RESUMEN
Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an indispensable component used in the treatment of acute lymphoblastic leukemia (ALL) and certain lymphoma entities. Native Erwinia chrysanthemi-derived ASNase (n-crisantaspase) has been approved as a second-line drug for treating patients exhibiting allergy syndromes to native and pegylated Escherichia coli-derived ASNase (EC-ASNase). However, it still induces hypersensitivity in at least 17 % of treated patients. In the present study, we investigated the pharmacological activity, immunogenicity and anti-leukemic activity of a new pegylated recombinant crisantaspase (PEG-r-crisantaspase). The results demonstrate that when compared to n-crisantaspase in vivo, PEG-r-crisantaspase maintains a complete depletion of plasma Asn for up to 72 h with a 50-fold lower dose. In mice receiving PEG-r-crisantaspase, specific antibodies against the enzyme were undetectable, indicating a lower immunogenicity of the pegylated enzyme. In vitro, PEG-r-crisantaspase exhibits similar cytotoxic effects (EC50 < 5 × 10(-4) U/mL for the most sensitive cell lines) to n-crisantaspase on various leukemia and lymphoma cells and was shown to be more efficient than EC-ASNase. Three repeated PEG-r-crisantaspase injections (2-20 U/Kg) prevented leukemia development in leukemia-bearing mice for 17 days and significantly prolonged animal survival to 7-12 days. Therefore, PEG-r-crisantaspase appears to be a promising drug candidate for ALL treatment and should be further explored in experimental and clinical trials.
Asunto(s)
Antineoplásicos , Asparaginasa , Animales , Anticuerpos/sangre , Antineoplásicos/química , Antineoplásicos/inmunología , Antineoplásicos/farmacología , Antineoplásicos/uso terapéutico , Asparaginasa/química , Asparaginasa/inmunología , Asparaginasa/farmacología , Asparaginasa/uso terapéutico , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dickeya chrysanthemi , Femenino , Humanos , Ratones , Polietilenglicoles/química , Leucemia-Linfoma Linfoblástico de Células Precursoras/sangre , Leucemia-Linfoma Linfoblástico de Células Precursoras/tratamiento farmacológico , Proteínas Recombinantes/química , Proteínas Recombinantes/inmunología , Proteínas Recombinantes/farmacología , Proteínas Recombinantes/uso terapéutico , Resultado del Tratamiento , Ensayos Antitumor por Modelo de XenoinjertoRESUMEN
Telomerase is essential for telomere maintenance, and its activation is thought to be a critical step in cellular immortalization and tumorigenesis. Human telomerase reverse transcriptase (hTERT) is a major component of telomerase activity. We show here that hTERT is expressed soon after lymphocyte activation and that its expression is inhibited by rapamycin, wortmannin, and FK506, which was the most potent inhibitor. These results suggest a potential role for the transcription factor nuclear factor of activated T cells (NFAT) in the regulation of hTERT expression. Five putative NFAT-binding sites were identified in the hTERT promoter. In luciferase assays, the hTERT promoter was activated by overexpressed NFAT1. Moreover, serial deletions revealed that the promoter activation was mainly due to a -40 NFAT1-binding site flanked by two SP1-binding sites. Mutation of the -40 NFAT-binding site caused a 53% reduction in the transcriptional activity of hTERT promoter. Simultaneous mutations of the -40 NFAT-responsive element together with one or both SP1-binding sites led to a more dramatic decrease in luciferase activity than single mutations, suggesting a functional synergy between NFAT1 and SP1 in hTERT transcriptional regulation. NFAT1 overexpression in MCF7 and Jurkat cell lines induced an increase in endogenous hTERT mRNA expression. Inversely, its down-regulation was induced by NFAT1 silencing. Furthermore, chromatin immunoprecipitation assay demonstrated that NFAT1 directly binds to two sites (-40 and -775) in the endogenous hTERT promoter. Thus, we show for the first time the direct involvement of NFAT1 in the transcriptional regulation of hTERT.
Asunto(s)
Regulación Enzimológica de la Expresión Génica/fisiología , Factores de Transcripción NFATC/metabolismo , Elementos de Respuesta/fisiología , Telomerasa/biosíntesis , Transcripción Genética/fisiología , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Células HeLa , Humanos , Inmunosupresores/farmacología , Células Jurkat , Activación de Linfocitos/efectos de los fármacos , Activación de Linfocitos/fisiología , Mutación , Factores de Transcripción NFATC/genética , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Telomerasa/genética , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND INFORMATION: miRNAs (microRNAs) are a class of non-coding RNAs that inhibit gene expression by binding to recognition elements, mainly in the 3' UTR (untranslated region) of mRNA. A single miRNA can target several hundred mRNAs, leading to a complex metabolic network. miR-16 (miRNA-16), located on chromosome 13q14, is involved in cell proliferation and apoptosis regulation; it may interfere with either oncogenic or tumour suppressor pathways, and is implicated in leukaemogenesis. These data prompted us to search for and validate novel targets of miR-16. RESULTS: In the present study, by using a combined bioinformatics and molecular approach, we identified two novel putative targets of miR-16, caprin-1 (cytoplasmic activation/proliferation-associated protein-1) and HMGA1 (high-mobility group A1), and we also studied cyclin E which had been previously recognized as an miR-16 target by bioinformatics database. Using luciferase activity assays, we demonstrated that miR-16 interacts with the 3' UTR of the three target mRNAs. We showed that miR-16, in MCF-7 and HeLa cell lines, down-regulates the expression of caprin-1, HMGA1a, HMGA1b and cyclin E at the protein level, and of cyclin E, HMGA1a and HMGA1b at the mRNA levels. CONCLUSIONS: Taken together, our data demonstrated that miR-16 can negatively regulate two new targets, HMGA1 and caprin-1, which are involved in cell proliferation. In addition, we also showed that the inhibition of cyclin E expression was due, at least in part, to a decrease in its mRNA stability.
Asunto(s)
Proteínas de Ciclo Celular/metabolismo , Proliferación Celular , Proteína HMGA1a/metabolismo , MicroARNs/metabolismo , Secuencia de Aminoácidos , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Línea Celular Tumoral , Regulación hacia Abajo , Regulación Neoplásica de la Expresión Génica , Proteína HMGA1a/química , Proteína HMGA1a/genética , Proteína HMGA1b/química , Proteína HMGA1b/genética , Proteína HMGA1b/metabolismo , Humanos , MicroARNs/química , MicroARNs/genética , Datos de Secuencia Molecular , Unión Proteica , Alineación de SecuenciaRESUMEN
p16(ink4a) is known to be a major inhibitor of cyclin-dependent kinases of G1-phase. Its accumulation is associated with replicative senescence. We analyzed to what extent the number of cell doublings may participate to p16(ink4a) expression in normal and malignant lymphocytes. p16(ink4a) expression, not found in normal quiescent B or T-lymphocytes, was observed after stimulation of B-lymphocytes (72 h) and T-lymphocytes (2 weeks) before the occurrence of replicative senescence markers such as senescence-associated-beta-galactosidase activity. Afterwards, in lymphocyte long-term cultures, the increase in p16(ink4a) followed the expression of features of cell ageing. In acute lymphoblastic leukemia, the analysis of the individual differences between peripheral blood and blood compartments (34 cases) showed a decrease in cell proliferation (p<0.005), in telomerase activity (p<0.0005), and in hTERT expression (p<0.04), associated with an increase of p16(ink4a) (p<0.035) in blood leukemic cells. These results support the hypothesis that (i) an increase in p16(ink4a) expression in normal lymphocytes is linked, in part, to the number of cell doublings before the occurrence of replicative senescence and (ii) this process is maintained in leukemic cell populations of numerous patients.
Asunto(s)
División Celular , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Regulación de la Expresión Génica/fisiología , Linfocitos/citología , Leucemia-Linfoma Linfoblástico de Células Precursoras/patología , Adolescente , Proliferación Celular , Senescencia Celular , Niño , Preescolar , Femenino , Humanos , Lactante , Masculino , Persona de Mediana Edad , Telomerasa/genética , Telomerasa/metabolismoRESUMEN
GA101, also known as obinutuzumab or Gazyva (Gazyvaro), is a glycoengineered type II humanized antibody that targets the CD20 antigen expressed at the surface of B-cells. This novel anti-CD20 antibody is currently assessed in clinical trials with promising results as a single agent or as part of therapeutic combinations for the treatment of B-cell malignancies. Detailed understanding of the mechanisms of GA101-induced cell death is needed to get insight into possible resistance mechanisms occurring in patients. Although multiple in vitro and in vivo mechanisms have been suggested to describe the effects of GA101 on B-cells, currently available data are ambiguous. The aim of our study was to clarify the cellular mechanisms involved in GA101-induced cell death in vitro, and more particularly the respective roles played by lysosomal and mitochondrial membrane permeabilization. Our results confirm previous reports suggesting that GA101 triggers homotypic adhesion and caspase-independent cell death, two processes that are dependent on actin remodeling and involve the production of reactive oxygen species. With respect to lysosomal membrane permeabilization (LMP), our data suggest that lack of specificity of available antibodies directed against cathepsin B may have confounded previously published results, possibly challenging current LMP-driven model of GA101 action mode.
Asunto(s)
Anticuerpos Monoclonales Humanizados/uso terapéutico , Anticuerpos Monoclonales/uso terapéutico , Catepsina B/inmunología , Reacciones Cruzadas/inmunología , Anticuerpos Monoclonales/inmunología , Anticuerpos Monoclonales/farmacología , Anticuerpos Monoclonales Humanizados/farmacocinética , Antígenos CD20/inmunología , Muerte Celular/efectos de los fármacos , Línea Celular Tumoral , Humanos , Membranas Intracelulares/metabolismo , Leucemia de Células B/tratamiento farmacológico , Lisosomas/ultraestructura , Membranas Mitocondriales/metabolismo , Permeabilidad/efectos de los fármacosRESUMEN
The transcriptional regulation of p16INK4a is essential for cellular aging and oncogenic stress response. This regulation involves p16INK4a transcriptional activators such as proteins Ets1 and 2 or E47. The binding of these proteins to INK4a promoter can be inhibited by proteins Id-1 or -4 after heterodimer formation. The transcriptional inhibition of p16INK4a includes also the transcriptional repression by Bmi-1, and an epigenetic regulation which appears complex and remains incompletely understood. Actually, INK4a promoter and exon1 present a CpG island which can be methylated on cytosines by DNA methyltransferases. This DNA methylation is preceded by the lysine 9 histone H3 methylation and by the deacetylation of histone H4 both involved in gene silencing. Indeed, RNA Helicase A might protect INK4a against methylation of CpG island. Furthermore, chromatin remodelling involving SWI/SNF complex, antagonist to Bmi-1, might activate INK4a expression. The analysis of INK4a regulation mechanisms and the comprehension of the epigenetic modulation of its expression may allow us to develop a rational use of new anti-neoplastic agents.
Asunto(s)
Envejecimiento/genética , Regulación de la Expresión Génica , Genes p16/fisiología , Neoplasias/genética , Animales , Epigénesis Genética , Humanos , Transcripción GenéticaRESUMEN
Bacterial L-asparaginase (ASNase), hydrolyzing L-asparagine (Asn), is an important drug for treating patients with acute lymphoblastic leukaemia (ALL) and natural killer (NK) cell lymphoma. Although different native or pegylated ASNase-based chemotherapy are efficient, disease relapse is frequently observed, especially in adult patients. The neo-synthesis of Asn by asparagine synthetase (AsnS) following ASNase treatment, which involves the amino acid response and mitogen-activated protein kinase kinase/extracellular signal-regulated kinase pathways, is believed to be the basis of ASNase-resistance mechanisms. However, AsnS expression has not emerged as an accurate predictive factor for ASNase susceptibility. The aim of this study was to identify possible ASNase sensitivity/resistance-related genes or pathways using a new asparaginase, namely a pegylated r-crisantaspase, with a focus on classic Asn-compensatory responses and cell death under conditions of Asn/L-glutamine limitation. We show that, for B-ALL cell lines, changes in the expression of apoptosis-regulatory genes (especially NFκB-related genes) are associated with ASNase susceptibility. The response of malignant NK cell lines to ASNase may depend on Asn-compensatory mechanisms and other cellular processes such as cleavage of BCL2A1, a prosurvival member of the Bcl-2 protein family. These results suggest that according to cellular context, factors other than AsnS can influence ASNase susceptibility.
Asunto(s)
Apoptosis/efectos de los fármacos , Asparaginasa/toxicidad , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Aspartatoamoníaco Ligasa/toxicidad , Línea Celular Tumoral , Humanos , Células Asesinas Naturales/citología , Células Asesinas Naturales/efectos de los fármacos , Células Asesinas Naturales/metabolismo , Linfoma/metabolismo , Linfoma/patología , Diana Mecanicista del Complejo 1 de la Rapamicina , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 3 Activada por Mitógenos/metabolismo , Complejos Multiproteicos/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/metabolismo , Leucemia-Linfoma Linfoblástico de Células Precursoras B/patología , Transducción de Señal/efectos de los fármacos , Serina-Treonina Quinasas TOR/metabolismo , eIF-2 Quinasa/metabolismoRESUMEN
The p16(INK4a) protein is considered to regulate the cell cycle progression mainly by inhibiting cyclin-dependent kinases (CDKs) 4 and 6 activity and leading to an arrest in G(0)/G(1). Here, we report that ectopic expression of p16(INK4a) in three p16-/pRb(Wt)/p53(Wt) human cancer cell lines MCF7, U2OS and U87 induces S-phase lengthening along with G(1) accumulation. S-phase lengthening is suggested by the discrepancy between the unchanged or even increased percentage of cells in S phase found by flow cytometry DNA content analysis and the drop of BrdU labelling, and demonstrated by IdU/BrdU double labelling. p16(INK4a) induces a profound decrease in the CDK4/6-mediated pRb phosphorylation on Ser-807/811, a downregulation of CDK2 and CDK1 protein expression independently of G(1) accumulation, and a decrease in Thr/Pro phosphorylation in part carried out by CDKs. In MCF7 cells, overexpression of the p16 G101W mutant, which is unable to inhibit CDK4/6 kinase activity and shows a modified subcellular localization, does not provoke the S-phase lengthening and the inhibition of Ser807/811-pRB and of Thr/Pro phosphorylation as wild-type p16(INK4a) does. Our results demonstrate that p16(INK4a) induces a S-phase lengthening independently of cellular origin. The CDK4/6 kinase activity inhibition together with the reduced expression of CDK2 and CDK1 acting downstream of G(1) phase may prevent cells from any possible kinasic compensatory mechanisms, and thus lead to a cell cycle progression inhibition.
Asunto(s)
Inhibidor p16 de la Quinasa Dependiente de Ciclina/metabolismo , Proteína de Retinoblastoma/metabolismo , Fase S , Proteína p53 Supresora de Tumor/metabolismo , Sustitución de Aminoácidos , Línea Celular Tumoral , Quinasa 2 Dependiente de la Ciclina/metabolismo , Quinasa 4 Dependiente de la Ciclina/antagonistas & inhibidores , Quinasa 4 Dependiente de la Ciclina/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Fase G1 , Humanos , Mutagénesis Sitio-Dirigida , FosforilaciónRESUMEN
Indirubin-3'-monoxime (IO) is a derivative of indirubin, an active compound of a traditional Chinese medicinal recipe used to treat various inflammatory and malignant diseases. The main in vitro targets of IO (i.e. cyclin dependent kinases, glycogen synthase kinase-3beta, Stat 3 and Aryl hydrocarbon receptor) are regulators of lymphocyte activation. We investigated the interest of IO and its derivative 6-bromo-indirubin-3'oxime (6BIO) for inhibiting the growth of malignant lymphoid cells. IO (1-20 microM) induced cell cycle inhibition and cell death in malignant B- (IM9, Reh6) and T- (Jurkat, CEM-T) lymphoid cell lines depending to cell type, doses, and duration of treatment. IO and 6BIO (10 microM) treatment for 24 and 48 h were compared: 6BIO treatment resulted in a stronger cytotoxicity and more profound inhibition of cell proliferation. Taken together, these results showed that IO and, moreover, its derivative 6BIO may be potent antiproliferative agents in malignant lymphoid cells.
Asunto(s)
Apoptosis/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Indoles/farmacología , Linfocitos/efectos de los fármacos , Oximas/farmacología , Antibióticos Antineoplásicos/farmacología , Autofagia/efectos de los fármacos , Western Blotting , Ciclo Celular/efectos de los fármacos , Proteínas de Ciclo Celular/metabolismo , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Ciclina E/metabolismo , Quinasa 6 Dependiente de la Ciclina/metabolismo , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/metabolismo , Inhibidor p27 de las Quinasas Dependientes de la Ciclina , Relación Dosis-Respuesta a Droga , Citometría de Flujo , Humanos , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células Jurkat , Linfocitos/metabolismo , Linfocitos/patología , Factores de TiempoRESUMEN
Normal lymphocytes represent examples of somatic cells that are able to induce telomerase activity when stimulated. As previously reported, we showed that, during lymphocyte long-term culture and repeated stimulations, the appearance of senescent cells is associated with telomere shortening and a progressive drop in telomerase activity. We further showed that this shortening preferentially occured at long telomeres and was interrupted at each stimulation by a transitory increase in telomere length. In agreement with the fact that telomere uncapping triggers lymphocyte senescence, we observed an increase in gamma-H2AX and 53BP1 foci as well as in the percentage of cells exhibiting DNA damage foci in telomeres. Such a DNA damage response may be related to the continuous increase of p16(ink4a) upon cell stimulation and cell aging. Remarkably, at each stimulation, the expression of shelterin genes, such as hTRF1, hTANK1, hTIN2, hPOT1 and hRAP1, was decreased. We propose that telomere dysfunction during lymphocyte senescence caused by iterative stimulations does not only result from an excessive telomere shortening, but also from a decrease in shelterin content. These observations may be relevant for T-cell biology and aging.
Asunto(s)
Linfocitos T/ultraestructura , Telómero/ultraestructura , Anciano , Animales , Ciclo Celular/fisiología , División Celular/fisiología , Células Cultivadas , Senescencia Celular/genética , Senescencia Celular/inmunología , Senescencia Celular/fisiología , Inhibidor p16 de la Quinasa Dependiente de Ciclina/biosíntesis , Inhibidor p16 de la Quinasa Dependiente de Ciclina/genética , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/biosíntesis , Inhibidor p21 de las Quinasas Dependientes de la Ciclina/genética , Regulación hacia Abajo , Histonas/sangre , Humanos , Inmunofenotipificación , Péptidos y Proteínas de Señalización Intracelular/genética , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Activación de Linfocitos , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Complejo Shelterina , Linfocitos T/citología , Linfocitos T/inmunología , Linfocitos T/metabolismo , Telomerasa/genética , Telomerasa/metabolismo , Telómero/metabolismo , Proteínas de Unión a Telómeros/biosíntesis , Proteínas de Unión a Telómeros/genética , Proteína 1 de Unión al Supresor Tumoral P53RESUMEN
Previously, we designed a ribozyme that targets the H-ras oncogene at the 12th codon mutation site (Chang et al., 1997). Ribozymes have antisense molecule and site-specific ribonuclease potential. In this study, an adenoviral vector was used to transduce the H-ras ribozyme into laryngeal cancer cells (HEp-2). This served to downregulate the H-ras gene expression in which this ribozyme performed antisense activity due to HEp-2 cells containing wild-type alleles in the 12th H-ras codon. Together, our data demonstrated that the recombinant adenovirus encoding H-ras ribozyme can be broadly regarded as a cytotoxic gene therapy in laryngeal cancer cells regardless of containing wild-type or mutant ras gene. In addition, the mechanism through which the H-ras ribozyme inhibited tumor growth was apoptosis and involved both caspase- and mitochondria-mediated pathways. The activators caspase-8 and -9 as well as the effector caspase-3 in the induction phase of apoptosis and the substrate PARP of caspase-3 in the execution phase were activated 48h following the H-ras ribozyme treatment. Mitochondrial events characterized by the production of superoxide anion and the release of cytochrome c started at 24h. Mitochondrial transmembrane potential loss occurred 48h after the ribozyme treatment. However, Bcl-2 delayed cytochrome c release to the cytosol, but it could not protect the apoptosis effect, suggesting that cytochrome c release from mitochondria may not play a role in H-ras ribozyme-induced apoptosis.