Different patterns of DNA fragmentation and degradation of nuclear matrix proteins during apoptosis induced by radiation, hyperthermia or etoposide.

Several nuclear matrix proteins are substrates for proteolytic cleavage during apoptosis. Using Western blotting, the temporal patterns of cleavage of three nuclear matrix proteins (lamin B, NUMA and the nucleoporin TPR) were compared in HL60 cells induced to undergo apoptosis after irradiation, heat shock or treatment with etoposide. Flow cytometry was used to compare the kinetics of post-cleavage degradation of lamin B, NUMA and TPR after irradiation, and to correlate DNA fragmentation with protein degradation in cells induced to undergo apoptosis with different agents. During radiation-induced apoptosis, cleavage and subsequent degradation of lamin B, NUMA and TPR occurred with different kinetics. Low-molecular-weight DNA fragmentation occurred subsequent to the initiation of NUMA cleavage, coincided with lamin B cleavage, but occurred before more extensive degradation of lamin B and NUMA. A similar sequence was observed for cells treated with etoposide. However, during heat-induced apoptosis, cleavage of lamin B and NUMA occurred much sooner compared to other agents, with NUMA cleaved into multiple fragments within 15 min after heating. We conclude that the hierarchical sequence and kinetics of degradative events contributing to nuclear disassembly during apoptosis are highly dependent on the inducing agent. Furthermore, the nuclear pore complex, like the nuclear lamina and internal nuclear matrix, is a target for proteolytic cleavage.

Degradation of the nuclear matrix is a common element during radiation-induced apoptosis and necrosis.

Human promyelocytic leukemia (HL60) cells were irradiated with 10 or 50 Gy of X rays and studied for up to 72 h postirradiation to determine the mode of death and assess changes in the nuclear matrix. After 50 Gy irradiation, cells were found to die early, primarily by apoptosis, while cells irradiated with 10 Gy died predominantly by necrosis. Disassembly of the nuclear lamina and degradation of the nuclear matrix protein lamin B occurred in cells undergoing radiation-induced apoptosis or necrosis. However, using Western blotting and a recently developed flow cytometry assay to detect changes in nuclear matrix protein content, we found that the kinetics and mechanisms of disassembly of the nuclear lamina are different for each mode of cell death. During radiation-induced apoptosis, cleavage and degradation of lamin B to a approximately 28-kDa fragment was detected in most cells within 4-12 h after irradiation. Measurements of dual-labeled apoptotic cells revealed that nonrandom DNA fragmentation was evident prior to or concomitant with breakdown of the nuclear lamina. Disassembly of the nuclear lamina during radiation-induced necrosis occurred much later (between 30-60 h after irradiation), and a different cleavage pattern of lamin B was observed. Degradation of the nuclear lamina was also inhibited in apoptosis-resistant BCL2-overexpressing HL60 cells exposed to 50 Gy until approximately 48 h after irradiation. These data indicate that breakdown of the nuclear matrix may be a common element in radiation-induced apoptosis and necrosis, but that the mechanisms and temporal patterns of breakdown of the nuclear lamina during apoptosis are distinct from those of necrosis.