RESUMO
Almost 30 years ago it was first appreciated that anti-apoptotic B-cell lymphoma-2 (BCL-2) prevents the induction of apoptosis not only in malignant cells, but also in normal cellular lineages. This critical observation has rapidly evolved from merely identifying new BCL-2 family members to understanding how their biochemical interactions trigger the cell death process, and, more recently, to pharmacological inhibition of anti-apoptotic BCL-2 function in disease. Indeed, the proper regulation of apoptosis is important in many aspects of life including development, homeostasis, and disease biology. To better understand these processes, scientists have used many tools to assess the contribution of individual anti-apoptotic BCL-2 family members. This review will focus on the prominent roles for BCL-2 and other pro-survival family members in promoting the development of mammals during early embryogenesis, neurogenesis, and hematopoiesis.
Assuntos
Apoptose , Desenvolvimento Embrionário , Proteínas Proto-Oncogênicas c-bcl-2/fisiologia , Animais , Proteínas Reguladoras de Apoptose/fisiologia , Hematopoese , Camundongos , Sistema Nervoso/embriologiaRESUMO
The ability to synchronize cells has been central to advancing our understanding of cell cycle regulation. Common techniques employed include serum deprivation; chemicals which arrest cells at different cell cycle phases; or the use of mitotic shake-off which exploits their reduced adherence. However, all of these have disadvantages. For example, serum starvation works well for normal cells but less well for tumor cells with compromised cell cycle checkpoints due to oncogene activation or tumor suppressor loss. Similarly, chemically-treated cell populations can harbor drug-induced damage and show stress-related alterations. A technique which circumvents these problems is counterflow centrifugal elutriation (CCE), where cells are subjected to two opposing forces, centrifugal force and fluid velocity, which results in the separation of cells on the basis of size and density. Since cells advancing through the cycle typically enlarge, CCE can be used to separate cells into different cell cycle phases. Here we apply this technique to primary acute lymphoblastic leukemia cells. Under optimal conditions, an essentially pure population of cells in G1 phase and a highly enriched population of cells in G2/M phases can be obtained in excellent yield. These cell populations are ideally suited for studying cell cycle-dependent mechanisms of action of anticancer drugs and for other applications. We also show how modifications to the standard procedure can result in suboptimal performance and discuss the limitations of the technique. The detailed methodology presented should facilitate application and exploration of the technique to other types of cells.
Assuntos
Contagem de Células/métodos , Ciclo Celular/fisiologia , Separação Celular/métodos , Centrifugação/métodos , Leucemia-Linfoma Linfoblástico de Células Precursoras/metabolismo , HumanosRESUMO
Purpose: BCR-ABL+ B-ALL leukemic cells are highly dependent on the expression of endogenous antiapoptotic MCL-1 to promote viability and are resistant to BH3-mimetic agents such as navitoclax (ABT-263) that target BCL-2, BCL-XL, and BCL-W. However, the survival of most normal blood cells and other cell types is also dependent on Mcl-1 Despite the requirement for MCL-1 in these cell types, initial reports of MCL-1-specific BH3-mimetics have not described any overt toxicities associated with single-agent use, but these agents are still early in clinical development. Therefore, we sought to identify approved drugs that could sensitize leukemic cells to ABT-263.Experimental Design: A screen identified dihydroartemisinin (DHA), a water-soluble metabolite of the antimalarial artemisinin. Using mouse and human leukemic cell lines, and primary patient-derived xenografts, the effect of DHA on survival was tested, and mechanistic studies were carried out to discover how DHA functions. We further tested in vitro and in vivo whether combining DHA with ABT-263 could enhance the response of leukemic cells to combination therapy.Results: DHA causes the downmodulation of MCL-1 expression by triggering a cellular stress response that represses translation. The repression of MCL-1 renders leukemic cells highly sensitive to synergistic cell death induced by ABT-263 in a mouse model of BCR-ABL+ B-ALL both in vitro and in vivo Furthermore, DHA synergizes with ABT-263 in human Ph+ ALL cell lines, and primary patient-derived xenografts of Ph+ ALL in culture.Conclusions: Our findings suggest that combining DHA with ABT-263 can improve therapeutic response in BCR-ABL+ B-ALL. Clin Cancer Res; 23(24); 7558-68. ©2017 AACR.
Assuntos
Compostos de Anilina/administração & dosagem , Proteínas de Fusão bcr-abl/genética , Leucemia de Células B/tratamento farmacológico , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Sulfonamidas/administração & dosagem , Animais , Protocolos de Quimioterapia Combinada Antineoplásica/administração & dosagem , Apoptose/efeitos dos fármacos , Artemisininas/administração & dosagem , Linhagem Celular Tumoral , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Leucemia de Células B/genética , Leucemia de Células B/patologia , Camundongos , Proteínas Proto-Oncogênicas c-bcl-2/genética , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Microtubule-targeting agents (MTA), such as the taxanes and vinca alkaloids, are used to treat a variety of cancers due to their ability to perturb microtubule dynamics. In cell culture, MTAs exert their anticancer effects primarily by causing mitotic arrest and cell death. However, accumulating indirect evidence suggests that MTAs may exert their cytotoxicity in human tumors by interfering with interphase microtubules. In this study, we sought to develop and characterize an experimental system in which to test the hypothesis that MTAs induce cell death during interphase. Primary adult acute lymphoblastic leukemia (ALL) cells treated with vincristine only weakly exhibited colocalization between mitotic and apoptotic markers and major characteristics of mitotic death, such as an increase in cells with 4N DNA content before the appearance of cells with <2N DNA content, suggesting a mixed response. Therefore, we separated ALL cells into distinct phases of the cell cycle by centrifugal elutriation, labeled cells with 5-ethynyl-2'-deoxyuridine (EdU), and then treated each population with vincristine. Cells isolated during G1 underwent cell death without evidence of EdU uptake, indicating that the cytotoxic effects of vincristine took place during G1 Conversely, cells isolated during S or G2-M phases underwent death following mitotic arrest. Thus, vincristine induces distinct death programs in primary ALL cells depending on cell-cycle phase, and cells in G1 are particularly susceptible to perturbation of interphase microtubules. Primary ALL cells may therefore provide a powerful model system in which to study the multimodal mechanisms underlying MTA-induced cell death. Cancer Res; 76(12); 3553-61. ©2016 AACR.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Apoptose/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Vincristina/farmacologia , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Humanos , Microtúbulos/química , Microtúbulos/efeitos dos fármacos , Mitose/efeitos dos fármacos , Leucemia-Linfoma Linfoblástico de Células Precursoras/patologia , Moduladores de Tubulina/farmacologiaRESUMO
Microtubule targeting agents (MTAs) characteristically promote phosphorylation and degradation of Mcl-1, and this represents a critical pro-apoptotic signal in mitotic death. While several phosphorylation sites and kinases have been implicated in mitotic arrest-induced Mcl-1 phosphorylation, a comprehensive biochemical analysis has been lacking. Contrary to previous reports suggesting that T92 phosphorylation by Cdk1 regulates Mcl-1 degradation, a T92A Mcl-1 mutant expressed in HeLa cells was phosphorylated and degraded with the same kinetics as wild-type Mcl-1 following vinblastine treatment. Similarly, when Mcl-1 with alanine replacements of all five putative Cdk sites (S64, T92, S121, S159, T163) was expressed, it was also phosphorylated and degraded in response to vinblastine. To analyze Mcl-1 phosphorylation in more detail, two-dimensional gel electrophoresis (2D-PAGE) was performed. While untreated cells expressed mainly unphosphorylated Mcl-1 with two minor phosphorylated species, Mcl-1 from vinblastine treated cells migrated during 2D-PAGE as a train of acidic spots representing nine or more phosphorylated species. Immunopurification and mass spectrometry of phosphorylated Mcl-1 derived from mitotically arrested HeLa cells revealed nine distinct sites, including several previously unreported. Mcl-1 bearing substitutions of all nine sites had a longer half-life than wild-type Mcl-1 under basal conditions, but still underwent phosphorylation and degradation in response to vinblastine treatment, and, like wild-type Mcl-1, was unable to protect cells from MTA treatment. These results reveal an unexpected complexity in Mcl-1 phosphorylation in response to MTAs and indicate that previous work has severely underestimated the number of sites, and thus encourage major revisions to the current model.
Assuntos
Pontos de Checagem do Ciclo Celular , Eletroforese em Gel Bidimensional , Mitose , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteômica/métodos , Neoplasias do Colo do Útero/metabolismo , Antineoplásicos/farmacologia , Proteína Quinase CDC2/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Cromatografia Líquida de Alta Pressão , Feminino , Células HeLa , Humanos , Cinética , Mitose/efeitos dos fármacos , Mutação , Proteína de Sequência 1 de Leucemia de Células Mieloides/química , Proteína de Sequência 1 de Leucemia de Células Mieloides/genética , Fosforilação , Proteólise , Espectrometria de Massas em Tandem , Moduladores de Tubulina/farmacologia , Neoplasias do Colo do Útero/tratamento farmacológico , Neoplasias do Colo do Útero/genética , Neoplasias do Colo do Útero/patologia , Vimblastina/farmacologiaRESUMO
BH3 mimetic drugs may be useful to treat acute lymphoblastic leukemia (ALL) but the sensitivity of primary tumor cells has not been fully evaluated. Here, B-lineage ALL cell cultures derived from a set of primary tumors were studied with respect to sensitivity to the BH3 mimetics ABT-263 and ABT-199 and to Bcl-2 dependence and function. These ALL cells each expressed high levels of Bcl-2 and exhibited great sensitivity to ABT-263 and ABT-199, which induced rapid apoptotic cell death. BH3 profiling indicated that the ALL cultures were Bcl-2 dependent. Coimmunoprecipitation studies revealed a multifaceted role for Bcl-2 in binding proapoptotic partners including Bax, Bak, Bik, and Bim. ABT-263 disrupted Bcl-2:Bim interaction in cells. Mcl-1 overexpression rendered ALL cells resistant to ABT-263 and ABT-199, with Mcl-1 assuming the role of Bcl-2 in binding Bim. Freshly isolated pediatric ALL blasts also expressed high levels of Bcl-2 and exhibited high sensitivity to Bcl-2 inhibition by the BH3 mimetic compounds. Overall, our results showed that primary ALL cultures were both more sensitive to BH3 mimetics and more uniform in their response than established ALL cell lines that have been evaluated previously. Furthermore, the primary cell model characterized here offers a powerful system for preclinical testing of novel drugs and drug combinations to treat ALL.
Assuntos
Compostos de Anilina/farmacologia , Antineoplásicos/farmacologia , Compostos Bicíclicos Heterocíclicos com Pontes/farmacologia , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamento farmacológico , Proteínas Proto-Oncogênicas c-bcl-2/metabolismo , Sulfonamidas/farmacologia , Apoptose/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Ensaios de Seleção de Medicamentos Antitumorais , Células HeLa , Humanos , Concentração Inibidora 50 , Proteína de Sequência 1 de Leucemia de Células Mieloides/metabolismo , Proteínas Proto-Oncogênicas c-bcl-2/antagonistas & inibidoresRESUMO
Microtubule inhibitors are widely used in cancer chemotherapy. These drugs characteristically induce mitotic arrest and cell death but the mechanisms linking the two are not firmly established. One of the problems is that cancer cells vary widely in their sensitivity to these agents, and thus comparison of data from different systems is difficult. To alleviate this problem we sought to molecularly induce mitotic death and study its mechanisms, by expressing non-degradable cyclin B (R42A) in HeLa cells. However, this approach failed to induce significant mitotic arrest, Cdk1 activation, or phosphorylation of anti-apoptotic Bcl-2 proteins, all characteristics of cells treated with microtubule inhibitors. Furthermore, cyclin B1-R42A induced rapid cell death, and when expressed in synchronized cells, cell death occurred in G1 phase. Decreasing the plasmid concentration reduced transfection efficiency but restored mitotic arrest and eliminated non-specific death. These results show that inappropriate overexpression of cyclin B1 causes non-specific cell death and suggest caution in its use for the study of mitotic events.