RESUMO
Elevated focal adhesion kinase (FAK) expression in human tumor cells has been correlated with an increased cell invasion potential. In cell culture, studies with FAK-null fibroblasts have shown that FAK function is required for cell migration. To determine the role of elevated FAK expression in facilitating epidermal growth factor (EGF)-stimulated human adenocarcinoma (A549) cell motility, antisense oligonucleotides were used to reduce FAK protein expression >75%. Treatment of A549 cells with FAK antisense (ISIS 15421) but not a mismatched control (ISIS 17636) oligonucleotide resulted in reduced EGF-stimulated p130(Cas)-Src complex formation, c-Jun NH(2)-terminal kinase (JNK) activation, directed cell motility, and serum-stimulated cell invasion through Matrigel. Because residual FAK protein in ISIS 15421-treated A549 cells was highly phosphorylated at the Tyr-397/Src homology (SH)2 binding site, expression of the FAK COOH-terminal domain (FRNK) was also used as an inhibitor of FAK function. Adenoviral-mediated infection and expression of FRNK promoted FAK dephosphorylation at Tyr-397, resulted in reduced EGF-stimulated JNK as well as extracellular-regulated kinase 2 (ERK2) kinase activation, inhibited matrix metalloproteinase-9 (MMP-9) secretion, and potently blocked both random and EGF-stimulated A549 cell motility. Equivalent expression of a FRNK (S-1034) point-mutant that did not promote FAK dephosphorylation also did not affect EGF-stimulated signaling or cell motility. Dose-dependent reduction in EGF-stimulated A549 motility was observed with the PD98059 MEK1 inhibitor and the batimastat (BB-94) inhibitor of MMP activity, but not with the SB203580 inhibitor of p38 kinase. Finally, comparisons between normal, FAK-null, and FAK-reconstituted fibroblasts revealed that FAK enhanced EGF-stimulated JNK and ERK2 kinase activation that was required for cell motility. These data indicate that FAK functions as an important signaling platform to coordinate EGF-stimulated cell migration in human tumor cells and support a role for inhibitors of FAK expression or activity in the control of neoplastic cell invasion.
Assuntos
Adenocarcinoma/enzimologia , Movimento Celular/fisiologia , Fator de Crescimento Epidérmico/antagonistas & inibidores , Proteínas Tirosina Quinases/antagonistas & inibidores , Adenocarcinoma/patologia , Movimento Celular/efeitos dos fármacos , Ativação Enzimática , Fator de Crescimento Epidérmico/farmacologia , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/fisiologia , Metaloproteinase 9 da Matriz/metabolismo , Inibidores de Metaloproteinases de Matriz , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Invasividade Neoplásica , Oligonucleotídeos Antissenso/genética , Oligonucleotídeos Antissenso/farmacologia , Fosforilação , Proteínas Tirosina Quinases/biossíntese , Proteínas Tirosina Quinases/genética , Proteínas Tirosina Quinases/fisiologia , Células Tumorais CultivadasRESUMO
Response to genotoxic stress can be considered as a multistage process involving initiation of cell-cycle arrest and maintenance of arrest during DNA repair. Although maintenance of G2/M checkpoints is known to involve Chk1, Chk2/Rad53 and upstream components, the mechanisms involved in its initiation are less well defined. Here we report that p38 kinase has a critical role in the initiation of a G2 delay after ultraviolet radiation. Inhibition of p38 blocks the rapid initiation of this checkpoint in both human and murine cells after ultraviolet radiation. In vitro, p38 binds and phosphorylates Cdc25B at serines 309 and 361, and Cdc25C at serine 216; phosphorylation of these residues is required for binding to 14-3-3 proteins. In vivo, inhibition of p38 prevents both phosphorylation of Cdc25B at serine 309 and 14-3-3 binding after ultraviolet radiation, and mutation of this site is sufficient to inhibit the checkpoint initiation. In contrast, in vivo Cdc25C binding to 14-3-3 is not affected by p38 inhibition after ultraviolet radiation. We propose that regulation of Cdc25B phosphorylation by p38 is a critical event for initiating the G2/M checkpoint after ultraviolet radiation.
Assuntos
Proteínas de Ciclo Celular/metabolismo , Fase G2/fisiologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Mitose/fisiologia , Fosfatases cdc25/metabolismo , Proteínas 14-3-3 , Animais , Fase G2/genética , Células HeLa , Humanos , Camundongos , Mitose/genética , Índice Mitótico , Fosforilação , Ligação Proteica , Serina/metabolismo , Tirosina 3-Mono-Oxigenase/metabolismo , Raios Ultravioleta , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
The p53 tumor suppressor protein plays a key role in the regulation of stress-mediated growth arrest and apoptosis. Stress-induced phosphorylation of p53 tightly regulates its stability and transcriptional activities. Mass spectrometry analysis of p53 phosphorylated in 293T cells by active Jun NH2-terminal kinase (JNK) identified T81 as the JNK phosphorylation site. JNK phosphorylated p53 at T81 in response to DNA damage and stress-inducing agents, as determined by phospho-specific antibodies to T81. Unlike wild-type p53, in response to JNK stimuli p53 mutated on T81 (T81A) did not exhibit increased expression or concomitant activation of transcriptional activity, growth inhibition, and apoptosis. Forced expression of MKP5, a JNK phosphatase, in JNK kinase-expressing cells decreased T81 phosphorylation while reducing p53 transcriptional activity and p53-mediated apoptosis. Similarly transfection of antisense JNK 1 and -2 decreased T81 phosphorylation in response to UV irradiation. More than 180 human tumors have been reported to contain p53 with mutations within the region that encompasses T81 and the JNK binding site (amino acids 81 to 116). Our studies identify an additional mechanism for the regulation of p53 stability and functional activities in response to stress.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteína Supressora de Tumor p53/química , Proteína Supressora de Tumor p53/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Divisão Celular , Linhagem Celular , Primers do DNA/genética , Estabilidade de Medicamentos , Fosfatases de Especificidade Dupla , Genes p53 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas Quinases JNK Ativadas por Mitógeno , MAP Quinase Quinase 4 , Espectrometria de Massas , Camundongos , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Fosfatases da Proteína Quinase Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/genética , Mutação , Neoplasias/genética , Neoplasias/metabolismo , Oligonucleotídeos Antissenso/farmacologia , Fosforilação , Proteínas Tirosina Fosfatases/metabolismo , Estresse Fisiológico/genética , Estresse Fisiológico/metabolismo , Treonina/química , Transcrição Gênica , Proteína Supressora de Tumor p53/genética , Raios UltravioletaRESUMO
Microtubule-damaging agents arrest cells at G(2)/M and induce apoptosis in association with phosphorylation of the anti-apoptotic proteins Bcl-2 and Bcl-X(L). Because microtubule inhibitors activate JNK, we sought to determine whether JNK was responsible for Bcl-2/Bcl-X(L) phosphorylation in KB-3 cells treated with vinblastine. Two major endogenous forms of JNK, p46(JNK1) and p54(JNK2), were present in KB-3 cells, and both isoforms were activated by vinblastine as determined by Mono Q chromatography. We used antisense oligonucleotides (AS) to specifically inhibit their expression. A combination of AS-JNK1 with AS-JNK2 inhibited by 80% vinblastine-induced phosphorylation of two known JNK substrates, c-Jun and ATF-2. In addition, AS-JNK1/2 inhibited vinblastine-induced phosphorylation of Bcl-2 by 85% and that of Bcl-X(L) by 65%. Stable expression of the JNK scaffold protein JIP-1 blocked vinblastine-induced phosphorylation of c-Jun and ATF-2, but did not affect Bcl-2/Bcl-X(L) phosphorylation, confirming a bifurcation in JNK signaling involving both nuclear and non-nuclear substrates. Vinblastine-induced phosphorylation of Raf-1 was unaffected by AS-JNK1/2 and was associated with loss of activity for MEK substrate in vitro and inactivation of ERK in vivo. These results provide evidence for a direct role of the JNK pathway in apoptotic regulation through Bcl-2/Bcl-X(L) phosphorylation.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteínas Proto-Oncogênicas c-raf/metabolismo , Vimblastina/farmacologia , Proteínas de Transporte/metabolismo , Ativação Enzimática , Humanos , Isoenzimas/metabolismo , Sistema de Sinalização das MAP Quinases , Proteína Quinase 8 Ativada por Mitógeno , Proteína Quinase 9 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/genética , Oligonucleotídeos Antissenso/farmacologia , Fosforilação , Células Tumorais Cultivadas , Proteína bcl-XRESUMO
The c-Jun N-terminal kinase/stress-activated protein kinase (JNK/SAPK) pathway is activated by numerous cellular stresses. Although it has been implicated in mediating apoptosis and growth factor signaling, its role in regulating cell growth is not yet clear. Here, the influence of JNK on basal (unstimulated) growth of human tumor glioblastoma T98G cells was investigated using highly specific JNK antisense oligonucleotides to inhibit JNK expression. Transient depletion of either JNK1 or JNK2 suppressed cell growth associated with an inhibition of DNA synthesis and cell cycle arrest in S phase. The growth-inhibitory potency of JNK2 antisense ((JNK)2 IC(50) = 0.14 micrometer) was greater than that of JNK1 antisense ((JNK)1 IC(50) = 0.37 micrometer), suggesting that JNK2 plays a dominant role in regulating growth of T98G cells. Indeed, JNK2 antisense-treated populations exhibited greater inhibition of DNA synthesis and accumulation of S-phase cells than did the JNK1 antisense-treated cultures, with a significant proportion of these cells detaching from the tissue culture plate. JNK2 (but not JNK1) antisense-treated cultures exhibited marked elevation in the expression of the cyclin-dependent kinase inhibitor p21(cip1/waf1) accompanied by inhibition of Cdk2/Cdc2 kinase activities. Taken together, these results indicate that JNK is required for growth of T98G cells in nonstress conditions and that p21(cip1/waf1) may contribute to the sustained growth arrest of JNK2-depleted T98G cultures.
Assuntos
Ciclo Celular/efeitos dos fármacos , Divisão Celular/fisiologia , Proteínas Quinases Ativadas por Mitógeno/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Transcrição Gênica/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , DNA de Neoplasias/biossíntese , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Glioblastoma , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Cinética , Proteína Quinase 8 Ativada por Mitógeno , Proteína Quinase 9 Ativada por Mitógeno , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fase S , Tionucleotídeos , Células Tumorais CultivadasRESUMO
Many diseases result from defects in cell signaling. Achieving an in-depth understanding of the complex mechanisms by which cells transduce extracellular signals into cellular responses in both normal and diseased systems is a crucial step in the discovery of more effective drugs to treat human diseases. Traditional approaches for studying cell signaling have some limitations. Antisense oligonucleotides represent a novel approach for studying signal transduction processes that offers significant advantages in terms of specificity and versatility. This article reviews the opportunities that antisense oligonucleotides offer for the study of signal transduction pathways and identification of inhibitors of these pathways for drug development.
Assuntos
Expressão Gênica/efeitos dos fármacos , Oligonucleotídeos Antissenso/farmacologia , Transdução de Sinais/efeitos dos fármacos , Animais , Expressão Gênica/fisiologia , Humanos , Oligonucleotídeos Antissenso/química , Oligonucleotídeos Antissenso/uso terapêutico , Transdução de Sinais/fisiologiaRESUMO
c-Jun N-terminal kinase (JNK) plays a critical role in coordinating the cellular response to stress and has been implicated in regulating cell growth and transformation. To investigate the growth-regulatory functions of JNK1 and JNK2, we used specific antisense oligonucleotides (AS) to inhibit their expression. A survey of several human tumor cell lines revealed that JNKAS treatment markedly inhibited the growth of cells with mutant p53 status but not that of cells with normal p53 function. To further examine the influence of p53 on cell sensitivity to JNKAS treatment, we compared the responsiveness of RKO, MCF-7, and HCT116 cells with normal p53 function to that of RKO E6, MCF-7 E6, and HCT116 p53(-/-), which were rendered p53 deficient by different methods. Inhibition of JNK2 (and to a lesser extent JNK1) expression dramatically reduced the growth of p53-deficient cells but not that of their normal counterparts. JNK2AS-induced growth inhibition was correlated with significant apoptosis. JNK2AS treatment induced the expression of the cyclin-dependent kinase inhibitor p21(Cip1/Waf1) in parental MCF-7, RKO, and HCT116 cells but not in the p53-deficient derivatives. That p21(Cip1/Waf1) expression contributes to the survival of JNK2AS-treated cells was supported by additional experiments demonstrating that p21(Cip1/Waf1) deficiency in HCT116 cells also results in heightened sensitivity to JNKAS treatment. Our results indicate that perturbation of JNK2 expression adversely affects the growth of otherwise nonstressed cells. p53 and its downstream effector p21(Cip1/Waf1) are important in counteracting these detrimental effects and promoting cell survival.
Assuntos
Apoptose , Proteínas Quinases Ativadas por Mitógeno , Proteínas Quinases/genética , Proteína Supressora de Tumor p53/genética , Apoptose/genética , Divisão Celular/genética , Sobrevivência Celular/genética , Feminino , Regulação Enzimológica da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Proteína Quinase 9 Ativada por Mitógeno , Proteínas Quinases/biossíntese , Transdução de Sinais/genética , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/metabolismoRESUMO
Erythropoietin (EPO), a major regulator of erythroid progenitor cells, is essential for the survival, proliferation, and differentiation of immature erythroid cells. To gain insight into the molecular mechanism by which EPO functions, we analyzed the activation of Jun N-terminal kinases (JNKs) and extracellular signal-regulated kinases (ERKs) in HCD-57 cells, a murine erythroid progenitor cell line that requires EPO for survival and proliferation. Withdrawal of EPO from the cell culture medium resulted in sustained activation of JNKs plus p38 MAP kinase, and inactivation of ERKs, preceding apoptosis of the cells. Addition of EPO to the EPO-deprived cells caused activation of ERKs accompanied by inactivation of JNKs and p38 MAP kinase and rescued the cells from apoptosis. Phorbol 12-myristate 13-acetate, which activated ERKs by a different mechanism, also suppressed the activation of JNKs and significantly retarded apoptosis of the cells caused by withdrawal of EPO. Furthermore, MEK inhibitor PD98059, which inhibited activation of ERKs, caused activation of JNKs, whereas suppression of JNK expression by antisense oligonucleotides and inhibition of p38 MAP kinase by SB203580 caused attenuation of the apoptosis that occurs upon withdrawal of EPO. Finally, the activation of JNKs and p38 MAP kinase and concurrent inactivation of ERKs upon withdrawal of EPO were also observed in primary human erythroid colony-forming cells. Taken together, the data suggest that activation of ERKs promotes cell survival, whereas activation of JNKs and p38 MAP kinase leads to apoptosis and EPO functions by controlling the dynamic balance between ERKs and JNKs.
Assuntos
Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Eritroblastos/patologia , Eritroblastos/fisiologia , Eritropoetina/farmacologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Animais , Linhagem Celular , Sobrevivência Celular/fisiologia , Humanos , Sistema de Sinalização das MAP Quinases/fisiologia , Camundongos , Transdução de Sinais/efeitos dos fármacos , Transdução de Sinais/fisiologia , Proteínas Quinases p38 Ativadas por MitógenoAssuntos
Artrite Experimental/imunologia , Transplante de Coração/imunologia , Imunossupressores/farmacologia , Linfonodos/imunologia , Transfusão de Linfócitos , Linfócitos/imunologia , Animais , Artrite Experimental/prevenção & controle , Peso Corporal/efeitos dos fármacos , Dexametasona/farmacologia , Feminino , Hiperplasia , Isoantígenos/imunologia , Linfonodos/patologia , Linfócitos/efeitos dos fármacos , Linfócitos/efeitos da radiação , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C3H , Compostos Orgânicos , Ratos , Ratos Endogâmicos Lew , Baço , Transplante HeterotópicoAssuntos
Fibrose/fisiopatologia , Proteoglicanas/fisiologia , Fator de Crescimento Transformador beta/fisiologia , Animais , Anticorpos Monoclonais , Colágeno/metabolismo , Decorina , Eletroforese , Proteínas da Matriz Extracelular , Glomerulonefrite/metabolismo , Glomerulonefrite/patologia , Humanos , Imuno-Histoquímica , Ligação Proteica , Proteoglicanas/análise , Proteoglicanas/farmacologia , Proteínas Recombinantes/metabolismo , CicatrizaçãoRESUMO
To investigate the role of interleukin 6 (IL-6) in adjuvant-induced arthritis, serum from adjuvant-immunized Lewis rats treated with cyclosporin, indomethacin, or saline was evaluated for IL-6 activity. Inflammation was quantitated by measuring paw volume. We found that an increase in serum IL-6 activity parallels the kinetics of paw edema development in adjuvant-immunized rats. Daily treatment with 5 mg cyclosporin A/kg prevented the increase in paw volume and held serum IL-6 activity to levels observed in untreated (normal) rats. Daily treatment with 1 mg indomethacin/kg resulted in a 50% reduction in serum IL-6 levels and a significant decrease (approximately 50%) in paw volume on Day 17 compared to saline-treated rats. Linear regression analysis confirmed the positive correlation between mean paw volume and mean serum IL-6 activity (R2 = 0.783, P less than 0.01 on Day 17) in normal, arthritic, and cyclosporin A- or indomethacin-treated groups. These results are consistent with a role for IL-6 in the pathology of arthritis and suggest that serum IL-6 activity may be a useful parameter for monitoring disease activity.
Assuntos
Artrite Experimental/sangue , Artrite/sangue , Ciclosporinas/uso terapêutico , Indometacina/uso terapêutico , Interleucina-6/sangue , Animais , Artrite Experimental/tratamento farmacológico , Modelos Animais de Doenças , Edema , Pé , Masculino , Mycobacterium tuberculosis , Ratos , Ratos Endogâmicos LewRESUMO
The single-anastomosis heart-lung transplantation method in the rat developed by Lee and associates has been examined and has proven to be simple and reproducible. Technical procedures are discussed emphasizing those procedures that require special caution to ensure successful graft function. The donor and recipient rat strain combination has been found to influence the success of the transplantation procedure. Rat strain combinations used in our laboratories resulted in different percentages of donor heart survival. Using Lewis rat (RT1 1/1) recipients, 68% of Brown Norway rat (RT1n/n) donor hearts and 97% of NBR rat (RT1 1/1) donor hearts survived. However, only 29% of Fischer rat (RT1 1/1) donor hearts survived longer than 2 days when transplanted into Lewis rat recipients. In our laboratories, the Lee method of single-anastomosis heart-lung transplantation has been used successfully to evaluate whether immunoregulatory compounds alter heart allograft rejection.