The use of recombinant adeno-associated viral vectors for the transduction of epithelial tumor cells.

Using hight-titer recombinant adeno-associated viral vectors (rAAV), we have investigated the feasibility of cancer vaccines from tumor explants. In a first set of experiments, rAAV vectors expressing firefly luciferase reporter genes were used to transduce different human tumor cell lines. At day three post transduction, all of the human tumor cell lines tested showed high levels of luciferase expression. To further evaluate rAAV-mediated gene transfer efficiency into primary tumor cells, we transduced freshly isolated tumor cells from malignant melanoma and ovarian carcinoma patients. As a remarkable result, reporter gene expression in primary tumor cells was significantly higher than in the tested established tumor cell lines. These data could also be reproduced with a rAAV/lacZ vector, since the portion of successfully transduced primary tumor was higher than 90%. Taken together, our data demonstrate that rAAV-mediated gene transfer is a very efficient method for the transduction of freshly isolated human tumor cells and may allow the generation of potent autologous cancer vaccines.

The mitochondrial aspartate/glutamate and ADP/ATP carrier switch from obligate counterexchange to unidirectional transport after modification by SH-reagents.

The influence of various SH-reagents on the aspartate/glutamate carrier was investigated in the reconstituted system. When liposomes carrying partially purified carrier protein were treated with 5,5'-dithiobis(2-nitrobenzoic acid) or N-ethylmaleimide, antiport activity was strongly reduced. Several mercury compounds exerted a dual effect. They completely blocked the antiport and, in addition, induced an efflux pathway for internal aspartate. The maximum rate of this unidirectional flux was comparable to the original antiport activity. Induction of efflux always was coupled to inhibition of antiport. Efflux was neither due to unspecific leakage of proteoliposomes nor to a possible contamination by porin, but depended on active carrier protein, as elucidated by the sensitivity to proteinases and protein-modifying reagents. Besides efflux of aspartate, HgCl2 and mersalyl also induced a slow efflux of ATP from liposomes carrying coreconstituted aspartate/glutamate and ADP/ATP carrier. The two efflux activities could be discriminated taking advantage of the differential effectiveness of several inhibitors and proteinases. Although basic carrier properties were changed by the applied mercurials (Dierks, T., Salentin, A. and Krämer, R. (1990) Biochim. Biophys. Acta 1028, 281), aspartate and ATP efflux could clearly be correlated with the aspartate/glutamate and the ADP/ATP carrier, respectively. When purifying these two translocators the respective efflux activity copurified with the antiporter, thus elucidating that the two different transport functions are mediated by the same protein. These results argue for a participation of the aspartate/glutamate and the ADP/ATP carrier in the generally observed increase of mitochondrial permeability after treatment with SH-reagents.

Characterization of woodchuck hepatitis virus DNA and RNA in the hepatocellular carcinomas of woodchucks.

Integration and transcription of woodchuck hepatitis virus DNA were studied by Southern and Northern blot analysis in 26 hepatocellular carcinomas and in adjacent nontumor tissue of woodchucks (Marmota monax). All liver tissue chronically infected with woodchuck hepatitis virus contained various amounts of episomal and replicative forms of woodchuck hepatitis virus DNA: episomal and replicative forms of woodchuck hepatitis virus DNA without integration were found in six tumors, episomal and integrated woodchuck hepatitis virus DNA was observed in 18 tumors and exclusively integrated woodchuck hepatitis virus DNA was found in two tumors. In most tumors and in all of the liver tissues chronically infected with woodchuck hepatitis virus, two major woodchuck hepatitis virus RNA species (3.7 and 2.1 kilobases) were detected. In tumors of two other animals (HW76 and HW89) with integrated woodchuck hepatitis virus DNA, only single major transcripts of 3.5 and 2.5 kilobases, respectively, were detected. Hybridization with subcloned woodchuck hepatitis virus DNA probes showed that both aberrant transcripts lacked the C gene and a part of the pre-S1 gene; characterization of corresponding integrated woodchuck hepatitis virus DNA sequences revealed that the C gene was deleted in one tumor, although not in the other. In agreement with the nucleic acid data, we found expression of core protein by Western blotting only in chronically infected liver tissue of these animals, but not in the corresponding tumors. Deletion of the C gene in mRNA may be due to deletion of this gene in the integrated sequences or due to transcriptional regulation.

Functional reconstitution of carrier proteins by removal of detergent with a hydrophobic ion exchange column.

A method has been developed for the functional reconstitution of membrane proteins in phospholipid vesicles. This method is an extension of a previously published procedure (Ueno, M., Tanford, C. and Reynolds, A. (1984) Biochemistry 23, 3070-3076) for the formation of unilamellar vesicles from mixed micelles of egg phosphatidylcholine and dodecyl octaoxyethylene ether. Mixed micelles are formed from detergent-solubilized protein and egg-yolk phospholipid vesicles. These micelles are subjected to repeated passage through small columns filled with Amberlite XAD-2 beads. Several carrier proteins from the inner mitochondrial membrane have been reconstituted in this way; experimental data are shown for the aspartate/glutamate carrier and the ADP/ATP carrier. Certain parameters proved to be important for optimal efficiency of reconstitution: the ratio of detergent/phospholipid in the mixed micelles, the concentration of phospholipid during the hydrophobic chromatography, the ratio of phospholipid/protein, (d) the ratio of detergent/Amberlite XAD 2 beads, the number of column passages, and the type of detergent. After optimization of these parameters, phospholipid vesicles with a diameter of about 150 nm were obtained. The main advantage of this procedure, however, lies in the fact that high amounts of membrane protein can be incorporated into the phospholipid vesicles, i.e. up to 15% (w/w).

Isolation and functional reconstitution of the aspartate/glutamate carrier from mitochondria.

The aspartate/glutamate carrier from beef heart mitochondria was solubilized by the detergent dodecyloctaoxyethylene ether (C12E8) in the presence of high concentrations of ammonium acetate. After separating the bulk amount of contaminating proteins by differential solubilization and by hydroxyapatite centrifugation chromatography, the aspartate/glutamate carrier was purified by high-performance liquid chromatography on hydroxyapatite. During the purification process, the aspartate/glutamate carrier as well as other transport proteins was identified by functional reconstitution. In sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis the purified aspartate/glutamate carrier protein appears as a protein band with an apparent molecular mass of 68 kDa. Small amounts of some contaminating proteins mainly at 31 kDa were also found. Since the ADP/ATP carrier has an apparent molecular mass of 31 kDa in SDS-gel electrophoresis, possible contamination by the nucleotide carrier was analyzed by immunological methods. The enrichment of the aspartate/glutamate carrier--based on functional reconstitution--was about 570-fold, the protein yield was 0.1%.

Activation of the ADP/ATP carrier from mitochondria by cationic effectors.

The ADP/ATP carrier from the mitochondrial inner membrane was found to be influenced by cationic substances from the hydrophilic surroundings. Under low-ionic-strength conditions, addition of these cationic effectors fully activated the reconstituted adenine nucleotide translocator. The list of activators included divalent cations, polyamines, peptides and cationic proteins. The minimum requirement for an activator to be effective was the presence of at least two positive net charges, regardless of the size of the molecule. Cationic molecules were not activating when an intramolecular charge compensation was possible or when the two charges were too far apart from one another. The affinity of these activators varied from several hundred microM (diaminoalkanes, divalent cations) to 1 microM (cytochrome c, spermine) and even down to a few nM (polylysine). The activation by cations was fully reversible and was not due to fusion processes. It was not mediated by an interaction with the anionic substrates ADP and ATP, nor by interaction with the liposomes. The stimulation could directly and functionally be correlated to the reconstituted carrier protein. Activation was not observed in intact mitochondria, but could be demonstrated when the outer mitochondrial membrane had been removed by treatment with digitonin. These mitoplasts were stimulated by polycations similar to the ADP/ATP carrier in the reconstituted system.