APOPTOSIS AND ACCIDENTAL CELL-DEATH IN CULTURED HUMAN KERATINOCYTES AFTER THERMAL-INJURY

The respective roles of apoptosis and accidental cell death after ther mal injury were evaluated in normal human epidermal keratinocytes. By coupling the LIVE/DEAD fluorescence viability assay with the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) m ethod and ultrastructural morphology, these two processes could be dis tinguished. Cells were grown on glass coverslips with a microgrid patt ern so that the results of several staining procedures performed seque ntially could be visualized in the same cells after heating at tempera tures of up to 72 degrees C for 1 second. After exposure to temperatur es of 58 to 59 degrees C, cells died predominantly by apoptosis; viabl e cells became TUNEL positive, indicating degradation of DNA. After ex posure to temperatures of 60 to 66 degrees C, both TUNEL-positive viab le cells and TUNEL-positive nonviable cells were observed, indicating that apoptosis and accidental cell death were occurring simultaneously . Cells died almost immediately after exposure to temperatures above 7 2 degrees C, presumably from heat fixation, The fluorescent mitochondr ial probe MitoTracker Orange indicated that cells undergoing apoptosis became TUNEL positive before loss of mitochondrial function. Nucleoso mal fragmentation of DNA analyzed by enzyme-linked immunosorbent assay and gel electrophoresis occurred after exposure to temperatures of 58 to 59 degrees C. The characteristic morphological findings of cells u ndergoing apoptosis, by transmission electron microscopy, included cel lular shrinkage, cytoplasmic budding, and relatively intact mitochondr ia. Depending on temperature and time of exposure, normal human epider mal. keratinocytes may die by apoptosis, accidental cell death, or hea t fixation.