Ibrado A M, Huang Y, Fang G, Liu L, Bhalla K
Department of Medicine, Winship Cancer Center, Emory University School of Medicine, Atlanta, Georgia 30322, USA.
Cancer Res. 1996 Oct 15;56(20):4743-8.
Ara-C has been shown to induce apoptosis of human acute myelogenous leukemia HL-60 cells. The DNA repair enzyme poly(ADP-ribose) polymerase (PARP) is known to be degraded during apoptosis. PARP as a substrate is cleaved by the Yama protease, encoded by the CPP32beta/Yama gene. Yama belongs to the interleukin 1beta converting enzyme/ced-3 family of cysteine proteases that are activated as a cascade, producing proteolytic cleavage of specific substrates that results in the morphological and biochemical features of apoptosis. In the present studies, we determined the effect of high intracellular levels of the antiapoptosis Bcl-2 or Bcl-xL protein on Yama protease activation and PARP degradation during Ara-C-induced apoptosis. For this, we utilized HL-60/Bcl-2, HL-60/Bcl-xL, or control HL-60/neo cells, which were created by transfection of the cDNA of the bcl-2, bcl-xL, or the neomycin-resistant genes, respectively. As compared to HL-60/neo, HL-60/Bcl-2 and HL-60/Bcl-xL cells have 5-fold greater expression of Bcl-2 and Bcl-xL, respectively. However, these cell lines have similar levels of p32Yama and PARP. Treatment with 10 or 100 microM Ara-C for 4 h produced DNA fragmentation and morphological features of apoptosis in HL-60/neo cells. This was associated with the cleavage and activation of p32Yama and PARP degradation but not with the induction of Yama mRNA. In contrast, in HL-60/Bcl-2 and HL-60/ Bcl-xL cells, Ara-C-induced p32Yama activation by its cleavage, PARP degradation and apoptosis were significantly inhibited. High Bcl-2 and Bcl-xL levels in these cells also inhibited Yama protease activity, PARP degradation, and apoptosis due to clinically relevant concentrations of etoposide and mitoxantrone. These results suggest that the activation of the Yama protease and PARP degradation are involved in Ara-C-, etoposide-, or mitoxantrone-induced apoptosis. In addition, they suggest that Bcl-2 and Bcl-xL antagonize drug-induced apoptosis by a mechanism that interferes in the activity of a key cysteine protease that is involved in the execution of apoptosis.
阿糖胞苷已被证明可诱导人急性髓性白血病HL - 60细胞凋亡。已知DNA修复酶聚（ADP - 核糖）聚合酶（PARP）在凋亡过程中会被降解。PARP作为底物被由CPP32β/Yama基因编码的Yama蛋白酶切割。Yama属于半胱氨酸蛋白酶的白细胞介素1β转换酶/ced - 3家族，该家族作为一个级联被激活，产生特定底物的蛋白水解切割，导致凋亡的形态学和生化特征。在本研究中，我们确定了细胞内高水平的抗凋亡蛋白Bcl - 2或Bcl - xL对阿糖胞苷诱导凋亡过程中Yama蛋白酶激活和PARP降解的影响。为此，我们使用了HL - 60/Bcl - 2、HL - 60/Bcl - xL或对照HL - 60/neo细胞，它们分别是通过转染bcl - 2、bcl - xL或新霉素抗性基因的cDNA构建的。与HL - 60/neo相比，HL - 60/Bcl - 2和HL - 60/Bcl - xL细胞中Bcl - 2和Bcl - xL的表达分别高5倍。然而，这些细胞系中p32Yama和PARP的水平相似。用10或100微摩尔/升阿糖胞苷处理4小时可使HL - 60/neo细胞产生DNA片段化和凋亡的形态学特征。这与p32Yama的切割和激活以及PARP降解相关，但与Yama mRNA的诱导无关。相反，在HL - 60/Bcl - 2和HL - 60/Bcl - xL细胞中，阿糖胞苷诱导的p32Yama通过其切割的激活、PARP降解和凋亡均被显著抑制。这些细胞中高表达的Bcl - 2和Bcl - xL也抑制了依托泊苷和米托蒽醌临床相关浓度诱导的Yama蛋白酶活性、PARP降解和凋亡。这些结果表明，Yama蛋白酶的激活和PARP降解参与了阿糖胞苷、依托泊苷或米托蒽醌诱导的凋亡。此外，它们表明Bcl - 2和Bcl - xL通过干扰参与凋亡执行的关键半胱氨酸蛋白酶的活性来拮抗药物诱导的凋亡。
