RESUMO
Hematopoietic stem and progenitor cells (HSPCs) represent the lifelong source of all blood cells and continuously regenerate the hematopoietic system through differentiation and self-renewal. The process of differentiation is initiated in the G1 phase of the cell cycle, when stem cells leave their quiescent state. During G1, the anaphase-promoting complex or cyclosome associated with the coactivator Cdh1 is highly active and marks proteins for proteasomal degradation to regulate cell proliferation. Following Cdh1 knockdown in HSPCs, we analyzed human and mouse hematopoiesis in vitro and in vivo in competitive transplantation assays. We found that Cdh1 is highly expressed in human CD34+ HSPCs and downregulated in differentiated subsets; whereas, loss of Cdh1 restricts myeloid differentiation, supports B cell development and preserves immature short-term HSPCs without affecting proliferation or viability. Our data highlight a role of Cdh1 as a regulator of balancing the maintenance of HSPCs and differentiation into mature blood cells.
Assuntos
Proteínas Cdh1/metabolismo , Diferenciação Celular/genética , Células-Tronco Hematopoéticas/citologia , Animais , Antígenos CD34/metabolismo , Células da Medula Óssea/citologia , Células da Medula Óssea/metabolismo , Proteínas Cdh1/antagonistas & inibidores , Proteínas Cdh1/genética , Proliferação de Células , Células-Tronco Hematopoéticas/metabolismo , Humanos , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Proto-Oncogênicas c-kit/deficiência , Proteínas Proto-Oncogênicas c-kit/genética , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Células-Tronco/citologia , Células-Tronco/metabolismoRESUMO
The E2F1 transcription factor enhances apoptosis by DNA damage in tumors lacking p53. To elucidate the mechanism of a potential cooperation between E2F1 and chemotherapy, whole-genome microarrays of chemoresistant tumor cell lines were performed focusing on the identification of cooperation response genes (CRG). This gene class is defined by a synergistic expression response upon endogenous E2F1 activation and drug treatment. Cluster analysis revealed an expression pattern of CRGs similar to E2F1 mono-therapy, suggesting that chemotherapeutics enhance E2F1-dependent gene expression at the transcriptional level. Using this approach as a tool to explore E2F1-driven gene expression in response to anticancer drugs, we identified novel apoptosis genes such as the tumor suppressor TIEG1/KLF10 as direct E2F1 targets. We show that TIEG1/KLF10 is transcriptionally activated by E2F1 and crucial for E2F1-mediated chemosensitization of cancer cells. Our results provide a broader picture of E2F1-regulated genes in conjunction with cytotoxic treatment that allows the design of more rational therapeutics.
Assuntos
Antineoplásicos/farmacologia , Fator de Transcrição E2F1/metabolismo , Fatores de Transcrição de Resposta de Crescimento Precoce/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Fatores de Transcrição Kruppel-Like/genética , Neoplasias/genética , Neoplasias/patologia , Morte Celular/efeitos dos fármacos , Análise por Conglomerados , Perfilação da Expressão Gênica , Humanos , Análise de Sequência com Séries de Oligonucleotídeos , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Células Tumorais CultivadasRESUMO
Targeting mitosis by taxanes is one of the most common chemotherapeutic approaches in various malignant solid tumors, but cancer cells may survive antimitotic treatment with attainable in vivo concentrations due to mitotic slippage with a residual activity of the ubiquitin ligase anaphase-promoting complex (APC/C) and a continuous slow ubiquitin-proteasome-dependent cyclin B-degradation leading to mitotic exit. Therefore, blocking cyclin B-proteolysis via additional proteasome (PI) or APC/C-inhibition may have the potential to enhance tumor cell eradication by inducing a more robust mitotic block and mitotic cell death. Here, we analyzed this approach in different cell lines and more physiological patient-derived xenografts (PDX) from lung and breast cancer. The sequential combination of paclitaxel with the PI bortezomib enhanced cell death, but in contrast to the hypothesis during interphase and not in mitosis in both lung and breast cancer. APC/C-inhibition alone or in sequential combination with paclitaxel led to strong mitotic cell death in lung cancer. But in breast cancer, with high expression of the anti-apoptotic regulator Mcl-1, cell death in interphase was induced. Here, combined APC/C- and Mcl-1-inhibition with or without paclitaxel was highly lethal but still resulted in interphase cell death. Taken together, the combination of antimitotic agents with a clinically approved PI or inhibitors of the APC/C and Mcl-1 is a promising approach to improve treatment response in different solid tumors, even though they act entity-dependent at different cell cycle phases.
RESUMO
The E3 ubiquitin ligase and tumor suppressor APC/CCdh1 is crucial for cell cycle progression, development and differentiation in many cell types. However, little is known about the role of Cdh1 in hematopoiesis. Here we analyzed Cdh1 expression and function in malignant hematopoiesis. We found a significant decrease of Cdh1 in primary acute myeloid leukemia (AML) blasts compared to normal CD34+ cells. Thus, according to its important role in connecting cell cycle exit and differentiation, decreased expression of Cdh1 may be a mechanism contributing to the differentiation block in leukemogenesis. Indeed, knockdown (kd) of Cdh1 in HL-60 cell line (AML with maturation, FAB M2) led to less differentiated cells and a delay in PMA-induced differentiation. Acute promyelocytic leukemia (APL, FAB M3) is an AML subtype which is highly vulnerable to differentiation therapy with all-trans retinoic acid (ATRA). Accordingly, we found that APL is resistant to a Cdh1-kd mediated differentiation block. However, further depletion of Cdh1 in APL significantly reduced viability of leukemia cells upon ATRA-induced differentiation. Thus, low Cdh1 expression may be important in AML biology by contributing to the differentiation block and response to therapy depending on differences in the microenvironment and the additional genetic background.
Assuntos
Caderinas/biossíntese , Leucemia Mieloide Aguda/metabolismo , Adulto , Idoso , Idoso de 80 Anos ou mais , Antígenos CD , Caderinas/antagonistas & inibidores , Caderinas/genética , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Feminino , Técnicas de Silenciamento de Genes , Células HL-60 , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/patologia , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/metabolismo , Leucemia Promielocítica Aguda/patologia , Masculino , Pessoa de Meia-Idade , Acetato de Tetradecanoilforbol/farmacologia , Tretinoína/farmacologia , Adulto JovemRESUMO
INTRODUCTION: Preclinical models, by definition, are an approximation of reality, and their use in developing anticancer drugs is eagerly explored. Positive clinical correlations have been identified with different test systems, in addition to limitations and a need to improve preclinical model systems. Predicting whether a potential new anticancer agent will have a positive therapeutic index in patients remains a challenge. AREAS COVERED: We here review the fundamental requirements and remarkable progress of preclinical in vitro and in vivo assays used to assess the therapeutic potential of experimental anticancer drugs in multiple myeloma (MM). In MM, the interaction with the bone marrow microenvironment (BMM) plays a crucial role in disease progression, including resistance to antimyeloma agents. In vitro and in vivo approaches are, therefore, discussed with respect to their ability to mimic the important characteristics of MM and its BMM. In general, MM models should parallel the biological, genetic, etiological, immunological and therapeutic properties of the human disease. EXPERT OPINION: All models discussed here have their defined strengths, but also limitations with respect to their predictive features. Understanding the preclinical models in a more profound way should lead to optimized clinical trials, thereby expanding the therapeutic arsenal and improving patient outcome further.
Assuntos
Antineoplásicos/uso terapêutico , Modelos Animais de Doenças , Avaliação Pré-Clínica de Medicamentos , Mieloma Múltiplo/tratamento farmacológico , Animais , HumanosRESUMO
Beta-catenin plays an important role in embryogenesis and carcinogenesis by controlling either cadherin-mediated cell adhesion or transcriptional activation of target gene expression. In many types of cancers nuclear translocation of beta-catenin has been observed. Our data indicate that during melanoma progression an increased dependency on the transcriptional function of beta-catenin takes place. Blockade of beta-catenin in metastatic melanoma cell lines efficiently induces apoptosis, inhibits proliferation, migration and invasion in monolayer and 3-dimensional skin reconstructs and decreases chemoresistance. In addition, subcutaneous melanoma growth in SCID mice was almost completely inhibited by an inducible beta-catenin knockdown. In contrast, the survival of benign melanocytes and primary melanoma cell lines was less affected by beta-catenin depletion. However, enhanced expression of beta-catenin in primary melanoma cell lines increased invasive capacity in vitro and tumor growth in the SCID mouse model. These data suggest that beta-catenin is an essential survival factor for metastatic melanoma cells, whereas it is dispensable for the survival of benign melanocytes and primary, non-invasive melanoma cells. Furthermore, beta-catenin increases tumorigenicity of primary melanoma cell lines. The differential requirements for beta-catenin signaling in aggressive melanoma versus benign melanocytic cells make beta-catenin a possible new target in melanoma therapy.