Assessment of caspase-4 released free AFC by RP-HPLC and fluorescence detection.

A simple RP-HPLC method based on fluorescence detection was developed for the quantitation of 7-amino-4-trifluoro methylcoumarin (AFC) in cell lysates from JEG-3 choriocarcinoma cells for determination of caspase-4 activity. In contrast to the established methods of AFC detection using a fluorescence microplate reader or using a fluorescence photometer, the separation of AFC-signals from interfering fluorescence signals by a reversed phase column affords more precise quantitation of released AFC. This can be important for analyses of cell lysates with low caspase activity or experimental series with marginal differences among samples. By applying this new method, a linear dynamic range of 40pmol/mL to 3nmol/mL with a correlation coefficient of 0.9996 was achieved. Due to the short retention time ( approximately 7min), the determination of AFC by RP-HPLC under isocratic conditions requires small amounts of samples (50microL injection volume), and allows increased sample throughput. This method should be easily applied with little or no modification to other caspase assays by using the same fluorophore.

Cell-specific RNA interference by peptide-inhibited-peptidase-activated siRNAs.

The use of chemically-synthesized short interfering RNAs (siRNAs) is the key method of choice to manipulate gene expression in mammalian cell cultures and in vivo. Several previous studies have aimed at inducing cell-specific RNA interference (RNAi) in order to use siRNA molecules as therapeutic reagents. Here, we used peptide-inhibited siRNAs that were activated after cleavage by cell-specific peptidases. We show that siRNAs with bound peptide at the antisense strand could be activated in target cells and were able to induce RNAi in a cell-specific manner. Green Fluorescent Protein (GFP) and Signal Transducer and Activator of Transcription (STAT)-3 gene expression were selectively reduced in a JEG-3 human choriocarcinoma cell line expressing the activating enzyme caspase-4, whereas the effect was absent in HEK cells which lacked the enzyme. In JEG-3 cells, reduction of STAT3 gene expression by conventional and peptide-inhibited siRNA led to a decrease in cell proliferation. This suggests that peptide-inhibited siRNAs provide improved cell specificity and offers new opportunities for their therapeutic use.