RESUMO
Constitutive nuclear factor (NF)-kappaB activation in haematological malignancies is caused in several cases by loss of function mutations within the coding sequence of NF-kappaB inhibitory molecules such as IkappaBalpha or p100. Hut-78, a truncated form of p100, constitutively generates p52 and contributes to the development of T-cell lymphomas but the molecular mechanism underlying this oncogenic potential remains unclear. We show here that MMP9 gene expression is induced through the alternative NF-kappaB-activating pathway in fibroblasts and also on Hut-78 or p52 overexpression in fibroblasts as well as in lymphoma cells. p52 is critical for Hut-78-mediated MMP9 gene induction as a Hut-78 mutant as well as other truncated NF-kappaB2 proteins that are not processed into p52 failed to induce the expression of this metalloproteinase. Conversely, MMP9 gene expression is impaired in p52-depleted HUT-78 cells. Interestingly, MLL1 and MLL2 H3K4 methyltransferase complexes are tethered by p52 on the MMP9 but not on the IkappaBalpha promoter, and the H3K4 trimethyltransferase activity recruited on the MMP9 promoter is impaired in p52-depleted HUT-78 cells. Moreover, MLL1 and MLL2 are associated with Hut-78 in a native chromatin-enriched extract. Thus, we identified a molecular mechanism by which the recruitment of a H3K4 histone methyltransferase complex on the promoter of a NF-kappaB-dependent gene induces its expression and potentially the invasive potential of lymphoma cells harbouring constitutive activity of the alternative NF-kappaB-activating pathway.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Metaloproteinase 9 da Matriz/biossíntese , Proteína de Leucina Linfoide-Mieloide/metabolismo , Subunidade p52 de NF-kappa B/farmacologia , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Sequência de Bases , Células Cultivadas , Proteínas de Ligação a DNA/fisiologia , Indução Enzimática/efeitos dos fármacos , Indução Enzimática/fisiologia , Células HeLa , Histona Metiltransferases , Histona-Lisina N-Metiltransferase , Humanos , Lisina/metabolismo , Metaloproteinase 9 da Matriz/genética , Metaloproteinase 9 da Matriz/fisiologia , Camundongos , Dados de Sequência Molecular , Complexos Multiproteicos/metabolismo , Complexos Multiproteicos/fisiologia , Proteínas Mutantes/farmacologia , Proteína de Leucina Linfoide-Mieloide/fisiologia , Subunidade p52 de NF-kappa B/química , Células NIH 3T3 , Proteínas de Neoplasias/fisiologia , Proteínas de Fusão Oncogênica/farmacologia , Proteínas Metiltransferases/metabolismo , Proteínas Metiltransferases/fisiologia , Homologia de Sequência de AminoácidosRESUMO
A high-performance liquid chromatographic method for the enantiomeric analysis of a mixture of an alpha-amino acid and the corresponding acid amide is described. Reversed-phase chromatography with copper(II) acetate and N,N-di-n-propyl-L-alanine in the mobile phase are used for the separation. For Val and Val-NH2, several parameters affecting retention and enantioselectivity were investigated. The results indicate that by manipulation of pH, ionic strength, temperature, concentration of CuII, N,N-di-n-propyl-L-alanine and ion-pairing reagent, good control of enantiomeric separation can be achieved. For alpha-amino acid amides a mechanism is proposed which may account for the retention and enantioselectivity. Examples of enantiomeric analysis of mixtures of alpha-amino acids and alpha-amino acid amides with aliphatic, aromatic and polar side-chains are given. The method can be used for the control of the enantiomeric purity of alpha-amino acids and the corresponding acid amides obtained by enantioselective synthesis.