A Role for Calcium in Regulating Apoptosis in Rat Thymocytes Irradiated in Vitro

Abstract

Thymus-derived lymphocytes undergo death after γ-irradiation via a pathway termed apoptosis, or programmed cell death. An early step in this pathway is the production of nucleosome-sized fragments of DNA. DNA fragmentation was used as the endpoint in these investigations to examine apoptosis in lymphocytes extracted from the rat thymus and irradiated in vitro. In unirradiated thymocytes the level of DNA fragmentation rose to 15% by the first hour of culture, where it remained approximately constant until the fifth hour. In contrast, thymocytes irradiated with a dose of 2·5 Gy exhibited a large and dramatic increase in DNA fragmentation beginning 2h postirradiation. DNA fragmentation measured 6h after irradiation was detected after as little as 0·25 Gy and reached a maximum of 90% with 10Gy. Metabolic control of DNA fragmentation after irradiation was evidenced by the suppression of DNA fragmentation when thymocytes were incubated with cyclohexamide or actinomycin D. When γ-irradiated thymocytes were incubated with the Ca²⁺ chelator EGTA, DNA fragmentation was reduced significantly. BAPTA-AM, a highly specific intracellular Ca²⁺ chelator, essentially eliminated DNA fragmentation in cells irradiated with 2·5Gy and, unlike EGTA, eliminated the background level of fragmentation in unirradiated samples. Therefore, our data are consistent with the possibility that Ca²⁺ serves as a second messenger to induce DNA fragmentation in irradiated thymocytes, suggesting a common pathway for cells prompted to enter apoptosis from seemingly dissimilar interval events.