Hormone-induced cell death. Purification ad properties of thymocytes undergoing apoptosis after glucocorticoid treatment

Abstract

A high proportion of cortical thymocytes obtained from suckling rats undergo apoptosis on exposure in vitro to the glucocorticoid methylprednisolone. The apoptotic cells can be separated from apparently normal thymocytes by isopyknic centrifugation on Percoll gradients. This experimental system provides homogeneous populations of apoptotic cells and thus permits a more incisive study of this physiologic mode of cell death than has been hitherto possible. It is shown that apoptosis involves a sharp but transient increase in buoyant density, concomitant with the appearance of characteristic morphologic changes in nucleus and cytoplasm. More than 80% fo the chromatin of apoptotic cells has a molecular weight sufficiently low to resist sedimentation at 27,000g and consists of short oligonucleosome chains, apparently as a result of endogenous endonuclease activity. By contrast, the chromatin of thymocytes that retain normal density (whether treated or control) is of high molecular weight. Apoptotic cells, unlike those of normal density, show little or no incorporation of nucleosides and amino acids into macromolecules. These investigators were unable to detect populations of cells intermediate between apoptotic and normal in buoyant density, morphologic characteristics, chromatin cleavage, or leucine incorporation, but evidence is presented suggesting that thymidine and uridine incorporation may fall prior to development of apoptosis.