A microscopic technique to study kinetics and concentration--response of drug-induced caspase-3 activation on a single cell level.

摘要

INTRODUCTION: Induction of apoptosis is perceived as the main intention of drug regimens for tumour therapy. Thus, the concentration- and time-dependence of drug-induced apoptosis should be carefully evaluated for experimental as well as for standard anti-tumour agents. A main feature of apoptosis is the activation of caspases which is a specific phenomenon of the individual cell. Since caspase-3 is one of the key enzymes we developed a fluorescence microscopy technique to detect caspase-3 activity on the single cell level. The results obtained with this technique were compared to a biochemical procedure investigating caspase-3 activation in a cell population. METHODS: For the single cell assay LoVo adenocarcinoma cells were stably transfected with the vector pCaspase3-Sensor. The activated caspase-3 cleaves the cytosolic fusion protein and its EYFP part translocates into the nucleus. Thus, each individual apoptotic cell displays a labelled nucleus and affected cells can be visually quantified by the use of a fluorescence microscope. To study kinetics and concentration-response of drug-induced caspase-3 activation we exposed cells towards trans-beta-nitrostyrene, a rapidly acting experimental agent, as well as towards 5-fluorouracil, a standard agent with slow pro-apoptotic kinetics. RESULTS: Viability tests confirmed a comparable cytotoxic sensitivity of the transfected LoVo(EYFP) and the parental non-transfected cells towards trans-beta-nitrostyrene, a rapidly acting experimental agent, as well as towards 5-fluorouracil, a standard agent with slow kinetics. When comparing both caspase-3 assays at the same time points and concentrations of both agents, the new microscopic assay proved to be more sensitive, especially at lower concentrations and at earlier time points. DISCUSSION: Thus, visual detection of caspase activation in each affected cells enabled a more careful evaluation of the concentration- and time-dependence of drug-induced apoptosis which should also be useful with other experimental or standard agents or with other tumour cells.