RESUMO
The chimaeric protein Bcr/Abl, the hallmark of chronic myeloid leukaemia, has been connected with several signalling pathways, such as those involving protein kinase B/Akt, JNK (c-Jun N-terminal kinase) or ERKs (extracellular-signal-regulated kinases) 1 and 2. However, no data about the p38 MAPK (mitogen-activated protein kinase) have been reported. Here, we present evidence showing that Bcr/Abl is able to modulate this signalling pathway. Transient transfection experiments indicated that overexpression of Bcr/Abl in 293T cells is able to activate p38 MAPK or induce p73 stabilization, suggesting that c-Abl and Bcr/Abl share some biological substrates. Interestingly, the control exerted by Bcr/Abl on the p38 MAPK pathway was not only mediated by the tyrosine kinase activity of Bcr/Abl, as the use of STI571 demonstrated. In fact, Bcr alone was able to induce p38 MAPK activation specifically through MKK3 (MAP kinase kinase 3). Supporting these observations, chronic myeloid leukaemia-derived K562 cells or BaF 3 cells stably transfected with Bcr/Abl showed higher levels of phosphorylated p38 MAPK compared with Bcr/Abl-negative cells. While Bcr/Abl-negative cells activated p38 MAPK in response to Ara-C (1-beta-D-arabinofuranosylcytosine), Bcr/Abl-positive cells were unable to activate p38 MAPK, suggesting that the p38 MAPK pathway is not sensitive to Abl-dependent stimuli in Bcr/Abl-positive cells. Our results demonstrate that the involvement of Bcr/Abl in the p38 MAPK pathway is a key mechanism for explaining resistance to Ara-C, and could provide a clue for new therapeutic approaches based on the use of specific Abl inhibitors.
Assuntos
Proteínas de Fusão bcr-abl/metabolismo , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Benzamidas , Linhagem Celular , Linhagem Celular Tumoral , Citarabina/antagonistas & inibidores , Citarabina/metabolismo , Citarabina/farmacologia , Proteínas de Ligação a DNA/metabolismo , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Proteínas de Fusão bcr-abl/fisiologia , Genes Supressores de Tumor , Humanos , Mesilato de Imatinib , Rim/química , Rim/citologia , Rim/embriologia , Rim/metabolismo , Proteínas Nucleares/metabolismo , Piperazinas/farmacologia , Proteínas Proto-Oncogênicas c-abl/metabolismo , Pirimidinas/farmacologia , Proteína Tumoral p73 , Proteínas Supressoras de Tumor , Células U937/enzimologia , Células U937/metabolismo , Células U937/patologiaRESUMO
p38 MAPK has been implicated in the response to cancer therapy. To determine whether the activation of p38 MAPK could be specific to cancer therapy, we investigated the activation of p38 MAPK in response to several chemotherapeutic agents, such as cisplatin, doxorubicin and taxol in several human cell lines. Activation of p38 MAPK was measured after exposure to several chemotherapeutic agents, using specific phosphoantibodies. Only cisplatin was able to activate p38 MAPK in all the cell lines tested. Furthermore, other platinum compounds such as transplatin and platinum (IV) chloride can induce activation of p38 MAPK. The kinetics of this activation is a key event in the biological role of p38 MAPK in response to cisplatin, as we conclude from the differences observed after treatment with transplatin and cisplatin. The p38 MAPK activation is independent of the origin or genetic alterations of the cell lines and seems to be mediated through both upstream activators MKK6 and MKK3. Although the isoforms alpha/beta are mainly activated, we also demonstrated that other members of the p38 MAPK family were susceptible to activation by cisplatin when they were overexpressed in 293 T. Finally, pretreatment with specific inhibitors (SB 203580 and SKF 86002) induces a resistant phenotype in response to cisplatin. Furthermore, low activation of this SAPK pathway correlates with a resistant phenotype as demonstrated in our experimental model of head and neck cancer. Therefore, we conclude that the p38 MAPK pathway is a specific target for cisplatin-based therapy with clinical implications.
Assuntos
Cisplatino/farmacologia , Proteínas Quinases Ativadas por Mitógeno , Animais , Antineoplásicos/farmacologia , Células COS , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Linhagem Celular , Sobrevivência Celular , Reagentes de Ligações Cruzadas/farmacologia , Relação Dose-Resposta a Droga , Relação Dose-Resposta à Radiação , Resistencia a Medicamentos Antineoplásicos , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Células HeLa , Humanos , Imidazóis/farmacologia , Cinética , MAP Quinase Quinase 3 , MAP Quinase Quinase 6 , Proteína Quinase 12 Ativada por Mitógeno , Proteína Quinase 13 Ativada por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fenótipo , Fosforilação , Plasmídeos/metabolismo , Compostos de Platina/farmacologia , Testes de Precipitina , Isoformas de Proteínas , Proteínas Tirosina Quinases/metabolismo , Piridinas/farmacologia , Transdução de Sinais , Tiazóis/farmacologia , Transfecção , Células Tumorais Cultivadas , Proteínas Quinases p38 Ativadas por MitógenoRESUMO
In order to investigate the molecular mechanisms implicated in the induction of chemo sensitivity by adenovirus E1a gene expression, we decided to investigate which signal transduction pathways could be affected by the E1a gene in Human Normal Fibroblast (IMR90). No effect was observed in SAPK pathways (p38MAPK and JNK), but E1a was able to affect the Akt activation mediated by insulin. This result was confirmed by transient transfection experiments performed in Cos-7 cells and also observed in other transformed cell lines such as A431. Furthermore, E1a expression induces a decrease in the basal status of Akt activity. Finally we demonstrated that E1a is able to block the Akt activation mediated by cisplatin and correlates with a sensitive phenotype. In summary, our data demonstrate that specific inhibition of the PI3K/Akt pathway mediates some of the biological properties of E1a such as induction of chemosensitivity.
Assuntos
Proteínas E1A de Adenovirus/genética , Antineoplásicos/farmacologia , Cisplatino/farmacologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/fisiologia , Transdução de Sinais , Animais , Células COS , Dano ao DNA , Resistencia a Medicamentos Antineoplásicos , Genes ras , Humanos , Insulina/farmacologia , Fosforilação , Proteínas Proto-Oncogênicas c-aktRESUMO
The gene mutated in ataxia telangiectasia, ATM, has been implicated in several cell functions such as cell cycle control and response to DNA damage and insulin. PKB/Akt has also been implicated in the cellular response to insulin, gamma-radiation, and cell cycle control. Interestingly, lack of PKB/Akt function in vivo is able to mimic some phenotypic abnormalities associated with ataxia telangiectasia (AT). Here we show that ATM is a major determinant of full PKB/Akt activation in response to insulin or gamma-radiation. This effect is mediated through the phosphatidylinositol 3-kinase domain of ATM that specifically affects Akt serine 473 phosphorylation. This conclusion was inferred from the results obtained in transient transfection assays using exogenous PKB/Akt and ATM in Cos cells. Moreover, the use of ATM inhibitors or small interfering RNA confirmed our observation. Further supporting these results, we also observed that biological responses tightly regulated by Akt, such as transcription factor of the forkhead family activity after insulin treatment or gamma-radiation response, were altered in cell lines derived from AT patients and knockout mice for ATM in which phosphorylation in serine 473 was almost abolished. This study proposes new clues in the search of the unknown PDK2 and new explanations for the radiosensitivity or insulin intolerance described more than 30 years ago in AT patients.