RESUMEN

AIM: Gemcitabine (GEM) enters normal and tumour cells via concentrative (CNT) and equilibrative nucleoside transporters (ENT) and is subsequently deaminated to the inactive difluorodeoxyurine (dFdU) by cytidine deaminase (CDA). The aim of our study was to ascertain whether the nucleoside transporter genotype and the CDA activity phenotype can predict total GEM plasma clearance. METHODS: Forty-seven patients received GEM 1000-1250mgm(-2) i.v. over 30min. Plasma concentrations of GEM and dFdU were measured and individual pharmacokinetic profiles were determined. CDA activity was measured ex vivo in plasma samples. The two most common hENT1 and hCNT1 polymorphisms were determined from genomic DNA. RESULTS: Multivariate analysis revealed that GEM plasma clearance (CL) was positively correlated with the end of infusion dFdU : GEM ratio (P < 0.0001), which is a marker of in vivo CDA activity. The ENT1 genotype characterized by high transport capacity (G/G) and age were inversely correlated with CL (P= 0.027 and 0.048, respectively). A strong correlation was found between end of infusion GEM concentration and area under the concentration-time curve from time 0 to infinity (AUC(0,∞)) (r(2) = 0.77). CONCLUSIONS: Our results confirm the role of CDA and age on the interindividual variability of GEM CL and show the contribution of the hENT1 genotype for the first time.

Asunto(s)

Antimetabolitos Antineoplásicos/farmacología , Citidina Desaminasa/genética , Tranportador Equilibrativo 1 de Nucleósido/genética , Neoplasias/tratamiento farmacológico , Factores de Edad , Anciano , Anciano de 80 o más Años , Citidina Desaminasa/metabolismo , Desoxicitidina/análogos & derivados , Tranportador Equilibrativo 1 de Nucleósido/metabolismo , Femenino , Genotipo , Humanos , Masculino , Persona de Mediana Edad , Análisis Multivariante , Neoplasias/genética , Polimorfismo Genético , Población Blanca , Gemcitabina

RESUMEN

Tumor growth is currently viewed as a phenomenon associated with neovascularization and sustained production of angiogenic factors, but whether a transient angiogenic switch may trigger tumor growth remains unclear. Here, we report that leukemia cells (MOLT-3) were poorly angiogenic and remained dormant when injected s.c. into immunodeficient mice. However, progressive growth of lymphoid tumors was invariably recorded when irradiated angiogenic cells from Kaposi's sarcoma (KS-IMM) were locally coinjected with MOLT-3 cells or administered later. The persistence of KS-IMM cells in vivo was tracked by flow cytometry and real-time PCR analysis, and it was limited to a few days, during which angiogenesis was induced and preceded tumor growth. The engraftment of other types of poorly tumorigenic cancer cells was also greatly improved by irradiated KS-IMM cells. Moreover, short-term treatment with angiogenic factors, including basic FGF or VEGF, either given as recombinant factors or delivered by retroviral vectors, also accelerated tumor growth. These findings may emphasize that tumor angiogenesis is a process requiring a higher amount of angiogenic factors for its induction than maintenance.

Asunto(s)

Neoplasias Experimentales/irrigación sanguínea , Neovascularización Patológica/etiología , Sarcoma de Kaposi/irrigación sanguínea , Animales , Línea Celular Tumoral , Factor 2 de Crecimiento de Fibroblastos/farmacología , Humanos , Ratones , Ratones Endogámicos NOD , Ratones SCID , Trasplante de Neoplasias , Proteínas Recombinantes/farmacología , Factores de Tiempo , Trasplante Heterólogo , Factor A de Crecimiento Endotelial Vascular/farmacología

RESUMEN

Ovarian cancer represents a suitable disease for gene therapy because of the containment of neoplastic cells in the peritoneal cavity even at advanced tumor stages. The aim of this study was to investigate whether intraperitoneal administration of a lentiviral vector encoding murine interferon-alpha (LV-IFN) could have therapeutic activity in a transplantable ovarian cancer model. Multiple injections of low amounts of LV-IFN into severe combined immunodeficiency (SCID) mice bearing IGROV-1 or OC316 ovarian cancer cells elicited remarkable antitumor activity, leading to prolongation of survival in the majority of animals. A definitive cure was obtained in animals bearing PD-OVA#1 tumors, generated by injecting tumor cells isolated from the ascitic fluid of a patient into SCID mice. Interferon-alpha levels were detected in the peritoneal fluids but not in the serum of treated mice, indicating that production of the cytokine is mainly local, by both tumor and normal cells of the host. Antitumor effects were associated with a remarkable decrease in the formation of hemorrhagic ascites, an increase in ischemic tumor necrosis, and a reduction in microvessel density. In conclusion, our findings show that intracavitary IFN-alpha gene therapy, using a lentiviral vector, provides strong antitumor effects in murine models of ovarian cancer and reinforces the evidence that angiogenesis inhibition is a promising strategy for the treatment of localized tumors.

Asunto(s)

Antineoplásicos/uso terapéutico , Técnicas de Transferencia de Gen , Terapia Genética/métodos , Interferón-alfa/genética , Interferón-alfa/uso terapéutico , Neovascularización Patológica , Neoplasias Ováricas/genética , Neoplasias Ováricas/patología , Animales , Antineoplásicos/administración & dosificación , Antineoplásicos/farmacocinética , Modelos Animales de Enfermedad , Femenino , Vectores Genéticos , Humanos , Infusiones Parenterales , Interferón-alfa/administración & dosificación , Interferón-alfa/farmacocinética , Lentivirus/genética , Ratones , Ratones SCID , Neoplasias Ováricas/veterinaria , Sobrevida

RESUMEN

Within the fascinating world of chemokines, C and CX3C chemokines have long been regarded as two minor components, even though they present unique features and show less redundancy than the other chemokine families. Nevertheless, the body of data on their expression and role in various inflammatory disorders has grown in the past few years. The C chemokine family is represented by two chemokines, XCL1/lymphotactin-alpha and XCL2/lymphotactin-beta, whereas the CX3C chemokine family contains only one member, called CX3CL1/ fractalkine. In this review, we present an overview on the structure, expression and signaling properties of these chemokines and their respective receptors and examine how they contribute to inflammation and the regulation of leukocyte trafficking, as well as their potential role in the pathophysiology of human inflammatory diseases. Taken together, these data expand the biological importance of C and CX3C chemokines from that of simple immune modulators to a much broader biological role, even though their precise commitment within the framework of immune responses has still to be determined.

Asunto(s)

Quimiocinas CX3C/fisiología , Quimiocinas C/fisiología , Inflamación/fisiopatología , Síndrome de Inmunodeficiencia Adquirida/fisiopatología , Animales , Arteriosclerosis/fisiopatología , Artritis Reumatoide/fisiopatología , Receptor 1 de Quimiocinas CX3C , Quimiocinas C/química , Quimiocinas C/genética , Quimiocinas CX3C/química , Quimiocinas CX3C/genética , Enfermedad de Crohn/fisiopatología , Expresión Génica , Glomerulonefritis por IGA/fisiopatología , Rechazo de Injerto/fisiopatología , Granuloma/fisiopatología , Humanos , Hipertensión Pulmonar/fisiopatología , Pulmón/fisiopatología , Proteínas de la Membrana/fisiología , Modelos Biológicos , Neoplasias/inmunología , Neoplasias/fisiopatología , Receptores de Quimiocina/fisiología

RESUMEN

To better characterize the cellular source of lymphotactin (XCL1), we compared XCL1 expression in different lymphocyte subsets by real-time PCR. XCL1 was constitutively expressed in both PBMC and CD4(+) cells, but its expression was almost 2 log higher in CD8(+) cells. In vitro activation was associated with a substantial increase in XCL1 expression in both PBMC and CD8(+) cells, but not in CD4(+) lymphocytes. The preferential expression of XCL1 in CD8(+) cells was confirmed by measuring XCL1 production in culture supernatants, and a good correlation was found between figures obtained by real-time PCR and XCL1 contents. XCL1 expression was mostly confined to a CD3(+)CD8(+) subset not expressing CD5, where XCL1 expression equaled that shown by gammadelta(+) T cells. Compared with the CD5(+) counterpart, CD3(+)CD8(+)CD5(-) cells, which did not express CD5 following in vitro activation, showed preferential expression of the alphaalpha form of CD8 and a lower expression of molecules associated with a noncommitted/naive phenotype, such as CD62L. CD3(+)CD8(+)CD5(-) cells also expressed higher levels of the XCL1 receptor; in addition, although not differing from CD3(+)CD8(+)CD5(+) cells in terms of the expression of most alpha- and beta-chemokines, they showed higher expression of CCL3/macrophage inflammatory protein-1alpha. These data show that TCR alphabeta-expressing lymphocytes that lack CD5 expression are a major XCL1 source, and that the contribution to its synthesis by different TCR alphabeta-expressing T cell subsets, namely CD4(+) lymphocytes, is negligible. In addition, they point to the CD3(+)CD8(+)CD5(-) population as a particular T cell subset within the CD8(+) compartment, whose functional properties deserve further attention.

Asunto(s)

Antígenos CD5 , Antígenos CD8/biosíntesis , Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/metabolismo , Quimiocinas C/biosíntesis , Subgrupos Linfocitarios/inmunología , Subgrupos Linfocitarios/metabolismo , Linfocinas/biosíntesis , Sialoglicoproteínas/biosíntesis , Adulto , Complejo CD3/biosíntesis , Linfocitos T CD4-Positivos/inmunología , Linfocitos T CD4-Positivos/metabolismo , Antígenos CD5/metabolismo , Células Cultivadas , Quimiocina CXCL1 , Quimiocinas C/sangre , Quimiocinas CC/biosíntesis , Quimiocinas CXC/biosíntesis , Niño , Preescolar , Humanos , Inmunofenotipificación , Péptidos y Proteínas de Señalización Intercelular , Cinética , Linfocinas/sangre , Persona de Mediana Edad , Sialoglicoproteínas/sangre , Subgrupos de Linfocitos T/inmunología , Subgrupos de Linfocitos T/metabolismo

RESUMEN

Local gene therapy could be a therapeutic option for ovarian carcinoma, a life-threatening malignancy, because of disease containment within the peritoneal cavity in most patients. Lentiviral vectors, which are potentially capable of stable transgene expression, may be useful to vehicle therapeutic molecules requiring long-term production in these tumors. To investigate this concept, we used lentiviral vectors to deliver the enhanced green fluorescent protein (EGFP) gene to ovarian cancer cells. Their efficiency of gene transfer was compared with that of a retroviral vector carrying the same envelope. In vitro, both vectors infected ovarian cancer cells with comparable efficiency under standard culture conditions; however, the lentiviral vector was much more efficient in transducing growth-arrested cells when compared with the retroviral vector. Gene transfer was fully neutralized by an anti-VSV-G antibody, and in vitro stability was similar. In vivo, the lentiviral vector delivered the transgene 10-fold more efficiently to ovarian cancer cells growing i.p. in SCID mice, as evaluated by real-time PCR analysis of the tumors. Confocal microscopy analysis of tumor sections showed a dramatic difference at the level of transgene expression, because abundant EGFP(+) cells were detected only in mice receiving the lentiviral vector. Quantitative analysis by flow cytometry confirmed this and indicated 0.05 and 5.6% EGFP(+) tumor cells after administration of the retroviral and lentiviral vector, respectively. Injection of ex vivo transduced tumor cells, sorted for EGFP expression, indicated that the lentiviral vector was considerably more resistant to in vivo silencing in comparison with the retroviral vector. Finally, multiple administrations of a murine IFN-alpha(1)-lentiviral vector to ovarian carcinoma-bearing mice significantly prolonged the animals' survival, indicating the therapeutic efficacy of this approach. These findings indicate that lentiviral vectors deserve attention in the design of future gene therapy approaches to ovarian cancer aimed at achieving long-term expression of therapeutic genes.

Asunto(s)

Adenoviridae/genética , Terapia Genética/métodos , Lentivirus/genética , Neoplasias Ováricas/terapia , Femenino , Expresión Génica , Técnicas de Transferencia de Gen , Vectores Genéticos/genética , Vectores Genéticos/farmacocinética , Proteínas Fluorescentes Verdes , Humanos , Interferón-alfa/biosíntesis , Interferón-alfa/genética , Proteínas Luminiscentes/biosíntesis , Proteínas Luminiscentes/genética , Neoplasias Ováricas/genética , Neoplasias Ováricas/metabolismo , Neoplasias Ováricas/virología , Distribución Tisular , Transcripción Genética , Transgenes , Células Tumorales Cultivadas

RESUMEN

The enhanced green fluorescent protein (EGFP) is increasingly used as a reporter gene in viral vectors for a number of applications. To establish a system to study the activity of cis-acting cellular regulatory sequences, we deleted the viral enhancer in EGFP-carrying retroviral vectors and replaced it with cell type-specific elements. In this study, we use this system to demonstrate the activity of the human CD2 lymphoid-specific and the Tie2 endothelial cell type-specific enhancers in cell lines and in primary cells transduced by retroviral vectors. Furthermore, we compare findings obtained with EGFP as the reporter gene to those obtained replacing EGFP with d2EGFP, an unstable variant of EGFP characterized by a much shorter half-life compared to EGFP, and by reduced accumulation in the cells. d2EGFP-carrying vectors were generated at titers which were not different from those generated by the corresponding vectors carrying EGFP. Moreover, the activity of a Moloney murine leukemia virus enhancer could be readily detected following transduction of target cells with either EGFP- or d2EGFP-carrying vectors. However, the activity of the relatively weak CD2 and Tie2 enhancers was exclusively detected using EGFP as the reporter gene. These findings indicate that enhancer replacement is a feasible and promising approach to address the function of cell type-specific regulatory elements in retroviral vectors carrying the EGFP gene.

Asunto(s)

Elementos de Facilitación Genéticos/fisiología , Regulación de la Expresión Génica , Células 3T3 , Animales , Secuencia de Bases , Línea Celular , Endotelio Vascular/citología , Endotelio Vascular/metabolismo , Elementos de Facilitación Genéticos/genética , Vectores Genéticos/genética , Proteínas Fluorescentes Verdes , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Ratones , Datos de Secuencia Molecular , Provirus/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Retroviridae/genética , Eliminación de Secuencia , Linfocitos T/citología , Linfocitos T/metabolismo , Transfección