OBJECTIVE— We studied how glucose and ATP-sensitive K + (K ATP ) channel modulators affect α-cell [Ca 2+ ] c . RESEARCH DESIGN AND METHODS— GYY mice (expressing enhanced yellow fluorescent protein in α-cells) and NMRI mice were used. [Ca 2+ ] c , the K ATP current (I KATP , perforated mode) and cell metabolism [NAD(P)H fluorescence] were monitored in single α-cells and, for comparison, in single β-cells. RESULTS— In 0.5 mmol/l glucose, [Ca 2+ ] c oscillated in some α-cells and was basal in the others. Increasing glucose to 15 mmol/l decreased [Ca 2+ ] c by ∼30% in oscillating cells and was ineffective in the others. α-Cell I KATP was inhibited by tolbutamide and activated by diazoxide or the mitochondrial poison azide, as in β-cells. Tolbutamide increased α-cell [Ca 2+ ] c , whereas diazoxide and azide abolished [Ca 2+ ] c oscillations. Increasing glucose from 0.5 to 15 mmol/l did not change I KATP and NAD(P)H fluorescence in α-cells in contrast to β-cells. The use of nimodipine showed that L-type Ca 2+ channels are the main conduits for Ca 2+ influx in α-cells. γ-Aminobutyric acid and zinc did not decrease α-cell [Ca 2+ ] c , and insulin, although lowering [Ca 2+ ] c very modestly, did not affect glucagon secretion. CONCLUSIONS— α-Cells display similarities with β-cells: K ATP channels control Ca 2+ influx mainly through L-type Ca 2+ channels. However, α-cells have distinct features from β-cells: Most K ATP channels are already closed at low glucose, glucose does not affect cell metabolism and I KATP , and it slightly decreases [Ca 2+ ] c . Hence, glucose and K ATP channel modulators exert distinct effects on α-cell [Ca 2+ ] c . The direct small glucose-induced drop in α-cell [Ca 2+ ] c contributes likely only partly to the strong glucose-induced inhibition of glucagon secretion in islets.
Recombinant adeno-associated virus (rAAV) vectors have emerged as vehicles for gene therapy. In addition, anti-neoplastic properties have been attributed to wild-type AAV. To take advantage of both features and to overcome technical problems associated with rAAV preparation, we developed a production method in which rAAV particles are amplified in an infectious cycle in the presence of wtAAV. This results in a 103−104-fold amplification of rAAV input particles. rAAV-GFP particles generated by this method were used to transduce ovarian cancer cell lines to evaluate their potential in ovarian cancer gene therapy, in comparison to a rAd-GFP vector. The transduction efficiency of NIH-OVCAR3, MDAH 2774 and SKOV3 cells with rAAV-GFP particles was low (< 1%) and did not improve by increasing the number of particles/cell. Repeated administration and continued exposure of NIH-OVCAR3 and MDAH 2774 improved transduction to over 3%. In contrast, these cell lines were more efficiently transduced by rAAV-GFP in the presence of adenovirus (~15%) and by rAd-GFP (> 50%). These results indicate that in contrast to rAd vectors, rAAV particles are not suitable for therapeutic gene transfer in ovarian cancer cells unless efficient help can be provided to mediate ss to ds DNA conversion.© 2001 Cancer Research Campaign http://www.bjcancer.com
Toxicity associated with plasmid/liposome transfection of eucaryote cells has been attributed to the inherent toxicity of cationic lipid formulations and also to bacterial contaminants of plasmid DNA preparations, such as lipopolysaccharides (LPS). Certain plasmid preparations were observed to trigger apoptosis in DNA/liposome transfected OVCAR3 human epithelial ovarian cancer cells. In contrast, AZ224 and SKOV3 cells were unaffected under the same conditions. Agarose gel electrophoresis with recovery of the plasmid DNA removed the toxic component, but not purification by phenol/chloroform extraction or isopicnic CsCl ultracentrifugation. The toxicity of individual preparations correlated with the concentration of bacterial LPS. However, polymixin B could not neutralise the toxicity and neither could the effect be reproduced by the addition of bacterial LPS to non-toxic plasmid preparations. Surprisingly, the conditioned medium of OVCAR3 cells undergoing apoptosis was found to kill non-transfected OVCAR3 cells but not AZ224 or SKOV3 cells. This observation illustrates the possibility that unpredictable contaminants of bacterial plasmid preparations are able to cause cell death in the context of plasmid/liposome transfection in a cell-type specific way. It emphasizes the importance of achieving maximal plasmid DNA purity when performing DNA transfection experiments that focus on cell survival.
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