RESUMO
Wnt signalling has an important role in cell fate determination, tissue patterning and tumorigenesis. Secreted antagonists of Wnt include Frizzled (Fz)-related proteins (FRPs), Cerberus, Wnt inhibitory factor (WIF) and Dickkopf (Dkk). FRPs, Cerberus and WIF have all been shown to act by binding and sequestering Wnt. We report a novel mechanism of Wnt-signalling inhibition by human Dkk-1. Dkk-1 demonstrated no interaction with Wnt but bound a single cell surface site with high affinity (K(D) = 0.39 nM). Its receptor was detectable in a complex with a relative molecular mass of 240,000 (M(r) 240K) with [(125)I] Dkk-1 by covalent affinity cross-linking. Wnt signalling through beta-catenin is mediated by the Fz receptor and a recently identified low-density-lipoprotein-receptor-related co-receptor, LRP6/Arrow. Overproduction of the 200K LRP6 protein, but not of Fz, strikingly increased Dkk-1 binding as well as the amount of the 240K cross-linked complex, which was shown to be composed of Dkk-1 and LRP6. Moreover, Dkk-1 function was completely independent of Fz but LRP6 dramatically interfered with the Dkk-1 inhibition of Wnt signalling. Thus, unlike Wnt antagonists, which exert their effects by molecular mimicry of Fz or Wnt sequestration through other mechanisms, Dkk-1 specifically inhibits canonical Wnt signalling by binding to the LRP6 component of the receptor complex.
Assuntos
Proteínas/farmacologia , Proteínas Proto-Oncogênicas/fisiologia , Receptores de LDL/metabolismo , Transdução de Sinais/efeitos dos fármacos , Transativadores , Proteínas de Peixe-Zebra , Linhagem Celular , Proteínas do Citoesqueleto/farmacologia , Interações Medicamentosas , Humanos , Immunoblotting , Peptídeos e Proteínas de Sinalização Intercelular , Cinética , Proteína-6 Relacionada a Receptor de Lipoproteína de Baixa Densidade , Ligação Proteica , Proteínas/metabolismo , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Transfecção , Proteínas Wnt , beta CateninaRESUMO
Protein tyrosine kinase blockers of the tyrphostin family inhibited the EGF-dependent proliferation of human and guinea pig keratinocytes grown in culture and induced their growth arrest. These blockers also significantly inhibited the growth of epidermal keratinocytes, but not of dermal cells, in whole skin organ culture from both guinea pig and human origin. The antiproliferative activity of these tyrphostins correlated quantitatively with their potency as inhibitors of EGF receptor autophosphorylation and the EGF-dependent protein phosphorylation of intracellular target proteins in the keratinocyte. Furthermore, no significant cell cytotoxicity or reduction in serine and threonine phosphorylation of many intracellular polypeptides were observed upon incubation of the cells with tyrphostins like AG213. The complete growth arrest induced by the tyrphostins is fully reversible and upon their removal the keratinocytes resumed their growth with the original growth rate. Because of the nontoxic nature of these compounds and their growth-arresting properties, we suggest their use as agents to treat hyperproliferative conditions of human skin.
Assuntos
Catecóis/farmacologia , Divisão Celular/efeitos dos fármacos , Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/fisiologia , Queratinócitos/citologia , Nitrilas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirfostinas , Animais , Células Cultivadas , Relação Dose-Resposta a Droga , Receptores ErbB/antagonistas & inibidores , Cobaias , Humanos , Técnicas In Vitro , Peso Molecular , Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fosforilação/efeitos dos fármacos , Fosfotirosina , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Protein tyrosine kinases (PTKs) regulate cell proliferation, cell differentiation, and signaling processes in the cells of the immune system. Uncontrolled signaling from receptor tyrosine kinases and intracellular tyrosine kinases can lead to inflammatory responses and to diseases such as cancer, atherosclerosis, and psoriasis. Thus, inhibitors that block the activity of tyrosine kinases and the signaling pathways they activate may provide a useful basis for drug development. This article summarizes recent progress in the development of PTK inhibitors and demonstrates their potential use in the treatment of disease.
Assuntos
Desenho de Fármacos , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirfostinas , Animais , Catecóis/farmacologia , Divisão Celular/efeitos dos fármacos , Humanos , Nitrilas/farmacologia , Transdução de Sinais/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
A systematic series of low molecular weight protein tyrosine kinase inhibitors were synthesized; they had progressively increasing affinity over a 2500-fold range toward the substrate site of epidermal growth factor (EGF) receptor kinase domain. These compounds inhibited EGF receptor kinase activity up to three orders of magnitude more than they inhibited insulin receptor kinase, and they also effectively inhibited the EGF-dependent autophosphorylation of the receptor. The most potent compounds effectively inhibited the EGF-dependent proliferation of A431/clone 15 cells with little or no effect on the EGF-independent proliferation of these cells. The potential use of tyrosine protein kinase inhibitors as antiproliferative agents is demonstrated.
Assuntos
Fator de Crescimento Epidérmico/farmacologia , Receptores ErbB/metabolismo , Proteínas Tirosina Quinases/antagonistas & inibidores , Ligação Competitiva , Divisão Celular/efeitos dos fármacos , Linhagem Celular , Estrutura Molecular , Peso Molecular , Fosforilação , Receptor de Insulina/metabolismo , Solubilidade , Relação Estrutura-AtividadeRESUMO
Septic shock results from excessive stimulation of the host immune system, especially macrophages, by lipopolysaccharide (LPS), or endotoxin, which resides on the outer membrane of bacteria. Protein tyrosine kinase inhibitors of the tyrphostin AG 126 family protect mice against LPS-induced lethal toxicity. The protection correlates with the ability of these agents to block LPS-induced production of tumor necrosis factor alpha (TNF-alpha) and nitric oxide in macrophages as well as LPS-induced production of TNF-alpha in vivo. Furthermore, this inhibitory effect correlated with the potency of AG 126 to block LPS-induced tyrosine phosphorylation of a p42MAPK protein substrate in the murine macrophage.
Assuntos
Compostos de Benzilideno/farmacologia , Lipopolissacarídeos/toxicidade , Macrófagos Peritoneais/efeitos dos fármacos , Óxido Nítrico/biossíntese , Nitrilas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Fator de Necrose Tumoral alfa/biossíntese , Tirfostinas , Animais , Bioensaio , Linhagem Celular , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Ensaio de Imunoadsorção Enzimática , Feminino , Ativação de Macrófagos , Macrófagos Peritoneais/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteína Quinase 1 Ativada por Mitógeno , Fosforilação , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fator de Necrose Tumoral alfa/análise , Fator de Necrose Tumoral alfa/toxicidadeRESUMO
Cross-linking of the surface antigen receptor on B lymphocytes has been demonstrated to lead to activation of phospholipase C (PLC) with subsequent increases in production of inositol phosphates and diacylglycerol. In turn, these second messengers increase cytosolic free calcium [( Ca2+]i) and activate the serine threonine phosphotransferase protein kinase C (PKC). These processes are thought to play a major role in B cell activation and proliferation. However, the mechanism linking the B lymphocyte antigen receptor to phospholipase C remains to be identified. We demonstrate herein that activation of the antigen receptor on human lymphocytes, in addition to activation of PLC, increases tyrosine phosphorylation of specific substrates. Tyrphostins, a new class of tyrosine kinase inhibitors which compete for substrate binding site of specific tyrosine kinases have recently been synthesized. Preincubation of B lymphocytes with two different tyrphostins blocked anti-IgM-induced proliferation, oncogene expression, tyrosine phosphorylation, increases in [Ca2+]i, and production of inositol phosphates. The same inhibitors were without effect on B cell proliferation induced by phorbol esters and cation ionophores which directly activate PKC and increase [Ca2+]i thus bypassing PLC. These findings strongly indicate that tyrphostins do not exhibit significant nonspecific toxicity and suggest that they act proximal to PLC. The ability of the tyrphostins to block increases in [Ca2+]i and inositol phosphate production, after activation of the B cell antigen receptor, indicates that a tyrosine kinase acts as an essential link between the B cell antigen receptor and PLC.
Assuntos
Linfócitos B/metabolismo , Proteínas Tirosina Quinases/metabolismo , Fosfolipases Tipo C/metabolismo , Western Blotting , Cálcio/metabolismo , Membrana Celular/enzimologia , Células Cultivadas , Ativação Enzimática , Expressão Gênica , Humanos , Técnicas In Vitro , Fosfatos de Inositol/metabolismo , Ativação Linfocitária/efeitos dos fármacos , Tonsila Palatina/citologia , Fosfoproteínas/metabolismo , Fosfotirosina , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-fos , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMO
Tumor necrosis factor-alpha (TNF) causes vasodilatation and a hyperdynamic state by activating nitric oxide (NO) synthesis. Tyrphostins, specific inhibitors of protein tyrosine kinase (PTK), block the signaling events induced by TNF and NO production. A hyperdynamic circulatory syndrome (HCS) is often observed in portal hypertension (PHT). TNF and NO seem to mediate these hemodynamic changes. The aim of this work was to study the effect of PTK inhibition on the systemic and portal hemodynamics, TNF and NO production, in cirrhotic rats with portal hypertension. Rats with liver cirrhosis induced by chronic inhalation of carbon tetrachloride were used. Animals were treated daily with tyrphostin AG 126 (alpha-cyano-(3-hydroxy-4-nitro) cinnamonitrile) or placebo for 5 d. Mean arterial pressure (MAP), heart rate (HR), and portal pressure (PP) were measured by indwelling catheters. Cardiac output (CI) and stroke volume (SV) were estimated by thermodilution, systemic vascular resistance (SVR) was calculated (MAP/CI), and portal systemic shunting (PSS) was quantitated using radioactive microspheres. Serum and mesenteric lymph node (MLN) TNF levels were measured using an immunoassay kit, and serum NOx was determined photometrically by its oxidation products. The AG 126-treated group showed a statistically significant increase in MAP and SVR, and decreases in CI, SV, MLN TNF, and serum NO oxidation products nitrite and nitrate (NOx) in comparison with the placebo-treated rats. No significant differences were noticed in HR, PP, PSS, or serum TNF. Significant correlations were observed between MAP and NOx, MAP and MLN TNF, PSS and NOx, and serum TNF and serum NOx. The HCS observed in PHT seems to be mediated, at least in part, by TNF and NO by the activation of PTKs and their signaling pathways. PTK activity inhibition ameliorates the hyperdynamic abnormalities that characterize animals with cirrhosis and PHT.
Assuntos
Ascite/fisiopatologia , Compostos de Benzilideno/administração & dosagem , Inibidores Enzimáticos/administração & dosagem , Hipertensão Portal/fisiopatologia , Cirrose Hepática Experimental/metabolismo , Cirrose Hepática Experimental/fisiopatologia , Óxido Nítrico/metabolismo , Nitrilas/administração & dosagem , Proteínas Tirosina Quinases/antagonistas & inibidores , Fator de Necrose Tumoral alfa/metabolismo , Tirfostinas , Animais , Ascite/metabolismo , Tetracloreto de Carbono , Hipertensão Portal/metabolismo , Cirrose Hepática Experimental/induzido quimicamente , Masculino , Ratos , Ratos Sprague-DawleyRESUMO
Tyrosine kinase-dependent cell signaling is postulated to be a pivotal control point in inflammatory responses initiated by bacterial products and TNF. Using a canine model of gram-negative septic shock, we investigated the effect of tyrosine kinase inhibitors (tyrphostins) on survival. Animals were infected intraperitoneally with Escherichia coli 0111: B4, and then, in a randomized, blinded fashion, were treated immediately with one of two tyrphostins, AG 556 (n = 40) or AG 126 (n = 10), or with control (n = 50), and followed for 28 d or until death. All animals received supplemental oxygen, fluids, and antibiotics. Tyrphostin AG 556 improved survival times when compared to controls (P = 0.05). During the first 48 h after infection, AG 556 also improved mean arterial pressure, left ventricular ejection fraction, cardiac output, oxygen delivery, and alveolar-arterial oxygen gradient compared to controls (all P < or = 0.05). These improvements in organ injury were significantly predictive of survival. Treatment with AG 556 had no effect on clearance of endotoxin or bacteria from the blood (both P = NS); however, AG 556 did significantly lower serum TNF levels (P = 0.03). These data are consistent with the conclusion that AG 556 prevented cytokine-induced multiorgan failure and death during septic shock by inhibiting cell-signaling pathways without impairing host defenses as determined by clearance of bacteria and endotoxin.
Assuntos
Inibidores Enzimáticos/farmacologia , Infecções por Escherichia coli/tratamento farmacológico , Insuficiência de Múltiplos Órgãos/prevenção & controle , Nitrilas/farmacologia , Peritonite/tratamento farmacológico , Fenóis/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Tirfostinas , Animais , Compostos de Benzilideno/farmacologia , Modelos Animais de Doenças , Cães , Infecções por Escherichia coli/complicações , Infecções por Escherichia coli/fisiopatologia , Coração/efeitos dos fármacos , Coração/fisiopatologia , Técnicas In Vitro , Pulmão/efeitos dos fármacos , Pulmão/fisiopatologia , Insuficiência de Múltiplos Órgãos/etiologia , Peritonite/complicações , Peritonite/fisiopatologia , Choque Séptico/complicações , Choque Séptico/tratamento farmacológico , Choque Séptico/fisiopatologia , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/metabolismo , gama-Glutamiltransferase/sangueRESUMO
PP1 has previously been described as an inhibitor of the Src-family kinases p56(Lck) and FynT. We have therefore decided to use PP1 to determine the functional role of Src in platelet-derived growth factor (PDGF)-induced proliferation and migration of human coronary artery smooth muscle cells (HCASMCs). A synthetic protocol for PP1/AGL1872 has been developed, and the inhibitory activity of PP1/AGL1872 against Src was examined. PP1/AGL1872 potently inhibited recombinant p60(c-src) in vitro and Src-dependent tyrosine phosphorylation in p60(c-srcF572)-transformed NIH3T3 cells. PP1/AGL1872 also potently inhibited PDGF-stimulated migration of HCASMCs, as determined in the modified Boyden chamber, as well as PDGF-stimulated proliferation of HCASMCs. Surprisingly, in addition to inhibition of Src kinase, PP1/AGL1872 was found to inhibit PDGF receptor kinase in cell-free assays and in various types of intact cells, including HCASMCs. PP1/AGL1872 did not inhibit phosphorylation of the vascular endothelial growth factor receptor KDR (VEGF receptor-2; kinase-insert domain containing receptor) in cell-free assays as well as in intact human coronary artery endothelial cells. In line with the insensitivity of KDR, PP1/AGL1872 had only a weak effect on vascular endothelial growth factor-stimulated migration of human coronary artery endothelial cells. On treatment of cells expressing different receptor tyrosine kinases, the activities of the epidermal growth factor receptor, fibroblast growth factor receptor-1, and insulin-like growth factor-1 receptor were resistant to PP1/AGL1872, whereas PDGF alpha-receptor was susceptible, albeit to a lesser extent than PDGF beta-receptor. These data suggest that the previously described tyrosine kinase inhibitor PP1/AGL1872 is not selective for the Src family of tyrosine kinases. It is also a potent inhibitor of the PDGF beta-receptor kinase but is not a ubiquitous tyrosine kinase inhibitor. PP1/AGL1872 inhibits migration and proliferation of HCASMCs probably by interference with 2 distinct tyrosine phosphorylation events, creating a novel and potent inhibitory principle with possible relevance for the treatment of pathological HCASMC activity, such as vascular remodeling and restenosis.
Assuntos
Inibidores Enzimáticos/farmacologia , Mitose/efeitos dos fármacos , Músculo Liso Vascular/efeitos dos fármacos , Fator de Crescimento Derivado de Plaquetas/farmacologia , Receptores do Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Quinases da Família src/antagonistas & inibidores , Células 3T3 , Animais , Becaplermina , Vasos Sanguíneos/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/citologia , Endotélio Vascular/fisiologia , Humanos , Linfocinas/farmacologia , Camundongos , Músculo Liso Vascular/citologia , Fator de Crescimento Derivado de Plaquetas/antagonistas & inibidores , Proteínas Proto-Oncogênicas c-sis , Proteínas Proto-Oncogênicas pp60(c-src)/antagonistas & inibidores , Pirazóis/farmacologia , Pirimidinas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptor beta de Fator de Crescimento Derivado de Plaquetas , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
DNA topoisomerase I (topo I) is a member of a group of essential nuclear enzymes which control and modify the topological state of DNA and is recognized as the target for anticancer drugs. During the course of the catalytic activity of topo I, a covalent bond is formed between a tyrosine group at the active site of the enzyme and a 3' phosphate group along the DNA backbone. This chemical reaction resembles the protein kinase-mediated tyrosine phosphorylation process. We assumed, therefore, that tyrphostins, potent and selective blockers of protein tyrosine kinases, might affect topo I activity. We found that of three derivatives of tyrphostins (AG-555, AG-18, and AG-213) that inhibited topo I activity in an in vitro assay, AG-555 was the most active. Examination of the mechanism by which these compounds act as topo I inhibitors revealed that AG-555 blocked the binding of this enzyme to the DNA due to its interaction with the topo I enzyme. We showed that its mode of action differed from that observed for camptothecin, a known topo I inhibitor. However, AG-555 did not affect the activity of other major DNA binding enzymes (i.e., DNA ligase, DNA polymerase I, and reverse transcriptase). This study suggests that tyrphostins may serve as a new class of topo I inhibitors, and these results also present additional explanations for their antiproliferative effect.
Assuntos
Antineoplásicos/farmacologia , Compostos de Benzilideno/farmacologia , Catecóis/farmacologia , Nitrilas/farmacologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Inibidores da Topoisomerase I , Tirfostinas , Células 3T3 , Animais , Camptotecina/farmacologia , DNA/metabolismo , DNA Topoisomerases Tipo I/metabolismo , CamundongosRESUMO
A novel class of tyrosine kinase blockers represented by the tyrphostins AG1295 and AG1296 is described. These compounds inhibit selectively the platelet-derived growth factor (PDGF) receptor kinase and the PDGF-dependent DNA synthesis in Swiss 3T3 cells and in porcine aorta endothelial cells with 50% inhibitory concentrations below 5 and 1 microM, respectively. The PDGF receptor blockers have not effect on epidermal growth factor receptor autophosphorylation; weak effects on DNA synthesis stimulated by insulin, by epidermal growth factor, or by a combination of both; and over an order of magnitude weaker blocking effect on fibroblast growth factor-dependent DNA synthesis. AG1296 potently inhibits signaling of human PDGF alpha- and beta-receptors as well as of the related stem cell factor receptor (c-Kit) but has no effect on autophosphorylation of the vascular endothelial growth factor receptor KDR or on DNA synthesis induced by vascular endothelial growth factor in porcine aortic endothelial cells. Treatment by AG1296 reverses the transformed phenotype of sis-transfected NIH 3T3 cells but has no effect on src-transformed NIH 3T3 cells or on the activity of the kinase p60c-src(F527) immunoprecipitated from these cells. These potent and selective compounds represent leads for the development of novel agents to combat tumors driven by PDGF or to inhibit PDGF action in other diseases in which PDGF plays a key role, such as restenosis.
Assuntos
Antineoplásicos/farmacologia , Catecóis/farmacologia , Transformação Celular Neoplásica/efeitos dos fármacos , Nitrilas/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores do Fator de Crescimento Derivado de Plaquetas/metabolismo , Tirfostinas , Células 3T3 , Animais , Divisão Celular/efeitos dos fármacos , DNA/biossíntese , Receptores ErbB/antagonistas & inibidores , Camundongos , Fosforilação , Fator de Crescimento Derivado de Plaquetas/farmacologia , Proteínas Proto-Oncogênicas , Proteínas Proto-Oncogênicas c-sisRESUMO
Human papilloma virus 16 (HPV 16) is associated with cervical cancer and is therefore considered a major health risk for women. Immortalization of keratinocytes induced by HPV infection is largely due to the binding of p53 and Rb by the the viral oncoproteins E6 and E7, respectively, and is driven to a large extent by a transforming growth factor alpha/amphiregulin epidermal growth factor receptor autocrine loop. In this study, we show that the growth of HPV 16-immortalized human keratinocytes can be blocked by a selective epidermal growth factor receptor kinase inhibitor, AG 1478, and by AG 555, a blocker of cyclin-dependent kinase 2 (Cdk2) activation. AG 1478 induces a massive increase in the Cdk2 protein inhibitors p27 and p21, whereas AG 555 appears to have a different mechanism of action, inhibiting the activation of Cdk2. Growth arrest induced by AG 1478 and AG 555 is accompanied by up to 20% of cells undergoing apoptosis. Following AG 1478 treatment but not AG 555 treatment, up to 50% of cells undergo terminal keratinocyte differentiation as determined by filaggrin expression and by the decline in the expression of cytokeratin 14. The growth-arresting properties of AG 1478 and AG 555 identifies them as possible lead antipapilloma agents.
Assuntos
Compostos de Benzilideno/farmacologia , Quinases Ciclina-Dependentes/antagonistas & inibidores , Inibidores Enzimáticos/farmacologia , Receptores ErbB/antagonistas & inibidores , Queratinócitos/efeitos dos fármacos , Nitrilas/farmacologia , Papillomaviridae , Quinazolinas/farmacologia , Tirfostinas , Anticorpos/farmacologia , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Linhagem Celular Transformada , Receptores ErbB/imunologia , Receptores ErbB/metabolismo , Proteínas Filagrinas , Humanos , Queratinócitos/citologia , Queratinócitos/virologia , Fosforilação/efeitos dos fármacosRESUMO
Tyrphostins are low molecular weight compounds that specifically inhibit protein tyrosine kinases. We studied the effects of tyrphostins on OCI-Ly8, a cell line derived from a patient with immunoblastic lymphoma that carries the t(14;18) translocation and overexpresses the B-cell lymphoma/leukemia-2 gene (bcl-2). To test the possibility that tyrphostins induce apoptosis in these cells, overcoming the protection rendered by bcl-2, we screened 16 tyrphostins representing different families at a concentration of 0.5-50 microM. We found that AG17 was the most potent in this regard. Cell cycle analysis demonstrated that AG17 induces arrest at the G1 phase followed by apoptosis with general reduction of the intracellular level of tyrosine-phosphorylated proteins. To further elucidate the mechanism of action of AG17, we investigated its effect on some of the key proteins that regulate the cell cycle. Bcl-2 and cdk2 protein levels were not altered with AG17, whereas cdk2 kinase activity, as well as p21 and p16 protein levels, were reduced markedly. These results suggest that the target of AG17 is inactivation of cdk2. Because lymphoma cells with the t(14;18) translocation and bcl-2 overexpression are resistant to chemotherapy, novel drugs selectively able to induce apoptosis in these cells could offer a new approach to the treatment of lymphoma patients.
Assuntos
Apoptose , Quinases relacionadas a CDC2 e CDC28 , Quinases Ciclina-Dependentes/metabolismo , Linfoma/metabolismo , Nitrilas/farmacologia , Fenóis/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Tirfostinas , Trifosfato de Adenosina/metabolismo , Proteínas de Transporte/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Quinase 2 Dependente de Ciclina , Inibidor p16 de Quinase Dependente de Ciclina , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Inibidores Enzimáticos/farmacologia , Fase G1 , Humanos , Inibidores da Síntese de Ácido Nucleico , Fosforilação/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
The HER-2/neu gene product, p185(neu), is a membrane-bound receptor with tyrosine kinase activity. High levels of p185(neu) is correlated with intrinsic chemoresistance of non-small cell lung cancer (NSCLC) cell lines. We investigated the effects of tyrphostin AG825, a selective tyrosine kinase inhibitor preferentially inhibiting HER-2/neu kinase, on the chemosensitivities and on the drug-induced cell cycle changes of NSCLC cell lines that expressed different levels of p185(neu). Compared to the low-p185(neu) expressing cell lines, we found that the high-p185(neu) expressing cell lines were more resistant to doxorubicin, etoposide, and cis-diamminedichloroplatinum(II) but more sensitive to AG825. AG825 was able to significantly enhance the chemosensitivities of the high-p185(neu) expressing cell lines, whereas it had little effect on the chemosensitivities of the low-p185(neu) expressing cells, with a few exceptions in which minor antagonistic effects were observed. Although high concentrations of AG825 could reduce the drug-induced G(2) arrest that was accompanied by the activation of phosphorylated p34(cdc2), we failed to find any remarkably differential effects of AG825 on drug-induced G(2), arrest and the accompanying phosphorylation status of p34(cdc2) of the high- and and the low-p185(neu) expressing cell lines. In summary, tyrphostin AG825 can enhance chemosensitivity in high- but not in low-p185(neu) expressing NSCLC cell lines. This differential effect cannot be explained by the alterations of drug-induced cell cycle changes by AG825. Our results provide a rationale to develop p185(neu)- specific tyrphostin and to test them in combination with anticancer agents in vivo and in clinical trials.
Assuntos
Compostos de Benzilideno/farmacologia , Carcinoma Pulmonar de Células não Pequenas/metabolismo , Neoplasias Pulmonares/metabolismo , Nitrilas/farmacologia , Receptor ErbB-2/biossíntese , Tirfostinas , Benzotiazóis , Carcinoma Pulmonar de Células não Pequenas/patologia , Divisão Celular/efeitos dos fármacos , Resistência a Medicamentos , Humanos , Neoplasias Pulmonares/patologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Células Tumorais CultivadasRESUMO
A number of growth factor receptor tyrosine kinases have been implicated in angiogenesis, including epidermal growth factor receptor, fibroblast growth factor receptor, platelet-derived growth factor receptor, Flk-1/KDR, Flt-1, Tie-1, and Tek/Tie-2. Flk-1/KDR, a receptor for vascular endothelial growth factor (VEGF), is expressed exclusively in endothelial cells. Using dominant-negative methods, Flk-1 was shown to play a role in angiogenesis and the growth of a variety of tumor types. Because of this, a drug discovery effort was established to identify Flk-1 kinase inhibitors. For initial screening, an ELISA in, a 96-well format was used to measure VEGF-induced Flk-1 tyrosine phosphorylation in whole cells. Compounds that inhibited ligand-induced receptor autophosphorylation were confirmed by antiphosphotyrosine immunoblotting. Inhibition of VEGF-stimulated DNA synthesis in human endothelial cells was also assessed. Inhibitors were further evaluated for their effects on vessel formation using the chorioallantoic membrane assay. Using these methods, antiangiogenesis compounds that inhibit Flk-1 tyrosine kinase activity, endothelial cell mitogenesis, and blood vessel formation in the chorioallantoic membrane assay have been found.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Receptores Proteína Tirosina Quinases/antagonistas & inibidores , Receptores de Fatores de Crescimento/antagonistas & inibidores , Células 3T3/efeitos dos fármacos , Células 3T3/enzimologia , Animais , Moléculas de Adesão Celular/análise , Divisão Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Células Cultivadas , Embrião de Galinha , Fatores de Crescimento Endotelial/farmacologia , Endotélio Vascular/efeitos dos fármacos , Endotélio Vascular/enzimologia , Ensaio de Imunoadsorção Enzimática , Humanos , Immunoblotting , Linfocinas/farmacologia , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Neovascularização Fisiológica/efeitos dos fármacos , Fosforilação , Receptores Proteína Tirosina Quinases/metabolismo , Receptores de Fatores de Crescimento/metabolismo , Receptores de Fatores de Crescimento do Endotélio Vascular , Estimulação Química , Fator A de Crescimento do Endotélio Vascular , Fatores de Crescimento do Endotélio VascularRESUMO
Members of the Wnt family induce mouse mammary tumors and partially transform mammary epithelial cells in culture. However, their mechanism of transformation remains to be elucidated. In NIH3T3 mouse embryo fibroblasts, a standard transformation model, Wnt-1 and Wnt-2 were shown to induce altered properties including increased saturation density and growth in soft agar. Such cells also exhibited increased cell-cell adhesiveness. However, unlike oncogenes such as PDGFB or ras, Wnt-1 and -2 failed to induce detectable transformed foci following transfection, and stable NIH3T3 transfectants lacked tumor forming capacity. Wnt-1 and -2 transfectants exhibited increased uncomplexed, cytosolic beta-catenin, which was not observed with PDGFB, ras or erbB2 transfectants. In transient transfection, Wnt-1 and -2 induced a rapid increase in cytosolic beta-catenin but no detectable increase in the phosphorylated activated forms of MAP kinase. In contrast, ras was a potent activator of MAP kinase but had no effect on free beta-catenin levels. These findings establish that both Wnt signaling and pattern of growth alterations differ from those of oncogenes which activate proliferative signaling pathways in NIH3T3 cells.
Assuntos
Divisão Celular , Transformação Celular Neoplásica , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais , Proteínas de Peixe-Zebra , Células 3T3 , Animais , Adesão Celular , Linhagem Celular Transformada , Humanos , Camundongos , Oncogenes , Proteínas Proto-Oncogênicas/genética , Transfecção , Proteínas Wnt , Proteína Wnt1 , Proteína Wnt2RESUMO
The human homologue of fz1 (Hfz1) was cloned from a cDNA library. Hfz1 was shown to couple to Wnt signal transduction pathways by its ability to enhance Wnt induced TCF dependent transcription in both autocrine and paracrine modes. Enhanced TCF dependent signaling was dose dependent with respect to both Wnt-3A and Hfz1. Moreover, Hfz1 deletion mutants with truncated carboxy termini showed markedly reduced capacity to enhance Wnt signal transduction. Specificity was demonstrated with respect to signal transduction by different Wnts. While Wnt-3a, -3, -1 and to a lesser extent Wnt-2 cooperated with Hfz1 in the paracrine assay for TCF dependent signaling, neither Wnt-4, -5a, -5b, -6, -7a nor -7b did so, despite similar levels of expression. However, coimmunoprecipitation of Hfz1 with both Wnt-3a and Wnt-5a indicated that TCF dependent signaling in response to Wnts is not determined solely by their ability to bind the receptor. All of these findings provide strong evidence that Hfz1 is a functional partner for certain Wnts in inducing TCF dependent transcription.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteínas/metabolismo , Receptores de Neurotransmissores/genética , Receptores de Neurotransmissores/metabolismo , Fatores de Transcrição/metabolismo , Proteínas de Peixe-Zebra , Linhagem Celular , Transformação Celular Neoplásica , Clonagem Molecular , Proteínas de Ligação a DNA/genética , Receptores Frizzled , Humanos , Fator 1 de Ligação ao Facilitador Linfoide , Biologia Molecular/métodos , Dados de Sequência Molecular , Mutação , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Receptores Acoplados a Proteínas G , Sensibilidade e Especificidade , Análise de Sequência , Transdução de Sinais , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção , Proteínas Wnt , Proteína Wnt-5a , Proteína Wnt2 , Proteína Wnt3 , Proteína Wnt3A , Proteína Wnt4RESUMO
Colorectal cancer (CRC) is one of the most frequent cancers in humans. It develops via a multistage process involving alterations of both protooncogenes and tumor suppressor genes. In the present report we determined the level of expression of several Wnt genes in CRC by RT-PCR and direct sequencing. While Wnt-1 was not detectably expressed in any colonic tissues, Wnt-5a gene was efficiently expressed both in nontumorous as well as in colonic tumor tissues. In contrast, the Wnt-2 gene, which was expressed at low levels in normal colon, exhibited overexpression in all tumor tissue samples at the different Dukes' stages of CRC progression, including premalignant polyps and liver metastases. Overexpression of the Wnt-2 gene occurred also in other digestive neoplasms such as gastric and esophageal carcinomas, as well as in diverticulitis associated with stenosis or pseudo-tumor.
Assuntos
Neoplasias Colorretais/genética , Regulação Neoplásica da Expressão Gênica , Proteínas Proto-Oncogênicas/genética , Proto-Oncogenes , Sequência de Bases , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Proteína Wnt2RESUMO
Constitutive activation of signal transducer and activator of transcription (STAT) proteins has been detected in a wide variety of human primary tumor specimens and tumor cell lines including blood malignancies, head and neck cancer, and breast cancer. We have previously demonstrated a high frequency of Stat3 DNA-binding activity that is constitutively-induced by an unknown mechanism in human breast cancer cell lines possessing elevated EGF receptor (EGF-R) and c-Src kinase activities. Using tyrosine kinase selective inhibitors, we show here that Src and JAK family tyrosine kinases cooperate to mediate constitutive Stat3 activation in the absence of EGF stimulation in model human breast cancer cell lines. Inhibition of Src or JAKs results in dose-dependent suppression of Stat3 DNA-binding activity, which is accompanied by growth inhibition and induction of programmed cell death. In addition, transfection of a dominant-negative form of Stat3 leads to growth inhibition involving apoptosis of breast cancer cells. These results indicate that the biological effects of the Src and JAK tyrosine kinase inhibitors are at least partially mediated by blocking Stat3 signaling. While EGF-R kinase activity is not required for constitutive Stat3 activation in breast cancer cells, EGF stimulation further increases STAT DNA-binding activity, consistent with an important role for EGF-R in STAT signaling and malignant progression. Analysis of primary breast tumor specimens from patients with advanced disease revealed that the majority exhibit elevated STAT DNA-binding activity compared to adjacent non-tumor tissues. Our findings, taken together, suggest that tyrosine kinases transduce signals through Stat3 protein that contribute to the growth and survival of human breast cancer cells in culture and potentially in vivo.