RESUMEN
O-linked N-acetylglucosamine transferase (OGT) critically regulates wide variety of biological processes such as gene expression, metabolism, stress response, signaling and proteostasis. In adult hematopoiesis, OGT is crucial for differentiation of B and T cells and the maintenance of hematopoietic stem cells (HSCs). However, a role for OGT in fetal liver (FL) hematopoiesis remains unknown. To investigate a role for OGT in FL hematopoiesis, we conditionally disrupted OGT in hematopoietic cells in developing FLs. Hematopoietic specific disruption of OGT resulted in embryonic lethality in late stage of gestation due to severe anemia and growth retardation. OGT loss led to profound reduction of differentiating erythroid cells and erythroid progenitors in FLs due to massive apoptosis. In addition, clonogenic capacity of FL cells was severely impaired by OGT loss. Interestingly, expression of BCL-XL, a well-known inhibitor of apoptosis in FL cells, dramatically decreased, and the levels of reactive oxygen species (ROS) were increased in OGT-deficient FL cells. Overexpression of Bcl-xL and reduction of ROS significantly restored the colony formation of OGT-deficient FL cells. This study revealed a novel role for OGT during embryogenesis, which ensures survival of FL hematopoietic cells partly by regulating Bcl-xL and oxidative phosphorylation.
Asunto(s)
N-Acetilglucosaminiltransferasas , Fosforilación Oxidativa , Ratones , Animales , Especies Reactivas de Oxígeno/metabolismo , Diferenciación Celular , N-Acetilglucosaminiltransferasas/genética , Hígado/metabolismoRESUMEN
Chronic pain induced by nerve damage due to trauma or invasion of cancer to the bone elicits severe ongoing pain as well as hyperalgesia and allodynia likely reflecting adaptive changes within central circuits that amplify nociceptive signals. The present study explored the possible contribution of the mesolimbic dopaminergic circuit in promoting allodynia related to neuropathic and cancer pain. Mice with ligation of the sciatic nerve or treated with intrafemoral osteosarcoma cells showed allodynia to a thermal stimulus applied to the paw on the injured side. Patch clamp electrophysiology revealed that the intrinsic neuronal excitability of ventral tegmental area (VTA) dopamine neurons projecting to the nucleus accumbens (N.Acc.) was significantly reduced in those mice. We used tyrosine hydroxylase (TH)-cre mice that were microinjected with adeno-associated virus (AAV) to express channelrhodopsin-2 (ChR2) to allow optogenetic stimulation of VTA dopaminergic neurons in the VTA or in their N.Acc. terminals. Optogenetic activation of these cells produced a significant but transient anti-allodynic effect in nerve injured or tumor-bearing mice without increasing response thresholds to thermal stimulation in sham-operated animals. Suppressed activity of mesolimbic dopaminergic neurons is likely to contribute to decreased inhibition of N.Acc. output neurons and to neuropathic or cancer pain-induced allodynia suggesting strategies for modulation of pathological pain states.
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Neoplasias Óseas/complicaciones , Neuronas Dopaminérgicas/patología , Hiperalgesia/etiología , Hiperalgesia/patología , Nervio Ciático/lesiones , Área Tegmental Ventral/patología , Animales , Neoplasias Óseas/fisiopatología , Dolor en Cáncer/etiología , Dolor en Cáncer/patología , Dolor en Cáncer/fisiopatología , Línea Celular Tumoral , Neuronas Dopaminérgicas/metabolismo , Hiperalgesia/fisiopatología , Ligadura , Masculino , Ratones Endogámicos C57BL , Neuralgia/patología , Núcleo Accumbens/patología , Núcleo Accumbens/fisiopatología , Nervio Ciático/patología , Nervio Ciático/fisiopatología , Área Tegmental Ventral/fisiopatologíaRESUMEN
Osteosarcoma is the most common type of primary bone tumor, and novel therapeutic approaches for this disease are urgently required. To identify effective agents, we screened a panel of Food and Drug Administration (FDA)-approved drugs in AXT cells, our newly established mouse osteosarcoma line, and identified calcitriol as a candidate compound with therapeutic efficacy for this disease. Calcitriol inhibited cell proliferation in AXT cells by blocking cell cycle progression. From a mechanistic standpoint, calcitriol induced endoplasmic reticulum (ER) stress, which was potentially responsible for downregulation of cyclin D1, activation of p38 MAPK, and intracellular production of reactive oxygen species (ROS). Knockdown of Atf4 or Ddit3 restored cell viability after calcitriol treatment, indicating that the ER stress response was indeed responsible for the anti-proliferative effect in AXT cells. Notably, the ER stress response was induced to a lesser extent in human osteosarcoma than in AXT cells, consistent with the weaker suppressive effect on cell growth in the human cells. Thus, the magnitude of ER stress induced by calcitriol might be an index of its anti-osteosarcoma effect. Although mice treated with calcitriol exhibited weight loss and elevated serum calcium levels, a single dose was sufficient to decrease osteosarcoma tumor size in vivo. Our findings suggest that calcitriol holds therapeutic potential for treatment of osteosarcoma, assuming that techniques to diminish its toxicity could be established. In addition, our results show that calcitriol could still be safely administered to osteosarcoma patients for its original purposes, including treatment of osteoporosis.
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Antineoplásicos/administración & dosificación , Neoplasias Óseas/tratamiento farmacológico , Calcitriol/administración & dosificación , Retículo Endoplásmico/metabolismo , Osteosarcoma/tratamiento farmacológico , Animales , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Retículo Endoplásmico/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Femenino , Humanos , Inyecciones Intraperitoneales , Ratones Endogámicos C57BL , Osteosarcoma/metabolismo , Especies Reactivas de Oxígeno/metabolismo , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
New therapeutic approaches are needed for osteosarcoma, which is the most common malignancy of the bone, especially for metastatic cases. Nintedanib is a potent, oral tyrosine kinase inhibitor approved for treating idiopathic pulmonary fibrosis, which blocks a variety of receptor signals, including fibroblast growth factor receptors, vascular endothelial growth factor receptors and platelet-derived growth factor receptors. The present study assessed the effect of nintedanib on previously developed mouse AXT osteosarcoma cells, and on AXT-derived osteosarcoma developed in C57BL/6 mice, which displays lethal tumors with osteoid formation and lung metastatic lesions that mimics human disease. In vitro analysis, including flow cytometry and immunoblotting, revealed that nintedanib inhibited AXT cell proliferation and cell cycle progression, induced apoptosis, and inactivated AKT and ERK1/2. Immunoblot analysis using tumor lysates demonstrated that nintedanib inhibited its target molecules in vivo. As a single agent, nintedanib decreased the size of primary AXT-derived osteosarcoma, and reduced circulating tumor cells and lung metastasis. Immunohistochemical findings indicated that nintedanib exerted antitumor activity mainly by inhibiting the formation of CD31-positive tumor vasculature, while αSMA-positive cells were still enriched in tumors after nintedanib treatment. In addition, nintedanib exhibited an anti-osteosarcoma effect on C57BL/6 severe combined immunodeficient mice in which T- and B-cell function is obsolete, suggesting that the antitumor effect of nintedanib was not attributable to antitumor immunity. Collectively, these findings indicated that nintedanib holds potential for treating osteosarcoma.
RESUMEN
Two types of mutations of a transcription factor CCAAT-enhancer binding protein α (C/EBPα) are found in leukemic cells of 5%-14% of acute myeloid leukemia (AML) patients: N-terminal mutations expressing dominant negative p30 and C-terminal mutations in the basic leucine zipper domain. Our results showed that a mutation of C/EBPα in one allele was observed in AML after myelodysplastic syndrome, while the 2 alleles are mutated in de novo AML. Unlike an N-terminal frame-shift mutant (C/EBPα-N(m))-transduced cells, a C-terminal mutant (C/EBPα-C(m))-transduced cells alone induced AML with leukopenia in mice 4-12 months after bone marrow transplantation. Coexpression of both mutants induced AML with marked leukocytosis with shorter latencies. Interestingly, C/EBPα-C(m) collaborated with an Flt3-activating mutant Flt3-ITD in inducing AML. Moreover, C/EBPα-C(m) strongly blocked myeloid differentiation of 32Dcl3 cells, suggesting its class II mutation-like role in leukemogenesis. Although C/EBPα-C(m) failed to inhibit transcriptional activity of wild-type C/EBPα, it suppressed the synergistic effect between C/EBPα and PU.1. On the other hand, C/EBPα-N(m) inhibited C/EBPα activation in the absence of PU.1, despite low expression levels of p30 protein generated by C/EBPα-N(m). Thus, 2 types of C/EBPα mutations are implicated in leukemo-genesis, involving different and cooperating molecular mechanisms.
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Proteína alfa Potenciadora de Unión a CCAAT/genética , Modelos Animales de Enfermedad , Leucemia Mieloide Aguda/etiología , Leucemia Mieloide Aguda/patología , Mutación/genética , Síndromes Mielodisplásicos/etiología , Síndromes Mielodisplásicos/patología , Adulto , Anciano , Anciano de 80 o más Años , Animales , Apoptosis , Western Blotting , Trasplante de Médula Ósea , Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Diferenciación Celular , Proliferación Celular , Ensayo de Cambio de Movilidad Electroforética , Femenino , Citometría de Flujo , Humanos , Leucemia Mieloide Aguda/terapia , Luciferasas/metabolismo , Masculino , Ratones , Persona de Mediana Edad , Síndromes Mielodisplásicos/terapia , Reacción en Cadena de la Polimerasa , Polimorfismo Conformacional Retorcido-Simple , ARN Mensajero/genética , Tasa de Supervivencia , Activación TranscripcionalRESUMEN
Regulating transition of hematopoietic stem cells (HSCs) between quiescent and cycling states is critical for maintaining homeostasis of blood cell production. The cycling states of HSCs are regulated by the extracellular factors such as cytokines and extracellular matrix; however, the molecular circuitry for such regulation remains elusive. Here we show that tissue inhibitor of metalloproteinase-3 (TIMP-3), an endogenous regulator of metalloproteinases, stimulates HSC proliferation by recruiting quiescent HSCs into the cell cycle. Myelosuppression induced TIMP-3 in the bone marrow before hematopoietic recovery. Interestingly, TIMP-3 enhanced proliferation of HSCs and promoted expansion of multipotent progenitors, which was achieved by stimulating cell-cycle entry of quiescent HSCs without compensating their long-term repopulating activity. Surprisingly, this effect did not require metalloproteinase inhibitory activity of TIMP-3 and was possibly mediated through a direct inhibition of angiopoietin-1 signaling, a critical mediator for HSC quiescence. Furthermore, bone marrow recovery from myelosuppression was accelerated by over-expression of TIMP-3, and in turn, impaired in TIMP-3-deficient animals. These results suggest that TIMP-3 may act as a molecular cue in response to myelosuppression for recruiting dormant HSCs into active cell cycle and may be clinically useful for facilitating hematopoietic recovery after chemotherapy or ex vivo expansion of HSCs.
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Ciclo Celular , Células Madre Hematopoyéticas/citología , Inhibidor Tisular de Metaloproteinasa-3/metabolismo , Angiopoyetina 1/metabolismo , Animales , Médula Ósea/metabolismo , Médula Ósea/patología , Línea Celular , Proliferación Celular , Eliminación de Gen , Hematopoyesis , Células Madre Hematopoyéticas/metabolismo , Leucopenia/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Inhibidor Tisular de Metaloproteinasa-3/genética , Regulación hacia ArribaRESUMEN
Novel therapeutic targets are needed to better treat osteosarcoma, which is the most common bone malignancy. We previously developed mouse osteosarcoma cells, designated AX (accelerated bone formation) cells from bone marrow stromal cells. AX cells harbor both wild-type and mutant forms of p53 (R270C in the DNA-binding domain, which is equivalent to human R273C). In this study, we showed that mutant p53 did not suppress the transcriptional activation function of wild-type p53 in AX cells. Notably, AXT cells, which are cells derived from tumors originating from AX cells, lost wild-type p53 expression, were devoid of the intact transcription activation function, and were resistant to doxorubicin. ChIP-seq analyses revealed that this mutant form of p53 bound to chromatin in the vicinity of the transcription start sites of various genes but exhibited a different binding profile from wild-type p53. The knockout of mutant p53 in AX and AXT cells by CRISPR-Cas9 attenuated tumor growth but did not affect the invasion of these cells. In addition, depletion of mutant p53 did not prevent metastasis in vivo. Therefore, the therapeutic potency targeting R270C (equivalent to human R273C) mutant p53 is limited in osteosarcoma. However, considering the heterogeneous nature of osteosarcoma, it is important to further evaluate the biological and clinical significance of mutant p53 in various cases.
Asunto(s)
Neoplasias Óseas , Osteosarcoma , Ratones , Animales , Humanos , Proteína p53 Supresora de Tumor/genética , Proteína p53 Supresora de Tumor/metabolismo , Línea Celular Tumoral , Osteosarcoma/metabolismo , Procesos Neoplásicos , Neoplasias Óseas/metabolismoAsunto(s)
Proteínas de Unión al ADN/genética , Mutación , Células Progenitoras Mieloides/metabolismo , Proteínas Proto-Oncogénicas/genética , Animales , Transformación Celular Neoplásica/genética , Transformación Celular Neoplásica/metabolismo , Proteínas de Unión al ADN/metabolismo , Dioxigenasas , Inmunofenotipificación , Ratones , Ratones Noqueados , Fenotipo , Proteínas Proto-Oncogénicas/metabolismoRESUMEN
STAT5 is a critical mediator of a variety of cytokine signaling whose transcriptional activity is regulated by associating with various proteins. During a search for STAT5-interacting proteins, we identified SHD1, a mammalian homologue of yeast gene Sac3, as a potential interacter. SHD1 was localized in the nucleus, and induced by cytokines that activate STAT5, such as erythropoietin, interleukin-2 (IL-2), or IL-3. SHD1 interacted specifically with STAT5A and STAT5B, and interestingly, it specifically repressed STAT5-dependent transcription in vitro without affecting the stability or phosphorylation of STAT5 protein. Gene disruption study revealed that T, B, or bone marrow cells from mice lacking SHD1 were hyperresponsive to T-cell-receptor engagement, or stimulation with various STAT5-activating cytokines. These results suggest that SHD1 is a novel cytokine-inducible negative feedback regulator of STAT5.
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Linfocitos B/metabolismo , Células de la Médula Ósea/metabolismo , Proteínas Represoras/metabolismo , Factor de Transcripción STAT5/metabolismo , Linfocitos T/metabolismo , Transcripción Genética/fisiología , Proteínas Supresoras de Tumor/metabolismo , Animales , Factor 1 de Ensamblaje de la Cromatina , Citocinas/farmacología , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Células HeLa , Humanos , Ratones , Ratones Mutantes , Fosforilación/efectos de los fármacos , Fosforilación/fisiología , Proteínas Represoras/genética , Factor de Transcripción STAT5/genética , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transcripción Genética/efectos de los fármacos , Proteínas Supresoras de Tumor/genéticaRESUMEN
Osteosarcoma is the most common high-grade malignancy of bone, and novel therapeutic options are urgently required. Previously, we developed mouse osteosarcoma AXT cells that can proliferate both under adherent and nonadherent conditions. Based on metabolite levels, nonadherent conditions were more similar to the in vivo environment than adherent conditions. A drug screen identified MEK inhibitors, including trametinib, that preferentially decreased the viability of nonadherent AXT cells. Trametinib inhibited the cell cycle and induced apoptosis in AXT cells, and both effects were stronger under nonadherent conditions. Trametinib also potently decreased viability in U2OS cells, but its effects were less prominent in MG63 or Saos2 cells. By contrast, MG63 and Saos2 cells were more sensitive to PI3K inhibition than AXT or U2OS cells. Notably, the combination of MAPK/ERK kinase (MEK) and PI3K inhibition synergistically decreased viability in U2OS and AXT cells, but this effect was less pronounced in MG63 or Saos2 cells. Therefore, signal dependence for cell survival and crosstalk between MEK-ERK and PI3K-AKT pathways in osteosarcoma are cell context-dependent. The activation status of other kinases including CREB varied in a cell context-dependent manner, which might determine the response to MEK inhibition. A single dose of trametinib was sufficient to decrease the size of the primary tumor and circulating tumor cells in vivo. Moreover, combined administration of trametinib and rapamycin or conventional anticancer drugs further increased antitumor activity. Thus, given optimal biomarkers for predicting its effects, trametinib holds therapeutic potential for the treatment of osteosarcoma.
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Neoplasias Óseas , Osteosarcoma , Animales , Apoptosis , Neoplasias Óseas/metabolismo , Línea Celular Tumoral , Proliferación Celular , Ratones , Quinasas de Proteína Quinasa Activadas por Mitógenos/metabolismo , Quinasas de Proteína Quinasa Activadas por Mitógenos/farmacología , Osteosarcoma/tratamiento farmacológico , Osteosarcoma/metabolismo , Fosfatidilinositol 3-QuinasasRESUMEN
O-linked N-acetylglucosamine (O-GlcNAc) transferase (OGT) is a unique enzyme introducing O-GlcNAc moiety on target proteins, and it critically regulates various cellular processes in diverse cell types. However, its roles in hematopoietic stem and progenitor cells (HSPCs) remain elusive. Here, using Ogt conditional knockout mice, we show that OGT is essential for HSPCs. Ogt is highly expressed in HSPCs, and its disruption induces rapid loss of HSPCs with increased reactive oxygen species and apoptosis. In particular, Ogt-deficient hematopoietic stem cells (HSCs) lose quiescence, cannot be maintained in vivo, and become vulnerable to regenerative and competitive stress. Interestingly, Ogt-deficient HSCs accumulate defective mitochondria due to impaired mitophagy with decreased key mitophagy regulator, Pink1, through dysregulation of H3K4me3. Furthermore, overexpression of PINK1 restores mitophagy and the number of Ogt-deficient HSCs. Collectively, our results reveal that OGT critically regulates maintenance and stress response of HSCs by ensuring mitochondrial quality through PINK1-dependent mitophagy.
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Células Madre Hematopoyéticas/metabolismo , Histonas/metabolismo , Mitocondrias/metabolismo , Mitofagia , N-Acetilglucosaminiltransferasas/metabolismo , Proteínas Quinasas/metabolismo , Acetilglucosamina/metabolismo , Animales , Apoptosis , Ciclo Celular , Línea Celular , Femenino , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , N-Acetilglucosaminiltransferasas/genética , Especies Reactivas de Oxígeno/metabolismo , Estrés FisiológicoRESUMEN
Roundabout (Robo) family proteins are immunoglobulin-type cell surface receptors that are expressed predominantly in the nervous system. The fourth member of this family, Robo4, is distinct from the other family members in that it is expressed specifically in endothelial cells. In this study, we examined the expression of Robo4 in hematopoietic stem cells (HSCs) and its possible role in HSC regulation. Robo4 mRNA was specifically expressed in murine HSCs and the immature progenitor cell fraction but not in lineage-positive cells or differentiated progenitors. Moreover, flow cytometry showed a correlation between higher expression of Robo4 and immature phenotypes of hematopoietic cells. Robo4(high) hematopoietic stem/progenitor cells presented higher clonogenic activity or long-term repopulating activity by colony assays or transplantation assays, respectively. A ligand for Robo4, Slit2, is specifically expressed in bone marrow stromal cells, and its expression was induced in osteoblasts in response to myelosuppressive stress. Interestingly, overexpression of Robo4 or Slit2 in HSCs resulted in their decreased residence in the c-Kit(+)Sca-1(+)Lineage(-)-side population fraction. These results indicate that Robo4 is expressed in HSCs, and Robo4/Slit2 signaling may play a role in HSC homeostasis in the bone marrow niche.
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Células Madre Hematopoyéticas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Receptores Inmunológicos/metabolismo , Nicho de Células Madre/metabolismo , Animales , Células de la Médula Ósea/citología , Células de la Médula Ósea/metabolismo , Diferenciación Celular , Ensayo de Unidades Formadoras de Colonias , Citometría de Flujo , Células Madre Hematopoyéticas/citología , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Ratones , Células Madre Multipotentes/citología , Células Madre Multipotentes/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Fenotipo , Receptores de Superficie Celular , Transducción de Señal , Estrés Fisiológico , Células del Estroma/citología , Células del Estroma/metabolismoRESUMEN
Acute myeloid leukemia (AML) or myelodysplastic syndromes (MDS) with both inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and monosomy 7 defines an extremely aggressive myeloid cancer whose molecular pathogenesis and optimal therapeutic strategy still remain unclear. We established a new MDS/AML cell line, YCU-AML1, and its patient-derived xenograft (PDX) model from a high-risk MDS patient who later transformed into AML harboring both t(3;3)(q21;q26.2) and monosomy 7. YCU-AML1 cells propagated in co-culture system with stromal cells in granulocyte macrophage colony-stimulating factor (GM-CSF)-dependent manner. CD34+ bone marrow cells derived from our PDX model showed high EVI1 and low GATA2 expression. Moreover, mutational profile of our MDS/AML model was consistent with recently published mutational spectrum of myeloid malignancies with inv(3)/t(3;3). These data suggest that YCU-AML1 cells and its MDS/AML model strongly mimics a high-risk human myeloid cancer with inv(3)(q21q26.2)/t(3;3)(q21;q26.2) and monosomy 7 in terms of both clinical phenotype and molecular basis. We believe our model can be used as a feasible tool to further explore molecular pathogenesis and novel treatment strategy of high-risk MDS/AML with t(3;3)(q21;q26.2) and monosomy 7.
RESUMEN
Wnt signaling has been implicated in the self-renewal of hematopoietic stem cells (HSCs). Secreted frizzled-related proteins (SFRPs) are a family of soluble proteins containing a region homologous to a receptor for Wnt, Frizzled, and are thought to act as endogenous modulators for Wnt signaling. This study examined the role of SFRPs in HSC regulation. Among the four family members, SFRP-1 and SFRP-2 are specifically induced in the bone marrow in response to myelosuppression, and immunostaining revealed that both proteins were expressed in osteoblasts. Interestingly, SFRP-1 reduced the number of multipotent progenitors in in vitro culture of CD34(-)KSL cells, while SFRP-2 did not. Furthermore, SFRP-1 compromised the long-term repopulating activity of HSCs, whereas SFRP-2 did not affect or even enhanced it in the same setting. These results indicate that although both SFRP-1 and SFRP-2 act as inhibitors for Wnt signaling in vitro, they differentially affect the homeostasis of HSCs.
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Células Madre Hematopoyéticas/fisiología , Péptidos y Proteínas de Señalización Intercelular/biosíntesis , Proteínas de la Membrana/biosíntesis , Osteoblastos/metabolismo , Proteínas Wnt/metabolismo , Animales , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Fluorouracilo/farmacología , Células Madre Hematopoyéticas/citología , Células Madre Hematopoyéticas/efectos de los fármacos , Homeostasis , Humanos , Ratones , Ratones Endogámicos C57BL , Proteína Wnt3RESUMEN
Previous studies using loss-of-function mutants revealed that CCAAT/enhancer-binding protein alpha (C/EBPalpha) and PU.1 are potential regulators for hematopoietic stem cells (HSCs). To gain further insight into the HSC regulation by C/EBPalpha or PU.1, we used transgenic mice expressing conditional forms of these transcription factors to examine whether their activation alone is sufficient for modulating HSC functions. The activation of C/EBPalpha or PU.1 in HSCs in vitro or in vivo led to their suppression of growth, decreased mixed colony formation, and impaired competitive repopulating activities because of their defective self-renewal. These effects were more prominently observed when C/EBPalpha was activated, and the differentiation capacity to megakaryocytic lineage was selectively impaired upon C/EBPalpha activation. Unexpectedly, the expression of Bmi-1 and HoxB4, well-known regulators for self-renewal of HSCs, was not affected by the activation of C/EBPalpha or PU.1, suggesting that they regulate HSC function through an as yet unknown mechanism. Our data suggest that the activation of C/EBPalpha or PU.1 is sufficient to repress stem cell capacities in HSCs, and their fine-tuned regulation is critical for HSC homeostasis.
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Proteína alfa Potenciadora de Unión a CCAAT/metabolismo , Células Madre Hematopoyéticas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Transactivadores/metabolismo , Animales , Apoptosis , Células de la Médula Ósea/citología , Diferenciación Celular , Linaje de la Célula , Proliferación Celular , Hematopoyesis , Proteínas de Homeodominio/metabolismo , Ratones , Ratones Transgénicos , Proteínas Nucleares/metabolismo , Complejo Represivo Polycomb 1 , Proteínas Represoras/metabolismo , Factores de Transcripción/metabolismo , TransgenesRESUMEN
Osteosarcoma is the most common type of primary bone tumor, novel therapeutic agents for which are urgently needed. To identify such agents, we screened a panel of approved drugs with a mouse model of osteosarcoma. The screen identified simvastatin, which inhibited the proliferation and migration of osteosarcoma cells in vitro Simvastatin also induced apoptosis in osteosarcoma cells in a manner dependent on inhibition of the mevalonate biosynthetic pathway. It also disrupted the function of the small GTPase RhoA and induced activation of AMP-activated protein kinase (AMPK) and p38 MAPK, with AMPK functioning upstream of p38 MAPK. Inhibitors of AMPK or p38 MAPK attenuated the induction of apoptosis by simvastatin, whereas metformin enhanced this effect of simvastatin by further activation of AMPK. Although treatment with simvastatin alone did not inhibit osteosarcoma tumor growth in vivo, its combination with a fat-free diet induced a significant antitumor effect that was enhanced further by metformin administration. Our findings suggest that simvastatin induces apoptosis in osteosarcoma cells via activation of AMPK and p38 MAPK, and that, in combination with other approaches, it holds therapeutic potential for osteosarcoma. Mol Cancer Ther; 16(1); 182-92. ©2016 AACR.
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Apoptosis/efectos de los fármacos , Neoplasias Óseas/metabolismo , Osteosarcoma/metabolismo , Transducción de Señal/efectos de los fármacos , Simvastatina/farmacología , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Neoplasias Óseas/genética , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Regulación Neoplásica de la Expresión Génica , Humanos , Metformina/farmacología , Ratones , Osteosarcoma/genética , Ensayos Antitumor por Modelo de Xenoinjerto , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Proteína de Unión al GTP rhoA/genética , Proteína de Unión al GTP rhoA/metabolismoRESUMEN
p27KIP1 is known as a regulator of cellular differentiation and apoptosis in human cancer cells. We have previously reported that human chronic myeloid leukemia (CML) KU812 and K562 cells show inhibited cellular proliferation in response to treatment with activin A, a member of TGF-beta superfamily. Apoptosis and erythroid differentiation can be induced in KU812 and K562 cells, respectively. We report herein that activin A induced the expression of p27KIP1 in CML cells along with the induction of cellular differentiation and apoptosis. There are putative binding sequences of erythroid-related transcription factor GATA-1 in the promoter region of the human p27KIP1 gene. Expression of GATA-1 protein in activin A-treated KU812 and K562 cells showed dissimilar regulation in these two cell lines. Induction of p27KIP1 was commonly observed, but it did not correspond to the expression levels of GATA-1 in either line of activin A-treated CML cells. In addition, ERK protein was rapidly and transiently activated with activin A in both types of CML cells, suggesting that phosphorylation of ERK is required for activin A signaling in CML cells. These results indicate that both p27KIP1 induction and regulation of GATA-1 play essential roles in activin A-induced erythroid differentiation and apoptosis.
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Activinas/fisiología , Apoptosis/fisiología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/fisiología , Factor de Transcripción GATA1/fisiología , Leucemia Mielógena Crónica BCR-ABL Positiva/patología , Activinas/genética , Activinas/metabolismo , Apoptosis/genética , Proteínas de Ciclo Celular/biosíntesis , Proteínas de Ciclo Celular/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/genética , Inhibidor p27 de las Quinasas Dependientes de la Ciclina/metabolismo , Factor de Transcripción GATA1/biosíntesis , Factor de Transcripción GATA1/genética , Humanos , Leucemia Mielógena Crónica BCR-ABL Positiva/genética , Leucemia Mielógena Crónica BCR-ABL Positiva/metabolismoRESUMEN
The fundamental mechanism how heterogeneous hepatic macrophage (Mφ) subsets fulfill diverse functions in health and disease has not been elucidated. We recently reported that CCR9+ inflammatory Mφs play a critical role in the course of acute liver injury. To clarify the origin and differentiation of CCR9+Mφs, we used a unique partial bone marrow (BM) chimera model with liver shielding for maintaining hepatic resident Mφs. First, irradiated mice developed less liver injury with less Mφs accumulation by Concanavalin A (Con A) regardless of liver shielding. In mice receiving further BM transplantation, CD11blowF4/80high hepatic-resident Mφs were not replaced by transplanted donors under steady state, while under inflammatory state by Con A, CCR9+Mφs were firmly replaced by donors, indicating that CCR9+Mφs originate from BM, but not from hepatic-resident cells. Regarding the mechanism of differentiation and proliferation, EdU+CCR9+Mφs with a proliferative potential were detected specifically in the inflamed liver, and in vitro study revealed that BM-derived CD11b+ cells co-cultured with hepatic stellate cells (HSCs) or stimulated with retinoic acids could acquire CCR9 with antigen-presenting ability. Collectively, our study demonstrates that inflammatory Mφs originate from BM and became locally differentiated and proliferated by interaction with HSCs via CCR9 axis during acute liver injury.
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Enfermedad Hepática Inducida por Sustancias y Drogas/patología , Concanavalina A/metabolismo , Factores Inmunológicos/metabolismo , Macrófagos/inmunología , Animales , Antígeno CD11b/análisis , Diferenciación Celular , Proliferación Celular , Concanavalina A/administración & dosificación , Factores Inmunológicos/administración & dosificación , Hígado/patología , Macrófagos/química , Macrófagos/clasificación , Ratones , Receptores CCR/análisisRESUMEN
A metal chelator, diphenylthiocarbazone (dithizone), has been reported to induce differentiation and apoptosis of the human myeloid leukemia cell line HL-60, however, very little is known about the mechanism of dithizone-induced apoptosis. Here, we report for the first time that dithizone can induce inhibition of cellular growth of retinoic acid (RA)-sensitive NB4 and RA-resistant UF-1 APL cells via induction of apoptosis but not differentiation. Treatment of NB4 cells with dithizone markedly-induced apoptosis, which was associated with the loss of mitochondrial transmembrane potentials (Delta Psi(m)) and activation of caspase-3 and -9. Further investigation of the RA-resistant UF-1 APL cells showed that dithizone-induced apoptosis to a lesser extent. However, neither dithizone alone nor in combination with all-trans RA induced the expression of myeloid differentiation antigen CD11b. Concomitantly, the degradation of PML/RARalpha fusion protein was not observed after treatment with dithizone alone, and the degradation was not enhanced by the combination of dithizone and all-trans RA. We conclude that dithizone, a metal chelator, induced apoptosis without differentiation in APL cells in association with Delta Psi(m) collapse and caspase-3 and -9 activation.
Asunto(s)
Apoptosis/efectos de los fármacos , Caspasas/fisiología , Quelantes/farmacología , Ditizona/farmacología , Leucemia Promielocítica Aguda/patología , Proteínas de Neoplasias/fisiología , Células Madre Neoplásicas/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Tretinoina/farmacología , Clorometilcetonas de Aminoácidos/farmacología , Caspasa 3 , Caspasa 9 , Inhibidores de Caspasas , Diferenciación Celular/efectos de los fármacos , Inhibidores de Cisteína Proteinasa/farmacología , Interacciones Farmacológicas , Resistencia a Antineoplásicos , Activación Enzimática/efectos de los fármacos , Membranas Intracelulares/efectos de los fármacos , Antígeno de Macrófago-1/biosíntesis , Potenciales de la Membrana/efectos de los fármacos , Mitocondrias/efectos de los fármacos , Proteínas de Neoplasias/antagonistas & inhibidores , Células Madre Neoplásicas/citología , Células Madre Neoplásicas/enzimología , Proteínas de Fusión Oncogénica/fisiología , Células Tumorales Cultivadas/citología , Células Tumorales Cultivadas/efectos de los fármacos , Células Tumorales Cultivadas/enzimologíaRESUMEN
Osteosarcoma is a malignant bone tumor in children and adolescents characterized by intrinsic therapeutic resistance. The IGF2 is expressed at elevated levels in osteosarcoma after treatment with chemotherapy, prompting an examination of its functional contributions to resistance. We found that continuous exposure to IGF2 or insulin in the absence of serum created a dormant growth state in osteosarcoma cells that conferred resistance to various chemotherapeutic drugs in vitro. Mechanistic investigations revealed that this dormant state correlated with downregulation of downstream signaling by the IGF1 receptor, heightened cell survival, enhanced autophagy, and the presence of extracellular glutamine. Notably, inhibiting autophagy or depleting glutamine was sufficient to increase chemotherapeutic sensitivity in osteosarcoma xenografts in mice. Clinically, we confirmed that IGF expression levels were elevated in human osteosarcoma specimens from patients who received chemotherapy. Together, our results suggest that activation of IGF or insulin signaling preserves the survival of osteosarcoma cells under chemotherapeutic stress, providing a drug-resistant population that may engender minimal residual disease. Attenuating this survival mechanism may help overcome therapeutic resistance in osteosarcoma.