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


CONCLUSIONS
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.
Werner
Jens Martin
JM
Institute of Pharmacology & Toxicology, University of Göttingen, Göttingen, Germany. jens.werner@klinik.uni-regensburg.de
Steinfelder
Hans Jürgen
HJ
D000230:Adenocarcinoma; D000970:Antineoplastic Agents / Q000008:administration & dosage / Q000494:pharmacology*; D017209:Apoptosis / Q000187:drug effects; D053148:Caspase 3 / Q000235:genetics / Q000378:metabolism*; D003110:Colonic Neoplasms; D004305:Dose-Response Relationship, Drug; D004789:Enzyme Activation / Q000187:drug effects; D005472:Fluorouracil / Q000008:administration & dosage / Q000494:pharmacology*; D005822:Genetic Vectors; D006801:Humans; D007700:Kinetics; D008856:Microscopy, Fluorescence / Q000379:methods*; D013343:Styrenes / Q000008:administration & dosage / Q000494:pharmacology*; D013997:Time Factors; D014162:Transfection; D014407:Tumor Cells, Cultured
D000970:Antineoplastic Agents; D013343:Styrenes; C011955:beta-nitrostyrene; D053148:Caspase 3; D005472:Fluorouracil