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1.
Angew Chem Int Ed Engl ; 63(18): e202314143, 2024 04 24.
Artigo em Inglês | MEDLINE | ID: mdl-38179812

RESUMO

Drug safety and efficacy due to premature release into the bloodstream and poor biodistribution remains a problem despite seminal advances in this area. To circumvent these limitations, we report drug cyclization based on dynamic covalent linkages to devise a dual lock for the small-molecule anticancer drug, camptothecin (CPT). Drug activity is "locked" within the cyclic structure by the redox responsive disulfide and pH-responsive boronic acid-salicylhydroxamate and turns on only in the presence of acidic pH, reactive oxygen species and glutathione through traceless release. Notably, the dual-responsive CPT is more active (100-fold) than the non-cleavable (permanently closed) analogue. We further include a bioorthogonal handle in the backbone for functionalization to generate cyclic-locked, cell-targeting peptide- and protein-CPTs, for targeted delivery of the drug and traceless release in triple negative metastatic breast cancer cells to inhibit cell growth at low nanomolar concentrations.


Assuntos
Antineoplásicos , Nanopartículas , Neoplasias , Camptotecina/química , Distribuição Tecidual , Antineoplásicos/química , Micelas , Proteínas , Sistemas de Liberação de Medicamentos , Nanopartículas/química , Liberação Controlada de Fármacos , Linhagem Celular Tumoral
2.
Bioconjug Chem ; 34(12): 2215-2220, 2023 12 20.
Artigo em Inglês | MEDLINE | ID: mdl-37962868

RESUMO

Bispecific antibodies as T cell engagers designed to display binding capabilities to both tumor-associated antigens and antigens on T cells are considered promising agents in the fight against cancer. Even though chemical strategies to develop such constructs have emerged, a method that readily converts a therapeutically applied antibody into a bispecific construct by a fully non-genetic process is not yet available. Herein, we report the application of a biogenic, tyrosine-based click reaction utilizing chemoenzymatic modifications of native IgG1 antibodies to generate a synthetic bispecific antibody construct that exhibits tumor-killing capability at picomolar concentrations. Control experiments revealed that a covalent linkage of the different components is required for the observed biological activities. In view of the highly potent nature of the constructs and the modular approach that relies on convenient synthetic methods utilizing therapeutically approved biomolecules, our method expedites the production of potent bispecific antibody constructs with tunable cell killing efficacy with significant impact on therapeutic properties.


Assuntos
Anticorpos Biespecíficos , Neoplasias , Humanos , Linfócitos T , Química Click , Neoplasias/tratamento farmacológico , Anticorpos Biespecíficos/química , Antígenos de Neoplasias/metabolismo
3.
Biomacromolecules ; 24(11): 4646-4652, 2023 11 13.
Artigo em Inglês | MEDLINE | ID: mdl-37792488

RESUMO

Thiol-reactive Michael acceptors are commonly used for the formation of chemically cross-linked hydrogels. In this paper, we address the drawbacks of many Michael acceptors by introducing pyridazinediones as new cross-linking agents. Through the use of pyridazinediones and their mono- or dibrominated analogues, we show that the mechanical strength, swelling ratio, and rate of gelation can all be controlled in a pH-sensitive manner. Moreover, we demonstrate that the degradation of pyridazinedione-gels can be induced by the addition of thiols, thus providing a route to responsive or dynamic gels, and that monobromo-pyridazinedione gels are able to support the proliferation of human cells. We anticipate that our results will provide a valuable and complementary addition to the existing toolkit of cross-linking agents, allowing researchers to tune and rationally design the properties of biomedical hydrogels.


Assuntos
Hidrogéis , Compostos de Sulfidrila , Humanos , Hidrogéis/química , Compostos de Sulfidrila/química , Reagentes de Ligações Cruzadas/química
4.
Chem Sci ; 14(14): 3752-3762, 2023 Apr 05.
Artigo em Inglês | MEDLINE | ID: mdl-37035695

RESUMO

Either as full IgGs or as fragments (Fabs, Fc, etc.), antibodies have received tremendous attention in the development of new therapeutics such as antibody-drug conjugates (ADCs). The production of ADCs involves the grafting of active payloads onto an antibody, which is generally enabled by the site-selective modification of native or engineered antibodies via chemical or enzymatic methods. Whatever method is employed, controlling the payload-antibody ratio (PAR) is a challenge in terms of multiple aspects including: (i) obtaining homogeneous protein conjugates; (ii) obtaining unusual PARs (PAR is rarely other than 2, 4 or 8); (iii) using a single method to access a range of different PARs; (iv) applicability to various antibody formats; and (v) flexibility for the production of heterofunctional antibody-conjugates (e.g. attachment of multiple types of payloads). In this article, we report a single pyridazinedione-based trifunctional dual bridging linker that enables, in a two-step procedure (re-bridging/click), the generation of either mAb-, Fab'-, or Fc-conjugates from native mAb, (Fab')2 or Fc formats, respectively. Fc and (Fab')2 formats were generated via enzymatic digestion of native mAbs. Whilst the same reduction and re-bridging protocols were applied to all three of the protein formats, the subsequent click reaction(s) employed to graft payload(s) drove the generation of a range of PARs, including heterofunctional PARs. As such, exploiting click reactivity and/or orthogonality afforded mAb-conjugates with PARs of 6, 4, 2 or 4 + 2, and Fab'- and Fc-conjugates with a PAR of 3, 2, 1 or 2 + 1 on-demand. We believe that the homogeneity, novelty and variety in accessible PARs, as well as the applicability to various antibody-conjugate formats enabled by our non-recombinant method could be a suitable tool for antibody-drug conjugates optimisation (optimal PAR value, optimal payloads combination) and boost the development of new antibody therapeutics (Fab'- and Fc-conjugates).

5.
ACS Cent Sci ; 9(3): 476-487, 2023 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-36968530

RESUMO

In recent years there has been rising interest in the field of protein-protein conjugation, especially related to bispecific antibodies (bsAbs) and their therapeutic applications. These constructs contain two paratopes capable of binding two distinct epitopes on target molecules and are thus able to perform complex biological functions (mechanisms of action) not available to monospecific mAbs. Traditionally these bsAbs have been constructed through protein engineering, but recently chemical methods for their construction have started to (re)emerge. While these have been shown to offer increased modularity, speed, and for some methods even the inherent capacity for further functionalization (e.g., with small molecule cargo), most of these approaches lacked the ability to include a fragment crystallizable (Fc) modality. The Fc component of IgG antibodies offers effector function and increased half-life. Here we report a first-in-class disulfide rebridging and click-chemistry-based method for the generation of Fc-containing, IgG-like mono- and bispecific antibodies. These are in the FcZ-(FabX)-FabY format, i.e., two distinct Fabs and an Fc, potentially all from different antibodies, attached in a homogeneous and covalent manner. We have dubbed these molecules synthetic antibodies (SynAbs). We have constructed a T cell-engager (TCE) SynAb, FcCD20-(FabHER2)-FabCD3, and have confirmed that it exhibits the expected biological functions, including the ability to kill HER2+ target cells in a coculture assay with T cells.

6.
Chem Sci ; 14(47): 13743-13754, 2023 Dec 06.
Artigo em Inglês | MEDLINE | ID: mdl-38075666

RESUMO

Reversible cysteine modification has been found to be a useful tool for a plethora of applications such as selective enzymatic inhibition, activity-based protein profiling and/or cargo release from a protein or a material. However, only a limited number of reagents display reliable dynamic/reversible thiol modification and, in most cases, many of these reagents suffer from issues of stability, a lack of modularity and/or poor rate tunability. In this work, we demonstrate the potential of pyridazinediones as novel reversible and tuneable covalent cysteine modifiers. We show that the electrophilicity of pyridazinediones correlates to the rates of the Michael addition and retro-Michael deconjugation reactions, demonstrating that pyridazinediones provide an enticing platform for readily tuneable and reversible thiol addition/release. We explore the regioselectivity of the novel reaction and unveil the reason for the fundamental increased reactivity of aryl bearing pyridazinediones by using DFT calculations and corroborating findings with SCXRD. We also applied this fundamental discovery to making more rapid disulfide rebridging agents in related work. We finally provide the groundwork for potential applications in various areas with exemplification using readily functionalised "clickable" pyridazinediones on clinically relevant cysteine and disulfide conjugated proteins, as well as on a hydrogel material.

7.
Adv Mater ; : e2300413, 2023 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-36905683

RESUMO

Semiconducting polymer nanoparticles (SPNs) are explored for applications in cancer theranostics because of their high absorption coefficients, photostability, and biocompatibility. However, SPNs are susceptible to aggregation and protein fouling in physiological conditions, which can be detrimental for in vivo applications. Here, a method for achieving colloidally stable and low-fouling SPNs is described by grafting poly(ethylene glycol) (PEG) onto the backbone of the fluorescent semiconducting polymer, poly(9,9'-dioctylfluorene-5-fluoro-2,1,3-benzothiadiazole), in a simple one-step substitution reaction, postpolymerization. Further, by utilizing azide-functionalized PEG, anti-human epidermal growth factor receptor 2 (HER2) antibodies, antibody fragments, or affibodies are site-specifically "clicked" onto the SPN surface, which allows the functionalized SPNs to specifically target HER2-positive cancer cells. In vivo, the PEGylated SPNs are found to have excellent circulation efficiencies in zebrafish embryos for up to seven days postinjection. SPNs functionalized with affibodies are then shown to be able to target HER2 expressing cancer cells in a zebrafish xenograft model. The covalent PEGylated SPN system described herein shows great potential for cancer theranostics.

8.
Chem Commun (Camb) ; 58(5): 645-648, 2022 Jan 13.
Artigo em Inglês | MEDLINE | ID: mdl-34747956

RESUMO

Herein we report a thiol-labile cysteine protecting group based on an unsaturated pyridazinedione (PD) scaffold. We establish compatibility of the PD in conventional solid phase peptide synthesis (SPPS), showcasing this in the on-resin synthesis of biologically relevant oxytocin. Furthermore, we establish the applicability of the PD protecting group towards both microwave-assisted SPPS and native chemical ligation (NCL) in a model system.


Assuntos
Técnicas de Síntese em Fase Sólida
9.
Methods Enzymol ; 644: 121-148, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32943142

RESUMO

Chemically modified proteins are increasingly being tested and approved as therapeutic products. Batch-to-batch homogeneity is crucial to ensure safety and quality of therapeutic products. Highly selective protein modification may be achieved using enzymatic routes. Microbial transglutaminase (mTG) is a robust, easy to use and well-established enzyme that is used at a very large scale in the food industry such that its efficacy and its safety for human consumption are well established. In the context of therapeutic protein modification, mTG should crosslink one or more glutamines on the target protein with an aminated moiety such as a solubilizer, a tracer or a cytotoxic moiety. mTG has the advantage of being unreactive toward the majority of surface-exposed glutamines on most proteins, reducing sample heterogeneity. The caveat is that there may be no reactive glutamine on the target protein, or else a reactive glutamine may be found in a location where its modification compromises function of the target protein. Here we describe the glutamine-walk (Gln-walk), a straightforward method to create a glutamine-substrate site that is reactive to mTG in a target protein. Iterative substitution of single amino acids to a glutamine is followed by facile identification of reactivity with mTG, where covalent labeling of the target with an aminated fluorophore allows visualization of the most reactive modified targets. The approach is empirical; knowledge of the target protein structure and functional regions facilitates application of the method.


Assuntos
Glutamina , Transglutaminases , Humanos , Proteínas , Especificidade por Substrato , Transglutaminases/metabolismo , Caminhada
10.
Oncogene ; 20(50): 7352-67, 2001 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-11704865

RESUMO

Cross-linking of the B cell antigen receptor (BCR) on immature WEHI 231 B cells results in G1 cell cycle arrest and apoptosis. Here we investigated the molecular mechanisms that are necessary and sufficient for these changes to occur. We show that BCR stimulation of WEHI 231 cells results in down-regulation of cyclin D2 and up-regulation of p27(Kip1), which are associated with pocket protein hypophosphorylation and E2F inactivation. Ectopic expression of p27(Kip1) by TAT-fusion protein or retroviral transduction is sufficient to cause G1 cell cycle arrest, followed by apoptosis. In contrast, over-expression of cyclin D2 overcomes the cell cycle arrest and apoptosis induced by anti-IgM, indicating that down-regulation of cyclin D2 is necessary for the cell cycle arrest and apoptosis activated by BCR stimulation. Thus, cyclin D2 and p27(Kip1) have opposing roles in these pathways and our data also suggest that cyclin D2 functions upstream of p27(Kip1) and the pRB pathway and therefore plays an essential part in integrating the signals from BCR with the cell cycle machinery. We next investigated which signal transduction pathways triggered by the BCR regulate cell proliferation and apoptosis via cyclin D2 and p27(Kip1). Inhibition of PI3-K signalling by LY294002 down-regulated cyclin D2 and up-regulated p27(Kip1) expression at both protein and RNA levels, mimicking the effects of BCR-stimulation. Furthermore, ectopic expression of a constitutively active form of AKT blocked the cell cycle arrest and apoptosis triggered by anti-IgM and also abrogated down-regulation of cyclin D2 and up-regulation of p27(Kip1) expression induced by BCR-engagement. These results indicate that BCR activation targets p27(Kip1) and cyclin D2 to mediate cell cycle arrest and apoptosis and that down-regulation of PI3-K/AKT activity post BCR stimulation is necessary for these to occur.


Assuntos
Apoptose/fisiologia , Linfócitos B/patologia , Proteínas de Ciclo Celular/fisiologia , Ciclinas/fisiologia , Proteínas de Ligação a DNA , Fase G1/fisiologia , Fosfatidilinositol 3-Quinases/fisiologia , Proteínas Serina-Treonina Quinases , Proteínas , Receptores de Antígenos de Linfócitos B/fisiologia , Transdução de Sinais/fisiologia , Proteínas Supressoras de Tumor/fisiologia , Anticorpos Anti-Idiotípicos/farmacologia , Linfócitos B/efeitos dos fármacos , Linfócitos B/metabolismo , Proteínas de Ciclo Celular/biossíntese , Proteínas de Ciclo Celular/genética , Cromonas/farmacologia , Ciclina D2 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/biossíntese , Ciclinas/genética , Fatores de Transcrição E2F , Inibidores Enzimáticos/farmacologia , Fase G1/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Regulação Neoplásica da Expressão Gênica/fisiologia , Humanos , Linfoma de Células B/patologia , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Morfolinas/farmacologia , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/fisiologia , Proteínas Nucleares/fisiologia , Inibidores de Fosfoinositídeo-3 Quinase , Fosfoproteínas/fisiologia , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-akt , RNA Mensageiro/biossíntese , RNA Neoplásico/biossíntese , Proteínas Recombinantes de Fusão/fisiologia , Proteína do Retinoblastoma/fisiologia , Proteína p107 Retinoblastoma-Like , Proteína p130 Retinoblastoma-Like , Fatores de Transcrição/antagonistas & inibidores , Células Tumorais Cultivadas/efeitos dos fármacos , Células Tumorais Cultivadas/metabolismo , Células Tumorais Cultivadas/patologia
11.
Int J Med Inform ; 84(11): 933-40, 2015 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-26342668

RESUMO

PURPOSE: To provide an overview of essential elements of good governance of data linkage for health-related research, to consider lessons learned so far and to examine key factors currently impeding the delivery of good governance in this area. Given the considerable hurdles which must be overcome and the changing landscape of health research and data linkage, a principled, proportionate, risk-based approach to governance is advocated. DISCUSSION: In light of the considerable value of data linkage to health and well-being, the United Kingdom aspires to design and deliver good governance in health-related research. A string of projects have been asking: what does good governance look like in data linkage for health research? It is argued here that considerable progress can and must be made in order to develop the UK's contribution to future health and wealth economies, particularly in light of mis-start initiatives such as care.data in NHS England. Discussion centres around lessons learned from previous successful health research initiatives, identifying those governance mechanisms which are essential to achieving good governance. CONCLUSION: This article suggests that a crucial element in any step-increase of research capability will be the adoption of adaptive governance models. These must recognise a range of approaches to delivering safe and effective data linkage, while remaining responsive to public and research user expectations and needs as these shift and change with time and experience. The targets are multiple and constantly moving. There is not--nor should we seek--a single magic bullet in delivering good governance in health research.


Assuntos
Confidencialidade , Registros Eletrônicos de Saúde , Ética em Pesquisa , Armazenamento e Recuperação da Informação , Animais , Confidencialidade/ética , Registros Eletrônicos de Saúde/legislação & jurisprudência , Registros Eletrônicos de Saúde/normas , Humanos , Armazenamento e Recuperação da Informação/ética , Armazenamento e Recuperação da Informação/métodos , Consentimento Livre e Esclarecido , Relações Interprofissionais , Pesquisa , Medicina Estatal , Reino Unido
15.
Aliment Pharmacol Ther ; 31(12): 1330-6, 2010 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20331577

RESUMO

BACKGROUND: Occult myeloproliferative disorders (MPD) are present in 25% of patients with chronic portal, splenic and mesenteric venous thrombosis (PSMVT). A somatic mutation of JAK2 (JAK2V617F) can be used to identify patients with latent MPD. AIM: We evaluated the prevalence and clinical significance of JAK2V617F in patients with chronic PSMVT. METHODS: Allele-specific polymerase chain reaction was performed to screen for JAK2V617F. RESULTS: Thirty-five patients were tested for JAK2V617F. The underlying pro-coagulant condition was MPD in seven of 35 (20.0%) patients; other aetiologies included hereditary thrombophilia (n = 5), chronic pancreatitis (n = 2), liver abscess (n = 1) and umbilical vein sepsis (n = 3). The remainder were labelled idiopathic, i.e. 17/35 (48.6%) patients. JAK2V617F was detected in 16/35 (45.7%) patients: seven of seven (100%) with MPD, two of 11 (18.1%) with non-MPD acquired conditions and seven of 17 (41.2%) with 'idiopathic' chronic PSMVT. Mean haemoglobin concentration (P = 0.04), haematocrit (P = 0.04), white cell count (P = 0.002) and platelet count (P = 0.05) were significantly higher in patients with JAK2V617F. None of the seven patients with latent MPD have progressed to overt MPD over median follow-up of 85 months. CONCLUSION: JAK2V617F occurs in 41% of patients with idiopathic chronic portal, splenic and mesenteric venous thrombosis, confirming the presence of latent myeloproliferative disorders, and should form part of the routine pro-coagulant screen.


Assuntos
Janus Quinase 2/genética , Veias Mesentéricas , Veia Porta , Veia Esplênica , Trombose Venosa/genética , Adolescente , Adulto , Idoso , Doença Crônica , Feminino , Seguimentos , Regulação da Expressão Gênica , Humanos , Masculino , Pessoa de Meia-Idade , Mutação , Prevalência , Adulto Jovem
16.
Open Med Inform J ; 3: 54-64, 2009 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-19738915

RESUMO

OBJECTIVES: This paper reports an evaluation of the properties of a generic electronic health record information model that were actually required and used when importing an existing clinical application into a generic EHR repository. METHOD: A generic EHR repository and system were developed as part of the EU Projects Synapses and SynEx. A Web application to support the management of anticoagulation therapy was developed to interface to the EHR system, and deployed within a north London hospital with five years of cumulative clinical data from the previous existing anticoagulation management application. This offered the opportunity to critique those parts of the generic EHR that were actually needed to represent the legacy data. RESULTS: The anticoagulation records from 3,226 patients were imported and represented using over 900,000 Record Components (i.e. each patient's record contained on average 289 nodes), of which around two thirds were Element Items (i.e. value-containing leaf nodes), the remainder being container nodes (i.e. headings and sub-headings). Each node is capable of incorporating a rich set of context properties, but in reality it was found that many properties were not used at all, and some infrequently (e.g. only around 0.5% of Record Components had ever been revised). CONCLUSIONS: The process of developing generic EHR information models, arising from research and embodied within new-generation interoperability standards and specifications, has been strongly driven by requirements. These requirements have been gathered primarily by collecting use cases and examples from clinical communities, and been added to successive generations of these models. A priority setting approach has not to date been pursued - all requirements have been received and almost invariably met. This work has shown how little of the resulting model is actually needed to represent useful and usable clinical data. A wider range of such evaluations, looking at different kinds of existing clinical system, is needed to balance the theoretical requirements gathering processes, in order to result in EHR information models of an ideal level of complexity.

17.
J Biol Chem ; 276(26): 23572-80, 2001 Jun 29.
Artigo em Inglês | MEDLINE | ID: mdl-11323429

RESUMO

Although it is evident that BCR-ABL can rescue cytokine-deprived hematopoietic progenitor cells from cell cycle arrest and apoptosis, the exact mechanism of action of BCR/ABL and interleukin (IL)-3 to promote proliferation and survival has not been established. Using the pro-B cell line BaF3 and a BaF3 cell line stably overexpressing BCR-ABL (BaF3-p210), we investigated the proliferative signals derived from BCR-ABL and IL-3. The results indicate that both IL-3 and BCR-ABL target the expression of cyclin Ds and down-regulation of p27(Kip1) to mediate pRB-related pocket protein phosphorylation, E2F activation, and thus S phase progression. These findings were further confirmed in a BaF3 cell line (TonB.210) where the BCR-ABL expression is inducible by doxycyclin and by using the drug STI571 to inactivate BCR-ABL activity in BaF3-p210. To establish the functional significance of cyclin D2 and p27(Kip1) expression in response to IL-3 and BCR-ABL expression, we studied the effects of ectopic expression of cyclin D2 and p27(Kip1) on cell proliferation and survival. Our results demonstrate that both cyclin D2 and p27(Kip1) have a role in BaF3 cell proliferation and survival, as ectopic expression of cyclin D2 is sufficient to abolish the cell cycle arrest and apoptosis induced by IL-3 withdrawal or by BCR-ABL inactivation, while overexpression of p27(Kip1) can cause cell cycle arrest and apoptosis in the BaF3 cells. Furthermore, our data also suggest that cyclin D2 functions upstream of p27(Kip1), cyclin E, and cyclin D3, and therefore, plays an essential part in integrating the signals from IL-3 and BCR-ABL with the pRB/E2F pathway.


Assuntos
Apoptose , Proteínas de Ciclo Celular/biossíntese , Ciclinas/biossíntese , Proteínas de Fusão bcr-abl/fisiologia , Células-Tronco Hematopoéticas/citologia , Interleucina-3/fisiologia , Proteínas Supressoras de Tumor , Animais , Benzamidas , Proteínas de Ciclo Celular/genética , Divisão Celular , Linhagem Celular , Sobrevivência Celular , Ciclina D2 , Inibidor de Quinase Dependente de Ciclina p27 , Ciclinas/genética , Ciclinas/fisiologia , Regulação para Baixo , Inibidores Enzimáticos/farmacologia , Proteínas de Fusão bcr-abl/antagonistas & inibidores , Proteínas de Fusão bcr-abl/genética , Fase G1 , Células-Tronco Hematopoéticas/efeitos dos fármacos , Mesilato de Imatinib , Interleucina-3/farmacologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Mensageiro/biossíntese , Proteína do Retinoblastoma/metabolismo , Transdução de Sinais , Transcrição Gênica
18.
J Immunol ; 167(10): 6021-30, 2001 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-11698483

RESUMO

Tumors produce a variety of immunosuppressive factors which can prevent the proliferation and maturation of a number of normal hemopoietic cell types. We have investigated whether primary acute myeloid leukemia (AML) cells have an effect on normal T cell function and signaling. Tumor cell supernatant (TSN) from AML cells inhibited T cell activation and Th1 cytokine production and also prevented activated T cells from entering the cell cycle. These effects occurred in the absence of AML cell-T cell contact. We have demonstrated that AML TSN contained none of the immunosuppressors described to date, namely gangliosides, nitric oxide, TGF-beta, IL-10, vascular endothelial growth factor, or PGs. Furthermore, IL-2 did not overcome the block, despite normal IL-2R expression. However, the effect was overcome by preincubation with inhibitors of protein secretion and abolished by trypsinization, indicating that the active substance includes one or more proteins. To determine the mechanism of inhibition, we have studied many of the major pathways involved in T cell activation and proliferation. We show that nuclear translocation of NFATc and NF-kappaB are markedly reduced in T cells activated in the presence of primary AML cells. In contrast, calcium mobilization and activation of other signal transduction pathways, namely extracellular signal-regulated kinase1/2, p38, and STAT5 were unaffected, but activation of c-Jun N-terminal kinase 1/2 was delayed. Phosphorylation of pRb by cyclin-dependent kinase 6/4-cyclin D and of p130 did not occur and c-Myc, cyclin D3, and p107 were not induced, consistent with cell cycle inhibition early during the transition from G(0) to G(1). Our data indicate that TSN generated by AML cells induces T cell immunosuppression and provides a mechanism by which the leukemic clone could evade T cell-mediated killing.


Assuntos
Proteínas de Ligação a DNA/antagonistas & inibidores , Leucemia Mieloide/imunologia , Ativação Linfocitária , Linfócitos T/imunologia , Evasão Tumoral , Transporte Ativo do Núcleo Celular , Doença Aguda , Núcleo Celular/metabolismo , Células Cultivadas , Meios de Cultivo Condicionados/farmacologia , Citocinas/biossíntese , Humanos , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/antagonistas & inibidores , NF-kappa B/metabolismo , Proteínas Proto-Oncogênicas c-myc/antagonistas & inibidores , Proteína do Retinoblastoma/antagonistas & inibidores , Transdução de Sinais , Células Tumorais Cultivadas
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