RESUMO
Eosinophils are key effector cells in allergic diseases. Here we investigated Mcl-1 (an anti-apoptotic protein) in experimental allergic airway inflammation using transgenic overexpressing human Mcl-1 mice (hMcl-1) and reducing Mcl-1 by a cyclin-dependent kinase inhibitor. Overexpression of Mcl-1 exacerbated allergic airway inflammation, with increased bronchoalveolar lavage fluid cellularity, eosinophil numbers and total protein, and an increase in airway mucus production. Eosinophil apoptosis was suppressed by Mcl-1 overexpression, with this resistance to apoptosis attenuated by cyclin-dependent kinase inhibition which also rescued Mcl-1-exacerbated allergic airway inflammation. We propose that targeting Mcl-1 may be beneficial in treatment of allergic airway disease.
Assuntos
Asma/genética , Eosinófilos/patologia , Regulação da Expressão Gênica , Hipersensibilidade/genética , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , RNA/genética , Animais , Apoptose , Asma/metabolismo , Asma/patologia , Líquido da Lavagem Broncoalveolar , Modelos Animais de Doenças , Eosinófilos/metabolismo , Feminino , Hipersensibilidade/metabolismo , Hipersensibilidade/patologia , Contagem de Leucócitos , Camundongos , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/biossínteseRESUMO
Rationale: Antimicrobial resistance challenges therapy of pneumonia. Enhancing macrophage microbicidal responses would combat this problem but is limited by our understanding of how alveolar macrophages (AMs) kill bacteria. Objectives: To define the role and mechanism of AM apoptosis-associated bacterial killing in the lung. Methods: We generated a unique CD68.hMcl-1 transgenic mouse with macrophage-specific overexpression of the human antiapoptotic Mcl-1 protein, a factor upregulated in AMs from patients at increased risk of community-acquired pneumonia, to address the requirement for apoptosis-associated killing. Measurements and Main Results: Wild-type and transgenic macrophages demonstrated comparable ingestion and initial phagolysosomal killing of bacteria. Continued ingestion (for ≥12 h) overwhelmed initial killing, and a second, late-phase microbicidal response killed viable bacteria in wild-type macrophages, but this response was blunted in CD68.hMcl-1 transgenic macrophages. The late phase of bacterial killing required both caspase-induced generation of mitochondrial reactive oxygen species and nitric oxide, the peak generation of which coincided with the late phase of killing. The CD68.hMcl-1 transgene prevented mitochondrial reactive oxygen species but not nitric oxide generation. Apoptosis-associated killing enhanced pulmonary clearance of Streptococcus pneumoniae and Haemophilus influenzae in wild-type mice but not CD68.hMcl-1 transgenic mice. Bacterial clearance was enhanced in vivo in CD68.hMcl-1 transgenic mice by reconstitution of apoptosis with BH3 mimetics or clodronate-encapsulated liposomes. Apoptosis-associated killing was not activated during Staphylococcus aureus lung infection. Conclusions: Mcl-1 upregulation prevents macrophage apoptosis-associated killing and establishes that apoptosis-associated killing is required to allow AMs to clear ingested bacteria. Engagement of macrophage apoptosis should be investigated as a novel, host-based antimicrobial strategy.
Assuntos
Apoptose/fisiologia , Macrófagos Alveolares/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fagocitose/genética , Fagossomos/fisiologia , Pneumonia Bacteriana , Animais , Apoptose/efeitos dos fármacos , Bactérias , Compostos de Bifenilo/farmacologia , Caspases/metabolismo , Ácido Clodrônico/farmacologia , Modelos Animais de Doenças , Haemophilus influenzae , Humanos , Macrófagos Alveolares/metabolismo , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Óxido Nítrico/metabolismo , Nitrofenóis/farmacologia , Piperazinas/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Staphylococcus aureus , Streptococcus pneumoniae , Sulfonamidas/farmacologiaRESUMO
RATIONALE: Chronic obstructive pulmonary disease (COPD) is characterized by impaired clearance of pulmonary bacteria. OBJECTIVES: The effect of COPD on alveolar macrophage (AM) microbicidal responses was investigated. METHODS: AMs were obtained from bronchoalveolar lavage from healthy donors or patients with COPD and challenged with opsonized serotype 14 Streptococcus pneumoniae. Cells were assessed for apoptosis, bactericidal activity, and mitochondrial reactive oxygen species (mROS) production. A transgenic mouse line in which the CD68 promoter ensures macrophage-specific expression of human induced myeloid leukemia cell differentiation protein Mcl-1 (CD68.hMcl-1) was used to model the molecular aspects of COPD. MEASUREMENTS AND MAIN RESULTS: COPD AMs had elevated levels of Mcl-1, an antiapoptotic B-cell lymphoma 2 family member, with selective reduction of delayed intracellular bacterial killing. CD68.hMcl-1 AMs phenocopied the microbicidal defect because transgenic mice demonstrated impaired clearance of pulmonary bacteria and increased neutrophilic inflammation. Murine bone marrow-derived macrophages and human monocyte-derived macrophages generated mROS in response to pneumococci, which colocalized with bacteria and phagolysosomes to enhance bacterial killing. The Mcl-1 transgene increased oxygen consumption rates and mROS expression in mock-infected bone marrow-derived macrophages but reduced caspase-dependent mROS production after pneumococcal challenge. COPD AMs also increased basal mROS expression, but they failed to increase production after pneumococcal challenge, in keeping with reduced intracellular bacterial killing. The defect in COPD AM intracellular killing was associated with a reduced ratio of mROS/superoxide dismutase 2. CONCLUSIONS: Up-regulation of Mcl-1 and chronic adaption to oxidative stress alter mitochondrial metabolism and microbicidal function, reducing the delayed phase of intracellular bacterial clearance in COPD.
Assuntos
Anti-Infecciosos/farmacologia , Macrófagos Alveolares/efeitos dos fármacos , Macrófagos Alveolares/metabolismo , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Doença Pulmonar Obstrutiva Crônica/metabolismo , Animais , Western Blotting , Lavagem Broncoalveolar , Modelos Animais de Doenças , Citometria de Fluxo , Humanos , Camundongos , Camundongos Transgênicos , Estresse Oxidativo/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/fisiopatologiaRESUMO
UNLABELLED: Viral infection results in the generation of massive numbers of activated effector CD8(+) T cells that recognize viral components. Most of these are short-lived effector T cells (SLECs) that die after clearance of the virus. However, a small proportion of this population survives and forms antigen-specific memory precursor effector cells (MPECs), which ultimately develop into memory cells. These can participate in a recall response upon reexposure to antigen even at protracted times postinfection. Here, antiapoptotic myeloid cell leukemia 1 (MCL1) was found to prolong survival upon T cell stimulation, and mice expressing human MCL1 as a transgene exhibited a skewing in the proportion of CD8(+) T cells, away from SLECs toward MPECs, during the acute phase of vaccinia virus infection. A higher frequency and total number of antigen-specific CD8(+) T cells were observed in MCL1 transgenic mice. These findings show that MCL1 can shape the makeup of the CD8(+) T cell response, promoting the formation of long-term memory. IMPORTANCE: During an immune response to a virus, CD8(+) T cells kill cells infected by the virus, and most die when the infection resolves. However, a small proportion of cells survives and differentiates into long-lived memory cells that confer protection from reinfection by the same virus. This report shows that transgenic expression of an MCL1 protein enhances survival of memory CD8(+) T cells following infection with vaccinia virus. This is important because it shows that MCL1 expression may be an important determinant of the formation of long-term CD8(+) T cell memory.
Assuntos
Linfócitos T CD8-Positivos/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Vaccinia virus/imunologia , Vacínia/imunologia , Animais , Linfócitos T CD8-Positivos/imunologia , Sobrevivência Celular , Modelos Animais de Doenças , Humanos , Memória Imunológica , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides/genéticaRESUMO
Abundant, sustained expression of prosurvival Mcl-1 is an important determinant of viability and drug resistance in cancer cells. The Mcl-1 protein contains PEST sequences (enriched in proline, glutamic acid, serine, and threonine) and is normally subject to rapid turnover via multiple different pathways. One of these pathways involves a phosphodegron in the PEST region, where Thr-163 phosphorylation primes for Ser-159 phosphorylation by glycogen synthase kinase-3. Turnover via this phosphodegron-targeted pathway is reduced in Mcl-1-overexpressing BL41-3 Burkitt lymphoma and other cancer cells; turnover is further slowed in the presence of phorbol ester-induced ERK activation, resulting in Mcl-1 stabilization and an exacerbation of chemoresistance. The present studies focused on Mcl-1 dephosphorylation, which was also found to profoundly influence turnover. Exposure of BL41-3 cells to an inhibitor of protein phosphatase 2A (PP2A), okadaic acid, resulted in a rapid increase in phosphorylation at Thr-163 and Ser-159, along with a precipitous decrease in Mcl-1 expression. The decline in Mcl-1 expression preceded the appearance of cell death markers and was not slowed in the presence of phorbol ester. Upon exposure to calyculin A, which also potently inhibits PP2A, versus tautomycin, which does not, only the former increased Thr-163/Ser-159 phosphorylation and decreased Mcl-1 expression. Mcl-1 co-immunoprecipitated with PP2A upon transfection into CHO cells, and PP2A/Aα knockdown recapitulated the increase in Mcl-1 phosphorylation and decrease in expression. In sum, inhibition of PP2A prevents Mcl-1 dephosphorylation and results in rapid loss of this prosurvival protein in chemoresistant cancer cells.
Assuntos
Linfoma de Burkitt/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteína Fosfatase 2/antagonistas & inibidores , Sítios de Ligação , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/genética , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos , Inibidores Enzimáticos/farmacologia , Expressão Gênica , Técnicas de Silenciamento de Genes , Humanos , Sistema de Sinalização das MAP Quinases , Toxinas Marinhas , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Ácido Okadáico/farmacologia , Oxazóis/farmacologia , Fosforilação/efeitos dos fármacos , Proteína Fosfatase 2/genética , Proteólise , Serina/química , Acetato de Tetradecanoilforbol/farmacologia , Treonina/químicaRESUMO
OBJECTIVE: To investigate the role of acyl-CoA:cholesterol acyltransferase 1 (ACAT1) in hematopoiesis. APPROACH AND RESULTS: ACAT1 converts cellular cholesterol to cholesteryl esters for storage in multiple cell types and is a potential drug target for human diseases. In mouse models for atherosclerosis, global Acat1 knockout causes increased lesion size; bone marrow transplantation experiments suggest that the increased lesion size might be caused by ACAT1 deficiency in macrophages. However, bone marrow contains hematopoietic stem cells that give rise to cells in myeloid and lymphoid lineages; these cell types affect atherosclerosis at various stages. Here, we test the hypothesis that global Acat1(-/-) may affect hematopoiesis, rather than affecting macrophage function only, and show that Acat1(-/-) mice contain significantly higher numbers of myeloid cells and other cells than wild-type mice. Detailed analysis of bone marrow cells demonstrated that Acat1(-/-) causes a higher proportion of the stem cell-enriched Lin(-)Sca-1(+)c-Kit(+) population to proliferate, resulting in higher numbers of myeloid progenitor cells. In addition, we show that Acat1(-/-) causes higher monocytosis in Apoe(-/-) mouse during atherosclerosis development. CONCLUSIONS: ACAT1 plays important roles in hematopoiesis in normal mouse and in Apoe(-/-) mouse during atherosclerosis development.
Assuntos
Acetil-CoA C-Acetiltransferase/deficiência , Aterosclerose/enzimologia , Células da Medula Óssea/enzimologia , Proliferação de Células , Hematopoese , Células-Tronco Hematopoéticas/enzimologia , Leucocitose/enzimologia , Acetil-CoA C-Acetiltransferase/genética , Animais , Antígenos Ly/metabolismo , Apolipoproteínas E/genética , Apolipoproteínas E/metabolismo , Aterosclerose/genética , Aterosclerose/imunologia , Biomarcadores/metabolismo , Linhagem da Célula , Células Cultivadas , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Feminino , Genótipo , Leucocitose/genética , Leucocitose/imunologia , Sistema de Sinalização das MAP Quinases , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Fenótipo , Células Precursoras de Linfócitos B/enzimologia , Proteínas Proto-Oncogênicas c-kit/metabolismo , Fatores de TempoRESUMO
Increasing the pool of cells at early T-cell developmental stages enhances thymopoiesis and is especially beneficial when T-cell production is compromised by radiation or aging. Within the immature double-negative (DN; CD4(-)CD8(-)) thymocyte subpopulation, the DN1 subset contains the most primitive cells including the rare early T-cell progenitors (ETPs). In the present study, a human MCL1 transgene, under the control of its endogenous promoter, resulted in enlargement of an undistorted thymus in C57/BL6 mice. Enlargement occurred in females but not males, being seen at 1 month of age and maintained during progression into adulthood as the thymus underwent involution. The small DN1 subset was expanded disproportionally (ETPs increasing from â¼0.016 to 0.03% of thymocytes), while more mature thymocytes were increased proportionally (1.5-fold) along with the stroma. DN1 cells from transgenic females exhibited increased viability with maintained proliferation, and their survival in primary culture was extended. Exposure of transgenic females to γ-irradiation also revealed an expanded pool of radioresistant DN1 cells exhibiting increased viability. While the viability of DN1 cells from transgenic males was equivalent to that of their non-transgenic counterparts directly after harvest, it was enhanced in culture-suggesting that the effect of the transgene was suppressed in the in vivo environment of the male. Viability was increased in ETPs from transgenic females, but unchanged in more mature thymocytes, indicating that primitive cells were affected selectively. The MCL1 transgene thus increases the viability and pool size of primitive ETP/DN1 cells, promoting thymopoiesis and radioresistance in peripubescent females and into adulthood.
Assuntos
Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Timócitos/citologia , Timo/crescimento & desenvolvimento , Animais , Sobrevivência Celular , Feminino , Humanos , Masculino , Camundongos , Camundongos Congênicos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas Proto-Oncogênicas c-bcl-2/deficiência , Linfócitos T/citologia , Linfócitos T/imunologia , Timócitos/imunologia , Timo/citologia , Timo/imunologia , Irradiação Corporal TotalRESUMO
Hepatocyte apoptosis contributes to liver injury and fibrosis after cholestatic injury. Our aim was to ascertain if the anti-apoptotic protein Mcl-1 alters liver injury or fibrosis in the bile duct-ligated mouse. Markers of apoptosis and fibrosis were compared in wild-type and transgenic mice expressing human Mcl-1 after bile duct ligation. Compared to hMcl-1 transgenic animals, ligated wild-type mice displayed a significant increase in TUNEL-positive cells and in caspase 3/7-positive hepatocytes. Consistent with apoptotic injury, the pro-apoptotic protein Bak underwent a conformational change to an activated form upon cholestatic injury, a change mitigated by hMcl-1 overexpression. Likewise, liver histology, number of bile infarcts, serum ALT values, markers of hepatic fibrosis, and animal survival were improved in bile duct-ligated mice transgenic for hMcl-1 as compared to wild-type mice. In conclusion, increased Mcl-1 expression plays a role in hepatoprotection upon cholestatic liver injury.
Assuntos
Apoptose , Colestase Intra-Hepática/fisiopatologia , Hepatócitos/fisiologia , Fígado/patologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Animais , Ductos Biliares/cirurgia , Biomarcadores/metabolismo , Colestase Intra-Hepática/patologia , Fibrose , Humanos , Ligadura , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células MieloidesRESUMO
Macrophages are critical effectors of bacterial clearance and must retain viability, despite exposure to toxic bacterial products, until key antimicrobial functions are performed. Subsequently, host-mediated macrophage apoptosis aids resolution of infection. The ability of macrophages to make this transition from resistance to susceptibility to apoptosis is important for effective host innate immune responses. We investigated the role of Mcl-1, an essential regulator of macrophage lifespan, in this switch from viability to apoptosis, using the model of pneumococcal-associated macrophage apoptosis. Upon exposure to pneumococci, macrophages initially upregulate Mcl-1 protein and maintain viability for up to 14 hours. Subsequently, macrophages reduce expression of full-length Mcl-1 and upregulate a 34-kDa isoform of Mcl-1 corresponding to a novel BH3-only splice variant, Mcl-1(Exon-1). Change in expression of Mcl-1 protein is associated with mitochondrial membrane permeabilization, which is characterized by loss of mitochondrial inner transmembrane potential and translocation of cytochrome c and apoptosis-inducing factor. Following pneumococcal infection, macrophages expressing full-length human Mcl-1 as a transgene exhibit a delay in apoptosis and in bacterial killing. Mcl-1 transgenic mice clear pneumococci from the lung less efficiently than nontransgenic mice. Dynamic changes in Mcl-1 expression determine macrophage viability as well as antibacterial host defense.
Assuntos
Apoptose , Macrófagos/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Streptococcus pneumoniae , Processamento Alternativo/genética , Processamento Alternativo/fisiologia , Animais , Apoptose/genética , Sobrevivência Celular/fisiologia , Regulação da Expressão Gênica , Humanos , Macrófagos/microbiologia , Macrófagos/patologia , Potenciais da Membrana , Camundongos , Camundongos Transgênicos , Mitocôndrias/metabolismo , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Pneumonia Pneumocócica/genética , Pneumonia Pneumocócica/metabolismo , Pneumonia Pneumocócica/patologia , Isoformas de Proteínas/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genéticaRESUMO
Members of the BCL2 gene family influence cell viability and can, therefore, affect the susceptibility of cancer cells to multiple chemotherapeutic agents. Thus, it is a challenge to devise approaches for inducing the death of tumor cells in which the expression of prosurvival family members is elevated or deregulated. BL41-3, a spontaneously derived subline of BL41 Burkitt lymphoma cells, was found to have amplified the prosurvival MCL1 gene (3-fold) and overexpressed the MCL1 protein. The level of MCL1 protein was 5-fold elevated compared with ML-1 cells expressing maximal MCL1 on exposure to phorbol-12-myristate-13- acetate. To assess whether this increase in MCL1 expression was associated with enhanced protection from cell death, cells were exposed to conditions of growth factor deprivation or to various cytotoxic agents. Whereas BL41-3 and BL41 cells exhibited similar growth rates in logarithmic phase, BL41-3 cells remained largely viable on reaching saturation phase in contrast to BL41 cells, which began to die. Similarly, the BL41-3 subline remained viable for an extended period under conditions of reduced serum. BL41-3 cells were also more resistant to the apoptosis-inducing effects of etoposide, camptothecin, and staurosporine (>3-fold more than BL41 cells). Unexpectedly, these cells exhibited enhanced sensitivity to 1-beta-D-arabinofuranosylcytosine, but only on exposure for an extended period (>10-fold more sensitive than BL41 cells with a 24-h but not a 6-h exposure). Thus, whereas cells expressing prosurvival BCL2 family members are frequently resistant to a variety of chemotherapeutic agents, the findings presented here, using a cell line exhibiting amplification and overexpression of MCL1, indicate that such cells may exhibit increased sensitivity to certain chemotherapeutic regimens.
Assuntos
Antimetabólitos Antineoplásicos/farmacologia , Linfoma de Burkitt/tratamento farmacológico , Linfoma de Burkitt/metabolismo , Citarabina/farmacologia , Inibidores Enzimáticos/farmacologia , Proteínas de Neoplasias/biossíntese , Estaurosporina/farmacologia , Apoptose/efeitos dos fármacos , Linfoma de Burkitt/genética , Linfoma de Burkitt/patologia , Camptotecina/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Meios de Cultura Livres de Soro , Resistência a Múltiplos Medicamentos , Resistencia a Medicamentos Antineoplásicos , Etoposídeo/farmacologia , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogênicas c-bcl-2/biossíntese , Proteínas Proto-Oncogênicas c-bcl-2/genética , Inibidores da Topoisomerase , Células Tumorais CultivadasRESUMO
Interactions between the novel histone deacetylase inhibitor LAQ824 and the cyclin-dependent kinase inhibitor roscovitine were examined in human leukemia cells. Pretreatment (24 hours) with a subtoxic concentration of LAQ824 (30 nmol/L) followed by a minimally toxic concentration of roscovitine (10 micromol/L; 24 hours) resulted in greater than additive effects on apoptosis in U937, Jurkat, and HL-60 human leukemia cells and blasts from three patients with acute myelogenous leukemia. These events were associated with enhanced conformational changes in Bax; mitochondrial release of cytochrome c, Smac/DIABLO, and apoptosis-inducing factor; and a marked increase in caspase activation. LAQ824/roscovitine-treated cells displayed caspase-dependent down-regulation of p21(CIP1) and Mcl-1 and a pronounced caspase-independent reduction in X-linked inhibitor of apoptosis (XIAP) expression. The lethality of this regimen was significantly attenuated by ectopic expression of XIAP, a nuclear localization signal-defective p21(CIP1) mutant, Mcl-1, and Bcl-2. Combined exposure to LAQ824 and roscovitine resulted in a significant reduction in XIAP mRNA levels and diminished phosphorylation of the carboxyl-terminal domain of RNA polymerase II. Notably, roscovitine blocked LAQ824-mediated differentiation. Finally, LAQ824 and roscovitine individually and in combination triggered an increase in generation of reactive oxygen species; moreover, coadministration of the free radical scavenger N-acetylcysteine prevented LAQ824/roscovitine-mediated mitochondrial injury and apoptosis. Collectively, these findings suggest that combined treatment of human leukemia cells with LAQ824 and roscovitine disrupts maturation and synergistically induces apoptosis, lending further support for an antileukemic strategy combining novel histone deacetylase and cyclin-dependent kinase inhibitors.
Assuntos
Antineoplásicos/farmacologia , Inibidores Enzimáticos/farmacologia , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Leucemia/tratamento farmacológico , Leucemia/enzimologia , Purinas/farmacologia , Apoptose , Fator de Indução de Apoptose/metabolismo , Western Blotting , Ciclo Celular , Linhagem Celular Tumoral , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Citocromos c/metabolismo , Citosol/metabolismo , Regulação para Baixo , Citometria de Fluxo , Células HL-60 , Humanos , Células Jurkat , Potenciais da Membrana , Mitocôndrias/patologia , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/metabolismo , Conformação Proteica , Inibidores de Proteínas Quinases/farmacologia , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Espécies Reativas de Oxigênio , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Roscovitina , Fatores de Tempo , Células U937 , Proteínas Inibidoras de Apoptose Ligadas ao Cromossomo X/metabolismoRESUMO
Mcl-1, an anti-apoptotic member of Bcl-2 family maintains cell viability during clonal expansion of CD8 T cells, but the cell intrinsic role of Mcl-1 in contraction of effectors or the number of memory CD8 T cells is unknown. Mcl-1 levels decline during the contraction phase but rebound to high levels in memory CD8 T cells. Therefore, by overexpressing Mcl-1 in CD8 T cells we asked whether limiting levels of Mcl-1 promote contraction of effectors and constrain CD8 T-cell memory. Mcl-1 overexpression failed to affect CD8 T-cell expansion, contraction or the magnitude of CD8 T-cell memory. Strikingly, high Mcl-1 levels enhanced mTOR phosphorylation and augmented the differentiation of terminal effector cells and effector memory CD8 T cells to the detriment of poly-cytokine-producing central memory CD8 T cells. Taken together, these findings provided unexpected insights into the role of Mcl-1 in the differentiation of effector and memory CD8 T cells.
Assuntos
Linfócitos T CD8-Positivos/citologia , Linfócitos T CD8-Positivos/imunologia , Diferenciação Celular , Memória Imunológica , Coriomeningite Linfocítica/imunologia , Vírus da Coriomeningite Linfocítica/fisiologia , Proteína de Sequência 1 de Leucemia de Células Mieloides/imunologia , Animais , Humanos , Coriomeningite Linfocítica/genética , Coriomeningite Linfocítica/fisiopatologia , Coriomeningite Linfocítica/virologia , Vírus da Coriomeningite Linfocítica/imunologia , Camundongos , Camundongos Endogâmicos C57BL , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Serina-Treonina Quinases TOR/genética , Serina-Treonina Quinases TOR/imunologiaRESUMO
BCL2 family members are subject to regulation at multiple levels, providing checks on their ability to contribute to tumorigenesis. However, findings on post-translational BCL2 phosphorylation in different systems have been difficult to integrate. Another antiapoptotic family member, MCL1, exhibits a difference in electrophoretic mobility upon phosphorylation induced by an activator of PKC (12-O-tetradecanoylphorbol 13-acetate; TPA) versus agents that act on microtubules or protein phosphatases 1/2A. A multiple pathway model is now presented, which demonstrates that MCL1 can undergo distinct phosphorylation events - mediated through separate signaling processes and involving different target sites - in cells that remain viable in the presence of TPA versus cells destined to die upon exposure to taxol or okadaic acid. Specifically, TPA induces phosphorylation at a conserved extracellular signal-regulated kinase (ERK) site in the PEST region (Thr 163) and slows turnover of the normally rapidly degraded MCL1 protein; however, okadaic acid and taxol induce ERK-independent MCL1 phosphorylation at additional discrete sites. These findings add a new dimension to our understanding of the complex regulation of antiapoptotic BCL2 family members by demonstrating that, in addition to transcriptional and post-transcriptional regulation, MCL1 is subject to multiple, separate, post-translational phosphorylation events, produced in living versus dying cells at ERK-inducible versus ERK-independent sites.
Assuntos
Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias/metabolismo , Sequência de Aminoácidos , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Sítios de Ligação , Células CHO , Carcinógenos , Linhagem Celular Tumoral , Sobrevivência Celular , Cricetinae , Cães , Relação Dose-Resposta a Droga , Ativação Enzimática , Inibidores Enzimáticos/farmacologia , Humanos , Sistema de Sinalização das MAP Quinases , Camundongos , Dados de Sequência Molecular , Proteína de Sequência 1 de Leucemia de Células Mieloides , Proteínas de Neoplasias/química , Ácido Okadáico/farmacologia , Paclitaxel/farmacologia , Mapeamento de Peptídeos , Fosforilação , Testes de Precipitina , Proteína Quinase C/metabolismo , Estrutura Terciária de Proteína , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Transdução de Sinais , Acetato de Tetradecanoilforbol , Treonina/química , Fatores de Tempo , TransfecçãoRESUMO
Enforced expression of the antiapoptotic Bcl-2 family protein Mcl-1 promotes lymphomagenesis in the mouse; however, the functional role of Mcl-1 in human B-cell lymphoma remains unclear. We demonstrate that Mcl-1 is widely expressed in malignant B-cells, and high-level expression of Mcl-1 is required for B-lymphoma cell survival, since transfection of Mcl-1-specific antisense oligodeoxynucleotides was sufficient to promote apoptosis in Akata6 lymphoma cells. Mcl-1 was efficiently cleaved by caspases at evolutionarily conserved aspartic acid residues in vitro, and during cisplatin-induced apoptosis in B-lymphoma cell lines and spontaneous apoptosis of primary malignant B-cells. Overexpression of the Mcl-1 cleavage product that accumulated during apoptosis was sufficient to kill cells. Therefore, Mcl-1 is an essential survival molecule for B-lymphoma cells and is cleaved by caspases to a death-promoting molecule during apoptosis. In contrast to Mcl-1, Bcl-2 and Bcl-XL were relatively resistant to caspase cleavage in vitro and in intact cells. Interfering with Mcl-1 function appears to be an effective means of inducing apoptosis in Mcl-1-positive B-cell lymphoma, and the unique sensitivity of Mcl-1 to caspase-mediated cleavage suggests an attractive strategy for converting it to a proapoptotic molecule.
Assuntos
Apoptose/fisiologia , Caspases/metabolismo , Proteínas de Ciclo Celular/metabolismo , Sobrevivência Celular/fisiologia , Linfoma de Células B/patologia , Proteínas Oncogênicas/metabolismo , Biópsia , Proteínas de Ciclo Celular/genética , Morte Celular , Linhagem Celular Tumoral , Humanos , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Proteínas Oncogênicas/genética , Fases de Leitura Aberta , Plasmídeos , Tionucleotídeos/farmacologiaRESUMO
Age-related thymic involution is characterized by a progressive regression in thymus size and a diminishment of thymic structure. A decrease in thymic compartments leads to the reduction of thymopoiesis. Thymic involution is closely associated with immunosenescence, a degeneration of the immune system primarily due to the alterations in T-cell composition. Strategies to improve the consequences of the aging thymus are currently under investigation. A wide array of knowledge has revealed a series of factors that are essential in the overall determination of thymic function and immune response. Evidence indicates that early programming of the thymus, sexual dimorphism, and the efficiency of specific T-cell progenitors and the thymic microenvironment are all crucial determinants of immune activity from early life through advanced ages. To fully understand the processes involved in age-related thymic involution, such determinants must be considered. The central purpose of this review is to emphasize previous and most recent evidence suggesting that these factors contribute to the influence of long-term immunity and ultimately shape the progression of thymic involution in advanced age.
RESUMO
The antiapoptotic Bcl-2 family member Mcl-1 is a PEST protein (containing sequences enriched in proline, glutamic acid, serine, and threonine) and is subject to rapid degradation via multiple pathways. Impaired degradation leading to the maintenance of Mcl-1 expression is an important determinant of drug resistance in cancer. Phosphorylation at Thr 163 in the PEST region, stimulated by 12-O-tetradecanoylphorbol acetic acid (TPA)-induced activation of extracellular signal-regulated kinase (ERK), is associated with Mcl-1 stabilization in BL41-3 Burkitt lymphoma cells. This contrasts with the observation that Thr 163 phosphorylation in normal fibroblasts primes glycogen synthase kinase (GSK3)-induced phosphorylation at Ser 159, producing a phosphodegron that targets Mcl-1 for degradation. In the present follow-up studies in BL41-3 cells, Mcl-1 degradation was found to be independent of the GSK3-mediated pathway, providing a parallel to emerging findings showing that Mcl-1 degradation through this pathway is lost in many different types of cancer. Findings in Mcl-1-transfected CHO cells corroborated those in BL41-3 cells in that the GSK3-targeted phosphodegron did not play a major role in Mcl-1 degradation, and a phosphomimetic T163E mutation resulted in marked Mcl-1 stabilization. TPA-treated BL41-3 cells, in addition to exhibiting Thr 163 phosphorylation and Mcl-1 stabilization, exhibited an â¼10-fold increase in resistance to multiple chemotherapeutic agents, including Ara-C, etoposide, vinblastine, or cisplatin. In these cancer cells in which Mcl-1 degradation is not dependent on the GSK3/phosphodegron-targeted pathway, ERK activation and Thr 163 phosphorylation are associated with pronounced Mcl-1 stabilization and drug resistance - effects that can be suppressed by inhibition of ERK activation.
Assuntos
Quinase 3 da Glicogênio Sintase/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Treonina/metabolismo , Animais , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Apoptose/genética , Western Blotting , Células CHO , Linhagem Celular Tumoral , Cisplatino/farmacologia , Cricetinae , Citarabina/farmacologia , Resistencia a Medicamentos Antineoplásicos/genética , Etoposídeo/farmacologia , Citometria de Fluxo , Quinase 3 da Glicogênio Sintase/genética , Humanos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Fosforilação , Estabilidade Proteica , Vimblastina/farmacologiaRESUMO
Mcl-1 is an antiapoptotic Bcl-2 family member that is highly regulated and when dysregulated contributes to cancer. The Mcl-1 protein is phosphorylated at multiple sites in response to different signaling events. Phosphorylations at Thr163 (by ERK) and Ser159 (by glycogen-synthase kinase 3beta) have recently been shown to slow and enhance, respectively, Mcl-1 protein turnover. Phosphorylation is also known to be stimulated at other, as-yet uncharacterized sites in the G2/M phase of the cell cycle. Using an S peptide-tagged Mcl-1 T163A mutant, Ser64 was identified as a novel Mcl-1 phosphorylation site by mass spectrometry. Immunoblotting demonstrated that phosphorylation at this site was maximal in cells in G2/M phase, was enhanced by tumor necrosis factor-alpha-related apoptosis-inducing ligand (TRAIL) treatment, was blocked by inhibitors of CDK (but not ERK or glycogen-synthase kinase 3beta), and was stimulated in vitro by CDK 1, CDK2, and JNK1. The half-life of a nonphosphorylatable S64A Mcl-1 mutant was indistinguishable from that of the wild type polypeptide. In contrast, this mutant failed to protect cells from TRAIL-mediated apoptosis, whereas reconstitution with the phosphomimetic S64E Mcl-1 mutant rendered cells TRAIL-resistant. This anti-apoptotic phenotype of the S64E Mcl-1 mutant was also associated with enhanced binding to the proapoptotic proteins Bim, Noxa, and Bak. A pharmacological CDK inhibitor that reduced Ser64 phosphorylation also sensitized cells to TRAIL cytotoxicity. Collectively, these observations not only identify G2/M-associated phosphorylation at Ser64 as a critical determinant of the antiapoptotic activity of Mcl-1 but also elucidate a novel mechanism by which CDK1/2 inhibitors can enhance the effectiveness of the cytotoxic cytokine TRAIL.
Assuntos
Apoptose , Proteínas de Neoplasias/fisiologia , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Serina/química , Sequência de Aminoácidos , Animais , Divisão Celular , Linhagem Celular Tumoral , Fase G2 , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Humanos , Dados de Sequência Molecular , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides , Peptídeos/química , Fenótipo , FosforilaçãoRESUMO
The antiapoptotic BCL2 family member MCL1 is normally up- and down-modulated in response to environmental signals and conditions, but is constitutively expressed in cancer where it promotes cell survival and drug resistance. A post-translational modification identified here, truncation at the N terminus, was found to act along with previously described ERK- and GSK3-induced phosphorylation events to regulate the turnover of the MCL1 protein and thus its availability for antiapoptotic effects. Although both N-terminally truncated and full-length MCL1 contain sequences enriched in proline, glutamic acid, serine, and threonine and were susceptible to proteasomal degradation, the truncated form decayed less rapidly and was maintained for an extended period in the presence of ERK activation. This was associated with extended cell survival because the truncated form of MCL1 (unlike those of BCL2 and BCLX) retained antiapoptotic activity. N-terminal truncation slightly increased the electrophoretic mobility of MCL1 and differed from the phosphorylation/band shift to decreased mobility, which occurs in the G2/M phase and was not found to affect MCL1 turnover. The N-terminally truncated form of MCL1 was expressed to varying extents in normal lymphoid tissues and was the predominant form present in lymphomas from transgenic mice and human tumor lines of B-lymphoid origin. The degradation versus stabilized expression of antiapoptotic MCL1 is thus controlled by N-terminal truncation as well as by ERK- and GSK3 (but not G2/M)-induced phosphorylation. These modifications may contribute to dysregulated MCL1 expression in cancer and represent targets for promoting its degradation to enhance tumor cell death.
Assuntos
Proteínas Reguladoras de Apoptose/genética , Divisão Celular , Fase G2 , Proteínas de Neoplasias/genética , Processamento de Proteína Pós-Traducional , Proteínas Proto-Oncogênicas c-bcl-2/genética , Animais , Linhagem Celular Tumoral , Células Cultivadas , Expressão Gênica , Humanos , Camundongos , Camundongos Transgênicos , Proteína de Sequência 1 de Leucemia de Células Mieloides , FosforilaçãoRESUMO
The antiapoptotic BCL2 family member MCL1 is rapidly upregulated upon exposure of ML-1 myeloid leukemia cells to either differentiation-inducing phorbol 12'-myristate 13'-acetate (PMA) or chemotherapeutic microtubule disrupting agents (MTDAs). This report examined how signaling for MCL1 upregulation is coupled to these two different phenotypic changes, and tested for upregulation in other hematopoietic cancers. With PMA, ERK stimulated MCL1 mRNA expression and ML-1 cell differentiation, and ERK additionally stabilized expression of the MCL1 protein. However, with MTDAs, transient ERK and ensuing JNK activation contributed to initial MCL1 upregulation and viability-retention, but sustained JNK activation eventually resulted in cell death. MCL1 was upregulated by PMA in THP-1 and U937 myeloid leukemia cells, but by MTDAs only in THP-1 cells. MCL1 expression was constitutively elevated in multiple myeloma cell lines, and was not affected by PMA/ERK or MTDAs. Thus, MCL1 expression level and sensitivity to regulation are important considerations in selecting approaches for targeting this antiapoptotic gene product to kill cancer cells.
Assuntos
Apoptose/efeitos dos fármacos , Leucemia Mieloide/fisiopatologia , Mieloma Múltiplo/fisiopatologia , Proteínas de Neoplasias/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Diferenciação Celular/fisiologia , Linhagem Celular Tumoral , Ativação Enzimática , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Regulação Neoplásica da Expressão Gênica , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Microtúbulos/efeitos dos fármacos , Proteína de Sequência 1 de Leucemia de Células Mieloides , Acetato de Tetradecanoilforbol/farmacologia , Moduladores de Tubulina/farmacologia , Células U937 , Vimblastina/farmacologia , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismoRESUMO
The constitutive androstane receptor (CAR) modulates xeno- and endobiotic hepatotoxicity by regulating detoxification pathways. Whether activation of CAR may also protect against liver injury by directly blocking apoptosis is unknown. To address this question, CAR wild-type (CAR+/+) and CAR knockout (CAR-/-) mice were treated with the CAR agonist 1,4-bis[2-(3,5-dichloropyridyloxy)] benzene (TCPOBOP) and then with the Fas agonist Jo2 or with concanavalin A (ConA). Following the administration of Jo2, hepatocyte apoptosis, liver injury, and animal fatalities were abated in TCPOBOP-treated CAR+/+ but not in CAR-/- mice. Likewise, acute and chronic ConA-mediated liver injury and fibrosis were also reduced in wild-type versus CAR(-/-) TCPOBOP-treated mice. The proapoptotic proteins Bak (Bcl-2 antagonistic killer) and Bax (Bcl-2-associated X protein) were depleted in livers from TCPOBOP-treated CAR+/+ mice. In contrast, mRNA expression of the antiapoptotic effector myeloid cell leukemia factor-1 (Mcl-1) was increased fourfold. Mcl-1 promoter activity was increased by transfection with CAR and administration of TCPOBOP in hepatoma cells, consistent with a direct CAR effect on Mcl-1 transcription. Indeed, site-directed mutagenesis of a putative CAR consensus binding sequence on the Mcl-1 promoter decreased Mcl-1 promoter activity. Mcl-1 transgenic animals demonstrated little to no acute liver injury after administration of Jo2, signifying Mcl-1 cytoprotection. In conclusion, these observations support a prominent role for CAR cytoprotection against Fas-mediated hepatocyte injury via a mechanism involving upregulation of Mcl-1 and, likely, downregulation of Bax and Bak.