RESUMO
Described herein is a method for assessing apoptosis in tissue culture cells that is facile, highly informative, readily quantifiable, and generally applicable. As in conventional DNA-based flow cytometric analysis, the approach utilizes fixed, propidium iodide-stained cells. However, this particular application employs correlated two-parameter analyses of log(10)DNA fluorescence signals versus log(10) side-scatter (SS) signals of cells undergoing apoptosis. The features and the advantages of this approach, which provides substantially more information than is otherwise available from conventional analysis, are demonstrated in experiments monitoring the effects of the antineoplastic agents cisplatinum (cisP) and camptothecin (CAM) on a variety of cultured cell types, including epithelial cells (SW480; human colon carcinoma), fibroblasts (rat2 and 3T3; normal fibroblast lines), and cells of myeloid origin (CCRF-CEM; human leukemia). The utility of the technique is demonstrated further in a series of experiments with the histidine analogue L-histidinol. These experiments reveal that L-histidinol is pro-apoptotic in CCRF-CEM cells, accentuates markedly the apoptotic response otherwise elicited by CAM in murine B16f10 melanoma cells and inhibits CAM-induced apoptosis in normal 3T3 fibroblasts.
