| Apoptosis is an active, energy-dependent form of cell death different from necrosis, which is passive and energy-independent, wherein cells respond catastrophically to external insults. The aim of this study was to investigate the changes that occur when leukemic T-lymphocytes (CEM and Jurkat) are subjected to low micromolar doses of a membrane-active protein cytotoxin (CTX). Since CTX causes permeability changes that occur rapidly but are typically complete within 4–6 hours, we investigated ten different features of cell death during the first 2.5 hours of exposure to CTX. Additionally, we investigated the effects of external calcium and calcium channel blockers upon these parameters.; Consistent with progression to apoptosis, CEM cells lost ∼80% of their ATP, while Jurkat cells lost only ∼16%. External glucose partially restored ATP, even in the presence of oligomycin. A fluorescent caspase substrate based on z-VAD-fmk showed that caspases were activated. Flow cytometry indicated significant shrinkage in cell sizes. Externalized phoshpatidylserine (PS) was observed on plasma membrane in both cell lines. As [CTX] increased, there was translocation of the pro-apoptotic protein bax from the cytoplasm to the mitochondria and release of cytochrome-c from mitochondria into cytoplasm. The amount of mitochondrial bcl-2 in both cell lines increased with CTX concentration over time. Changes in the cell cycle did not occur. In only CEM cells, a loss of mitochondrial transmembrane potential (Δψm) occurred. Two characteristics typical of apoptosis noticeably absent were condensation of nuclear chromatin and formation of apoptotic bodies (DNA sub G0/G 1). These features, however, may occur later in the process of cell death. Consistent with necrosis, cells become permeable to calcium, ethidium bromide, propidium iodide, and trypan blue. Membrane blebbing occurred in both cell types, and at 6 μM CTX, extensive loss of membrane integrity and cell lysis was observed. External calcium (5 mM) inhibited only 30% of CTX binding, yet protected both cell lines from apoptosis and necrosis; nickel protected only against necrosis; verapamil did not protect against either form of cell death. We believe that low doses of CTX cause cells to initiate apoptotic processes, which become overwhelmed by subsequent damage to the plasma membrane, resulting in necrotic cell death. |
