In Vitro Models for Biochemical Investigations on Programmed Cell Death

Abstract

Isolated rat thymocytes treated with methylprednisolone (MPS), cultured human synovial (McCoy's) cells exposed to cold shock and human mammary adenocarcinoma (BT-20) cells treated with Tumour Necrosis Factor (TNF), all showed DNA fragmentation and the morphological and biochemical features of cell death characteristic of a process known as apoptosis.

DNA fragmentation in MPS-treated thymocytes and in cold shock-exposed McCoy's cells was preceded by a marked increase in cytosolic free Ca²⁺ concentration resulting either from a sustained influx of Ca²⁺ from the extracellular medium or from the release of Ca²⁺ from intracellular pools. On the other hand, in TNF-treated BT-20 cells, DNA fragmentation was not associated with any early, marked increase in the cytosolic free Ca²⁺ level. In all three models, however, DNA fragmentation was efficiently prevented by intracellular Ca²⁺ chelators, calmodulin inhibitors and activators of protein kinase C (PKC). These results suggest that the activation of common mechanisms involving Ca²⁺ and PKC may be essential for the development of apoptosis.