Subcellullar Localization Of Caspase-3 Activation Correlates With Changes In Apoptotic Morphology In Molt-4 Leukemia Cells Exposed To X-Ray Irradiation

Caspase-3 is a critical effector caspase for apoptosis, which cleaves proteins, including cytoskeletal and associated proteins, kinases, and members of the Bcl-2 Family of apoptosis-related proteins. This leads to changes in apoptotic morphology, such as membrane externalization and cytoplasm and nuclear condensation. It has been reported that procaspase-3 is activated in the cytosol. However, it remains obscure how caspase-3 activation correlates to serial changes in cell morphology during apoptosis. The current study was therefore undertaken to assess the relationship between caspase-3 activation and its subcellular localization and alterations in apoptotic morphology in MOLT-4 human leukemia cells exposed to X-ray irradiation. Fluorescence labeled inhibitor of caspases (FLICA) was used to detect caspase-3 activity in apoptotic cells in this project; cell morphology and caspase-3 sub-localization were determined by confocal microscopy. Our data showed that MOLT-4 cells presented typical morphological changes in apoptosis, such as membrane reversion, DNA fragmentation, and Formation of apoptotic cell bodies Following 10 Gray (Gy) of X-ray irradiation. Caspase-3 was activated 2 h after X-ray irradiation, and its activity increased markedly after 4-6-h exposure. Membrane reversion in MOLT-4 leukemia cells was detected by Annexin V assay at 4 h following X-ray irradiation, 2 h after the elevated caspase-3 activity was measured. Cytologically, activation of caspase-3 was first observed close to the inside surface 4 the cellular membrane, then transferred to the cytoplasm, and finally translocated to the nuclear re-ion. We conclude that caspase-3 is activated in MOLT-4 cells following exposure to X-rays, and that the enhanced caspase-3 activity and its sub-localization shifting is correlated to changes in apoptotic morphology. The spatial shift of activated caspase-3 in X-ray-induced apoptotic MOLT-4 leukemia cells is a process of crucial importance for apoptosis.