RESUMEN
Targeting retroviral vectors to tumor vasculature is an important goal of cancer gene therapy. In this study, we report a novel targeting approach wherein IgG-binding peptides were inserted into the Moloney murine leukemia virus (MuLV) envelope (env) protein. The modifications on the viral env included replacement of the entire receptor binding region of the viral env with protein A (or ZZ) domains. The truncated env incorporating IgG-binding motifs (known as proteins) provided the targeting function, while the co-expressed wild-type (WT) env protein enabled viral fusion and cell entry. An anti-human VEGF receptor (Flk-1/KDR) antibody served as a molecular bridge, directing the retroviral vector to the endothelial cell. Hence, the IgG-targeted vectors bound to the Flk-1/KDR antibody which in turn bound to VEGF receptors on Kaposi sarcoma, KSY1, endothelial cells. The net effect was increased viral fusion and infectivity of IgG-bound retroviral vectors when compared to non-targeted vectors bearing WT env alone. These data provide the proof of concept that IgG-binding vector/VEGF receptor antibody complexes may be used to enhance retroviral gene delivery to activated endothelial cells.
Asunto(s)
Anticuerpos/inmunología , Proteínas Tirosina Quinasas Receptoras/inmunología , Receptores de Factores de Crecimiento/inmunología , Receptores de IgG/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Sitios de Unión/genética , Western Blotting , Línea Celular , Productos del Gen env/genética , Productos del Gen env/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/inmunología , Humanos , Inmunohistoquímica , Ratones , Virus de la Leucemia Murina de Moloney/genética , Receptores de IgG/metabolismo , Receptores de Factores de Crecimiento Endotelial Vascular , Retroviridae/genética , Proteína Estafilocócica A/genética , Proteína Estafilocócica A/metabolismo , Transfección/métodos , Células Tumorales CultivadasRESUMEN
Tumor invasion and associated angiogenesis evoke a remodeling of extracellular matrix components. Retroviral vectors bearing auxiliary matrix-targeting motifs (ie., collagen-binding polypeptides) accumulate at sites of newly exposed collagen, thus promoting tumor site-specific gene delivery. In this study, we assessed the antitumor effects of serial portal vein infusions of matrix-targeted vectors bearing a mutant cyclin G1 (dnG1) construct in a nude mouse model of liver metastasis. The size of tumor foci was dramatically reduced in dnG1 vector-treated mice compared with that in control vector- or PBS-treated animals (P = 0.0002). These findings represent a definitive advance in the development of targeted injectable vectors for metastatic cancer.
Asunto(s)
Ciclinas/genética , Neoplasias Hepáticas/secundario , Neoplasias Pancreáticas/patología , Neoplasias Pancreáticas/terapia , Células 3T3 , Animales , Línea Celular , Ciclina G , Ciclina G1 , Ciclinas/fisiología , Terapia Genética , Vectores Genéticos , Humanos , Infusiones Intravenosas , Neoplasias Hepáticas/patología , Neoplasias Hepáticas/prevención & control , Ratones , Ratones Desnudos , Vena Porta , RetroviridaeRESUMEN
There are numerous examples of the regular segregation of achiasmate chromosomes at meiosis I in Drosophila melanogaster females. Classically, the choice of achiasmate segregational partners has been thought to be independent of homology, but rather made on the basis of availability or similarities in size and shape. To the contrary, we show here that heterochromatic homology plays a primary role in ensuring the proper segregation of achiasmate homologs. We observe that the heterochromatin of chromosome 4 functions as, or contains, a meiotic pairing site. We show that free duplications carrying the 4th chromosome pericentric heterochromatin induce high frequencies of 4th chromosome nondisjunction regardless of their size. Moreover, a duplication from which some of the 4th chromosome heterochromatin has been removed is unable to induce 4th chromosome nondisjunction. Similarly, in the absence of either euchromatic homology or a size similarity, duplications bearing the X chromosome heterochromatin also disrupt the segregation of two achiasmate X chromosome centromeres. Although heterochromatic regions are sufficient to conjoin nonexchange homologues, we confirm that the segregation of heterologous chromosomes is determined by size, shape, and availability. The meiotic mutation Axs differentiates between these two processes of achiasmate centromere coorientation by disrupting only the homology-dependent mechanism. Thus there are two different mechanisms by which achiasmate segregational partners are chosen. We propose that the absence of diplotene-diakinesis during female meiosis allows heterochromatic pairings to persist until prometaphase and thus to co-orient homologous centromeres. We also propose that heterologous disjunctions result from a separate and homology-independent process that likely occurs during prometaphase. The latter process, which may not require the physical association of segregational partners, is similar to those observed in many insects, in Saccharomyces cerevisiae and in C. elegans males. We also suggest that the physical basis of this process may reflect known properties of the Drosophila meiotic spindle.
Asunto(s)
Drosophila melanogaster/genética , Meiosis , No Disyunción Genética , Animales , Femenino , Cromosoma X/fisiologíaRESUMEN
The present investigation employed a balanced placebo design to examine the effects of alcohol versus the belief that one has consumed alcohol, i.e. alcohol expectancy, on error production while operating a driving simulator. The male subjects employed were social drinkers, having no history of alcohol abuse. The alcoholic beverage consisted of vodka and tonic in the ratio of 1:5, resulting in a mean blood alcohol level of .064%. The placebo beverage consisted of water and tonic, also in the ration 1:5. Principal findings were that alcohol ingestion had a debilitating effect on certain measures of driving behavior (operation of brakes, steering), whereas the belief that one had consumed alcohol had no discernable effects. The results are discussed in relation to other findings using the balanced placebo design. It was concluded that reckless driving under the influence of alcohol, is at least partly a result of the pharmological effects of ethanol.