The morphological and spectral phenotype of apoptosis in HeLa cells varies following exposure to UV-C and the addition of inhibitors of ICE and CPP32.

Abstract

Numerous extra- and intracellular factors, including UV radiation, can initiate a programme of cell death by apoptosis. While apoptosis is commonly defined morphologically, the relationships between morphology and molecular events are not well established. To investigate these relationships in HeLa cells, eight morphometric criteria for cell proliferation and damage and 10 criteria for apoptotic phenotype were examined using light microscopy, and corroborated by ultrastructure and spectral imaging. They were identified (1) during a time course after irradiation with 0, 10 or 30 J/m2 UV-C; (2) after separation of apoptotic from normal cells on a Percoll gradient; and (3) after irradiation with UV-C plus perturbation of the apoptotic pathway by treatment with inhibitors of two caspases, ICE and CPP32. The number of cells in apoptosis increased in a dose-dependent manner after UV-C treatment. Centrifugation of irradiated cells on a Percoll gradient increased the collection of apoptotic cells tenfold. The stereotypical apoptotic phenotype, in which cells have deep cytoplasmic blebbing and highly condensed DNA, comprised only a few percent of all apoptosis, and was rarely seen in groups receiving caspase inhibitors. The most common apoptotic phenotype was a rounded cell with large spherical nucleolus and associated DNA. After treatment with UV-C plus inhibitors the apoptotic index was decreased by about 30% compared to UV-C radiation alone. These apoptotic cells had dark spherical cytoplasm with small blebs, greatly increased numbers of cytoplasmic ribosomes, abundant nucleolar material with a large separate granular component, and chromatin condensed at the nuclear membrane. Using the technique of spectral imaging, it was found that the spectrum obtained from the granular component of the nucleolus, which was elevated in apoptotic cells treated with UV-C plus inhibitors, was similar to the dense accumulation of ribosomes in the apoptotic cytoplasm. The data indicate that spectral imaging may be a useful tool for identifying and characterizing variations in the apoptotic process, and that the caspase inhibitors used here do not completely abolish UV-C induced apoptosis, but rather alter its incidence and progression.