RESUMEN
Metabolic pathways play important roles in proliferation and differentiation of malignant cells. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr), a precursor in purine biosynthesis and a well-established activator of AMP-activated protein kinase (AMPK), induces widespread metabolic alterations and is commonly used for dissecting the role of metabolism in cancer. We have previously reported that AICAr promotes differentiation and inhibits proliferation of myeloid leukemia cells. Here, using metabolic assays, immunoblotting, flow cytometry analyses, and siRNA-mediated gene silencing in leukemia cell lines, we show that AICAr-mediated differentiation was independent of the known metabolic effects of AMPK, including glucose consumption, but instead depends on the activation of the DNA damage-associated enzyme checkpoint kinase 1 (Chk1) induced by pyrimidine depletion. LC/MS/MS metabolomics analysis revealed that AICAr increases orotate levels and decreases uridine monophosphate (UMP) levels, consistent with inhibition of UMP synthesis at a step downstream of dihydroorotate dehydrogenase (DHODH). AICAr and the DHODH inhibitor brequinar had similar effects on differentiation markers and S-phase arrest, and genetic or pharmacological Chk1 inactivation abrogated both of these effects. Our results delineate an AMPK-independent effect of AICAr on myeloid leukemia differentiation that involves perturbation of pyrimidine biosynthesis and activation of the DNA damage response network.
Asunto(s)
Diferenciación Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Leucemia Promielocítica Aguda/metabolismo , Pirimidinas/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Línea Celular Tumoral , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/genética , Dihidroorotato Deshidrogenasa , Humanos , Leucemia Promielocítica Aguda/genética , Leucemia Promielocítica Aguda/fisiopatología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-CH/metabolismo , Ribonucleósidos/genética , Ribonucleósidos/metabolismo , Puntos de Control de la Fase S del Ciclo CelularRESUMEN
BACKGROUND: All-trans retinoic acid (ATRA)-based treatment of acute promyelocytic leukemia (APL) is the most successful pharmacological treatment of acute myeloid leukemia (AML). Recent development of inhibitors of mutated isocitrate dehydrogenase and dihydroorotate dehydrogenase (DHODH) has revived interest in differentiation therapy of non-APL AML. Our previous studies demonstrated that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) induced differentiation of monocytic cell lines by activating the ATR/Chk1 via pyrimidine depletion. In the present study, the effects of AICAr on the viability and differentiation of primary AML blasts isolated from bone marrow of patients with non-APL AML were tested and compared with the effects of DHODH inhibitor brequinar and ATRA. METHODS: Bone marrow samples were obtained from 35 patients and leukemia blasts were cultured ex vivo. The cell viability was assessed by MTT assay and AML cell differentiation was determined by flow cytometry and morphological analyses. RNA sequencing and partial data analysis were conducted using ClusterProfiler package. Statistical analysis was performed using GraphPad Prism 6.0. RESULTS: AICAr is capable of triggering differentiation in samples of bone marrow blasts cultured ex vivo that were resistant to ATRA. AICAr-induced differentiation correlates with proliferation and sensitivity to DHODH inhibition. RNA-seq data obtained in primary AML blasts confirmed that AICAr treatment induced downregulation of pyrimidine metabolism pathways together with an upregulation of gene set involved in hematopoietic cell lineage. CONCLUSION: AICAr induces differentiation in a subset of primary non-APL AML blasts, and these effects correlate with sensitivity to a well-known, potent DHODH inhibitor.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Biomarcadores de Tumor/metabolismo , Crisis Blástica/tratamiento farmacológico , Médula Ósea/efectos de los fármacos , Diferenciación Celular , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Ribonucleósidos/farmacología , Aminoimidazol Carboxamida/farmacología , Biomarcadores de Tumor/genética , Crisis Blástica/genética , Crisis Blástica/metabolismo , Crisis Blástica/patología , Médula Ósea/metabolismo , Médula Ósea/patología , Estudios de Casos y Controles , Proliferación Celular , Humanos , Leucemia Mieloide Aguda/genética , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , RNA-Seq , Células Tumorales CultivadasRESUMEN
Several studies have demonstrated the activation of phosphoinositide-specific phospholipase C (Plc) in nuclei of mammalian cells during synchronous progression through the cell cycle, but the downstream targets of Plc-generated inositol 1,4,5-trisphosphate are poorly described. Phospholipid signaling in the budding yeast Saccharomyces cerevisiae shares similarities with endonuclear phospholipid signaling in mammals, and many recent studies point to a role for inositol phosphates, including InsP(5), InsP(6), and inositol pyrophosphates, in mediating the action of Plc. In this study, we investigated the changes in inositol phosphate levels in α-factor-treated S. cerevisiae, which allows cells to progress synchronously through the cell cycle after release from a G(1) block. We found an increase in the activity of Plc1 early after release from the block with a concomitant increase in the levels of InsP(7) and InsP(8). Treatment of cells with the Plc inhibitor U73122 prevented increases in inositol phosphate levels and blocked progression of cells through S phase after pheromone arrest. The enzymatic activity of Kcs1 in vitro and HPLC analysis of [(3)H]inositol-labeled kcs1Δ cells confirmed that Kcs1 is the principal kinase responsible for generation of pyrophosphates in synchronously progressing cells. Analysis of plc1Δ, kcs1Δ, and ddp1Δ yeast mutants further confirmed the role that a Plc1- and Kcs1-mediated increase in pyrophosphates may have in progression through S phase. Our data provide genetic, metabolic, and biochemical evidence that synthesis of inositol pyrophosphates through activation of Plc1 and Kcs1 plays an important role in the signaling response required for cell cycle progression after mating pheromone arrest.
Asunto(s)
Difosfatos/metabolismo , Fosfatos de Inositol/metabolismo , Péptidos/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato)/genética , Fase S/efectos de los fármacos , Proteínas de Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/genética , Fosfolipasas de Tipo C/genética , Núcleo Celular , Estrenos/farmacología , Fase G1/efectos de los fármacos , Fase G1/genética , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Factor de Apareamiento , Inhibidores de Fosfodiesterasa/farmacología , Fosfotransferasas (Aceptor del Grupo Fosfato)/metabolismo , Pirrolidinonas/farmacología , Fase S/genética , Saccharomyces cerevisiae/efectos de los fármacos , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/antagonistas & inhibidores , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal/efectos de los fármacos , Coloración y Etiquetado , Tritio , Fosfolipasas de Tipo C/antagonistas & inhibidores , Fosfolipasas de Tipo C/metabolismoRESUMEN
Low-dose cytarabine (LDAC) is a standard therapy for elderly acute myeloid leukemia (AML) patients unfit for intensive chemotherapy. While high doses of cytarabine induce cytotoxicity, the precise mechanism of action of LDAC in AML remains elusive. In vitro studies have demonstrated LDAC-induced differentiation; however, such differentiation is seldom observed in vivo. We hypothesize that this discrepancy may be attributed to the influence of bone marrow (BM) stromal cells on AML cells. Thus, this study aimed to investigate the impact of BM stromal cells on LDAC-induced differentiation of AML cell lines and primary samples. Our results demonstrate that the presence of MS-5 stromal cells prevented LDAC-induced cell cycle arrest, DNA damage signaling and differentiation of U937 and MOLM-13 cell lines. Although transcriptomic analysis revealed that the stroma reduces the expression of genes involved in cytokine signaling and oxidative stress, data obtained with pharmacological inhibitors and neutralizing antibodies did not support the role for CXCL12, TGF-ß1 or reactive oxygen species. The presence of stromal cells reduces LDAC-induced differentiation in primary samples from AML-M4 and myelodysplastic syndrome/AML patients. In conclusion, our study demonstrates that BM stroma reduces differentiation of AML induced by LDAC. These findings provide insights into the limited occurrence of terminal differentiation observed in AML patients, and suggest a potential explanation for this observation.
RESUMEN
Bendamustine is an alkylating agent classified into the group of nitrogen mustard analogues, synthesized almost sixty years ago. It was registered in former East Germany in 1971 and approved by the US Food and Drug Administration in 2008 for treatment of chronic lymphocytic leukemia and indolent Bcell nonHodgkin lymphoma. Considering its beneficial properties in the therapy of relapsed or refractory hematological malignancies, synergistic effects with other antineoplastic agents and increasing recent reports on its immunomodulatory effects, bendamustine has once again gained its justified attention. The uniqueness of bendamustinemediated effects should be observed keeping in mind its distinctive structure with structural similarities to both alkylating agents and purine analogs. In the present review, the current knowledge on the use of bendamustine in oncology, its pharmacokinetics, mechanism of action and toxicity was summarized. In addition, its immunemodulating effects that have not been fully elucidated so far are emphasized, hoping to encourage further investigations of this unique drug.
Asunto(s)
Antineoplásicos , Leucemia Linfocítica Crónica de Células B , Linfoma de Células B , Compuestos de Mostaza Nitrogenada , Antineoplásicos/farmacología , Clorhidrato de Bendamustina/farmacología , Clorhidrato de Bendamustina/uso terapéutico , Humanos , Leucemia Linfocítica Crónica de Células B/tratamiento farmacológico , Linfoma de Células B/tratamiento farmacológico , Compuestos de Mostaza Nitrogenada/química , Compuestos de Mostaza Nitrogenada/farmacología , Compuestos de Mostaza Nitrogenada/uso terapéuticoRESUMEN
Acute myeloid leukemia (AML) is characterized by arrested differentiation making differentiation therapy a promising treatment strategy. Recent success of inhibitors of mutated isocitrate dehydrogenase (IDH) invigorated interest in differentiation therapy of AML so that several new drugs have been proposed, including inhibitors of dihydroorotate dehydrogenase (DHODH), an enzyme in pyrimidine synthesis. Cytarabine, a backbone of standard AML therapy, is known to induce differentiation at low doses, but the mechanism is not completely elucidated. We have previously reported that 5-aminoimidazole-4-carboxamide ribonucleoside (AICAr) and brequinar, a DHODH inhibitor, induced differentiation of myeloid leukemia by activating the ataxia telangiectasia and Rad3-related (ATR)/checkpoint kinase 1 (Chk1) via pyrimidine depletion. In this study, using immunoblotting, flow cytometry analyses, pharmacologic inhibitors and genetic inactivation of Chk1 in myeloid leukemia cell lines, we show that low dose cytarabine induces differentiation by activating Chk1. In addition, cytarabine induces differentiation ex vivo in a subset of primary AML samples that are sensitive to AICAr and DHODH inhibitor. The results of our study suggest that leukemic cell differentiation stimulated by low doses of cytarabine depends on the activation of Chk1 and thus shares the same pathway as pyrimidine synthesis inhibitors.
Asunto(s)
Citarabina , Leucemia Mieloide Aguda , Diferenciación Celular , Quinasa 1 Reguladora del Ciclo Celular (Checkpoint 1)/metabolismo , Citarabina/farmacología , Citarabina/uso terapéutico , Dihidroorotato Deshidrogenasa , Inhibidores Enzimáticos/farmacología , Humanos , Leucemia Mieloide Aguda/genética , Pirimidinas/uso terapéuticoRESUMEN
All-trans retinoic acid (ATRA)-based therapy for acute promyelocytic leukemia (APL), a subtype of acute myeloid leukemia (AML), is the most successful example of differentiation therapy. Although ATRA can induce differentiation in some non-APL AML cell lines and primary blasts, clinical results of adding ATRA to standard therapy in non-APL AML patients have been inconsistent, probably due to use of different regimens and lack of diagnostic tools for identifying which patients may be sensitive to ATRA. In this study, we exposed primary blasts obtained from non-APL AML patients to ATRA to test for differentiation potential in vitro. We observed increased expression of differentiation markers, indicating a response to ATRA, in four out of fifteen primary AML samples. Three samples in which CD11b increased in response to ATRA had an inversion of chromosome 16 as well as the CBFB-MYH11 fusion gene, and the fourth sample was from a patient with KMT2A-rearranged, therapy-related AML. In conclusion, we identified a subgroup of non-APL AML patients with inv(16) and CBFB-MYH11 as the most sensitive to ATRA-mediated differentiation in vitro, and our results can help identify patients who may benefit from ATRA treatment.
Asunto(s)
Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Crisis Blástica/genética , Crisis Blástica/patología , Inversión Cromosómica/genética , Cromosomas Humanos Par 16/genética , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/genética , Tretinoina/farmacología , Tretinoina/uso terapéutico , Antígeno CD11b/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Subunidad beta del Factor de Unión al Sitio Principal/genética , Fusión Génica/efectos de los fármacos , Reordenamiento Génico/genética , N-Metiltransferasa de Histona-Lisina/genética , Humanos , Proteína de la Leucemia Mieloide-Linfoide/genética , Cadenas Pesadas de Miosina/genéticaRESUMEN
5-Aminoimidazole-4-carboxamide ribonucleoside (AICAr) has been one of the most commonly used pharmacological modulators of AMPK activity. The majority of early studies on the role of AMPK, both in the physiological regulation of metabolism and in cancer pathogenesis, were based solely on the use of AICAr as an AMPK-activator. Even with more complex models of AMPK downregulation and knockout being introduced, AICAr remained a regular starting point for many studies focusing on AMPK biology. However, there is an increasing number of studies showing that numerous AICAr effects, previously attributed to AMPK activation, are in fact AMPK-independent. This review aims to give an overview of the present knowledge on AMPK-dependent and AMPK-independent effects of AICAr on metabolism, hypoxia, exercise, nucleotide synthesis, and cancer, calling for caution in the interpretation of AICAr-based studies in the context of understanding AMPK signaling pathway.
Asunto(s)
Aminoimidazol Carboxamida/análogos & derivados , Hipoglucemiantes/farmacología , Proteínas Quinasas/metabolismo , Ribonucleótidos/farmacología , Quinasas de la Proteína-Quinasa Activada por el AMP , Aminoimidazol Carboxamida/farmacología , Animales , Carcinogénesis/efectos de los fármacos , Ciclo Celular/efectos de los fármacos , Metabolismo Energético/efectos de los fármacos , Humanos , Miocitos Cardíacos/efectos de los fármacosRESUMEN
Although the class II phosphoinositide 3-kinase enzymes PI3K-C2alpha and PI3K-C2beta act acutely downstream of cell surface receptors they have also been localized to nuclei in mammalian cells. As with the class I PI3K enzymes, the relationship between the pools of enzyme present in cytoplasm and nuclei remains poorly understood. In this study we test the hypothesis that PI3K-C2beta translocates to nuclei in response to growth factor stimulation. Fractionating homogenates of quiescent cells revealed that less than 5% of total PI3K-C2beta resides in nuclei. Stimulation with epidermal growth factor sequentially increased levels of this enzyme, firstly in the cytosol and secondly in the nuclei. Using detergent-treated nuclei, we showed that PI3K-C2beta co-localized with lamin A/C in the nuclear matrix. This was confirmed biochemically, and a phosphoinositide kinase assay showed a statistically significant increase in nuclear PI3K-C2beta levels and lipid kinase activity following epidermal growth factor stimulation. C-terminal deletion and point mutations of PI3K-C2beta demonstrated that epidermal growth factor-driven translocation to the nucleus is dependent on a sequence of basic amino acid residues (KxKxK) that form a nuclear localization motif within the C-terminal C2 domain. Furthermore, when this sequence was expressed as an EGFP (enhanced green fluorescent protein) fusion protein, it translocated fluorescence into nuclei with an efficiency dependent upon copy number. These data demonstrate that epidermal growth factor stimulates the appearance of PI3K-C2beta in nuclei. Further, this effect is dependent on a nuclear localization signal present within the C-terminal C2 domain, indicating its bimodal function regulating phospholipid binding and shuttling PI3K-C2beta into the nucleus.
Asunto(s)
Núcleo Celular/efectos de los fármacos , Núcleo Celular/enzimología , Factor de Crecimiento Epidérmico/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Secuencia de Aminoácidos , Línea Celular , Membrana Celular/efectos de los fármacos , Membrana Celular/enzimología , Fosfatidilinositol 3-Quinasas Clase II , Citosol/efectos de los fármacos , Citosol/enzimología , Proteínas Fluorescentes Verdes , Humanos , Laminas/metabolismo , Modelos Biológicos , Datos de Secuencia Molecular , Señales de Localización Nuclear/metabolismo , Matriz Nuclear/efectos de los fármacos , Matriz Nuclear/enzimología , Fosfatidilinositol 3-Quinasas/química , Transporte de Proteínas/efectos de los fármacosRESUMEN
Differentiation therapy of acute promyelocytic leukemia with all-trans retinoic acid represents the most successful pharmacological therapy of acute myeloid leukemia (AML). Numerous studies demonstrate that drugs that inhibit mechanistic target of rapamycin (mTOR) and activate AMP-kinase (AMPK) have beneficial effects in promoting differentiation and blocking proliferation of AML. Most of these drugs are already in use for other purposes; rapalogs as immunosuppressants, biguanides as oral antidiabetics, and 5-amino-4-imidazolecarboxamide ribonucleoside (AICAr, acadesine) as an exercise mimetic. Although most of these pharmacological modulators have been widely used for decades, their mechanism of action is only partially understood. In this review, we summarize the role of AMPK and mTOR in hematological malignancies and discuss the possible role of pharmacological modulators in proliferation and differentiation of leukemia cells.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Leucemia Mieloide Aguda/tratamiento farmacológico , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Quinasas Activadas por AMP/antagonistas & inhibidores , Proteínas Quinasas Activadas por AMP/química , Aminoimidazol Carboxamida/análogos & derivados , Aminoimidazol Carboxamida/metabolismo , Aminoimidazol Carboxamida/uso terapéutico , Ensayos Clínicos como Asunto , Humanos , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Metformina/metabolismo , Metformina/uso terapéutico , ARN Interferente Pequeño/metabolismo , ARN Interferente Pequeño/uso terapéutico , Ribonucleósidos/metabolismo , Ribonucleósidos/uso terapéutico , Transducción de Señal/efectos de los fármacos , Sirolimus/análogos & derivados , Sirolimus/metabolismo , Sirolimus/uso terapéutico , Serina-Treonina Quinasas TOR/químicaRESUMEN
Phosphatidylinositol-specific phospholipase C (PI-PLC) is activated in cell nuclei during the cell cycle progression. We have previously demonstrated two peaks of an increase in the nuclear PI-PLC activities in nocodazole-synchronized HL-60 cells. In this study, the activity of nuclear PI-PLC was investigated in serum-stimulated HL-60 cells. In serum-starved HL-60 cells, two peaks of the activity of nuclear PI-PLC were detected at 30 min and 11 h after the re-addition of serum with no parallel increase in PLC activity in cytosol, postnuclear membranes or total cell lysates. An increase in the serine phosphorylation of b splicing variant of PI-PLCbeta(1) was detected with no change in the amount of PI-PLCbeta(1b) in nuclei isolated at 30 min and 11 h after the addition of serum. PI-PLC inhibitor ET-18-OCH(3) and MEK inhibitor PD 98059 completely abolished serum-mediated increase at both time-points. The addition of inhibitors either immediately or 6 h after the addition of serum had inhibitory effects on the number of cells entering S phase. These results demonstrate that two waves of nuclear PI-PLCbeta(1b) activity occur in serum-stimulated cells during G(1) phase of the cell cycle and that the later increase in the PLC activity is equally important for the progression into the S phase.
Asunto(s)
Núcleo Celular/enzimología , Fase G1 , Fosfatidilinositol Diacilglicerol-Liasa/metabolismo , Suero/metabolismo , Animales , Bovinos , Núcleo Celular/efectos de los fármacos , Inhibidores Enzimáticos/farmacología , Fase G1/efectos de los fármacos , Células HL-60 , Humanos , Fosfatidilinositol Diacilglicerol-Liasa/antagonistas & inhibidores , Fosfoinositido Fosfolipasa C , Fosfoserina/metabolismo , Fracciones Subcelulares/efectos de los fármacos , Fracciones Subcelulares/enzimología , Factores de TiempoRESUMEN
Pharmacological modulators of AMP-dependent kinase (AMPK) have been suggested in treatment of cancer. The biguanide metformin and 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR) have been reported to inhibit proliferation of solid tumors and hematological malignancies, but their role in differentiation is less explored. Our previous study demonstrated that AICAR alone induced AMPK-independent expression of differentiation markers in monocytic U937 leukemia cells, and no such effects were observed in response to metformin. The aim of this study was to determine the mechanism of AICAR-mediated effects and to test for the possible role of autophagy in differentiation of leukemia cells. The results showed that AICAR-mediated effects on the expression of differentiation markers were not mimicked by A769662, a more specific direct AMPK activator. Long-term incubation of U937 cells with AICAR and other differentiation agents, all-trans-retinoic acid (ATRA) and phorbol 12-myristate 13-acetate, increased the expression of the autophagy marker LC3B-II, and these effects were not observed in response to metformin. Western blot and immunofluorescence analyses of U937 cells treated with bafilomycin A1 or transfected with mRFP-GFP-LC3 proved that the increase in the expression of LC3B-II was due to an increase in autophagy flux, and not to a decrease in lysosomal degradation. 3-Methyladenine inhibited the expression of differentiation markers in response to all inducers, but had stimulatory effects on autophagy flux at dose that effectively inhibited the production of phosphatidylinositol 3-phosphate. The small inhibitory RNA-mediated down-modulation of Beclin 1 and hVPS34 had no effects on AICAR and ATRA-mediated increase in the expression of differentiation markers. These results show that AICAR and other differentiation agents induce autophagy flux in U937 cells and that the effects of AICAR and ATRA on the expression of differentiation markers do not depend on the normal levels of key proteins of the classical or canonical autophagy pathway.
RESUMEN
In this study, the activity of nuclear phosphatidylinositol-specific phosholipase C (PI-PLC) was investigated in HL-60 cells blocked at G(2)/M phase by the addition of nocodazole, and released into medium as synchronously progressing cells. Two peaks of an increase in the nuclear PI-PLC activities were detected; an early peak reached a maximum at 1 h after release from the nocodazole block, and a second increase was detected at 8.5 h after the release. Immunoprecipitation studies indicated that the increase in the activity was due to the activation of the nuclear PI-PLC-beta(1). Western blot analysis demonstrated no changes in the level of both a and b splicing variants of PI-PLC-beta(1) in the nuclei of cells isolated at either 1 h or 8.5 h after the block. However, an increase in the serine-phosphorylation of PI-PLC-beta(1b) was detected in the nuclei of HL-60 cells isolated at 1 and 8.5 h after the block, and the presence of MEK-inhibitor PD98059 completely inhibited both the serine phosphorylation and the increase in the PI-PLC activities in vitro. The presence of PI-PLC inhibitor prevented the progression of HL-60 cells through the G(1) into S phase of the cell cycle. These results demonstrate that two peaks of nuclear PI-PLC activities, which are due to a PD98059-sensitive phosphorylation of nuclear PLC-beta(1b) on serine, occur at the G(2)/M and late G(1) phase and are necessary for the progression of the cells through the cell cycle.
Asunto(s)
Isoenzimas/metabolismo , Nocodazol/farmacología , Fosfolipasas de Tipo C/metabolismo , Ciclo Celular/efectos de los fármacos , Núcleo Celular/efectos de los fármacos , Núcleo Celular/metabolismo , Activación Enzimática/efectos de los fármacos , Flavonoides/farmacología , Fase G1 , Fase G2 , Células HL-60 , Humanos , Fosfolipasa C beta , Fosforilación/efectos de los fármacosRESUMEN
In the nuclear matrix harvested 20 h after partial hepatectomy, an increase in immunoprecipitable PI3K-C2beta activity is observed, which is sensitive to wortmannin (10 Mm) and shows strong preference for PtdIns over PtdIns(4)P as a substrate. On western blots PI3K-C2beta revealed a single immunoreactive band of 180 kD, whereas 20 h after partial hepatectomy gel shift of 18kDa was noticed in the nuclear matrix, suggesting that observed activation of enzyme is achieved by proteolysis. As it is know that PI3K-C2alpha is associated with nuclear speckles [Didichenko SA, Thelen M. Phosphatidylinositol 3-kinase C2alpha contains a nuclear localization sequence and associates with nuclear speckles. J Biol Chem 2001;276:48135-42.], the data presented in this report show that in the nuclear matrix PI3K-C2beta is activated during the compensatory liver growth, which clearly demonstrates that different class II PI3K enzymes have different subnuclear localization and therefore might have different intranuclear functions.
Asunto(s)
Núcleo Celular/enzimología , Hígado/enzimología , Hígado/crecimiento & desarrollo , Fosfatidilinositol 3-Quinasas/metabolismo , Subunidades de Proteína/metabolismo , Animales , Activación Enzimática/fisiología , Masculino , Ratas , Ratas WistarRESUMEN
Synthesis of inositol pyrophosphates through activation of Kcs1 plays an important role in the signalling response required for cell cycle progression after mating pheromone arrest. Overexpression of Kcs1 doubled the level of inositol pyrophosphates when compared to wild type cells and 30 min following the release from α-factor block further increase in inositol pyrophosphates was observed, which resulted that cells overexpressing Kcs1 reached G2/M phase earlier than wild type cells. Similar effect was observed in ipk1Δ cells, which are unable to synthesize IP6-derived inositol pyrophosphates (IP7 and IP8) but will synthesize IP5-derived inositol pyrophosphates (PP-IP4 and (PP)2-IP3). Although ipk1Δ cells have shorter telomeres than wild type cells, overexpression of Kcs1 in both strains have similar effect on cell cycle progression. As it is known that PP-IP4 regulates telomere length through Tel1, inositol polyphosphates, cell cycle and telomere length were determined in tel1Δ cells. The release of the cells from α-factor block and overexpression of Kcs1 in tel1Δ cells produced similar effects on inositol pyrophosphates level and cell cycle progression when compared to wild type cells, although tel1Δ cells possesses shorter telomeres than wild type cells. It can be concluded that telomere length does not affect cell cycle progression, since cells with short telomeres (ipk1Δ and tel1Δ) progress through cell cycle in a similar manner as wild type cells and that overexpression of Kcs1 in cells with either short or normal telomeres will increase S phase progression without affecting telomere length. Furthermore, IP5-derived inositol pyrophosphates can compensate for the loss of IP6-derived inositol pyrophosphates, in modulating S phase progression of the cell cycle.
Asunto(s)
Ciclo Celular , Fosfatos de Inositol/metabolismo , Saccharomyces cerevisiae/citología , Telómero/metabolismo , División Celular , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Telómero/genéticaRESUMEN
The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells blocked by aphidicolin at G(1)/S boundary and allowed to progress synchronously through the cell cycle. The activity of immunoprecipitated PI3K-C2beta in the nuclei and nuclear envelopes showed peak activity at 8 h after release from the G(1)/S block, which correlates with G(2)/M phase of the cell cycle. In the nuclei and nuclear envelopes isolated from HL-60 cells at 8 h after release from G(1)/S block, a significant increase in the level of incorporation of radiolabeled phosphate into phosphatidylinositol 3-phosphate (PtdIns(3)P) was observed with no change in the level of radiolabeled PtdIns(4)P, PtdIns(4,5)P(2) and PtdIns(3,4,5)P(3). On Western blots, PI3K-C2beta revealed a single immunoreactive band of 180 kDa, whereas in the nuclei and nuclear envelopes isolated at 8 h after release, the gel shift of 18 kDa was observed. When nuclear envelopes were treated for 20 min with mu-calpain in vitro, the similar gel shift and increase in PI3K-C2beta activity was observed which was completely inhibited by pretreatment with calpain inhibitor calpeptin. The presence of PI3K inhibitor LY 294002 completely abolished the calpain-mediated increase in the activity of PI3K-C2beta but did not prevent the gel shift. When HL-60 cells were released from G(1)/S block in the presence of either calpeptin or LY 294002, the activation of nuclear PI3K-C2beta was completely inhibited. These results demonstrate the calpain-mediated activation of the nuclear PI3K-C2beta during G(2)/M phase of the cell cycle in HL-60 cells.
Asunto(s)
Ciclo Celular/fisiología , Núcleo Celular/metabolismo , Complemento C2/biosíntesis , Membrana Nuclear/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Afidicolina , Calpaína/farmacología , Cromonas/farmacología , Activación de Complemento/efectos de los fármacos , Complemento C2/análisis , Complemento C2b , Dipéptidos/farmacología , Inhibidores Enzimáticos/farmacología , Citometría de Flujo , Células HL-60 , Humanos , Morfolinas/farmacología , Membrana Nuclear/química , Fosfatidilinositoles/metabolismoRESUMEN
Arsenic trioxide (ATO) has potent clinical activity in the treatment of patients with acute promyelocytic leukemia (APL), but is much less efficacious in acute myeloid leukemia (AML) lacking t(15;17) translocation. Recent studies have indicated that the addition of mammalian target of rapamycin (mTOR) inhibitors may increase the sensitivity of malignant cells to ATO. The aim of the present study was to test for possible synergistic effects of ATO and rapamycin at therapeutically achievable doses in non-APL AML cells. In HL-60 and U937 cell lines, the inhibitory effects of low concentrations of ATO and rapamycin were synergistic and more pronounced in U937 cells. The combination of drugs increased apoptosis in HL-60 cells and increased the percentage of cells in G(0)/G(1) phase in both cell lines. In U937 cells, rapamycin alone increased the activity of mitogen-activated protein kinase/extracellular signal-regulated kinase (MAPK/ERK) and the addition of ATO decreased the level of phosphorylated ERK, Ser473 phosphorylated Akt and anti-apoptotic Mcl-1 protein. Primary AML cells show high sensitivity to growth-inhibitory effects of rapamycin alone or in combination with ATO. The results of the present study reveal the mechanism of the synergistic effects of two drugs at therapeutically achievable doses in non-APL AML cells.
Asunto(s)
Antineoplásicos/farmacología , Arsenicales/farmacología , Leucemia Mieloide Aguda/genética , Óxidos/farmacología , Sirolimus/farmacología , Translocación Genética , Anciano , Apoptosis/efectos de los fármacos , Trióxido de Arsénico , Ciclo Celular/efectos de los fármacos , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Cromosomas Humanos Par 15 , Cromosomas Humanos Par 17 , Sinergismo Farmacológico , Femenino , Células HL-60 , Humanos , Leucemia Mieloide Aguda/tratamiento farmacológico , Leucemia Mieloide Aguda/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismoRESUMEN
Phospholipase C (PLC) was purified from the membrane-depleted rat liver nuclei. About 60% of the total PLC-activity corresponded to beta1b isoform, 30% to PLC-gamma1 and less than 10% to PLC-delta1. PLC-beta1b and -gamma1 were found in the nuclear matrix, while PLC-delta1 was detected in the chromatin. Two peaks of an increase in the total PLC-activity were detected occurring at 6 and 20 h after partial hepatectomy. An early increase in PLC-beta1b activity in the nuclear matrix was associated with serine phosphorylation of the enzyme, while the later increase paralleled the increase in the amount of protein. The increase in the PLC-gamma1 activity measured at 6 and 20 h after partial hepatectomy was associated with tyrosine phosphorylation of the enzyme. The activity of PLC-delta1 and the amount of the protein found in the chromatin was increased only at 20 h after partial hepatectomy.
Asunto(s)
Núcleo Celular/enzimología , Regeneración Hepática/fisiología , Hígado/enzimología , Fosfolipasas de Tipo C/metabolismo , Animales , Fraccionamiento Celular , Activación Enzimática , Hepatectomía , Membranas Intracelulares/enzimología , Isoenzimas/metabolismo , Cinética , Masculino , Ratas , Ratas WistarRESUMEN
The activity of nuclear phosphoinositide 3-kinase C2beta (PI3K-C2beta) was investigated in HL-60 cells induced to differentiate along granulocytic or monocytic lineages. A significant increase in the activity of immunoprecipitated PI3K-C2beta was observed in the nuclei and nuclear envelopes isolated from all-trans-retinoic acid (ATRA)-differentiated cells which was inhibited by the presence of PI3K inhibitor LY 294002. High-performance liquid chromatography analysis of inositol lipids showed an increased incorporation of radiolabelled phosphate in both PtdIns(3)P and PtdIns(3,4,5)P(3) with no changes in the levels of PtdIns(4)P, PtdIns(3,4)P(2) and PtdIns(4,5)P(2). Western blot analysis of the PI3K-C2beta immunoprecipitates with anti-P-Tyr antibody revealed a significant increase in the level of the immunoreactive band corresponding to PI3K-C2beta in the nuclei and nuclear envelopes isolated from ATRA-differentiated cells.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Núcleo Celular/enzimología , Fosfatidilinositol 3-Quinasas/metabolismo , Tretinoina/farmacología , Western Blotting , Activación Enzimática , Células HL-60 , Humanos , Fosforilación , Pruebas de PrecipitinaRESUMEN
Adenosine monophosphate (AMP)-activated kinase (AMPK) modulators have been shown to exert cytotoxic activity in hematological malignancies, but their role in the differentiation of acute myeloid leukemia (AML) is less explored. In this study, the effects of AMPK agonists on all-trans retinoic acid (ATRA)-mediated differentiation of acute promyelocytic leukemia (APL) and non-APL AML cell lines were investigated. The results show that AMPK agonists inhibit the growth of myeloblastic HL-60, promyelocytic NB4 and monocytic U937 cells. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR), an AMPK activator, enhances ATRA-mediated differentiation of NB4 cells. In U937 cells, AICAR alone induces the expression of cell surface markers associated with mature monocytes and macrophages. In both cell lines, AICAR increases the activity of mitogen-activated protein kinase (MAPK), and the presence of a MAPK inhibitor reduces the expression of differentiation markers. These results reveal beneficial effects of AICAR in AML, including differentiation of non-APL AML cells.