RESUMO
Drugs with prolonged on-target residence times often show superior efficacy, yet general strategies for optimizing drug-target residence time are lacking. Here we made progress toward this elusive goal by targeting a noncatalytic cysteine in Bruton's tyrosine kinase (BTK) with reversible covalent inhibitors. Using an inverted orientation of the cysteine-reactive cyanoacrylamide electrophile, we identified potent and selective BTK inhibitors that demonstrated biochemical residence times spanning from minutes to 7 d. An inverted cyanoacrylamide with prolonged residence time in vivo remained bound to BTK for more than 18 h after clearance from the circulation. The inverted cyanoacrylamide strategy was further used to discover fibroblast growth factor receptor (FGFR) kinase inhibitors with residence times of several days, demonstrating the generalizability of the approach. Targeting of noncatalytic cysteines with inverted cyanoacrylamides may serve as a broadly applicable platform that facilitates 'residence time by design', the ability to modulate and improve the duration of target engagement in vivo.
Assuntos
Acrilamidas/farmacocinética , Linfócitos B/efeitos dos fármacos , Cianoacrilatos/farmacocinética , Inibidores de Proteínas Quinases/farmacocinética , Proteínas Tirosina Quinases/antagonistas & inibidores , Acrilamidas/síntese química , Tirosina Quinase da Agamaglobulinemia , Animais , Linfócitos B/enzimologia , Linfócitos B/patologia , Linhagem Celular Tumoral , Cristalografia por Raios X , Cianoacrilatos/síntese química , Dasatinibe , Feminino , Expressão Gênica , Humanos , Ligantes , Simulação de Acoplamento Molecular , Inibidores de Proteínas Quinases/síntese química , Estrutura Terciária de Proteína , Proteínas Tirosina Quinases/química , Proteínas Tirosina Quinases/genética , Pirimidinas/farmacocinética , Ratos , Ratos Sprague-Dawley , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Células Sf9 , Spodoptera , Relação Estrutura-Atividade , Especificidade por Substrato , Tiazóis/farmacocinética , Fatores de TempoRESUMO
OBJECTIVE: To test the hypothesis that rapamycin inhibits induced microvascular hyperpermeability directly in vivo. METHODS: Male golden Syrian hamsters (80-120 g) were treated with either rapamycin (at 0.1, 0.5, 2, and 10 mg/kg i.p.) or vehicle at 24 hours and at 1 hour prior to preparation of the cheek pouch. Caveolin-1 scaffolding (1 mg/kg; positive inhibitory control) was injected i.p. 24 hours prior to the experiment. 10(-8) M vascular endothelial growth factor (VEGF) or 10(-7) M platelet-activating factor (PAF) were topically applied to the cheek pouch. Microvascular permeability and arteriolar diameter were assessed using integrated optical intensity (IOI) and vascular wall imaging, respectively. RESULTS: Rapamycin at 0.1 and 0.5 mg/kg significantly reduced VEGF-stimulated mean IOI from 63.0 +/- 4.2 to 9.7 +/- 5.0 (85% reduction, P < 0.001) and 3.6 +/- 2.7 (95% reduction, P < 0.001), respectively. Rapamycin at 2 mg/kg also lowered VEGF-stimulated hyperpermeability (40% reduction, P < 0.05). However, 10 mg/kg rapamycin increased VEGF-induced microvascular hyperpermeability. Rapamycin at 0.5 mg/kg attenuated VEGF-induced vasodilation and PAF-induced hyperpermeability, but did not inhibit PAF-induced vasoconstriction. CONCLUSIONS: At therapeutically relevant concentrations, rapamycin inhibits VEGF- and PAF-induced microvascular permeability. This inhibition is (i) a direct effect on the endothelial barrier, and (ii) independent of arteriolar vasodilation. Rapamycin at 10 mg/kg stimulates effectors that increase microvascular permeability.
Assuntos
Permeabilidade Capilar/efeitos dos fármacos , Microcirculação/efeitos dos fármacos , Sirolimo/farmacologia , Fator A de Crescimento do Endotélio Vascular/farmacologia , Animais , Arteríolas/efeitos dos fármacos , Arteríolas/fisiologia , Velocidade do Fluxo Sanguíneo/efeitos dos fármacos , Bochecha , Cricetinae , Masculino , Mesocricetus , Microcirculação/fisiologia , Mucosa Bucal/irrigação sanguínea , Mucosa Bucal/efeitos dos fármacos , Fator de Ativação de Plaquetas/antagonistas & inibidores , Fator de Ativação de Plaquetas/farmacologia , Fator A de Crescimento do Endotélio Vascular/antagonistas & inibidores , Vasodilatação/efeitos dos fármacosRESUMO
PURPOSE: Mutations associated with resistance to kinase inhibition are an important mechanism of intrinsic or acquired loss of clinical efficacy for kinase-targeted therapeutics. We report the prospective discovery of ErbB2 mutations that confer resistance to the small-molecule inhibitor lapatinib. EXPERIMENTAL DESIGN: We did in vitro screening using a randomly mutagenized ErbB2 expression library in Ba/F3 cells, which were dependent on ErbB2 activity for survival and growth. RESULTS: Lapatinib resistance screens identified mutations at 16 different ErbB2 amino acid residues, with 12 mutated amino acids mapping to the kinase domain. Mutations conferring the greatest lapatinib resistance cluster in the NH2-terminal kinase lobe and hinge region. Structural computer modeling studies suggest that lapatinib resistance is caused by multiple mechanisms; including direct steric interference and restriction of conformational flexibility (the inactive state required for lapatinib binding is energetically unfavorable). ErbB2 T798I imparts the strongest lapatinib resistance effect and is analogous to the epidermal growth factor receptor T790M, ABL T315I, and cKIT T670I gatekeeper mutations that are associated with clinical drug resistance. ErbB2 mutants associated with lapatinib resistance transformed NIH-3T3 cells, including L755S and T733I mutations known to occur in human breast and gastric carcinomas, supporting a direct mechanism for lapatinib resistance in ErbB2-driven human cancers. The epidermal growth factor receptor/ErbB2/vascular endothelial growth factor receptor inhibitor EXEL-7647 was found to inhibit almost all lapatinib resistance-associated mutations. Furthermore, no ErbB2 mutations were found to be associated with EXEL-7647 resistance and lapatinib sensitivity. CONCLUSIONS: Taken together, these data suggest potential target-based mechanisms of resistance to lapatinib and suggest that EXEL-7647 may be able to circumvent these effects.
Assuntos
Antineoplásicos/farmacologia , Transformação Celular Neoplásica , Mutação , Inibidores de Proteínas Quinases/farmacologia , Quinazolinas/farmacologia , Receptor ErbB-2/antagonistas & inibidores , Receptor ErbB-2/genética , Sobrevivência Celular , Resistencia a Medicamentos Antineoplásicos , Humanos , Lapatinib , Fosforilação , Conformação Proteica , Receptor ErbB-2/químicaRESUMO
Aberrant signaling of the FGF/FGFR pathway occurs frequently in cancers and is an oncogenic driver in many solid tumors. Clinical validation of FGFR as a therapeutic target has been demonstrated in bladder, liver, lung, breast, and gastric cancers. Our goal was to develop an irreversible covalent inhibitor of FGFR1-4 for use in oncology indications. An irreversible covalent binding mechanism imparts many desirable pharmacological benefits including high potency, selectivity, and prolonged target inhibition. Herein we report the structure-based design, medicinal chemistry optimization, and unique ADME assays of our irreversible covalent drug discovery program which culminated in the discovery of compound 34 (PRN1371), a highly selective and potent FGFR1-4 inhibitor.
Assuntos
Antineoplásicos/farmacologia , Neoplasias/tratamento farmacológico , Piridonas/farmacologia , Pirimidinas/farmacologia , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/síntese química , Antineoplásicos/farmacocinética , Linhagem Celular Tumoral , Cães , Desenho de Fármacos , Estabilidade de Medicamentos , Feminino , Humanos , Absorção Intestinal , Macaca fascicularis , Masculino , Piridonas/administração & dosagem , Piridonas/síntese química , Piridonas/farmacocinética , Pirimidinas/administração & dosagem , Pirimidinas/síntese química , Pirimidinas/farmacocinética , Ratos Sprague-Dawley , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 2 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 3 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Receptor Tipo 4 de Fator de Crescimento de Fibroblastos/antagonistas & inibidores , Solubilidade , Relação Estrutura-AtividadeRESUMO
An increasing number of cancers are known to harbor mutations, translocations, or amplifications in the fibroblast growth factor receptor (FGFR) family of kinases. The FGFR inhibitors evaluated in clinical trials to date have shown promise at treating these cancers. Here, we describe PRN1371, an irreversible covalent inhibitor of FGFR1-4 targeting a cysteine within the kinase active site. PRN1371 demonstrated strong FGFR potency and excellent kinome-wide selectivity in a number of biochemical and cellular assays, including in various cancer cell lines exhibiting FGFR alterations. Furthermore, PRN1371 maintained FGFR inhibition in vivo, not only when circulating drug levels were high but also after the drug had been cleared from circulation, indicating the possibility of sustained FGFR inhibition in the clinic without the need for continuous drug exposure. Durable tumor regression was also obtained in multiple tumor xenografts and patient-derived tumor xenograft models and was sustained even using an intermittent dosing strategy that provided drug holidays. PRN1371 is currently under clinical investigation for treatment of patients with solid tumors. Mol Cancer Ther; 16(12); 2668-76. ©2017 AACR.
Assuntos
Piridonas/uso terapêutico , Pirimidinas/uso terapêutico , Receptores de Fatores de Crescimento de Fibroblastos/antagonistas & inibidores , Animais , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Camundongos , Piridonas/farmacologia , Pirimidinas/farmacologia , Transdução de Sinais , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
PAK1 is a protein kinase downstream of the small GTPases Rac and Cdc42 that previous work has implicated in endothelial cell migration via modulation of cell contraction. The first proline-rich region of PAK that binds to an SH3 domain from the adapter protein NCK was responsible for these dominant-negative effects. To test the role of PAK in angiogenesis, we prepared a peptide in which the proline-rich region was fused to the polybasic sequence from the HIV Tat protein to facilitate entry into cells. We show that the short peptide selectively binds NCK, whereas a mutant peptide does not. Treatment of cells with the PAK peptide but not the control peptide disrupts localization of PAK. This peptide specifically inhibited endothelial cell migration and contractility similarly to full-length dominant-negative PAK. In an in vitro tube-forming assay, the PAK peptide specifically blocked formation of multicellular networks. In an in vivo chick chorioallantoic membrane assay, the PAK peptide specifically blocked angiogenesis. These results, therefore, suggest a role for PAK in angiogenesis.
Assuntos
Movimento Celular/fisiologia , Endotélio Vascular/fisiologia , Neovascularização Fisiológica/fisiologia , Proteínas Oncogênicas/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Proteínas Adaptadoras de Transdução de Sinal , Movimento Celular/efeitos dos fármacos , Células Cultivadas , Endotélio Vascular/efeitos dos fármacos , Produtos do Gene tat/genética , Produtos do Gene tat/fisiologia , Humanos , Mutação , Neovascularização Fisiológica/efeitos dos fármacos , Peptídeos/genética , Peptídeos/farmacologia , Ligação Proteica , Proteínas Recombinantes de Fusão/farmacologia , Quinases Ativadas por p21RESUMO
The Seventh World Congress for Microcirculation, organized by the Australian and New Zealand Microcirculation Society, was held on 19-22 August 2001 in Sydney, New South Wales, Australia.
Assuntos
Microcirculação , Austrália , Nova Zelândia , Sociedades MédicasRESUMO
OBJECTIVE: This study evaluated the relative roles of the vascular endothelial growth factor (VEGF) receptors KDR and Flt-1 in the mediation of altered gene expression elicited by VEGF. METHODS AND RESULTS: We used mutants of VEGF selective for the KDR and Flt-1 receptors to differentiate gene expression patterns mediated by wild-type VEGF (VEGFwt) in human umbilical vein endothelial cells. RNA was extracted from cells treated for 24 hours with 1 nmol/L of each ligand, and gene expression was monitored by using oligonucleotide arrays (Affymetrix U95A). We report that activation of KDR was sufficient to upregulate all the genes induced by VEGFwt. In contrast, there were no genes selectively upregulated by the Flt-selective mutant. However, high concentrations of the Flt-selective mutant could augment the expression of some genes induced by submaximal concentrations of VEGFwt but not the KDR-selective mutant. CONCLUSIONS: The binding of VEGF to its receptor, KDR, is necessary and sufficient to induce the gene expression profile induced by this growth factor. Furthermore, in human umbilical vein endothelial cells, the Flt-1 receptor appears to act as a decoy receptor, tempering the response to lower concentrations of VEGF.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/química , Endotélio Vascular/metabolismo , Regulação da Expressão Gênica/fisiologia , Peptídeos e Proteínas de Sinalização Intercelular/fisiologia , Linfocinas/fisiologia , Veias Umbilicais/química , Veias Umbilicais/metabolismo , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/fisiologia , Células Cultivadas , Fatores de Crescimento Endotelial/genética , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Perfilação da Expressão Gênica/métodos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/genética , Humanos , Peptídeos e Proteínas de Sinalização Intercelular/genética , Peptídeos e Proteínas de Sinalização Intercelular/farmacologia , Linfocinas/genética , Linfocinas/farmacologia , Mutação/genética , Mutação/fisiologia , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase/métodos , Receptores CXCR4/biossíntese , Fatores de Tempo , Veias Umbilicais/citologia , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/fisiologia , Fator A de Crescimento do Endotélio Vascular , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 1 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genética , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fatores de Crescimento do Endotélio VascularRESUMO
The objective of this study was to use gene expression data from well-defined cell culture models, in combination with expression data from diagnostic samples of human diseased tissues, to identify potential therapeutic targets and markers of disease. Using Affymetrix oligonucleotide array technology, we identified a common profile of genes upregulated during endothelial morphogenesis into tubelike structures in three in vitro models of angiogenesis. Rigorous data selection criteria were used to identify a list of over 1,000 genes whose expression was increased more than twofold over baseline at either 4, 8, 24, 40 or 50 h. To further refine and prioritize this list, we used standard bioinformatic algorithms to identify potential transmembrane and secreted proteins. We then overlapped this gene set with genes upregulated in colon tumors vs. normal colon, resulting in a subset of 128 genes in common with our endothelial list. We removed from this list those genes expressed in 6 different colon tumor lines, resulting in a list of 24 putative, vascular-specific angiogenesis-associated genes. Three genes, gp34, stanniocalcin-1 (STC-1), and GA733-1, were expressed at levels 10-fold or more in colon tumors compared with normal mucosa. We validated the vascular-specific expression of one of these genes, STC-1, by in situ hybridization. The ability to combine in vitro and in vivo data sets should permit one to identify putative angiogenesis target genes in various tumors, chronic inflammation, and other disorders where therapeutic manipulation of angiogenesis is a desirable treatment modality.
Assuntos
Bases de Dados Genéticas , Perfilação da Expressão Gênica/métodos , Neovascularização Patológica/genética , Neovascularização Fisiológica/genética , Animais , Linhagem Celular , Endotélio Vascular/química , Endotélio Vascular/metabolismo , Endotélio Vascular/fisiologia , Marcação de Genes/métodos , Humanos , Masculino , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Ratos , Ratos Sprague-Dawley , Células Tumorais Cultivadas , Veias UmbilicaisRESUMO
Mechanical forces modulate endothelial cell functions through several mechanisms including regulation of gene transcription. In the present study, gene transcription by human umbilical vein endothelial cells (HUVEC) either maintained under control pressure (that is, standard cell culture conditions equivalent to 0.15 mmHg sustained hydrostatic pressure) or exposed to 60/20 mmHg sinusoidal pressures at 1 Hz were compared using Affymetrix GeneChip microarrays to identify cellular/molecular mechanisms associated with endothelial cell responses to cyclic pressure. Cyclic pressure selectively affected transcription of 14 genes that included a set of mechanosensitive proteins involved in hemostasis (tissue plasminogen activator), cell adhesion (integrin-alpha2), and cell signaling (Rho B, cytosolic phospholipase A2), as well as a unique subset of cyclic pressure-sensitive genes such as vascular endothelial growth factor (VEGF)-C and transforming growth factor (TGF)-beta2. The present study also provided first evidence that VEGF-C, the most highly induced gene under 60/20 mmHg, mediated HUVEC proliferation in response to this cyclic pressure. Cyclic pressure is, therefore, a mechanical force that modulates endothelial cell functions (such as proliferation) by activating a specific transcriptional program.
Assuntos
Fatores de Crescimento Endotelial/fisiologia , Endotélio Vascular/metabolismo , Transcrição Gênica , Divisão Celular , Células Cultivadas , Fatores de Crescimento Endotelial/genética , Endotélio Vascular/efeitos dos fármacos , Perfilação da Expressão Gênica , Regulação da Expressão Gênica , Humanos , Periodicidade , Pressão , RNA Mensageiro/biossíntese , Estresse Mecânico , Fator C de Crescimento do Endotélio VascularRESUMO
DNA microarrays were used to measure the time course of gene expression during skeletal muscle damage and regeneration in mice following femoral artery ligation (FAL). We found 1,289 known sequences were differentially expressed between the FAL and control groups. Gene expression peaked on day 3, and the functional cluster "inflammation" contained the greatest number of genes. Muscle function was depressed for 3 days postligation, but returned to normal by day 7. Decreased muscle function was accompanied by reduced expression of genes involved in mitochondrial energy production, muscle contraction, and calcium handling. The induction of MyoD on day 1 denoted the beginning of muscle regeneration and was followed by the reemergence of the embryonic forms of muscle contractile proteins, which peaked at day 7. Transcriptional analysis indicated that the ischemic skeletal muscle may transition through a functional adaptation stage with recovery of contractile force prior to full regeneration. Several members of the insulin-like growth factor axis were coordinately induced in a time frame consistent with their playing a role in the regenerative process.
Assuntos
Isquemia/genética , Músculo Esquelético/metabolismo , Músculo Esquelético/fisiologia , Regeneração , Doença Aguda , Animais , Citocinas/biossíntese , Citocinas/genética , Artéria Femoral/cirurgia , Perfilação da Expressão Gênica , Isquemia/metabolismo , Isquemia/patologia , Cinética , Ligadura , Extremidade Inferior , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Contração Muscular , Músculo Esquelético/patologia , Miosinas/biossíntese , Miosinas/genética , Análise de Sequência com Séries de Oligonucleotídeos , RNA Mensageiro/biossíntese , Receptores de Citocinas/biossíntese , Receptores de Citocinas/genética , Somatomedinas/biossíntese , Somatomedinas/genética , Transcrição GênicaRESUMO
Fish stanniocalcin (STC) inhibits uptake of calcium and stimulates phosphate reabsorption. To determine the role of the highly homologous mammalian protein, STC-1, we created and characterized transgenic mice that express STC-1 under control of a muscle-specific promoter. STC-1 transgenic mice were smaller than wild-type littermates and had normal growth plate cartilage morphology but increased cartilage matrix synthesis. In STC-1 mice, the rate of bone formation, but not bone mineralization, was decreased. Increased cortical bone thickness and changes in trabeculae number, density, and thickness in STC-1 mice indicated a concomitant suppression of osteoclast activity, which was supported by microcomputed tomography analyses and histochemistry. Skeletal muscles were disproportionately small and showed altered function and response to injury in STC-1 mice. Electron microscopy indicated that muscle mitochondria were dramatically enlarged in STC-1 mice. These changes in STC-1 mice could not be explained by deficits in blood vessel formation, as vascularity in organs and skeletal tissues was increased as was induction of vascularity in response to femoral artery ligation. Our results indicate that STC-1 can affect calcium homeostasis, bone and muscle mass and structure, and angiogenesis through effects on osteoblasts, osteoclasts, myoblasts/myocytes, and endothelial cells.
Assuntos
Osso e Ossos/anatomia & histologia , Osso e Ossos/fisiologia , Glicoproteínas/fisiologia , Hormônios/fisiologia , Músculo Esquelético/anatomia & histologia , Músculo Esquelético/fisiologia , Animais , Composição Corporal , Constituição Corporal , Densidade Óssea , Desenvolvimento Ósseo , Matriz Óssea/metabolismo , Calcificação Fisiológica , Cálcio/sangue , Cartilagem/metabolismo , Feminino , Expressão Gênica , Glicoproteínas/genética , Crescimento/genética , Lâmina de Crescimento/anatomia & histologia , Hormônios/genética , Masculino , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Neovascularização Fisiológica , Osteoclastos/fisiologia , Crânio/diagnóstico por imagem , Tomografia Computadorizada por Raios XRESUMO
Hepatocyte growth factor (HGF) and vascular endothelial cell growth factor (VEGF) are two potent endothelial mitogens with demonstrated angiogenic activities in animal models of therapeutic angiogenesis. Several recent studies suggest that these growth factors may act synergistically, although the mechanism of this interaction is not understood. Changes in the gene expression profile of human umbilical vein endothelial cells treated with HGF, VEGF or the combination of the two were analyzed with high-density oligonucleotide arrays, representing approximately 22000 genes. Notably, the genes significantly up- and downregulated by VEGF versus HGF exhibited very little overlap, indicating distinct signal transduction pathways. The combination of HGF and VEGF markedly increased the number of significantly up- and downregulated genes. At 4 h, the combination of the two growth factors induced a number of chemokine and cytokines and their receptors (IL-8, IL-6, IL-11, CCR6, CXCR1,CXC1 and IL17RC), numerous genes involved in growth factor signal transduction (egr-1, fosB, grb10, grb14,MAP2K3,MAP3K8, MAPKAP2,MPK3, DUSP4 and DUSP6), as well as a number of other growth factors (PDGFA, BMP2, Hb-EGF, FGF16, heuregulin beta 1, c-kit ligand, angiopoietin 2 and angiopoietin 4 and VEGFC). In addition, the VEGF receptors neuropilin-1 and flt-1 were also upregulated. At 24 h, a clear 'cell cycle' signature is noted, with the upregulated expression of various cell cycle control proteins and gene involved in the regulation of mitosis and mitotic spindle assembly. The receptor for HGF, c-met, is also upregulated. These data are consistent with the hypothesis that the combination of HGF and VEGF results in the cooperative upregulation of a number of different molecular pathways leading to a more robust proliferative response, that is, growth factor(s), receptors, molecules involved in growth factor signal transduction, as well as, at later time points, upregulation of the necessary cellular proteins required for cells to escape cell cycle arrest and enter the cell cycle.
Assuntos
Células Endoteliais/metabolismo , Perfilação da Expressão Gênica/métodos , Fator de Crescimento de Hepatócito/metabolismo , Sondas Moleculares/química , Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator de Crescimento de Hepatócito/química , Fator de Crescimento de Hepatócito/genética , Humanos , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Fatores de Crescimento do Endotélio Vascular/química , Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Vascular endothelial cells sense and respond to pressure by molecular mechanism(s) which, to date, remain poorly understood. The present study investigated basic fibroblast growth factor (bFGF) signaling as a putative mechanotransduction pathway involved in the proliferative responses of human umbilical vein endothelia cells (HUVECs) to 60/20 mm Hg cyclic pressure at 1 Hz for 24 h. Under these conditions, the enhanced proliferative response of these HUVECs was not associated with an increased synthesis/release of bFGF, but involved rapid (within 30 min from the onset of exposure to pressure) tyrosine phosphorylation of the bFGF receptor, FGFR-2. Furthermore, monoclonal antibodies to either bFGF or FGFR-2 attenuated the increased proliferation of HUVECs exposed to 60/20 mm Hg cyclic pressure. HUVECs proliferation under 60/20 mm Hg at 1 Hz cyclic pressure is, therefore, dependent upon bFGF and involves FGFR-2 activation.
Assuntos
Divisão Celular/fisiologia , Células Endoteliais/fisiologia , Fator 2 de Crescimento de Fibroblastos/fisiologia , Receptores de Fatores de Crescimento de Fibroblastos/fisiologia , Transdução de Sinais/fisiologia , Anticorpos Monoclonais/imunologia , Células Endoteliais/citologia , Endotélio Vascular/fisiologia , Fator 2 de Crescimento de Fibroblastos/imunologia , Humanos , Fosforilação , Receptores de Fatores de Crescimento de Fibroblastos/imunologia , Tirosina/metabolismoRESUMO
BACKGROUND: Many proteins that are dysregulated or mutated in cancer cells rely on the molecular chaperone HSP90 for their proper folding and activity, which has led to considerable interest in HSP90 as a cancer drug target. The diverse array of HSP90 client proteins encompasses oncogenic drivers, cell cycle components, and a variety of regulatory factors, so inhibition of HSP90 perturbs multiple cellular processes, including mitogenic signaling and cell cycle control. Although many reports have investigated HSP90 inhibition in the context of the cell cycle, no large-scale studies have examined potential correlations between cell genotype and the cell cycle phenotypes of HSP90 inhibition. METHODOLOGY/PRINCIPAL FINDINGS: To address this question, we developed a novel high-content, high-throughput cell cycle assay and profiled the effects of two distinct small molecule HSP90 inhibitors (XL888 and 17-AAG [17-allylamino-17-demethoxygeldanamycin]) in a large, genetically diverse panel of cancer cell lines. The cell cycle phenotypes of both inhibitors were strikingly similar and fell into three classes: accumulation in M-phase, G2-phase, or G1-phase. Accumulation in M-phase was the most prominent phenotype and notably, was also correlated with TP53 mutant status. We additionally observed unexpected complexity in the response of the cell cycle-associated client PLK1 to HSP90 inhibition, and we suggest that inhibitor-induced PLK1 depletion may contribute to the striking metaphase arrest phenotype seen in many of the M-arrested cell lines. CONCLUSIONS/SIGNIFICANCE: Our analysis of the cell cycle phenotypes induced by HSP90 inhibition in 25 cancer cell lines revealed that the phenotypic response was highly dependent on cellular genotype as well as on the concentration of HSP90 inhibitor and the time of treatment. M-phase arrest correlated with the presence of TP53 mutations, while G2 or G1 arrest was more commonly seen in cells bearing wt TP53. We draw upon previous literature to suggest an integrated model that accounts for these varying observations.
Assuntos
Compostos Azabicíclicos/farmacologia , Ciclo Celular , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Ensaios de Triagem em Larga Escala/métodos , Ácidos Ftálicos/farmacologia , Benzoquinonas/farmacologia , Ciclo Celular/efeitos dos fármacos , Proteínas de Ciclo Celular/metabolismo , Linhagem Celular , Citometria de Fluxo , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Lactamas Macrocíclicas/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Fatores de Tempo , Quinase 1 Polo-LikeRESUMO
This review addresses a rapidly growing area of vascular biology, i.e. genomic variations in vascular genes that underlie different human phenotypes. Two of the most important molecular in vascular biology, endothelial nitric oxide synthase (eNOS) and vascular endothelial cell growth factor (VEGF)are discussed. Variations in the eNOS gene have been correlated with a number of human diseases including hypertension, coronary vasospasm, smoking dependent risk of coronary disease, myocardial infarction and placental disruption. Similarly, variations in the VEGF gene have been associated with increased risk of various cancers, DiGeorge syndrome, psoriasis, diabetic renal disease and amyotropic lateral sclerosis. Understanding the molecular basis of these genetic variations and how they contribute to the pathophysiology provides new and important insights into human disease.
Assuntos
Óxido Nítrico Sintase/genética , Fator A de Crescimento do Endotélio Vascular/genética , Doenças Cardiovasculares/genética , Variação Genética , Humanos , Óxido Nítrico Sintase Tipo IIIRESUMO
Stanniocalcin was originally described as a hormone with calcitonin-like actions in fish. During the last decade, mammalian forms of stanniocalcin have been identified, and this discovery has led to important advances in our understanding of this enigmatic polypeptide hormone. This review briefly covers some early studies on stanniocalcin in fish and then provides a more in-depth look at some of the more intriguing, new aspects of its functions in mammals. The roles of stanniocalcin in renal function, metabolism, angiogenesis, pregnancy and lactation, bone formation, and neural protection are discussed, along with new information relating to its receptor-mediated sequestration and accumulation in target cell organelles.
Assuntos
Glicoproteínas/fisiologia , Sequência de Aminoácidos , Animais , Feminino , Peixes , Glicoproteínas/química , Glicoproteínas/genética , Glicoproteínas/história , História do Século XIX , História do Século XX , Humanos , Mamíferos , Camundongos , Camundongos Transgênicos , Mitocôndrias , Dados de Sequência Molecular , GravidezRESUMO
The present study investigated the proliferative and apoptotic responses of human umbilical vein endothelial cells (HUVECs) to well-defined, sinusoidal pressures (60/20, 100/60, and 140/100 mm Hg/mm Hg) at 1 Hz for up to 24 h under Media 199 containing either 1% FBS and 0.04% bovine brain extract (BBE) (low serum/growth factor conditions) or 10% FBS and 0.4% BBE (normal serum/growth factor conditions). Controls were HUVEC maintained under 0.2 mm Hg sustained pressure, but otherwise, similar experimental conditions. Under low serum/growth factor conditions, exposure of HUVEC to 60/20 mm Hg/mm Hg cyclic pressure at 1 Hz for time periods up to 24 h resulted in increases in total cell population density, apoptosis, and DNA synthesis. Under normal serum/growth factor conditions, exposure of HUVEC to either 60/20 or 100/60 mm Hg/mm Hg cyclic pressures resulted in increased DNA synthesis but did not significantly affect cell density or the apoptotic index. A reduced rate of cell death was observed in HUVEC under low serum/growth factor conditions after exposure to 140/100 mm Hg/mm Hg. Under normal serum/growth factor conditions. HUVEC exposed to 140/100 mm Hg/mm Hg cyclic pressure exhibited reduced DNA synthesis. Endothelial cells. therefore, sense and respond to physiologic levels of cyclic pressure by modifying cell proliferation and apoptosis in a mean-pressure-selective manner.
Assuntos
Apoptose/fisiologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Mecanotransdução Celular/fisiologia , Contagem de Células , Células Cultivadas , DNA/biossíntese , Humanos , Periodicidade , Pressão , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Veias Umbilicais/citologia , Veias Umbilicais/fisiologiaRESUMO
Recent advances in gene expression profiling have led to the development of comprehensive databases which can be queried in various manners. In the present report, we have taken a list of genes previously associated with angiogenesis, either in in vivo or in in vitro models, and queried a commercial database established by GeneLogic to determine the relative expression of these candidate genes in normal kidneys and in renal cell carcinomas (RCC). We identified a number of genes, including CXCR4, matrix metalloproteinase 9, thrombospondin 2, and vascular endothelial growth factor, that were highly expressed in RCC versus normal tissue. One gene, hevin, appears to be selectively upregulated in RCC in contrast to downregulation of this gene in lung and colon tumors. This approach provides a powerful means to identify potential markers of tumor vascularization.