RESUMO
PURPOSE: The microtubule-stabilizing agent patupilone (epothilone B, EPO906) and the tyrosine kinase inhibitor imatinib (STI571, Glivec) which primarily inhibits Bcr-Abl, PDGF and c-Kit tyrosine kinase receptors, were combined in vivo to determine if any interaction would occur with respect to antitumour effect and tolerability using rat C6 glioma xenografted into nude mice. METHODS: Patupilone and imatinib were administered alone or in combination at suboptimal doses. Imatinib treatment (orally once daily) was initiated 4 days after s.c. injection of rat C6 glioma cells into athymic nude mice and patupilone administration (i.v. once per week) was started 3 or 4 days after imatinib treatment. RESULTS: As a single agent, imatinib was inactive in the regimens selected (100 mg/kg: T/C 86% and 116%; 200 mg/kg: T/C 68% and 84%; two independent experiments), but well tolerated (gain in body weight and no mortalities). Patupilone weekly monotherapy demonstrated dose-dependent antitumour effects (1 mg/kg: T/C 67% and 70%; 2 mg/kg: T/C 32% and 63%; 4 mg/kg: T/C 3% and 46%). As expected, dose-dependent body weight losses occurred (final body weight changes at 1 mg/kg were -7% and -3%; at 2 mg/kg were -23% and -13%; and at 4 mg/kg were -33% and -15%). Combining 2 mg/kg patupilone and 200 mg/kg per day imatinib in one experiment produced a non-statistically significant trend for an improved antitumour effect over patupilone alone (combination, T/C 9%), while in the second experiment, enhancement was seen with the combination and reached statistical significance versus patupilone alone (combination, T/C 22%; P=0.008). Reduction of the imatinib dose to 100 mg/kg per day resulted in no enhancement of antitumour activity in combination with 2 mg/kg patupilone. Reduction of the patupilone dose to 1 mg/kg resulted in a reduced antitumour effect, and only a trend for synergy with either imatinib dose (combination, T/C 46% and 40%). Pooling the data from the two experiments confirmed a significant synergy for the combination of 2 mg/kg patupilone and 200 mg/kg per day imatinib (P=0.032), and a trend for synergy at the 1 mg/kg patupilone dose. Reduction in the imatinib dose to 100 mg/kg per day resulted only in additivity with either dose of patupilone. Body weight losses were dominated by the effect of patupilone, since no greater body weight loss was observed in the combination groups. CONCLUSION: Combining patupilone with high-dose imatinib produced an increased antitumour effect without affecting the tolerability of treatment in a relatively chemoresistant rat C6 glioma model. Such results indicate that further evaluation is warranted, in particular to elucidate possible mechanisms of combined action.
Assuntos
Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Epotilonas/administração & dosagem , Glioma/tratamento farmacológico , Piperazinas/administração & dosagem , Pirimidinas/administração & dosagem , Animais , Benzamidas , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Mesilato de Imatinib , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Transplante de Neoplasias , RatosRESUMO
Epothilones are naturally occurring 16-membered macrolides with the ability to promote tubulin polymerization in vitro and to stabilize preformed microtubules against Ca(2+)- or cold-induced depolymerization. At the cellular level, interference with microtubule functionality results in potent inhibition of cancer cell proliferation at nM to even sub-nM concentrations. Most significantly, epothilones, unlike paclitaxel (Taxol), are equally active against drug-sensitive and multidrug-resistant cell lines in vitro and epothilone B has also shown potent in vivo antitumor activity in Taxol-resistant human tumor models. Epothilone B is currently undergoing Novartis-sponsored Phase II clinical trials. In addition to naturally occurring epothilones, numerous synthetic and semi-synthetic analogs have been prepared since the absolute stereochemistry of epothilone B was first disclosed in mid-1996 and their in vitro biological activity has been determined. These studies have generated a wealth of SAR data in a remarkably short period of time, given the complexity of the synthetic targets pursued. One of these analogs, BMS-247550, is presently in Phase II clinical trials by Bristol-Myers Squibb. In a first part this review is intended to provide a summary of the basic features of the in vitro biological profile of epothilones A and B, including emerging data on potential cellular epothilone resistance mechanisms. The second and third part will feature a comprehensive discussion of the epothilone SAR as it has emerged from the work of various (industrial and academic) laboratories across the world, including our own, with regard to effects on tubulin polymerization, in vitro antiproliferative activity, and in vivo antitumor activity.
Assuntos
Antineoplásicos/química , Antineoplásicos/farmacologia , Epotilonas/química , Epotilonas/farmacologia , Tubulina (Proteína)/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Humanos , Mutação , Relação Estrutura-Atividade , Tubulina (Proteína)/genética , Células Tumorais CultivadasRESUMO
The design, chemical synthesis, and biological evaluation of a series of cyclopropyl and cyclobutyl epothilone analogues (3-12, Figure 1) are described. The synthetic strategies toward these epothilones involved a Nozaki-Hiyama-Kishi coupling to form the C15-C16 carbon-carbon bond, an aldol reaction to construct the C6-C7 carbon-carbon bond, and a Yamaguchi macrolactonization to complete the required skeletal framework. Biological studies with the synthesized compounds led to the identification of epothilone analogues 3, 4, 7, 8, 9, and 11 as potent tubulin polymerization promoters and cytotoxic agents with (12R,13S,15S)-cyclopropyl 5-methylpyridine epothilone A (11) as the most powerful compound whose potencies (e.g. IC(50) = 0.6 nM against the 1A9 ovarian carcinoma cell line) approach those of epothilone B. These investigations led to a number of important structure-activity relationships, including the conclusion that neither the epoxide nor the stereochemistry at C12 are essential, while the stereochemistry at both C13 and C15 are crucial for biological activity. These studies also confirmed the importance of both the cyclopropyl and 5-methylpyridine moieties in conferring potent and potentially clinically useful biological properties to the epothilone scaffold.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Compostos de Epóxi/síntese química , Compostos de Epóxi/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Tiazóis/síntese química , Tiazóis/farmacologia , Carcinoma de Células Escamosas/tratamento farmacológico , Ensaios de Seleção de Medicamentos Antitumorais , Feminino , Humanos , Neoplasias Ovarianas/tratamento farmacológico , Tubulina (Proteína)/metabolismo , Células Tumorais CultivadasRESUMO
The phosphonium salt 35, representing one of the two principal subunits of the epothilones, was prepared from propargyl alcohol via heptenone 22. A Wittig reaction of the phosphorane from 35 with aldehyde 33, obtained from aldol condensation of ketone 27 with aldehyde 28, afforded 37. Seco acid 42 derived from 37 underwent lactonization to give cis-9,10-dehydroepothilone D (43) which was selectively reduced with diimide to yield epothilone D (4) and, after epoxidation, epothilone B (2). An alternative route to epothilone D employed alkyne 39, obtained from 33, in a Castro-Stephens reaction with allylic bromide 34 to furnish enyne 40. The latter was semi-hydrogenated to provide 37. Alkyne 46, prepared from alcohol 45, was converted to trans-vinylstannane 47 which, in a Stille coupling with allylic chloride 50, gave 51. Seco acid 52 derived from 51 underwent lactonization to give trans-9,10-dehydroepothilone D (54). Bioassay data comparing the antiproliferative activity and tubulin polymerization of 43 and 54 with epothilone B (2), epothilone D (4), and paclitaxel (7) showed that the synthetic analogues were less potent than their natural counterparts, although both retain full antiproliferative activity against a paclitaxel-resistant cell line. No significant difference in potency was noted between cis analogue 43 and its trans isomer 54.
Assuntos
Epotilonas , Compostos de Epóxi/síntese química , Macrolídeos/síntese química , Tiazóis/síntese química , Animais , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Compostos de Epóxi/farmacologia , Humanos , Macrolídeos/farmacologia , Suínos , Tiazóis/farmacologia , Tubulina (Proteína)/efeitos dos fármacos , Tubulina (Proteína)/metabolismo , Células Tumorais CultivadasRESUMO
Naturally occurring epothilones have been synthesized starting from enantiomerically pure aldol compounds 9-11, which were obtained by antibody catalysis. Aldolase antibody 38C2 catalyzed the resolution of (+/-)-9 by enantioselective retro-aldol reaction to afford 9 in 90% ee at 50 % conversion. Compounds 10 and 11 were obtained in more than 99% ee at 50% conversion by resolution of their racemic mixtures using newly developed aldolase antibodies 84G3, 85H6 or 93F3. Compounds 9, 10 and 11 were resolved in multigram quantities and then converted to the epothilones by metathesis processes, which were catalyzed by Grubbs' catalysts.
Assuntos
Antibacterianos/síntese química , Anticorpos Catalíticos/metabolismo , Antineoplásicos/síntese química , Epotilonas , Compostos de Epóxi/síntese química , Tiazóis/síntese química , Antibióticos Antineoplásicos/síntese química , Frutose-Bifosfato Aldolase/metabolismo , Haptenos/metabolismo , Macrolídeos , Espectroscopia de Ressonância Magnética , Estrutura Molecular , Myxococcales/metabolismo , EstereoisomerismoRESUMO
Three monoclonal aldolase antibodies (84G3, 85H6, and 93F3), generated against a beta-diketone hapten (II) by the reactive immunization technique, catalyzed highly enantioselective retro-aldol reactions of the racemic thiazole aldols 13-20. Antibody 84G3 (0.0004-0.005 mol%) was used to resolve (+/-)-13-(+/-)-18 to afford compounds 13-18 in multigram quantities. Multiple 13-alkyl analogues of epothilone (7-12) and their trans isomers ((E)-7-(E)-12) were synthesized starting from thiazole aldols 13-18. Construction of the trisubstituted olefin moiety in compounds 7-12 and (E)-7-(E)-12 was catalyzed by Grubbs' catalyst (X). Initial biological testing with compounds 7-10 and their trans isomers showed that compounds 9, 10, and (E)-10 have appreciable tubulin polymerization and antiproliferative activities that approached those of epothilone C. The most active compound, (E)-9, even displayed potencies comparable to those observed for epothilones A and D. Interestingly, all trans analogues were more potent than their corresponding cis isomers. While introduction of an alkyl group at C-13 in the cis series led to an overall reduction in biological activity (compared to epothilone C), appropriate modification of the thiazole moiety (replacement of the 2-methyl substituent by a 2-methylthio group) was able to compensate for this loss. These results are encouraging in view of the expectation that epoxidations of these compounds should further increase their cellular activities. Thus, compounds 9, 10, and (E)-9 and (E)-10 represent highly promising candidates for further studies.
Assuntos
Anticorpos/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Epotilonas , Macrolídeos/síntese química , Macrolídeos/farmacologia , Tiazóis/química , Aldeído Liases/química , Aldeído Liases/imunologia , Alquilação , Animais , Antineoplásicos/química , Catálise , Humanos , Lactonas/síntese química , Macrolídeos/química , Espectroscopia de Ressonância Magnética , Conformação Molecular , Estereoisomerismo , Suínos , Tubulina (Proteína)/biossíntese , Tubulina (Proteína)/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
BACKGROUND: Numerous analogs of the antitumor agents epothilones A and B have been synthesized in search of better pharmacological profiles. Insights into the structure-activity relationships within the epothilone family are still needed and more potent and selective analogs of these compounds are in demand, both as biological tools and as chemotherapeutic agents, especially against drug-resistant tumors. RESULTS: A series of pyridine epothilone B analogs were designed, synthesized and screened. The synthesized compounds exhibited varying degrees of tubulin polymerization and cytotoxicity properties against a number of human cancer cell lines depending on the location of the nitrogen atom and the methyl substituent within the pyridine nucleus. CONCLUSIONS: The biological screening results in this study established the importance of the nitrogen atom at the ortho position as well as the beneficial effect of a methyl substituent at the 4- or 5-position of the pyridine ring. Two pyridine epothilone B analogs (i.e. compounds 3 and 4) possessing higher potencies against drug-resistant tumor cells than epothilone B, the most powerful of the naturally occurring epothilones, were identified.
Assuntos
Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Epotilonas , Compostos de Epóxi/química , Compostos de Epóxi/farmacologia , Piridinas/síntese química , Piridinas/farmacologia , Tiazóis/química , Tiazóis/farmacologia , Antineoplásicos/química , Antineoplásicos/uso terapêutico , Divisão Celular/efeitos dos fármacos , Desenho de Fármacos , Avaliação Pré-Clínica de Medicamentos , Resistencia a Medicamentos Antineoplásicos , Compostos de Epóxi/síntese química , Compostos de Epóxi/uso terapêutico , Humanos , Concentração Inibidora 50 , Macrolídeos/síntese química , Macrolídeos/química , Macrolídeos/farmacologia , Macrolídeos/uso terapêutico , Modelos Moleculares , Estrutura Molecular , Piridinas/química , Piridinas/uso terapêutico , Relação Estrutura-Atividade , Tiazóis/síntese química , Tiazóis/uso terapêutico , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Células Tumorais CultivadasAssuntos
Antineoplásicos/farmacologia , Epotilonas , Compostos de Epóxi/farmacologia , Microtúbulos/efeitos dos fármacos , Tiazóis/farmacologia , Animais , Antineoplásicos/uso terapêutico , Apoptose , Ciclo Celular/efeitos dos fármacos , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Resistência a Múltiplos Medicamentos , Tolerância a Medicamentos , Compostos de Epóxi/uso terapêutico , Compostos de Epóxi/toxicidade , Humanos , Lactonas/química , Estrutura Molecular , Paclitaxel/farmacologia , Relação Estrutura-Atividade , Tiazóis/química , Tiazóis/uso terapêutico , Tiazóis/toxicidade , Tubulina (Proteína)/química , Tubulina (Proteína)/metabolismo , Células Tumorais CultivadasRESUMO
1. Extracellular ATP and UTP have been reported to activate a nucleotide receptor (P2Y2-receptor) that mediates arachidonic acid release with subsequent prostaglandin formation, a reaction critically depending on the activity of a cytosolic phospholipase A2. In addition, extracellular nucleotides trigger activation of the classical mitogen-activated protein kinase (MAPK) cascade and cell proliferation as well as of the stress-activated protein kinase (SAPK) cascade. 2. In this study, we report that ATP and UTP are also able to activate the p38-MAPK pathway as measured by phosphorylation of the p38-MAPK and its upstream activators MKK3/6, as well as phosphorylation of the transcription factor ATF2 in a immunocomplex-kinase assay. 3. Time courses reveal that ATP and UTP induce a rapid and transient activation of the p38-MAPK activity with a maximal activation after 5 min of stimulation which declined to control levels over the next 20 min. 4. A series of ATP and UPT analogues were tested for their ability to stimulate p38-MAPK activity. UTP and ATP were very effective analogues to activate p38-MAPK, whereas ADP and gamma-thio-ATP had only moderate activating effects. 2-Methyl-thio-ATP, beta gamma-imido-ATP, AMP, adenosine and UDP had no significant effects of p38-MAPK activity. In addition, the extracellular nucleotide-mediated effect on p38-MAPK was almost completely blocked by 1 mM of suramin, a putative P2-purinoceptor antagonist. 5. In summary, these results demonstrate for the first time that extracellular nucleotides are able to activate the MKK3/6- p38-MAPK cascade most likely via the P2Y2-receptor. Moreover, this finding implies that all three MAPK subtypes are signalling candidates for extracellular nucleotide-stimulated cell responses.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Mesângio Glomerular/enzimologia , Proteínas Quinases Ativadas por Mitógeno , Nucleotídeos/farmacologia , Estresse Fisiológico/enzimologia , Fator 2 Ativador da Transcrição , Trifosfato de Adenosina/farmacologia , Animais , Western Blotting , Bovinos , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Inibidores Enzimáticos/farmacologia , Proteínas de Ligação ao GTP/fisiologia , Mesângio Glomerular/citologia , Mesângio Glomerular/efeitos dos fármacos , Imidazóis/farmacologia , Isoenzimas/metabolismo , Fosforilação , Piridinas/farmacologia , Ratos , Receptores Purinérgicos P2/fisiologia , Receptores Purinérgicos P2Y2 , Suramina/farmacologia , Fatores de Transcrição/metabolismo , Uridina Trifosfato/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoAssuntos
Antineoplásicos/síntese química , Compostos Aza/síntese química , Epotilonas , Macrolídeos/síntese química , Antineoplásicos/farmacologia , Compostos Aza/farmacologia , Biotransformação , Humanos , Macrolídeos/metabolismo , Macrolídeos/farmacologia , Tubulina (Proteína)/metabolismo , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismoRESUMO
A series of new epothilone B and D analogues incorporating fused hetero-aromatic side chains have been prepared. The synthetic strategy is based on olefin 3 as the common intermediate and allows variation of the side-chain structure in a highly convergent and stereoselective manner. Epothilone analogues 1a-d and 2a-d are more potent inhibitors of cancer cell proliferation than the corresponding parent epothilones B or D.
Assuntos
Antineoplásicos/síntese química , Epotilonas , Compostos de Epóxi/síntese química , Tiazóis/síntese química , Antineoplásicos/farmacologia , Divisão Celular/efeitos dos fármacos , Resistência a Medicamentos , Compostos de Epóxi/farmacologia , Humanos , Concentração Inibidora 50 , Paclitaxel , Estereoisomerismo , Tiazóis/farmacologia , Células Tumorais CultivadasRESUMO
Treatment of renal mesangial cells with the vasoconstrictor angiotensin II stimulates a concentration-dependent increase in stress-activated protein kinase (SAPK) activity as measured by phosphorylation of the substrate c-Jun. Time course studies reveal a transient SAPK activation by angiotensin II which is maximal after 5-10 min of stimulation and rapidly declines thereafter to basal levels within 30 min. Using the highly selective angiotensin II AT1 receptor antagonist valsartan, a concentration-dependent inhibition of angiotensin II-induced SAPK activity is observed, clearly implying the AT1-receptor in this angiotensin II-mediated response. To further elucidate the mechanism involved in angiotensin II-induced SAPK activation, cells were treated with different inhibitors. Genistein, a tyrosine kinase inhibitor, greatly blocks (by 90%) the angiotensin II response, whereas pertussis toxin only partially inhibits angiotensin II-activated SAPK activity (by 76%). A highly potent protein kinase C inhibitor [3-[1-[3-(amidinothio)propyl-1H-indoyl-3-yl]-3-(1-methyl-1H- indoyl-3-yl) maleimide methane sulfonate], Ro 31-8220, as well as protein kinase C depletion from the cells by prolonged phorbol ester pretreatment, fail to inhibit the angiotensin II-induced SAPK activation. In summary these results suggest that angiotensin II AT1-receptor is able to activate the SAPK cascade in mesangial cells by a pathway independent of protein kinase C, but requiring both pertussis-toxin-sensitive and -insensitive G-proteins and tyrosine kinase activation.
Assuntos
Angiotensina II/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Mesângio Glomerular/efeitos dos fármacos , Receptores de Angiotensina/metabolismo , Angiotensina I/metabolismo , Animais , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Mesângio Glomerular/enzimologia , Mesângio Glomerular/fisiologia , Indóis/farmacologia , Fosforilação , Proteína Quinase C/antagonistas & inibidores , RatosRESUMO
It has been widely accepted that the oncogene product bcl-2 protects mammalian cells from programmed cell death (apoptosis). The molecules and signalling pathways upon which bcl-2 acts are, however, still ill-defined. Recently, bcl-2 was shown to interact with c-raf-1 in vitro. Furthermore, an active form of c-raf-1 delayed apoptosis induced by trophic factor deprivation and enhanced the death-suppressive function of bcl-2 when co-expressed. This has led to the hypothesis that bcl-2 communicates cell-death protection via a raf-dependent signal transduction pathway. Here we show, by various immunological and biochemical methods, that bcl-2 does not stably associate with c-raf-1 in cellular extracts prepared from fibroblasts before or after treatment with agents that induce apoptosis. Unexpectedly, bcl-2 function is entirely maintained, if not improved, when raf-dependent signalling is experimentally abrogated. In fact, bcl-2 allows the stable overexpression of a kinase-defective dominant-negative raf mutant that usually interferes with cell viability and/or proliferation. Our results indicate that bcl-2 does not require c-raf-1 kinase activity and an associated mitogen-activated protein kinase signalling pathway for its survival function. This property may be exploited to dissect cellular events that are dependent or independent of c-raf-1 kinase activity.
Assuntos
Apoptose , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Brefeldina A , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Divisão Celular , Sobrevivência Celular , Células Cultivadas , Cloranfenicol O-Acetiltransferase/biossíntese , Clonagem Molecular , AMP Cíclico/farmacologia , Ciclopentanos/farmacologia , Inibidores Enzimáticos/farmacologia , Fibroblastos , Humanos , Cinética , Proteínas Proto-Oncogênicas c-raf , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Transdução de Sinais , Estaurosporina/farmacologia , TransfecçãoRESUMO
1. Extracellular adenosine 5'-triphosphate (ATP) and uridine 5'-triphosphate (UTP) have been shown to activate a nucleotide receptor (P2U receptor) in rat mesangial cells that mediates phosphoinositide and phosphatidylcholine hydrolysis by phospholipases C and D, respectively. This is followed by an increased activity of the mitogen-activated protein kinase cascade and cell proliferation. Here we show that ATP and UTP potently stimulate the stress-activated protein kinase pathway and phosphorylation of the transcription factor c-Jun. 2. Both nucleotides stimulated a rapid (within 5 min) and concentration-dependent activation of stress-activated protein kinases as measured by the phosphorylation of c-Jun in a solid phase kinase assay. 3. When added at 100 microM the rank order of potency of a series of nucleotide analogues for stimulation of c-Jun phosphorylation was UTP > ATP = UDP = ATP gamma S > 2-methylthio-ATP > beta gamma-imido-ATP = ADP > AMP = UMP = adenosine = uridine. Activation of stress-activated protein kinase activity by ATP and UTP was dose-dependently attenuated by suramin. 4. Down-regulation of protein kinase C-alpha, -delta and -epsilon isoenzymes by 24 h treatment of the cells with 12-O-tetradecanoylphorbol 13-acetate did not inhibit ATP- and UTP-induced activation of c-Jun phosphorylation. Furthermore, the specific protein kinase C inhibitors, CGP 41251 and Ro 31-8220, did not inhibit nucleotide-stimulated c-Jun phosphorylation, suggesting that protein kinase C is not involved in ATP- and UTP-triggered stress-activated protein kinase activation. 5. Pretreatment of the cells with pertussis toxin or the tyrosine kinase inhibitor, genistein, strongly attenuated ATP- and UTP-induced c-Jun phosphorylation. Furthermore, N-acetyl-cysteine completely blocked the activation of stress-activated protein kinase in response to extracellular nucleotide stimulation. 6. In summary, these results suggest that ATP and UTP trigger the activation of the stress-activated protein kinase module in mesangial cells by a pathway independent of protein kinase C but requiring a pertussis toxin-sensitive G-protein and tyrosine kinase activation.
Assuntos
Trifosfato de Adenosina/farmacologia , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Mesângio Glomerular/efeitos dos fármacos , Proteínas Quinases Ativadas por Mitógeno , Uridina Trifosfato/farmacologia , Acetilcisteína/farmacologia , Sequência de Aminoácidos , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , Proteínas Quinases Dependentes de Cálcio-Calmodulina/química , Células Cultivadas , Ativação Enzimática , Genisteína , Mesângio Glomerular/citologia , Mesângio Glomerular/enzimologia , Isoflavonas/farmacologia , Proteínas Quinases JNK Ativadas por Mitógeno , Dados de Sequência Molecular , Toxina Pertussis , Proteína Quinase C/metabolismo , Ratos , Fatores de Virulência de Bordetella/farmacologia , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The serine/threonine-specific protein kinase Raf-1 plays a key role in mitogenic signal transduction by coupling Ras to the mitogen-activated protein (MAP) kinase cascade. Ras-mediated translocation to the plasma membrane represents a crucial step in the process of serum-stimulated Raf-1 kinase activation. The exact role of the multisite phosphorylation in Raf regulation, however, is not clear. We have previously reported that the mobility shift-associated hyperphosphorylation of Raf correlates with a reduction of serum-stimulated Raf kinase activity (Wartmann, M., and Davis, R. J. (1994) J. Biol. Chem. 269, 6695-6701). Here we show that incubation of serum-starved CHO cells with D609, a purported inhibitor of phosphatidylcholine-specific phospholipase C, also results in a mobility shift of Raf-1 that is due to hyperphosphorylation on sites identical to those observed following mitogen stimulation. Subcellular fractionation analyses revealed that D609-induced mobility shift-associated hyperphosphorylation was paralleled by a decreased membrane association of Raf-1. Similar results were obtained in an in vitro reconstitution system. Furthermore, PD98059, a specific inhibitor of activation of the MAP kinase kinase MEK, prevented D609-induced Raf hyperphosphorylation and restored the amount of membrane-bound Raf to control levels. Taken together, these data suggest that mobility shift-associated hyperphosphorylation of Raf-1, by virtue of reducing the amount of plasma membrane-bound Raf-1, represents a negative feedback mechanism contributing to the desensitization of the MAP kinase signaling cascade.
Assuntos
Proteínas de Membrana/metabolismo , Proteínas Serina-Treonina Quinases/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Animais , Hidrocarbonetos Aromáticos com Pontes/farmacologia , Células CHO , Cricetinae , Ativação Enzimática , Flavonoides/farmacologia , Humanos , Norbornanos , Fosforilação , Proteínas Proto-Oncogênicas c-raf , Tiocarbamatos , Tionas/farmacologia , Fosfolipases Tipo C/antagonistas & inibidoresRESUMO
Prolactin binds to a member of the cytokine receptor superfamily. The cytoplasmic domain of the prolactin receptor (PrlR) displays no enzymatic activity yet prolactin treatment leads to the induction of protein tyrosine phosphorylation. PrlR is associated with JAK2, a protein tyrosine kinase whose activity is stimulated following receptor dimerization. JAK2 subsequently phosphorylates PrlR and other cellular proteins which are recruited to the activated receptor complex. Among the JAK2 substrates is the transcription factor Stat5 whose phosphorylation mediates the transcriptional activation of beta-casein gene expression. In this review we discuss the prolactin induced signaling pathways which mediate differentiation of the mammary gland.
Assuntos
Mama/citologia , Glândulas Mamárias Animais/citologia , Proteínas do Leite , Prolactina/fisiologia , Proteínas Proto-Oncogênicas , Animais , Diferenciação Celular , Proteínas de Ligação a DNA/fisiologia , Células Epiteliais/fisiologia , Feminino , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Janus Quinase 2 , Sistema de Sinalização das MAP Quinases , Proteína Tirosina Fosfatase não Receptora Tipo 11 , Proteína Tirosina Fosfatase não Receptora Tipo 6 , Proteínas Tirosina Fosfatases/fisiologia , Proteínas Tirosina Quinases/fisiologia , Receptores da Prolactina/fisiologia , Fator de Transcrição STAT5 , Transativadores/fisiologiaRESUMO
HC11 mammary epithelial cells have been used to characterize molecular events involved in the regulation of milk protein gene expression. Treatment of HC11 cells with the lactogenic hormones prolactin, insulin, and glucocorticoids results in transcription of the beta-casein gene. Prolactin induces a signaling event which involves tyrosine phosphorylation of the mammary gland factor, Stat5, a member of the family of signal transducers and activators of transcription (Stat). Here we show that HC11 cells express two Stat5 proteins, Stat5a and Stat5b. Phosphopeptide and phosphoamino acid analysis of Stat5a and Stat5b immunoprecipitated from phosphate-labeled HC11 cells revealed that both proteins were constitutively phosphorylated on serine. Lactogenic hormone treatment resulted in the appearance of a tyrosine-phosphorylated peptide in both Stat5 proteins. Consistent with this observation, a Western blot analysis of Stat5a and Stat5b showed that lactogenic hormones induced a rapid, transient increase in phosphotyrosine which paralleled the binding of Stat5 to its cognate recognition sequence in the beta-casein gene promoter. Lactogenic hormone treatment of the HC11 cells also led to a rapid activation of the mitogen-activated protein (MAP) kinase pathway. We examined the role of this pathway in beta-casein transcription using a specific MAP kinase kinase inhibitor, PD98059. Concentrations of PD98059 which completely abrogated lactogen-induced MAP kinase activation did not affect the phosphorylation state of Stat5, its DNA binding activity, or transcriptional activation of a beta-casein reporter construct. This indicates that the MAP kinase pathway does not contribute to lactogenic hormone induction of the beta-casein gene.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Caseínas/genética , Proteínas de Ligação a DNA/metabolismo , Insulina/farmacologia , Glândulas Mamárias Animais/metabolismo , Proteínas do Leite , Prolactina/farmacologia , Transativadores/metabolismo , Transcrição Gênica/efeitos dos fármacos , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/antagonistas & inibidores , DNA/metabolismo , Inibidores Enzimáticos/farmacologia , Epitélio/metabolismo , Feminino , Flavonoides/farmacologia , Glândulas Mamárias Animais/citologia , Proteína Quinase 1 Ativada por Mitógeno , Fosforilação , Regiões Promotoras Genéticas , Fator de Transcrição STAT5RESUMO
Anandamide is an endogenous ligand for delta 9-tetrahydrocannabinol (THC) receptors. Incubation of cultured cells with anandamide or THC causes increased arachidonic acid release and eicosanoid biosynthesis. Here we demonstrate that the MAP kinase signal transduction pathway contributes to this response. Treatment of WI-38 fibroblasts with anandamide causes increased MAP kinase activity and increased phosphorylation of the arachidonate-specific cytoplasmic phospholipase A2 (cPLA2). Significantly, MAP kinase phosphorylates and activates cPLA2 [Lin, et al., Cell, 72 (1993) 269-278]. The MAP kinase signal transduction pathway may therefore mediate the effects of anadamide on cPLA2 activation and arachidonic acid release.
Assuntos
Ácidos Araquidônicos/farmacologia , Canabinoides/farmacologia , Proteínas Quinases/metabolismo , Transdução de Sinais , Ácido Araquidônico/metabolismo , Linhagem Celular , Citoplasma/metabolismo , Endocanabinoides , Ativação Enzimática/efeitos dos fármacos , Humanos , Fosfolipases A/metabolismo , Fosfolipases A2 , Fosforilação , Alcamidas Poli-InsaturadasRESUMO
Mitogen-activated protein kinase kinase (MKK) phosphorylates and activates mitogen-activated protein kinase (MAPK) in response to stimulation of various eukaryotic signaling pathways. Conversely, a recent report showed that MAPK phosphorylates MKK in vitro [Matsuda, S., Gotoh, Y., and Nishida, E. (1993) J. Biol. Chem. 268, 3277-3281]. To gain insight into the function of this feedback phosphorylation, we identified the major sites targeted for phosphorylation by MAPK and examined whether such a modification plays a role in regulating the basal and stimulated MKK activities. Two phosphopeptides generated by tryptic digestion of MAPK-phosphorylated MKK were identified by electrospray ionization mass spectrometry. Cyanogen bromide cleavage also yielded two phosphopeptides whose sequence overlapped with the tryptic phosphopeptides. Both sets of phosphopeptides contained candidate MAPK target sites at Thr292 and Thr386 that fit the consensus sequence ProXThr*Pro. Replacement of either Thr292 or Thr386 with alanine by site-directed mutagenesis reduced the phosphate incorporation respectively to 32 or 75% that of wild type MKK. Replacement of both threonine residues with alanine reduced phosphate incorporation to 2.5% that of wild type enzyme. Comparison of MAPK-phosphorylated vs. unphosphorylated MKK showed no significant differences in basal or Raf-1-stimulated MKK activity. We conclude that the phosphorylation of MKK at Thr292 and Thr386 does not interfere with catalysis in vitro.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Mutagênese Sítio-Dirigida , Proteínas Quinases/metabolismo , Sequência de Aminoácidos , Animais , Sítios de Ligação , Células CHO , Cricetinae , Ativação Enzimática , Humanos , Espectrometria de Massas , Quinases de Proteína Quinase Ativadas por Mitógeno , Dados de Sequência Molecular , Mapeamento de Peptídeos , Fosforilação , Proteínas Quinases/genética , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/farmacologia , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/farmacologia , Proteínas Proto-Oncogênicas c-raf , Coelhos , Ratos , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Raf is a mitogen-stimulated protein kinase that functions as a component of the signaling cascade that leads to the stimulation of mitogen-activated protein kinase. Here we show that the native structure of Raf is a large multi-subunit protein complex with an apparent mass of 300-500 kDa that interacts with Ras and the mitogen-activated protein kinase kinase Mek. Analysis of the structure of the Raf complex demonstrates that it contains a single Raf protein kinase together with the molecular chaperones hsp90 and p50. The Raf-hsp90-p50 complex was observed in starved cells and in cells activated with serum or phorbol ester. Thus, changes in complex formation with hsp90 and p50 are not required for activation of the Raf protein kinase. However, Raf activation caused by Ras was associated with the translocation of the cytoplasmic Raf-hsp90-p50 complex to the cell membrane. Significantly, it is only the membrane-bound complex that exhibits increased protein kinase activity. Thus, the Ras-activated Raf protein kinase functions as a membrane-bound multi-subunit complex.