RESUMEN
CK2 is a ubiquitously expressed, constitutively active Ser/Thr protein kinase, which is considered the most pleiotropic protein kinase in the human kinome. Such a pleiotropy explains the involvement of CK2 in many cellular events. However, its predominant roles are stimulation of cell growth and prevention of apoptosis. High levels of CK2 messenger RNA and protein are associated with CK2 pathological functions in human cancers. Over the last decade, basic and translational studies have provided evidence of CK2 as a pivotal molecule driving the growth of different blood malignancies. CK2 overexpression has been demonstrated in nearly all the types of hematological cancers, including acute and chronic leukemias, where CK2 is a key regulator of signaling networks critical for cell proliferation, survival and drug resistance. The findings that emerged from these studies suggest that CK2 could be a valuable therapeutic target in leukemias and supported the initiation of clinical trials using CK2 antagonists. In this review, we summarize the recent advances on the understanding of the signaling pathways involved in CK2 inhibition-mediated effects with a particular emphasis on the combinatorial use of CK2 inhibitors as novel therapeutic strategies for treating both acute and chronic leukemia patients.
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Quinasa de la Caseína II/metabolismo , Leucemia/tratamiento farmacológico , Leucemia/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , Inhibidores de Proteínas Quinasas/uso terapéutico , Animales , Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Humanos , Transducción de Señal/efectos de los fármacosRESUMEN
Constitutively active casein kinase 2 (CK2) signaling is a common feature of T-cell acute lymphoblastic leukemia (T-ALL). CK2 phosphorylates PTEN (phosphatase and tensin homolog) tumor suppressor, resulting in PTEN stabilization and functional inactivation. Downregulation of PTEN activity has an impact on PI3K/Akt/mTOR signaling, which is of fundamental importance for T-ALL cell survival. These observations lend compelling weight to the application of CK2 inhibitors in the therapy of T-ALL. Here, we have analyzed the therapeutic potential of CX-4945-a novel, highly specific, orally available, ATP-competitive inhibitor of CK2α. We show that CX-4945 treatment induced apoptosis in T-ALL cell lines and patient T lymphoblasts. CX-4945 downregulated PI3K/Akt/mTOR signaling in leukemic cells. Notably, CX-4945 affected the unfolded protein response (UPR), as demonstrated by a significant decrease in the levels of the main UPR regulator GRP78/BIP, and led to apoptosis via upregulation of the ER stress/UPR cell death mediators IRE1α and CHOP. In vivo administration of CX-4945 to a subcutaneous xenotransplant model of human T-ALL significantly delayed tumor growth. Our findings indicate that modulation of the ER stress/UPR signaling through CK2 inhibition could be exploited for inducing apoptosis in T-ALL cells and that CX-4945 may be an efficient treatment for those T-ALLs displaying upregulation of CK2α/PI3K/Akt/mTOR signaling.
Asunto(s)
Antineoplásicos/uso terapéutico , Quinasa de la Caseína II/antagonistas & inhibidores , Naftiridinas/uso terapéutico , Proteínas de Neoplasias/metabolismo , Leucemia-Linfoma Linfoblástico de Células T Precursoras/tratamiento farmacológico , Transducción de Señal , Respuesta de Proteína Desplegada , Animales , División Celular , Chaperón BiP del Retículo Endoplásmico , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Proteínas de Neoplasias/química , Fenazinas , Leucemia-Linfoma Linfoblástico de Células T Precursoras/patologíaRESUMEN
The phosphatidylinositol 3-kinase (PI3K) and the mammalian target of rapamycin (mTOR) are two major signaling molecules in the PI3K/Akt/mTOR signal transduction cascade. This pathway is a key regulator of a wide range of physiological cell processes which include proliferation, differentiation, survival, metabolism, exocytosis, motility, and autophagy. However, aberrantly upregulated PI3K/Akt/mTOR signaling characterizes many types of cancers where it negatively influences response to therapeutic treatments. Therefore, targeting PI3K/Akt/mTOR signaling with small molecule inhibitors could improve cancer patient outcome. The PI3K/Akt/mTOR signaling network is activated in acute leukemias of both myelogenous and lymphoid lineage, where it correlates with poor prognosis and enhanced drug-resistance. The catalytic sites of PI3K and mTOR share a high degree of sequence homology. This feature has allowed the synthesis of ATP-competitive compounds that targeted the catalytic site of both PI3K and mTOR (e.g. PI-103, NVP-BEZ235). In preclinical settings, dual PI3K/mTOR inhibitors displayed a much stronger cytotoxicity against leukemic cells than either PI3K inhibitors or allosteric mTOR inhibitors, such as rapamycin and its derivatives (rapalogs). At variance with rapamycin/rapalogs, dual PI3K/mTOR inhibitors targeted both mTOR complex 1 and mTOR complex 2, and inhibited the rapamycin-resistant phosphorylation of eukaryotic initiation factor 4E-binding protein 1, resulting in a marked inhibition of oncogenetic protein translation in leukemic cells. Hence, they strongly reduced the proliferation rate and induced an important apoptotic response. Here, we reviewed the evidence documenting that dual PI3K/mTOR inhibitors represent a promising option for future targeted therapies of leukemic patients.
RESUMEN
OBJECTIVE: To examine prospectively the reliability of ultrasound-trained obstetricians performing a first-trimester fetal cardiac scan with high-frequency transabdominal probes, by confirming normal or abnormal heart anatomy, in pregnancies referred for increased nuchal translucency thickness (NT). METHODS: Trained obstetric operators assessed the fetal heart in 133 fetuses with increased NT (> 95th centile) at 11-14 weeks of gestation. A high-frequency transabdominal probe was used to confirm or refute normal cardiac anatomy rather than to establish a specific diagnosis. Following this preliminary screening by the ultrasound-trained obstetrician, specialized fetal echocardiographers rescanned the fetal heart in order to confirm the accuracy of the obstetric operators' findings and to establish a diagnosis in abnormal cases. Fetal cardiologists repeated the examinations at 20 and 32 weeks of pregnancy. Postnatal follow-up lasted 2 years. Twelve fetuses with normal karyotype and normal anatomy were lost to follow-up. RESULTS: A total of 121 fetuses with increased NT between 11 and 14 weeks' gestation were studied. Congenital heart disease (CHD) was detected in 20/121 (16.5%) fetuses. In addition, there were three with mild ventricular disproportion, the right ventricle being larger than the left, considered as a minor non-specific cardiac abnormality. CHD was associated with chromosomal anomalies in 12/20 (60%) cases. Among the 121 fetuses, there was agreement between ultrasound-trained obstetricians and fetal cardiologists in 116 (95.9%) of the cases, and the ultrasound-trained obstetricians correctly identified 18 cases with major cardiac defects. However, there was disagreement in five cases: two with small ventricular septal defects and three with ventricular disproportion. CONCLUSIONS: Our results provide evidence that obstetricians, trained to study the heart in the second trimester, can also differentiate reliably between normal and abnormal heart findings in the first trimester, when using a high-frequency transabdominal ultrasound probe.
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Trastornos de los Cromosomas/diagnóstico por imagen , Corazón Fetal/diagnóstico por imagen , Cardiopatías Congénitas/diagnóstico por imagen , Medida de Translucencia Nucal/métodos , Adolescente , Adulto , Trastornos de los Cromosomas/embriología , Trastornos de los Cromosomas/genética , Femenino , Corazón Fetal/anomalías , Corazón Fetal/anatomía & histología , Edad Gestacional , Humanos , Obstetricia , Embarazo , Primer Trimestre del Embarazo , Estudios Prospectivos , Reproducibilidad de los Resultados , Adulto JovenRESUMEN
Alkylphospholipids and alkylphosphocholines (APCs) are promising antitumor agents, which target the plasma membrane and affect multiple signal transduction networks. We investigated the therapeutic potential of erucylphosphohomocholine (ErPC3), the first intravenously applicable APC, in human acute myelogenous leukemia (AML) cells. ErPC3 was tested on AML cell lines, as well as AML primary cells. At short (6-12 h) incubation times, the drug blocked cells in G2/M phase of the cell cycle, whereas, at longer incubation times, it decreased survival and induced cell death by apoptosis. ErPC3 caused JNK 1/2 activation as well as ERK 1/2 dephosphorylation. Pharmacological inhibition of caspase-3 or a JNK 1/2 inhibitor peptide markedly reduced ErPC3 cytotoxicity. Protein phosphatase 2A downregulation by siRNA opposed ERK 1/2 dephosphorylation and blunted the cytotoxic effect of ErPC3. ErPC3 was cytotoxic to AML primary cells and reduced the clonogenic activity of CD34(+) leukemic cells. ErPC3 induced a significant apoptosis in the compartment (CD34(+) CD38(Low/Neg) CD123(+)) enriched in putative leukemia-initiating cells. This conclusion was supported by ErPC3 cytotoxicity on AML blasts showing high aldehyde dehydrogenase activity and on the side population of AML cell lines and blasts. These findings indicate that ErPC3 might be a promising therapeutic agent for the treatment of AML patients.
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Apoptosis/efectos de los fármacos , Ácidos Erucicos/farmacología , Leucemia Mieloide Aguda/tratamiento farmacológico , MAP Quinasa Quinasa 4/metabolismo , Fosforilcolina/análogos & derivados , Células Precursoras de Linfocitos B/efectos de los fármacos , Proteína Fosfatasa 2/metabolismo , Animales , Western Blotting , Ciclo Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Células Cultivadas , Citometría de Flujo , Técnica del Anticuerpo Fluorescente , Humanos , Técnicas para Inmunoenzimas , Leucemia Mieloide Aguda/metabolismo , Leucemia Mieloide Aguda/patología , Ratones , Proteína Quinasa 1 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/antagonistas & inhibidores , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilcolina/farmacología , Células Precursoras de Linfocitos B/metabolismo , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/genética , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de SeñalRESUMEN
Glycogen storage disease type IV (GSD IV, or Andersen disease) is an autosomal recessive disorder due to the deficiency of 1,4-alpha-glucan branching enzyme (or glycogen branching enzyme, GBE1), resulting in an accumulation of amylopectin-like polysaccharide in muscle, liver, heart and central and peripheral nervous system. Typically, the presentation is in childhood with liver involvement up to cirrhosis. The neuromuscular form varies in onset (congenital, perinatal, juvenile and adult) and in severity. Congenital cases are rare, and fewer than 20 cases have been described and genetically determined so far. This form is characterized by polyhydramnios, neonatal hypotonia, and neuronal involvement; hepatopathy is uncommon, and the babies usually die between 4 weeks and 4 months of age. We report the case of an infant who presented severe hypotonia, dilatative cardiomyopathy, mild hepatopathy, and brain lateral ventricle haemorrhage, features consistent with the congenital form of GSD IV. He died at one month of life of cardiorespiratory failure. Muscle biopsy and heart and liver autoptic specimens showed many vacuoles filled with PAS-positive diastase-resistant materials. Electron-microscopic analysis showed mainly polyglucosan accumulations in all the tissues examined. Postmortem examination showed the presence of vacuolated neurons containing this abnormal polysaccharide. GBE1 biochemical activity was virtually absent in muscle and fibroblasts, and totally lacking in liver and heart as well as glycogen synthase activity. GBE1 gene sequence analysis revealed a novel homozygous nonsense mutation, p.E152X, in exon 4, correlating with the lack of enzyme activity and with the severe neonatal involvement. Our findings contribute to increasing the spectrum of mutation associated with congenital GSD IV.
Asunto(s)
Codón sin Sentido , Sistema de la Enzima Desramificadora del Glucógeno/deficiencia , Sistema de la Enzima Desramificadora del Glucógeno/genética , Enfermedad del Almacenamiento de Glucógeno Tipo IV/diagnóstico , Enfermedad del Almacenamiento de Glucógeno Tipo IV/genética , Secuencia de Bases , Encéfalo/enzimología , Encéfalo/patología , Análisis Mutacional de ADN , Resultado Fatal , Enfermedad del Almacenamiento de Glucógeno Tipo IV/enzimología , Homocigoto , Humanos , Recién Nacido , Hígado/enzimología , Hígado/patología , Masculino , Microscopía Electrónica de Transmisión , Músculo Esquelético/patología , Miocardio/enzimología , Miocardio/patologíaRESUMEN
OBJECTIVES: Among the many pathological aspects of Down syndrome, brain hypoplasia and mental retardation have been recently ascribed to defective proliferation of neural precursors during central nervous system development. By analogy, other features of Down syndrome, such as heart defects, gastrointestinal abnormalities, craniofacial dystrophy and reduced growth rate could be related, at least in theory, to similar proliferation impairment in peripheral tissues. MATERIALS AND METHODS: In order to test this hypothesis, we evaluated cell proliferation in peripheral tissues of the Ts65Dn mouse, one of the animal models most commonly used to investigate Down syndrome. RESULTS: In fibroblast cultures from neonatal Ts65Dn mice, we found that cell proliferation was notably impaired. While length of the cell cycle was similar in fibroblasts from Ts65Dn and control mice, the number of actively proliferating cells was significantly smaller in Ts65Dn mice. Moreover, fibroblasts from Ts65Dn animals exhibited limited population-doubling capacity, decreased proliferative lifespan and premature senescence. Analysis of cell proliferation in the skin of neonates, in vivo, showed that in Ts65Dn mice, cell proliferation was significantly reduced compared to control mice. CONCLUSIONS: Our results suggest that defective proliferation may be a generalized feature of trisomic mice. In view of the genetic and phenotypic similarities between Ts65Dn mice and individuals with Down syndrome, proliferation impairment in various organs may also occur in subjects with Down syndrome. Thus, perturbation of a basic developmental function, cell proliferation, may be a critical determinant that contributes to the many aspects of pathology of this condition.
Asunto(s)
Modelos Animales de Enfermedad , Síndrome de Down/patología , Animales , Animales Recién Nacidos , Apoptosis/genética , Peso Corporal/genética , Ciclo Celular/genética , Proliferación Celular , Senescencia Celular/genética , ADN/metabolismo , Femenino , Fibroblastos/citología , Fibroblastos/metabolismo , Fibroblastos/patología , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Mutantes , Poliploidía , Piel/patología , beta-Galactosidasa/metabolismoRESUMEN
AIM: This study had the following aims: to verify whether children living in different environmental areas present a different development degree of the functional prerequisites of psychomotricity; to test whether a targeted psychomotricity education program could favourably modify the potential differences which may be observed; to investigate the relationship, if any, between the anthropometric differences and the functional prerequisites of psychomotricity. METHODS: One hundred and sixty-five Italian children, 83 males and 82 females, 6-7 years old were enrolled in this study. Based on the provenance area, the children were subdivided into two groups: the urban one (N=85) and the rural one (N=80). Both groups underwent an initial psychomotor assessment including standardised psychomotor tests aimed at evidencing the general dynamic coordination ability and the static and dynamic balance capacity of every child. RESULTS: The findings of this research point out that children living in an urban setting selectively showed a lower degree of balance development, if compared to children living in rural areas; a targeted psychomotor education program favourably modified the differences in the balance development between the two examined groups, up to their disappearance. In the urban group the body mass index had a trend towards a negative relationship with balance development. CONCLUSION: Children grown up in an urban environment showed a delay in balance development, if compared to children of the same age grown up in rural areas. This study also clearly proves that such a delay may be regained by means of a targeted psychomotor education program.
Asunto(s)
Antropometría , Desarrollo Infantil , Ambiente , Desempeño Psicomotor , Población Rural , Población Urbana , Niño , Femenino , Humanos , Italia , MasculinoRESUMEN
A significant impediment to the success of cancer chemotherapy is the occurrence of multidrug resistance, which, in many cases, is attributable to overexpression of membrane transport proteins, such as the 170-kDa P-glycoprotein (P-gp). Also, upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt-signaling pathway is known to play an important role in drug resistance, and has been implicated in the aggressiveness of a number of different cancers, including T-acute lymphoblastic leukemia (T-ALL). We have investigated the therapeutic potential of the novel Akt inhibitor, perifosine (a synthetic alkylphospholipid), on human T-ALL CEM cells (CEM-R), characterized by both overexpression of P-gp and constitutive upregulation of the PI3K/Akt network. Perifosine treatment induced death by apoptosis in CEM-R cells. Apoptosis was characterized by caspase activation, Bid cleavage and cytochrome c release from mitochondria. The proapoptotic effect of perifosine was in part dependent on the Fas/FasL interactions and c-Jun NH(2)-terminal kinase (JNK) activation, as well as on the integrity of lipid rafts. Perifosine downregulated the expression of P-gp mRNA and protein and this effect required JNK activity. Our findings indicate that perifosine is a promising therapeutic agent for treatment of T-ALL cases characterized by both upregulation of the PI3K/Akt survival pathway and overexpression of P-gp.
Asunto(s)
Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Apoptosis/efectos de los fármacos , Caspasas/metabolismo , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Leucemia-Linfoma de Células T del Adulto/patología , Fosforilcolina/análogos & derivados , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/genética , Antineoplásicos Fitogénicos/farmacología , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Western Blotting , Supervivencia Celular/efectos de los fármacos , Citocromos c/metabolismo , Regulación hacia Abajo , Resistencia a Múltiples Medicamentos , Resistencia a Antineoplásicos , Ensayo de Cambio de Movilidad Electroforética , Activación Enzimática , Citometría de Flujo , Humanos , Técnicas para Inmunoenzimas , Proteínas Quinasas JNK Activadas por Mitógenos/antagonistas & inhibidores , Proteínas Quinasas JNK Activadas por Mitógenos/genética , Leucemia-Linfoma de Células T del Adulto/metabolismo , Microdominios de Membrana/metabolismo , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilcolina/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/farmacología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Tumorales Cultivadas , Vinblastina/farmacologíaRESUMEN
The serine/threonine kinase Akt, a downstream effector of phosphatidylinositol 3-kinase (PI3K), is known to play an important role in antiapoptotic signaling and has been implicated in the aggressiveness of a number of different human cancers including acute myelogenous leukemia (AML). We have investigated the therapeutic potential of the novel Akt inhibitor, perifosine, on human AML cells. Perifosine is a synthetic alkylphospholipid, a new class of antitumor agents, which target plasma membrane and inhibit signal transduction networks. Perifosine was tested on THP-1 and MV 4-11 cell lines, as well as primary leukemia cells. Perifosine treatment induced cell death by apoptosis in AML cell lines. Perifosine caused Akt and ERK 1/2 dephosphorylation as well as caspase activation. In THP-1 cells, the proapoptotic effect of perifosine was partly dependent on the Fas/FasL system and c-jun-N-kinase activation. In MV 4-11 cells, perifosine downregulated phosphorylated Akt, but not phosphorylated FLT3. Moreover, perifosine reduced the clonogenic activity of AML, but not normal, CD34(+) cells, and markedly increased blast cell sensitivity to etoposide. Our findings indicate that perifosine, either alone or in combination with existing drugs, might be a promising therapeutic agent for the treatment of those AML cases characterized by upregulation of the PI3K-Akt survival pathway.
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Apoptosis/efectos de los fármacos , Leucemia Mieloide Aguda/tratamiento farmacológico , Fosforilcolina/análogos & derivados , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Western Blotting , Proliferación Celular/efectos de los fármacos , Ensayo de Unidades Formadoras de Colonias , Proteína Ligando Fas/genética , Proteína Ligando Fas/metabolismo , Citometría de Flujo , Humanos , Inmunoprecipitación , Leucemia Mieloide Aguda/metabolismo , MAP Quinasa Quinasa 4/metabolismo , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación/efectos de los fármacos , Fosforilcolina/farmacología , Proteínas Proto-Oncogénicas c-akt/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Transducción de Señal , Células Tumorales Cultivadas , Proteína Letal Asociada a bcl/metabolismo , Receptor fas/metabolismo , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
OBJECTIVE: The fetal heart is not studied routinely in the first trimester because of technical and time limitations. Our aim was to assess the feasibility of performing a fetal cardiac study in pregnancies referred for nuchal translucency (NT) screening, using high-frequency linear transabdominal transducers with a specific ultrasound preset. METHODS: A single trained operator assessed the fetal heart in pregnancies with a fetal crown-rump length (CRL) of 60-84 mm that had been referred for NT screening. A 15- or 6-MHz transabdominal linear transducer with a specific preset suitable mainly for color-flow mapping was used to confirm or refute normal cardiac anatomy rather than to establish a specific diagnosis. Fetuses having an increased risk for congenital heart disease were referred to a tertiary center for a further examination within 1 week. This group consisted of all fetuses with NT > 95(th) centile and those in which a family history or the initial heart scan increased the risk. RESULTS: A total of 608 fetuses with a median CRL of 65 mm was examined between 2003 and 2005. A cardiac scan was performed successfully in 456 (75%) using a 15-MHz linear transducer alone, and the additional use of a 6-MHz transducer allowed diagnostic images to be obtained in a further 152. Normal cardiac anatomy was assessed confidently within 10 min in 517/608 (85%) pregnancies; in 85 (14%) a longer time was needed and six patients were rescheduled within 2 weeks because of non-diagnostic images at the initial scan. In 571/608 (94%) the risk for congenital heart disease (CHD) was not increased and the heart was considered normal at initial echocardiography; this was confirmed by later scans and at postnatal follow-up. In 37/608 (6%) fetuses the risk for CHD was increased (35 for NT > 95(th) centile and two for family history). In this group normal heart anatomy was described in 34 fetuses and confirmed by subsequent specialist echocardiography. Cardiac defects were suspected in three fetuses (all with increased NT) and confirmed by a fetal cardiologist in each case. CONCLUSIONS: A trained operator can perform a fetal heart study during the NT screening test using transabdominal high-resolution transducers in an acceptable length of time.
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Enfermedades Fetales/diagnóstico por imagen , Corazón Fetal/diagnóstico por imagen , Cardiopatías Congénitas/diagnóstico por imagen , Medida de Translucencia Nucal/métodos , Adulto , Largo Cráneo-Cadera , Estudios de Factibilidad , Femenino , Enfermedades Fetales/epidemiología , Corazón Fetal/anatomía & histología , Cardiopatías Congénitas/epidemiología , Humanos , Tamizaje Masivo/métodos , Embarazo , Primer Trimestre del Embarazo , Sensibilidad y Especificidad , Factores de Tiempo , Ultrasonografía Prenatal/métodosRESUMEN
A high incidence of relapses following induction chemotherapy is a major hindrance to patient survival in acute myelogenous leukemia (AML). There is strong evidence that activation of the phosphoinositide 3 kinase (PI3K)/Akt signaling network plays a significant role in rendering AML blasts drug resistant. An important mechanism underlying drug resistance is represented by overexpression of membrane drug transporters such as multidrug resistance-associated protein 1 (MRP1) or 170-kDa P-glycoprotein (P-gp). Here, we present evidence that MRP1, but not P-gp, expression is under the control of the PI3K/Akt axis in AML blasts. We observed a highly significant correlation between levels of phosphorylated Akt and MRP1 expression in AML cells. Furthermore, incubation of AML blasts with wortmannin, a PI3K pharmacological inhibitor, resulted in lower levels of phosphorylated Akt, downregulated MRP1 expression, and decreased Rhodamine 123 extrusion in an in vitro functional dye efflux assay. We also demonstrate that wortmannin-dependent PI3K/Akt inhibition upregulated p53 protein levels in most AML cases, and this correlated with diminished MRP1 expression and enhanced phosphorylation of murine double minute 2 (MDM2). Taken together, these data suggest that PI3K/Akt activation may lead to the development of chemoresistance in AML blasts through a mechanism involving a p53-dependent suppression of MRP1 expression.
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Resistencia a Antineoplásicos/genética , Regulación Leucémica de la Expresión Génica/fisiología , Leucemia Mieloide/patología , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/biosíntesis , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Proto-Oncogénicas c-akt/fisiología , Miembro 1 de la Subfamilia B de Casetes de Unión a ATP/metabolismo , Enfermedad Aguda , Adulto , Anciano , Anciano de 80 o más Años , Androstadienos/farmacología , Neoplasias Óseas/patología , Línea Celular Tumoral/efectos de los fármacos , Línea Celular Tumoral/metabolismo , Femenino , Colorantes Fluorescentes/metabolismo , Regulación Leucémica de la Expresión Génica/efectos de los fármacos , Regulación Leucémica de la Expresión Génica/genética , Genes p53 , Humanos , Células Jurkat/efectos de los fármacos , Células Jurkat/metabolismo , Leucemia Mieloide/genética , Leucemia Mieloide/metabolismo , Leucemia Promielocítica Aguda/patología , Leucemia-Linfoma de Células T del Adulto/patología , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Masculino , Persona de Mediana Edad , Proteínas Asociadas a Resistencia a Múltiples Medicamentos/genética , Proteínas de Neoplasias/biosíntesis , Proteínas de Neoplasias/genética , Células Madre Neoplásicas/efectos de los fármacos , Células Madre Neoplásicas/metabolismo , Osteosarcoma/patología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Fosforilación , Procesamiento Proteico-Postraduccional , Proteínas Proto-Oncogénicas c-mdm2/biosíntesis , Proteínas Proto-Oncogénicas c-mdm2/genética , Rodamina 123/metabolismo , Proteína p53 Supresora de Tumor/biosíntesis , WortmaninaRESUMEN
A combination of 8-methoxypsoralen and ultraviolet-A radiation (320-400 nm) (PUVA) is used for the treatment of T cell-mediated disorders, including chronic graft-versus-host disease, autoimmune disorders, and cutaneous T-cell lymphomas. The mechanisms of action of this therapy, referred to as extracorporeal phototherapy, have not been fully elucidated. PUVA is known to induce apoptosis in T lymphocytes collected by apheresis, however no information is available concerning the underlying signaling pathways which are activated by PUVA. In this study, we found that PUVA treatment of Jurkat cells and human T lymphocytes up-regulates the p38 MAPK pathway but not the p42/44 MAPK or the SAPK/JNK signaling networks. The use of a pharmacological inhibitor selective for the p38 MAPK pathway, SB203580, allowed us to demonstrate that this network exerts an antiapoptotic effect in PUVA-treated Jurkat cells and T lymphocytes from healthy donors. Moreover, the effect of SB203580 was not due to a down-regulation of the Akt survival pathway which was not activated in response to PUVA. These results may suggest that p38 MAPK-dependent signaling is very important for the regulation of survival genes after exposure to PUVA. Since the therapeutic effect of PUVA seems to depend, at least in part, on apoptosis, further studies on the apoptosis signaling networks activated by this treatment might lead to the use of signal transduction modulators in combination with PUVA, to increase the efficacy of this form of therapy.
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Apoptosis/efectos de los fármacos , Apoptosis/efectos de la radiación , Metoxaleno/farmacología , Linfocitos T/efectos de los fármacos , Linfocitos T/efectos de la radiación , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismo , Células Cultivadas , Activación Enzimática , Humanos , Células Jurkat , Rayos UltravioletaRESUMEN
A major factor undermining successful cancer treatment is the occurrence of resistance to conventional treatments such as chemotherapy and ionizing radiation. Evidence accumulated over the recent years has indicated the phosphoinositide 3-kinase/Akt signal transduction pathway as one of the major factors implicated in cancer resistance to conventional therapies. Indeed, the phosphoinositide 3-kinase/Akt axis regulates the expression and/or function of many anti-apoptotic proteins which strongly contributes to cancer cell survival. As a result, small molecules designed to specifically target key components of this signaling network are now being developed for clinical use as single therapeutic agents and/or in combination with other forms of therapy to overcome resistance. Initially, the phosphoinositide 3-kinase/Akt signal transduction pathway has been mainly investigated in solid tumors. Recently, however, this network has also been recognized as an important therapeutic target in human leukemias. Specific inhibition of this signalling pathway may be a valid approach to treat these diseases and increase the efficacy of standard types of therapy.
Asunto(s)
Resistencia a Antineoplásicos/fisiología , Leucemia/enzimología , Fosfatidilinositol 3-Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/fisiología , Proteínas Proto-Oncogénicas/fisiología , Humanos , Isoenzimas/fisiología , Proteínas Quinasas/fisiología , Proteínas Serina-Treonina Quinasas/antagonistas & inhibidores , Proteínas Serina-Treonina Quinasas/genética , Proteínas Proto-Oncogénicas/antagonistas & inhibidores , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas c-akt , Transducción de Señal/fisiología , Serina-Treonina Quinasas TORRESUMEN
Apart from the lipids present in the nuclear envelope, the nucleus also contains lipids which are located further inside and are resistant to treatment with nonionic detergents. Evidence is being accumulated on the importance of internal nuclear lipid metabolism. Nuclear lipid metabolism gives rise to several lipid second messengers that function within the nucleus. Moreover, it is beginning to emerge that nuclear lipids not only act as precursors of bioactive second messengers but may be directly involved in regulation of nuclear structure and gene expression. Over the last 10 years, especially the role of the inositol lipid cycle in nuclear signal transduction has been extensively studied. This cycle is activated following a variety of stimuli and is regulated independently from the inositide cycle located at the plasma membrane. However, the nucleus contain other lipids, such as phosphatidylcholine, sphingomyelin, fatty acids and eicosanoids. There are numerous reports which suggest that these classes of nuclear lipids may play roles in the nucleus as important as those of phosphoinositides. This review aims at highlighting the most important aspects regarding the metabolism and signaling activities of nuclear phosphatidylcholine, sphingomyelin, fatty acids and eicosanoids.
Asunto(s)
Núcleo Celular/metabolismo , Lípidos/fisiología , Transducción de Señal , Animales , Diglicéridos/metabolismo , Regulación de la Expresión Génica , Humanos , Lípidos/química , Modelos Biológicos , Fosfatidilcolinas/química , Fosfolipasa D/química , Fosfolipasas A/metabolismo , Esfingomielina Fosfodiesterasa/metabolismo , Esfingomielinas/químicaRESUMEN
The serine/threonine protein kinase Akt, a downstream effector of phosphoinositide 3-kinase (PI3K), plays a pivotal role in tumorigenesis because it affects the growth and survival of cancer cells. Several laboratories have demonstrated that Akt inhibits transcriptional activation of a number of related forkhead transcription factors now referred to as FoxO1, FoxO3, and FoxO4. Akt-regulated forkhead transcription factors are involved in the control of the expression of both the cyclin-dependent kinase (cdk) inhibitor p27(Kip1) and proapoptotic Bim protein. Very little information is available concerning the importance of the PI3K/Akt pathway in HL60 human leukemia cells. Here, we present our findings showing that the PI3K/Akt axis regulates cell cycle progression of HL60 cells through multiple mechanisms also involving the control of FoxO1 and FoxO3. To this end, we took advantage of a HL60 cell clone (HL60AR cells) with a constitutively activated PI3K/Akt axis. When compared with parental (PT) HL60 cells, HL60AR cells displayed higher levels of phosphorylated FoxO1 and FoxO3. In AR cells forkhead factors localized predominantly in the cytoplasm, whereas in PT cells they were mostly nuclear. AR cells proliferated faster than PT cells and showed a lower amount of the cdk inhibitor p27(Kip1), which was mainly found in the cytoplasm and was hyperphosphorylated on threonine residues. AR cells also displayed higher levels of cyclin D1 and phosphorylated p110 Retinoblastoma protein. The protein levels of cdk2, cdk4, and cdk6 were not altered in HL60AR cells, whereas the activities of both ckd2 and cdk6 were higher in AR than in PT cells. These results show that in HL60 cells the PI3K/Akt signaling pathway may be involved in the control of the cell cycle progression most likely through mechanisms involving the activation of forkhead transcription factors.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Ciclo Celular/fisiología , Ciclina D1/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Serina-Treonina Quinasas , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Androstadienos/farmacología , Proteínas de Ciclo Celular/genética , Núcleo Celular/enzimología , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Inhibidores Enzimáticos/metabolismo , Inhibidores Enzimáticos/farmacología , Técnica del Anticuerpo Fluorescente , Fase G1/fisiología , Regulación Neoplásica de la Expresión Génica , Células HL-60 , Humanos , Proteínas Proto-Oncogénicas c-akt , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/genética , WortmaninaRESUMEN
TRAIL is a member of the tumor necrosis factor superfamily which induces apoptosis in cancer but not in normal cells. Akt1 promotes cell survival and blocks apoptosis. The scope of this paper was to investigate whether a HL60 human leukemia cell clone (named AR) with constitutively active Akt1 was resistant to TRAIL. We found that parental (PT) HL60 cells were very sensitive to a 6 h incubation in the presence of TRAIL and died by apoptosis. In contrast, AR cells were resistant to TRAIL concentrations as high as 2 microg/ml for 24 h. Two pharmacological inhibitors of PI3K, Ly294002 and wortmannin, restored TRAIL sensitivity of AR cells. AR cells stably overexpressing PTEN had lower Akt1 activity and were sensitive to TRAIL. Conversely, PT cells stably overexpressing a constitutive active form of Akt1 became TRAIL resistant. TRAIL activated caspase-8 but not caspase-9 or -10 in HL60 cells. We did not observe a protective effect of Bcl-X(L) or Bcl-2 against the cytotoxic activity of TRAIL, even though TRAIL induced cleavage of BID. There was a close correlation between TRAIL sensitivity and intranuclear presence of the p50 subunit of NF-kappaB. Higher levels of the FLICE inhibitory protein, cFLIP(L), were observed in TRAIL-resistant cells. Both the cell permeable NF-kappaB inhibitor SN50 and cycloheximide lowered cFLIP(L)expression and restored sentivity of AR cells to TRAIL. Our results suggest that Akt1 may be an important regulator of TRAIL sensitivity in HL60 cells through the activation of NF-kappaB and up-regulation of cFLIP(L) synthesis.
Asunto(s)
Apoptosis/efectos de los fármacos , Proteínas Portadoras/metabolismo , Péptidos y Proteínas de Señalización Intracelular , Glicoproteínas de Membrana/toxicidad , FN-kappa B/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas , Factor de Necrosis Tumoral alfa/toxicidad , Proteínas Reguladoras de la Apoptosis , Proteína Reguladora de Apoptosis Similar a CASP8 y FADD , Cromonas/farmacología , Citosol/efectos de los fármacos , Citosol/fisiología , Resistencia a Antineoplásicos , Inhibidores Enzimáticos/farmacología , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Glicoproteínas de Membrana/farmacocinética , Mitocondrias/efectos de los fármacos , Mitocondrias/fisiología , Morfolinas/farmacología , Inhibidores de las Quinasa Fosfoinosítidos-3 , Proteínas Proto-Oncogénicas c-akt , Ligando Inductor de Apoptosis Relacionado con TNF , Factor de Necrosis Tumoral alfa/farmacocinéticaRESUMEN
The nuclear matrix is defined as the residual framework after the removal of the nuclear envelope, chromatin, and soluble components by sequential extractions. According to several investigators the nuclear matrix provides the structural basis for intranuclear order. However, the existence itself and the nature of this structure is still uncertain. Although the techniques used for the visualization of the nuclear matrix have improved over the years, it is still unclear to what extent the isolated nuclear matrix corresponds to an in vivo existing structure. Therefore, considerable skepticism continues to surround the nuclear matrix fraction as an accurate representation of the situation in living cells. Here, we summarize the experimental evidence in favor of, or against, the presence of a diffuse nucleoskeleton as a facilitating organizational nonchromatin structure of the nucleus.
Asunto(s)
Núcleo Celular/fisiología , Matriz Nuclear/fisiología , Animales , Núcleo Celular/metabolismo , Núcleo Celular/ultraestructura , Humanos , Matriz Nuclear/metabolismo , Matriz Nuclear/ultraestructura , Proteínas Nucleares/metabolismo , Fijación del TejidoRESUMEN
OBJECTIVE: To evaluate the placental histopathology findings in women with systemic lupus erythematosus or antiphospholipid syndrome delivered preterm. METHODS: We performed a case-control study comparing clinical outcomes and placental histopathology of 18 consecutive singleton pregnancies with systemic lupus erythematosus (n = 9) or antiphospholipid syndrome (n = 9) delivered between 24 and 37 weeks, and 54 controls matched for gestational age and type of preterm delivery (spontaneous or indicated). Placental examinations were performed by a single pathologist, and placental lesions were grouped into four categories: uteroplacental vascular pathology and related villous lesions; coagulation-related damage; chronic inflammation; and acute inflammatory lesions. Statistical analysis included the Mantel-Haenzsel or Fisher's exact test, and logistic regression, with a value of p < 0.05 or an odds ratio (OR) with 95% confidence intervals (Cl) not inclusive of unity considered significant. RESULTS: Lupus anticoagulant was positive in ten out of 18 cases and medium or high positive IgG anticardiolipin antibodies in seven out of 18. Antenatal treatment included corticosteroids (n = 9), low-dose aspirin (n = 15) and heparin (n = 8). Rates of necrotizing enterocolitis (33% vs. 0%, p < 0.001) and of perinatal mortality (33% vs. 9%, p = 0.02) were significantly different between cases and controls, and rates of birth weight < 10th centile approached statistical significance. Uteroplacental vascular lesions (OR 3.7, 95% CI 1.1, 11.7) and coagulation-related damage (OR 16.8, 95% CI 3.9, 72.6) were significantly more common among cases than controls, and rates of chronic inflammatory lesions approached significance. CONCLUSIONS: Cases of systemic lupus erythematosus and antiphospholipid syndrome delivered preterm are associated with a significant increase in placental vascular and coagulation-related lesions, which are reflected clinically by higher rates of perinatal mortality, necrotizing enterocolitis, and small-for-gestational age neonates.
Asunto(s)
Síndrome Antifosfolípido/inmunología , Autoanticuerpos/inmunología , Lupus Eritematoso Sistémico/inmunología , Enfermedades Placentarias/inmunología , Complicaciones del Embarazo/inmunología , Enfermedades Vasculares/inmunología , Adulto , Síndrome Antifosfolípido/complicaciones , Autoanticuerpos/sangre , Estudios de Casos y Controles , Femenino , Feto/inmunología , Humanos , Lupus Eritematoso Sistémico/complicaciones , Trabajo de Parto Prematuro/etiología , Enfermedades Placentarias/etiología , Embarazo , Resultado del Embarazo , Enfermedades Vasculares/etiologíaRESUMEN
Dual site antibody-base immunoassays are commonly used in clinical laboratories to quantify the CT serum concentrations as a specific and sensitive marker of medullary thyroid carcinoma (MTC). Heterophile antibodies can interfere with these assays, however, and cause erroneous results. In order to avoid this interference, immobilized and conjugated antibodies from two different animal species or immunoreactive antibody fragments, as well as the addition of non-immune globulins, are generally included among the assay reagents. We describe the case of a 73-year-old man affected by a multinodular goiter, who showed high basal CT plasma levels as measured by a monoclonal antibody based IRMA. The finding of negative results for the presence of MTC at fine needle aspiration (FNA) and the mild increase observed in plasma CT during a pentagastrin (Pg) stimulation test, suggested that the high CT levels might depend on a cross-reaction with heterophilic antibodies. In fact, after the addition of the heterophilic blocking tube (HBT) to each specimen, the CT levels markedly decreased by more than 80% (average decrease+/-SE= 87.6+/-2.668%). Such a decrease strongly suggests that in our case the routinely used F(ab')2 fragments were unable to eliminate all of the interference and that the elevated serum CT levels might have been caused by human heterophilic antibodies. In conclusion, these results indicate a novel cause of CT false positivity, suggesting that high serum CT levels, when combined with a slight increase during Pg stimulation, should be critically interpreted in view of the possible presence of heterophilic antibodies in the specimens.