Kimura C, Zhao Q L, Kondo T, Amatsu M, Fujiwara Y
Department of Radiation Biophysics and Genetics, Kobe University School of Medicine, Japan.
Exp Cell Res. 1998 Mar 15;239(2):411-22. doi: 10.1006/excr.1997.3912.
Ultraviolet light (UV) induced rapid apoptosis of U937 leukemia cells, concurrent with DNA fragmentation and cleavage of poly(ADP-ribose)polymerase (PARP) by activated caspase-3. The in vitro reconstitution of intact HeLa S3 nuclei and apoptotic U937 cytosolic extract (CE) revealed that (i) Ca2+/Mg(2+)-dependent, Zn(2+)-sensitive endonuclease activated in the apoptotic CE induced DNA ladder in HeLa nuclei at pH 6.8-7.4, (ii) activated caspase-3 cleaved PARP in HeLa nuclei, and (iii) when the apoptotic CE was treated with the caspase-3 inhibitor (1 microM Ac-DEVD-CHO) or the caspase-1 inhibitor (10 microM Ac-YVAD-CHO), the former, but not the latter, caused a 50% inhibition of DNA fragmentation and the complete inhibition of PARP cleavage in HeLa nuclei. Similarly, Ac-DEVD-CHO (100 microM) inhibited apoptosis and DNA ladder by 50% and PARP cleavage completely in UV-irradiated U937 cells, but Ac-YVAD-CHO (100 microM) did not. Thus, UV-induced apoptosis of U937 cells involves the Ca2+/Mg(2+)-dependent endonuclease pathway and the caspase-3-PARP cleavage-Ca2+/Mg(2+)-dependent endonuclease pathway. The former pathway produced directly 50% of apoptotic DNA ladder, and the latter involved activated caspase-3 and PARP cleavage, followed by formation of the remaining 50% DNA ladder by the activated endonuclease. In UV-irradiated B-cell lines, further, p53-dependent increase of Bax resulted in a greater caspase-3 activation compared to its absence. However, UV-induced activation of JNK1 and p38 was not affected by the caspase-1 and -3 inhibitors in U937 cells, so that caspases-1 and -3 do not function upstream of JNK1 and p38.
紫外线（UV）可诱导U937白血病细胞快速凋亡，同时伴有DNA片段化以及活化的caspase-3对聚（ADP-核糖）聚合酶（PARP）的切割。完整的HeLa S3细胞核与凋亡的U937细胞胞质提取物（CE）的体外重组显示：（i）凋亡的CE中活化的Ca2+/Mg(2+)依赖性、Zn(2+)敏感的核酸内切酶在pH 6.8 - 7.4条件下可诱导HeLa细胞核内出现DNA梯状条带；（ii）活化的caspase-3可切割HeLa细胞核内的PARP；（iii）当凋亡的CE用caspase-3抑制剂（1 μM Ac-DEVD-CHO）或caspase-1抑制剂（10 μM Ac-YVAD-CHO）处理时，前者而非后者可导致HeLa细胞核内DNA片段化受到50%的抑制以及PARP切割完全被抑制。同样，Ac-DEVD-CHO（100 μM）可使紫外线照射的U937细胞凋亡和DNA梯状条带受到50%的抑制，并完全抑制PARP切割，但Ac-YVAD-CHO（100 μM）则无此作用。因此，紫外线诱导的U937细胞凋亡涉及Ca2+/Mg(2+)依赖性核酸内切酶途径以及caspase-3 - PARP切割 - Ca2+/Mg(2+)依赖性核酸内切酶途径。前一途径直接产生50%的凋亡DNA梯状条带，后一途径涉及活化的caspase-3和PARP切割，随后由活化的核酸内切酶形成剩余50%的DNA梯状条带。此外，在紫外线照射的B细胞系中，与无p53时相比，p53依赖性的Bax增加导致caspase-3活化程度更高。然而，紫外线诱导的JNK1和p38活化不受U937细胞中caspase-1和 - 3抑制剂的影响，因此caspase-1和 - 3不在JNK1和p38的上游发挥作用。
