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

Abstract

Thymus-derived lymphocytes undergo death after gamma-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 2 h postirradiation. DNA fragmentation measured 6 h after irradiation was detected after as little as 0.25 Gy and reached a maximum of 90% with 10 Gy. 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 gamma-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 2.5 Gy 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.

Relationship between clonogenic radiosensitivity, radiation-induced apoptosis and DNA damage/repair in human colon cancer cells

“…They concluded that apoptosis measured within 24 h after irradiation could account for only a fraction of the total clonogenic cell kill, and consequently that mitotic death must be responsible for a significant proportion of the observed cytotoxicity. Several studies suggest that an enhanced apoptotic response results in greater sensitivity (Story et al, 1992;Russell et al, 1995); however, there are also reports showing that interference with apoptosis does not affect radiosensitivity (Agaki et al, 1993). One possible explanation for this is that there may be a poor correlation between apoptotic cell frequency of attached cells and clonogenic cell survival (Guo et al, 1999).…”

Alterations of Extracellular Calcium Elicit Selective Modes of Cell Death and Protease Activation in SH‐SY5Y Human Neuroblastoma Cells

“…An overload of Ca2+, contributes directly to excitotoxicity-induced necrosis (Munir et a]., 1995;Ikeda et al, 1996;Gwag et al, 1997) but can also lead to apoptosis (Furuya et al, 1994;Zhu and Loh, 1995) depending on the cell type. Chelation of extracellular Ca" (Ca",) protects against apoptosis induced by specific agents in thymocytes (McConkey et al, 1989;Story …”

T Cells Deficient in Inositol 1,4,5-Trisphosphate Receptor Are Resistant to Apoptosis

“…Pharmacological elevation of [Ca 2ϩ ] i with calcium ionophores (56) or the endoplasmic reticulum Ca 2ϩ -ATPase inhibitor thapsigargin can initiate apoptosis (18). The antideath protein Bcl-2 reduces calcium release from the endoplasmic reticulum via an unknown mechanism (26), and chelation of intracellular calcium inhibits apoptosis (32,47). Thus, it would appear that elevation of [Ca 2ϩ ] i is a generalized trigger for apoptosis.…”

“…Increases in [Ca 2ϩ ] i have been shown to trigger apoptosis (19), and inducers of apoptosis that lead to increases in [Ca 2ϩ ] i include glucocorticoids (20,32), ␥-irradiation (47), and ␣CD3 (46). Pharmacological elevation of [Ca 2ϩ ] i with calcium ionophores (56) or the endoplasmic reticulum Ca 2ϩ -ATPase inhibitor thapsigargin can initiate apoptosis (18).…”