Dynamics of caspase-3 activation and inhibition in embryonic micromasses evaluated by a photon-counting chemiluminescence approach.

Caspases are key enzymatic components of the intracellular apoptotic machinery, and their role in mammalian systems is often studied using fluoromethylketone (FMK) inhibitors. Despite many advantages of such approach, efficiency of the inhibitor and membrane permeability speed are often questioned. This work therefore focuses on an exact evaluation of caspase-3 FMK inhibition dynamics in camptothecin-induced mesenchymal micromasses. Two parameters of caspase-3 FMK inhibitor were investigated: first, the stability of the inhibitory potential in the time course of cultivation and, simultaneously, the dynamics of caspase-3 FMK inhibition after camptothecin-induced apoptosis peak. A photon-counting chemiluminescence approach was applied for quantification of active caspase-3. The sensitivity of the photon-counting method allowed for evaluation of active caspase-3 concentration in femtogram amounts per cell. The inhibitor penetrated the cells within the first minute after its application, and the peak of caspase-3 started to decline to the blank level after 30 min. The inhibitory effect of the FMK inhibitor was unchanged during the entire 48 h of cultivation.