RESUMO
Heterozygosity for a mutant dysfunctional C1 inhibitor protein, a member of the serine proteinase inhibitor (serpin) superfamily, results in type II hereditary angioneurotic oedema. We identified a "hinge" region mutation in C1 inhibitor with a Val to Glu replacement at P14 Val-432. Recombinant C1 inhibitors P10 Ala-->Thr and P14Val-->Glu did not form stable complexes with fluid phase C1s or kallikrein. The P14 Val-->Glu mutant, however, was cleaved to a 96K form by C1s, while the P10 Ala-->Thr mutant was not. The recombinant P10 mutant also did not complex with C1s, kallikrein or beta-factor Xlla-Sepharose. The two mutations, therefore, result in dysfunction by different mechanisms: in one (P14 Val-->Glu), the inhibitor is converted to a substrate, while in the other (P10 Ala-->Thr), interaction with target protease is blocked.
Assuntos
Angioedema/genética , Proteínas Inativadoras do Complemento 1/genética , Mutação Puntual , Alanina , Sequência de Aminoácidos , Angioedema/sangue , Animais , Sequência de Bases , Linhagem Celular , Células Cultivadas , Códon/genética , Proteínas Inativadoras do Complemento 1/química , Proteínas Inativadoras do Complemento 1/metabolismo , Complemento C1s/metabolismo , Fibroblastos/metabolismo , Glutamatos , Ácido Glutâmico , Heterozigoto , Humanos , Calicreínas/metabolismo , Dados de Sequência Molecular , Estrutura Secundária de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Treonina , Transfecção , ValinaRESUMO
OBJECTIVE: This study aimed to evaluate the expression level of anti-apoptotic Bcl-2 family proteins in B and T cells in patients with systemic lupus erythematosus (SLE) and rheumatoid arthritis (RA) in relation to disease activity and the effect of various Bcl-2 family inhibitors (BH3 mimetics) as potential treatment. METHODS: We included 14 SLE patients, 12 RA patients, and 13 healthy controls to study anti-apoptotic Bcl-2, Bcl-XL, and Mcl-1 expression and cell survival in different B and T cell subsets using stimulation assays and intracellular flow cytometry. Effect of various BH3 mimetics was assessed by cell viability analyses. RESULTS: In SLE, significant differences in Bcl-2 family members were confined to the B cell compartment with decreased induction of Bcl-XL (p ≤ 0.05) and Mcl-1 (p ≤ 0.001) upon CpG stimulation. In RA, we did not observe any differences in expression levels of Bcl-2 family proteins. Expression patterns did not correlate with disease activity apart from decreased induction of Mcl-1 in B cells in active SLE. After in vitro stimulation with CpG, plasmablasts were more viable after treatment with three different BH3 mimetics compared to naïve or memory B cells in control and patient cells. After activation, Mcl-1 inhibition was most effective in reducing plasmablast and T cell viability, however, less in patients than controls. CONCLUSION: Our study provides evidence for the increased differential expression pattern of Bcl-2 family members in B and T cell subsets of patients with SLE compared to controls. Tested BH3 mimetics showed higher efficacy in controls compared to both autoimmune diseases, though nonsignificant due to low patient numbers.
Assuntos
Artrite Reumatoide , Lúpus Eritematoso Sistêmico , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Apoptose , Linfócitos T/metabolismoRESUMO
Seliciclib (R-roscovitine) is a cyclin-dependent kinase inhibitor in clinical development. It triggers apoptosis by inhibiting de novo transcription of the short-lived Mcl-1 protein, but it is unknown how this leads to Bax/Bak activation that is required for most forms of cell death. Here, we studied the effects of seliciclib in B-cell chronic lymphocytic leukemia (B-CLL), a malignancy with aberrant expression of apoptosis regulators. Although seliciclib-induced Mcl-1 degradation within 4 h, Bax/Bak activation occurred between 16 and 20 h. During this period, no transcriptional changes in apoptosis-related genes occurred. In untreated cells, prosurvival Mcl-1 was engaged by the proapoptotic proteins Noxa and Bim. Upon drug treatment, Bim was quickly released. The contribution of Noxa and Bim as a specific mediator of seliciclib-induced apoptosis was demonstrated via RNAi. Significantly, 16 h after seliciclib treatment, there was accumulation of Bcl-2, Bim and Bax in the 'mitochondria-rich' insoluble fraction of the cell. This suggests that after Mcl-1 degradation, the remaining apoptosis neutralizing capacity of Bcl-2 is gradually overwhelmed, until Bax forms large multimeric pores in the mitochondria. These data demonstrate in primary leukemic cells hierarchical binding and crosstalk among Bcl-2 members, and suggest that their functional interdependence can be exploited therapeutically.
Assuntos
Apoptose , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas de Neoplasias/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Purinas/farmacologia , Idoso , Idoso de 80 Anos ou mais , Proteínas Reguladoras de Apoptose/metabolismo , Proteína 11 Semelhante a Bcl-2 , Linhagem Celular Tumoral , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Masculino , Proteínas de Membrana/metabolismo , Pessoa de Meia-Idade , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidores , Interferência de RNA , Roscovitina , Proteína Killer-Antagonista Homóloga a bcl-2/metabolismo , Proteína X Associada a bcl-2/metabolismoRESUMO
The last exon of the C1-1NH gene was screened for point mutations in 36 unrelated hereditary angioedema patients. Mutations were found in eight patients, predicting changes in the short COOH-terminal region which anchors the reactive site loop on its COOH-terminal side. The effects of each of these mutations were examined in transiently transfected Cos-7 cells. Complete intracellular retention or degradation was observed with substitutions in the COOH-terminal strands 4B or 5B: Leu459-->Pro, Leu459-->Arg, and Pro467-->Arg were all blocked at early stages of intracellular transport, but differences in the immunofluorescence patterns indicated that a significant fraction of the Leu459-->Pro and of the Pro467-->Arg proteins reached a compartment distinct from the endoplasmic reticulum. In line with previous findings with alpha 1-antitrypsin, chain termination within strand 5B resulted in rapid degradation. Mutant Val451-->Met, in strand 1C, and mutant Pro476-->Ser, replacing the invariant proline near the COOH terminus, yielded reduced secretion, but these extracellular proteins were unable to bind the target protease C1s. Presence of low levels of both dysfunctional proteins in patient plasmas defies the conventional classification of C1 inhibitor deficiencies as type I or type II. These data point to a key role of certain residues in the conserved COOH-terminal region of serpins in determining the protein foldings compatible with transport and proper exposure of the reactive site loop.
Assuntos
Angioedema/genética , Proteínas Inativadoras do Complemento 1/genética , Éxons/genética , Mutação Puntual , Serpinas/genética , Sequência de Aminoácidos , Angioedema/classificação , Sequência de Bases , Transporte Biológico , Clonagem Molecular , Complemento C1/metabolismo , Proteínas Inativadoras do Complemento 1/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Ligação Proteica , Seleção Genética , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Relação Estrutura-AtividadeRESUMO
Proteolytic inactivation of serine protease inhibitors (serpins) by neutrophil elastase (HNE) is presumed to contribute to the deregulation of plasma cascade systems in septic shock. Here, we report a supplementary approach to construct serpins, in our case C1 inhibitor, that are resistant to catalytic inactivation by HNE. Instead of shifting the specificity of alpha 1-antitrypsin towards the proteases of the contact activation and complement systems, we attempted to obtain a C1 inhibitor species which resists proteolytic inactivation by HNE. 12 recombinant C1 inhibitor variants were produced with mainly conservative substitutions at the cleavage sites for HNE, 440-Ile and/or 442-Val. Three variants significantly resisted proteolytic inactivation, both by purified HNE, as well as by activated neutrophils. The increase in functional half-life in the presence of FMLP-stimulated cells was found to be 18-fold for the 440-Leu/442-Ala variant. Inhibitory function of these variants was relatively unimpaired, as examined by the formation of stable complexes with C1s, beta-Factor XIIa, kallikrein, and plasmin, and as determined by kinetic analysis. The calculated association rate constants (k(on)) were reduced twofold at most for C1s, and appeared unaffected for beta-Factor XIIa. The effect on the k(on) with kallikrein was more pronounced, ranging from a significant ninefold reduction to an unmodified rate. The results show that the reactive centre loop of C1 inhibitor can be modified towards decreased sensitivity for nontarget proteases without loss of specificity for target proteases. We conclude that this approach extends the possibilities of applying recombinant serpin variants for therapeutic use in inflammatory diseases.
Assuntos
Proteínas Inativadoras do Complemento 1/metabolismo , Neutrófilos/metabolismo , Elastase Pancreática/metabolismo , Proteínas Recombinantes/sangue , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , Células Clonais , Proteínas Inativadoras do Complemento 1/genética , Fator XIIa/metabolismo , Fibrinolisina/metabolismo , Variação Genética , Humanos , Calicreínas/metabolismo , Cinética , Elastase de Leucócito , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Oligodesoxirribonucleotídeos , TransfecçãoRESUMO
B-cell chronic lymphocytic leukaemia (B-CLL) is a slowly progressing malignancy of CD5(+) B cells, for which at present no curative treatment is available. In our current study, we apply a novel bridging reagent to redirect cytomegalovirus (CMV)-specific cytotoxic T lymphocytes (CTL) to target B-CLL. A streptavidin-fused anti-CD20 single-chain variable fragment (scFv) is used in combination with biotinylated MHC class I molecules containing CMV pp65 peptide (HLA/CMV). We demonstrate that B-CLL cells coated with this CD20-HLA/CMV complex can be lysed by autologous CMV-specific CTL with similar efficiency as B-CLL cells directly loaded with CMV peptide. Killing is HLA restricted and occurs at scFv CD20 concentrations of >/=100 ng ml(-1) and HLA/CMV concentrations of >/=20 ng ml(-1). Furthermore, complex-coated B-CLL cells induce both proliferation and cytokine production (interferon gamma, tumour necrosis factor alpha and macrophage inflammatory protein-1 beta) in CMV-specific CD8(+) T cells. Hereby, a necessary step towards possible application of CD20-HLA/CMV complexes for immunotherapy of B-cell malignancies is constituted.
Assuntos
Antígenos CD20/imunologia , Citomegalovirus/imunologia , Antígenos HLA/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Peptídeos/imunologia , Linfócitos T Citotóxicos/imunologia , Antígenos CD20/biossíntese , Proliferação de Células , Citocinas/biossíntese , Testes Imunológicos de Citotoxicidade , Humanos , Leucemia Linfocítica Crônica de Células B/diagnóstico , Complexos Multiproteicos/imunologia , Células Tumorais CultivadasRESUMO
The clinical success of B-cell receptor (BCR) signaling pathway inhibitors in chronic lymphocytic leukemia (CLL) is attributed to inhibition of adhesion in and migration towards the lymph node. Proliferation of CLL cells is restricted to this protective niche, but the underlying mechanism(s) is/are not known. Treatment with BCR pathway inhibitors results in rapid reductions of total clone size, while CLL cell survival is not affected, which points towards inhibition of proliferation. In vitro, BCR stimulation does not induce proliferation of CLL, but triggering via Toll-like receptor, tumor necrosis factor or cytokine receptors does. Here, we investigated the effects of clinically applied inhibitors that target BCR signaling, in the context of proliferation triggered either via CD40L/IL-21 or after CpG stimulation. CD40L/IL-21-induced proliferation could be inhibited by idelalisib and ibrutinib. We demonstrate this was due to blockade of CD40L-induced ERK-signaling. Targeting JAKs, but not SYK, blocked CD40L/IL-21-induced proliferation. In contrast, PI3K, BTK as well as SYK inhibition prevented CpG-induced proliferation. Knockdown experiments showed that CD40L/IL-21 did not co-opt upstream BCR components such as CD79A, in contrast to CpG-induced proliferation. Our data indicate that currently applied BTK/PI3K inhibitors target antigen-independent proliferation in CLL, and suggest that targeting of JAK and/or SYK might be clinically useful.
Assuntos
Antígenos de Neoplasias/imunologia , Leucemia Linfocítica Crônica de Células B/imunologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Fosfotransferases/antagonistas & inibidores , Biomarcadores , Antígenos CD40/imunologia , Antígenos CD40/metabolismo , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Interleucinas/metabolismo , Janus Quinases/metabolismo , Leucemia Linfocítica Crônica de Células B/genética , NF-kappa B/metabolismo , Fosfotransferases/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Receptores de Antígenos de Linfócitos B/metabolismo , Fatores de Transcrição STAT/metabolismo , Transdução de Sinais/efeitos dos fármacos , Células Tumorais CultivadasRESUMO
Protective interactions with bystander cells in micro-environmental niches, such as lymph nodes (LNs), contribute to survival and therapy resistance of chronic lymphocytic leukemia (CLL) cells. This is caused by a shift in expression of B-cell lymphoma 2 (BCL-2) family members. Pro-survival proteins B-cell lymphoma-extra large (BCL-XL), BCL-2-related protein A1 (BFL-1) and myeloid leukemia cell differentiation protein 1 (MCL-1) are upregulated by LN-residing T cells through CD40L interaction, presumably via nuclear factor (NF)-κB signaling. Macrophages (Mφs) also reside in the LN, and are assumed to provide important supportive functions for CLL cells. However, if and how Mφs are able to induce survival is incompletely known. We first established that Mφs induced survival because of an exclusive upregulation of MCL-1. Next, we investigated the mechanism underlying MCL-1 induction by Mφs in comparison with CD40L. Genome-wide expression profiling of in vitro Mφ- and CD40L-stimulated CLL cells indicated activation of the phosphoinositide 3-kinase (PI3K)-V-Akt murine thymoma viral oncogene homolog (AKT)-mammalian target of rapamycin (mTOR) pathway, which was confirmed in ex vivo CLL LN material. Inhibition of PI3K-AKT-mTOR signaling abrogated MCL-1 upregulation and survival by Mφs, as well as CD40 stimulation. MCL-1 can be regulated at multiple levels, and we established that AKT leads to increased MCL-1 translation, but does not affect MCL-1 transcription or protein stabilization. Furthermore, among Mφ-secreted factors that could activate AKT, we found that induction of MCL-1 and survival critically depended on C-C motif chemokine receptor-1 (CCR1). In conclusion, this study indicates that two distinct micro-environmental factors, CD40L and Mφs, signal via CCR1 to induce AKT activation resulting in translational stabilization of MCL-1, and hence can contribute to CLL cell survival.
Assuntos
Leucemia Linfocítica Crônica de Células B/patologia , Macrófagos/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Receptores CCR1/metabolismo , Animais , Apoptose/fisiologia , Ligante de CD40/genética , Ligante de CD40/metabolismo , Linhagem Celular Tumoral , Sobrevivência Celular/fisiologia , Regulação para Baixo , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Macrófagos/metabolismo , Camundongos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Células NIH 3T3 , Receptores CCR1/genética , Transdução de Sinais , Linfócitos T/metabolismo , Linfócitos T/patologia , TransfecçãoRESUMO
We investigated the apoptosis gene expression profile of chronic lymphocytic leukemia (CLL) cells in relation to (1) normal peripheral and tonsillar B-cell subsets, (2) IgV(H) mutation status, and (3) effects of cytotoxic drugs. In accord with their noncycling, antiapoptotic status in vivo, CLL cells displayed high constitutive expression of Bcl-2 and Flip mRNA, while Survivin, Bid and Bik were absent. Paradoxically, along with these antiapoptotic genes CLL cells had high-level expression of proapoptotic BH3-only proteins Bmf and Noxa. Treatment of CLL cells with fludarabine induced only the proapoptotic genes Bax and Puma in a p53-dependent manner. Interestingly, the degree of Puma induction was more pronounced in cells with mutated IgVH genes. Thus, disturbed apoptosis in CLL is the net result of both protective and sensitizing aberrations. This delicate balance can be tipped via induction of Puma in a p53-dependent matter, the level of which may vary between groups of patients with a different tendency for disease progression.
Assuntos
Antineoplásicos/farmacologia , Leucemia Linfocítica Crônica de Células B/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proteína Supressora de Tumor p53/metabolismo , Fosfato de Vidarabina/análogos & derivados , Fosfato de Vidarabina/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Apoptose/fisiologia , Proteínas Reguladoras de Apoptose , Resistencia a Medicamentos Antineoplásicos , Perfilação da Expressão Gênica/métodos , Regulação Leucêmica da Expressão Gênica , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-bcl-2/genética , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/genética , Regulação para Cima/efeitos dos fármacosRESUMO
Survival of chronic lymphocytic leukemia (CLL) cells is mainly driven by interactions within the lymph node (LN) microenvironment with bystander cells such as T cells or cells from the monocytic lineage. Although the survival effect by T cells is largely governed by the TNFR ligand family member CD40L, the exact mechanism of monocyte-derived cell-induced survival is not known. An important role has been attributed to the TNFR ligand, a proliferation-inducing ligand (APRIL), although the exact mechanism remained unclear. Since we detected that APRIL was expressed by CD68+ cells in CLL LN, we addressed its relevance in various aspects of CLL biology, using a novel APRIL overexpressing co-culture system, recombinant APRIL, and APRIL reporter cells. Unexpectedly, we found, that in these various systems, APRIL had no effect on survival of CLL cells, and activation of NF-κB was not enhanced on APRIL stimulation. Moreover, APRIL stity mulation did not affect CLL proliferation, neither as single stimulus nor in combination with known CLL proliferation stimuli. Furthermore, the survival effect conveyed by macrophages to CLL cells was not affected by transmembrane activator and CAML interactor-Fc, an APRIL decoy receptor. We conclude that the direct role ascribed to APRIL in CLL cell survival might be overestimated due to application of supraphysiological levels of recombinant APRIL.
RESUMO
The phosphoinositide 3-kinases (PI3Ks) are critical components of the B-cell receptor (BCR) pathway and have an important role in the pathobiology of chronic lymphocytic leukemia (CLL). Inhibitors of PI3Kδ block BCR-mediated cross-talk between CLL cells and the lymph node microenvironment and provide significant clinical benefit to CLL patients. However, the PI3Kδ inhibitors applied thus far have limited direct impact on leukemia cell survival and thus are unlikely to eradicate the disease. The use of inhibitors of multiple isoforms of PI3K might lead to deeper remissions. Here we demonstrate that the pan-PI3K/mammalian target of rapamycin inhibitor SAR245409 (voxtalisib/XL765) was more pro-apoptotic to CLL cells--irrespective of their ATM/p53 status--than PI3Kα or PI3Kδ isoform selective inhibitors. Furthermore, SAR245409 blocked CLL survival, adhesion and proliferation. Moreover, SAR245409 was a more potent inhibitor of T-cell-mediated production of cytokines, which support CLL survival. Taken together, our in vitro data provide a rationale for the evaluation of a pan-PI3K inhibitor in CLL patients.
Assuntos
Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Inibidores de Fosfoinositídeo-3 Quinase , Quinoxalinas/farmacologia , Sulfonamidas/farmacologia , Serina-Treonina Quinases TOR/antagonistas & inibidores , Adolescente , Adulto , Antígenos CD40/fisiologia , Adesão Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Citocinas/biossíntese , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Pessoa de Meia-Idade , Purinas/farmacologia , Quinazolinonas/farmacologia , Linfócitos T/imunologiaRESUMO
The identification of specific amino acid residues involved in protein-protein interaction is fundamental to understanding structure-function relationships. Supported by mathematical calculations, we designed a high-density mutagenesis procedure for the generation of a mutant library of which a limited number of random clones would suffice to exactly localize amino acid residues essential for a particular protein-protein interaction. This goal was achieved experimentally by consecutive cycles of DNA shuffling, under error prone conditions, each followed by exposure of the target protein on the surface of phages to screen and select for correctly folded, functional mutants. To validate the procedure, human plasminogen activator inhibitor 1 (PAI-1) was chosen, because its 3D structure is known, many experimental tools are available and it may serve as a model protein for structure-function studies of serine proteinases and their inhibitors (serpins). After five cycles of DNA shuffling and selection for t-PA binding, analysis of 27 randomly picked clones revealed that PAI-1 mutants contained an average of 9.1 amino acid substitutions distributed over 114 different positions, which were preferentially located at the surface of the protein. This limited collection of mutant PAI-1 preparations contained multiple mutants defective in binding to three out of four tested anti-PAI-1 monoclonal antibodies. Alignment of the nucleotide sequence of defective clones permitted assignment of single dominant amino acid residues for binding to each monoclonal antibody. The importance of these residues was confirmed by testing the properties of single point mutants. From the position of these amino acid residues in the 3D structure of PAI-1 and the effects of the corresponding monoclonal antibodies on t-PA-PAI-1 interaction, conclusions can be drawn with respect to this serpin-serine proteinase interaction.
Assuntos
DNA Recombinante/genética , Mutagênese/genética , Biblioteca de Peptídeos , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Clonagem Molecular , Mapeamento de Epitopos , Epitopos/imunologia , Meia-Vida , Humanos , Camundongos , Modelos Moleculares , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/imunologia , Mutação Puntual/genética , Ligação Proteica , Alinhamento de Sequência , Relação Estrutura-Atividade , Ressonância de Plasmônio de Superfície , Ativador de Plasminogênio Tecidual/metabolismoRESUMO
Plasminogen activator inhibitor type 1 (PAI-1) is a member of the serine protease inhibitor (serpin) superfamily. Its highly mobile reactive-center loop (RCL) is thought to account for both the rapid inhibition of tissue-type plasminogen activator (t-PA), and the rapid and spontaneous transition of the unstable, active form of PAI-1 into a stable, inactive (latent) conformation (t(1/2) at 37 degrees C, 2.2 hours). We determined the amino acid residues responsible for the inherent instability of PAI-1, to assess whether these properties are independent and, consequently, whether the structural basis for inhibition and latency transition is different. For that purpose, a hypermutated PAI-1 library that is displayed on phage was pre-incubated for increasing periods (20 to 72 hours) at 37 degrees C, prior to a stringent selection for rapid t-PA binding. Accordingly, four rounds of phage-display selection resulted in the isolation of a stable PAI-1 variant (st-44: t(1/2) 450 hours) with 11 amino acid mutations. Backcrossing by DNA shuffling of this stable mutant with wt PAI-1 was performed to eliminate non-contributing mutations. It was shown that the combination of mutations at positions 50, 56, 61, 70, 94, 150, 222, 223, 264 and 331 increases the half-life of PAI-1 245-fold. Furthermore, within the limits of detection the stable mutants isolated are functionally indistinguishable from wild-type PAI-1 with respect to the rate of inhibition of t-PA, cleavage by t-PA, and binding to vitronectin. These stabilizing mutations constitute largely reversions to the stable "serpin consensus sequence" and are located in areas implicated in PAI-1 stability (e.g. the vitronectin-binding domain and the proximal hinge). Collectively, our data provide evidence that the structural requirements for PAI-1 loop insertion during latency transition and target proteinase inhibition can be separated.
Assuntos
Mutagênese/genética , Biblioteca de Peptídeos , Inibidor 1 de Ativador de Plasminogênio/química , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Ativador de Plasminogênio Tecidual/antagonistas & inibidores , Animais , Sequência Consenso , Meia-Vida , Humanos , Cinética , Camundongos , Modelos Moleculares , Inibidor 1 de Ativador de Plasminogênio/genética , Inibidor 1 de Ativador de Plasminogênio/isolamento & purificação , Ligação Proteica , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismo , Ressonância de Plasmônio de Superfície , Termodinâmica , Ativador de Plasminogênio Tecidual/metabolismo , Vitronectina/metabolismoRESUMO
In acute myeloid leukaemia (AML), alterations in apoptotic pathways are crucial for treatment outcome, resulting either in refractoriness or in minimal residual disease (MRD). The apoptosis characteristics of MRD cells may differ from those at diagnosis and thereby determine the adequacy of further treatment. Such characteristics are largely unknown, since studies hereto are hampered by minimal cell availability. This study explores the applicability of the recently described RT-Multiplex Ligation-dependent Probe Amplification (RT-MLPA) for gene expression analysis of small amounts of RNA obtained from MRD cells. Reproducibility and dilution experiments showed that the relative expression of 37 apoptosis-related genes starting with only 1000 cells could be measured with 12% variation; for 100 cells, 31/37 genes could still be quantified, though expression variation increased. In practice 100-1000 leukemic cells can be obtained from bone marrow samples with clinically relevant MRD percentages of 0.01-0.1. Procedures often necessary to obtain AML blasts, that is, FACS-sorting, freeze-thawing or combinations are possible, provided that selected viable nonapoptotic cells are used. Concluding, RT-MLPA allows accurate gene expression profiling of MRD cells. This method will help to gain insight into the processes of MRD emergence and persistence in AML, which may ultimately guide new therapeutic strategies in AML.
Assuntos
Perfilação da Expressão Gênica , Leucemia Mieloide/genética , Neoplasia Residual/genética , Reação em Cadeia da Polimerase/métodos , Doença Aguda , Humanos , Leucemia Mieloide/diagnóstico , Neoplasia Residual/diagnóstico , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Sensibilidade e EspecificidadeRESUMO
The apoptotic pathway activated by chimeric anti-CD20 monoclonal antibodies (rituximab, IDEC.C2B8) was analyzed using the Burkitt lymphoma cell line Ramos. Crosslinking of CD20 (CD20XL) induced apoptosis in Ramos cells, which involved loss of mitochondrial membrane potential (Deltapsi(m)), the release of cytochrome-c (cyt-c), and activation of caspases-9 and -3. Nevertheless, several lines of evidence showed that the apoptotic outcome did not depend on these events. First, under circumstances where Ramos cells display resistance to either CD95- or B cell receptor (BCR)-induced apoptosis, CD20XL-induced apoptosis was not affected, pointing to a distinct pathway. Second, the broad-spectrum caspase inhibitor zVAD-fmk prevented processing of caspase-9, -3 and PARP as well as DNA fragmentation, but did not block apoptosis as measured by annexin V staining, cell size and membrane integrity. Lastly, Bcl-2 overexpression blocked cyt-c release and the decrease in Deltapsi(m), and completely prevented CD95- or BCR-mediated apoptosis; however, it did not affect CD20XL-induced cell death. We conclude that although CD20XL can initiate the mitochondrial apoptosis pathway, CD20-induced apoptosis does not necessarily require active caspases and cannot be blocked by Bcl-2. Since most chemotherapeutic drugs require the activation of caspases to exert their cytotoxicity, these findings provide an important rationale for the use of CD20 mAbs in chemoresistant malignancies.
Assuntos
Anticorpos Monoclonais/farmacologia , Antígenos CD20/imunologia , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Linfócitos B/efeitos dos fármacos , Caspases/metabolismo , Mitocôndrias/efeitos dos fármacos , Clorometilcetonas de Aminoácidos/farmacologia , Anticorpos Monoclonais Murinos , Linfócitos B/patologia , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/metabolismo , Linfoma de Burkitt/patologia , Inibidores de Caspase , Inibidores de Cisteína Proteinase/farmacologia , Grupo dos Citocromos c/metabolismo , Ativação Enzimática , Humanos , Immunoblotting , Marcação In Situ das Extremidades Cortadas , Potenciais da Membrana , Mitocôndrias/enzimologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Rituximab , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologiaRESUMO
The treatment of chronic lymphocytic leukemia (CLL) with inhibitors targeting B cell receptor signaling and other survival mechanisms holds great promise. Especially the early clinical success of Ibrutinib, an irreversible inhibitor of Bruton's tyrosine kinase (BTK), has received widespread attention. In this review we will focus on the fundamental and clinical aspects of BTK inhibitors in CLL, with emphasis on Ibrutinib as the best studied of this class of drugs. Furthermore, we summarize recent laboratory as well as clinical findings relating to the first cases of Ibrutinib resistance. Finally, we address combination strategies with Ibrutinib, and attempt to extrapolate its current status to the near future in the clinic.
Assuntos
Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Inibidores de Proteínas Quinases/uso terapêutico , Proteínas Tirosina Quinases/antagonistas & inibidores , Pirazóis/uso terapêutico , Pirimidinas/uso terapêutico , Receptores de Antígenos de Linfócitos B/antagonistas & inibidores , Adenina/análogos & derivados , Tirosina Quinase da Agamaglobulinemia , Anticorpos Monoclonais/uso terapêutico , Humanos , Piperidinas , Inibidores de Proteínas Quinases/efeitos adversos , Pirazóis/efeitos adversos , Pirimidinas/efeitos adversos , Transdução de Sinais/efeitos dos fármacosRESUMO
The ATM-p53 DNA-damage response (DDR) pathway has a crucial role in chemoresistance in CLL, as indicated by the adverse prognostic impact of genetic aberrations of TP53 and ATM. Identifying and distinguishing TP53 and ATM functional defects has become relevant as epigenetic and posttranscriptional dysregulation of the ATM/p53 axis is increasingly being recognized as the underlying cause of chemoresistance. Also, specific treatments sensitizing TP53- or ATM-deficient CLL cells are emerging. We therefore developed a new ATM-p53 functional assay with the aim to (i) identify and (ii) distinguish abnormalities of TP53 versus ATM and (iii) enable the identification of additional defects in the ATM-p53 pathway. Reversed transcriptase multiplex ligation-dependent probe amplification (RT-MLPA) was used to measure ATM and/or p53-dependent genes at the RNA level following DNA damage using irradiation. Here, we showed that this assay is able to identify and distinguish three subgroups of CLL tumors (i.e., TP53-defective, ATM-defective and WT) and is also able to detect additional samples with a defective DDR, without molecular aberrations in TP53 and/or ATM. These findings make the ATM-p53 RT-MLPA functional assay a promising prognostic tool for predicting treatment responses in CLL.
Assuntos
Proteínas Mutadas de Ataxia Telangiectasia/genética , Regulação Leucêmica da Expressão Gênica , Reação em Cadeia da Polimerase Multiplex/métodos , Mutação , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Proteína Supressora de Tumor p53/genética , Antineoplásicos/farmacologia , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Bioensaio , Dano ao DNA , Doxorrubicina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Epigênese Genética , Raios gama , Humanos , Leucemia Linfocítica Crônica de Células B/tratamento farmacológico , Leucemia Linfocítica Crônica de Células B/genética , Leucemia Linfocítica Crônica de Células B/metabolismo , Leucemia Linfocítica Crônica de Células B/patologia , RNA Neoplásico/genética , Sensibilidade e Especificidade , Proteína Supressora de Tumor p53/metabolismo , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
Mutations or deletions in TP53 or ATM are well-known determinants of poor prognosis in chronic lymphocytic leukemia (CLL), but only account for approximately 40% of chemo-resistant patients. Genome-wide sequencing has uncovered novel mutations in the splicing factor sf3b1, that were in part associated with ATM aberrations, suggesting functional synergy. We first performed detailed genetic analyses in a CLL cohort (n=110) containing ATM, SF3B1 and TP53 gene defects. Next, we applied a newly developed multiplex assay for p53/ATM target gene induction and measured apoptotic responses to DNA damage. Interestingly, SF3B1 mutated samples without concurrent ATM and TP53 aberrations (sole SF3B1) displayed partially defective ATM/p53 transcriptional and apoptotic responses to various DNA-damaging regimens. In contrast, NOTCH1 or K/N-RAS mutated CLL displayed normal responses in p53/ATM target gene induction and apoptosis. In sole SF3B1 mutated cases, ATM kinase function remained intact, and γH2AX formation, a marker for DNA damage, was increased at baseline and upon irradiation. Our data demonstrate that single mutations in sf3b1 are associated with increased DNA damage and/or an aberrant response to DNA damage. Together, our observations may offer an explanation for the poor prognosis associated with SF3B1 mutations.
Assuntos
Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/genética , Mutação , Fosfoproteínas/genética , Ribonucleoproteína Nuclear Pequena U2/genética , Apoptose , Proteínas Mutadas de Ataxia Telangiectasia/metabolismo , Estudos de Coortes , Dano ao DNA , Análise Mutacional de DNA , Doxorrubicina/farmacologia , Citometria de Fluxo , Deleção de Genes , Genoma Humano , Histonas/metabolismo , Humanos , Imidazóis/farmacologia , Piperazinas/farmacologia , Prognóstico , Fatores de Processamento de RNA , Receptor Notch1/genética , Proteína Supressora de Tumor p53/genética , Vidarabina/análogos & derivados , Vidarabina/farmacologiaRESUMO
Large random hexa- and decapenta-peptide libraries were constructed and displayed on the surface of the filamentous phagemid pComb8. Panning of the hexa-peptide library on immobilized plasminogen activator inhibitor 1 (PAI-1) specifically selected a minor fraction of concatemers, indicating that binding to PAI-1 requires an extended amino acid sequence. Accordingly, the decapenta-peptide library exclusively yielded PAI-1 binding peptides of 15 amino acid residues. None of these phage-bound peptides prevented the interaction between PAI-1 and its target serine protease urokinase (u-PA). To isolate peptides that block the interaction between PAI-1 and u-PA, phages bound to immobilized PAI-1 were eluted by incubation with u-PA. Remarkably, this procedure resulted in elution of a unique phage type that harbors a concatemer of decapentamers, consisting of 49 amino acid residues with no obvious similarity to the primary sequence of PAI-1 or u-PA.
Assuntos
Biblioteca de Peptídeos , Peptídeos/metabolismo , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Sequência de Aminoácidos , Bacteriófagos , Sítios de Ligação , Humanos , Dados de Sequência Molecular , Homologia de Sequência de Aminoácidos , Ativador de Plasminogênio Tipo Uroquinase/metabolismoRESUMO
In the lymph node (LN) environment, chronic lymphocytic leukemia (CLL) cells display increased NF-κB activity compared with peripheral blood CLL cells, which contributes to chemoresistance. Antagonists of cellular inhibitor of apoptosis proteins (cIAPs) can induce apoptosis in various cancer cells in a tumor necrosis factor-α (TNFα)-dependent manner and are in preclinical development. Smac-mimetics promote degradation of cIAP1 and cIAP2, which results in TNFR-mediated apoptosis via formation of a ripoptosome complex, comprising RIPK1, Fas-associated protein with death domain, FLICE-like inhibitory protein and caspase-8. CD40 stimulation of CLL cells in vitro is used as a model to mimic the LN microenvironment and results in NF-κB activation and TNFα production. In this study, we investigated the response of CLL cells to smac-mimetics in the context of CD40 stimulation. We found that treatment with smac-mimetics results in cIAP1 and cIAP2 degradation, yet although TNFα is produced, this did not induce apoptosis. Despite the presence of all components, the ripoptosome complex did not form upon smac-mimetic treatment in CLL cells. Thus, CLL cells seem to possess aberrant upstream NF-κB regulation that prevents ripoptosome formation upon IAP degradation. Unraveling the exact molecular mechanisms of disturbed ripoptosome formation may offer novel targets for treatment in CLL.