmonary microvessel counts were also elevated in CBDL rats. CXCR4 inhibition reduced pulmonary c-kit+ cells and microvessel counts and improved arterial oxygenation within 3weeks in CBDL rats. The pulmonary prosurvival signals and pro-angiogenic activity of c-kit+ cells were also down-regulated in AMD3100-treated rats. In conclusion, AMD3100 treatment attenuated pulmonary angiogenesis in CBDL rats and prevented the development of HPS via reductions in pulmonary c-kit+ cells and inhibition of the prosurvival signals. Our study provides new insights in HPS treatment. Copyright © 2017. Published by Elsevier B.V.

c-Jun controls the efficiency of MAP kinase signaling by transcriptional repression of MAP kinase phosphatases

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sprowles, Amy; Robinson, Dan; Wu Yimi

2005-08-15

The mammalian JNK signaling pathway regulates the transcriptional response of cells to environmental stress, including UV irradiation. This signaling pathway is composed of a classical MAP kinase cascade; activation results in phosphorylation of the transcription factor substrates c-Jun and ATF2, and leads to changes in gene expression. The defining components of this pathway are conserved in the fission yeast S. pombe, where the genetic studies have shown that the ability of the JNK homolog Spc1 to be activated in response to UV irradiation is dependent on the presence of the transcription factor substrate Atf1. We have used genetic analysis tomore » define the role of c-Jun in activation of the mammalian JNK signaling pathway. Our results show that optimal activation of JNK requires the presence of its transcription factor substrate c-Jun. Mutational analysis shows that the ability of c-Jun to support efficient activation of JNK requires the ability of Jun to bind DNA, suggesting a transcriptional mechanism. Consistent with this, we show that c-Jun represses the expression of several MAP kinase phosphatases. In the absence of c-Jun, the increased expression of MAP kinase phosphatases leads to impaired activation of the ERK, JNK, and p38 MAP kinases after pathway activation. The results show that one function of c-Jun is to regulate the efficiency of signaling by the ERK, p38, and JNK MAP kinases, a function that is likely to affect cellular responses to many different stimuli.« less

Regulation of Ca(2+)/calmodulin-dependent protein kinase kinase alpha by cAMP-dependent protein kinase: I. Biochemical analysis.

PubMed

Okuno, S; Kitani, T; Fujisawa, H

2001-10-01

Ca(2+)/calmodulin-dependent protein kinases (CaM-kinases) I and IV are activated upon phosphorylation of their Thr(177) and Thr(196), respectively, by the upstream Ca(2+)/calmodulin-dependent protein kinases CaM-kinase kinase alpha and beta, and deactivated upon dephosphorylation by protein phosphatases such as CaM-kinase phosphatase. Recent studies demonstrated that the activity of CaM-kinase kinase alpha is decreased upon phosphorylation by cAMP-dependent protein kinase (PKA), and the relationship between the inhibition and phosphorylation of CaM-kinase kinase alpha by PKA has been studied. In the present study, we demonstrate that the activity of CaM-kinase kinase alpha toward PKIV peptide, which contains the sequence surrounding Thr(196) of CaM-kinase IV, is increased by incubation with PKA in the presence of Ca(2+)/calmodulin but decreased in its absence, while the activity toward CaM-kinase IV is decreased by incubation with PKA in both the presence and absence of Ca(2+)/calmodulin. Six phosphorylation sites on CaM-kinase kinase alpha, Ser(24) for autophosphorylation, and Ser(52), Ser(74), Thr(108), Ser(458), and Ser(475) for phosphorylation by PKA, were identified by amino acid sequence analysis of the phosphopeptides purified from the tryptic digest of the phosphorylated enzymes. The presence of Ca(2+)/calmodulin suppresses phosphorylation on Ser(52), Ser(74), Thr(108), and Ser(458) by PKA, but accelerates phosphorylation on Ser(475). The changes in the activity of the enzyme upon phosphorylation appear to occur as a result of conformational changes induced by phosphorylation on several sites.

[Development of ELISA-kit of quantitative analysis for Zearalenone].

PubMed

Wang, Yu-ping; Ji, Rong; Jiang, Tao; Kang, Wei-jun

2006-03-01

To develope a rapid, sensitive, quantitative ELISA-kit for Zearalenone and determine zearalenone in cereals. On the base of monoclonal antibodies against ZEN, apply indirect ELISA to study the performance parameter of the kit. The limited concentration of detection of the ELISA-kit was 1ng/ml, linear range was 1-200 ng/ml, the linear equation was Y = 0.99 - 0.40 x (R2 = 0.99). The inhibition concentration of 50% against ZEN was 16.3 ng/ml. The average recovery rate of spiked corn and wheat was 96.5% and 95.5%, respectively, the coefficient of variant was 13.2% and 10.9%, respectively. The kit can be stored at 4 degrees C over 6 months. The cross reaction rate with the other mycotoxins was less than 1%, and coefficient of variant within-laboratory and between-laboratory was less than 15% and less than 20%, respectively. Detecting the VICAM sample with ELISA method and HPLC method, the results were within the range of the sample, and there was no statistic difference between the two methods. This ELISA-kit was quick, sensitive, stable and specific and can be used to determine ZEN in cereals.

c-Kit Mutation and Localization Status as Response Predictors in Mast Cell Tumors in Dogs Treated with Prednisone and Toceranib or Vinblastine.

PubMed

Weishaar, K M; Ehrhart, E J; Avery, A C; Charles, J B; Elmslie, R E; Vail, D M; London, C A; Clifford, C A; Eickhoff, J C; Thamm, D H

2018-01-01

KIT inhibitors, such as toceranib (TOC), and vinblastine (VBL) have not been prospectively compared in the treatment of macroscopic mast cell tumors (MCTs). Also, it is unknown whether VBL or TOC is superior for treating MCT without c-kit mutations. To determine the value of KIT genotyping and localization in treatment decisions for dogs with macroscopic MCT. We hypothesized that c-kit mutated MCT would have a better response to TOC than VBL. Eighty-eight client-owned dogs with macroscopic MCT. Prospective, randomized trial. Dogs were randomized to TOC (2.75 mg/kg EOD) or VBL (2.5 mg/m 2 weekly × 4 then EOW) by KIT localization and c-kit mutation status using an adaptive randomization scheme. Sixty dogs were allocated to TOC and 28 to VBL. Of the dogs receiving TOC, 20% had c-kit mutations, compared to 30% receiving VBL (P = 0.74). Overall response rates were 46% (TOC) and 30% (VBL) (odds ratio = 1.56 [0.62-3.92]; P = 0.28). Median progression-free survival (PFS) for dogs receiving VBL was 78 days (7-1,521) and for TOC 95.5 (14-990); hazard ratio (HR) = 1.34 [0.72-2.50]; P = 0.36. Median overall survival (OS) was 241.5 days (10-1,521) for the VBL group and 159 (20-990) for the TOC group; HR = 0.80 ([0.45-1.41]; P = 0.44). Neither PFS nor OS was significantly different between treatment groups. As the proportion of dogs with c-kit mutations was not different between treatment groups in this population of dogs, c-kit mutation status did not predict treatment response. Copyright © 2017 The Authors. Journal of Veterinary Internal Medicine published by Wiley Periodicals, Inc. on behalf of the American College of Veterinary Internal Medicine.

Recent Advances of Colony-Stimulating Factor-1 Receptor (CSF-1R) Kinase and Its Inhibitors.

PubMed

El-Gamal, Mohammed I; Al-Ameen, Shahad K; Al-Koumi, Dania M; Hamad, Mawadda G; Jalal, Nouran A; Oh, Chang-Hyun

2018-01-17

Colony stimulation factor-1 receptor (CSF-1R), which is also known as FMS kinase, plays an important role in initiating inflammatory, cancer, and bone disorders when it is overstimulated by its ligand, CSF-1. Innate immunity, as well as macrophage differentiation and survival, are regulated by the stimulation of the CSF-1R. Another ligand, interlukin-34 (IL-34), was recently reported to activate the CSF-1R receptor in a different manner. The relationship between CSF-1R and microglia has been reviewed. Both CSF-1 antibodies and small molecule CSF-1R kinase inhibitors have now been tested in animal models and in humans. In this Perspective, we discuss the role of CSF-1 and IL-34 in producing cancer, bone disorders, and inflammation. We also review the newly discovered and improved small molecule kinase inhibitors and monoclonal antibodies that have shown potent activity toward CSF-1R, reported from 2012 until 2017.

The heterozygous R1441C mutation of leucine-rich repeat kinase 2 gene in a Chinese patient with Parkinson disease: A five-year follow-up and literatures review.

PubMed

Peng, Fang; Sun, Yi-Min; Chen, Chen; Luo, Su-Shan; Li, Da-Ke; Wang, Yi-Xuan; Yang, Ke; Liu, Feng-Tao; Zuo, Chuan-Tao; Ding, Zheng-Tong; An, Yu; Wu, Jian-Jun; Wang, Jian

2017-02-15

Leucine-rich repeat kinase 2 gene (LRRK2) was recognized associated with both familial and sporadic Parkinson Disease (PD). Seven missense mutations (G2019S, R1441C, R1441G, R1441H, Y1699C, I2020T, N1437H) of it have been confirmed disease- causing. They were common among Caucasian PD patients, but rarely reported in Asian, especially in Chinese Han population. We aimed to identify the frequencies of these seven mutations of LRRK2 in Chinese early-onset PD (EOPD) patients and analyze the phenotypes. One hundred and thirty seven EOPD patients were enrolled for genetic testing. The seven disease-causing mutations of LRRK2 were carried out by target sequencing using Illumina HiSeq 2000 Sequencer. The identified variants were further confirmed by Sanger sequence. The clinical materials were investigated retrospectively. Only one patient (0.73%) was found carrying pathogenetic LRRK2 mutation of R1441C. The age at onset of the female patient was 44. She manifested typical motor symptoms of PD and responded well to levodopa therapy. Longitudinal evaluation showed progression of motor symptoms and depression but no cognitive impairment. The dopamine transporter (DAT) imaging via [11C]-2β-carbomethoxy-3β-(4-fluorophenyl) tropan (CFT) and Positron emission computed tomography (PET) revealed typical dopamine transporter uptake reduction. The LRRK2 R1441C mutation was found in a Chinese EOPD patient for the first time. The manifestations of LRRK2-R1441C carriers were indistinguishable from sporadic PD patients. Copyright © 2016 Elsevier B.V. All rights reserved.

TCDD AND EGF AFFECT MAPK PATHWAY ACTIVATION IN MURINE EMBRYONIC PALATE

EPA Science Inventory

Palatal fusion occurs on GD 14-15 in the mouse, accompanied by a decrease in EGF receptor (EGFR) at the medial edge of the palatal shelves. 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) induces cleft palate and maintains EGF and EGF receptor (EGFR) expression levels in the medial ed...

Function-specific intracellular signaling pathways downstream of heparin-binding EGF-like growth factor utilized by human trophoblasts.

PubMed

Jessmon, Philip; Kilburn, Brian A; Romero, Roberto; Leach, Richard E; Armant, D Randall

2010-05-01

Heparin-binding EGF-like growth factor (HBEGF) is expressed by trophoblast cells throughout gestation. First-trimester cytotrophoblast cells are protected from hypoxia-induced apoptosis because of the accumulation of HBEGF through a posttranscriptional autocrine mechanism. Exogenous application of HBEGF is cytoprotective in a hypoxia/reoxygenation (H/R) injury model and initiates trophoblast extravillous differentiation to an invasive phenotype. The downstream signaling pathways induced by HBEGF that mediate these various cellular activities were identified using two human first-trimester cytotrophoblast cell lines, HTR-8/SVneo and SW.71, with similar results. Recombinant HBEGF (1 nM) induced transient phosphorylation of MAPK3/1 (ERK), MAPK14 (p38), and AKT within 15 min and JNK after 1-2 h. To determine which downstream pathways regulate the various functions of HBEGF, cells were treated with specific inhibitors of the ERK upstream regulator MEK (U0126), the AKT upstream regulator phosphoinositide-3 (PI3)-kinase (LY294002), MAPK14 (SB203580), and JNK (SP600125), as well as with inactive structural analogues. Only SB203580 specifically prevented HBEGF-mediated rescue during H/R, while each inhibitor attenuated HBEGF-stimulated cell migration. Accumulation of HBEGF at reduced oxygen was blocked only by a combination of U0126, SB203580, and SP600125. We conclude that HBEGF advances trophoblast extravillous differentiation through coordinate activation of PI3 kinase, ERK, MAPK14, and JNK, while only MAPK14 is required for its antiapoptotic activity. Additionally, hypoxia induces an autocrine increase in HBEGF protein levels through MAPK14, JNK or ERK. These experiments reveal a complexity of the intracellular signaling circuitry that regulates trophoblast functions critical for implantation and placentation.

Repeated dose intramuscular injection of the CIMAvax-EGF vaccine in Sprague Dawley rats induces local and systemic toxicity.

PubMed

Mancebo, A; Casacó, A; González, B; Ledón, N; Sorlozabal, J; León, A; Gómez, D; González, Y; Bada, A M; González, C; Arteaga, M E; Ramírez, H; Fuentes, D

2012-05-09

CIMAvax-EGF consists of a human recombinant epidermal growth factor (EGF), coupled to P64k, a recombinant carrier protein from N. meningitis, and Montanide ISA 51 as adjuvant. The vaccine immunization induces a specific antibody production, inhibiting the EGF/EGF-R interaction through EGF deprivation. The objective of this study was to assess the CIMAvax-EGF toxicity in Sprague Dawley rats after intramuscular administration of repeated doses (6 months) and at the same time to determine if rat is a relevant species for studying CIMAvax-EGF vaccine. Rats were randomly distributed into four groups: control, Montanide ISA 51, treated with 1× and 15× of human total dose of the antigen. Animals were immunized weekly during 9 weeks, plus 9 immunizations every 14 days. Rats were inspected daily for clinical signs. Body weight, food consumption, and rectal temperature were measured during the administration of doses. Blood samples were collected for hematological, serum biochemical determinations and EGF titles at the beginning, three months and at the end of experimentation. Gross necropsy and histological examination of tissues were performed on animals at the end of the assay. Vaccine provoked the apparition of antibodies against EGF in the rats, demonstrating rat species relevance in these studies. Body weight gain, food and water consumption were not affected. CIMAvax-EGF and Montanide ISA 51 produced local damage at the administration site, showing multiple cysts and granulomas. Both vaccine-treated groups showed neutrophil elevation, besides an AST increase probably related to the damage at the administration site. Rectal temperature was found to be significantly higher in 15× treated group after immunizations, probably induced by the inflammatory process at the injection site. In summary, the clinical pathology findings together with the body temperature results, appear to be caused by the inflammatory reaction at the administration site of the vaccine, mainly

A Frameshift Mutation in KIT is Associated with White Spotting in the Arabian Camel

PubMed Central

Holl, Heather; Isaza, Ramiro; Mohamoud, Yasmin; Ahmed, Ayeda; Almathen, Faisal; Youcef, Cherifi; Gaouar, Semir; Antczak, Douglas F.; Brooks, Samantha

2017-01-01

While the typical Arabian camel is characterized by a single colored coat, there are rare populations with white spotting patterns. White spotting coat patterns are found in virtually all domesticated species, but are rare in wild species. Theories suggest that white spotting is linked to the domestication process, and is occasionally associated with health disorders. Though mutations have been found in a diverse array of species, fewer than 30 genes have been associated with spotting patterns, thus providing a key set of candidate genes for the Arabian camel. We obtained 26 spotted camels and 24 solid controls for candidate gene analysis. One spotted and eight solid camels were whole genome sequenced as part of a separate project. The spotted camel was heterozygous for a frameshift deletion in KIT (c.1842delG, named KITW1 for White spotting 1), whereas all other camels were wild-type (KIT+/KIT+). No additional mutations unique to the spotted camel were detected in the EDNRB, EDN3, SOX10, KITLG, PDGFRA, MITF, and PAX3 candidate white spotting genes. Sanger sequencing of the study population identified an additional five KITW1/KIT+ spotted camels. The frameshift results in a premature stop codon five amino acids downstream, thus terminating KIT at the tyrosine kinase domain. An additional 13 spotted camels tested KIT+/KIT+, but due to phenotypic differences when compared to the KITW1/KIT+ camels, they likely represent an independent mutation. Our study suggests that there are at least two causes of white spotting in the Arabian camel, the newly described KITW1 allele and an uncharacterized mutation. PMID:28282952

Identification of human cyclin-dependent kinase 8, a putative protein kinase partner for cyclin C.

PubMed Central

Tassan, J P; Jaquenoud, M; Léopold, P; Schultz, S J; Nigg, E A

1995-01-01

Metazoan cyclin C was originally isolated by virtue of its ability to rescue Saccharomyces cerevisiae cells deficient in G1 cyclin function. This suggested that cyclin C might play a role in cell cycle control, but progress toward understanding the function of this cyclin has been hampered by the lack of information on a potential kinase partner. Here we report the identification of a human protein kinase, K35 [cyclin-dependent kinase 8 (CDK8)], that is likely to be a physiological partner of cyclin C. A specific interaction between K35 and cyclin C could be demonstrated after translation of CDKs and cyclins in vitro. Furthermore, cyclin C could be detected in K35 immunoprecipitates prepared from HeLa cells, indicating that the two proteins form a complex also in vivo. The K35-cyclin C complex is structurally related to SRB10-SRB11, a CDK-cyclin pair recently shown to be part of the RNA polymerase II holoenzyme of S. cerevisiae. Hence, we propose that human K35(CDK8)-cyclin C might be functionally associated with the mammalian transcription apparatus, perhaps involved in relaying growth-regulatory signals. Images Fig. 2 Fig. 3 PMID:7568034

Comparison of presumptive blood test kits including hexagon OBTI.

PubMed

Johnston, Emma; Ames, Carole E; Dagnall, Kathryn E; Foster, John; Daniel, Barbara E

2008-05-01

Four presumptive blood tests, Hexagon OBTI, Hemastix(R), Leucomalachite green (LMG), and Kastle-Meyer (KM) were compared for their sensitivity in the identification of dried bloodstains. Stains of varying blood dilutions were subjected to each presumptive test and the results compared. The Hexagon OBTI buffer volume was also reduced to ascertain whether this increased the sensitivity of the kit. The study found that Hemastix(R) was the most sensitive test for trace blood detection. Only with the reduced buffer volume was the Hexagon OBTI kit as sensitive as the LMG and KM tests. However, the Hexagon OBTI kit has the advantage of being a primate specific blood detection kit. This study also investigated whether the OBTI buffer within the kit could be utilized for DNA profiling after presumptive testing. The results show that DNA profiles can be obtained from the Hexagon OBTI kit buffer directly.

Unraveling the spectrum of KIT mutations in gastrointestinal stromal tumors: An Indian Tertiary Cancer Center Experience.

PubMed

Pai, Trupti; Bal, Munita; Shetty, Omshree; Gurav, Mamta; Ostwal, Vikas; Ramaswamy, Anant; Ramadwar, Mukta; Desai, Sangeeta

2017-01-01

Primary mutations in the KIT gene are the driving force for gastrointestinal stromal tumors (GIST) tumorigenesis. Predictive role of KIT mutation status aids oncologists in patient management. There is a paucity of comprehensive data on the frequency of mutations in the KIT gene in GIST affecting Indian patients. The aims of this study were to determine the frequency and spectrum of molecular alterations affecting the KIT gene and assess their association with clinicopathologic features in a cohort of patients of GIST. Morphological and immunohistochemically confirmed GIST cases ( n = 114) accessioned from August 2014-June 2015 were analyzed for mutations in KIT exons 9, 11, 13, and 17 and subjected to Sanger sequencing onto the ABI 3500 Genetic Analyzer. The sequences were analyzed using sequence analysis software: SeqScape ® and Chromas Lite. KIT mutations were seen in 70% of cases and the majority of KIT mutations involved exon 11 (57%), followed by exon 9 (10%), exon 13 (3%), and exon 17 (1%). Most common exon 11 mutations were in-frame deletions (61.4%) followed by substitution mutations (19.3%). Exon 9 mutations showed identical duplication of Ala-Tyr at codons 502-503. Simultaneous mutations affecting exon 11 and 13 were discovered. Novel variations, namely, p.Q556E (c.1666C>G), p.Q556dup (c.1666_1668dupCAG), p.K558_V559delinsS (c.1672_1677delAAGGTTinsAGT), p.Y503_F504insTY (c.1509_1510insACCTAT), and p.K642R (c.1925A>G) involving exons 11, 9, and 13, respectively, were observed. First study with complete analysis of all 4 exons of KIT (exons 9, 11, 13, and 17) in Indian GIST patients. Along with well-described KIT mutations, several rare double mutations as well as novel alterations were reported in this series.

Kit signaling inhibits the sphingomyelin-ceramide pathway through PLC gamma 1: implication in stem cell factor radioprotective effect.

PubMed

Maddens, Stéphane; Charruyer, Alexandra; Plo, Isabelle; Dubreuil, Patrice; Berger, Stuart; Salles, Bernard; Laurent, Guy; Jaffrézou, Jean-Pierre

2002-08-15

Previous studies demonstrated that Kit activation confers radioprotection. However, the mechanism by which Kit signaling interferes with cellular response to ionizing radiation (IR) has not been firmly established. Based on the role of the sphingomyelin (SM) cycle apoptotic pathway in IR-induced apoptosis, we hypothesized that one of the Kit signaling components might inhibit IR-induced ceramide production or ceramide-induced apoptosis. Results show that, in both Ba/F3 and 32D murine cell lines transfected with wild-type c-kit, stem cell factor (SCF) stimulation resulted in a significant reduction of IR-induced apoptosis and cytotoxicity, whereas DNA repair remained unaffected. Moreover, SCF stimulation inhibited IR-induced neutral sphingomyelinase (N-SMase) stimulation and ceramide production. The SCF inhibitory effect on SM cycle was not influenced by wortmannin, a phosphoinositide-3 kinase (PI3K) inhibitor. The SCF protective effect was maintained in 32D-KitYF719 cells in which the PI3K/Akt signaling pathway is abolished due to mutation in Kit docking site for PI3K. In contrast, phospholipase C gamma (PLC gamma) inhibition by U73122 totally restored IR-induced N-SMase stimulation, ceramide production, and apoptosis in Kit-activated cells. Moreover, SCF did not protect 32D-KitYF728 cells (lacking a functional docking site for PLC gamma 1), from IR-induced SM cycle. Finally, SCF-induced radioprotection of human CD34(+) bone marrow cells was also inhibited by U73122. Altogether, these results suggest that SCF radioprotection is due to PLC gamma 1-dependent negative regulation of IR-induced N-SMase stimulation. Beyond the scope of Kit-expressing cells, it suggests that PLC gamma 1 status could greatly influence the post-DNA damage cellular response to IR, and perhaps, to other genotoxic agents.

FGF/EGF signaling regulates the renewal of early nephron progenitors during embryonic development.

PubMed

Brown, Aaron C; Adams, Derek; de Caestecker, Mark; Yang, Xuehui; Friesel, Robert; Oxburgh, Leif

2011-12-01

Recent studies indicate that nephron progenitor cells of the embryonic kidney are arranged in a series of compartments of an increasing state of differentiation. The earliest progenitor compartment, distinguished by expression of CITED1, possesses greater capacity for renewal and differentiation than later compartments. Signaling events governing progression of nephron progenitor cells through stages of increasing differentiation are poorly understood, and their elucidation will provide key insights into normal and dysregulated nephrogenesis, as well as into regenerative processes that follow kidney injury. In this study, we found that the mouse CITED1(+) progenitor compartment is maintained in response to receptor tyrosine kinase (RTK) ligands that activate both FGF and EGF receptors. This RTK signaling function is dependent on RAS and PI3K signaling but not ERK. In vivo, RAS inactivation by expression of sprouty 1 (Spry1) in CITED1(+) nephron progenitors results in loss of characteristic molecular marker expression and in increased death of progenitor cells. Lineage tracing shows that surviving Spry1-expressing progenitor cells are impaired in their subsequent epithelial differentiation, infrequently contributing to epithelial structures. These findings demonstrate that the survival and developmental potential of cells in the earliest embryonic nephron progenitor cell compartment are dependent on FGF/EGF signaling through RAS.

Blockade of epidermal growth factor receptor signaling in tumor cells and tumor-associated endothelial cells for therapy of androgen-independent human prostate cancer growing in the bone of nude mice.

PubMed

Kim, Sun-Jin; Uehara, Hisanori; Karashima, Takashi; Shepherd, David L; Killion, Jerald J; Fidler, Isaiah J

2003-03-01

We determined whether blockade of the epidermal growth factor receptor (EGF-R) signaling pathway by oral administration of the EGF-R tyrosine kinase inhibitor (PKI 166) alone or in combination with injectable Taxol inhibits the growth of PC-3MM2 human prostate cancer cells in the bone of nude mice. Male nude mice implanted with PC-3MM2 cells in the tibia were treated with oral administrations of PKI 166 or PKI 166 plus injectable Taxol beginning 3 days after implantation. The incidence and size of bone tumors and destruction of bone were determined by digitalized radiography. Expression of epidermal growth factor (EGF), EGF-R, and activated EGF-R in tumor cells and tumor-associated endothelial cells was determined by immunohistochemistry. Oral administration of PKI 166 or PKI 166 plus injectable Taxol reduced the incidence and size of bone tumors and destruction of bone. Immunohistochemical analysis revealed that PC-3MM2 cells growing adjacent to the bone expressed high levels of EGF and activated EGF-R, whereas tumor cells in the adjacent musculature did not. Moreover, endothelial cells within the bone tumor lesions, but not in uninvolved bone or tumors in the muscle, expressed high levels of activated EGF-R. Treatment with PKI 166 and more so with PKI 166 plus Taxol significantly inhibited phosphorylation of EGF-R on tumor and endothelial cells and induced significant apoptosis and endothelial cells within tumor lesions. These data indicate that endothelial cells exposed to EGF produced by tumor cells express activated EGF-R and that targeting EGF-R can produce significant therapeutic effects against prostate cancer bone metastasis.

Upregulation of miR-181c contributes to chemoresistance in pancreatic cancer by inactivating the Hippo signaling pathway.

PubMed

Chen, Meiyuan; Wang, Min; Xu, Simiao; Guo, Xingjun; Jiang, Jianxin

2015-12-29

The Hippo signaling pathway plays a crucial role in regulating tissue homeostasis, organ size, tumorigenesis and cancer chemoresistance when deregulated. Physiologically, the Hippo core kinase cassette that consists of mamma-lian STE20-like protein kinase 1/2 (MST1/2), and large tumour suppressor 1/2 (LATS1/2), together with the adaptor proteins Salvador homologue 1 (SAV1) and MOB kinase activator 1 (MOB1), tightly restricts the activities of homologous oncoproteins Yes-associated protein (YAP) and transcriptional co-activator with PDZ-binding motif (TAZ) to low levels. However, how the Hippo kinase cassette core components are simultaneously inhibited, to exhibit constitutively inactivated Hippo signaling and activated YAP/TAZ in cancer remains puzzling. Herein, we reported that miR-181c directly repressed MST1, LATS2, MOB1 and SAV1 expression in human pancreatic cancer cells. Overexpression of miR-181c induced hyperactivation of the YAP/TAZ and enhanced expression of the Hippo signaling downstream genes CTGF, BIRC5 and BLC2L1, leading to pancreatic cancer cell survival and chemoresistance in vitro and in vivo. Importantly, high miR-181c levels were significantly correlated with Hippo signaling inactivation in pancreatic cancer samples, and predicted a poor patient overall survival. These findings provide a novel mechanism for Hippo signaling inactivation in cancer, indicating not only a potentially pivotal role for miR-181c in the progression of pancreatic cancer, but also may represent a new therapeutic target and prognostic marker.

Rho kinase regulates the survival and transformation of cells bearing oncogenic forms of KIT, FLT3 and BCR-ABL

PubMed Central

Mali, Raghuveer Singh; Ramdas, Baskar; Ma, Peilin; Shi, Jianjian; Munugalavadla, Veerendra; Sims, Emily; Wei, Lei; Vemula, Sasidhar; Nabinger, Sarah C.; Goodwin, Charles B.; Chan, Rebecca J.; Traina, Fabiola; Visconte, Valeria; Tiu, Ramon V.; Lewis, Timothy A.; Stern, Andrew M.; Wen, Qiang; Crispino, John D.; Boswell, H. Scott; Kapur, Reuben

2011-01-01

Summary We show constitutive activation of Rho kinase (ROCK) in cells bearing oncogenic forms of KIT, FLT3 and BCR-ABL, which is dependent on PI3K and Rho GTPase. Genetic or pharmacologic inhibition of ROCK in oncogene bearing cells impaired their growth as well as the growth of acute myeloid leukemia patient derived blasts and prolonged the life span of mice bearing myeloproliferative disease. Downstream from ROCK, rapid dephosphorylation or loss of expression of myosin light chain resulted in enhanced apoptosis, reduced growth and loss of actin polymerization in oncogene bearing cells leading to significantly prolonged life span of leukemic mice. In summary, we describe a pathway involving PI3K/Rho/ROCK/MLC which may contribute to myeloproliferative disease and/or acute myeloid leukemia in humans. PMID:21907926

Resveratrol modulates MED28 (Magicin/EG-1) expression and inhibits epidermal growth factor (EGF)-induced migration in MDA-MB-231 human breast cancer cells.

PubMed

Lee, Ming-Fen; Pan, Min-Hsiung; Chiou, Yi-Siou; Cheng, An-Chin; Huang, Han

2011-11-09

Resveratrol and pterostilbene exhibit diverse biological activities. MED28, a subunit of the mammalian Mediator complex for transcription, was also identified as magicin, an actin cytoskeleton Grb2-associated protein, and as endothelial-derived gene (EG-1). Several tumors exhibit aberrant MED28 expression, whereas the underlying mechanism is unclear. Triple-negative breast cancers, often expressing epidermal growth factor (EGF) receptor (EGFR), are associated with metastasis and poor survival. The objective of this study is to compare the effect of resveratrol and pterostilbene and to investigate the role of MED28 in EGFR-overexpressing MDA-MB-231 breast cancer cells. Pretreatment of resveratrol, but not pterostlbene, suppressed EGF-mediated migration and expression of MED28 and matrix metalloproteinase (MMP)-9 in MDA-MB-231 cells. Moreover, overexpression of MED28 increased migration, and the addition of EGF further enhanced migration. Our data indicate that resveratrol modulates the effect of MED28 on cellular migration, presumably through the EGFR/phosphatidylinositol 3-kinase (PI3K) signaling pathway, in breast cancer cells.