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Apoptosis induced by calcein acetoxymethyl ester in the human histiocytic lymphoma cell line U-937 GTB

2000; Elsevier BV; Volume: 60; Issue: 12 Linguagem: Inglês

10.1016/s0006-2952(00)00494-9

1873-2968

Gunnar Liminga, Petra Martinsson, Bertil Jönsson, Peter Nygren, Rolf Larsson,

Protein Kinase Regulation and GTPase Signaling

Effects of calcein acetoxymethyl ester (calcein/AM) on macromolecular synthesis, mitochondrial membrane potential, and mode of death were studied in U-937 GTB lymphoma cells. This was accomplished by measurements of (14)C-labeled thymidine and leucine incorporation, 5,5',6,6'-tetrachloro-1,1',3, 3'-tetraethylbenzimidazolyl carbocyanine iodide (JC-1) and caspase-3 activity measurements, TdT-mediated dUTP nick end labeling (TUNEL) staining, morphology, and a newly developed assay of apoptosis detection, the microculture kinetic assay (MiCK). This assay, based on absorbance measurements of cells, has been reported to reflect morphological changes in apoptosis. At 2.5 microg/mL, rapid inhibition of DNA and protein synthesis resembling that of the known inhibitors, aphidicholin and cycloheximide, was observed. Decreased mitochondrial membrane potential was evident after 1 hr of exposure and was followed by an increase in caspase-3 activity, while at 6 hr 30% of cells appeared positive with TUNEL staining. After 12 hr of exposure, viability was less than 5% as judged by morphological examination. In the MiCK assay, calcein (2.5 microg/mL) gave a rapid rise in absorbance after 3.5 hr of exposure with a peak at 5 hr, indicating maximum extent of apoptosis at that time. This was similar to the pattern generated for etoposide and doxorubicin. The results indicate that calcein, similar to cytotoxic drugs, induces a strong apoptotic response within hours of exposure.