A recombinant MVMp of the fibrotropic strain of minute virus of mice (MVMp) expressing the chloramphenicol acetyltransferase reporter gene was used to infect a series of biologically relevant cultured cells, normal or tumor-derived, including normal melanocytes versus melanoma cells, normal mammary epithelial cells versus breast adenocarcinoma cells, and normal neurons or astrocytes versus glioma cells. As a reference cell system we used normal human fibroblasts versus the SV40-transformed fibroblast cell line NB324K. After infection, we observed good expression of the reporter gene in the different tumor cell types, but only poor expression if any in the corresponding normal cells. We also constructed a recombinant MVMp expressing the green fluorescent protein reporter gene and assessed by flow cytometry the efficiency of gene transduction into the different target cells. At a multiplicity of infection of 30, we observed

Recombinant adenoviruses were used for the expression of human amyloid precursor protein (APP) of Alzheimer's disease in primary cultures of rat cortical neurons and astrocytes. The catabolic pathways of human APP were studied 3 to 4 days after infection, when the equilibrium of APP production was reached. Although the expression of human wild type APP (WtAPP) by rat neurons induced the production of both extracellular and intraneuronal amyloid peptide (A␤), A␤ was not detected in the culture medium of rat astrocytes producing human WtAPP. Because a low ␤-secretase activity was previously reported in rodent astrocytes, we wondered whether modifications of the APP amino acid sequence at the ␤-secretase clipping site would modify the astrocytic production of A␤. Interestingly, rat astrocytes produced high amounts of A␤ after expression of human APP carrying a double amino acid substitution responsible for Alzheimer's disease in a large Swedish family (SwAPP). In both rat cortical neurons and astrocytes, the ␤-secretase cleavage of the human SwAPP occurred very early in the secretion process in a cellular compartment in which a different sorting of SwAPP and WtAPP seems unlikely. These results suggest that human WtAPP and SwAPP could be processed by different ␤-secretase activities.Alzheimer's disease is the most common form of dementia. The major hallmarks of this neurodegenerative disorder are intraneuronal neurofibrillary tangles as well as senile plaques containing an extracellular amyloid core associated with reactive astrocytes (1). The amyloid peptide (A␤) 1 is the major constituent of the amyloid deposits (2) and is derived from the amyloid precursor protein (APP) (3). The processing of human APP has been widely studied in transfected cells (4,5). These cellular models have identified different proteolytic pathways for the processing of APP. A portion of APP is cleaved by ␣-secretase within the A␤ region. This non-amyloidogenic pathway, which precludes the formation of full-length A␤, occurs during the processing of APP to the plasma membrane as early as in the trans-Golgi network (6, 7). This cleavage releases the N-terminal ectodomain of APP containing the first 17 amino acids of A␤ into the culture medium of transfected cells.Another fraction of newly synthesized APP appears at the plasma membrane. Following endocytosis of this transmembrane APP, the cleavage by ␤-secretase at the N terminus of A␤ generates a C-terminal fragment of APP that contains the entire A␤ sequence. A second protease activity called ␥-secretase cleaves this C-terminal fragment of APP to release the full-length amyloid peptide, which is detected in the extracellular medium (8).In transfected cells, the cellular trafficking of APP plays a key role in the choice between the two catabolic pathways of the protein, since the non-amyloidogenic pathway occurs during the processing of APP to the plasma membrane, whereas endocytosis of APP is required for A␤ production (9). Because the intracellular trafficking of APP in transfected cells c...

The toxicity of the suicide HSVtk gene approach is known to be targeted to DNA synthesis and, consequently, to dividing cells. This system is therefore useful for the treatment of brain tumors which contain dividing cells surrounded by a quiescent normal tissue. Adenoviruses are efficient vectors for the transfer of the HSVtk gene into the tumor but this can lead to the transduction of quiescent cells. In this study, we focused on the toxicity of the HSVtk/ganciclovir treatment for the two main cell types of the normal brain: astrocytes and neurons. Astrocytes and neurons in primary culture were infected by an adenoviral vector bearing the HSVtk gene (Ad.tk) and cells were exposed to different concentrations of ganclclovir. After 5 days of treatment, an MTT test measured a dramatic decrease in cell viability for treated astrocytes while a small decrease in cell viability was observed for neurons treated in the same experimental conditions. The differential toxicity of the HSVtk/ganciclovir treatment was also observed in cocultures of astrocytes and neurons: an immunocytochemical analysis of the treated cells showed major morphological modifications for astrocytes but not for neurons. Furthermore, our data suggest that a bystander effect is able to kill all the astrocytes while neurons from the same culture remain unaffected.

The present study involving adenovirus-mediated gene transfer to dog eyes provides an essential basis for gene therapy treatment of uveal melanoma-bearing patients.

Using a rat C6 brain tumor model, we studied the antitumor effects of Herpes simplex virus type 1 thymidine kinase (HSV-tk) gene transfer followed by ganciclovir treatment. C6 glioma cells were transfected in vitro with the HSV-tk gene, and tested for their sensitivity to ganciclovir. Although there was no surviving cell at a 30 microM ganciclovir concentration, unmodified C6 cells were not affected by the drug. For in vivo experiments, intracerebral tumors were induced in rats by stereotactic injection of 10(4) HSV-tk-modified C6 cells. Ten days later, the animals were treated with intraperitoneal injections of ganciclovir for 21 days. The tumors evolution was evaluated by high resolution magnetic resonance imaging. In 33% of the rats, the signal intensity of the tumors became heterogeneous, with development of highly hyperintense areas, and a complete tumor regression was subsequently noted. Histological examination of successfully treated tumors revealed progressive necrosis with formation of cysts. The survival time of the HSV-tk/ganciclovir treated animals was consistently increased, all rats surviving more than 30 days and 33% of them being still alive after 80 days.