RESUMEN
Protein kinases play key roles in oncogenic signaling and are a major focus in the development of targeted cancer therapies. Imatinib, a BCR-Abl tyrosine kinase inhibitor, is a successful front-line treatment for chronic myelogenous leukemia (CML). However, resistance to imatinib may be acquired by BCR-Abl mutations or hyperactivation of Src family kinases such as Lyn. We have used multiplexed kinase inhibitor beads (MIBs) and quantitative mass spectrometry (MS) to compare kinase expression and activity in an imatinib-resistant (MYL-R) and -sensitive (MYL) cell model of CML. Using MIB/MS, expression and activity changes of over 150 kinases were quantitatively measured from various protein kinase families. Statistical analysis of experimental replicates assigned significance to 35 of these kinases, referred to as the MYL-R kinome profile. MIB/MS and immunoblotting confirmed the over-expression and activation of Lyn in MYL-R cells and identified additional kinases with increased (MEK, ERK, IKKα, PKCß, NEK9) or decreased (Abl, Kit, JNK, ATM, Yes) abundance or activity. Inhibiting Lyn with dasatinib or by shRNA-mediated knockdown reduced the phosphorylation of MEK and IKKα. Because MYL-R cells showed elevated NF-κB signaling relative to MYL cells, as demonstrated by increased IκBα and IL-6 mRNA expression, we tested the effects of an IKK inhibitor (BAY 65-1942). MIB/MS and immunoblotting revealed that BAY 65-1942 increased MEK/ERK signaling and that this increase was prevented by co-treatment with a MEK inhibitor (AZD6244). Furthermore, the combined inhibition of MEK and IKKα resulted in reduced IL-6 mRNA expression, synergistic loss of cell viability and increased apoptosis. Thus, MIB/MS analysis identified MEK and IKKα as important downstream targets of Lyn, suggesting that co-targeting these kinases may provide a unique strategy to inhibit Lyn-dependent imatinib-resistant CML. These results demonstrate the utility of MIB/MS as a tool to identify dysregulated kinases and to interrogate kinome dynamics as cells respond to targeted kinase inhibition.
Asunto(s)
Leucemia/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Benzamidas/farmacología , Línea Celular , Supervivencia Celular/efectos de los fármacos , Cromatografía de Afinidad , Dasatinib , Resistencia a Antineoplásicos/genética , Proteínas de Fusión bcr-abl , Humanos , Mesilato de Imatinib , Immunoblotting , FN-kappa B/genética , FN-kappa B/metabolismo , Piperazinas/farmacología , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Pirimidinas/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tiazoles/farmacología , Familia-src Quinasas/antagonistas & inhibidores , Familia-src Quinasas/genética , Familia-src Quinasas/metabolismoRESUMEN
Hyperthyroid patients develop left ventricular hypertrophy associated with alterations of several cardiac parameters such as heart rate, cardiac output, cardiac contraction and hemodynamic overload leading to cardiac complications. Although cardiac hypertrophy and contractile abnormality occur, interstitial fibrosis in the heart usually does not take place in hyperthyroid condition. Therefore, in the present study, the mechanism regulating myocardial extracellular matrix (ECM) remodeling in hyperthyroid condition was investigated. Cardiac hypertrophy was developed in Sprague-Dawley rats by administration of 3,5,3'-triiodo-L-thyronine (triiodothyronine, 8 microg/100g BW, ip, SID) and glucocorticosteroid, dexamethasone (DEX, 35 microg/100g BW, po, SID), which is also an inducer of hypertrophy for 15 days. Heart/Body weight ratio and atrial and brain natriuretic peptide mRNAs were significantly increased in both triiodothyronine- and DEX-treated rats compared to control. Collagens-I and -III deposition in the left ventricular sections was reduced in triiodothyronine-treated rats, whereas in DEX-treated animals those were increased compared to control. While mRNA and protein levels of procollagens-I and -III were increased with triiodothyronine (p<0.01), the levels of mature collagens-I and -III were decreased. The levels of the mature collagens were increased with DEX compared to control. MMP-1 activity in the serum and left ventricle was higher with reduced levels of TIMPs-3 and -4 in the left ventricle of triiodothyronine-treated rats. The results suggest that accelerated breakdown of collagens-I and -III by MMP-1 due to suppression of the endogenous TIMPs plays an important role in regulating the ECM in myocardium of hyperthyroid rat.