THE MORPHOLOGICAL AND SPECTRAL PHENOTYPE OF APOPTOSIS IN HELA-CELLS VARIES FOLLOWING EXPOSURE TO UV-C AND THE ADDITION OF INHIBITORS OF ICEAND CPP32

Numerous extra-and intracellular factors; including UV radiation, can initiate a programme of cell death by apoptosis. While apoptosis is co mmonly defined morphologically, the relationships between morphology a nd molecular events are not well established. To investigate these rel ationships in HeLa cells, eight morphometric criteria for cell prolife ration and damage and 10 criteria for apoptotic phenotype were examine d using light microscopy, and corroborated by ultrastructure and spect ral imaging. They were identified (1) during a time course after irrad iation with 0, 10 or 30 J/m(2) UV-C; (2) after separation of apoptotic from normal cells on a Percoll gradient; and (3) after irradiation wi th UV-C plus perturbation of the apoptotic pathway by treatment with i nhibitors of two caspases, ICE and CPP32. The number of cells in apopt osis increased in a dose-dependent manner after UV-C treatment. Centri fugation of irradiated cells on a Percoll gradient increased the colle ction of apoptotic cells tenfold. The stereotypical apoptotic phenotyp e, in which cells have deep cytoplasmic blebbing and highly condensed DNA, comprised only a few percent of all apoptoses, and was rarely see n in groups receiving caspase inhibitors. The most common apoptotic ph enotype was a rounded cell with large spherical nucleolus and associat ed DNA. After treatment with UV-C plus inhibitors the apoptotic index was decreased by about 30% compared to UV-C radiation alone. These apo ptotic cells had dark spherical cytoplasm with small blabs, greatly in creased numbers of cytoplasmic ribosomes, abundant nucleolar material with a large separate granular component, and chromatin condensed at t he nuclear membrane. Using the technique of spectral imaging, it was f ound that the spectrum obtained from the granular component of the nuc leolus, which was elevated in apoptotic cells treated with UV-C plus i nhibitors, was similar to the dense accumulation of ribosomes in the a poptotic cytoplasm. The data indicate that spectral imaging may be a u seful tool for identifying and characterizing variations in the apopto tic process, and that the caspase inhibitors used here do not complete ly abolish UV-C induced apoptosis, but rather alter its incidence and progression.